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。     

毛竹茎秆伸长过程中赤霉素生物合成、降解和信号转导关键基因的鉴定及表达分析

赤霉素(Gibberellin)是一类非常重要的植物激素,在高等植物生命活动的整个周期都起着重要的调控作用。从毛竹Phyllostachys edulis基因组中共鉴定出23个赤霉素途径基因,包括赤霉素生物合成相关的8个GA20ox和1个GA3ox基因、降解相关的8个GA2ox基因、参与赤霉素感知的2个GID1基因以及信号转导的2个GID2基因和2个DELLA基因。拟南芥、水稻和毛竹的系统进化树和保守基序分析显示赤霉素的合成代谢与信号转导在这些物种中是高度保守的。利用外源赤霉素处理毛竹种子和幼苗,发现赤霉素能显著提高种子的萌发率和幼苗的茎秆伸长,并且有着最佳的作用浓度。在GA3处理后,毛竹体内赤霉素生物合成基因GA20ox和GA3ox表达量均下调而降解活性赤霉素的GA2ox基因表达量上调;赤霉素受体GID1和正调控基因GID2的转录水平显著提高而负调控基因DELLA的表达受到抑制。这些基因在竹笋茎秆的不同形态学位置表达差异明显,大部分赤霉素生物合成与降解的相关基因GA20ox、GA3ox和GA2ox以及赤霉素受体GID1和正调控基因GID2都在竹笋的形态学上端大量表达,而赤霉素信号转导的阻遏基因DELLA在笋体形态学底端大量积累而顶端基本不表达。     

拟南芥GA2ox基因家族启动子分析

赤霉素是一类重要的植物激素,它参与调节植物生长发育的各个阶段。真核基因的表达受多种因素的调控,其中启动子在转录水平上的调节作用至关重要,但是目前对拟南芥GA2ox基因家族上游顺式元件的详细分析较少。本研究分析了AtGA2ox基因家族染色体定位、进化关系、蛋白模体以及顺式元件。分析结果表明：AtGA2ox基因家族分为2个亚类;具有相对集中的染色体分布：具有相似的内含子和外显子结构;AtGA2ox基因家族成员进化关系越相近,模体和顺式元件则越保守且分布位置更接近。PLACE和Plant CARE以及DMB分析显示,AtGA2ox基因家族存在许多保守元件,受多因素的调控,在此家族的上游调控区域普遍存在组织和器官特异性表达元件、光响应元停、激素响应元件以及其他环境响应元件。基因表达谱分析结果表明,在激素诱导下,AtGA2ox基因家族均有响应。这些元件不仅作为正调控元件,诱导部分AtGA2ox基因的表达,而且还能作为负调控元件．抑制其他AtGA2ox基因的表达。     

烟草GA合成调控转录因子RSG应答甲醇和乙醇刺激的分子机理初探

为了考察甲醇或乙醇促进植物生长与赤霉素(GA)的合成关系,该研究在MS固体培养基中培养并添加外源GA和GA合成抑制剂多效唑(PAC),分析其对2mmol/L甲醇或乙醇促进烟草生长的影响及GA合成调控转录因子RSG(for repression of shoot growth)应答甲醇或乙醇刺激的分子机理。结果显示:(1)外源添加GA可增强甲醇或乙醇对烟草生长的刺激作用,而添加PAC却抑制甲醇和乙醇对烟草生长的刺激作用。(2)14-3-3蛋白与RSG结合抑制RSG进入细胞核及其转录调控活性;甲醇和乙醇诱导烟草14-3-3基因的转录和表达,对RSG蛋白表达也有诱导作用。(3)甲醇和乙醇可降低14-3-3蛋白与RSG的相互作用,同时增强RSG与GA20ox1启动子的结合。研究表明,甲醇和乙醇刺激烟草的生长可能通过增加RSG表达,且减弱RSG与14-3-3蛋白的结合来增加RSG细胞核定位作用,从而增强RSG与GA20ox1启动子的结合,最终增加GA的合成,从而促进烟草的生长,这可能是甲醇和乙醇促进烟草生长的一种重要的分子机制。     

拟南芥CKI1基因上游转录调控因子筛选及鉴定

拟南芥CKI1(cytokininindependent 1)是双组分信号系统中一个组氨酸激酶蛋白,通过作用于下游组氨酸磷酸转移蛋白激活双组分信号通路,在调控胚囊中央细胞命运分化和发育过程中具有重要作用。然而目前对于CKI1基因上游转录调控因子还知之甚少。本研究分析了不同长度的CKI1启动子在拟南芥胚囊中的活性,并利用酵母单杂交技术对CKI1上游转录调控因子进行了筛选和鉴定。结果表明,位于内含子区域中的F5/R2片段表现出与CKI1启动子全长相一致的表达活性。进一步选取3个串联重复的F5/R2片段用于构建诱饵表达载体,同时,选取拟南芥雌蕊构建cDNA文库,通过酵母单杂交筛选获得226个阳性克隆。去除低质量及冗余重复的序列后共获得66条可读序列,其中8条序列对应的基因编码具有DNA结合功能的蛋白。研究结果为进一步揭示CKI1基因的转录调控机制提供了重要参考信息。     

OsGA3ox通过合成不同活性GA调控水稻育性及株高

赤霉素(gibberellin,GA)是一种重要的激素,参与调控植物多种生长发育过程。GA生物合成通路已基本阐明,其中赤霉素3β羟化酶(gibberellin 3β-hydroxylase,GA3ox)是多种活性GA合成的关键酶。水稻中有2个GA3ox基因(OsGA3ox1和OsGA3ox2),其生理功能虽有初步研究,但它们在合成活性GA调控水稻发育过程中是如何分工协作尚不清楚。本研究通过CRISPR/Cas9技术获得基因编辑突变体ga3ox1和ga3ox2,发现ga3ox1花粉育性显著下降,而ga3ox2株高显著变矮,表明OsGA3ox1是花粉正常发育必需的,而OsGA3ox2是茎叶伸长必需的。组织表达分析表明,OsGA3ox1主要在未开的花中表达,OsGA3ox2主要在未伸长的叶中表达。进一步对野生型(WT)和两个ga3ox突变体未开的花、未伸长的叶及根中的GA进行检测分析,发现OsGA3ox1在花中催化GA9形成GA7与花粉育性密切相关;OsGA3ox2在未伸长的叶中催化GA20形成GA1调控株高;OsGA3ox1在根中催化GA19形成GA20,调控GA3的生成。总之,OsGA3...     

酚类化合物代谢中受UV-C辐照调控的转录因子的筛选

SlPAL5基因是酚类化合物代谢的关键基因。UV-C辐照可以有效提高番茄果实中酚类化合物的含量。因此研究调控SlPAL5基因表达的转录因子,对于进一步阐明UV-C诱导番茄果实酚类化合物合成的调控机制具有重要意义。文中通过构建番茄酵母单杂交文库,利用酵母单杂交技术筛选调控酚类化合物合成关键基因SlPAL5表达的转录因子。通过测序和Blast同源性分析得到转录因子SlERF7,并证实SlERF7可以与SlPAL5的启动子相互作用。另外,UV-C辐照可以显著提高SlERF7的表达水平。结果表明受UV-C辐照诱导的SlERF7可能参与了SlPAL5的转录调控,为研究UV-C诱导番茄果实酚类化合物合成的调控机制提供了基础。     

白桦E-box元件的载体构建及与BplMYB46转录因子的互作分析

MYB转录因子是植物中最大的转录因子家族成员之一,主要参与植物次生代谢调控、激素和环境因子的应答,在植物的生长发育中起着至关重要的作用。已有研究发现,E-box的核心序列为CANNTG(N:A/G/C/T),是一类与光响应和苯丙氨酸生物合成途径相关的元件。在前期研究中,先构建顺式作用元件库,然后以转录因子为中心的酵母单杂交技术筛选发现,白桦的BplMYB46转录因子能够与核心序列为CAAATG的E-box顺式作用元件结合。但是,是否能与E-box的其他核心序列结合还不清楚。本研究将每一种E-box顺式作用元件的核心序列分别进行双链DNA的复性并连接到pHIS2载体上,然后通过酵母单杂交技术筛选与BplMYB46转录因子能够特异结合的E-box顺式作用元件。结果显示,当E-box的核心序列第3个碱基为A/T/C、第四个碱基为A/G/C时,酵母单菌落能在三缺培养基TDO/3AT上生长,表明与BplMYB46转录因子结合的E-box顺式作用元件的特异性序列为CA(A/T/C)(A/G/C)TG。为后续通过BplMYB46转录因子与E-box顺式作用元件的结合来分析BplMYB46转录因子对下游基因的调控,以及为筛选优良下游基因改良白桦的遗传性状奠定数据基础。     

高等植物赤霉素3-β-双氧化酶基因研究进展

赤霉素3-β-双氧化酶(GA3ox)是赤霉素(GA)生物合成过程中的关键酶之一,直接作用于活性GA的生成,在植物的生长发育过程中发挥着重要作用。GA3ox已在拟南芥、烟草、高粱和杨树等多种植物中得到克隆,在植物个体的不同生长阶段、不同组织中GA3ox的表达量均存在着差异,这主要是由物种内在的遗传因素所决定的,同时环境因子和外源激素处理对GA3ox表达也有着显著的影响。通过对基因突变体的研究,揭示出GA3ox突变导致GA的生物合成受阻而间接影响植物的生长,导致植物茎秆、花、种子、果、根等部位性状的改变,包括植株矮化、结籽困难等。过量表达GA3ox基因促进GA的生成,诱导细胞的分裂与分化,促进根尖的径向伸长、种子萌发等。主要从GA3ox的基因克隆与表达模式及其功能效应两方面综述了其在高等植物中研究进展,通过对GA3ox在不同植物中的相关功能研究,探讨其在植物生长发育过程中的具体作用,以期为了解GA3ox对植物生长发育的调控机制提供参考,也有助于在生产实践中更好地利用GA3ox开展基因工程研究,定向培育植物新品种。     

受赤霉素调节的拟南芥F-box基因筛选分析

赤霉素(Gibberellins,GAs)作为一种植物激素,对植物的生长发育具有重要调控作用,但其作用机制有待进一步完善。F-box蛋白是SCF复合体的一个亚基,通过特异性识别底物来调控植物的生长发育。本研究采用生物信息学方法,通过分析拟南芥基因芯片数据库提供的数据筛选到38个受GA调节的候选F-box基因,并对其中6个基因进行了实时荧光定量PCR验证。Plant CARE分析显示,其中30个基因的启动子区具有GA响应元件、以及IAA、ABA、光、温度干旱胁迫、或生物钟相关的顺式作用元件。通过分析Bio Grid数据库提供的相互作用对象,发现其中18个候选F-box蛋白与GA2ox1,GA3ox1和GA3ox3具有相互作用关系。基因表达谱分析表明,这些候选F-box基因在拟南芥各个组织器官中都有不同程度的表达,对IAA、ABA、光、温度干旱等胁迫或不同光周期都具有一定的响应。为深入研究GA调控植物生长发育的分子机制提供了重要线索。     

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.     

Distinct and overlapping roles of two gibberellin 3-oxidases in Arabidopsis development

Gibberellin (GA) 3-oxidase, a class of 2-oxoglutarate-dependent dioxygenases, catalyzes the conversion of precursor GAs to their bioactive forms, thereby playing a direct role in determining the levels of bioactive GAs in plants. Gibberellin 3-oxidase in Arabidopsis is encoded by a multigene family consisting of at least four members, designated AtGA3ox1 to AtGA3ox4. It has yet to be investigated how each AtGA3ox gene contributes to optimizing bioactive GA levels during growth and development. Using quantitative real-time PCR analysis, we have shown that each AtGA3ox gene exhibits a unique organ-specific expression pattern, suggesting distinct developmental roles played by individual AtGA3ox members. To investigate the sites of synthesis of bioactive GA in plants, we generated transgenic Arabidopsis that carried AtGA3ox1-GUS and AtGA3ox2-GUS fusions. Comparisons of the GUS staining patterns of these plants with that of AtCPS-GUS from previous studies revealed the possible physical separation of the early and late stages of the GA pathway in roots. Phenotypic characterization and quantitative analysis of the endogenous GA content of ga3ox1 and ga3ox2 single and ga3ox1/ga3ox2 double mutants revealed distinct as well as overlapping roles of AtGA3ox1 and AtGA3ox2 in Arabidopsis development. Our results show that AtGA3ox1 and AtGA3ox2 are responsible for the synthesis of bioactive GAs during vegetative growth, but that they are dispensable for reproductive development. The stage-specific severe GA-deficient phenotypes of the ga3ox1/ga3ox2 mutant suggest that AtGA3ox3 and AtGA3ox4 are tightly regulated by developmental cues; AtGA3ox3 and AtGA3ox4 are not upregulated to compensate for GA deficiency during vegetative growth of the double mutant.     

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.     

Analyses of GA20ox- and GID1-over-expressing aspen suggest that gibberellins play two distinct roles in wood formation

Gibberellins (GAs) are involved in many aspects of plant development, including shoot growth, flowering and wood formation. Increased levels of bioactive GAs are known to induce xylogenesis and xylem fiber elongation in aspen. However, there is currently little information on the response pathway(s) that mediate GA effects on wood formation. Here we characterize an important element of the GA pathway in hybrid aspen: the GA receptor, GID1. Four orthologs of GID1 were identified in Populus tremula  ×  P. tremuloides ( PttGID1.1–1.4 ). These were functional when expressed in Arabidopsis thaliana , and appear to present a degree of sub-functionalization in hybrid aspen. PttGID1.1 and PttGID1.3 were over-expressed in independent lines of hybrid aspen using either the 35S promoter or a xylem-specific promoter ( LMX5 ). The 35S : PttGID1 over-expressors shared several phenotypic traits previously described in 35S:AtGA20ox1 over-expressors, including rapid growth, increased elongation, and increased xylogenesis. However, their xylem fibers were not elongated, unlike those of 35S:AtGA20ox1 plants. Similar differences in the xylem fiber phenotype were observed when PttGID1.1 , PttGID1.3 or AtGA20ox1 were expressed under the control of the LMX5 promoter, suggesting either that PttGID1.1 and PttGID1.3 play no role in fiber elongation or that GA homeostasis is strongly controlled when GA signaling is altered. Our data suggest that GAs are required in two distinct wood-formation processes that have tissue-specific signaling pathways: xylogenesis, as mediated by GA signaling in the cambium, and fiber elongation in the developing xylem.     

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.     

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.     

16,17-dihydro gibberellin A5 competitively inhibits a recombinant Arabidopsis GA 3beta-hydroxylase encoded by the GA4 gene

Ring D-modified gibberellin (GA) A5 and A20 derivatives are structurally similar to GA20 and GA9 (the precursors to growth-active GA1 and GA4) and, when applied to higher plants, especially grasses, can reduce shoot growth with concomitant reductions in levels of growth-active GAs and increases in levels of their immediate 3-deoxy precursors. The recombinant Arabidopsis GA 3beta-hydroxylase (AtGA3ox1) protein was used in vitro to test a number of ring D-modified GA structures as possible inhibitors of AtGA3ox1. This fusion protein was able to 3beta-hydroxylate the 3-deoxy GAs, GA9 and GA20, to GA4 and GA1, respectively, and convert the 2,3-didehydro GA, GA5, to its 2,3-epoxide, GA6. Michaelis-Menten constant (Km) values of 1.25 and 10 microM, respectively, were obtained for the GA9 and GA20 conversions. We utilized the enzyme's ability to convert GA20 to GA1 in order to test the efficacy of GA5, 16,17-dihydro GA5 (dihydro GA5), and a number of other ring D-modified GAs as inhibitors of AtGA3ox activity. For the exo-isomer of dihydro GA5, inhibition increased with the dose of dihydro GA5, with Lineweaver-Burk plots showing that dihydro GA5 changed only the Km of the enzyme reaction, not the V(max), giving a dissociation constant of the enzyme-inhibitor complex (Ki) of 70 microM. Other ring D-modified GA derivatives showed similar inhibitory effects on GA1 production, with 16,17-dihydro GA20-13-acetate being the most effective inhibitor. This behavior is consistent with dihydro GA5, at least, functioning as a competitive substrate inhibitor of AtGA3ox1. Finally, the recombinant AtGA3ox1 fusion protein may be a useful screening tool for other effective 3beta-hydroxylase inhibitors, including naturally occurring ones.     

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.