Cloning of gibberellin 3 beta-hydroxylase cDNA and analysis of endogenous gibberellins in the developing seeds in watermelon

We have isolated Cv3h, a cDNA clone from the developing seeds of watermelon, and have demonstrated significant amino acid homology with gibberellin (GA) 3 beta-hydroxylases. This cDNA clone was expressed in Escherichia coli as a fusion protein that oxidized GA(9) and GA(12) to GA(4) and GA(14), respectively. The Cv3h protein had the highest similarity with pumpkin GA 2 beta,3 beta-hydroxylase, but did not possess 2 beta-hydroxylation function. RNA blot analysis showed that the gene was expressed primarily in the inner parts of developing seeds, up to 10 d after pollination (DAP). In the parthenocarpic fruits induced by treatment with 1-(2-chloro-4-pyridyl)-3-phenylurea (CPPU), the embryo and endosperm of the seeds were undeveloped, whereas the integumental tissues, of maternal origin, showed nearly normal development. Cv3h mRNA was undetectable in the seeds of CPPU-treated fruits, indicating that the GA 3 beta-hydroxylase gene was expressed in zygotic cells. In our analysis of endogenous GAs from developing seeds, GA(9) and GA(4) were detected at high levels but those of GA(20) and GA(1) were very low. This demonstrates that GA biosynthesis in seeds prefers a non-13-hydroxylation pathway over an early 13-hydroxylation pathway. We also analyzed endogenous GAs from seeds of the parthenocarpic fruits. The level of bioactive GA(4 )was much lower there than in normal seeds, indicating that bioactive GAs, unconnected with Cv3h, exist in integumental tissues during early seed development.     

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.     

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.     

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.     

Distribution of gibberellins and expressional analysis of GA 20-oxidase genes of morning glory during fruit maturation

Gibberellins A1/3 (GA1/3) and GA20 appeared earlier in surrounding tissues (pericarps/carpel/placenta) than in developing seeds of morning glory. The content of GA1/3 became higher in seeds than in the surrounding tissues at 9 days after anthesis (DAA), while that of GA20 stayed lower in seeds even at 12 DAA, suggesting the possibility that GA20 was translocated into seeds from the surrounding tissues and converted to GA1/3. The site of biosynthesis of GA20 in the fruits was determined by RNA-blotting and in situ hybridization of GA 20-oxidase genes (InGA20ox1, InGA20ox2). InGA20ox1 was not expressed in the surrounding tissues but in seeds, while no signal due to InGA20ox2 was detected in neither tissue. The expression of InGA20ox1 started in the seed coat near the hilum and spread in the seed coat like those of GA 3-oxidase and GA-inducible alpha-amylase genes. These observations suggest that GA biosynthesis is tissue-specifically and time-dependently regulated in the fruit of morning glory.     

Gibberellin induces alpha-amylase gene in seed coat of Ipomoea nil immature seeds

Two full-length cDNAs encoding gibberellin 3-oxidases, InGA3ox1 and InGA3ox2, were cloned from developing seeds of morning glory (Ipomoea nil (Pharbitis nil) Choisy cv. Violet) with degenerate-PCR and RACEs. The RNA-blot analysis for these clones revealed that the InGA3ox2 gene was organ-specifically expressed in the developing seeds at 6-18 days after anthesis. In situ hybridization showed the signals of InGA3ox2 mRNA in the seed coat, suggesting that active gibberellins (GAs) were synthesized in the tissue, although no active GA was detected there by immunohistochemistry. In situ hybridization analysis for InAmy1 (former PnAmy1) mRNA showed that InAmy1 was also synthesized in the seed coat. Both InGA3ox2 and InAmy1 genes were expressed spatially overlapped without a clear time lag, suggesting that both active GAs and InAmy1 were synthesized almost simultaneously in seed coat and secreted to the integument. These observations support the idea that GAs play an important role in seed development by inducing alpha-amylase.     

High temperature-induced abscisic acid biosynthesis and its role in the inhibition of gibberellin action in Arabidopsis seeds

Suppression of seed germination at supraoptimal high temperature (thermoinhibiton) during summer is crucial for Arabidopsis (Arabidopsis thaliana) to establish vegetative and reproductive growth in appropriate seasons. Abscisic acid (ABA) and gibberellins (GAs) are well known to be involved in germination control, but it remains unknown how these hormone actions (metabolism and responsiveness) are altered at high temperature. Here, we show that ABA levels in imbibed seeds are elevated at high temperature and that this increase is correlated with up-regulation of the zeaxanthin epoxidase gene ABA1/ZEP and three 9-cis-epoxycarotenoid dioxygenase genes, NCED2, NCED5, and NCED9. Reverse-genetic studies show that NCED9 plays a major and NCED5 and NCED2 play relatively minor roles in high temperature-induced ABA synthesis and germination inhibition. We also show that bioactive GAs stay at low levels at high temperature, presumably through suppression of GA 20-oxidase genes, GA20ox1, GA20ox2, and GA20ox3, and GA 3-oxidase genes, GA3ox1 and GA3ox2. Thermoinhibition-tolerant germination of loss-of-function mutants of GA negative regulators, SPINDLY (SPY) and RGL2, suggests that repression of GA signaling is required for thermoinibition. Interestingly, ABA-deficient aba2-2 mutant seeds show significant expression of GA synthesis genes and repression of SPY expression even at high temperature. In addition, the thermoinhibition-resistant germination phenotype of aba2-1 seeds is suppressed by a GA biosynthesis inhibitor, paclobutrazol. We conclude that high temperature stimulates ABA synthesis and represses GA synthesis and signaling through the action of ABA in Arabidopsis seeds.     

Isolation and expression of GA 2-oxidase2 in tomato.

GA 2-oxidases, a key enzyme involves GA biosynthesis, catalyze the degradation of active C(19)-Gibberellins (GAs) through 2-hydroxylation yields inactive GA product. Searching public tomato database, the putative GA2ox2 sequences were assembled. We isolated a full-length GA2ox2 cDNA with primers designed from the assembled sequence. This gene was designed as SlGA2ox2 (GenBank accession No. EF017805). The full-length GA2ox2 gene contained a complete open reading frame (ORF) of 1203 bp, which encoded 322 amino acid residues. Amino acid sequence homology analysis of SlGA2ox2 showed an 88% identity with NtGA2ox2 in tobacco. And alignments of SlGA2ox2 with other known GA2ox from Arabidopsis, Pea, Adzuki Bean, Winter Squash etc indicate low similarity of 47-70%. Semi-quantitative RT-PCR analysis showed a specific expression profile of SlGA2ox2 in different tissues, which mainly expressed in flowers and traces were detected in roots, stems, leaves and immature fruits.     

Gibberellin 3-oxidases in developing embryos of the southern wild cucumber, Marah macrocarpus

Immature seeds of the southern wild cucumber, Marah macrocarpus, are a rich source of gibberellins (GAs) and were used in some of the earliest experiments on GA biosynthesis. The main biologically active GAs in developing embryos and endosperm of M. macrocarpus are GA(4) and GA(7), which have been shown previously to be formed from GA(9) in separate pathways, GA(4) being formed directly by 3β-hydroxylation, while GA(7) is produced in two steps via 2,3-didehydroGA(9). In order to identify the enzymes responsible for these conversions, three cDNA clones encoding functionally different GA 3-oxidases, MmGA3ox1, -2 and -3, were obtained from young immature M. macrocarpus embryos. Their biochemical functions were determined by expression of the cDNAs in Escherichia coli and incubation of cell lysates with (14)C-labelled substrates. MmGA3ox1 and MmGA3ox3 converted GA(9) to GA(4) as sole product, while MmGA3ox2 produced several products, including GA(4), 2,3-didehydroGA(9), 2,3-epoxyGA(9), GA(20) and GA(5), these last two products requiring 13-hydroxylation of GA(9) and 2,3-didehydroGA(9), respectively. MmGA3ox1 converted 2,3-didehydroGA(9) to GA(7), while MmGA3ox3 converted this substrate to the 2,3-epoxide, and MmGA3ox2 also formed the epoxide, but also GA(5.) Thus, formation of GA(7) requires the sequential activities of MmGA3ox2 and MmGA3ox1, while MmGA3ox3 is not involved in GA(7) production. The enzymes catalysed similar reactions when incubated with 13-hydroxylated GAs, although with reduced efficiencies. The 13-hydroxylase activity of MmGA3ox2 may be responsible for the production of GA(1) and GA(3), which are present at low levels in developing M. macrocarpus seeds.     

烟草赤霉素2-氧化酶基因NtGA2ox1的克隆、表达及亚细胞定位分析

赤霉素2-氧化酶(GA2ox)通过2-β-羟基化作用产生失活的赤霉素,进而调节植物体内的赤霉素的活性水平。前期,本研究在烟草侧枝发育突变体转录组数据中,发现一个赤霉素2-氧化酶基因,其表达水平与野生型相比存在显著差异,命名为NtGA2ox1。为了更好地研究该基因在烟草侧枝发育中的作用,本研究从普通烟草中分离克隆了NtGA2ox1基因。通过测序分析该基因的编码及全长序列,发现NtGA2ox1基因含有2个外显子和1个内含子,编码一条长度为379个氨基酸的序列。同源进化分析表明,该基因在多种植物中存在同源序列,特别是茄科植物。组织特异性表达分析发现,NtGA2ox1基因在烟草的各个生长阶段均有表达,其中,在花和根中表达量较高。同时,激光共聚焦显微镜结果表明,YFP-NtGA2ox1融合蛋白在细胞质和细胞核中有很强的荧光信号,表明NtGA2ox1蛋白很可能定位于细胞核和细胞质中。本研究为进一步研究赤霉素调控烟草侧枝发育提供了理论依据。     

AtGA3ox2, a key gene responsible for bioactive gibberellin biosynthesis, is regulated during embryogenesis by LEAFY COTYLEDON2 and FUSCA3 in Arabidopsis

Embryonic regulators LEC2 (LEAFY COTYLEDON2) and FUS3 (FUSCA3) are involved in multiple aspects of Arabidopsis (Arabidopsis thaliana) seed development, including repression of leaf traits and premature germination and activation of seed storage protein genes. In this study, we show that gibberellin (GA) hormone biosynthesis is regulated by LEC2 and FUS3 pathways. The level of bioactive GAs is increased in immature seeds of lec2 and fus3 mutants relative to wild-type level. In addition, we show that the formation of ectopic trichome cells on lec2 and fus3 embryos is a GA-dependent process as in true leaves, suggesting that the GA pathway is misactivated in embryonic mutants. We next demonstrate that the GA-biosynthesis gene AtGA3ox2, which encodes the key enzyme AtGA3ox2 that catalyzes the conversion of inactive to bioactive GAs, is ectopically activated in embryos of the two mutants. Interestingly, both beta-glucuronidase reporter gene expression and in situ hybridization indicate that FUS3 represses AtGA3ox2 expression mainly in epidermal cells of embryo axis, which is distinct from AtGA3ox2 pattern at germination. Finally, we show that the FUS3 protein physically interacts with two RY elements (CATGCATG) present in the AtGA3ox2 promoter. This work suggests that GA biosynthesis is directly controlled by embryonic regulators during Arabidopsis embryonic development.