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。     

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.     

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.     

Interaction of the parathyroid hormone receptor with the 14-3-3 protein

The receptor for parathyroid hormone (PTH) and PTH-related protein (PTHrP) regulates calcium homeostasis, bone remodeling and skeletal development. 14-3-3 proteins bind to signaling proteins and act as molecular scaffolds and regulators of subcellular localization. We show that the parathyroid hormone receptor (PTHR) interacts with 14-3-3 and the proteins colocalize within the cell. 14-3-3 interacts with the C-terminal tail of the receptor containing a consensus 14-3-3 binding motif, but additional binding sites are also used. Protein kinase-A treatment of the receptor and especially the C-terminal tail reduces 14-3-3 binding. The expressed C-terminal tail is primarily localized in the nucleus, supporting the function of a putative nuclear localization signal that could be involved in the previously described nuclear localization of PTHR. The observed interaction between PTHR and the 14-3-3 protein implies that 14-3-3 could contribute to regulation of PTHR signaling.     

In vivo Binding of Gibberellin A(1) in Dwarf Pea Epicotyls

Binding of [(3)H]gibberellin A(1) (GA(1)) to extracts of dwarf pea epicotyls was investigated using sliced pea epicotyls (0.5-1.0 millimeter thick) that had been incubated in a solution containing [(3)H]GA(1) at 0 C for 3 days. Gel filtration of a 100,000g supernatant indicated binding to a high (HMW) and an intermediate molecular weight (IMW) fraction with estimated molecular weights of 6 x 10(5) daltons and 4 to 7 x 10(4) daltons, respectively. The bound (3)H-activity was [(3)H]GA(1) and not a metabolite as deduced by thin layer chromatography. The bound label did not sediment during centrifugation at 100,000g for 2 hours; also, binding was not disrupted after treatment of a combined HMW and IMW fraction with DNase, RNase, or phospholipase A or C, but it was disrupted by protease or heat treatment. These facts suggest that binding of [(3)H]GA(1) was occurring to a soluble protein(s). [(3)H]GA(1) bound to a combined HMW and IMW fraction was not susceptible to changes in pH, nor could it be exchanged with a variety of GAs tested under in vitro conditions. Under in vivo equilibrium conditions, biologically active GAs, such as GA(1), GA(3), GA(4), GA(5), GA(7), and keto GA(1), could reduce the level of [(3)H]GA(1) binding, whereas inactive GAs, such as iodo GA(1) methyl ester, GA(8), GA(13), GA(26), and non-GAs, such as (+/-)abscisic acid, had no effect. By varying the concentration of [(3)H]GA(1) in the incubation medium, the specific binding of [(3)H]GA(1) appeared to be due to two classes of binding sites having estimated K(d) of 6 x 10(-8) molar and 1.4 x 10(-6) molar. The concentrations of the two sites were estimated to be 0.45 picomole per gram and 4.04 picomoles per gram on a fresh weight and 0.1 picomole per milligram and 0.9 picomole per milligram on a soluble protein basis, respectively.     

The NADPH-cytochrome P450 reductase gene from Gibberella fujikuroi is essential for gibberellin biosynthesis

The fungus Gibberella fujikuroi is used for the commercial production of gibberellins (GAs), which it produces in very large quantities. Four of the seven GA biosynthetic genes in this species encode cytochrome P450 monooxygenases, which function in association with NADPH-cytochrome P450 reductases (CPRs) that mediate the transfer of electrons from NADPH to the P450 monooxygenases. Only one cpr gene (cpr-Gf) was found in G. fujikuroi and cloned by a PCR approach. The encoded protein contains the conserved CPR functional domains, including the FAD, FMN, and NADPH binding motifs. cpr-Gf disruption mutants were viable but showed a reduced growth rate. Furthermore, disruption resulted in total loss of GA(3), GA(4), and GA(7) production, but low levels of non-hydroxylated C(20)-GAs (GA(15) and GA(24)) were still detected. In addition, the knock-out mutants were much more sensitive to benzoate than the wild type due to loss of activity of another P450 monooxygenase, the detoxifying enzyme, benzoate p-hydroxylase. The UV-induced mutant of G. fujikuroi, SG138, which was shown to be blocked at most of the GA biosynthetic steps catalyzed by P450 monooxygenases, displayed the same phenotype. Sequence analysis of the mutant cpr allele in SG138 revealed a nonsense mutation at amino acid position 627. The mutant was complemented with the cpr-Gf and the Aspergillus niger cprA genes, both genes fully restoring the ability to produce GAs. Northern blot analysis revealed co-regulated expression of the cpr-Gf gene and the GA biosynthetic genes P450-1, P450-2, P450-4 under GA production conditions (nitrogen starvation). In addition, expression of cpr-Gf is induced by benzoate. These results indicate that CPR-Gf is the main but not the only electron donor for several P450 monooxygenases from primary and secondary metabolism.     

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.     

Metabolic enzymes as targets for 14-3-3 proteins

The 14-3-3 proteins are binding proteins that have been shown to interact with a wide array of enzymes involved in primary biosynthetic and energy metabolism in plants. In most cases, the significance of binding of the 14-3-3 protein is not known. However, most of the interactions are phosphorylation-dependent and most of the known binding partners are found in the cytosol, while some may also be localized to plastids and mitochondria. In this review, we examine the factors that may regulate the binding of 14-3-3s to their target proteins, and discuss their possible roles in the regulation of the activity and proteolytic degradation of enzymes involved in primary carbon and nitrogen metabolism.     

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.