Modification of gibberellin production and plant development in Arabidopsis by sense and antisense expression of gibberellin 20-oxidase genes

Gibberellin (GA) 20-oxidase catalyses consecutive steps late in GA biosynthesis in plants. In Arabidopsis, the enzyme is encoded by a gene family of at least three members (AtGA20ox1, AtGA20ox2 and AtGA20ox3) with differential patterns of expression. The genes are regulated by feedback from bioactive GAs, suggesting that the enzymes may be involved in regulating GA biosynthesis. To investigate this, we produced transgenic Arabidopsis expressing sense or antisense copies of each of the GA 20-oxidase cDNAs. Over-expression of any of the cDNAs gave rise to seedlings with elongated hypocotyls; the plants flowered earlier than controls in both long and short days and were 25% taller at maturity. GA analysis of the vegetative rosettes showed a two- to threefold increase in the level of GA4, indicating that GA 20-oxidase normally limits bioactive GA levels. Plants expressing antisense copies of AtGA20ox1 had short hypocotyls and reduced rates of stem elongation. This was reflected in reduced levels of GA4 in both rosettes and shoot tips. In short days, flowering was delayed and the reduction in the rate of stem elongation was greater. Antisense expression of AtGA20ox2 had no apparent effects in long days, but stem growth in one transgenic line grown in short days was reduced by 20%. Expression of antisense copies of AtGA20ox3 had no visible effect, except for one transgenic line that had short hypocotyls. These results demonstrate that GA levels and, hence, plant growth and development can be modified by manipulation of GA 20-oxidase expression in transgenic plants.     

Expression of gibberellin 2-oxidase 4 from Arabidopsis under the control of a senescence-associated promoter results in a dominant semi-dwarf plant with normal flowering

Gibberellin (GA), a plant hormone, is involved in many aspects of plant growth and development both in vegetative and reproductive phases. GA2-oxidase plays a key role in the GA catabolic pathway to reduce bioactive GAs. We produced transgenic Arabidopsis plants expressing GA2-oxidase 4 (AtGA2ox4) under the control of a senescenceassociated promoter (SEN1). As we hypothesized, transgenic plants (SEN1::AtGA2ox4) exhibited a dominant semi-dwarf phenotype with a decrease of bioactive GAs (e.g., GA4 and GA1) up to two-fold compared to control plants. Application of bioactive GA3 resulted in increased shoot length, indicating that the GA signaling pathway functions normally in the SEN1::AtGA2ox4 plants. Expressions of other members of GA2-oxidase family, such as AtGA2ox1, AtGA2ox3, AtGA2ox6, and AtGA2ox8, were decreased slightly in the flower and silique tissues while GA biosynthetic genes (e.g., AtGA20ox1, AtGA20ox2 and AtGA3ox1) were not significantly changed in the SEN::AtGA2ox4 plants. Using proteome profiling (2-D PAGE followed by MALDI-TOF/MS), we identified 29 protein spots that were increased in the SEN1::AtGA2ox4 plants, but were decreased to wild-type levels by GA3 treatment. The majority were found to be involved in photosynthesis and carbon/energy metabolism. Unlike the previous constitutive over-expression of GA2-oxidases, which frequently led to floral deformity and/or loss of fertility, the SEN1::AtGA2ox4 plants retained normal floral morphology and seed production. Accordingly, the expressions of FT and CO genes remained unchanged in the SEN1::AtGA2ox4 plants. Taken together, our results suggest that the dominant dwarf trait carried by SEN1::AtGA2ox4 plants can be used as an efficient dwarfing tool in plant biotechnological applications.     

Contribution of gibberellin deactivation by AtGA2ox2 to the suppression of germination of dark-imbibed Arabidopsis thaliana seeds

Gibberellin levels in imbibed Arabidopsis thaliana seeds are regulated by light via phytochrome, presumably through regulation of gibberellin biosynthesis genes, AtGA3ox1 and AtGA3ox2, and a deactivation gene, AtGA2ox2. Here, we show that a loss-of-function ga2ox2 mutation causes an increase in GA(4) levels and partly suppresses the germination inability during dark imbibition after inactivation of phytochrome. Experiments using 2,2-dimethylGA(4), a GA(4) analog resistant to gibberellin 2-oxidase, in combination with ga2ox2 mutant seeds suggest that the efficiency of deactivation of exogenous GA(4) by AtGA2ox2 is dependent on light conditions, which partly explains phytochrome-mediated changes in gibberellin effectiveness (sensitivity) found in previous studies.     

Expression of gibberellin 20-oxidase1 (AtGA20ox1) in Arabidopsis seedlings with altered auxin status is regulated at multiple levels

Bioactive gibberellins (GAs) affect many biological processes including germination, stem growth, transition to flowering, and fruit development. The location, timing, and level of bioactive GA are finely tuned to ensure that optimal growth and development occur. The balance between GA biosynthesis and deactivation is controlled by external factors such as light and by internal factors that include auxin. The role of auxin transport inhibitors (ATIs) and auxins on GA homeostasis in intact light-grown Arabidopsis thaliana (L.) Heynh. seedlings was investigated. Two ATIs, 1-N-naphthylthalamic acid (NPA) and 1-naphthoxyacetic acid (NOA) caused elevated expression of the GA biosynthetic enzyme AtGA20-oxidase1 (AtGA20ox1) in shoot but not in root tissues, and only at certain developmental stages. It was investigated whether enhanced AtGA20ox1 gene expression was a consequence of altered flow through the GA biosynthetic pathway, or was due to impaired GA signalling that can lead to enhanced AtGA20ox1 expression and accumulation of a DELLA protein, Repressor of ga1-3 (RGA). Both ATIs promoted accumulation of GFP-fused RGA in shoots and roots, and this increase was counteracted by the application of GA(4). These results suggest that in ATI-treated seedlings the impediment to DELLA protein degradation may be a deficiency of bioactive GA at sites of GA response. It is proposed that the four different levels of AtGA20ox1 regulation observed here are imposed in a strict hierarchy: spatial (organ-, tissue-, cell-specific) > developmental > metabolic > auxin regulation. Thus results show that, in intact auxin- and auxin transport inhibitor-treated light-grown Arabidopsis seedlings, three other levels of regulation supersede the effects of auxin on AtGA20ox1.     

Distinct cell-specific expression patterns of early and late gibberellin biosynthetic genes during Arabidopsis seed germination

Gibberellins (GAs) are biosynthesized through a complex pathway that involves several classes of enzymes. To predict sites of individual GA biosynthetic steps, we studied cell type-specific expression of genes encoding early and late GA biosynthetic enzymes in germinating Arabidopsis seeds. We showed that expression of two genes, AtGA3ox1 and AtGA3ox2, encoding GA 3-oxidase, which catalyzes the terminal biosynthetic step, was mainly localized in the cortex and endodermis of embryo axes in germinating seeds. Because another GA biosynthetic gene, AtKO1, coding for ent-kaurene oxidase, exhibited a similar cell-specific expression pattern, we predicted that the synthesis of bioactive GAs from ent-kaurene oxidation occurs in the same cell types during seed germination. We also showed that the cortical cells expand during germination, suggesting a spatial correlation between GA production and response. However, promoter activity of the AtCPS1 gene, responsible for the first committed step in GA biosynthesis, was detected exclusively in the embryo provasculature in germinating seeds. When the AtCPS1 cDNA was expressed only in the cortex and endodermis of non-germinating ga1-3 seeds (deficient in AtCPS1) using the AtGA3ox2 promoter, germination was not as resistant to a GA biosynthesis inhibitor as expression in the provasculature. These results suggest that the biosynthesis of GAs during seed germination takes place in two separate locations with the early step occurring in the provasculature and the later steps in the cortex and endodermis. This implies that intercellular transport of an intermediate of the GA biosynthetic pathway is required to produce bioactive GAs.     

Overexpression of a novel class of gibberellin 2-oxidases decreases gibberellin levels and creates dwarf plants

Degradation of active C(19)-gibberellins (GAs) by dioxygenases through 2beta-hydroxylation yields inactive GA products. We identified two genes in Arabidopsis (AtGA2ox7 and AtGA2ox8), using an activation-tagging mutant screen, that encode 2beta-hydroxylases. GA levels in both activation-tagged lines were reduced significantly, and the lines displayed dwarf phenotypes typical of mutants with a GA deficiency. Increased expression of either AtGA2ox7 or AtGA2ox8 also caused a dwarf phenotype in tobacco, indicating that the substrates for these enzymes are conserved. AtGA2ox7 and AtGA2ox8 are more similar to each other than to other proteins encoded in the Arabidopsis genome, indicating that they may constitute a separate class of GA-modifying enzymes. Indeed, enzymatic assays demonstrated that AtGA2ox7 and AtGA2ox8 both perform the same GA modification: 2beta-hydroxylation of C(20)-GAs but not of C(19)-GAs. Lines containing increased expression of AtGA2ox8 exhibited a GA dose-response curve for stem elongation similar to that of the biosynthetic mutant ga1-11. Double loss-of-function Atga2ox7 Atga2ox8 mutants had twofold to fourfold higher levels of active GAs and displayed phenotypes associated with excess GAs, such as early bolting in short days, resistance to the GA biosynthesis inhibitor ancymidol, and decreased mRNA levels of AtGA20ox1, a gene in the GA biosynthetic pathway.     

AGF1, an AT-hook protein, is necessary for the negative feedback of AtGA3ox1 encoding GA 3-oxidase

Negative feedback is a fundamental mechanism of organisms to maintain the internal environment within tolerable limits. Gibberellins (GAs) are essential regulators of many aspects of plant development, including seed germination, stem elongation, and flowering. GA biosynthesis is regulated by the feedback mechanism in plants. GA 3-oxidase (GA3ox) catalyzes the final step of the biosynthetic pathway to produce the physiologically active GAs. Here, we found that only the AtGA3ox1 among the AtGA3ox family of Arabidopsis (Arabidopsis thaliana) is under the regulation of GA-negative feedback. We have identified a cis-acting sequence responsible for the GA-negative feedback of AtGA3ox1 using transgenic plants. Furthermore, we have identified an AT-hook protein, AGF1 (for the AT-hook protein of GA feedback regulation), as a DNA-binding protein for the cis-acting sequence of GA-negative feedback. The mutation in the cis-acting sequence abolished both GA-negative feedback and AGF1 binding. In addition, constitutive expression of AGF1 affected GA-negative feedback in Arabidopsis. Our results suggest that AGF1 plays a role in the homeostasis of GAs through binding to the cis-acting sequence of the GA-negative feedback of AtGA3ox1.     

与AtGA20ox1启动子结合的转录因子筛选分析

赤霉素(gibberellin,GA)在植物生长发育的各个时期发挥重要作用。GA20-氧化酶(Gibberellin20-oxidase,GA20ox)是赤霉素生物合成途径中关键的限速酶,因此研究调控GA20ox基因表达的转录因子对进一步阐述赤霉素生物合成及其调控具有重要意义。本研究通过酵母单杂交技术利用AtGA20ox1启动子筛选拟南芥转录因子库,筛选获得转录因子RAP2.4f;酵母单杂交和X-gal显色结果进一步证实RAP2.4能与AtGA20ox1启动子结合;CPRG定量分析发现RAP2.4f与AtGA20ox1启动子结合作用强;双荧光素酶检测结果显示RAP2.4f对AtGA20ox1的启动子活性具有抑制作用。这些研究结果表明,RAP2.4f可能参与调控AtGA20ox1的转录。     

The involvement of gibberellin 20-oxidase genes in phytochrome-regulated petiole elongation of Arabidopsis

Long day (LD) exposure of rosette plants causes rapid stem/petiole elongation, a more vertical growth habit, and flowering; all changes are suggestive of a role for the gibberellin (GA) plant growth regulators. For Arabidopsis (Arabidopsis thaliana) L. (Heynh), we show that enhancement of petiole elongation by a far-red (FR)-rich LD is mimicked by a brief (10 min) end-of-day (EOD) FR exposure in short day (SD). The EOD response shows red (R)/FR photoreversibility and is not affected in a phytochrome (PHY) A mutant so it is mediated by PHYB and related PHYs. FR photoconversion of PHYB to an inactive form activates a signaling pathway, leading to increased GA biosynthesis. Of 10 GA biosynthetic genes, expression of the 20-oxidase, AtGA20ox2, responded most to FR (up to a 40-fold increase within 3 h). AtGA20ox1 also responded but to a lesser extent. Stimulation of petiole elongation by EOD FR is reduced in a transgenic AtGA20ox2 hairpin gene silencing line. By contrast, it was only in SD that a T-DNA insertional mutant of AtGA20ox1 (ga5-3) showed reduced response. Circadian entrainment to a daytime pattern provides an explanation for the SD expression of AtGA20ox1. Conversely, the strong EOD/LD FR responses of AtGA20ox2 may reflect its independence of circadian regulation. While FR acting via PHYB increases expression of AtGA20ox2, other GA biosynthetic genes are known to respond to R rather than FR light and/or to other PHYs. Thus, there must be different signal transduction pathways, one at least showing a positive response to active PHYB and another showing a negative response.     

Potential sites of bioactive gibberellin production during reproductive growth in Arabidopsis

Gibberellin 3-oxidase (GA3ox) catalyzes the final step in the synthesis of bioactive gibberellins (GAs). We examined the expression patterns of all four GA3ox genes in Arabidopsis thaliana by promoter-beta-glucuronidase gene fusions and by quantitative RT-PCR and defined their physiological roles by characterizing single, double, and triple mutants. In developing flowers, GA3ox genes are only expressed in stamen filaments, anthers, and flower receptacles. Mutant plants that lack both GA3ox1 and GA3ox3 functions displayed stamen and petal defects, indicating that these two genes are important for GA production in the flower. Our data suggest that de novo synthesis of active GAs is necessary for stamen development in early flowers and that bioactive GAs made in the stamens and/or flower receptacles are transported to petals to promote their growth. In developing siliques, GA3ox1 is mainly expressed in the replums, funiculi, and the silique receptacles, whereas the other GA3ox genes are only expressed in developing seeds. Active GAs appear to be transported from the seed endosperm to the surrounding maternal tissues where they promote growth. The immediate upregulation of GA3ox1 and GA3ox4 after anthesis suggests that pollination and/or fertilization is a prerequisite for de novo GA biosynthesis in fruit, which in turn promotes initial elongation of the silique.     

Dissection of GA 20-oxidase members affecting tomato morphology by RNAi-mediated silencing

GA 20-oxidase is a key enzyme involved in gibberellin (GA) biosynthesis. In tomato, the GA 20-oxidase gene family consists of three members: GA20ox1, GA20ox2, and GA20ox3. To investigate the roles of these three genes in regulating plant growth and development, we used RNA interference technology to generate three kinds of transgenic tomato plants with suppressed expression of each three individual genes. Suppression of GA20ox1 or GA20ox2 resulted in shorter stems, a decreased length of internodes, and small dark green leaves while plants with decreased expression of GA20ox3 had no visible changes on stems and leaves. The plants of the three transgenic lines can flower and set fruits normally, but the seeds from these plants germinated slower than that from the normal plants. Decreased levels of endogenous GAs were detected in the apex of the three transgenic lines. These results demonstrate that the three GA 20-oxidase genes play different roles in the control of plan vegetative growth, but show no effects on flower and fruit development.Equal contribution authors: J. Xiao and H. Li.     

拟南芥AtGA3OX1和AtGA3OX2基因影响茎秆次生细胞壁增厚的分子机理

赤霉素不仅对植物的种子萌发、叶片伸展和开花结果有重要的影响, 而且在茎秆的发育过程中扮演关键的角色。它的生物合成受到多种酶的调控, 其中赤霉素3-氧化酶(GA3OX)是关键的限速酶, 备受重视。拟南芥AtGA3OX 基因由4个成员组成, 其中A3OX1 和 AtGA3OX2 基因在茎中超量表达, 可能与茎的发育有关。目前, 尚未见到AtGA3OX1、AtGA3OX2基因调控次生细胞壁增厚的报道。文章以拟南芥AtGA3OX1 和 AtGA3OX2 基因双突变体atga3ox1atga3ox2为材料, 系统研究了AtGA3OX1和AtGA3OX2 基因对次生细胞壁的影响。结果表明：同时突变 AtGA3OX1和AtGA3OX2基因不仅显著抑制了茎秆次生细胞壁纤维细胞的增厚(对导管细胞没有影响), 而且也明显降低了次生细胞壁3个组分(纤维素、半纤维素和木质素)的含量。利用实时荧光定量PCR (qRT-PCR) 进一步分析次生细胞壁3个组分生物合成基因及相关的转录因子的表达情况, 结果显示这些基因在双突变体中均受到显著影响, 表明拟南芥AtGA3OX1和 AtGA3OX2 基因可能是通过调控这些转录因子进而调控了次生细胞壁的加厚。研究结果为基因工程调控拟南芥AtGA3OX1、AtGA3OX2 基因(或其他物种同源基因), 进而增强粮食作物抗倒伏性和提高能源植物纤维生物质量提供了理论依据。     

Ectopic expression of pumpkin gibberellin oxidases alters gibberellin biosynthesis and development of transgenic Arabidopsis plants

Immature pumpkin (Cucurbita maxima) seeds contain gibberellin (GA) oxidases with unique catalytic properties resulting in GAs of unknown function for plant growth and development. Overexpression of pumpkin GA 7-oxidase (CmGA7ox) in Arabidopsis (Arabidopsis thaliana) resulted in seedlings with elongated roots, taller plants that flower earlier with only a little increase in bioactive GA4 levels compared to control plants. In the same way, overexpression of the pumpkin GA 3-oxidase1 (CmGA3ox1) resulted in a GA overdose phenotype with increased levels of endogenous 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, overexpression 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 unexpected GA34 levels. Severe dwarfed plants were obtained by overexpression of the pumpkin GA 2-oxidase1 (CmGA2ox1) in Arabidopsis. This dramatic change in phenotype 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-encoding genes to modulate the GA plant hormone pool and alter plant stature and development.     

Where do gibberellin biosynthesis and gibberellin signaling occur in rice plants?

To identify where gibberellin (GA) biosynthesis and signaling occur, we analyzed the expression of four genes involved in GA biosynthesis, GA 20-oxidase1 and GA 20-oxidase2 (OsGA20ox1 and OsGA20ox2), and GA 3-oxidase1 and GA 3-oxidase2 (OsGA3ox1 and OsGA3ox2), and two genes involved in GA signaling, namely, the gene encoding the alpha-subunit of the heterotrimeric GTP-binding protein (Galpha), and SLENDER RICE1 (SLR1), which encodes a repressor of GA signaling. At the vegetative stage, the expression of OsGA20ox2, OsGA3ox2, Galpha, and SLR1 was observed in rapidly elongating or dividing organs and tissues, whereas the expression of OsGA20ox1 or OsGA3ox1 could not be detected. At the inflorescence or floral stage, the expression of OsGA20ox2, OsGA3ox2, Galpha, and SLR1 was also observed in the shoot meristems and stamen primordia. The overlapping expression of genes for GA biosynthesis and signaling indicates that in these tissues and organs, active GA biosynthesis occurs at the same site as does GA signaling. In contrast, no GA-biosynthesis genes were expressed in the aleurone cells of the endosperm; however, the two GA-signaling genes were actively expressed, indicating that the aleurone does not produce bioactive GAs, but can perceive GAs. The expression of OsGA20ox1 and OsGA3ox1 was observed only in the epithelium of the embryo and the tapetum of the anther. Based on the specific expression pattern of OsGA20ox1 and OsGA3ox1 in these tissues, we discuss the unique nature of the epithelium and the tapetum in terms of GA biosynthesis. The epithelium and the tapetum are considered to be an important source of bioactive GAs for aleurone and other organs of the flower, respectively.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.