Molecular cloning and photoperiod-regulated expression of gibberellin 20-oxidase from the long-day plant spinach.

Spinach (Spinacia oleracea L.) is a long-day (LD) rosette plant in which stem growth under LD conditions is mediated by gibberellins (GAs). Major control points in spinach are the later steps of sequential oxidation and elimination of C-20 of C20-GAs. Degenerate oligonucleotide primers were used to obtain a polymerase chain reaction product from spinach genomic DNA that has a high homology with GA 20-oxidase cDNAs from Cucurbita maxima L. and Arabidopsis thaliana Heynh. This polymerase chain reaction product was used as a probe to isolate a full-length cDNA clone with an open reading frame encoding a putative 43-kD protein of 374 amino acid residues. When this cDNA clone was expressed in Escherichia coli, the fusion protein catalyzed the biosynthetic sequence GA53-->GA44-->GA19-->GA20 and GA19-->GA17. This establishes that in spinach a single protein catalyzes the oxidation and elimination of C-20. Transfer of spinach plants from short day (SD) to LD conditions caused an increase in the level of all GAs of the early-13-hydroxylation pathway, except GA53, with GA20, GA1, and GA8 showing the largest increases. Northern blot analysis indicated that the level of GA 20-oxidase mRNA was higher in plants in LD than in SD conditions, with highest level of expression in the shoot tips and elongating stems. This expression pattern of GA 20-oxidase is consistent with the different levels of GA20, GA1, and GA8 found in spinach plants grown in SD and LD conditions.     

Feedback regulation of GA5 expression and metabolic engineering of gibberellin levels in Arabidopsis.

The gibberellin (GA) 20-oxidase encoded by the GA5 gene of Arabidopsis directs GA biosynthesis to active GAs, whereas that encoded by the P16 gene of pumpkin endosperm leads to biosynthesis of inactive GAs. Negative feedback regulation of GA5 expression was demonstrated in stems of Arabidopsis by bioactive GAs but not by inactive GA. In transgenic Arabidopsis plants overexpressing P16, there was a severe reduction in the amounts of C20-GA intermediates, accumulation of large amounts of inactive GA25 and GA17, a reduction in GA4 content, and a small increase in GA1. However, due to feedback regulation, expression of GA5 and GA4, the gene coding for the subsequent 3beta-hydroxylase, was greatly increased to compensate for the effects of the P16 transgene. Consequently, stem height was only slightly reduced in the transgenic plants.     

Expression of a gibberellin 2-oxidase gene around the shoot apex is related to phase transition in rice

A major catabolic pathway for gibberellin (GA) is initiated by 2beta-hydroxylation, a reaction catalyzed by GA 2-oxidase. We have isolated and characterized a cDNA, designated Oryza sativa GA 2-oxidase 1 (OsGA2ox1) from rice (Oryza sativa L. cv Nipponbare) that encodes a GA 2-oxidase. The encoded protein, produced by heterologous expression in Escherichia coli, converted GA(1), GA(4), GA(9), GA(20), and GA(44) to the corresponding 2beta-hydroxylated products GA(8), GA(34), GA(51), GA(29), and GA(98), respectively. Ectopic expression of the OsGA2ox1 cDNA in transgenic rice inhibited stem elongation and the development of reproductive organs. These transgenic plants were deficient in endogenous GA(1). These results indicate that OsGA2ox1 encodes a GA 2-oxidase, which is functional not only in vitro but also in vivo. OsGA2ox1 was expressed in shoot apex and roots but not in leaves and stems. In situ hybridization analysis revealed that OsGA2ox1 mRNA was localized in a ring at the basal region of leaf primordia and young leaves. This ring-shaped expression around the shoot apex was drastically decreased after the phase transition from vegetative to reproductive growth. It was absent in the floral meristem, but it was still present in the lateral meristem that remained in the vegetative phase. These observations suggest that OsGA2ox1 controls the level of bioactive GAs in the shoot apical meristem; therefore, reduction in its expression may contribute to the early development of the inflorescence meristem.     

Gibberellins and the photoperiodic control of stem elongation in the long-day plant Agrostemma githago L.

Agrostemma githago is a long-day rosette plant in which transfer from short days (SD) to long days (LD) results in rapid stem elongation, following a lag phase of 7–8 d. Application of gibberellin A₂₀ (GA₂₀) stimulated stem elongation in plants under SD, while 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride (AMO-1618, an inhibitor of GA biosynthesis) inhibited stem elongation in plants exposed to LD. This inhibition of stem elongation by AMO-1618 was overcome by simultaneous application of GA₂₀, indicating that GAs play a role in the photoperiodic control of stem elongation in this species. Endogenous GA-like substances were analyzed using reverse-phase high-performance liquid chromatography and the d-5 corn (Zea mays L.) assay. Three zones with GA-like activity were detected and designated, in order of decreasing polarity, as A, B, and C. A transient, 10-fold increase in the activity of zone B occurred after 8–10 LD, coincident with the transition from lag phase to the phase of rapid stem elongation. After 16 LD the activity in this zone had returned to a level similar to that under SD, even though the plants were elongating rapidly by this time. However, when AMO-1618 was applied to plants after 11 LD, there was a rapid reduction in the rate of stem elongation, indicating that continued GA biosynthesis was necessary following the transient increase in activity of zone B, if stem elongation was to continue under LD. It was concluded that control of stem elongation in A. githago involves more than a simple qualitative or quantitative change in the levels of endogenous GAs, and that photoperiodic induction alters both the sensitivity to GAs and the rate of turnover of endogenous GAs.Abbreviations AMO-1618 2-isopropyl-4-dimethylamino-5-methylphenyl-1-piperidine-carboxylate methyl chloride - GA(s) gibberellin(s) - LD long day(s) - LDP long-day plant(s) - SD short day(s)     

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.     

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.     

Changes in GA 20-oxidase gene expression strongly affect stem length, tuber induction and tuber yield of potato plants

Gene StGA20ox1 encoding potato GA 20-oxidase is expressed to relatively high levels in leaves and regulated by daylength. To investigate whether this gene is involved in photoperiodic regulation of tuber formation, we have obtained transgenic potato plants expressing sense and antisense copies of the StGA20ox1 cDNA. Over-expression of this cDNA resulted in taller plants that required a longer duration of a short day photoperiod (SD) to tuberize. Tubers from these plants had a decreased time of dormancy and developed sprouts with elongated internodes. Plants expressing antisense copies of the StGA20ox1 cDNA had shorter stems, a decreased length of the internodes and tuberized earlier than control plants, showing increased tuber yields. Antisense inhibition of this gene had no visible effect on the time of dormancy of the tubers, although at the end of dormancy these formed sprouts with shortened internodes. Decreased levels of endogenous GA20 and GA1 were detected in the apex and first leaves of the antisense lines. These results demonstrate the involvement of the GA 20-oxidase activity encoded by StGA20ox1 in the control of stem elongation and in tuber induction but not in tuber dormancy, indicating that the latter may be regulated by another member of the gene family.     

Overexpression of 20-Oxidase Confers a Gibberellin-Overproduction Phenotype in Arabidopsis

In the gibberellin (GA) biosynthesis pathway, 20-oxidase catalyzes the oxidation and elimination of carbon-20 to give rise to C₁₉-GAs. All bioactive GAs are C₁₉-GAs. We have overexpressed a cDNA encoding 20-oxidase isolated from Arabidopsis seedlings in transgenic Arabidopsis plants. These transgenic plants display a phenotype that may be attributed to the overproduction of GA. The phenotype includes a longer hypocotyl, lighter-green leaves, increased stem elongation, earlier flowering, and decreased seed dormancy. However, the fertility of the transgenic plants is not affected. Increased levels of endogenous GA₁, GA₉, and GA₂₀ were detected in seedlings of the transgenic line examined. GA₄, which is thought to be the predominantly active GA in Arabidopsis, was not present at increased levels in this line. These results suggest that the overexpression of this 20-oxidase increases the levels of some endogenous GAs in transgenic seedlings, which causes the GA-overproduction phenotype.     

Gibberellins and the photoperiodic control of leaf growth in Poa pratensis

The role of gibberellins (GAs) in photoperiodic control of leaf elongation in Poa pratensis was studied by both application of exogenous GAs and analysis of endogenous GAs. Leaf elongation was strongly increased under long day (LD, 24 h) conditions at both 9 and 21°C, leaf length at 9°C LD being similar to that in plants grown in short days (SD, 8 h) at 21°C. However, even at 21°C leaf elongation was enhanced by LD. Exogenous GA₁ could completely compensate for LD at both 9 and 21°C. Gibberellins A₂₀, A₁₉ and A₄₄ could also partly replace LD, but they were significantly less active than GA₁, GA₅₃ was inactive when applied to plants grown at 9°C in SD and exhibited only marginal activity at 9°C LD and 21°C SD. The total level of GAs of the early 13-hydroxylation pathway (A₅₃, A₄₄, A₁₉, A₂₀ and A₁) increased rapidly when plants were transferred from SD to LD at 9°C. After transfer from 9 to 21°C, there was an increase in GA levels at both LD and SD, followed by a decrease under LD conditions. In all cases, GA₁₉ was the predominant GA, accounting for 60 to 80% of the analysed GAs. Levels of the bioactive GA₁ were low and increased transiently by LD four days after transfer from SD to LD. At both temperatures, the ratio GA₁₉ to GA₂₀ and GA₂₀ to GA₁ at 9°C was enhanced by LD compared with SD. Taken together, these results support the hypothesis that photoperiodic regulation of leaf elongation in Poa pratensis is GA-mediated, and they indicate a photoperiodic control of oxidation of GA₅₃ to GA₄₄ and GA₁₉ to GA₂₀, and perhaps also of 3β-hydroxylation of GA₂₀ to GA₁.     

Gibberellins and Stem Growth as Related to Photoperiod in Silene armeria L

Stem growth and flowering in the long-day plant Silene armeria L. are induced by exposure to a minimum of 3 to 6 long days (LD). Stem growth continues in subsequent short days (SD), albeit at a reduced rate. The growth retardant tetcyclacis inhibited stem elongation induced by LD, but had no effect on flowering. This indicates that photoperiodic control of stem growth in Silene is mediated by gibberellins (GA). The objective of this study was to analyze the effects of photoperiod on the levels and distribution of endogenous GAs in Silene and to determine the nature of the photoperiodic after-effect on stem growth in this plant. The GAs identified in extracts from Silene by full-scan combined gas chromatography-mass spectrometry (GC-MS), GA₁₂, GA₅₃, GA₄₄, GA₁₇, GA₁₉, GA₂₀, GA₁, GA₂₉, and GA₈, are members of the early 13-hydroxylation pathway. All of these GAs were present in plants under SD as well as under LD conditions. The GA₅₃ level was highest in plants in SD, and decreased in plants transferred to LD conditions. By contrast, GA₁₉, GA₂₀, and GA₁ initially increased in plants transferred to LD, and then declined. Likewise, when Silene plants were returned from LD to SD, there was an increase in GA₅₃, and a decrease in GA₁₉, GA₂₀, and GA₁ which ultimately reached levels similar to those found in plants kept in SD. Thus, measurements of GA levels in whole shoots of Silene as well as in individual parts of the plant suggest that the photoperiod modulates GA metabolism mainly through the rate of conversion of GA₅₃. As a result of LD induction, GA₁ accumulates at its highest level in shoot tips which, in turn, results in stem elongation. In addition, LD also appear to increase the sensitivity of the tissue to GA, and this effect is presumably responsible for the photoperiodic after-effect on stem elongation in Silene.     

Vernalization and Gibberellin Physiology of Winter Canola (Endogenous Gibberellin (GA) Content and Metabolism of [3H]GA1 and [3H]GA20

Winter canola (Brassica napus cv Crystal) is an oilseed crop that requires vernalization (chilling treatment) for the induction of stem elongation and flowering. To investigate the role of gibberellins (GAs) in vernalization-induced events, endogenous GA content and the metabolism of [3H]GAs were examined in 10-week vernalized and nonvernalized plants. Shoot tips were harvested 0, 8, and 18 d postvernalization (DPV), and GAs were purified and quantified using 2H2-internal standards and gas chromatography-selected ion monitoring. Concentrations of GA1, GA3, GA8, GA19, and GA20 were 3.1-, 2.3-, 7.8-, 12.0-, and 24.5-fold higher, respectively, in the vernalized plants at the end of the vernalization treatment (0 DPV) relative to the nonvernalized plants. Thermoregulation apparently occurs prior to GA19 biosynthesis, since vernalization elevated the concentration of all of the monitored GAs. [3H]GA20 or [3H]GA1 was applied to the shoot tips of vernalized and nonvernalized plants, and after 24 h, plants were harvested at 6, 12, and 15 DPV. Following high-performance liquid chromatography analyses, vernalized plants showed increased conversion of [3H]GA20 to a [3H]GA1-like metabolite and reduced conversion of [3H]GA1 or [3H]GA20 to polar 3H-metabolites, putative glucosyl conjugates. These results demonstrate that vernalization influences GA content and GA metabolism, with GAs serving as probable regulatory intermediaries between chilling treatment and subsequent stem growth.     

棉花GA 20-氧化酶基因转毛白杨的研究

以毛白杨为材料,研究了超量表达赤霉素合成酶基因(GA20-氧化酶基因)对毛白杨根、茎和叶的生长以及组织结构的影响,结果显示,表达GA20-氧化酶基因能显著提高毛白杨茎的生长,但是对根系的生长带来负面影响.同时,超量表达GA20-氧化酶基因促进了毛白杨茎木质部的生长,抑制了韧皮部和皮层的生长.研究结果表明GA20-氧化酶基因在毛白杨遗传改良中具有一定的应用价值.     

Decreased GA1 Content Caused by the Overexpression of OSH1 Is Accompanied by Suppression of GA 20-Oxidase Gene Expression

We previously reported that overexpression of the rice homeobox gene OSH1 led to altered morphology and hormone levels in transgenic tobacco (Nicotiana tabacum L.) plants. Among the hormones whose levels were changed, GA₁ was dramatically reduced. Here we report the results of our analysis on the regulatory mechanism(s) of OSH1 on GA metabolism. GA₅₃ and GA₂₀, precursors of GA₁, were applied separately to transgenic tobacco plants exhibiting severely changed morphology due to overexpression of OSH1. Only treatment with the end product of GA 20-oxidase, GA₂₀, resulted in a striking promotion of stem elongation in transgenic tobacco plants. The internal GA₁ and GA₂₀ contents in OSH1-transformed tobacco were dramatically reduced compared with those of wild-type plants, whereas the level of GA₁₉, a mid-product of GA 20-oxidase, was 25% of the wild-type level. We have isolated a cDNA encoding a putative tobacco GA 20-oxidase, which is mainly expressed in vegetative stem tissue. RNA-blot analysis revealed that GA 20-oxidase gene expression was suppressed in stem tissue of OSH1-transformed tobacco plants. Based on these results, we conclude that overexpression of OSH1 causes a reduction of the level of GA₁ by suppressing GA 20-oxidase expression.     

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.     

The Control of Apical Bud Growth and Senescence by Auxin and Gibberellin in Genetic Lines of Peas

Pea (Pisum sativum L.) lines G2 (dwarf) and NGB1769 (tall) (Sn Hr) produce flowers and fruit under long (LD) or short (SD) days, but senesce only under LD. Endogenous gibberellin (GA) levels were inversely correlated with photoperiod (over 9-18 h) and senescence: GA20 was 3-fold and GA1 was 10- to 11-fold higher in flowering SD G2 shoots, and the vegetative tissues within the SD apical bud contained 4-fold higher levels of GA20, as compared with the LD tissues. Prefloral G2 plants under both photoperiods had GA1 and GA20 levels similar to the flowering plants under LD. Levels of indole-3-acetic acid (IAA) were similar in G2 shoots in LD or SD; SD apical bud vegetative tissues had a slightly higher IAA content. Young floral buds from LD plants had twice as much IAA as under SD. In NGB1769 shoots GA1 decreased after flower initiation only under LD, which correlated with the decreased growth potential. We suggest that the higher GA1 content of G2 and NGB1769 plants under SD conditions is responsible for the extended vegetative growth and continued meristematic activity in the shoot apex. This and the increased IAA level of LD floral buds may play a role in the regulation of nutrient partitioning, since more photosynthate partitions of reproductive tissue under LD conditions, and the rate of reproductive development in LD peas is faster than under SD.     

Regulation of flowering in the long-day grass Lolium temulentum by gibberellins and the FLOWERING LOCUS T gene

Seasonal control of flowering often involves leaf sensing of daylength coupled to time measurement and generation and transport of florigenic signals to the shoot apex. We show that transmitted signals in the grass Lolium temulentum may include gibberellins (GAs) and the FLOWERING LOCUS T (FT) gene. Within 2 h of starting a florally inductive long day (LD), expression of a 20-oxidase GA biosynthetic gene increases in the leaf; its product, GA(20), then increases 5.7-fold versus short day; its substrate, GA(19), decreases equivalently; and a bioactive product, GA(5), increases 4-fold. A link between flowering, LD, GAs, and GA biosynthesis is shown in three ways: (1) applied GA(19) became florigenic on exposure to LD; (2) expression of LtGA20ox1, an important GA biosynthetic gene, increased in a florally effective LD involving incandescent lamps, but not with noninductive fluorescent lamps; and (3) paclobutrazol, an inhibitor of an early step of GA biosynthesis, blocked flowering, but only if applied before the LD. Expression studies of a 2-oxidase catabolic gene showed no changes favoring a GA increase. Thus, the early LD increase in leaf GA(5) biosynthesis, coupled with subsequent doubling in GA(5) content at the shoot apex, provides a substantial trail of evidence for GA(5) as a LD florigen. LD signaling may also involve transport of FT mRNA or protein because expression of LtFT and LtCONSTANS increased rapidly, substantially (>80-fold for FT), and independently of GA. However, because a LD from fluorescent lamps induced LtFT expression but not flowering, the nature of the light response of FT requires clarification.