Epigenetic regulation of APT3 seems to explain the masculinized phenotype of a floral mutant of Listán Prieto

Abstract

While most cultivated grapevines (Vitis vinifera L.) possess perfect flowers, their wild ancestors were dioecious. Genomic analyses of commercial cultivars and wild grapevines have identified the structure of the sex-determining region, where an ancient recombination event enabled the expression of genes required for hermaphrodite flower formation. To preserve their agronomic properties, cultivated grapevines are clonally propagated; consequently, somatic mutations represent the primary source of genetic variability. The accession Criolla Chica Nº2 (CCN2), a floral somatic mutant of Criolla Chica (the Argentine synonym for cv. Listán Prieto) is characterized by predominantly masculinized flowers as compared to Criolla Chica Ballista (a Listán Prieto accession with hermaphrodite flowers -CCBA-). Exogenous application of cytokinins to early-stage CCN2 inflorescences induced the reversion of the phenotypic mutation. To elucidate the molecular mechanisms underlying this mutant phenotype, we sequenced and annotated the cv. Listán Prieto genome. Using Listán Prieto as reference genome, we evaluated differential gene expression between CCN2 and CCBAin inflorescences at a pre-anthesis stage (E-L 14 stage). Among the differentially expressed genes (DEGs), cytokinin dehydrogenase 3 and adenine phosphoribosyltransferase-3 (APT3) were significantly upregulated in CCN2. APT3 is associated with male flower development and is proposed to interfere with cytokinin metabolism and to be responsible for pistil development suppression. To further investigate the cause of this upregulation, Nanopore long-reads from the CCN2 and CCBA accessions were obtained and used to analyze structural variants (SVs) and methylation profiles. None of the identified SVs were directly associated with the phenotype of interest. However, we identified a differentially methylated region upstream of the APT3 gene on the hermaphrodite haplotype, where CCN2 showed significant hypermethylation. We propose that this epigenetic modification increases APT3 expression, interfering with the cytokinin metabolism and the female organs’ development, ultimately resulting in the masculinized phenotype observed in CCN2.

References

Alañón, N. et al. (2023). Identification of a stable epi-allele associated with flower development and low bunch compactness in a somatic variant of Tempranillo Tinto. IVES Conference Series, ICGWS 2023.

Aliquó, G. et al., (2017). Identity and parentage of some South American grapevine cultivars present in Argentina. Aust J Grape Wine Res 23, 452–460.

Oroño, T. et al. (2024). Phenotypical impact of a floral somatic mutation in the cultivar Listán Prieto. IVES Conference Series, Open GPB 2024.

Acknowledgements

This work was supported by European Commission’s Horizon 2020 Marie Sklodowska-Curie-Research and Innovation Staff Exchange programme “(vWISE) Vine and Wine Innovation through Scientific Exchange” and Agencia Nacional de Promoción Científica y Tecnológica (ANPCyT) PICT-2021-00039.