| Tomato(Solanum lycopersicum Mill),a typical climacteric berry fruit.One of the most obvious changes in fruit ripening process of tomato is the change of fruit color.Ethylene plays a key role in the ripening process of tomato fruit.WRKY transcription factor is one of the largest family of regulatory transcription factors in plants.Knowledge of WRKY transcription factors in plant development and senescence are still mainly in Arabidopsis,tobacco,rice and other model plants.Although people have conducted extensive research on the ripening mechanism of tomato fruit,there are still many areas that are not clear enough.So far,studies on the involvement of WRKY transcription factors in the regulation of tomato fruit growth and color changes in fruits have rarely been reported.The participation of WRKYs in tomato fruit ripening regulation is still not clear.This study is based on the previous research of our group.and further investigates the regulatory pathways of WRKY16,WRKY17,WRKY53,WRKY54.The mutant tomato rin,nor,gr,nr and wild-type Hongyan tomato were used as materials,to explore the relevant physiological indices and the expression of the ripening-related genes during tomato ripening process.Q-PCR method,yeast one hybridization,and Dual luciferase system were applied to analyze expression patterns of multiple tomato WRKY family members SlWRKY16,SlWRKY17,SlWRKY53,SlWRKY54 genes,and the regulation of key ripening transcription factors on these genes,so as to provide information for the study of SlWRKY transcription factor related regulatory mechanisms.The main results were as follows:(1)Green mature fruit of wild type Hongyan quickly turned red and softened after ethylene initiation of the ripening.Exogenous ethylene treatment can promote the ripening process of Hongyan fruit.There was no significant change in hardness and color of rin and gr green ripe tomato fruits treated with ethylene.nr green ripe tomato fruit treated with ethylene can also play a role in the later stages of process.Based on the above analysis,thephysiological effects of ethylene treatment on these mutants can be roughly divided into the following categories.Insensitive to ethylene,exogenous ethylene can not be restored after ripening,such as rin and gr.Exogenous ethylene can restore a large degree of fruit maturation.if not,exogenous ethylene can be partially matured,such as nr.(2)WRKY16 and WRKY17 and WRKY53 were induced by ethylene in Hongyan fruits.WRKY54 was weakly induced by ethylene.WRKY16,WRKY17,genes were up-regulated in rin,nor,nr and gr mutants.WRKY53 was up-regulated in all but gr mutations,and WRKY54 was not up-regulated in all but in nor mutants.(3)The tobacco transient expression results showed that under the action of RIN transcription factors,WRKY16,WRKY17 and WRKY53 all showed positive regulation,and the activity increased.while the activity of WRKY54 decreased.Under the effect of NOR transcription factor,the activity of WRKY16,WRKY17 and WRKY53 also increased,while the regulation of WRKY54 was close to the control,indicating that the NOR transcription factor had no significant effect on the activity of WRKY54.WRKY promoter activity reporter vector and ERF1 over expression vector were co-transformed into tobacco leaves.The expression of WRKY16,WRKY17,WRKY53 was up-regulated.and the activity of WRKY54 was decreased,indicating that the transcription factor ERF1 had a significant regulation on WRKY16,WRKY17,WRKY53.(4)Yeast one-hybrid results showed that RIN,NOR and ERF1 all interacted with WRKY17.and RIN,ERF1 and WRKY53 interacted.This shows that RIN and ERF1 have regulatory effects on WRKY17 and WRKY53,while NOR only controls WRKY17.The regulatory mechanisms of other transcription factors and WRKY remain to be further studied.The above results indicate that the SlWRKY genes is closely related to the process of tomato ripening.The SlWRKY17,SlWRKY53 genes play important roles in the process of the regulation.The regulatory mechanisms of SlWRKY16,SlWRKY54 are still needed to be further explored. |
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