Function Analysis Of The Foxtail Millet(Setaria Italica) Gene,SiASR4,in Response To Drought And Salt Stresses Of Plants

Drought and salt stresses are major environment factors that affect the growth and development of plants and limit crop productivity.In order to resist the influence of the external environment,plants have evolved a series of physiological and molecular mechanisms that respond to stresses.Foxtail millet(Setaria italica),an important grain crop in China,has rich and balanced nutrients.Because of its excellent drought tolerance,short life cycle and small diploid genome size,it is an ideal model system to study the molecular biology of abiotic stresses.With the generation of the genetic map,completion of the genome sequencing and establishment of the genetic transformation,the researches on molecular biology of foxtail millet have been further promoted.The abscisic acid-,stress,and ripening-induced(ASR)proteins are types of plant-specific proteins,which involve in stress resistance,senescence,fruit maturation and pollen development.In this study,eight ASR genes were identified in foxtail millet.Expression profile analysis showed that these genes induced by drought,salt and ABA.Because of the degree of induction of SiASR4 were higher and SiASR4 encoded a protein with high-lysine content(18.66%),we chose it for further study.qRT-PCR assay showed that SiASR4 was expressed in leaf,stem,root and mature seed,and the transcript levels were highest in mature seed,followed by root.SiASR4 was located in the cell nucleus,cytoplasm and cytomembrane and it lacked transcriptional activation activity in yeast and tobacco cells.Heterologous expression of SiASR4 in Arabidopsis thaliana improved the tolerance of drought and salt stresses.ABA and H2O2 induced stomatal closure was greater in transgenic plants than that in wide-type plants,which indicated that SiASR4 played a role in drought stress by regulating stomatal movement.Under drought and salt stress,ROS accumulation in transgenic Arabidopsis was reduced.In addition,heterologous expression of SiASR4 in maize significantly increased tolerance to salt and drought stresses.To investigate the function of SiASR4,we obtained SiASR4 overexpressing(SiASR4-OE)and SiASR4 RNA interference(SiASR4-RNAi)foxtail millet by Agrobacterium infection method.Further analysis showed that overexpression of SiASR4 in foxtail millet enhanced the tolerance of drought and salt stresses at seed germination and seedling stage,and under salt stress,ROS accumulation was reduced.SiASR4 RNAi plants had no significant difference compared with wide-type plants.In transgenic Arabidopsis and overexpressing foxtail millet,the expression of stress-relevant genes such as SOS1,LTP,CAT and SOD were increased.The EMSA and yeast one-hybird assays showed that SiARDP could bind the DRE element in SiASR4 promoter region in vitro and vivo.In SiARDP overexpression plants,the transcription level of SiASR4 was increased significantly.Previous study showed that overexpression of SiARDP in foxtail millet enhanced the tolerance of drought,but the tolerance of salt stress was not enhanced.These results indicated that SiASR4 was regulated by SiARDP under drought stress.Under salt stress,SiASR4 may be regulated by other transcription factors.In addition,SiASR4 homologues SiASR3 and SiASR9 were also cloned in this study.SiASR3 and SiASR9 were located in the cell membrane,cytoplasm and nucleus in tobacco epidermal cells,and they have no transcriptional activation activity in yeast.Heterologous expression of SiASR3 and SiASR9 inArabidopsis thaliana enhanced the tolerance of drought and salt stresses.The activity of SiASR3 and SiASR9 was regulated by post-translationally.In summary,SiASR4 played an important role in plant resistance to drought and salt stresses.The expression of SiASR4 was regulated by the DREB transcription factor SiARDP and participated in the response of plants to abiotic stresses through the ABA signaling pathway.