The Mechanism Of Abscisic Acid And Transcription Factor SlAREB1 In Regulating Saline-Alkaline And Low-Temperature Tolerance In Tomato

Tomato（Solanum lycopersicum L.）has great demand due to the favor of consumers as an important facility-cultivated vegetable.During the process of protected facility cultivation,the aggravated soil salinization owing to the unreasonable cultivation methods and the low temperature caused by the limitation of facility heating equipment in winter,which have seriously affected the yield and quality of tomato.As a result,saline-alkaline and low temperature stresses have become the major factors restricting the development of tomato facility industry.Abscisic acid（ABA）plays an important role in response to stress as a critical phytohormone and its signal transduction pathway is also induced by stress.Among them,AREB/ABF are core transcription factors involved in stress response and signal transduction in the ABA signaling pathway.However,the regulatory mechanisms of AREB/ABF transcription factors in tomato under saline-alkaline and low temperature stresses are still unknown.In this research,‘Ailsa Craig’and ABA-deficient mutant‘notabilis’were taken as experimental materials to clear the effect of ABA under saline-alkaline and low temperature stress.Moreover,plants overexpressing SlAREB1 were constructed in the background of‘Ailsa Craig’to explore the regulatory mechanism of SlAREB1 under saline-alkaline and low temperature stresses,which providing a theoretical basis to obtain stress-tolerance varieties through genetic engineering technology.The main findings are as following:1.ABA is involved in improving the saline-alkaline stress tolerance of tomato seedlings.The expression of Sl NCED2 which was ABA synthesis gene and the accumulation of endogenous ABA were induced by saline-alkaline stress.Moreover,saline-alkaline stress also induced the expression of SlAREB1 and Sl Sn RK2.2/2.3/2.6 genes which were the key of ABA signaling pathway.The ABA content decreases in ABA-deficient mutant‘notabilis’tomato seedlings,which become more sensitive to saline-alkaline stress,however,the exogenous spraying of ABA improved the tolerance of tomato seedlings to saline-alkaline stress.Compared with the control,exogenous ABA reduced REC and MDA content in tomato leaves under saline-alkaline stress,and improved the RWC of tomato leaves.Exogenous ABA also increased the content of chlorophyll a in tomato leaves under saline-alkaline stress,and promoted the accumulation of soluble sugars and proline.Moreover,ABA treatment promoted the expression of Sl SOD,Sl POD and Sl CAT genes,reduced the content of ROS such as H₂O₂ and O₂^·-,and alleviated the damage of oxidative stress caused by saline-alkaline stress.On the other hand,ABA is involved in improving the adaptability of tomato seedlings to different low-temperature stresses.The expression of Sl NCED2 gene and the content of endogenous ABA were induced under two different low-temperature stress conditions（15/8°C and 4/4°C）.Exogenous ABA reduced the accumulation of ROS such as H₂O₂ and O₂^·-by increasing the activities of Sl SOD,Sl POD and Sl CAT,thus reducing the accumulation of MDA.Under sub-low temperature（15/8℃）condition,the SlAREB1 gene was significantly induced by ABA signal pathway,and the expression of Sl Sn RK2.6/OST1 gene was also induced.There was protein-protein interaction between Sl Sn RK2.6/OST1 and SlAREB1 in vitro and in vitro.2.The AREB/ABF-type transcription factors of tomatoes are involved in responding to ABA,saline-alkaline and low temperature stress.Four AREB/ABF-type transcription factors in tomatoes were found by means of screening and identification of gene family,of which possessed b ZIP conserved domains and another four highly conserved regions（C1,C2,C3,C4）with putative phosphorylation site.It was found that various acting elements with response stresses and hormones by analyzing the promoter cis-acting elements of four AREB/ABF-type transcription factors.Through expression analysis,it was found that the expression of four AREB/ABF genes was induced by ABA,saline-alkaline and low-temperature stress,of which SlAREB1 was particularly significant in response to ABA and abiotic stress.With 477 amino acid residues and a total molecular weight of 47.98 k Da.SlAREB1 protein is an unstable hydrophilic protein with no transmembrane structure.The transcription factor SlAREB1 respond to ABA-activated signal pathways and possess 1-phosphatidylinositol-4-kinase activity.The prediction results of interacting proteins indicated that there was interaction between SlAREB1 protein with Sn RK2 protein kinase,E3 ubiquitin protein ligase and CBF1 transcription factor.Moreover,the results of subcellular localization manifested that SlAREB1 was localized in the cell nucleus.3.The transcription factor SlAREB1 strengthened the tolerance of tomatoes under saline-alkaline stress through increasing antioxidant enzyme activity.Overexpression of SlAREB1 increases the sensitivity of tomato seeds to ABA.Compared with the wild-type tomato seedlings,overexpression of SlAREB1 improved the content of RWC in tomato leaves under saline-alkaline stress,reduced the accumulation of MDA,alleviated the degradation of chlorophyll a,chlorophyll b and carotenoids and thus improved the ratio of Fv/Fm to enhance the saline-alkaline tolerance of tomato seedlings.More importantly,overexpression of SlAREB1 increased the activity of SOD and improved the antioxidant capacity of tomatoes,thus reducing oxidative stress resulted from H₂O₂ and O₂^?-.Moreover,the results of yeast two-hybrid screening library and interaction verification in vivo and vitro shown that the transcription factor SlAREB1 directly affected the activity of Sl Mn-SOD by interacting with the protein of Sl Mn-SOD,thereby elevating the antioxidation capacity of tomato seedlings and enhancing tolerance to saline-alkaline stress.4.The transcription factor SlAREB1 improves the tolerance of tomatoes to low-temperature stress by promoting the synthesis of anthocyanins.Compared with the wild-type tomato seedlings,overexpression of SlAREB1 improved the sub-low temperature（15/8℃）tolerance of tomato seedlings by increasing the ratio of Fv/Fm and enhancing total antioxidant capacity.Under sub-low temperature stress,the overexpression of SlAREB1promoted the expression of anthocyanin synthesis genes Sl DFR and Sl3’5’H and the accumulation of anthocyanins in tomato leaves,whereas silencing SlAREB1 reduced the biosynthesis and accumulation of anthocyanins.The transcription factor SlAREB1interacted with the ABRE cis-action element in the promoter region of Sl DFR and Sl3’5’H genes,and positively regulated the expression of Sl DFR and Sl F3’5’H genes under sub-low temperature stress.Therefore,it participates in the regulatory network of anthocyanin biosynthesis induced by sub-low temperature stress.