No abstract

Drought, elevated air temperature, and high evaporative demand are increasingly frequent during summer in grape growing areas like the Mediterranean basin, limiting grapevine productivity and berry quality. The foliar exogenous application of kaolin, a radiation-reflecting inert mineral, has proven effective in mitigating the negative impacts of these abiotic stresses in grapevine and other fruit crops, however, little is known about its influence on the composition of the grape berry and on key molecular mechanisms and metabolic pathways notably important for grape berry quality parameters. Here, we performed a thorough molecular and biochemical analysis to assess how foliar application of kaolin influences major secondary metabolism pathways associated with berry quality-traits, leading to biosynthesis of phenolics and anthocyanins, with a focus on the phenylpropanoid, flavonoid (both flavonol- and anthocyanin-biosynthetic) and stilbenoid pathways. In grape berries from different ripening stages, targeted transcriptional analysis by qPCR revealed that several genes involved in these pathways—VvPAL1, VvC4H1, VvSTSs, VvCHS1, VvFLS1, VvDFR, and VvUFGT—were more expressed in response to the foliar kaolin treatment, particularly in the latter maturation phases. In agreement, enzymatic activities of phenylalanine ammonia lyase (PAL), flavonol synthase (FLS), and UDP-glucose:flavonoid 3-O-glucosyltransferase (UFGT) were about two-fold higher in mature or fully mature berries from kaolin-treated plants, suggesting regulation also at a transcriptional level. The expression of the glutathione S-transferase VvGST4, and of the tonoplast anthocyanin transporters VvMATE1 and VvABCC1 were also all significantly increased at véraison and in mature berries, thus, when anthocyanins start to accumulate in the vacuole, in agreement with previously observed higher total concentrations of phenolics and anthocyanins in berries from kaolin-treated plants, especially at full maturity stage. Metabolomic analysis by reverse phase LC-QTOF-MS confirmed several kaolin-induced modifications including a significant increase in the quantities of several secondary metabolites including flavonoids and anthocyanins in the latter ripening stages, probably resulting from the general stimulation of the phenylpropanoid and flavonoid pathways.

Hormones play an important role in fruit ripening and in response to biotic stress. Nevertheless, analyses of hormonal profiling during plant development and defense are scarce. In this work, changes in hormonal metabolism in grapevine (Vitis vinifera) were compared between a susceptible (Trincadeira) and a tolerant (Syrah) variety during grape ripening and upon infection with Botrytis cinerea. Infection of grapes with the necrotrophic pathogen Botrytis cinerea leads to significant economic losses worldwide. Peppercorn-sized fruits were infected in the field and mock-treated and infected berries were collected at green, veraison and harvest stages for hormone analysis and targeted qPCR analysis of genes involved in hormonal metabolism and signaling. Results indicate a substantial reprogramming of hormonal metabolism during grape ripening and in response to fungal attack. Syrah and Trincadeira presented differences in the metabolism of abscisic acid (ABA), indole-3-acetic acid (IAA) and jasmonates during grape ripening that may be connected to fruit quality. On the other hand, high basal levels of salicylic acid (SA), jasmonates and IAA at an early stage of ripening, together with activated SA, jasmonates and IAA signaling, likely enable a fast defense response leading to grape resistance/ tolerance towards B. cinerea. The balance among the different phytohormones seems to depend on the ripening stage and on the intraspecific genetic background and may be fundamental in providing resistance or susceptibility. In addition, this study indicated the involvement of SA and IAA in defense against necrotrophic pathogens and gains insights into possible strategies for conventional breeding and/or gene editing aiming at improving grape quality and grape resistance against Botrytis cinerea.

The newly-identified SWEETs are high-capacity, low-affinity sugar transporters with important roles in numerous physiological mechanisms where sugar efflux is critical. SWEETs are desirable targets for manipulation by pathogens and their expression may be transcriptionally reprogrammed during infection. So far, few plant SWEET transporters have been functionally characterized, especially in grapevine. In this study, in the Botrytissusceptible variety "Trincadeira," we thoroughly analyzed modifications in the gene expression profile of key SWEET genes in Botrytis cinerea-infected grape berries. VvSWEET7 and VvSWEET15 are likely to play an important role during fruit development and Botrytis infection as they are strongly expressed at the green and mature stage, respectively, and were clearly up-regulated in response to infection. Also, B. cinerea infection down-regulated VvSWEET17a expression at the green stage, VvSWEET10 and VvSWEET17d expression at the veraison stage, and VvSWEET11 expression at the mature stage. VvSWEET7 was functionally characterized by heterologous expression in Saccharomyces cerevisiae as a low-affinity, high-capacity glucose and sucrose transporter with a K m of 15.42 mM for glucose and a K m of 40.08 mM for sucrose. VvSWEET7-GFP and VvSWEET15-GFP fusion proteins were transiently expressed in Nicotiana benthamiana epidermal cells and confocal microscopy allowed to observe that both proteins clearly localize to the plasma membrane. In sum, VvSWEETs transporters are important players in sugar mobilization during grape berry development and their expression is transcriptionally reprogrammed in response to Botrytis infection.