Biology subjects

Castellarin, S. D.

Publications and source records attributed to Castellarin, S. D..

4 recordsLinked to original sources

bioRxiv2026

TPS Expression, Not Copy Number, Explains Aroma Diversity among Grapevine (Vitis vinifera L.) Cultivars

Terpenoids are major contributors to grapevine berry and wine aroma, with mono- and sesquiterpenoids exhibiting substantial variation among cultivars. However, the genetic basis of cultivar-specific terpenoid profiles remains only partially understood due to gene duplications that expanded the terpenoid synthase (TPS) gene family across different cultivars. Here, we compared haplotype-resolved genome assemblies for seven cultivars to analyze copy number variation and expression profiles of terpenoid biosynthesis genes and terpenoid accumulation throughout flower and berry development. These assemblies resolved duplicated TPS loci at the haplotype level, revealing extensive copy number variation across cultivars and between haplotypes within a cultivar, with up to 44 mono-TPS genes on a single haplotype. We identified eight specific monoterpenoids as key drivers of cultivar-specific profiles in ripe berries. Total TPS transcript abundance correlated strongly with total terpenoid accumulation over time. In contrast, TPS copy number was largely decoupled from both expression and terpenoid accumulation, indicating that expression regulation, rather than gene dosage, underlies aroma differences among cultivars. Transcript-metabolite network analysis revealed both expected correlations between specific TPS genes and putative products as well as novel associations. We functionally validated selected candidate TPS in Nicotiana benthamiana, characterizing -farnesene, {beta}-ocimene, and multi-product -terpineol synthases. The in vivo characterization demonstrated that minor sequence variations can alter catalytic activity and product profile. Together, these findings show that the extensive duplication of TPS genes in grapevine does not itself determine terpenoid output; instead, cultivar-specific aroma arises from how these genes are expressed and from sequence differences that shape enzyme activity.

plant biology↗

bioRxiv2024

Grape ripening speed slowed down using natural variation

Understanding ripening patterns and governing ripening speed are central aspects of grapevine (Vitis vinifera) berry biology owing to the importance of grape ripeness in winemaking. Despite this, the genetic control of ripening is largely unknown. Here, we report a major quantitative trait locus that controls ripening speed, expressed as speed of sugar accumulation. A haplotype originating from the species Vitis riparia halves maximum speed regardless of crop levels and berry sizes. The sequence of events that are normally completed at the onset of ripening in a two-week period known in viticulture as veraison are taking place at a slower speed, thereby attaining ripeness under milder weather conditions in late summer. V. vinifera cultivars show limited phenotypic variation for ripening speed and no selective sweep in the causal genomic region that could derive from domestication or improvement. Closely related species make up for the lack of standing variation, supplying major effect alleles for adapting grape cultivars to climate change. HIGHLIGHT / SIGNIFICANCE STATEMENTReducing the speed of fruit ripening genetically is a means for adapting the grape berry developmental program to the changing needs of the wine industry and in response to global warming. We identified a haplotype in a wild grape species that slows down the speed of ripening in progenies of Vitis vinifera by limiting the speed of sugar accumulation throughout the duration of ripening, a condition of great importance for winemakers to harvest their grapes at the desired level of technological ripeness.

plant biology↗

bioRxiv2021

The grape MYB24 mediates the coordination of light-induced terpene and flavonol accumulation in response to berry anthocyanin sunscreen depletion

The presence of naturally-occurring color mutants in plants has permitted the identification of many regulatory genes implicated in the synthesis of discrete metabolic compounds, mostly anthocyanins and carotenoids. Conversely, transcription factors that coordinate more than one specialized metabolic pathway seem challenging to screen from a forward genetics perspective. We explored the relationship between different branches of the phenylpropanoid and isoprenoid pathways while examining an infrequent berry skin color variegation in grapevine. Red and white berry skin sections were compared at the genetic, transcriptomic and metabolomic levels showing that, as in most cultivated white grape varieties, the uncolored skin section convened the non-functional alleles of the anthocyanin regulators MYBA1 and MYBA2, explaining the lack of pigments. In contrast, light-responsive flavonols and monoterpenes increased in anthocyanin-depleted areas. We disclosed an enrichment of the flavonol, terpene and carotenoid pathways among up-regulated genes from white-skin sections, accompanied by increased expressions of flavonol regulators and the still uncharacterized MYB24 gene. We used DAP-seq to examine the in vitro binding of affinity-purified MYB24 protein to genomic DNA and demonstrated its binding in the promoter regions of terpene (22) and carotenoid (6) genes, in addition to more than 30 photosynthesis and light-response genes, including the flavonol-regulator HY5 homologue (HYH). We confirmed the activation of TPS35 and HYH promoter:luciferase reporters in the presence of MYB24 and the grape bHLH MYC2, all of which correlate in their higher expression in white skin variegated sections. The integration of several datasets allowed to define a list of high confidence targets, suggesting MYB24 as a modulator of light responses including the synthesis of flavonoids (flavonols) and isoprenoids (terpenes, and putatively carotenoids). The correspondence between MYB24 and monoterpenes in all conditions surveyed implies that this regulatory network is broadly triggered towards berry ripening, and that the absence of anthocyanin sunscreens accelerates its activation most likely in a dose-dependent manner due to increased radiation exposure.

plant biology↗

bioRxiv2019

Minimally invasive, pressure probe based sampling allows for in-situ gene expression analyses in plant cells.

BackgroundGene expression analyses are conducted using multiple approaches and increasingly research has been focused on assessing gene expression at the level of a tissue or even single-cells. To date, methods to assess gene expression at the single-cell in plant tissues have been semi-quantitative, require tissue disruption, and/or involve laborious, possibly artifact-inducing manipulation. In this work, we used grape berries (Vitis vinifera L. Zinfandel) as a model in order to examine the validity and reproducibility of an in-situ gene expression analysis method combining a cell pressure probe (CPP) with quantitative PCR (qPCR).\n\nResultsWe developed a method to directly assess gene expression levels via qPCR from cellular fluids sampled in-situ with a CPP. Cellular fluids, with volumes in the picoliter range, were collected from intact berries with a CPP at various depths across skin and mesocarp tissues. The expression of a key anthocyanin biosynthetic gene, UDP-glucose: flavonoid 3-O-glucosyltransferase (VviUFGT), was analyzed as a test case since its expression is restricted to cells producing anthocyanins in grape berry skins during ripening. The method identifies samples contaminated with significant levels of genomic DNA by amplifying a region of VviUFGT that spans an intron. Therefore false positives were discarded which occurred in 28% of the samples tested. Shallow probing of skin cells showed high VviUFGT expression as expected while deeper probing of mesocarp cells resulted in no VviUFGT expression.\n\nConclusionsThe clear correspondence of VviUFGT expression to the targeted cell samples suggests that the in-situ gene expression analysis using a CPP is reliable and does not result in contamination as the probe moves through tissues. This method can be paired to single-cell transcriptomic analyses in the future. We conclude that this technique represents a minimally invasive method of sampling plant cells in-situ which creates an opportunity for the analysis of cellular level, spatiotemporal responses in heterogeneous plant tissues.

plant biology↗