Contrasting Responses of Two Grapevine Cultivars to Drought: The Role of Non-structural Carbohydrates in Xylem Hydraulic Recovery.

Xylem embolism is one of the possible outcomes of decreasing xylem pressure when plants face drought. Recent studies have proposed a role for non-structural carbohydrates (NSCs) in osmotic pressure generation, required for refilling embolized conduits. Potted cuttings of grapevine Grenache and Barbera, selected for their adaptation to different climatic conditions, were subjected to a drought stress followed by re-irrigation. Stem embolism rate and its recovery were monitored in vivo by X-ray micro-computed tomography (micro-CT). The same plants were further analyzed for xylem conduit dimension and NSC content. Both cultivars significantly decreased Ψpd in response to drought and recovered from xylem embolism after re-irrigation. However, although the mean vessel diameter was similar between the cultivars, Barbera was more prone to embolism. Surprisingly, vessel diameter was apparently reduced during recovery in this cultivar. Hydraulic recovery was linked to sugar content in both cultivars, showing a positive relationship between soluble NSCs and the degree of xylem embolism. However, when starch and sucrose concentrations were considered separately, the relationships showed cultivar-specific and contrasting trends. We showed that the two cultivars adopted different NSC-use strategies in response to drought, suggesting two possible scenarios driving conduit refilling. In Grenache, sucrose accumulation seems to be directly linked to embolism formation and possibly sustains refilling. In Barbera, maltose/maltodextrins could be involved in a conduit recovery strategy via the formation of cell-wall hydrogels, likely responsible for the reduction of conduit lumen detected by micro-CT.

Do the ends justify the means? Impact of drought progression rate on stress response and recovery in Vitis vinifera.

Plants are frequently exposed to prolonged and intense drought events. To survive, species must implement strategies to overcome progressive drought while maintaining sufficient resources to sustain the recovery of functions. Our objective was to understand how stress rate development modulates energy reserves and affects the recovery process. Grenache Vitis vinifera cultivar was exposed to either fast-developing drought (within few days; FDD), typical of pot experiments, or slow-developing drought (few weeks, SDD), more typical for natural conditions. FDD was characterized by fast (2-3 days) stomatal closure in response to increased stress level, high abscisic acid (ABA) accumulation in xylem sap (>400 µg L-1 ) without the substantial changes associated with stem priming for recovery (no accumulation of sugar or drop in xylem sap pH). In contrast, SDD was characterized by gradual stomatal closure, low ABA accumulation (<100 µg L-1 ) and changes that primed the stem for recovery (xylem sap acidification from 6 to 5.5 pH and sugar accumulation from 1 to 3 g L-1 ). Despite FDD and SDD demonstrating similar trends over time in the recovery of stomatal conductance, they differed in their sensitivity to xylem ABA. Grenache showed near-isohydric and near-anisohydric behavior depending on the rate of drought progression, gauging the risk between hydraulic integrity and photosynthetic gain. The isohydry observed during FDD could potentially provide protection from large sudden swings in tension, while transitioning to anisohydry during SDD could prioritize the maintenance of photosynthetic activity over hydraulic security.

Chemical inhibition of xylem cellular activity impedes the removal of drought-induced embolisms in poplar stems - new insights from micro-CT analysis.

In drought-stressed plants a coordinated cascade of chemical and transcriptional adjustments occurs at the same time as embolism formation. While these processes do not affect embolism formation during stress, they may prime stems for recovery during rehydration by modifying apoplast pH and increasing sugar concentration in the xylem sap. Here we show that in vivo treatments modifying apoplastic pH (stem infiltration with a pH buffer) or reducing stem metabolic activity (infiltration with sodium vanadate and sodium cyanide; plant exposure to carbon monoxide) can reduce sugar accumulation, thus disrupting or delaying the recovery process. Application of the vanadate treatment (NaVO3, an inhibitor of many ATPases) completely halted recovery from drought-induced embolism for up to 24 h after re-irrigation, while partial recovery was observed in vivo in control plants using X-ray microcomputed tomography. Our results suggest that stem hydraulic recovery in poplar is a biological, energy-dependent process that coincides with accumulation of sugars in the apoplast during stress. Recovery and damage are spatially coordinated, with embolism formation occurring from the inside out and refilling from the outside in. The outside-in pattern highlights the importance of xylem proximity to the sugars within the phloem to the embolism recovery process.

Priming xylem for stress recovery depends on coordinated activity of sugar metabolic pathways and changes in xylem sap pH.

Some plant species are capable of significant reduction of xylem embolism during recovery from drought despite stem water potential remains negative. However, the functional biology underlying this process is elusive. We subjected poplar trees to drought stress followed by a period of recovery. Water potential, hydraulic conductivity, gas exchange, xylem sap pH, and carbohydrate content in sap and woody stems were monitored in combination with an analysis of carbohydrate metabolism, enzyme activity, and expression of genes involved in sugar metabolic and transport pathways. Drought resulted in an alteration of differential partitioning between starch and soluble sugars. Upon stress, an increase in the starch degradation rate and the overexpression of sugar symporter genes promoted the efflux of disaccharides (mostly maltose and sucrose) to the apoplast. In turn, the efflux activity of the sugar-proton cotransporters caused a drop in xylem pH. The newly acidic environment induced the activity of apoplastic invertases leading to the accumulation of monosaccharides in the apoplast, thus providing the main osmoticum necessary for recovery. During drought and recovery, a complex network of coordinated molecular and biochemical signals was activated at the interface between xylem and parenchyma cells that appeared to prime the xylem for hydraulic recovery.

The pitfalls of in vivo imaging techniques: evidence for cellular damage caused by synchrotron X-ray computed micro-tomography.

Synchrotron X-ray computed micro-tomography (microCT) has emerged as a promising noninvasive technique for in vivo monitoring of xylem function, including embolism build-up under drought and hydraulic recovery following re-irrigation. Yet, the possible harmful effects of ionizing radiation on plant tissues have never been quantified. We specifically investigated the eventual damage suffered by stem living cells of three different species exposed to repeated microCT scans. Stem samples exposed to one, two or three scans were used to measure cell membrane and RNA integrity, and compared to controls never exposed to X-rays. Samples exposed to microCT scans suffered serious alterations to cell membranes, as revealed by marked increase in relative electrolyte leakage, and also underwent severe damage to RNA integrity. The negative effects of X-rays were apparent in all species tested, but the magnitude of damage and the minimum number of scans inducing negative effects were species-specific. Our data show that multiple microCT scans lead to disruption of fundamental cellular functions and processes. Hence, microCT investigation of phenomena that depend on physiological activity of living cells may produce erroneous results and lead to incorrect conclusions.

Acclimation of Pistacia integerrima trees to frost in semi-arid environments depends on autumn's drought.

Main conclusion Cold acclimation is revealed through induced stem respiration during pre-winter frost of native Pistacia integerrima trees in continental semi-arid environments. Semi-arid environments challenge vegetation by simultaneous abiotic stresses. In this study, we examine the combined effects of water stress and frost on the physiology of Pistacia integerrima stems. This species is native to semi-arid environments where drought and frost frequently co-occur. We quantified carbohydrates and proline in P. integerrima stems responding to frost and experiencing water potentials between -0.2 and -1.8 MPa. We report that dehydrated trees (i.e., Ψstem <=-1 MPa) had more soluble sugars and proline than the well-watered trees (-0.2 MPa). The dehydrated trees also froze at lower temperatures and were less damaged by freezing. Interestingly, we observed a significant increase in stem CO2 efflux at near-freezing temperatures that could be linked to frost protection. This novel finding challenges current paradigm of plant respiration-kinetics which predicts, according to Arrhenius equation, lower respiration rates during frost. Our results support the notion that drought and frost are analogous stresses that can independently activate corresponding physiological processes in trees and amplify protection. This inevitable stress response 'collaboration' may be the key to understanding how non-dormant perennial plants survive the highly variable weather patterns of early winters in semi-arid environments.

The functional role of xylem parenchyma cells and aquaporins during recovery from severe water stress.

Xylem parenchyma cells [vessel associated cells (VACs)] constitute a significant fraction of the xylem in woody plants. These cells are often closely connected with xylem vessels or tracheids via simple pores (remnants of plasmodesmata fields). The close contact and biological activity of VACs during times of severe water stress and recovery from stress suggest that they are involved in the maintenance of xylem transport capacity and responsible for the restoration of vessel/tracheid functionality following embolism events. As recovery from embolism requires the transport of water across xylem parenchyma cell membranes, an understanding of stem-specific aquaporin expression patterns, localization and activity is a crucial part of any biological model dealing with embolism recovery processes in woody plants. In this review, we provide a short overview of xylem parenchyma cell biology with a special focus on aquaporins. In particular we address their distributions and activity during the development of drought stress, during the formation of embolism and the subsequent recovery from stress that may result in refilling. Complemented by the current biological model of parenchyma cell function during recovery from stress, this overview highlights recent breakthroughs on the unique ability of long-lived perennial plants to undergo cycles of embolism-recovery related to drought/rewetting or freeze/thaw events.

The tomato plastidic fructokinase SlFRK3 plays a role in xylem development.

Plants have two kinds of fructokinases (FRKs) that catalyze the key step of fructose phosphorylation, cytosolic and plastidic. The major cytosolic tomato FRK, SlFRK2, is essential for the development of xylem vessels. In order to study the role of SlFRK3, which encodes the only plastidic FRK, we generated transgenic tomato (Solanum lycopersicon) plants with RNAi suppression of SlFRK3 as well as plants expressing beta-glucoronidase (GUS) under the SlFRK3 promoter. GUS staining indicated SlFRK3 expression in vascular tissues of the leaves and stems, including cambium, differentiating xylem, young xylem fibers and phloem companion cells. Suppression of SlFRK3 reduced the stem xylem area, stem and root water conductance, and whole-plant transpiration, with minor effects on plant development. However, suppression of SlFRK3 accompanied by partial suppression of SlFRK2 induced significant growth-inhibition effects, including the wilting of mature leaves. Grafting experiments revealed that these growth effects are imposed primarily by the leaves, whose petioles had unlignified, thin-walled xylem fibers with collapsed parenchyma cells around the vessels. A cross between the SlFRK2-antisense and SlFRK3-RNAi lines exhibited similar wilting and anatomical effects, confirming that these effects are the result of the combined suppression of SlFRK3 and SlFRK2. These results demonstrate a role of the plastidic SlFRK3 in xylem development and hydraulic conductance.

Accumulation of sugars in the xylem apoplast observed under water stress conditions is controlled by xylem pH.

Severe water stress constrains, or even stops, water transport in the xylem due to embolism formation. Previously, the xylem of poplar trees was shown to respond to embolism formation by accumulating carbohydrates in the xylem apoplast and dropping xylem sap pH. We hypothesize that these two processes may be functionally linked as lower pH activates acidic invertases degrading sucrose and inducing accumulation of monosaccharides in xylem apoplast. Using a novel in vivo method to measure xylem apoplast pH, we show that pH drops from ~6.2 to ~5.6 in stems of severely stressed plants and rises following recovery of stem water status. We also show that in a lower pH environment, sugars are continuously accumulating in the xylem apoplast. Apoplastic carbohydrate accumulation was reduced significantly in the presence of a proton pump blocker (orthovanadate). These observations suggest that a balance in sugar concentrations exists between the xylem apoplast and symplast that can be controlled by xylem pH and sugar concentration. We conclude that lower pH is related to loss of xylem transport function, eventually resulting in accumulation of sugars that primes stems for recovery from embolism when water stress is relieved.

Changes in Air CO2 Concentration Differentially Alter Transcript Levels of NtAQP1 and NtPIP2;1 Aquaporin Genes in Tobacco Leaves.

The aquaporin specific control on water versus carbon pathways in leaves is pivotal in controlling gas exchange and leaf hydraulics. We investigated whether Nicotiana tabacum aquaporin 1 (NtAQP1) and Nicotiana tabacum plasma membrane intrinsic protein 2;1 (NtPIP2;1) gene expression varies in tobacco leaves subjected to treatments with different CO2 concentrations (ranging from 0 to 800 ppm), inducing changes in photosynthesis, stomatal regulation and water evaporation from the leaf. Changes in air CO2 concentration ([CO2]) affected net photosynthesis (Pn) and leaf substomatal [CO2] (Ci). Pn was slightly negative at 0 ppm air CO2; it was one-third that of ambient controls at 200 ppm, and not different from controls at 800 ppm. Leaves fed with 800 ppm [CO2] showed one-third reduced stomatal conductance (gs) and transpiration (E), and their gs was in turn slightly lower than in 200 ppm- and in 0 ppm-treated leaves. The 800 ppm air [CO2] strongly impaired both NtAQP1 and NtPIP2;1 gene expression, whereas 0 ppm air [CO2], a concentration below any in vivo possible conditions and specifically chosen to maximize the gene expression alteration, increased only the NtAQP1 transcript level. We propose that NtAQP1 expression, an aquaporin devoted to CO2 transport, positively responds to CO2 scarcity in the air in the whole range 0-800 ppm. On the contrary, expression of NtPIP2;1, an aquaporin not devoted to CO2 transport, is related to water balance in the leaf, and changes in parallel with gs. These observations fit in a model where upregulation of leaf aquaporins is activated at low Ci, while downregulation occurs when high Ci saturates photosynthesis and causes stomatal closure.

Down-regulation of plasma intrinsic protein1 aquaporin in poplar trees is detrimental to recovery from embolism.

During their lifecycles, trees encounter multiple events of water stress that often result in embolism formation and temporal decreases in xylem transport capacity. The restoration of xylem transport capacity requires changes in cell metabolic activity and gene expression. Specifically, in poplar (Populus spp.), the formation of xylem embolisms leads to a clear up-regulation of plasma membrane protein1 (PIP1) aquaporin genes. To determine their role in poplar response to water stress, transgenic Populus tremula × Populus alba plants characterized by the strong down-regulation of multiple isoforms belonging to the PIP1 subfamily were used. Transgenic lines showed that they are more vulnerable to embolism, with 50% percent loss of conductance occurring 0.3 MPa earlier than in wild-type plants, and that they also have a reduced capacity to restore xylem conductance during recovery. Transgenic plants also show symptoms of a reduced capacity to control percent loss of conductance through stomatal conductance in response to drought, because they have a much narrower vulnerability safety margin. Finally, a delay in stomatal conductance recovery during the period of stress relief was observed. The presented results suggest that PIP1 genes are involved in the maintenance of xylem transport system capacity, in the promotion of recovery from stress, and in contribution to a plant's control of stomatal conductance under water stress.

Threats to xylem hydraulic function of trees under 'new climate normal' conditions.

Climate models predict increases in frequency and intensity of extreme environmental conditions, such as changes to minimum and maximum temperatures, duration of drought periods, intensity of rainfall/snowfall events and wind strength. These local extremes, rather than average climatic conditions, are closely linked to woody plant survival, as trees cope with such events over long lifespans. While the xylem provides trees with structural strength and is considered the most robust part of a tree's structure, it is also the most physiologically vulnerable as tree survival depends on its ability to sustain water supply to the tree crown under variable environmental conditions. Many structural, functional and biological tree properties evolved to protect xylem from loss of transport function because of embolism or to restore xylem transport capacity following embolism formation. How 'the new climate normal' conditions will affect these evolved strategies is yet to be seen. Our understanding of xylem physiology and current conceptual models describing embolism formation and plant recovery from water stress, however, can provide insight into near-future challenges that woody plants will face. In addition, knowledge of species-specific properties of xylem function may help guide mitigation of climate change impacts on woody plants in natural and agricultural tree communities.

Analysis of xylem sap from functional (nonembolized) and nonfunctional (embolized) vessels of Populus nigra: chemistry of refilling.

It is assumed that the refilling of drought-induced embolism requires the creation of an osmotic gradient between xylem parenchyma cells and vessel lumens to generate the water efflux needed to fill the void. To assess the mechanism of embolism repair, it is crucial to determine if plants can up-regulate the efflux of osmotically active substances into embolized vessels and identify the major components of the released osmoticum. Here, we introduce a new approach of sap collection designed to separate water from nonembolized (functional) and embolized (nonfunctional) vessels. This new approach made possible the chemical analysis of liquid collected from both types of vessels in plants subjected to different levels of water stress. The technique also allowed us to determine the water volumes in nonfunctional vessels as a function of stress level. Overall, with the increase of water stress in plants, the osmotic potential of liquid collected from nonfunctional vessels increased while its volume decreased. These results revealed the presence of both sugars and ions in nonfunctional vessels at elevated levels in comparison with liquid collected from functional vessels, in which only traces of sugars were found. The increased sugar concentration was accompanied by decreased xylem sap pH. These results provide new insight into the biology of refilling, underlining the role of sugar and sugar transporters, and imply that a large degree of hydraulic compartmentalization must exist in the xylem during the refilling process.

Alkaline soil primes the recovery from drought in Populus nigra plants through physiological and chemical adjustments.

Perennial plants are frequently exposed to severe and prolonged drought, and when the balance between water transport and transpirational demand is compromised trees are in danger of embolism formation. To maintain the physiological balance, plants can rely on mechanisms to quickly recover the lost xylem hydraulic capacity and reduce the prolonged impact on photosynthetic activity upon rehydration. Among factors helpful for plants to sustain acclimation and adaptation responses to drought and promote recovery, maintaining an optimal nutritional status is crucial for plant survival. This study aimed to investigate the physiological and biochemical responses under drought and recovery of Populus nigra plants grown in soil with impaired nutrient bioavailability obtained by adding calcium oxide (CaO) to the substrate. Although the CaO treatment did not affect plant growth, in well-watered conditions, treated poplars displayed an impaired inorganic ions profile in tissues. Under drought, although CaO-treated and untreated plants showed similar physiological responses, the former closed the stomata earlier. During water stress relief, the CaO-treated poplars exhibited a faster stomatal opening and a higher capacity to restore xylem hydraulic conductivity compared to not-treated plants, probably due to the higher osmolyte accumulation during drought. The content of some inorganic ions (e.g, Ca2+ and Cl-) was also higher in the xylem sap collected from stressed CaO-treated plants, thus contributing to increase the osmotic gradient necessary for the recovery. Taken together, our results suggest that CaO treatment promotes a faster and more efficient plant recovery after drought due to a modulation of ions homeostasis.

Responses to Drought Stress in Poplar: What Do We Know and What Can We Learn?

Poplar (Populus spp.) is a high-value crop for wood and biomass production and a model organism for tree physiology and genomics. The early release, in 2006, of the complete genome sequence of P. trichocarpa was followed by a wealth of studies that significantly enriched our knowledge of complex pathways inherent to woody plants, such as lignin biosynthesis and secondary cell wall deposition. Recently, in the attempt to cope with the challenges posed by ongoing climate change, fundamental studies and breeding programs with poplar have gradually shifted their focus to address the responses to abiotic stresses, particularly drought. Taking advantage from a set of modern genomic and phenotyping tools, these studies are now shedding light on important processes, including embolism formation (the entry and expansion of air bubbles in the xylem) and repair, the impact of drought stress on biomass yield and quality, and the long-term effects of drought events. In this review, we summarize the status of the research on the molecular bases of the responses to drought in poplar. We highlight how this knowledge can be exploited to select more tolerant genotypes and how it can be translated to other tree species to improve our understanding of forest dynamics under rapidly changing environmental conditions.

Transcriptome response to embolism formation in stems of Populus trichocarpa provides insight into signaling and the biology of refilling.

The mechanism of embolism repair in transpiring plants is still not understood, despite significant scientific effort. The refilling process is crucial to maintaining stem transport capacity and ensuring survival for plants experiencing dynamic changes in water stress. Refilling air-filled xylem vessels requires an energy and water source that can only be provided by adjacent living parenchyma cells. Here, we report an analysis of the transcriptome response of xylem parenchyma cells after embolism formation in Populus trichocarpa trees. Genes encoding aquaporins, ion transporters, and carbohydrate metabolic pathways were up-regulated, and there was a significant reduction in the expression of genes responding to oxidative stress. Thus, a novel view of the plant response to embolism emerges that suggests a role for oxygen in embolized vessels as a signal triggering xylem refilling and for the activity of cation transport as having a significant role in the generation of the energy gradient necessary to heal embolized vessels. These findings redefine current hypotheses surrounding the refilling phenomenon and provide insight into the complexity of the biological response to the seemingly simple physical event of xylem embolism formation.

The dynamics of embolism refilling in abscisic acid (ABA)-deficient tomato plants.

Plants are in danger of embolism formation in xylem vessels when the balance between water transport capacity and transpirational demand is compromised. To maintain this delicate balance, plants must regulate the rate of transpiration and, if necessary, restore water transport in embolized vessels. Abscisic acid (ABA) is the dominant long-distance signal responsible for plant response to stress, and it is possible that it plays a role in the embolism/refilling cycle. To test this idea, a temporal analysis of embolism and refilling dynamics, transpiration rate and starch content was performed on ABA-deficient mutant tomato plants. ABA-deficient mutants were more vulnerable to embolism formation than wild-type plants, and application of exogenous ABA had no effect on vulnerability. However, mutant plants treated with exogenous ABA had lower stomatal conductance and reduced starch content in the xylem parenchyma cells. The lower starch content could have an indirect effect on the plant's refilling activity. The results confirm that plants with high starch content (moderately stressed mutant plants) were more likely to recover from loss of water transport capacity than plants with low starch content (mutant plants with application of exogenous ABA) or plants experiencing severe water stress. This study demonstrates that ABA most likely does not play any direct role in embolism refilling, but through the modulation of carbohydrate content, it could influence the plant's capacity for refilling.

The C4 protein of tomato yellow leaf curl Sardinia virus primes drought tolerance in tomato through morphological adjustments.

Viruses can interfere with the ability of plants to overcome abiotic stresses, indicating the existence of common molecular networks that regulate stress responses. A begomovirus causing the tomato yellow leaf curl disease was recently shown to enhance heat tolerance in tomato and drought tolerance in tomato and Nicotiana benthamiana and experimental evidence suggested that the virus-encoded protein C4 is the main trigger of drought responses. However, the physiological and molecular events underlying C4-induced drought tolerance need further elucidation. In this study, transgenic tomato plants expressing the tomato yellow leaf curl Sardinia virus (TYLCSV) C4 protein were subjected to severe drought stress, followed by recovery. Morphometric parameters, water potential, gas exchanges, and hormone contents in leaves were measured, in combination with molecular analysis of candidate genes involved in stress response and hormone metabolism. Collected data proved that the expression of TYLCSV C4 positively affected the ability of transgenic plants to tolerate water stress, by delaying the onset of stress-related features, improving the plant water use efficiency and facilitating a rapid post-rehydration recovery. In addition, we demonstrated that specific anatomical and hydraulic traits, rather than biochemical signals, are the keynote of the C4-associated stress resilience. Our results provide novel insights into the biology underpinning drought tolerance in TYLCSV C4-expressing tomato plants, paving the way for further deepening the mechanism through which such proteins tune the plant-virus interaction.

Sensing embolism in xylem vessels: the role of sucrose as a trigger for refilling.

Refilling of embolized vessels requires a source of water and the release of energy stored in xylem parenchyma cells. Past evidence suggests that embolism presence can trigger a biological response that is switched off upon successful vessel refilling. As embolism formation is a purely physical process and most biological triggers rely on chemical sensors, we hypothesized that accumulation of osmotic compounds in walls of embolized vessels are involved in the embolism sensing mechanism. Analysis of Populus trichocarpa's response to infiltration of sucrose, monosaccharides, polyethylene glycol and potassium chloride into the xylem revealed that only presence of sucrose resulted in a simultaneous physiological and molecular response similar to that induced by embolism. This response included reduction of the starch pool in xylem parenchyma cells and significant correlation of gene expression from aquaporins, amylases and sugar transporter families. The work provides evidence of the ability of plants to sense embolism and suggests that sucrose concentration is the stimulus that allows plants to trigger a biological response to embolism.

Grapevine TPS (trehalose-6-phosphate synthase) family genes are differentially regulated during development, upon sugar treatment and drought stress.

Trehalose-6-phosphate synthase (TPS) performs the first step in the biosynthetic pathway of trehalose-6-phosphate and trehalose. These two molecules play key roles in the control of carbon allocation and of stress responses in plants. We investigated the organization of the TPS gene family and its developmental and environmental expression regulation in grapevine, a major horticultural crop. We identified three novel genes in the family, and assessed the expression of the 11 family members in tissues and developmental phases. Two potentially biosynthetic TPS isoforms belonging to Class I were preferentially expressed in leaf (VvTPS1_A) and in fruit (VvTPS1_B) respectively. Sucrose treatment induced expression of VvTPS1_B, but not of VvTPS1_A, and a progressive decrease of sucrose concentration. Expression of a few Class II genes was affected by sucrose treatment. Application of osmotic stress by withdrawing irrigation also induced a decrease in sucrose and an increase of glucose content, and down-regulation of the VvTPS1_A gene. We discuss the possible role of these potential biosynthetic TPS genes. Subgroups of TPS genes, including both Class I and ClassII isoforms, followed a co-expression pattern in different conditions, suggesting that Class II TPS proteins may directly or indirectly interact with TPS biosynthetic genes. Our results pave the way for clarification of the role of TPS isoforms in grapevine responses to environmental stress.