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Perennial plants maintain xylem hydraulic functionality to sustain photosynthetic capacity upon periods of severe drought. Under water stress, plants adjust their xylem pH and gene expression priming stem by accumulation of sugars and ions in the apoplast for xylem functional recovery. Here, we studied if application of ortho-vanadate solution, aimed to inhibit the metabolic activity of wood parenchyma cells and block proton pumps, can affect the xylem sap properties and hinder or delay the recovery process after soil rehydration. Poplar trees were used for in vivo analysis of embolism using X-ray microCT. Despite restoration of water potential, the ortho-vanadate treated rehydrated poplars revealed a significant impediment of in removal of drought-induced embolisms, while control plants showed almost full recovery from embolism. These findings indicate that embolism removal is an energy dependent process that requires accumulation of sugars in the apoplast, while visual observations indicate that process is spatially coordinated, with embolism formation accruing from inside out and recovery from outside in; thus underlining the importance of xylem proximity to phloem (sugar source).

"To study the allocation kinetics of carbon in the different sinks competing in grapevines, a plant growth chamber for stable isotope labeling has been set in an environmental control system (light intensity, temperature and relative humidity of the air, pot water content) in order to simulate different climate scenarios. Basing on pulse-chasing isotopic strategy, the isotope acted as a tracer of carbon phloem flows. Furthermore, an open-air plant/soil growth system consisting in twelve independent plant/pot balloons with computing-adjustable air flows allowing continuous gas exchange detection between plants / soil and atmosphere has been set. The two platforms have been coordinated and used before, during and after induction of drought stress. Photosynthetic assimilation, stomatal regulation and respiration have been checked in the various phases to size 13CO2 enrichment flows in the chamber for stable isotope labeling. Maximum assimilation ranged from 9 to 12 ?mol m-2 s-1, reduced by stomatal control 3 o 4 times at the end of the drought period. Saturation of net photosynthesis occurred around 1000 ?mol m-2 s-1 PPFD: thereafter photoinhibition impairment started in old leaves, whereas young leaves experienced half of maximum assimilation without drastic photoinhibition. An in-vineyard parallel trial of measurements was used to calibrate and reference pot measurements. "

Our study highlights that leaf hydraulic capacitance (Ch), as well as leaf hydraulic resistance (Rh) are influenced by the constitutive over-expression of VvPIP2;4N aquaporin. Ch 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 are well known to control leaf hydraulic resistance (Rh) but their role in the control of Ch is unknown. Here, we assess Rh and Ch on detached grapevines leaves (cv. Brachetto) wild type (WT) 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 the dark, in light non-transpirative conditions, light-transpirative conditions and transpirative condition adding abscisic acid (ABA). Pressurizing leaves in the OE line, the recorded Rh and Ch were respectively lower and higher than those in the WT. The same results were obtained in the dark recovery treatment. In the presence of light, either when leaves transpired or not (by depressing vapour pressure deficit), the described effects disappeared. The change in these hydraulic traits did not affect the kinetics of desiccation of detached leaves in the dark in air, in OE plants compared to WT. The effect of aquaporins on Ch is reported here for the first time and may involve a modulation of cell reflexion coefficient.

In a pot experiment the following grapevines were grown in a calcareous soil: Pinot blanc own-rooted and self-grafted, grafted on SO 4 and on 3309 C; SO 4 own-rooted, self-grafted and grafted on Pinot blanc; 3309 C own-rooted, self-grafted and grafted on Pinot blanc. The occurrence of chlorosis was strongly affected by graft combinations. In self-grafted 3309 C plants the specific conductivity was significantly reduced as compared to own-rooted ones. Own-rooted SO 4 plants showed the highest specific conductivity, associated with the highest rate of shoot growth and leaf chlorophyll content., VITIS - Journal of Grapevine Research, Vol. 39 No. 3 (2000): Vitis

We constitutively overexpressed VvPIP2;4N in Vitis vinifera L. 'Brachetto' and in the resulting transgenic plants we analysed i) the expression of endogenous and transgenic VvPIP2;4N and of four other aquaporins, ii) whole-plant, root, and leaf ecophysiological parameters, and iii) 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 ABA, take over control of water flow. On detached leaves, hydraulic capacitance (Ch) and resistance (Rh) were assessed following recovery dynamics after leaf dehydration. The dehydration was imposed by submitting the leaves at 0.5 to 1 MPa overpressure through a Scholander bomb. The recovery after dehydration was checked in dark, in light non-transpirative condition, and in light-transpirative condition. In leaves processed in dark conditions, Rh decreased and Ch increased compared to wild type plants, suggesting that in transgenic leaves, trans-cellular pathways activated during dehydration superimposed on apoplastic ones, saving water inside cells, as in/out cell-to-apoplasm water movements were aquaporin-controlled. Upon light, either when leaves transpired or not (either by depressing vapor pressure deficit or by feeding leaves with abscisic acid, causing stomatal closure) the described effects disappeared. We conclude that light activates leaf aquaporins masking the transgene effect on controlling leaf Ch and Rh, and confirming that the in-vivo leaf hydraulics is not affected by transgenic aquaporin VvPIP2;4N overexpression.

Grapevine rupestris stem pitting-associated virus (GRSPaV) is a virus that infects grapevines and establishes a compatible interaction in Vitis vinifera without the development of macroscopic phenotypic alterations. Some unexpected responses are induced in V. vinifera by GRSPaV, including overlapping responses to drought and salinity stress. In the frame of a CNR project (Progetto Premiale AQUA), we hypothesised an interaction between GRSPaV and drought and we subjected GRSPaV-free and infected plants to water stress under controlled conditions. By investigating ecophysiological parameters, we show that GRSPaV activates in V. vinifera a physiological state that induces resilience to drought. Indeed, infected plants under controlled water stress conditions show (i) a high rate of photosynthesis and stomatal conductance; (ii) low hydraulic resistance to water transport; and (iii) more growth than GRSPaV-free plants. The molecular basis of these virus-grapevine-drought interactions is still poorly understood; however, it is reasonable to hypothesise an involvement of RNA silencing, which is a natural defence against invading viruses in plants. Micro(mi)RNAs have fundamental roles in plant development and adaptation to stresses through post transcriptional control of several physiological pathway intermediates. The analysis of four libraries of small RNAs (i.e., from GRSPaV-free and infected plants, both in well watered and stress conditions) allowed us to highlight some known miRNAs and novel miRNA candidates that could be correlated with the physiological modifications in plants upon water stress treatment. Targets of known (i.e. miR396, miR164, miR156, miR3633) and novel miRNAs (miRC121, miRC129, miRs409712_2) annotated as players in drought stress adaptations were indeed validated. The relevance of miRNAs in the interaction between GRSPaV infection and water stress is not defined only by their tissue abundance but mainly by their activity on targeted mRNAs. In infected plants, the high photosynthesis reported above was accompanied by higher stomatal conductance, which was also linked to modifications in leaf morphology (high stomatal density and cell number). In infected grapevines, we observed the regulation of several miRNAs and their targets involved in leaf development (miR156, miR164, miR319, miR394, miR396). The grapevine is a woody plant that has been cultivated and selected together with its viruses for centuries. Our data support the idea of mutual adaptation between GRSPaV and grapevine, resulting in beneficial effects for the host under water stress conditions. Therefore, GRSPaV might represent the first example of a plant virus that is more appropriately defined by the categories of 'conditional mutualism' and 'beneficial virus' proposed by Roossinck (2011).

Grapevine rupestris stem pitting-associated virus (GRSPaV) is one of the most prevalent viruses that infects grapevines and belongs to the genus Foveavirus, in the Betaflexiviridae family. We have shown previously that plants of V. vinifera cv Bosco infected by the GRSPaV-1 sequence variant exerted a very moderate decrease in physiological efficiency and yield. Importantly, this response overlapped with molecular responses to water and salinity stress. These considerations prompted us to investigate possible interactions between GRSPaV and abiotic stress. The molecular basis of these interactions is still poorly understood; however, it is reasonable to hypothesise an involvement of RNA silencing, which is a natural defence against invading viruses in plants. Both miRNAs and siRNAs are involved in several developmental processes in defence responses to biotic and abiotic stresses and in genome stability maintenance, as reviewed in many studies. We analysed the ecophysiological and molecular interactions between GRSPaV infection and drought in grapevine. During the progressive drying of soil, the stomatal conductance (gs) and net photosynthesis (Pn) of both infected and GRSPaV-free sets of plants decreased in response to water stress; however, this response was delayed in infected plants Prolonged drought conditions are known to reduce or block growth. However, in both irrigated and water stress conditions, infected plants showed a greater leaf area, height, and stem growth diameter compared to GRSPaV-free plants. In the small endogenous RNAs libraries produced from leaves of infected and GRSPaV-free plants under WW and SWS, we identified members of almost all known miRNAs and 25 novel miRNA candidates. The analysis of the data revealed that the accumulation of some known miRNAs was clearly associated with GRSPaV infection. In addition, when GRSPaV-infected plants were subjected to water stress, the ratio of miRNA accumulation between infected and GRSPaV-free grapevines changed significantly. Interestingly, this study clearly shows that the sanitary status of the plants may play a determinant role in miRNA regulation during abiotic stress. Our data support the idea of mutual adaptation between GRSPaV and grapevine, resulting in beneficial effects for the host under water stress conditions. Therefore, GRSPaV might represent the first example of a plant virus that is more appropriately defined by the categories of 'conditional mutualism' and 'beneficial virus'.

Hydraulic capacitance (Ch) and resistance (Rh) were assessed on detached leaves sampled from grapevines overexpressing the aquaporin gene VvPIP2;4N by following recovery dynamics after leaf dehydration. The dehydration was imposed by submitting the leaves at 0.5 to 1 MPa overpressure through a Scholander bomb. The recovery after dehydration was checked in dark, in light non-transpirative condition, and in light-transpirative condition. In leaves processed in dark conditions, Rh decreased and Ch increased compared to wild type plants, suggesting that in transgenic leaves, trans-cellular pathways activated during dehydration superimposed on apoplastic ones, saving water inside cells, as in/out cell-to-apoplasm water movements were aquaporin-controlled. Upon light, either when leaves transpired or not (either by depressing vapor pressure deficit or by feeding leaves with abscisic acid, causing stomatal closure) the described effects disappeared. We conclude that light activates leaf aquaporins masking the transgene effect on controlling leaf Ch and Rh, and confirming that the in-vivo leaf hydraulics is not affected by transgenic aquaporin VvPIP2;4N overexpression.

Flavescence dorée (FD) is among the major grapevine diseases causing highmanagement costs; curative methods against FD are unavailable. In FD-infected plants, decrease in photosynthesis is usually recorded, but deregulation in stomatal control of leaf gas exchange during FD infection and recovery is unknown. We measured the seasonal time course of gas exchange rates in two cultivars ('Barbera' and 'Nebbiolo') during the term of 1 year when grapevines experienced a water stress and another with no drought, with difference in gas exchange rates in response to FD infection and recovery as assessed by symptom observation and phytoplasma detection through PCR analysis. Chlorophyll fluorescence was also evaluated at the time of maximum symptom severity in 'Barbera', the cultivar showing the most severe stress response to FD infection, causing the highest damage in vineyards of north-western Italy. In FD-infected plants, net photosynthesis and transpiration gradually decreased during the season, more during the no drought year than during drought. During recovery, healthy (PCR negative) plants infected 2 years before, but not those infected an year before, regained the gas exchange performances to the level as measured before infection. The relationships between stomatal conductance and the residual leaf intercellular CO2 concentration (ci) discriminated healthy versus FD-infected and recovered plants; at the same ci, FD-infected leaves had higher non-photochemical quenching than healthy ones. We conclude that metabolic, not stomatal, leaf gas exchange limitation in FD-infected and recovered grapevines is the basis of plant response to FD disease. In addition,we also suggest that such response is dependent upon water stress, by showing that water stress superimposes on FD infection in terms of stomatal and metabolic non-stomatal limitations to carbon assimilation.

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 take commonly 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, including 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 increases observed in abscisic acid cellular levels could represent a signal triggering the activation of responses to rehydration after stress.

After overexpressing the grape aquaporin gene VvPIP2;4N in Vitis vinifera L. Brachetto we analysed i) the expression of /VvPIP2;4N /and five other aquaporins, ii) whole-plant, root, and leaf ecophysiological parameters, and iii) leaf abscisic acid content. The availability of six different transgenic grapevine lines with different /VvPIP2;4N /transcript levels allowed us to study the correlation between expression of this aquaporin and water transport processes at the whole-plant level. Expression of transgenic /VvPIP2;4N/ inhibited neither the expression of the endogenous gene nor that of other PIP aquaporins in both root and leaf. The expression level of VvPIP2;4N (endogenous + transgene) was inversely correlated to root hydraulic resistance. Partitioning of plant hydraulic resistance at the organ level showed that leaf resistance was not affected, despite the fact that the transgene was expressed in both roots and shoots. 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, but not leaf, hydraulics play a substantial role in the regulation of water flow in irrigated grapevine plants, while they have a minor role upon drought, probably because other signals, such as ABA, take over control of water flow

Downward-growing grapevine shoots have smaller and more frequent vessels and as a consequence a lower hydraulic conductivity. In a first experiment, grapevine shoot growth orientation was manipulated in order to test whether downward shoot orientation negatively affects vessel growth in the apex via a shortage of water and nutrients. The orientation of the central shoot portion in vines was inverted by two consecutive 135° bends, the first one downwards and the second one upwards, resulting in double-bent N-shaped vines; the plant central downward shoot portion was of different length in the experimental treatments, in order to induce increasing reductions of shoot conductivity. These treatments effectively reduced shoot conductivity and water flow, but had no effects on vessel development and frequency in the apex. In a second experiment, auxin concentration was assessed in shoots of upward and downward growing plants. IAA concentration at the apical internodes was higher in downward oriented shoots than in shoots growing upwards. In addition, a higher density and a lower vessel diameter were observed in the lower shoot side of the shoot than in the upper, suggesting an increased accumulation of auxin in the lower shoot side. These results suggest that the downward orientation induces accumulation of auxin in the apex which in turn affects the density and the size of the xylem vessels, causing reduction of hydraulic conductivity.

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.