Spontaneous somatic mutations produce changes in berry skin color in grapevine. VviMybA1 and VviMybA2 transcription factors induce anthocyanin biosynthesis pathway through the regulation of UDP-glucose: flavonoid 3-O-glucosyltransferase (UFGT) expression. The lack of berry color in white-skinned cultivars is caused by an insertion of Gret1 retro-transposable element in the VviMybA1 promoter region preventing gene expression, and a two-nucleotide deletion in VviMybA2 coding sequence producing a null frameshift (Walker et al., 2007). Nevertheless, occasionally, anthocyanin pigmentation capacity is partially recovered from white-skinned cultivars that then produce red berries (Azuma et al., 2009). Here we have studied the cases of Albariño and Verdejo red-berried somatic variants appearing spontaneously in the field. We used transcriptomics and targeted UPLC-QqQ-MS/MS metabolomics to reveal the molecular cause of color recovery in these somatic variant berries at véraison stage. RNA-sequencing analysis identified 39 and 225 genes that were differentially expressed in the berry skin of Albariño and Verdejo red-berried variants compared to their respective white-berried ancestors (FDR2 fold-change). In both colored mutants, flavonoid biosynthesis pathway was activated, including the upregulation of UFGT, flavonone-3- hydroxylase, and chalcone synthase genes. The anthocyanins cyanidin-3-glucoside, cyanidin-3-(6-coum)-glucoside and cyanidin-3-(6-acetyl)-glucoside, were detected in the berry skin of both red Albariño and Verdejo, respectively. Additionally, Albariño red berries also contained detectable amounts of delphinidin-3-glucoside and peonidin-3- glucoside, and of cyanidin-3-arabinoside at trace levels explaining the more intense red color observed in this variant. In addition, we identified the molecular genetic alterations responsible for color recovery. The red Albariño variant had recovered VviMybA1 expression as a consequence of a recombination between LTRs of Gret1. This recombination left behind a SOLO LTR leading to a partially recovered expression of the gene. On the other hand, RNA-seq alignments indicated non-synonymous singlenucleotide polymorphisms and a possible homologous recombination within VviMybA2 exon 3, which may recover the reading frame and protein function in Verdejo. This is the first time that a berry color recovery is related to a VviMybA2 regain of function,enabling to determine the specific role of the two major transcriptional factors (VviMybA1 and VviMybA2) regulating grape anthocyanin biosynthesis.

Resumen del trabajo presentado en la XXIV Reunión de la Sociedad Española de Biología de Plantas y el XVII Congreso Hispano-Luso de Biología de Plantas, celebrado en modalidad online del 7 al 9 de Julio de 2021

Thanks to Miguel Angulo and Miguel Angel Fernández Recio for technical assistance. The Spanish MICINN BIO2017-86375 and PID2019-108851RBC22 supported this project