Unraveling the Physiological Mechanisms Underlying the Intracultivar Variability of Water Use Efficiency in Vitis vinifera "Grenache".

Selecting genotypes with a better capacity to respond and adapt to soil water deficits is essential to achieve the sustainability of grapevine cultivation in the context of increasing water scarcity. However, cultivar changes are very poorly accepted, and therefore it is particularly interesting to explore the intracultivar genetic diversity in water use efficiency (WUE). In previous studies, the cultivar "Grenache" has shown up to 30% variability in WUE. This research aimed to confirm the intracultivar variability and to elucidate the traits underlying this variability in the response to a water deficit by analyzing the growth rates, water relations, osmotic potential, leaf morphology, leaf gas exchange and carbon isotope discrimination in nine "Grenache" genotypes grown in pots during two seasons. The results showed lower differences in WUE and carbon isotope ratio than in previous field studies, but fairly good consistency in genotype ranking. Leaf mass area and osmotic potential did not underlie differences in stem water potential and in stomatal conductance. Overall, stomatal regulation and photosynthetic capacity seem to underlie differences in WUE among genotypes with an important environmental influence. These results confirm the ability to select clones with higher WUE and present an opportunity for the genetic improvement of WUE in grapevines.

Intracultivar genetic diversity in grapevine: Water use efficiency variability within cv. Grenache.

The selection of genotypes best adapted to environmental conditions has traditionally focused on agronomic and grape composition parameters. However, to classify the genotypes most adapted to climate change conditions, the aim must be to focus on the ecophysiological responses that will ultimately determine their performance. The variability in water use efficiency of 13 Grenache genotypes over three-seasons was assessed under field conditions at leaf, grape and plant level. Results showed a significant effect of genotype at all three levels, and despite the large interannual variability there was a remarkable consistency among levels. Furthermore, using genotype-specific regressions it was possible to identify significant differences in the intrinsic water use efficiency response of each genotype as a function of the vine water status. The relationship between net photosynthesis and stomatal conductance, as well as carbon isotope discrimination in grapes, were also confirmed as reliable physiological indicators for selecting grapevine genotypes to future environmental conditions. Therefore, the proposed multi-level methodology was useful to quantify the intracultivar variability and the identification of more and less efficient genotypes within Grenache.

Plant water status and genotype affect fruit respiration in grapevines.

An understanding of fruit gas exchange is necessary to determine the carbon balance in grapevines, but little attention has been paid to the relationships among fruit respiration, plant water status and genetic variability. The effect of plant water status and genotype on cluster respiration was studied over two seasons (2013 and 2014) under field conditions using a whole cluster respiration chamber. Whole cluster CO2 fluxes were measured in growing grapevines at hard-green, veraison and ripening stages under irrigated and non-irrigated conditions, and under light and dark conditions in two grapevine varieties, Tempranillo and Grenache. A direct relationship between cluster CO2 efflux and plant water status was found at hard-green stage. Genotype influenced the fruit CO2 efflux that resulted in higher carbon losses in Tempranillo than in Grenache. Fruit respiration rates decreased from the first berry developmental stages to ripening stage. The integration of fruit respiration rates under light and dark conditions showed the magnitude of fruit carbon losses and gains as well as interesting variety and environmental conditions effects on those processes.

Nano and Micro Unmanned Aerial Vehicles (UAVs): A New Grand Challenge for Precision Agriculture?

By collecting data at spatial and temporal scales that are inaccessible to satellite and field observation, unmanned aerial vehicles (UAVs) are revolutionizing a number of scientific and management disciplines. UAVs may be particularly valuable for precision agricultural applications, offering strong potential to improve the efficiency of water, nutrient, and disease management. However, some authors have suggested that the UAV industry has overhyped the potential value of this technology for agriculture, given that it is difficult for non-specialists to operate UAVs as well as to process and interpret the resulting data. Here, we analyze the barriers to applying UAVs for precision agriculture, which range from regulatory issues to technical requirements. We then evaluate how new developments in the nano- and micro-UAV (NAV and MAV, respectively) markets may help to overcome these barriers. Among the possible breakthroughs that we identify is the ability of NAV/MAV platforms to directly quantify plant traits using methods (e.g., object-oriented classification) that require less image calibration and interpretation than spectral index-based approaches. We suggest that this potential, when combined with steady improvements in sensor miniaturization, flight precision, and autonomy as well as cloud-based image processing, will make UAVs a tool with much broader adoption by agricultural managers in the near future. If this wider uptake is realized, then UAVs have real potential to improve agriculture's resource-use efficiency. © 2020 by John Wiley & Sons, Inc.

Leaf growth rate and nitrogen content determine respiratory costs during leaf expansion in grapevines.

Respiration processes are well recognized as fundamental for the plant carbon balance, but little attention has been paid to the relationships among respiration rates, environment and genetic variability. This can be of particular interest to understand the differences in net carbon balances in crops as grapevines. Night respiration (Rn ) and its associated growth (Rg ) and maintenance (Rm ) components were evaluated during leaf expansion in two grapevine cultivars (Tempranillo cv. and Garnacha cv.) that differ in their plant growth pattern and carbon balance. Simultaneously, leaf traits as leaf mass area, nitrogen (N) and carbon (C) content were evaluated in order to relate to the respiratory processes and the leaf growth. The results showed the differences in respiration rates associated with the leaf expansion pattern. Tempranillo developed leaves with higher leaf area and lower dry weight per leaf unit than Garnacha. Although differences between cultivars were observed in terms of growth costs in expanding leaves, the maintenance costs were similar for both cultivars. Also, a significant linear regression was found between respiration rates and N content in expanding and mature leaves. The results indicate that differences in structure and nitrogen content of expanding leaves may lead to respiratory differences between cultivars. These results also demonstrate the importance of respiratory cost components in carbon balance calculations in grapevines.

Whole-Plant Water Use in Field Grown Grapevine: Seasonal and Environmental Effects on Water and Carbon Balance.

Water scarcity is a main challenge in vineyards sustainability in most of the grapevine areas now and even more in near future due to climatic change perspectives. In consequence, water use efficiency (WUE) measurements are of the highest interest to improve the sustainability of this crop. The vast majority of WUE measurements relays on measurements of leaf carbon and water fluxes at leaf-level. However, less data are available at the whole-plant level, and for the moment those data are not totally coincident with conclusions reached at leaf scale. In this study, we used whole-plant chambers able to enclose an entire plant of 12 years old to measure at the same time water and carbon fluxes under realistic field grown conditions. The main objectives were to identify the technical issues interfering the whole-plant measurements and track the environmental and other abiotic factors that can affect water and carbon balance, i.e., WUE at the whole-plant scale. To achieve those objectives, we measured whole-plant water and carbon fluxes in grapevine exposed to two different water regimes at three phenological stages [pea size (July), ripening (August), and harvest (September)]. In September, measurements were repeated under high CO2 to also check its effect at the whole-plant scale. The results indicate that water and carbon fluxes are well coordinated under both water availability treatments. Under drought conditions, both fluxes were drastically reduced, but surprisingly the estimated WUE resulted not improved but decreased, contrarily to what is shown at the leaf scale. The phenology (September) also strongly decreased both water and carbon fluxes when compared to measurements in July. We hypostatized that harvest load respiration rates could have an important weight on the whole-plant net carbon exchange (NCE). Finally, high CO2 measurements, after correction for leaks, indicated an increase of whole-plant NCE as well as increased whole-plant WUE, as expected. Several technical issues were identified, like 1/instability of [CO2] during the night period that prevent robust estimation of whole-plant respiration and 2/condensation during last night and sun-rise hours which may affect the estimation of daily plant transpiration.

Combined drought and virus infection trigger aspects of respiratory metabolism related to grapevine physiological responses.

In the Mediterranean region, grapevines usually deal with drought during their summer growth season. Concurrently, grapevines are hosts to a large number of viruses from which grapevine leafroll associated virus-3 is one of the most widespread and provokes considerable economic losses in many vineyards. However, information concerning grapevine metabolic responses to the combination of drought and viral infection is scarce. Gas-chromatography coupled to mass-spectrometry based metabolite profiling was used in combination with growth analysis, viral loads and gas exchange data to perform an integrative study of the effects of individual and combined stress in two Majorcan grapevine varieties at two experimental years. Metabolic responses of both varieties to the combination of water stress and virus infection were specific and not predicted from the sum of single stress responses. Correlations between respiration, biomass and key metabolites highlight specific adjustments of respiratory and amino acid metabolism possibly underlying the maintenance of carbon balance and growth in grapevines under stress combination.

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.

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.

The Application of Leaf Ultrasonic Resonance to Vitis vinifera L. Suggests the Existence of a Diurnal Osmotic Adjustment Subjected to Photosynthesis.

The main objective of this study was to apply the air-coupled broad-band ultrasonic spectroscopy in attached transpiring leaves of Vitis vinifera L. to monitor changes in leaf water potential (Ψ) through the measurements of the standardized value of the resonant frequency associated with the maximum transmitance (f/fo). With this purpose, the response of grapevine to a drought stress period was investigated in terms of leaf water status, ultrasounds, gas exchange and sugar accumulation. Two strong correlations were obtained between f/fo and Ψ measured at predawn (pd) and at midday (md) with different slopes. This fact implied the existence of two values of Ψ for a given value of f/fo, which was taken as a sign that the ultrasonic technique was not directly related to the overall Ψ, but only to one of its components: the turgor pressure (P). The difference in Ψ at constant f/fo (δ) was found to be dependent on net CO2 assimilation (A) and might be used as a rough estimator of photosynthetic activity. It was then, the other main component of Ψ, osmotic potential (π), the one that may have lowered the values of md Ψ with respect to pd Ψ by the accumulation of sugars associated to net CO2 assimilation. This phenomenon suggests the existence of a diurnal osmotic adjustment in this species associated to sugars production in well-watered plants.

Exploring the genetic variability in water use efficiency: Evaluation of inter and intra cultivar genetic diversity in grapevines.

Genetic improvement of crop Water Use Efficiency (WUE) is a general goal because the increasing water scarcity and the trend to a more sustainable agriculture. For grapevines, this subject is relevant and need an urgent response because their wide distribution in semi-arid areas. New cultivars are difficult to introduce in viticulture due to the narrow dependency of consumer appreciation often linked to a certain particular wine taste. Clones of reputed cultivars would presumably be more accepted but little is known on the intra-cultivar genetic variability of the WUE. The present work compares, on the basis of two field assays, the variability of intrinsic water use efficiency (WUEi) in a large collection of cultivars in contrast with a collection of clones of Tempranillo cultivar. The results show that clonal variability of WUEi was around 80% of the inter-cultivar, thus providing a first assessment on the opportunity for clonal selection by WUE. Plotting the WUEi data against stem water potential or stomatal conductance it was possible to identify cultivars and clones out of the confidence intervals of this linear regression thus with significantly higher and lower WUEi values. The present results contribute to open the expectative for a genetic improvement of grapevine WUE.

Interactive effects of grapevine leafroll-associated virus 3 (GLRaV-3) and water stress on the physiology of Vitis vinifera L. cv. Malvasia de Banyalbufar and Giro-Ros.

Among several biotic and abiotic stress combinations, interaction between drought and pathogen is one of the most studied combinations in some crops but still not in grapevine. In the present work, we focused on the interaction effects of biotic (GLRaV-3) and abiotic (drought) stresses on grapevine photosynthetic metabolism on two cultivars (cvs. 'Malvasia de Banyalbufar and Giro-Ros'). Non-infected and GLRaV-3 infected potted plants were compared under water stress conditions (WS) and well-watered (WW) conditions. Under WW condition, the results showed that photosynthesis (AN) in both cultivars was decreased by the presence of GLRaV-3. The stomatal conductance (gs) was the main factor for decreasing AN in Malvasia, meanwhile reductions in Giro-Ros were closely related to decreases in gm. The observed differences in gm between both cultivars might result from variation in their leaf anatomical, Giro-Ros having higher values of gm and leaf porosity (in all treatments). Moderate water deficit resulted in a closure of stomata and a decrease in gm accompanied by a decrease in AN in both cultivars. The maximum velocity of carboxylation (Vcmax) and electron transport rate (Jmax) were also reduced under water stress. Moreover, the combined stress resulted in a reduction of most physiological parameters compared to healthy irrigated plants. However, no considerable differences were found between non-infected and virus infected (GLRaV-3) plants under water stress. Most of the results could be explained by the difference of virus concentration between cultivars and treatments.

Plasticity of vulnerability to leaf hydraulic dysfunction during acclimation to drought in grapevines: an osmotic-mediated process.

Previous studies have reported correlation of leaf hydraulic vulnerability with pressure-volume parameters related to cell turgor. This link has been explained on the basis of the effects of turgor on connectivity among cells and tissue structural integrity, which affect leaf water transport. In this study, we tested the hypothesis that osmotic adjustment to water stress would shift the leaf vulnerability curve toward more negative water potential (Ψ leaf ) by increasing turgor at low Ψ leaf . We measured leaf hydraulic conductance (K leaf ), K leaf vulnerability [50 and 80% loss of K leaf (P50 and P80 ); |Ψ leaf | at 50 and 80% loss of K leaf , respectively), bulk leaf water relations, leaf gas exchange and sap flow in two Vitis vinifera cultivars (Tempranillo and Grenache), under two water treatments. We found that P50 , P80 and maximum K leaf decreased seasonally by more than 20% in both cultivars and watering treatments. However, K leaf at 2 MPa increased threefold, while osmotic potential at full turgor and turgor loss point decreased. Our results indicate that leaf resistance to hydraulic dysfunction is seasonally plastic, and this plasticity may be mediated by osmotic adjustment.

Effects of long-term individual and combined water and temperature stress on the growth of rice, wheat and maize: relationship with morphological and physiological acclimation.

This study evaluates the long-term individual and combined effects of high temperature (HT) and water deficit (WD) stress on plant growth, leaf gas-exchange and water use efficiency in cultivars of the three most important crops worldwide, rice, wheat and maize. Total plant biomass (Bt ) accumulation decreased under all treatments, being the combined HT-WD treatment the most detrimental in all three species. Although decreases in Bt correlated with adjustments in biomass allocation patterns (i.e. the leaf area ratio), most of the variation observed in Bt was explained by changes in leaf gas exchange parameters. Thus, integrated values of leaf carbon balance obtained from daily course measurements of photosynthesis and respiration were better predictors of plant growth than the instantaneous measurements of leaf gas exchange. Leaf water use efficiency, assessed both by gas exchange and carbon isotope measurements, was negatively correlated with Bt under WD, but not under the combined WD and HT treatment. A comparative analysis of the negative effects of single and combined stresses on the main parameters showed an additive component for WD and HT in rice and maize, in contrast to wheat. Overall, the results of the specific cultivars included in the study suggest that the species native climate plays a role shaping the species acclimation potential to the applied stresses. In this regard, wheat, originated in a cold climate, was the most affected species, which foretells a higher affectation of this crop due to climate change.

Opportunities for improving leaf water use efficiency under climate change conditions.

WUEi (intrinsic water use efficiency) is a complex (multi)-trait, that depends on several physiological processes, driving plant productivity and its relation with a changing environment. Climatic change predictions estimate increases in temperature and drought in the semi-arid regions, rendering improved water use efficiency is a mandatory objective to maintain the current global food supply. The aims of this review were (i) to identify through a meta-analysis the leaf traits mostly related to intrinsic water use efficiency (WUEi, the ratio between A - net photosynthesis and gs - stomatal conductance), based on a newly compiled dataset covering more than 200 species/varieties and 106 genus of C3 plants (ii) to describe the main potential targets for WUEi improvement via biotechnological manipulations and (iii) to introduce emergent and innovative technologies including UAVs (Unmanned Aerial Vehicles) to scale up levels from leaf to whole plant water status. We confirmed that increases in gm/gs and Vcmax/gs ratios are systematically related with increases in WUEi maintained across species, habitats, and environmental conditions. Other emergent opportunities to improve WUEi are described such as the relationship between photosynthesis and respiration and their link with metabolomics. Finally, we outline our hypothesis that we are observing the advent of a "smart" agriculture, wherein new technologies, such as UAVs equipped with remote sensors will rapidly facilitate an efficient water use regulating the irrigation schedule and determination, under field conditions, of cultivars with improved water use efficiency. We, therefore, conclude that the multi-disciplinary challenge toward WUE has only just begun.

Is stomatal conductance optimized over both time and space in plant crowns? A field test in grapevine (Vitis vinifera).

Crown carbon gain is maximized for a given total water loss if stomatal conductance (gs ) varies such that the marginal carbon product of water (∂A/∂E) remains invariant both over time and among leaves in a plant crown, provided the curvature of assimilation rate (A) versus transpiration rate (E) is negative. We tested this prediction across distinct crown positions in situ for the first time by parameterizing a biophysical model across 14 positions in four grapevine crowns (Vitis vinifera), computing optimal patterns of gs and E over a day and comparing these to the observed patterns. Observed water use was higher than optimal for leaves in the crown interior, but lower than optimal in most other positions. Crown carbon gain was 18% lower under measured gs than under optimal gs . Positive curvature occurred in 39.6% of cases due to low boundary layer conductance (gbw ), and optimal gs was zero in 11% of cases because ∂A/∂E was below the target value at all gs . Some conclusions changed if we assumed infinite gbw , but optimal and measured E still diverged systematically in time and space. We conclude that the theory's spatial dimension and assumption of positive curvature require further experimental testing.

Rapid hydraulic recovery in Eucalyptus pauciflora after drought: linkages between stem hydraulics and leaf gas exchange.

In woody plants, photosynthetic capacity is closely linked to rates at which the plant hydraulic system can supply water to the leaf surface. Drought-induced embolism can cause sharp declines in xylem hydraulic conductivity that coincide with stomatal closure and reduced photosynthesis. Recovery of photosynthetic capacity after drought is dependent on restored xylem function, although few data exist to elucidate this coordination. We examined the dynamics of leaf gas exchange and xylem function in Eucalyptus pauciflora seedlings exposed to a cycle of severe water stress and recovery after re-watering. Stomatal closure and leaf turgor loss occurred at water potentials that delayed the extensive spread of embolism through the stem xylem. Stem hydraulic conductance recovered to control levels within 6 h after re-watering despite a severe drought treatment, suggesting an active mechanism embolism repair. However, stomatal conductance did not recover after 10 d of re-watering, effecting tighter control of transpiration post drought. The dynamics of recovery suggest that a combination of hydraulic and non-hydraulic factors influenced stomatal behaviour post drought.

Variation in Rubisco content and activity under variable climatic factors.

The main objective of the present review is to provide a compilation of published data of the effects of several climatic conditions on Rubisco, particularly its activity, state of activation, and concentration, and its influence on leaf gas exchange and photosynthesis. The environmental conditions analyzed include drought, salinity, heavy metals, growth temperature, and elevated [O3], [CO2], and ultraviolet-B irradiance. The results show conclusive evidence for a major negative effect on activity of Rubisco with increasing intensity of a range of abiotic stress factors. This decrease in the activity of Rubisco is associated with down-regulation of the activation state of the enzyme (e.g., by de-carbamylation and/or binding of inhibitory sugar phosphates) in response to drought or high temperature. On the contrary, the negative effects of low temperature, heavy metal stress (cadmium), ozone, and UV-B stress on Rubisco activity are associated with changes in the concentration of Rubisco. Notably, in response to all environmental factors, the regulation of in vivo CO2 assimilation rate was related to Rubisco in vitro parameters, either concentration and/or carboxylation, depending on the particular stress. The importance of the loss of Rubisco activity and its repercussion on plant photosynthesis are discussed in the context of climate change. It is suggested that decreased Rubisco activity will be a major effect induced by climate change, which will need to be considered in any prediction model on plant productivity in the near future.

Diffusional conductances to CO2 as a target for increasing photosynthesis and photosynthetic water-use efficiency.

A key objective for sustainable agriculture and forestry is to breed plants with both high carbon gain and water-use efficiency (WUE). At the level of leaf physiology, this implies increasing net photosynthesis (A N) relative to stomatal conductance (g s). Here, we review evidence for CO2 diffusional constraints on photosynthesis and WUE. Analyzing past observations for an extensive pool of crop and wild plant species that vary widely in mesophyll conductance to CO2 (g m), g s, and foliage A N, it was shown that both g s and g m limit A N, although the relative importance of each of the two conductances depends on species and conditions. Based on Fick's law of diffusion, intrinsic WUE (the ratio A N/g s) should correlate on the ratio g m/g s, and not g m itself. Such a correlation is indeed often observed in the data. However, since besides diffusion A N also depends on photosynthetic capacity (i.e., V c,max), this relationship is not always sustained. It was shown that only in a very few cases, genotype selection has resulted in simultaneous increases of both A N and WUE. In fact, such a response has never been observed in genetically modified plants specifically engineered for either reduced g s or enhanced g m. Although increasing g m alone would result in increasing photosynthesis, and potentially increasing WUE, in practice, higher WUE seems to be only achieved when there are no parallel changes in g s. We conclude that for simultaneous improvement of A N and WUE, genetic manipulation of g m should avoid parallel changes in g s, and we suggest that the appropriate trait for selection for enhanced WUE is increased g m/g s.

Importance of leaf anatomy in determining mesophyll diffusion conductance to CO2 across species: quantitative limitations and scaling up by models.

Foliage photosynthetic and structural traits were studied in 15 species with a wide range of foliage anatomies to gain insight into the importance of key anatomical traits in the limitation of diffusion of CO2 from substomatal cavities to chloroplasts. The relative importance of different anatomical traits in constraining CO2 diffusion was evaluated using a quantitative model. Mesophyll conductance (g m) was most strongly correlated with chloroplast exposed surface to leaf area ratio (S c/S) and cell wall thickness (T cw), but, depending on foliage structure, the overall importance of g m in constraining photosynthesis and the importance of different anatomical traits in the restriction of CO2 diffusion varied. In species with mesophytic leaves, membrane permeabilities and cytosol and stromal conductance dominated the variation in g m. However, in species with sclerophytic leaves, g m was mostly limited by T cw. These results demonstrate the major role of anatomy in constraining mesophyll diffusion conductance and, consequently, in determining the variability in photosynthetic capacity among species.