Dissecting the genetic, physiological and metabolic mechanisms of grapevine resilience to heat stress

Grapevine (Vitis spp.) is one of the most widely cultivated perennial fruit crops in the world and its economic relevance is mainly related to wine production. In recent years, the increased frequency of extreme phenomena such as heat waves has been acknowledged as one of the most significant climate variables negatively affecting grape yield and berry composition, with consequences also on wine quality. Thus, studying the physiological, metabolic and genetic factors that are involved in grapevine response to high temperatures is essential to improve the knowledge of mechanisms underlying thermotolerance, aiming to support plant breeding innovation and the development of new management strategies in viticulture. In this work, a segregating population obtained from the crossing of ‘Rhine Riesling’ and ‘Cabernet Sauvignon’ was studied in the field with a multidisciplinary approach. The progeny (around 120 genotypes) was evaluated for phenological traits affected by changing temperatures, in particular bud burst, flowering and véraison, while physiological response to heat stress was assessed in various hot summer days by measuring chlorophyll fluorescence kinetics and stomatal conductance. Measures were collected in the early morning as control and in the afternoon during hot hours. Phenotypic data were then used in combination with a high-density linkage map (average distance between adjacent markers 0.78 cM), previously developed using genotypic information from 139 individuals, to perform QTL analysis. Based on physiological responses to high temperatures, selected individuals showing contrasting behaviour, together with parental lines, were further studied in controlled conditions. In the field, in fact, plants may be subjected to combined stresses and changes in environmental conditions may heavily influence plants response. With the experiment in controlled condition, on the other hand, plants were stressed at higher temperatures, compared to the ones registered in the field, by maintaining all the other sources of variability constant. In the growth chamber plants were studied for their physiological response to heat stress by using the same approach adopted in the field. To better understand mechanisms involved in grapevine adaptation to heat stress conditions, individuals with contrasting behaviour were studied also for their metabolome modifications, both in the field and in controlled conditions. Volatile organic compounds (VOCs) were investigated with an untargeted approach applying conventional methods of analysis. Accumulation of VOCs in grapevine leaves was analysed using gas chromatography coupled with mass spectrometry (GC-MS) after a pre-concentration with a solid-phase micro-extraction (SPME) approach. On the other hand, VOCs emission during stress was investigated in controlled conditions thanks to the use of the Closed-Loop Stripping Analysis (CLSA) which allows the collection of VOCs directly emitted by plants. Analysis was then performed with GC-MS. Metabolic alterations of non-volatile compounds were examined with an untargeted analysis using high-performance liquid chromatography coupled with a high-resolution mass spectrometer equipped with an electrospray soft ionization (HPLC-HR-ESI-MS). In this work a metabolomic workflow was developed, starting from sample collection and extraction to sample analysis and data interpretation. The analytical method developed allowed the preliminary evaluation of leaf metabolome alterations due to stress factors. In fact, the use of a weak cation-exchange mixed mode column, in combination with a data dependent acquisition mode, allowed a first wide screening of both primary and secondary metabolites resulting in a good compromise for metabolic fingerprinting. QTL analysis on the segregating population allowed the identification of several QTLs, related to both phenological and physiological traits, with the discovery of interesting putative candidate genes for grapevine resilience to changing temperatures. This is the first time that a similar approach has been applied to a perennial fruit crop by analysing chlorophyll fluorescence and leaf transpiration traits related to heat stress. On the other hand, the multidisciplinary approach allowed the fine characterization of Rhine Riesling and Cabernet Sauvignon response to high temperatures, both in controlled and field conditions, a tentative classification of ‘tolerant’ and ‘susceptible’ progeny individuals and the identification of metabolic pathways altered during heat stress in the susceptible plants. Together with the implementation of a novel metabolomic workflow based on HPLC-HR-ESI-MS, this work represents a novelty in studies on grapevine response to changing temperatures, as it considered not only the berry metabolism but the resilience of the plants itself, paving the way for future studies on thermotolerance.