WRKY transcription factors in the response of table grapes (cv. Autumn Royal) to high CO2 levels and low temperature

WRKY transcription factors are regulated by biotic and abiotic stress in Vitis vinifera, although little is known about their role in grape berries. The latest V. vinifera reference genome (PN40024 12 x .v2, VCOST.v3 annotation) gave us a good opportunity to perform an analysis of VviVVRKYs, identifying 61 genes. Our study examines whether the application of high CO2 levels for 3 d to maintain table grape quality activated the gene expression of 15 WRKYs in the skin and pulp of grapes (cv. Autumn Royal) stored at 0 degrees C. Results showed that the induction of VviVVRKYs gene expression by CO2 was tissue dependent, being this effect mainly observed in the pulp where 9 out of the 15 genes analyzed were activated by the gaseous treatment. The expression analysis of genes associated with ethylene (ACC synthasel (ACS1) and ACC oxidasel (ACOI)) and ABA (9-cis-epoxycarotenoid dioxygenasel (NCED1)) biosynthesis indicated that the expression of ACOI in the skin of non-treated samples and in the pulp of CO2 -treated ones correlated with the changes observed in VviWRKYs. In addition, we studied the role of WRKYs in the modulation of genes encoding pathogenesis-related proteins (PRs) in table grapes. Results showed there was a significant positive correlation between VVRKYs modulated by CO2 in the skin and pulp with a beta-1,3-glucanase (Vcgnsl) and a thaumatin (VvTL1), respectively. Through electrophoretic mobility shift assay (EMSA) we observed that VviWRKYIIa_3 recombinant protein was able to in vitro bind to the W-box present in the promoter of the three PRs analyzed while VviWRKYIII_3 was only able to specifically bind to the Vcgnsl probe. Our results support the fact that high CO2 treatment is an active process that requires the activation of transcription factors, such as those of the WRKY family, which could participate in the molecular events involved in maintaining table grape quality during postharvest.