Cloning And Function Characteriztion Of Genes Involved In Regulated Ascorbate Synthesis In Tomato

Tomato is one of the most important vegetable crops in the world,while it is a model plant for fruit research.The level of ascorbic acid is an important nutrient quality trait.At present,the pathway of ascorbic acid biosynthesis is clear,but the study of its regulation has just begun.Function characterization of ascorbic acid regulation mechanism from tomato is very important in palnt research.In tomato(Solanum lycopersicum),SIGMP3 and SIGME1 play an important role in the D-Man/L-Gal pathway.In this study,we isolated two baits from SlGMP3 and SIGME1 promoters sequences,and obtained three genes(S1HZ24,S1IAA9 and S1NFY)from cDNA library of Y1H.SlHZ24 and SlIAA9 regulated SIGMP3 expression,and SlNFYA regulated SIGME1 expression.We analyzed the mechanism of regulating ascorbate motabolism at physiological and biochemical and molecular level by transgenic lines of these genes.The main results are as follows:1.Transcription factor SIHZ24 regulated SIGMP3 expression and affected ascorbate accumulationThere are flour GMPs in tomato,and SlGMP3 is very important in ascorbate biosynthesis.Here,it was revealed by a yeast one-hybrid assay that a tomato(Slanum lycopersicum)HD-Zip I family transcription factor,SlHZ24,binds to the promoter of an AsA biosynthetic gene,SlGMP3.Both the transient expression system and the electrophoretic mobility shift assay(EMSA)showed that SlHZ24 binds to a regulatory cis-element in the SIGMP3 promoter,and further overexpression of SIHZ24 in transgenic tomato lines resulted in increased SIGMP3 expression AsA levels,on the contrast,suppressing expression of the gene using RNA interference(RNAi)had the opposite effect.These data suggest that SIHZ24 can positively regulate AsA accumulation,and in support of this it was shown that SIGMP3 expression increased in the SlHZ24-overexpressing lines and declined in SlHZ24-RNAi lines.SlHZ24 also affected the expression of other genes in the D-mannose/L-galactose(D-Man/L-Gal)AsA biosynthesis pathway,such as SIGMP4,SIGME2,SIGGP and SlcAPX.The EMSA indicated that SlHZ24 bound to the promoters of SlGME2 and SIGGP,indicating a multi-targeted regulation of AsA biosynthesis.Finally,SlHZ24-overexpressing plants showed less sensitivity to oxidative stress and so we conclude that SlHZ24 promotes AsA biosynthesis,which in turn enhances oxidative stress tolerance.2.Regulation factor SlIAA9 regulated the expression of SIGMP3 and affected ascorbate content in tomatoSlIAA9 is belonged to Aux/IAA transcription factor through yeast one-hybrid by SlGMP3 promoter.Ascorbate content of leaves and fruits decreased in SlIAA9-RNAi lines.The exprssions of AsA biosynthesis-related genes were down-regulated.The expression of these genes in entire mutant(SlIAA9 mutent)was no significant difference compared with wild-type.The content of ascorbate in AC and entire was significantly decreased under IAA-treated.But the content in SlIAA9-RNAi lines was no significant difference compared with water-treated.The transient expression system showed that SlIAA9 did not bind to SlGMP3 promoter.The yeast one-hybrid assay indicated that SlARF8 could recognize and bind to SIGMP3 promoter.These data showed SlIAA9 might regulate the accumulation of ascorbate by S1ARF8 binding to SIGMP3 promoter..3.Regulation factor SlNFYA negatively regulated SlGME1 expression and affected ascorbate accumulationWe utilized a SlGME1 promoter as a bait to screen a tomato cDNA Y1H library and obaited a transcription factor SlNFYA,which was belonged to NF-Y family and bound to CCAAT-box.Transient expression system showed that SINFYA could recognize and bind to the SlGME1 promoter,and the binding region was located on-1792--1787 bp of the SIGMEl promoter sequence upstream of the translation initiation codon(ATG).There were negatively correlated the expression of SlGME1 and SINFYA in tomato tissues.Overexpression of SlNFYA in transgenic lines resulted in decreased ascorbate levels,and accompanied by the dwarf plants,the leaves turn dark green,flower abnormality traits.Ascorbate content was no significant diffence between AC and SlNFYA-RNAi lines.The expression of SlGME1 significantly increased in the SlNFYA-RNAi lines compared with wild-type.These data indicated that SlNFYA might negatively regulated SlGME1 expression.SlNFYA also affected the expression of genes in ascorbate metabolism pathway,such as SlGGP and SlcAPX.These date indicated that SlNFYA negatively regulated the expression of SlGME1,which led to decreased ascorbate content.