Cloning And Functional Characterization Of TaASR1in Response To Abiotic Stresses In Wheat (Triticum Aestivum L.)

Wheat (Triticum aestivum L.) is the foremost staple food crop in the world which provides both calories and proteins to over35%of the human population. Wheat is the second most important staple crops in China. The term’abiotic stress’includes numerous stresses caused by complex environmental conditions, e.g. strong light, UV, high and low temperatures, freezing, drought, salinity, heavy metals and hypoxia. Abiotic stresses lead to serious damage for plants, especially for crop yield and quality. Therefore, understanding the molecular mechanisms of abiotic stress responses is necessary for genetic improvement of stress resistance in wheat. Recently, Abscisic acid ABA-, stress-, and ripening-induced proteins (ASR) have been reported to increase tolerance to abiotic stresses in various species. However, the mechanisms for its action remain unclear and no research on ASR in wheat has been reported. The perpose of present study is to investigate whether wheat ASR genes can confer drought stress tolerance and if so what is mechanisms underlying the tolerance? Firstly, we analysed the relationship between ASR family gene and osmotic stress in model wheat variety Chinese Spring and cloned a candidate gene TaASRl. Furthermore, we characterized the function of TaASR1in abiotic stresses by using genetic, physiological and biochemical and molecular approavhes. The main results are as follows.1. The relationship between the ESTs of ASR gene family and osmotic stressFrom the DFCI wheat gene index database,16ASR EST sequences were acquired. RT-PCR analysis showed that expression of BE414974(group1), TC418737I (group2), CJ554788(group3) and CK154393(group6) was induced by PEG treatment. Notably, group1exhibited higher expression in normal conditions and was strongly up-regulated after PEG treatment. Therefore group1was selected as a candidate for further functional characterization.2. TaASRl cloning and bioinformatics analysisEmploying RACE-PCR the full-length cDNA of ASR gene, designated as TaASR1, was cloned using mRNA extracted from the leaves of wheat seedlings as template. TaASR1cDNA comprised of544bp with a414bp open reading frame (ORF). The deduced TaASR1protein contained137amino acid residues. Based on amino acid sequence alignment, two highly conserved regions were observed in TaASR1. Phylogenetic analysis indicated that16ASR proteins branched out into separate monocot and dicotyledon groups with TaASR1very close to OsASR and ZmASR2in monocot group.3. The expression analysis of TaASRlTaASR1was expressed in all tissues including root, stem, leaf, stamen, pistil and lemma with higher expression in root, stem, leaf and lemma. TaASRl was induced by osmotic stress, ABA and H2O2. TaASR1transcript induction by MeJA, SA and auxin was marginal and occurred much later. Moreover, the upregulation of TaASRl by PEG possibly involves ABA and H2O2signaling.4. Analysis of drought stress tolerance in TaASR1-overexpressing tobacco and WT plantsTo further investigate the role of TaASR1in drought/osmotic stress tolerance, transgenic tobacco plants over-expressing TaASRl under the control of CaMV35S promoter were generated. Over-expression of TaASRl in tobacco resulted in increased drought/osmotic tolerance, which was demonstrated that transgenic lines had lesser malondialdehyde (MDA), ion leakage (IL) and reactive oxygen species (ROS), but higher relative water content (RWC) and superoxide dismutase (SOD) and catalase (CAT) activities than wild type (WT) under drought stress.5. Analysis of osmotic stress tolerance in TaASR1-overexpressing tobacco and WT plantsSeeds germination rate and root length were higher and longer in transgenic lines than in WT and Vector Control (VC) treated with150or300mM mannitol. Over-expression of TaASR1reduced ROS accumulation by enhancing the SOD and CAT activities under osmotic stress. Stress related genes selected include NtSOD, NtCAT and NtPOX involved in ROS detoxification, NtNCED1involved in ABA biosynthesis, NtERD10C, NtERD10D and NtLEA5related to stress defense, the regulatory gene NtDREB3and lipid-transfer protein genes NtLTP1and TobLTPl. Under normal conditions, expression of NtERDIOC, NtERD10D and NtNCED1were higher in OE12than in WT. Although expression levels of all tested genes were up-regulated by osmotic stress, they were higher in transgenic plants than in WT except for the NtPOX under osmotic stress.6. Analysis of oxidative stress tolerance in TaASRl-overexpressing tobacco and WT plantsTobacco plants were subjected to30μM methyl viologen treatment. This resulted in severe cotyledon bleaching or chlorosis in WT and VC plants than transgenic plants. In addition, transgenic lines displayed higher chlorophyll, lower H2O2contents and higher activity and expression of SOD and CAT than WT and VC.7. Transcriptional activity of TaASRlYeast strain AH109was transformed with the fusion plasmids pGBKT7-TaASR1, pGBKT7-TaASR1-N, pGBKT7-TaASR1-C and pGBKT7(Control)(Fig.12A) and the growth status of transformants was evaluated. Yeast cells containing pGBKT7-TaASR1and pGBKT7-TaASR1-N grew well in SD medium lacking histidine. Moreover, in the presence of X-gal, yeast cells that grew well on the SD medium without histidine turned blue.8. Subcellular location of TaASRlFluorescence of the35S::TaASR1-GFP chimera was associated with cellular nucleus in onion epidermal cells, suggesting a nuclear localization, In the root cells of plants transformed with TaASR1-GFP, fluorescence was also observed in the nuclei.9. Overexpression of TaASRl in wheatThe positive transgenic wheat plants were aquired by using gene gun method.In conclusion, the findings of this study demonstrated that TaASRl function as a transcription factor in abiotic stress tolerance. TaASRl conferred drought, osmotic and oxdidative stress tolerance through regulating the expression of stress-, and defense-associated genes and enhancing the antioxidant system, thus preventing plants from oxidative damage.