GA3ox expression influences plant growth in pea · Canada Discovery Grants to JAO and RPP, and the...

1

GA3ox expression influences plant growth in pea

Jocelyn A. Ozga (corresponding author) 4-10 Agriculture/Forestry Centre Dept. of Agricultural, Food and Nutritional Science University of Alberta Edmonton, Alberta, Canada T6G 2P5 Phone: 780-492-2653 Fax: 780-492-4265 e-mail: [email protected] Research Area/Associate Editor: Biochemistry and Metabolism/ Julian Hibberd (Cambridge) This work provides insight into the fundamental understanding of GA biosynthesis and

catabolism regulation at the plant tissue and organ level, and the importance of

timing/localization of gene expression for the production of bioactive GA, and thereby plant

growth.

Plant Physiology Preview. Published on August 26, 2013, as DOI:10.1104/pp.113.225987

Copyright 2013 by the American Society of Plant Biologists

www.plantphysiol.orgon March 16, 2019 - Published by Downloaded from Copyright © 2013 American Society of Plant Biologists. All rights reserved.

http://www.plantphysiol.org

2

GA3ox gene expression patterns influence GA biosynthesis, growth, and development in pea

Dennis M. Reinecke†1, Aruna D. Wickramarathna†1, Jocelyn A. Ozga*1, Leonid V. Kurepin2, Alena L. Jin1, Allen G. Good3, Richard P. Pharis2

1 Plant BioSystems, Department of Agricultural, Food and Nutritional Science University of Alberta, Edmonton, Alberta, Canada T6G 2P5

2 Department of Biological Sciences, University of Calgary, Calgary, AB, Canada T2N 1N4 3 Department of Biological Sciences, University of Alberta, Edmonton, Alberta, Canada T6G 2E9 † These authors contributed equally to this manuscript. *Corresponding Author



3

Footnotes:

This research was supported by the Natural Sciences and Engineering Research Council of

Canada Discovery Grants to JAO and RPP, and the Alberta Agriculture Research Institute Grant

to JAO. ADW also received a Commonwealth Scholarship supporting his graduate studies.

Jocelyn A. Ozga (corresponding author; e-mail: [email protected])



4

ABSTRACT

Gibberellins (GAs) are key modulators of plant growth and development. PsGA3ox1 (LE)

encodes a GA 3β-hydroxylase which catalyzes the conversion of GA20 to biologically active

GA1. To further clarify the role of GA3ox expression during pea (Pisum sativum) plant growth

and development, we generated transgenic pea lines (in a lele background) with CaMV-35S-

driven expression of PsGA3ox1 (LE). PsGA3ox1 transgene expression led to higher GA1

concentrations in a tissue-specific and developmentally-specific manner, altering GA

biosynthesis and catabolism gene expression, and plant phenotype. PsGA3ox1 transgenic plants

had longer internodes, tendrils and fruits, larger stipules, and displayed delayed flowering,

increased apical meristem life, and altered vascular development, relative to the null controls.

Transgenic PsGA3ox1 overexpression lines were then compared to lines where endogenous

PsGA3ox1 (LE) was introduced, by a series of backcrosses, into the same genetic background

(BC LEle). Most notably, the BC LEle plants had substantially longer internodes containing

much greater GA1 levels than the transgenic PsGA3ox1 plants. Induction of expression of the

GA deactivation gene PsGA2ox1 appears to make an important contribution to limiting the

increase of internode GA1 to modest levels for the transgenic lines. In contrast, PsGA3ox1 (LE)

expression driven by its endogenous promoter was coordinated within the internode tissue to

avoid feed-forward regulation of PsGA2ox1, resulting in much greater GA1 accumulation. These

studies further our fundamental understanding of the regulation of GA biosynthesis and

catabolism at the tissue and organ level, and demonstrate that the timing/localization of GA3ox

expression within an organ affects both GA homeostasis and GA1 levels, and thereby growth.



5

INTRODUCTION

It is now possible to alter the levels of bioactive GAs in plants through transgenic

modification of the expression of GA biosynthesis and catabolism genes, in some cases

reproducing the effects obtained by the application of chemical growth regulators (Hedden and

Phillips, 2000a). For targeting changes in phenotype of specific tissues, it is important to

understand how GA biosynthesis and catabolism are regulated at both the tissue and organ level,

and also across developmental stages. Attempts to modulate the expression of GA biosynthesis

and catabolism genes through transgenic approaches in various species have been reported (see

Phillips, 2004) and data suggest that the successful regulation of levels of bioactive GAs

involves modifications in the expression of genes in the later part of the GA pathway (Fleet et

al., 2003; Swain et al. 2005). Results from the use of GA biosynthesis mutants and also studies

on GA biosynthesis inhibitors suggest, for pea (Pisum sativum L.) and Arabidopsis, that

feedback regulation of transcript abundance by high concentrations of a bioactive GA also

appear to be specific to the later part of the GA biosynthesis pathway (Fig. 1; Ayele et al., 2006:

Davidson et al., 2005: Hedden and Phillips, 2000b).

Overexpression of Arabidopsis GA20ox homologs which encode the multi-functional

enzymes (GA 20-oxidases) that catalyze the GA12 � GA15 � GA24 � GA9 biosynthesis steps,

can yield higher levels of GA9, which in turn can be converted by a GA 3-oxidase to GA4 (the

main bioactive GA in Arabidopsis). The results of this GA20ox overexpression are Arabidopsis

plants with longer hypocotyls and stems, earlier flowering, and the production of seed with a

reduced period of dormancy (Huang et al., 1998; Coles et al., 1999). Similarly, Biemelt et al.

(2004) reported that stem growth, rate of photosynthesis and biomass production in tobacco were

all enhanced when AtGA20ox1 was overexpressed. Over expression of citrus GA20ox

(CcGA20ox1) in tobacco also resulted in a shift from the early 13-hydroxylation pathway of GA

biosynthesis to the early non-13-hydroxylation pathway, leading to GA4 becoming the

predominant bioactive GA in the transgenic tobacco plants (Vidal et al., 2001). In rice, over

expression of OsGA20ox1 resulted in internode elongation that was about 2-fold greater than the

elongation of the wild-type line (Oikawa et. al., 2004).

For hybrid aspen (Populus tremula x P. tremuloides), over expression of the AtGA20ox1

transgene resulted in transgenic lines with appreciably increased levels of C20 GAs as well as

increased levels of bioactive GA1 and GA4. There was also increased growth in tree stem heights



6

and diameters, larger leaves, more numerous and longer xylem fibers, and increased stem

biomass (Eriksson et al., 2000). The implication for the hybrid aspen was that the GA 20-

oxidase-catalyzed steps in GA biosynthesis were rate-limiting in the non-transgenic trees. In a

subsequent study, over expression of AtGA3ox1 (which encodes for a GA 3-oxidase) in

transgenic hybrid aspen did not lead to major increases in GA1 or GA4, or to increases in tree

growth or morphology. Based on these latter results it was hypothesized that the GA 3-oxidase

step in the non-transgenic hybrid aspen trees was not rate-limiting (Israelsson et. al., 2004). In

contrast, Radi et al. (2006) reported that over expression of pumpkin CmGA3ox1 in Arabidopsis

resulted in transgenic plants with elevated levels of GA4, as well as increased hypocotyl,

internode and leaf growth, and earlier flowering, all relative to wild-type plants. From these

results, Radi et al. (2006) concluded that GA 3-oxidation is a rate-limiting step in GA plant

hormone biosynthesis that controls plant development. Over expression of the pea gene that

encodes for GA 3-oxidase (PsGA3ox1) in tobacco plants resulted in modest increases in

hypocotyl and stem length, and larger leaves, and was associated with a small but significant

increase of bioactive GA1 (Gallego-Giraldo et al., 2008). However, since transgenic over

expression of GA20ox had a greater effect on plant height and active GA levels than transgenic

GA3ox overexpression in tobacco, and the phenotype of hybrid GA3ox/GA20ox over expression

tobacco lines was relatively similar to the GA20ox overexpressor parental line, Gallego-Giraldo

et al. (2008) concluded that GA 3-oxidases are non-rate limiting enzymes in tobacco. These

researchers also observed large increases in transcript abundance of the GA catabolism genes

NtGA2ox3 and NtGA2ox5 (between 10- and 60-fold) in the PsGA3ox1-overexpressed transgenic

lines of tobacco. These latter data suggest that enhanced GA catabolism (2β-hydroxylation) may

be associated with elevated GA3ox transcript levels and the associated increase in bioactive GA.

Pea, a large-seeded non-bolting legume, can serve as a model species for comparison to other

studied species such as Arabidopsis, tobacco and spinach, which are small-seeded species that

exhibit a rosette habit while vegetative., but undergo rapid internode elongation (bolting) under

certain environmental conditions that then leads to flowering. The role for GAs in the regulation

of internode elongation in pea has been well established by the use of PsGA3ox1 mutants which

lead to GA deficiency (Ingram et al., 1984; Ross et al., 1989; Lester et al., 1997; Martin et al.,

1997). Of equal interest is the characterization of GA1 biosynthesis over-production pea

mutants, though pea GA1 biosynthesis mutants with an intact GA signal transduction pathway



7

that lead to overproduction of GA1 have not yet been identified. Given the variation in response

to GA3ox overexpression among the plant species cited above, and the lack of information on

timing and localization of transgenic GA3ox expression in these studies, it was of interest to

investigate whether internode elongation and/or other plant phenotypes are affected by higher

GA1 levels caused by over expression of PsGA3ox1 in pea. Furthermore, to determine if the

localization of PsGA3ox1 transcripts within a tissue affects GA homeostasis, we used two

approaches, transgenic and backcrossing, to determine if increasing PsGA3ox1 transcript

abundance can alter GA homeostasis, and plant vegetative and reproductive morphology in this

species. The effects of modifying PsGA3ox1 (LE gene) transcript abundance using these two

approaches in a similar genetic background were then compared at the phenotypic, molecular,

chemical and biochemical levels. These studies further the fundamental understanding of GA

biosynthesis and catabolism regulation at the plant tissue and organ level, and across

development. Furthermore, this work provides insight into the importance of timing/localization

of GA3ox gene expression for the production of bioactive GA, and highlights the importance of

understanding gene expression dynamics when attempting to modulate the expression of GA

biosynthesis and catabolism genes to bring about specific phenotype changes through transgenic

approaches.

RESULTS AND DISCUSSION

Transgenic lines

We selected 2 transgenic lines (TG1 and TG2) that constitutively expressed PsGA3ox1

(LE; a fully functional wild-type GA 3β-hydroxylase gene; Lester et al., 1997) in a semi-dwarf

lele pea line (‘Carneval’; le-1; single base-pair mutation in PsGA3ox1; Lester et al., 1997) based

on morphological characteristics (node and numbering system given in Supplementary Fig. S1).

Their transgenic nulls (C1 and C2; lines where the transgene had segregated out at the T2

generation) were also characterized and they were found to be morphologically similar to each

other in shoot height, total number of internodes, and number and length of lateral branches

(LSD P≥0.5, data not shown); therefore, one (C1) was chosen as the transgenic null control line

in the various comparisons. Characterization of a third transgenic line (TG3) and its transgenic

null control (C3, which varied from C1 null in phenotype) is described in Supplementary Figs.

S2-S5.



8

CaMV-35S constitutive promoter expression in pea tissues

PsGA3ox1 driven by the CaMV-35S promoter was expressed constitutively in all cell types

of expanding internodes, tendrils, stipules and pericarps as suggested by the expression of the

marker gene, β-glucuronidase (GUS), driven by the CaMV-35S constitutive promoter

(Supplementary Fig. S6). The low abundance and labile nature of the 2-oxoglutarate-dependent

dioxygenases occurring late in the GA biosynthesis pathway including GA 3-oxidase make it

difficult to confirm that PsGA3ox1 transcript abundance leads to increases in GA 3-oxidase

protein levels. However, precedence for the close correlation between the mRNA abundance of

a GA 2-oxoglutarate-dependent dioxygenase (GA 20-oxidase) with the levels of the protein was

established by Lee and Zeevaart (2007) in the photoperiodic regulation of SoGA20ox1

expression in spinach.

Transgenic PsGA3ox1 over expression increases internode elongation and modifies GA

metabolism

TG1 produced significantly (P<0.05) longer internodes relative to its null control (C1) in

all internodes from 1 to 15 (Fig. 2A), with an average internode length 22% greater than C1. A

similar pattern of increased internode length was observed in TG2 and TG3, where the average

increase in internode length was 13% (TG2; Fig. 2A) and 8% (TG3; Supplementary Fig. S2A)

relative to their respective null controls. These increased internode lengths are consistent with the

expression of the PsGA3ox1 transgene and also with higher levels of total PsGA3ox1 (transgene

+ endogenous gene) transcript (Table I; Supplementary Fig. S3C and D). The CaMV-35S-driven

expression of the PsGA3ox1 (LE) gene in ‘Carneval’ (le) consistently resulted in an increase in

biosynthetic flux for endogenous GA20 � bioactive GA1 � biologically inactive GA8 (the

immediate catabolite of GA1) for TG1 (Table II), the line with the highest levels of transgene

expression (Table I; TG1 relative to C1).

Elongating internode transcript levels of the endogenous biosynthesis gene PsGA20ox1

(encodes for GA 20-oxidase, which catalyzes the GA53 � GA44 � GA19 � GA20 biosynthesis

steps; Fig. 1), either increased (compared to C1) or were maintained at similar levels (compared

to NTC1) in the presence of PsGA3ox1 transgene (Table I), while GA20 levels in both TG1 and

TG2 plants were maintained at levels similar to those seen for C1 (Table II). In contrast,



9

endogenous GA20 in tobacco apical shoot parts (Gallego-Giraldo et al. 2008) and both GA20 and

GA9 in hybrid aspen internodes (Israelsson et. al., 2004), markedly decreased in transgenic

GA3ox1-overexpression lines, suggesting that for both tobacco and aspen the transgenic GA3ox

over expression quickly depleted the substrate pool of upstream precursor GAs. Furthermore,

up-regulation of NtGA20ox1 was not observed in the transgenic PsGA3ox1-overexpression

tobacco lines (Gallego-Giraldo et al., 2008).

GA1 levels are low in the lele pea genotypes due to the presence of the PsGA3ox1 (le) gene

that contains a point mutation that dramatically reduces the enzyme activity of the GA 3-oxidase

that it encodes (Lester et al., 1997; Martin et al., 1997). This leads to the accumulation of GA20,

the substrate for GA 3-oxidase in pea lele shoot tissue (Ross et al., 1992). Furthermore,

PsGA20ox1 gene expression is induced in response to low levels of GA1 in the lele genotypes as

part of a feedback regulatory mechanism (Martin et al., 1996; Hedden and Phillips, 2000b).

These events lead to the accumulation of high levels of GA20 in the lele genotypes (see C1 which

is in a lele genetic background, Table II). Our results, then, imply that high levels of endogenous

PsGA20ox1 (Table I) and production of GA20 in the internodes (Table II) circumvent depleting

the GA20 substrate pool that is needed for 3β-hydroxylation to bioactive GA1 in the PsGA3ox1

transgenic pea lines. This is in contrast to GA20 depletion observed in GA3ox overexpression

lines of tobacco (Gallego-Giraldo et al. 2008) and hybrid aspen (Israelsson et. al., 2004).

In elongating internodes, transgene PsGA3ox1 expression also led to increased transcript

abundance of the catabolic GA 2β-hydroxylation step gene, PsGA2ox1 (this gene is primarily

responsible for conversion of GA20 to GA29; Table I; Supplementary Fig. S3E), and this

increased PsGA2ox1 transcript abundance was positively correlated with elevated internode

GA29 levels for TG1 and TG2 (Table II). Increased GA catabolism in elongating internodes of

the PsGA3ox1-overexpression lines is considered to be indicative of the feed-forward regulation

of GA biosynthesis that is induced by increased levels of a bioactive GA (Thomas et al., 1999).

Although up-regulation of the expression of two GA catabolic genes, NtGA2ox3 and NtGA2ox5,

was observed in the apical shoots of 7 d-old tobacco plants when PsGA3ox1 was over expressed

(Gallego-Giraldo et al., 2008), whether 2β-hydroxylation of GA20 or GA9 (to GA29 or GA51,

respectively) actually increased was not determined.

There was no consistent effect of transgenic PsGA3ox1-over expression on PsGA2ox2

transcript levels (Table I). However, higher transcript abundance of the GA1 deactivating gene,



10

PsGA2ox2 in TG2 internodes was observed (Table I), and this may explain the lower GA1 levels

(Table II) observed in TG2, relative to TG1. Also, PsGA3ox2 transcripts (data not shown) were

not detected in any of our transgenic or control lines within the elongating internode tissues at

the chosen nodal position.

Transgenic PsGA3ox1 over expression increases apical meristem life and delays transition

to a floral meristem

Zhu and Davies (1997) showed that apical bud vigor and increased longevity in the G2 pea

genotype was correlated with an increased level of GA20 and GA1 in the apical buds. Consistent

with the elevated flux through the GA biosynthesis pathway that was seen for the internodes, we

also observed, for two of the transgenic lines, an increased apical meristem life (more nodes per

plant) and a delayed apical meristem transition from the vegetative phase to the reproductive

phase (see Table III for TG1 and also see TG3 compared to C3; Supplementary Table S1; the

highest number of nodes was with TG1, the transgenic line with the highest level of PsGA3ox1

expression, Table I), as also noted by Kelly and Davies (1986) for G2 peas in short days, a

condition associated with higher levels of GA20 and GA1 (Proebsting et al., 1978; Zhu and

Davies, 1997). Proebsting et al. (1978) demonstrated that application of GA3 to the apical buds

of G2 peas maintained apical growth, and that apical vigor observed in short days in these peas

was correlated with the presence of a growth-active GA later shown by Zhu and Davies (1997) to

be GA1. ‘Carneval’ responds similarly to GA3 application (Wickramarathna, 2009). The increase

in shoot apical meristem longevity caused by increased levels of a bioactive GA may be

attributed to increased mitotic activity in the shoot apex, as exogenously applied GA3 can

increase the mitotic activity in the shoot apex of Silene and Perilla (for review, see Pharis and

King, 1985; Besnard-Wibaut et al., 1983), and an increase in the number of cells in pea (Yang et

al., 1996). Furthermore, it has been observed that high concentrations of applied bioactive GAs

can not only stimulate vegetative growth (Dennis, 1973), but also lengthen the morphological

age to flowering in some plant genera (Fuschia, Hieracium and Fragaria; for review, see Pharis

and King, 1985). However, this response is not ubiquitous across genera, as increased flux

through the GA biosynthesis pathway generated by GA20ox overexpression (Huang et al., 1998;

Coles et al., 1999) or GA3ox overexpression (Radi et al., 2006) led to earlier flowering in

Arabidopsis.



11

Transgenic PsGA3ox1 over expression changes internode vascular anatomy

Internodes at the chronologically early nodal positions were longer (TG1 113% and TG2

40% at internode 2) and thinner (smaller in cross-sectional area and diameter; 9-24% smaller at

internode 2) in the transgenic PsGA3ox1-over expressing lines than in the C1 line (Fig. 2A;

Supplementary Table S2 and Fig. 3A). This phenotype is reminiscent of the sln pea mutant,

where a null mutation in the GA catabolism gene, PsGA2ox1, leads to higher levels of GA20 in

the mature seed, and likely increased transport of the GA20 from the large cotyledonary pool to

the embryo axis in the imbibed seed (where it can be converted to bioactive GA1). It is

hypothesized that the resultant higher bioactive GA1 levels in the embryo axis are responsible for

the very long, slender internodes seen during early seedling growth of sln (Reid et al., 1992;

Ross et al., 1993; Lester et al., 1999; Ayele et al., 2012). Fagoaga et al (2007) also observed a

decrease in stem diameters of citrus when CcGA20ox1 was over expressed. However, transgenic

(AtGA20ox1) hybrid aspen exhibited increased average stem diameter, likely due to GA1 and

GA4 effects on growth of cambial region xylem and phloem tissues (Eriksson et al., 2000). For

TG1 the cross-sectional area ratio of cortex to vascular stele tissue was similar to C1 at internode

2 (Supplementary Table S2), indicating that the GA1-induced decrease in stem cross-sectional

area reflects similar reductions in cortical and vascular stele regions. Also, within the vascular

stele of TG1’s internode 2, the phloem-enriched regions were reduced, while the cross-sectional

area of the xylem vessel elements was increased, both relative to C1 (Fig. 3B; Supplementary

Table S2). Higher CaMV-35S-driven PsGA3ox1 expression in developing xylem vessel

elements, compared to other cell types (see Supplemental Fig. S6), is likely a primary factor in

elevating GA1 concentration, resulting in the larger diameter xylem vessel elements. Biemelt et

al. (2004) observed an increase in the number of xylem vessel elements when AtGA20ox1, driven

by the CaMV-35S promoter, was expressed in tobacco stems, and a decrease in number of xylem

vessel elements when AtGA2ox1, driven by the CaMV-35S promoter, was expressed in the stem

tissue of tobacco. Tissue-specific localization of GAs and expression of GA-biosynthetic and

signaling genes in wood-forming tissues of hybrid aspen indicated that the late stages of GA

biosynthesis occur in expanding xylem, and this suggests that bioactive GAs play a role in the

elongation of the hybrid aspen xylem (Israelsson et al., 2005). Our data also strongly suggest that

bioactive GA1 is involved in increasing xylem vessel element diameters in pea.



12

Interestingly, in the hybrid aspen stems a gradient of GAs were observed across the

vascular tissue (phloem-cambium-xylem; Israelsson et al., 2005). High levels of the immediate

C-2 deoxy precursors to bioactive GA4 and GA1 (GA9 and GA20, respectively) and low levels of

GA1 and GA4 per se were found in the phloem tissue, whereas high levels of all four GAs were

observed in the expanding xylem tissue (Israelsson et al., 2005). Expression of transgenic

PsGA3ox1 in all tissues of pea internode 2 would likely lead to higher levels of GA1 in both the

phloem and xylem tissues of the pea stem. If similar GA pools exist in the phloem and xylem

tissues in pea stems as demonstrated for the hybrid aspen stems, it is possible that higher than

normal (supra-optimal) levels of GA1 in the phloem is associated with the reduced phloem tissue

growth as seen at this internode in TG1 and TG2 compared to C1 (Fig. 3B; Supplementary Table

S2).

Tendril petiole development

To date, little is known about GA biosynthesis and catabolism pathways in tendrils.

‘Carneval’ is semi-leafless (af; afila), where leaflets are replaced by tendrils of normal anatomy.

We assessed tendril petioles, for consistency of tissue type within the tendril. We found that

transcript abundance of the endogenous GA biosynthetic genes PsGA20ox1 and PsGA3ox1

(control; Fig. 4A, B, F and G) decreased, while the endogenous GA catabolic genes PsGA2ox1

and PsGA2ox2 both increased in the tendril petioles as they matured (control; Fig. 4H-K).

Concomitantly, a decrease in endogenous levels of GA20, GA1, GA8 and GA29 was observed in

the tendril petioles as they matured from 40 to 80% full length (control; Fig. 5). Smith et al.

(1992) also observed that younger pea petioles contained higher endogenous levels of GA20,

GA1, GA8 and GA29 than mature petioles. These data are indicative of an overall decreased flux

through the GA biosynthesis pathway (decreased production of GA20 and GA1, resulting in lower

substrate levels of these GAs for the production of GA29 and GA8 by GA 2-oxidases) in tendril

petioles as they complete their development. PsGA2ox1 transcript abundance was lower in the

tendril petioles (more than 10-fold lower at similar growth stages in controls; Fig 4H) than in the

internodes (Table I), suggesting that PsGA2ox1 plays a more important role in maintaining GA

homeostasis in the internodes than this tendril tissue.

Ross et al. (2003) reported that mature pea shoot tissues (leaves, tendrils and internode

tissues were combined) contained lower levels of GA20 and GA1 compared to young apical bud



13

tissues. Data from [14C]GA20 metabolism studies (Ross et al., 2003) suggested that the low GA20

and GA1 levels observed in the mature shoot tissues were due to rapid 2β-hydroxylation of these

GAs. Our expression data, along with endogenous GA analysis, suggest that the lower levels of

GA20 and GA1, as the tendril petioles mature, are likely the result of both a decrease in GA

biosynthesis capacity (reduced GA 20- and GA 3-oxidase activity) as well as an increase in GA

deactivation (enhanced GA 2-oxidase activity).

Transgenic PsGA3ox1 over expression increases tendril petiole length and modifies GA

metabolism

Tendril petiole length in TG1, TG2 and TG3 was on average 12% greater than in the null

controls (C1, Fig. 2C; C3, Supplementary Fig. S2B). The higher PsGA3ox1 transcript abundance

during the earlier developmental stages (stages 1 and 2; Fig. 4E-G), concomitant with increased

GA1 levels (Fig. 5B) in the rapidly elongating tendrils (for the transgenic lines) at 40% of final

size (stage 2) indicates that transgenic PsGA3ox1 over expression increased the GA20 � GA1

biosynthetic flux, with the elevated tissue GA1 concentration stimulating tendril petiole growth.

It should be noted that PsGA3ox2 transcripts were not detected in tendril petioles (stage 1) of

either the transgenic or control lines (data not shown). Endogenous PsGA20ox1 and PsGA20ox2

transcript levels were elevated in TG1 and TG2 tendril petioles, relative to that seen in C1 (at

stage 3; 80% full size; Fig. 4A-D). This is analogous to the situation seen for endogenous

PsGA20ox1 transcript abundance in TG1 and C1 internodes (Table I). Also, GA20 concentrations

did not decline in the tendril petioles of the transgenic lines (TG1and TG2) at either 40% or 80%

expansion, relative to C1 at the same stage of development (Fig. 5A). These data suggest that

the elevated transcript levels of endogenous tendril petiole PsGA20ox and production of GA20,

also circumvented depletion of GA20 substrate for subsequent 3β-hydroxylation to GA1 in the

PsGA3ox1 overexpression transgenic lines (as opposed to GA20 depletion observed in GA3ox

overexpression lines of tobacco and hybrid aspen).

Our results, then, imply that high levels of endogenous PsGA20ox1 (Table I) and

production of GA20 (Table II) circumvent depleting the GA20 substrate pool that is needed for

3β-hydroxylation to bioactive GA1 in the PsGA3ox1 transgenic pea lines. This is in contrast to

GA20 depletion observed in GA3ox overexpression lines of tobacco (Gallego-Giraldo et al. 2008)

and hybrid aspen (Israelsson et. al., 2004).



14

Transcript abundance of the GA catabolic gene, PsGA2ox2, was consistently higher from

stage 2 to 4 (Fig. 4J and K) in the tendril petioles of both of TG1 and TG2 (and also higher at

stage 2 in TG3; Supplementary Fig. S4F), all relative to the null controls (C1 or C3). These data,

along with overall lower transcript abundance of PsGA2ox1 (Fig. 4H and I) than PsGA2ox2 (Fig.

4J and K; note the 100-fold higher y-axis range for PsGA2ox2), are consistent with the increased

levels of expression of PsGA2ox2 having a primary role in the GA 2-oxidase-catalyzed

deactivation of GA1 in tendril petioles, and suggest that a feed forward regulation of GA

biosynthesis by increased concentrations of GA1 is occurring in this tissue. Further to this

conclusion, there was an influence of transgenic PsGA3ox1 over expression on the tendril

petiole’s GA 2β-hydroxylated metabolite profile, i.e. late in tendril development (80% expanded)

higher GA29 levels were observed in tendril petioles of both TG1 and TG2, and higher GA8

levels were observed in tendril petioles of TG1, all relative to C1 (Fig. 5C and D). Finally, in

both the transgenic and control lines, levels of GA20, GA1, GA8 and GA29 decreased in the tendril

petioles with increasing maturity and the slowing of tendril petiole growth (Fig. 5; stage 2

compared to stage 3).

Stipule development

The pea cultivar used in our study (‘Carneval’) is semi-leafless; it has stipule leaves but

lacks leaflets. Transcript levels of the two GA biosynthesis genes, PsGA20ox1 and PsGA20ox2,

were high during early stipule expansion, but then decreased as the stipules matured (C1; Fig. 6A

and C; C3, Supplemental Fig. S5A and B). The decrease in PsGA20ox transcript abundance

coincided with a 7.8-fold decrease in GA20 as the stipules expanded from 40 to 80% full size

(Fig. 7A, C1). The stipule PsGA20ox expression profile is similar to that seen for PsGA20ox1 in

the tendril petioles (C1; Fig. 4A and B). However, developmental regulation of endogenous

PsGA3ox1 expression in the stipules differed appreciably from that seen for the tendril petioles.

Endogenous PsGA3ox1 transcript levels were low at stage 1 (20% expanded), peaked at stages 2

and 3 (40 to 80% expanded), then decreased as stipules reached maturity (C1; Fig. 6F; C3,

Supplemental Fig. S5D). No PsGA3ox2 transcripts were detected in stage 1 stipules (data not

shown). Markedly higher levels of GA1 (6- to10-fold) were observed in rapidly expanding

stipules for C1 (40% expanded), relative to elongating internodes or tendril petioles at a similar



15

growth stage (see C1; Table II; Figs.5B and 7B). The concentration of GA1 decreased more than

5-fold as the stipules continued to expand from 40% to 80% of expected final size (see C1; Fig.

7B). A 4-fold increase in the transcript abundance of the catabolic gene PsGA2ox1 occurred

early in the expansion of the stipules (20% to 40% expansion), and PsGA2ox1 levels remained at

similar levels from 40% to the fully expanded stage (C1, Fig. 6H; Supplementary Table S3).

With the marked reduction in GA20 substrate for 2 β-hydroxylation, GA29 levels decreased 3.6-

fold as the stipules expanded from 40 to 80% final size (C1, Fig. 7D). A similar 4-fold increase

in PsGA2ox2 transcript abundance occurred early in stipule expansion (C1; 20% to 40%

expansion; Supplementary Table S3). PsGA2ox2 transcript abundance increased 1.7-fold as the

stipules expanded from 40% to 80% full size, and then dramatically increased (12.6-fold) as the

stipules expanded from 80% of final size to full size, (C1, Fig. 6J; C3, Supplementary Fig. S5F;

note that the scale for PsGA2ox2 is 1000-fold greater than PsGA2ox1; Supplementary Table S3).

The level of GA8, the product of 2 β-hydroxylation of GA1 (GA1 is the preferred substrate for the

GA 2-oxidase coded for by PsGA2ox2) declined modestly (1.6-fold) as the stipules expanded

from 40 to 80% final size (C1; Fig. 7C).

Leaflets and stipules are leaves that vary in shape and location on the pea plant (stipules

are attached directly to main stem at the node; leaflets are attached to the leaf petiole).

Interestingly, although both are leaves, the regulation of PsGA2ox1 and PsGA2ox2 in the stipules

of ‘Carneval’ dramatically differ from than that reported for the leaflets of ls-1 or LS (Elliott et

al., 2001; Ross et al., 2003). Elliott et al. (2001) using Northern blot analysis observed reduced

expression of PsGA2ox1 in fully expanded leaflets of the pea GA1-deficient ls-1 mutant

compared to its near isogenic wildtype LS. Furthermore, PsGA2ox2 transcripts were not

detected in the fully expanded leaflets of GA-deficient ls-1 or wild-type LS lines. Ross et al.

(2003) suggested, based on [14C]GA20 metabolism and the PsGA2ox expression data (Elliott et

al., 2001), that the lower GA20 and GA1 levels observed in mature pea leaf tissues were mainly

due to rapid 2β-hydroxylation of these GAs by PsGA2ox1. Our gene expression data across

stipule development, along with endogenous GA analysis, suggest that the lower levels of GA20

and GA1, as the stipules mature, are mainly the result of a decrease in GA biosynthesis capacity

(reduced PsGA20ox1 and PsGA20ox2 [stage 2 to maturity] and PsGA3ox1 [stage 3 to 4]

transcript abundance [C1; Fig. 6A, C, and F]) suggesting reduced GA 20- and GA 3-oxidase

activity). Furthermore, the dramatic increase in PsGA2ox2 transcript abundance as the stipules



16

complete their growth (from 80% to 100% leaf expansion, stage 3 to 4; C1, Fig. 6J) suggests that

GA1 deactivation (enhanced GA 2-oxidase activity) by the GA 2-oxidase encoded by PsGA2ox2

may act as the primary mechanism to ensure minimal levels of bioactive GA1 in this tissue at

maturity (at least in the lele genetic background). A similar dramatic increase in PsGA2ox2

transcript abundance was observed in developing pea embryo axes as they shifted from GA

biosynthesis (to the growth-active GA1) to catabolic GA metabolism (GA1 deactivation) during

mid seed development. This is very likely the mechanism for limiting pea embryo axis growth

and allowing embryo maturation to proceed (Nadeau et al., 2011).

Transgenic PsGA3ox1 over expression increases stipule size and modifies GA metabolism

All three transgenic lines had larger stipules (approximately 15% larger at nodal position 8;

Fig. 2D; Supplementary Fig. S2C) and they also had higher stipule PsGA3ox1 transcript levels

relative to the control lines (Fig. 6F and G; Supplementary Fig. S5D). Although similar GA1

levels were observed in the stipules of the transgenic and control lines, higher GA8 levels were

detected in the rapidly expanding transgenic stipule tissue (stage 2; Fig. 7B and C). This suggests

that an increased flux of GA1 � GA8 occurred in the transgenic stipules during their expansion

phase of growth. An increased leaf area was also observed when PsGA3ox1 or CmGA3ox1 were

overexpressed in tobacco (Gallego-Giraldo et al., 2008) and Arabidopsis (Radi et al., 2006),

respectively.

In contrast to transcript levels observed in the internode and tendril tissues, minimal to no

increase in endogenous PsGA20ox transcript abundance was seen for stipule tissue (Fig. 6A-D;

Supplementary Fig. S5A and B). In fact, a reduced PsGA20ox1 transcript abundance was

observed for TG1, relative to C1 (Fig. 6A). Lower GA20 levels were also observed in the stipules

at 40% stipule expansion for the PsGA3ox1 over expression lines (TG1 and TG2), relative to C1

(Fig. 7A, stage 2). Interestingly, although over expression of transgenic PsGA3ox1 similarly

increased stipule leaf area in all three transgenic lines (compare node 8, Fig. 2D, Supplementary

Fig. S2C), expression of the GA deactivation gene PsGA2ox1 varied across stipule development

among the independent transgenic lines (Fig. 6H and I; Supplementary Fig. S5E). Additionally,

over expression of transgenic PsGA3ox1 had minimal to no effect on the PsGA2ox2 expression

profile for the stipules (Fig. 6J and K; Supplementary Fig. S5F). Overall, stipule GA29 levels for

the transgenics (TG1 and TG2) were either lower (40% expanded) or similar (80% expanded) to

GA29 levels of C1 (Fig. 7D). These GA gene expression and metabolism profiles in the



17

PsGA3ox1 over expression lines further support that a decrease in GA biosynthesis capacity, as

opposed to an increased PsGA2ox1gene expression, lowers GA20 and GA1 levels limiting stipule

expansion as the stipules mature.

Transgenic PsGA3ox1 over expression increases pericarp length and modifies reproductive

morphology

PsGA3ox1 (LE) plays an important role in pea plant height (internode length; Lester et al.,

1997; Martin et al., 1997), though its role in fruit development has been less clear. Santes et al.

(1993) investigated the effect of the le mutation on the growth and GA content of developing

fruits in near-isogenic lines of Pisum sativum line 205+ (LELE; PsGA3ox1) and 205- (lele).

Although GA1 concentration was lower in young seeds (2.7 times) and pericarps (7 times) of lele,

compared to levels in LELE at 6 days after anthesis (DAA), the growth of pods (in length) and

seeds (in weight) was not affected by the le mutation. However, the levels of GA20 and GA29

were substantially elevated in lele compared to LELE in both tissues (Santes et al., 1993). These

results suggest that 3ß-hydroxylation of GA20 to GA1 is reduced in pericarps and seeds by the le

mutation, even though it did not affect the general fruit phenotype, bringing into question a

possible causal role of PsGA3ox1 (LE) in the production of GA1 during fruit development in

pea.

In pea, normal pericarp growth requires the presence of seeds, and seed number is

positively correlated with pea pericarp growth (Ozga et al., 1992). Removal or destruction of the

seeds at 2 to 3 DAA results in the slowing of pericarp growth and subsequently results in

abscission (Ozga et al., 1992). Signaling molecules originating from the seeds, such as auxin (4-

Cl-IAA; Ozga and Reinecke, 2003; Ozga et al., 2009), may be responsible for continued fruit

development by maintaining hormone levels in the surrounding tissue.

The transcript abundance of endogenous PsGA3ox1 is low in young pea pericarp tissue

compared to other organ tissues (Ozga et al., 2003). Data suggest that pollination, the presence

of developing seeds, and bioactive auxin (4-Cl-IAA, which is present in the developing seeds)

maintain and/or stimulate the synthesis of pea pericarp PsGA3ox1 mRNA message. It is

hypothesized that GA1 is synthesized from the pool of GA20 present in pre-pollinated pericarps

by the pericarp GA 3ß-hydroxylase that is at least partially encoded by PsGA3ox1; and this pulse

of GA1 stimulates initial fruit set and development (Ozga et al., 2003). Consistent with

PsGA3ox1 playing a role in the production of GA1 for fruit set and development, we found that



18

young pericarps of the transgenic PsGA3ox1 over expression line TG1 contained higher

PsGA3ox1 transcript levels (12.6-fold higher) and produced longer pericarps relative to the

control line C1, when pericarps containing a similar number of seeds were compared (Tables III

and IV). Parthenocarpic fruit development in the transgenic PsGA3ox1-overexpression lines was

not observed under normal growing conditions. Transgenic PsGA3ox1 over expression also

markedly increased pericarp endogenous PsGA2ox1 transcript abundance (15-fold increase;

Table IV), suggesting that the GA 2-oxidase encoded by PsGA2ox1 plays an important role for

deactivating bioactive GA1 in the pericarp at this early stage of development. Overall, these data

support that pericarp PsGA3ox1 is important for the production of GA1 for pericarp growth.

Furthermore, pericarp GA1, along with other seed-derived factors, drive both fruit set and

pericarp development.

The TG1 line also exhibited significantly more fruit abortion prior to first fruit set, relative

to the control (C1; Table III). In previous work, pea seed PsGA3ox1 mRNA levels were

observed to peak just following pollination (0 DAA), then markedly decrease at 1 DAA (Ozga et

al., 2003). It has been documented that high concentrations of applied GA3 can inhibit

embryogenesis in excised embryos of Phaseolus coccineus (Yeung and Sussex, 1979; discussed

in Pharis and King, 1985). Since the presence of viable seeds is required for pericarp growth in

pea (Ozga et al., 1992), embryo abortion due to supra-optimal levels of bioactive GA in the

ovule tissues could have led to more fruit abortion for TG1, in comparison with C1.

Comparisons of transgenic PsGA3ox1 over expression lines to lines with PsGA3ox1 (LE)

introgressed into the pea genome by backcrossing

For comparison to the transgenic lines, a pea line was created that was near isogenic for LE

through backcrossing the maternal dwarf parent, ‘Carneval’ (le) with a tall ‘Alaska’ (LE) male

parent. Then the F1 progeny that produced a tall plant phenotype (maternal parent) were again

backcrossed to ‘Carneval’ (le) for a total of 5 (line BC5 LEle) to 7 (line BC7 LEle) backcross

generations. Interestingly, PsGA3ox1 (LE) introgressed into the ‘Carneval’ genetic background

by crossing had a much greater effect on increasing internode length than was seen for the

transgenic PsGA3ox1 lines TG1, TG2, or TG3 (Fig. 2, compare B for BC5 LEle with A for TG1

and TG2; also see Supplementary Fig. S2A for TG3; similar internode lengths across generations

BC5, BC6, and BC7 were observed for LEle and lele lines, respectively, data not shown).

Comparable internode length differences, as observed between the BC LEle and BC lele lines,



19

were also noted for pea lines that were near isogenic for LELE (205+, tall line) and lele (205-,

dwarf line; Ross et al., 1992), though in a genetic background different than ‘Carneval’. The

difference in internode lengths can be attributed to accumulation of much higher GA1 levels (43-

fold higher) in BC LEle than in the transgenic line (TG1; Table II).

Gene expression studies were completed to determine if the transcript abundance of

PsGA3ox1 and other genes in the GA biosynthesis and catabolic pathways differed between the

transgenic LE and BC LEle lines. Our current data suggest that the cellular and/or temporal

perturbation of PsGA3ox1 transcript abundance (5-fold increase of total PsGA3ox1 ) in the

internode tissues of TG1 compared to its control (C1; Table I) initiated in response to

CaMV35S-driven PsGA3ox1 (LE) expression (across all cell types of the internode), induced a

feed-forward regulation of GA1 levels by increasing PsGA2ox1 transcript abundance. Since a

mutant with a nonfunctional PsGA2ox1 gene product [slender (sln) pea mutant; Lester et al.,

1999; Martin et al., 1999] exhibits longer internodes (up to 60% longer compared to SLN at the

morphologically earlier internodes at a similar nodal position as assayed in our study; Reid et al.,

1992), and higher GA1 levels, we conclude that the induction of the GA deactivation gene

PsGA2ox1 expression in the transgenic lines makes an important contribution to limiting the

increase in GA1 levels to only modest levels (Table II).

In contrast, in the BC LEle line, our data suggest that under the coordination of regulatory

mechanisms evolved in conjunction with its endogenous promoter, tissue-specific expression of

the fully functional PsGA3ox1 LE gene led to markedly higher internode GA1 levels. As a result

of high internode GA1 content, feedback regulation of PsGA20ox1 and PsGA3ox1 gene

expression (Martin et al., 1996 and 1997; Hedden and Thomas, 2012) was minimized in the BC

LEle line compared to the BC lele line (lower total PsGA3ox1 and PsGA20ox1 transcript

abundance in BC Lele compared to its control, BC lele; Table I). Lower PsGA20ox1 transcript

levels and increased flux of GA20 through GA1, are main contributors to lower GA20

accumulation in internodes of BC Lele compared to BC lele (Table II). Furthermore, our data

suggest that the endogenous promoter-driven PsGA3ox1(LE) expression pattern circumvents

feed-forward regulation of PsGA2ox1 transcript abundance in the internode tissue (PsGA2ox1

expression was not induced in the internode tissue in the BC LEle line when it is compared to the

BC lele line; Table I). In contrast, a 2-fold increase in the internode transcript abundance of the

GA catabolic gene PsGA2ox2 did occur in the BC LEle line, when compared to the BC lele line



20

(Table I). It should be noted that PsGA2ox2 transcript abundance was unaffected with

CaMV35S-driven PsGA3ox1 overexpression (compare TG1 with C1; Table I). The C19-GA 2-

oxidases can be divided into two subgroups on the basis of amino acid sequence, PsGA2ox1

belonging to the type I subgroup and PsGA2ox2 to the type II subgroup (Lee and Zeevaart,

2005). Consistent with other reports that the transcript abundance of these two pea GA2ox genes

are differentially regulated by hormones and specific environmental signals (Ayele et al., 2006;

O’Neill and Ross, 2002; Stavang et al., 2005; Elliott et al., 2001), our data suggest that the

transcript abundance of PsGA2ox1 and PsGA2ox2 are differentially regulated as a mechanism to

maintain GA homeostasis within the elongating pea internode.

Interestingly, effects of LE on apical meristem life in the BC LEle line were similar to

those effects seen for TG1 and TG2 lines (Table III, compare nodes per plant in BC LEle to its

isogenic line BC lele and also compare transgenic PsGA3ox1-overexpressor line TG1with C1).

Here, we conclude that it is the increased GA1 biosynthesis capacity that is associated with the

increased apical meristem life in the BC LEle line.

A delay in the timing of floral initiation of 4 to 5 nodes occurred for the PsGA3ox1-

overexpression line TG1 (Table III, compare with C1) and of 1 node for TG3 (Supplementary

Table S1, compare with C3). This delayed flowering response was not observed for the BC LEle

lines (Table III, compare with BC5 lele). These data suggest that the GA1-induced delay in the

floral initiation process in the shoot apex is influenced primarily by the location of GA1, i.e.,

cellular-specific expression pattern of PsGA3ox1 (LE).

Santes et al. (1993) reported that the growth of pericarp (in length) and seeds (in weight)

was not affected by the le mutation; however, the effect of seed number on pericarp elongation

was not investigated. We found the BC LEle line did produced significantly longer pericarps

relative to the BC lele line plants, but only when there was a low seed number per pericarp (3

seeds; Table III). The transgenic line (TG1) produced significantly longer pericarps when 3 to 6

seeds were present in the fruit, compared to C1 plants with comparable seed numbers (Table III).

We conclude that the increase in pericarp PsGA3ox1 transcript abundance observed for TG1

(relative to C1; Table IV) was sufficient to raise the baseline levels of GA1 to a level that

stimulated pericarp elongation in fruits with 3 to 6 seeds.

We also monitored ABA levels in the internodes of the transgenic PsGA3ox1-

overexpressor lines, in the BC LEle and BC lele lines, and in their control lines to determine if



21

GA1 differences influenced the levels of ABA. As internode ABA levels did not vary between

any of these lines (Table II), our data provide no evidence for GA1 regulation of ABA levels in

pea internodes. Ross et al. (2011) also could find no evidence for regulation of GA metabolism

by ABA in pea internodes.

Summary

Using pea as a model legume system, our data demonstrate that there is a dynamic and

tissue- and organ-specific nature to GA biosynthesis and catabolism pathways, one that can

explain a wide range of morphological and physiological phenotypes in both vegetative and

reproductive tissues. For both tendril petioles and stipules, there was increased flux occurring in

the GA biosynthesis pathway over development leading to the production of GA20 and GA1, the

latter GA being the ‘effector’ that stimulates tissue elongation or expansion. However, the

different developmental and tissue-specific GA biosynthesis and catabolism gene expression

patterns of the tendril petioles (having a long cylindrical-shape), when compared to the stipules

(a leaf-shaped organ) likely reflect the regulation of GA biosynthesis genes in a manner that

helps coordinate the different growth patterns required for these organs. The tendril petiole GA

biosynthesis gene expression pattern is consistent with the presence of sufficient GA 20-oxidase

(for production of GA20 substrate) and also sufficient GA 3-oxidase for conversion of GA20 to

bioactive GA1 at moderate levels (2.6 to 1.3 ng gdwt-1) through the mid and later stages of organ

development (40 to 80% of expected full length). In contrast, the stipule GA gene expression

pattern is consistent with providing a higher GA 20-oxidase capacity earlier in organ

development (1.4-fold higher GA20 levels by 40% of full expansion; stage 2), thereby producing

a pool of GA20 substrate for use by GA 3-oxidase during mid development. Thus, GA20 to GA1

conversion peaks at the mid stage of stipule organ development (12.2 ng gdwt-1). As both

tendrils and stipules completed their development, the flux through the GA biosynthesis pathway

declined. Our data suggest that this decline in GA biosynthesis capacity was both the result of

reduced GA biosynthesis gene expression and increased GA deactivation gene (PsGA2ox)

expression across tendril and stipule development.

In this paper, we have also, through the production and characterization of PsGA3ox1

overexpression transgenic lines, identified lines that produce elevated levels of PsGA3ox1

transcripts and increased GA1 levels or increased flux through the GA biosynthesis pathway. In



22

so doing, we have shown that increasing GA1 in this manner can modify both vegetative and

reproductive plant phenotype. Our data also support the conclusion that CaMV-35S-driven

constitutive expression of PsGA3ox1 across cell types, within a tissue, increases the expression

levels of endogenous GA catabolic PsGA2ox genes in a tissue-specific manner. Increased

expression of these GA2ox genes is thus indicative of a feed-forward regulation of GA

catabolism in response to increased tissue concentrations of GA1.

We then compared the unique phenotypes (lines) produced by transgenic PsGA3ox1

overexpression to lines where PsGA3ox1 (LE) was introduced, by a series of back crosses, into

the same genetic background. In the internodes of lines where PsGA3ox1 (LE) expression was

driven by its endogenous promoter, markedly lower PsGA3ox1 transcript abundance (per

internode) was correlated with much greater production of GA1 than that observed in the

internodes of lines where PsGA3ox1 (LE) expression was driven by the CaMV-35S constitutive

promoter. We conclude that the induction of expression of the GA deactivation gene PsGA2ox1

likely makes an important contribution to limiting the increase in internode GA1 to modest levels

for the transgenic lines. In contrast, PsGA3ox1 (LE) expression driven by its endogenous

promoter was coordinated within the internode tissue to avoid feed-forward regulation of

PsGA2ox1, and this resulted in a much greater GA1 accumulation in this organ.

Overall, our findings do not support that GA 3-oxidase is a non-rate limiting enzyme in the

GA biosynthesis pathway as suggested by previous studies. Instead, our data support that the

timing and/or location of expression of GA3ox within an organ are important mechanisms to

avoid excessive deactivation of GA1 during tissue expansion. The specific localization of GA

gene transcripts and GAs within the cell-types of each organ would be very informative in

further understanding the role of GAs in the growth and development of these tissues.

MATERIALS AND METHODS

Plant material and plant transformation

Transgenic pea lines were generated to overexpress PsGA3ox1 (LE; a fully functional

wild-type GA 3β-hydroxylase gene; GenBank accession number AF001219) in Pisum sativum L.

cv. Carneval by Agrobacterium tumefaciens-mediated transformation of PsGA3ox1 fused to a



23

CaMV-35S constitutive promoter. ‘Carneval’ is a semi-dwarf (le-1; single base-pair mutation in

PsGA3ox1) and semi-leafless (af; afila, leaflets are replaced by tendrils of normal anatomy) field

pea cultivar. This cultivar has white flowers and yellow cotyledons at maturity. Plant

transformation vectors were constructed to stably transform PsGA3ox1 in sense orientation into

pea. The binary plasmid pCGN1559 was modified for plant transformation by inserting a

duplicate-enhancer CaMV-35S promoter sequence proximal to the PsGA3ox1 gene sequence as

diagramed in Supplementary Fig. S7A. The binary plasmid was transferred into a disarmed A.

tumefaciens strain EHA101 and used for plant transformation. Then, for determining CaMV-

35S promoter expression patterns in various pea tissues, the binary plasmid pPBI-3010

containing a CaMV-35S promoter proximal to the GUS gene (Supplementary Fig. S7B) was

inserted into a disarmed A. tumefaciens strain EHA101 and used for plant transformation (see

supplementary section for details). The A. tumefaciens-mediated transformation and plant

regeneration process was similar to that described earlier (Schroeder et al. 1993; Grant et al.,

1995; Bean et al., 1997), with modifications (see Supplementary Materials and Methods).

Independently transformed T1 lines were tested for the presence of the NPTII marker gene by

PCR, and T2 seeds from the NPTII-positive lines were selected for homozygosity using a

kanamycin selection (see Supplementary Materials and Methods). Confirmation of stable gene

transformation into the plant genome (Supplementary Figs. S8 and S9) and the presence of

transgene PsGA3ox1 mRNA (Supplementary Figs. S10 and S11) was performed as described in

the Supplementary Materials and Methods.

Morphological, biochemical and molecular characterizations were performed on three

selected independent homozygous transgenic lines (TG1, TG2 and TG3). These lines segregated

at the T2 generation consistently with carrying one copy of the transgenic PsGA3ox1 (LE) gene

per haploid genome (assessed by screening the T2 generation for the kanamycin selection gene,

nptII). There were also three transformation control lines (C1, C2 and C3, where the transgene

had segregated out; null controls) and a wild-type line (NT-C; non-transformed control, it did not

undergo the tissue-culture process). All transgenic and transgenic control null lines were from

generations T4 to T6.

To create a line similar to ‘Carneval’ (LE), ‘Carneval’ (le), as the maternal parent, was

crossed with ‘Alaska’ (LE) and plants which produced a tall plant phenotype in subsequent

generations (maternal parent), were backcrossed to ‘Carneval’ (le) for a total of 5 to 7 backcross



24

generations. The line designated BC5 LEle produced tall shoots (longer internodes) and the line

designated BC5 lele produced shorter shoots (shorter internodes) when seed of the 5th backcross

generation was germinated (the BC6 and BC7 LEle, and BC6 and BC7 lele lines produced

similar phenotypes to the BC5 lines).

Growth conditions for phenotype characterization

Mature air-dried seeds were planted at a depth of approximately 2.5 cm into Sunshine Mix

#4 (Sungro Horticulture, Seba Beach, AB) potting medium in 1.5 L plastic pots (one seed per

pot). Pots were arranged in a completely randomized design in a growth chamber and grown at

19oC/17oC (day/night) with a 16/8-h photoperiod under cool-white fluorescent lights

(F54/I5/835/HO fluorescent bulbs, Phillips, Holland; 366 µE m2 s-2). Lateral shoots were

removed as they emerged in order to facilitate maximum extension of the main stem.

Morphological assessment

We recorded the total number of internodes at plant maturity (the first internode was

defined as the one between the cotyledon attachment and the first stem node; see Supplementary

Fig. S1). The length of each internode was measured at maturity from position 1 to 20, as were

the number of nodes to first flower, and the number of fruits aborted before the first fruit was set.

The area of the larger stipule of each pair at specific nodes was measured at maturity using a leaf

area meter (LI-3100 Area Meter, LI-COR, Inc. Lincoln, Nebraska, USA). The petiole length of

tendrils at selected nodes was measured at maturity from the point of attachment to the main

stem to the first tendril branching point. The data presented are from one experiment consisting

of eight to 10 plants per line, i.e. biological replications. These morphological assessment

experiments have been repeated twice over time with TG1 and C1 lines, and two to four times at

selected internodes and nodes with all lines. The morphological data were analyzed using the

General Linear Model of SAS 9.1 statistical software (SAS Institute Inc., Cary, NC, USA) using

a completely randomized design. Means were separated by LSD at P≤0.05.

Tissue harvest for gene expression and GA analyses

Three different vegetative organs (internode, tendril petiole and stipule) were harvested for

gene expression and GA analyses. Internodes from position 8 (between nodes 7 and 8) were



25

harvested when they were approximately 15-20% of final length. Tendril petioles were harvested

at 4 different maturity stages: 10-15% of final length (stage 1), 35-40% of final length (stage 2),

75-80% of final length (stage 3) and at maturity (stage 4). Stipules were also harvested at 4

different maturity stages: 15-20% of final size (stage 1), 35-40% of final size (stage 2), 75-80%

of final size (stage 3) and at maturity (stage 4). All tendril and stipule tissues for analysis at

stages 2, 3 and 4 were collected from node eight. In order to obtain enough tissue for RNA

isolation from the smallest development stage (stage 1), tendril and stipule tissues from nodes 7

and 8 were pooled. To study the gene expression patterns in the fruit, pericarps (without seeds)

were harvested when they were at 2 DAA. The gene expression data presented are results from

one experiment consisting of three biological replicates for each tissue and stage. Each replicate

thus contained tissues from two individual plants. In addition, two to three independent gene

expression experiments using internodes, tendrils, and stipules of selected growth stages were

also run for comparison.

For GA analyses, internodes from position 8 (between nodes 7 and 8; Supplementary Fig.

S1) were harvested when they were approximately 50% of final length; tendrils and stipules at

node eight were also harvested when at approximately 40% and 80% of final size. The data are

from one experiment, where two biological replicates per tissue were collected (each replicate

usually contained tissues from nearly two hundred plants). Tissues were immediately frozen in

liquid nitrogen upon harvesting and stored at -80oC until RNA was extracted, or freeze-dried

prior to analysis of endogenous GAs.

Light microscopy

Internodes from position 2 (between nodes 1 and 2) were harvested at maturity for

histological studies from pea seedlings of two transgenic lines (TG1 and TG2) and the control

line C1 (transgenic null control line 1). Internode sections (5 mm sections from the uppermost

part of the internode) were harvested into a fixing solution containing 2.5% (v/v) glutaraldehyde

in 0.1M phosphate buffer at pH 7.5, embedded in paraffin, sectioned into 7 µm thick cross-

sections, mounted on slides, and stained with 1% (w/v) safranin O and 0.25% (w/v) fast green

FCF. The cross-sectional areas were estimated as described in the Supplemental Materials and

Methods section. For histochemical analysis of GUS activity, fresh pea tissues (internode,

tendril petiole, and stipule leaf tissues) were harvested, then immediately submerged in GUS



26

staining buffer containing 1 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronide (BIOSYNTH,

Switzerland), 100 mM sodium phosphate (pH 7.5), 0.5 mM potassium ferricyanide, 0.5 mM

potassium ferrocyanide, and 10 mM EDTA (Jefferson et al. 1987). The tissues were incubated at

37oC for 5 to 6 h; rinsed in 70% ethanol; then fixed in FAA (4% formaldehyde, 5% acetic acid,

50% ethanol) for 2 days under vacuum. The tissues were then embedded in paraffin, sectioned

into 10 µm thick cross-sections, mounted on slides, and micrographs obtained as described in the

Supplemental Materials and Methods section. Fruits at 2 DAA were cut in the longitudinal

direction (exposing cut surfaces of the dorsal and ventral pericarp walls) then immediately

emerged into the GUS staining buffer under vacuum and incubated for 30 min. The fruits were

then incubated at 37oC for 27 h. The fruit GUS staining pattern was observed under a ZEISS

stereo microscope after rinsing the tissue with 70% ethanol.

RNA extraction and qRT-PCR gene expression analysis

Plant tissues were finely ground in liquid nitrogen and total RNA was isolated using a

modified TRIzol (Invitrogen) protocol (Ayele et al., 2006) as described in the Supplemental

Materials and Methods section. For qRT-PCR, primers and probe for the transgene PsGA3ox1

quantifying amplicon (TPsGA3ox1-130) were designed using Primer Express software (version

3, Applied Biosystems, Foster City, CA, USA) to produce a transgene-specific quantifying

amplicon: forward primer: 5’-AAC ATT TCA ACA AAG CAC TCT CAT CT-3’; reverse

primer: 5’-GGG CTG CAG GAA TTC GAT ATC-3’; Probe: 5’-AGT GTC CAA GTG GGC

TA-3’ (Supplementary Fig. S12). The amplicon spans the 3’ coding region of PsGA3ox1 (LE)

through to a small remnant vector sequence situated prior to the NOS terminator sequence, thus

providing specificity for the transgenic PsGA3ox1 transcript. The qRT-PCR product produced by

the TPsGA3ox1-130 primers was separated on a 0.8% (w/v) agarose gel, purified from the gel

using a Qiagen gel elution kit, and directly sequenced using the Applied Biosystems 3.1 Dye

terminator method at the Molecular Biology Facility, University of Alberta.

The TPsGA3ox1-130 primers produced a single product with the desired length

(Supplementary Fig. S13) and sequencing of the fragment further confirmed the specificity of the

primers for transgenic PsGA3ox1 mRNA. Primers and probes for the target gene quantifying

amplicon PsGA3ox1-87 [used for total PsGA3ox1 (endogenous+ transgene) transcript

quantitation] and for the reference gene amplicon, 18S-62 (used for pea 18S rRNA transcript



27

quantitation) were designed as described by Ozga et al. (2003). Primers and probes for the target

gene quantifying amplicon GA2ox1-73 (used for PsGA2ox1 quantification), GA2ox2-83 (used

for PsGA2ox2 quantification), PsGA20ox1-104 (used for PsGA20ox1 quantification), and

GA20ox2-88 (used for PsGA20ox2 quantification) were designed as described by Ayele et al.

(2006). Primers and probes for the PsGA3ox2 quantitation (PsGA3ox2-104) were designed as

described by Ozga et al. (2009).

Quantitative real-time RT-PCR assays were performed on a model 7700 sequence detector

(Applied Biosystems) using a TaqMan One-Step RT-PCR Master Mix reagent kit (Applied

Biosystems) as described by Ayele et al. (2006, and the Supplemental Materials and Methods

section). The relative transcript abundance of the target genes in the individual plant sample was

determined by 2-ΔCt (Livak and Schmittgen, 2001) where ΔCt was the difference between the Ct

of the target sample and the average Ct of the reference sample. Transcript levels of PsGA20ox1,

PsGA20ox2, transgenic PsGA3ox1, total (transgenic + endogenous) PsGA3ox1, PsGA2ox1, and

PsGA2ox2 were standardized across genes, tissues and developmental stages using the lowest

sample average Ct value (38; from sample TG1 tendril stage 2, PsGA20ox2). At least two, more

often three plants were used as biological replicates for these assays. The pea 18S small subunit

nuclear ribosomal RNA gene was used as a loading control to estimate variation in input total

RNA concentration across all samples. The coefficient of variation (CV) of the 18S rRNA

amplicon Ct value across all samples was 2.7% and, therefore, the target amplicon mRNA values

were not normalized to the 18S signal (Livak and Schmittgen, 2001, Ozga et al., 2009).

Analysis of endogenous hormone levels

Endogenous GAs and ABA in the elongating internodes (approximately 50% of the final

size), and GAs in developing tendrils (approximately 40% and 80% of the final size) and

developing stipules (approximately 40% and 80% of the final size) were identified and

quantified using a gas chromatograph connected to a mass spectrometer using the selected ion

monitoring mode with stable-isotope labeled internal standards (method adapted from Kurepin et

al. 2007 as described in the Supplemental Materials and Methods section).

ACKNOWLEDGMENTS



28

We would like to thank Natalie Labbe for construction of the PsGA3ox1plasmid vector, Dr.

John Mahon (National Research Council of Canada, Saskatoon) for providing the 35S-GUS

plasmid vector, and many past and current members of the Ozga-Reinecke lab that worked on

aspects of pea transformation and transgenic characterization.

SUPPLEMENTAL MATERIAL

Supplementary Figure S1. Node and internode numbering scheme.

Supplementary Figure S2: Vegetative phenotypic comparison of transgenic TG3 and its

transgenic null.

Supplementary Figure S3. Internode GA biosynthesis and catabolism gene expression profiles

of transgenic TG3 and its transgenic null.

Supplementary Figure S4. Tendril petiole GA biosynthesis and catabolism gene expression

profiles of transgenic TG3 and its transgenic null.

Supplementary Figure S5. Stipule GA biosynthesis and catabolism gene expression profiles of

transgenic TG3 and its transgenic null.

Supplementary Figure S6: Representative micrographs of CaMV-35S-driven GUS expression

in pea tissues.

Supplementary Figure S7: Diagram of the CaMV 35S-PsGA3ox1 (LE) and the CaMV 35S-

GUS plasmid constructs

Supplementary Figures S8 and S9: Confirmation of the stable integration of transgenic

PsGA3ox1 into host DNA.

Supplementary Figures S10 and S11: Confirmation of the presence of transgenic PsGA3ox1

transcript in transformed pea lines.

Supplementary Figures S12 and S13: Confirmation of qRT-PCR quantifying amplicon

TPsGA3ox1-130.

Supplementary Table S1: Reproductive phenotype comparison of the transgenic TG3 and its

transgenic null lines.

Supplementary Table S2: Cross-sectional areas of internode 2 in transgenic TG1 and TG2, and

null control (C1) lines.

Supplementary Table S3: PsGA2ox transcript abundance in pea stipules over development of

TG1, TG2 and C1 lines.



29

SupplementaryProtocols:

Transformation and selection protocol

Confirmation of stable gene transformation into plant genome

Confirmation of transgene PsGA3ox1 mRNA

Light microscopy

RNA extraction

qRT-PCR gene expression analysis

Analysis of endogenous hormone levels



30

LITERATURE CITED

Ayele BT, Ozga JA, Reinecke DM (2006) Regulation of GA biosynthesis genes during

germination and young seedling growth of pea (Pisum sativum L.). J Plant Growth Regul

25: 219-232

Ayele BT, Ozga JA, Wickramarathna AD, Reinecke DM (2012) Gibberellin metabolism and

transport during germination and young seedling growth of pea (Pisum sativum L.). J Plant

Growth Regul 31: 235–252

Bean SJ, Gooding PS, Mullineaux PM (1997) A simple system for pea transformation. Plant

Cell Rep 16: 513-519

Besnard-Wibaut C, Noin M, Zeevaart JAD (1983) Mitotic activities and levels of nuclear

DNA in the apical meristem of Silene armeria (Strain S1.2) following application of

gibberellin A3. Plant Cell Physiol 24: 1269-1279

Biemelt S, Tschiersch H, Sonnewald U (2004) Impact of altered gibberellin metabolism on

biomass accumulation, lignin biosynthesis and photosynthesis of transgenic tobacco plants.

Plant Physiol 135: 254-265

Coles JP, Phillips AL, Croker SJ, Garcia-Lepe R, Lewis MJ, Hedden P (1999) Modification

of gibberellin production and plant development in Arabidopsis by sense and antisense

expression of gibberellin 20-oxidase genes. Plant J 17: 547-556

Davidson SE, Swain SM, Reid JB (2005) Regulation of the early GA biosynthesis pathway in

pea. Planta 222: 1010-1019

Dennis FG (1973) Physiological control of fruit set and development with growth regulators.

Acta Hort 34: 251-259

Elliott RC, Ross JJ, Smith JJ, Lester DR, Reid JB (2001) Feed-forward regulation of

gibberellin deactivation in pea. J Plant Growth Regul 20:87-94

Eriksson M, Israelsson M, Olsson O, Moritz T (2000) Increased gibberellin biosynthesis in

transgenic trees promotes growth, biomass production and xylem fiber length. Nature

Biotech 18: 784-788

Fagoaga C, Tadeo FR, Iglesias DJ, Huerta L, Lliso I, Vidal AM, Talon M, Navarro L,

Gracia-Martinez JL, Pena L (2007) Engineering of gibberellin levels in citrus by sense



31

and antisense overexpression of a GA 20-oxidase gene modifies plant architecture. J Exp

Bot 58: 1407-1420

Fleet CM, Yamaguchi S, Hanada A, Kawaide H, David CJ, Kamiya Y, Sun T-p (2003)

Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene

production but no increase in bioactive gibberellins. Plant Physiol 132: 830-839

Gallego-Giraldo L, Ubeda-Tomas S, Gisbert C, Garcia-Martinez JL, Moritz T, Lopez-Diaz

I (2008) Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes

with different gibberellin sensitivity. Plant Cell Physiol 49: 679-690

Grant JE, Cooper PA, McAra AE, Frew TJ (1995) Transformation of peas (Pisum sativum L.)

using immature cotyledons. Plant Cell Rep 15: 254-258

Hedden P, Phillips AL (2000a) Manipulation of hormone biosynthetic genes in transgenic

plants. Curr Opin Biotech 11: 130-137

Hedden P, Phillips AL (2000b) Gibberellin metabolism: new insights revealed by the genes.

Trends Plant Sci 5: 523-530

Hedden P, Thomas SG (2012) Gibberellin biosynthesis and its regulation. Biochem J: 444: 11-

25

Huang S, Raman AS, Ream JE, Fujiwara H, Cerny RE, Brown SM (1998) Overexpression

of 20-oxidase confers a gibberellin-over production phenotype in Arabidopsis. Plant Physiol

118: 773-781

Ingram TJ, Reid JB, Murfet IC, Gaskin P, Willis CL, MacMillan J (1984) Internode length

in Pisum: The Le gene controls the 3β-hydroxylation of gibberellin A20 to gibberellin A1.

Planta 160: 455-463

Israelson M, Sundberg B, Moritz T (2005) Tissue-specific localization of gibberellins and

expression of gibberellin-biosynthetic and signaling genes in wood forming tissues in aspen.

Plant J 44: 494-504

Israelsson M, Mellerowicz E, Chono M, Gullberg J, Moritz T (2004) Cloning and

overproduction of gibberellin 3-oxidase in hybrid aspen trees. Effects on gibberellin

homeostasis and development. Plant Physiol 135: 221-230

Kelly MO, Davies PJ (1986) Genetic and photoperiodic control of the relative rates of

reproductive and vegetative development in peas. Ann Bot 58: 13-21



32

Kurepin LV, Emery RJN, Pharis RP, Reid DM (2007) Uncoupling light quality from light

irradiance effects in Helianthus annuus shoots: putative roles for plant hormones in leaf and

internode growth. J Exp Bot 58: 2145-2157

Lee DJ, Zeevaart JAD (2005) Molecular cloning of GA 2-oxidase3 from spinach and its

ectopic expression in Nicotiana sylvestris. Plant Physiol 138: 243-254

Lee DJ, Zeevaart JAD (2007) Regulation of gibberellin 20-oxidase1 expression in spinach by

photoperiod. Planta 226: 35-44

Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time

quantitative PCR and the 2-ΔΔCt method. Methods 25: 402-408

Lester DR, Ross JJ, Davies PJ, Reid JB (1997) Mendel’s stem length gene (Le) encodes a

gibberellin 3β-hydroxylase. Plant Cell 9: 1435-1443

Lester DR, Ross JJ, Smith JJ, Elliott RC, Reid JB (1999) Gibberellin 2-oxidation and the SLN

gene of Pisum sativum. Plant J 19: 65-73

Martin DN, Proebsting WM, Parks TD, Dougherty WG, Lange T, Lewis MJ, Gaskin P,

Hedden P (1996) Feed-back regulation of gibberellin biosynthesis and gene expression in

Pisum sativum L. Planta 200:159-166

Martin DN, Proebsting WM, Hedden P (1997) Mendel’s dwarfing gene: cDNAs from the Le

alleles and function of the expressed proteins. Proc Natl Acad Sci USA 94: 8907–8911

Martin DN, Proebsting WM, Hedden P (1999) The SLENDER gene of pea encodes a

gibberellin 2-oxidase. Plant Physiol 121: 775–781

Nadeau CD, Ozga JA, Kurepin LV, Jin A, Pharis RP, Reinecke DM (2011) Tissue-specific

regulation of gibberellin biosynthesis in developing pea seeds. Plant Physiol 156: 897-912

Oikawa T, Koshioka M, Kojima K, Yoshida H, Kawata M (2004) A role of OsGA20ox1,

encoding an isoform of gibberellin 20-oxidase, for regulation of plant stature in rice. Plant

Mol Biol 55: 687-700

O’Neill DP, Ross JJ (2002) Auxin regulation of the gibberellin pathway in pea. Plant Physiol

130: 1974-1982

Ozga JA, Brenner ML, Reinecke DM (1992) Seed effects on gibberellin metabolism in pea

pericarp. Plant Physiol 100: 88-94

Ozga JA, Reinecke DM (2003) Hormonal interactions in fruit development. J Plant Growth

Regul 22: 73-81



33

Ozga JA, Reinecke DM, Ayele BT, Ngo P, Nadeau C, Wickramarathna AD (2009)

Developmental and hormonal regulation of gibberellin biosynthesis and catabolism in pea

fruit. Plant Physiol 150: 448-462

Ozga JA, Yu J, Reinecke DM (2003) Pollination-, development-, and auxin-specific regulation

of gibberellin 3β-hydroxylase gene expression in pea fruit and seeds. Plant Physiol 131:

1137-1146

Pharis RP, King RW (1985) Gibberellins and reproductive development in seed plants. Annu

Rev Plant Physiol 36: 517-568

Phillips AL (2004) Genetic and transgenic approaches to improving crop performance. In PJ

Davies, ed, Plant hormones: biosynthesis, signal transduction, action! Ed 3. Springer,

Dordrecht, The Netherlands, pp 582-609

Proebsting WM, Davies PJ, Marx GA (1978) Photoperiod-induced changes in gibberellin

metabolism in relation to apical growth and senescence in genetic lines of peas (Pisum

sativum L.). Planta 141: 231-238

Proebsting WM, Hedden P, Lewis MJ, Croker SJ, Proebsting LN (1992) Gibberellin

concentration and transport in genetic lines of pea. Plant Physiol 100:1354-1360

Radi A, Lange T, Niki T, Koshioka M, Lange MJP (2006) Ectopic expression of pumpkin

gibberellin oxidases alters gibberellin biosynthesis and development of transgenic

Arabidopsis plants. Plant Physiol 140: 528-536

Reid JB, Ross JJ, Swain SM (1992) Internode length in Pisum. A new slender mutant with

elevated levels of C19 gibberellins. Planta 188: 462-467

Ross JJ, Davidson SE, Wolbang CM, Bayly-Stark E, Smith JJ, Reid JB (2003)

Developmental regulation of the gibberellin pathway in pea shoots. Funct Plant Biol 30: 83-

89

Ross JJ, Reid JB, Dungey HS (1992) Ontogenetic variation in levels of gibberellin A1 in Pisum.

Implications for the control of stem elongation. Planta 186: 166-171

Ross JJ, Reid JB, Gaskin P, MacMillan J (1989) Internode length in Pisum. Estimation of

GA1 levels in genotypes Le, le and led. Physiol Plant 76: 173-176.

Ross JJ, Weston DE, Davidson SE, Reid JB (2011) Plant hormone interactions : how complex

are they? Physiol Plant 141: 299-309.



34

Ross JJ, Reid JB, Swain SM (1993) Control of stem elongation by gibberellin A1: Evidence

from genetic studies including the slender mutant sln. Aust J Plant Physiol 20: 585-599.

Santes CM, Hedden P, Sponsel VM, Reid JB, Garcia-Martinez JL (1993) Expression of the

le mutation in young ovaries of Pisum sativum and its effect on fruit development. Plant

Physiol 101: 759-764.

Schroeder HE, Schotz AH, Wardley-Richardson T, Spencer D, Higgins TJV (1993)

Transformation and regeneration of two cultivars of pea (Pisum sativum L.). Plant Physiol

101: 751-757

Smith V A, Knatt CJ, Gaskin P, Reid JB (1992) The distribution of gibberellins in vegetative

tissues of Pisum sativum L. I. Biological and biochemical consequences of the le mutation.

Plant Physiol 99: 368–371

Stavang JA, Lindgard B, Erntsen A, Lid SE, Moe R, Olsen JE (2005) Thermoperiodic stem

elongation involves transcriptional regulation of gibberellins deactivation in Pea. Plant

Physiol 138: 2344-2353

Swain SM, Sing DP, Helliwell CA, Poole AT (2005) Plants with increased expression of ent-

kaurene oxidase are resistant to chemical inhibitors of this gibberellin biosynthesis enzyme.

Plant Cell Physiol 46: 284-291

Thomas SG, Phillips AL, Hedden P (1999) Molecular cloning and functional expression of

gibberellin 2-oxidases, multifunctional enzymes involved in gibberellin deactivation. Proc

Natl Acad Sci USA 96: 4698-4703

Vidal AM, Gisbert C, Talon M, Primo-Millo E, Lopez-Diaz I, Garcia-Martinez JL (2001)

The ectopic over-expression of a citrus gibberellin 20-oxidase enhances the non 13-

hydroxylation pathway of gibberellin biosynthesis and induces an extremely elongated

phenotype in tobacco. Physiol Plant 112: 251-260

Wickramarathna AD (2009) Characterization of gibberellin overexpression lines in pea. PhD

Thesis, University of Alberta, Edmonton, Alberta, Canada.

Yang T, Davies PJ, Reid JB (1996) Genetic dissection of the relative roles of auxin and

gibberellin in the regulation of stem elongation in intact light-grown peas. Plant Physiol

110: 1029-1034

Yeung EC, Sussex IM (1979) Embryogeny of Phaseolus coccineus: The suspensor and the

growth of the embryo-proper in vitro. Z Pflanzenphysiol 91: 423-433



35

Zhu Y-X, Davies PJ (1997) The control of apical bud growth and senescence by auxin and

gibberellin in genetic lines of peas. Plant Physiol 113: 631-637

FIGURE LEGENDS Figure 1: The latter part of the early 13-hydroxylation GA biosynthesis and catabolism pathway

in pea. GA12 is 13-hydroxylated to GA53; carbon 20 is sequentially oxidized by a GA 20-oxidase

(coded for by PsGA20ox genes) from GA53 to GA44, to GA19 and finally to GA20. GA20 is then

oxidized by a 3β-hydroxylase (coded for by PsGA3ox genes) to GA1 (a bioactive GA). GA20 and

GA1 can each be oxidized by a 2β-hydroxylase (coded for by PsGA2ox genes) to GA29 and GA8,

respectively, the latter conversion inactivating GA1.

Figure 2: Phenotypic comparison of pea transgenic TG1 and TG2, and transgenic null control

(C1) lines (in ‘Carneval’le), and also in backcross generation 5 (BC5) LEle and BC5 lele, and

non-transformed ‘Carneval’ le control (NT-C) lines. (A) Internode length at maturity of

transgenic and transgenic null control lines from internode 1 to 20. (B) Internode length at

maturity of BC5 LEle and BC5 lele, and non-transformed ‘Carneval’ le control (NT-C) lines

from internode 1 to 20. (C) Tendril petiole length at maturity (from the point of attachment to the

stem to the first tendril branchlet) of transgenic and transgenic null control lines at different

nodal positions. (D) Stipule area at maturity of transgenic and transgenic null control lines at

different nodal positions. Data are means ± SE, n= 8 to 10. Letters indicate significant

differences among tendril length and stipule area within nodal position by LSD at P<0.05.

Figure 3: Representative light micrographs of mature internodes at position 2 for the transgenic

lines TG1 and TG2, and the null control C1 line. Stem transverse cross-sections at 2.5x

magnification (A) and at 10x magnification focusing on the vascular stele region of the stem (B).

cfb, cortical fiber bundle; co, cortex; cvb, cortical vascular bundle; la, lacunae; ph, phloem; vs,

vascular stele; xy, xylem.

Figure 4: GA biosynthesis and catabolism gene expression profiles in the tendril petioles of pea

transgenic TG1and TG2 and transgenic null control (C1) lines across development. Relative



36

transcript abundance of PsGA20ox1 (A and B), PsGA20ox2 (C and D), transgenic PsGA3ox1

(E), total PsGA3ox1 (transgenic + endogenous; F and G), PsGA2ox1 (H and I), and PsGA2ox2 (J

and K) in pea tendril petioles. Tendril petioles were harvested at 10-15% of final length (stage

1), 35-40% of final length (stage 2), 75-80% of final length (stage3), and at maturity (stage 4).

Transcript levels were compared across all genes and lines using the Ct value of 38 as a

reference for normalization. Data are means ± SE of two to three biological replicates. Y-axis

scales in graphs are consistent across the same gene.

Figure 5: Endogenous GA concentrations in pea tendril petioles. GA20 (A), GA1 (B), GA8 (C),

and GA29 (D) levels were quantified in elongating tendril petioles at approximately 40% (stage

2) and 80% full length (stage 3) at nodal position 8 of the transgenic lines TG1 and TG2, and the

transgenic null control line C1. Data are means ± SE, n= 2, with one exception, 40% full length

tendrils of TG1, n=1.

Figure 6: GA biosynthesis and catabolism gene expression profiles in the stipules of pea

transgenic TG1and TG2 and transgenic null control (C1) lines across development. Relative

transcript abundance of PsGA20ox1 (A and B), PsGA20ox2 (C and D), transgenic PsGA3ox1

(E), total PsGA3ox1 (transgenic + endogenous; F and G), PsGA2ox1 (H and I), PsGA2ox2 (J and

K) is shown in pea stipules harvested at 15-20% of final size (stage 1), 35-40% of final size

(stage 2), 75-80% of final size (stage3) and at maturity (stage 4). Transcript levels were

compared across all genes and lines using the Ct value of 38 as a reference for normalization.

Data are means ± SE of two to three biological replicates. Y-axis scales in graphs are consistent

across the same gene.

Figure 7: Endogenous GA concentrations in pea stipules. GA20 (A), GA1 (B), GA8 (C), and GA29

(D) levels were quantified in stipules at approximately 40% (stage 2) and 80% full size (stage 3)

at nodal position 8 of transgenic lines TG1 and TG2, and the transgenic null control line C1.

Data are means ± SE, n= 1 for 40% full size samples, n= 2 for 80% full size samples.



37

Table I. Relative transcript abundance of late GA biosynthesis and catabolism genes in elongating internode 8 (harvested at 15-20% of final length) for transgenic (TG1 and TG2), transgenic null (C1), backcrossed (BC LEle and BC lele), and non-transformed control (NTC1 and NTC2) lines.

TG1 a TG2 a C1 a NTC1 a BC LEle b BC lele b NTC2 b

PsGA20ox1 44,620 c ±

4862d 66,610 ± 11,920

25,110 ± 2297

45,070 ± 4696

11,310 ± 234

45,440 ± 3677

42,040 ± 4045

PsGA20ox2 3.3 ± 0.3 186 ± 13 23.1 ± 2.7 43.7 ± 5.1 1.4 ± 0.1 0.3 ± 0.1 20.7 ± 1.4

Total PsGA3ox1 (transgenic

+endogenous)

106,000 ± 7621

34,770 ± 2230

22,850 ± 1687

29,960 ± 3181

13,050 ± 948

35,490 ± 2711

30,430 ± 1740

PsGA3ox1 (transgenic)

4819 ± 354 856 ± 142 nde npf np np np

PsGA3ox2 nag na na na nd nd nd

PsGA2ox1 5393 ± 656 4527 ± 416 1284 ± 125 926 ±136 181 ± 23 166 ± 5 106 ± 19

PsGA2ox2 2638 ± 187 6376 ± 1036 2966 ± 155 2983 ± 236 2,757 ± 191 1,256 ± 87 704 ± 10

a The TG1, TG2, C1 and NTC1 pea lines (all in a lele genetic background) were grown together under the same environmental conditions using a completely randomized design and relative transcript abundance was assessed in these lines in the same qRT-PCR experiments. b The BC LEle, BC lele (where 6 backcross generations to ‘Carneval’ (le) were performed to generate these lines, BC6) and NTC2 pea lines were grown together under the same environmental conditions using a completely randomized design and relative transcript abundance was assessed in these lines in the same qRT-PCR experiments. c Transcript levels were standardized across genes using the Ct value of 38 as reference for normalization. d Data are means ± SE, n= 3 to 4; e not detected; f np, not present in this line; g na, not available; as PsGA3ox2 transcripts were not detected in previous transgenic internode samples, the abundance of this gene was not assessed in these samples.

w

ww

.plantphysiol.orgon M

arch 16, 2019 - Published by

Dow

nloaded from

Copyright ©

2013 Am

erican Society of P

lant Biologists. A

ll rights reserved.


38

Table II. Endogenous GA and ABA concentrations in elongating internodes (harvested at approximately 50 % full length; internode 8 on stem) of transgenic PsGA3ox1 overexpression lines TG1 and TG2, and the transformation null line (C1), and the backcross lines BC LEle and BC lele.

TG1a TG2 C1 BC LEleb BC leleb

GA1 1.8c ± 0.4d 1.0 ± 0.2 1.3 ± 0.3 77.4 ± 8.9 3.6 ± 1.1

GA8 95.7 ± 15.6 50.3 ±3.0 45.4 ± 3.6 430.8 ± 56.0 43.8 ± 8.7

GA19 nae na na 463.6 ± 17.3 115.6 ±11.0

GA20 630.7 ± 106.4 608.1 ± 256.4 650.3 ± 151.0 52.9 ± 1.5 226.3 ± 13.9

GA29 577.9 ± 20.6 567.4 ± 62.3 427.7 ± 13.9 130.3 ± 2.5 377.7 ± 25.6

GA44 na na na 124.9 ± 15.2 50.2 ± 7.9

ABA 107.9 ± 9.5 115.3 ±2.5 111.4 ± 5.9 94.5 ± 6.9 93.7 ± 5.1

a The transgenic lines TG1 and TG2, and transformation null line C1 are in a lele genetic

background. b The BC LEle and BC lele pea lines, represent lines backcrossed 7 times (BC 7) to ‘Carneval’ (le). c Hormone concentrations are expressed as ng gdw-1

d Data are means ± SE, n=2. e Not available



39

Table III: Number of nodes per plant, nodes to first flower, number of fruits aborted before first fruit set and length of pericarps at maturity of ‘Carneval’ le PsGA3ox1-overexpression transgenic (TG1 and TG2), transgenic null control (C1), backcrossed (BC LEle and BC lele), and non-transformed ‘Carneval’ le control (NTC) lines.

Lines TG1 TG2 C1 BC LElez BC lelez NTC

Nodes per plant 47.4±1.3yax 39.2±1.0b 37.6±0.6bc 35.4±0.6e 27.9±0.5g 30.5±0.9f

Nodes to first flower 30.1±0.1a 25.5±0.0b 25.7±0.0b 26.5±0.6e 26.7±1.0e naw

Fruits aborted before first fruit

set 5.9±1.0a 3.2±0.1b 1.3±0.1bc na na na

Pericarp lengthv (3 seedsu) 61.5±3.2a 49.6±2.5b 47.8±2.1b 52.4±1.4e 47.9±1.3f 44.4±2.1f

Pericarp length (4 seeds) 62.0±1.4a 54.5±1.9b 57.5±2.3b 58.7±1.4e 53.8±2.2ef 53.0±2.4f

Pericarp length (5 seeds) 68.4±1.4a 62.1±3.8b 60.6±2.3b 64.3±1.2e 60.3±1.8e 59.2±3.0e

Pericarp length (6 seeds) 75.7±1.8a 68.5±1.5b 65.6±1.3b 70.1±12e 68.1±2.4e 65.8±2.7e

z The BC LEle and BC lele pea lines represent lines backcrossed 5 times (BC 5) to ‘Carneval’ (le). y Data are means ± SE, n = 8 to 10 for all parameters except for pericarp length, where n =7 to 16 from 7 to 10 individual plants. x Means followed by different letters (a, b, c) indicate significant difference among TG1, TG2 and C1 lines within parameter by LSD, P<0.05; Means followed by different letters (e, f, g) indicate significant difference among the BC LEle, BC lele, and the NTC lines within parameter by LSD, P<0.05. w na = not available v Pericarp length at maturity (mm) u Number of seeds per fruit



40

Table IV: Relative transcript abundance of late GA biosynthesis and catabolism genes in 2 DAA pericarps of the TG1 and C1 linesa.

Gene TG1 C1 PsGA20ox1 9637 ± 1781b 6841 ± 568 PsGA20ox2 2.2 ± 0.4 0.9 ± 0.0 Total PsGA3ox1 604 ± 59 48 ± 3 Transgenic PsGA3ox1 161 ± 25 ndc PsGA3ox2 nd nd PsGA2ox1 4840 ± 1069 313 ± 31 PsGA2ox2 98 ± 27 299 ± 47

a Transcript levels were standardize across genes using the Ct value of 38 as the reference for normalization. b Data are means ± SE, n= 3. c not detected.



1

Figure 1: The latter part of the early 13-hydroxylation GA biosynthesis and catabolism pathway in pea. GA12 is 13-hydroxylated to GA53; carbon 20 is sequentially oxidized by a GA 20-oxidase (coded for by PsGA20ox genes) from GA53 to GA44, to GA19 and finally to GA20. GA20 is then oxidized by a 3β-hydroxylase (coded for by PsGA3ox genes) to GA1 (a bioactive GA). GA20 and GA1 can each be oxidized by a 2β-hydroxylase (coded for by PsGA2ox genes) to GA29 and GA8, respectively, the latter conversion inactivating GA1.



2

B

Internode number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Inte

rnod

e le

ngth

(m

m)

0

5

10

15

20

25

30

35

40

45

TG1TG2C1

A B

Node 8

Node 10

Node 16

Node 25

Ten

dril

peti

ole

leng

th (

mm

)

60

70

80

90

100

110

120TG1TG2C1

a

a

b

aba

b

aab

ba

ab

bc

C

Node 6Node 8

Stip

ule

leaf

are

a (c

m2 )

4

6

8

10

12TG1TG2C1

aab

b

a a

b

D

Internode number

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20

Inte

rnod

e le

ngth

(m

m)

0

10

20

30

40

50

60

70

80

90

100

110

BC5 LEBC5 leNT-C

B

Figure 2: Phenotypic comparison of pea transgenic TG1 and TG2, and transgenic null control (C1) lines (in ‘Carneval’le), and also in backcross generation 5 (BC5) LEle and BC5 lele, and non-transformed ‘Carneval’ le control (NT-C) lines. (A) Internode length at maturity of transgenic and transgenic null control lines from internode 1 to 20. (B) Internode length at maturity of BC5 LEle and BC5 lele, and non-transformed ‘Carneval’ le control (NT-C) lines from internode 1 to 20. (C) Tendril petiole length at maturity (from the point of attachment to the stem to the first tendril branchlet) of transgenic and transgenic null control lines at different nodal positions. (D) Stipule area at maturity of transgenic and transgenic null control lines at different nodal positions. Data are means ± SE, n= 8 to 10. Letters indicate significant differences among tendril length and stipule area within nodal position by LSD at P<0.05.



3

Figure 3: Representative light micrographs of mature internodes at position 2 for the transgenic

lines TG1 and TG2, and the null control C1 line. Stem transverse cross-sections at 2.5x

magnification (A) and at 10x magnification focusing on the vascular stele region of the stem (B).

cfb, cortical fiber bundle; co, cortex; cvb, cortical vascular bundle; la, lacunae; ph, phloem; vs,

vascular stele; xy, xylem.



4

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-3

)0

40

80

120

160

A

PsGA20ox1

1 2 3 4

0

40

80

120

160

B

PsGA20ox1

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-2

)

0

4

8

12

16D

1 2 3 4

0

4

8

12

16PsGA20ox2 PsGA20ox2C

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-2

)

0

25

50

75

100

Trans PsGA3ox1

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-3

)

0

40

80

120

160F

Total PsGA3ox1

1 2 3 4

Tra

nsc

rip

t Lev

el

0

100

200

300

400TG1C1

Development stage of tendril petioles

1 2 3 4

0

60

120

180

240

D

E

1 2 3 4

0

100

200

300

400TG2C1

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-2

)

0

60

120

180

240TG1C1

H I

J K

PsGA2ox1 PsGA2ox1

PsGA2ox2 PsGA2ox2

TG2C1

TG1TG2

TG1C1

TG2C1

TG1C1

TG2C1

TG1C1

1 2 3 4

0

40

80

120

160

TG2C1

G

Total PsGA3ox1

Figure 4: GA biosynthesis and catabolism gene expression profiles in the tendril petioles of pea transgenic TG1and TG2 and transgenic null control (C1) lines across development. Relative transcript abundance of PsGA20ox1 (A and B), PsGA20ox2 (C and D), transgenic PsGA3ox1 (E), total PsGA3ox1 (transgenic + endogenous; F and G), PsGA2ox1 (H and I), and PsGA2ox2 (J and K) in pea tendril petioles. Tendril petioles were harvested at 10-15% of final length (stage 1), 35-40% of final length (stage 2), 75-80% of final length (stage3), and at maturity (stage 4). Transcript levels were compared across all genes and lines using the Ct value of 38 as a reference for normalization. Data are means ± SE of two to three biological replicates. Y-axis scales in graphs are consistent across the same gene.



5

TG1 TG2 C1

GA

29 (

ng

gd

wt-1

)

0

200

400

600

800Stage 2Stage 3D

TG1 TG2 C1

GA

1 (n

g g

dw

t-1 )

0.0

1.5

3.0

4.5

6.0Stage 2Stage 3

B

TG1 TG2 C1

GA

8 (n

g g

dw

t-1)

0

20

40

60

80Stage 2Stage 3

C

TG1 TG2 C1

GA

20 (

ng

gd

wt-1

)

0

300

600

900

1200Stage 2Stage 3

A

Figure 5: Endogenous GA concentrations in pea tendril petioles. GA20 (A), GA1 (B), GA8

(C), and GA29 (D) levels were quantified in elongating tendril petioles at approximately

40% (stage 2) and 80% full length (stage 3) at nodal position 8 of the transgenic lines TG1

and TG2, and the transgenic null control line C1. Data are means ± SE, n= 2, with one

exception, 40% full length tendrils of TG1, n=1.



6

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-3

)0

40

80

120

160

A

PsGA20ox1

1 2 3 4

0

40

80

120

160

B

PsGA20ox1

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-2

)

0

4

8

12

16D

1 2 3 4

0

4

8

12

16PsGA20ox2 PsGA20ox2C

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-2

)

0

25

50

75

100

Trans PsGA3ox1

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-3

)

0

40

80

120

160F

Total PsGA3ox1

1 2 3 4

Tra

nsc

rip

t Lev

el

0

100

200

300

400TG1C1

Development stage of tendril petioles

1 2 3 4

0

60

120

180

240

D

E

1 2 3 4

0

100

200

300

400TG2C1

1 2 3 4

Tra

nsc

rip

t Lev

el (x

10-2

)

0

60

120

180

240TG1C1

H I

J K

PsGA2ox1 PsGA2ox1

PsGA2ox2 PsGA2ox2

TG2C1

TG1TG2

TG1C1

TG2C1

TG1C1

TG2C1

TG1C1

1 2 3 4

0

40

80

120

160

TG2C1

G

Total PsGA3ox1

Figure 6: GA biosynthesis and catabolism gene expression profiles in the stipules of pea transgenic TG1and TG2 and transgenic null control (C1) lines across development. Relative transcript abundance of PsGA20ox1 (A and B), PsGA20ox2 (C and D), transgenic PsGA3ox1 (E), total PsGA3ox1 (transgenic + endogenous; F and G), PsGA2ox1 (H and I), PsGA2ox2 (J and K) is shown in pea stipules harvested at 15-20% of final size (stage 1), 35-40% of final size (stage 2), 75-80% of final size (stage3) and at maturity (stage 4). Transcript levels were compared across all genes and lines using the Ct value of 38 as a reference for normalization. Data are means ± SE of two to three biological replicates. Y-axis scales in graphs are consistent across the same gene.



7

TG1 TG2 C1

GA

20 (

ng

gdw

t -1 )

0

300

600

900

1200 Stage 2Stage 3

A

TG1 TG2 C1

GA

1 (n

g g

dwt-

1 )

0

4

8

12

16Stage 2Stage 3

B

TG1 TG2 C1

GA

8 (n

g g

dw

t-1 )

0

20

40

60

80Stage 2Stage 3

C D

TG1 TG2 C1

GA

29 (n

g g

dw

t-1)

0

200

400

600

800Stage 2Stage 3

D

Figure 7: Endogenous GA concentrations in pea stipules. GA20 (A), GA1 (B), GA8 (C), and

GA29 (D) levels were quantified in stipules at approximately 40% (stage 2) and 80% full

size (stage 3) at nodal position 8 of transgenic lines TG1 and TG2, and the transgenic null

control line C1. Data are means ± SE, n= 1 for 40% full size samples, n= 2 for 80% full

size samples.



GA3ox expression influences plant growth in pea · Canada Discovery Grants to JAO and RPP, and the...

Documents

Transcript of GA3ox expression influences plant growth in pea · Canada Discovery Grants to JAO and RPP, and the...