Functional Analysis Of The Cystatin Gene MpCYS4 Gene In Response To Nitrogen Deficiency In Malus

Cysteine proteases are involved in protein degradation andnitrogen recycling during leaf senescence and programmed cell death of seed development.Cystatins(CYS)are well-characterized protease inhibitors whichcan regulating plant growth and development by degrading cysteine proteases.CYS geneshave been identified in many plants,including Arabidopsis thaliana,riceand soybean,and theirbiological functions have been checked in different species.Apple is a nitrogen loving plant,and nitrogen stress can seriously affect the growth and development,fruit quality and yield of apple.However,few studies have been carried out on apple CYSgenes,and little information is reported during nutritional stress process.Our research grouphas identified the members of apple CYSgene family,cloned the MpCYS4 gene and obtained the transgenic M.26 that overexpressing MpCYS4 gene.In this study,we investigated the gene function of MpCYS4 under low nitrogen stress by means of tissue culture,sand culture and hydroponics.The main results obtained are as follows:1?Plant growth was significantly inhibited by nitrogen deficiency.Nitrogen deficiency inbihited biomass,plant height,stem diameter,chlorophyll content,and net photosynthesis of apple seedlings which showedtypical symptom of nitrogendeficiency.Based on previous research on the subject,six apple CYSgenes were selectedto analyze their expression under nitrogen stress.These genes(Md CYS1,Md CYS8,Md CYS15,Md CYS20,Md CYS21,Md CYS26)were induced duringthe process of nitrogen stress,In addition,according to previous studies on CYS involvement in nitrogenstress,Md CYS21 was selected for further functional identification.2?Studied on MpCYS4 response to nitrogen stress using tissue culture.The vigourof transgenic plants wasstronger than wild typeafter low nitrogen treatment and nitrogen-free treatment,and the number of root,root mean length,root surface area of transgenic plants were significantly larger than those of the wild-type.It was suggested that the overexpression of MpCYS4 enhanced the resistance of M.26 to nitrogen stress.3?Studied on MpCYS4 response to low nitrogen stress under sandy conditions.Under low nitrogen conditions,photosynthetic pigment content,net photosynthetic rate andbiomasses of roots as well as shoots of transgenic apple were significantly larger than that of wild type.However,the rate of root/shoot was lower in transgenic apple in nitrogen stress.In addition,the accumulation of H2O2 in the leaves was significantly decreased,and the content of nitrogen in mature leaves was greater in transgenic apple.4?Studied on MpCYS4 response to low nitrogen stress under hydroponicsconditions.The plants showed typical symptoms of nitrogen deficiency.Under low nitrogen conditions,biomasses of roots as well asshootsand the photosynthetic pigment content of transgenic plants were significantly larger.The accumulation of H2O2 was significantly lower in transgenic plants.The activities of antioxidant enzymes,such as SOD,POD,CAT and APX,were significantlylower in wild apple.Additionally,Md NPF6.8,Md NPF6.9,Md NRT2.4 and Md NRT2.7 were significantly up-regulatedin the leaves of transgenic plants.In conclusion,the function analysis of MpCYS4 response to nitrogen stress indcates that MpCYS4 play an important role in resistance to nitrogen stress.