Study On The Heat Response Mechanism And Expression And Function Analysis Of ERECTA Gene Family In Grape Seedlings

With global warming and frequent extreme high-temperature events,heat stress has become an important factor affecting the agricultural production.In Northwest China,such as Xinjiang,Gansu and Ningxia,sufficient sunlight,large diurnal temperature variation,and little rain in summer confer this region an excellent producing area for wine grapes.The growing season of grapevines is from June to August.High temperature in summer can inhibit the growth of grapevines,and affect the synthesis and accumulation of sugars,acids,anthocyanins and aroma substances in berries,thus adversely affecting the quality and style of wines.In this study,Vitis vinifera L.‘Cabernet Sauvignon’ and Vitis davidii Foex.‘Junzi 1’cuttings were used as test materials for short-time continuous heat treatment(35°C,40°C and45°C treatment for 48 h)and long-time intermittent heat treatment(45 °C treatment for 6 h per day,25 d in total).The physiological response,cellular structure,transcriptome profiling and functional genes(ERECTA gene family)of grapevines were studied in order to uncover their physiological basis and molecular mechanism in response to heat stress.This study laid a theoretical foundation for improving grapevine heat resistance and using cultivation measures to relieve heat damage,and also provided basis for screening high quality grape germplasm resources suitable for arid and semi-arid regions.The main results of this study were as follows:(1)The study on the physiological responses of grape under heat stress.The results of short-time continuous and long-time intermittent heat treatment showed that the thermotolerance of Cabernet Sauvignon was higher than Junzi 1.After heat treatment,the changing range of relative electrolyte leakage,relative water content,Fo,Fv/Fm and Pn in Junzi 1 was higher than in Cabernet Sauvignon.SOD,POD and ABA play important roles in heat resistance of Cabernet Sauvignon.During long-time intermittent heat treatment,the2-hexenal content was increased in leaves of Cabernet Sauvignon and Junzi 1.(2)The study on anatomical and subcellular structure of grape under heat stress.After heat treatment,the thicknesses of palisade tissues,spongy tissues and leaves of Cabernet Sauvignon and Junzi 1 increased significantly;the shape of palisade tissue cells became irregular;the space between spongy tissue cells became larger.Scanning electron microscopy observations revealed that heat treatment resulted in irregular shape of epidermal cells,wrinkling of cell planes and reducing the degree of stomatal opening,but had no significant effects on stomatal width and density.By transmission electron microscopy,we found that after heat treatment chloroplast became larger and rounder,chloroplast membrane collapsed,and a large number of big starch grains appeared.In addition,many osmiophilic granules were observed on the chloroplast of Junzi 1.The chloroplast thermal stability of Cabernet sauvignon was higher than that in Junzi 1.(3)The study on the molecular mechanism of grape in response to heat stress(H)and heat and drought combined stress(H+D)by transcriptome data.There were 2960 and 5090 differentially expressed genes in H and H+D treatment,respectively,in which 1192 genes were co-upregulated and 888 genes were down-regulated.Twelve highly expressed HSPs were screened,including 9 small HSPs,and HSP17.6C,HSP21.7 and HSP90-1 were specifically expressed in H+D treatment.Fifteen antioxidant enzyme genes were up-regulated,including 7 enzyme genes involved in glutathione metabolism.There were 18 receptor-like kinase genes up-regulated,including 11 LRR-RLK genes.A total of 42 highly expressed genes of transcription factors were screened,including 9 MYBs,7 AP2-EREBPs,4 NACs,4HSFs and other family genes,in which DREB1 D,DREB1F and HSF30 expressed at a high level.The number of alternative splicing events of exon skipping and multiple exons skipping increased after H and H+D treatment,and 12 and 18 highly expressed genes of transcription factors occurred alternative splicing,respectively.(4)Identification,cloning and expression analysis of grape ERECTA gene family.Two members of ERECTA gene family,Vv ERECTA and Vv ERL2,were identified in the grape genome,and their full-length c DNAs and promoter sequences were cloned.Subcellular localization results showed that these two genes were located on the cell membrane.The expression levels of Vv ERECTA and Vv ERL2 were high in young leaves,summer buds and flowers.Vv ERECTA was down-regulated during berry development,but the expression level of Vv ERL2 was not affected.Me JA,SA,ABA,MT and BR could induce the expression of Vv ERECTA and Vv ERL2 in leaves and berries.High temperature,drought,downy mildew and powdery mildew increased the expression levels of these two genes,while low temperature suppressed their expression.Under heat stress,the expression level of Vv ERECTA or Vv ERL2 was positively correlated with the content of ABA in leaves.(5)The function verification of the grape ERECTA in Arabidopsis thaliana.Overexpression of Vv ERECTA in Arabidopsis increased the thermotolerance of transgenetic seedling and plant.The plant height,leave number,mesophyll cell width and lengths of leaves,pedicels and pods in transgenic plant were higher than those in wild type.