LOOKING FOR CONSERVED REGULATORY FUNCTIONS IN PETUNIA AND GRAPEVINE. PART II: WRKY AND bHLH TRANSCRIPTION FACTORS.

As a part of an extensive project aimed to functionally analyze grapevine regulatory genes putatively involved in flavonoid and/or vacuolar acidification pathways we tested one WRKY and two bHLH transcription factors by heterologous expression in petunia. The coding sequence of VvWRKY19, the grape closest homolog to petunia PhPH3 and Arabidopsis AtTTG2 , was isolated, fused to the constitutive promoter 35S and transformed into a ph3 petunia mutant line. Analyses of transgenic plants revealed that VvWRKY19 is the true orthologue of PhPH3, being able to completely fulfill its function. By exploring the last released grape genome prediction we found a gene with high similarity to petunia PhJAF13 and other known bHLH factors regulating flavonoid synthesis in species ifferent from grapevine. Molecular analysis of VvJAF13 revealed that it displays all the basic features for participating to the MYB-bHLH-WD regulatory complex and to be a candidate regulator of the flavonoid pathway. In order to gain information about the function of VvJAF13 and of the another previously partially characterized grape bHLH (VvMYC1), we ectopically expressed them in a petunia an1 unstable mutant line. VvMYC1 was able to restore the wild type petal pigmentation and pH confirming that it is a functional horthologue of PhAN1. On the other hand VvJAF13 was unable to restore the wild type phenotype and, instead, caused the complete absence of pigmented revertant sectors in petals. To better understand the function of VvWRKY19 and VvJAF13 the stable transformation of Vitis vinifera cv Shiraz has been performed with the aim to silence or overexpress them. The phenotypic and molecular characterization of transformants is underway.