The Regulation of Ethylene-responsive SLWRKYS on the Color Change during Tomato Fruit Ripening

WRKY transcription factors are specifically and widely present in planta. They have been found playing important roles in plant responses to biotic and abiotic stresses as well as developmental regulation. Tomato is a typical climacteric fruit, where rapid respiration and ethylene production, chlorophyll degradation and lycopene accumulation occur during fruit ripening process. To study the role of SlWRKY family transcription factors in regulation of fruit ripening, tomato fruit ripening was manipulated by treatment with ethylene and 1-MCP, an ethylene receptor inhibitor. Based on the similarities to 24 ethylene responsive WRKY genes from Arabidopsis, rice, Gossypium hirsutum and Brassica napus, 23 SlWRKY genes were selected from the whole tomato SlWRKY gene family that includes 81 members. The expression profiling of these SlWRKYs was analyzed in the ripening system along with 8 fruit ripening related genes, including 5 color change related genes. Eight SlWRKYs genes displayed over-lapping up-regulation expression patterns as the ripening related genes and were detected to localize in nucleus, and showed potential transcriptional activation in yeasts. Yeast One-Hybrid and in vivo regulation assays in tobacco leaves showed that SlWRKY16, 17, 22, 25, 31, 33, 53, 54 were found to bind and activate the promoters of the color change related genes, SlPPH (PHEOPHYTINASE), SlPAO (PHEOPHORBIDE A OXYGENASE), SlPSY1 (PHYTOENE SYNTHASE1) and SlPDS (PHYTOENE DESATURASE). Furthermore, yeast Two-Hybrid assay indicated that obvious interaction may occur between WRKY16, 17, 33, 53, 54 and with other ripening regulators including, SlRIN, SlERF2b and SlERF7. Our study provides for the first time direct evidence that the WRKY transcription factors may function in fruit ripening, particularly in color change, and may connect to the intricate regulatory network with other ripening key regulators.