Characterization of Endogenous Levels of Brassinosteroids and Related Genes in Grapevines

Abstract

Agronomic breeding practices for grapevines (Vitis vinifera L.) include the application of growth regulators in the field. Brassinosteroids (BRs) are a family of sterol-derived plant hormones that regulate several physiological processes and responses to biotic and abiotic stress. In grapevine berries, the production of biologically active BRs, castasterone and 6-deoxocastasterone, has been reported. In this work, key BR genes were identified, and their expression profiles were determined in grapevine. Bioinformatic homology analyses of the Arabidopsis genome found 14 genes associated with biosynthetic, perception and signaling pathways, suggesting a partial conservation of these pathways between the two species. The tissue- and development-specific expression profiles of these genes were determined by qRT-PCR in nine different grapevine tissues. Using UHPLC-MS/MS, 10 different BR compounds were pinpointed and quantified in 20 different tissues, each presenting specific accumulation patterns. Although, in general, the expression profile of the biosynthesis pathway genes of BRs did not directly correlate with the accumulation of metabolites, this could reflect the complexity of the BR biosynthesis pathway and its regulation. The development of this work thus generates a contribution to our knowledge about the presence, and diversity of BRs in grapevines.

Keywords: UHPLC-MS/MS; brassinosteroids; development; grapevine.

Figure 1

Brassinosteroid biosynthesis routes. The different routes of biosynthesis of brassinosteroids are depicted. Dashed lines indicate multiple enzymatic steps. Compounds detected in this work are highlighted in orange. Diagram based on [33,34,35,51,52,53].

Figure 2

Quantification of brassinosteroids (pmol/g DW) in cv. Cabernet sauvignon grapevine tissues during development. Brassinosteroids were measured by UHPLC-MS/MS, and results were expressed as a heat map. Three biological samples and three technical replicates were measured, with each biological replicate corresponding to a pool of tissue collected from five different plants.

Figure 3

Expression levels of genes involved in brassinosteroid biosynthesis in cv. Cabernet sauvignon grapevines. Expression data was normalized against the reference gene, VviUBI. Bars are the mean ± standard deviation of three biological and three technical replicates. Each biological replicate corresponds to a pool of tissue collected from five different plants.

Figure 4

Expression levels of genes involved in brassinosteroid perception and signaling in cv. Cabernet sauvignon grapevines. Expression data was normalized against the reference gene, VviUBI. Bars are the mean ± standard deviation of three biological and three technical replicates. Each biological replicate corresponds to a pool of tissue collected from five different plants.