Functional Analysis Of Cuticle Development Regulated Gene WOOLLY And Carotenoids Accumulation Regulated Gene SLBBX20 In Tomato

Tomato is one of the most important vegetable crops and occupies an important place in the vegetable industry.The major nutrients of tomato fruits were carotenoids,which have strong antioxidant abilities and help us combat the damage of the reactive oxygen.Tomato is a popular fruit and vegetable,and how to modify the nutrional quality of tomato fruits has always been the research hotspot.The storage and freshness of tomato fruits is an important production problem.Cuticular development play an important role in limiting nonstomatal water loss of tomato fruits.Function characterization of cuticle biosynthesis pathway in tomato fruit is very important.In previous researches,we found that Woolly and SlBBX20 may regulate the cuticular development and carotenoids synthesis of tomato fruits,respectively.In this paper,we studied the function of these two genes using the transgenic technology,yeast one/two hybrid,EMSA and Co-IP assays and so on.The main results are as follows:?.The cuticle regulated gene Woolly in tomato:1.The cuticular wax amount of LA3186 leaves and fruits was increased,compared to the wild-type.Moreover,the expression of wax biosynthetic genes was upregulated in LA3186.The wax accumulation in the leaves and fruits of Wo-OE transgenic plants were accelerated.Wo could bound to the L1-box motif in the CER6 promoter.Wo regulated the wax biosynthesis of tomato by directly binding to the promoter of CER6.2.The interaction between Wo and SlMYB31 was confirmed by Y2H,BIFC and COIP assays.The wax load in the SlMYB31-OE leaves and fruits was significantly increased compared to the wild type,but greatly reduced in the SlMYB31-RNAi lines.The fruits of SlMYB31-RNAi were brighter than the wild type and lost water quickly.The wax amount in the leaves of Wo-OE/SlMYB31-RNAi and SlMYB31-OE/Wo-RNAi double mutant plants was less than Wo-OE and SlMYB31-OE,respectively.These data demonstrated that Wo and SlMYB31 regulated the cuticular wax synthesis co-operatively.?.The carotenoids synthesis regulated gene SlBBX20:1.The SlBBX20-OE plants were with dark green leaves and fruits.The levels of carotenoids in leaves and fruits from SlBBX20 overexpression lines were higher than that of the wild type plants.2.SlBBX20 can activate the expression of PSY1,encoding a key enzyme in carotenoid biosynthesis,by directly binding its promoter,which results in the elevated levels of carotenoids in SlBBX20 overexpression lines.3.SlBBX20 is a substrate of the CUL4-DDB1-DET1 E3 ligase.Interactions between SlBBX20 and DE-ETIOLATED 1(SlDET1)lead to the ubiquitination and 26S proteasome-mediated degradation of SlBBX20.4.We suggest that two factors underpin the elevated levels of carotenoids in the SlBBX20-OE fruit:increased expression of the PSY1 gene and elevated chloroplast content at the green fruit stage.