The Drought-tolerance Function Of Ck?-ECS In Caragana Korshinskii Kom

Drought is the main cause of desertification and sandification,especially in the northwest region in China.Drought stress would destroy the redox homeostasis in plants,also influenced the growth and development.Leaf is the main organ where assimilation and transpiration is taking place,and stomatal movement is one of the major way to adapt long-term drought.?-glutamylcysteine synthetase(?-ECS)is rate-limiting enzyme of biosynthesis of glutathione.Glutathione is an important mercaptan compound and plays a key role in response to stress.Glutathione affects the auxin exporter and regulates the distribution of auxin in the root.Moreover,research shows that stomatal development is also affected by auxin,thus affecting the plant's resistance to stress.Caragana korshinskii Kom.is a predominant leguminous shrub species that distributed in arid and semiarid of China,which has strong adaptation to environmental changes,and it is used as a plant of soil conservation,windbreak and sand fixation in arid areas.At present,little studys were reported about the effect of Ck?-ECS gene on drought resistance at the molecular level.Thus,this study focused on the function of Ck?-ECS in auxin singnal pathway and stomata development.This work has an important theoretical and practical significance for the long-term drought tolerance of plants in arid areas.The major results as following:(1)The samples were collected from the three areas(AS,SM,DS)where precipitation is decrease successively.We found that stomatal density was positively correlated with precipitation which indicated that the reduced stomatal density of C.korshinskii is adapted to drought.Under PEG6000 treatments,primary root growth was increased,however,plant height and leaf area was decreased compared with control.These results suggested that C.korshinskii response to drought stresses through changing root and underground development.According to qPT-PCR analysis,the relative genes of stomatal development CkFAMA and CkSTOMAGEN were decreased.Auxin relative genes of CkAXR3 and CkARF5 were accumulated under drought stresses.In addtion,the expression of Ck?-ECS and its activity were significantly increased.These results suggested that under drought stress,induced Ck?-ECS expression by drought,upregulated the auxin genes expression and decreased the expression of stomatal development genes.(2)The cDNA of Ck?-ECS is 1527 bp,encoding 508 amino acids with 57.59 kDa.Overexpression of Ck?-ECS in Arabidopsis by using the floral dip method,then screened and identified the transgenic plants by RT-PCR and Western Blotting.(3)The primary root was longer compared with WT.The leaf area area and width of overexpression plants were decreased.The stomatal density and aperture were reduced.The ?-ECS activity was significantly enhanced compared with WT.The expression of AtAXR3 and AtARF5 in transgenic lines were higher than that of WT,indicated that overexpression of Ck?-ECS influenced the genes expression of auxin signaling pathway.Also,the mRNA level of AtFAMA and AtSTOMAGEN were decreased,while the AtEPF1 was upregulated.All of results suggested that Ck?-ECS gene may negative regulate stomatal development by affecting auxin signaling pathway.(4)Under drought stress,overexpressing plants were better,relative water content were higher than that of WT plants.Transgenic plants have decreased water loss rate and MDA content.These results demonstrated that the water retention capacity was elevated in overexpressing Ck?-ECS plants,thus and improve the drought tolerance.In conclusion,the Ck?-ECS gene could influence stomatal development by auxin signaling pathway,Ck?-ECS negative regulate to reduce stomatal density,and improve drought tolerance.