The mycorrhizal root-shoot axis elicits Coffea arabica growth under low phosphate conditions.

Coffee is one of the most traded commodities world-wide. As with 70% of land plants, coffee is associated with arbuscular mycorrhizal (AM) fungi, but the molecular bases of this interaction are unknown. We studied the mycorrhizal phenotype of two commercially important Coffea arabica cultivars ('Typica National' and 'Catimor Amarillo'), upon Funnelliformis mosseae colonisation grown under phosphorus limitation, using an integrated functional approach based on multi-omics, physiology and biochemistry. The two cultivars revealed a strong biomass increase upon mycorrhization, even at low level of fungal colonisation, improving photosynthetic efficiency and plant nutrition. The more important iconic markers of AM symbiosis were activated: We detected two gene copies of AM-inducible phosphate (Pt4), ammonium (AM2) and nitrate (NPF4.5) transporters, which were identified as belonging to the C. arabica parental species (C. canephora and C. eugenioides) with both copies being upregulated. Transcriptomics data were confirmed by ions and metabolomics analyses, which highlighted an increased amount of glucose, fructose and flavonoid glycosides. In conclusion, both coffee cultivars revealed a high responsiveness to the AM fungus along their root-shoot axis, showing a clear-cut re-organisation of the major metabolic pathways, which involve nutrient acquisition, carbon fixation, and primary and secondary metabolism.

Technical advances for measurement of gas exchange at the whole plant level: Design solutions and prototype tests to carry out shoot and rootzone analyses in plants of different sizes.

To measure gas exchange at the whole plant (WP) level, design solutions were provided and prototypes of gas-exchange systems (GESs) were tested to carry out shoot and rootzone analyses in plants of different sizes. A WP-GES for small herbaceous plants was tested on the ability to maximize the net assimilation rate of CO2 in lettuce plants grown either under blue-red light or upon full spectrum artificial light. A WP-GES for large woody plants was tested during an experiment describing the drought stress inhibition of grapevine transpiration and photosynthesis. Technical advances pointed to optimize: i) the choice of cuvette material and its technical configuration to allow hermetic isolation of the interface shoot-rootzone, to avoid contamination between the two compartments, and to allow climate control of both shoot and rootzone cuvettes, ii) accurate measurements of the mass air-flow entering both cuvettes, and iii) an adequate homogenization of the cuvette air volume for stable and accurate detection of CO2 and H2O concentration in cuvettes before and after CO2 and H2O contamination of the air volumes exerted by plant organs.

Photosynthetic recovery in drought-rehydrated grapevines is associated with high demand from the sinks, maximizing the fruit-oriented performance.

To understand how grapevine sinks compete with each other during water stress and subsequent rehydration, carbon (C) allocation patterns in drought-rehydrated vines (REC) at the beginning of fruit ripening were compared with control vines maintained under drought (WS) or fully irrigated (WW). In the 30 days following rehydration, the quantity and distribution of newly fixed C between leaves, roots and fruits was evaluated through 13 CO2 pulse-labeling and stable isotope ratio mass spectrometry. REC plants diverted the same percentage of fixed C towards the berries as the WS plants, although the percentage was higher than that of WW plants. Net photosynthesis (measured simultaneously with root respiration in a multichamber system for analysis of gas exchange above- and below-ground) was approximately two-fold greater in REC compared to WS treatment, and comparable or even higher than in WW plants. Maximizing C assimilation and delivery in REC plants led to a significantly higher amount of newly fixed C compared to both control treatments, already 2 days after rehydration in root, and 2 days later in the berries, in line with the expression of genes responsible for sugar metabolism. In REC plants, the increase in C assimilation was able to support the requests of the sinks during fruit ripening, without affecting the reserves, as was the case in WS. These mechanisms clarify what is experienced in fruit crops, when occasional rain or irrigation events are more effective in determining sugar delivery towards fruits, rather than constant and satisfactory water availabilities.

Spray-induced gene silencing targeting a glutathione S-transferase gene improves resilience to drought in grapevine.

Along with the ongoing climate change, drought events are predicted to become more severe. In this context, the spray-induced gene silencing (SIGS) technique could represent a useful strategy to improve crop stress resilience. A previous study demonstrated that the Arabidopsis mutants for a glutathione S-transferase (GST) gene had increased abscisic acid (ABA) levels and a more activated antioxidant system, both features that improved drought resilience. Here, we used SIGS to target a putative grape GST gene (VvGST40). Then, ecophysiological, biochemical and molecular responses of 'Chardonnay' cuttings were analysed during a drought and recovery time-course. Gas exchange, ABA and t-resveratrol concentration as well as expression of stress-related genes were monitored in not treated controls, dsRNA-VvGST40- and dsRNA-GFP- (negative control of the technique) treated plants, either submitted or not to drought. VvGST40-treated plants revealed increased resilience to severe drought as attested by the ecophysiological data. Analysis of target metabolites and antioxidant- and ABA-related transcripts confirmed that VvGST40-treated plants were in a priming status compared with controls. SIGS targeting an endogenous gene was successfully applied in grapevine, confirming the ability of this technique to be exploited not only for plant protection issues but also for functional genomic studies.

The Potential of the Synthetic Strigolactone Analogue GR24 for the Maintenance of Photosynthesis and Yield in Winter Wheat under Drought: Investigations on the Mechanisms of Action and Delivery Modes.

Strigolactones (SLs) have been implicated in many plant biological and physiological processes, including the responses to abiotic stresses such as drought, in concert with other phytohormones. While it is now clear that exogenous SLs may help plants to survive in harsh environmental condition, the best, most effective protocols for treatment have not been defined yet, and the mechanisms of action are far from being fully understood. In the set of experiments reported here, we contrasted two application methods for treatment with a synthetic analog of SL, GR24. A number of morphometric, physiological and biochemical parameters were measured following foliar application of GR24 or application in the residual irrigation water in winter wheat plants under irrigated and drought stress conditions. Depending on the concentration and the method of GR24 application, differentiated photosynthesis and transpiration rate, stomatal conductance, leaf water potential, antioxidant enzyme activities and yield in drought conditions were observed. We present evidence that different methods of GR24 application led to increased photosynthesis and yield under stress by a combination of drought tolerance and escape factors, which should be considered for future research exploring the potential of this new family of bioactive molecules for practical applications.

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.

A novel strigolactone-miR156 module controls stomatal behaviour during drought recovery.

miR156 is a conserved microRNA whose role and induction mechanisms under stress are poorly known. Strigolactones are phytohormones needed in shoots for drought acclimation. They promote stomatal closure ABA-dependently and independently; however, downstream effectors for the former have not been identified. Linkage between miR156 and strigolactones under stress has not been reported. We compared ABA accumulation and sensitivity as well as performances of wt and miR156-overexpressing (miR156-oe) tomato plants during drought. We also quantified miR156 levels in wt, strigolactone-depleted and strigolactone-treated plants, exposed to drought stress. Under irrigated conditions, miR156 overexpression and strigolactone treatment led to lower stomatal conductance and higher ABA sensitivity. Exogenous strigolactones were sufficient for miR156 accumulation in leaves, while endogenous strigolactones were required for miR156 induction by drought. The "after-effect" of drought, by which stomata do not completely re-open after rewatering, was enhanced by both strigolactones and miR156. The transcript profiles of several miR156 targets were altered in strigolactone-depleted plants. Our results show that strigolactones act as a molecular link between drought and miR156 in tomato, and identify miR156 as a mediator of ABA-dependent effect of strigolactones on the after-effect of drought on stomata. Thus, we provide insights into both strigolactone and miR156 action on stomata.

The Accumulation of miRNAs Differentially Modulated by Drought Stress Is Affected by Grafting in Grapevine.

Grapevine (Vitis vinifera) is routinely grafted, and rootstocks inducing drought tolerance represent a source for adapting vineyards to climate change in temperate areas. Our goal was to investigate drought stress effects on microRNA (miRNA) abundance in a drought-resistant grapevine rootstock, M4 (Vitis vinifera × Vitis berlandieri), compared with a commercial cultivar, Cabernet Sauvignon, using their autografts and reciprocal grafts. RNA extracted from roots and leaves of droughted and irrigated plants of different graft combinations was used to prepare cDNA libraries for small RNA sequencing and to analyze miRNAs by quantitative real-time polymerase chain reaction (RT-qPCR). Measurements of leaf water potential, leaf gas exchange, and root hydraulic conductance attested that, under irrigation, M4 reduced water loss in comparison with cultivar Cabernet Sauvignon mostly through nonhydraulic, root-specific mechanisms. Under drought, stomatal conductance decreased at similar levels in the two genotypes. Small RNA sequencing allowed the identification of 70 conserved miRNAs and the prediction of 28 novel miRNAs. Different accumulation trends of miRNAs, observed upon drought and in different genotypes and organs, were confirmed by RT-qPCR Corresponding target transcripts, predicted in silico and validated by RT-qPCR, often showed opposite expression profiles than the related miRNAs. Drought effects on miRNA abundance differed between the two genotypes. Furthermore, the concentration of drought-responsive miRNAs in each genotype was affected by reciprocal grafting, suggesting either the movement of signals inducing miRNA expression in the graft partner or, possibly, miRNA transport between scion and rootstock. These results open new perspectives in the selection of rootstocks for improving grapevine adaptation to drought.

Factors influencing stomatal conductance in response to water availability in grapevine: a meta-analysis.

The main factors regulating grapevine response to decreasing water availability were assessed under statistical support using published data related to leaf water relations in an extensive range of scion and rootstock genotypes. Matching leaf water potential (Ψleaf ) and stomatal conductance (gs ) data were collected from peer-reviewed literature with associated information. The resulting database contained 718 data points from 26 different Vitis vinifera varieties investigated as scions, 15 non-V. vinifera rootstock genotypes and 11 own-rooted V. vinifera varieties. Linearised data were analysed using the univariate general linear model (GLM) with factorial design including biological (scion and rootstock genotypes), methodological and environmental (soil) fixed factors. The first GLM performed on the whole database explained 82.4% of the variability in data distribution having the rootstock genotype the greatest contribution to variability (19.1%) followed by the scion genotype (16.2%). A classification of scions and rootstocks according to their mean predicted gs in response to moderate water stress was generated. This model also revealed that gs data obtained using a porometer were in average 2.1 times higher than using an infra-red gas analyser. The effect of soil water-holding properties was evaluated in a second analysis on a restricted database and showed a scion-dependant effect, which was dominant over rootstock effect, in predicting gs values. Overall the results suggest that a continuum exists in the range of stomatal sensitivities to water stress in V. vinifera, rather than an isohydric-anisohydric dichotomy, that is further enriched by the diversity of scion-rootstock combinations and their interaction with different soils.

Low levels of strigolactones in roots as a component of the systemic signal of drought stress in tomato.

Strigolactones (SL) contribute to drought acclimatization in shoots, because SL-depleted plants are hypersensitive to drought due to stomatal hyposensitivity to abscisic acid (ABA). However, under drought, SL biosynthesis is repressed in roots, suggesting organ specificity in their metabolism and role. Because SL can be transported acropetally, such a drop may also affect shoots, as a systemic indication of stress. We investigated this hypothesis by analysing molecularly and physiologically wild-type (WT) tomato (Solanum lycopersicum) scions grafted onto SL-depleted rootstocks, compared with self-grafted WT and SL-depleted genotypes, during a drought time-course. Shoots receiving few SL from the roots behaved as if under mild stress even if irrigated. Their stomata were hypersensitive to ABA (likely via a localized enhancement of SL synthesis in shoots). Exogenous SL also enhanced stomata sensitivity to ABA. As the partial shift of SL synthesis from roots to shoots mimics what happens under drought, a reduction of root-produced SL might represent a systemic signal unlinked from shootward ABA translocation, and sufficient to prime the plant for better stress avoidance.

VvPIP2;4N aquaporin involvement in controlling leaf hydraulic capacitance and resistance in grapevine.

Hydraulic capacitance (C) in a plant tissue buffers the xylem tension, storing and releasing water and has been highlighted in recent years as an important factor that affects water relations such as drought tolerance and embolism formation. Aquaporins (AQPs) are well known to control leaf hydraulic resistance (Rh) but their role in the control of C is unknown. Here, we assess Rh and C on detached grapevines wild-type (WT) (cv. Brachetto) leaves and over-expressing the aquaporin gene VvPIP2;4N (OE). For this purpose, we developed a new method inspired from the pressure-volume curve technique and the rehydration-kinetic-method, which allowed us to monitor the dynamics of dehydration and rehydration in the same leaf. The recovery after dehydration was measured in dark, light non-transpirative conditions, light-transpirative conditions and light-transpirative condition adding abscisic acid. Pressurizing to dehydrate leaves in the OE line, the recorded Rh and C were respectively lower and higher than those in the WT. The same results were obtained in the dark recovery by rehydration treatment. In the presence of light, either when leaves transpired or not (by depressing vapor pressure deficit), the described effects disappeared. The change in Rh and C did not affect the kinetics of desiccation of detached leaves in dark in air, in OE plants compared to WT ones. Our study highlighted that both Rh and C were influenced by the constitutive over-expression of VvPIP2;4N. The effect of AQPs on C is reported here for the first time and may involve a modulation of cell reflection coefficient.

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.

Novel functional microRNAs from virus-free and infected Vitis vinifera plants under water stress.

MicroRNAs (miRNAs) are small non-coding RNAs that regulate the post-transcriptional control of several pathway intermediates, thus playing pivotal roles in plant growth, development and response to biotic and abiotic stresses. In recent years, the grapevine genome release, small(s)-RNAseq and degradome-RNAseq together has allowed the discovery and characterisation of many miRNA species, thus rendering the discovery of additional miRNAs difficult and uncertain. Taking advantage of the miRNA responsiveness to stresses and the availability of virus-free Vitis vinifera plants and those infected only by a latent virus, we have analysed grapevines subjected to drought in greenhouse conditions. The sRNA-seq and other sequence-specific molecular analyses have allowed us to characterise conserved miRNA expression profiles in association with specific eco-physiological parameters. In addition, we here report 12 novel grapevine-specific miRNA candidates and describe their expression profile. We show that latent viral infection can influence the miRNA profiles of V. vinifera in response to drought. Moreover, study of eco-physiological parameters showed that photosynthetic rate, stomatal conductance and hydraulic resistance to water transport were significantly influenced by drought and viral infection. Although no unequivocal cause-effect explanation could be attributed to each miRNA target, their contribution to the drought response is discussed.

Osmotic stress represses strigolactone biosynthesis in Lotus japonicus roots: exploring the interaction between strigolactones and ABA under abiotic stress.

MAIN CONCLUSION: Strigolactone changes and cross talk with ABA unveil a picture of root-specific hormonal dynamics under stress. Strigolactones (SLs) are carotenoid-derived hormones influencing diverse aspects of development and communication with (micro)organisms, and proposed as mediators of environmental stimuli in resource allocation processes; to contribute to adaptive adjustments, therefore, their pathway must be responsive to environmental cues. To investigate the relationship between SLs and abiotic stress in Lotus japonicus, we compared wild-type and SL-depleted plants, and studied SL metabolism in roots stressed osmotically and/or phosphate starved. SL-depleted plants showed increased stomatal conductance, both under normal and stress conditions, and impaired resistance to drought associated with slower stomatal closure in response to abscisic acid (ABA). This confirms that SLs contribute to drought resistance in species other than Arabidopsis. However, we also observed that osmotic stress rapidly and strongly decreased SL concentration in tissues and exudates of wild-type Lotus roots, by acting on the transcription of biosynthetic and transporter-encoding genes and independently of phosphate abundance. Pre-treatment with exogenous SLs inhibited the osmotic stress-induced ABA increase in wild-type roots and down-regulated the transcription of the ABA biosynthetic gene LjNCED2. We propose that a transcriptionally regulated, early SL decrease under osmotic stress is needed (but not sufficient) to allow the physiological increase of ABA in roots. This work shows that SL metabolism and effects on ABA are seemingly opposite in roots and shoots under stress.

Gene expression in vessel-associated cells upon xylem embolism repair in Vitis vinifera L. petioles.

In this work, the involvement of vessel-associated cells in embolism recovery was investigated by studying leaf petiole hydraulics and expression profiles of aquaporins and genes related to sugar metabolism. Two different stress treatments were imposed onto grapevines to induce xylem embolism: one involved a pressure collar applied to the stems, while the other consisted of water deprivation (drought). Embolism formation and repair were monitored during stress application and release (recovery). At the same time, stomatal conductance (g(s)), leaf water potential (Ψ(leaf)) and leaf abscisic acid (ABA) concentration were measured. For each treatment, gene transcript levels were assessed on vessel-associated cells (isolated from leaf petioles by laser microdissection technique) and whole petioles. Both treatments induced severe xylem embolism formation and drops in g s and Ψ (leaf) at a lesser degree and with faster recovery in the case of application of the pressure collar. Leaf ABA concentration only increased upon drought and subsequent recovery. Transcripts linked to sugar mobilisation (encoding a ß-amylase and a glucose-6-P transporter) were over-expressed upon stress or recovery, both in vessel-associated cells and whole petioles. However, two aquaporin genes (VvPIP2;1 and VvPIP2;4N) were activated upon stress or recovery only in vessel-associated cells, suggesting a specific effect on embolism refilling. Furthermore, the latter gene was only activated upon drought and subsequent recovery, suggesting that either severe water stress or ABA is required for its regulation.

Soil water-holding capacity mediates hydraulic and hormonal signals of near-isohydric and near-anisohydric Vitis cultivars in potted grapevines.

Grapevine (Vitis vinifera L.) expresses different responses to water stress, depending not only on genotype, but also on the influence of vineyard growing conditions or seasonality. Our aim was to analyse the effects on drought response of two grapevine cultivars growing on two soils, one water draining (WD) containing sand 80% volume and the other water retaining (WR), with no sand. Under these two different water-holding capacities Syrah, displaying a near-anisohydric response to water stress, and Cabernet Sauvignon (on the contrary, near-isohydric) were submitted to water stress in a pot trial. Xylem embolism contributed to plant adaptation to soil water deprivation: in both cultivars during late phases of water stress, however, in Syrah, already at moderate early stress levels. By contrast, Syrah showed a less effective stomatal control of drought than Cabernet Sauvignon. The abscisic acid (ABA) influenced tightly the stomatal conductance of Cabernet Sauvignon on both pot soils. In the near-anisohydric variety Syrah an ABA-related stomatal closure was induced in WR soil to maintain high levels of water potential, showing that a soil-related hormonal root-to-shoot signal causing stomatal closure superimposes on the putatively variety-induced anisohydric response to water stress.

Carotenoid cleavage dioxygenase 7 modulates plant growth, reproduction, senescence, and determinate nodulation in the model legume Lotus japonicus.

Strigolactones (SLs) are newly identified hormones that regulate multiple aspects of plant development, infection by parasitic weeds, and mutualistic symbiosis in the roots. In this study, the role of SLs was studied for the first time in the model plant Lotus japonicus using transgenic lines silenced for carotenoid cleavage dioxygenase 7 (LjCCD7), the orthologue of Arabidopsis More Axillary Growth 3. Transgenic LjCCD7-silenced plants displayed reduced height due to shorter internodes, and more branched shoots and roots than the controls, and an increase in total plant biomass, while their root:shoot ratio remained unchanged. Moreover, these lines had longer primary roots, delayed senescence, and reduced flower/pod numbers from the third round of flower and pod setting onwards. Only a mild reduction in determinate nodule numbers and hardly any impact on the colonization by arbuscular mycorrhizal fungi were observed. The results show that the impairment of CCD7 activity in L. japonicus leads to a phenotype linked to SL functions, but with specific features possibly due to the peculiar developmental pattern of this plant species. It is believed that the data also link determinate nodulation, plant reproduction, and senescence to CCD7 function for the first time.

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 grapevine root-specific aquaporin VvPIP2;4N controls root hydraulic conductance and leaf gas exchange under well-watered conditions but not under water stress.

We functionally characterized the grape (Vitis vinifera) VvPIP2;4N (for Plasma membrane Intrinsic Protein) aquaporin gene. Expression of VvPIP2;4N in Xenopus laevis oocytes increased their swelling rate 54-fold. Northern blot and quantitative reverse transcription-polymerase chain reaction analyses showed that VvPIP2;4N is the most expressed PIP2 gene in root. In situ hybridization confirmed root localization in the cortical parenchyma and close to the endodermis. We then constitutively overexpressed VvPIP2;4N in grape 'Brachetto', and in the resulting transgenic plants we analyzed (1) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, (2) whole-plant, root, and leaf ecophysiological parameters, and (3) leaf abscisic acid content. Expression of transgenic VvPIP2;4N inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. Under well-watered conditions, transgenic plants showed higher stomatal conductance, gas exchange, and shoot growth. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. The leaf component of total plant hydraulic resistance was low and unaffected by overexpression of VvPIP2;4N. Upon water stress, the overexpression of VvPIP2;4N induced a surge in leaf abscisic acid content and a decrease in stomatal conductance and leaf gas exchange. Our results show that aquaporin-mediated modifications of root hydraulics play a substantial role in the regulation of water flow in well-watered grapevine plants, while they have a minor role upon drought, probably because other signals, such as abscisic acid, take over the control of water flow.

Recovery from water stress affects grape leaf petiole transcriptome.

Fast and efficient recovery from water stress is a key determinant of plant adaptation to changing meteorological conditions modulating transpiration, i.e. air temperature and humidity. We analysed transcriptomic responses during rehydration after water stress in grapevine leaf petioles, where embolism formation and repair commonly take place, and where metabolic changes related to embolism recovery are expected to be particularly important. We compared gene expression of recovering plants with irrigated controls, upon high and low transpiration conditions, using cDNA microarrays. In parallel, we assessed the daily dynamics of water relations, embolism formation and repair, and leaf abscisic acid concentration. In recovering plants, the most affected gene categories were secondary metabolism, including genes linked to flavonoid biosynthesis; sugar metabolism and transport, and several aquaporin genes. The physiological dynamics of recovery were lower and the number of differentially expressed probes was much lower upon low transpiration than found in actively transpiring grapevines, suggesting the existence of a more intense metabolic reorganization upon high transpiration conditions and of a signal eliciting these responses. In plants recovering under high transpiration, abscisic acid concentrations significantly increased, and, in parallel, transcripts linked to abscisic acid metabolism and signalling (ABA-8'-hydroxylase, serine-threonine kinases, RD22 proteins) were upregulated; a trend that was not observed upon low transpiration. Our results show that recovery from water stress elicits complex transcriptomic responses in grapevine. The increase observed in abscisic acid cellular levels could represent a signal triggering the activation of responses to rehydration after stress.