生长素介导OsRGP1和0sSuS参与水稻地上部基部向重性弯曲生长

在水稻幼苗被放平后,地上部重力响应部位（地上部基部）的上半部生长比下半部生长慢,从而导致地上部基部发生向上弯曲．发生不对称生长的向重性反应。通过抑制差减杂交（SSH）实验发现．水稻地上部基部向重性弯曲生长过程中,反复糖基化多肽OsRGP1和蔗糖合成酶OsSuS在基部上下部有不对称表达。我们还采用Realtime PCR开展进一步研究。实验结果显示OsRGP1和0sSMS的表达受到IAA的调控。通过生物信息学分析也发现OsRGPl和OsSuS的启动子上存在着生长素的调控元件．生长素极性运输抑制剂TIBA处理能够消除OsRGP1和OsSuS在向重性过程中的不对称表达．这进一步说明向重性过程中不对称分布的生长素介导了OsRGP1和OsSuS基因的不对称表达。另外．向重性反应过程中,检测到基部下半部的己糖浓度增加。因此,OsRGP1的不对称表达可能有助于下半部细胞壁多糖的积累,进而促进了下半部细胞壁的扩张。OsSuS不对称表达也有可能会引起己糖在上下半部的不对称分布。己糖和细胞壁多糖在下半部的积累可能在下部细胞扩张从而导致向重性弯曲生长中起一定的作用。     

Gravistimulation leads to asymmetry of both auxin and gibberellin levels in barley pulvini

The auxin indole-3-acetic acid (IAA) is known to promote the biosynthesis of active gibberellins (GAs) in barley ( Hordeum vulgare ). We therefore investigated the possibility that this interaction might contribute to the gravitropic response of barley leaf sheath pulvini. Barley plants at the inflorescence stage were gravistimulated for varying times, and the pulvini were then separated into upper and lower halves for quantification of IAA and GAs by GC-MS. Consistent with the Cholodny–Went theory, the lower portion contained more IAA than did the upper portion. This difference was detected as early as 2.5 h after the start of gravistimulation, and bending was also observed at this stage. At later time points tested (6 h and 24 h), but not at 2.5 h or 3 h, the higher auxin content of the lower half was associated with a higher level of GA₁, the main bioactive GA in barley. Consistent with that result, the expression of Hv3ox2 , which encodes a key enzyme for the conversion of GA₂₀ to GA₁, was higher in the lower side than in the upper, after 6 h. It is suggested that in gravistimulated leaf sheath pulvini, auxin accumulates in the lower side, leading to a higher level of GA₁, which contributes to the bending response. Further evidence that GAs play a role in the gravitropic response was obtained from GA-related mutants, including the elongated sln1c mutant, in which GA signalling is constitutive. Pulvinar bending in the sln1c mutant was greater than in the wild-type. This result indicates that in the lower side of the gravistimulated pulvinus, the relatively high level of bioactive GA facilitates, but does not mediate, the bending response.     

OsEXPA4 and OsRWC3 are involved in asymmetric growth during gravitropic bending of rice leaf sheath bases

Expression of an expansin gene ( OsEXPA4 ) and a plasma membrane aquaporin ( OsRWC3 ) were differentially induced in the abaxial (lower) side of rice leaf sheath bases during gravitropism. Expression of both of these genes was induced by GA₃. β-glucuronidase (GUS) activity in transgenic rice expressing a GUS reporter gene under the control of the gibberellin (GA)-responsive OsRWC3 promoter was investigated during gravitropic curvature and found to be enhanced in the abaxial side of graviresponding leaf sheath bases. Ancymidol, a GA biosynthesis inhibitor, reduced the expression gradient of OsEXPA4 and OsRWC3 . The osmotic potential was transiently lower on the abaxial side of gravistimulated leaf sheath bases, and the gravitropic curvature was inhibited by HgCl₂ and phloretin, two known inhibitors of aquaporins. These data suggest that asymmetric GA redistribution following gravistimulation results in the localized expression of OsEXPA4 and OsRWC3 , that in turn might bring about differential cell wall loosening, cell expansion and water influx via aquaporins, thus leading to gravitropic bending.     

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.     

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.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.     

OsGA2ox5, a Gibberellin Metabolism Enzyme,Is Involved in Plant Growth,the Root Gravity Response and Salt Stress

Gibberellin (GA) 2-oxidases play an important role in the GA catabolic pathway through 2β-hydroxylation. There are two classes of GA2oxs, i.e., a larger class of C₁₉-GA2oxs and a smaller class of C₂₀-GA2oxs. In this study, the gene encoding a GA 2-oxidase of rice, Oryza sativa GA 2-oxidase 5 (OsGA2ox5), was cloned and characterized. BLASTP analysis showed that OsGA2ox5 belongs to the C₂₀-GA2oxs subfamily, a subfamily of GA2oxs acting on C₂₀-GAs (GA₁₂, GA₅₃). Subcellular localization of OsGA2ox5-YFP in transiently transformed onion epidermal cells revealed the presence of this protein in both of the nucleus and cytoplasm. Real-time PCR analysis, along with GUS staining, revealed that OsGA2ox5 is expressed in the roots, culms, leaves, sheaths and panicles of rice. Rice plants overexpressing OsGA2ox5 exhibited dominant dwarf and GA-deficient phenotypes, with shorter stems and later development of reproductive organs than the wild type. The dwarfism phenotype was partially rescued by the application of exogenous GA₃ at a concentration of 10 µM. Ectopic expression of OsGA2ox5 cDNA in Arabidopsis resulted in a similar phenotype. Real-time PCR assays revealed that both GA synthesis-related genes and GA signaling genes were expressed at higher levels in transgenic rice plants than in wild-type rice; OsGA3ox1, which encodes a key enzyme in the last step of the bioactive GAs synthesis pathway, was highly expressed in transgenic rice. The roots of OsGA2ox5-ox plants exhibited increased starch granule accumulation and gravity responses, revealing a role for GA in root starch granule development and gravity responses. Furthermore, rice and Arabidopsis plants overexpressing OsGA2ox5 were more resistant to high-salinity stress than wild-type plants. These results suggest that OsGA2ox5 plays important roles in GAs homeostasis, development, gravity responses and stress tolerance in rice.     

Differences and similarities in the photoregulation of gibberellin metabolism between rice and dicots

In rice seedlings, elongation of leaf sheaths is suppressed by light stimuli. The response is mediated by two classes of photoreceptors, phytochromes and cryptochromes. However, it remains unclear how these photoreceptors interact in the process. Our recent study using phytochrome mutants and novel cryptochrome RNAi lines revealed that cryptochromes and phytochromes function cooperatively, but independently to reduce active GA contents in seedlings in visible light. Blue light captured by cryptochrome 1 (cry1a and cry1b) induces robust expression of GA 2-oxidase genes (OsGA2ox4-7). In parallel, phytochrome B with auxiliary action of phytochrome A mediates repression of GA 20-oxidase genes (OsGA20ox2 and OsGA20ox4). The independent effects cumulatively reduce active GA contents, leading to a suppression of leaf sheath elongation. These regulatory mechanisms are distinct from phytochrome B function in dicots. We discuss reasons why the distinct system appeared in rice, and advantages of the rice system in early photomorphogenesis.     

Efficacy of microarray profiling data combined with QTL mapping for the identification of a QTL gene controlling the initial growth rate in rice

Seedling vigor, which is controlled by many quantitative trait loci (QTLs), is one of several important agronomic traits for direct-seedling rice systems. However, isolating these QTL genes is laborious and expensive. Here, we combined QTL mapping and microarray profiling to identify QTL genes for seedling vigor. By performing QTL mapping using 82 backcross inbred lines (BILs) of the Koshihikari (japonica) and Habataki (indica) cultivars for the rice initial growth, we identified two QTLs, early-stage plant development1/2 (qEPD1 and qEPD2), whose Koshihikari alleles promote plant height and/or leaf sheath length. Phenotypic analysis of the two substituted lines carrying the Habataki qEPD1 or qEPD2 allele revealed that qEPD2 functioned more dominantly for the initial growth of rice. From the microarray experiment, 55 and 45 candidate genes were found in the qEPD1 and qEPD2 genomic regions, which are expressed differentially between each substitution line (SL) and Koshihikari. Gibberellin 20 oxidase-2 (OsGA20ox2), which is identical to Semi Dwarf1 (SD1), was included among the 55 candidate genes of qEPD1, whereas its paralog, OsGA20ox1, was included among the 45 candidate genes of qEPD2. Consistently, introduction of the Koshihikari OsGA20ox1 allele into SL(qEPD2) increaseed its plant height and leaf sheath length significantly relative to the introduction of the Habataki OsGA20ox1 allele. Therefore, microarray profiling could be useful for rapidly screening QTL candidate genes. We concluded that OsGA20ox1 and OsGA20ox2 (SD1) function during the initial growth of rice, but OsGA20ox1 plays a dominant role in increasing plant height and leaf sheath length at the initial growth stage.     

The alpha-amylase induction in endosperm during rice seed germination is caused by gibberellin synthesized in epithelium

We recently isolated two genes (OsGA3ox1 and OsGA3ox2) from rice (Oryza sativa) encoding 3beta-hydroxylase, which catalyzes the final step of active gibberellin (GA) biosynthesis (H. Itoh, M. Ueguchi-Tanaka, N. Sentoku, H. Kitano, M. Matsuoka, M. Kobayashi [2001] Proc Natl Acad Sci USA 98: 8909-8914). Using these cloned cDNAs, we analyzed the temporal and spatial expression patterns of the 3beta-hydroxylase genes and also an alpha-amylase gene (RAmy1A) during rice seed germination to investigate the relationship between GA biosynthesis and alpha-amylase expression. Northern-blot analyses revealed that RAmy1A expression in the embryo occurs before the induction of 3beta-hydroxylase expression, whereas in the endosperm, a high level of RAmy1A expression occurs 1 to 2 d after the peak of OsGA3ox2 expression and only in the absence of uniconazol. Based on the analysis of an OsGA3ox2 null mutant (d18-Akibare dwarf), we determined that 3beta-hydroxylase produced by OsGA3ox2 is important for the induction of RAmy1A expression and that the OsGA3ox1 product is not essential for alpha-amylase induction. The expression of OsGA3ox2 was localized to the shoot region and epithelium of the embryo, strongly suggesting that active GA biosynthesis occurs in these two regions. The synthesis of active GA in the epithelium is important for alpha-amylase expression in the endosperm, because an embryonic mutant defective in shoot formation, but which developed epithelium cells, induced alpha-amylase expression in the endosperm, whereas a mutant defective in epithelium development did not.     

Changes in starch and inositol 1,4,5-trisphosphate levels and auxin transport are interrelated in graviresponding oat (Avena sativa) shoots

This study was conducted to unravel a mechanism for the gravitropic curvature response in oat (Avena sativa) shoot pulvini. For this purpose, we examined the downward movement of starch-filled chloroplast gravisensors, differential changes in inositol 1,4,5-trisphosphate (IP(3)) levels, transport of indole-3-acetic acid (IAA) and gravitropic curvature. Upon gravistimulation, the ratio for IAA levels in lower halves versus those in upper halves (L/U) increased from 1.0 at 0 h and reached a maximum value of 1.45 at 8 h. When shoots were grown in the dark for 10 d, to deplete starch in the chloroplast, the gravity-induced L/U of IAA was reduced to 1.0. N-naphthylphthalamic acid (NPA) and 2,3,5-triiodobenzoic acid (TIBA), both auxin transport inhibitors, significantly reduced the amount of gravitropic curvature and gravity-induced lateral IAA transport, but did not reduce the gravity-induced late change in the L/U ratio of IP(3) levels. U73122, a specific phospholipase C (PLC) inhibitor, decreased gravity-induced curvature. Because U73122 reduced the ratio of L/U of IAA imposed by gravistimulation, it is clear that IAA transport is correlated with changes in IP(3) levels upon gravistimulation. These results indicate that gravistimulation-induced differential lateral IAA transport may result from the onset of graviperception in the chloroplast gravisensors coupled with gravity-induced asymmetric changes in IP(3) levels in oat shoot pulvini.     

IAA和GA3在调控豌豆黄化苗茎切段伸长生长中的相互作用

IAA和GA3均能促进豌豆黄化苗茎切段的伸长。IAA效应可以为GA的合成抑制剂S-3307抑制,GA3效应同样也为IAA的运输抑制剂TIBA所抑制,并且分别再施用GA3和IAA后,抑制效应又能有所解除。观察顶端切半茎切段的结果表明,IAA主要促进茎切段表皮细胞的伸长,而GA3可能主要促进内部组织细胞的伸长。观察切段横纵切片的结果则显示,IAA促进皮层细胞的伸长和增大,而GA3只促进皮层细胞的伸长。这些结果说明两者是通过不同的作用部位和方式共同调节豌豆茎切段伸长生长的。     

A role of <Emphasis Type="Italic">OsGA20ox1</Emphasis>, encoding an isoform of gibberellin 20-oxidase,for regulation of plant stature in rice

Gibberellin (GA) 20-oxidase (GA20ox) is a key enzyme that normally catalyzes the penultimate steps in GA biosynthesis. One of the GA20ox genes in rice (Oryza sativaL.), OsGA20ox2 (SD1), is well known as the Green Revolution gene, and loss-of function mutation in this locus causes semi-dwarfism. Another GA20ox gene, OsGA20ox1, has also been identified, but its contribution to plant stature has remained unclear because no suitable mutants have been available. We isolated a mutant, B142, tagged with a T-DNA containing three CaMV 35S promoters, which showed a tall, GA-overproduction phenotype. The final stature of the B142 mutant reflects internode overgrowth and is approximately twice that of its wild-type parent. This mutant responds to application of both GA3 and a GA biosynthesis inhibitor, indicating that it is a novel tall mutant of rice distinct from GA signaling mutants such as slr1. The integrated T-DNAs, which contain three CaMV 35S promoters, are located upstream of the OsGA20ox1 open reading frame (ORF) in the B142 mutant genome. Analysis of mRNA and the endogenous GAs reveal that biologically active GA level is increased by up-regulation of the OsGA20ox1 gene in B142. Introduction of OsGA20ox1 cDNA driven by 35S promoter into the wild type phenocopies the morphological characteristics of B142. These results indicate that the elongated phenotype of the B142 mutant is caused by up-regulation of the OsGA20ox1 gene. Moreover, the final stature of rice was reduced by specific suppression of the OsGA20ox1 gene expression. This result indicates that not only OsGA20ox2 but also OsGA20ox1 affects plant stature.     

Gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature interaction

Soybean is an important oilseed crop grown globally. However, two examples of environmental stresses that drastically regulate soybean growth are low light and high-temperature. Emerging evidence suggests a possible interconnection between these two environmental stimuli. Low light and high-temperature as individual factors have been reported to regulate plant hypocotyl elongation. However, their interactive signal effect on soybean growth and development remains largely unclear. Here, we report that gibberellins (GAs) and auxin are required for soybean hypocotyl elongation under low light and high-temperature interaction. Our analysis indicated that low light and high-temperature interaction enhanced the regulation of soybean hypocotyl elongation and that the endogenous GA₃, GA₇, indole-3-acetic acid (IAA), and indole-3-pyruvate (IPA) contents significantly increased. Again, analysis of the effect of exogenous phytohormones and biosynthesis inhibitors treatments showed that exogenous GA, IAA, and paclobutrazol (PAC), 2, 3, 5,-triiodobenzoic acid (TIBA) treatments significantly regulated soybean seedlings growth under low light and high-temperature interaction. Further qRT-PCR analysis showed that the expression level of GA biosynthesis pathway genes (GmGA3ox1, GmGA3ox2 and GmGA3) and auxin biosynthesis pathway genes (GmYUCCA3, GmYUCCA5 and GmYUCCA7) significantly increased under (i) low light and high-temperature interaction and (ii) exogenous GA and IAA treatments. Altogether, these observations support the hypothesis that gibberellins and auxin regulate soybean hypocotyl elongation under low light and high-temperature stress interaction.     

Transcriptional regulation of gibberellin metabolism genes by auxin signaling in Arabidopsis

Auxin and gibberellins (GAs) overlap in the regulation of multiple aspects of plant development, such as root growth and organ expansion. This coincidence raises questions about whether these two hormones interact to regulate common targets and what type of interaction occurs in each case. Auxins induce GA biosynthesis in a range of plant species. We have undertaken a detailed analysis of the auxin regulation of expression of Arabidopsis (Arabidopsis thaliana) genes encoding GA 20-oxidases and GA 3-oxidases involved in GA biosynthesis, and GA 2-oxidases involved in GA inactivation. Our results show that auxin differentially up-regulates the expression of various genes involved in GA metabolism, in particular several AtGA20ox and AtGA2ox genes. Up-regulation occurred very quickly after auxin application; the response was mimicked by incubations with the protein synthesis inhibitor cycloheximide and was blocked by treatments with the proteasome inhibitor MG132. The effects of auxin treatment reflect endogenous regulation because equivalent changes in gene expression were observed in the auxin overproducer mutant yucca. The results suggest direct regulation of the expression of GA metabolism genes by Aux/IAA and ARF proteins. The physiological relevance of this regulation is supported by the observation that the phenotype of certain gain-of-function Aux/IAA alleles could be alleviated by GA application, which suggests that changes in GA metabolism mediate part of auxin action during development.     

Genetic manipulation of gibberellin metabolism in transgenic rice

The 'green revolution' was fueled by the introduction of the semi-dwarf trait into cereal crop cultivars. The semi-dwarf cultivars--which respond abnormally to the plant growth hormone gibberellin (GA)--are more resistant to wind and rain damage and thus yield more grain when fertilized. To generate dwarf rice plants using a biotechnological approach, we modified the level of GA by overproduction of a GA catabolic enzyme, GA 2-oxidase. When the gene encoding GA 2-oxidase, OsGA2ox1, was constitutively expressed by the actin promoter, transgenic rice showed severe dwarfism but failed to set grain because GA is involved in both shoot elongation and reproductive development. In contrast, OsGA2ox1 ectopic expression at the site of bioactive GA synthesis in shoots under the control of the promoter of a GA biosynthesis gene, OsGA3ox2 (D18), resulted in a semi-dwarf phenotype that is normal in flowering and grain development. The stability and inheritance of these traits shows the feasibility of genetic improvement of cereal crops by modulation of GA catabolism and bioactive GA content.     

Expression of novel rice gibberellin 2-oxidase gene is under homeostatic regulation by biologically active gibberellins

We have cloned two genes for gibberellin (GA) 2-oxidase from rice (Oryza sativa L.). Expression of OsGA2ox2 was not observed. The other gene, OsGA2ox3, was expressed in every tissue examined and was enhanced by the application of biologically active GA. Recombinant OsGA2ox3 protein catalyzed the metabolism of GA₁ to GA₈ and GA₂₀ to GA₂₉-catabolite. These results indicate that OsGA2ox3 is involved in the homeostatic regulation of the endogenous level of biologically active GA in rice. Electronic Publication