Functional Study Of Tomato SlMYB75 And SlNAC6 Transcription Factors In Fruit Ripening And Stress Responses

Tomato(Solanum lycopersicum)is an important economic crop that widely loved by consumers around the world,and also the best model plant to study the development and ripening of fleshy fruit.Both MYB and NAC transcription factors are one of the largest transcription factor family in plants,of which the NAC transcription factor are unique to plants.Currently,though some progress have been made on the study of the two transcription factor families,whereas these studies are mainly limited in model plant Arabidopsis,and some of the transcription factors are demonstrated to play important roles in plant growth and development,ripening and stress response processes.In the present thesis,we use tomato as the research material to study the function roles of SlMYB75 and SlNAC6 in tomato fruit ripening and stress response by using bioinformatics,genetics,cytology,molecular biology and physiology and biochemistry methods.The main results obtained in this thesis are as follows:(1)Sequence alignment analysis showed SlMYB75 is the highest homologous gene of the key MYB transcription factor that control the biosynthesis of anthocyanins in blood orange.The SlMYB75 gene can be induced by various stress conditions or exogenous hormones,which is consistent with the various cis-elements that related to stresses or hormones exist on its promoter.qPCR analysis showed the background expression level of SlMYB75 in tomato fruit organ is very low,but overexpression of a single SlMYB75 gene(SlMYB75-OE)can induce accumulation of abundant amounts of anthocyanins in various tissues of tomato,and obtain a remarkable purple tomato fruit,whose anthocyanins content at red ripening stage could increase to 1.86 mg g~-11 fresh weight.In addition,SlMYB75-OE fruit exhibit a delay of ripening when compared to wild type(WT)fruit.(2)In addition to the significantly increase of anthocyanins content,several fruit quality attributes involved in fruit ripening process are also significantly improved in SlMYB75-OE tomato,including flavonoids,phenolics,soluble sugar and aroma volatiles,especially some of the terpenes being more than 10 times higher levels than those in the WT fruit.Consistently,transcriptome profiling revealed that several corresponding metabolic pathways,such as phenylalanine pathway and aroma volatile related pathway in SlMYB75-OE fruit are also greatly affected.Yeast one-hybrid and dual-luciferase assays indicated SlMYB75 transcription factor has a regulatory function that interact with MYBPLANT and MYBPZM elements,and that may promote aroma volatile accumulation partly through directly regulating the expression of downstream aroma biosynthesis genes(LOXC,AADC2 and TPS genes).(3)SlMYB75-OE transgenic purple tomato contains three main anthocyanin compontents,and the compontent that occupies the highest content is preliminary identified as petunidin-3-o-(p-coumaroyl)-rutinoside-5-o-glucoside.The antioxidant ability of SlMYB75-OE fruit is greatly enhanced,which results in a significantly prolong of shelf life and greatly increase of resistance to Botrytis cinerea.(4)Phylogenetic tree showed that SlNAC6 is clustered to the same evolutionary branch with two stress-related NAC transcription factors.The promoter of SlNAC6 gene contain several ABA and stress response cis-elements,and SlNAC6 gene can be induced by PEG stress and exogenous application of ABA.SlNAC6 is a nuclear-localization protein with a highly expression level in leaf tissue.(5)The plants overexpression of SlNAC6 gene(SlNAC6-OE)display decreased leaf stomatal conductance,decreased leaf water loss rate,delayed seed germination and retarded seedling growth,and these parameters are also affected to some extent in SlNAC6-interference(SlNAC6-RNAi)lines.Further studies revealed that down-regulation or overexpression of SlNAC6 gene can decrease or increace the transcript levels of ABA biosynthesis pathway genes(NCED1,NCED2 and ABA2 genes,etc.)separately,thereby affecting the endogenous ABA content.(6)In compared to WT plants,PEG stress caused more serious damage to SlNAC6-RNAi lines with displaying lower relative water content,proline content and antioxidant enzymes activity,as well as higher hydrogen peroxide content and malondialdehyde content,and the transcript levels of multi stress-related genes(P5CS,SOD and POD genes,etc.)are significantly decreased.Consistently,overexpression of SlNAC6 effectively improved the tolerance of tomato plants to PEG stress,and these parameters showed opposite changes in SlNAC6-OE lines.This suggest SlNAC6transcription factor plays positively roles in tomato plants response to drought stress.In addition,SlNAC6-OE lines display an earlier ripening when compared to WT fruit.In conclusion,this thesis confirms that a single SlMYB75 transcription factor plays excellent roles in producing anthocyanins-enriched tomato fruit,improving several fruit quality attributes involved in fruit ripening process and promoting diease resistance and storage capacity of postharvest fruit,and confirming that the SlNAC6 transcription factor plays important roles in tomato response to drought stress and fruit ripening process.These results lay a foundation for clarifying the function characteristics of SlMYB75 and SlNAC6 transcription factors in plants,and also provide important references for improving the quality and agronomic traits of tomato or other Solanaceae crops through breeding or genetic engineering techniques.