The rapidly increasing world population and the scarcity of suitable land for agricultural food production together with a changing climate will ultimately put pressure on grape-producing areas for the use of land and the input of resources. For most grape-producing areas, the predicted developments in climate will be identical to becoming more marginal for quality production and/or to be forced to improve resource management. This will have a pronounced impact on grapevine physiology, biochemistry and ultimately production methods. Research in the entire area of stress physiology, from the gene to the whole plant and vineyard level (including soils) will need to be expanded to aid in the mitigation of arising problems.
In this review, we elaborate on some key issues in environmental stress physiology such as efficient water use to illustrate some of the challenges, current limitations and future possibilities of certain experimental techniques and/or data interpretations. Key regulatory mechanisms in the control of stomatal conductance are treated in some detail and several future research directions are outlined. Diverse physiological aspects such as the functional role of aquaporins, the importance of mesophyll conductance in leaf physiology, night-time water use and respiration under environmental constraints are discussed. New developments for improved resource management (mainly water) such as the use of remote sensing and thermal imagery technologies are also reviewed. Specific cases where our experimental systems are limited or where research has been largely discontinued (i.e. stomatal patchiness) are treated and some promising new developments, such as the use of coupled structural functional models to assess for environmental stress effects on a whole-plant or canopy level are outlined. Finally, the status quo and research challenges around the ‘CO2-problem’ are presented, an area which is highly significant for the study of ‘the future’ of the grape and wine industry, but where substantial financial commitment is needed.
maximum rate of carboxylation of Rubisco; A net CO2 assimilation rate
vapour pressure deficit
integrated water use efficiency
water use efficiency
intrinsic water use efficiency
instantaneous water use efficiency
Grapevines are cultivated in six out of seven continents, between latitudes 4° and 51° in the Northern Hemisphere (NH) and between 6° and 45° in the Southern Hemisphere (SH) across a large diversity of climates (oceanic, warm oceanic, transition temperate, continental, cold continental, Mediterranean, subtropical, attenuated tropical, arid and hyperarid climates) (Peguy 1970, Tonietto and Carbonneau 2004). Accordingly, the range and magnitude of environmental factors differ considerably from region to region and so do the principal environmental constraints for grape production. Problems of low winter temperatures have limited grape cultivation in the past in areas with continental climates in Eastern Europe, Asia and North America. Low temperatures during the growing season have prevented the extension of grape-growing in regions approximately beyond the 12°C temperature isotherm (April–October (NH), October–April (SH)) (Jones et al. 2005a). The effects of hot temperatures, on the contrary, are less clear with respect to the distribution of grapevine cultivation areas. In general, the 22°C temperature isotherm is considered limiting for wine grape production (Jones 2007a, Schultz and Jones 2008), but many areas in the tropics are much warmer than this (Tonietto and Carbonneau 2004) and detrimental effects of high temperatures may be largely mitigated if water supply is sufficient and/or if humidity is high. Within the existing production areas, water shortage is probably the most dominant environmental constraint (Williams and Matthews 1990), and even in moderate temperate climates, grapevines often face some degree of drought stress during the growing season (Morlat et al. 1992, van Leeuwen and Seguin 1994, Gaudillère et al. 2002, Gruber and Schultz 2009).
The primary and global challenge for the grape and wine industry of the future will be climate change because its direct (temperature, precipitation, CO2 concentration, etc.) and indirect consequences (resource management, energy efficiency, sustainability in production and consumer acceptance, etc.) will affect all facets of the industry. Climate change has already caused significant warming in most grape-growing areas of the world during the last approximately 55 years (Jones et al. 2005a,b). The degree of warming varies strongly between regions, with large observed differences within each climate maturity grouping (cool, intermediate, warm, hot), ranging form 0.1°C during the growing season for Burgundy, to about 4°C for the Northern Rhone Valley (Jones et al. 2005a). Warming will continue, and depending on the baseline scenario (Intergovernmental Panel on Climate Change (IPCC) 2008) and the model used to make regional predictions up to 2050, temperatures may increase between somewhat less than 1°C for the growing season in South Africa, to near 3°C in Eastern Washington, Southern Portugal (Jones et al. 2005a) and some areas in Australia (Webb et al. 2007, 2008).
Predictions of the total annual amount of precipitation and its annual and regional distribution are much more uncertain (IPCC 2008). However, according to many experts, water and its availability and quality will be the main pressures on, and issues for, societies and the environment (IPCC 2008). Because of rising temperatures and solar radiation in many places, and decreasing and/or more irregular precipitation patterns, climate change will exacerbate soil degradation and desertification (IPCC 2008). Desertification is often accompanied by soil salinization which today affects 7% of the global total land area and 20–50% of the global irrigated farmland (IPCC 2008). Irrigation in agriculture already accounts for about 70% of the total water use worldwide, and the irrigated surface area has increased linearly since 1960. Driven by apparent changes in the climate conditions in viticultural areas previously entirely rain-fed, there is already an increasing interest in irrigation. However, population growth is predicted to reach between 8.7 billion (by 2050) in the most conservative estimation to about 15 billion (by 2100) in a A2 ‘worst case’ scenario (IPCC baseline scenarios 2007). This will cause a general increase in water demand on a global scale, and will become a problem for agricultural water use in light of sharp increases in water consumption of the urban, industrial and environmental sectors (Fereres and Soriano 2007). Some fresh water basins in the world termed ‘water-stressed’ by the IPCC (2008), that is water availability decreases below 1000 m3/capita/yr or withdrawal to average run-off increases above a ratio of 0.4, are partly congruent to areas where grapes are currently cultivated on a larger scale (e.g. the Murray–Darling River basin in Australia). These developments will put enormous pressure on irrigated land not directly devoted to food production with the combined consequences (temperature and water) that grape cultivation will be partly displaced from traditional areas (Schultz and Jones 2008) and will be forced to use more marginal land under environmental conditions previously termed less suitable.
Therefore, more than ever before, there is a need to understand the physiological mechanisms and the genetic background underlying the interactions between plants and the environment, and to pay attention to research fields which will be pivotal for the development of sustainable concepts under changing conditions. Because of the large array of possible issues under this rather wide topic, this review will not be able to address every aspect in adequate detail. Focus will be on some key subjects and on the challenges and limitations the research community is currently facing.
Efficient water use
Water use efficiency (WUE)
Regional estimates on changes in water availability under future climate conditions suggest that for most grape-growing regions, the propensity for water deficiency will increase during the growing season (IPCC 2008). Together with increasing competition for irrigation water, WUE in viticulture will be an important issue of the future. Therefore, to secure a sustainable and effective use of water, more information on the physiological and genetic basis of WUE is needed (Chaves and Oliveira 2004). There is evidence for variation in WUE among species and cultivars in grapevines (Düring and Scienza 1980, Eibach and Alleweldt 1984, Chaves et al. 1987, Bota et al. 2001, Schultz 2003a, Flexas et al. 2004, Souza et al. 2005b, Soar et al. 2006a, Flexas et al. 2008) and some of the aspects of genetic variation of WUE have been elucidated in Arabidopsis thaliana as a model plant (Masle et al. 2005, Nilson and Assmann 2007, Lake and Woodward 2008). Despite demonstrated interest in WUE, it can be defined in different ways and the physiological basis of its regulation is not fully understood. WUE depends on complex interactions between environmental factors and physiological mechanisms such as stomatal behaviour, photosynthetic capacity, and leaf and plant anatomy (for a review see Bacon 2004). Additionally, all mechanisms that tend to maintain plant survival or a certain productivity under conditions of limited water supply or high evaporative demand (or adverse conditions in general) come at certain ‘costs’, which, from an agricultural point of view, will either reduce dry matter production or competitive ability (Jones 1992). In that sense, high WUE genotypes may not be the ideal compromise between drought tolerance and economic performance.
WUE – challenges and limitations of measurements and data interpretation. WUE is often determined from single leaf gas-exchange measurements, relating net CO2 assimilation rate (A) either to stomatal conductance for water vapour (g), termed intrinsic water use efficiency (WUEi) (Osmond et al. 1980) (Eqn 1),
or A to leaf transpiration rate (E), termed instantaneous water use efficiency WUEinst (Eqn 2).
Less frequently used as WUEi is WUEinst or the integrated WUE (Wp) (Farquhar et al. 1989) which includes losses of carbon beause of respiration at night or from non-photosynthetic organs such as roots and water loss at night because of incomplete stomatal closure or high cuticular conductance (Eqn 3). Data like these are most relevant but are confined to pot studies and are reflected in ratios such as dry matter produced per unit of water lost (i.e. Düring and Scienza 1980, Gibberd et al. 2001).
where ΦC = carbon loss via respiration
where ΦE = unproductive water loss
More recently, the analyses of 13C carbon isotope discrimination (Δ13C) has become popular and widespread for the evaluation of water use in plants and grapevines under field conditions (Gaudillère et al. 2002, Medrano et al. 2005, Souza et al. 2005b, Chaves et al. 2007). In contrast to gas-exchange techniques that provide measurements of photosynthesis rates at a single point in time, leaf carbon isotope signature (δ13C) integrates the ratio of intercellular (Ci) to atmospheric CO2 concentration (Ca) for longer periods of time (Condon et al. 2004). The basis of the biochemical discrimination (Δ) against 13C in C3 plants lies within the primary carboxylating enzyme, ribulose-1,5-bisphosphate carboxylase-oxygenase (Rubisco) which discriminates against 13C because of the intrinsically lower reactivity of 13C compared with 12C (Farquhar et al. 1982).
Variations in Δ13C are used to analyse temporal and spatial trends in plant–carbon water relations. In the most frequently used version of the Farquhar et al. (1982) model – the linear (or reduced) form relates Δ13C linearly to Ci/Ca (Eqn 4).
With a and b' being isotopic fractionation coefficients (a = 4.4‰, fractionation during CO2 diffusion through stomata, O'Leary 1981; b' = 27‰, fractionation associated with reactions by Rubisco and phosphoenol pyruvate (PEP)-carboxylase, Farquhar and Richards 1984). The linear relationship between Ci/Ca and Δ13C can be used to calculate WUEi (A/g) because Ci/Ca reflects the balance between A and stomatal conductance for CO2 (gc) and because gc and stomatal conductance for water vapour (g) are related by a constant factor (g = 1.6 gc) (Farquhar et al. 1980) (Eqn 5)
Inherit in Equations 4 and 5 are, that if Ci/Ca decreases, then Δ13C decreases and WUEi will increase. If we relate the linear model of carbon isotope discrimination in an analogous manner to WUEinst, where leaf transpiration rate, E, is a function of g and the leaf to air vapour pressure deficit (LAVPD) (kPa), (Eqn 6) (Farquhar and Richards 1984)
which implies that WUEinst will vary with Ci (affecting Δ13C) and LAVPD because Ca is essentially constant. It also implies that because evaporative demand is considered in WUEinst, but not in WUEi, both can show different trends (opposite directions) while these can still be consistent with the same trend in Δ13C (Seibt et al. 2008).
VPD response and signalling mechanisms
Research has shown that stomatal conductance of grapevines is sensitive to the vapour pressure deficit of the air (VPD) (kPa), (i.e. Düring 1987, Soar et al. 2006a,b, Poni et al. 2009) closely related to LAVPD, which will directly affect WUEinst (g↓, E↑) and WUEi (g↓). Figure 1 shows the relationships between WUEinst and WUEi with LAVPD for four grapevine varieties grown in pots (30L, 3-year old vines) under a wide range of climatic conditions (photosynthetic photon flux density, PPFD 750–1750 µmol/m2/s; air temperature, Tair, 19°C–37°C) during different drought and recovery experiments (pre-dawn water potential ψPD, −0.15 to −1.45 MPa) over a period of several weeks (Chouzouri and Schultz, unpublished data). When all data are pooled, there is a clear relationship of WUEinst to LAVPD, with WUEinst decreasing with increasing LAVPD, while there is none for WUEi. This is consistent with the optimisation theory (Cowan 1977) and is important because (i) it indicates that LAVPD negatively affects WUEinst over a wide range of plant water status; and (ii) because this is not apparent in WUEi, measurements conducted under high LAVPD and water deficit may show opposite tendencies in these two parameters. This becomes clear from diurnal gas-exchange data of a field experiment with the variety Syrah (Shiraz), where LAVPD was high during a continuous dry down over several months (Figure 2). As expected, WUEi was higher for stressed than for irrigated plants throughout most of the day at any level of water deficit (Figure 2(a),(b),(c)). However, WUEinst was either not different between irrigated and non-irrigated plants (Figure 2(d)) or lower in the stressed plants when water deficit became more severe (Figure 2(e),(f)) and stomatal closure caused LAVPD to rise substantially (Figure 2(j)–(l)) because of increases in leaf temperature (Figure 2(g),(i)).
There is uncertainty whether a pronounced stomatal response to VPD during water deficit is beneficial for improving WUE, but there are clear differences in this response between varieties (Düring 1987, Soar et al. 2006b) and rootstock/scion combinations (Soar et al. 2006a, Koundouras et al. 2008). Water stress intensified the VPD response in some rootstock/scion combinations but had the opposite effect on others (Soar et al. 2006a). Irrespective of the irrigation strategy applied, g decreased in response to rising VPD under partial root zone drying (PRD) (Loveys et al. 2004) and regulated deficit irrigation (RDI) (Patakas et al. 2005, Pou et al. 2008) which would allow the plant to better control E. However, depending on the intensity of the closure response and the extent of the subsequent rise in temperature and LAVPD, WUEinst may be reduced (as in Figure 2), which has been observed in other studies on grapevines (Naor et al. 1994, Naor and Bravdo 2000, Koundouras et al. 2008) and trees (Jifon and Syvertsen 2003, Baldocchi and Bowling 2003). In this context, it is important to realise, that a reduction in WUEinst may occur despite Δ13C data suggesting the opposite (Koundouras et al. 2008, Seibt et al. 2008). Figure 3 illustrates this using different scenarios and combining Equations 4, 5 and 7. At the same carbon isotope signature indicating an increase in WUEi with decreasing Δ13C, WUEinst may increase, stay constant or even decrease (Figure 3(b)) depending on the development in LAVPD (Figure 3(a)). It is recognised that this model ignores the contribution of changes in mesophyll conductance with drought and/or temperature increase and photorespiration (Farquhar et al. 1982, Flexas et al. 2002, 2007) or even decreased activities of photosynthetic enzymes under severe stress (Flexas et al. 2006). Incorporating these aspects into a more comprehensive model can yield similar results (Baldocchi and Bowling 2003, Seibt et al. 2008). Added in Figure 3 are some data from Gibberd et al. (2001) obtained in a pot study of 19 different grapevine genotypes, where different groups were separated depending on obtained relationships between WUE on the whole plant level (dry matter produced/amount of water transpired, e.g. close to Eqn 3) and Δ13C under well-watered conditions. Functional groups which exhibited a greater decrease in g had lower Δ13C values but gained little in WUE (Figure 3B), probably because increasing leaf temperature and LAVPD increased E and did offset decreases in g. This effect will strengthen with decreasing wind-speed, decreasing water availability, increasing leaf size, increasing ambient temperature and denser canopies, where the latter four could be expected to be more important in the future because of warming and rising ambient CO2 concentrations. All the listed factors and their impact on leaf and canopy temperature are also playing a role in the use of new proxy and remote sensing techniques for stress monitoring and irrigation management (Jones et al. 2002, Möller et al. 2007, see section Canopy (leaf) temperature and imaging technologies).
Differences in the responsiveness of stomata to environmental factors, such as VPD and/or water deficit, seem to be modulated by abscisic acid (ABA), either through the increased ability of the xylem to supply ABA (Loveys 1984a, Loveys et al. 2004), increases in xylem pH affecting ABA ionization and general metabolism (Stoll et al. 2000) or through differential gene expression in the ABA biosynthetic pathway involved in regulating varietal responses to VPD (Soar et al. 2006a). However, absolute control of g by ABA is disputed, and hydraulic signalling may also play a role (Schultz 2003a, Rodrigues et al. 2008) and both factors may actually partly depend on each other in the regulation and recovery of g during and after a water stress (Lovisolo et al. 2008a). Depending on the degree of stress, the rate and degree of recovery may be very different. Flexas et al. (2004, 2006) and Galmés et al. (2007) found that grapevines subjected to a mild deficit (i.e. maximum g above 100 mmol/m2/s) fully recovered within a day after rewatering, whereas more severely stressed plants recovered slowly during the following week and did not reach pre-stress levels in A and g.
Nitric oxide (NO) may be another signalling molecule (aside of or together with ABA) involved in stomatal responses to drought. NO has been shown to be involved in many biological events (Quan et al. 2008), and the synthesis of NO by nitric oxide synthase and nitrate reductase in response to drought may elicit the increase of anti-oxidant enzymes which can scavenge water stress-induced hydrogen peroxide (Sang et al. 2008) or may mediate ABA-induced closure of stomata (Neil et al. 2003). Little is known about the relationships between drought and NO accumulation in grapevines, but Patakas and Zotos (2005) found a rapid increase in NO-induced fluorescence close to the stomates in leaves of stressed plants.
Isotopic signatures in leaves and fruits
For grapevine leaves from plants exposed to different irrigation regimes, even the relationships between WUEi and Δ13C may be weak (Souza et al. 2005b, Chaves et al. 2007), but there are reasonable relationships of WUEi with Δ13C of the fruit (Medrano et al. 2005, Souza et al. 2005b, Chaves et al. 2007). These differences have been interpreted as being related to the fact that sugar accumulation starts late in the season and results from current leaf photosynthates, which are produced during the water deficit as compared with the isotopic signature of leaves which are formed earlier during the season (Gaudillère et al. 2002). There could also be more post-photosynthetic fractionation processes (namely respiration) in berries, which might result in differences in the carbon isotope composition of the two organs (Badeck et al. 2005). Despite good correlations to plant water status during berry ripening (i.e. van Leeuwen et al. 2001, Gaudillère et al. 2002), so far the final evidence is lacking that fruit Δ13C is an indicator of crop scale water use efficiency (i.e biomass produced per unit water used, Jones (1992), because correlations to WUEi may not reflect relationships on the whole-canopy level.
Aside from the isotopic discrimination of carbon during photosynthesis and respiration, it may also be useful in future studies to measure the isotopic enrichment of oxygen (18O‰) occurring in leaves during the evaporation of water. The enrichment will increase with a decrease in g, an increase in leaf temperature, a decrease in relative humidity (or an increase in VPD and LAVPD) (Figure 4), a decrease in E, and as such may allow the distinction between effects of soil water deficit and evaporative demand on WUE (Farquhar et al. 2007). This response to relative humidity on the leaf level can also be retraced in grape juice (Tardaguila et al. 1997) and wines coming from areas with different evaporative demand where isotopic enrichment can be related to the prevailing conditions 30 days before harvest (Figure 4) (Hermann and Voerkelius 2008).
Varietal differences in stress responses (isohydric versus anisohydric)
There is clear evidence that varieties differ in their physiological response to stress in their response velocity (Düring and Scienza 1980), adaptive mechanisms (i.e. Düring and Loveys 1982, Loveys 1984b, Schultz 1996, Bota et al. 2001), and effectiveness in regulating gas-exchange (i.e. Chaves et al. 1987, Escalona et al. 1999, Flexas et al. 2002, Schultz 2003a, Souza et al. 2005a,b). A fortunate choice of varieties to study has led to the illustration of these differences in a field experiment with the varieties Grenache and Syrah, whose stomatal behaviour under water deficit led to a classification termed ‘near-isohydric’ and ‘anisohydric’ in reference to Stocker (1956) (Schultz 2003a). Subsequent studies have shown, that these differences in g were also triggered in response to VPD (Soar et al. 2006a), and correlated with differential expression of key genes in the ABA biosynthetic pathway in leaves but not roots (Soar et al. 2006a). The isohydric – anisohydric classification leaves sufficient space to test other varieties, whether they can fit these categories or can enlarge the existing spectrum in order to quantify a general type of WUE, which may be important in irrigated viticulture in the future. However, recent findings in an outdoor ‘heating experiment’ (40°C for three consecutive days) showed that anisohydric Syrah vines up-regulated g, E, and A in response to temperature (Soar et al. 2009) at a common VPD. This may be a positive adaptation in terms of heat tolerance through increased evaporative cooling at the expense of long-term transpiration efficiency (Soar et al. 2009).
The continuum between classical isohydry and anisohydry was well illustrated in a study by Turner et al. (1984) with nine woody and herbaceous species, where the response of E to VPD varied largely. Despite of the correlation to ABA physiology (Soar et al. 2006a), the underlying mechanisms for isohydric or anisohydric behaviour are little understood, and there is evidence that plant hydraulic conductance may play a decisive role (Franks et al. 2007). This conclusion was based on the fact that even in plants capable of strongly down-regulating g as a reaction to water deficit (Franks et al. 2007) it could not be prevented that midday leaf water potential fell to levels where xylem air embolisms may have become the dominant factor in determining water flow and where a functional correlation between embolism formation and leaf gas-exchange existed (Domec et al. 2006, Maherali et al. 2006). Nevertheless, Alsina et al. (2007) did not find a correlation between drought tolerance and vulnerability to xylem embolisms of different grapevine varieties, whereas other data from pot and field experiments showed a correlation (Schultz 2003a, Chouzouri and Schultz 2005, Lovisolo et al. 2008b). The solution to the dilemma in terms of experimental set up may be to test varieties in their combined response of gas-exchange to environmental stresses using the Ball-Woodrow-Berry model (Ball et al. 1987) (Figure 5(a),(b)) and to accompany its application with both ABA and hydraulic conductance measurements to gain clarity. The advantage of the model is that it incorporates the composite sensitivity of stomata to assimilation rate, CO2 concentration, humidity and temperature (Ball et al. 1987), is applicable to grapevines (Schultz et al. 1999, Schultz 2003b), and gives an indication of overall stomatal sensitivity to these factors (Figure 5(a)) (Eqn 8):
where g0 is a residual stomatal conductance to H2O vapour (A→0 when light intensity approaches 0), and k is a constant representing stomatal sensitivity (Tenhunen et al. 1990, Harley and Tenhunen 1991) (Figure 5(a)). Measurements of relative humidity, rh, and CO2 partial pressure (Ca) outside the leaf boundary layer can be used to drive the model. Because this relationship is purely empirical, k and (A [rh/Ca]) are dimensionless (Ball et al. 1987). While the use of rh has been criticised as being non-mechanistic (Aphalo and Jarvis 1993), the model has been successfully coupled to assimilation models of individual perennial crops (Katul et al. 2000), including grapevines (Schultz 2003b) or entire terrestrial ecosystems (Lloyd et al. 1995, Wang et al. 2007, Mo et al. 2008). The constant representing stomatal sensitivity, k, i.e. the slope of the relationship between g and (A [rh/Ca]) is sensitive to water deficit (Figure 5(a)) and can be used to distinguish the sensitivities of g of different varieties (Figure 5(b)). Note, the smaller k, the higher is the stomatal sensitivity (Harley and Tenhunen 1991), thus Figure 5(b) shows that stomates of Grenache exhibit a higher sensitivity (smaller k) than Syrah at similar water potentials.
Specific physiological aspects
The differences between varieties in the response scheme to drought and/or VPD may be mediated by aquaporins (AQP) (Sade et al. 2009, Vandeleur et al. 2009). AQP are members of the mayor membrane intrinsic protein family, can act as water channels and can regulate cell-to-cell water transport (Maurel et al. 2008). So far, 35 AQP have been identified in Arabidopsis (Johanson et al. 2001) and 28 in the Vitis genome (Fouquet et al. 2008). Under short-term water deficit, gene expression for AQP in grapevines was stronger in roots than in leaves, particularly under moderate stress levels (Galmés et al. 2007). This may be an indication that the function of some AQP is more important in roots than in leaves, which would be consistent with the idea that they facilitate water transport (Luu and Maurel 2005), although a relation of leaf hydraulic conductance to AQP expression has also been found (Cochard et al. 2007). However, the role of AQP in regulating plant water status is a complex issue, because different subfamilies and subclasses may be up- or down-regulated or remain unchanged depending on the degree of water deficit and/or the time during the stress period (Galmés et al. 2007). Lovisolo and Schubert (2006) showed that AQP may be involved in the recovery of grapevine shoot water relations after embolisms were formed during water stress. However, it remains unclear if this effect was more important in the regulation of water flow in roots or shoots.
A recent study exploring the nature of the isohydric and anisohydric response pattern of different grapevine cultivars suggested that physiological and anatomical differences in the roots played a major role in water transport (Vandeleur et al. 2009). In this study, substantial differences in root hydraulic conductance under water deficit and recovery could be related to the differential expression of the two most highly expressed plasma membrane intrinsic protein (PIP) AQP (VvPIP1;1 and VvPIP2;2). In the anisohydric cultivar (in this case Chardonnay), root hydraulic conductance was up-regulated under water deficit and so was VvPIP1;1, whereas in the isohydric variety (Grenache) there was no up-regulation indicating that water transport across roots was regulated to match transpirational demand (Vandeleur et al. 2009) and possibly stomatal conductance (Galmés et al. 2007). In a functional study on AQP in different Vitis spp. used as rootstocks, Lovisolo et al. (2008b) also found differences in their role in root hydraulic conductance and susceptibility to embolism formation. However, expression patterns in AQP genes not necessarily correlate always with physiological parameters, such as hydraulic conductance (Galmés et al. 2007), because the complexity of responses on the metabolic, cellular, organ or whole plant level may mask such a correlation. Nevertheless, the involvement of AQP in the regulation of isohydric/anisohydric behaviour has also been demonstrated for different lines of tomatoes (Solanum lycopersicum) (Sade et al. 2009).
There may be differences in this component between genotypes because of differences in leaf anatomy currently unknown or in the response of gi to factors such as variations in Ci (Flexas et al. 2007) and temperature (Bernacchi et al. 2002). If confirmed, they may offer a way to improve WUE in the future (Flexas et al. 2009, this issue). Because gi also changes with CO2 concentration, it is important to investigate possible differences between genotypes, because gi will play a crucial role in the photosynthetic performance under future CO2 concentrations (Flexas et al. 2007, Flexas et al. 2008). The possibility of genetic differences was already indicated by variations in the CO2 specificity factor of Rubisco in the absence and presence of water deficit (Bota et al. 2001).
Night-time water use and Respiration
Night-time water use and dark respiration also affect daily WUE (see Eqn 3), and changes in plant water status will alter both, yet there is only little information available on both processes for grapevines.
Incomplete stomatal closure during the night has been observed in a diverse range of C3 and C4 species, among these are many horticultural and crop species (for a review see Caird et al. 2007). This can lead to substantial night-time transpirational water loss even in dessert species were water is a strongly limiting factor (Snyder et al. 2003). The magnitude of water transpired during the night depends on the cuticular conductance, g, and VPD. It can account for E values between 5 and 15% of daytime rates, sometimes even more, and has been detected using a variety of techniques, from single leaf gas exchange measurements to whole plant sap flow, and field scale lysimeters (Caird et al. 2007). Green et al. (1989) reported that 20–30% of total daily transpiration could occur at night in kiwi and apple orchards in New Zealand under certain conditions. There may be a genetic attribute involved, because successions of Arabidopsis thaliana showed different night g in a common environment which correlated with the average VPD of their native habitat (Caird et al. 2007). While E during the night responds positively to VPD, night-time g may show a similar negative response as observed during the day (Bucci et al. 2004). There may be some benefits to the plant by not fully closing its stomata including better nutrient availability because of continuous mass flow or a better carbon balance because of high early morning g when VPD and temperature are still low (Caird et al. 2007). There is little information available on night-time water losses of grapevines, but it could potentially be important for regions with frequently high wind velocities (i.e southern France, California coast) (Chu et al. 2009) and high night-time temperatures and VPD. In fact, Schmid (1997) observed that high wind velocities correlated with high night-time sap flow rates of field-grown Riesling grapevines in Germany. This effect on whole plant E was much stronger later during the season (September, Northern Hemisphere) than during mid-summer (July–August). The seasonal difference in this study was attributed to a partial loss of stomatal control and seemed unrelated to soil water content (Schmid 1997). However, most studies have shown that water deficit and salt stress will cause night-time g to decrease (Caird et al. 2007).
Data presented in Figure 6, are among the few examples available where the night-time flux of water and its dependence on temperature (Figure 6(a)) and VPD (Figure 6(b)) has been measured for grapevines (Schmid 1997). The strong response to both environmental factors indicates that these phenomena should be studied specifically in warmer and dryer areas. The relationship to temperature was very similar to that of leaf respiration measured concomitantly in a gas exchange cuvette (Figure 6(a), Schmid 1997).
Apart from photosynthesis, plant respiration is the other basic component of plant productivity and it is remarkable how little information on respiratory responses of grapevines to environmental factors there is. Dark respiration rate (R) has been shown to change with temperature (Figure 6(a)), leaf age and the temperature response coefficient, Q10, and depends on phenology and canopy position (sun and shade leaves) (Schultz 1991). Leaves are generally responsible for most of whole plant above-ground respiration in grapevines (Palliotti et al. 2004). Under water stress, the response seems to depend on the plant species and the plant organ investigated (Flexas et al. 2005) and on the degree of stress exposed to (Flexas et al. 2006). In a recent review on this subject, Atkin and Macherel (2009) reported that root and whole plant respiration were almost always reduced, whereas the response of mature leaves was different, with about two-thirds of the reviewed studies showing a reduction in R and most of the remainder showing no differences with a few reports on increasing R under severe water deficit. For grapevines, results are also not clear. Escalona et al. (1999) did not find significant effects of water stress on leaf respiration for the varieties Manto Negro and Tempranillo, whereas Gómez del Campo et al. (2004) observed a decrease in R (leaves) and some differences between varieties. They also found that the relationship of respiration rate to photosynthesis first decreased before it increased when water stress became severe. However, because the measurement temperature was not constant in that study, these observations are difficult to compare with others. Figure 7 shows an example from a field trial, where R was measured at a leaf temperature of 20°C before or after determining ψPD for the varieties Grenache and Syrah during a progressive dry-down. R decreased for both varieties but somewhat faster for Grenache than for Syrah (Figure 7(a)), whereas the ratio of R/Amax was similar for both declining at first and increasing at more severe water deficits (Figure 7(b)) corroborating the results of Gómez del Campo et al. (2004). These results are not totally in line with the general view that A is much more sensitive than R to water stress irrespective of the degree of water deficit (Atkin and Macherel 2009). It would be interesting to determine the response of R of different varieties to increasing temperatures in order to determine the acclimation potential for future climate developments because substantial differences have been observed with other plants (Atkin et al. 2008).
Irrigation systems are crucial in translating genetic and physiological information on how WUE is responding to the environment into suitable application systems. There are various deficit irrigation techniques currently investigated, such as RDI (i.e. Matthews et al. 1987, Bravdo and Naor 1996, McCarthy et al. 2002, Girona et al. 2006), PRD (i.e. Dry et al. 1996, Loveys et al. 2004, Santos et al. 2005, Rodrigues et al. 2008) or sustained deficit irrigation (SDI) (Fereres and Soriano 2007) to achieve a more efficient use of water and better crop quality. A recent survey on the efficiency of these techniques stated that there was no significant advantage of one over the other (Sadras 2009), but the current challenge remains to find the best suited parameters for scheduling irrigation irrespective of the system used. In most cases, a certain percentage of potential (reference) crop evapo-transpiration is used, but this can result in severe water stress during short time periods of temperature extremes (Goodwin and Jerie 1992), so some form of knowledge about soil and/or plant water status is necessary.
Because ψPD is the parameter least affected by diurnal variations in weather conditions, this parameter seems best suited for cool and variable climates with intermittent summer rainfall (Gruber and Schultz 2009), whereas ψST and ψM seem suitable for more stable climates (Naor 1998, Choné et al. 2001, Williams and Araujo 2002, Möller et al. 2007). Nevertheless, both respond directly to the prevailing environmental conditions (solar radiation, temperature, VPD) and to phenological stage (Matthews et al. 1987, Möller et al. 2007, Olivo et al. 2009), so it seems difficult to determine suitable thresholds for irrigation. Recent trials on the use of thermal and visible imagery of grapevines using the crop water stress index (CWSI) for irrigation management showed that while the relationship CWSI to g remained unchanged throughout the season, CWSI to ψST varied (Möller et al. 2007).
There is a need to develop more dynamic scheduling systems and to fine tune further the balance between water use, mineral nutrient uptake and fruit quality (Keller 2005). There is also an obvious tendency to use red varieties in many irrigation trials related to wine quality, possibly because of more obvious responses in fruit composition (i.e. phenolic compounds in general), and consequently a lack of understanding with respect to white varieties. However, the future will force many regions to apply deficit irrigation strategies irrespective of the colour of their grapes.
Canopy (leaf) temperature and imaging technologies
The increase in leaf temperature with increasing water deficit is a response which may be somewhat problematic in measurements on individual leaves. For instance, in order to measure WUEi and WUEinst in the field, leaf measurements need to be conducted under saturating light to make them comparable between treatments, thus leaves will be exposed to the sun. Under these conditions the leaf energy balance will change, increasing leaf temperature and LAVPD (Fuchs 1990) and decreasing g which may drive WUEinst and WUEi in opposite directions (i.e. Schulze and Hall 1982). Such an ‘indirect’ contribution may have also influenced the data presented in Figure 2 and many other studies.
The sensitivity of leaf temperature to changes in g, and hence the utility of thermal imaging, depends on the absorbed radiation, boundary layer conductance (leaf size or canopy density and wind speed) and air humidity. Figure 8, adapted from Jones et al. (2002), illustrates how the modelled difference between wet and dry leaves varies as a function of absorbed radiation and wind speed. It is evident that the sensitivity, i.e. the temperature difference, increases with radiation absorbed and with decreasing wind speed.
It is uncertain how important the variability in leaf temperature across a canopy is with respect to the assessment of CWSI in an application for irrigation management (Jones et al. 2002, Möller et al. 2007). The variability in g increases substantially below g values of about 100 mmol/m2/s and should be reflected in leaf temperature if no adjustment in leaf angle occurs (Möller et al. 2007). Although restricted CO2 diffusion across leaves is the most dominant cause for decreased photosynthetic rates under water stress, metabolic impairment of about 15% of total A limitation becomes apparent at or below 100 mmol/m2/s, irrespective of the species tested (Flexas et al. 2006). In non-irrigated field-grown grapevines in a production situation, g largely resides below 100 mmol/m2/s during extended periods of the growing season in different canopy sections and throughout the day when leaves are measured in their natural position (for examples see Schultz et al. 2001, Schultz 2003b). Although this type of measurement is not a common practice, the results seem more relevant to whole canopies, and may be more compatible with the use of imaging technologies.
It is also uncertain how estimates of CSWI relate to how efficiently water is used in a whole canopy situation, because leaves are exposed to a wide array of sunlight intensity at any time during the day and WUE of entire plants may differ from the WUE of individual leaves. A recent model analyses on different sections of tree canopies has shown that WUEi was highest and WUEinst was lowest where VPD and light exposure and subsequent temperature were highest (Seibt et al. 2008). If integrated over the whole canopy, these effects can lead to a reduction in plant WUE during water deficit at an unchanged Δ13C even at relative moderate water deficits (ψPD near −0.4 MPa) as recently shown for grapevines with measurements of whole plant gas-exchange (Poni et al. 2009). Thus, assessing the variability in leaf temperature across a canopy and the relative change in temperature caused by different water supply is an important area of current research to exploit this response for the use of deficit irrigation strategies (Grant et al. 2007, Möller et al. 2007).
Measuring the physiological status of a plant using non-invasive remote sensing techniques is challenging and a radiation-footprint is central to many biochemical and biophysical properties of a grapevine. Thus, radiation interaction is measured for a wide range of purposes, and remote sensing data are recorded both from satellite or aircraft sensors and from ground based measurements. The techniques available are used to track for vineyard variability (Bramley and Hamilton 2004), to monitor vineyard canopy density (Johnson et al. 1996, Hall et al. 2002, Zarco-Tejada et al. 2005), to trace nutrient deficiencies (Johnson 2001, Martin et al. 2007) or to act as a management support system prior to harvest (Johnson et al. 2001). Furthermore, simultaneous signals for different physiological indicators have been introduced to predict berry phenolics and colour (Lamb et al. 2004, Agati et al. 2007) to estimate the photosynthetic light use efficiency (Cerovic et al. 2002, Rascher and Pieruschka 2008) or plant water status (Seelig et al. 2008). These approaches already show great potential to monitor the physiological status non-invasively. Nevertheless, scaling up from leaf-level to a complex three dimensional canopy structure will remain challenging in future.
Aside from the vines themselves, cover crops also play a role in the carbon and water balance of a vineyard, yet their impact is largely neglected although they have an additional effect on the turnover rate of organic matter in the soil, another possible source of CO2 during climate warming (Litton and Giardina 2008). It seems important to somewhat refocus research on cover crops because, depending on the region/climate type combination, these plants will have a substantial effect on sustainability in both vineyard mechanisation and their additional role in affecting the carbon and water balance of vineyards. Figure 9 shows some recent results on comparing C3 and C4 species in studies on the suitability of cover crops in terms of improving vineyard WUE (Uliarte and Schultz unpublished). This research area will gain importance because open soil cultivation will increase CO2 release because of organic matter degradation and may not be a sustainable solution in light of increased precipitation intensity and increased risk for erosion (IPCC 2008). Additionally, competition for resources such as water between cover crop and vines will increase in the future and thus will need to be minimised by carefully selecting adapted species (Olmstead et al. 2001) and by considering both carbon and water resource efficiency (Lopes et al. 2004).
Limitations of our experimental systems
Water status and its measurements
There are limitations and contradictions in some experimental systems we use. It is sometimes difficult to relate results obtained on potted plants to data obtained in the field, but pot studies are a necessary compromise to investigate certain phenomena. However, there are some observations which should be addressed in the future because they may hold some relevant physiological answers. For example, several studies on the factors controlling g in grapevines have reported substantial effects at ψPD's in the vicinity of −0.2 MPa (i.e. Lovisolo et al. 2002, Pellegrino 2003, Pou et al. 2008, and some results from our group), which is considered no stress for field-grown grapevines. In such cases, there is a need to investigate why these changes in physiology can occur at these deficit levels in pots while there is not even a response in growth or g under field conditions (Escalona et al. 1999, Schultz 2003a). If water potential is equilibrating with the wettest soil layer, and moisture distribution in pots is not uniform, a potential disequilibrium between soil and plant water potential can occur resulting in large variations of potential plant responses (Donovan et al. 2003). In these cases, enclosing two tubes consisting of porous tissue and containing sand into the pot medium and placing them at opposite sides ensures homogeneous water distribution (Pellegrino 2003) and good relations between plant and soil water potential (Chouzouri and Schultz 2005).
In addition to these problems, Jones (2007b), in a recent review, outlined the common lack of application of adequate measures of plant and soil water status in many physiological and agronomic studies on drought tolerance, breeding and the management of irrigation systems. He particularly criticised the omission of necessary water-status measurements in the majority of published molecular studies dealing with effects of drought on gene expression or effects of transgenes on the performance under water stress in general. Although his survey was not on studies related to grapevines, his suggestion to ‘enhance inputs from environmental plant physiologists to improve the value of molecular studies’ would certainly benefit both sides (Jones 2007b).
Some aspects of physiological research on grapevines have been ignored in the recent past, although they may intuitively be important. For instance, heterogeneous stomatal closure across leaves under water deficit, salt stress, high light and low humidity leads to erroneous estimates of Ci. This has been known to occur for a long time (Laisk 1983, Downton et al. 1988, Mott and Buckley 1998, Mott and Peak 2007) and there was a period when this phenomenon was actively researched and demonstrated to occur in grapevine leaves (i.e. Downton et al. 1988, Downton et al. 1990, Düring 1992, Düring and Loveys 1996) and where Ci values derived from gas-exchange measurements could not be published as a consequence. However, despite its demonstrated occurrence in hundreds of species (Beyschlag and Eckstein 1998) and the fact that patchiness appears to reduce WUE, the absence of any apparent physiological function has caused this to remain a little studied phenomenon (Mott and Peak 2007). In more recent studies on the importance of mesophyll conductance in the limitation of photosynthesis under water stress, patchiness was measured (Flexas et al. 2002) but was considered unimportant, and others have doubted that there is any significance to it (Lawlor and Tezera 2009). Yet, significant patchiness has been shown to occur in grapevine leaves below g values of about 120 mmol m−2 s−1 with a linear decrease in the number of open patches with decreasing g (Düring and Loveys 1996). Thus, it should not be ignored that there may be substantial variability between varieties in the occurrence of patchiness which could also be related to their drought or salt resistance. A recent hypothesis states that patchy patterns of stomatal opening and closing are similar to structures and behaviours in locally connected networks of dynamic units that perform complex tasks and may as such be a form of communication across leaves (Mott and Peak 2007). Combining image analysing technologies and programes such as thermal and fluorescence imagery may allow the study of these phenomenon on a finer scale in the future (Mott and Peak 2007).
Structural and physiological models
One possibility to integrate physiological responses on a single leaf level into canopy or even stand scale responses are coupled structural-functional models, which have been developed for annual species (maize, Fournier and Andrieu 1999), trees (peach, Allen et al. 2005) and recently grapevines (Grenache and Syrah, Louarn et al. 2008). These models can integrate structural components of a canopy, such as shape, orientation and location of plant organs which influence light interception and thus canopy energy balance with functional properties such as stomatal aperture, photosynthetic capacity and photomorphogenesis or other metabolic processes. Because vineyard canopy structure, functioning and management are important in the formation of yield and quality (i.e. Smart et al. 1982, Reynolds and Wardle 1989, Carbonneau and Cargnello 2003, Gladstone and Dokoozlian 2003, Downey et al. 2004), these type of models, in the future, may serve to deduce management decisions with respect to yield and quality production, and go well beyond simple descriptions of canopy architecture (leaf area density distribution) and light harvesting (Schultz 1995). Louarn et al. (2008) have used a combination of approaches to construct virtual canopies of two varieties, Grenache and Syrah, with four common spur-pruned canopy systems (Gobelet, bilateral free cordon system, and two bilateral cordon system with vertical shoot orientation differing in the number of catch wires). They employed a limited number of parameters to describe the volume occupied by a shoot (turbid-medium-like envelope) and combined this with results from random samplings for the position of individual shoot organs (leaves as discrete geometric polygons) within this volume to generate individual shoots with individual leaf positions and orientations. Coupled to a set of descriptors of plant architecture, bud location and shoot orientation and angle, complex three dimensional canopies were regenerated (Figure 10(a)–(d)).
A particular advantage of this more statistical approach as compared to earlier three dimensional descriptions of grapevine canopies (Mabrouk et al. 1997, Mabrouk and Sinoquet 1998) was the improved integration of inter-plant variability and the implementation of varietal specific parameters (Louarn et al. 2008). This type of model allows for a more accurate simulation of light interception and can be coupled to the mechanistic gas-exchange model of Farquhar et al. (1980) (Louarn et al. 2005) which has been fully parameterised for grapevines (Schultz 2003b) (Figure 10(e)). If research development continues, in the future, this type of approach will allow the simulation of the response of entire vineyards to changes in environmental conditions such as water deficit or salt stress and may be able to give some answers to the impact of climate change and the mitigation possibilities in terms of canopy structure and management.
The CO2 problem
In a recent editorial for the New Phytologist titled ‘an inconvenient truth’ with reference to the Academy award for the best documentary film by former US Vice President Al Gore, Woodward (2007) described and analysed the dilemma between practical experiments with elevated CO2 concentrations and the need to understand and predict the future responses of plants in the field. Aside from the fact that increasing CO2 concentrations will impact on global temperature, CO2 itself is generally beneficial to plant growth, although the response strongly varies between species (Long et al. 2004). Because stomata are sensitive to CO2 but photosynthesis increases in response to it, increased biomass production at reduced water losses is expected (Ainsworth and Rogers 2007, Woodward 2007). Additionally, even a reduced plant sensitivity to the pollutant ozone may be a consequence (Morgan et al. 2006). However, Woodward (2007) continued that CO2 enrichment experiments usually do not mimic the gradual increase in CO2 plants are experiencing in the field but rather follow a step-up approach, and possible differences in plant responses to these approaches are unknown. Additionally, CO2 enrichment is not usually accompanied by warming as would be predicted by climate models because of ‘the problem of securing long-term funding which is a bothersome limitation to a more general approach’ (Woodward 2007). Recent results from models including the physiological impact of CO2 on plants (more biomass, reduced g) suggest that rising CO2 will increase the temperature driven water evaporation from the oceans resulting in an increased absolute water vapour content of the air. However, the decrease in evapotranspiration over land (because of g↓) would still lead to an overall decrease in relative humidity and to an increased evaporative demand according to current knowledge (Boucher et al. 2009). Plant surfaces should then heat up more because of stomatal closure which is similar to findings from field-based warming/CO2 experiments (Musil et al. 2005) and adds to the complexity of expected responses difficult to trace and simulate in experiments.
It is exactly this complexity which necessitates a more global approach to setting up experimental systems to study the response of grapevines to the combined increase in temperature and CO2. Few studies have investigated the response of grapevines to CO2 either in small free air carbon dioxide enrichment systems (Bindi et al. 1995, Bindi et al. 2001a) or in open top chambers (Gonçalves et al. 2009), but these could only describe the impact of increasing CO2 concentration in the absence of rising Tair. Nevertheless, the generally predicted increase in biomass was confirmed, yet the effects on water consumption remained unclear (Bindi et al. 1995, Bindi et al. 2001a). These experiments also showed that fruit sugar concentration should increase and acidity levels decrease under elevated CO2 (Bindi et al. 2001b), but the response of other components contributing to flavour and aroma of grapes were heterogeneous and indicated a significant ‘chamber effect’, with plants grown outside responding differently than plants in open top chambers with or without elevated CO2 (Gonçalves et al. 2009).
Data from single leaf experiments on grapevines (Figure 11) showed that the degree of stomatal closure induced by CO2 matches the average closure response of about −20% in g (Figure 11(c)) analysed for different functional groups based on a literature survey and assuming Ca would rise from 380 to 560 µmol mol−1 (Figure 11(c),(e)) (Ainsworth and Rogers 2007). The concomitant rise in WUEinst could even be larger than this depending on LAVPD (Figure 11(c),(d)) and considering that respiration rate may also be suppressed by elevated CO2 (Smart 2004). However, studies on individual leaves (or woody pieces in the case of Smart 2004) may not be representative of whole plant field experiments and the need to study the effects of elevated CO2 and temperature in combination may be illustrated by the following example. Alonso et al. (2008) separated the effects of a doubling in CO2 concentration and a 4°C increase in temperature on the photosynthetic apparatus of wheat in a field study with open top chambers over two successive years. Elevated CO2 alone decreased the maximum rate of carboxylation of Rubisco (Vcmax) and so did an increase in temperature without changes in CO2. However, an increase in temperature under elevated CO2 increased Vcmax particularly at high measurement temperatures demonstrating the complex interactions between just these two environmental factors (Alonso et al. 2009). In an early experiment investigating the effects of elevated CO2 (900 ppm) in combination with a heat therapy (35–40°C) of several weeks up to several months on grapevine functioning, Kriedemann et al. (1976) observed substantial changes in leaf anatomy with a much larger spongy mesophyll and an enrichment in the number of chloroplasts. Vine growth rate more than doubled and dry matter distribution was altered in favour of greater root growth. Photosynthetic capacity was enhanced, photorespiration depressed and high CO2 mitigated the high temperature effect, similar to the more recent study on wheat mentioned above (Alonso et al. 2008).
We thank Prof. H.G. Jones, University of Dundee, Dundee, UK, Dr. Gaëtan Louarn, INRA Centre Poitou-Charentes, Lusignan, France and Dr. Joachim Schmid, Forschungsanstalt Geisenheim, Germany for providing published and unpublished material. Financial support was provided by the ‘Forschungsschwerpunkt Umweltstress und nachhaltige Produktion’, Forschungsanstalt Geisenheim, Germany.