Tomato SlNAC3 Regulates Fruit Ripening By Delaying Fruit Softening And Altering Carotenoid Biosynthesis

Fruits are the main source for human and animals. The development and maturation process of fruits are specific for plants, so the investigation into them have important theoretical and socio-ecnomic value. During the process of fruit maturation, a variety of changes occur with the fruit quality and the nutrient components. Although the characteristics of maturation among different species are varied, the differences can be broadly described as color, texture, flavor, aroma and nutrition. Plants attract organisms to help themselves to spread the seeds via the aroma.Fruits can be divided into two types, climacteric fruit and non-climacteric one. The former has a significant climacteric peak in the initial stage of ripening. Ethylene accumulation for the fruit maturity is required for the climacteric fruits, such as tomato, banana and apple, and not required for non-climacteric fruit, for example strawberrie, grape and citrus. Till now, the studies about maturation process at the molecular level have focused on the regulatory pathway of ethylene, but the regulatory process and the regulatory mechanism before the ethylene induction are poorly investigated.Recent researches showed that fruit maturity is under the control of multiple of genes. A large number of genes express at the beginning of fruit ripening. The expression of these genes determine the biochemical changes in fruit color, texture and flavor. It is very difficult to know the role of individual gene, so identifying some key factors in the regulation pathway is more effective.In this study a new NAC member SINAC3 was analyzed. The gene expression pattern, subcellular localization and transcriptional activation activity were analyzed, and investigation into the transformed tomato revealed the gene’s function in fruit development and maturation at molecular level. The main results are as follows:1. The structure analysis of the target gene. The alignment analysis among NAC amino acid revealed that the sequences showed highly conserved at N-terminal, while varied for the C-terminal.2. Phylogenetic analysis. Constructed phylogenetic tree using the sequences of NACs from tomato, Arabidopsis, petunia and other species. The result showed that SINAC3 was involved in the group with NOR and OsNAC3. It revealed that SINAC3 might function in tomato ripening and stress responses. 3. Transient expression of CaMV35S::SlNAC3-GFP fusion construct in onion epidermal cells showed that SlNAC3 localized in the nucleus.4. The expression profile analysis of target gene. SlNAC3 had higher levels of expression in tomato flowers and fruits, especially in mature green fruit and breaker fruit, low expression in tomato stems and leaves. During all the stages of fruit development and maturation SINAC3 have different levels of expression, and SINAC3 expresses not only in the fruit peel and pulp tissues but also in the seeds.5. Stress expression. SlNAC3 showed down-regulated exposed to ABA, drought and NaCl treatment.6. Obtained over-expression and RNAi suppression transgenic tomato by transformation. The RNAi transgenic tomato showed lycopene and total carotenoid synthesis inhibited, while lutein andβ-carotene significantly increased in fruit and the fruit revealed orange colour.7. The cell wall degradation related genes showed significantly decreased in RNAi transgenic tomato. The fruits had no climacteric peak or respiration emission and also improved fruit firmness.8. The fruit ripening process of RNAi transgenic tomato was significantly delayed. Normally tomato fruit reaches the stage of red ripe at 45 DPA while RNAi transgenic tomato remained to be orange and immature at 70 DPA.9. The pollen viability of RNAi transgenic flower showed significantly decreased and the seed development showed symptoms of abortion. The ultrastructure analysis of RNAi transgenic seeds using paraffin section and transmission electron microscopy showed that undetectable starch grains in the endosperm and irregular cells.10. Microarray results showed that the expression of cell wall metabolism related genes showed significantly inhibited in RNAi transgenic tomato.