The Mechanism Of 5-aminolevulinic Acid In Regulating Tomato Cold Tolerance

Tomato（Solanum lycopersicum L.）is an important thermophilic vegetable crop.However,facility grown tomatoes often suffer from low temperature damage during winter and early spring in China,which seriously affects their normal growth and development and reduces yield and quality.Therefore,exploring ways to alleviate the tomato damage under low temperature stress are especially important for achieving safe production of tomato in winter,which has important scientific and practical significance.In this paper,tomato was used as a test material to study the regulation of exogenous 5-aminolevulinic acid（ALA）on redox balance,endogenous hormone levels,chlorophyll synthesis and transcription level in tomato seedling leaves under low temperature stress.Determing the signaling molecules involved in ALA regulation of tomato redox balance.Exploring the ALA induced differentially expressed genes by transcriptome analysis,and finally determining the roles of the key gene,glutathione-S-transferase gene（GSTU43）,in the anti-oxidation and chlorophyll synthesis of tomato under low temperature stress by virus-induced gene silencing technology（VIGS）.The main results obtained were as follows:1.Exogenous application of appropriate concentration of ALA can enhance the low temperature tolerance,and improve the antioxidant capacity of different varieties of tomato.Low temperature caused oxidative stress damage in two varieties of tomato seedlings.?Jinpeng No 1‘tomato was more sensitive to low temperature than?Zhongza No 9‘tomato variety.In summary,the tomato plants pretreated with 6 mL 25 mg·L^-1 ALA significantly enhanced tomato low temperature tolerance.2.H₂O₂,JA,NO,GSH and AsA are involved in ALA regulated antioxidation of tomato under low temperature stress.Exogenous application of ALA induced the expression of Respiratory burst oxidase homolog 1（RBOH1）gene and the increasement of apoplastic H₂O₂ content.H₂O₂ was crucial and may be the initial step for perception and signaling transmission,which then improved the ratio of reduced glutathione and ascorbic acid（GSH and AsA）.GSH and AsA may then interact with H₂O₂ signaling,resulting in enhanced antioxidant capacity in tomato plants at low temperatures.In addition,ALA induced H₂O₂ and Jasmonic acid（JA）displayed independent but synergistic roles in regulating tomato antioxidation.Nitric oxide（NO）may act downstream of H₂O₂ along with JA to regulate antioxidant enzyme gene expression and increase tomato cold tolerance.3.Exogenous application of ALA alleviates low temperature caused chlorophyll synthesis disorder and promotes the process of chlorophyll synthesis in tomato seedling leaves;these processes are regulated by non-transcriptional level.Low temperature treatment reduced the content of chlorophyll and most of its precursors in tomato seedling leaves,but the content of porphobilinogen（PBG）and uroporphyrinogen III（UROIII）and chlorophyll a/b increased.Under low temperature stress,the contents of PBG and UROIII in ALA pretreated tomato seedling leaves were significantly lower than those in low temperature treatment alone,while the chlorophyll content and other precursors were significantly increased.The regulation of chlorophyll synthesis enzyme genes by low temperature or exogenous ALA treatment was inconsistent with the change of precursor‘s contents.In conclusion,exogenous ALA can promote the coordinated chlorophyll synthesis of tomato seedling leaves under low temperature stress,and the regulation of chlorophyll synthesis may be at the post-transcriptional or translational level,not the level of transcription.4.GST plays key roles in the ALA regulated antioxidation and chlorophyll synthesis of tomato under low temperature stress.Exogenous ALA induced the up-regulation of glutathione-S-transferase（GSTs）,glutamate decarboxylase（GADs）and cell wall synthase（XTHs,tXET and UDPGH）genes in tomato seedling leaves under low temperature stress.Among them,GSTU43 gene and protein expression and GST activity were significantly induced.Silencing GSTU43 gene by VIGS technology reduced the activities of GST,superoxide dismutase（SOD）,catalase（CAT）,ascorbate peroxidase（APX）and glutathione reductase（GR）in tomato seedling leaves,aggravated the degree of membrane lipid peroxidation and excessively increased the content of porphyrins in leaves under low temperature.Exogenous ALA pretreated GSTU43 silenced plants increased these antioxidant enzyme activities in leaves,alleviated the membrane lipid peroxidation and decreased the excessive content of porphyrins.In conclusion,exogenous ALA can induce up-regulation of GSTU43 gene and GST enzyme activity.Ascorbate-glutathione cycle（AsA-GSH）played a synergistic role with GST,regulated the redox state of plants,and enhanced the low temperature tolerance of tomato plants.This latter effect reduced the degree of membrane lipid peroxidation,which was essential for the coordinated synthesis of chlorophyll.