Regulatory Mechanisms Of Transcription Factor SlNAP1 In Controlling Tomato Fruit Ripening By GA

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.NAC transcription factor is one of the largest transcription factor family in plants,of which the NAC transcription factor are unique to plants.Currently,though some progresses have been made on the study of the NAC transcription factor,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 study,we use tomato ‘Micro Tom’ as the research material to functional study of Gibberellin(GA)mediated by SlNAP1 transcription factor during tomato fruit ripening by using bioinformatics,physiology and biochemistry and molecular biology methods.The main results obtained in this study are as follows:(1)Alignment analysis,evolutionary relationship analysis and RT-q PCR showed that SlNAP1,SlNAP2,SlNAC2,SlNAC36 and SlNAC72 is the high expression during fruit ripening stage in tomato.It is further confirmed that the genes play a positive role in the tomato fruits ripening by VIGS.(2)The subcellular localization result reveals that SlNAP1 is nuclear transcription factor.In addition,SlNAP1 knockout mutants slnap1-5 and slnap1-16 were obtained by CRISPR/Cas9.We find that chlorophyll degradation,carotenoids accumulation,ethylene release and degree of fruit softening are significantly decreased in the mature green and breaker fruits of slnap1-5 and slnap1-16 mutants during fruit ripening compared to WT.These results suggest that SlNAP1 improves tomato fruit ripening.(3)The endogenous GA content is significantly increased in the mature green and breaker fruits when SlNAP1 is momentarily silenced.Compared to WT,the endogenous GA content of knockout mutants slnap1-5 and slnap1-16 aslo is significantly increased.When GA and its synthesis inhibitor PAC were exogenously applied to the mature green and breaker fruits of WT,slnap1-5 and slnap1-16 knockout mutants,we found that GA delays tomato fruits ripening by decreasing the content of chlorophyll and carotenoids,ethylene release and the degree of fruit softening.However,the effect of PAC treatment is the opposite.Compared to WT,the effect of GA/PAC was more significant in slnap1-5 and slnap1-16 knockout mutants.The result further firmed that SlNAP1 may be mediate GA to regulate tomato fruit ripening.(4)SlNAP1 positively regulates the expression levels of GA degradation-related genes GA2ox1 and GA2ox5 promoter,negatively regulates the expression levels of GA synthesis-related genes GA3ox1 and GA3ox2 promoter by Y1 H and DLR.Simultaneously,SlNAP1 interacts GA receptor protein GID4.The crosstalk GID4 with SlNAP1 is enhanced by GA and weakened by PAC.In conclusion,SlNAP1 improves tomato fruit ripening.SlNAP1 directly binding to the promoter of GA2ox1,GA2ox5,GA3ox1 and GA3ox2 genes and interacts GA receptor protein GID4 during this process,thereby inhibiting GA synthesis.Therefore,SlNAP1 promotes tomato fruits ripening by accelerating chlorophyll degradation,carotenoids accumulation,ethylene production and fruits softening.