Global transcriptome and gene co-expression network analyses reveal regulatory and non-additive effects of drought and heat stress in grapevine.

Despite frequent co-occurrence of drought and heat stress, the molecular mechanisms governing plant responses to these stresses in combination have not often been studied. This is particularly evident in non-model, perennial plants. We conducted large scale physiological and transcriptome analyses to identify genes and pathways associated with grapevine response to drought and/or heat stress during stress progression and recovery. We identified gene clusters with expression correlated to leaf temperature and water stress and five hub genes for the combined stress co-expression network. Several differentially expressed genes were common to the individual and combined stresses, but the majority were unique to the individual or combined stress treatments. These included heat-shock proteins, mitogen-activated kinases, sugar metabolizing enzymes, and transcription factors, while phenylpropanoid biosynthesis and histone modifying genes were unique to the combined stress treatment. Following physiological recovery, differentially expressed genes were found only in plants under heat stress, both alone and combined with drought. Taken collectively, our results suggest that the effect of the combined stress on physiology and gene expression is more severe than that of individual stresses, but not simply additive, and that epigenetic chromatin modifications may play an important role in grapevine responses to combined drought and heat stress.

Regulation of anthocyanin and sugar accumulation in grape berry through carbon limitation and exogenous ABA application.

To optimize vineyard management practices to adapt viticulture to climate change, knowledge of the regulation mechanism of metabolite accumulation under carbon source limitation and abscisic acid (ABA) application in grapes should be deepened. Here, carbon source limitations were imposed by reducing leaf area from 12 to 2 leaves per vine (at pea sized stage, - 2L-P; or one week prior to veraison - 2L-V) and phloem girdling between the second and third leaf from bottom to top (one week prior to veraison - 12L-girdling) were compared for their effects on berry composition. All three modalities significantly reduced sugar, anthocyanin and ABA content in comparison with berries under sufficient carbon supply (12 leaves per vine - 12L), with 2L-V being the greatest. Allowing leaf area to partially recover (2L-R) or berry ABA application (400 mg. L-1) one week before veraison increased the ratio of anthocyanin to sugar under source limitation. Combined with the analysis of berry metabolites and transcript abundances, our results indicate that source limitation and exogenous ABA co-regulated anthocyanins content through differential gene expression.

GABA signalling modulates stomatal opening to enhance plant water use efficiency and drought resilience.

The non-protein amino acid γ-aminobutyric acid (GABA) has been proposed to be an ancient messenger for cellular communication conserved across biological kingdoms. GABA has well-defined signalling roles in animals; however, whilst GABA accumulates in plants under stress it has not been determined if, how, where and when GABA acts as an endogenous plant signalling molecule. Here, we establish endogenous GABA as a bona fide plant signal, acting via a mechanism not found in animals. Using Arabidopsis thaliana, we show guard cell GABA production is necessary and sufficient to reduce stomatal opening and transpirational water loss, which improves water use efficiency and drought tolerance, via negative regulation of a stomatal guard cell tonoplast-localised anion transporter. We find GABA modulation of stomata occurs in multiple plants, including dicot and monocot crops. This study highlights a role for GABA metabolism in fine tuning physiology and opens alternative avenues for improving plant stress resilience.

Gene body demethylation increases expression and is associated with self-pruning during grape genome duplication.

A colchicine-induced autotetraploid grapevine exhibiting potentially valuable agronomic traits for grape production and breeding, including self-pruning, was identified. This study investigated DNA methylation variation and its role in gene expression during self-pruning in the autotetraploid grapevine. We used RNA-Seq to estimate differentially expressed genes between diploid and autotetraploid grapevine shoot tips. The genes showing increases in the autotetraploid were mainly related to stress response pathways, whereas those showing decreases in the autotetraploid were related to biological metabolism and biosynthesis. Whole-genome bisulfite sequencing was performed to produce single-base methylomes for the diploid and autotetraploid grapevines. Comparison between the methylomes revealed that they were conserved in CG and CHG contexts. In the autotetraploid grapevine, hypodifferentially methylated regions (DMRs) and hyper-DMRs in the gene body increased or decreased gene expression, respectively. Our results indicated that a hypo-DMR in the ACO1 gene body increased its expression and might promote self-pruning. This study reports that hypo-DMRs in the gene body increase gene expression in plants and reveals the mechanism underlying the changes in the modifications affecting gene expression during genome duplication. Overall, our results provide valuable information for understanding the relationships between DNA methylation, gene expression, and autotetraploid breeding in grape.

A whole canopy gas exchange system for the targeted manipulation of grapevine source-sink relations using sub-ambient CO2.

BACKGROUND: Elucidating the effect of source-sink relations on berry composition is of interest for wine grape production as it represents a mechanistic link between yield, photosynthetic capacity and wine quality. However, the specific effects of carbohydrate supply on berry composition are difficult to study in isolation as leaf area or crop adjustments can also change fruit exposure, or lead to compensatory growth or photosynthetic responses. A new experimental system was therefore devised to slow berry sugar accumulation without changing canopy structure or yield. This consisted of six transparent 1.2 m3 chambers to enclose large pot-grown grapevines, and large soda-lime filled scrubbers that reduced carbon dioxide (CO2) concentration of day-time supply air by approximately 200 ppm below ambient. RESULTS: In the first full scale test of the system, the chambers were installed on mature Shiraz grapevines for 14 days from the onset of berry sugar accumulation. Three chambers were run at sub-ambient CO2 for 10 days before returning to ambient. Canopy gas exchange, and juice hexose concentrations were determined. Net CO2 exchange was reduced from 65.2 to 30 g vine- 1 day- 1, or 54%, by the sub-ambient treatment. At the end of the 10 day period, total sugar concentration was reduced from 95 to 77 g L- 1 from an average starting value of 23 g L- 1, representing a 25% reduction. Scaling to a per vine basis, it was estimated that 223 g of berry sugars accumulated under ambient supply compared to 166 g under sub-ambient, an amount equivalent to 50 and 72% of total C assimilated. CONCLUSIONS: Through supply of sub-ambient CO2 using whole canopy gas exchange chambers system, an effective method was developed for reducing photosynthesis and slowing the rate of berry sugar accumulation without modifying yield or leaf area. While in this case developed for further investigations of grape and wine composition, the system has broader applications for the manipulation and of study of grapevine source-sink relations.

Fast Phenomics in Vineyards: Development of GRover, the Grapevine Rover, and LiDAR for Assessing Grapevine Traits in the Field.

This paper introduces GRover (the grapevine rover), an adaptable mobile platform for the deployment and testing of proximal imaging sensors in vineyards for the non-destructive assessment of trunk and cordon volume and pruning weight. A SICK LMS-400 light detection and ranging (LiDAR) radar mounted on GRover was capable of producing precise (±3 mm) 3D point clouds of vine rows. Vineyard scans of the grapevine variety Shiraz grown under different management systems at two separate locations have demonstrated that GRover is able to successfully reproduce a variety of vine structures. Correlations of pruning weight and vine wood (trunk and cordon) volume with LiDAR scans have resulted in high coefficients of determination (R² = 0.91 for pruning weight; 0.76 for wood volume). This is the first time that a LiDAR of this type has been extensively tested in vineyards. Its high scanning rate, eye safe laser and ability to distinguish tissue types make it an appealing option for further development to offer breeders, and potentially growers, quantified measurements of traits that otherwise would be difficult to determine.

Functional differences in transport properties of natural HKT1;1 variants influence shoot Na+ exclusion in grapevine rootstocks.

Under salinity, Vitis spp. rootstocks can mediate salt (NaCl) exclusion from grafted V. vinifera scions enabling higher grapevine yields and production of superior wines with lower salt content. Until now, the genetic and mechanistic elements controlling sodium (Na+ ) exclusion in grapevine were unknown. Using a cross between two Vitis interspecific hybrid rootstocks, we mapped a dominant quantitative trait locus (QTL) associated with leaf Na+ exclusion (NaE) under salinity stress. The NaE locus encodes six high-affinity potassium transporters (HKT). Transcript profiling and functional characterization in heterologous systems identified VisHKT1;1 as the best candidate gene for controlling leaf Na+ exclusion. We characterized four proteins encoded by unique VisHKT1;1 alleles from the parents, and revealed that the dominant HKT variants exhibit greater Na+ conductance with less rectification than the recessive variants. Mutagenesis of VisHKT1;1 and TaHKT1.5-D from bread wheat, demonstrated that charged amino acid residues in the eighth predicted transmembrane domain of HKT proteins reduces inward Na+ conductance, and causes inward rectification of Na+ transport. The origin of the recessive VisHKT1;1 alleles was traced to V. champinii and V. rupestris. We propose that the genetic and functional data presented here will assist with breeding Na+ -tolerant grapevine rootstocks.

Abscisic Acid Down-Regulates Hydraulic Conductance of Grapevine Leaves in Isohydric Genotypes Only.

Plants evolved different strategies to cope with water stress. While isohydric species maintain their midday leaf water potential (ΨM) under soil water deficit by closing their stomata, anisohydric species maintain higher stomatal aperture and exhibit substantial reductions in ΨM It was hypothesized that isohydry is related to a locally higher sensitivity of stomata to the drought-hormone abscisic acid (ABA). Interestingly, recent lines of evidence in Arabidopsis (Arabidopsis thaliana) suggested that stomatal responsiveness is also controlled by an ABA action on leaf water supply upstream from stomata. Here, we tested the possibility in grapevine (Vitis vinifera) that different genotypes ranging from near isohydric to more anisohydric may have different sensitivities in these ABA responses. Measurements on whole plants in drought conditions were combined with assays on detached leaves fed with ABA. Two different methods consistently showed that leaf hydraulic conductance (Kleaf) was down-regulated by exogenous ABA, with strong variations depending on the genotype. Importantly, variation between isohydry and anisohydry correlated with Kleaf sensitivity to ABA, with Kleaf in the most anisohydric genotypes being unresponsive to the hormone. We propose that the observed response of Kleaf to ABA may be part of the overall ABA regulation of leaf water status.

The Synthesis and Accumulation of Resveratrol Are Associated with Veraison and Abscisic Acid Concentration in Beihong (Vitis vinifera × Vitis amurensis) Berry Skin.

Resveratrols are polyphenolic secondary metabolites that can benefit human health, and only occur in a few plant families including Vitaceae. It has been reported that abscisic acid (ABA) can induce veraison (the onset of grape berry ripening) and may induce the accumulation of resveratrol in berry skin. However, the relationships between ABA, veraison, the accumulation of anthocyanins and the accumulation of resveratrol in the berry are poorly understood. This study attempted to answer this question through an investigation of the effect of applied ABA and fluridone (a synthetic inhibitor of ABA) on the biosynthesis and accumulation of ABA, anthocyanin, and resveratrol in Beihong (Vitis vinifera × Vitis amurensis) berry skin. Under natural conditions, resveratrol concentration was very low before 91 DAA (days after anthesis), i.e., 2 weeks after veraison, however, it increased sharply from this point to 126 DAA (maturity). Exogenous ABA applications all resulted in an increase in berry skin ABA and anthocyanin concentration, irrespective of the developmental stage at which the treatment occurred (20 and 10 days pre-veraison, veraison or 7 days post-veraison), thereby advancing veraison. In contrast, resveratrol concentration increased only when ABA was applied at 10 days pre-veraison or at veraison. As a result, the accumulation of resveratrol was associated with veraison in grape berry skin and this accumulation, together with that of anthocyanins, was associated with ABA concentration. The response of resveratrol biosynthesis in the berry skin to manipulation of ABA varied during berry development and was less sensitive to ABA than the response of anthocyanin biosynthesis.

VitiCanopy: A Free Computer App to Estimate Canopy Vigor and Porosity for Grapevine.

Leaf area index (LAI) and plant area index (PAI) are common and important biophysical parameters used to estimate agronomical variables such as canopy growth, light interception and water requirements of plants and trees. LAI can be either measured directly using destructive methods or indirectly using dedicated and expensive instrumentation, both of which require a high level of know-how to operate equipment, handle data and interpret results. Recently, a novel smartphone and tablet PC application, VitiCanopy, has been developed by a group of researchers from the University of Adelaide and the University of Melbourne, to estimate grapevine canopy size (LAI and PAI), canopy porosity, canopy cover and clumping index. VitiCanopy uses the front in-built camera and GPS capabilities of smartphones and tablet PCs to automatically implement image analysis algorithms on upward-looking digital images of canopies and calculates relevant canopy architecture parameters. Results from the use of VitiCanopy on grapevines correlated well with traditional methods to measure/estimate LAI and PAI. Like other indirect methods, VitiCanopy does not distinguish between leaf and non-leaf material but it was demonstrated that the non-leaf material could be extracted from the results, if needed, to increase accuracy. VitiCanopy is an accurate, user-friendly and free alternative to current techniques used by scientists and viticultural practitioners to assess the dynamics of LAI, PAI and canopy architecture in vineyards, and has the potential to be adapted for use on other plants.

ABA-mediated responses to water deficit separate grapevine genotypes by their genetic background.

BACKGROUND: ABA-mediated processes are involved in plant responses to water deficit, especially the control of stomatal opening. However in grapevine it is not known if these processes participate in the phenotypic variation in drought adaptation existing between genotypes. To elucidate this question, the response to short-term water-deficit was analysed in roots and shoots of nine Vitis genotypes differing in their drought adaptation in the field. The transcript abundance of 12 genes involved in ABA biosynthesis, catabolism, and signalling were monitored, together with physiological and metabolic parameters related to ABA and its role in controlling plant transpiration. RESULTS: Although transpiration and ABA responses were well-conserved among the genotypes, multifactorial analyses separated Vitis vinifera varieties and V. berlandieri x V. rupestris hybrids (all considered drought tolerant) from the other genotypes studied. Generally, V. vinifera varieties, followed by V. berlandieri x V. rupestris hybrids, displayed more pronounced responses to water-deficit in comparison to the other genotypes. However, changes in transcript abundance in roots were more pronounced for Vitis hybrids than V. vinifera genotypes. Changes in the expression of the cornerstone ABA biosynthetic gene VviNCED1, and the ABA transcriptional regulator VviABF1, were associated with the response of V. vinifera genotypes, while changes in VviNCED2 abundance were associated with the response of other Vitis genotypes. In contrast, the ABA RCAR receptors were not identified as key components of the genotypic variability of water-deficit responses. Interestingly, the expression of VviSnRK2.6 (an AtOST1 ortholog) was constitutively lower in roots and leaves of V. vinifera genotypes and higher in roots of V. berlandieri x V. rupestris hybrids. CONCLUSIONS: This study highlights that Vitis genotypes exhibiting different levels of drought adaptation differ in key steps involved in ABA metabolism and signalling; both under well-watered conditions and in response to water-deficit. In addition, it supports that adaptation may be related to various mechanisms related or not to ABA responses.

Anthocyanin biosynthesis is differentially regulated by light in the skin and flesh of white-fleshed and teinturier grape berries.

MAIN CONCLUSION: Light exclusion reduces the concentration and modifies the composition of grape anthocyanins, by altering the expression of genes involved in anthocyanin biosynthesis and transport, in a cultivar- and tissue-specific manner. Unlike most grapes, teinturier grapes accumulate anthocyanins both in skin and flesh. However, the concentration and composition of anthocyanins in both tissues differ, providing a valuable system to study tissue-specific regulation of anthocyanin synthesis. Furthermore, little is known about the mechanisms controlling the sensitivity of anthocyanin accumulation to light. Here, light was excluded from Gamay (white-fleshed) and Gamay Fréaux (teinturier mutant) berries throughout berry development. Under light-exposed conditions, the skin of Gamay Fréaux accumulated the highest level of anthocyanins, followed by the skin of Gamay, while the pulp of Gamay Fréaux had much lower anthocyanins than the skins. Network analysis revealed the same order on the number of significant correlations among metabolites and transcripts in the three colored tissues, indicating a higher connectivity that reflects a higher efficiency of the anthocyanin pathway. Compared to light conditions, light exclusion reduced the total amount of anthocyanins, most severely in the skin of Gamay and to a lesser extent in the flesh and skin of Gamay Fréaux. Coordinated decrease in the transcript abundance of structural, regulatory and transporter genes by light exclusion correlated with the reduced anthocyanin concentration in a cultivar- and tissue-specific manner. Moreover, light exclusion increased the ratio of dihydroxylated to trihydroxylated anthocyanins, in parallel with F3'H and F3'5'H transcript amounts. Sugars and ABA only play a limited role in the control of anthocyanin synthesis in the berries, in contrast with what has been described in cell suspensions. This study provides novel insights into the regulation of anthocyanin in wild type and teinturier cultivars.

Root biomass in the upper layer of the soil profile is related to the stomatal response of wheat as the soil dries.

Terminal drought is a common abiotic stress affecting wheat yield in Mediterranean-type environments. As terminal drought develops, top layers of the soil profile dry, exposing the upper part of the root system to soil water deficit while deeper roots can still access soil water. Since open stomata rapidly exhausts available soil water, reducing stomatal conductance to prolong availability of soil water during grain filling may improve wheat yields in water-limited environments. It was hypothesised that genotypes with more root biomass in the drying upper layer of the soil profile accumulate more abscisic acid in the leaf and initiate stomatal closure to regulate water use under terminal drought. The wheat cultivar Drysdale and the breeding line IGW-3262 were grown in pots horizontally split into two segments by a wax-coated layer that hydraulically isolated the top and bottom segments, but allowed roots to grow into the bottom segment. Terminal drought was induced from anthesis by withholding water from (i) the top segment only (DW) and (ii) the top and bottom segments (DD) while both segments in well-watered pots (WW) were maintained at 90% pot soil water capacity. Drysdale, initiated stomatal closure earlier than IGW-3262, possibly due to higher signal strength generated in its relatively larger proportion of roots in the drying top segment. The relationship between leaf ABA and stomatal conductance was strong in Drysdale but weak in IGW-3262. Analysis of ABA metabolites suggests possible differences in ABA metabolism between these two genotypes. A higher capability of deeper roots to extract available water is also important in reducing the gap between actual and potential yield.

Expression of ABA synthesis and metabolism genes under different irrigation strategies and atmospheric VPDs is associated with stomatal conductance in grapevine (Vitis vinifera L. cv Cabernet Sauvignon).

The influence of different levels of irrigation and of variation in atmospheric vapour pressure deficit (VPD) on the synthesis, metabolism, and transport of abscisic acid (ABA) and the effects on stomatal conductance were examined in field-grown Cabernet Sauvignon grapevines. Xylem sap, leaf tissue, and root tissue were collected at regular intervals during two seasons in conjunction with measurements of leaf water potential (Ψleaf) and stomatal conductance (gs). The different irrigation levels significantly altered the Ψleaf and gs of the vines across both seasons. ABA abundance in the xylem sap was correlated with gs. The expression of genes associated with ABA synthesis, NCED1 and NCED2, was higher in the roots than in the leaves throughout and highest in the roots in mid January, a time when soil moisture declined and VPD was at its highest. Their expression in roots was also inversely related to the levels of irrigation and correlated with ABA abundance in the roots, xylem sap, and leaves. Three genes encoding ABA 8'-hydroxylases were isolated and their identities confirmed by expression in yeast cells. The expression of one of these, Hyd1, was elevated in leaves when VPD was below 2.0-2.5 kPa and minimal at higher VPD levels. The results provide evidence that ABA plays an important role in linking stomatal response to soil moisture status and that changes in ABA catabolism at or near its site of action allows optimization of gas exchange to current environmental conditions.

Changes in nutritional value of cyanogenic trifolium repens grown at elevated atmospheric CO2.

Global food security in a changing climate depends on both the nutritive value of staple crops as well as their yields. Here, we examined the direct effect of atmospheric carbon dioxide on the toxicity of the important pasture crop, Trifolium repens L. (clover). Shoots of T. repens contain cyanogenic glycosides that break down to release toxic hydrogen cyanide when damaged. The ability of animals to tolerate cyanogenic compounds is dependent, in part, on their overall protein intake. We grew T. repens communities at ambient and approximately twice-ambient CO(2) in a controlled environment greenhouse experiment. We found that the ratio of total cyanogenic glycosides to total protein ratio was nearly two times higher in leaves of T. repens grown at elevated CO(2). This study highlights the importance of assessing the nutritive value of this and other plants in response to rising CO(2) so that steps can be taken to address any adverse consequences for herbivores.

Nitrogen in cell walls of sclerophyllous leaves accounts for little of the variation in photosynthetic nitrogen-use efficiency.

Photosynthetic rate per unit nitrogen generally declines as leaf mass per unit area (LMA) increases. To determine how much of this decline was associated with allocating a greater proportion of leaf nitrogen into cell wall material, we compared two groups of plants. The first group consisted of two species from each of eight genera, all of which were perennial evergreens growing in the Australian National Botanic Gardens (ANBG). The second group consisted of seven Eucalyptus species growing in a greenhouse. The percentage of leaf biomass in cell walls was independent of variation in LMA within any genus, but varied from 25 to 65% between genera. The nitrogen concentration of cell wall material was 0.4 times leaf nitrogen concentration for all species apart from Eucalyptus, which was 0.6 times leaf nitrogen concentration. Between 10 and 30% of leaf nitrogen was recovered in the cell wall fraction, but this was independent of LMA. No trade-off was observed between nitrogen associated with cell walls and the nitrogen allocated to ribulose 1.5-bisphosphate carboxylase/oxygenase (Rubisco). Variation in photosynthetic rate per unit nitrogen could not be explained by variation in cell wall nitrogen.

Phosphorus availability and elevated CO2 affect biological nitrogen fixation and nutrient fluxes in a clover-dominated sward.

The response of biological nitrogen fixation (BNF) to elevated CO(2) was examined in white clover (Trifolium repens)-dominated swards under both high and low phosphorus availability. Mixed swards of clover and buffalo grass (Stenotaphrum secundatum) were grown for 15 months in 0.2 m2 sand-filled mesocosms under two CO2 treatments (ambient and twice ambient) and three nutrient treatments [no N, and either low or high P (5 or 134 kg P ha(-1)); the third nutrient treatment was supplied with high P and N (240 kg N ha(-1))]. Under ambient CO2, high P increased BNF from 410 to 900 kg ha(-1). Elevated CO2 further increased BNF to 1180 kg ha(-1) with high P, but there was no effect of CO2 on BNF with low P. Allocation of N belowground increased by approx. 50% under elevated CO2 irrespective of supplied P. The results suggest that where soil P availability is low, elevated CO2 will not increase BNF, and pasture quality could decrease because of a reduction in aboveground N.