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IDENTIFICATION AND EXPRESSION OF INVERTASE GENES IN POPULUS

By

PHILIP N. BOCOCK

A DISSERTATION PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT

OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY

UNIVERSITY OF FLORIDA

2007

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© 2007 Philip N Bocock

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To my wife, Traci

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ACKNOWLEDGMENTS

I would like to thank my advisor, Dr. John Davis, who has helped to deepen my knowledge

of molecular biology and has taught me how to ask questions. Without his endless help, I would

not be here. I would also like to acknowledge my committee members: Drs. Curt Hannah, Karen

Koch, Tim Martin and Gary Peter for their valuable advice throughout this undertaking. Also, I

would especially like to thank Drs. Li-Fen Huang and Karen Koch whose collaboration have

made this project possible. I am also grateful to other faculty, staff, and graduate students for

their help and encouragement, especially Alison Morse, Kathy Smith, Chris Dervinis, Mike

Reed, Gogce Kayihan, Gustavo Ramirez, Rocio Diaz, John Mayfield and Diego Fajardo.

Without the support of my family this endeavor would have never been accomplished. I

must especially thank my wife, Traci, and our two children, Isaiah and Edward, for their love,

encouragement and support that have gotten me through this education.

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TABLE OF CONTENTS page

ACKNOWLEDGMENTS ...............................................................................................................4

LIST OF TABLES...........................................................................................................................8

LIST OF FIGURES .........................................................................................................................9

ABSTRACT...................................................................................................................................11

CHAPTER

1 INTRODUCTION AND LITERATURE REVIEW ..............................................................13

Poplar as a Model ...................................................................................................................13 Carbon Transport and Utilization ...........................................................................................13 Sucrose....................................................................................................................................14 Sucrose Cleaving Enzymes.....................................................................................................14 Acid Invertase.........................................................................................................................15

Proteinaceous Inhibitors of Acid Invertase .....................................................................16 Structure of Acid Invertases ............................................................................................18

Neutral/Alkaline Invertase (Cytosolic Invertase) ...................................................................19 Regulation of Invertase:..........................................................................................................19

Sugars ..............................................................................................................................19 Hormones ........................................................................................................................20 Wounding and Pathogens ................................................................................................20 Environmental Stimuli.....................................................................................................21

Invertase Investigations in Transgenic Plants.........................................................................22 Research Objectives................................................................................................................23

2 EVOLUTION AND DIVERSITY OF INVERTASE GENES IN Populus trichocarpa .......27

Introduction.............................................................................................................................27 Materials and Methods ...........................................................................................................29

Plant Material ..................................................................................................................29 BLAST Searches and DNA Annotation..........................................................................30 Construction of Sequence Similarity Trees.....................................................................30 Identification of Gene Duplication..................................................................................30 Isolation of RNA and RT-PCR Assay.............................................................................31 Quantitative RT-PCR ......................................................................................................31 Genomic DNA Isolation..................................................................................................32 Microarray Design and Analysis .....................................................................................32

Results.....................................................................................................................................33 Identification of Poplar Invertase Genes .........................................................................33 Structure of the Poplar Invertase Genes ..........................................................................34 Expression of the Poplar Invertase Gene Family ............................................................35

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Evolutionary Development of the Poplar Invertase Family ............................................35 Putative Evidence for Origins of PtVIN1 ........................................................................39

Discussion...............................................................................................................................39

3 RECIPROCAL SUGAR REGULATION IS CONSERVED AMONG VACUOLAR INVERTASAES OF POPLAR, ARABIDOPSIS, MAIZE AND RICE .................................56

Introduction.............................................................................................................................56 Materials and Methods ...........................................................................................................58

Plant Material ..................................................................................................................58 Sequence Alignments and Similarity Trees ....................................................................59 RNA Extraction ...............................................................................................................59 Quantitative RT-PCR in Poplar and Pine........................................................................59 Quantitative RT-PCR in Arabidopsis ..............................................................................60 Sugar Treatments for Poplar, Arabidopsis, Pine and Rice ..............................................61 Light-Dark Treatments for Poplar, Pine and Arabidopsis...............................................61

Results.....................................................................................................................................62 Predicting Gene Orthology Using Protein Sequence Similarity .....................................62 A Chromosome Duplication Event is Responsible for the Two Member VIN Family

in Poplar and Arabidopsis ............................................................................................64 Sugar Response Demonstrates Conservation of Gene Function in VINs ........................65 Reciprocal Response in Light..........................................................................................66

Discussion...............................................................................................................................67

4 OVEREXPRESSION OF YEAST INVERTASE IN POPLAR ............................................76

Introduction.............................................................................................................................76 Materials and Methods ...........................................................................................................78

Plant Material, Transgenesis, and Growth Conditions ....................................................78 Vector Construction.........................................................................................................78 Construction of Similarity Trees .....................................................................................79 Isolation of RNA, Generation of cDNA and Real-Time PCR Assay..............................79 Genomic DNA Isolation and PCR Amplification ...........................................................80 Metabolic Profiling: Extraction, Separation and Identification ......................................81 Construction and Experimental Design of Grafts ...........................................................81 Measurement of Photosynthesis and Respiration............................................................82 Protein Extraction............................................................................................................83 Total Invertase Activity Assay ........................................................................................84 Detection of Invertase Activity in Native Polyacrylamide Gels .....................................84 Sugar and Starch Determination......................................................................................84

Results and Discussion ...........................................................................................................85 Construction of Overexpressing Yeast Invertase Vectors...............................................85 Expression of SUC2 in Poplar.........................................................................................86 Yeast Invertase Active in Cytosol ...................................................................................87 Metabolic Profiling Reveals Alterations in CwSUC2 and VacSUC2 Transgenic

Lines.............................................................................................................................88

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Metabolic Profiling Reveals Alterations in Sugar Accumulation in CytSUC2 Transgenic Lines..........................................................................................................89

Whole-Plant Phenotypes are not Apparent......................................................................90 Concluding Remarks .......................................................................................................90

5 CONCLUSIONS ..................................................................................................................102

APPENDIX

A REPRESSION OF INVERTASE IN POPULUS .................................................................106

Introduction...........................................................................................................................106 Materials and Methods .........................................................................................................107

Plant Material, Transgenesis, and Growth Conditions ..................................................107 Vector Construction.......................................................................................................107 Genomic DNA Isolation and PCR Amplification .........................................................108

Results...................................................................................................................................109 Construction of RNAi Vector........................................................................................109 Insertion of Target Gene Sequence ...............................................................................109 Further Work .................................................................................................................110

B REGULATION OF INVERTASE: A"SUITE" OF TRANSCRIPTIONAL AND POST-TRANSCRIPTIONAL MECHANISMS..............................................................................115

Abstract.................................................................................................................................115 Introduction...........................................................................................................................116 Compartmentalization of Vacuolar Invertases in Precursor Protease Vesicles (PPV).........117 Wall-Associated Kinase (WAK) ..........................................................................................119 Other Kinases Affecting Invertases ......................................................................................120 Differential Sugar Regulation of Invertases .........................................................................121 RNA Turn Over and DST.....................................................................................................124 Invertase Inhibitors ...............................................................................................................125 Summary...............................................................................................................................127 Acknowledgements...............................................................................................................128

LIST OF REFERENCES.............................................................................................................131

BIOGRAPHICAL SKETCH .......................................................................................................149

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LIST OF TABLES

Table page 2-1 Nomenclature and chromosomal location of 24 poplar invertase genes ...............................44

2-2 Primers used in RT-qPCR and RT-PCR................................................................................49

3-1 Percent similarity of predicted VINs from poplar, Arabidopsis, rice and maize ..................71

4-1 Metabolites altered in CwSUC2 and VacSUC2 overexpressing plants .................................99

4-2 Metabolites altered in CytSUC2 overexpressing plants ......................................................100

4-3 Summary of phenotypic experiments performed. ...............................................................101

A-1 Primer sequences used for ~200 bp target sequence cloning. ............................................113

A-2 Summary of RNAi constructs made ...................................................................................114

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LIST OF FIGURES

Figure page 1-1 Sucrose synthesis, transport and metabolism in source and sink cells..................................24

1-2 Protein inhibitors of cell wall invertases share strong sequence similarity with inhibitors of pectin methylesterase ....................................................................................25

1-3 Representation of cell wall and vacuolar isoforms of carrot invertase..................................26

2-1 Exon-intron structures of predicted invertase genes..............................................................45

2-2 Invertase amino acid similarity trees .....................................................................................46

2-3 Expression of poplar invertase genes ....................................................................................47

2-4 Evolutionary development of the poplar acid invertase family.............................................48

2-5 Amino acid alignment of poplar invertases...........................................................................55

3-1 Protein similarity tree of VINs ..............................................................................................72

3-2 Phylogenetic representation of relevant plant species...........................................................73

3-3 Conservation of specific VIN isoform transcript repression under sugar treatments across taxa..........................................................................................................................74

3-4 Conservation of reciprocal regulation of VIN transcript under dark and light treatments across taxa..........................................................................................................................75

4-1 Schematic representation of yeast invertase overexpressing constructs targeted to the cytosol, cell wall and vacuole ............................................................................................93

4-2 S. cerevisiae SUC2 in relationship to other plant invertases.................................................95

4-3 Relative expression of double 35S driven SUC2 in root, stem and leaf................................96

4-4 Relative expression of SUC2 transcript in overexpressing transgenic events in leaf............97

4-5 CytSUC2 transgenic plants display increased invertase activity due to presence of transgene ............................................................................................................................98

A-1 Schematic representation of pCAPT vector used for RNAi silencing ...............................111

A-2 Schematic representation of RNAi directed constructs ......................................................112

B-1 Recent additions to mechanisms controlling invertases .....................................................129

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B-2 Protein inhibitors of cell wall invertases share strong sequence similarity with inhibitors of pectin methylesterase ..................................................................................130

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Abstract of Dissertation Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy

IDENTIFICATION AND EXPRESSION OF INVERTASE GENES IN POPULUS

By

Philip N. Bocock

December 2007

Chair: John M. Davis Major: Plant Molecular and Cellular Biology

Invertase (EC 3.2.1.26) plays a key role in carbon utilization as it catalyzes the irreversible

hydrolysis of sucrose into glucose and fructose. These sugars can act as both metabolic fuel and

as signaling compounds directly affecting resource allocation in the plant and indirectly

influencing the expression of genes responsive to shifts in hexose and sucrose availability. The

invertase family in plants is composed of two sub-families thought to have distinct evolutionary

origins and can be distinguished by their pH optima for activity: acid invertases and

neutral/alkaline invertases. The acid invertases apparently originated in eubacteria and are

targeted to the cell wall and vacuole, while neutral/alkaline invertases apparently originated in

cyanobacteria and function in the cytosol. The recently sequenced genome of Populus

trichocharpa (Torr. & Gray) allowed us to identify the genes encoding invertase in this woody

perennial. Here we describe the identification of eight acid invertase genes; three of which

belong to the vacuolar targeted group (PtVIN1-3), and five of which belong to the cell wall

targeted group (PtCIN1-5). Similarly, we report the identification of 16 neutral/alkaline invertase

genes (PtNIN1-16). Expression analyses using whole genome microarrays and RT-PCR reveal

evidence for expression of all invertase family members. An examination of the microsyntenic

regions surrounding the poplar invertase genes reveals extensive colinearity with Arabidopsis

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invertases. We also find evidence for expression of a novel intronless vacuolar invertase

(PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the

genome. To our knowledge, this is the first intronless invertase found in plants. The response of

two poplar vacuolar invertases (PtVIN2 and -3) to exogenous sugar treatments was examined and

compared to those of the Arabidopsis, maize and rice vacuolar invertase orthologs. We found

that PtVIN2 and -3 exhibit a reciprocal response to both light and exogenous sugar treatments

whereby PtVIN2 expression is repressed under both light and high sugar conditions while

PtVIN3 is induced under the same conditions. This reciprocal response has been previously

documented in other plant systems including Arabidopsis, maize and rice. An examination of the

microsyntenic chromosomal regions containing vacuolar invertase reveals extensive colinearity

between Arabidopsis and poplar, but does not include rice and maize. The conserved colinear

structure of the chromosomal segments containing the vacuolar invertases in Arabidopsis and

poplar, coupled with the conserved reciprocal responses of these vacuolar invertases to sugar in

Arabidopsis, poplar, maize and rice, has led to the hypothesis that the reciprocal nature of this

sugar response arose in an ancient genome duplication event that occurred prior to the monocot

and eudicot divergence on the evolutionary tree. To better understand the role of invertase in

sucrose export and sink development, yeast invertase (SUC2) was ectopically expressed in a

Populus tremula x P. alba hybrid. Despite the efficacy of this transgenic approach in other plant

systems resulting in whole-plant shifts in carbon allocation and dramatic changes in sugar

accumulation; transgenic poplars showed no whole-plant carbon allocation shifts. Metabolic

analyses revealed that while most of the transgenic poplar lines did not exhibit the expected

alterations in sugar accumulation, intermediates in the glyoxylate and the tricarboxylic acid

cycles did fluctuate relative to the non-transgenic controls.

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CHAPTER 1 INTRODUCTION AND LITERATURE REVIEW

Current population growth combined with increasing development and urbanization

worldwide is putting strain on the terrestrial ecosystem’s main carbon sink, forests. One way to

combat this increasing demand of decreasing forest resources is to manipulate carbon

sequestration patterns in trees in order to direct carbon allocation into the most desirable organs

such as stems, to meet industrial demands, or roots to help increase long term carbon storage

capacity in the soil. Unfortunately, these mechanisms are not well understood. Carbon

sequestration is not part of a single pathway, but a culmination of many pathways dealing with

carbon absorption through photosynthesis, carbon loss through respiration, carbon transport

through the vascular system, carbon partitioning within a cell, and carbon allocation into long-

term storage compounds such as lignin.

Poplar as a Model

The perennial tree, poplar, has emerged as a model species for physiology and genetics

research. Poplar is amenable to transformation, can be clonally propagated and has a haploid

genome size of 480 million base pairs which is roughly four times larger than Arabidopsis, has

been sequenced and encodes approximately 45,000 genes (Bradshaw and Stettler, 1995;

Bradshaw et al., 2000; Tuskan et al., 2006). Poplar also is an economically important tree on the

global market with interest from industry on its improvement.

Carbon Transport and Utilization

Plants utilize carbon by precisely partitioning the reduced carbon obtained through

photosynthesis into different locations within the cell and subsequently allocating it throughout

the plant (Figure 1-1). Photosynthesis reduces carbon dioxide into sugars that can be ushered into

glycolysis and the tricarboxylic acid cycle for the production of ATP and NADH, or the sugars

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can be used as a source of carbon for the synthesis of primary and secondary metabolites

essential for the growth and development of the plant. Alternatively, these sugars can be

converted into starches, triacyl glycerides or polypeptides for long-term storage (Sturm, 1999).

These same sugars are also used to synthesize sucrose, the primary sugar used for carbon

transport in higher plants. Sucrose can be transiently stored in the vacuole and then exported to

the non-photosynthesizing sink tissues via the phloem. In source cells, sucrose is synthesized in

the cytosol, stored for later use in the vacuole, travels to neighboring cells through

plasmodesmata, and eventually enters the phloem via plasmodesmata or from the surrounding

apoplast (Turgeon and Hepler, 1989; Grusak et al., 1996).

Sucrose

Sucrose and its glucose and fructose cleavage products are primary molecules in

carbohydrate translocation, sugar signaling, and osmotic maintenance. Sucrose is a non-reducing

disaccharide composed of an α1-β2 linked glucose and fructose. The cleavage of a single sucrose

molecule in solution results in two molecules of hexoses thereby doubling the osmotic potential

of the solution. By compartmentalizing sucrose and/or the hexoses in various cellular

compartments, osmotic gradients are realized, providing the basis for short-distance transport

from cell to cell, as well as long-distance transport from organ to organ via the phloem as first

proposed by Münch (1930). Sucrose and its component hexoses also provide sugar signals that

trigger numerous biological pathways playing roles in cell division, expansion, differentiation

and maturation (Koch, 2004).

Sucrose Cleaving Enzymes

Plants possess two enzymes that have the capability of cleaving sucrose: sucrose synthase

(EC 2.4.1.13) and invertase (EC 3.2.1.26). Sucrose synthase is a glycosyl transferase catalyzing a

reversible reaction converting sucrose into UDP-glucose and fructose in the presence of UDP.

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Invertase, in contrast, hydrolyzes sucrose into glucose and fructose in an irreversible reaction.

The free energy of these two reactions are –4.18 KJ and –27.6 KJ, respectively (Ap Rees, 1984).

The invertase family can be grouped according to two properties: cellular location and pH

optima for activity (Winter and Huber, 2000). The first group, known as the acid invertases, has

a pH optimum of between 4.5 and 5.0 and consists of an insoluble, extracellular, cell wall-bound

form and a soluble form located in the lumen of the vacuole (Haouazine-Takvorian et al., 1997;

Sherson et al., 2003). The second group, known as the neutral/alkaline invertases, has a pH

optimum of about 7.0-7.8. The neutral/alkaline invertases are entirely soluble and appear to be

located in the cytosol (Chen and Black, 1992; Van den Ende and Van Laere, 1995).

Acid Invertase

Acid invertases are β-fructofuranosidases that hydrolyze not only sucrose, but also other β-

fructose containing oligosaccharides such as raffinose and stachyose. The acid invertase Km for

sucrose is in the low mM range and activity can be inhibited by Tris buffer, heavy metal ions

such as Hg2+ and Ag2+, as well as other divalent cations such as Mg2+, Ca2+, and Zn2+ (Krausgrill

et al., 1996; Tymowska-Lalanne and Kreis, 1998b).

Acid invertases have also been shown in a variety of species to be glycosylated. Some

examples are in radish (Faye and Ghorbel, 1983), maize (Doehlert and Felker, 1987), carrot

(Lauriere et al., 1988; Stommel and Simon, 1990; Unger et al., 1992), and tobacco (Weil and

Rausch, 1990). In yeast, there are two isoforms of acid invertase that are encoded by a single

gene, but arise from differential splicing of exons. One isoform is glycosylated and targeted to

the extracellular space, while the other is non-glycosylated and is located in the cytosol. This

glycosylation seems to have no effect on the enzymatic properties of acid invertase (Faye et al.,

1981), but is instead required for its transport to the secretory pathway where it is directed to the

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apoplastic and vacuolar spaces (Carlson and Botstein, 1982; Perlman et al., 1982; Bergh et al.,

1987). In contrast to yeast, the invertase isoforms in plants are encoded by different genes.

Invertase can range in molecular mass from a 48 kD monomeric form found in radish

(Faye et al., 1981) to a 450 kD oligomeric form found in lily pollen (Singh and Knox, 1984),

however, most molecular masses are around 60 kD. Analyses of some invertases under

denaturing conditions on SDS gels show the presence of proteolytic fragments. A 70 kD

vacuolar invertase monomer from mung bean hypocotyls was processed into a 30 kD N-terminal

fragment and a 38 kD C-terminal fragment (Arai et al., 1991). In carrot, a 68 kD monomer of

vacuolar invertase was processed into an N-terminal fragment of 43 kD and a C-terminal

fragment of 25 kD (Unger et al., 1992; Unger et al., 1994). The role of this fragmentation is

unknown, but under native conditions these fragments are tightly associated.

Proteinaceous Inhibitors of Acid Invertase

Cell wall and vacuolar invertase activity can be regulated by a family of proteinaceous

inhibitors known as cell wall inhibitor of fructosidase (CIF) and vacuolar inhibitor of

fructosidase (VIF), or collectively as C/VIF (Rausch and Greiner, 2004). Although CIF are cell

wall invertase inhibitors, they are broadly active against both cell wall and vacuolar invertases.

In contrast, VIF inhibition is specific to vacuolar invertases. Neither of the inhibitors affect

fungal invertases indicating a minimal role for these interactions in pathogen defense (Greiner et

al., 1998; Greiner et al., 1999; Link et al., 2004). The C/VIF related protein family is not highly

conserved, and in Arabidopsis, sequence identities range from roughly 20 % to 40 % for

approximately 14 family members (Rausch and Greiner, 2004). The C/VIF related protein family

also contains pectin methylesterase inhibitors (PMEI). PMEI are indistinguishable from the

C/VIF by sequence alone and retain nearly identical structures to the C/VIF (Giovane et al.,

2004; Hothorn et al., 2004a; Hothorn et al., 2004b; Di Matteo et al., 2005).

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Important implications arise from the capacity of these related proteinaceous inhibitors to

distinguish between their targets. Arabidopsis has only eight putative acid invertases (six cell

wall and two vacuolar), but more than 60 pectin methylesterase (PME)-related genes based on

sequence similarity (Micheli, 2001; Sherson et al., 2003; Rausch and Greiner, 2004; De Coninck

et al., 2005). X-ray crystollagraphy has revealed that CIF is conformationally stable over a broad

pH and temperature range, however, the invertase/inhibitor complex is only stable at an acidic

pH. This indicates that binding is determined not by conformational changes, but rather by pH-

induced changes at the interface of the invertase/inhibitor complex (Hothorn et al., 2004a;

Hothorn and Scheffzek, 2006). In contrast, the highly similar structure of the PMEI was found to

undergo large structural rearrangements (Figure 1-2) under these same conditions suggesting

PMEI uses a different mode of binding than does the CIF (Hothorn et al., 2004b). PMEI contains

a flexible α-hairpin that is important both in dimer formation and in binding of PME (Hothorn et

al., 2004b). Chimeral domain swap experiments of the α-hairpin domain between PMEI and CIF

indicate that this domain of PMEI is necessary and sufficient for activity against PME; however,

the corresponding α-hairpin in NtCIF is not sufficient for invertase inhibition (Hothorn et al.,

2004b). The approximately 28 amino acid residues encoding this α-hairpin may be key in

distinguishing these two classes of inhibitors (Hothorn et al., 2004b).

Analysis of PMEI and CIF crystal structures not only clarifies interactions between these

inhibitors and their targets, but also aids our understanding of how distinct functions can arise for

proteins sharing very similar structures and ancestry. It is worth noting, however, that the

crystallographic analyses were performed on the cell wall invertase inhibitor and thus do not

explain the apparently narrower substrate specificity of the vacuolar invertase inhibitor. These

data indicate that the VIF may use a different mode of action than the CIF. It is also of interest

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that two proteins in Arabidopsis previously annotated based on sequence similarity as cell wall

invertases (AtcwINV3&6), may actually be fructan exohydrolases (FEH). FEH protein

sequences are nearly identical to those of demonstrated acid invertase family members, yet the

recombinant enzymes are completely inactive against sucrose (De Coninck et al., 2005). It has

yet to be determined if the C/VIF family can distinguish between FEH and the known invertase

and PME substrates. Given the level of sequence diversity within the inhibitor family it seems

likely that biochemical and structural analyses will be required to resolve these questions.

Structure of Acid Invertases

Acid invertases are initially synthesized as pre-proteins containing a leader sequence that is

cleaved upon entry into the secretory pathway, the mature peptide sequence, and in the case of

the vacuolar invertases, a short C-terminal extension (Figure 1-3). The leader sequence consists

of two parts: a signal peptide and an N-terminal extension of unknown function, but is thought to

play a role in protein folding, targeting, or activity (Sturm, 1999). The signal peptide is required

for entry into the endoplasmic reticulum and from there to the apoplast (Blobel, 1980) or to the

vacuole if the required vacuolar targeting domain is present (Neuhaus and Rogers, 1998). The

vacuolar targeting domain is thought to be located either in the C-terminal extension or in the N-

terminal propeptide which is longer in vacuolar invertases than in cell wall invertases.

Plant acid invertases have two common features in their sequences. Towards the N-teminal

end of the protein is the β-fructosidase domain, NDPN, which is usually encoded by a mini-exon

of only 9 nucleotides. Towards the C-terminal end of the protein is another conserved sequence

consisting of the amino acids WECXDF. In potato, Bournay (1996) observed that under cold

stress, the mini-exon of the potato pCD111 cell wall invertase encoding the NDPN domain is

skipped in an alternative splicing event.

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Neutral/Alkaline Invertase (Cytosolic Invertase)

Neutral/alkaline invertases are located in the cytosol and have a pH optimum in the neutral

to slightly alkaline range. In contrast to the acid invertases, neutral/alkaline invertases are not

glycosylated and hydrolyze only sucrose indicating that they are not fructofuranosidases.

Neutral/alkaline invertase activity is strongly inhibited by its reaction products; however, activity

is not inhibited by heavy metal ions indicating a different catalytic site than that of the acid

invertases. To date, neutral/alkaline invertases have not been found in association with a protein

inhibitor.

Regulation of Invertase

Sugars

Sugars play important roles in plants not only as fuel for metabolism, but they also

generate osmotic pressure and act as signaling molecules for various metabolic pathways. Sugars

have been shown to act as repressors of genes related to sugar acquisition and mobilization as

well as activators of genes related to sugar storage and utilization (Koch, 1996; Rolland et al.,

2002; Halford and Paul, 2003). As one of only two enzymes able to cleave sucrose, invertase is

an important player in sugar signaling. The majority of acid invertases have been shown to be

sugar induced rather than repressed (Roitsch, 1999; Roitsch and Ehness, 2000). This is consistent

with their proposed roles in carbon use versus carbon acquisition (Koch, 1996; Rolland et al.,

2002; Koch, 2004). However, some isoforms of vacuolar invertase have been shown to be

inhibited by sugars. This has been the case in tomato where transcripts for vacuolar invertase

TIV1 were shown to be reduced after treatment with 20 mM glucose (Godt and Roitsch, 1997b).

In maize, transcript as well as activity for vacuolar invertase Ivr1 was also shown to be repressed

in the presence of 4 % glucose (Xu et al., 1996).

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Hormones

Phytohormones play a central role in controlling growth, differentiation and development

in plants. As such, these hormones are particularly involved in regulating sink strength (Kuiper,

1993) and carbohydrate partitioning (Brenner and Cheikh, 1995). Invertases have been shown to

be regulated by the full set of phytohormones (Roitsch et al., 2003). In most cases this is related

to an increased demand for carbohydrates as a result of the hormone stimulated growth. In a

promoter study of the Lin5 invertase promoter linked to a reporter gene in tomato, it was shown

that gibberellic acid, auxin, and ABA all increased GUS activity indicating induction of this

isoform by these hormones (Proels et al., 2003). Work has also been done in maize

demonstrating that ABA increases the accumulation of the vacuolar invertase Ivr2 activity

(Trouverie et al., 2003). Brassinosteroids (Goetz et al., 2000), zeatin (Godt and Roitsch, 1997b),

ethylene (Linden et al., 1996) and cytokinin (Ehness and Roitsch, 1997) have all also been

shown to alter transcript accumulation of various invertase isoforms in plants.

Wounding and Pathogens

The wounding of plants is a common occurrence that can result in water loss as well as

pathogen infection. Response to wounding therefore often involves mechanisms intended to

counter osmotic stress as well as pathogen attack (Reymond and Farmer, 1998; Reymond et al.,

2000). As invading organisms alter sugar levels and effectively increase the sink of the tissue

being invaded, increases in invertase activity and expression logically follows. It is often difficult

to distinguish the invertase activity of the invading pathogen from that of the host plant (Ruffner

et al., 1992). In 1990, it was shown that carrot extracellular invertase transcript accumulates

dramatically after bacterial infection in the roots and leaves as well as after mechanical

wounding (Sturm and Chrispeels, 1990a). Results obtained from studying infection by biotrophic

fungi (Fotopoulos et al., 2003), necrotrophic fungi (Benhamou et al., 1991) and viruses (Herbers

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et al., 2000) all show increases in invertase activity. Wounding has been shown in numerous

systems to correlate with increases in invertase activity and transcript level. An increase in either

one or both of these has been demonstrated in carrot (Sturm and Chrispeels, 1990a; Ramloch-

Lorenz et al., 1993), red goosefoot (Ehness and Roitsch, 1997), tomato (Ohyama et al., 1998),

sugar beet (Rosenkranz et al., 2001) and pea (Zhang et al., 1996).

Environmental Stimuli

The partitioning and allocation of photoassimilates can be affected by several

environmental factors such as temperature, gravity, light, wounding, drought and nutrient

availability (Wardlaw, 1990). As would be expected, these stimuli have all been demonstrated to

alter invertase transcript abundance and/or activity levels. Cold temperatures have stimulated

invertase transcripts that were undetectable at higher temperatures in potato tubers (Pressey and

Shaw, 1966) and Jerusalem artichoke tubers (Goupil et al., 1988). In sweet potato (Huang et al.,

1999) and tulip (Balk and de Boer, 1999), cold temperatures were shown to dramatically increase

the activity of invertase. Alternative splicing has also been shown to occur in potato resulting in

the elimination of the β-fructosidase motif (Bournay et al., 1996). Invertase transcripts are also

induced and peak at one hour after gravistimulation (Kaufman et al., 1985; Wu et al., 1993a; Wu

et al., 1993b). Far-red light also increases cell wall invertase activity in radish (Zouaghi and

Rollin, 1976) and wheat (Krishnan et al., 1985). Salinity generally causes a reduction in sink

enzyme activities. These in turn could contribute to observed increases in sucrose of source

leaves and decreases in photosynthesis from both feedback inhibition and sugar-repression of

photosynthetic genes. Experiments in tomato revealed that in a salt sensitive strain, growth and

photosynthesis were not positively correlated as they were in the salt tolerant strain. In this salt

sensitive strain, growth and photosynthesis were both negatively correlated with glucose,

fructose and sucrose accumulation in young and old leaves, suggesting a blockage in their use for

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growth. In the salt tolerant variety, transient increases in these same sugars were accompanied by

increases in acid invertase activity (Balibrea et al., 2000).

Invertase Investigations in Transgenic Plants

Invertase’s irreversible reaction combined with its location in a variety of cellular

compartments makes it a key component in the carbon utilization strategy of the plant and thus

an ideal target for manipulation to understand carbon allocation and partitioning.

Several groups working with tomato (Dickinson et al., 1991), tobacco (Sonnewald et al.,

1991) and potato (Heineke et al., 1992) all saw very similar results after overexpressing the yeast

invertase, SUC2, in the apoplast, cytosol, and vacuole. These invertase overexpressors all had the

common theme of turning source tissues into sink tissues and reducing the sucrose available for

export. All plants demonstrated stunted growth and reduced root formation, including a reduction

in the number of tubers produced in the case of potato. These tubers also had an increase in the

protein to starch ratio. The mature (source) leaves in all three plants accumulated starches in

addition to simple sugars, while in the case of the cytosolic overexpressors, these accumulations

were also seen in the young (sink) leaves. Leaves were curved indicating rapid cell expansion or

division. The plants also demonstrated bleaching and necrotic regions that appeared to be linked

to the source state of the leaf. These lesions began at the leaf margin and moved inward while

also being accompanied by a reduction in photosynthesis.

The cell wall and vacuolar invertases have been repressed in several plant species,

including tomato (Ohyama et al., 1995), potato (Zrenner et al., 1996), and carrot (Tang and

Sturm, 1999). However, the results from these experiments were more varied than those of the

invertase overexpressors, although the theme of increased sucrose content accompanied by a

decrease in hexoses pervaded. In tomato these alterations in sugar accumulations appeared in

both the fruit and leaves. In addition to altered sugar levels, the fruit had elevated rates of

23

ethylene evolution. The wounding induced activity of invertase was also suppressed in the

transgenic plants.

The results from repressing the cell wall and vacuolar invertases in carrot are probably the

most interesting. Growth was shown to be altered at very early stages of development. The

transgenic, cotyledon-stage embryos were still masses of cells while the control plants had

already developed two to three leaves and one primary root. However, when these plantlets were

grown on media supplemented with hexoses, their growth returned to normal. After maturation

and transfer to soil, the plants expressing the cell wall antisense cDNA had a much bushier

appearance and accumulated more sucrose and starch than the controls. The taproot size was also

reduced and contained lower levels of carbohydrates than that of the controls. The dry weight

ratio of leaf to root shifted from 1:3 in the control to 17:1 in the cell wall antisense transgenics.

The vacuolar antisense plants also had increased numbers of leaves, although the tap root

developed normally, yet slightly smaller. The leaf to shoot ratio in these plants was 1.5:1.

Research Objectives

The purpose of this research was to investigate invertase contributions to carbon allocation

and partitioning in a woody perennial. To achieve this goal, the invertase family was identified

and annotated using the sequenced genome of Populus trichocarpa. Expression characteristics of

the invertase family were then examined across multiple tissues as well as through various

exogenous treatments including auxin, nitrogen-supplemented fertilizer and wounding. The

response of two poplar vacuolar invertases (PtVIN2 and -3) to exogenous sugar treatments was

examined and compared to the sugar responses of Arabidopsis, maize and rice. A reverse genetic

approach was also employed by ectopic expression of yeast invertase in transgenic poplar. In

conjunction, RNAi was used to target endogenous poplar invertases for down-regulation.

24

NIN

VIN

CIN

CIN

LIGHT

NIN

VIN

CIN

CIN

NIN

VIN

CIN

CIN

LIGHT

Figure 1-1. Sucrose synthesis, transport and metabolism in source and sink cells as well as invertase localization. This cartoon demonstrates apoplastic phloem loading in the source leaf and apoplastic or symplastic phloem unloading in the sink tissue. In this scheme, sucrose (S) is synthesized in the cytosol from triose-phosphates (TP). Sucrose then enters the apoplast where it is actively transported into the phloem by a sucrose proton symporter. This phloem loading step can be affected by activity levels of the apoplastically located cell wall invertase (CIN) which hydrolyzes sucrose into glucose (G) and fructose (F). Sucrose can be unloaded from the phloem either symplastically or apoplastically. From the apoplast, sucrose can enter the sink cell directly via a sucrose proton symporter, or be hydrolyzed by CIN into glucose and fructose. In the sink cells, sucrose can be cleaved by sucrose synthase (SUSY) into UDP-glucose (UDPG) and fructose, or hydrolyzed by a neutral/alkaline invertase (NIN). Sucrose can also enter the vacuole where it can then be hydrolyzed by a vacuolar invertase (VIN). After phosphorylation by hexokinase (HK), the hexoses can enter respiration. Figure adapted from Rausch et al. (2004).

25

Figure 1-2. Protein inhibitors of cell wall invertases (CIF, cell wall inhibitor of fructosidase) share strong sequence similarity with inhibitors of pectin methylesterase (PMEI, pectin methylesterase inhibitor), but crystal structures indicate important differences in flexibility. The top panel depicts CIF. The oval denotes an α-hairpin thought responsible for binding specificity of CIF and PMEI. The α-hairpin in CIF is rigid at all pHs and temperatures tested. The bottom panel shows PMEI in three different conformations that demonstrate the flexibility of the PMEI α-hairpin. (Hothorn et al., 2004a; Hothorn et al., 2004b; Hothorn and Scheffzek, 2006). Images were constructed by Protein Explorer (Martz, 2002). Figure adapted from Huang et al. (2007).

26

Figure 1-3. Representation of cell wall and vacuolar isoforms of carrot invertase (Sturm, 1999).

27

CHAPTER 2 EVOLUTION AND DIVERSITY OF INVERTASE GENES IN POPULUS TRICHOCARPA

Introduction

Invertase (EC 3.2.1.26), also known as a β-fructofuranosidase, catalyzes the irreversible

hydrolysis of sucrose into glucose and fructose, indicating this enzyme has a key role in carbon

utilization. These sugars are synthesized in photosynthesizing source leaves and transported to

non-photosynthesizing sink tissues. Sucrose is the primary form of sugar transport in most

plants, establishing this disaccharide and its glucose and fructose components as central to plant

growth and development.

The invertase family is composed of two smaller sub-families distinguished by their pH

optima for activity and are thought to have distinct evolutionary origins in plants (Winter and

Huber, 2000). The acid invertase sub-family is targeted to either the cell wall or vacuole

(Haouazine-Takvorian et al., 1997; Sherson et al., 2003) and is believed to have originated from

respiratory eukaryotes and aerobic bacteria (Sturm and Chrispeels, 1990b). The neutral/alkaline

invertases appear to be localized to the cytosol (Chen and Black, 1992; Van den Ende and Van

Laere, 1995) and are found in cyanobacteria, where the family is thought to have originated

(Vargas et al., 2003), green algae and plants. The presence of these two sub-families reflect the

hypothesized origin of green algae and higher plants; the endosymbiotic event in which a

cyanobacteria invaded a non-photosynthetic, respiratory eukaryote (Mereschkowsky, 1905;

Margulis, 1981; Margulis and Sagan, 2003).

Members of the acid invertase sub-family share enzymatic and biochemical properties as

well as sequence similarity. Both vacuolar and cell wall targeted isoforms are β-

fructofuranosidases that can hydrolyze fructose-containing compounds other than sucrose, such

as raffinose and stachyose. Acid invertases are closely related to a class of enzymes known as

28

fructan exohydrolases (FEH). FEHs are difficult to distinguish from acid invertases by sequence

alone and are thought to have evolved from acid invertases even though FEHs exclusively break

down fructans and cannot hydrolyze sucrose (Van den Ende et al., 2002). It was recently

discovered that two previously annotated cell wall invertases from Arabidopsis (AtcwINV3 and

AtcwINV6) may, in fact, be FEHs (De Coninck et al., 2005).

Much less is known about the neutral/alkaline invertase sub-family due to purification

difficulties and low, unstable enzymatic activity (Sturm and Tang, 1999; Roitsch and Gonzalez,

2004). This sub-family likely has a different mode of action than the acid invertase sub-family

since sucrose is the sole substrate and they are not inhibited by heavy metals, elements that

strongly inhibit the acid invertases (Roitsch and Gonzalez, 2004).

An important driver of species origination and diversification is gene duplication.

Duplication makes it possible for a gene to acquire new functions without losing the function of

the progenitor gene (Kramer et al., 1998; Lynch and Conery, 2000; Sankoff, 2001; Becker and

Theissen, 2003; Irish, 2003; Litt and Irish, 2003; Zahn et al., 2005). Gene duplication can occur

in tandem, through the duplication of a chromosomal segment, an entire chromosome, or through

genome duplication (Otto and Whitton, 2000; Wendel, 2000; Adams and Wendel, 2005).

Chromosomal duplications result in conserved, colinear locus order that can be used to infer

ancestry of loci of interest both within and between species (Lynch and Conery, 2000).

Poplar is known to have undergone at least two genome duplication events in its

evolutionary history (Sterck et al., 2005; Tuskan et al., 2006). The first genomic duplication

event occurred prior to the divergence of Arabidopsis and poplar and is known as the “eurosid”

duplication event (Bowers et al., 2003; Blanc and Wolfe, 2004; De Bodt et al., 2005; Zahn et al.,

2005; Tuskan et al., 2006). The second genome duplication event occurred after the divergence

29

of Arabidopsis and poplar but prior to the divergence of the Populus and Salix genera and is

referred to as the “salicoid” event (Tuskan et al., 2006). Arabidopsis is known to have undergone

at least one genomic duplication event after the divergence with poplar (Bowers et al., 2003;

Blanc and Wolfe, 2004; De Bodt et al., 2005; Zahn et al., 2005; Tuskan et al., 2006). By

knowing the timing and location of genomic duplication events, the evolutionary development of

a variety of gene families can be predicted in these species.

The recently released sequence of the poplar genome (Tuskan et al., 2006) opens the door

for systematic analysis of metabolically important gene families in a model tree. Here, I report

the identification of the poplar invertase gene family and show expression data from both

microarray and reverse transcription-polymerase chain reaction (RT-PCR) experiments. I show

that acid and neutral/alkaline invertase genes are regulated in vegetative or floral organs. An

examination of the microsyntenic regions surrounding the poplar invertase genes reveals

extensive colinearity with Arabidopsis invertases and allows orthologous and paralogous

relationships among genes to be inferred.

Materials and Methods

Plant Material

Rooted softwood cuttings of Populus trichocarpa (Torr. & Gray) (genotype “Nisqually-1”)

were planted in 8 L pots and placed in a fan- and pad-cooled greenhouse with natural light

augmented with full spectrum fluorescent lighting during the winter to give a day length of 15 h.

Plants were grown on an ebb-and-flow flood bench system with a daily supply of Peters

Professional® 20-10-20 water-soluble fertilizer diluted to a final concentration of 4 mM

nitrogen. Plants were grown until 80 cm tall at which point microarray and quantitative RT-PCR

(RT-qPCR) experiments were performed. Floral organs used in RT-PCR experiments were

30

harvested from a Populus deltoides growing locally on the University of Florida-Gainesville

campus.

BLAST Searches and DNA Annotation

The Populus acid and neutral/alkaline invertase gene families were identified using the

tBLASTn function of the Joint Genome Institute’s database (http://www.jgi.doe.gov/) and the

previously described genes AtvacINV1 and -2 (At1g62660, At1g12240), AtcwINV1-6

(At3g13790, At3g52600,At1g55120, At2g36190, At3g13784, At5g11920), from Arabidopsis

and InvDC1-5 (Accession #s X69321, X78424, X78423, Y18707, Y18706) from carrot (Lee and

Sturm, 1996; Sturm, 1996; Sherson et al., 2003). The resulting nucleotide sequences were

annotated using a combination of protein alignments with known Arabidopsis and carrot

invertases as well as the predictions of GENSCAN (http://genes.mit.edu/GENSCAN.html)

(Burge and Karlin, 1997).

Construction of Sequence Similarity Trees

Predicted amino acid sequences were aligned using CLUSTALW

(http://clustalw.genome.jp) to construct similarity trees using the TREEVIEW program (Page,

1996). PAUP (Swofford, 1993) was used for bootstrap analysis with 100,000 iterations.

Identification of Gene Duplication

Gene duplications were identified as “recent” or “ancient” by Tuskan et al. (2006) where

“recent” and “ancient” refer to the “salicoid” and “eurosid” duplication events, respectively.

Briefly, the poplar and Arabidopsis genomes were reconstructed into conserved syntenic

segments that were subsequently compared with a variant of the algorithm described by Hokamp

et al. (2003). Tandem duplications were defined as neighboring gene models with high sequence

identity on a chromosomal segment.

31

Isolation of RNA and RT-PCR Assay

Total RNA was extracted using standard methods (Chang, 1993), DNase-treated and

purified on RNAeasy QIAGEN columns (Valencia, CA). Purified RNA (5 μg) was used to

synthesize cDNA using a mixture of 500 ng oligo-dT, 100 ng random primers, and M-MLV-RT

as per manufacturer’s instructions (Invitrogen, Carlsbad, CA), with the exception that the DTT

was excluded. PCR reactions were run with a single step at 94 ˚C for 3 min, and then 35 cycles

of 94 ˚C (30 s), 57 ˚C (30 s), and 72 ˚C (4 min), and a single step at 72 ˚C for 10 min. Actin was

used to verify integrity of cDNA template. All primers are listed in Table 2-2.

Quantitative RT-PCR

Gene expression was analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and

Mx3000P thermo-cycler (Stratagene) as per manufacturer’s instructions. Briefly, 1 μl of the

synthesized cDNA and 0.15 μl of a 0.25 μM solution of each primer were used for each 50 μl

RT-qPCR reaction. Primers were designed using NetPrimer (Premier Biosoft International, Palo

Alto, CA) software and synthesized by Invitrogen. Each real-time PCR reaction was performed

in triplicate (technical replicates) on four individual plants (biological replicates) and carried out

for 40 cycles with annealing, extension, and melting temperatures of 55 °C, 72 °C, and 95 °C,

respectively. Melting curves were generated to check the specificity of the amplified fragments.

In the case of PtVIN2, an extension temperature of 79 °C with the fluorescence reading taken at

the end of the run was used to correct for a spurious primer-dimer amplicon. Changes (n-fold) in

gene expression relative to the geometric mean (Vandesompele et al., 2002) of three control

genes encoding actin, ubiquitin and ubiquitin_L (Brunner et al., 2004) were determined using the

program DART-PCRv1.0 (Peirson et al., 2003).

32

Genomic DNA Isolation

Genomic DNA was isolated from poplar shoot tip tissue. Approximately 250 mg of tissue

was ground in liquid nitrogen and added to a buffer containing 0.3 M sucrose, 10 mM Tris (pH

7.9), 1 mM EDTA, and 4 mg/mL diethyldithiocarbamic acid. Samples were spun (20,800 rcf)

and pellets resuspended in a buffer containing 100 mM Tris (pH 7.9), 500 mM NaCl, 20 mM

EDTA, 1 % SDS, 0.1 % 2-mercaptoethanol, and 100 μg/ml proteinase K. Samples were

incubated at 65 ˚C for 1 h, spun (20,800 rcf) and supernatant extracted with chloroform.

Isopropanol was used to precipitate the DNA at room temperature, spun (20,800 rcf), and

resuspended in Tris-EDTA buffer. One μl RNase A was added to DNA sample and incubated at

37 ˚C for 30 min. The chloroform extraction was repeated, followed by ethanol precipitation in

presence of sodium acetate at -80 °C for 1 h. Samples were spun for 10 min (20,800 rcf), air

dried and resuspended in Tris-EDTA buffer. Ten ng of genomic DNA was used in PCR reactions

as previously described.

Microarray Design and Analysis

Poplar whole-genome 60-mer oligonucleotide microarrays (three different 60-mer probes

per gene model) were designed by NimbleGen (Madison, WI) in collaboration with Oak Ridge

National Laboratory. Labeling, hybridization and scanning were carried out by NimbleGen using

standard procedures (Quesada et al. unpublished data). Briefly, signal intensity detected for each

probe was log2-transformed, normalized, and contrasted to a set of 20 negative control probes. A

mixed-model analysis of variance (ANOVA) was applied to each individual probeset with gene

as a fixed effect, and probe as a random effect. P-values were adjusted for false discovery rate

(Benjamini and Hochberg, 1995), with the modifications reported by Storey and Tibshirani

(Storey and Tibshirani, 2003). All the analyses described were carried out using SAS and JMP

software (SAS Institute, Cary, NC). For contrasting the transcript abundance between treatments,

33

individual probes that gave no signal on any array based on comparison to the negative controls

were excluded, and a complete mixed ANOVA model was used that included gene and tissue

type as fixed effects, while probe ID, plant, and the interaction of tissue by plant were treated as

random effects. Microarray data have been deposited in the Gene Expression Omnibus (GEO)

database (www.ncbi.nlm.nih.gov/geo/); accession number GSE6422.

Results

Identification of Poplar Invertase Genes

I used Arabidopsis and carrot invertase genes as queries to identify 24 putative invertase

genes (Table 2-1), eight in the acid invertase sub-family and 16 in the neutral/alkaline invertase

sub-family, and followed the rice invertase nomenclature (Hirose et al., 2002; Ji et al., 2005).

The distinct origin of the acid and neutral/alkaline sub-families (Sturm, 1999; Vargas et al.,

2003) is reflected in their intron/exon structures (Figure 2-1) and amino acid alignments (Figure

2-5). The acid invertases can be further subdivided into two well-supported clades, “α” and “β”,

which are inferred to be cell wall and vacuolar targeted, respectively (Figure 2-2A). This

inference is based on sequence similarity to the Arabidopsis invertases. The neutral/alkaline

invertases also subdivide into two clades (α and β) that are supported both by bootstrap analysis

(Figure 2-2B) and intron/exon structure (Figure 2-1B), however the functional implications of

this sub-division are not clear.

Four (PtNIN13-16) of the 16 neutral/alkaline invertases are encoded by seemingly

incomplete ORFs (data not shown). PtNIN13 is missing ORFs for the first and last exons. In the

case of PtNIN14 and -15, a portion of the ORF encoding the third exon is missing as well as

ORFs corresponding to the first and last exons. Finally, PtNIN16 contains a short ORF encoding

a portion of the third exon. In all cases, genomic sequence was examined for several Kb in either

direction until neighboring genes were identified in order to verify that the sequences were truly

34

missing. Evidence of gene expression was obtained for all four of these “partial” genes using one

or more of the methods listed in Table 2-1. Further work will need to be performed to understand

their evolution.

Structure of the Poplar Invertase Genes

The acid invertase sub-family is encoded by seven exons whose locations are generally

conserved in plants (Tymowska-Lalanne and Kreis, 1998b). This family also contains PtVIN1

which contains no introns. All eight genes encode the motifs NDPNG and WECXDF, which are

essential for catalytic activity and are conserved in this gene family (Sturm and Chrispeels,

1990b) (Figure 2-5A). Except for PtVIN1, the NDPNG motif is partially encoded by a mini-exon

encoding the tripeptide DPN, one of the smallest known exons in plants (Bournay et al., 1996).

There are several key features that distinguish the β clade of the poplar acid invertases

from the α clade with a highly significant bootstrap value of 100 %. The first two features are N-

terminal and C-terminal extensions, both proposed to play a role in targeting to the vacuole

(Sturm, 1999). Third is the conserved WECXDF domain that contains one of the three

carboxylate groups required for activity (Pons et al., 1998; Alberto et al., 2004); the X in this

domain is a proline in the α clade and a valine in the β clade (Figure 2-5A).

The neutral/alkaline invertase sub-family also clusters into α and β clades based on amino

acid alignments (Figure 2-2B, Figure 2-5B) that are distinct and well supported (Figure 2-2B).

The members of the α clade (PtNIN1-6) are encoded by six exons with conserved locations

(Figure 2-1B), whereas the six members of the β clade (PtNIN7-12) are encoded by four exons

(Figure 2-1B). The different intron/exon structures and the different number of exons between

the clades, suggests that the α and β clades arose from different ancestral genes.

35

Expression of the Poplar Invertase Gene Family

Expression of the poplar invertase family in mature leaves, young leaves, nodes,

internodes, and roots was examined using whole genome microarrays where evidence for

expression was detected for 16 of the 24 genes (Figure 2-3A). RT-qPCR with gene specific

primers designed against PtCIN3, PtVIN3, PtNIN3 and PtNIN9 was then used to validate the

microarray data using the same samples that were analyzed in the microarrays (Figure 2-3B).I

then obtained evidence for transcript-level regulation of PtNIN7, -10, -11 and -16 in additional

microarray analyses of responses to nitrogen availability (PtNIN7 and -11) (data not shown) and

exogenous auxin (PtNIN10 and -16) (data not shown).

Analyses of microarrays provided evidence for expression of all but four invertase genes

(PtCIN1 and -2, PtVIN1, PtNIN13). One possible explanation for this result is that these four

invertase genes are not transcribed in the organ(s) examined. Alternatively, the gene may have

been transcribed in the organ(s) examined but transcript abundance was not distinguishable from

background on the arrays. To test the first hypothesis, RT-PCR was carried out on a larger

number of poplar organs. I detected transcripts for PtCIN1 and -2 in floral organs and not organs

used in the array analysis (Figure 2-3C). This is consistent with previous results in tomato (Godt

and Roitsch, 1997a), carrot (Lorenz et al., 1995), and Arabidopsis (Tymowska-Lalanne and

Kreis, 1998a; Sherson et al., 2003) where certain cell wall invertases are expressed solely in

floral organs. I detected transcripts for PtVIN1 and PtNIN13 using RT-PCR in nearly all organs

tested (Figure 2-3C). This indicates that the transcripts of these two genes were below the

detection level of the arrays.

Evolutionary Development of the Poplar Invertase Family

Determining orthology and paralogy between genes can be a useful tool in elucidating

gene function. The shared evolutionary history between Arabidopsis and poplar, as well as the

36

extensive research previously conducted in the Arabidopsis invertase gene family, makes

Arabidopsis invertases ideal candidates to compare with poplar invertases in order to determine

orthology. Amino acid alignments between poplar and Arabidopsis invertases (Figure 2-2,

Figure 2-5) reveal little about orthology since the Arabidopsis invertases are more similar to each

other than they are to their presumed poplar orthologs. Consequently, I examined the

chromosomal segments encoding invertases to infer orthology since the linear order of ORFs are

often conserved after duplication events.

I examined paralogous regions of the poplar genome, as defined by Tuskan et al. (2006),

containing the neutral/alkaline invertase sub-family members and found that five of the 16 poplar

neutral/alkaline invertases appear to have originated in the recent salicoid genome duplication

event (Table 2-1). No evidence was found for tandem duplications (defined as identical, adjacent

genes on the chromosome), or for expansion of the neutral/alkaline family as a result of the more

ancient eurosid genome duplication event that occurred in the common ancestor of poplar and

Arabidopsis. Therefore, the salicoid duplication event can explain the growth of the poplar

neutral/alkaline invertase family relative to the Arabidopsis gene family (16 versus 9 members,

respectively).

To establish orthology between the poplar acid invertase sub-family and the more widely

studied Arabidopsis acid invertase sub-family, I compared the genomic organization of the two

species’ acid invertase sub-families and found substantial conservation of microsyntenic regions

(Figure 2-4A). These conserved, microsyntenic regions suggest that the common ancestor to

poplar and Arabidopsis contained two cell wall invertases and one vacuolar invertase (Figure 2-

4B). I hypothesize that these three progenitor invertases (VINa, CINa, and CINb) underwent a

37

series of duplication events prior to and subsequent to the speciation event between poplar and

Arabidopsis, as outlined in Figure 2-4B.

Vacuolar invertases PtVIN2 and -3 lie within conserved, colinear chromosomal segments

(Figure 2-4B), indicating that a chromosomal duplication event gave rise to these two invertases.

Similarly, the two Arabidopsis vacuolar invertases (AtvacINV1 and -2) reside in a conserved,

colinear arrangement (Figure 2-4A), also indicative of a chromosomal duplication event.

Conservation can also be observed across the poplar/Arabidopsis species divide. For example, a

nuclear apical meristem (NAM) family ORF lies downstream of the vacuolar invertases on

relevant chromosomal segments in both species (Figure 2-4A). In addition, loci encoding a

thylakoid luminal related protein, a 3-oxoacyl-ACP synthase III protein and a flavin containing

monoxygenase are located on colinear chromosomal segments with both the poplar and

Arabidopsis vacuolar invertases (Figure 2-4A). The colinearity of multiple loci across

Arabidopsis and poplar suggests that a genomic duplication event of a progenitor vacuolar

invertase and surrounding loci occurred in a common ancestor of poplar and Arabidopsis giving

rise to the two vacuolar invertases (Figure 2-4A, B).

Two additional chromosomal duplication events can be identified through colinearity. The

conservation of loci encoding a kinesin motor related protein, ubiquitin-2, and a 40S ribosomal

protein S9 can be seen surrounding AtcwINV2 and -4 as well as PtCIN3 and the tandem pair

PtCIN1 and -2. The conservation of this chromosomal segment between Arabidopsis and poplar

is again indicative of a duplication event giving rise to these genes prior to the Arabidopsis and

poplar speciation event (Figure 2-4B). Because the presence of the tandem pair PtCIN1 and -2

occurs only in poplar, duplication likely occurred after the poplar and Arabidopsis speciation

event.

38

The microsyntenic segment containing the poplar cell wall invertases PtCIN4 and -5 is

more difficult to interpret (Figure 2-4B). This chromosomal segment contains a locus encoding a

C2H2 zinc finger family protein that is shared on two chromosomal segments in Arabidopsis that

also contain AtcwINV3 and the tandem pair AtcwINV1 and -5. This poplar segment also contains

a DEAD box RNA helicase downstream of the tandem invertase pair that is conserved on the

AtcwINV3 chromosomal segment but absent from the tandem AtcwINV1 and -5 chromosomal

segment. The simplest explanation of the development of these invertases is a tandem

duplication event prior to the poplar/Arabidopsis speciation event (duplication of CINa, Figure

2-4B). Following the speciation event, a chromosomal duplication event occurred only in

Arabidopsis to give rise to a tandem pair; AtcwINV3 and the other member which was lost.

Poplar-specific innovations include a tandem duplication giving rise to the PtCIN1 and -2

pair (Table 2-1, Figure 2-4) and the appearance of PtVIN1, the third poplar vacuolar invertase,

described in more detail in the next section. I hypothesize PtVIN1 arose from a processed PtVIN2

mRNA that was inserted in trans- into the poplar genome and therefore is not colinear with any

other invertase.

Arabidopsis innovations include AtcwINV3, which apparently arose from a duplication of

the chromosomal segment containing AtcwINV1 and -5. This duplication was subsequently

followed by the loss of one of the proposed two new invertases resulting in the single AtcwINV3.

The sixth and final cell wall invertase in Arabidopsis, AtcwINV6, poses a bit of a conundrum.

The lack of colinearity with other regions of the Arabidopsis genome and its isolation on the

protein similarity tree (Figure 2-2A) makes it difficult to derive a putative progenitor. I

hypothesize that AtcwINV6 arose from a gene duplication event followed by functional

divergence, as this is the most common mode of gene family diversification. However, because

39

genes are frequently rearranged and/or lost after chromosomal duplication events, the absence of

colinearity cannot rule out a chromosomal duplication event. Some other technique will need to

be employed to define the origin of AtcwINV6.

Putative Evidence for Origins of PtVIN1

In its recent evolutionary history, poplar has acquired an innovation in the acid invertase

family. PtVIN1 is an intronless invertase that clusters with the β group, indicating vacuolar

targeting (Figure 2-1, 2-2). An intronless invertase, to my knowledge, has not previously been

reported. This gene retains all of the characteristics relevant to the acid invertase family with the

exception of introns (Figure 2-5A).

PtVIN1 consists of a single 1.6 Kb ORF with several possible upstream TATAA boxes and

a 3’ polyadenylation signal (data not shown). I verified the sequence obtained from the Joint

Genome Institute’s database by designing gene specific primers (Table 2-2) flanking the entire

ORF of PtVIN1 and cloning and sequencing PCR products amplified from both purified P.

trichocarpa genomic DNA as well as cDNA generated from DNase-treated RNA. PtVIN1

transcript was detected in all tissues examined (Figure 2-3C).

I speculate that PtVIN1 arose relatively recently in poplar evolutionary history as a

processed transcript of PtVIN2 (86 % identity at the nucleotide level and 77 % identity at the

amino acid level) that was reverse transcribed and reinserted into the genome. This phenomenon

is unusual but not unprecedented with examples being found in the alcohol dehydrogenase gene

family in the genus Leavenworthia (Charlesworth et al., 1998), as well as numerous examples in

human, rat, and dog (Coulombe-Huntington and Majewski, 2007).

Discussion

In my study of the poplar genome, I identified 24 putative invertase genes: eight acid and

16 neutral/alkaline invertases. The poplar genome encodes ca. 45,000 genes which, when

40

compared to ca. 27,000 genes in Arabidopsis represents an approximately 1.6-fold increase in

gene number (Tuskan et al., 2006). This total gene number expansion is not the result of unique

poplar genes, but rather the expansion of specific gene families (Tuskan et al., 2006).

Interestingly, the expansion of poplar’s invertase gene family relative to Arabidopsis and rice is

not consistent between the two invertase sub-families. Arabidopsis contains 17 members: eight

acid and nine neutral/alkaline invertases (Vargas et al., 2003; Ji et al., 2005), whereas the rice

genome encodes 19 invertase genes: eleven acid and eight neutral/alkaline invertases (Ji et al.,

2005). Thus, the overall increase in poplar invertase gene numbers is driven primarily by an

increase in the neutral/alkaline invertases.

One explanation for the expansion of the neutral/alkaline invertase sub-family in poplar

may lie in the apparent lack of an active phloem loading step in this group of woody perennial

plants. Turgeon and Medville (1998) suggest that in willow, and likely poplar (both are members

of the Salicaceae), photoassimilate accumulation in the source phloem is accomplished via an

uninterrupted, symplastically connected sucrose concentration gradient between the mesophyll

cells and the sieve element-companion cell complex (SE-CCC). This is in contrast to both

apoplastic phloem loaders and symplastic phloem loaders where the solute concentration in the

SE-CCC is dramatically higher than the solute concentration in the surrounding apoplastic space

and mesophyll cytoplasm. Turgeon and Medville (1998) propose that regulation of the sucrose

concentration gradient from the mesophyll cells to the SE-CCC in poplar could be maintained in

the cytoplasm and/or vacuole. This could implicate both neutral/alkaline invertases (thought to

be cytoplasmically located) and vacuolar invertases as key components of this pathway. The

growth of the poplar neutral/alkaline invertase subfamily occurred in the recent genomic

duplication event (salicoid) that took place prior to the willow/poplar speciation event (Tuskan et

41

al., 2006). This makes it likely that willow also contains the expanded neutral/alkaline invertase

sub-family.

Of the 16 neutral/alkaline invertases identified in poplar, four (PtNIN13-16) are missing

significant portions of their coding regions. The sequences found in these four neutral/alkaline

invertases retain conserved intron/exon splice sites, conserved sequence motifs and the ability to

be transcribed (data not shown). This transcriptional evidence appears to rule out these genes

from being defined as “pseudogenes” under traditional definitions of the term in which

pseudogenes are transcriptionally and translationally silent and therefore not subject to selection

(Li et al., 1981). However, there are examples of transcribed pseudogenes in eukaryotic

organisms including human, mouse, silk moth, Arabidopsis and liverwort (Balakirev et al.,

2003). McCarrey and Riggs (1986) propose that pseudogenes could act as negative regulators of

transcription of their progenitor genes by providing antisense RNA to hybridize with the sense

RNA of the progenitor. Troyanovsky and Leube (1994) identified cis elements in the promoter

region of a pseodogene derived from human cytokeratin 17 that can interact with distal elements

in the promoter of the functional gene to regulate transcriptional activity. Thus, PtNIN13-16 may

have biologically relevant functions in gene regulation, as opposed to invertase enzyme activity,

and may still be subjected to selection.

ClustalW and bootstrap analysis of the acid invertase families of poplar and Arabidopsis

clearly divide the acid invertases into the α clade (cell wall invertases) and the β clade (vacuolar

invertases). It has recently been reported that two of six cell wall invertases in Arabidopsis may

not be invertases, but rather fructan exohydrolases (FEHs) (De Coninck et al., 2005). FEH

protein sequences are nearly identical to those of demonstrated acid invertase proteins but FEH

does not use sucrose as a substrate (De Coninck et al., 2005). As I did not test recombinant cell

42

wall invertase activity in this work, I cannot rule out that one or more of the five PtCIN genes

described here may encode an FEH.

To my knowledge, this work is the first report of a plant with more than two vacuolar

invertases. Analysis of the microsyntenic regions surrounding these vacuolar invertases in poplar

and Arabidopsis identified colinearity that I use to postulate that these genes arose from an

ancient gene duplication event in a common ancestor of these two species. In contrast, I found no

colinearity in the microsyntenic region containing PtVIN1 with regions in Arabidopsis or

elsewhere in the poplar genome. This, along with the lack of introns in PtVIN1, leads me to

hypothesize that PtVIN1 arose from a processed PtVIN2 mRNA that was inserted in trans- into

the poplar genome.

The absence of an intronless vacuolar invertase in any other organism studied to date

suggests that PtVIN1 arose recently in evolutionary history. It will be interesting to see if this

invertase anomaly extends to Populus species other than the sequenced Populus trichocarpa

genome. Work performed by Coulombe-Huntington and Majews (2007) on intron loss in

mammalian systems found that genes susceptible to intron loss tend to be involved in

housekeeping functions and expressed at high levels. This is consistent with the high expression

patterns of PtVIN2, the putative progenitor to the intronless PtVIN1. Because PtVIN1 has

retained all the features necessary for transcription, such as an intact TATAA binding site and no

indels or stop codons, it appears that there has been some level of selection in favor of the

expression of PtVIN1. The hypothesis that PtVIN1 originated as a reverse transcribed, processed

transcript is supported by the concomitant loss of all six introns from the presumed progenitor

locus PtVIN2.

43

One distinguishing feature of the expression of the invertase family is the floral specificity

of certain cell wall isoforms. Tomato Lin7 is floral specific with highest expression in the stamen

(Godt and Roitsch, 1997b). In carrot, InvDC2 was expressed solely in floral buds (Lorenz et al.,

1995) and in Arabidopsis, AtcwINV2 was found to be floral specific (Tymowska-Lalanne and

Kreis, 1998a). Poplar is a dioecious tree and, as I only collected floral material from a female

tree, I did not examine invertase expression in the male floral organs. Even so, I identified two

floral specific cell wall isoforms in PtCIN1 and -2.

The recently released assembly of the poplar genome (Tuskan et al., 2006) opens the door

for analyses of metabolically important gene families in a model tree. In this study of the

invertase gene family, I identified 24 putative gene family members and obtained evidence for

expression of all members. A better understanding of the roles that individual invertase isoforms

play in carbon utilization will be important in dissecting the functional implications of invertase

gene family evolution and diversity.

44

Table 2-1. Nomenclature and chromosomal location of 24 poplar invertase genes. Gene name Predicted compartment JGIv1.1 gene model name Linkage group location Expression Support Type of gene duplication Duplicate gene

Acid invertasesPtCIN1 Cell wall gw1.XVI.2453.1 16: 5729453 R tandem PtCIN2PtCIN2 Cell wall gw1.XVI.2454.1 16: 5739912 E, R tandem PtCIN1PtCIN3 Cell wall estExt_fgenesh4_pg.C_LG_VI1370 6: 13897519 E, M, R n/aPtCIN4 Cell wall estExt_fgenesh4_pg.C_LG_VI1536 6: 15233568 E, M, R tandem PtCIN5PtCIN5 Cell wall eugene3.00061607 6: 15229483 M tandem PtCIN4PtVIN1 Vacuole fgenesh4_pm.C_LG_III000407 3: 11913972 R insertion PtVIN2PtVIN2 Vacuole estExt_fgenesh4_pg.C_LG_III0902 3: 108433683 E, M, R ancient PtVIN3PtVIN3 Vacuole estExt_Genewise1_v1.C_LG_XV2841 15: 9251445 E, M, R ancient PtVIN2

Neutral/alkaline invertasesPtNIN1 Cytosol estExt_Genewise1_v1.C_LG_VIII2120 8: 6388303 E, M, R n/aPtNIN2 Cytosol eugene3.00130058 13: 512039 E, M, R recent PtNIN5PtNIN3 Cytosol gw1.VIII.2341.1 8: 1096197 E, M, R recent PtNIN4PtNIN4 Cytosol gw1.X.3512.1 10: 20965604 E, M, R recent PtNIN3PtNIN5 Cytosol gw1.131.249.1 scfld131: 616042 E, M, R recent PtNIN2PtNIN6 Cytosol gw1.66.49.1 scfld66: 160259 E, M, R n/aPtNIN7 Cytosol estExt_fgenesh4_pg.C_LG_V1531 5:16687404 E, M, R n/aPtNIN8 Cytosol eugene3.00190739 19: 9274739 E, M, R recent PtNIN12PtNIN9 Cytosol fgenesh4_pg.C_LG_IV001415 4: 15428994 E, M, R recent PtNIN11PtNIN10 Cytosol fgenesh4_pm.C_LG_II000804 2: 13356400 M, R recent PtNIN13PtNIN11 Cytosol gw1.IX.1371.1 9: 2392685 E, M, R recent PtNIN9PtNIN12 Cytosol eugene3.00410102 scfld41: 1148890 E, M, R recent PtNIN8

PtNIN13 a Cytosol gw1.XIV.1765.1 14: 3660215 R recent PtNIN10PtNIN14 b Cytosol gw1.XIV.4326.1 14: 11708730 M n/aPtNIN15 b Cytosol gw1.376.8.1 scfld376: 31617 E, M, R n/aPtNIN16 c Cytosol eugene3.00170140 17: 1427133 M n/a

aMissing first and last exon; bMissing first, last, and portions of third exons; cContains only portion of third exon. Type of predicted duplication event (if any) is noted. E: EST support; M: Microarray support; R: RT-PCR support; n/a: not assigned.

45

A PtVIN1

PtVIN2

PtCIN5

1Kb1Kb1Kb1Kb

PtCIN1

PtVIN3

PtCIN4

PtCIN2

PtCIN3

B PtNIN1

PtNIN2

PtNIN8

PtNIN9

PtNIN7

PtNIN10

PtNIN11

PtNIN12

PtNIN3

PtNIN5

PtNIN4

PtNIN6

1Kb1Kb

PtNIN1

PtNIN2

PtNIN8

PtNIN9

PtNIN7

PtNIN10

PtNIN11

PtNIN12

PtNIN3

PtNIN5

PtNIN4

PtNIN6

1Kb1Kb

Figure 2-1. Exon-intron structures of predicted invertase genes with complete exons (20 of 24). PtNIN13-16 are not represented here as the complete sequence could not be determined. A) Acid invertases. B) Neutral/alkaline invertases. Exons whose junctions have been verified (using EST and/or sequencing data) are represented by solid black boxes, exons whose junctions have not been verified are represented by empty boxes. Introns are represented by black lines.

46

A

α

β

59 100

95

54

85

100

100

100

100

100

100

100

100

PtCIN4

PtCIN1PtCIN2

PtCIN3

PtCIN5

AtvacINV2

(At1G12240)

PtVIN3

PtVIN1

PtVIN2

AtvacINV1

(At1G62660)

AtcwINV6(FEH)

(At5G11920)

AtcwINV3(FEH)

(At1G55120)

AtcwINV5

(At3G13784)

AtcwINV1

(At3G13790)

AtcwINV4

(At2G36190)

AtcwINV2

(At3G52600)

B

αβ

100

100

51

8665 65

100

100

69

93

93

10083

100

100

100

51

PtNIN10 PtNIN7

At1G35580

At4G09510

PtNIN8

PtNIN12

At1G72000At1G22650

At5G22510

PtNIN6

PtNIN4

PtNIN3

At3G065600

At3G05820 At1G56560

PtNIN1

PtNIN5

PtNIN2

PtNIN9PtNIN11 At4G34860

PtNIN1 PtNIN5

58

αβ

100

100

51

8665 65

100

100

69

93

93

10083

100

100

100

51

PtNIN10 PtNIN7

At1G35580

At4G09510

PtNIN8

PtNIN12

At1G72000At1G22650

At5G22510

PtNIN6

PtNIN4

PtNIN3

At3G065600

At3G05820 At1G56560

PtNIN1

PtNIN5

PtNIN2

PtNIN9PtNIN11 At4G34860

PtNIN1 PtNIN5

αβ

100

100

51

8665 65

100

100

69

93

93

10083

100

100

100

51

PtNIN10 PtNIN7

At1G35580

At4G09510

PtNIN8

PtNIN12

At1G72000At1G22650

At5G22510

PtNIN6

PtNIN4

PtNIN3

At3G065600

At3G05820 At1G56560

PtNIN1

PtNIN5

PtNIN2

PtNIN9PtNIN11 At4G34860

PtNIN1 PtNIN5

58

Figure 2-2. Invertase amino acid similarity trees based on full length sequences. A) Protein similarity tree of eight poplar and eight Arabidopsis acid invertases. α denotes cell wall invertases (PtCIN1-5) and β denotes vacuolar invertases (PtVIN1-3). B) Protein similarity tree of 12 poplar and 9 Arabidopsis neutral/alkaline invertases. The α clade (PtNIN1-6) and β clade (PtNIN7-12) are marked. PtNIN13-16 are not included. Bootstrap values are reported as a percentage of 100,000 repetitions. Branch lengths denote protein similarity.

47

A

PtCIN3

PtNIN6

PtNIN8

PtNIN9

PtNIN12

PtNIN14

PtNIN15

PtNIN1

PtNIN2

PtNIN3

PtNIN4

PtNIN5

PtCIN5

PtVIN2

PtVIN3

PtCIN4

Mat

ure

leaf

Youn

g le

af

Inte

rnod

eNo

de

Root

α

β

α

β

B PtCIN3

0

0.2

0.4

0.6

0.8

1

1.2

ML YL R IN N

PtVIN3

0

0.2

0.4

0.6

0.8

1

1.2

ML YL R IN N

PtNIN3

0

0.2

0.4

0.6

0.8

1

1.2

ML YL R IN N

PtNIN9

0

0.2

0.4

0.6

0.8

1

1.2

ML YL R IN N C

Imm

atur

e St

em

PtNIN13

Roo

t

Xyle

m

Phlo

em

Leaf

Shoo

t Tip

Cat

kin

Pre

Zygo

tic F

lora

l

Post

Zyg

otic

Flo

ral

PtCIN1

PtCIN2

Actin

PtVIN1

P. trichocarpa P. deltoides

Figure 2-3. Expression of poplar invertase genes. A) Heat map showing relative expression of poplar invertase genes in five organs. If transcript for a gene was significantly detected at an FDR of 10 % (contrasted with the array negative control probes) in at least one organ, then data from all organs was included on the heat map. Dark blue denotes high expression and light blue denotes low expression. B) RT-qPCR validation of microarray data. Relative transcript levels detected by RT-qPCR (black bars) and microarray analysis (grey bars) in mature leaves (ML), young leaves (YL), roots (R), internodes (IN), and nodes (N). In order to compare the microarray and RT-qPCR platforms, RNA was re-extracted from the same tissues used in the microarray experiment. First strand cDNA was then synthesized and used as template in the RT-qPCR reactions. The highest and lowest relative transcript estimates were assigned values of 1 and 0, respectively. Intermediate values were then adjusted relative to their difference between the highest and lowest transcript estimate. C) Expression of PtCIN1,2, PtVIN1 and PtNIN13 in various organs using cDNA generated from P. trichocarpa (vegetative organs) and P. deltoides (floral organs) as template. Contrast and resolution of images were adjusted in order to maximize visibility of bands. Actin cDNA was amplified to verify integrity of the RT-PCR template in each reaction.

48

A

AtcwIVR5AtcwIVR1 AtcwIVR5AtcwIVR1

AtcwIVR3AtcwIVR3

PtCIN5PtCIN4

24 Kb

PtVIN3PtVIN3

AtvacINV1AtvacINV1

AtvacINV2AtvacINV2

32 Kb

PtVIN2

45 Kb

AtcwIVR2

AtcwIVR4

PtCIN3PtCIN3

4 Kb4 Kb

PtCIN2 PtCIN1

22 Kb 11.6 Kb

AtcwIVR5AtcwIVR1 AtcwIVR5AtcwIVR1

AtcwIVR3AtcwIVR3

PtCIN5PtCIN4

24 Kb

PtVIN3PtVIN3

AtvacINV1AtvacINV1

AtvacINV2AtvacINV2

32 Kb

PtVIN2

45 Kb

AtcwIVR2AtcwIVR2

AtcwIVR4AtcwIVR4

PtCIN3PtCIN3

4 Kb4 Kb

PtCIN2 PtCIN1

22 Kb 11.6 Kb

NAM

Thylakoid luminalprotein related

Multicopper oxidase

Flavin containingmonoxygenase

CC-NBS-LRR classdisease resistance

3-oxoacyl-ACPsynthase III

Trehalose-6-phosphatephosphatase

Kinesin motor related

40S ribosomalprotein S9

UBQ2

F-box family Endomembrane protein 70C2H2 zinc finger family protein

PX domaincontaining protein

DEAD box RNA helicase

B

VINa

VINa

VINa

CINa

CINbCINaCINa

CINb

CINb

Chromosomal duplication Tandem duplication Chromosomal duplication

Speciation event

Poplar Arabidopsis

Tandem duplication

VINa

VINa

CINa CINb

CINb

VINa

VINa

CINa CINb

CINb

PtVIN1

PtVIN2

PtCIN4

PtCIN3

PtCIN1 PtCIN2

PtVIN3

AtvacINV1

AtvacINV2 AtcwINV1

AtcwINV6(unknownancestry)

AtcwINV2

AtcwINV4

Trans-reinsertion ofprocessed transcript

Chromosomal duplication& subsequent loss of

gene pair

AtcwINV3

AtcwINV5

PtCIN5

CINa

CINa CINa

VINa

VINa

VINa

CINa

CINbCINaCINa

CINb

CINb

Chromosomal duplication Tandem duplication Chromosomal duplication

Speciation event

Poplar Arabidopsis

Tandem duplication

VINa

VINa

CINa CINb

CINb

VINa

VINa

CINa CINb

CINb

PtVIN1

PtVIN2

PtCIN4

PtCIN3

PtCIN1 PtCIN2

PtVIN3

AtvacINV1

AtvacINV2 AtcwINV1

AtcwINV6(unknownancestry)

AtcwINV2

AtcwINV4

Trans-reinsertion ofprocessed transcript

Chromosomal duplication& subsequent loss of

gene pair

AtcwINV3

AtcwINV5

PtCIN5

CINa

CINa CINa

Figure 2-4. Evolutionary development of the poplar acid invertase family. A) Colinear chromosomal segments containing poplar invertase ORFs (checkered) and Arabidopsis invertase ORFs (solid). Conserved ORFs between chromosomal segments are depicted with identical shading; PX, phox; NAM, nuclear apical meristem; ACP, acyl carrier protein. B) Cartoon depicting hypothesized development of the acid invertase sub-family in poplar and Arabidopsis.

49

Table 2-2. Primers used in RT-qPCR and RT-PCR. Gene name Primer sequence Tm (°C) Amplicon size (base pairs)PtVIN1_RTqPCR_3' TCCTGGCCTCAAAATTTGC 58 216PtVIN1_RTqPCR_5' GCCCTGGTTCCACCTATAGTT 58PtVIN2_RTqPCR_3' CGCATCTTCGTCTTCTTGTG 57 189PtVIN2_RTqPCR_5' CCCAGCTATAGTCCTGCCC 57PtVIN3_RTqPCR_3' GCTTGGAAAATGACTCTGTAGGTC 59 197PtVIN3_RTqPCR_5' ATACACTCCCTTGCTAGACAACC 57PtCIN3_RTqPCR_3' TTGGTAGAATAGATGGTATAGCCCC 61 224PtCIN3_RTqPCR_5' TGTTAAAGTTTCTCCCAGTCTTGG 60PtNIN3_RTqPCR_3' TGGGGCGAGCATCTCCT 59 148PtNIN3_RTqPCR_5' TGCTGATGGTTTTGACATGTTC 58PtNIN8_RTqPCR_3' GTTTCAACAATGAGCAAGCG 57 211PtNIN8_RTqPCR_5' GATTTATCTCTAGCAGAAACTCCAG 56PtNIN9_RTqPCR_3' GGCATGAAGGCGTTTAGTG 56 393PtNIN9_RTqPCR_5' CACGATCCTGTCAGGAACAG 56PtNIN10_RT_PCR_3' GCTTCCCAACATATCTGCCG 60 824PtNIN10_RT_PCR_5' TCGCCCGGAGGTACAGAAT 60PtNIN13_RT_PCR_3' GCTCCCCTACATACCTGCCA 60 629PtNIN13_RT_PCR_5' TCGCCCAGAAGTGCAGAGA 60PtNIN14_RT_PCR_3' TGTTATTTAATTCAGTGAAATTCAGCA 60 334PtNIN14_RT_PCR_5' GTTCTATATGACTTGCATCGTCAAAAT 61PtNIN15_RT_PCR_3' TGTTATTTAATTGAGTGAAATTCAGCC 61 241PtNIN15_RT_PCR_5' CTCTATGACTCGCATCGTCAAAAG 61PtNIN16_RT_PCR_3' CTAGCCATGAAGGAATTTGATCTTC 61 201PtNIN16_RT_PCR_5' ATGCTCTTTGTCAATGATGGAAC 59

50

A PtCIN1 1 ------------------------------------------------------------ PtCIN2 1 ------------------------------------------------------------ PtCIN3 1 ------------------------------------------------------------ PtCIN4 1 ------------------------------------------------------------ PtCIN5 1 ------------------------------------------------------------ PtVIN1 1 MADTSPLLPFSHSLGP-----------GSTYSSIRWRSKIVLLLVFSGLFLVPLIVSIAS PtVIN2 1 MADPSPLLPVSNSLEPSYSPAPEGAVSAGCPATHLRRSKKVLIAVFSGLLVVSLILATIN PtVIN3 1 -MDTNPSHTSSDPPYTPLLD-------NPSPARIRRPFNGFAAILASLIFLLSLVALIIN PtCIN1 1 -------------------------MMAMPHTLSVLALFALLFVLTNNGAEASHKIYSEY PtCIN2 1 --------------------------MAMPNTLSVLALFALFFVLSNNGAEASHKIYPQF PtCIN3 1 --------------------------MALLKFLPVLALFALLFVLSNNGVEASHKIYLRY PtCIN4 1 --------------------------MEILAVFLVGLCCVLQSSGIEVEALENNGCQNFQ PtCIN5 1 --------------------------MEISVIWVVGFCVLLVDHGVQASHQSSR------ PtVIN1 50 NDNGFKQHVQYLQEDDQNVSFSPPKETTKPQILRPG--SRGVSAGVSEKANVNLKGAQEK PtVIN2 61 NNN-GGRHVQYHSQEDEDASLATPKEMAKPETLLPAGYSRGVSAGVSEKANVNLKGAQVK PtVIN3 53 QSQ------ESLPEQNQNRSPSTPRPTESFSKPEPR----GVAQGVSPKSNPSFFSDKVS < * A> < B > <B1 PtCIN1 36 QTLSVENVNQVHRTGYHFQPPRHWINDPNAPMYYKGLYHLFYQYNPKGAVWG-NIVWAHS PtCIN2 35 QTLSVENVNQVHRTGYHFQPPRNWINDPNAPMYYKGLYHLFYQYNPKGAVWG-NIVWAHS PtCIN3 35 QSLSVDKVKQTHRTGYHFQPPKNWINDPNGPLYYKGLYHLFYQYNPKGAVWG-NIVWAHS PtCIN4 35 PHTVMMQEKQSYRTSFHFQPPRNWLNDPNGPMWYKGVYHLFYQYNPYGALFGDFMIWAHS PtCIN5 29 ---NLQETDQPYRTGYHFQPPKNWMNDPNGPMYYKGVYHLFYQYNPDGAVWG-NIIWAHS PtVIN1 108 GYPQNDSMLSWQRTSFHFQPEKNWMNDPNGPLYYKGWYHFFYQHNPHAAVWG-DIVWGHA PtVIN2 120 DYPWNNSMLSWQRTAFHFQPEENWMNDPNGPLYYKGWYHFFYQYNPHAAVWG-DIVWGHA PtVIN3 103 -YNWTNAMFSWQRTAYHFQPEKNWMNDPDGPLFHKGWYHLFYQYNPDSAVWG-NITWGHA > < C > PtCIN1 95 VSKDLINWESLEPAIYPSKWFDNYGCWSGSATVLPNGEPVIFYTGIVDKNNSQIQNYAVP PtCIN2 94 VSKDLINWESLEPALYPSKWFDNYGCWSGSATILPNGEPVIFYTGIADKNNSQIQNYAVP PtCIN3 94 VSKDLINWESLEPAIYPSKWFDNYGCWSGSATILPNGEPVIFYTGIVDENNRQIQNYAVP PtCIN4 95 VSYDLINWIHLNHALCPTEPYDINSCWSGSATILPGKGPVILYTGIDAN-HCQVQNMAMP PtCIN5 85 VSYDLVNWVHIDHAIYPTQPSDINGCWSGSTTILPGEKPAILYTGIDTK-NHQVQNLAVP PtVIN1 167 VSRDLINWFHLPLAIVSDEWFDINGVWTGSATILLNGKIVMLYT-GSTNESVQVQNLAYP PtVIN2 179 VSKDLIHWLHLPLAMVADKWYDKNGVWTGSATILPDGKIVMLYT-GSTNESVQVQNLAYP PtVIN3 161 VSTDLIHWLYLPFAMVPDHWYDINGVWTGSATLLPDGQIMMLYT-GSTNESVQVQNLAYP < * D> PtCIN1 155 ANLSDPYLREWVKPDDNPIVNPDANVNGSAFRDPTTAWWADG-HWRILIGSRRKHRG-VA PtCIN2 154 ANLSDPYLREWVKPDDNPIVNPDVSVNGSAFRDPTTAWWADG-HWRILIGSRRNHVG-VA PtCIN3 154 ANSSDPYLREWVKPDDNPIVYPDPSVNASAFRDPTTAWRVDG-HWRILIGSKKRDRG-IA PtCIN4 154 KNLSDPFLEEWIKFAQNPIMTPPDGVEGNNFRDPTTAWLSHDGKWSVIIGSWNNNQG-MA PtCIN5 144 KNLSDPLLKEWKKSPYNPLMTPIDGIDPDLYRDPTTAWQGPDKIWRVIVGSQINGHG-RA PtVIN1 226 ADHNDPLLLKWVKYSGNPVLVSPPGIDPNDFRDPTTAWYTSEGKWRITIGSKANNTG-IA PtVIN2 238 ADHDDPLLLKWVKYSGNPVLVPPPGIGAKDFRDPTTAWKTSEGKWRIIIGSKINKTG-IA PtVIN3 220 ANLSDPLLIDWVKYPNNPVITPPNGTETDEFRDPTTAWMGPDGTWRITIGSRHNKSIGLS

51

<* E > PtCIN1 213 YLYRSKDFKKWVKAKHPLHSVQGTGMWECPDFYPVSLSGENGLDPSVMGQNVKHVLKVSL PtCIN2 212 YLYRSRDLKKWAKTKHPLHSVQRTGMWECPDFFPVSSFGENGLDPSVNGQNVKHALKVSL PtCIN3 212 YLYRSLDFKKWFKAKHPLHSVQGTGMWECPDFFPVSLSSEDGLDTSVGGSNVRHVLKVSL PtCIN4 213 ILYRSEDFFNWTKYQDPLYSTERTGMWECPDFYPVSVNSTDGVDTSVLNAGVKHVMKASF PtCIN5 203 ILYRSKDFVNWTRIDSPLHSSGKTEMWECPDFFPVSTSSTNGVDTSSQDKSTKHVLKASF PtVIN1 285 LVYDTEDFINFKLSG-VLHGVPGTGMWECVDFYPVSKTGQNGLDTSANGPHVKHVVKTSL PtVIN2 297 LVYDTEDFINYELLSGILHGVPKTGMWECVDFYPVSKTGQNGLDTSVNGPQVKHVIKTSL PtVIN3 280 LVYQTSNFTTYELLEGVLHAVPGTGMWECVDFYPVAINGSTGLDTSAYGAGIKHVLKASL < F > PtCIN1 273 DMTRYEYYTMGTYDKKKDKYFPDEGLVDGWAGLRLDYGNFYASKTFFDPSTNRRILWGWA PtCIN2 272 DLTRYEYYTLGTYDNKKEKYFPDEGLVDGWAGLRLDYGNFYASKTFFDPSTNRRILWGWV PtCIN3 272 DLTRYEYYTIGTYDEKKDRYYPDEALVDGWAGLRYDCGNFYASKTFFDPSTNRRILWGWA PtCIN4 273 NS--HDYYMIGTYVPEIEKYIPDNDFTGTGMDLRYDHGKFYASKTFFDSVKNRRILWGWV PtCIN5 263 NH--HDYYILGSYMPENDKFSVETNFMDSGVDLRYDYGKFYASKTFFDGAMNRRILWGWI PtVIN1 344 DDVRKDSYALGTYDDKTGKWYPDNPEIDVGIGIMLDYGMFYASKTFYDQDKGRRVLWGWV PtVIN2 357 DDDRHDYYALGTYADKVGKWYPDNPEIDVGIGIRYDYGIFYASKTFYDQSKGRRVLWGWI PtVIN3 340 DDTKRDHYAIGVYDPVTDKWTPDNPKEDVGIGLQVDYGRYYASKTFYDQNTQRRILWGWI PtCIN1 333 NESDDPQKDKDKGWAGIQLIPRKVWLD-PSGKQLLQWPVAELEKLRGHNVQLSNQMLDQG PtCIN2 332 NESDAVQQDTDKGWAGILLIPRKVWLD-PSGKQLLQWPVAELEKLRGHNVQLSNQMLDQG PtCIN3 332 NESDSVQQDKNKGWAGIQLIPRRVWLD-PSGKQLLQWPVAELEKLRSHNVQLRNQKLYQG PtCIN4 331 NESDSIEDDMDKGWSGLQSIPRHIWLD-RSGKQLVQWPIEEINKLHGKKVSFLDKKIDSE PtCIN5 321 NESDSESDDIKKGWSGLQSIPRTVLLS-KNGKQIVQWPVKEIEKLRSKNVSFHDKKLKSG PtVIN1 404 AESDTEVDDVKKGWASLQGIPRTILLDTKTSSNLLQWPVEEVERLRLKGKEFNNIEVKTG PtVIN2 417 GESDSEVADVKKGWASLQGIPRTVVLDTKTGSNLLQWPVEEVESLRLKSKNFNNIEVKAG PtVIN3 400 NETDTETDDLDKGWASVQTIPRKVLYDNKTGTNILQWPVEEIEGLRLRSTDFTEIVVGPG PtCIN1 392 NHVEVKVITAAQADVDVTFSFSSLDKAEPFDPKWAKLDALDVCAQKGSKDPGGLGPFGLL PtCIN2 391 NHVEVKVITAAQADVDVTFSFSSLDKAEPFDPKWAKLDALDVCAQKGSKAPGGLGPFGLL PtCIN3 391 YHVEVKGITAAQADVDVTFSFPSLDKAEPFDPKWAKLDALDVCAQKGSKAQGGLGPFGLL PtCIN4 390 SIFEVQGITAAQADVEVVFELPELQETEFLN--LTAVDPQLLCSDANASIKGRLGPFGLL PtCIN5 380 SVLEVPGITASQADVDVSFELLNLEDAEILD--PSWTDPQLLCSQKKASVRGKLGPFGLL PtVIN1 464 SVMPLELDGATQLDIAAEFELDKKALESTAESNVDFSCSTSGG----AAQRGALGPFGLL PtVIN2 477 SAVPLELDGATQLDIVAEFELDRKAIERTAESNVEFSCSTNGG----ASHRGALGPFGLL PtVIN3 460 SVVPLDIGQATQLDIFAEFEIEIISETKHEKY------GCSGG----AVDRSALGPFGLL PtCIN1 452 TLASENLEEFTPVFFRVFKAADK-HKVLLCSDAR-------SSSLGKELYKPSFAGFVDV PtCIN2 451 TLASENLEEFTPVFFRVFKAVDK-HKVLLCSDARRFLASLNSSSLGEELYKPSFAGFVDV PtCIN3 451 TLASEKLEEFTPVFFRVFKAADK-HKVLLCSDAR-------SSSLGVGLYKPPFAGFVDV PtCIN4 448 TLATKDLTEQTAIFFRIFKGLKG-YVVLMCSDQS-------RSALRDEVDKTTYGAFIDI PtCIN5 438 AFATKDLKEQTAIYFRIFRSNHK-YIVLMCSDQS-------RSSVREELDKTTYGAFVDM PtVIN1 520 VLADDSLAEHTSVYFYVAKGNNGTHKTFFCTDQS-------RSSVANDVKKEIYGSYVPV PtVIN2 533 VLADDDLTEYTPVYFFVAKGNNGSLKTFFCTDQS-------RSSVANDVRKEIYGSYVPV PtVIN3 510 VVADQTLSELTPIFFRPVNTTEGIVETYFCADET-------RFVSQIDLLNSVYGSTVPV

52

PtCIN1 504 DLTDKKLSLRSLIDHSVVESFGAGGRIAISSRVYPTIAVFENAHLYVFNNGSETITVENL PtCIN2 510 DLTDKKLSLRSLIDHSVVESFGAGGRTAITSRVYPTIAVFEKAHLYVFNNGSETITVENL PtCIN3 503 DLTDKKLTLRSLIDHSVVESFGAGGRTVITSRVYPIIAVFDKAHLFVFNNGSETVTVETL PtCIN4 500 DPQRENISLR-SLDHSIIESFGGEGRACITNRVYPKLAIQEEARLFIFNNGTLSVTISSL PtCIN5 490 DPRHEIITLRSLIDHSIVESFGGEGRACITTRAYAKLAIHKQAYLFAFNNGTSSVKISRL PtVIN1 573 LE-GEKLSVRILVDHSIVESFAQGGRTVITSRVYPTRAIYGAARLFLFNNAIEATVTSSL PtVIN2 586 LE-GEKLSVRILVDHSIIESFAQGGRTCITSRVYPTRAIYGSARLFLFNNATEAGVTSSL PtVIN3 563 FT-DEKFQMRVLVREIDLLSFAQGGRRVITSRIYPTKAIYGDARLFLFNNATGVNVKATL PtCIN1 564 NAWSMNTPVMNVPVKS--------- PtCIN2 570 NAWSMNLPVMNVPIKNRGGENPRNE PtCIN3 563 NAWSMKVPVMNVPVKS--------- PtCIN4 559 NAWSMNKAQINHKENFI-------- PtCIN5 550 NAWSMKNAQIVSTTKRRKPHL---- PtVIN1 632 KIWQMNSAFIRRYSNEQ-------- PtVIN2 645 KIWNMNSAFIRPYSNEQQ------- PtVIN3 622 KIWELNSAFIHPFLFDQN------- B PtNIN9 1 -MSSLDGDVSQNGSLKSVDAHPALAEIEDLDFSRILDKPPRPLNMERQRSCDER-----S PtNIN11 1 -MSSINVDVSLKGSLRNAETLCDMAEIEEMDFSRIFDRPPRPLNMDRQRSCDER-----S PtNIN8 1 -----MDATKETVGLMNGSSVWSISEMDDIDFSRLSDKPK--LNIERKRSFDERSLSELS PtNIN12 1 -----MDGTKEMGGLRNVSSVCSISEMDDFDLSRLLDKPK--LNIERQRSFDERSLSELS PtNIN7 1 MSPIAAMDVCQNASVKNFEAAGSIFEIDSEFLR--LSDKPRPVNVERKRSFDERSFS--- PtNIN10 1 ------------------------------------------------------------ PtNIN2 1 ------------------------------------------------------------ PtNIN5 1 -----MRPSCRFFLSKKNRVFFNLHHSLTSNLSGNQFNFEKNKQFFTYPFRILGSRTIFK PtNIN1 1 ------------------------------------------------------------ PtNIN3 1 -----MATSDAVLQVLSGAGPRSFSSDLCFNNLDLAFR-SKHIKYVKKRASRHMKMLECS PtNIN4 1 -----MATTEAILQVLSGAGPCVFSSDPCFRSSDLTFSSKLHIKRVKKRASRCMKMFECS PtNIN6 1 -----MATSKTVLQVLSGGLPCPHRFDLSFGGLNSVLSICSDVKRRKNIGLVYKKLNNGM PtNIN9 55 LNELFG-VPLLSPRPSSRAESNFRLIDHLDGLYSPGRRSGFN------------------ PtNIN11 55 LSELSTGLPIPSPRPSSRVENNFRLIDHLNCLPSPGRRSGFN------------------ PtNIN8 54 IGLARG-------------------IDNFETTNSPGGRSGFN------------------ PtNIN12 54 IGLARG-------------------IDTFETTYSPGGRSGFN------------------ PtNIN7 56 -------------------ENSFRIIDHLENLSPAGRRSGFN------------------ PtNIN10 1 ------------------------------------------------------------ PtNIN2 1 ------------------------------------------------------------ PtNIN5 56 EAQKSFCAPYISFGQSRLITGDFRGASIVASVASQVRKFSTSVETRVNDNNFERIYVQNG PtNIN1 1 ------------------------------------------------------------ PtNIN3 55 SVQQNCIGKHWFKRSGDGDLSVNATIKRLQLLRCKCQKAERVSGVTTEGGNGTWFVDSAK PtNIN4 56 NVLQNGIGNHWFKGLGDRDRSVNATINRLQLLRCKGPQAERVSGVTE-GGNGTWFVDGAN PtNIN6 56 RLLGKCRSRGVG---AVTSRGKVKCIDRWESMRCKCQKAESFGGATANEWSPVSLPVNGV

53

PtNIN9 96 --------------------------------------------------TPRSQYG--F PtNIN11 97 --------------------------------------------------TPLSQFG--V PtNIN8 77 --------------------------------------------------TPASSARNSF PtNIN12 77 --------------------------------------------------TPASSTRNSF PtNIN7 79 --------------------------------------------------TPR---SCGF PtNIN10 1 ------------------------------------------------------------ PtNIN2 1 ------------------------------------------------------------ PtNIN5 116 IGIKPLVVER----------------------------------------IDKDENVLGD PtNIN1 1 ------------------------------------------------------------ PtNIN3 115 TLNLNGAVN--TPGVLELGDTQQLMREKEVLTSNGSANKEEESLATNGAVGTGRDASRKV PtNIN4 115 TLNQNGAVTGEHTDCFGAWDAQQLTREKEGFASKAALNQEKESLATNGAVGTGRDASPKV PtNIN6 113 HG------------------------------------------ATNIFEKGSFALKGNE PtNIN9 104 ETHPAVAEAWDALRRSLVVFRGQPVGTIAALDNT-GEQLNYDQVFVRDFVPSALAFLMNG PtNIN11 105 ETHPTVAEAWEALRRSLVYFRGEPVGTIAALDNS-EEQVNYDQVFVRDFVPSALAFLMNG PtNIN8 87 EPHPMVADAWEALRRSLVFFRGQPVGTIAAYDHASEEVLNYDQVFVRDFVPSALAFLMNG PtNIN12 87 EPHPMVADAWEALRRSLVYFRGQPVGTIAAYDHASEEVLNYDQVFVRDFVPSALAFLMNG PtNIN7 86 ESHPMVVDAWESLRRTLVYFRSQPVGTIAALDHS-VEELNYDQVFVRDFVPSALAFLMNG PtNIN10 1 ----MVDEAWERLNKSYVYFKGKPVGTLAAMDTS-ADALNYNQVFVRDFVPTGLACLMKE PtNIN2 1 ------------------MYCGSPVGTVAANDPGDKMPLNYDQVFVRDFVPSALAFLLRG PtNIN5 136 EESRIGVLVDDCESVNRENLDGGQEVEIVSPKREESEIEKEAWKLLNDAVVMYCGSPVGT PtNIN1 1 ------------------------------------------------------------ PtNIN3 173 SVDPTEEEAWELLRDSVVHYCGSPIGTIAANDPTSSSVLNYDQVFIRDFIPSGIAFLLKG PtNIN4 175 SVDPIEEEAWELLRNSMVYYCGSPIGTIAANDPTSSSVLNYDQVFIRDFIPSGIAFLLKG PtNIN6 131 ETQSIEEEAWDLLRASVVCYCGNPIGTIAANDPNSTSILNYDQVFIRDFIPSGIAFLLKG A PtNIN9 163 --EPEIVKNFILKTLRLQSWEKKIDRFHLGEGVMPASFKVLHDPVRNSE------TLMAD PtNIN11 164 --EPEIVKNFILKTLRLQSWEKKIDRFQLGEGVMPASFKVLHDPVTHNE------TLMAD PtNIN8 147 --EPEIVKQFLLKTLHLQGWEKRIDRFKLGEGAMPASFKVLHDPIRKTD------SLVAD PtNIN12 147 --EPDIVKHFLLKTLYLQGWEKRIDRFKLGEGAMPASFKVLHDPIRKTD------SLVAD PtNIN7 145 --EHEVVRNFLLKTLHLQSREKMVDQFKLGAGVMPASFKVLHHPDRNIE------TLMAD PtNIN10 56 PPEPEIVRNFLLKTLHLQGLEKRVDNFTLGEGVLPASFKVLYDSDLEKE------TLLVD PtNIN2 43 --EGEIVKNFLLHALQLQSWEKTVDCYSPGQGLMPASFKVRTVPLD--D-NNLEEVLDPD PtNIN5 196 --VAANDPGDKMPLNYDQSWEKTVDCYSPGQGLMPASFKVRTVPLD--D-SKFEEVLDPD PtNIN1 1 ---MEIVKNFLLHTLQLQSWEKTVDCYSPGQGLMPASFKVKTVPLDGSD-GGFEEVLDPD PtNIN3 233 --EYDIVRNFLLHTLQLQSWEKTMDCHSPGQGLMPASFKVRTFPLDGDD-SATEEVLDPD PtNIN4 235 --EYDIVRNFLLHTLQLQSWEKTMDCHSPGQGLMPASFKVRTVRLDGDDDFATEEVLDPD PtNIN6 191 --EYDIVRNFILYTLQLQSWEKTMDCYSPGQGLMPASFKVRTVPLDSED-SATEEVLDAD * B PtNIN9 215 FGESAIGRVAPVDSGFWWIFLLRAYTKSTGDTSLAEMPECQKGMRLILSLCLSEGFDTFP PtNIN11 216 FGESAIGRVAPVDSGFWWIFLLRAYTKSTGDTSLAEKPECQKGMRLILSLCLSEGFDTFP PtNIN8 199 FGESAIGRVAPVDSGFWWIILLRAYTKSTGDLSLAETPECQKGMRLILTLCLSEGFDTFP PtNIN12 199 FGESAIGRVAPVDSGFWWIILLRAYTKSTGDLSLAERPECQKGMKLILTLCLSEGFDTFP PtNIN7 197 FGESAIGRVAPVDSGFWWIILLRAYTKSTGDSSLAEMPECQRGMRLILNLCLSEGFDTFP PtNIN10 110 FGASAIGRVAPVDSGFWWIILLRSYIKRTRDYALLDRPEVQNGMKLILKLCLSDGFDTFP PtNIN2 98 FGESAIGRVAPVDSGLWWIILLRAYGKLTGDYALQERVDVQTGIKLILNLCLADGFDMFP PtNIN5 251 FGESAIGRVAPVDSGLWWIILLRAYGKLTGDYALQERVDVQTGIKLILNLCLTDGFDMFP PtNIN1 57 FGESAIGRVAPVDSGLWWIILLRAYGKITGDYALQERVDVQTGIRLGLNLCLSDGFDMFP PtNIN3 290 FGEAAIGRVAPVDSGLWWIILLRAYGKCSGDLSVQERIDVQTGIKMILRLCLADGFDMFP PtNIN4 293 FGEAAIGRVAPVDSGLWWIILLRAYGKCSGDLSLQERIDVQTGIKMILRLCLADGFDMFP PtNIN6 248 FGEAAIGRVAPVDSGLWWIILLRAYGKCSGDLSVQERVDVQTGMKMILRLCLADGFDMFP

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C * D E F PtNIN9 275 TLLCADGCCMIDRRMGVYGYPIEIQALFFMALRCALLLLKQDEEGKEFVERITKRLHALS PtNIN11 276 TLLCADGCCMVDRRMGVYGYPIEIQALFFMALRCALLLLKQDEEGNEFVERITKRLHALS PtNIN8 259 TLLCADGCSMIDRRMGIYGYPIEIQALFFMALRSACSLLKHDEEGKECIERIVKRLHALS PtNIN12 259 TLLCADGCSMIDRRMGIYGYPIEIQALFFMALRSASSMLKHDQEGNEFIERIVKRLHALS PtNIN7 257 TLLCADGCCMIDRRMGVYGYPIEIQALFFMALRCALILLKQDDEGKEFVDRVATRLHALS PtNIN10 170 TLLCADGCSMIDRRMGIYGYPIEIQALFYFALRCAKQMLKPELDGKEFIERIEKRITALS PtNIN2 158 SLLVTDGSCMIDRRMGIHGHPLEIQALFYSALRSSREMLVVNDGSKNLVRAINNRLSALS PtNIN5 311 SLLVTDGSCMIDRRMGIHGHPLEIQ------------MIVVNDGSKNLVRAINNRLSALS PtNIN1 117 TLLVTDGSCMIDRRMGIHGHPLEIQALFYSALRCAREMLIVNDETKNLVAAINNRLSALS PtNIN3 350 TLLVTDGSCMIDRRMGIHGHPLEIQALFYSALLCAKEMLAPEDGSADLLRALNNRLVALS PtNIN4 353 TLLVTDGSCMIDRRMGIHGHPLEIQALFYSALLCAREMLAPEDGSADLIRALNNRLVALS PtNIN6 308 TLLVTDGSCMIDRRMGIHGHPLEIEALFYSALLCAREMLAPEDGSADLIRALNNRLVALS G H IJ PtNIN9 335 FHMRSYYWIDLKQLNDIYRYKTEEYSHTAVNKFNVIPDSLPEWIFDFMPVHGGYFIGNVS PtNIN11 336 FHMRSYYWIDLKQLNDIYRYKTEEYSHTAVNKFNVIPDSLPEWIFDFMPVRGGYFIGNVS PtNIN8 319 YHMRSYFWLDFQQLNDIYRYKTEEYSHTAVNKFNVIPDSIPDWVFDFMPTRGGYFIGNVS PtNIN12 319 YHMRSYFWLDFQQLNDIYRYKTEEYSHTAVNKFNVIPDSIPDWVFDFMPIRGGYFIGNVS PtNIN7 317 YHMRNYFWLDMKQLNDIYRYKTEEYSHTAVNKFNVMPDSLPDWVFDFMPTRGGYFIGNVS PtNIN10 230 YHIQTYYWLDFTQLNNIYRYKTEEYSHTAVNKFNVIPESIPDWVFDFMPLRGGYLIGNVS PtNIN2 218 FHIREYYWVDMRKINEIYRYKTEEYSTEATNKFNIYPEQIPSWLMDWIPEEGGYLIGNLQ PtNIN5 359 FHIREYYWVDMNKINVIYRYKTEEYSTEATNKFNIYPEQIPSWLMDWIPEEGGYLIGNLQ PtNIN1 177 FHIREYYWVDMRKINEIYRYNTEEYSTDAVNKFNIYPDQIPSWLVDWIPEEGGYLIGNLQ PtNIN3 410 FHIREYYWIDLRKLNEIYRYKTEEYSYDAVNKFNIYPDQVSPWLVEWMPNQGGYLIGNLQ PtNIN4 413 FHIREYYWIDLRKLNEIYRYKTEEYSYDAVNKFNIYPDQISPWLVEWMPNQGGYLIGNLQ PtNIN6 368 FHIREYYWIDLKKLNEIYRYTTEEYSYDAVNKFNIYPDQIPPWLVEFMPNKGGYLIGNLQ K PtNIN9 395 PAKMDFRWFCLGNCIAILSSLATPEQSTAIMDLIESRWEELVGEMPLKVIYPAIESHEWR PtNIN11 396 PARMDFRWFCLGNCIAILSSLATPEQSTAIMDLIESRWEELVGEMPLKVIYPAIESHEWR PtNIN8 379 PARMDFRWFALGNCIAILSSLATHEQAMAIMDLIEARWEELVGEMPLKIAYPAIESHEWR PtNIN12 379 PARMDFRWFALGNCIAILSSLATHEQAMAIMDLIEARWEELVGEMPLKIAYPAIESHEWR PtNIN7 377 PARMDFRWFCLGNCVAILSSLATPEQASAIMDLIESRWEELVGEMPLKICYPALESHEWR PtNIN10 290 PARMDFRWFLVGNCVAILSSLVTPAQATAIMDLVEERWEDLIGEMPLKITYPALEGHEWR PtNIN2 278 PAHMDFRFFTLGNLWSVVSSLGTPKQNEAVLNLIESKWDDLVGNMPLKICYPALESEDWR PtNIN5 419 PAHMDFRFFTLGNLWSVISSLGTPKHNEAILNLIEAKWDDLVGNMPLKICYPALEHEDWR PtNIN1 237 PAHMDFRFFTLGNLWAIVSSLGTSKQNEGILNLIEARWDDLMGHMPLKICYPALEYEEWR PtNIN3 470 PAHMDFRFFSLGNIWSVVSGLATRDQSNAILDLIEAKWSDLVADMPLKICYPALEGQEWQ PtNIN4 473 PAHMDFRFFSLGNIWSIVSGLATRDQSNAILDFIEAKWSDLIADMPLKICYPALEGQEWQ PtNIN6 428 PAHMDFRFFTLGNLWSIVSSLATLDQSHAILDLIEAKWAELVAEMPIKICYPALEGQEWR L M PtNIN9 455 IVTGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQIARRAIELAETRLVKDNWPEYY PtNIN11 456 IVTGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQIARRAIELAETRLIKDNWPEYY PtNIN8 439 IVTGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQIARKAIDLAETRLLKDSWPEYY PtNIN12 439 IVTGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQIARKAIDLAETRLLKDGWPEYY PtNIN7 437 TVTGCDPKNTRWSYHNGGSWPVLLWLLTAACIKTGRPQIARRAIELAESRLSKDHWPEYY PtNIN10 350 LVTGFDPKNTRWSYHNGGSWPMLLWLLSAACIKVGRPQIAKRAIELAEQRLSKDGWPEYY PtNIN2 338 IITGSDPKNTPWSYHNGGSWPTLLWQFTLACMKMDRMELAQKAIALAEKRLQVDHWPEYY PtNIN5 479 IITGSDPKNTPWSYHNGGSWPTLLWQFTLACIKMNRVELAQKAIALAEKRLQVDHWPEYY PtNIN1 297 IITGSDPKNTPWSYHNGGSWPTLLWQFTLACIKMGKPELAQKAIALAETRLSMDQWPEYY PtNIN3 530 IITGSDPKNTPWSYHNAGSWPTLLWQLTVACIKMNRPEIAARAVDIAEKRISRDKWPEYY PtNIN4 533 IITGSDPKNTPWSYHNAGSWPTLLWQLTAACIKMNRPELAARAVEIAEKRISRDKWPEYY PtNIN6 488 IVTGSDPKNTAWSYHNGGSWPTLLWQLTVACIKMNRPEIAERAVQLVERRISRDKWPEYY

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N O PtNIN9 515 DGKLGRFVGKQARKFQTWSIAGYLVAKMLLEDPSHLGMVALEEDKQMKPPMRRSHSWTF- PtNIN11 516 DGKLGRFIGKQARKSQTWSIAGYLVAKMMLEDPSHLGTVALEEDKQMKPPIRRSNSWT-- PtNIN8 499 DGKLGRYIGKQARKYQTWSIAGYLVAKMMLEDPSHLGMISLEEDKQMNPVLKRSSSWTC- PtNIN12 499 DGKLGRYVGKQARKYQTWSIAGYLVAKMMLEDPSHLGMISLEEDRQMKPVLRRSSSWTC- PtNIN7 497 DGKLGLYVGKQARKFQTWSIAGYLVAKMMLEDPSHLGMISLEEDKQITHLVKRSASWTC- PtNIN10 410 DGKTGRYVGKQARKYQTWSIAGYLVAKMMVENPSNLLMISLEEDKKSARSRLTRSNSTSF PtNIN2 398 DTRSGKFIGKQSRLYQTWTVAGFLTSKVLLENPEKASLLFWDEDYDLLEFCVCGLNTSGR PtNIN5 539 DTRTGKFIGKQSRLYQTWTVAGFLTSKILLENPQRASLLFWDEDYELLEICVCGLNTSGR PtNIN1 357 DTRSGRFIGKQSRLFQTWTISGFLTSKMLLENPDKASLLFLEEDYELLEICVCALSKTGR PtNIN3 590 DTKKARFIGKQARLFQTWSIAGYLVAKLLLADPSAARMLVTDEDPELVNAFSCMISSNPR PtNIN4 593 DTKKARFIGKQAHLFQTWSIAGYLVAKLLLADPSAARMLVMDEDPELVSAFSCMISTHPR PtNIN6 548 DTKRARFIGKQAHLFQTWSISGYLVAKLFLANPSAAKIFVNEEDPELVN---ALISANPR PtNIN9 -------------- PtNIN11 -------------- PtNIN8 -------------- PtNIN12 -------------- PtNIN7 -------------- PtNIN10 470 -------------- PtNIN2 458 KRCSRVAARSQILV PtNIN5 599 KRCSRGAAKSQILV PtNIN1 417 KKCSRFAARSQILV PtNIN3 650 RKRGQKNSKKPFIV PtNIN4 653 RNRGQKNSKKTFMV PtNIN6 605 RKRARKIFKQPFIV Figure 2-5. Amino acid alignment of poplar invertases. A) Amino acid alignment of acid

invertases. Black shading corresponds to identical residues, grey shading corresponds to similar residues. A-F denote highly conserved motifs as defined by Pons et al. (1998) which are thought to play roles in activity. Asterisks mark completely conserved residues thought to be involved in substrate binding or catalysis (Alberto et al. 2004). B) Amino acid alignment of neutral/alkaline invertases. A-O correspond to amino acids that are consistently different between α and β group invertases. Asterisks show amino acids most likely to correspond to active residues for neutral/alkaline invertases, assuming equivalence with the catalytic residues of unsaturated glucuronyl hydrolase (UGL) from Bacillus sp. (Itoh et al. 2004). Black shading corresponds to identical residues, grey shading corresponds to similar residues. Alignment produced with CLUSTALW and figure produced with BOXSHADE.

56

CHAPTER 3 RECIPROCAL SUGAR REGULATION IS CONSERVED AMONG VACUOLAR

INVERTASES OF POPLAR, ARABIDOPSIS, MAIZE AND RICE

Introduction

Sugars play an essential role in plant cell growth and development as they act as fuel for

metabolism, generate osmotic pressure for short and long distance transport and act as signaling

molecules for various metabolic pathways (Koch, 1996; Rolland et al., 2002; Halford and Paul,

2003). Sucrose is the primary form of sugar transport in most plants, establishing this

disaccharide and its glucose and fructose cleavage products as central to plant growth and

development.

Invertase (EC 3.2.1.26), also known as β-fructofuranosidase, irreversibly hydrolyzes

sucrose into glucose and fructose, and is positioned to play a central role in both carbon

metabolism and sugar signaling. Several invertase isozymes have been identified from plant

species as either soluble (readily extractable from cytosol or vacuole), or insoluble (bound to cell

wall components). Vacuolar and cell wall invertases show optimal activity at an acidic pH and

are also called acid invertases. The mature acid invertases are glycosylated and located in the

cellular compartment their name implies (e.g. cell wall or vacuolar).

Vacuolar invertases (VIN), like their cell wall localized family members, are thought to

have evolved from respiratory eukaryotes and aerobic bacteria (Sturm and Chrispeels, 1990a).

VINs have been identified and characterized to various levels in many different plant species

including, but not limited to, Arabidopsis, carrot, maize, poplar, rice, tobacco and tomato

(Tymowska-Lalanne and Kreis, 1998b; Ji et al., 2005; Bocock et al., 2007). To my knowledge,

with the exception of poplar (containing three), all plants studied to date encode only two VINs.

Of poplar’s three VINs, one contains no introns and is thought to be a recent evolutionary

57

innovation stemming from a processed transcript inserted in trans back into the genome (Bocock

et al., 2007).

VIN transcripts have been found to be reciprocally regulated by sugar. In maize, Ivr1 was

found to be transcriptionally repressed after treatment with exogenous glucose under the same

conditions that induced transcription of Ivr2 (Xu et al., 1996). In tomato, the VIN, TIV1, was also

found to be repressed after treatment with exogenous sugar, however, induction of the second

tomato VIN under the same conditions was not shown (Godt and Roitsch, 1997a).

An important driver of species origination and diversification is gene duplication.

Duplication makes it possible for a gene to acquire new functions without losing that of the

progenitor gene (Kramer et al., 1998; Lynch and Conery, 2000; Sankoff, 2001; Becker and

Theissen, 2003; Irish, 2003; Litt and Irish, 2003; Zahn et al., 2005). Gene duplication can occur

in tandem, through the duplication of a chromosomal segment, an entire chromosome, or through

genome duplication (Otto and Whitton, 2000; Wendel, 2000; Adams and Wendel, 2005).

Genome duplications give rise to copies of linked genes in a particular order that can be used to

infer ancestry of loci of interest both within and between species (Lynch and Conery, 2000).

Poplar, Arabidopsis, maize and rice are all known to have undergone multiple genome

duplication events in their evolutionary history. Poplar and Arabidopsis have each undergone a

genome duplication event that post-dates their divergence on the evolutionary tree (Bowers et

al., 2003; Sterck et al., 2005; Tuskan et al., 2006). Poplar and Arabidopsis also share at least two

genome duplication events that pre-date their evolutionary divergence and are referred to as the β

and γ genome duplication events (Bowers et al., 2003; De Bodt et al., 2005; Tuskan et al., 2006).

On the monocot side of the evolutionary tree, maize is known to have undergone two genome

duplication events, one post- and the other pre-dating its divergence with rice (Blanc and Wolfe,

58

2004). The γ event referred to previously is thought to have occurred prior to the monocot-

eudicot divergence. Some evidence suggests that the γ event may even pre-date the gymnosperm

divergence, although this conclusion requires more data (Bowers et al., 2003). The timing and

location of genomic duplication events can be utilized to understand the evolutionary

development of a variety of gene families.

In this work, the sequenced genomes of poplar, Arabidopsis, and rice are compared in

order to test possible scenarios for the evolutionary development of the VIN family. I also take

advantage of the unique reciprocal sugar regulation that occurs in this family to analyze the

functional conservation of VIN in monocots and eudicots. This evidence indicates that the two

VINs so common throughout the plant world arose at, or prior to the γ genome duplication event.

I also examined the reciprocal sugar response in Pinus taeda, a gymnosperm, but was unable to

validate the presence of this reciprocal response.

Materials and Methods

Plant Material

Populus trichocarpa (genotype “Nisqually-1”) and Pinus taeda were grown in 8 L pots in

a fan- and pad-cooled greenhouse with natural light augmented with full spectrum fluorescent

lighting during the winter to give a day length of 15 h. Greenhouse temperatures ranged from 20-

35 °C. At noon, light intensity in the greenhouse averaged 500-700 µE/m2/min PAR, which is

one-half the light intensity outside the greenhouse. Plants were grown on an ebb-and-flow flood

bench system with a daily supply of Peters Professional® 20-10-20 water-soluble fertilizer

diluted to a final concentration of 4 mM nitrogen. Plants were grown to a height of 60-100 cm

prior to experimentation.

Arabidopsis plant material was prepared as described by Huang (2006). Briefly,

Arabidopsis thaliana (Col-0) seeds were sterilized and soaked in water for 4-5 d at 4°C in the

59

dark. Seeds were sown on half-strength Murashige and Skoog (MS) medium, pH 5.8

(GIBCOBRL, U.S.A.) solidified with 0.16% (w/v) phytagel. The typical sugar supplement for

enhanced plant growth was omitted. Plantlets were cultured under cycles of 12 h light/dark at 25

°C. All plant material was immediately frozen in liquid nitrogen after harvesting, and stored at –

80 °C prior to RNA extraction.

Sequence Alignments and Similarity Trees

Predicted amino acid sequences were aligned using CLUSTALW

(http://clustalw.genome.jp) to construct similarity trees using the TREEVIEW program (Page,

1996). PAUP (Swofford, 1993) was used for bootstrap analysis with 100,000 iterations.

RNA Extraction

Total RNA was extracted from poplar and pine leaves using standard methods (Chang,

1993), DNase-treated and purified on RNAeasy QIAGEN columns (Valencia, CA). Total RNA

was extracted from Arabidopsis plants and rice embryos using the RNeasy Plant Mini Kit

(QIAGEN) and DNase treated (DNA-free Kit, Ambion, Austin, TX) according to manufacturers’

instructions.

Quantitative RT-PCR in Poplar and Pine

Poplar and pine cDNA was synthesized from purified RNA (5 μg) using a mixture of 500

ng oligo-dT, 100 ng random primers, and M-MLV-RT as per manufacturer’s instructions

(Invitrogen, Carlsbad, CA), with the exception that the DTT was excluded. Gene expression was

analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and Mx3000P thermo-cycler

(Stratagene) as per manufacturer’s instructions. Briefly, 1 μl of synthesized cDNA and 0.15 μl of

a 0.25 μM solution of each primer were used for each 50 μl RT-qPCR reaction. Primers were

designed using NetPrimer (Premier Biosoft International, Palo Alto, CA) software and

synthesized by Invitrogen. Primer sequences are as follows: PtVIN1 forward, 5’-

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GCCCTGGTTCCACCTATAGTT-3’; reverse, 5’-TCCTGGCCTCAAAATTTGC-3’ (yields 216

bp product); PtVIN2 forward, 5’-CCCAGCTATAGTCCTGCCC-3’; reverse, 5’-

CGCATCTTCGTCTTCTTGTG-3’ (yields 189 bp product); PtVIN3 forward, 5’-

ATACACTCCCTTGCTAGACAACC-3’; reverse, 5’-GCTTGGAAAATGACTCTGTAGGTC-

3’(yields 197 bp product); PtaedaVIN1 forward, 5’-TGATTCCCGACCGCTGG-3’; reverse, 5’-

ATTAGCCTCCGACTTCACCC-3’ (yields 185 bp product); PtaedaVIN2 forward, 5’-

GGGCTGCGGTATGATTATGG-3’; reverse, 5’-CTGGAGCAGCACATTCTCG-3’ (yields 261

bp product). Each real-time PCR reaction was performed in triplicate (technical replicates) on

four individual plants (biological replicates) in poplar and six individual plants in pine and

carried out for 40 cycles with annealing, extension, and melting temperatures of 55 °C, 72 °C,

and 95 °C, respectively. Melting curves were generated to check the specificity of the amplified

fragments. In the case of PtVIN2, an extension temperature of 79 °C with the fluorescence

reading taken at the end of the run was used to correct for a spurious primer-dimer amplicon.

Changes (n-fold) in gene expression relative to the geometric mean (Vandesompele et al., 2002)

of three control genes encoding actin, ubiquitin and ubiquitin_L (Brunner et al., 2004) were

determined using the program DART-PCRv1.0 (Peirson et al., 2003).

Quantitative RT-PCR in Arabidopsis

Real time quantitative RT-PCR in Arabidopsis was conducted as described by Huang

(2006). Briefly, 200 ng of RNA were used in 25 μl reactions with Taq-Man one-step RT-PCR

Master Mix reagents (Applied Biosystems, Foster City, CA) according to manufacturer’s

instructions.

61

Sugar Treatments for Poplar, Arabidopsis, Pine and Rice

Mature leaves (LPI 12) from four greenhouse grown poplars (biological replicates, one leaf

per plant, n=4) were covered in aluminum foil for three to five days. Leaves were then excised

and taken to a dark room where aluminum foil was removed. Leaves were lightly rubbed with a

carborundum suspension to remove the cuticle and subsequently cut in half (lengthwise). The

two leaf halves were placed in 1 % (w/v) mannitol (negative control) and 1 % (w/v) glucose,

respectively, and incubated in the dark at room temperature on a shaker. After a 16 h incubation

period, leaves were frozen in liquid nitrogen and RNA extracted.

Sugar treatments in Arabidopsis were conducted on two week old plants that were placed

in the dark for three days, after which they were transferred to 1/2 strength liquid MS minimal

media supplemented with 1 % (w/v) mannitol or glucose and incubated in the dark at room

temperature on a shaker. After a 16 h incubation period, plants were harvested.

Greenhouse grown Pinus taeda branches containing fully mature needles were wrapped in

aluminum foil for five days. Branches were then excised and taken to a dark room where

aluminum foil was removed. Needles were removed and then lightly rubbed with a carborundum

suspension to remove the cuticle. Needles from a single branch were then divided in half

between solutions of 1 % (w/v) mannitol and 1 % (w/v) glucose, respectively, and incubated in

the dark at room temperature on a shaker. After a 16 h incubation period, needles were frozen in

liquid nitrogen and RNA extracted. Needles were used from six different plants (biological

replicates, n=6).

Light-Dark Treatments for Poplar, Pine and Arabidopsis

Mature leaves (LPI 12) from four greenhouse grown poplars (biological replicates, n=4)

were covered in aluminum foil for five days. After the fifth day, half of the leaves were

uncovered and exposed to sunlight for approximately 5 h. At noon, wrapped and exposed leaves

62

were harvested and RNA extracted. Greenhouse grown Pinus taeda branches containing fully

mature needles from six plants (biological replicates, n=6) were wrapped in aluminum foil for

five days. After the fifth day, half of the branches were uncovered and exposed to sunlight for

approximately 5 h. At noon, needles from the wrapped and exposed branches were harvested and

the RNA extracted. Treatments in Arabidopsis were conducted on two week old plants that were

placed in the dark for three days, after which half were exposed to light for approximately 5 h

before plants were harvested and RNA extracted.

Results

Predicting Gene Orthology Using Protein Sequence Similarity

The hypothesis that the reciprocal regulation of VIN, first identified in the maize VINs Ivr1

(ZmIVR1) and Ivr2 (ZmIVR2),(Xu et al., 1996) is conserved not only in monocots, but also in

eudicots was tested. To my knowledge, all plants studied to date contain two VINs, with the

exception of poplar, which contains three. Poplar’s PtVIN1 is thought to be a very recent

innovation specific to poplar since it has no introns and likely arose through a processed

transcript of PtVIN2 inserting into the genome in trans (Bocock et al., 2007). I predict that the

poplar VINs PtVIN2 and -3 (not PtVIN1) are the likely othologs of ZmIVR1 and -2. Arabiodopsis

and rice both contain only two VINs and are referred to here as AtvacIVR1, AtvacIVR2, OsVIN1

and OsVIN2, respectively (Haouazine-Takvorian et al., 1997; Ji et al., 2005).

Nucleotide and protein sequences were obtained from the various databases (Joint Genome

Institute, poplar; NCBI, Arabidopsis and maize; TIGR, rice and maize). I then translated the

nucleotide sequences and constructed amino acid alignments between the four species (data not

shown). I discovered that ZmIVR2 (accession # CAD91358) as it exists in NCBI, is missing

information on the N-terminal end. In order to obtain more complete sequence, I searched the

TIGR Maize database. I found that ZmIVR2 corresponds to TIGR contig AZM5_84630.

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Subsequent analysis of the contig in GENESCAN (http://genes.mit.edu/GENSCAN.html) (Burge

and Karlin, 1997) allowed me to identify an additional 42 amino acids on the N-terminal end.

Based on amino acid alignments with the other VINs, I estimate that there are still roughly 140

amino acids missing from the N-terminal end (data not shown).

To obtain VIN sequences for pine, I searched for Pinus taeda ESTs on NCBI using

AtvacIVR1 and -2 as queries. The ESTs were then aligned and assembled using the Sequencher

program (Gene Codes, Ann Arbor, MI). This technique resulted in two, distinct contigs referred

to hereafter as PtaedaVIN1 and PtaedaVIN2. The lack of a third contig resulting from the

assembled ESTs indicates that pine, like the other plant species mentioned above, encodes only

two VINs. The nucleotide sequences were translated into amino acid sequences and protein

alignments revealed that PtaedaVIN1 and -2 are missing roughly 130 and 270 amino acids from

their N-terminal ends, respectively (data not shown).

The amino acid sequences were then analyzed in an attempt to identify the vacuolar

orthologs between species. As can be seen in Table 3-1, OsVIN1 and ZmIVR1 appear to be

orthologs as they are more similar to each other (82 % similarity) than they are to their family

members within each species. This pattern is repeated between OsVIN2 and ZmIVR2 (88 %

similarity) indicating that these two genes are also orthologous, based on sequence similarity. In

the case of poplar, Arabidopsis and pine, orthology cannot be determined. In Arabidopsis and

pine, the VINs are most similar to their family member within the species rather than their

putative orthologs in other species. In contrast, the poplar VINs in comparison to each other are

less similar than compared to any other VIN (Table 3-1).

These alignments were then used to generate a bootstrapped protein similarity tree (Figure

3-1). As expected from percent similarity, OsVIN1 and ZmIVR1 cluster together as do OsVIN2

64

and MzIVR2. However, the poplar, Arabidopsis and pine VINs tend to cluster within each

species rather than with their orthologs (Figure 3-1). Using this data, it was predicted that

ZmIVR1 and OsVIN1 are orthologs whose transcripts are sugar repressed while ZmIVR2 and

OsVIN2 encode sugar induced transcripts. It is not straightforward to predict orthology between

poplar, Arabidopsis and pine using this approach due to the lack of obvious pairwise clustering

of protein sequences among species.

A Chromosome Duplication Event is Responsible for the Two-Member VIN Family in Poplar and Arabidopsis

Large scale chromosomal or genome duplications are responsible for the growth of many

gene families (Sankoff, 2001). Chromosomal segments arising from genome duplication events

often retain a conserved linear order of loci. Conservation of loci order on a chromosomal

segment is not only evidence of gene family expansion through large scale chromosomal

duplication, but it also can serve as another method to be used in predicting orthology, and

therefore function. Figure 3-2A depicts the locations of several putative large scale or genomic

chromosomal duplications overlayed on an evolutionary tree outlining the development of

poplar, Arabidopsis, maize, pine and rice.

In a previous work, I examined the chromosomal segments containing the VINs in poplar

and Arabidopsis and identified neighboring open reading frames (ORF) that were used to

establish colinearity between the species (Bocock et al., 2007). In summary, I showed that poplar

and Arabidopsis VINs lie within conserved, colinear chromosomal segments (Figure 3-2B)

(Bocock et al., 2007). These data indicate that a chromosomal duplication event is responsible

for the development of the two-member VIN family in poplar and Arabidopsis. Conservation of

colinearity between the two species indicates that the duplication event must have occurred prior

to poplar and Arabidopsis divergence, likely in duplication event β or γ (Figure 3-2A).

65

I hypothesized that the duplication event giving rise to the two member VIN family

occurred prior to the eudicot and monocot divergence, which would explain the conserved two

member VIN family size in maize and rice. To address this question, I examined the

chromosomal segments containing the VINs in maize and rice to identify neighboring ORFs that

could be used to establish colinearity between maize, rice and the eudictos. Unfortunately, I was

unsuccessful in identifying neighboring ORFs in maize as the contigs containing ZmIVR1 and -2

were not large enough to contain any ORFs other than the VINs. In rice, I was able to identify

numerous ORFs upstream and downstream of OsVIN1 and -2 and compare these to the eudicots.

No evidence of colinearity was found between rice and the eudicots. This finding does not

support the hypothesis, however it also does not necessarily contradict the hypothesis as it is

quite common for small insertions, deletions and rearrangements to occur subsequent to genome

duplication events (Sankoff, 2001). If the VINs truly arose from the γ event, then there have been

approximately 221-300+ million years (Blanc and Wolfe, 2004) for rearrangements to occur and

obscure the duplication event.

Sugar Response Demonstrates Conservation of Gene Function in VINs

Based on amino acid sequence similarity, I hypothesized that OsVIN1 and ZmIVR1 are

orthologs and likely retain the same transcript repression to exogenous sugar. Similarly, I

predicted OsVIN2 and ZmIVR1 to be orthologous and retain the same transcript induction to

exogenous sugar. In poplar and Arabidopsis, the link is a bit more confusing. I was able to use

conservation of colinearity to establish chromosomal duplication as the likely originator of the

two VINs, however I was unable to use it to establish orthology.

I next examined the response of the VINs to exogenous sugar treatment to address the

above questions. Leaves from greenhouse-grown poplars were wrapped in foil for approximately

three to five days in order to clear the leaves of any carbohydrate reserves. The leaves were then

66

excised, treated with a suspension of carborundum to remove the cuticle, and incubated

overnight in solutions of either 1 % mannitol (to serve as an osmotic and wounding control) or

glucose in the dark. Transcripts for PtVIN1,2 and -3 were then assayed. I found that, like

ZmIVR1, transcripts for PtVIN2 were repressed in the presence of sugar (Figure 3-4A).

Transcripts for PtVIN3 were induced in the presence of sugar (data not shown). PtVIN1, the

intronless poplar invertase discussed earlier showed no sugar response at all. Transcripts for

PtVIN1 were also found to be two orders of magnitude less abundant than its presumed

progenitor, PtVIN2 (data not shown). I speculate that PtVIN1 lost at least some of the regulatory

features of PtVIN2 when it re-inserted into the genome.

Huang (2006) examined Arabidopsis plants for this sugar response. Whole plants grown on

sugar free media were transferred to the dark for approximately three days to clear the

carbohydrate reserves. The plants were then transferred to 1 % mannitol or glucose in the dark.

After incubation in the solutes, transcripts for AtvacIVR1 and -2 were assayed. Similar to

ZmIVR1 and PtVIN2, transcripts for AtvacIVR2 were found to be repressed (Figure 3-4A).

AtvacIVR1 was found to be induced under these conditions (data not shown).

In a similar fashion, Huang (unpublished data) treated rice embryos with 3 % sucrose,

which in poplar, Arabidopsis and maize incurs the same VIN response as glucose (data not

shown). OsVIN1 transcripts were found to be down-regulated as expected from OsVIN1’s

similarity to ZmIVR1 (Figure 3-4A).

Reciprocal Response in Light

Exogenous sugar treatments serve as an in vitro assay to mimic photosynthesis conditions

in vivo by recreating the high sugar environments associated with active photosynthesis. I

hypothesized that the VINs repressed by sugar would also be repressed by light, while the sugar

induced VINs would be induced by light. Leaves of greenhouse grown poplar were covered for

67

three to five days to clear the leaves of any starch reserves. Half of the foil covered leaves were

then uncovered and exposed to light. At noon on the same day, the covered and light exposed

leaves were harvested and transcripts associated with PtVIN2 and -3 were assayed. Similarly,

Arabidopsis plants grown on ½ strength MS media lacking sugars were placed in the dark for

three to five days. Half of the plants were then moved to the light and allowed to photosynthesize

for approximately 5 h. The plants were then harvested and transcripts for AtvacIVR1 and -2 were

assayed. As expected, light was found to repress transcripts for PtVIN2 and AtvacIVR2 while

inducing transcripts for PtVIN3 and AtvacIVR1 (Figure 3-3).

Discussion

In this study, I utilized newly released sequence information to establish orthology

between VINs in poplar, Arabidopsis, maize, pine and rice. I was able to establish clear orthology

between ZmIVR1 and OsVIN1 as well as ZmIVR2 and OsVIN2 using available nucleotide data,

however establishing orthology between the other species proved more difficult.

Through an examination of gene colinearity on chromosomal segments encoding the VIN

loci, I was able to establish that a chromosomal duplication gave rise to the two-member VIN

family in poplar and Arabidopsis. I was unable to establish conservation of colinearity between

the eudicots and rice, however a lack of sequence information from maize made it impossible to

compare the two monocots. The fact that the maize and rice genomes both appear to encode only

two VINs makes it likely that the chromosome duplication event giving rise to the two VINs in

the eudicots occurred prior to the eudicot-monocot divergence in the γ event. Convergent

evolution in the monocots could serve as an alternate explanation for the appearance of the two-

member VIN family in the monocots. This can only be ruled out by identifying a conservation of

colinearity on the VIN chromosomal regions between rice and another monocot, such as maize or

sorghum, as more genomic sequence becomes available. The grasses retain considerable

68

conservation of colinearity (Bennetzen and Ma, 2003), so it is not unreasonable to expect that the

predicted colinear regions will be identified.

It will be more difficult to test colinearity between the eudicot lines and those of the

monocot lines due to the extent of time and evolutionary change since their divergence. The

conservation of function described in this paper, however does go a long way in establishing

orthology between the monocot and eudicot VINs. While the conservation of reciprocal sugar

response cannot rule out the explanation of convergent evolution, the likelihood that only two

VIN family members would be encoded by such a diverse array of plant genomes and also retain

the same functions for such a long evolutionary time frame makes the convergent evolution

hypothesis extremely unlikely.

This work also describes the identification of two EST contigs from the gymnosperm,

Pinus taeda, that are highly similar to previously described VINs in other species. I name these

two contigs PtaedaVIN1 and PtaedaVIN2. These two putative VINs had good EST coverage for

nearly the entire coding region. As I was unable to find any ESTs that assembled into a third VIN

contig, I believe that PtaedaVIN1 and -2 are either the only two VINs encoded by the Pinus taeda

genome, or, at the very least, the only two significantly expressed VINs in this species. In order

to identify conservation of VIN function between the gymnosperms and angiosperms, I

conducted the all the same sugar and light/dark experiments as done for poplar and Arabidopsis.

I was unable to identify the same reciprocal response that was seen in poplar and Arabdidopsis.

It is unclear whether this is because the P. taeda VINs do not have the reciprocal regulation

feature as do the angiosperms, or that conditions tested did not effectively reveal these responses.

The possibility remains that additional experimentation could define a differential sugar

responsiveness in pine under somewhat different conditions.

69

In this study, I show that the reciprocal sugar regulation of VINs first identified in maize

(Xu et al., 1996) is conserved in at least three eudicot species. Sugar treatments were conducted

on poplar and Arabidopsis and demonstrate the reciprocal response in these two species. I also

demonstrate that sugar repressed OsVIN1 transcripts, as predicted based on its sequence

similarity to the sugar-repressed ZmIVR1. A search of the literature also revealed that TIV1, one

of tomato’s two vacuolar invertases is also repressed after treatment with glucose (Godt and

Roitsch, 1997a). Collectively, these data help to establish this sugar response as highly conserved

throughout plants.

Poplar and Arabidopsis plants were subjected to a light/dark experiment intended to mimic

the exogenous sugar treatment experiments. The purpose of this is two-fold. First, demonstrating

a commonality between exogenous sugar applications and light treatments helps to establish the

sugar-induced VINs as “feast” genes. Feast genes are those that are induced in response to sugars

and are thought to play roles in storage processes and carbon utilization. These genes would also

be up-regulated in actively photosynthesizing cells (Koch, 1996). This helps us to place these

sugar up-regulated genes in a more general context regarding their role in plant growth and

development. The second purpose of establishing the commonality of transcript response

between the light/dark treatment and the exogenous sugar treatment would be the ease of the

assay. For plants that are less amenable to experimental manipulation than poplar and

Arabidopsis, the light/dark treatment provides another tool for testing the reciprocal VIN

response.

Advances in genome sequencing are rapidly increasing available genomic sequence. This

abundance of data is allowing new types of questions to be asked as well as application of

functional data to the larger story of plant evolution. In this study of the VIN gene family, I have

70

added to our knowledge of the response of VINs to sugar and light treatments. I have tied this

into a larger, evolutionary story and discussed how this gene family has developed with the

evolution of plants. Hopefully this work has achieved its goal of aiding our understanding of how

plants process and utilize carbohydrates obtained through photosynthesis.

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Table 3-1. Percent similarity of predicted VINs from poplar, Arabidopsis, rice and maize. Protein names are in bold with the corresponding gene model names below.

PtVIN2 PtVIN3 AtvacIVR1 AtvacIVR2 OsVIN1 OsVIN2 MzIVR1 MzIVR2 PtaedaVIN1 PtaedaVIN2PtVIN2 69 77 76 77 69 72 76 74 74estExt_fgenesh4_pg.C_LG_III0902PtVIN3 69 71 71 67 71 70 71 70estExt_Genewise1_v1.C_LG_XV2841AtvacIVR1 85 70 67 69 71 73 71At1g62660AtvacIVR2 70 70 69 72 73 71At1g12240OsVIN1 77 82 76 74 73Os04g45290.1OsVIN2 74 88 70 69Os02g01590MzIVR1 76 73 75P49175MzIVR2 69 70AZM5_84630PtaedaVIN1 89

PtaedaVIN2

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AtvacIVR2 AtvacIVR1

PtVIN2

PtVIN3

OsVIN1

OsVIN2

ZmIVR1

ZmIVR2PtaedaVIN2

PtaedaVIN1

100

100

100

100

9886

91

Figure 3-1. Protein similarity tree of VINs. Bootstrap values are reported as a percentage of

100,000 repetitions. Branch lengths denote protein similarity with the exception of ZmIVR2, PtaedaVIN1 and -2 where the first ~140-230 amino acids have not yet been sequenced.

73

A

Poplar Arabidopsis Maize Rice

α

γ

β

Pine

?

B

4 Kb

no apical meristem(NAM) family protein

PtVIN3

Multicopper oxidaseTrehalose-6-phosphate phosphatase

no apical meristem (NAM) family protein

AtvacINV1

disease resistance protein(CC-NBS-LRR class)

3-oxoacyl-[acyl-carrier-protein] synthase III

flavin-containingmonooxygenasefamily protein

flavin-containingmonooxygenasefamily protein

disease resistance protein(CC-NBS-LRR class)

no apical meristem (NAM) family protein

AtvacINV2

Thylakoidlumenal protein-related

no apical meristem (NAM) family protein

flavin-containingmonooxygenasefamily protein

PtVIN2

Multicopper oxidase

Trehalose-6-phosphate phosphatase45 Kb45 Kb

3-oxoacyl-[acyl-carrier-protein] synthase III 32 Kb Thylakoid

lumenal protein-related

Figure 3-2. A) Phylogenetic representation of relevant plant species. Stars represent large scale

duplication events as proposed by Blanc and Wolfe (2004), Van de Peer (2004), and Sterck et al. (2005). Greek letters denote duplication events as described by Bowers et al. (2003). There is some uncertainty as to the location of the γ event relative to the divergence of the gymnosperms and angiosperms, this is denoted by “?”. B) Microcolinearity between poplar and Arabidopsis VINs. Boxes depict genes. Poplar VINs are checkered, and Arabidopsis VINs are solid, all other members of a particular gene family are depicted with matching patterns. Labels appear over the first appearance of a certain gene family member. Panel B adapted from Bocock et al. (2007).

74

A

PtVIN2

0

20

40

60

80

100

120

Mannitol Glucose

% m

RN

A

Actin

0%

OsVIN1

3%

SucRice embryos

AtvacIVR2

0

20

40

60

80

100

120

Mannitol Glucose

% m

RN

A

Figure 3-3. Conservation of specific VIN isoform transcript repression under sugar treatments

across taxa. A) PtVIN2, AtvacIVR2 and OsVIN1 transcripts are repressed by sugar. Bars represent the mean level of transcript; error bars denote SEM; n=4 and n=3 for PtVIN2 and AtvacIVR2, respectively. Rice panel contributed by L-F Huang. Suc, sucrose.

75

A PtVIN3

0

20

40

60

80

100

120

Dark Light

% m

RN

A

PtVIN2

0

20

40

60

80

100

120

Dark Light

% m

RN

A

B

AtvacIVR2

0

20

40

60

80

100

120

Dark Light

% m

RN

A

AtvacIVR1

0

20

40

60

80

100

120

Dark Light

% m

RN

A

Figure 3-4. Conservation of reciprocal regulation of VIN transcript under dark and light

treatments across taxa. A) Poplar’s PtVIN2 is repressed by light while PtVIN3 is induced by light. Bars represent the mean level of transcript; error bars denote standard error (SEM); n=3. B) Arabidopsis’ AtvacIVR2 is repressed by light while AtvacIVR1 is induced by light. Bars represent the mean level of transcript; error bars denote SEM; n=3. Panel B contributed by L-F Huang (2006).

76

CHAPTER 4 OVEREXPRESSION OF YEAST INVERTASE IN POPLAR

Introduction

Plants fix carbon and obtain energy through photosynthesis whereby sunlight is harnessed

and used to reduce and fix carbon obtained from CO2. As photosynthesis does not occur in all

plant cells, carbohydrates synthesized in the photosynthesizing “source” cells must be

transported to the non-photosynthesizing “sink” cells. The primary products of photosynthesis

are starch and sucrose. While starch serves primarily as a means of storage, sucrose plays a

central role in the transport of photoassimilates throughout the plant.

Sucrose, as the primary form of sugar transport in most plants, acts as a major player in

plant growth and development. Sucrose is a non-reducing disaccharide made of a molecule of

glucose covalently bonded to a molecule of fructose, and is found in multiple compartments

within the cell. In source cells, sucrose is synthesized in the cytosol, can be stored in the vacuole

for later use, can travel to neighboring cells through plasmodesmata, and eventually enters the

phloem via plasmodesmata or from the surrounding apoplast depending on whether the plant is a

symplastic or apoplastic loader of phloem (Turgeon, 1989; Grusak et al., 1996).

The composition of sucrose from two hexoses means sucrose plays roles not only as a

transport molecule, but also in osmotic maintenance and sugar signaling. The cleavage of a

single sucrose molecule in solution results in two molecules of hexoses thereby doubling the

osmotic potential of the solution. By cleaving sucrose and compartmentalizing the resulting

hexoses in various cellular compartments, osmotic gradients are created providing the basis not

only for short-distance transport from cell to cell, but also for long-distance transport from organ

to organ via the phloem as first proposed by Münch (1930). Sucrose and its component hexoses

77

provide sugar signals triggering numerous biological pathways playing roles in cell division,

expansion, differentiation and maturation (Koch, 2004).

The enzymes that cleave sucrose are influential players of plant metabolism and

development. Invertase (EC 3.2.1.26) is one of only two enzymes able to cleave sucrose.

Invertase, also known as β-fructofuranosidase, hydrolyzes sucrose into two hexoses, glucose and

fructose, in an irreversible reaction. Invertase is found in multiple cellular compartments and can

be divided into three sub-families base on cellular localization. Invertases localized to the

vacuole and apoplast are also known as acid invertases based upon their pH optimum for activity

(Haouazine-Takvorian et al., 1997; Sherson et al., 2003). The third sub-family of invertase is

localized to the cytosol and are also known as neutral/alkaline invertases due to their pH

optimum (Chen and Black, 1992; Van den Ende and Van Laere, 1995).

Several groups in the past have utilized transgenic forms of plants to study the role of

invertase in sucrose translocation. Invertase derived from yeast has been overexpressed in the

apoplast in Arabidopsis (Von Schaewen et al., 1990), potato (Heineke et al., 1992), tobacco

(Sonnewald et al., 1991) and tomato (Dickinson et al., 1991). Overexpression in this

compartment resulted in numerous growth defects summarized by stunted growth, inhibition of

photosynthesis, accumulation of leaf starch, and necrotic lesions on leaves followed by overall

yellowing of the leaf (Von Schaewen et al., 1990; Dickinson et al., 1991; Sonnewald et al.,

1991; Heineke et al., 1992). Sonnewald et al. (1991) also overexpressed yeast derived invertase

in the vacuolar and cytosolic compartments of tobacco resulting in similar phenotypes to the

apoplastic overexpressing tobacco plants.

Here I describe the overexpression of yeast invertase in Populus, a deciduous, perennial

tree species. While the previously mentioned work has done much to establish the role of

78

invertase in carbon allocation and partitioning, it is still unknown what roles invertase may play

in a deciduous, perennial plant. The deciduous, perennial nature of poplar requires numerous

cycles of sugar movement and carbon sequestration in storage organs such as the root and stem

in the winter, followed by remobilization of these stored carbon compounds the following spring.

It is also thought that poplar may employ a unique mode of sucrose movement into the phloem

of source leaves analogous to that of poplar’s closely related cousin, willow (both members of

Salicaceae) (Turgeon and Medville, 1998). A reverse genetic approach was utilized here to

directly manipulate invertase levels in three subcellular compartments, so that the effects of

ectopic invertase expression could be assessed in poplar.

Materials and Methods

Plant Material, Transgenesis, and Growth Conditions

Hybrid poplar clone, INRA 717-1-B4 (P. tremula x P. alba) was placed into sterile culture

prior to Agrobacterium-mediated transformation (Leple et al., 1992). Individual clones from

independent lines were clonally propagated as softwood cuttings under mist, transferred to 8 L

pots and grown to a height of 60-100 cm prior to experimentation in a fan- and pad-cooled

greenhouse with natural light augmented with full spectrum fluorescent lighting during the

winter to give a day length of 15 h (Lawrence et al., 1997). Greenhouse temperatures ranged

from 20-35 °C. At noon, the light intensity in the greenhouse averaged 500-700 µE/m2/min PAR,

which is one-half the light intensity outside the greenhouse. Plants were grown on an ebb-and-

flow flood bench system with a daily supply of Peters Professional® 20-10-20 water-soluble

fertilizer diluted to a final concentration of 4 mM nitrogen.

Vector Construction

The cell wall targeted SUC2 construct (CwSUC2) was obtained by a generous donation

from Uwe Sonnewald (Institut für Biologie der Universität Erlangen-Nürnberg, Erlangen,

79

Germany). The cytosolic targeted SUC2 construct (CytSUC2) was PCR amplified without the N-

terminal signal peptide from the CwSUC2 construct using the primer sequences: CytSUC2

forward, 5’-CACCATGACAAACGAAACTAGCGATAG-3’; CytSUC2 reverse, 5’-

CAGGTAACTGGGGTCGGGAGAA-3’. The vacuolar targeted SUC2 construct (VacSUC2) was

designed by adding a vacuolar targeting domain from tobacco chitinase to the 3’ end of the

CwSUC2 construct in two PCR steps. The first step used the primer sequences: CwSUC2

forward, 5’-CACCATGGATGTTCACAAGGAAGTTA-3’; VacSUC2 reverse_step_1, 5’-

GTCCAACAAACCATTACCTTTTACTTCCCTTACTTGGAACTTGTCAAT-3’. The second

step used the CwSUC2 forward primer again, and the primer VacSUC2 reverse_step_2, 5’-

TCACATCGTATCTACCAAGTCCAACAAACCATTACCTTTTACTTCC-3’. Cycling

parameters for all PCR reactions were 95 °C for 5 min to activate DNA polymerase, then 35

cycles of 95 °C for 30 s, 60 °C for 30 s and 72 °C for 2 min, followed by a final step of 72 °C for

5 min. The above PCR amplicons were cloned into the pENTR/D-TOPO vector (Invitrogen,

Carlsbad, CA) and then sub-cloned into Gateway destination vectors by LR recombination

reaction for expression in E. coli and Agrobacterium. The Gateway destination vectors were

kindly donated by G. Tuskan (Oak Ridge National Laboratory, Oak Ridge, TN).

Construction of Similarity Trees

Amino acid sequences were aligned using CLUSTALW (http://clustalw.genome.jp) to

construct similarity trees using the TREEVIEW program (Page, 1996). The PAUP (Swofford,

1993) program was used for bootstrap analysis with 1,000 iterations.

Isolation of RNA, Generation of cDNA and Real-Time PCR Assay

Total RNA was extracted using standard methods (Chang, 1993), DNase-treated and

purified in RNAeasy QIAGEN columns (Valencia, CA). Purified RNA (5 μg) was used to

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synthesize cDNA using a mixture of 500 ng oligo-dT, 100 ng random hexamer primers, and M-

MLV-RT as per manufacturer’s instructions (Invitrogen), with the exception that the DTT was

excluded.

Gene expression was analyzed using the SYBR Green kit (Stratagene, La Jolla, CA) and

Mx3000P thermo-cycler (Stratagene) as per manufacturer’s instructions. Briefly, each reaction

was run in triplicate and contained 1 μl of synthesized cDNA along with 0.15 μl of each 0.25 μM

primer in a final reaction volume of 50 μl. Primers were designed using NetPrimer (Premier

Biosoft International) software and synthesized by Invitrogen. Primer sequences were as follows:

SUC2 forward, 5’-TTTGAGTTGGTTTACGCTG-3’; SUC2 reverse, 5’-

TATTTTACTTCCCTTACTTGG-3’ (428 bp product). Cycling parameters were 95 °C for 10

min to activate DNA polymerase, then 40 cycles of 95 °C for 30 s, 46 °C for 30 s and 72 °C for 1

min. Melting curves were generated to check the specificity of the amplified fragments. Changes

(n-fold) in gene expression relative to the geometric mean (Vandesompele et al., 2002) of three

control genes encoding actin, ubiquitin and ubiquitin_L (Brunner et al., 2004) were determined

using the program DART-PCRv1.0 (Peirson et al., 2003).

Genomic DNA Isolation and PCR Amplification

To confirm transgene presence, genomic DNA was isolated from poplar leaves using the

Plant DNAeasy Kit (Qiagen) as per manufacturer’s instructions. Approximately 25 ng of DNA

was used as template for PCR. Transgene presence was confirmed by PCR using nptII-specific

primers with the following sequences: forward, 5’-ATCCATCATGGCTGATGCAATGCG-3’;

reverse, 5’-CCATGATATTCGGCAAGCAGGCAT-3’ (253 bp of T-DNA insertion). Cycling

parameters were 30 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min. Amplicons

were then separated on 1 % (w/v) agarose gels and stained with ethidium bromide.

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Metabolic Profiling: Extraction, Separation and Identification

For metabolic profiling by GC-MS, three biological replicates for each of the selected

transgenic lines (CwSUC2-2, -14, CytSUC2-18, -50 and VacSUC2-20, -30) and a non-transgenic

(NT) control line were randomized in the greenhouse and grown to a final height of 1 m prior to

the harvesting and pooling of LPI 10-12 leaf tissues. Metabolites were analyzed in pooled leaf

tissues using GC-MS where chromatogram peaks were evaluated via their retention times and

mass spectra as described in Morse et al. (2007).

Construction and Experimental Design of Grafts

Plants were grown under normal conditions until approximately 1 m in height and were

then used as the source for rootstock and scion buds. A bud from scion material was attached to

the rootstock with Parafilm approximately 5 cm above the soil surface. The lateral shoots that

emerged from the rootstock after grafting were removed to facilitate growth of the scion. After

the grafts took (approximately 2 weeks), Parafilm was removed as well as all plant material

above the graft and leaves below the graft inducing the scion bud to break. The scion was

maintained as the only growing stem on the plant and measurements were taken regularly during

growth. Grafting combinations were conducted such that each combination had three replicates

(in some cases, one replicate was lost; these are noted). Grafting combinations are as follows

(scion/rootstock): CwSUC2-2/NT (n=2), CwSUC2-2/CwSUC2-2 (n=2), CwSUC2-14/NT,

CwSUC2-14/CwSUC2-14 (n=2), VacSUC2-20/NT, VacSUC2-20/VacSUC2-20 (n=2), VacSUC2-

30/NT (n=2), VacSUC2-30/VacSUC2-30, NT/CwSUC2-2, NT/CwSUC2-14, NT/VacSUC2-20,

NT/VacSUC2-30, NT/NT. Plants were randomized in the greenhouse and grown to a final height

of approximately 1 m.

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Measurement of Photosynthesis and Respiration

Photosynthesis parameters were measured on a mature leaf (LPI 12) from each plant of the

grafted plants described previously. Light saturated net photosynthesis was measured with a

portable photosynthesis system (Li-6400, Li-Cor, Lincoln, NE) equipped with a red/blue LED

light source under the following chamber conditions: chamber [CO2] 380 μmol mol-1,

photosynthetic photon flux density 2000 μmol m-2 s-1, leaf temperature 18 to 22 °C, vapor

pressure deficit < 1.5 kPa, air flow rate 500 μmol s-1. Measurements were recorded when the sum

of the coefficients of variation for [CO2], [H2O], and flow rate for a 30 s running period dropped

below 0.3 %. Immediately following the light-saturated net photosynthesis measurement, a net

photosynthesis versus leaf internal CO2 concentration (A-Ci) curve was generated by taking

measurements at nine additional levels of reference [CO2] ranging from 50 μmol mol-1 to 1800

μmol mol-1, with all other chamber conditions remaining constant. The parameters for maximum

rate of carboxylation of Rubisco (Vcmax) and maximum rate of electron transport (Jmax) were

derived from the A-Ci curves with a program (Photosyn Assistant, Dundee Scientific, Dundee,

UK) that fits a model proposed by Farquhar et al. (1980) and modified by von Caemmerer and

Farquhar (1981), Sharkey (1985), Harley and Sharkey (1991) and Harley et al. (1992).

Parameters were adjusted to a standard temperature of 20°C using the methods described in

Walcroft et al. (1997). Statistical differences were assessed by ANOVA using the GLM

procedure of the SAS Version 8.0 statistical software package (SAS Institute, Cary, NC).

To measure foliage respiration, four biological replicates for each of the transgenic lines

CytSUC2-18 and -50 and a NT control line were randomized in the greenhouse and grown to a

final height of approximately 1 m on an ebb-and-flow flood bench system with a daily supply of

Peters Professional® 20-10-20 water soluble fertilizer diluted to a final concentration of 10 mM

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nitrogen. At noon, plants were moved to a dark growth chamber maintained at 35 °C while

respiration measurements were conducted. Measurements were recorded on both a sink (LPI 5)

and source leaf (LPI 12) from each plant. Respiration was measured with the same

photosynthesis system used for photosynthesis measurements. The photosynthesis leaf chamber

conditions were as follows: chamber [CO2] 380 μmol mol-1, leaf temperature 34 to 36 °C, air

flow rate 200 μmol s-1. Measurements were recorded when the sum of the coefficients of

variation for [CO2], [H2O], and flow rate for a 30 s running period dropped below 0.3 %.

Statistical differences were subjected to ANOVA using the Mixed procedure of the SAS Version

8.0 statistical software package (SAS Institute).

Protein Extraction

Poplar leaves were frozen and ground in liquid nitrogen. Total protein was extracted by

sonicating 300 mg ground tissue for 30 s in 1 ml of cold extraction buffer containing 50 mM

Hepes-KOH, pH 7.4, 2 mM EDTA, 2 mM EGTA, 5 mM DTT, 100 µM PMSF and 0.3 %

DIECA. Supernatant was collected after centrifugation at 3,220 g for 20 min at 4 °C and is

referred to as the soluble fraction. The pellet was then washed four times with 5 ml extraction

buffer and resuspended in 2.5 ml extraction buffer containing 1 M NaCl and incubated overnight

at 4 °C. Supernatant was collected after centrifugation at 3,220 g for 20 min at 4 °C and is

referred to as the insoluble fraction. Soluble and insoluble fractions were desalted with extraction

buffer using a 30 kD cutoff Centricon (Millipore, Billerica, MA) in order to concentrate protein,

remove the NaCl from the insoluble fraction and remove the invertase inhibitor from both

fractions. Proteins were quantified using a BCA Protein Assay Kit (Pierce, Bonn, Germany) as

per manufacturer’s instructions for the microplate procedure.

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Total Invertase Activity Assay

Total protein extracts were assayed for total invertase activity using a modification of the

method of Arnold (1965). Briefly, protein extracts (25 µl of soluble or insoluble fractions) were

added to 155 µl of 60 mM phosphate-citrate buffer, pH 4.5. Assays were brought up to 30 °C and

at time zero, 20 µl of 1 M sucrose was added. Reactions were stopped at 30 min by adding 600

µl of a modified Sumner’s reagent containing 1 % (w/v) 3,5 dinitrosalicylic acid, 0.05 % (w/v)

Na2SO3, 1 % (w/v) NaOH and 0.2 % (v/v) phenol. Samples were then incubated at 95 °C for 10

min and absorbance read at 540 nm.

Detection of Invertase Activity in Native Polyacrylamide Gels

SUC2 activity was detected using a native invertase activity gel assay (Gabriel and Wang,

1969). Briefly, protein extracts in 100 mM Tris-phosphate, pH 6.7, 0.1 % (w/v) bromophenol

blue, 10 % (v/v) glycerol, and 0.1 % (v/v) Triton X-100 were loaded on a 10 % (w/v)

polyacrylamide gel. The gel and running buffers were 100 mM Tris-phosphate, pH 6.7. After

running overnight at 40 V and 4 °C, gels were incubated in a 100 mM sucrose, 100 mM NaOAc,

pH 5.0 solution for 30 min at 30 °C. After a quick rinse in distilled water, gels were developed

by incubating in a 95 °C solution of 500 mM NaOH containing 0.1 % (w/v) 2,3,5-

triphenyltetrazolium chloride, giving rise to red bands at positions of invertase activity.

Sugar and Starch Determination

For soluble sugar (glucose, fructose and sucrose) determinations, ground, frozen tissue

samples were extracted using double distilled water and clarified via the Carrez method to

remove proteins. Glucose, fructose and sucrose were calculated from the spectrophotometric

measurement of NADPH production using a D-glucose/D-fructose sugar assay kit as per

manufacturer’s instructions (Boehringer Mannheim, Germany). Starch was solubilized from

85

ground, frozen samples using dimethylsulfoxide and hydrochloric acid and assayed using a

Boehringer Mannheim starch assay kit as per manufacturer’s instructions.

Results and Discussion

Construction of Overexpressing Yeast Invertase Vectors

To further define the role of invertase in the different cellular compartments (apoplast,

cytosol and vacuole) in poplar, I created three constructs designed to target and express the yeast

invertase gene, SUC2, in those compartments via Agrobacterium-mediated transformation

(Figure 4-1). A plasmid containing a cell wall targeted SUC2 was received through a generous

donation from Uwe Sonnewald (Institut für Biologie der Universität Erlangen-Nürnberg,

Erlangen, Germany). This construct contains a fusion of SUC2 (nucleotides 64-1765) (Taussig

and Carlson, 1983) with the signal peptide of potato proteinase inhibitor II (nucleotides 1-230)

(Keil et al., 1986). The gene fusion was PCR amplified and recombined into an overexpression

vector using the Gateway (Invitrogen) cloning system and is hereafter referred to as CwSUC2. In

order to create a vacuolar targeted SUC2 gene fusion, the vacuolar targeting domain from

tobacco chitinase (Neuhaus et al., 1991) was fused to the C-terminal end of the CwSUC2

construct and is hereafter referred to as VacSUC2. To direct accumulation of SUC2 in the

cytosol, the coding region of SUC2 was PCR amplified without any signal peptide from the

original CwSUC2 construct and subsequently recombined into the overexpression vector and is

hereafter referred to as CytSUC2. All constructs were driven by two, identical constitutive

CaMV 35S promoters linked in tandem and terminated with the OCS terminator (Figure 4-1).

Yeast invertase was used in these constructs for three reasons: first, SUC2 is known to be

active over a broad pH range (Goldstein and Lampen, 1975), which is important considering the

pH differences between the acidic vacuole and apoplast as compared to the more neutral cytosol.

Secondly, this invertase is not inhibited by the endogenous invertase inhibitors present in plants

86

(Greiner et al., 1998; Greiner et al., 1999; Link et al., 2004; Huang et al., 2007). Lastly, SUC2

has been expressed in a variety of plants to date including Arabidopsis (Von Schaewen et al.,

1990), potato (Heineke et al., 1992), tobacco (Von Schaewen et al., 1990; Sonnewald et al.,

1991; Ding et al., 1993; Sonnewald et al., 1993; Tomlinson et al., 2004) and tomato (Dickinson

et al., 1991), with significant results.

The two major sub-families of plant invertases are the acid and neutral/alkaline invertases.

The acid invertases are thought to have evolved from respiratory eukaryotes and aerobic bacteria

(Sturm and Chrispeels, 1990a) while the neutral/alkaline invertases are thought to have

originated from cyanobacteria (Vargas et al., 2003). SUC2, coming from yeast, a eukaryote,

indicates that SUC2 is more closely related to the plant acid invertase sub-family than the

neutral/alkaline sub-family. The acidic pH optimum for activity of SUC2 also supports this

conclusion. To further address this, amino acid alignments of SUC2 with endogenous poplar

invertases were examined (Figure 4-2A). Additionally, a protein similarity tree was constructed

using full length sequences of invertases included from Arabidopsis, carrot, corn, poplar, potato,

tomato, various micro-organisms, as well as levanase and inulinase (related enzymes provided as

outliers) (Figure 4-2B). These data indicate that although SUC2 may be evolutionarily more

closely related to the acid invertases than the neutral/alkaline invertases, amino acid sequences

have diverged such that SUC2 sequence is as different from the acid invertases as it is from the

neutral/alkaline invertases (Figure 2B). This lack of sequence similarity from the plant invertases

is likely the reason behind the lack of SUC2 inhibition from the endogenous plant invertase

inhibitors.

Expression of SUC2 in Poplar

In order to verify the constitutive nature of the double 35S (2x35S) promoter in poplar, I

analyzed expression of the 2x35S::SUC2 construct in three different organs with RT-qPCR.

87

Plants representing three separate transgenic events were tested in order to average out any line

specific effects. I found that the transgene driven under the double 35S promoter is expressed at

highest levels in leaf (Figure 4-3). Expression levels of the transgene in root and stem are

approximately 50 % of the level of expression of that in leaf (Figure 4-3). RNA from leaf

material was extracted and transgene expression levels analyzed in order to identify the highest

expressing lines of each cellular compartment directed construct (Figure 4-4). The two highest

expressing lines from each compartment were then selected for further experimental analysis.

Yeast Invertase Active in Cytosol

To test that the accumulation of SUC2 transcripts translate to increases in invertase

activity, total protein was extracted and fractionated according to solubility from leaf. The

insoluble fraction yielded no detectable increase in total invertase activity in any of the

transgenic lines examined (data not shown). In the soluble fraction, as much as two fold

increases of total invertase activity were detected in the CytSUC2-18 line (Figure 4-5A), but,

unexpectedly, not in any of the VacSUC2 or CwSUC2 constructs (data not shown). This indicates

that the SUC2 transcript accumulation seen in the cell wall and vacuolar targeted constructs

either did not translate into increased activity or that the increase occurred in the wrong cellular

compartment.

One explanation for the lack of total invertase activity increases in the CwSUC2 and

VacSUC2 transgenic lines could be that the endogenous plant invertases were down-regulated in

order to compensate for the exogenous increases in invertase activity. To address this, an

invertase assay was employed that does not detect plant invertase activity, but can detect SUC2

activity (Sonnewald et al., 1991). Total protein extracts from the insoluble and soluble fractions

were separated by electrophoresis on a non-denaturing polyacrylamide gel and then assayed for

activity (see Materials and Methods). Activity was detected only in the soluble fraction of lines

88

CytSUC2-18 and -50 (Figure 4-5B). This indicates that the total soluble invertase activity

increases seen in Figure 4-5A are a result of the expression of ectopic SUC2. This also indicates

that the lack of activity seen in the cell wall and vacuolar constructs is due to the lack of an

active form of SUC2 in the targeted compartment and not necessarily due to down-regulation of

plant invertase activity.

Metabolic Profiling Reveals Alterations in CwSUC2 and VacSUC2 Transgenic Lines

To determine whether SUC2 expression altered endogenous levels of sugars and any of the

metabolic pathways they feed, leaf tissue from three biological replicates from each of the

selected transgenic lines and NT controls were subjected to metabolite analysis using gas

chromatography-mass spectrometry (GC-MS) with electron impact ionization. As was expected

due to lack of detectable invertase activity alterations, the CwSUC2 and VacSUC2 transgenic

lines showed no significant changes in the levels of sucrose, glucose or fructose. Surprisingly, 12

metabolites were found to be significantly altered in at least one of those transgenic lines (Table

4-1). For three of those metabolites, the exact structures could not be determined and are referred

to by their retention index followed by their key mass/charge (m/z) ratios. Many of the other nine

metabolites can be found in, or are associated with, both the glyoxylate and TCA cycles. This

would indicate that even though I could not detect an increase in invertase activity, metabolic

shifts were still occurring in these transgenic poplars. If the glyoxylate cycle is indeed stimulated

in the CwSUC2 and VacSUC2 transgenic lines, this could account for my inability to detect shifts

in glucose and fructose as one end product of the glyoxylate cycle is glucose. As the glyoxylate

cycle occurs in specialized peroxisomes called glyoxysomes, it is possible that the metabolic

consequences of SUC2 expression are restricted to cellular compartments that represent a small

proportion of the total metabolite pool measured in these studies. One possible way to test this

89

hypothesis would be to isolate peroxisomes and evaluate metabolic shifts in these specific

compartments.

Metabolic Profiling Reveals Alterations in Sugar Accumulation in CytSUC2 Transgenic Lines

As would be expected from the increased invertase activity that accumulated in the

CytSUC2 transgenic lines (Figure 4-5), metabolite analysis revealed alterations in the levels of

sucrose as well as its hydrolysis products: glucose and fructose (Table 4-2). The transgenic line

containing the greatest increases of invertase activity (CytSUC2-18) also expressed the greatest

reduction in sucrose levels by nearly 30 %. CytSUC2-50 also revealed reduced levels of sucrose

(20 % reduction), however this was not found to be a statistically significant shift and could be

explained by the lower levels of invertase activity evident in line -50 compared to line -18

(Figure 4-5).

One would expect that increases in invertase activity would result not only in lower levels

of sucrose, but also in increased levels of glucose and fructose since these compounds would be

produced by sucrose hydrolysis. Surprisingly, this was not the case for the CytSUC2 lines where

analyses of both -18 and -50 revealed reductions in glucose and fructose levels (Table 4-2).

Interestingly, the greatest decreases in hexose abundance occurred in line -50, which had the

lowest invertase activity of the two lines. This may indicate that sucrose levels are directly

related to alterations in invertase activity, whereas observed hexose reductions are indirectly

linked to invertase activity. Also of note are the 50 % reductions in malic and glyceric acid levels

in the two CytSUC2 lines (Table 4-2).

One possible explanation for the reductions in hexoses as well as glyceric and malic acids

may lie in increased respiration rates. To test this hypothesis, CytSUC2-18 and -50 plants were

propagated and grown along with NT control plants with a high nutrient fertilizer in a

90

greenhouse. Plants were then moved to a warm (35 °C) growth chamber and kept in the dark

while leaf respiration rates were measured using a portable photosynthesis system from both

source (LPI 12) and sink (LPI 5) leaves. Respiration rates remained unchanged between the

CytSUC2 lines and the NT control plants (data not shown).

Whole-Plant Phenotypes Are Not Apparent

Phenotypes of clonally propagated SUC2 and NT plants were compared at whole-plant, -

organ and molecular-levels (Table 4-3). I detected no differences in propagation efficiency,

growth rate, plant architecture or plant size when grown under controlled greenhouse conditions

(data not shown). One strategy to increase whole-plant differences was to graft transgenic

material onto non-transgenic. This allows me to specifically target SUC2 expression to the root

and/or crown of the tree thereby increasing the sink of the roots while not affecting the ability of

the source organs from photosynthesizing and exporting sucrose. CwSUC2 and VacSUC2 lines

(scion) were grafted onto NT rootstock as well as NT plants (scion) onto CwSUC2 and VacSUC2

rootstock. Control grafts were also performed for all lines (transgenic onto itself, and NT onto

itself) and whole plant phenotypes including growth rates, plant architecture and plant size were

measured as well as photosynthetic rates. In all cases, the transgene was found to have no

detectable effect relative to the NT control (data not shown). Unfortunately the CytSUC2 plants

were excluded from this experiment due to problems in propagation that were unrelated to the

transgenic construct per se.

Concluding Remarks

My results indicate that it was possible to successfully transform poplar with the yeast

invertase gene, SUC2. In contrast to the many other plant species transformed with this gene (see

Introduction) I saw no whole-plant altering phenotypes. This is likely due to the relatively low

level of induction as compared to what was seen in other plant species. In the transformant

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containing the highest SUC2 activity (CytSUC2-18), I saw approximately a two-fold induction in

total activity (Figure 4-5A). In contrast, tobacco and potato transgenics expressing the same

SUC2 gene saw as much as 50- and 100-fold increases in activity, respectively (Sonnewald et al.,

1991; Heineke et al., 1992). Even with the modest increases in invertase activity seen in poplar, a

subtle metabolic phenotype could be detected in transgenic lines CytSUC2-18 and -50. While the

reduction in sucrose is to be expected, the reduction in glucose and fructose is counter-intuitive.

Increased respiration could be one explanation of the decrease in hexoses I observed. This

hypothesis would also be supported by the more dramatic reductions observed in malic acid.

Respiration was measured, but no significant changes were detected. This may simply be

because respiration was not altered significantly enough to be measurable. Alternatively,

respiration may not have been altered at all, in which case the metabolic shifts observed may

have resulted from some other metabolic pathway.

The SUC2 constructs targeted to the cell wall and vacuole pose a conundrum. While

transcripts were detected for the CwSUC2 and VacSUC2 transgenes, invertase activity was not

detected either in the total activity assay or in the native gel assay. This would seemingly

indicate that the transcript simply did not result in detectable levels of mature, active protein.

However, a metabolic phenotype was evident in these transgenics. Since no increase in total

activity was found, one potential explanation for this metabolic phenotype may reside in altered

workloads of endogenous invertase to exogenous yeast invertase. The increase in activity

resulting from the transgene may have led to a compensatory down-regulation of the plants’

endogenous invertase activity. Minimal changes to sugar levels would thus have been likely. The

lack of detectable activity in the native gel makes this a tenuous hypothesis, however. This

hypothesis could only be correct if the yeast invertase underwent some sort of post-translational

92

modification in the cell’s secretory system rendering the yeast invertase inactive in the native gel

assay, much like the undetectable plant invertases. If this were true, the in vivo increases in

activity must be quite small. Collectively, my data indicate that the transgenic poplar lines did

give rise to metabolic phenotypes, but that these were not straightforward to interpret. Also,

these transgenics did not show detectable whole-plant phenotypes potentially due to the

relatively subtle increases of SUC2 dependent invertase activity.

93

SP SUC2

Vacuolar targeted invertase

ChitinaseVTD

35S35S

ATGTAGSP SUC2

Vacuolar targeted invertase

ChitinaseVTD

35S35S

ATGTAG

SP SUC2

Cell wall targeted invertase

35S35S

ATG

TAGSP SUC2

Cell wall targeted invertase

35S35S

ATG

TAG

SUC2

Cytosolic targeted invertase

35S35S

ATGTAGSUC2

Cytosolic targeted invertase

35S35S

ATGTAG

Figure 4-1. Schematic representation of yeast invertase overexpressing constructs

targeted to the cytosol, cell wall and vacuole. Block arrows represent 35S promoter, solid black box represents SUC2 coding region, brick filled box represents the signal peptide (SP) taken from potato proteinase inhibitor II, cross-hatched box represents the vacuolar targeting domain (VTD) taken from tobacco chitinase.

94

A PtVIN2 1 WQRTAFHFQPEENWMNDPNGPLYYKG--WYHFFYQYNPHAAVW-GDIVWGHAVSKDLIHW PtCIN1 1 VHRTGYHFQPPRHWINDPNAPMYYKG--LYHLFYQYNPKGAVW-GNIVWAHSVSKDLINW PtNIN2 1 -------------AANDPGDKMPLNY--DQVFVRDFVPS-----ALAFLLRG--EGEIVK PtNIN10 1 -------------AAMDTSADALNY---NQVFVRDFVPT-----GLACLMKEPPEPEIVR S_cerSUC2 1 ---------PNKGWMNDPNGLWYDEKDAKWHLYFQYNPNDTVWGTPLFWGHATSDDLTNW PtVIN2 58 LHLPLAMVADKWYDKNGVWTGSATILPDGKIVMLYTGSTN-ESVQVQNLAYPADHDDPLL PtCIN1 58 ESLEPAIYPSKWFDNYGCWSGSATVLPNGEPVIFYTGIVDKNNSQIQNYAVPANLSDPYL PtNIN2 39 NFLLHALQLQSWEKTVDCYSPGQGLMP--------------ASFKVRTVPLDDNNLEEVL PtNIN10 40 NFLLKTLHLQGLEKRVDNFTLGEGVLP--------------ASFKV---LYDSDLEKETL S_cerSUC2 52 EDQPIAIAPK--RNDSGAFSGSMVVDYN-------------NTSGFFNDTIDPRQRCVAI PtVIN2 117 LKWVKYSGNPVLVPPPGIGAKDFRDPTTAWKTSEGKWRIIIGSKINKTGIALVYDTEDFI PtCIN1 118 REWVKPDDNPIVNPDANVNGSAFRDPTTAWWADG-HWRILIGSRRKHRGVAYLYRSKDFK PtNIN2 85 DPDFGESAIGRVAP-----------------VDSGLWWIILLRAYGKLTGDYALQER--- PtNIN10 83 LVDFGASAIGRVAP-----------------VDSGFWWIILLRSYIKRTRDYALLDR--- S_cerSUC2 97 WTYNTPESEEQYIS---------------YSLDGGYTFTEYQKNPVLAANSTQFRDPKVF PtVIN2 177 NYELLSGILHGVPKTGMWECVDFYPVSKTGQNGLDTSVNGPQVKHVIKTS-LDDDRHDYY PtCIN1 177 KWVKAKHPLHSVQGTGMWECPDFYPVSLSGENGLDPSVMGQNVKHVLKVS-LDMTRYEYY PtNIN2 125 ------VDVQTGIKLILNLCLADGFDMFPSLLVTDGSCMIDRRMGIHGHP-LEIQALFYS PtNIN10 123 ------PEVQNGMKLILKLCLSDGFDTFPTLLCADGCSMIDRRMGIYGYP-IEIQALFYF S_cerSUC2 142 WYEPSQKWIMTAAKSQDYKIEIYSSDDLKSWKLESAFANEGFLGYQYECPGLIEVPTEQD PtVIN2 236 ALGTYADKVGKWYPDNPEIDVGIGIRYDYGIFYASKTFYDQSKGRRVLWGWIGESDSEVA PtCIN1 236 TMGTYDKKKDKYFPDEGLVDGWAGLRLDYGNFYASKTFFDPSTNRRILWGWANESDDPQK PtNIN2 178 ALRSSREMLVVNDGSKNLVRAINNRLSALSFHIREYYWVDMRKINEIYRYKTEEYSTEAT PtNIN10 176 ALRCAKQMLKPELDGKEFIERIEKRITALSYHIQTYYWLDFTQLNNIYRYKTEEYSHTAV S_cerSUC2 202 PSKSYWVMFISINPGAPAGGSFNQYFVGSFNGTHFEAFDNQSRVVDFGKDYYALQTFFNT PtVIN2 296 DVKKGWASLQGIPRTVVLDTKTGSNLLQWPVEEVESLRLKSKNFNNIEVKAGSAVPLELD PtCIN1 296 DKDKGWAGIQLIPRKVWLD-PSGKQLLQWPVAELEKLRGHNVQLSNQMLDQGNHVEVKVI PtNIN2 238 NKFN--IYPEQIPSWLMDWIPEEGGYLIGNLQPAHMDFRFFTLGNLWSVVSSLGTPKQNE PtNIN10 236 NKFN--VIPESIPDWVFDFMPLRGGYLIGNVSPARMDFRWFLVGNCVAILSSLVTPAQAT S_cerSUC2 262 DPTYGSALGIAWASNWEYSAFVPTNPWRSSMSLVRKFSLNTEYQANPETELINLKAEPIL PtVIN2 356 GATQLDIVAEFELD----RKAIERTAESNVEFSCSTNGGASHRGALGPFGLLVLADDDLT PtCIN1 355 TAAQADVDVTFSFSSLDKAEPFDPKWAKLDALDVCAQKGSKDPGGLGPFGLLTLASENLE PtNIN2 296 AVLNLIESKWDDLVG-----------NMPLKICYPALESEDWRIITGSDPKNTPWSYHNG PtNIN10 294 AIMDLVEERWEDLIG-----------EMPLKITYPALEGHEWRLVTGFDPKNTRWSYHNG S_cerSUC2 322 NISNAGPWSRFATNT-------TLTKANSYNVDLSNSTGTLEFELVYAVNTTQTISKSVF PtVIN2 412 EYTPVYFFVAKGNNGSLKTFFCTDQSRSSVANDVRKEIYGSYVPVLEGEKLS----VRIL PtCIN1 415 EFTPVFFRVFKAADKHKVLLCSDARSSSLGKELYKPSFAGFVDVDLTDKKLS----LRSL PtNIN2 345 GSWPTLLWQFTLACMKMDRMELAQKAIALAEKRLQVDHWPEYYDTRSGKFIG----KQSR PtNIN10 343 GSWPMLLWLLSAACIKVGRPQIAKRAIELAEQRLSKDGWPEYYDGKTGRYVG----KQAR S_cerSUC2 375 ADLSLWFKGLEDPEEYLRMGFEVSASSFFLDRGNSKVKFVKENPYFTNRMSVNNQPFKSE

95

PtVIN2 468 VDHSIIESFAQGGRTCITSRVYPTRAIYGSARLFLFNNAT-EAGVTSSLKIWNMNSAFIR PtCIN1 471 IDHSVVESFGAGGRIAISSRVYPTIAVFEKAHLYVFNNGS-ETITVKNLNAWSMNTPVMN PtNIN2 401 LYQTWTVAGFLTSKVLLENPEKASLLFWDEDYDLLEFCVC-GLNTSGRKRCSRVAARSQI PtNIN10 399 KYQTWSIAGYLVAKMMVENPSNLLMISLEEDKKSARSRLT-RSNSTSF------------ S_cerSUC2 435 NDLSYYKVYGLLDQNILELYFNDGDVVSTNTYFMTTGNALGSVNMTTGVDNLFYIDKFQV PtVIN2 527 PYSNEQQ PtCIN1 530 VPVKS-- PtNIN2 460 LV----- PtNIN10 ------- S_cerSUC2 495 REVK--- B

tomatoTIV1tomatoTIV1

AT4G09510AT4G09510AT1G35580AT1G35580

PtNIN12PtNIN12PtNIN8PtNIN8

AT1G22650AT1G22650

AtcwINV1AtcwINV1

AT3G06500AT3G06500

InulinaseInulinase

carrotInvcarrotInv Dc4Dc4PtVIN3PtVIN3

AT1G72000AT1G72000

AtcwINV6AtcwINV6maizecwIVR4maizecwIVR4

AT4G34860AT4G34860PtNIN9PtNIN9

PtNIN11PtNIN11

maizecwIVR2maizecwIVR2maizecwIVR3maizecwIVR3

PtCIN5PtCIN5

PtCIN4PtCIN4

PtCIN2PtCIN2PtCIN1PtCIN1

PtCIN3PtCIN3

maizecwIVR1maizecwIVR1

tomatoLin8tomatoLin8tomatoLin7tomatoLin7

carrotInvcarrotInv Dc3Dc3carrotInvcarrotInv Dc2Dc2

carrotInvcarrotInv Dc1Dc1

AtcwINV3AtcwINV3AtcwINV5AtcwINV5

tomatoLin5tomatoLin5potatoIVRGEpotatoIVRGEtomatoLin6tomatoLin6

S cerevisiaeSUC2S cerevisiaeSUC2

100100

100100

100100

9393

100100

100100II

IIIIII

IIII

IVIV

Figure 4-2. S. cerevisiae SUC2 in relationship to other plant invertases. A) Amino acid

alignment of SUC2 and select poplar invertases. Black shading corresponds to identical residues, grey shading corresponds to similar residues. Alignment produced with CLUSTALW and figure produced with BOXSHADE. B) Amino acid similarity tree using full length sequences. Depicts phylogenetic relationship of S. cerevisiae SUC2 (in bold) with selected invertases from Arabidopsis, carrot, corn, poplar, potato and tomato. Invertases from various micro-organisms are also included along with the closely related enzymes, levanase and inulinase, provided as outliers. Clades I-IV represent vacuolar, cell wall, α-neutral/alkaline and β-neutral/alkaline invertases, respectively. α- and β- designations of the neutral/alkaline invertases represent two distinct clades as described by Bocock et al. (2007). Bootstrap values of the major clades are depicted and reported as a percentage of 1,000 repetitions. Branch lengths denote protein similarity.

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Double 35S driven SUC2 expression

0

20

40

60

80

100

120

140

root stem leaf

Organ

Rel

ativ

e tra

nscr

ipt (

%)

Figure 4-3. Relative expression of double 35S driven SUC2 in root, stem and leaf. Bars represent the mean level of SUC2 transcript across three separate transgenic events; error bars denote standard error, n=3.

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A Relative expression of CytSUC2

0

20

40

60

80

100

120

18 50 16 2 21 25 17 28 31 12 6 9 36 NT 19

Transgenic line

Rel

ativ

e tra

nscr

ipt (

%)

B

Relative expression of CwSUC2

0

20

40

60

80

100

120

140

2 14 25 26 28 11 3 10 NT

Transgenic line

Rela

tive

tran

scri

pt (%

)

C Relative expression of VacSUC2

-20

0

20

40

60

80

100

120

140

160

30 20 29 15 21 38 27 36 24 25 34 NT 7

Transgenic line

Rela

tive

trans

crip

t (%

)

Figure 4-4. Relative expression of SUC2 transcript in overexpressing transgenic events in leaf. A) Cytosolic targeted construct. B) Cell wall targeted construct. C) Vacuolar targeted construct. In all panels, bars represent mean level of SUC2 transcript; error bars denote standard error when available, n=3.

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A Total invertase activity (soluble

fraction)

0

20

40

60

80

100

120

140

NT Cyt-18 Cyt50

Transgenic line

Act

ivity

(µm

ol s

uc

hydr

olyz

ed *

min

-1 *

mg

prot

ein

-1)

B

Cyt

18-1

Cyt

18-2

Cyt

50-1

Cyt

50-2

NT

NT

Cyt

18-1

Cyt

18-2

Cyt

50-1

Cyt

50-2

NT

NT

IS S

Cyt

18-1

Cyt

18-2

Cyt

50-1

Cyt

50-2

NT

NT

Cyt

18-1

Cyt

18-2

Cyt

50-1

Cyt

50-2

NT

NT

IS S

Figure 4-5. CytSUC2 transgenic plants display increased invertase activity due to presence of transgene. A) Soluble proteins were extracted from a source leaf (LPI 12) and assayed for invertase activity. Bars represent mean invertase activity, error bars denote standard error, n=2. B) Protein extracts (20 µg total protein each lane) were separated using non-denaturing gel electrophoretic conditions and assayed for activity.Top panel: No invertase activity is detected in NT plants or in the insoluble fraction (IS). Activity can be seen (red bands) in the soluble fraction (S) of CytSUC2 lines -18 and -50 in both biological reps. Bottom panel: Coomassie stain of gel to show loading.

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Table 4-1. Metabolites altered in CwSUC2 and VacSUC2 overexpressing plants expressed as a ratio of μg/g FW (glucose equivalents) of transgenic/NT controls.

VacSUC2-20/NT VacSUC2-30/NT CwSUC2-2/NT CwSUC2-14/NT5 Carbon-sugar alcohol 1.08 1.18* 1.17* 1.18†

Iditol 1.11 1.29* 1.21* 1.16Glucoside (m/z 434) 1.08 1.55† 1.21 1.3716.25-273 (Unknown compound) 0.74 0.56 0.52† 0.45†

alpha-keto-Glutaric acid 1.11 1.27 1.63* 2.43*Maleic acid 1.13 1.25 1.42* 1.33Citric acid 1.21 1.25 1.39* 0.7910.79-184 (Unknown compound) 1.08 0.58* 0.35* 0.28*Fumaric acid 1.43 2.48 1.58* 1.32Serine 0.88 0.87 0.89 0.69*Carbamoyl-phosphate 0.97 0.62† 0.35* 0.35*15.7-221 327 (Unknown compound) 0.5* 0.62* 0.51* 0.42**=significant at 0.05†=significant at 0.10

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Table 4-2. Metabolites altered in CytSUC2 overexpressing plants expressed as a ratio of μg/g FW (sorbitol equivalents) of transgenic/NT controls.

CytSUC2-18/NT CytSUC2-50/NTFructose 0.83* 0.80*Sucrose 0.71* 0.82Myoinositol 0.68* 0.96Glucose 0.75 0.70†

Galactose 0.65† 0.61†

Glyceric acid 0.52† 0.57†

Malic acid 0.52† 0.52†

*=significant at 0.05†=significant at 0.10

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Table 4-3. Summary of phenotypic experiments performed. Trait Tested Plants Tested Significant Changes Found

PCR verification of transgene Cyt, Cw, Vac Cyt, Cw, VacTranscriptional changes Cyt, Cw, Vac Cyt, Cw, VacInvertase activity Cyt, Cw, Vac CytSugars (fructose, glucose, sucrose) (root, stem, leaf) Cyt, Cw, Vac Cyt (leaf)Metabolites Cyt, Cw, Vac Cyt, Cw, VacRootability Cyt, Cw, Vac noGrowth rates Cyt, Cw, Vac noStarch (leaf, root and stem) Cyt, Cw, Vac noRespiration Cyt noGrowth rates after grafting Cw, Vac noPhotosynthetic rates after grafting Cw, Vac no

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CHAPTER 5 CONCLUSIONS

As a first step in examining the role invertase plays in carbon allocation and partitioning,

the invertase family was identified in Populus trichocarpa, a woody, perennial tree species

whose genome was recently sequenced (Tuskan et al., 2006) (Chapter 2). The identification of

eight acid invertase genes; three of which belong to the vacuolar targeted group (PtVIN1-3), and

five of which belong to the cell wall targeted group (PtCIN1-5) is described. Similarly, I report

the identification of 16 neutral/alkaline invertase genes (PtNIN1-16). Expression analyses using

whole genome microarrays and RT-PCR revealed evidence for expression of all invertase family

members. Evidence is also reported for expression of a novel intronless vacuolar invertase

(PtVIN1), which apparently arose from a processed PtVIN2 transcript that re-inserted into the

genome. An examination of the microsyntenic regions surrounding the poplar invertase genes

revealed extensive colinearity with Arabidopsis invertases, indicating strong evolutionary

conservation in the development of the invertase family between the two species. To further

determine if the evolutionary conservation of the invertase family, demonstrated by conserved

colinearity, extended to the functional level, vacuolar invertases were selected as a case study

(Chapter 3). Vacuolar invertases have a well documented reciprocal response to sugar treatment

in which one invertase is up-regulated while the other is down-regulated after sugar treatment in

maize (Xu et al., 1996). The conserved, colinear structure of the chromosomal regions

containing these vacuolar invertase genes in Arabidopsis and poplar did not extend to maize and

rice. However, the reciprocal response was found to occur in all four plant species. One

explanation for this is that the function of these genes has been conserved throughout evolution.

An alternative hypothesis is that this reciprocal response is an example of parallel evolution that

arose independently in the different plant lineages. Finally, invertase activity in poplar was

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manipulated with transgenesis by ectopically expressing yeast invertase in three cellular

compartments (apoplast, cytosol and vacuole) (Chapter 4). This doubled invertase activity in the

cytosol, but did not alter invertase activity in other cellular compartments. Subtle shifts in carbon

partitioning within the cell were observed, but no detectable alterations in carbon allocation

between tissues. Specific invertase genes identified in Chapter 2 were also targeted for repression

via RNAi (Appendix A).

The overall objective of determining contributions by invertase to carbon allocation and

partitioning in a woody perennial was met in the following ways:

• Poplar contains three vacuolar invertases, one of which is a novel, intronless gene found only in poplar.

• Poplar contains five cell wall invertases. • Poplar contains 16 neutral/alkaline invertases that clearly divide into two, structurally

distinct groups and represents a significant expansion in gene number over that of Arabidopsis and rice.

• Poplar and Arabidopsis acid invertases share substantial similarities in their chromosomal

arrangement indicating that chromosomal duplication can explain much of the development of the invertase family.

• Poplar and Arabidopsis vacuolar orthologs identified through shared colinearity both retain

a conserved, reciprocal response to exogenous sugar treatment that is also found in the monocots, maize and rice.

• Invertase activity in poplar can be increased through the introduction of exogenous yeast

invertase. • An increase in invertase activity shifts carbon partitioning within the cell.

This research has opened up several new questions that would be valuable to be addressed

in future research. The first question is to address how invertase fits into the hypothesized lack of

active phloem loading model proposed by Turgeon and Medville (1998) for poplar and similar

species. One possibility could be that the expansion of the neutral/alkaline invertase sub-family

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in poplar may contribute to the movement of photoassimilates through the mesophyll cells and

into the phloem.

A second question that arises from this research deals with the significance and

development of the intronless PtVIN1. It is unknown when in poplar’s evolutionary history

PtVIN1 arose. When this question is addressed, it may also help us to understand the function of

this intronless gene.

The third question deals with the activity of the cell wall invertase sub-family members. It

has recently been reported that two of the six cell wall invertases in Arabidopsis may not be

invertases, but rather fructan exohydrolases (FEHs) (De Coninck et al., 2005). FEH protein

sequences are nearly identical to those of demonstrated acid invertase proteins, but FEH does not

use sucrose as a substrate (De Coninck et al., 2005). It is likely that at least one poplar cell wall

invertase is in fact an FEH, but analysis of recombinant cell wall invertase activity was outside

the scope of research presented here.

The fourth question is why the poplars ectopically expressing yeast invertase lack whole-

plant phenotypes. Several groups overexpressed yeast derived invertase in the apoplast of

Arabidopsis (Von Schaewen et al., 1990), potato (Heineke et al., 1992), tobacco (Sonnewald et

al., 1991) and tomato (Dickinson et al., 1991). Sonnewald et al. (1991) also overexpressed yeast-

derived invertase in the vacuolar and cytosolic compartments of tobacco. In all cases,

overexpression resulted in numerous growth defects that included stunted growth, inhibition of

photosynthesis, accumulation of leaf starch and formation of necrotic lesions on leaves followed

by overall yellowing of the leaf (Von Schaewen et al., 1990; Dickinson et al., 1991; Sonnewald

et al., 1991; Heineke et al., 1992). The lack of phenotypes might be due to the relatively modest

induction of activity (two-fold) as compared to these other plants. This may have resulted from

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inadvertent selection and propagation of the most vigorous transgenic plants, and hence those

least markedly altered.

Fifth, and finally, valuable work could be performed on poplar plants with a reduction in

invertase activity. As described in Appendix A, transgenic poplars were produced encoding

constructs designed to repress specific endogenous invertase genes through the use of RNAi.

These plants have been confirmed to express the transgene, however they have not been screened

for reduced expression of the targeted invertase gene or even a reduction in overall invertase

activity. The phenotype of these lines is normal and thus the cost of intensive phenotypic

screening must be weighed against the likely benefit of the approach.

The purpose of this research was to investigate invertase contributions to carbon allocation

and partitioning in a woody perennial. To achieve this goal, the invertase family was identified

and annotated using the sequenced genome of Populus trichocarpa. Several novel features were

identified in the poplar invertase family. These include a three-member vacuolar invertase sub-

family, an expressed, intronless vacuolar invertase as well as an expansion of the neutral/alkaline

invertase family relative to that of Arabidopsis and rice. The response of two poplar vacuolar

invertases (PtVIN2 and -3) to exogenous sugar treatments was examined. A reciprocal regulation

of transcript first documented in maize was also found to occur, similar to that of Arabidopsis

and rice. A reverse genetic approach was also employed by ectopic expression of yeast invertase

in transgenic poplar resulting in a subtle shift in carbon partitioning as shown by metabolic

analyses. In conjunction, RNAi was used to target endogenous poplar invertases for down-

regulation.

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APPENDIX A REPRESSION OF INVERTASE IN POPULUS

Introduction

The cell wall and vacuolar invertases have been repressed in several plant species,

including tomato (Ohyama et al., 1995), potato (Zrenner et al., 1996), and carrot (Tang et al.,

1999). The results from these experiments were more varied than those of the invertase

overexpressors, although the theme of increased sucrose content accompanied by a decrease in

hexoses pervaded. In tomato these alterations in sugar accumulations appeared in both the fruit

and leaves. In addition to altered sugar levels, the fruit also had elevated rates of ethylene

evolution. In carrot, growth was shown to be altered at very early stages of development. The

transgenic, cotyledon-stage embryos were still masses of cells while the control plants had

already developed two to three leaves and one primary root. However, when these plantlets were

grown on media supplemented with hexoses, their growth returned to normal. After maturation

and transfer to soil, the plants expressing the cell wall anti-sense cDNA had a significantly

increased shoot to root ratio and accumulated more sucrose and starch than the controls. The

vacuolar anti-sense plants had increased numbers of leaves, while tap root size was slightly

decreased.

Here I describe the design of vectors aimed at endogenous invertase repression via RNAi

in Populus, a deciduous, perennial tree species. While the previously mentioned work has done

much to establish the role of invertase in carbon allocation and partitioning, it is still unknown

what roles invertase may play in a deciduous, perennial plant system. The deciduous, perennial

nature of poplar requires numerous cycles of sugar movement and carbon sequestration in

storage organs such as the root and stem in the winter, followed by remobilization of these stored

carbon compounds the following spring. It is also thought that poplar may employ a unique

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mode of sucrose movement into the phloem of source leaves analogous to that of poplar’s

closely related cousin, willow (both members of Salicaceae) (Turgeon and Medville, 1998).

These added levels of complexity that can be seen by utilizing the model tree, poplar, may offer

us new insights into the function of invertase.

Materials and Methods

Plant Material, Transgenesis and Growth Conditions

Hybrid poplar clone, INRA 717-1-B4 (P. tremula x P. alba) was placed into sterile culture

prior to Agrobacterium-mediated transformation (Leple et al., 1992) on media supplemented

with glucose and fructose. Individual clones from independent lines were clonally propagated as

softwood cuttings under mist, transferred to 8 L pots and grown to a height of 60-100 cm prior to

experimentation in a fan- and pad-cooled greenhouse with natural light augmented with full

spectrum fluorescent lighting during the winter to give a day length of 15 h (Lawrence et al.,

1997). Greenhouse temperatures ranged from 20-35 °C. At noon, the light intensity in the

greenhouse averaged 500-700 µE/m2/min PAR, which is one-half the light intensity outside the

greenhouse. Plants were grown on an ebb-and-flow flood bench system with a daily supply of

Peters Professional® 20-10-20 water-soluble fertilizer diluted to a final concentration of 4 mM

nitrogen.

Vector Construction

The pCAPT binary vectors for RNAi was constructed as described by Filichkin et al.

(2007). Briefly, the vector was constructed using the pART27 backbone (Gleave, 1992). The

GATEWAY Conversion System (Invitrogen) was used to incorporate the proper recombination

sites and genes for positive and negative selection. This consists of an attR recombination site

flanking a ccdB gene and a chloramphenicol resistance gene. The vector contains spectinomycin

and kanamycin (NPTII) resistance genes for selection in bacteria and plants, respectively.

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Approximately 200 bp target fragments of acid invertase genes from poplar (primer sequences

listed in Table A-1) were PCR amplified using primers with tails encoding attB recombination

sites. GATEWAY entry clones were created using BP Clonase (Invitrogen) mediated

recombination. The entry clones were finally cloned into the pCAPT binary vector using LR

Clonase (Invitrogen) according to manufacturer’s instructions. All constructs were sequenced.

Genomic DNA Isolation and PCR Amplification

To clone target gene sequences and transgene presence, genomic DNA was isolated from

poplar shoot tip tissue. Approximately 250 mg of tissue was ground in liquid nitrogen and added

to a buffer containing 0.3 M sucrose, 10 mM Tris (pH 7.9), 1 mM EDTA, and 4 mg/mL

diethyldithiocarbamic acid. Samples were spun (20,800 rcf) and pellets resuspended in a buffer

containing 100 mM Tris (pH 7.9), 500 mM NaCl, 20 mM EDTA, 1 % SDS, 0.1 % 2-

mercaptoethanol, and 100 μg/ml proteinase K. Samples were incubated at 65 ˚C for 1 h, spun

(20,800 rcf) and supernatant extracted with chloroform. Isopropanol was used to precipitate the

DNA at room temperature, spun (20,800 rcf), and resuspended in Tris-EDTA buffer. One μl

RNase A was added to DNA sample and incubated at 37 ˚C for 30 min. The chloroform

extraction was repeated, followed by ethanol precipitation in presence of sodium acetate at -80

°C for 1 h. Samples were spun for 10 min (20,800 rcf), air dried and resuspended in Tris-EDTA

buffer. Approximately 25 ng of genomic DNA was used as template for PCR. Cycling

parameters were 95 °C for 5 min to activate DNA polymerase, then 35 cycles of 95 °C for 30 s,

annealing temperature for 30 s and 72 °C for 1 min followed by a final step at 72 °C for 5 min.

The annealing temperature was set to five degrees below the melting temperature for the primer

pair (Tm listed in Table A-1). Amplicons were then separated on 1 % (w/v) agarose gels and

stained with ethidium bromide. Transgene presence was confirmed by PCR using nptII-specific

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primers with the following sequences: forward, 5’-ATCCATCATGGCTGATGCAATGCG-3’;

reverse, 5’-CCATGATATTCGGCAAGCAGGCAT-3’ (253 bp of T-DNA insertion). Cycling

parameters were 30 cycles of 94 °C for 1 min, 58 °C for 1 min and 72 °C for 1 min. Amplicons

were then separated on 1 % (w/v) agarose gels and stained with ethidium bromide.

Results

Construction of RNAi Vector

To repress endogenous poplar acid invertase genes, I used a binary vector, pCAPT,

graciously donated by Stephen DiFazio (West Virginia University, Morgantown, WV) (Figure

A-1). The pCAPT vector is designed for the cloning and insertion of a target sequence. A single

GATEWAY cassette (Invitrogen) is located upstream of an inverted repeat of the octapine

synthase (OCS) terminator. Transcription is controlled by two CaMV 35S promoters linked in

tandem. This transcript results in a hairpin formed by the inverted repeat of the OCS terminator.

Transitive silencing of the inserted target gene fragment then occurs via RNAi (Filichkin et al.,

2007).

Insertion of Target Gene Sequence

The acid invertase gene family was selected to be used for RNAi directed gene silencing.

Using annotated sequences described in Chapter 2, nucleotide alignments were constructed using

the eight acid invertases in poplar. Two gene specific primer pairs per gene were designed such

that a ~200 bp fragment from both the N-terminal and C-terminal ends of the gene could be PCR

amplified from genomicDNA (Figure A-2; Table A-1). These fragments were then cloned into

the pCAPT binary vector which was then sequenced to verify insertion in the proper orientation.

The pCAPT binary containing the target gene insert was then grown in Agrobactierium and used

to transform poplar trees as outlined in the Materials and Methods. Shoot tips from regenerated

transgenic poplars were used as a source of genomic DNA to screen for the presence of the

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transgene using primers directed against the NPTII reporter gene as outlined in the Materials and

Methods. Table A-2 contains a complete list of all pCAPT vectors and regenerated transgenic

poplars that were successfully made.

Further Work

The next step in this project is to screen the approximately 30 separate transgenic events

per construct in order to verify the effectiveness of the RNAi vector and rank the transgenic

plants according to target gene transcript levels. Transcripts from the other acid invertase family

members then need to be quantified to check for the specificity of the RNAi mediated gene

silencing as well as to screen for compensatory increases in related gene transcripts in case of

functional redundancy. Total invertase activity should then be assayed to verify that a

successfully silenced invertase gene results in decreased invertase activity. Phenotyping could

then be done where one would expect increases in available sucrose, decreases in available

hexoses, as well as whole-plant phenotypes such as alterations in growth rates.

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RB 35S gfp R1 R2 OCS-R PIV2 OCS-F nos5’ NPTII nos3’ LB

Gene Fragment

Figure A-1. Schematic representation of pCAPT vector (16041 bp) used for RNAi silencing. R1

and R2, attR recombination sites flanking a ccdB gene and a chloramphenicol resistance gene from the GATEWAY vector conversion cassette (Invitrogen). RB and LB, right and left T-DNA borders, respectively; 35S, double CaMV 35S promoter linked in tandem; gfp, fragment of GFP coding sequence; PIV2, potato intron; OCS-R and OCS-F, octopine synthase terminator fragments in reverse (R) and forward (F) orientations, respectively; NPTII, neomycin phosphotransferase II; gene fragment, location of targeted invertase sequences. Diagram not to scale, adapted from (Filichkin et al., 2007).

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Spacer

~200 base pair oligo OCS OCS

PtCIN2 ~3.6kb

5’ construct

3’ construct

35S35S

Spacer

~200 base pair oligo OCS OCS

35S35S

Figure A-2. Schematic representation of RNAi directed constructs. Endogenous PtCIN2 demonstrates strategy of RNAi construct design. PtCIN2 exons are represented by black boxes and introns by black lines. The 5’ and 3’ ~200 bp-oligos to be targeted by RNAi constructs are marked by black lines below their respective locations. The RNAi constructs containing the ~200 bp-oligos are depicted below the PtCIN2 schematic. Grey block arrows represent 35S promoter, solid black box represents endogenous ~200bp-oligo to be targeted by RNAi machinery, dotted block arrows represent OCS terminator oriented in opposing directions and separated by a short potato intron (grey box) in order to form hairpin in vivo. Figure not drawn to scale.

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Table A-1. Primer sequences used for ~200 bp target sequence cloning. Primer Name Primer Sequence Tm (°C)PtCIN1 NT forward CACCATGGGCTATGCCACACAC 61PtCIN1 NT reverse CGTTGATCCAGTGCCTTGGAGG 66PtCIN1 CT forward CACCGAGAGTTTTGGAGC 53PtCIN1 CT reverse AACATTCATGACAGGCGT 51PtCIN2 NT forward CACCATGGCTATATGCCAAACAC 61PtCIN2 NT reverse GTTGATCCAGTTCCTAGGAGGCT 61PtCIN2 CT forward CACCGAGAGTTTTGGAGC 53PtCIN2 CT reverse TTCATTGCGTGGATTCTCTC 56PtCIN3 NT forward CACCATGGCTTTGTTAAAGTTTCTC 62PtCIN3 NT reverse GTTGATCCAGTTCTTAGGAGGCTG 61PtCIN3 CT forward CACCGAAAGTTTTGGAG 48PtCIN3 CT reverse GACATTCATCACAGGCAC 48PtCIN4 NT forward CACCTTCTTGGTTGGTTTATGC 59PtCIN4 NT reverse GGTTCTATATGACTGCTTTTCTTGC 59PtCIN4 CT forward CACCGAGAGTTTTGGTGGG 58PtCIN4 CT reverse TTGATTTGGGCTTTGTTCATG 59PtCIN5 NT forward CACCGGAGATATCTGTTATTTGG 58PtCIN5 NT reverse ATTCATCCAGTTTTTAGGAGGTTG 59PtCIN5 CT forward CACCTAGTTGAGAGTTTTGGTGGT 60PtCIN5 CT reverse CTAAAGGTGTGGCTTCCTTCG 59PtVIN2 NT forward CACCCTTCCAGTTTCCAATTCCTTA 65PtVIN2 NT reverse AACAAAGTCTCGGGTTTCGC 61PtVIN2 CT forward CACCCGTTGCTGAGTTTGAGTTA 62PtVIN2 CT reverse TGAGGCTGCCATTATTTCCTTTG 63PtVIN3 NT forward CACCCCCACCATACACTCCCTTG 67PtVIN3 NT reverse TGGTGATACCCCTTGAGCCACTCC 68PtVIN3 CT forward CACCAGTTTTGCTCAAGGAG 56PtVIN3 CT reverse TTGGTCAAATAGGAAAGGATGG 59

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Table A-2. Summary of RNAi constructs made. NT and CT, N-terminal and C-terminal constructs, respectively; X denotes possession of the RNAi construct (pCAPT) and transgenic plant expressing the construct.

Gene pCAPT Transgenic PlantPtCIN1 , NT X X

CT XPtCIN2 , NT X

CT X XPtCIN3 , NT X

CT X XPtCIN4 , NT X

CT XPtCIN5 , NT X X

CT X XPtVIN2 , NT XPtVIN3 , NT X X

CT X X

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APPENDIX B REGULATION OF INVERTASE: A “SUITE” OF TRANSCRIPTIONAL AND POST-

TRANSCRIPTIONAL MECHANISMS

This appendix contains a review that resulted from a collaboration between Dr. Li-Fen

Huang, Dr. Karen Koch and myself. It was published in Functional Plant Biology. While I took

part in the construction and editing of the entire manuscript, some parts I did not write. I did not

write the PPV compartmentalization, differential sugar regulation, or RNA turnover sections. I

also originate the idea for the first figure. All other parts are my original work.

Abstract

Recent evidence indicates that several mechanisms can alter invertase activity and, thus,

affect sucrose metabolism and resource allocation in plants. One of these mechanisms is the

compartmentalization of at least some vacuolar invertases in precursor protease vesicles (PPV),

where their retention could control timing of delivery to vacuoles and hence activity. PPV are

small, ER-derived bodies that sequester a subset of vacuolar-bound proteins (such as invertases

and protease precursors) releasing them to acid vacuoles in response to developmental or

environmental signals. Another newly-identified effector of invertases is wall-associated kinase

2 (WAK2), which can regulate a specific vacuolar invertase in Arabidopsis (AtvacINV1) and

alter root growth when osmolyte supplies are limiting. WAKs are ideally positioned to sense

changes in the interface between the cell wall and plasma membrane (such as turgor), because

the N-terminus of each WAK extends into the cell wall matrix (where a pectin association is

hypothesised) and the C-terminus has a cytoplasmic serine/threonine kinase domain (signaling).

Still other avenues of invertase control are provided by a diverse group of kinases and

phosphatases, consistent with input from multiple sensing systems for sugars, pathogens, ABA

and other hormones. Mechanisms of regulation may also vary for the contrasting sugar responses

of different acid invertase transcripts. Some degree of hexokinase involvement and distinctive

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kinetics have been observed for the sugar-repressed invertases, but not for the more common,

sugar-induced forms examined thus far. An additional means of regulation for invertase gene

expression lies in the multiple DST (Down STream) elements of the 3’- untranslated region for

the most rapidly repressed invertases. Similar sequences were initially identified in small auxin-

up RNAs (SAUR) where they mediate rapid mRNA turnover. Finally, the invertase inhibitors,

cell wall- and vacuolar inhibitors of fructosidase (CIF and VIF, respectively) are

indistinguishable by sequence alone from pectin methylesterase inhibitors (PMEI); however,

recent evidence suggests binding specificity may be determined by flexibility of a short, N-

terminal region. These recently characterized processes increase the suite of regulatory

mechanisms by which invertase – and, thus, sucrose metabolism and resource partitioning – can

be altered in plants.

Introduction

The capacity to use sucrose is essential to most plant cells, because this sugar is typically

the long-distance transport form for carbohydrates. Invertase (EC 3.2.1.26) irreversibly

hydrolyses sucrose into glucose and fructose, and is thus positioned to play a central role in both

carbon metabolism and sugar signaling. Glucose and fructose are hexoses that serve as an energy

source for respiration, provide metabolites for synthetic processes, generate osmotic pressure in

growing tissues and function as metabolic signals affecting the expression of many downstream

genes (Koch, 2004). Given the importance of sucrose cleavage and the diverse roles for products

of invertase activity, it is not surprising that multiple avenues have developed for the regulation

of the invertases.

Several invertase isozymes have been identified from plant species as either soluble

(readily extractable from cytosol or vacuole), or insoluble (bound to cell wall components).

Vacuolar and cell wall invertases show optimal activity at an acidic pH and thus are also called

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acid invertases. The mature acid invertases are glycosylated and located in the cellular

compartment their name implies (e.g. cell wall or vacuolar).

Invertase is regulated at transcriptional and post-translational levels by many different

factors including hormones, sugars, pathogens, oxygen availability and proteinaceous inhibitors

(Zeng et al., 1999; Long et al., 2002; Link et al., 2004; Trouverie et al., 2004; Voegele et al.,

2006). However, important new methods of control have also been identified by recent work on

the invertase path of sucrose use. These are reviewed here and include protein trafficking,

signaling cascades, transcript turnover and inhibitor binding.

Compartmentalization of Vacuolar Invertases in Precursor Protease Vesicles (PPV)

A new level of regulation has been revealed for vacuolar invertase activity by recent

evidence for their localization in a class of minute vesicles called precursor protease vesicles

(PPV) (Rojo et al., 2003). These organelles surround the large, central vacuole, as well as the

smaller, protein-storage vacuoles (Chrispeels and Herman, 2000; Hayashi et al., 2001; Rojo et

al., 2003). The PPV are best known for their roles as storage sites for precursor proteases that are

later released into vacuoles, where the low pH activates maturation of the protease precursors

(Chrispeels and Herman, 2000; Hayashi et al., 2001; Schmid et al., 2001). These vesicles are

plant-specific ER bodies, formed from dilated cisternae of the ER and surrounded with

ribosomes (Chrispeels and Herman, 2000; Hayashi et al., 2001). PPVcontain a distinct

population of proteins that include protein disulfide isomerase (PDI), binding protein [(BiP)

HSP-70] (Schmid et al., 2001), PYK10 β-glucosidase (Matsushima et al., 2003) and precursor

proteins for diverse cysteine proteases such as responsive to dehydration-21 (RD21) (Hayashi et

al., 2001), cysteine endopeptidase [CysEP (from castor bean)] (Schmid et al., 2001) and vacuolar

processing enzyme gamma (VPEγ) (Chrispeels and Herman, 2000; Rojo et al., 2003).

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Presence of vacuolar invertases in PPV is supported by localization of at least one vacuolar

invertase to these compartments in Arabidopsis (Rojo et al., 2003). Collectively, the PPV

contents, including the vacuolar invertase(s), are released into the vacuolar lumen by a fusion of

these organelles with the tonoplast. The process is distinct from the autophagic engulfment of

other vesicles by the vacuole (Chrispeels and Herman, 2000; Hayashi et al., 2001). When the

PPV contents enter acidic vacuoles, the acid invertase moves into a low-pH, high-sucrose

environment optimal for its activity.

Previous evidence indicates that before vacuolar fusion, PPV are not acidic enough to

support appreciable acid invertase activity. A low pH would also activate maturation of the

VPEγ cysteine protease (Hayashi et al., 2001; Schmid et al., 2001) and, thus, rapidly degrade the

invertase. The AtvacINV2 vacuolar invertase is indeed a target of VPEγ after this protease

matures in the acidic central vacuole (Rojo et al., 2003). However, the rate of proteolytic

degradation of invertase by VPEγ is modest in the vacuole and probably a result of dilution in

the larger compartment. It is feasible that co-retention of both the VPEγ protease and vacuolar

invertase in PPV could be a mechanism of retaining their activities in reserve for later purposes.

Once the PPV fuses with the central vacuole, action of the maturing protease on the invertase

would limit the duration of any increases in sucrose-cleaving activity that arose from the release

of PPV contents into the acidic vacuole. Such pulse-type mechanisms of regulation facilitate fine

control, and are evident for some of the sucrose synthases (Hardin et al., 2003), and for

increments of starvation-induced autophagy (Rose et al., 2006).

The impact of vacuolar invertase retention by PPV could vary considerably. The portion of

total invertase in these organelles at any given time could be significant under some

circumstances and limited in others (Rojo et al., 2003). It is also unclear whether all vacuolar

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invertases follow a PPV path to acid vacuoles and whether this might vary with conditions or

plant development. In Arabidopsis, one or both vacuolar invertases may be involved, because the

proteins are virtually identical (Rojo et al., 2003; Carter et al., 2004). Still another factor is likely

to be the timing and extent of PPV fusions with acid vacuoles. This can occur during senescence

(Schmid et al., 2001; Rojo et al., 2003), salt stress (Hayashi et al., 2001) and possibly during

diverse, pre-senescent phases of development (Rojo et al., 2003). Recent evidence has indicated

PPV may contribute to pathogen responses (Rojo et al., 2004) and aspects of seed development

such as seed coat formation (Nakaune et al., 2005), seed protein properties (Gruis et al., 2004)

and remobilization of seed structures including the megagametophyte (He and Kermode, 2003),

endosperm (Schmid et al., 2001) and nucellar tissue (Greenwood et al., 2005).

Wall-Associated Kinase (WAK)

Recent work has shown that a wall-associated kinase (WAK) can play a role in the

regulation of vacuolar invertase thus establishing a cross-compartmental link between WAK and

vacuolar invertase(s) (Kohorn et al., 2006b).AT-DNA null allele of WAK2 reduced vacuolar

invertase activity in Arabidopsis roots by 62% and decreased levels of a specific vacuolar

invertase transcript (AtvacINV1) by over 50%. The wak2–1 null allele also reduced plant growth

under low nutrient conditions. Unlike effects on vacuolar invertase, the cell wall isoforms were

unaltered by the wak2–1 null allele, indicating either that other WAKs may regulate cell wall

invertases, or that WAK-based signals mediate only vacuolar forms (Kohorn et al., 2006b). This

work further supports the role of vacuolar invertases in resource partitioning as well as turgor

maintenance and cell wall expansion (Koch, 2004).

Wall-associated kinases proteins are hypothesized to serve as sensors of cellular turgor, the

extent of cell wall loosening, and/or the degree of cell expansion (Zhang et al., 2005; Kohorn et

al., 2006b). WAKs are ideally positioned to do so, because they span the plasma membrane

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(Figure B-1). Each of the five WAK genes encodes a transmembrane protein with an active

cytoplasmic serine/threonine kinase domain on the C-terminus and a distinctive extracellular

domain on the N-terminus. The extracellular domain is similar to the vertebrate epidermal

growth factor-like domain. Evidence indicates this extracellular domain may be bound to pectin

in the cell wall, which would provide a link to the extracellular matrix (He et al., 1999; Wagner

and Kohorn, 2001; Decreux and Messiaen, 2005; Kohorn et al., 2006a). This physical tie

between the extracellular matrix and the cytoplasm could provide an anchor, or reference point

enabling the WAK to play a role in sensing or transmitting information on the status of the cell

wall relative to the plasma membrane. Such information could be invaluable to adjustment of

cell expansion or turgor. Different members of the WAK family are expressed at organ junctions,

in shoot and root apical meristems, in expanding leaves, and in response to environmental stimuli

such as wounding and pathogen attack (Wagner and Kohorn, 2001). Antisense reduction of

WAK1–5 protein levels also established the necessity of these genes for expansion of leaf cells

(cellular division was unaffected) (Lally et al., 2001; Wagner and Kohorn, 2001).

The WAK family appears to be part of a larger, 22-member WAK-like (WAKL) family in

Arabidopsis and is widespread in the plant kingdom (Verica and He, 2002). Protein gel blots

usingWAK1 antibodies indicate related proteins in pea, tobacco and maize (He et al., 1996; Gens

et al., 2000). WAKL expressed sequence tags have also been identified in tomato, soybean and

rice (Verica and He, 2002), but a functional relationship between WAK and WAKL has yet to be

shown.

Other Kinases Affecting Invertases

Invertase can be activated by mycorrhization, pathogen infection and wounding, as well as

various hormones (Sturm and Chrispeels, 1990a; Benhamou et al., 1991; Ehness et al., 1997;

Hall and Williams, 2000; Blee and Anderson, 2002; Pan et al., 2005). Diverse kinases appear to

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regulate activity of both cell wall and vacuolar invertases in many of these instances (Ehness et

al., 1997; Pan et al., 2005). The serine/threonine kinase inhibitor, staurosporine, prevents the

accumulation of invertase transcripts that typically occurs during defence responses (Ehness et

al., 1997). In contrast, the same inhibitor, and also two other serine/threonine kinase inhibitors

(K252a and H7), increase the extent to which ABA induces both vacuolar and cell wall

invertases (Pan et al., 2005). However, the tyrosine protein kinase inhibitor, quercetin, strongly

suppresses the ABA induction of acid invertases (Pan et al., 2005) indicating that multiple types

of kinases with differing modes of regulation are involved in modulating invertase activity.

Phosphatases have also been implicated in the regulation of invertases (Ehness et al., 1997;

Pan et al., 2005). Pan et al. (2005) demonstrated that expression and activity of both vacuolar

and cell wall invertases increased in response to ABA and acid phosphatase. These data, in

conjunction with the kinase inhibitor data, indicate that reversible protein phosphorylation is

playing some role in the ABA signaling network (Pan et al., 2005). The phosphatase inhibitor,

endothall, also induces cell wall invertase transcripts when added to cell suspensions of

Chenopodium rubrum (Ehness et al., 1997). This work on invertase is consistent with other

ongoing analyses of both ABA and defense-signaling cascades (Roitsch et al., 2003; Roitsch and

Gonzalez, 2004).

Differential Sugar Regulation of Invertases

Despite the widespread use of invertase expression as a sugar response marker in yeast

(Ahuatzi et al., 2004; Kig et al., 2005), the regulation of these genes has been difficult to dissect

in plants. Not only are there many more individual invertase genes (Tymowska-Lalanne and

Kreis, 1998a; Sturm, 1999; Sherson et al., 2003), but they are also differentially regulated (Xu et

al., 1996; Tymowska-Lalanne and Kreis, 1998a; Huang, 2006), and sugar response mechanisms

vary (Roitsch et al., 1995; Xiao et al., 2000; Huang, 2006). All plant species studied to date have

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two vacuolar invertases (Haouazine-Takvorian et al., 1997), with several cell wall invertases.

Fully-sequenced genomes indicate six putative cell wall invertases in Arabidopsis (Tymowska-

Lalanne and Kreis, 1998a; Sherson et al., 2003) and nine in rice (Ji et al., 2005). Additional

complexity has been introduced by recent evidence that two of the putative cell wall invertase

sequences may encode fructan exohydrolases (a closely-related enzyme) (De Coninck et al.,

2005). Similar invertase family structures are also evident in poplar, maize, potato and tomato

(Fridman and Zamir, 2003; Huang, 2006); P. N. Bocock, unpubl. data).

The majority of these invertases are sugar-induced rather than repressed (Roitsch, 1999;

Roitsch and Ehness, 2000), which is consistent with their roles in carbon use v. carbon

acquisition by plants (Koch, 1996; Rolland et al., 2002; Koch, 2004). Both vacuolar and cell wall

invertases enhance use of carbohydrate resources by cleaving imported sucrose at sites of growth

or storage (Winter and Huber, 2000; Koch, 2004). These invertases can also amplify sugar

signals to other genes that respond to changes in hexose availability (Koch, 2004).

The identities of sugars that induce invertases remain unclear, however. Evidence thus far

indicates limited involvement of the classic hexokinase-mediated sensing system in up-

regulation of invertases by sugars. Neither overexpression of the key hexokinase gene, AtHXK1,

nor its antisense reduction reportedly affects expression of a sugar-induced AtcwINV1 (β-fruct1)

gene for cell wall invertase in Arabidopsis (Xiao et al., 2000). Non-hexokinase mechanisms of

sugar sensing may play more widespread roles in sugar-induced gene expression than previously

recognized (Purcell et al., 1998; Rolland et al., 2002; Sinha et al., 2002; Lalonde et al., 2004;

Halford, 2006). Although signaling systems differ between plants and yeast, the hexokinase-

based sensing in yeast is largely linked to sugar repression rather than induction (Johnston, 1999;

Palomino et al., 2005; Kim et al., 2006), and a balance between the two is hypothesized to

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mediate responses (Ronen and Botstein, 2006). In plants, mechanisms of sugar sensing have thus

far been studied mainly by following responses of genes down-regulated by glucose (e.g.

photosynthesis, seed germination) (Smeekens, 2000; Rolland et al., 2002; Rolland and Sheen,

2005). Another consideration is that genes known to be sucrose- rather than hexose-responsive

are often expressed in or near the vascular system (Rook et al., 1998), as are many invertases

(Andersen et al., 2002; Wachter et al., 2003).

Some acid invertase genes are sugar-repressed. This is less common, but occurs

consistently in all species examined thus far. The singular repression of one vacuolar invertase

by sugars is conserved across gene families in maize (Xu et al., 1996), tomato (Godt and

Roitsch, 1997b), rice (Huang, 2006) and poplar (P. N. Bocock, unpubl. data). At least some

degree of involvement has been indicated for the hexokinase-mediated sensing system in this

repression. Contributions from this mechanism are supported by data from metabolizeable and

non-metabolizeable glucose analogues tested in Arabidopsis for their capacity to repress the

AtvacINV2 vacuolar invertase (Huang, 2006). Another feature of invertase repression by sugars

is its rapid down-regulation, with responses evident in minutes rather than hours (Huang, 2006).

Still another factor may be the demonstrated compartmentalization of the sugar-repressed

AtvacINV2 invertase protein in the precursor protease vesicles [see section on PPV (Rojo et al.,

2003)], implying a possible relationship between the PPV and the observed sugar responses.

Finally, additional insights into the sugar repression of some invertases may lie in their

respective roles. Demands for osmotic constituents (two hexoses from one sucrose) may well

dominate sucrose partitioning in response to specific developmental and/or stress signals.

Expression of at least one vacuolar invertase under these conditions could be highly

advantageous, and need not be viewed as separate from its role in sucrose import. Sucrose

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entering cells is often cleaved first in the vacuole, thus giving vacuolar invertase a prominent role

in sucrose partitioning (Koch, 2004). Theoretically, expression of a starvation-tolerant invertase

could confer an import priority for certain cells or tissues under stress, as well as favor

immediate allocation of incoming resources to osmotic constituents. A similar scenario could

hold for key aspects of development that rely heavily on osmotic constituents for cellular

expansion. Data thus far are consistent with contributions by both the sugar-induced and -

repressed vacuolar invertases to import-based osmotic support of expansion sinks. Examples

include root elongation in Arabidopsis (Stessman, 2004; Huang, 2006; Kohorn et al., 2006b;

Sergeeva et al., 2006), petiole growth in sugar beet (Gonzalez et al., 2005), expansion of ovaries

and silks in maize (Andersen et al., 2002), enlargement of Agrobacterium-induced galls

(Wachter et al., 2003) and growth of diverse fruits and vegetables such as tomatoes, carrot roots

and newly-forming potato tubers (Koch and Zeng, 2002).

RNA Turn Over and DST

At least one mechanism of post-transcriptional regulation of invertase is also indicated for

the sugar-repressed vacuolar invertases. In both rice and Arabidopsis, the 3’ untranslated regions

of the sugar-repressed genes OsVIN1 and AtvacINV2 carry apparent downstream (DST) elements

implicated in rapid turnover of plant mRNAs (Huang, 2006). The DST are highly conserved and

can mediate sequence-specific decay of short-lived mRNA in vivo (Newman et al., 1993;

Sullivan and Green, 1996). The DST are especially notable for their role in rapid destabilization

of small auxin-up RNAs (SAUR) (Newman et al., 1993; Gil and Green, 1996; Johnson et al.,

2000; Feldbrugge et al., 2002).

Additional evidence is also consistent with altered mRNA longevity for the sugar-

repressed form of vacuolar invertase. Although glucose rapidly decreases mRNA levels of the

AtvacINV2 vacuolar invertase in vivo (within 30 min), the promoter alone shows a contrasting

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induction by sugars (Huang, 2006). When transcription is blocked by cordycepin, however, a

glucose-enhanced rate of decay is evident for AtvacINV2 mRNA (Huang, 2006). Glucose-based

destabilization of mRNAs thus seems a likely contributor to the rapid repression observed for the

AtvacINV2 vacuolar invertase (Huang, 2006).

Invertase Inhibitors

Cell wall and vacuolar invertase activity can be regulated by a family of proteinaceous

inhibitors known as cell wall inhibitor of fructosidase (CIF) and vacuolar inhibitor of

fructosidase (VIF), or collectively as C/VIF reviewed by Rausch and Greiner (2004). Although

CIF are cell wall invertase inhibitors, they are broadly active against both cell wall and vacuolar

invertases. In contrast, VIF inhibition is specific to vacuolar invertases. Neither of the inhibitors

affect fungal invertases indicating a minimal role for these interactions in pathogen defense

(Greiner et al., 1998; Greiner et al., 1999; Link et al., 2004). The C/VIF related protein family is

not highly conserved, and in Arabidopsis, sequence identities range from roughly 20 to 40% for

~14 family members (Rausch and Greiner, 2004). The C/VIF related protein family also contains

pectin methylesterase inhibitors (PMEI). PMEI are indistinguishable from the C/VIF by

sequence alone and retain nearly identical structures to the C/VIF (Giovane et al., 2004; Hothorn

et al., 2004a; Hothorn et al., 2004b; Di Matteo et al., 2005).

Important implications arise from the capacity of these related proteinaceous inhibitors to

distinguish between their targets. Arabidopsis has only eight putative acid invertases (six cell

wall and two vacuolar), but more than 60 pectin methylesterase (PME)-related genes based on

sequence similarity (Micheli, 2001; Sherson et al., 2003; Rausch and Greiner, 2004; De Coninck

et al., 2005). X-ray crystollagraphy has revealed that CIF is conformationally stable over a broad

pH and temperature range, however, the invertase/inhibitor complex is only stable at an acidic

pH. This indicates that binding is determined not by conformational changes, but rather by pH-

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induced changes at the interface of the invertase/inhibitor complex (Hothorn et al., 2004a;

Hothorn and Scheffzek, 2006). In contrast, the highly similar structure of the PMEI was found to

undergo large structural rearrangements (Figure B-2) under these same conditions suggesting

PMEI uses a different mode of binding than does the CIF (Hothorn et al., 2004b). PMEI contains

a flexible α-hairpin that is important both in dimer formation and in binding of PME (Hothorn et

al., 2004b). Chimeral domain swap experiments of the α-hairpin domain between PMEI and CIF

indicate that this domain of PMEI is necessary and sufficient for activity against PME; however,

the corresponding α-hairpin in NtCIF is not sufficient for invertase inhibition (Hothorn et al.,

2004b). The ~28 amino acid residues encoding this α-hairpin may be key in distinguishing these

two classes of inhibitors (Hothorn et al., 2004b).

Analysis of PMEI and CIF crystal structures not only clarifies interactions between these

inhibitors and their targets, but also aids our understanding of how distinct functions can arise for

proteins sharing very similar structures and ancestry. It is worth noting, however, that the

crystallographic analyses were performed on the cell wall invertase inhibitor and thus do not

explain the apparently narrower substrate specificity of the vacuolar invertase inhibitor. These

data indicate that the VIF may use a different mode of action than the CIF. It is also of interest

that two proteins in Arabidopsis previously annotated based on sequence similarity as cell wall

invertases (AtcwINV3&6), may actually be fructan exohydrolases (FEH).FEH protein sequences

are nearly identical to those of demonstrated acid invertase family members, yet the recombinant

enzymes are completely inactive against sucrose (De Coninck et al., 2005). It has yet to be

determined if the C/VIF family can distinguish between the FEH and the known invertase and

PME substrates, and given the level of sequence diversity within the inhibitor family it seems

likely that biochemical and structural analyses will be required to resolve these questions.

127

Summary

Compartmentalization of vacuolar invertase in PPV introduces a new dimension to

regulation of invertases in vivo (Rojo et al., 2003; Koch, 2004). The PPV sequester at least some

of the vacuolar invertase(s) (Rojo et al., 2003) and other proteins (especially protease precursors)

(Chrispeels and Herman, 2000; Hayashi et al., 2001) for later release into acid vacuoles. It is

currently unclear whether these invertases are active inside PPV before vesicle fusion with

acidic, sucrose-containing vacuoles.

Wall-associated kinases can regulate vacuolar invertases (Kohorn et al., 2006b). At least

one of them (WAK2) regulates a specific vacuolar invertase (AtvacINV1) that predominates in

Arabidopsis roots. IfWAK2 is dysfunctional, vacuolar invertase activity drops to less than 50%

in roots, and growth is impaired under low-osmolyte conditions. The WAK kinases are ideally

positioned to serve as status sensors for the interface between the plasma membrane and cell

wall, because each WAK has an N-terminus in the extracellular matrix (possibly associated with

pectin), and a C-terminal serine/threonine kinase domain in the cytoplasm (signaling capacity).

Diverse kinases and phosphatases have been implicated in the regulation of invertases,

which is consistent with the range of signaling networks that affect them. These extend from

sugar and ABA sensing, to interactions with pathogens and symbionts.

Contrasting responses to sugars within the invertase gene family may also be mediated by

different mechanisms. Some degree of hexokinase involvement and distinctive kinetics have

been observed for the sugar-repressed invertases (Huang, 2006), but not for the more common,

sugar-induced forms examined thus far (Xiao et al., 2000).

DST elements implicated in mRNA turnover also lie in the 3’ untranslated region of the

most rapidly repressed invertases. Their sequences resemble those in SAUR where they mediate

mRNA destabilization.

128

The invertase inhibitors, CIF and VIF share high sequence similarity with PMEI, but

evidence from crystal structures and chimeral proteins suggests that binding specificity may be

determined by flexibility of a short, N-terminal region.

Acknowledgements

We thank the National Science Foundation, Metabolic Biochemistry Program, GrantAward

No. MCB-0080282; the Department of Energy,Office of Science, Office of Biological and

Environmental Research, Grant Award No. DE-AC05–00OR22725; and the University of

Florida Experiment Station for funding.

129

Cellwall

Extracellulardomain

Transmembranedomain

Nucleus

PPVs

Plasma membrane

VPEγ

Vacuole

Vacuolar invertase

STK domain

WAK

Cellwall

Extracellulardomain

Transmembranedomain

Nucleus

PPVs

Plasma membrane

VPEγ

Vacuole

Vacuolar invertase

STK domain

WAK

Figure B-1. Recent additions to mechanisms controlling invertases include the wall associated kinases (WAK), precursor protease vessicles (PPV), and vacuolar processing enzymes (VPE). At least one WAK (WAK2) can regulate activity of a specific vacuolar invertase (AtvacINV1) in Arabidopsis. The WAK are ideally positioned to detect alterations in the interface between the cell wall and plasma membrane (e.g. turgor or expansion), because their extracellular terminus resides in the cell wall matrix (possibly anchored in pectin) and their cytoplasmic tail includes a kinase domain. This kinase can apparently trigger cascades regulating transcription of vacuolar invertase and other effectors of its activity. Also, at least some vacuolar invertase(s) reach the vacuole through PPV, where they can be sequestered for extended periods. Timing of activity may be determined by when the enzyme is released to an acidic, sucrose-storing environment. The PPV also releases protease precursors such as the vacuolar processing enzyme [VPEγ (▲)], into the acidic vacuole which then act upon vacuolar invertases (●) to limit the duration of their activity.

130

PMEIOpen conformation

Closed conformation

CIF

XX

PMEIOpen conformation

Closed conformation

CIF

XXXX

Figure B-2. Protein inhibitors of cell wall invertases (CIF, cell wall inhibitor of fructosidase) share strong sequence similarity with inhibitors of pectin methylesterase (PMEI, pectin methylesterase inhibitor), but crystal structures indicate important differences in flexibility. The top panel depicts CIF. The oval denotes an α-hairpin thought responsible for binding specificity of CIF and PMEI. The α-hairpin in CIF is rigid at all pHs and temperatures tested. The bottom panel shows PMEI in three different conformations that demonstrate the flexibility of the PMEI α-hairpin. (Hothorn et al., 2004a; Hothorn et al., 2004b; Hothorn and Scheffzek, 2006). Images were constructed by Protein Explorer (Martz, 2002).

131

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BIOGRAPHICAL SKETCH

Philip Bocock was born May 20, 1979 in Wilmington, Delaware. He was raised in Austin,

Texas where he attended Westwood High School. He graduated high school in 1997 and entered

Texas A&M University in September of the same year. He earned his Bachelor of Science in

biochemistry and genetics accompanied with a minor in forest science in May of 2001.

In the fall of 2001, Philip began work on a PhD in the Plant Molecular and Cellular

Biology Program at the University of Florida. At the University of Florida, Philip entered the

laboratory of Dr. John Davis and began conducting research on carbon allocation and

partitioning using the model tree, Populus. Under the tutelage of Dr. John Davis, Philip

identified the members of the poplar invertase gene family and subsequently employed a variety

of techniques to seek to understand the role this enzymatic family plays in carbon allocation and

partitioning in poplar. The work detailed in this dissertation was entirely conducted in the

laboratory of Dr. John Davis.