Running head: Expression of pumpkin GA-oxidases in ...Dec 29, 2005 · Abeer Radi1, Theo Lange1, Tomoya Niki2, Masaji Koshioka2, and Maria João Pimenta Lange1 1Institut für Pflanzenbiologie

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Running head: Expression of pumpkin GA-oxidases in Arabidopsis

Author for correspondence:

Dr. Maria João Pimenta Lange

Institut für Pflanzenbiologie der Technischen Universität Braunschweig

Mendelssohnstr. 4

D-38106 Braunschweig

Germany

Telephone: +49-531-391-5880

Fax: +49-531-391-8180

e-mail: [email protected]

Research Area: Development and Hormone Action

Plant Physiology Preview. Published on December 29, 2005, as DOI:10.1104/pp.105.073668

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Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and

development of transgenic Arabidopsis plants

Abeer Radi1, Theo Lange1, Tomoya Niki2, Masaji Koshioka2, and Maria João Pimenta

Lange1

1Institut für Pflanzenbiologie der Technischen Universität Braunschweig, Mendelssohnstr.

4, D-38106 Braunschweig, Germany

2National Institute of Floricultural Science, 2-1 Fujimoto, Tsukuba, Ibaraki 305-8519, Ja-

pan



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Footnotes:

This work was supported by grants from the DFG priority program “molecular analysis of

phytohormone action” (La880/4-3) and by a fellowship from the Egyptian government to

A.R.

Corresponding author: Maria João Pimenta Lange, Fax: +49-531-391-8180, e-mail:

[email protected]



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Immature pumpkin (Cucurbita maxima) seeds contain Gibberellin (GA) oxidases with

unique catalytic properties resulting in GAs of unknown function for plant growth

and development. Over-expression of pumpkin GA 7-oxidase (CmGA7ox) in Arabi-

dopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants

that flower earlier with only a little increase in bioactive GA4 levels compared to con-

trol plants. In the same way, over-expression of the pumpkin GA 3-oxidase1

(CmGA3ox1) resulted in a GA-overdose phenotype with increased levels of endoge-

nous GA4. This indicates that, in Arabidopsis, 7-oxidation and 3-oxidation are rate

limiting steps in GA plant hormone biosynthesis that control plant development. With

an opposite effect, over-expression of pumpkin seed specific GA 20-oxidase1

(CmGA20ox1) in Arabidopsis resulted in dwarfed plants that flower late with reduced

levels of GA4, and increased levels of physiological inactive GA17 and GA25, and, un-

expected, GA34 levels. Severe dwarfed plants were obtained by over-expression of the

pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phe-

notype was accompanied by a considerable decrease in the levels of bioactive GA4 and

an increase in the corresponding inactivation product GA34 in comparison to control

plants. In this study we demonstrate the potential of four pumpkin GA-oxidase encod-

ing genes to modulate the GA plant hormone pool and, by this, alter plant stature and

development.



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The gibberellin (GA) plant hormones are known for playing an important role in many as-

pects of plant growth and development, including germination, stem growth, flowering, and

fruit development (Hedden and Proebsting, 1999; Richards et al., 2001; Olszewski et al.,

2002). The GA biosynthetic pathway has been characterized and the genes encoding most

of the GA biosynthetic enzymes have been cloned in Arabidopsis (Arabidopsis thaliana)

and other species (Lange, 1998; Hedden and Phillips, 2000a). Gibberellin biosynthesis in

plants can be divided into three major parts according to the type of the enzymes involved

and their subcellular localization (Lange, 1998; Hedden and Phillips, 2000a). The first part

takes place in plastids and results in the formation of ent-kaurene from the precursor trans-

geranylgeranyldiphosphate (GGDP) by the action of diterpene cyclases. This part of the

pathway is common to all plant systems that have been studied. The second part of the

pathway takes place at the endoplasmic reticulum and cytochrome-P450-dependent

monooxygenases are involved in the conversion of ent-kaurene to GA12-aldehyde and

GA12. The final part of GA-biosynthesis involves soluble 2-oxoglutarate-dependent dioxy-

genases and leads to the production of the GA plant hormone and inactive GAs. GA dioxy-

genases are often multifunctional with broad substrate specificity, resulting in many side

reactions and numerous GAs (Hedden and Kamiya, 1997; Lange, 1998; Hedden and Phil-

lips, 2000a).

In Arabidopsis, two pathways are described diverging from GA12 to GA plant hormones: A

non-13-hydroxylation pathway leading to GA4 and a 13-hydroxylation pathway leading to

GA1 (Fig. 1). These steps are catalyzed by GA 20-oxidase and GA 3-oxidase enzymes,

each encoded by small multigene families (Sponsel and Hedden, 2004). Eight GA-2 oxi-

dases have been identified in Arabidopsis with specificity for either C19- (Thomas et al.,

1999) or C20-GAs (Schomburg et al., 2003).

Developing pumpkin (Cucurbita maxima) seeds express a set of GA-oxidases with unique

catalytic properties that have not been identified in other plant species to date and that syn-

thesize GAs of unknown function in plant development (Fig. 1; Lange, 1998). A multifunc-

tional GA 7-oxidase from pumpkin (CmGA7ox) oxidizes GA12-aldehyde to GA12 and, less

efficiently, GA12 to GA14 (Fig. 1; Lange, 1997; Frisse et al., 2003). The seed specific GA



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20-oxidase1 from pumpkin (CmGA20ox1) is, like other GA 20-oxidases, multifunctional

with broad substrate specificity but produces mainly C20-GAs (e.g. GA25) instead of C19-

GAs (e.g. GA9, Fig.1; Lange, 1994, 1998; Lange et al., 1994; Frisse et al., 2003). Unlike in

most plant species that have been investigated so far, in pumpkin GA 3-oxidase1

(CmGA3ox1, formerly 2β,3β-hydroxylase; Lange et al., 1997) is a bifunctional enzyme. In

addition to its 3-oxidation catalytic properties that can lead to the formation of GA plant

hormones (e.g. GA4, Fig. 1) it also exhibit 2-oxidation catalytic function (Lange et al.,

1997). Moreover, 3-oxidases from other plant species act mainly on C19-GAs but

CmGA3ox1 prefers C20-GAs as the substrate (Fig. 1; Lange et al., 1997; Hedden, 1999).

Finally, the pumpkin GA 2-oxidase1 (CmGA2ox1) shares very high sequence identity with

an unidentified dioxygenase from Marah macrocarpus and the recombinant protein uses

C19-GAs as the substrate (Fig. 1; MacMillan et al., 1997; Frisse et al., 2003).

The multiple roles of GAs and the large number of enzymes and genes involved in the bio-

synthetic pathway suggest that regulation of GA levels in planta is likely to be rather com-

plex (Hedden and Phillips, 2000a). Transgenic plants over-expressing genes of the GA bio-

synthetic pathway have been produced to investigate their effects on GA biosynthesis, GA

homeostasis and plant morphology. Furthermore, this approach can be of benefit for con-

trolling plant stature in agriculture and horticulture (Phillips, 2004).

Up-regulation of early steps of the pathway has been achieved by over-expressing AtCPS

and AtKS in Arabidopsis and resulted in accumulation of early intermediates of the biosyn-

thetic pathway but cause no changes in plant morphology and levels of active GAs showing

the ability of plants in maintaining GA homeostasis (Fleet et al., 2003). However, GA-

overdose morphologies were obtained by over-expression of GA 20-oxidases in Arabidop-

sis (Huang et al., 1998; Coles et al., 1999) and potato (Carrera et al., 2000). Similarly, over-

expression of Arabidopsis GA 20-oxidase in hybrid aspen (Eriksson et al., 2000) and over-

expression of a GA 20-oxidase from citrus or Arabidopsis in tobacco plants (Vidal et al.,

2001; Biemelt et al., 2004) resulted in elongated phenotypes associated with GA overpro-

duction. In hybrid aspen and Arabidopsis, over-expression of an Arabidopsis GA 3-oxidase

resulted in no major changes in morphology (Israelsson et al., 2004; Phillips, 2004) and in



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the case of hybrid aspen, the authors suggest that 20-oxidation is the limiting biosynthetic

step for GA-controlled shoot elongation.

The ‘green revolution’ that originated an increased yield in cereal crop cultivars resulted

from the introduction of dwarfed varieties (Peng et al., 1999; Spielmeyer et al., 2002;

Monna et al., 2002; Sasaki et al., 2002). Ectopic over-expression of the seed-specific GA

20-oxidase1 from pumpkin (CmGA20ox1), that produces mainly inactive GA products,

might result in a reduction of bioactive GAs by diverting the pathway to the tricarboxylic

acids (Fig. 1) and therefore originate dwarf phenotypes. This has been achieved success-

fully in lettuce plants were CmGA20ox1 was introduced under a very strong promoter cas-

sette and dwarfed lettuce plants were obtained (Niki et al., 2001). However, in Arabidopsis

and Solanum dulcamara this strategy to reduce GA contents and produce dwarfed plants

was not achieved, weakening the usefulness of this approach to alter GA levels and reduce

plant height in other plant species (Xu et al., 1999; Curtis et al., 2000). The over-expression

of CmGA20ox1 resulted in semi-dwarfed plants in Solanum dulcamara and slight reduction

in plant height in Arabidopsis being suggested that a feed-back control of endogenous GA

20-oxidase gene (Solanum dulcamara and Arabidopsis) and GA 3-oxidase (Arabidopsis)

compensate the effect of CmGA20ox1 transgene (Xu et al., 1999; Curtis et al., 2000). GA 2-

oxidases are catabolic enzymes and can potentially be used to decrease GA levels and cre-

ate dwarfed phenotypes. Over-expression of GA 2-oxidase genes in Arabidopsis, tobacco,

rice and poplar resulted in the expected dwarfed phenotypes (Schomburg et al., 2003; Bie-

melt et al., 2004; Sakamoto et al., 2003; Busov et al., 2003) but the developmental role of

GA 2-oxidases in plants is not well understood.

To our knowledge, no attempt had been made to over-express the multifunctional

CmGA7ox from pumpkin and investigate its potential regulatory function in controlling the

levels of bioactive GAs. Here we discuss the feasibility of increasing bioactive GAs and

alter plant morphology changing the flux through the pathway by over-expressing

CmGA7ox in Arabidopsis. Moreover, we also show that over-expression of the bifunc-

tional CmGA3ox1 in Arabidopsis results in increased plant height and increased GA4 lev-

els, despite the enzyme’s preferences for oxidizing C20-GAs instead of C19-GAs.



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In the present work, we obtained dwarfed Arabidopsis plants and divert GA precursors into

inactive forms by over-expressing CmGA20ox1 gene using a very strong promoter cassette

(Niki et al., 2001) and confirm the importance of this approach to control plant stature. In

Arabidopsis GA 2-oxidase is encoded by a gene family (Thomas et al., 1999; Hedden and

Phillips, 2000a; Schomburg et al., 2003) but from pumpkin, up to now, only one active GA

2-oxidase gene has been cloned (CmGA2ox1; Frisse et al., 2003). Contrarily to the GA 2-

oxidases that have been over-expressed so far in Arabidopsis (Schomburg et al., 2003), the

CmGA2ox1 uses C19-GAs as substrates instead of C20-GAs. To understand its role in plant

development, we over-expressed CmGA2ox1 in Arabidopsis resulting in extreme dwarfed

phenotypes and reduction of active GA levels.

Results

Generation of transgenic Arabidopsis plants expressing pumpkin GA-oxidases

Arabidopsis plants were transformed with constructs containing sense copies of

CmGA20ox1 and sense or antisense copies of CmGA7ox, CmGA3ox1 or CmGA2ox1. The

transgenic plants were selected in the presence of kanamycin and the integration of the

pumpkin GA-oxidase sense or antisense copies of ten lines were analyzed by polymerase

chain reaction (PCR; data not shown). The antisense lines were used together with the wild-

type plants as controls. From the ten lines, three homozygous (T4) lines showing altered

phenotypes were chosen and the expression of the pumpkin GA-oxidases estimated by

quantitative reverse transcriptase (RT-) PCR (Table I).

Transgenic lines expressing CmGA7ox (S13.1, S8.9, and S12.8) and CmGA3ox1 (S1.3,

S19.4, and S17.7) were slender, while transgenic lines expressing CmGA20ox1 (S17.2) and

CmGA2ox1 (S9.7, S5.5 and S12.9) were dwarf. Lines S10.8 and S2.2 expressed semi-

dwarfed phenotypes, despite the fact that in both lines no CmGA20ox1 transcripts were de-

tectable (Table I; data not shown). Transcripts of the four pumpkin GA-oxidase encoding

genes were not found in either wild-type Arabidopsis plants nor antisense lines (data not

shown). Lines with highest expression levels of each pumpkin GA-oxidase gene showed



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always the strongest phenotype and were choosen for further investigation (Fig. 2A, 2B, 2C

and Table II).

Arabidopsis plants over-expressing CmGA7ox or CmGA3ox1 show accelerated develop-

ment

Arabidopsis 14-day old seedlings over-expressing CmGA7ox or CmGA3ox1 showed altered

root shapes when compared to wild-type seedlings (Fig. 2A). The seedlings of CmGA7ox

over-expressors showed one thin long primary root with very few lateral roots while the

seedlings of CmGA3ox1 over-expressors developed many thick lateral roots. Seedlings

over-expressing CmGA3ox1 showed also enlarged leaves and an increased number of

trichomes when compared to seedlings over-expressing CmGA7ox or wild-type seedlings

(Fig. 2A). Arabidopsis plants over-expressing CmGA7ox or CmGA3ox1 showed slender

phenotypes when compared to plants transformed with antisense copies of the respective

genes or to wild-type plants (Fig. 2B, Table II). The strongest CmGA7ox over-expressing

line according to the RT-PCR results (S12.8; Table I) was chosen for phenotypical charac-

terization. Line S12.8 had an increase of about 50% in final height, with a similar increase

in internode length, developed two times more siliques, and flowered earlier when com-

pared to wild-type plants or to the antisense line AS15.9 (Fig. 2B, Table II). Line S17.7

showed the strongest over-expression of CmGA3ox1 by RT-PCR (Table I) and developed

longer shoots, longer internodes, and flowered much earlier than wild-type plants or plants

transformed with antisense copies of the respective gene (line AS5.9). However, compared

to line S12.8 (CmGA7ox overexpressor), in line S17.7 branching was more frequent, and

the total number of siliques increased (Table II).

Over-expression of CmGA20ox1 or CmGA2ox1 in Arabidopsis results in retarded devel-

opment

In order to get T4 homozygous Arabidopsis plants expressing CmGA20ox1 or CmGA2ox1,

selection in the presence of kanamycin and correct segregations ratios could be obtained

only in the presence of GA3. Therefore to compare phenotypes, seeds of CmGA20ox1 and

CmGA2ox1 expressing lines as well as seeds of CmGA2ox1 antisense line and a second set



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of wild-type plants (WTa) were all germinated in MS media containing 10-6M GA3. Arabi-

dopsis plants expressing CmGA20ox1 or CmGA2ox1 were dwarfed with slightly darker

green leaves compared to wild-type plants or plants transformed with antisense copies of

CmGA2ox1 (Fig. 2C). Line S17.2 showed the strongest expression of CmGA20ox1 and was

therefore subjected to more extensive phenotype characterization. S17.2 plants showed re-

duced development, flowered later and were much shorter reaching only 28% of the final

height of similarly grown wild-type plants (Fig. 2C and Table II). The number of siliques of

the dwarf plants was reduced to only 29%, compared to wild-type plants grown under the

same conditions (Table II). Expression of CmGA2ox1 in Arabidopsis resulted in severely

dwarfed phenotypes (Fig. 2C). Line S12.9 expressed the strongest transcript levels as de-

termined by RT-PCR (Table I) and they reached only 12% of the final height of their re-

spective antisense line (AS7.7) grown under similar conditions (Table II). These plants

showed a reduced development and flowered very late (Fig. 2C, Table II). The dwarf plants

had an extremely low number of siliques, only about 17% of the respective antisense line

(Table II).

Analysis of endogenous GA levels in Arabidopsis plants over-expressing pumpkin GA-

oxidases

Endogenous GA levels were determined by combined gas chromatography-mass spec-

trometry selected ion monitoring (GC-MS SIM) in the transgenic Arabidopsis lines ex-

pressing the highest levels of pumpkin GA-oxidases to identify which steps of the GA bio-

synthetic pathway were affected in these plants (Table III). Similarly, endogenous GA lev-

els were also determined in control Arabidopsis plants (plants expressing antisense copies

of pumpkin GA-oxidases and wild-type plants). Arabidopsis slender plants expressing

CmGA7ox had a three to four times increase in GA12 levels in comparison to control plants

but only a very slight increase in biological active GA4 and catabolic GA34 levels. GA-

levels of the early 3-hydroxylated pathway (GA14, GA36, GA37), and of precursors of the

13-hydroxylated pathway (GA53, GA44) decreased. No changes of levels for the other GAs

were observed. In contrast, Arabidopsis slender plants expressing CmGA3ox1 showed in-

creased GA4 levels as well as increased levels of the correspondent inactivation product



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GA34 and no changes in other GA levels when compared to their respective control plants

(Fig. 1 and Table III). The Arabidopsis dwarfed plants expressing CmGA20ox1 showed re-

duced levels of the bioactive GA4, increased levels of the respective inactivation product

GA34 and increased levels of the tricarboxylic C20-GAs, GA25 and, particularly, GA17, when

compared to wild-type plants (Table III, WTa). The dwarfed plants showed also a slight de-

crease in GA12-ald and GA12, and an increase in GA36 (Table III). The level of the bioactive

GA4 was considerably reduced in the severe dwarfed Arabidopsis plants expressing

CmGA2ox1 accompanied by an increase in the respective inactivation product GA34, when

compared to their respective control wild-type plants (WTa) or antisense line. The dwarfed

plants showed also a slight decrease in GA12 and GA36 levels (Table III).

Discussion

Genes of the GA biosynthetic pathway have been over-expressed in plants to investigate

their effects on GA biosynthesis, GA homeostasis and plant morphology (Phillips, 2004).

Developing pumpkin seeds express GA dioxygenases with unique catalytic properties re-

sulting in GAs of unknown function for plant development. In order to investigate their po-

tential role for modulation of GA biosynthesis, pushing the flux through the pathway and

possibly leading to an increase in bioactive GAs, we over-expressed pumpkin GA-oxidases

in Arabidopsis.

Over-expression ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase (KS)

alone or in combination in Arabidopsis resulted in high accumulation of ent-kaurene and

ent-kaurenoic acid (1,000-times more), GA12 (about 10-times), and GA24 levels (about 4-

times) compared to wild-type plants (Fleet et al., 2003). Surprisingly, no changes in bioac-

tive GA-levels or plant morphology were observed. The authors propose that P450 ent-

kaurenoic acid oxidase (KAO), producing GA12, may be limiting for production of middle

and later GA intermediates. In contrast, Arabidopsis plants expressing CmGA7ox (although

in a different ecotype) show accelerated development, have longer stems and internodes,

increase in number of siliques, and flower earlier than wild type Arabidopsis plants



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(Fig.2B, 2C and Table II). All these phenotypic changes took place despite a three- to four-

fold increase of GA12 levels, with no considerable changes in GA4 levels, the primary ac-

tive GA in Arabidopsis (Talon et al., 1990; Cowling et al., 1998; Table III). This apparent

discrepancy might be due to variations in GA-levels between various types of tissues and

during different stages of plant developmental. For instance over-expression of GA 20-

oxidase genes in Arabidopsis have been reported to result in GA-overproduction pheno-

types with no consistent differences in GA4 and GA1 levels between shoot tips of the trans-

genic lines and wild-type plants, while a two- to three-fold increase in GA4 levels was ob-

served when the rosette leaves of the transgenic lines were analyzed (Coles et al., 1999). In

the case of 14-day old Arabidopsis CmGA7ox over-expressors (using intact seedlings for

the GA-analysis), again GA4-levels did not increase considerably compared to wild-type

seedlings (data not shown). Therefore, in Arabidopsis GA levels might be tightly regulated

with only small changes in the GA plant hormone pool sufficient for modulating plant

growth and development. No increase of early 3-hydroxylated GA levels (GA14, GA37,

GA36) were observed, indicating that 3-hydroxylation side activity of CmGA7ox that has

been identified with recombinant CmGA7ox (Lange, 1997; Frisse et al., 2003) had no ma-

jor impact in GA-biosynthesis of the transgenic Arabidopsis line. However, the existence of

yet unidentified GA biosynthetic pathways in Arabidopsis that play a role in plant devel-

opment can not be excluded.

In hybrid aspen, over-expression of a GA 3-oxidase from Arabidopsis resulted in no major

changes in morphology and in only small changes of bioactive GA1 and GA4 levels (Is-

raelsson et al., 2004). The authors conclude that, in hybrid aspen, 20-oxidation rather than

3-oxidation is the limiting step in the formation of GA1 and GA4 and that expression of GA

3-oxidases alone does not increase the flux toward bioactive GAs. Moreover, Phillips

(2004) reported that, in Arabidopsis, over-expression of GA 3-oxidase does not effect plant

development. However, our results demonstrate that ectopic over-expression of the seed

specific CmGA3ox1 in Arabidopsis leads to dramatic changes in plant growth and devel-

opment (Fig. 2A, 2B and Table II). Arabidopsis seedlings over-expressing CmGA3ox1 have

elevated hypocotyl and leaf growth and an increased number of trichomes compared to



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wild-type plants which are all known typical GA effects (Olszewski et al., 2002; Perazza et

al., 1998). Adult over-expressors of CmGA3ox1 develop similar to plants over-expressing

CmGA7ox. They show a slender phenotype, and they flower earlier compared to wild type

plants or plants expressing antisense copies of CmGA3ox1. Moreover, the number of

siliques observed per plant was higher in CmGA3ox1 even compared to CmGA7ox over-

expressors, suggesting that flower formation and/or seed set are favored in these lines (Ta-

ble II). In spite of the fact that CmGA3ox1 prefers C20-GAs as the substrate the phenotypic

changes are accompanied by a two-fold increase in bioactive GA4 levels and a slight in-

crease in the correspondent inactivation product GA34 in the CmGA3ox1 over-expressors

which would account for the obtained morphological changes (Fig.1 and Table III).

Root and shoot organs react differently to GA levels; e.g. for normal root growth much

lower concentration of GAs are required than for normal shoot growth (Tanimoto, 1990).

Root morphology of transgenic Arabidopsis seedlings over-expressing pumpkin CmGA7ox

and CmGA3ox1 has changed dramatically (Fig. 2A). CmGA7ox over-expressors develop

much longer primary roots compared to wild-type plants, indicating that a little increase in

endogenous GA-levels of these plants favor root elongation. However, CmGA3ox1 trans-

genic lines develop more lateral roots that are thicker compared to wild type plants (Fig.

2A), indicating that a considerable increase of endogenous GA-levels does not help root

elongation but lateral root formation. Recently, Fu and Harberd (2003) found that GAs

regulate root growth by repressing GAI and RGA, two DELLA proteins involved in GA-

signaling (Fleet and Sun, 2005). However, little is known of how GA-signaling components

modulate GA-biosynthesis (Richards et al., 2001; Olszewski et al., 2002; Sun and Gubler,

2004; Thomas and Sun, 2004; Fleet and Sun, 2005). Moreover, other plant hormones, e.g.

auxins, are known to interact with the components of the GA-biosynthetic pathway that af-

fects plant development, e.g. the development of lateral roots (Casimiro et al., 2003; Fu and

Harberd, 2003; Achard et al., 2003; Fleet and Sun, 2005).

Seed-specific GA 20-oxidase1 from pumpkin (CmGA20ox1) encodes an enzyme with

unique catalytic GA 20-oxidation properties: It is the only known GA 20-oxidase that pro-

duces mainly tricarboxylic C20-GAs (e.g. GA25, GA17), that have no known physiological



14

function, rather than C19-GAs (e.g. GA9, GA20) that serve as precursors in GA plant hor-

mone synthesis (Fig. 1; Lange, 1994, 1998; Lange et al., 1994; Frisse et al., 2003). There-

fore, over-expression of Cm20ox1 might be a useful strategy for reduction of bioactive GAs

in planta (Hedden and Phillips, 2000b). This hypothesis has been tested already by several

groups using different plant species. In lettuce over-expression of CmGA20ox1 resulted in

dwarfed plants with reduced levels of GA1 and GA4 and increased levels of GA17 and GA25

(Niki et al., 2001). However, in Arabidopsis and Solanum dulcamara, over-expression of

CmGA20ox1 resulted in only a slight reduction of plant height associated with a semi-

dwarfed phenotype (Xu et al., 1999; Curtis et al., 2000). In the case of Arabidopsis

CmGA20ox1 over-expressing plants showed reduced levels of GA4 and no clear effect on

GA1 levels. In the case of Solanum dulcamara, GA4 levels were unaltered in stems and in-

creased in leaves, and GA1 levels were reduced. It was suggested that a feedback type of

regulation, resulting in increased transcript levels of the endogenous GA 20-oxidase encod-

ing gene (Arabidopsis and Solanum dulcamara) and GA 3-oxidase encoding gene (Arabi-

dopsis), was responsible for the limited success in reducing plant height. We re-investigated

over-expression of CmGA20ox1 in Arabidopsis plants using a strong promoter cassette

similar to the one that drove expression of CmGA20ox1 in lettuce (Niki et al., 2001) and

obtained Arabidopsis plants with severe dwarfed phenotypes. As in transgenic lettuce,

flowering was delayed and seed number was dramatically reduced in the dwarfed Arabi-

dopsis plants (Table II). However, in contrast to what was reported for lettuce, we found

that seed germination was affected and transgenic seeds were not able to germinate in the

absence of applied GA3 (data not shown). The transgenic Arabidopsis accumulated tricar-

boxylic GA25 and GA17, and had reduced levels of the bioactive GA4 similar to the findings

of Xu et al. (2004). In addition, increased endogenous levels of GA34 indicate a surprising

increase of 2-oxidation activity in the transgenic plants.

Genetic manipulation of catabolic GA 2-oxidases offers another suitable strategy for modu-

lating plant development (Hedden and Phillips, 2000b). Dwarfed phenotypes with de-

creases GA levels in planta have been achieved by over-expression of GA 2-oxidases in

several plant species, including tobacco, rice and poplar (Biemelt et al., 2004; Sakamoto et



15

al., 2003; Busov et al., 2003). In Arabidopsis GA 2-oxidases are encoded by a complex

gene family (Thomas et al., 1999; Sponsel and Hedden, 2004; Schomburg et al., 2003).

Over-expression of two of them that hydroxylate C20- rather that C19-GA precursors, re-

sulted in dwarfed phenotypes in Arabidopsis (Schomburg et al., 2003). Recombinant

pumpkin CmGA2ox1 hydroxylates C19-GAs, and by this, inactivates efficiently GA plant

hormones, including GA1 and GA4 (Frisse et al., 2003). Expression studies indicate that

CmGA2ox1 transcript levels are most abundant in roots of pumpkin plants (Lange et al.,

2005). To test the influence of this gene on modulating Arabidopsis plant growth and its

GA hormone pool, we used a strong promoter cassette (Niki et al., 2001) to express consti-

tutively CmGA2ox1 in Arabidopsis. The transgenic plants express extremely dwarfed phe-

notypes and seeds of those lines are unable to germinate in the absence of exogenous ap-

plied GA. Compared to control plants, CmGA2ox1 over-expressors show severe reduced

stem elongation and flower late, with dramatic decrease in the siliques number (Fig. 2C,

Table II). Rice plants over-expressing GA 2-oxidase constitutively, by the action of the ac-

tin promoter, showed severe dwarfism and failed to set grain. However, ectopic expression

of the same gene in shoots under the control of the promoter of the GA biosynthesis gene,

OsGA3ox2, resulted in semi-dwarfed phenotypes that are normal in flowering and grain

development (Sakamoto et al., 2003). The dramatic changes observed in the phenotype of

CmGA2ox1 over-expressors were accompanied by a severe reduction of the GA4 levels

and, as expected, in an increase of the correspondent inactivation product GA34.

The four pumpkin GA-oxidases utilized in this study offer a set of tools for manipulating

GA biosynthesis and for regulation of plant development that might gain enormous benefits

for designing optimized agriculture and horticulture important plant species. Our results

demonstrate that, by over-expression of the CmGA7ox and CmGA3ox1, it becomes possible

to increase GA levels and elevate plant development in Arabidopsis. With an opposite ef-

fect, over-expression of CmGA20ox1 and CmGA2ox1 in Arabidopsis decreases GA levels

resulting in severe dwarfed phenotypes. Moreover, our results demonstrate the usefulness

of over-expressing CmGA20ox1 under the control of a strong promoter cassette, and, by

this, offering an attractive alternative strategy for reducing GA content and modulating



16

plant development. GA 20-oxidation steps have been shown to limit production of bioac-

tive GAs with associated GA-phenotypes (Huang et al., 1998; Carrera et al., 1999; Coles et

al., 1999; Eriksson et al., 2000; Vidal et al., 2001; Biemelt et al., 2004). Our results indicate

that, in addition, GA 7-oxidase and 3-oxidase also catalyze rate limiting steps of the GA

biosynthetic pathway in Arabidopsis. It is possible that local modulation of GA levels in the

over-expressor lines account for the differences in plant development. As reviewed by

Sponsel and Hedden (2004), expression studies on rice genes involved in GA-biosynthesis

and signaling suggest that bioactive GAs are produced at their site of action. Further studies

are necessary to understand the impact of the sites of GA biosynthesis, perception, and sig-

naling as well as cross-talk with other plant hormones.

Materials and Methods

Plant material and growth conditions

Arabidopsis thaliana ecotype Columbia was used in all experiments. Seeds were sown on

soil and stratified at 4 °C for 2 to 3 days before transfer to a growth chamber under long

day conditions: 16 h light (approximately 120 µmol m-2s-1) and 8 h dark. The temperature

was kept at 22 °C and 20 °C during the light and dark periods, respectively. For plate

growth assays, seeds were sterilized and plated on 0.8% plant agar in 0.5x MS media

(Duchefa, Haarlem, The Netherlands) containing, when appropriate, 50 µg ml-1 kanamycin

and 10-6M GA3 . The seeds were stratified and grown as above and transferred to soil after

2 to 4 weeks. For RT-PCR analysis the rosette leaves of 8-week-old plants (for wild-type,

CmGA7ox and CmGA3-ox1 transgenic lines) or the rosette leaves of 9-week-old plants (for

CmGA20ox1 and CmGA2ox1 transgenic lines) were collected and frozen immediately in

liquid nitrogen. For GA quantification, the aerial part of 7-week old wild-type and trans-

genic plants was harvested and frozen immediately in liquid nitrogen.



17

Plasmid constructs and Plant transformation

To enhance the CmGA20ox1 expression, a construct containing a strong promoter cassette

and a translational enhancer (E12-35-Ω) was used as described by Mitsuhara et al. (1996)

and Niki et al. (2001). The constructs for expression of sense or antisense copies of

CmGA7ox, CmGA3ox1 or CmGA2ox1 were similar to the one used for expressing

CmGA20ox1 but the sense or antisense copies of the different cDNAs were cloned at an

unique EcoRI site of a multi cloning site of the vector modified from pBE2113 as described

by Mitsuhara et al. (1996) replacing CmGA20ox1.

The constructs carrying the sense or antisense copies of the different pumpkin GA-oxidases

were introduced in Arabidopsis wild-type plants via Agrobacterium tumefaciens-mediated

transformation using the floral dip method (Clough and Bent, 1998). To identify transgenic

plants, seeds from the dipped plants were grown on MS media plates supplemented with 50

µg ml-1 kanamycin. The seeds of the kanamycin resistant plants were further analyzed for

kanamycin resistance and segregation. T2 seedlings of CmGA20ox1 and CmGA2ox1 sense

lines showed a segregation ratio of 3:1 only when GA3 was added to the MS plates. There-

fore, T2 and further generation seeds of sense lines of CmGA20-ox1 and sense and an-

tisense CmGA2-ox1 lines were all germinated in the presence of 10-6 M GA3. A second set

of wild-type plants (WTa) was generated where the seeds were germinated in the presence

of 10-6 M GA3 and used as a control in the experiments involving CmGA20ox1 and

CmGA2ox1 lines. The presence of every transgene was checked by PCR in ten of the T2

sense lines and five of the T2 antisense lines that showed a segregation ratio of 3:1. Spe-

cific pumpkin GA-oxidases primers, identical to the ones described below for the RT-PCR

experiments, and a vector specific primer 5’-CTACAACTACATCTAGAGG-3’ were used

respectively as reverse and forward primers in the PCR experiments. After scoring at T3,

three homozygous sense lines and two homozygous antisense lines for every gene were

taken to generate T4 homozygous plants used for phenotype, biochemical and molecular

characterization.



18

Quantitative RT-PCR

Transcript levels of CmGA7ox, CmGA20ox1 and CmGA3ox1 (previously named 2β,3β-

hydroxylase) were quantified as described previously by Lange et al. (1997), except that 50

ng of total RNA was reverse transcribed using RevertAidTMH Minus 1st Strand cDNA Syn-

thesis Kit (MBI Fermentas, Germany). For the quantification of CmGA2ox1, three specific

oligonucleotides were synthesized based on its cDNA sequence: CmGA2ox1 F (5’-CTC

TGC AGC ATT CTA CTC TGG GAT TCC-3’), CmGA2ox1 R (5’-GGC CCA CCG AAG

TAG ATC ATT GAA ACC -3’) and CmGA2ox1 RT (5’-AGA TGT TCG AAT CC-3’). For

the preparation of the internal RNA standard, pBluescript SK plasmid containing the

CmGA2ox1 cDNA was digested with HindIII that released a 448 bp fragment. The vector

was re-ligated and used for RNA synthesis. The annealing temperature used for PCR was

60 °C.

Quantification of endogenous Gibberellins

For quantitative determination of endogenous GAs of frozen plant tissue from aerial part (2

g fresh weight) was spiked with 17,17-d2-GA standards (2 ng each; from Professor L.

Mander, Australian National University, Canberra, Australia) and pulverized under liquid

nitrogen. Samples ware extracted, purified, derivatized, and analyzed by gas chromatogra-

phy mass spectrometry using selected ion monitoring as described elsewhere (Lange et al.

2005).

Acknowledgments

We thank Anja Liebrandt for technical assistance.



19

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25

Figure legends

Figure 1. The third part of the GA biosynthetic pathway leading to the formation of bioac-

tive GAs in Arabidopsis. Biosynthetic steps resulting from the over-expression of pumpkin

GA-oxidases: a, 7-oxidase (CmGA7ox); b, seed-specific 20-oxidase1 (CmGA20ox1); c,

seed-specific 3-oxidase1 (CmGA3ox1); and d, 2-oxidase1 (CmGA2ox1). Main pathways

are indicated by thick arrows. Boxed GAs, bioactive GAs. Metabolic relationships are dis-

cussed in the text.

Figure 2. Over-expression of pumpkin GA-oxidases in Arabidopsis: effects on plant devel-

opment. A, Phenotypes of 14-d-old seedlings grown in MS media. Wild-type seedlings

(left) compared to seedlings expressing sense copies of CmGA7ox (line S12.8, middle) or

expressing sense copies of CmGA3ox1 (line S17.7, right). Bar = 1 cm. B, Phenotypes of 7-

week-old plants transferred to soil after 28 days in MS media. Non transformed plants

(WT; left) compared to different antisense (AS) and sense (S) lines transformed with

CmGA7ox (middle) or CmGA3ox1 (right). C, Phenotypes of 7-week-old plants transferred

to soil after 28 days in MS media containing 10-6 M GA3. Non transformed plants (WTa;

left) compared to different antisense (AS) and sense (S) lines transformed with

CmGA20ox1 (middle) or CmGA2ox1 (right).



26

Table I. Levels of pumpkin GA-oxidase mRNAs in Arabidopsis over-expression lines as determined by

quantitative RT-PCR.

Transcript levels (µg.g-1)

CmGA7ox CmGA20ox1 CmGA3ox1 CmGA2ox1

Lines S13.1 S8.9 S12.8 S10.8 S2.2 S17.2 S1.3 S19.4 S17.7 S9.7 S5.5 S12.9

60 80 100 n.d.a n.d.a 10 20 100 1000 20 90 125

an.d., not detectable.

https://plantphysiol.orgD

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ovember 11, 2020. - Published by

Copyright (c) 2020 A

merican Society of Plant B

iologists. All rights reserved.


27

Table II. Phenotypic characteristics of wild-type (WT) and transgenic Arabidopsis plants expressing pumpkin GA-oxidases. AS,

antisense lines used as a control. S, sense lines.

Results are shown as mean ± SE (n=5).

WT

CmGA7ox

CmGA3ox1

WTa

CmGA20ox1a

CmGA2ox1a

AS15.9 S12.8 AS5.9 S17.7 S17.2 AS7.7 S12.9

Final heightb (cm) 35.0± 2.4 36.7± 1.8 50.8±0.6 30.7±1.0 52.6±2.0 38.9±2.4 11±0.8 39.3±2.2 4.84±0.4

Internode lengthb,c (cm) 1.38 ± 0.1 1.70 ± 0.3 2.42±0.1 1.30±0.1 2.44±0.2 1.40±0.1 0.90±0.1 1.90±0.1 0.68±0.1

Siliques bd (#) 22.0±1.8 27.2±0.7 43.8±0.7 22.4±1.0 54.2±1.5 36.4±0.5 10.4±2.6 40.2±1.0 6.8±1.8

Flowering timee (d) 42.6 ± 1.0 41.4 ± 0.5 34.2±1.5 42.6±0.5 30±1.6 41.2±0.4 50.4±1.3 42.0±1.4 57.0±0.9

aPlants were transferred to soil after 28 days in MS media containing 10-6 M GA3. bPlants nine-week old. cThe first internode of the main

inflorescence. dNumber per plant. eWhen the first flower appears.


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28

Table III. GA-levels (ng per plant) in 7-week old wild-type (WT) and transgenic Arabidopsis plants expressing sense (S) or an-

tisense (AS) copies of CmGA7ox, CmGA20ox1, CmGA3ox1, or CmGA2ox1 gene. Measurements have been repeated at least

once with similar results.

WT WTa 7ox 20ox1a 3ox1 2ox1a

AS15.9 S12.8 S17.2 AS5.9 S17.7 AS7.7 S12.9

GA12ald. 0.37 0.31 0.52 0.64 0.17 0.38 0.24 0.20 0.16

GA12 2.24 1.64 3.24 9.06 1.27 2.14 2.64 1.36 1.09

GA15 0.52 0.30 0.51 0.39 0.39 0.46 0.39 0.32 0.42

GA24 1.34 1.06 2.12 1.45 0.96 1.55 1.65 1.03 0.91

GA9 0.14 0.15 0.11 0.18 0.13 0.18 0.19 0.11 0.08

GA25 0.11 0.08 0.09 0.15 0.18 0.11 0.13 0.08 0.09

GA4 0.32 0.27 0.32 0.35 0.18 0.29 0.54 0.22 0.09

GA34 0.54 0.37 0.41 0.57 1.24 0.50 0.61 0.34 0.45

GA53 0.34 0.17 0.44 0.24 0.18 0.34 0.33 0.17 0.15

GA44 0.09 0.04 0.06 0.05 0.10 0.06 0.05 0.05 0.07

GA20 n.d.b 0.01 0.01 0.01 n.d.b 0.01 0.01 0.01 n.d.b

GA17 0.09 n.d.b n.d.b 0.04 0.51 0.01 0.03 n.d.b 0.04

GA1 0.01 0.01 0.01 0.01 0.01 0.01 0.01 0.01 n.d.b

GA8 -c -c -c -c 0.01 0.01 -c 0.01 0.01

GA14 0.01 n.d.b 0.07 n.d.b n.d.b n.d.b n.d.b n.d.b n.d.b


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29

Table II, continued.

GA36 0.92 0.48 1.32 0.82 0.87 1.13 0.91 0.55 0.31

GA37 0.03 n.d.b 0.01 n.d.b 0.01 0.01 0.01 n.d.b n.d.b

aPlants have been transferred to soil after 28 days in MS media containing 10-6 M GA3. bNo dilution of internal standard. cNot de-

termined.


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30

GA12-ald.

GA15

GA24

GA9

GA34

GA12 GA53

GA44

GA19

GA20

GA8

GA1GA4

GA25 GA17

GA14-ald. c

a

GA14

c

GA37

GA36

c

c

ab

b

b

b

c

d

b

b

b

b

c

d

CHOCOOH

723

2013

Figure 1



Figure 2 31

S8.9 13.1 15.9

10.8 2.2

5.9

S1.3

WT

CmGA7ox lines CmGA3ox1 lines

WTa CmGA20ox line CmGA2ox1 lines

S5.5

B

C

S5.5

AS15.9 S17.7

S17.2 AS7.7 S12.9

S12.8 AS5.9

A

WT

CmGA3ox1 line S17.7

CmGA7ox line S12.8



Documents

Running head: Expression of pumpkin GA-oxidases in ...Dec 29, 2005 · Abeer Radi1, Theo Lange1, Tomoya Niki2, Masaji Koshioka2, and Maria João Pimenta Lange1 1Institut für Pflanzenbiologie