Functional Analysis Of Hormone And Inositol Phosphates Metabolism Related Genes To Strss Resistance In Rice

Abiotic stresses such as dehydration, salinity, and extreme temperatures are major limiting factors for plant growth and reproduction. Plant responses to abiotic stresses are coordinated by arrays of genes in association with growth and developmental processes. Rice(Oryza Sativa L.) is one of the most important crops worldwide and its production need favorite environmental conditions including sufficient but not excess water and optimal temperature. However, the availability of fresh water resource and suitable temperature is often a problem for rice production, which threaten the sustainable development of agriculture in the world. Therefore, great effort has been spent on the discovery of potentially useful genes in improvement of stress resistance of crops in the past decade, and a number of studies suggest that the change of some important regulatory or functional genes can significantly improve stress resistance in some transgenic plants.Abscisic acid (ABA), ethylene, auxin (or IAA), and inositol1,3,4-trisphosphate (IP3) are signal molecules playing critical role in developmental programs and environmental responses through complex signalling and metabolism networks. However, crosstalk between these signal molecules in stress responses remains largely unknown. To identify critical genes for abiotic stresses resistance in rice, we screened T-DNA insertion mutants or overexpressing lines of some stress-induced candidate genes. Then, we continued to characterize the functions of four genes based on two drought-sensitive mutants (dsm2, dsm3), a drought-resistant mutant (osetoll), overexpression lines of OsGH3-2and some ABA deficient mutants. The main results are as follows:The drought sensitive phenotype of mutant dsm2was caused by a T-DNA insertion in a gene encoding a putative β-carotene hydroxylase (BCH). BCH is predicted for the biosynthesis of zeaxanthin, a carotenoid precursor of abscisic acid (ABA). The amounts of zeaxanthin and ABA were significantly reduced in two allelic dsm2mutants after drought stress compared with the wild type. Under drought stress conditions, the mutant leaves lost water faster than the wild type and the photosynthesis rate, biomass, and grain yield were significantly reduced, whereas malondialdehyde level and stomata aperture were increased in the mutant. The mutant is also hypersensitive to oxidative stresses. The mutant had significantly lower maximal efficiency of photosystem II photochemistry and nonphotochemical quenching capacity than the wild type, indicating photoinhibition in photosystem Ⅱ and decreased capacity for eliminating excess energy by thermal dissipation. Overexpression of DSM2in rice resulted in significantly increased resistance to drought and oxidative stresses and increases of the xanthophylls and nonphotochemical quenching. Some stress related ABA responsive genes were up-regulated in the overexpression line. DSM2is a chloroplast protein, and the response of DSM2to environmental stimuli is distinctive from the other two BCH members in rice. We conclude that the DSM2gene significantly contributes to control of the xanthophyll cycle and ABA synthesis, both of which play critical roles in the establishment of drought resistance in rice.The drought-and salt-hypersensitive phenotype of mutant dsm3was caused by a T-DNA insertion in a gene encoding a putative inositol1,3,4-trisphosphate5/6-kinase previously named OsITPK2with unknown function. Under drought stress conditions, the mutant had significantly less accumulation of osmolytes such as proline and soluble sugar and showed significantly reduced root volume, spikelet fertility, biomass, and grain yield; however, malondialdehyde level was increased in the mutant. Interestingly, overexpression of DSM3(OsITPK2) in rice resulted in drought-and salt-hypersensitive phenotypes and physiological changes similar to those in the mutant. Inositol trisphosphate (IP3) level was decreased in the overexpressors under normal condition and drought stress. A few genes related to osmotic adjustment and reactive oxygen species scavenging were down-regulated in the mutant and overexpression lines. The expression level of DSM3promoter-driven β-glucuronidase (GUS) reporter gene in rice was induced by drought, salt and abscisic acid. Protoplast transient expression assay indicated that DSM3is an endoplasmic reticulum protein. Sequence analysis revealed six putative ITPKs in rice. Transcript level analysis of OsITPK genes revealed that they had different tempo-spatial expression patterns, and the responses of DSM3to abiotic stresses, including drought, salinity, cold, and high temperature, were distinct from the other five members in rice. These results together suggest that DSM3/OsITPK2is an important member of the OsITPK family for stress responses, and an optimal expression level is essential for drought and salt tolerance in rice.A drought-resistant mutant osetoll was caused by impairing of a gene encoding a putative E3ubiquitin ligase. Two allelic mutants (Zhonghua11background) of this gene, osetoll-1and osetoll-2, showed increased resistance to drought stress at panicle development stage as indicated by higher seed setting rate. Interestingly, osetoll-1mutant showed significantly lower growth rate under submergence stress at both the seedling and panicle development stages. Overexpression of OsETOL1in rice resulted in phenotype reverse to that of the mutants. Tempo-spatial expression pattern analysis revealed higher transcript level of OsETOL1in matured tissue at panicle development stages. OsETOL1gene was strongly induced by abiotic stress, and the responses of OsETOL1to stresses are distinctive from other OsETOL1homologues in rice. Further analyses suggested that carbohydrate catabolism-related genes and intermediate metabolites were changed in osetol1, implying that OsETOL1may function as a regulator of energy metabolism. Yeast two-hybrid analysis found OsETOL1can interact with OsACS2, a key enzyme for ethylene production, and the interaction was further confirmed by BiFC. Furthermore, in the osacs2mutant and OsETOL1overexpression line, the ACC and ethylene content was significantly decreased. Exogenous application of10μM. ACC can restore the phenotypes of osacs2and OsETOL1overexpression line, both with ACC-deficient phenotype under submergence, implying that the negative regulation of ethylene synthesis by OsETOL1is conserved in ETOL family. However, genetic and phylogenetic analyses suggested both functional redundancy and specificity exist in of the ETOL family from Arabidopsis and rice.The function OsGH3-2in abiotic stress tolerance was also investigated. OsGH3-2, encoding an enzyme catalyzing IAA conjugation to amino acids, was found to be involved in the modulation of ABA level and stress tolerance. The expression of OsGH3-2is induced by drought but is suppressed by cold. Overexpression of OsGH3-2in rice caused significant morphological aberrations related to IAA-deficiency, such as dwarfism, smaller leaves, and fewer crown roots and root hairs. The overexpression line showed significantly reduced carotene, ABA, and free-IAA levels, greater stomata aperture and faster water loss, and was hypersensitive to drought stress. However, the overexpression line showed increased cold tolerance, which was due to the combined effects of reduced free-IAA content, alleviated oxidative damage and decreased membrane penetrability. Furthermore, expression levels of some ABA synthesis-and stress-related genes were significantly changed in the overexpression line. Suppression of OsGH3-2caused no significant changes in the phytohormone levels or stress tolerance, which is likely due to functional redundancy of the OsGH3family. We conclude that OsGH3-2modulates both endogenous free-IAA and ABA homeostasis and differentially affects drought and cold tolerance in rice. we report that ABA deficient mutants phsl phs2phs3phs4and PDS-RNAi transgenic rice impaired the biosynthesis of IAA, under drought condition phs3-l and PDS-RNAi transgenic rice shown higher stomata aperture and wiled easier compared to the wild type at both seedling and panicle developmental stage. Interestingly, these ABA and IAA deficient lines were cold resistance, which was due to the combined effects of reduced IAA content, alleviated oxidative damage and decreased membrane penetrability. Furthermore, we measure fluridone (an ABA biosynthesis inhibitor) treated rice, and found ABA and IAA content were declined, due to the change of auxin synthesis and metabolism genes similar to the ABA deficient mutants. In addtion, we use exogenous IAA can restore the dwarf of phs3-l and PDS-RNAi transgenic rice, however, exogenous ABA can’t restore the phenotype. So far, the crosstalk between ABA and IAA at the biosynthesis level had never been reported for functions in development and abiotic response. So our research may be very useful for understanding the crosstalk between ABA and IAA, and differentially affects drought and cold tolerance in rice.