Functional Analysis Of Fruit Color And Texture Regulated Genes SlSGR1and NsLTP In Tomato

Tomato (Solanum lycopersicum) is a very popular fruit and vegetable worldwide. As one of the world’s most important horticultural crop, it plays an important position in China’s vegetable industry. Moreover, tomato is a model plant for fruit ripening and mature research.Both the color and texture of tomato fruit are important commodities traits, and their formation process contains variety of complex physiological process and involves many biologically active substances, thus the tomato fruit color and texture have always been the research hotspot in the field of ripe fruit. The important antioxidant nutrients such as lycopene become the main coupler substance when tomato fruit color gradually turns red. Lycopene can be intaken by human daily diet, and palys an important roles in human anti-aging, anti-cancer efficacy academia widely recognized. The softening is a significant feature when tomato fruits matured, and it is also the reason of tomato fruit being direct edible and the key factor to affect tomato storage. Here, we described two gene SISGR1and nsLTP which involved in the formation of pigment accumulation and texture of tomato fruit using transgenic technology. The main results are as follows:I. The color regulated gene SISGR1in tomato fruit:1. SISGRl-KNAi transgenic fruits had an abnormal deep red color at the red ripe stage as compared to Wild type. Knock out of SISGR1in tomato significantly increases the lycopene and β-carotene contents at different ripening stages. As the fruits turned red, the concentration of lycopene and β-carotene increased significantly in transgenic fruits which was4.2-fold and9-fold as high as in the wt fruits, respectively.2. On visual observations, knockout of SISGR1by RNAi leads the thylakoid stacks in chloroplast of fruits disappeared early than wild type fruits during the ripening process and the globular increased noticeably at different ripen stages.3. In order to investigate the molecular regulation mechanism of S1SGR1in carotenoid accumulation in tomato fruit, a full-length SISGR1as a bait was used to screen a tomato fruit specific cDNA Y2H library. The result showed that S1SGR1regulates tomato lycopene accumulation by directly interacting with and inhibiting the activity of a key carotenoid synthetic enzyme PHYTOENE SYNTHASE1(S1PSY1). Via direct protein-protein interaction S1SGR1mediates the accumulation of lycopene during tomato fruit maturation. We confirmed this inhibitory activity in bacteria engineered to produce lycopene where introduction of SlSGRl dramatically reduced lycopene biosynthesis.4. Tomato fruits from SISGRl-RNAi lines had a lower H2O2content.The results also indicated that SlSGRl activity influences ethylene signal transduction via altered expression of ethylene receptor genes and ethylene-induced genes PG2a, PE and EXP1. Pericarp texture was more firm and fruit shelf-life significantly extended in SlSGRl repressed tomatoes.Ⅱ. The fruit texture regulated gene nsLTP in tomato fruit:1. Pericarp cells of tomato fruits tightly packed and PG enzyme activity decreased in nsLTP-RNAi lines.According to the observation by scanning electron microscope (SEM), transgenic fruit had better intact cell morphology and a lower level of cell wall degradation. Double fluorescent protein experiments show that tomato nsLTP protein had an interaction with PG2A and exercised its biological function by regulating PG enzymatic activity.2. The waxes layer of tomato cuticular with inhibited nsLTP gene expression are thicker than wild-type. Using GC-MS analysis of fruit surface wax ingredients, we found10kinds of the waxy components exist significant differences, hexane and31alkoxy content in transgenic tomato fruit was significantly increased, while another8alkanes, such as dodecane, n-pentane, n-heptane, pentadecane, subericalkoxy, n-octadecane, etc. only in the wild-type to be detected.3. CYP86and MS2, the key enzymes in tomato fruit wax biosynthetic pathway, had an expression patterns change in transgenic fruit during different ripen stage. The yeast two-hybrid results show the presences of nsLTP can interaction with a HD-Zip IV transcription factor Cutin2, which is a participation in the tomato fruit wax transport. BiFC results show that the interactions were positive.4. Tomato fruits from nsLTP-RNAi lines had lower water content and exhibited water loss during postharvest. Transgenic tomato fruits had an enhanced Biotic stress capability when compared to the wild type. Botrytis cinerea inoculation experiments showed that the transgenic tomato fruit has good resistance to gray mold, three days after inoculation of Botrytis cinerea plaque was significantly smaller than that of the control, gray fungal hyphae proliferation was inhibited.