Characteristic Analysis And Heat Resistance Identification Of SlGRAS4 And SlPYL4 Gene In Tomato

Tomato（Solanum lycopersicum L.）is an annual herbaceous plant belonging to the Solanum of Solanaceae,with strong adaptability and a wide range of cultivation.Tomato is a thermophilic vegetable,optimum growth temperature of which is between15℃～28℃.With the increase of global temperature in recent years,high temperature stress has gradually become one of the main abiotic stresses that affects the growth and development of tomato.Plants activate the complex transcriptional regulatory network to adapt to high temperature stress.Therefore,transcription factors play an important role in the regulation of tomato heat resistance.GRAS and PYR/PYL/RCAR transcription factors respond to a variety of abiotic stresses,but there is little research on their regulatory mechanism involved in tomato high temperature tolerance.Two heat stress-responsive genes SlGRAS4and SlPYL4 were found after analyzing the results of RNA-seq based on our previous studies.However,their bioinformatics characteristics and function responding to high temperature are not yet understood.First of all,gene characteristics,evolutionary relationship and promoter sequence of SlGRAS4 and SlPYL4 were identified using bioinformatics methods.The expression level of SlGRAS4 and SlPYL4 responding to abiotic stress and hormone treatments were detected using q RT-PCR with tomato‘LA2093’and‘LA1698’as plant materials.Afterwards,the function of SlGRAS4responding to high-temperature were analyzed using VIGS,and the related physiological indexes and gene expression level were measured with tomato‘LA2093’as plant materials.Finally,the function of SlPYL4 responding to high-temperature were detected using VIGS and overexpression transformation techniques,and the related physiological indicators and gene expression level were detected with tomato‘LA1698’and‘Ailsa Craig’as materials.The main results were listed:1.The results showed SlGRAS4 protein has highest homology with Nicotiana tabacum Nt GRAS1,sly PYL4 protein has narest relationship with Solanum tuberosum stu PYL4-like protein.Abscisic acid（ABA）and salicylic acid（SA）responding elements were detected in promoter region of SlGRAS4 and SlPYL4.The SlGRAS4 and SlPYL4 were induced by heat,cold,salt,PEG,ABA and SA treatments.2.Under high temperature stress,as compared with Ve plants,VSlGRAS4 plants were more sensitive to heat stress with significantly reduced photosystem II maximum photochemical quantum yield（F_v/F_m）,superoxide dismutase（SOD）and peroxidase（POD）activities,as well as significantly increased relative conductivity（REL）and hydrogen peroxide（H₂O₂）content.Moreover,the expression level of Hsf A1b and Hsf90（high temperature signal transduction key genes）,ZAT10 and ZAT12（ROS signal transduction related gene）,Cu Zn SOD,Fe SOD,APX1,APX2 and CAT（ROS scavenging enzyme code genes）were also significantly down-regulated in VSlGRAS4 plants.The results indicated that SlGRAS4 can enhance heat tolerance of tomato by improving the expression level of high-temperature and ROS response genes.3.Under high temperature stress,as compared with Ve plants,VSlPYL4 plants were easy to wither which showed significantly increased REL and contents of H₂O₂,as well as significantly reduced F_v/F_mand activity of ROS scavenging enzymes include POD,CAT,APX.On the contrary,as compared with WT,OESlPYL4 lines plant showed better phenotype with significantly reduced REL and H₂O₂content,as well as significantly increased F_v/F_mand POD,CAT,APX activities.In addition,the expression level of Hsf A1a,Hsf A1b（high temperature signal transduction key genes）,ZAT12（ROS signal transduction related gene）,POD,CAT,APX（ROS scavenging enzyme encoding genes）,and ABF2,ABF4（ABA signal transduction related genes）in VSlPYL4 plants were significantly lower than that in Ve plants,which were significantly higher in OE lines than that in WT lines.These indicated that SlPYL4 positively regulated tomato heat tolerance by regulating the expression of high-temperature and ROS response genes,and promoting ABA signal transduction related gene expression.