Nitrogen sources differentially affect respiration, growth, and carbon allocation in Andean and Lowland ecotypes of Chenopodium quinoa Willd.

Chenopodium quinoa Willd. is a native species that originated in the High Andes plateau (Altiplano) and its cultivation spread out to the south of Chile. Because of the different edaphoclimatic characteristics of both regions, soils from Altiplano accumulated higher levels of nitrate (NO3-) than in the south of Chile, where soils favor ammonium (NH4 +) accumulation. To elucidate whether C. quinoa ecotypes differ in several physiological and biochemical parameters related to their capacity to assimilate NO3- and NH4 +, juvenile plants of Socaire (from Altiplano) and Faro (from Lowland/South of Chile) were grown under different sources of N (NO3- or NH4 +). Measurements of photosynthesis and foliar oxygen-isotope fractionation were carried out, together with biochemical analyses, as proxies for the analysis of plant performance or sensitivity to NH4 +. Overall, while NH4 + reduced the growth of Socaire, it induced higher biomass productivity and increased protein synthesis, oxygen consumption, and cytochrome oxidase activity in Faro. We discussed that ATP yield from respiration in Faro could promote protein production from assimilated NH4 + to benefit its growth. The characterization of this differential sensitivity of both quinoa ecotypes for NH4 + contributes to a better understanding of nutritional aspects driving plant primary productivity.

The Lack of Alternative Oxidase 1a Restricts in vivo Respiratory Activity and Stress-Related Metabolism for Leaf Osmoprotection and Redox Balancing Under Sudden Acute Water and Salt Stress in Arabidopsis thaliana.

In plants salt and water stress result in an induction of respiration and accumulation of stress-related metabolites (SRMs) with osmoregulation and osmoprotection functions that benefit photosynthesis. The synthesis of SRMs may depend on an active respiratory metabolism, which can be restricted under stress by the inhibition of the cytochrome oxidase pathway (COP), thus causing an increase in the reduction level of the ubiquinone pool. However, the activity of the alternative oxidase pathway (AOP) is thought to prevent this from occurring while at the same time, dissipates excess of reducing power from the chloroplast and thereby improves photosynthetic performance. The present research is based on the hypothesis that the accumulation of SRMs under osmotic stress will be affected by changes in folial AOP activity. To test this, the oxygen isotope-fractionation technique was used to study the in vivo respiratory activities of COP and AOP in leaves of wild-type Arabidopsis thaliana plants and of aox1a mutants under sudden acute stress conditions induced by mannitol and salt treatments. Levels of leaf primary metabolites and transcripts of respiratory-related proteins were also determined in parallel to photosynthetic analyses. The lack of in vivo AOP response in the aox1a mutants coincided with a lower leaf relative water content and a decreased accumulation of crucial osmoregulators. Additionally, levels of oxidative stress-related metabolites and transcripts encoding alternative respiratory components were increased. Coordinated changes in metabolite levels, respiratory activities and photosynthetic performance highlight the contribution of the AOP in providing flexibility to carbon metabolism for the accumulation of SRMs.

Leaf physiological traits of plants from the Qinghai-Tibet Plateau and other arid sites in China: Identifying susceptible species and well-adapted extremophiles.

Extreme environments, such as deserts and high-elevation ecosystems, are very important from biodiversity and ecological perspectives. However, plant physiology at those sites has been scarcely studied, likely due to logistic difficulties. In the present study, leaf physiological traits in native plants were analyzed from arid zones across an elevational transect in Western China, from Turpan Basin to the Qinghai-Tibet Plateau (QTP) at Delingha. The aim of this study was to use leaf physiological traits to help identifying potentially threatened species and true extremophiles. Physiological measurements in the field, and particularly in situ measurements of gas exchange and chlorophyll fluorescence, have been determined to be useful to determine the current state of plants at a given environment. Using this approach plus a combination of leaf traits, several species performing particularly well at the QTP were identified, e.g. Hedysarum multijugum, as well as at Manas drylands, e.g. Peganum harmala and Setaria viridis. On the other hand, several species showed marked signs of severe stress, in particular a very low photosynthetic rate over its potential maximum, as well as other negative traits, like low water and/or nitrogen-use-efficiency, which should be considered in conservation plans. Interestingly, all C4 species studied except Setaria viridis were among the most stressed species. Despite their higher water use efficiency and drought-tolerance reputation, they presented a much larger photosynthesis depression than most C3 species. This is an intriguing and interesting observation that deserves further studies.

Different Metabolic Roles for Alternative Oxidase in Leaves of Palustrine and Terrestrial Species.

The alternative oxidase pathway (AOP) is associated with excess energy dissipation in leaves of terrestrial plants. To address whether this association is less important in palustrine plants, we compared the role of AOP in balancing energy and carbon metabolism in palustrine and terrestrial environments by identifying metabolic relationships between primary carbon metabolites and AOP in each habitat. We measured oxygen isotope discrimination during respiration, gas exchange, and metabolite profiles in aerial leaves of ten fern and angiosperm species belonging to five families organized as pairs of palustrine and terrestrial species. We performed a partial least square model combined with variable importance for projection to reveal relationships between the electron partitioning to the AOP (τa) and metabolite levels. Terrestrial plants showed higher values of net photosynthesis (AN) and τa, together with stronger metabolic relationships between τa and sugars, important for water conservation. Palustrine plants showed relationships between τa and metabolites related to the shikimate pathway and the GABA shunt, to be important for heterophylly. Excess energy dissipation via AOX is less crucial in palustrine environments than on land. The basis of this difference resides in the contrasting photosynthetic performance observed in each environment, thus reinforcing the importance of AOP for photosynthesis.

Cytochrome c Deficiency Differentially Affects the In Vivo Mitochondrial Electron Partitioning and Primary Metabolism Depending on the Photoperiod.

Plant respiration provides metabolic flexibility under changing environmental conditions by modulating the activity of the nonphosphorylating alternative pathways from the mitochondrial electron transport chain, which bypass the main energy-producing components of the cytochrome oxidase pathway (COP). While adjustments in leaf primary metabolism induced by changes in day length are well studied, possible differences in the in vivo contribution of the COP and the alternative oxidase pathway (AOP) between different photoperiods remain unknown. In our study, in vivo electron partitioning between AOP and COP and expression analysis of respiratory components, photosynthesis, and the levels of primary metabolites were studied in leaves of wild-type (WT) plants and cytochrome c (CYTc) mutants, with reduced levels of COP components, under short- and long-day photoperiods. Our results clearly show that differences in AOP and COP in vivo activities between WT and cytc mutants depend on the photoperiod likely due to energy and stress signaling constraints. Parallel responses observed between in vivo respiratory activities, TCA cycle intermediates, amino acids, and stress signaling metabolites indicate the coordination of different pathways of primary metabolism to support growth adaptation under different photoperiods.

Decreased Levels of Thioredoxin o1 Influences Stomatal Development and Aperture but Not Photosynthesis under Non-Stress and Saline Conditions.

Salinity has a negative impact on plant growth, with photosynthesis being downregulated partially due to osmotic effect and enhanced cellular oxidation. Redox signaling contributes to the plant response playing thioredoxins (TRXs) a central role. In this work we explore the potential contribution of Arabidopsis TRXo1 to the photosynthetic response under salinity analyzing Arabidopsis wild-type (WT) and two Attrxo1 mutant lines in their growth under short photoperiod and higher light intensity than previous reported works. Stomatal development and apertures and the antioxidant, hormonal and metabolic acclimation are also analyzed. In control conditions mutant plants displayed less and larger developed stomata and higher pore size which could underlie their higher stomatal conductance, without being affected in other photosynthetic parameters. Under salinity, all genotypes displayed a general decrease in photosynthesis and the oxidative status in the Attrxo1 mutant lines was altered, with higher levels of H2O2 and NO but also higher ascorbate/glutathione (ASC/GSH) redox states than WT plants. Finally, sugar changes and increases in abscisic acid (ABA) and NO may be involved in the observed higher stomatal response of the TRXo1-altered plants. Therefore, the lack of AtTRXo1 affected stomata development and opening and the mutants modulate their antioxidant, metabolic and hormonal responses to optimize their adaptation to salinity.

Biochemical and mesophyll diffusional limits to photosynthesis are determined by prey and root nutrient uptake in the carnivorous pitcher plant Nepenthes × ventrata.

BACKGROUND AND AIMS: Carnivorous plants can enhance photosynthetic efficiency in response to prey nutrient uptake, but the underlying mechanisms of increased photosynthesis are largely unknown. Here we investigated photosynthesis in the pitcher plant Nepenthes × ventrata in response to different prey-derived and root mineral nutrition to reveal photosynthetic constrains. METHODS: Nutrient-stressed plants were irrigated with full inorganic solution or fed with four different insects: wasps, ants, beetles or flies. Full dissection of photosynthetic traits was achieved by means of gas exchange, chlorophyll fluorescence and immunodetection of photosynthesis-related proteins. Leaf biochemical and anatomical parameters together with mineral composition, nitrogen and carbon isotopic discrimination of leaves and insects were also analysed. KEY RESULTS: Mesophyll diffusion was the major photosynthetic limitation for nutrient-stressed Nepenthes × ventrata, while biochemistry was the major photosynthetic limitation after nutrient application. The better nutrient status of insect-fed and root-fertilized treatments increased chlorophyll, pigment-protein complexes and Rubisco content. As a result, both photochemical and carboxylation potential were enhanced, increasing carbon assimilation. Different nutrient application affected growth, and root-fertilized treatment led to the investment of more biomass in leaves instead of pitchers. CONCLUSIONS: The study resolved a 35-year-old hypothesis that carnivorous plants increase photosynthetic assimilation via the investment of prey-derived nitrogen in the photosynthetic apparatus. The equilibrium between biochemical and mesophyll limitations of photosynthesis is strongly affected by the nutrient treatment.

The alternative oxidase pathway is involved in optimizing photosynthesis in Medicago truncatula infected by Fusarium oxysporum and Rhizoctonia solani.

Phytopathogen infection alters primary metabolism status and plant development. The alternative oxidase (AOX) has been hypothesized to increase under pathogen attack preventing reductions, thus optimizing photosynthesis and growth. In this study, two genotypes of Medicago truncatula, one relatively resistant (Jemalong A17) and one susceptible (TN1.11), were infected with Fusarium oxysporum and Rhizoctonia solani. The in vivo foliar respiratory activities of the cytochrome oxidase pathway (COP) and the alternative oxidase pathway (AOP) were measured using the oxygen isotope fractionation. Gas exchange and photosynthesis-related parameters were measured and calculated together with antioxidant enzymes activities and organic acids contents. Our results show that the in vivo activity of AOX (valt ) plays a role under fungal infection. When infected with R. solani, the increase of valt in A17 was concomitant to an increase in net assimilation, in mesophyll conductance, to an improvement in the maximum velocity of Rubisco carboxylation and to unchanged malate content. However, under F. oxysporum infection, the induced valt was accompanied by an enhancement in the antioxidant enzymes, superoxide dismutase (SOD; EC1.15.1.1), catalase (CAT; EC1.11.1.6) and guaiacol peroxidase (GPX; EC1.11.1.7), activities and to an unchanged tricarboxylic acid cycle intermediates. These results provide new insight into the role of the in vivo activity of AOX in coordinating primary metabolism interactions that, partly, modulate the relative resistance of M. truncatula to diseases caused by soil-borne pathogenic fungi.

Low-temperature tolerance of the Antarctic species Deschampsia antarctica: A complex metabolic response associated with nutrient remobilization.

The species Deschampsia antarctica (DA) is one of the only two native vascular species that live in Antarctica. We performed ecophysiological, biochemical, and metabolomic studies to investigate the responses of DA to low temperature. In parallel, we assessed the responses in a non-Antarctic reference species (Triticum aestivum [TA]) from the same family (Poaceae). At low temperature (4°C), both species showed lower photosynthetic rates (reductions were 70% and 80% for DA and TA, respectively) and symptoms of oxidative stress but opposite responses of antioxidant enzymes (peroxidases and catalase). We employed fused least absolute shrinkage and selection operator statistical modelling to associate the species-dependent physiological and antioxidant responses to primary metabolism. Model results for DA indicated associations with osmoprotection, cell wall remodelling, membrane stabilization, and antioxidant secondary metabolism (synthesis of flavonols and phenylpropanoids), coordinated with nutrient mobilization from source to sink tissues (confirmed by elemental analysis), which were not observed in TA. The metabolic behaviour of DA, with significant changes in particular metabolites, was compared with a newly compiled multispecies dataset showing a general accumulation of metabolites in response to low temperatures. Altogether, the responses displayed by DA suggest a compromise between catabolism and maintenance of leaf functionality.

Exploring molecular evolution of Rubisco in C3 and CAM Orchidaceae and Bromeliaceae.

BACKGROUND: The CO2-concentrating mechanism associated to Crassulacean acid metabolism (CAM) alters the catalytic context for Rubisco by increasing CO2 availability and provides an advantage in particular ecological conditions. We hypothesized about the existence of molecular changes linked to these particular adaptations in CAM Rubisco. We investigated molecular evolution of the Rubisco large (L-) subunit in 78 orchids and 144 bromeliads with C3 and CAM photosynthetic pathways. The sequence analyses were complemented with measurements of Rubisco kinetics in some species with contrasting photosynthetic mechanism and differing in the L-subunit sequence. RESULTS: We identified potential positively selected sites and residues with signatures of co-adaptation. The implementation of a decision tree model related Rubisco specific variable sites to the leaf carbon isotopic composition of the species. Differences in the Rubisco catalytic traits found among C3 orchids and between strong CAM and C3 bromeliads suggested Rubisco had evolved in response to differing CO2 concentration. CONCLUSIONS: The results revealed that the variability in the Rubisco L-subunit sequence in orchids and bromeliads is composed of coevolving sites under potential positive adaptive signal. The sequence variability was related to δ13C in orchids and bromeliads, however it could not be linked to the variability found in the kinetic properties of the studied species.

Cytochrome respiration pathway and sulphur metabolism sustain stress tolerance to low temperature in the Antarctic species Colobanthus quitensis.

Understanding the strategies employed by plant species that live in extreme environments offers the possibility to discover stress tolerance mechanisms. We studied the physiological, antioxidant and metabolic responses to three temperature conditions (4, 15, and 23°C) of Colobanthus quitensis (CQ), one of the only two native vascular species in Antarctica. We also employed Dianthus chinensis (DC), to assess the effects of the treatments in a non-Antarctic species from the same family. Using fused LASSO modelling, we associated physiological and biochemical antioxidant responses with primary metabolism. This approach allowed us to highlight the metabolic pathways driving the response specific to CQ. Low temperature imposed dramatic reductions in photosynthesis (up to 88%) but not in respiration (sustaining rates of 3.0-4.2 µmol CO2  m-2  s-1 ) in CQ, and no change in the physiological stress parameters was found. Its notable antioxidant capacity and mitochondrial cytochrome respiratory activity (20 and two times higher than DC, respectively), which ensure ATP production even at low temperature, was significantly associated with sulphur-containing metabolites and polyamines. Our findings potentially open new biotechnological opportunities regarding the role of antioxidant compounds and respiratory mechanisms associated with sulphur metabolism in stress tolerance strategies to low temperature.

The Lack of Mitochondrial Thioredoxin TRXo1 Affects In Vivo Alternative Oxidase Activity and Carbon Metabolism under Different Light Conditions.

The alternative oxidase (AOX) constitutes a nonphosphorylating pathway of electron transport in the mitochondrial respiratory chain that provides flexibility to energy and carbon primary metabolism. Its activity is regulated in vitro by the mitochondrial thioredoxin (TRX) system which reduces conserved cysteines residues of AOX. However, in vivo evidence for redox regulation of the AOX activity is still scarce. In the present study, the redox state, protein levels and in vivo activity of the AOX in parallel to photosynthetic parameters were determined in Arabidopsis knockout mutants lacking mitochondrial trxo1 under moderate (ML) and high light (HL) conditions, known to induce in vivo AOX activity. In addition, 13C- and 14C-labeling experiments together with metabolite profiling were performed to better understand the metabolic coordination between energy and carbon metabolism in the trxo1 mutants. Our results show that the in vivo AOX activity is higher in the trxo1 mutants at ML while the AOX redox state is apparently unaltered. These results suggest that mitochondrial thiol redox systems are responsible for maintaining AOX in its reduced form rather than regulating its activity in vivo. Moreover, the negative regulation of the tricarboxylic acid cycle by the TRX system is coordinated with the increased input of electrons into the AOX pathway. Under HL conditions, while AOX and photosynthesis displayed similar patterns in the mutants, photorespiration is restricted at the level of glycine decarboxylation most likely as a consequence of redox imbalance.

Anatomical constraints to nonstomatal diffusion conductance and photosynthesis in lycophytes and bryophytes.

Photosynthesis in bryophytes and lycophytes has received less attention than terrestrial plant groups. In particular, few studies have addressed the nonstomatal diffusion conductance to CO2 gnsd of these plant groups. Their lower photosynthetic rate per leaf mass area at any given nitrogen concentration compared with vascular plants suggested a stronger limitation by CO2 diffusion. We hypothesized that bryophyte and lycophyte photosynthesis is largely limited by low gnsd . Here, we studied CO2 diffusion inside the photosynthetic tissues and its relationships with photosynthesis and anatomical parameters in bryophyte and lycophyte species in Antarctica, Australia, Estonia, Hawaii and Spain. On average, lycophytes and, specially, bryophytes had the lowest photosynthetic rates and nonstomatal diffusion conductance reported for terrestrial plants. These low values are related to their very thick cell walls and their low exposure of chloroplasts to cell perimeter. We conclude that the reason why bryophytes lie at the lower end of the leaf economics spectrum is their strong nonstomatal diffusion conductance limitation to photosynthesis, which is driven by their specific anatomical characteristics.

PGPR Reduce Root Respiration and Oxidative Stress Enhancing Spartina maritima Root Growth and Heavy Metal Rhizoaccumulation.

The present study aims to unravel ecophysiological mechanisms underlying plant-microbe interactions under natural abiotic stress conditions, specifically heavy metal pollution. Effect of plant growth promoting rhizobacteria (PGPR) bioaugmentation on Spartina maritima in vivo root respiration and oxidative stress was investigated. This autochthonous plant is a heavy metal hyperaccumulator cordgrass growing in one of the most polluted estuaries in the world. The association with native PGPR is being studied with a view to their biotechnological potential in environmental decontamination. As a novelty, the oxygen-isotope fractionation technique was used to study the in vivo activities of cytochrome oxidase (COX) and alternative oxidase (AOX) pathways. Inoculated plants showed decreased antioxidant enzymatic activities and in vivo root respiration rates. The reduction in respiratory carbon consumption and the stress alleviation may explain the increments observed in S. maritima root biomass and metal rhizoaccumulation after inoculation. For the first time, plant carbon balance and PGPR are interrelated to explain the effect of rhizobacteria under abiotic stress.

Phosphorus concentration coordinates a respiratory bypass, synthesis and exudation of citrate, and the expression of high-affinity phosphorus transporters in Solanum lycopersicum.

Plants exhibit respiratory bypasses (e.g., the alternative oxidase [AOX]) and increase the synthesis of carboxylates in their organs (leaves and roots) in response to phosphorus (P) deficiency, which increases P uptake capacity. They also show differential expression of high-affinity inorganic phosphorus (Pi) transporters, thus avoiding P toxicity at a high P availability. The association between AOX and carboxylate synthesis was tested in Solanum lycopersicum plants grown at different soil P availability, by using plants grown under P-sufficient and P-limiting conditions and by applying a short-term (24 hr) P-sufficient pulse to plants grown under P limitation. Tests were also performed with plants colonized with arbuscular mycorrhizal fungi, which increased plant P concentration under reduced P availability. The in vivo activities of AOX and cytochrome oxidase were measured together with the concentration of carboxylates and the P concentration in plant organs. Gene transcription of Pi transporters (LePT1 and LePT2) was also studied. A coordinated response between plant P concentration with these traits was observed, indicating that a sufficient P availability in soil led to a suppression of both AOX activity and synthesis of citrate and a downregulation of the transcription of genes encoding high-affinity Pi transporters, presumably to avoid P toxicity.

An In Vivo Perspective of the Role(s) of the Alternative Oxidase Pathway.

Despite intense research on the in vitro characterization of regulatory factors modulating the alternative oxidase (AOX) pathway, the regulation of its activity in vivo is still not fully understood. Advances concerning in vivo regulation of AOX based on the oxygen-isotope fractionation technique are reviewed, and regulatory factors that merit future research are highlighted. In addition, we review and discuss the main biological functions assigned to the plant AOX, and suggest future experiments involving in vivo activity measurements to test different hypothesized physiological roles.

Integrative field scale phenotyping for investigating metabolic components of water stress within a vineyard.

BACKGROUND: There is currently a high requirement for field phenotyping methodologies/technologies to determine quantitative traits related to crop yield and plant stress responses under field conditions. METHODS: We employed an unmanned aerial vehicle equipped with a thermal camera as a high-throughput phenotyping platform to obtain canopy level data of the vines under three irrigation treatments. High-resolution imagery (< 2.5 cm/pixel) was employed to estimate the canopy conductance (gc ) via the leaf energy balance model. In parallel, physiological stress measurements at leaf and stem level as well as leaf sampling for primary and secondary metabolome analysis were performed. RESULTS: Aerial gc correlated significantly with leaf stomatal conductance (gs ) and stem sap flow, benchmarking the quality of our remote sensing technique. Metabolome profiles were subsequently linked with gc and gs via partial least square modelling. By this approach malate and flavonols, which have previously been implicated to play a role in stomatal function under controlled greenhouse conditions within model species, were demonstrated to also be relevant in field conditions. CONCLUSIONS: We propose an integrative methodology combining metabolomics, organ-level physiology and UAV-based remote sensing of the whole canopy responses to water stress within a vineyard. Finally, we discuss the general utility of this integrative methodology for broad field phenotyping.

Measurements of Electron Partitioning Between Cytochrome and Alternative Oxidase Pathways in Plant Tissues.

Plant respiration is characterized by the existence of the alternative oxidase pathway (AOP) that competes with cytochrome oxidase pathway (COP) for the electrons of the ubiquinone pool of the mitochondrial electron transport chain, thus reducing ATP synthesis. The oxygen (O2) isotope fractionation technique is the only available to determine the electron partitioning between the two pathways and their in vivo activities in plant tissues. In this chapter, the basis of the O2 isotope fractionation technique and its derived calculations are carefully explained together with a detailed description of the dual-inlet isotope ratio mass spectrometry (DI-IRMS) system and the protocol developed at the University of Balearic Islands. The key advantages of the DI-IRMS over other systems are highlighted as well as the potential problems of this technique. Among these problems, those associated with leakage, diffusion, and inhibitor treatments are noted and solutions to prevent, detect, and repair these problems are detailed.

Respiratory ATP cost and benefit of arbuscular mycorrhizal symbiosis with Nicotiana tabacum at different growth stages and under salinity.

Growth and maintenance partly depend on both respiration and ATP production during oxidative phosphorylation in leaves. Under stress, ATP is needed to maintain the accumulated biomass. ATP production mostly proceeds from the cytochrome oxidase pathway (COP), while respiration via the alternative oxidase pathway (AOP) may decrease the production of ATP per oxygen consumed, especially under phosphorus (P) limitation and salinity conditions. Symbiosis with arbuscular mycorrhizal (AM) fungi is reputed by their positive effect on plant growth under stress at mature stages of colonization; however, fungal colonization may decrease plant growth at early stages. Thus, the present research is based on the hypothesis that AM fungus colonization will increase both foliar respiration and ATP production at mature stages of plant growth while decreasing them both at early stages. We used the oxygen-isotope-fractionation technique to study the in vivo respiratory activities and ATP production of the COP and AOP in AM and non-AM (NM) tobacco plants grown under P-limiting and saline conditions in sand at different growth stages (14, 28 and 49days). Our results suggest that AM symbiosis represents an ATP cost detrimental for shoot growth at early stages, whilst it represents a benefit on ATP allowing for faster rates of growth at mature stages, even under salinity conditions.

Combined effect of virus infection and water stress on water flow and water economy in grapevines.

Water limitation is one of the major threats affecting grapevine production. Thus, improving water-use efficiency (WUE) is crucial for a sustainable viticulture industry in Mediterranean regions. Under field conditions, water stress (WS) is often combined with viral infections as those are present in major grape-growing areas worldwide. Grapevine leafroll-associated virus 3 (GLRaV-3) is one of the most important viruses affecting grapevines. Indeed, the optimization of water use in a real context of virus infection is an important topic that needs to be understood. In this work, we have focused our attention on determining the interaction of biotic and abiotic stresses on WUE and hydraulic conductance (Kh ) parameters in two white grapevine cultivars (Malvasia de Banyalbufar and Giró Ros). Under well-watered (WW) conditions, virus infection provokes a strong reduction (P < 0.001) in Kpetiole in both cultivars; however, Kleaf was only reduced in Malvasia de Banyalbufar. Moreover, the presence of virus also reduced whole-plant hydraulic conductance (Khplant ) in 2013 and 2014 for Malvasia de Banyalbufar and in 2014 for Giró Ros. Thus, the effect of virus infection on water flow might explain the imposed stomatal limitation. Under WS conditions, the virus effect on Kplant was negligible, because of the bigger effect of WS than virus infection. Whole-plant WUE (WUEWP ) was not affected by the presence of virus neither under WW nor under WS conditions, indicating that plants may adjust their physiology to counteract the virus infection by maintaining a tight stomatal control and by sustaining a balanced carbon change.