ARBORKNOX2 Gene Regulation of Secondary Growth in Populus.

Du, Juan

USDA Forest Service

Institute of Forest Genetics

Davis, CA

Dr. Andrew Groover lab.

ARBORKNOX2 Gene Regulation of Secondary Growth

Research Outline

3

ARK2 Expression pattern analysis

ARK2 functional analysis using transgenic poplar

32

Identifying genes and processes regulated by ARK2

41 Introduction of secondary growth and Class I KNOX gene

34

Secondary Growth is a Continual Developmental Process

• Vascular cambium cells process periclinal division, their daughter cells are recruited into secondary xylem (wood) or phloem (bark) (Esau 1965; Esau 1977).

Phloem

Cambium

Xylem

The Vascular Cambium Shares Fundamental Features With Meristematic Cells of Shoot Apical Meristem

(Groover 2005)

Meristematicmaintenance

PatterningPolarity

Recruitment &differentiation

Shoot Apex Stem

We hypothesize that genes which regulate SAM also regulate vascular cambium development.

KNOX Transcription Factors Regulate the Shoot Apical Meristem

• KNAT1 is a class I KNOX gene family member• KNAT1 regulates meristem maintenance and differentiation.

M.Byrne

ARK2 is a Populus ortholog of Arabidopsis KNAT1 .(G.Chuck et al 1996)

ARK2 is Expressed During Secondary Growth

• ARK2 is expressed in both shoot apex and stem tissue in poplar

Rel

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xpre

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Whole Mount In Situ Hybridization

Cambium

ARK2-Antisense 5X ARK2-Sense 5X

Procambium

Cambium;Developing phloem fibers;Developing xylem cells;

Internode 1

Internode 3

Internode 5

Internode last

ARK2 Gene Regulation During secondary growth by

trans-genetics approach

Gain-of function Loss –of function

Over expression (35sCAMV)

RNA interferenceArtificial micro-RNA

ARK2 c35s-37 amicroRNA2RNAi23

ARK2 Knockdown Transgenics Show Early Secondary Growth

Internode 7 X10 Internode 7 X40

WT

ark2

ARK2 Expression is Down-regulated in ark2 Knockdown Plants

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ARK2 Overexpression Changes in Plant Architecture (2 month old plants)

shorter internodes, altered leaf shape and leaf vein

WT 717 35S:ARK2 35S:ARK2WT 717

In Transition to Secondary Growth, 35S:ARK2 has Increased Procambium Tissue and Early Cambium Formation

Wild Type

35S:ARK2

procambium

cambium

Internode 1 10X Internode 1 40X

Later in Development, 35S:ARK2 Has an Wider Cambium Region, and Reduced or Delayed Cell Differentiation

Wild Type

35S:ARK2

Internode Last 10X Internode Last 40X

Cambium

ARK2 Expression Is Up-regulated in 35S:ARK2 R

elat

ive

Exp

ress

ion

Genes Misexpressed In 35S:ARK2 Stems

217 probes from annotated genes show 3 fold or greater expression differences in 35S:ARK2 stems

HormoneTranscriptionWallOther

Genes Misexpressed in Both 35S:ARK2 and miRNA:ark2 Stems --Transcription Factors

Homeobox protein knotted-1 like 1 (KNAT1) HD-Zip transcription factor PHBHD-Zip transcription factor REV BEL1-like homeodomain 3 protein

myb family transcription factor (MYB4) (MYB86) Axial regulator YABBY1TCP family transcription factor

Transcription Factors Expression in 35S:ARK2, Wild Type, miRNA:ark2

01

2345

67

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ARK2 REV PHB BEL MYB4 YAB1 YAB1-3

35S:ARK2Wild Type

miRNA: ark2

Genes Misexpressed in Both 35S:ARK2 and miRNA:ark2 Stems --Hormone Related Genes

•auxin efflux carrier protein (PIN1)•auxin-responsive AUX/IAA family protein•auxin-responsive GH3 protein•IAA-amino acid hydrolase 1 (ILR1)

•ethylene response factor 1 (ERF1)•ethylene-responsive element-binding factor 2 (ERF2)•gibberellin-regulated family protein•gibberellin 20-oxidase

Hormone Related Genes Expression in 35S:ARK2, Wild Type, miRNA:ark2

0

1

2

3

4

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35S:ARK2

Wild Type

miRNA: ark2

AP2 GH3 GAR

Genes Misexpressed in Both 35S:ARK2 and miRNA:ark2 Stems --Cell Wall and Lignin Synthesis

•cellulose synthase•expansin family protein (EXPR3)•fasciclin-like arabinogalactan-protein (FLA11)•glycogenin glucosyltransferase•xyloglucan:xyloglucosyl transferase•Laccase

•ferulate-5-hydroxylase (F5H)•caffeoyl-CoA 3-O-methyltransferase (CCoAOMT)•4-coumarate--CoA ligase (4CL)•cinnamic acid 4-hydroxylase (C4H)•cinnamyl-alcohol dehydrogenase (CAD)

Lignin Genes Expression in 35S:ARK2, Wild Type, miRNA:ark2

0

0.5

1

1.5

2

2.5

3

3.5

Rel

ativ

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xpre

ssio

n 35S:ARK2

Wild Type

miRNA: ark2

C4H 4CL CAD C3H F5H CCoAOMT LAC

Lignification is Altered in 35S:ARK2

Wild Type35S:ARK2

18.9 2.014.3 0.9

Acid

inso

luble*

Acid

Solu

ble*

*mg/100mg

A marked reduction of polymerized lignin in the stem.

ARK2 Regulation of the Cambium Differentiation and Secondary Growth

Mer

iste

mM

eris

tem

Mai

nten

ance

Mai

nten

ance

Cell

& Ti

ssue

Cell

& Ti

ssue

Dif

fere

ntia

tion

Dif

fere

ntia

tion

ARK2

Image from Dr David Webb

• ARK2 negatively regulates differentiation of meristem cells, and promotes indeterminate cell fate.

•ARK2 negatively regulates lignin biosynthesis and secondary cell wall synthesis process.

•ARK2 shows overlapping mechanisms regulate the shoot apical meristem and the vascular cambium

Institute of Forest GeneticsAndrew Groover

Gayle DupperMarcel Robischon

Cecilia Osorio

University British ColumbiaShawn Mansfield

Acknowledgements

ARK2 Knockdown transgenics shows early secondary growth and thicker secondary cell wall in phloem fiber cells and

xylem tissue

WT

RNAiark2

Internode 4 Internode 7 Internode 7

ARK2 expression is down-regulated in ark2 knockdown plants

RNAi

• RNAi原理示意图：紫 色为正义RNA断，蓝 色为反义RNA断，绿 色为目标信使RNA， dsRNA触发了高效的 基因沉默机制并极大 降低了靶mRNA水 平，达到干扰目的

miRNA• MicroRNAs (miRNAs) constitute a large group of

endogenous single stranded small RNAs (19-24 nucleotides) that have negative regulatory function on gene expression. They are processed from longer non-coding RNAs with characteristic fold- back structures by double-strand specific RNases of the Dicer family. Upon processing, they are incorporated in the RNA induced silencing complex RISC by binding to its main component, an Argonaute protein. MiRNAs serve as the specificity components of RISC, since they base-pair to target nucleic acids, mostly mRNAs, in the cytoplasm. Subsequent regulatory events include target mRNA cleavage (often followed by degradation of cleavage products), which is mainly triggered by plant miRNAs, and translational inhibition, as mostly seen in animals. Target sites in plant miRNAs normally share high sequence complementarity to the respective miRNA and are often in coding regions, whereas in animals, target sites are often only partially complementary to their target sites and are mostly located in the 3'UTR of target genes

ARBORKNOX2 Gene Regulation of Secondary Growth --Talk Outline

3

ARK2 Expression pattern analysis

ARK2 functional analysis using transgenic poplar

32

Identifying genes and processes regulated by ARK2

- qRT-PCR analysis- In situ hybridization analysis

- Gain of function and Loss of function transgenetic plants- Histology to check secondary growth in stem- Real-Time PCR and Microarray to check genes expression

- Microarray data

41 Introduction of secondary growth and Class I KNOX gene

34

miRNA:ark2 transgenics

longer internodes