Drought escape involves the ability of the plant to complete the whole life cycle before severe water constraint occurs. Drought tolerance with low plant water potential involves desiccation tolerance and the maintenance of turgor, mainly by osmotic adjustment. Drought tolerance with high plant water potential involves a reduction of water loss and an increase in water uptake, which is a way to avoid drought (Chaves and Oliveira 2004). Grapevines do not fall under the drought escape mechanism. Most of the grapevines cultivated around the world are located in a Mediterranean type of climate, meaning that most of the vegetative and reproductive growth occurs under moderate to severe water constraints if irrigation is not applied. Grapevine roots and rootstocks present drought tolerance mechanisms related to low and high plant water potential (Figure 4, Tables 2-4) involving drought responses, such as stomatal closure, decrease of cell growth and photosynthesis, activation of respiration, and accumulation of osmolytes and proteins (Shinozaki and Yamaguchi-Shinozaki 2007). In addition, grapevine rootstocks can affect leaf area and root development depending on the vigour inducing capacity (Gambetta et al. 2012) affecting the canopy water demand and supply. During dry hot seasons, higher vigour rootstocks can explore root zones to an extent greater than low vigour rootstocks (Bauerle et al. 2008b) and as a consequence can access water from deeper soil layers (a drought avoidance strategy). Gambetta et al. (2012) found that the higher canopy water demand due to the effect of rootstocks that promote scion vigour appears to be balanced by adjustments in root hydraulic conductivity through fine root hydraulic conductivity and higher root surface area. The mechanisms involved can develop in different time scales, from minutes to months. For example, an adjustment to stomatal conductance can occur within minutes or less, whereas osmotic adjustment and the response to ABA can occur in hours and adaptations in terms of root system development can take several days or weeks (Passioura 1996).
Although many genes related to drought response have been identified, their physiological relevance is not always known (Chaves et al. 2003). Drought-tolerance characteristics are controlled by many genes, known as quantitative traits (Bartels and Sunkar 2005), which will complicate the understanding of the plant response to water deficits at a molecular level. QTLs are regions within genomes that contain genes associated with a particular quantitative trait (Jones et al. 1997). Recently, a study carried out on QTLs identified one genomic region of the grapevine rootstock that was related to water extraction capacity and scion transpiration and acclimation (Marguerit et al. 2012). This finding supports previous hypotheses that rootstocks differ in their ability to provide water to the scion and that chemical signalling, primarily ABA, and hydraulic signalling via aquaporins regulate stomatal conductance.
Control of water loss
Many studies have shown that rootstocks can modify their leaf gas exchange capability in response to water deficit conditions (Candolfi-Vasconcelos et al. 1994, Düring 1994, Bica et al. 2000, Padgett-Johnson et al. 2000). Such responses, however, could vary according to different rootstock/scion combinations (Keller et al. 2012), as well as the level of water deficit experienced (Soar et al. 2006). The effect of rootstock on the photosynthetic capacity of the scion appears to increase under higher water constraint conditions (Soar et al. 2006). Under well-watered conditions, it has been reported that the scion genotype predominates the determination of transpiration efficiency, i.e. the CO2 assimilation to H2O transpiration ratio compared with the rootstock (Gibberd et al. 2001, Virgona et al. 2003). In the absence of root-to-shoot signals, differences in the leaf anatomy of the scion might play a more relevant role in the regulation of photosynthesis, as they can present different mesophyll conductance to CO2 (gm), i.e. the capacity for CO2 diffusion inside leaves (Flexas et al. 2008). It has been shown that differences in leaf anatomical properties associated with differences in gm explained the differences in photosynthesis between two pine species (Peguero-Pina et al. 2012). In relation to grapevines, it has been suggested that the level of gm could be related to the carboxylation efficiency of the specific genotype (Düring 2003). Furthermore, it has been shown that grapevine shoots have some ability to regulate ABA concentration under conditions of low water constraints, independent of root-to-shoot signalling (Soar et al. 2004).
Water losses could also be reduced by limiting transpiration through the regulation of stomatal conductance. Under conditions of water constraint, drought-sensitive rootstocks induce a lower stomatal conductance of the scion, leading to a higher reduction in photosynthetic carbon assimilation rates compared with that of drought-tolerant rootstocks (Alsina et al. 2011). Stomatal density and stomatal size determine the possible maximum stomatal conductance (Franks and Beerling 2009). The control of stomatal movement is mediated by changes in guard cell turgor, cytoskeleton organisation, membrane transport and gene expression (Hetherington 2001). Many mechanisms for stomatal regulation have been postulated, such as changes in hydraulic conductivity (Schultz 2003, Christmann et al. 2007), abscisic acid synthesis (Davies et al. 2005, Dodd 2005, Jiang and Hartung 2008) and alkalinisation of the xylem pH (Davies et al. 2002, 2005). Grapevine roots are responsible for sensing the soil water deficit and sending a signal to the shoots, thereby primarily regulating shoot growth and water use (Lovisolo et al. 2010).
Chemical signalling is based on evidence that stomatal closure is well correlated with soil water deficits, whereas it only correlates weakly with leaf water potential (Comstock 2002). Abscisic acid is one of the most studied hormones and is considered to be the most important in root-to-shoot water deficit signalling (Davies et al. 2005, Schachtman and Goodger 2008). This does not, however, rule out the possibility that other compounds are involved (Schachtman and Goodger 2008). It has been confirmed that ABA is synthesised in the roots in response to drought (Lovisolo et al. 2002). Following this, ABA is transported via the xylem to the aerial parts of the plant, where it regulates stomatal functioning and the activity of shoot meristems (Jiang and Hartung 2008). In V. vinifera, there are two genes, VvNCED and VvZEP, that have been described putatively to be involved in the ABA biosynthetic pathway (Soar et al. 2004) in response to soil water deficit in the roots (Seo and Koshiba 2002). Soar et al. (2006) have suggested that a difference in concentration in xylem ABA among rootstocks is not due to their ability to synthesise ABA but primarily due to a difference in water constraints experienced by the rootstock genotypes caused by variable water uptake capacity. The intensity of the root-to-shoot ABA signal is regulated at four anatomical levels: (i) the rhizosphere; (ii) the root cortex; (iii) the stem; and (iv) the leaves (Jiang and Hartung 2008). In V. riparia × V. labrusca, the intensity of the root-sourced ABA signal is intensified along its way, due in part to a higher xylem pH at higher node positions, resulting in a lower stomatal conductance of leaves at higher nodes compared with that of lower nodes on the stem (Li et al. 2011). Consequently, the stomatal conductance of leaves at higher nodes along the stem is lower compared with that of leaves at lower nodes. Cytokinins (CKs), which are synthesised mainly in the roots (Aloni et al. 2005), have been described as an antagonist to ABA in stomatal closure (Dodd 2005). In V. vinifera, zeatin and zeatin riboside have been found to be reduced by partial root zone drying (Stoll et al. 2000). Nevertheless, there still are many questions concerning the role of CKs in stomatal behaviour, as it is not clear which CKs will be affected by drought stress and, more so, which transport forms should be measured in the xylem (Davies et al. 2005, Schachtman and Goodger 2008).
Hydraulic signalling is based on the fact that plants would probably not survive in the absence of root-to-shoot signalling, which responds to changes in hydraulic conductivity and the failure of water transport due to cavitation and embolism (Comstock 2002). Furthermore, it is argued that, within the hydraulic continuum of the root system, the information concerning water availability can be transmitted to the leaves to control stomatal functioning (Christmann et al. 2007). Nevertheless, the mechanisms involved are still under debate (Buckley 2005). Using Arabidopsis mutants that are deficient in ABA biosynthesis and defective in ABA signalling, it was demonstrated that water constraint-induced stomatal closure requires hydraulic as well as ABA signals (Christmann et al. 2007). It was concluded that the generation of the hydraulic signal is not dependent on ABA biosynthesis and/or ABA signalling, which proves that the hydraulic signal precedes the ABA signal. It was found that own-rooted grapevine cultivars that differ in their response to soil water deficits via differences in the regulation of the leaf water potential also vary in their root response to water soil deficits in terms of aquaporin expression (Vandeleur et al. 2009). This finding suggests a close relationship between root water transport and shoot transpiration. Domec and Johnson (2012) suggested that whole-plant hydraulic conductance is driven by leaf hydraulic conductance under no water deficit and by root hydraulic conductance under water deficit.
The relative importance of chemical and hydraulic signalling in the control of stomatal functioning is debatable (Chaves et al. 2010). Some grapevine studies have concluded that hydraulic signals play a dominant role when water deficits occur (Rodrigues et al. 2008), whereas others have shown that the control is primarily due to ABA signalling and that hydraulic signalling plays a secondary role (Pou et al. 2008). Only hydraulic signalling, however, is involved during recovery from water deficits (Pou et al. 2008). Hydraulic and chemical signalling are considered to be the most important mechanisms in the regulation of stomatal conductance, and these signals probably function in an integrated way (Comstock 2002, Rodrigues et al. 2008).
Our results showed that stomatal density and size, i.e. number of stomata per unit area, are affected by water constraints and light and that the same scion grafted onto different rootstock cultivars can have different stomatal densities and sizes (Figures 5 and 6). Soil water deficit induced a response in the stomatal development that resulted in a reduction of the pore diameter (Figure 5). Leaves growing in an environment with a lower light intensity, i.e. lower ratio of red light (660 nm) to far-red light (730 nm) (R/FR) ratio, had a lower stomatal density but bigger pore diameter than leaves growing under full sun exposure (Figure 6). These results might have implications in the interaction of vigour induced by the rootstock (canopy microclimate) and canopy water demand. Significant differences in stomatal density and size were observed on Pinotage leaves grafted onto different rootstocks, where plants grafted onto 140 Ruggeri presented lower stomatal density but bigger pore diameter than those grafted onto 110 Richter and 1103 Paulsen (Figure 6). Scienza and Boselli (1981) found that rootstocks considered drought tolerant have lower stomatal density in their leaves in comparison with that of rootstocks considered drought sensitive. The mechanisms involved in stomatal development, as affected by rootstock, cannot be explained at this stage. It is hypothesised, however, that differences in hydraulic conductance between rootstocks affect the plant water status, thereby affecting leaf growth, and that they consequently cause variability in stomatal density and size that is closely related to leaf gas exchange and water use efficiency (Xu and Zhou 2008).
Figure 5. Stomata on the abaxial leaf surface of cv. Pinotage, growing under greenhouse conditions, grafted onto 99 Richter (a) subjected to water constraints and (b) without water constraints. Average stomatal density (pores/mm2) of 109.5 ± 6.2 and 95.7 ± 6.2 for water constraints and without water constraints, respectively. Average guard cell length (μm) of 13.5 ± 0.24 and 12.6 ± 0.24 for water constraints and without water constraints, respectively (150 × magnification, panels a and b; scale bar represents 20 μm) (Dr Ignacio Serra, unpublished data).
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Figure 6. Stomata on the abaxial leaf surface of cv. Pinotage, growing under field conditions, grafted onto 1103 Paulsen (a) subjected to water constraints and fully exposed to sunlight, (b) subjected to water constraints and shaded; compared to the same scion grafted onto 140 Ruggeri, (c) subjected to water constraints and fully exposed to sunlight and (d) subjected to water constraints and shaded. Average stomatal density (pores/mm2) of 119.1 ± 6.3, 91.0 ± 6.3 (Pinotage/1103 P), 113.8 ± 6.3 and 96.3 ± 6.3 (Pinotage/140 Ru), fully exposed to sunlight and shaded, respectively. Average guard cell length (μm) of 13.2 ± 0.31, 20.0 ± 0.31 (Pinotage/1103 P), 16.0 ± 0.31 and 17.2 ± 0.31 (Pinotage/140 Ru), fully exposed to sunlight and shaded, respectively (150 × magnification, panels a, b, c and d; scale bar represents 20 μm) (Dr Ignacio Serra, unpublished data).
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