Identification of grapevine Pectate Lyase genes altering Berry Texture during ripening

Vitis vinifera L. is one of the most important crops worldwide due to its economic relevance in winemaking and as a fresh fruit (table grapes). Several parameters determine consumer acceptance in the table grape market, including berry size, aroma, flavour, and texture. While consumers perceive berry texture as an indicator of fruit freshness, for producers, the firmness is a critical trait influencing resistance to pathogen invasion and postharvest spoilage. Firm berries resist mechanical damage during transportation and storage, enhancing fruit quality and shelf life. Producers frequently apply plant growth regulators to optimise textural traits in table grapes, particularly gibberellic acid, to improve berry size and firmness. Also, postharvest management strategies, such as cold storage and sulfur dioxide treatments, are commonly employed to mitigate mould growth and prolong fruit quality. Although these approaches have demonstrated effectiveness, table grape breeders continue to seek new varieties with improved textural properties to reduce production costs while ensuring high market acceptance. During grape ripening, significant textural changes occur, leading to berry softening. This complex process is the result of the loosening and partial disassembly of the cell wall. The primary cell wall in dicotyledonous plants comprises approximately 30-35% cellulose, 10% hemicellulose, and 35-40% pectins. Cellulose microfibrils, which are composed of β-1,4-glucan chains, provide tensile strength and serve as the structural backbone of the cell wall. These microfibrils are cross-linked by hemicelluloses, with xyloglucan being the predominant of these polysaccharides in dicots. Pectins form a heterogeneous matrix embedding the cellulose fibrils, and they are classified into three major groups: homogalacturonan (HG), a linear polymer of α-(1,4)-linked D-galacturonic acid, and the heterogeneous polymers, rhamnogalacturonan-I (RG-I), and rhamnogalacturonan-II (RG-II). Pectins are the primary constituents of the middle lamella, the outermost region of the cell wall, where they play a crucial role in tricellular junctions (TCJ) during softening. The depolymerisation of pectin in the TCJ weakens the adhesion between the cells, ultimately contributing to overall fruit softening. Several enzymes mediate cell wall modifications during ripening, particularly those targeting highly methylesterified pectin. Pectin methylesterases (PMEs) regulate the degree of methylesterification of HG. Following demethylesterification, two pathways are possible: (i) HG chains can cross-link with calcium ions, forming an egg-box motif that promotes cell wall stiffening, or (ii) HG can be hydrolysed by polygalacturonases (PGs) and/or cleaved by pectate lyases (PLs), leading to pectin degradation and tissue softening. Previous studies have investigated the role of specific genes or gene families in regulating fruit firmness, particularly pectate lyases. For instance, a knock-out study in tomato (Solanum lycopersicum) demonstrated that the loss-of-function mutation of the main SlPL in the cv. Ailsa Craig, resulted in increased fruit firmness. In contrast, the knock-out of SlPG had no significant impact on firmness. Given these findings, the present study aimed to identify and characterise the Vitis vinifera pectate lyase (VviPL) genes involved in berry softening during ripening, particularly those responsible of berry texture. The study was structured around three specific objectives: (i) identifying the VviPL gene family and selecting candidate genes for functional characterisation based on their potential role in regulating texture in table grapes; (ii) evaluating VviPL gene expression in table grape varieties exhibiting contrasting firmness; and (iii) conducting functional analyses of selected VviPL genes to assess their impact on texture at full ripening. The first part of the project involved a genome-wide analysis using the latest annotation of the V. vinifera PN40024 genome assembly. This analysis identified 17 VviPL genes, one more than previously reported in the literature. To select VviPL genes for functional characterisation, publicly available transcriptomic datasets were analysed to examine VviPL expression patterns in table grape varieties contrasting for firmness during ripening. The in silico analysis identified two candidate genes, VviPL14 and VviPL16, as the most upregulated after veraison (E-L 35) in soft genotypes. To validate VviPL14 and VviPL16 function, gene expression profiles were analysed in genotypes with contrasting firmness at three developmental stages, in both the skin and flesh, separately:veraison (E-L 35), mid-ripening (E-L 36), and full ripening (E-L 38). The commercial cultivar ‘Allison’ was selected as a representative firm variety, whereas ‘Misket Dunavski’ and ‘Rosina’ were chosen as soft table grape varieties, in order to perform analysis in contrasting phenotypes. The gene expression of VviPL14 and VviPL16 were higher at mid-ripening (E-L 36) in ‘Misket Dunavski’ and ‘Rosina’ compared to ‘Allison’, supporting their potential role in determining berry softening. Berries of ‘Allison’ (cv. with firmer berries) and ‘Misket Dunavski’ (cv. with softer berries) were immunohistochemically and biochemically analysed to further investigate the role of pectin modifications in berry texture. Immunohistochemical staining with LM7 (recognising partially methylesterified pectin) and LM15 (identifying a xyloglucan motif, XXXG) revealed stronger signals in ‘Misket Dunavski’, suggesting a higher degree of pectin depolimerisation in the softer genotype. Additionally, monosaccharide composition analysis demonstrated that ‘Allison’ had a higher content of galacturonic acid units compared to ‘Misket Dunavski’, further supporting the role of pectin degradation in berry texture differences. Therefore, VviPL14 and VviPL16 were selected as promising candidates for functional characterisation by means of two approaches: knock-out via CRISPR-Cas9 and knock-down via RNA interference (RNAi) by delivery of dsRNA (double-stranded RNA) in planta, using A. tumefaciens as the carrier. VviPL14 and VviPL16 knock-out lines were generated through Agrobacterium tumefaciens-mediated transformation of the ‘Sugraone’ embryogenic callus. A greenhouse phenotyping indicated that VviPL16 knock-out plants exhibited a slower growth rate and reduced internode length compared to wild-type plants. Notably, berries obtained from one knock-out VviPL16 line displayed a preliminary trend toward increased firmness compared to wild-type berries, suggesting a pivotal role in the berry texture of this gene in ‘Sugraone’. For the knock-down approach, RNAi constructs targeting VviPL14 and VviPL16 were delivered via Agrobacterium tumefaciens-mediated agroinfiltration in mature ‘Rosina’ vines, between veraison (EL-35) and mid-ripening (EL-36). These phenological stages were selected previously in the gene expression analysis where they showed a high transcript level of VviPL14 and VviPL16. At full ripening, berries treated with A.t. carrying VviPL14 and VviPL16 RNAi vectors exhibited higher firmness, with a 15.7% and 6.2% increase, respectively, compared to control berries. In conclusion, this study identified VviPL14 and VviPL16 as pectate lyase genes involved in grape berry softening and texture determination. The expression and functional analyses suggest their role in regulating texture during ripening in grapevine, with knock-down and knock-out experiments confirming their impact on firmness. These findings contribute to the understanding of cell wall dynamics in grapevine and offer potential targets for breeding programs or biotechnological strategies aimed at improving textural properties in table grapes.