Abstract
The Australian wine industry is currently under pressure to sustain its profitability due to climate change. Therefore, there is a pressing need to explore grapevine genetic diversity and identify superior clones with improved drought resistance. We previously characterised more than 15,000 dry-farmed (for over 65 years) Cabernet Sauvignon clones in a vineyard and identified three drought-tolerant (DT) clones, which can maintain significantly higher intrinsic water use efficiency (WUEi) under limited soil moisture than drought-sensitive (DS) clones. To understand whether DT clones grown under multi-decadal cyclical drought can prime their vegetatively-propagated clonal progenies for future drought events, in this study, all DT and DS vegetative progenies were propagated with commercial clones in the glasshouse. Their physiological and molecular responses were investigated under well-watered and two recurrent drought (D1 and D2) conditions. We observed that concentration of a natural priming agent, γ-amino butyric acid (GABA), were significantly higher in all DT progenies relative to other progenies under drought. Both commercial and DT progenies exhibited improved gas exchange, photosynthetic performance and WUEi under recurrent drought events relative to DS progenies. Our results suggest that DT progenies have adapted to be in a “primed state” to withstand future drought events.
Introduction
The viticulture industry, which consists of wine, raisin and table grape production is the largest fruit industry in Australia. Currently, Australia is the world’s fifth-largest wine exporter and in 2019, the wine sector contributed $45.4 bn to the Australian economy [1]. Due to recent extreme drought events, heat waves and bushfires, Australian grapevine production reduced 20% in 2020, and the smallest vintage was recorded: 1.4–1.5 M tons compared to the average 1.75 M tons [2]. Given that the incidence and severity of drought events are predicted to increase in the future [3], there is an increasing need to select and/or breed superior grapevine cultivars and clones, which could perform better in dry climates.
Grapevine planting materials were first introduced to Australia from Europe in 1832. There was a massive expansion of genetic resources in Australia in 1960s due to mass introduction of Phylloxera-free clones with superior agronomic and oenological performances from overseas [4]. A clone is referred to as a vegetatively propagated population of vines originated from a single parental vine and clonal progenies are generally considered to be genetically identical to their parental vine. However, over time diverse notable phenotypic variations can emerge due to progressive accumulation of spontaneous mutations [5] or epigenetic modifications [6,7]. Since the beginning of the viticulture in Australia, clonal selection was employed as the main tool in grapevine improvement programs to preserve elite clones with desirable characteristics [8,9]. Australian old vineyards still preserve extensive genetic diversity of European selections, but intravarietal genetic variability has not been completely explored in Australia [9]. Therefore, exploring the superior genotypes hidden within early introductions would pave the wave for improving complex traits such as drought tolerance and water use efficiency (WUE).
Drought tolerance is a highly complex trait, which is determined by genotype and environmental interactions [10-12]. Although over the past decades, our knowledge on short-term drought acclimation responses has increased, a comprehensive picture of how key plant physiological processes are regulated under prolong cyclical drought episodes is largely unknown. Stomatal closure is one of the earlier responses to water deficit. It has long been known that root-derived stress signalling molecules such as abscisic acid (ABA) sense subtle changes of soil moisture and transduce the signal to upper parts of the plant for inducing stomatal closure [13-16]. However, there is still controversy regarding the proposed role of ABA [17]. Recent studies have demonstrated that aquaporin (AQP)-mediated hydraulic signals and chemical signals such as γ-amino butyric acid (GABA) and CLAVATA3/embryo-surrounding region-related (CLE) small peptides also contribute to long-distance communications in response to drought stress [18,19]. Stomatal closure is crucial in preventing excessive transpirational water loss, however it dramatically reduces photosynthesis due to limitation in CO2 influx [20]. When field-grown grapevines undergo severe water stress, photosynthesis is further constrained by reduction in mesophyll conductance (gm), biochemical limitations and reactive oxygen species (ROS)-mediated photooxidative damage [21,22]. A considerable body of evidence has recently indicated that prolonged exposure of a plant to mild to moderate stress conditions can effectively stimulate faster and stronger tolerance to subsequent stress events through the acquisition of a “stress memory” [23-25]. Interestingly, in some instances, priming-induced “stress memory” has shown to be inherited to seed-derived offspring [26,27]. Recent work has discovered certain priming elicitors that can trigger natural defence mechanisms in a metabolically cost-effective manner. For instance, several studies have shown that plants accumulate γ-amino butyric acid (GABA), a non-protein amino acid, at the onset of the stress [28,29]. GABA is a metabolite and stress signalling molecule which is synthesized from glutamate in the cytosol [30,31] and metabolised through the GABA shunt pathway in both cytosol and mitochondria [32]. Increased concentrations of GABA under drought stress has shown to induce stomatal closure via activation of anion channel, aluminium-activated malate transporters (ALMTs) [33]. Accumulation of GABA under stress conditions also helps activating plant’s innate defence potential and pre-conditioning of the plant to next drought event through synthesising osmolytes [34], enhancing photochemical efficiency and WUE and ROS detoxification [35-37].
The overarching aim of this study was to understand whether woody perennial crops such as grapevine grown under multi-decadal cyclical drought can prime their vegetatively propagated clonal progenies for future drought events. As the first step towards understanding this, we characterised more than 15,000 dry-farmed Cabernet Sauvignon clones that were planted in 1954 as a mass selection of unknown clones in a South Australian vineyard. Due to the extended period of dry-farming, over 65 years, these vines are ideal for unravelling long-term drought adaptation mechanisms. In our pilot field trial, we identified several drought-tolerant (DT) clones which can maintain significantly higher intrinsic water use efficiency (WUEi) under limited soil moisture and drought-sensitive (DS) clones [38]. We hypothesized that these superior DT clones may have already primed due to long-term exposure to limited soil moisture and thereby may have the ability to successfully transmit drought acclimation strategies to their subsequent clonal progenies. In this study, we examined this hypothesis by evaluating the drought acclimation responses of their clonal progenies under two recurrent cyclic drought events in the glasshouse. To the best of our knowledge, the results obtained here are the first demonstration which provides novel insights into transgenerational and intragenerational drought priming mechanisms in grapevine clones.
Materials and Methods
Plant Materials
The drought-tolerant (DT) and drought-sensitive (DS) clones were selected based on differences in WUEi observed under field conditions as described in Pagay et al. (2022) [38]. Five cuttings each were vegetatively propagated from previously selected three drought-tolerant (DT) and two drought-sensitive (DS) dry-farmed clones along with three commercial clones (G9V3, CW44 and SA125) at the Yalumba Nursery (Nuriootpa, SA, Australia). Propagated vines were re-potted in 4.5 L pots with a mixture of 50% University of California soil Mix (61.5 L of sand, 38.5 L of peat moss, 50g of calcium hydroxide, 90g of calcium carbonate, and 100g of Nitrophoska (12:5:1, N: P: K plus trace elements) and 50% perlite and vermiculite mix (50:50) at the Plant Research Centre (Waite Campus, University of Adelaide, SA, Australia). In order to facilitate similar growth rates, all vines were pruned to four nodes and incubated in a dark cool room at 4°C for at least 25 days. All clones were grown under the glasshouse conditions with 16 h photoperiod. Two overhead supplemental light sources were turned on from 6:00 to 20:00 every day to maintain uniform light distribution and intensity independently of external environmental conditions. Temperature and relative humidity were continuously recorded using data loggers (Tinytag Plus 2, Gemini, UK) in the glasshouse. After budburst, the vines were treated with 1.6 mL/L of Megamix® (13:10:15 N: P: K plus trace elements) every second week. When they reached 20-leaf stage, they were pruned to a similar leaf area.
Drought cycles
In order to compare drought stress responses between pre-selected DT and DS clones, all 15 vines from 3 DT clones were grouped and compared with DS clonal group which consisted of 10 vines and commercial clonal group with 15 vines. Each group will be mentioned as DT, DS and commercial clonal progenies hereafter. Nine vines from each DT and commercial clonal progenies and 6 from DT progeny (3 vines/clone) were subjected to two cyclic drought events (treatment group) and remaining vines (6 from DT and commercial and 4 from DS progenies) were included in the well-watered treatment (control group). All vines were randomly positioned within each group. Vines in the control group were watered daily to achieve an equivalent pot weight. Soil moisture was measured daily using a Teros-10 soil triple sensor (METER, WA, USA). For evaluating clonal performances upon drought stress, water supply was withheld for the treatment group until soil moisture reached 4% volumetric soil water content (VSW) or less. If the soil moisture reduced under 4%, the vines were watered to the equivalent pot weight until the drought cycle was finished. Then they were rewatered to field capacity and until stomatal conductance retuned to similar values displayed by control vines (Figure 1a). After full recovery from D1 (21 days after rewatering), second drought cycle was imposed by withholding water as described above. Measurements and leaf tissue sampling were taken before drought stress (Day 0), at the peak of the 1st drought cycle (7 days after withholding water-D1), after full recovery from D1 (21 days after rewatering (day 0 of the second drought cycle), and at the peak of the 2nd drought cycle (4 days after withholding water-D2).
Midday stem water potential (Ψs) and leaf Gas exchange measurements
Midday stem water potential and in vivo gas exchange parameters were measured using a Scholander-type pressure chamber (Model 1505, PMS Instruments, Albany, NY USA) and LI-6400XT (LI-COR Inc., Lincoln, NE, USA) respectively as described in Pagay et al. (2022) [38].
Chlorophyll fluorescence
Chlorophyll fluorescence parameters were measured with a LI-6400XT equipped with a leaf chamber fluorometer (model: 6400-40, LI-COR Inc., Lincoln, NE, USA) in dark- and light-adapted fully expanded leaves positioned in the bottom, middle and top levels per vine. In light-adapted leaves, the steady-state fluorescence yield (s) was measured. Then a saturating white light pulse (8000 μmol m-2 s-1) was applied for 0.8 s to achieve the light-adapted maximum fluorescence (Fm′). The actinic light was then turned off, and far-red illumination was applied (2 μmol m−2 s−1) to measure the light-adapted initial fluorescence (F0′). Vines were kept overnight in darkness for dark-adapted measurements and basal fluorescence (F0) and maximum fluorescence emission (Fm) were measured by illuminating leaves to weak modulating beam at 0.03 μmol m-2s-1 and saturating white light pulses of 8000 μmol m-2 s-1, respectively. Non-photochemical quenching (NPQ), photochemical quenching coefficient (qP), and actual photochemical efficiency of PSII (ΦPSII) were calculated as: NPQ = (Fm−Fm′) / Fm′ [39], qP = (Fm′– Fs)/(Fm′– F0′) [40], ΦPSII =(Fm′–Fs)/Fm′ [39]. Electron transport rate (ETR) was calculated according to Rahimzadeh-Bajgiran, et al. [41] using the following equations. Where, ΔF/Fm′ is the PSII photochemical efficiency, PPFD is the photosynthetic photon flux density incident on the leaf, 0.5 is a factor that assumes equal distribution of energy between the two photosystems, and 0.84 is the assumed leaf absorptance [42,43]. Mesophyll conductance (gm) was calculated using a theoretical value of non-respiratory compensation point G*=42.9 μmol mol-1 (ppm) CO2 [44].
Abscisic Acid (ABA) quantification
Approximately 30 μl of Xylem sap was extracted from leaves at the end of Ψs measurements by increasing the balancing pressure by 0.2-0.4 MPa. Sap samples were snap frozen in liquid nitrogen and subsequently transferred to a −80°C freezer until analysis. ABA abundance in xylem sap (ABAxyl) was analysed by liquid chromatography/mass spectrometry (LC MS/MS, Agilent 6410) [12].
Analysis of Ascorbate peroxidase (APX) enzyme activity
0.5 g of frozen leaf samples were suspended in 2 ml of 0.1 M sodium phosphate buffer (pH 7.0) and incubated for 10 min on ice. Samples were centrifuged at 12,000 g for 15 min and 10 µL of the supernatant from each sample was mixed with 290 µL of assay mixture consisting of 0.5 mM ascorbic acid, 0.1 mM EDTA-Na2 and 0.1 mM H2O2 solutions prepared in 0.05 M sodium phosphate buffer (pH 7.0). The APX activity was determined by measuring the absorbance at 290 nm, in a FLUOstar Omega plate reader (BMG LABTECH GmbH, Ortenbery, Germany). The decrease in absorbance corresponded to oxidation of ascorbic acid. One enzyme unit was defined as 1 mol of ascorbic acid oxidized per minute at 290 nm [45].
Quantification of endogenous GABA concentrations
Leaf samples were snap frozen in liquid nitrogen and 0.1g of frozen ground samples were used for GABA extraction. GABA quantification was conducted according to the protocol described in Ramesh, et al. [46].
Gene expression analysis by Quantitative real-time PCR
Grapevine leaves that were used in Ψs measurements were snap frozen and stored at −80 0C freezer. RNA was extracted using Spectrum plant total RNA kit (Sigma, USA) according to the manufacturer’s instructions, and contaminated DNA was removed according to On-column DNase digestion protocol (Sigma, USA). Total RNA was quantified with a UV spectrophotometer and quality of RNA was assessed by gel-electrophoresis. For cDNA synthesis, 1 μg of total RNA was reverse transcribed using the iScript cDNA synthesis kit (Bio-Rad, CA, USA). In Quantitative real-time PCR (qPCR), 1 μL of 1/10 diluted cDNA was amplified in a reaction containing, 5 μL KAPA SYBR® FAST Master Mix (2X) Universal (Kapa Biosystems Inc., MA, USA), and 100 nM of gene-specific primers. qPCR primer sequences for two aquaporin (AQP) genes (VvTIP2;1 and VvPIP1;1) and two stable housekeeping genes, VvELF and VvUbi were obtained from Shelden, et al. [47]. The amplification was conducted in a QuantStudio 12K Flex Real-Time PCR system (ThermoFisher Scientific, USA) according to the following conditions: one cycle of 3 min at 95 °C followed by 40 cycles of 16 s at 95 °C, and 20 s at 60 °C. To ensure single-product amplification, melt curve analysis was performed by heating the PCR products from 60°C to 95 °C at a ramp rate of 0.05 °C s-1. A two-round normalization of qPCR data was carried out by geometric averaging of multiple control genes as described by Vandesompele, et al. [48] and Burton, et al. [49].
Statistical analysis
In order to understand the long-term drought acclimation responses of dry-farmed clones, raw data from all 3 DT clonal progenies were pooled and compared with pooled raw data of DS clonal progenies and commercial clones separately. Raw data were statistically analysed by two-way ANOVA by fitting a mixed effects model using GraphPad Prism 9 software (GraphPad, CA, US). Tukey’s multiple comparisons test was performed with individual variances for each comparison. Differences were considered to be statistically significant when P ≤ 0.05.
Results
To investigate whether field-grown DT clones have the ability to successfully transmit drought acclimation strategies to their subsequent clonal progenies, we attempted to propagate five vegetative cuttings from three DT and DS clones at Yalumba Nursery (Nuriootpa, SA, Australia). Even though, all three DT clones were successful propagated, only two DS clones were regenerated successfully.
Variations in soil moisture depletion during two dehydration cycles
In order to identify drought stress responses of dry-farmed clonal progenies, vines in the treatment group were subjected to two recurrent drought cycles in the glasshouse (Figure 1a). Once irrigation was withheld, the soil moisture content progressively decreased in all potted vines during D1 cycle. In comparison, a steeper decline was observed during the second drought cycle leading up to D2 (Figure 1a, b, c). This rapid reduction of soil moisture leading to D2 could be attributed to increased vapour pressure deficit (VPD) caused by slightly higher day/night temperature and marked reduction in relative humidity (Figure 1d, Table 1). Soil moisture content decreased more rapidly in commercial clones relative to other clones at D1, and DS clones maintained the highest soil moisture (Figure 1b). However, no marked differences were observed between clones during the second drought event (Figure 1c).
Effect of differential mid-day stem water potential (Ψs) and gas exchange on photosynthetic performances of dry-farmed clonal progenies under multiple drought events
In the control group, all irrigated clones had Ψs ∼ −0.4 MPa prior to drought stress for cycle 1, but prior to cycle 2, Ψs was significantly lower, between −0.6 & −0.8 MPa (Figure 2a). In drought-treated vines, Ψs was drastically reduced to −1.2 and −1.1 MPa at D1 and D2, respectively, indicating a moderate to high drought stress condition, with similar Ψs values between clones. None of the clones showed wilting symptoms or chlorosis of leaves even under these low Ψs values. All clones recovered after rewatering (Figure 2a).
In order to understand stomatal responses to soil drying, leaf gas exchange parameters were analysed in all clones under drought conditions. It is important to note that, well-watered vines as well as drought treated vines before commencement of the drought stress, displayed low gs values which ranged from 0.02 to 0.08 mol H2O m-2 s-1 and low AN (from 2 to 7 μmol CO2 m-2 s-1) irrespective of abundant soil moisture (Figure 2b, c). As those vines vigorously grew without any sign of water stress (leaf chlorosis or root growth restrictions) throughout the experiment, we speculated that low gas exchange values might be because of the low light intensities provided during vine growth and measurements (<50 and 1000 μmol m-2 s-1 respectively). Differences in gs was similar between clones at D1 (Figure 2b). For instance, when clones were exposed to the first drought event, gs decreased in commercial, DS and DT clones by 78%, 83% and 74%, respectively compared to the clones in the control group. All clones completely recovered upon rewatering. During the second drought cycle, DS clones showed further decline in gs (93%) relative to recovery, whereas it was significantly higher in both commercial and DT clones relative to DS clones and at D1 (Figure 2b).
Even though in the control group, all well-irrigated clones exhibited similar plant water status and gs, it was consistently observed that AN of commercial clones was significantly higher relative to other clones at early developmental stages. However, over time DS and DT clones also increased AN to a similar level as commercial clones, therefore no statistical differences were apparent between clones at the latter developmental stages (Figure 2c). At D1, AN steeply decreased to near zero in all drought-stressed clones. Although AN of DS clones was also drastically reduced to near zero at D2, in commercial and DT clones, reduction of AN was approximately 66% and 58% respectively (Figure 2c). Leaf WUEi which represents the ratio of AN versus gs, was similar in all clones before imposing the drought stress (Figure 2d). At the first drought cycle, WUEi was marginally reduced in commercial and DT clones by 44% and 29% respectively, but 83% significant reduction was observed in DS clones. All clones displayed basal level of WUEi upon rewatering. When the recovered vines were exposed to the second drought event, commercial clones exhibited a 29% increase in WUEi, whereas it was marginally decreased in DS clones (26%), while, an 83% significant increase in WUEi was observed in DT clones. Intriguingly, DT clones exhibited significantly higher WUEi relative to both commercial and DS clones at D2 (Figure 2d).
Variations in mesophyll conductance and chlorophyll fluorescence parameters in dry-farmed Cabernet clonal progenies under prolonged drought stress
In comparison to the previously reported gm values (0.1-0.15 mol CO2 m-2 s-1) in Cabernet Sauvignon [21], 68% reduction of gm was observed in all clones at D1, but when the second drought stress was imposed, it was further depleted in both commercial and DS clones by 82% and 84% respectively (Figure 3a). In contrast, gm values in DT clones were significantly higher relative to other two clones at D2 (Figure 3a). Drought stress provokes remarkable changes in the photosystem II activity and chlorophyll fluorescence traits. Under non-stressed conditions, the maximum efficiency of the photosystem II (PSII) photochemistry (Fv/Fm) and fraction of open PSII reaction centres (qP) have reported to be around 0.75-0.80 [40,50] in Cabernet Sauvignon. Additionally, thermal dissipation of excess light energy (non-photochemical quenching; NPQ) and actual photochemical efficiency of PSII (ΦPSII) are approximately 0.5 and 0.2, respectively [40].
In comparison to those reported values, in our study, Fv/Fm remained unchanged at 0.74-0.75 in all clones at both D1 and D2, and differences could not be detected between clones (Figure 3b). Marginal decrease in ΦPSII was observed only in DS and commercial clones upon exposure to both drought events, but in DT clones ΦPSII remained unchanged at 0.2 (Figure 3c). All clones demonstrated substantial decline in qP at both drought cycles. For instance, at D1, DS clones exhibited a marked reduction in qP (62.6%) (Fig. 3d), whereas both commercial and DT clones displayed only 54.7% and 48.8% decline respectively. qP was similar between clones at D2 (Figure 3d). Dissipation of excess thermal energy within chlorophyll-containing complexes via non-photochemical quenching (NPQ), helps prevent the likelihood of formation of ROS [51]. During both drought cycles, NPQ was significantly higher in all clones relative to non-stressed conditions [40] (Figure 3e). During the first drought cycle, commercial clones displayed significantly higher NPQ, relative to dry-farmed clones. At the second drought stress, increase in NPQ was also observed in DS clones, however in DT clones NPQ remained at relatively lower level. It was interesting to note that electron transport rate (ETR) was significantly higher in DT clones relative to commercial and DS clones at both drought events (Figure 3f).
Water stress-induced changes in expression of AQPs (VvTIP2;1 and VvPIP1;1) in leaves
To understand inter-clonal variation in aquaporin-mediated hydraulic regulatory mechanisms, we analysed expressions of AQP genes encoding a tonoplast intrinsic protein, VvTIP2;1, and a plasma membrane intrinsic protein, VvPIP1;1 under drought stress conditions. Aquaporin transcript abundance differed significantly between clones in response to dehydration. For instance, before clones were exposed to drought stress, VvTIP2;1 transcript abundance was significantly higher in both DS and DT clones relative to commercial clones. Irrespective of the treatment, commercial clones exhibited basal level of VvTIP2;1 expression. However, at D1, VvTIP2;1 expression was significantly down-regulated in both DS and DT clones and it remained constant in both clones at recovery. At D2, the transcript abundance of VvTIP2;1 in DS clones was significantly lower relative to both commercial and DT clones (Figure 4a). VvPIP1;1 expression was similar in all clones before exposure to drought stress whereas it was slightly down-regulated in both commercial and DT clones at D1. However, significant up-regulation of VvPIP1;1 was observed in DS clones at D1. At the recovery stage, both commercial and DS clones displayed basal level of VvPIP1;1 expression, but DT clones exhibited statistically non-significant increase in VvPIP1;1 expression. At D2, VvPIP1;1 transcript abundance was markedly increased in commercial clones. In contrast, it was decreased in both DS and DT clones at D2 (Figure 4b).
Drought-mediated changes in ROS detoxification
To investigate whether exposure to prolonged drought stress enhances antioxidative mechanisms in Cabernet clones, ascorbate peroxidase (APX) enzyme activity were evaluated. It is interesting to note that APX activity increased significantly in commercial and DS clones at D1, but it did not change in DT clones at both drought events. Upon rewatering, APX activity remained higher in commercial clones, however both DS and DT clones displayed basal level of APX activity. At the second drought event, commercial and DT clones exhibited basal level of APX enzyme activity at D2. Unfortunately, APX activity in DS clones could not be reliably detected at D2 due to sample cross-contamination (Figure 5).
Variations in ABA in the xylem sap (ABAxyl) and GABA accumulation in leaf tissues
As large body of evidence has demonstrated that significant increase in ABA in leaves is highly correlated with the abundance of ABAxyl, we investigated the drought-mediated changes in ABAxyl in all Cabernet clones. ABAxyl concentration increased significantly in all clones during both drought treatments and no marked differences were observed between clones. It returned to the basal levels upon rewatering (Figure 6a). Before imposition of the drought stress, similar GABA levels were detected in all three clones. In line with previous studies [28,29,34,35], 2-fold and 12-fold increase in GABA concentration was observed in DS and DT clones respectively at D1. However, in commercial clones, leaf GABA levels did not change in response to soil moisture content. After rewatering and at D2, basal GABA concentrations were detected in all clones (Figure 6b).
Discussion
As for many commercial grapevine clones, unique range of viticultural and oenological traits could exist between Cabernet dry-farmed clones used in our study due to an accumulation of somatic mutations over long period of time. However, so far, no studies have been conducted to explore the genetic variations that underlies their phenotypic differences. Previously we reported that all superior shallow-rooted DT clones, identified through our preliminary field trial exhibited significantly higher WUEi under limited soil moisture relative to all selected deep-rooted DS clones grown with more soil moisture [38]. This observation tempted us to speculate that irrespective of their potential individual genotypic and phenotypic diversity, all DT clones may have primed in a still unknown mechanism to perform better under limited soil moisture than all DS clones. In order to test this hypothesis, all DT, DS and commercial clonal progenies were grouped separately and their physiological and molecular responses were evaluated under two recurrent cyclical drought conditions. In support of our hypothesis, Zamorano et al. (2021) [24] recently demonstrated that previous season drought stress can significantly improve photosynthesis rate and WUEi in field-grown grapevines under recurrent drought events. Findings of our glasshouse study further confirm the fact that field-grown DT clones have a greater ability to transmit WUEi and other drought adaptation mechanisms to their subsequent clonal progenies under recurrent cyclical drought episodes relative to DS clones. In our study, transgenerational drought adaptation capability of the progeny of field-grown grapevine clones is reflected at the first drought cycle whereas their intragenerational adaptation is represented under the second drought event. Based on our findings, we propose simplified models which represents differential molecular and physiological mechanisms underpinning transgenerational and intragenerational drought stress priming in DT and DS clonal progenies and commercial clones (Figure 6).
Differential water transport capacities of grapevine clones under drought stress
During the first drought cycle, even though all clones displayed similar water flux through stomata (gs) and Ψs, soil moisture content depleted more rapidly in commercial clones relative to DT and DS clones (Figure 1b, 2a, 2b). At the second drought event, all clones were able to maintain similar Ψs irrespective of the higher evaporative demand (Figure 1c, 2a, 2b). Given that stomatal regulation of transpiration has a positive correlation with leaf hydraulic conductance (Kleaf) [52] and root hydraulic conductivity (Lpr) [53,54], this observation tempted us to speculate that commercial clones may have higher Kleaf and root water uptake capacity (Kroot) to facilitate higher water transport than DT and DS clones.
Whole-plant water transport occurs through three routes i.e., apoplastic (through cell walls), symplastic (through plasmadesmata) and transcellular (across cell membrane) [54]. Some of the AQP isoforms belonging to the major intrinsic family (MIP) are considered as the key intra/intercellular water and ion channels which regulate transcellular or radial water transport in both leaves and roots [47,55,56]. The function and regulation of AQP are highly variable among distinct isoforms [47,54]. Previous studies have shown that water permeability of VvTIPs is higher than VvPIPs particularly to allow the cells to recruit the vacuolar space for water storage [53,56]. As VvTIP2;1 expression in leaves was found to be well correlated with gs and Kleaf [57], we examined its expression patterns in grapevine leaf tissues. Additionally, changes in transcript abundance of VvPIP1;1 was also analysed because VvPIP1;1 expression positively correlates with Kleaf in isohydric grapevine varieties.
In line with previous studies [47,57], VvTIP2;1 expression pattern seems to have a close association with gs under two drought events. Significant upregulation of VvPIP1;1 was detected in DS and commercial clones at D1 and D2 respectively and DT clones exhibited statistically non-significant increase at the recovery (Figure 4a). In line with our findings, previous studies have also shown that VvPIP1;1 expression significantly increases [47,58] or stably expressed [57] in different grapevine varieties under drought stress. Collectively, our study suggests that at D1, AQP-driven Kleaf is likely to be downregulated in DT clones, and upregulated in DS clones while commercial clones maintain constant Kleaf. At the second drought stress, Kleaf may have been upregulated in commercial clones and down-regulated in both DT and DS clonal progenies. Higher soil moisture content in DT clones relative to commercial clones under two drought events can be explained by the reduction of water uptake. As isohydric varieties such as Cabernet Sauvignon are highly vulnerable to embolism, reduction of Kleaf has been proposed as a favourable mechanism to prevent building up the xylem tension which leads to cavitation [59,60]. Significantly higher and lower soil moisture percentages in DS and commercial clones relative to other clones at D2 can primarily be attributed to changes in plant hydraulics. As AQP expression does not directly correlate with the soil drying patterns of DS and commercial clones at D1, we speculate that differences in leaf area might have influenced their soil drying rates under drought stress. However, future studies are needed to confirm the exact roles of AQP in regulating Lpr in dry-farmed grapevine clones. Collectively, our findings suggest that DT clones may have adapted to consume less amount of water upon dehydration, whereas commercial clones may require relatively higher amount of water for maintaining plant metabolism under water stress.
Differential stomatal-regulatory mechanisms exist in dry-farmed and commercial grapevine clones under drought
During our experimental conditions, all clones exhibited near zero gsat D1. In response to second drought event, gswas further declined in DS clones whereas DT and commercial clones showed significant improvement in gs (Figure 2b). In order to understand whether differential stomatal responses among clones is related to different sensitivities to key chemical and hydraulic stomatal mediators, we examined changes in concentrations of ABAxyland GABA in leaf tissues in addition to AQP expression under two drought events.
In line with previous studies [61,62], ABAxylwas significantly increased in all clones during both drought events (Figure 6a). It has long been known that ABA induces stomatal closure either directly via the activation of ion channels or indirectly via restricting radial water flow from the xylem by down-regulating AQP expression [63,64]. Given the changes in AQP-mediated Kleafand GABA concentrations in leaves, it can be proposed that at D1, stomatal closure in DS clones might have induced by an additive effects of chemical (ABA- and GABA-driven) mechanisms whereas both chemical and hydraulic mechanisms are likely to be exist in DT clones (Figure 7). At D2, even though both ABA- and AQP-mediated stomatal regulatory mechanisms seem to exist in both DS and DT clones, significant reduction in gs was observed only in DS clones. Interestingly, commercial clones do not seem to have hydraulic and GABA-dependant stomatal regulation under both drought events. Even though, increased accumulation of ABAxylhas shown to downregulate gs in commercial clones at D1, similar gs reduction was not detected at D2 (Figure 2b). Therefore, further investigations are required to understand whether there is another internal stimuli which prevents stomatal closure in both DT and commercial clones under recurrent drought events (Figure 7). For instance, recent work in Arabidopsis have shed light on CLAVATA3/embryo-surrounding region-related (CLE) peptides as novel messengers which are involved in root-to-shoot signalling for regulating stomatal aperture movements under drought [18,65]. However, no detailed investigations have been conducted in grapevine to understand their specific functions in stomatal regulation under long-term drought stress.
Effect of differential hydraulic, stomatal and non-stomatal regulatory mechanisms on photosynthetic performance of dry-farmed clones
Our findings suggest that DT and commercial clones are able to improve plant photosynthetic performances to adapt faster to prolonged drought episodes than DS clones (Figure 2b). Overall, our results indicate that the significant decline in AN in all clones at D1 and in DS clones and D2 are likely to be associated with a decrease in gs and qP. Interestingly, under prolong drought stress, improved photosynthetic performances of both commercial and DT clones can be explained as a result of improved CO2 diffusion and activation of their photoprotective mechanisms.
Under steady state conditions, major metabolic processes such as photosynthesis generate highly toxic reactive oxygen species (ROS), but the potential cytotoxicity is minimized by activating ROS detoxification mechanisms. However, when plants are exposed to drought, the delicate equilibrium between ROS production and scavenging is perturbed due to limitations on CO2 assimilation [66]. Different grapevine varieties possess various photoprotective mechanisms to cope with drought-mediated photoinhibition [67,68]. For instance, enhanced electron transport rate (ETR) facilitates channelling of majority of excess electrons to photosystems [40,42]. Additionally, the absorbed excess light energy can be dissipated as heat through non-photochemical quenching (NPQ) [69]. Comprehensive studies demonstrating intervarietal clonal differences in photo-protective mechanisms are still scarce. Our study demonstrate that such diversity is still exist between grapevine clones upon dehydration.
Previous studies have shown that APX plays a vital role in removing hydrogen peroxide (H2O2), which is the primary photosynthesis-associated ROS [70]. In line with previous studies, APX activity was significantly increased only in both commercial and DS clones at D1, but DT clones did not show increase in APX activity under both drought cycles (Figure 5). Significantly lower NPQ and higher ETR in DT clones at both drought events also imply that DT clones may have low level of acute oxidative stress relative to other clones (Figure 3e, f). As previous studies have shown that in mitochondria GABA is catabolised into Succinate, which acts as an electron donor to the mitochondrial electron transport, we speculated that increased GABA accumulation in DT clones may help redirecting excess photochemical energy through mitochondrial electron transport system to enhance cellular respiration and therefore, DT clones may have reduced ROS-mediated photo-oxidative damages at D1 relative to both DS and commercial clones. It is interesting to further investigate whether increased electron transport in the light harvesting complex is also mediated by GABA. Even though, commercial clones do not possess GABA-mediated ROS-detoxification mechanisms, they seemed to maintain cellular homeostasis by activating antioxidation system and non-photochemical quenching under drought. However, increased APX activity and NPQ and low ETR in DS clones would indicate that 2-fold accumulation of GABA may not be sufficient to provide complete protection against drought-induced photooxidative damages in DS clones (Figure 3e, f, 5).
The GABA is also considered as an important priming agent which allows plants to adapt faster and stronger to subsequent stress events, however, its priming-associated mechanisms are largely unknown. Given the fact that DT clones had similar level of APX activity, NPQ and ETR at both D1 and D2, our study in part suggests that GABA may also play a crucial role in intragenerational priming in DT clones to enhance their long-term drought resilience. However, some fundamental questions remain unanswered. If GABA is deemed bona fide priming elicitor, does it mediate stomatal regulation, photochemical efficiency and oxidative stress tolerance in grapevine clones? (Fig. 6) Are epigenetic modifications such as DNA methylation, histone modification or chromosomal remodelling also contribute to stress priming? Our future studies will elucidate whether long-term drought adaptation of DT clones is caused by genetic or epigenetic modifications. Ultimately, the fundamental insights obtained from this study will pave the way for future research aiming towards understanding whether we can “stress-memory”-associated candidate genes/epigenes to develop primed-grapevine varieties for a range of changing environments. Additionally, this study will also be beneficial for the grapevine industry, to develop/select drought resilient genotypes and clones suitable for grapevine breeding programs.
Funding
This research was supported by the University of Adelaide, South Australia.
Acknowledgements
The authors would like to thank Dr. Catherine M. Kidman at Wynns Coonawarra Estate and Yalumba Nursery for providing plant materials and their in-kind support and Annette Betts for assisting with the ABA analyses.