Drought and salinity are major abiotic stresses to crop production. Here, we show that overexpression of stress responsive gene SNAC1 (STRESS-RESPONSIVE NAC 1) significantly enhances drought resistance in transgenic rice (22-34% higher seed setting than control) in the field under severe drought stress conditions at the reproductive stage while showing no phenotypic changes or yield penalty. The transgenic rice also shows significantly improved drought resistance and salt tolerance at the vegetative stage. Compared with WT, the transgenic rice are more sensitive to abscisic acid and lose water more slowly by closing more stomatal pores, yet display no significant difference in the rate of photosynthesis. SNAC1 is induced predominantly in guard cells by drought and encodes a NAM, ATAF, and CUC (NAC) transcription factor with transactivation activity. DNA chip analysis revealed that a large number of stress-related genes were up-regulated in the SNAC1-overexpressing rice plants. Our data suggest that SNAC1 holds promising utility in improving drought and salinity tolerance in rice.Oryza sativa ͉ abscisic acid ͉ stomata ͉ dehydration P oor water management, increased competition for limited water resources, and the uncertain threats associated with global warming all highlight the looming water crisis that threatens agricultural productivity worldwide. In China alone, the estimated annual loss of national economy from water shortage alone reaches Ͼ$25 billion (1). In addition to altered water management practices, the ability to enhance the tolerance of crops to drought and salinity stress, particularly at the most sensitive reproductive stage of growth, can have a potentially huge impact on productivity in the years to come.Plants can develop numerous physiological and biochemical strategies to cope with adverse conditions (2, 3). The major events of plant response to dehydration stresses are perception and transduction of the stress signals through signaling components, resulting in activation of a large number of stress-related genes and synthesis of diverse functional proteins that finally lead to various physiological and metabolic responses (4-6). Well characterized proteins involved in the protection of plant cells from dehydration stress damage include molecule chaperons, osmotic adjustment proteins (7), ion channels (8), transporters (9), and antioxidation or detoxification proteins (10). The expression of these functional proteins is largely regulated by specific transcription factors (4, 11).More than 30 families of transcription factors have been predicted for Arabidopsis (12). Members of DREB or CBF, MYB, bZIP, and zinc-finger families have been well characterized with roles in the regulation of plant defense and stress responses (4-6, 13, 14). Most of these transcription factors regulate their target gene expression through binding to the cognate cis-elements in the promoters of the stress-related genes. Two well characterized dehydration stress-related cis-elements bound by transcription factors are...

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.

Many stress responsive genes have been reported with an effect on improving stress resistance in model plants under greenhouse conditions. Towards identification of genes for drought resistance breeding, seven well documented genes (CBF3, SOS2, NCED2, NPK1, LOS5, ZAT10, and NHX1) in stress resistance were selected in this study and transformed into rice cultivar Zhonghua 11 under the control of constitutive promoter Actin1 and stress-inducible promoter of a rice HVA22 homolog, and transgenic rice were tested for drought resistance under field conditions. A total of 1598 independent transgenic T0 plants were generated. The percentages of single copy and expression of the transgenes were 36.7% and 57.6%, respectively. For each gene construct, 30 T1 families with expression of transgene were selected for drought resistance testing at the reproductive stage in field, and 10 of them were tested in PVC pipes with a defined stress protocol at the same stage. Relative yield and relative spikelet fertility were used as two major criteria to evaluate drought resistance performance because significantly decreased yield was observed in the T1 generation. Transgenic families of eight constructs (HVA22P:CBF3, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, Actin1:NHX1, and HVA22P:NHX1) showed significantly higher RY than wild-type (WT) under both drought stress field and PVC tube conditions. Transgenic families of 9 constructs (HVA22P:SOS2 and CBF3, LOS5, ZAT10, and NHX1 by both promoters) showed significantly higher relative spikelet fertility than WT in the field or PVC pipes. In the field drought resistance testing of T2 families derived from the T1 families with relatively lower yield decrease, transgenic families of seven constructs (HVA22P:CBF3, Actin1:NPK1, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, and HVA22P:ZAT10) showed significantly higher yield per plant than WT, and families of nine constructs (Actin1:CBF3, HVA22P:CBF3, HVA22P:SOS2, HVA22P:NPK1, Actin1:LOS5, HVA22P:LOS5, Actin1:ZAT10, HVA22P:ZAT10, and Actin1:NHX1) had higher spikelet fertility than WT. In general, LOS5 and ZAT10 showed relatively better effect than the other five genes in improving drought resistance of transgenic rice under field conditions. The results and experience obtained from this study could be a useful reference for drought resistance engineering in rice.

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.

Drought stress is one of the major adverse environmental factors reducing plant growth. With the aim to elucidate the underlying molecular basis of rice response to drought stress, comparative transcriptome analysis was conducted between drought susceptible rice cultivar Zhenshan97 and tolerant cultivar IRAT109 at the seedling stage. 436 genes showed differential expression and mainly enriched in the Gene Ontology (GO) terms of stress defence. A large number of variations exist between these two genotypes including 2564 high-quality insertion and deletions (INDELs) and 70,264 single nucleotide polymorphism (SNPs). 1041 orthologous gene pairs show the ratio of nonsynonymous nucleotide substitution rate to synonymous nucleotide substitutions rate (Ka/Ks) larger than 1.5, indicating the rapid adaptation to different environments during domestication. GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of positive selection genes suggested that photosynthesis represents the most significant category. The collocation of positively selected genes with the QTLs of photosynthesis and the different photosynthesis performance of these two cultivars further illuminate the crucial function of photosynthesis in rice adaptation to drought stress. Our results also provide fruitful functional markers and candidate genes for future genetic research and improvement of drought tolerance in rice.