Gibberellin-regulated XET is differentially induced by auxin in rice leaf sheath bases during gravitropic bending

The asymmetric distribution of auxin plays a fundamental role in plant gravitropism, yet little is understood about how its lateral distribution stimulates growth. In the present work, the asymmetric distribution not only of auxin, but also that of gibberellins (GAs), was observed in rice leaf sheath bases following gravistimulation. Gravistimulation induced the transient accumulation of greater amounts of both IAA and GA in the lower halves of the leaf sheath bases of rice seedlings. OsGA3ox1, a gene of active GA synthesis, was differentially induced by gravistimulation. Furthermore, 2,3,5-tri-iodobenzoic acid (TIBA), an inhibitor of auxin transport, substantially decreased the asymmetric distribution of IAA and the gradient of OsGA3ox1 expression. Externally applied GA(3) restored the gravitropic curvature of rice leaf sheaths inhibited by either TIBA or by ancymidol, a GA synthesis inhibitor. The expression of XET (encoding xyloglucan endotransglycosylase) was differentially induced in the lower halves of gravistimulated leaf sheath bases and was also up-regulated by exogenous IAA and GA(3). Both ancymidol and TIBA decreased the gradient of XET expression. These data suggest that the asymmetric distribution of auxin effected by gravistimulation induced a gradient of GAs via asymmetric expression of OsGA3ox1 in rice leaf sheath bases, and hence caused the asymmetric expression of XET. Cell wall loosening in the curvature site of the leaf sheath triggered by the expression of XET would contribute to gravitropic growth.     

Organ and cellular localization of asparagine synthetase in rice plants

DNA gel blot analysis suggested that asparagine synthetase (AS; EC 6.3.5.4) occurred as a single gene in rice. A fusion protein consisting of 17 kDa tagged-region from pET32a(+) expression plasmid and 42 kDa N-terminal region of rice AS was first expressed in Escherichia coli. The resulting polypeptide was purified and a mono-specific antibody for rice AS was prepared after affinity-purification with the antigen. Immunoblotting revealed a high content of AS protein in the leaf sheath at the second position from the fully expanded top leaf and in grains at the middle stage of ripening. Accumulation of mRNA for AS was also observed in these organs. During the ripening of the spikelets, the AS protein contents increased during the first 21 days after flowering, then declined rapidly. Immunolocalization analysis revealed signals for AS protein in the companion cells of vascular bundles of leaf sheath and phloem-parenchyma cells, nucellar projection, and nucellar epidermis of dorsal vascular bundles of grains.     

Identification and characterization of a gibberellin-regulated protein,which is ASR5, in the basal region of rice leaf sheaths

Gibberellins (GAs) regulate growth and development in higher plants. To identify GA-regulated proteins during rice leaf sheath elongation, a proteomic approach was used. Proteins from the basal region of leaf sheath in rice seedling treated with GA₃ were analyzed by fluorescence two-dimensional difference gel electrophoresis. The levels of abscisic acid-stress-ripening-inducible 5 protein (ASR5), elongation factor-1 beta, translationally controlled tumor protein, fructose-bisphosphate aldolase and a novel protein increased; whereas the level of RuBisCO subunit binding-protein decreased by GA₃ treatment. ASR5 out of these six proteins was significantly regulated by GA₃ at the protein level but not at the mRNA level in the basal region of leaf sheaths. Since this protein is regulated not only by abscisic acid but also by GA₃, these results indicate that ASR5 might be involved in plant growth in addition to stress in the basal regions of leaf sheaths.     

Characterization of a xyloglucan endotransglucosylase gene that is up-regulated by gibberellin in rice

Xyloglucan endotransglucosylases/hydrolases (XTHs) that mediate cleavage and rejoining of the beta (1-4)-xyloglucans of the primary cell wall are considered to play an important role in the construction and restructuring of xyloglucan cross-links. A novel rice (Oryza sativa) XTH-related gene, OsXTH8, was cloned and characterized after being identified by cDNA microarray analysis of gibberellin-induced changes in gene expression in rice seedlings. OsXTH8 was a single copy gene; its full-length cDNA was 1,298 bp encoding a predicted protein of 290 amino acids. Phylogenetic analysis revealed that OsXTH8 falls outside of the three established subfamilies of XTH-related genes. OsXTH8 was preferentially expressed in rice leaf sheath in response to gibberellic acid. In situ hybridization and OsXTH8 promoter GUS fusion analysis revealed that OsXTH8 was highly expressed in vascular bundles of leaf sheath and young nodal roots where the cells are actively undergoing elongation and differentiation. OsXTH8 gene expression was up-regulated by gibberellic acid and there was very little effect of other hormones. In two genetic mutants of rice with abnormal height, the expression of OsXTH8 positively correlated with the height of the mutants. Transgenic rice expressing an RNAi construct of OsXTH8 exhibited repressed growth. These results indicate that OsXTH8 is differentially expressed in rice leaf sheath in relation to gibberellin and potentially involved in cell elongation processes.     

Analysis of embryonic proteome modulation by GA and ABA from germinating rice seeds

The phytohormones gibberellic acid (GA) and abscisic acid (ABA) play essential and often antagonistic roles in regulating plant growth, development, and stress responses. Using a proteomics-based approach, we examined the role of GA and ABA in the modulation of protein expression levels during seed germination. Rice seeds were treated with GA (200 microM), ABA (10 microM), ABA followed by GA, GA followed by ABA, and water as a control and then incubated for 3 days. The embryo was dissected from germinated seeds, and proteins were subjected to 2-DE. Approximately, 665 total protein spots were resolved in the 2-D gels. Among them, 16 proteins notably modulated by either GA or ABA were identified by MALDI-TOF MS. Northern analyses demonstrated that expression patterns of 13 of these 16 genes were consistent with those of the proteome analysis. Further examination of two proteins, rice isoflavone resuctase (OsIFR) and rice PR10 (OsPR10), using Western blot and immunolocalization, revealed that both are specifically expressed in the embryo but not in the endosperm and are dramatically downregulated by ABA.     

Comparison of two proteomics techniques used to identify proteins regulated by gibberellin in rice

Proteomics has become an essential methodology for large-scale analysis of proteins in various fields of plant biology. We compared two proteomics techniques, two-dimensional liquid chromatography (2D-LC) and fluorescence two-dimensional difference gel electrophoresis (2D-DIGE), for their ability to identify proteins regulated by gibberellin (GA) in rice. Two-week-old rice seedlings were treated with or without 5 microM GA3 for 48 h and proteins extracted from the basal region of the leaf sheath. After separation of the proteins by the two techniques, the amino acid sequences of GA3-responsive proteins were analyzed using a protein sequencer and mass spectrometry. 2D-LC and 2D-DIGE were able to resolve 1248 protein fractions and 1500 proteins, respectively. Out of these, 2D-LC identified 9 proteins that were up-regulated and 9 that were down-regulated by GA treatment; 2D-DIGE identified 4 up-regulated and 4 down-regulated proteins. The two techniques detected overlapping sets of proteins. For example, cytosolic glyceraldehyde-3-phosphate dehydrogenase and photosystem II oxygen-evolving complex protein were identified as GA3-regulated proteins by both methods. In addition, these two methods uncovered GA3-regulated unknown proteins which had not been reported previously, and novel proteins which are not detected in 2D-PAGE followed by Coomassie brilliant blue staining. These results suggest that these two methods are among some of the very useful tools for detecting proteins that may function in various physiological and developmental processes in plants.     

Microarray and proteomic analysis of brassinosteroid- and gibberellin-regulated gene and protein expression in rice

Brassinosteroid (BR) and gibberellin (GA) are two groups of plant growth regulators essential for normal plant growth and development. To gain insight into the molecular mechanism by which BR and GA regulate the growth and development of plants, especially the monocot plant rice, it is necessary to identify and analyze more genes and proteins that are regulated by them. With the availability of draft sequences of two major types, japonica and indica rice, it has become possible to analyze expression changes of genes and proteins at genome scale. In this review, we summarize rice functional genomic research by using microarray and proteomic approaches and our recent research results focusing on the comparison of cDNA microarray and proteomic analyses of BR- and GA-regulated gene and protein expression in rice. We believe our findings have important implications for understanding the mechanism by which BR and GA regulate the growth and development of rice.     

Proteome approach to characterize the methylmalonate-semialdehyde dehydrogenase that is regulated by gibberellin

Proteins regulated by gibberellin (GA) in rice were determined by proteome analysis. Proteins extracted from suspension culture cells of slr1, a constitutive GA response mutant of rice, were separated by two-dimensional polyacrylamide gel electrophoresis, and three proteins were greatly accumulated in the mutant. The most up-regulated protein was methylmalonate-semialdehyde dehydrogenase (MMSDH), and the amount of protein was 7-fold that of wild type. In this study, the function of MMSDH in rice was analyzed. MMSDH gene expression in suspension culture cells, roots, and leaf sheaths ofslr1 was higher than that in its wild-type. MMSDH expression in wild-type roots was increased by exogenous GA(3). Analyzed by in situ hybridization, MMSDH mRNA was expressed in root primordia of slr1, where cells are undergoing growth. MMSDH gene expression in the root zone of tissue differentiation was higher than in the elongation zone or meristem. Transgenic rice expressing antisense MMSDH showed that its seminal roots were thinner than that of control, and that the leaf sheath elongation was slightly inhibited compared to control. Concentrations of TCA cycle metabolites were decreased in the antisense plants as compared with the control plants, suggesting that acetyl-CoA was reduced in the antisense plants. These results suggest that one of the regulations by GA signal transduction including SLR1 is the expression of MMSDH, and that MMSDH may play a role in root development and leaf sheath elongation in rice.     

A proteomic approach to analyze salt-responsive proteins in rice leaf sheath

To examine the response of rice to salt stress, changes in protein expression were analyzed using a proteomic approach. To investigate dose- and time-dependent responses, rice seedlings were exposed to 50, 100 and 150 mM NaCl for 6 to 48 h. Proteins were extracted from leaf sheath and separated by two-dimensional polyacrylamide gel electrophoresis. Eight proteins showed 1- to 3-fold up-regulation in leaf sheath, in response to 50 mM NaCl for 24 h. Among these, three proteins were unidentified (LSY081, LSY262 and LSY363) while five proteins were identified as fructose bisphosphate aldolases, photosystem II (PSII) oxygen evolving complex protein, oxygen evolving enhancer protein 2 (OEE2) and superoxide dismutase (SOD). The maximum expression levels of seven proteins were at 24 h. Their expression declined after 48 h of 50 mM NaCl treatment. In contrast, SOD maintained its elevated expression throughout these conditions. The increased expression of proteins seen in the 50 mM NaCl treatment group was less pronounced in the groups receiving 100 or 150 mM NaCl for 24 h. The expression of SOD was a common response to cold, drought, salt and abscisic acid (ABA) stresses while the expression of LSY081, LSY363 and OEE2 was enhanced by salt and ABA stresses. LSY262 was expressed in leaf sheath and root, while fructose bisphosphate aldolases, PSII oxygen evolving complex protein and OEE2 were expressed in leaf sheath and leaf blade. LSY363 was expressed in leaf sheath but was below the level of detection in leaf blade and root. These results indicate that specific proteins expressed in specific regions of rice show a coordinated response to salt stress.     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced by cold and gibberellin in rice leaf sheath

Calcium-dependent protein kinases (CDPKs) play an important role in rice signal transduction, but the precise role of each individual CDPK is still largely unknown. Recently, a full-length cDNA encoding OsCDPK13 from rice seedling was isolated. To characterize the function of OsCDPK13, its responses to various stresses and hormones were analyzed in this study. OsCDPK13 accumulated in 2-week-old leaf sheath and callus, and became phosphorylated in response to cold and gibberellin (GA). OsCDPK13 gene expression and protein accumulation were up-regulated in response to GA₃ treatment, but suppressed in response to abscisic acid and brassinolide. Antisense OsCDPK13 transgenic rice lines were shorter than the vector control lines, and the expression of OsCDPK13 was lower in dwarf mutants of rice than in wild type. Furthermore, OsCDPK13 gene expression and protein accumulation were enhanced in response to cold, but suppressed under salt and drought stresses. Sense OsCDPK13 transgenic rice lines had higher recovery rates after cold stress than vector control rice. The expression of OsCDPK13 was stronger in cold-tolerant rice varieties than in cold-sensitive ones. The results suggest that OsCDPK13 might be an important signaling component in the response of rice to GA and cold stress.     

Proteomic analysis of jasmonic acid-regulated proteins in rice leaf blades

Jasmonates play a critical role in plant defense against pathogens through regulation of the expression of defense-related genes. To study the role of jasmonic acid (JA) in the rice self-defense mechanism, a proteomic approach was applied. When 3-week-old rice cv. Java 14 was treated with 100 microM JA for 3 days, numerous necrotic brown spots were observed on the leaf blade. Three-week-old rice was treated with JA and proteins from cytosolic and membrane fractions of leaf blade were separated by two-dimensional polyacrylamide gel electrophoresis. A total of 305 proteins were detected in both cytosolic and membrane fractions. When rice plant was treated with 100 microM JA for 2 days, 12 proteins were up-regulated and 2 proteins were down-regulated. Out of them, 8 proteins were changed in dose dependence manner, while 4 proteins were changed in a time course manner. Among them, pathogenesis-related protein 5 (PR5) and probenazole inducible protein 1 (PBZ1) were significantly induced by 100 microM JA for 2 days. These results suggest that PR5 and PBZ1 are important proteins expressed down-stream of JA signals in rice cv. Java 14.     

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.     

Promotion of leaf sheath growth by gibberellic acid in a dwarf mutant of rice

The mechanism of gibberellin (GA)-induced leaf sheath growth was examined using a dwarf mutant of rice (Oryza sativa L. cv. Tan-ginbozu) treated in advance with an inhibitor of GA biosynthesis. Gibberellic acid (GA₃) enhanced the growth of the second leaf sheath, but auxins did not. Measurement of the mitotic index and cell size revealed that cell elongation rather than cell division is promoted by GA₃. Gibberellic acid increased the extensibility of cell walls in the elongation zone of the leaf sheath. It also increased the total amount of osmotic solutes including sugars in the leaf sheath, but did not increase the osmotic concentration of the cell sap, due to an accompanying increase in cell volume by water absorption. In the later stage of GA₃-induced growth, starch granules completely disappeared from leaf sheath cells, whereas dense granules remained in control plants. These findings indicate that GA enhances cell elongation by increasing wall extensibility, osmotic concentration being kept unchanged by starch degradation. Received: 28 August 1997 / Accepted: 16 October 1997     

Characterization of a histidine- and alanine-rich protein showing interaction with calreticulin in rice

Calreticulin (CRT) is a major calcium-sequestering protein in the endoplasmic reticulum and has been implicated in a variety of cellular functions. To analyze the function of CRT in rice, a yeast two-hybrid protein interaction assay was used for identifying interacting proteins. Fourteen of 17 interacting cDNA clones found coded for a novel histidine- and alanine-rich protein (OsHARP) of 342 amino acid residues. The mRNA expression level of OsHARP was up-regulated in rice seedlings treated with gibberellin (GA), but not ABA and showed a similar pattern as OsCRT mRNA. Rice plants transformed with the OsHARP promoter-GUS construct showed GUS staining in the basal parts of leaf sheaths, and although GUS activity increased when treated with GA₃, it was not as high an increase as when mRNA was analyzed. To elucidate the role of OsHARP in leaf sheath elongation, antisense OsHARP transgenic rice lines were constructed. The antisense OsHARP transgenic rice plants were consistently shorter than the vector control under normal conditions. To examine whether OsHARP expression would affect other proteins, basal leaf sheaths from antisense OsHARP transgenic rice plants were analyzed using proteomic techniques. In antisense transgenic-rice OsHARP plants, OsCRT was down-regulated and the levels of 20 other proteins were changed compared to the pattern of the vector control. These results signify an important role of HARP in rice leaf sheath cell division or elongation and suggest that CRT may interact with HARP during certain stages of development.