Plants respond to adverse environments by initiating a series of signaling processes that often involves diverse protein kinases, including calcineurin B-like protein-interacting protein kinases (CIPKs). In this study, putative CIPK genes (OsCIPK01-OsCIPK30) in the rice (Oryza sativa) genome were surveyed for their transcriptional responses to various abiotic stresses. The results showed that 20 OsCIPK genes were differentially induced by at least one of the stresses, including drought, salinity, cold, polyethylene glycol, and abscisic acid treatment. Most of the genes induced by drought or salt stress were also induced by abscisic acid treatment but not by cold. A few CIPK genes containing none of the reported stress-responsive cis-elements in their promoter regions were also induced by multiple stresses. To prove that some of these stress-responsive OsCIPK genes are potentially useful for stress-tolerance improvement, three CIPK genes (OsCIPK03, OsCIPK12, and OsCIPK15) were overexpressed in japonica rice 'Zhonghua 11'. Transgenic plants overexpressing the transgenes OsCIPK03, OsCIPK12, and OsCIPK15 showed significantly improved tolerance to cold, drought, and salt stress, respectively. Under cold and drought stresses, OsCIPK03-and OsCIPK12-overexpressing transgenic plants accumulated significantly higher contents of proline and soluble sugars than the wild type. Putative proline synthetase and transporter genes had significantly higher expression level in the transgenic plants than in the wild type. The differentially induced expression of OsCIPK genes by different stresses and the examples of improved stress tolerance of the OsCIPK transgenic rice suggest that rice CIPK genes have diverse roles in different stress responses and some of them may possess potential usefulness in stress-tolerance improvement of rice.

Late embryogenesis abundant (LEA) proteins have been implicated in many stress responses of plants. In this report, a LEA protein gene OsLEA3-1 was identified and over-expressed in rice to test the drought resistance of transgenic lines under the field conditions. OsLEA3-1 is induced by drought, salt and abscisic acid (ABA), but not by cold stress. The promoter of OsLEA3-1 isolated from the upland rice IRAT109 exhibits strong activity under drought- and salt-stress conditions. Three expression constructs consisting of the full-length cDNA driven by the drought-inducible promoter of OsLEA3-1 (OsLEA3-H), the CaMV 35S promoter (OsLEA3-S), and the rice Actin1 promoter (OsLEA3-A) were transformed into the drought-sensitive japonica rice Zhonghua 11. Drought resistance pre-screening of T(1) families at anthesis stage revealed that the over-expressing families with OsLEA3-S and OsLEA3-H constructs had significantly higher relative yield (yield under drought stress treatment/yield under normal growth conditions) than the wild type under drought stress conditions, although a yield penalty existed in T(1) families under normal growth conditions. Nine homozygous families, exhibiting over-expression of a single-copy of the transgene and relatively low yield penalty in the T(1) generation, were tested in the field for drought resistance in the T(2) and T(3) generations and in the PVC pipes for drought tolerance in the T(2) generation. Except for two families (transformed with OsLEA3-A), all the other families (transformed with OsLEA3-S and OsLEA3-H constructs) had higher grain yield than the wild type under drought stress in both the field and the PVC pipes conditions. No significant yield penalty was detected for these T(2 )and T(3) families. These results indicate that transgenic rice with significantly enhanced drought resistance and without yield penalty can be generated by over-expressing OsLEA3-1 gene with appropriate promoters and following a bipartite (stress and non-stress) in-field screening protocol.

A full-length cDNA gene, designated Oryza sativa chymotrypsin inhibitor-like 1 (OCPI1), was characterized in rice. The predicted protein of OCPI1 shows very high sequence identity to reported chymotrypsin inhibitors from various plant species. Northern-blot analysis showed that the expression of OCPI1 was strongly induced by dehydration stresses and abscisic acid (ABA). The expression of beta-glucuronidase (GUS) reporter gene under the control of OCPI1 promoter transformed into rice was strongly induced by drought and salt stresses. Interestingly, strong dehydration stress-induced GUS activity was also detected in the transgenic rice containing the reverse sequence of OCPI1 promoter fused to GUS gene, suggesting of a bidirectional transcriptional activity in the OCPI1 promoter. OCPI1 gene was over-expressed in japonica cv. Zhonghua 11 and transgenic plants containing single copy of transgene were tested for drought resistance at reproductive stage. The positive transgenic plants (OCPI1 was over-expressed) had significantly higher grain yield and seed setting rate than the wild type and the negative transgenic control (no over-expression of the transgene) under the severe drought stress conditions, whereas the potential yield of transgenic plants under normal growth conditions was not affected. Chymotrypsin-inhibitor activity assay showed that the crude protein of the positive transgenic plants had stronger inhibitory activity than the negative control. Transgenic plants had less decrease of total proteins than the wild type under drought stress. Taken together, these data indicate that OCPI1 might potentially be useful in the genetic improvement of drought resistance in rice.

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