Metabolism and Biological Activity of Gibberellin A4 in Vegetative Shoots of Zea mays,Oryza sativa,and Arabidopsis thaliana

[17-13C,3H]Gibberellin A4 (GA4) was injected into the shoots of tall (W23/L317), dwarf-1 (d1), and dwarf-5 (d5) Zea mays L. (maize); tall (cv Nipponbare), dwarf-x (dx), and dwarf-y (dy) Oryza sativa L. (rice); and tall (ecotype Landsberg erecta), ga4, and ga5 Arabidopsis thaliana (L.) Heynh. [13C]GA4 and its metabolites were identified from the shoots by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA4 was metabolized to GA1 in all nine genotypes. GA4 was also metabolized in some of the genotypes to 3-epi-GA1, GA2, 2[beta]-OH-GA2, 3-epi-GA2, endo-GA4, 16[alpha], 17-H2-16, 17-(OH)2-GA4, GA34, endo-GA34, GA58, 15-epi-GA63, GA71, and 16-epi-GA82. No evidence was found for the metabolism of GA4 to GA7 or of GA4 to GA3. The bioactivities of GA4 and GA1 were determined using the six dwarf mutants for assay. GA4 and GA1 had similar activities for the maize and rice mutants. For the Arabidopsis mutants, GA4 was more active than GA1 at low dosages; GA4 was less active than GA1 at higher dosages.     

Azospirillum brasilense and Azospirillum lipoferum hydrolyze conjugates of GA20 and metabolize the resultant aglycones to GA1 in seedlings of rice dwarf mutants

Azospirillum species are plant growth-promotive bacteria whose beneficial effects have been postulated to be partially due to production of phytohormones, including gibberellins (GAs). In this work, Azospirillum brasilense strain Cd and Azospirillum lipoferum strain USA 5b promoted sheath elongation growth of two single gene GA-deficient dwarf rice (Oryza sativa) mutants, dy and dx, when the inoculated seedlings were supplied with [17,17-2H2]GA20-glucosyl ester or [17,17- 2H2]GA20-glucosyl ether. Results of capillary gas chromatography-mass spectrometry analysis show that this growth was due primarily to release of the aglycone [17,17-2H2]GA20 and its subsequent 3beta-hydroxylation to [17,17-2H2]GA1 by the microorganism for the dy mutant, and by both the rice plant and microorganism for the dx mutant.     

Gibberellin biosynthesis: metabolic evidence for three steps in the early 13-hydroxylation pathway of rice

[14C4]GA53, [14C4]GA44, and [2H2/14C4]GA19 were injected separately into seedlings of rice (Oryza sativa) using a dwarf mutant (d35) that has low levels of endogenous gibberellins (GAs). After 8 h incubation, the shoots were extracted and the labeled metabolites were identified by full-scan gas chromatography mass spectrometry (GC-MS) and Kovats retention indices (KRIs). Our results document the metabolic sequence, GA53-->GA44-->GA19-->GA20 and the presence of endogenous GA53, GA44, GA19, GA20 and GA1. Previous metabolic studies have shown the presence of the step, GA20-->GA1 in rice. Taken together, the data establish in vegetative shoots of rice the presence of the early 13-hydroxylation pathway, a pathway that originates from GA12 and leads to bioactive GA1.     

Gibberellin Metabolism in Maize (The Stepwise Conversion of Gibberellin A12-Aldehyde to Gibberellin A20

The stepwise metabolism of gibberellin A12-aldehyde (GA12-aldehyde) to GA20 is demonstrated from seedling shoots of maize (Zea mays L.). The labeled substrates [13C,3H]GA12-aldehyde, [13C,3H]GA12, [14C4]GA53, [14C4/2H2]GA44, and [14C4/2H2]GA19 were fed individually to dwarf-5 vegetative shoots. Both [13C,3H]GA12-aldehyde and [13C,3H]GA12 were also added individually to normal shoots. The labeled metabolites were identified by full-scan gas chromatography-mass spectrometry and Kovats retention indices. GA12-aldehyde was metabolized to GA53-aldehyde, GA12, GA53, GA44, and GA19; GA12 was metabolized to 2[beta]-hydroxy-GA12, GA53, 2[beta]-hydroxyGA53, GA44, 2[beta]-hydroxyGA44, and GA19; GA53 was metabolized to GA44, GA19, GA20, and GA1; GA44 was metabolized to GA19; and GA19 was metabolized to GA20. These results, together with previously published data from this laboratory, document the most completely defined gibberellin pathway for the vegetative tissues of higher plants.     

赤霉素和光对拟南芥种子萌发和幼苗生长的影响

以拟南芥的赤霉素 (GA)缺陷型突变体ga 1,ga 2 ,ga 3和GA不敏感型突变体ga i为材料 ,研究了光和 4种GA对拟南芥种子萌发和幼苗生长影响的相互关系。结果表明 :(1)烯效唑对ga i种子萌发的抑制在光下可明显被GA恢复 ,而在黑暗中GA的作用不明显。 (2 )在光下低浓度的外源GA3 可使ga 1,ga 2和ga 3的种子萌发 ,而在黑暗中同样浓度的GA3 则难以使种子萌发。 (3)光可以降低种子萌发所需求的GA的剂量。 (4 )ga i和ga 1的幼苗的呼吸代谢有明显差异。以上结果说明 :光对拟南芥种子萌发的促进主要是提高了种子对GA反应的敏感性而不是增加GA的生物合成     

Azospirillum spp. metabolize [17,17-2H2]gibberellin A20 to [17,17-2H2]gibberellin A1 in vivo in dy rice mutant seedlings

Azospirillum spp. are endophytic bacteria with beneficial effects on cereals--effects partially attributed to gibberellin production by the microorganisms. Azospirillum lipoferum and Azospirillum brasilense inoculated to rice dy mutant reversed dwarfism in seedlings incubated with [17,17-2H2]GA20 with formation of [17,17-2H2]GA1, showing the in vivo capacity to perform the 3beta-hydroxylation. When prohexadione-Ca, an inhibitor of late steps in gibberellin biosynthesis, was added to the culture medium, no complementation was observed and no [17,17-2H2]GA1 was produced. The latter suggests that the bacterial operating enzyme may be a 2-oxoglutarate-dependent dioxygenase, similar to those of plants.     

The New Rga Locus Encodes a Negative Regulator of Gibberellin Response in Arabidopsis Thaliana

We have identified a new locus involved in gibberellin (GA) signal transduction by screening for suppressors of the Arabidopsis thaliana GA biosynthetic mutant ga1-3. The locus is named RGA for repressor of ga1-3. Based on the recessive phenotype of the digenic rga/ga1-3 mutant, the wild-type gene product of RGA is probably a negative regulator of GA responses. Our screen for suppressors of ga1-3 identified 17 mutant alleles of RGA as well as 10 new mutant alleles at the previously identified SPY locus. The digenic (double homozygous) rga/ga1-3 mutants are able to partially repress several defects of ga1-3 including stem growth, leaf abaxial trichome initiation, flowering time, and apical dominance. The phenotype of the trigenic mutant (triple homozygous) rga/spy/ga1-3 shows that rga and spy have additive effects regulating flowering time, abaxial leaf trichome initiation and apical dominance. This trigenic mutant is similar to wild type with respect to each of these developmental events. Because rga/spy/ga1-3 is almost insensitive to GA for hypocotyl growth and its bolting stem is taller than the wild-type plant, the combined effects of the rga and spy mutations appear to allow GA-independent stem growth. Our studies indicate that RGA lies on a separate branch of the GA signal transduction pathway from SPY, which leads us to propose a modified model of the GA response pathway.     

能源植物小桐子赤霉素合成代谢及信号转导相关基因的鉴定及序列分析

赤霉素(gibberellin,GA)是一类非常重要的植物激素,在植物种子萌发、茎干伸长、叶片生长、腺毛发育、花粉成熟、开花诱导和果实成熟等生长发育过程中都发挥着重要的作用。GA在一年生草本植物中可以促进开花,而在大多数多年生木本植物中则抑制成花诱导。为了更好地研究赤霉素在木本油料能源植物小桐子(Jatropha curcas)开花调控方面的作用机理,我们对小桐子整个基因组中参与GA合成代谢和信号转导的全部基因进行了鉴定和序列分析。这些基因包括6个多基因家族编码的蛋白,即GA2氧化酶(GA2-oxidase,GA2ox)、GA3氧化酶(GA3-oxidase,GA3ox)、GA20氧化酶(GA20-oxidase,GA20ox)、GID1(GIBBERELLIN INSENSITIVE DWARF1)、DELLAs和F-box蛋白,以及2个单基因编码的蛋白,EL1(EARLY FLOWERING1)和SPY(SPINDLY)。采用拟南芥和水稻中已经鉴定的上述基因编码的蛋白序列在小桐子基因组序列数据库和本实验的小桐子转录组数据库中进行BLASTP分析,找到17个同源蛋白的全长序列,并将其与28个拟南芥的、16个水稻的、24个葡萄的和22个蓖麻的同源蛋白构建系统发育树进行比对分析。结果表明,小桐子中参与赤霉素合成代谢及信号转导的大多数基因与蓖麻和葡萄同源基因的相似度更高。     

The ELONGATED gene of Arabidopsis acts independently of light and gibberellins in the control of elongation growth

A novel elongated mutant has been isolated from EMS-mutagenized populations of the Arabidopsis thaliana ga4 mutant. After backcrossing with the Landsberg erecta ( Ler ) wild-type (WT) followed by selling, the mutant phenotype was identified in the GA4 background. Seedlings of the mutant, which has been named elg (elongated), are characterized by elongated hypocotyls and petioles, leaves that are narrow and somewhat epinastic and early flowering. Allelism tests with the hy1–hy5 mutants indicate that elg is not allelic with any of these long-hypocotyl mutants. From linkage analyses, the location of elg on chromosome 4, between cer2 and ap2 has been established. The pleiotropic phenotype of elg seedlings is suggestive of a disruption of phytochrome and/or gibberellin (GA) function. Although the elg mutant displays a light-dependent long-hypocotyl phenotype, elg seedlings retain a full range of photomorphogenic responses and the elg mutation acts additively with the photomorphogenic mutants phyB, hy1 and hy2 . This suggests that ELG acts independently of phytochrome action. The elg mutation partially suppresses the effect of GA-deficiency on elongation growth, and, although elg ga1 seedlings are more elongated than ga1 seedlings, both genotypes respond in the same way to applied GA. That applied GA and the elg mutation interact additively suggests that ELG acts independently of GA action.     

Gibberellin A(3) Is Biosynthesized from Gibberellin A(20) via Gibberellin A(5) in Shoots of Zea mays L

[17-¹³C,³H]-Labeled gibberellin A₂₀ (GA₂₀), GA₅, and GA₁ were fed to homozygous normal (+/+), heterozygous dominant dwarf (D8/+), and homozygous dominant dwarf (D8/D8) seedlings of Zea mays L. (maize). ¹³C-Labeled GA₂₉, GA₈, GA₅, GA₁, and 3-epi-GA₁, as well as unmetabolized [¹³C]GA₂₀, were identified by gas chromatography-selected ion monitoring (GC-SIM) from feeds of [17-¹³C, ³H]GA₂₀ to all three genotypes. ¹³C-Labeled GA₈ and 3-epi-G₁, as well as unmetabolized [¹³C]GA₁, were identified by GC-SIM from feeds of [17-¹³C, ³H]GA₁ to all three genotypes. From feeds of [17-¹³C, ³H]GA₅, ¹³C-labeled GA₃ and the GA₃-isolactone, as well as unmetabolized [¹³C]GA₅, were identified by GC-SIM from +/+ and D8/D8, and by full scan GC-MS from D8/+. No evidence was found for the metabolism of [17-¹³C, ³H]GA₅ to [¹³C]GA₁, either by full scan GC-mass spectrometry or by GC-SIM. The results demonstrate the presence in maize seedlings of three separate branches from GA₂₀, as follows: (a) GA₂₀ → GA₁ → GA₈; (b) GA₂₀ → GA₅ → GA₃; and (c) GA₂₀ → GA₂₉. The in vivo biogenesis of GA₃ from GA₅, as well as the origin of GA₅ from GA₂₀, are conclusively established for the first time in a higher plant (maize shoots).     

Decapitation Reduces the Metabolism of Gibberellin A20 to A1 in Pisum sativum L., Decreasing the Le/le Difference

When the metabolism of [13C,3H]gibberellin (GA)20 in Pisum sativum L. was investigated using decapitated plants and stem sections, no evidence was obtained for the recently postulated inhibitor of GA20 3[beta]-hydroxylase (V.A. Smith [1992] Plant Physiol 99: 372-377). Instead, the results are consistent with the hypothesis that the mutation le reduces GA1 production by altering the structure or level of the 3[beta]-hydroxylase.     

The Gibberellin Status of lip1, a Mutant of Pea That Exhibits Light-Independent Photomorphogenesis

Dark-grown seedlings of the lip1 (light independent photomorphogenesis) mutant of Pisum sativum L. display many features of de-etiolated growth and are similar in many respects to wild-type (WT) seedlings grown in the light. The involvement of gibberellins (GAs) with the mutant phenotype was examined by applying GA1 and GA20 to the mutant and WT, and by quantifying endogenous GA1, GA8, GA19, GA20, and GA29 levels in the two genotypes. These experiments were conducted in both the light and the dark. In neither environment could GA application restore elongation in the mutant to that in GA-treated WT plants. Quantification of GAs provided further evidence that the mutant phenotype is not attributable to a deficiency in endogenous GA1. However, dark-grown lip1 seedlings contained lower levels of GA19 and higher levels of GA20 than dark-grown WT plants, whereas in the light, the effect of the mutation on the ratio of GA19 to GA20 was reversed. Thus, there appears to be a complex interaction between the lip1 mutation, the light regime, and the step GA19 to GA20.     

Cryptochrome and Phytochrome Cooperatively but Independently Reduce Active Gibberellin Content in Rice Seedlings under Light Irradiation

In contrast to a wealth of knowledge about the photoregulation of gibberellin metabolism in dicots, that in monocots remains largely unclear. In this study, we found that a blue light signal triggers reduction of active gibberellin content in rice seedlings with simultaneous repression of two gibberellin 20-oxidase genes (OsGA20ox2 and OsGA20ox4) and acute induction of four gibberellin 2-oxidase genes (OsGA2ox4-OsGA2ox7). For further examination of the regulation of these genes, we established a series of cryptochrome-deficient lines through reverse genetic screening from a Tos17 mutant population and construction of knockdown lines based on an RNA interference technique. By using these lines and phytochrome mutants, we elucidated that cryptochrome 1 (cry1), consisting of two species in rice plants (cry1a and cry1b), is indispensable for robust induction of the GA2ox genes. On the other hand, repression of the GA20ox genes is mediated by phytochromes. In addition, we found that the phytochromes also mediate the repression of a gibberellin 3-oxidase gene (OsGA3ox2) in the light. These results imply that, in rice seedlings, phytochromes mediate the repression of gibberellin biosynthesis capacity, while cry1 mediates the induction of gibberellin inactivation capacity. The cry1 action was demonstrated to be dominant in the reduction of active gibberellin content, but, in rice seedlings, the cumulative effects of these independent actions reduced active gibberellin content in the light. This pathway design in which different types of photoreceptors independently but cooperatively regulate active gibberellin content is unique from the viewpoint of dicot research. This redundancy should provide robustness to the response in rice plants.     

Gibberellins and Gravitropism in Maize Shoots : Endogenous Gibberellin-Like Substances and Movement and Metabolism of [3H]Gibberellin A20

[³H]Gibberellin A₂₀ (GA₂₀) of high specific radioactivity (49.9 gigabecquerel per millimole) was applied equilaterally in a ring of microdrops to the internodal pulvinus of shoots of 3-week-old gravistimulated and vertical normal maize (Zea mays L.), and to a pleiogravitropic (prostrate) maize mutant, lazy (la). All plants converted the [³H]GA₂₀ to [³H]GA₁⁻ and [³H]GA₂₉-like metabolites as well as to several metabolites with the partitioning and chromatographic behavior of glucosyl conjugates of [³H]GA₁, [³H]GA₂₉, and [³H]GA₈. The tentative identification of these putative [³H]GA glucosyl conjugates was further supported by the release of the free [³H]GA moiety after cleavage with cellulase. Within 12 hours of the [³H]GA₂₀ feed, there was a significantly higher proportion of total radioactivity in lower than in upper halves of internode and leaf sheath pulvini in gravistimulated normal maize. Further, there was a significantly higher proportion of putative free GA metabolites of [³H]GA₂₀, especially [³H]GA₁, in the lower halves of normal maize relative to upper halves. The differential localization of the metabolites between upper and lower halves was not apparent in the pleiogravitropic mutant, la. Endogenous GA-like substances were also examined in gravistimulated maize shoots. Forty-eight hours after gravistimulation of 3-week-old maize seedlings, endogenous free GA-like substances in upper and lower leaf sheath and internode pulvini halves were extracted, chromatographed, and bioassayed using the `Tanginbozu' dwarf rice microdrop assay. Lower halves contained consistently higher total levels of GA-like activity. The qualitative elution profile of GA-like substances differed consistently, upper halves containing principally a GA₂₀-like substance and lower halves containing mainly GA₁-like and GA₁₉-like substances. Gibberellins A₁ (10 nanograms per gram) and A₂₀ (5 nanograms per gram) were identified from these lower leaf sheath pulvini by capillary gas chromatography-selected ion monitoring. Results from all of these experiments are consistent with a role for GAs in the differential shoot growth that follows gravitropism, although the results do not eliminate the possibility that the redistribution of GAs results from the gravitropic response.     

Differential regulation of trichome formation on the adaxial and abaxial leaf surfaces by gibberellins and photoperiod in Arabidopsis thaliana (L.) Heynh.

In wild-type (WT) Columbia and Landsberg erecta ecotypes of Arabidopsis thaliana (L.) Heynh., trichomes are present on the adaxial surfaces of all rosette leaves but are absent from the abaxial surfaces of the first-formed leaves. We have determined that both long-day (LD) photoperiod and gibberellin (GA) stimulate trichome formation. WT plants grown in LD conditions produce the first abaxial trichome on earlier leaves than plants grown in short-day (SD) conditions. Photoperiod sensitivity of abaxial trichome formation on WT plants develops gradually over time, reaching the maximum sensitivity about 24 d after germination. Application of gibberellic acid to WT plants growing in SD conditions accelerates the onset of abaxial trichomes. Conversely, application of 20 to 80 mg L-1 paclobutrazol, a GA biosynthesis inhibitor, to wild-type plants suppresses trichome initiation on the abaxial epidermis. The GA-deficient mutants ga1-5 and ga4-1 and the GA-insensitive mutant gai-1 exhibit delayed onset of abaxial trichomes when grown in LD conditions. The null mutant ga1-3 produces completely glabrous leaves when grown in SD conditions. Application of gibberellic acid to glabrous ga1-3 plants consistently induces earlier formation of trichomes on the adaxial epidermis than on the abaxial epidermis, demonstrating a difference between the adaxial and abaxial surfaces in their response to GA with regard to trichome formation.     

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.     

Gibberellin requirement for Arabidopsis seed germination is determined both by testa characteristics and embryonic abscisic acid

The mechanisms imposing a gibberellin (GA) requirement to promote the germination of dormant and non-dormant Arabidopsis seeds were analyzed using the GA-deficient mutant ga1, several seed coat pigmentation and structure mutants, and the abscisic acid (ABA)-deficient mutant aba1. Testa mutants, which exhibit reduced seed dormancy, were not resistant to GA biosynthesis inhibitors such as tetcyclacis and paclobutrazol, contrarily to what was found before for other non-dormant mutants in Arabidopsis. However, testa mutants were more sensitive to exogenous GAs than the wild-types in the presence of the inhibitors or when transferred to a GA-deficient background. The germination capacity of the ga1-1 mutant could be integrally restored, without the help of exogenous GAs, by removing the envelopes or by transferring the mutation to a tt background (tt4 and ttg1). The double mutants still required light and chilling for dormancy breaking, which may indicate that both agents can have an effect independently of GA biosynthesis. The ABA biosynthesis inhibitor norflurazon was partially efficient in releasing the dormancy of wild-type and mutant seeds. These results suggest that GAs are required to overcome the germination constraints imposed both by the seed coat and ABA-related embryo dormancy.