Clusters of grapevine genes for a burning world

Abstract

Extreme events associated with climate change increasingly threaten agriculture. Experimenting on a grapevine diversity panel suddenly exposed to a record heatwave in South France, we observed varietal responses ranging from complete tolerance to severe burning. We uncovered a handful of genomic regions associated with extreme heat tolerance, showing that we may leverage genetic diversity for breeding perennial fruit crops capable of withstanding heatwaves. Extreme heatwaves are proliferating with climate change, yet their impacts on plants are barely documented, especially in perennials that populate orchards and forests (Teskey et al., 2015; Breshears et al., 2021). As breeding programmes for perennial plants typically take several decades, we urgently need to explore whether the current genetic diversity contains alleles that could be mined to improve tolerance to extreme heat (Wolkovich et al., 2018). However, conducting genetic analyses for heat tolerance in perennials is challenging. On the one hand, experimental facilities with controlled conditions are generally not designed for studying perennial plants at the genetic diversity scale. Moreover, controlled conditions rarely reproduce the short-wave radiation of the sun that critically contributes to raising tissue temperature, driving the damaging production of reactive oxygen species (Jagadish et al., 2021). On the other hand, extreme heatwaves have remained erratic episodes so far, making it difficult to have plant diversity panels ready to be exposed to natural episodes and free of confounding effects like plant phenology (Driedonks et al., 2016), intra-field heterogeneity (Costa et al., 2018) or co-occurrence of drought (Tricker et al., 2018). Hence, the genetic analyses for extreme heat tolerance are currently limited to a few annual crop species grown in natural (Chen et al., 2017; McNellie et al., 2018) or glasshouse (Wu et al., 2022) conditions. On 28 June 2019, we had the ‘chance’ to go through a record heatwave while we were growing a grapevine diversity panel of 279 cultivars (Nicolas et al., 2016) in a common garden experiment in Montpellier, at the heart of the Mediterranean area in South France. An incoming mass of hot air from the Sahara desert originated the all-time highest temperature observed in France, and overheated a large part of Europe (Sousa et al., 2020). This heatwave generated c. 35% yield losses on damaged farms in the viticultural region around Montpellier (Reluy et al., 2022). In our experimental vineyard with young potted vines sharing a similar vegetative stage, air temperature peaked at 45.2°C (Fig. 1a; Supporting Information Table S1), 10.8°C above the mean maximal temperature of the period over the 2009–2023 records. Three-dimensional modelling of energy balance suggests that canopy temperature reached 47.3°C in the shade and up to 53.8°C in the sun (Figs 1b,c, S1a). A few hours later, part of the vines was literally burnt – a symptom termed ‘leaf firing’, whereby tissue death quickly follows exposure to heat (Chen et al., 2017; McNellie et al., 2018). Leaf firing was noticeably cultivar-dependent: while some cultivars displayed severe leaf firing (especially the sunlit leaves close to the heated soil, consistent with the leaf temperature profile obtained from the model), others showed no visible symptoms (Figs 1d,e, S2). This suggests that current grapevine diversity holds a genetic potential useful to breed for plants adapted to extreme heat. We scored the proportion and intensity of leaf firing of each genotype, obtained the firing magnitude as proportion × intensity and performed genome-wide association studies (GWAS) on each trait using one single-locus (MM4LMM) and two multi-locus (MLMM, varbvs) methods. We found six regions with at least one associated single-nucleotide polymorphism (SNP; Figs 1f, S3; Table S2), which we named the Burned Leaves After heatwave and Zonal Sun Exposure (BLAZE) loci. Most associations were significant both with MM4LMM and MLMM, and among the top SNPs by inclusion probability with varbvs. Each individual SNP explained between 7% and 10% of the genotypic variation, while the key set of SNPs jointly explained up to 22% for the firing magnitude. BLAZE5.1 was significant for all traits. BLAZE13.1 and BLAZE13.2 were separated by only 1.1 Mb and were significant only for the magnitude, with both components being close to the significance threshold. The three remaining loci, BLAZE6.1, BLAZE10.1 and BLAZE14.1, contained associated SNPs that were significant or almost significant for the proportion and/or the magnitude. Cumulating favourable alleles made it more likely for a cultivar to achieve thermotolerance, yet there was large variation in the relationship (Fig. S4a). Moreover, BLAZE10.1, BLAZE13.1, BLAZE13.2 and BLAZE14.1 were most frequently found together in their homozygous favourable form (85% of cultivars bearing homozygous favourable alleles for at least three of the loci), making it difficult to quantify their individual effect (Fig. S4). We then tested whether some of these SNPs co-localized with associations for morphological traits known to correlate with heat tolerance across species. For instance, small leaf size and high leaf mass per area (LMA, closely linked to leaf thickness) have been reported to correlate with enhanced heat tolerance/avoidance across 20 broadleaf evergreen tree or shrub species (Marchin et al., 2022). This is in line with biophysical analyses showing that small leaves have a thinner boundary layer, favouring heat dissipation (Leigh et al., 2017), while thicker leaves have a higher capacity to buffer temperature variations (Leigh et al., 2012). Are these traits reliable proxies of heat tolerance at the intra-specific level? Here, while leaf size loosely correlated with the firing proportion, LMA positively correlated with all firing symptoms (Fig. S2), at odds with inter-specific correlations. Moreover, the associations we detected for leaf size and LMA did not co-localize with BLAZE associations (Fig. S5). This suggests that genetic variation for heat tolerance in this grapevine panel was driven by other traits. Alternatively, leaf temperature could be reduced by minimizing the absorption of solar radiation (through leaf optical properties, leaf orientation or shoot architecture), or by enhancing latent heat loss through leaf transpiration, also known as evaporative cooling. Evaporative cooling is a powerful strategy to limit leaf temperature under hot environments (Drake et al., 2018). Simulating an enhancement or reduction in evapotranspiration within biologically relevant ranges (Materials and Methods) resulted in a −2.9°C or +1.9°C change in mean leaf temperature during the heatwave compared with the default simulation, respectively (Fig. S1), highlighting evaporative cooling as a potent lever for reducing leaf firing. Evapotranspiration occurs at the leaf surface through the stomatal pores and, to a lesser extent, the cuticle. Surprisingly, however, we found no candidate gene obviously linked to stomata or cuticle properties around the detected SNPs (Fig. 2; Table S3): We only noticed the E3 ubiquitin ligase COP1 (93 kb from BLAZE10.1) and the ras-related small GTP-binding protein RabE1C (50 kb from BLAZE13.2), both involved in abscisic acid-induced stomatal closure in Arabidopsis (Chen et al., 2021a, 2021b), but they were not the best candidates in their region (to be described later). This unexpected output may yet be explained by the high risk of hydraulic failure during extreme heatwaves, as the extensive water flow required to support evaporative cooling could be readily disrupted if soil water becomes limited or if the hydraulic conductances on the path for leaf water supply are not large enough to meet evaporative demand (Cochard, 2021). Beyond temperature regulation, a myriad of processes potentially underlie heat tolerance such as membrane stabilization, scavenging of reactive oxygen species or osmoprotection (Pettenuzzo et al., 2022). Heat shock proteins (HSP) and heat shock transcription factors (HSF) are known as central actors in plant response to heat. Recently in grapevine, allelic variations at HSFA2 and HSFB1 from the species Vitis davidii and Vitis quinquangularis were found to confer higher thermotolerance compared with that of the cultivated Vitis vinifera (Chen et al., 2023; Liu et al., 2023). Here, within the list of candidate genes born from our pure Vitis vinifera panel, we found a 15.4 kDa class V HSP (69 kb from BLAZE10.1, Fig. 2c) and one HSF named HSFB4b (55 kb from BLAZE6.1, Fig. 2b). The latter, however, was also close (50 kb) to a cluster of flavonoid 3′,5′-hydroxylases (F3′5′Hs; Falginella et al., 2010). These cytochrome P450 enzymes (CYP75A family) are involved in the synthesis of anthocyanins, which have sunscreen and antioxidant properties. This cytochrome P450 cluster on chromosome 6 is the largest in grapevine, gathering 35 members of the CYP75 and CYP79 families (Ilc et al., 2018). Interestingly, on the ohnologous chromosome 13, BLAZE13.1 localized 5-kb upstream from another cluster of cytochromes P450 (Fig. 2d), mostly from the CYP79 family that is responsible for the production of oxime derivatives precursors of cyanogenic glucosides (Ilc et al., 2018). They were annotated as tryptophan N-monooxygenase CYP79A68 or phenylalanine N-monooxygenase CYP79D16-like. In Prunus mume, CYP79D16 produces phenylacetaldoxime, a toxic intermediate in the amygdalin pathway (Yamaguchi et al., 2014). We also noticed that the cluster at BLAZE13.1 included one CYP716B1, tandem of which was found at BLAZE10.1 as well. The CYP716 family is involved in the biosynthesis of triterpenoid saponins (Miettinen et al., 2017), which are generally able to permeabilize and perforate plasma membranes (Mugford & Osbourn, 2013). Whether toxic compounds accumulate upon heat stress in grapevine leaves of sensitive cultivars to induce apoptosis remains an avenue to be explored. The additional regions revealed other promising candidate genes (Fig. 2; Table S3). Noticeably, BLAZE5.1, associated with all firing traits (Fig. 2a), was located within a large cluster of 24 ankyrin repeat-containing proteins orthologues of Arabidopsis INCREASED TOLERANCE TO NaCl 1 (ITN1) and ANKYRIN-LIKE1 (ANK1). Arabidopsis ITN1 promotes the production of reactive oxygen species under salt stress (Sakamoto et al., 2008), making it credible that one of its grapevine orthologues plays a similar role during heat shocks. The two remaining BLAZE loci also mapped to genomic regions containing gene clusters, but other genes therein were more likely candidates for firing traits. First, BLAZE13.2 (Fig. 2e) fell a few kb away from ZAT12, a zinc finger protein that prevents leaf burning upon excessive light in Arabidopsis (Iida et al., 2000) and shows a pivotal role in many stresses, including heat and oxidative stress (Davletova et al., 2005). Second, BLAZE14.1 (Fig. 2f) was 56-kb downstream of a gene coding for phosphatidylinositol 4-kinase gamma 5 (PI4Kγ5), which engages in the generation of phosphatidylinositol 4,5-bisphosphate (PIP2), a messenger involved in the signalling of heat stress (Mishkind et al., 2009). Our real-life study demonstrates that cultivated grapevine possesses strong genetic variation for its canopy response to extreme heat, which could not have been detected using morphological proxies, and discloses a suite of promising markers to breed a perennial plant for extreme heat tolerance. The prioritized candidate genes deserve further investigation to decipher the underlying mechanisms. The relevance of BLAZE loci should also be assessed in productive vineyards where berries are subjected to sunburn (Gambetta et al., 2021), and under conditions of water deficit that could reveal genetic links between leaf firing and transpiration, which were not apparent here. A diversity panel of 279 cultivars of Vitis vinifera L. was designed to maximize genetic diversity and minimize relatedness among cultivated grapevine while capturing the main low structure in three genetic pools (Nicolas et al., 2016). In winter 2018, cuttings of each cultivar were obtained from the Vassal-Montpellier Grapevine Biological Resource Center (Marseillan-Plage, France). In spring 2018, 20 own-rooted plants per cultivar were then transplanted into individual 3-l pots containing a 30 : 70 (v/v) mixture of loamy soil and organic compost and cultivated at the Pierre Galet experimental vineyard of Institut Agro Montpellier (France), fitted with a drip fertigation system and a weather station. Plants were organized along eight double rows (Fig. 1d). For each cultivar, all 20 plants were grouped, with 10 plants facing south-west and 10 plants facing north-east. While a randomized design would have been ideal, we chose to group the plants by cultivar for practical reasons (i.e. to facilitate plant management and phenology assessment). Each row had the same geometry and both extreme rows (1 and 8) were surrounded by several rows of other potted vines with similar architecture, thus limiting spatial effects. Plants were well-irrigated, and one leafy axis was selected on each plant. Inflorescences were removed if present, and the leafy axis was topped at a height of 2 m. In winter 2019, plants were spur-pruned and then managed as in the previous season. Ten plants of each genotype were scored for the presence of inflorescences, and only 20% of the genotypes carried inflorescences on at least one plant. In these genotypes, flowering occurred between 12 and 27 May. Flowers were then eliminated to avoid potential bias. By the end of June, all plants had been topped at c. 2 m and had started growing lateral shoots. Thus, all plants shared similar vegetative stage, which was far from bud break and unaffected by reproductive development of the current year or by autumn senescence. This contrasts with Monocot species, where it is virtually impossible to synchronize phenological stages. Moreover, in Monocots, developing leaves are known to be more susceptible to leaf firing than mature leaves (Chen et al., 2017; McNellie et al., 2018), making phenology a critical factor. In our grapevine system, young leaves were less prone to heating (due to their increased distance to the soil), and leaf firing was observed predominantly on proximal, mature leaves. Thus, phenology was unlikely to be a confounding factor here. Irrigation was adjusted weekly based on meteorological conditions and shoot development. On the day of the heatwave, each plant received 880 ml of drip irrigation in three deliveries (morning, midday and evening). was carried in the of on The proportion and intensity of leaf firing were scored for all 279 cultivars of the diversity panel on 2 the was based on a (Fig. for leaf firing proportion and one for leaf firing intensity were for the group of 10 plants of each cultivar in each orientation or of leaf firing was scored from to as the proportion of leaves at least burnt within the of leaf firing was scored as or 5 to the of the firing within one leaf no 1 only leaf were up to 5 the leaf was We further the magnitude of symptoms at the canopy as the of proportion and to the end of we leaves for each cultivar in to their Leaves were as and generally between the and from the They were and if to avoid and then using a Leaf area was then obtained through a in Leaf was 1 in a and LMA was as the between and analyses were in 2020). For leaf area and the best of genetic were for in this were tested with a effect of the genotype and two and orientation or For both the showed that for orientation only were as for both orientation and Moreover, genotypic was was included or Thus, we selected the same including genotype and orientation effects for of both traits S4). components were to 2 2 2 with 2 the genotypic 2 the and the mean of per were high for both morphological traits Table S4). 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