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.     

A new <Emphasis Type="Italic">Em</Emphasis>-like protein from <Emphasis Type="Italic">Lactuca sativa</Emphasis>, <Emphasis Type="Italic">LsEm1</Emphasis>, enhances drought and salt stress tolerance in <Emphasis Type="Italic">Escherichia coli</Emphasis> and rice

Late embryogenesis abundant (LEA) proteins are closely related to abiotic stress tolerance of plants. In the present study, we identified a novel Em-like gene from lettuce, termed LsEm1, which could be classified into group 1 LEA proteins, and shared high homology with Cynara cardunculus Em protein. The LsEm1 protein contained three different 20-mer conserved elements (C-element, N-element, and M-element) in the C-termini, N-termini, and middle-region, respectively. The LsEm1 mRNAs were accumulated in all examined tissues during the flowering and mature stages, with a little accumulation in the roots and leaves during the seedling stage. Furthermore, the LsEm1 gene was also expressed in response to salt, dehydration, abscisic acid (ABA), and cold stresses in young seedlings. The LsEm1 protein could effectively reduce damage to the lactate dehydrogenase (LDH) and protect LDH activity under desiccation and salt treatments. The Escherichia coli cells overexpressing the LsEm1 gene showed a growth advantage over the control under drought and salt stresses. Moreover, LsEm1-overexpressing rice seeds were relatively sensitive to exogenously applied ABA, suggesting that the LsEm1 gene might depend on an ABA signaling pathway in response to environmental stresses. The transgenic rice plants overexpressing the LsEm1 gene showed higher tolerance to drought and salt stresses than did wild-type (WT) plants on the basis of the germination performances, higher survival rates, higher chlorophyll content, more accumulation of soluble sugar, lower relative electrolyte leakage, and higher superoxide dismutase activity under stress conditions. The LsEm1-overexpressing rice lines also showed less yield loss compared with WT rice under stress conditions. Furthermore, the LsEm1 gene had a positive effect on the expression of the OsCDPK9, OsCDPK13, OsCDPK15, OsCDPK25, and rab21 (rab16a) genes in transgenic rice under drought and salt stress conditions, implying that overexpression of these genes may be involved in the enhanced drought and salt tolerance of transgenic rice. Thus, this work paves the way for improvement in tolerance of crops by genetic engineering breeding.     

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.     

Expression of TERF1 in rice regulates expression of stress-responsive genes and enhances tolerance to drought and high-salinity

Drought and high-salinity are the important constraints that severely affect plant development and crop yield worldwide. It has been established that ethylene response factor (ERF) proteins play important regulatory roles in plant response to abiotic and biotic stresses. Our previous researches have revealed that transgenic tobacco over-expressing TERF1 (encoding a tomato ERF protein) showed enhanced tolerance to abiotic stress. Here, we further investigate the function of TERF1 in transgenic rice. Compared with the wild-type plants, overexpression of TERF1 resulted in an increased tolerance to drought and high-salt in transgenic rice. And the enhanced tolerance may be associated with the accumulation of proline and the decrease of water loss. Furthermore, TERF1 can effectively regulate the expression of stress-related functional genes Lip5, Wcor413-l, OsPrx and OsABA2, as well as regulatory genes OsCDPK7, OsCDPK13 and OsCDPK19 under normal growth conditions. Our analyses of cis-acting elements show that there exist DRE/CRT and/or GCC-box existing in TERF1 targeted gene promoters. Our results revealed that ectopic expression of TERF1 in rice caused a series of molecular and physiological alterations and resulted in the transgenic rice with enhanced tolerance to abiotic stress, indicating that TERF1 might have similar regulatory roles in response to abiotic stress in tobacco and rice. Shumei Gao, Haiwen Zhang and Yun Tian contributed equally to this work.     

Rice 14-3-3 protein (GF14e) negatively affects cell death and disease resistance

Plant 14-3-3 proteins regulate important cellular processes, including plant immune responses, through protein-protein interactions with a wide range of target proteins. In rice (Oryza sativa), the GF14e gene, which encodes a 14-3-3 protein, is induced during effector-triggered immunity (ETI) associated with pathogens such as Xanthomonas oryzae pv. oryzae (Xoo). To determine whether the GF14e gene plays a direct role in resistance to disease in rice, we suppressed its expression by RNAi silencing. GF14e suppression was correlated with the appearance of a lesion-mimic (LM) phenotype in the transgenic plants at 3 weeks after sowing. This indicates inappropriate regulation of cell death, a phenotype that is frequently associated with enhanced resistance to pathogens. GF14e-silenced rice plants showed high levels of resistance to a virulent strain of Xoo compared with plants that were not silenced. Enhanced resistance was correlated with GF14e silencing prior to and after development of the LM phenotype, higher basal expression of a defense response peroxidase gene (POX22.3), and accumulation of reactive oxygen species (ROS). In addition, GF14e-silenced plants also exhibit enhanced resistance to the necrotrophic fungal pathogen Rhizoctonia solani. Together, our findings suggest that GF14e negatively affects the induction of plant defense response genes, cell death and broad-spectrum resistance in rice.     

OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against Magnaporthe grisea

Calcium-dependent protein kinases are important decoders of calcium signals in plants, which are involved in plant immunity. We report isolation and functional characterization of a pathogen-responsive OsCPK20 gene in rice. The expression of OsCPK20 in rice was significantly induced following treatment with a Magnaporthe grisea elicitor. Overexpression of constitutively active OsCPK20 in Arabidopsis enhanced the resistance to infection with Pseudomonas syringae pv. tomato, associated with elevated expression of both SA- and JA-related defense genes. Similarly, transgenic rice plants containing constitutively active OsCPK20 exhibited enhanced resistance to blast fungus M. grisea. The enhanced resistance in the transgenic Arabidopsis and rice was associated with activated expression of both SA- and JA-related defense genes. We also found that OsCPK20 was significantly induced by drought stress, indicating that OsCPK20 might be involved in plant response to drought stress. Taken together, our results indicate that rice OsCPK20 positively regulates Arabidopsis resistance against Pseudomonas syringae pv. tomato and rice resistance against M. grisea, and that it may enhance disease resistance by activating both SA- and JA-dependent defense responses.     

The modified rice αAmy8 promoter confers high-level foreign gene expression in a novel hypoxia-inducible expression system in transgenic rice seedlings

Expression of α-amylase genes in rice is induced not only by sugar starvation and gibberellin (GA) but also by O₂ deficiency. Promoters of two rice α-amylase genes, αAmy3 and αAmy8, have been shown to direct high-level production of recombinant proteins in rice suspension cells and germinated seeds. In the present study, we modified the cis-acting DNA elements within the sugar/GA response complex (SRC/GARC) of αAmy8 promoter. We found that addition of a G box and duplicated TA box leads to high-level expression of αAmy8 SRC/GARC and significantly enhances αAmy8 promoter activity in transformed rice cells and germinated transgenic rice seeds. We also show that these modifications have drastically increased the activity of αAmy8 promoter in rice seedlings under hypoxia. Our results reveal that the G box and duplicated TA box may play important roles in stimulating promoter activity in response to hypoxia in rice. The modified αAmy8 promoter was used to produce the recombinant human epidermal growth factor (hEGF) in rice cells and hypoxic seedlings. We found that the bioactive recombinant hEGF are stably produced and yields are up to 1.8 % of total soluble protein (TSP) in transformed rice cells. The expression level of synthetic hEGF containing preferred rice codon usage comprises up to 7.8 % of TSP in hypoxic transgenic seedlings. Our studies reveal that the modified αAmy8 promoter can be applicable in establishing a novel expression system for the high-level production of foreign proteins in transgenic rice cells and seedlings under hypoxia.     

The rice 14-3-3 gene family and its involvement in responses to biotic and abiotic stress.

14-3-3 proteins function as major regulators of primary metabolism and cellular signal transduction in plants. However, their involvement in plant defense and stress responses is largely unknown. In order to better address functions of the rice 14-3-3/GF14 proteins in defense and abiotic stress responses, we examined the rice GF14 family that comprises eight numbers. The phylogenetic comparison with the Arabidopsis 14-3-3 family revealed that the majority of rice GF14s might have evolved as an independent branch. At least four rice GF14 genes, GF14b, GF14c, GF14e and Gf14f were differentially regulated in the interactions of rice-Magnaporthe grisea and rice-Xanthomonas oryzae pv. oryzae, and the incompatible interactions stronger induced the genes than the compatible interactions. These GF14 genes were also induced by the defense compounds, benzothiadiazole, methyl jasmonate, ethephon and hydrogen peroxide. Similarly, they were differentially regulated by salinity, drought, wounding and abscisic acid. Tissue-specific analysis and expression of GF14-YFP fusions revealed that the four GF14 isoforms were expressed with tissue specificity and accumulated differentially in the cytoplasm and nucleus. Our current study provides fundamental information for the further investigation of the rice GF14 proteins.     

Effect of localized reduction of gibberellins in different tobacco organs on drought stress tolerance and recovery

Drought resistance is increased in plants by the absence of the hormone gibberellic acid (GA) or by a lack of GA sensitivity. We studied the effects of tissue-specific reduction in GA levels on drought tolerance, on recovery from drought stress, and on primary and secondary growth using transgenic tobacco plants expressing the GA-inactivating gene PtGA2ox ₁ (GA 2-oxidase) specifically in leaves, stems, or roots. Localized reduction of bioactive GA₁ levels was achieved by tissue-specific expression of the PtGA2ox ₁ gene in leaves using the rbcs promoter (LD plants), in roots using the TobRB7 promoter (RD plants), and in stems using the LMX5 promoter (SD plants). In response to drought stress, all transgenic tobacco plants exhibited reduced primary and secondary growth and increased drought tolerance with a corresponding reduction in malondialdehyde levels, higher relative water content, increased proline and sugar content, and elevated peroxidase, superoxide dismutase, and catalase activities relative to wild-type plants. The highest level of drought tolerance and the most rapid recovery from stress was achieved by localized reduction of GA₁ in the roots of the RD transgenic plants. In addition, although the total bioactive GA₁ content in RD and LD plants was essentially identical, the heights of LD plants were significantly greater and drought tolerance was significantly less than in RD plants. It is possible that the site of gibberellin-related gene expression plays an important role in the balance between growth and drought tolerance.     

OsDi19‐4 acts downstream of OsCDPK14 to positively regulate ABA response in rice

The drought‐induced 19 protein family consists of several atypical Cys2/His2‐type zinc finger proteins in plants and plays an important role in abiotic stress. In this study, we found that overexpressing OsDi19‐4 in rice altered the expression of a series of abscisic acid (ABA)‐responsive genes, resulting in strong ABA‐hypersensitive phenotypes including ABA‐induced seed germination inhibition, early seedling growth inhibition and stomatal closure. On the contrary, OsDi19‐4 knockdown lines were less sensitive to ABA. Additionally, OsCDPK14 was identified to interact with OsDi19‐4 and be responsible for the phosphorylation of OsDi19‐4, and the phosphorylation of OsDi19‐4 was further enhanced after the treatment of ABA. Apart from these, OsDi19‐4 was shown to directly bind to the promoters of OsASPG1 and OsNAC18 genes, two ABA‐responsive genes, and regulate their expression. Transient expression assays confirmed the direct regulation role of OsDi19‐4, and the regulation was further enhanced by the increased phosphorylation of OsDi19‐4 after the treatment of ABA. Taken together, these data demonstrate that OsDi19‐4 acts downstream of OsCDPK14 to positively regulate ABA response by modulating the expression of ABA‐responsive genes in rice.     

CaPUB1, a Hot Pepper U-box E3 Ubiquitin Ligase,Confers Enhanced Cold Stress Tolerance and Decreased Drought Stress Tolerance in Transgenic Rice (Oryza sativa L.)

Abiotic stresses such as drought and low temperature critically restrict plant growth, reproduction, and productivity. Higher plants have developed various defense strategies against these unfavorable conditions. CaPUB1 (Capsicum annuum Putative U-box protein 1) is a hot pepper U-box E3 Ub ligase. Transgenic Arabidopsis plants that constitutively expressed CaPUB1 exhibited drought-sensitive phenotypes, suggesting that it functions as a negative regulator of the drought stress response. In this study, CaPUB1 was over-expressed in rice (Oryza sativa L.), and the phenotypic properties of transgenic rice plants were examined in terms of their drought and cold stress tolerance. Ubi:CaPUB1 T3 transgenic rice plants displayed phenotypes hypersensitive to dehydration, suggesting that its role in the negative regulation of drought stress response is conserved in dicot Arabidopsis and monocot rice plants. In contrast, Ubi:CaPUB1 progeny exhibited phenotypes markedly tolerant to prolonged low temperature (4°C) treatment, compared to those of wild-type plants, as determined by survival rates, electrolyte leakage, and total chlorophyll content. Cold stress-induced marker genes, including DREB1A, DREB1B, DREB1C, and Cytochrome P450, were more up-regulated by cold treatment in Ubi:CaPUB1 plants than in wild-type plants. These results suggest that CaPUB1 serves as both a negative regulator of the drought stress response and a positive regulator of the cold stress response in transgenic rice plants. This raises the possibility that CaPUB1 participates in the cross-talk between drought and low-temperature signaling pathways.     

OsGL1-3 is Involved in Cuticular Wax Biosynthesis and Tolerance to Water Deficit in Rice

Cuticular wax covers aerial organs of plants and functions as the outermost barrier against non-stomatal water loss. We reported here the functional characterization of the Glossy1(GL1)-homologous gene OsGL1-3 in rice using overexpression and RNAi transgenic rice plants. OsGL1-3 gene was ubiquitously expressed at different level in rice plants except root and its expression was up-regulated under ABA and PEG treatments. The transient expression of OsGL1-3–GFP fusion protein indicated that OsGL1-3 is mainly localized in the plasma membrane. Compared to the wild type, overexpression rice plants exhibited stunted growth, more wax crystallization on leaf surface, and significantly increased total cuticular wax load due to the prominent changes of C₃₀–C₃₂ aldehydes and C₃₀ primary alcohols. While the RNAi knockdown mutant of OsGL1-3 exhibited no significant difference in plant height, but less wax crystallization and decreased total cuticular wax accumulation on leaf surface. All these evidences, together with the effects of OsGL1-3 on the expression of some wax synthesis related genes, suggest that OsGL1-3 is involved in cuticular wax biosynthesis. Overexpression of OsGL1-3 decreased chlorophyll leaching and water loss rate whereas increased tolerance to water deficit at both seedling and late-tillering stages, suggesting an important role of OsGL1-3 in drought tolerance.     

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.