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.     

Cold stress-induced calcium-dependent protein kinase(s) in rice (Oryza sativa L.) seedling stem tissues

Ca²⁺-dependent protein kinases (CDPKs) play an important role in plant signal transduction. Protein kinase(s) activities induced by 5°C cold stress in rice (Oryza sativa L.) seedlings were investigated in both leaf and stem tissues in an early (up to 45 min) and late (up to 12 h) response study. The leaf had 37-, 47- and 55-kDa protein kinase activities, and the stem had 37-, 47- and 55-kDa protein kinase activities. A 16-kDa protein showed constitutive kinase activity in the rice seedling leaf and stem. It was further identified that the 47-kDa protein kinase activity induced by cold in both the cytosolic and membrane fractions of the stem was strictly Ca²⁺-dependent. This CDPK activitiy increased in the presence of the Ca²⁺ ionophore A23187 in stem segments, whereas it was decreased by the Ca²⁺ channel blocker, LaCl₃, and the Ca²⁺ chelator, EGTA. The general protein kinase inhibitor, staurosporine, completely inhibited this CDPK activity in vitro, and both W7, a calmodulin antagonist, and H7, a protein kinase C inhibitor, could only partially decrease this activity. The protein phosphatase inhibitor, okadaic acid, increased CDPK activity. This CDPK activity was also induced by salt, drought stress and the phytohormone abscicic acid. Among the 18 rice varieties tested, this cold-induced 47-kDa CDPK activity was stronger in the cold-tolerant varieties than in the sensitive ones. Received: 13 August 1999 / Accepted: 24 January 2000     

OsCDPK13, a calcium-dependent protein kinase gene from rice,is induced in response to cold and gibberellin

Ca²⁺-dependent protein kinases (CDPKs) (EC 2.7.1.37) are the predominant Ca²⁺-regulated serine/threonine protein kinase in plants and their genes are encoded by a multigene family. CDPKs are important components in signal transduction, but the precise role of each individual CDPK is still largely unknown. A CDPK gene designated as OsCDPK13 was cloned from rice seedlings and it showed a high level of sequence similarities to rice and other plant CDPK genes. OsCDPK13 contains all conserved regions found in CDPKs. It was a single copy gene and was highly expressed in root and leaf sheath tissues of rice seedlings. OsCDPK13 expression was increased in leaf sheath segments treated with gibberellin or subjected to cold stress. The results in this investigation, together with our previous studies, suggest that OsCDPK13 may be an important signaling component in rice seedlings under cold stress condition and in response to gibberellin.     

Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice

Calcium acts as a messenger in various signal transduction pathways in plants. Calcium-dependent protein kinases (CDPKs) play important roles in regulating downstream components in calcium signaling pathways. In rice, the CDPKs constitute a large multigene family consisting of 29 genes, but the biological functions and functional divergence or redundancy of most of these genes remain unclear. Using a mini-scale full-length cDNA overexpressor (FOX) gene hunting system, we generated 250 independent transgenic rice plants overexpressing individual rice CDPKs (CDPK FOX-rice lines). These CDPK FOX-rice lines were screened for salt stress tolerance. The survival rate of the OsCPK21-FOX plants was higher than that of wild-type (WT) plants grown under high salinity conditions. The inhibition of seedling growth by abscisic acid (ABA) treatment was greater in the OsCPK21-FOX plants than in WT plants. Several ABA- and high salinity-inducible genes were more highly expressed in the OsCPK21-FOX plants than in WT plants. These results suggest that OsCPK21 is involved in the positive regulation of the signaling pathways that are involved in the response to ABA and salt stress.     

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.     

Involvement of a Ca(2+)-dependent protein kinase component downstream to the gibberellin-binding phosphoprotein, RuBisCO activase, in rice.

Previously, we reported the identification of a gibberellin (GA)-binding protein in rice using ligand binding assay that was homologous to RuBisCO activase (Komatsu et al., FEBS Lett. 384, 167-171, 1996). Here, we provide an evidence for the involvement of protein kinases components downstream to the GA-binding phosphoprotein, RuBisCO activase in rice. Ca(2+)-dependent protein kinase activity was studied in subcellular fractions of leaf sheath from transgenic rice containing sense and antisense constructs of RuBisCO activase. In-gel kinase assay using histone III-S as a substrate showed constitutive induction of a 46- and 48-kDa Ca(2+)-dependent protein kinase activity in the sense transgenic plants. Kinase activities of these proteins were significantly reduced in the presence of uniconazole, a potent GA biosynthesis inhibitor, but one of them was strongly promoted by GA(3) treatment in transgenic plants carrying a smaller subunit of RuBisCO activase (OsrcaA1) compared to the larger subunit OsrcaA2. Also, in vitro phosphorylation studies using two-dimensional polyacrylamide gel showed changes in the degree of phosphorylation of several proteins in OsrcaA1- and OsrcaA2-sense transgenic rice. These studies suggest the presence of two independent cytosolic Ca(2+)-dependent protein kinase signaling components downstream to the GA-binding protein in rice suggesting their role in GA signaling.     

Down-Regulation of OsSPX1 Causes High Sensitivity to Cold and Oxidative Stresses in Rice Seedlings

Rice SPX domain gene, OsSPX1, plays an important role in the phosphate (Pi) signaling network. Our previous work showed that constitutive overexpression of OsSPX1 in tobacco and Arabidopsis plants improved cold tolerance while also decreasing total leaf Pi. In the present study, we generated rice antisense and sense transgenic lines of OsSPX1 and found that down-regulation of OsSPX1 caused high sensitivity to cold and oxidative stresses in rice seedlings. Compared to wild-type and OsSPX1-sense transgenic lines, more hydrogen peroxide accumulated in seedling leaves of OsSPX1-antisense transgenic lines for controls, cold and methyl viologen (MV) treatments. Glutathione as a ROS scavenger could protect the antisense transgenic lines from cold and MV stress. Rice whole genome GeneChip analysis showed that some oxidative-stress marker genes (e.g. glutathione S-transferase and P450s) and Pi-signaling pathway related genes (e.g. OsPHO2) were significantly down-regulated by the antisense of OsSPX1. The microarray results were validated by real-time RT-PCR. Our study indicated that OsSPX1 may be involved in cross-talks between oxidative stress, cold stress and phosphate homeostasis in rice seedling leaves.     

A novel interaction between calreticulin and ubiquitin-like nuclear protein in rice

Calreticulin (CRT), a major Ca²⁺-sequestering protein, has beenimplicated in a variety of cellular functions such as Ca²⁺ storage,signaling and chaperone activity within the cytoplasm and endoplasmicreticulum. To investigate the biological role of CRT in rice,21 partial cDNAs, encoding proteins that interacted with riceCRT in a yeast two-hybrid interaction-cloning system, were characterizedand the nucleotide sequences were found to be identical to eachother. A full-length cDNA of 3.5 kb, obtained from ricegenomic sequence data and 5' RACE, codes for a novel proteinof 966 amino acid residues and was designated as CRTintP (CRTinteracting protein). Primary sequence analysis of CRTintP showedno sequence homology with the known functional proteins; however,a potential ubiquitin-like domain at the N-terminal togetherwith a putative leucine zipper, a nuclear localization signaland several sites for serine/threonine kinases were evident.Cellular localization of CRTintP demonstrated its role in directinggreen fluorescent protein to the nucleus in onion epidermalcells. Northern and immunoblot analysis showed increased expressionof CRT and CRTintP in response to cold stress. Co-immunoprecipitationusing anti-CRT antibodies confirmed the existence of the CRT-CRTintPcomplex in vivo in the stressed leaf tissue, suggesting theirpotential role in regulating stress response. ⁴ Corresponding author: E-mail, skomatsu{at}affrc.go.jp; Fax, +81-298-38-7464.     

Production and characterization of auxin-insensitive rice by overexpression of a mutagenized rice IAA protein

Since auxin was first isolated and characterized as a plant hormone, the underlying molecular mechanism of auxin signaling has been elucidated primarily in dicot plants represented by Arabidopsis. In monocot plants, the molecular mechanism of auxin signaling has remained unclear, despite various physiological experiments. To understand the function and mechanism of auxin signaling in rice ( Oryza sativa ), we focused on the IAA gene, a well-studied gene in Arabidopsis that serves as a negative regulator of auxin signaling. We found 24 IAA gene family members in the rice genome. OsIAA3 is one of these family members whose expression is rapidly increased in response to auxin. We produced transgenic rice harboring m OsIAA3 - GR , which can overproduce mutant OsIAA3 protein containing an amino acid change in domain II to cause a gain-of-function phenotype, by treatment with dexamethasone. The transgenic rice was insensitive to auxin and gravitropic stimuli, and exhibited short leaf blades, reduced crown root formation, and abnormal leaf formation. These results suggest that , in rice, auxin is important for development and its signaling is mediated by IAA genes.     

Interaction of N-acetylglutamate kinase with a PII-like protein in rice

PII protein in bacteria is a sensor for 2-oxoglutarate and a transmitter for glutamine signaling. We identified an OsGlnB gene that encoded a bacterial PII-like protein in rice. Yeast two-hybrid analysis showed that an OsGlnB gene product interacted with N-acetylglutamate kinase 1 (OsNAGK1) and PII-like protein (OsGlnB) itself in rice. In cyanobacteria, NAGK is a key enzyme in arginine biosynthesis. Transient expression of OsGlnB cDNA or OsNAGK1 cDNA fused with sGFP in rice leaf blades strongly suggested that the PII-like protein as well as OsNAGK1 protein is located in chloroplasts. Both OsGlnB and OsNAGK1 genes were expressed in roots, leaf blades, leaf sheaths and spikelets of rice, and these two genes were coordinately expressed in leaf blades during the life span. Thus, PII-like protein in rice plants is potentially able to interact with OsNAGK1 protein in vivo. This finding will provide a clue to the precise physiological function of PII-like protein in rice.     

Over-expression of a single Ca2+-dependent protein kinase confers both cold and salt/drought tolerance on rice plants

A rice gene encoding a calcium-dependent protein kinase (CDPK), OsCDPK7, was induced by cold and salt stresses. To elucidate the physiological function of OsCDPK7, we generated transgenic rice plants with altered levels of the protein. The extent of tolerance to cold and salt/drought stresses of these plants correlated well with the level of OsCDPK7 expression. Therefore, OsCDPK7 was shown to be a positive regulator commonly involved in the tolerance to both stresses in rice. Over-expression of OsCDPK7 enhanced induction of some stress-responsive genes in response to salinity/drought, but not to cold. Thus, it was suggested that the downstream pathways leading to the cold and salt/drought tolerance are different from each other. It seems likely that at least two distinct pathways commonly use a single CDPK, maintaining the signalling specificity through unknown post-translational regulation mechanisms. These results demonstrate that simple manipulation of CDPK activity has great potential with regard to plant improvement.     

Proteomic analysis of bacterial blight defence signalling pathway using transgenic rice overexpressing thaumatin-like protein

Rice overexpressed thaumatin-like protein gene and the proteins from the leaf blades of 2-week-old transgenic rice seedlings were fractionated into cytosolic and membrane fractions, and separated by two-dimensional polyacrylamide gel electrophoresis and stained with Commassie brilliant blue. Among of 440 detected proteins, 5 proteins were up-regulated and 5 proteins were down-regulated by the overexpression of thaumatin-like protein. In the sense thaumatin-like protein transgenic rice and/or in rice inoculated with Xanthomonas oryzae pv. oryzae (Xo7435), 2-cys peroxiredoxin, thaumatin-like protein and glycine cleavage H protein were up-regulated, while oxygen evolving complex protein 2 was down-regulated. These results suggest that thaumatin-like protein-mediated disease resistance of rice against bacterial blight disease is the results of changes in proteins related to oxidative stress and energy metabolism in addition to changes in proteins related to defence.     

Field evaluation and risk assessment of transgenic indica basmati rice

We report the first field trial of different transgenic lines of Indica Basmati rice (B-370) expressing cry1Ac and cry2A genes. Different transgenic lines were grown under field conditions for two consecutive years, according to RCBD and Split Plot Design respectively. All the biosafety measures were taken into consideration. Sixty neonate larvae of yellow stem borer were artificially infested into each plant in three installments. Data was recorded in terms of dead hearts and white heads at vegetative and flowering stage respectively. Transgenic lines exhibited inherent ability to protect rice plants from target insects (p<0.01). Natural infestations of rice skipper and rice leaf folder were also observed and transgenic plants were statistically superior to their untransformed counterparts. Green house whole plant bioassays were done by infesting two 2^nd instar larvae of rice leaf folder per tiller. Transgenics were 96% more resistant than untransformed control plants. The presence of cry genes was observed with Dot blot, PCR and Southern blot analysis, while ELISA and Western blot analysis confirmed the expression of Cry proteins. All lines expressed higher level of Cry proteins when compared with commercially released cultivars of Bt cotton, maize and potato. It was also observed that although toxin titer substantially decreased with increasing age of the plants, it remained well within the limits to kill the target insects. Morphological studies showed significant variation for days to maturity, plant height and panicle length. Cooking qualities of seeds harvested from these lines were compared with the untransformed control. The transgenic lines had no effect on non-target insects (insects belonging to orders other than diptera and lepidoptera) and germination of three local varieties of wheat. Chances of gene spread were calculated at a level of 0.18% cross pollination in experimental lines.     

Overexpression of The Calcium-Dependent Protein Kinase OsCDPK2 in Transgenic Rice is Repressed by Light in Leaves and Disrupts Seed Development

Independent transgenic rice lines overexpressing the rice CDPK isoform OsCDPK2 were generated by particle bombardment. High levels of OsCDPK2 were detected in leaves removed from etiolated plants, as well as in stems and flowers. However, there was no overexpression in green leaves that had been exposed to light, confirming that OsCDPK2 protein stability was subject to light regulation. The morphological phenotype of transgenic plants producing high levels of recombinant OsCDPK2 was normal until the onset of seed development. Flowers developed normally, producing well-shaped ovaries and stigmas, and mature anthers filled with pollen grains. However, seed formation in these plants was strongly inhibited, with only 3–7% of the flowers producing seeds. Seed development was arrested at an early stage. We discuss these data with respect to the possible requirement for specific CDPK isoforms during rice seed 4.4ptdevelopment.     

Phosphorylation upon cold stress in rice (Oryza sativa L.) seedlings

The response of plants to cold stress is not well understood at the biochemical level, although it has been studied extensively at the ecological level. To investigate whether protein phosphorylation may play an important role in cold stress, we exposed rice seedlings to low temperatures, prepared protein extracts from the leaves and incubated these in the presence of [γ-³²P]ATP. The proteins were then separated by two-dimensional polyacrylamide gel electrophoresis. While several proteins were found to be phosphorylated upon cold stress one protein, pp35, which has an isoelectric point of 8.0, was more phosphorylated than the others. The pp35 protein was found to be phosphorylated when rice seedlings were incubated for 6 h at 5°C before the leaf protein extract was prepared and radioactive labeling was performed. The pp35 was, however, significantly more phosphorylated in cold-tolerant rice varieties. Antibodies were raised against purified pp35 in adult rabbits. Using this pp35 antibody, which can recognize the RuBisCO large-chain subunit (LSU), and from amino acid sequencing of pp35, we were able to identify and confirm the pp35 protein as the fragment of RuBisCO LSU (EC 4.1.1.39). Phosphorylation of the RuBisCO LSU may be important in cold tolerance. Received: 7 July 1998 / Accepted: 19 December 1998     

Overexpression of a western white pine PR10 protein enhances cold tolerance in transgenic Arabidopsis

The PmPR10-1.10 protein from western white pine is known to be associated with frost hardiness, and up-regulated by seasonal cold acclimation and biotic and abiotic stresses. To gain insight into the molecular basis of cold hardiness, we investigated the potential physiological role of PmPR10-1.10 by gene overexpression in transgenic Arabidopsis plants. A binary vector was constructed for PmPR10-1.10 synthesis in higher plants and transgenic Arabidopsis lines were generated by Agrobacterium-mediated transformation. Following Western protein blot analysis confirming target protein production, transgenic Arabidopsis lines were tested for cold tolerance by electrolyte leakage analysis post treatment of different freezing temperatures. Our results demonstrate that accumulation of PmPR10-1.10 protein resulted in significantly greater freezing tolerance in transgenic plants than in wild type plants. This indicates that the transfer and selection of cold acclimation proteins like PmPR10-1.10 may be a breeding strategy for the development of freezing tolerance in conifers.     

Low temperature treatment at the young microspore stage induces protein changes in rice anthers

Male reproductive development in rice is very sensitive to various forms of environmental stresses including low temperature. A few days of cold treatment (<20 degrees C) at the young microspore stage induce severe pollen sterility and thus large grain yield reductions. To investigate this phenomenon, anther proteins at the early stages of microspore development, with or without cold treatment at 12 degrees C, were extracted, separated by two-dimensional gel electrophoresis, and compared. The cold-sensitive cultivar Doongara and the relatively cold-tolerant cultivar HSC55 were used. The abundance of 37 anther proteins was changed more than 2-fold after 1, 2, and 4 days of cold treatment in cv. Doongara. Among them, one protein was newly induced, 32 protein spots were up-regulated, and four protein spots were down-regulated. Of these 37 protein spots, we identified two anther-specific proteins (putative lipid transfer protein and Osg6B) and a calreticulin that were down-regulated and a cystine synthase, a beta-6 subunit of the 20 S proteasome, an H protein of the glycine cleavage system, cytochrome c oxidase subunit VB, an osmotin protein homologue, a putative 6-phosphogluconolactonase, a putative adenylate kinase, a putative cysteine proteinase inhibitor, ribosomal protein S12E, a caffeoyl-CoA O-methyltransferase, and a monodehydroascorbate reductase that were up-regulated. Identification of these proteins is available upon request. Accumulation of these proteins did not vary greatly after cold treatment in panicles of cv. Doongara or in the anthers of the cv. HSC55. The newly induced protein named Oryza sativa cold-induced anther protein (OsCIA) was identified as an unknown protein. The OsCIA protein was detected in panicles, leaves, and seedling tissues under normal growth conditions. Quantitative real time RT-PCR analysis of OsCIA mRNA expression showed no significant change between low temperature-treated and untreated plants. A possible regulatory role for the newly induced protein is proposed.     

新城疫病毒融合蛋白在转基因水稻叶片中的表达及其免疫原性

利用转基因植物作为生物反应器表达抗原蛋白具有广阔的应用前景。以新城疫病毒融合蛋白（NDVF）基因1.7kb全长编码区序列为外源基因与组成型表达的玉米泛素蛋白基因（Ubi）启动子和农杆菌胭脂碱合成酶基因（nos）终止子组成嵌合基因,构建了适宜于农杆菌介导转化水稻的转化载体pUNDV,经根癌农杆菌介导的遗传转化方法将由Ubi动子驱动的NDVF嵌合基因导入水稻细胞中,经潮霉素抗性筛选,共再生获得了6个独立的转基因株系。PCR分析结果表明NDVF基因已整合到水稻基因组中。ELISA和Western blot分析结果证实NDVF蛋白在部分转基因水稻叶片组织中获得表达,其中植株F5叶片组织中具有较高的表达水平。将F5叶片可溶性总蛋白皮下注射免疫BALB／c小鼠,结果表明能够诱导小鼠产生一定水平的NDVF蛋白特异抗体。