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The classical view that the drought-related hormone ABA simply acts locally at the guard cell level to induce stomatal closure is questioned by differences between isolated epidermis and intact leaves in stomatal response to several stimuli. We tested the hypothesis that ABA mediates, in addition to a local effect, a remote effect in planta by changing hydraulic regulation in the leaf upstream of the stomata.
By gravimetry, porometry to water vapour and argon, and psychrometry, we investigated the effect of exogenous ABA on transpiration, stomatal conductance and leaf hydraulic conductance of mutants described as ABA-insensitive at the guard cell level.
We show that foliar transpiration of several ABA-insensitive mutants decreases in response to ABA. We demonstrate that ABA decreases stomatal conductance and down-regulates leaf hydraulic conductance in both the wildtype Col-0 and the ABA-insensitive mutant ost2-2.
We propose that ABA promotes stomatal closure in a dual way via its already known biochemical effect on guard cells and a novel, indirect hydraulic effect through a decrease in water permeability within leaf vascular tissues. Variability in sensitivity of leaf hydraulic conductance to ABA among species could provide a physiological basis to the isohydric or anisohydric behaviour.
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Leaves of plants are furnished with stomata made of pores surrounded by pairs of adjacent guard cells that tightly regulate the pore aperture. By facilitating gas diffusion, open stomata allow CO2 to reach sites of photosynthesis, but simultaneously let water vapour exit the leaf to the atmosphere. Hence, by opening or closing, stomata regulate not only the carbon assimilation but also the leaf water status. Fascinatingly, stomatal movements also involve water relations at the cell level: actively pumped osmotica regulate turgor pressure in the guard cells, which modulates the aperture of the central pore. Thus, it is not surprising that a key feature of plants’ adaptation to their environment is leaf hydraulics (Sack & Holbrook, 2006), that is, all leaf characteristics that determine their capacity to transport water to the evaporating sites. Accordingly, evolutionary processes have led to the selection of regulatory mechanisms at several levels in the leaf water pathway upstream to stomata, including xylem, vascular parenchyma, bundle sheath and mesophyll.
Among the numerous environmental factors affecting stomatal aperture and hence stomatal conductance for water vapour (gs), water availability in the soil and atmospheric vapour pressure dominate in dry conditions. A decrease in soil water potential (Ψsoil) or an increase in atmospheric vapour pressure deficit (VPD) induces a hydraulic cascade of water potential drops in the plant, leading to a decrease in gs, although the precise physical mechanism by which gs and water potential are coordinated remains a matter of debate (Buckley, 2005; Peak & Mott, 2011). A biochemical mediation also participates in stomatal closure when plants are submitted to drought stress and can trigger stomatal closure even in absence of any change in leaf water potential (Ψleaf) (Gollan et al., 1986). ABA, as the major mediator involved in this response, is synthesized in roots (Simonneau et al., 1998) and shoots (Christmann et al., 2005, 2007) and is transported to the guard cells where it induces stomatal closure, depending on the leaf capacity to compartmentalize and metabolize ABA (Davies & Zhang, 1991; Trejo et al., 1993, 1995; Wilkinson & Davies, 2002). Guard cell response to ABA is triggered by a molecular cascade where the binding of ABA to its receptors (PYR/PYL/RCARs) releases the inhibition of PP2Cs (notably ABI1 and ABI2) on SnRK2s (notably OST1), which in turn initiates many ABA responses, including the activation of the efflux ion channels (in particular SLAC1), ultimately leading to water efflux and stomatal closure (Kim et al., 2010). This biochemical effect of ABA on gs combines with hydraulic effect as though low Ψleaf sensitizes stomata to ABA, leading to stomatal closure under water stress (Tardieu & Davies, 1992, 1993).
Interestingly, Shatil-Cohen et al. (2011) reported that xylem-fed ABA reduces leaf hydraulic conductance for water in the liquid phase (Kleaf), in particular by decreasing water permeability in the vascular bundle sheath cells. This emergent role of perivascular tissues in the sensitivity of leaf hydraulics to ABA questions the classical view of ABA solely acting at the guard cell level to induce stomatal closure. The mesophyll is already known for its capacity to catabolize ABA, thus decreasing the apparent sensitivity of stomata to exogenous ABA in intact leaves compared with isolated epidermis (Trejo et al., 1993, 1995). Moreover, conflicting results have been found between isolated epidermis and intact leaves with a higher sensitivity of stomata in leaves compared with isolated epidermis in response to humidity, light and CO2 (Mott et al., 2008; Shope et al., 2008; Mott, 2009; Sibbernsen & Mott, 2010). This suggests that signals originated from the leaf mesophyll partly govern stomatal responses to a variety of stimuli in intact leaves.
In this study, we examined the hypothesis that, in planta, ABA remotely controls stomatal response by changing hydraulic regulation in the leaf upstream of stomata. We investigated the effect of exogenous ABA on transpiration, gs and Kleaf in Arabidopsis mutants, previously described as being ABA-insensitive according to assays based on epidermal peels. We show that ABA decreases stomatal transpiration as well as Kleaf of the intact ABA-insensitive mutants. We propose a conceptual model in which ABA closes stomata in adverse hydraulic conditions via its biochemical effect on the guard cells, but also via a hydraulic feedback triggered by a drop in Kleaf, presumably through a decrease in water permeability within the leaf vascular tissues.
Materials and Methods
Plant material and growth conditions
A set of mutants of Arabidopsis thaliana (L.) Heynh. was selected for their stomatal insensitivity to ABA as evidenced in assays using epidermal peels. The ABA-insensitive mutants abi1-1 and abi2-1 (Koornneef et al., 1984) are dominant negative mutations of ABI1 and ABI2, two homologue protein phosphatases type 2C (PP2Cs) involved in ABA transduction pathway (Leung et al., 1997). The slac1-1 mutant is impaired in a guard cell channel whose anion efflux activity normally triggers stomatal closure when activated by OST1 in response to ABA (Negi et al., 2008; Vahisalu et al., 2008). In the mutants ost2-1 and ost2-2, the plasma membrane H+-ATPase AHA1 is constitutively activated, which prevents ABA-mediated stomatal closure (Merlot et al., 2007). The wildtype controls were chosen according to the mutant background: Columbia-0 (Col-0) for ost2-2 and slac1-1, and Landsberg erecta (Ler) for abi1-1, abi2-1 and ost2-1.
For gravimetry experiments, plants were grown as in Pantin et al. (2011) in pots filled with a mixture of loamy soil and organic compost in growth chambers with a 10 h photoperiod. Each pot was weighed and well-watered daily so that soil water content was maintained at a value corresponding to a predawn water potential of –0.3 MPa (Hummel et al., 2010). For porometry and measurements of leaf hydraulic conductance, plants were grown with an 8 h photoperiod in water-saturated loam as to obtain larger leaf lamina.
Water vapour and argon porometry
For water vapour porometry, stomatal conductance (gs) was measured using a Li-Cor 6400 gas analyser system (Li-Cor Inc., Lincoln, NE, USA) equipped with a clamp-on leaf cuvette (modified 6400-40 Leaf Chamber Fluorometer; Li-Cor, Inc.).
For simultaneous water vapour and argon (Ar) porometry, we developed a gas exchange setup involving two Li-Cor 6400 systems. One analyser system was equipped with a top-side leaf cuvette only, while the other one was only equipped with a corresponding bottom-side cuvette. By clamping the corresponding bottom and top cuvettes over a leaf that fully filled the 2-cm-diameter aperture of the cuvettes, gas composition was independently controlled and monitored for each leaf side using the appropriate Li-Cor 6400 system. In the top cuvette, gas exchange was performed using background air supply (containing Ar at natural abundance), whereas in the lower cuvette, gas exchange was performed in a nitrox atmosphere (79% N2 and 21% O2 background). The rate of Ar diffusion through the leaf was measured by monitoring the Ar appearing in the gas flow exiting the lower cuvette using online isotope-ratio mass spectrometry (IRMS, VG 14-80, Middlewich, UK). The rate of transpiration was measured separately for the upper and the lower sides of the leaf using both Li-Cor analysers. Leaf temperature was monitored using remote infrared thermometry of the lower leaf side in the bottom cuvette. Calculation of leaf conductances to Ar and water (gAr and gw, respectively) is described in the Supporting Information, Notes S1.
These measurements were performed on detached leaves. Leaves were excised, and after re-cutting under water their petioles were maintained in degassed water. The leaf blade was immediately enclosed in the cuvette and gs was allowed to reach a steady state (at least 20 min). For ABA treatment, the water supply was substituted by a degassed 50 μM ABA solution to feed the leaf through the petiole. Leaf temperature was controlled at 23.5 °C, and leaf-to-air VPD was maintained below 0.8 kPa. A 10% blue and 90% red illumination was set to provide 500 μmol m−2 s−1 photosynthetically active radiation irradiance using an LED array on the top-side cuvette.
Leaf hydraulic conductance
Kleaf was measured on detached leaves according to the principles of the evaporative flux method (Sack et al., 2002), that is, calculated as the transpiration flux (E) divided by the water potential gradient, ΔΨ, between the xylem at the leaf entry (Ψxylem) and the bulk leaf (Ψleaf). Contrary to other methods where the flow is forced during the experiment, leading water to travel in non-physiological ways, this method is the closest to what occurs during in vivo transpiration (Sack et al., 2002). Transpiration was measured using a Li–Cor 6400 gas exchange system equipped with the 2 × 3 cm2 aperture standard leaf cuvette, and then two discs per leaf were immediately punched and inserted in a sealed chamber carrying a thermocouple (C-52, Wescor, Logan, UT, USA) connected to a wet bulb depression psychrometer (Psypro, Wescor, Logan, UT, USA). Ψleaf was determined after 5 h equilibration by averaging the values obtained for the two discs. Because petioles were dipped in degassed water or ABA solution with near-zero osmolarity, Ψxylem was close to zero, so that ΔΨ = –Ψleaf.
Leaf preparation, ABA treatment and gas exchange measurements were performed as described for the porometry experiments, except that leaf-to-air VPD was maintained within 0.8 and 1.0 kPa, leaf temperature was 20.7 ± 0.3 °C, and illumination was supplied using a white light source similar to that used in the growth chamber.
Transpiration by gravimetry
For gravimetry experiments, fully expanded leaves were excised throughout the daytime, except during the first hour to avoid nonsteady-state transpiration (Hosy et al., 2003; Lebaudy et al., 2008). Water was prevented from leaking from the petiole using impermeable glue (Henkel, Düsseldorf, Germany). The fresh mass of each leaf was recorded immediately after excision and 10 min later using a balance of 10−5 g precision (Sartorius, Goettingen, Germany) connected to a computer. Leaves were repositioned in the growth chamber during this 10 min period. For the ABA treatment, 0.5 ml of a (±) ABA (Fluka, Buchs, Switzerland) solution was sprayed on the rosette. After waiting 2 h for the ABA solution to evaporate, excision and transpiration assays were performed as described earlier. At the end of each measurement, leaves were scanned and leaf area determined using ImageJ (Rasband, 2009). The transpiration rate was computed from the slope of the fresh mass loss per unit leaf area and time. The relationship between mass loss and time is linear for this time window in Arabidopsis (e.g. Hosy et al., 2003), meaning that stomatal response (closure or opening) can be neglected in these conditions.
Statistical analyses were performed with R 2.6.2 (R Development Core Team, 2008). The Kruskal–Wallis test at the 5% alpha level was used for comparison of means.
Stomata of ABA-insensitive mutants close in response to ABA
Stomatal conductance response to xylem-fed exogenous ABA was monitored in detached leaves of ABA-insensitive mutants. Applying a 50 μM ABA solution, gs decreased rapidly in the wildtype plants, and also in all of the investigated ABA-insensitive mutants: abi1-1, abi2-1, ost2-1, ost2-2, and slac1-1 (Fig. 1a,b). Hence, all mutants described as ABA-insensitive on isolated epidermis displayed a significant decrease in gs in response to ABA. Argon porometry was used to probe the stomatal origin of the ABA-induced decrease in leaf conductance to water. By creating an Ar gradient between the upper side and the lower side of the leaf and monitoring Ar diffusion through stomata, we were able to trace stomatal conductance independently of the fluxes of water vapour (Moreshet et al., 1968). The effect of ABA on leaf conductance for Ar diffusion through the leaf (gAr) and the equivalent conductance estimated from the water vapour exchanges (gw) were largely similar for both the wildtype and ost2-2 (Fig. 1b,c). This unambiguously shows that stomatal closure is involved in ABA-induced decrease of leaf conductance to water vapour and that other processes are likely to be of minor importance (i.e. change of cuticular conductance to water vapour, variation of water vapour pressure at the evaporating sites in the mesophyll; see Farquhar & Raschke, 1978).
The ABA effect on stomata in ABA-insensitive mutants was confirmed in fully expanded leaves detached from intact rosettes by monitoring the transpiration rate gravimetrically. A preliminary response curve performed on Col-0 and ost2-2 indicated that transpiration of Col-0 was significantly affected by ABA for concentrations ≥ 10 μM, while > 100 μM were required to observe a significant decrease in ost2-2, a saturating concentration for Col-0 (Fig. 2a). We therefore applied the highest concentration tested (400 μM) to measure the response to ABA in other genotypes. In line with the results obtained using xylem-fed excised leaves, ABA sprayed on the intact rosette was able to trigger a decrease in transpiration in all tested genotypes, including ost2-2, abi1-1 and abi2-1 (Fig. 2b). In conclusion, ABA taken up by the leaf, either externally applied to the rosette or internally via the petiole, is able to induce stomatal closure in the so-called ABA-insensitive mutants.
ABA induces a similar decrease in leaf hydraulic conductance in Col-0 and ost2-2
Following Shatil-Cohen et al. (2011), we hypothesized that ABA induced a decrease in leaf hydraulic conductance, thereby causing ABA-induced stomatal closure in the ABA-insensitive mutants. To test this hypothesis, we measured Kleaf in Col-0 and ost2-2 during the course of an independent dedicated experiment. Gas exchange measurements confirmed that gs of ost2-2 as well as Col-0 were sensitive to ABA (Fig. 3a). Accordingly, transpiration decreased in both genotypes (Fig. 3b). At constant Kleaf, it would be expected that Ψleaf becomes less negative as a result of the decrease in transpiration. By contrast, Ψleaf tends to become more negative in response to ABA, yet no significant difference could be detected at the 5% threshold (Fig. 3c). This underlined the fact that Kleaf decreases in response to ABA in both genotypes (Fig. 3d). Interestingly, the ABA-induced decrease in Kleaf was similar in Col-0 (−37%) and ost2-2 (−35%). This suggests that ABA decreases Kleaf through a shared mechanism, unravelling an important and novel transduction pathway involved in plant response to drought stress.
Stomata embody the compromise between the conflicting needs for H2O conservation and CO2 capture, the basic molecules for plant growth (Pantin et al., 2012). In this study, we provide new insights into the connected roles of ABA and leaf hydraulics in stomatal regulation.
ABA-induced stomatal closure in planta involves two distinct signals
Our experiments clearly show that stomatal transpiration of mutants described as ABA-insensitive, as based on epidermal peels, decreases in response to ABA in planta (Figs 1-3). Leymarie et al. (1998) also observed that transpiration of abi1-1 and abi2-1 was affected when ABA was added in the root medium of intact plants. This ABA effect in single mutants could be partly attributed to a functional redundancy between both proteins, which share strong homologies (Leung et al., 1997). This could also explain why ABA slightly decreases stomatal aperture on epidermal peels of abi1-1 via still active ABI2 (Merlot et al., 2002), and of abi2-1 via still active ABI1 (Merlot et al., 2001). However, the double mutant abi1-1 abi2-1 also displayed a decrease in gs when fed with ABA (Leymarie et al., 1998). Furthermore, slac1-1 and ost2-2 stomata were totally insensitive to ABA up to the highest concentration (10 and 100 μM, respectively) tested on epidermal peels, well over the physiological range (Merlot et al., 2007; Negi et al., 2008; Vahisalu et al., 2008). To reconcile these observations on epidermal peels with our results in intact plants, we propose that ABA in planta controls stomatal conductance via an additional signalling pathway originating in the leaf internal tissues and circumventing the pathway described so far at the guard cell level.
Vascular tissues are likely to play a central role in the alternative signal, which is triggered by ABA within the leaf internal tissues and propagates to the stomata. We propose that this regulation involves translocation or synthesis of ABA in vascular parenchyma, which impairs water permeability of bundle sheath cells (Shatil-Cohen et al., 2011). This, in turn, could trigger a drop in water potential that could propagate hydraulically to the mesophyll to finally reach stomata and reduce transpiration, which feeds back on leaf water potential. Four lines of evidence are in agreement with this proposed model. First, vascular ABA metabolism is crucial in response to drought (Christmann et al., 2005, 2007; Endo et al., 2008) and high air humidity (Okamoto et al., 2009). Secondly, impairment in leaf water status provoked by high light intensity triggers ABA biosynthesis in vascular parenchyma and signalling events in neighbouring bundle sheath cells (Galvez-Valdivieso et al., 2009). Thirdly, our results not only confirm the ABA-induced decrease of Kleaf in Col-0, as already shown (Shatil-Cohen et al., 2011), but are also evidence of an effect of the same magnitude in the ABA-insensitive ost2-2 (Fig. 3). Fourthly, the fact that a 50 μM ABA solution has no significant effect on stomata of ost2-2 when sprayed on the leaf surface (Fig. 2a) but has a significant effect when xylem-fed (Figs 1a, 3a) indicates that the ABA-induced hydraulic decrease in gs is perceived in the internal tissues of the leaf. This is consistent with higher ABA concentrations required to observe a decrease in gs on sprayed leaves of ost2-2 (Fig. 2a), and also with the results of Shatil-Cohen et al. (2011) showing no effect on Kleaf of Col-0 when a 10 μM ABA solution is smeared directly on the leaf surface. Thus, ABA has a dual effect on stomata: a direct biochemical action on guard cells and an indirect hydraulic action through a decrease in leaf water permeability triggered within vascular tissues.
A new model accounting for the dual ABA action on stomata
We synthesized these results in a conceptual model (Fig. 4), which accounts for the different roles of ABA and hydraulics in regulating gs. In the daytime, light causes stomata to open. Transpiration increases and then Ψleaf decreases, which triggers ABA synthesis in vascular tissues and in guard cells. This is suggested by low but significant ABA concentrations in vascular tissues and stomata of Arabidopsis cotyledons in the absence of water stress (Christmann et al., 2005) and also by ABA2 and AAO3 localization in turgid plants (Koiwai et al., 2004; Endo et al., 2008). This production of ABA induced by low Ψleaf during the day could prevent stomata from reaching their maximal opening, through the transduction network in guard cells partly involving ABI1, ABI2 and the effectors OST2 and SLAC1 (Kim et al., 2010), finally lowering any risk of cavitation. Stomata are ultimately regulated by a hydraulic mechanism that follows the balance between epidermis and guard cell turgor pressures (Franks et al., 1998). In conditions where stomata are expected to close, opposing, wrong-way responses resulting from the back-pressure of epidermal cells are frequently observed, that is, a transient stomatal opening under low air humidity. However, the capacity of stomata to adjust their turgor by changes in osmotic potential makes the feed-forward response dominate in the steady state (Buckley & Mott, 2002; Buckley et al., 2003; Buckley, 2005). This could partly explain why aba1, abi1-1 and abi2-1 have a wildtype response to a decrease in humidity (Assmann et al., 2000), although ABA synthesis and signalling are involved in the stomatal response to low humidity (Xie et al., 2006). Under either soil or atmospheric water stress, ABA concentration rises in vascular parenchyma, because ABA is imported from the roots or synthesized locally (Christmann et al., 2005, 2007; Endo et al., 2008). Vascular ABA translocates to the guard cells to promote stomatal closure (Christmann et al., 2005). An indirect, remote mechanism allows the closure to be amplified. Vascular ABA decreases Kleaf putatively by inactivating bundle sheath aquaporins such as the plasma membrane intrinsic proteins (PIPs; Shatil-Cohen et al., 2011; Fig. 3), through a transduction pathway distinct from the network already described in guard cells. Further studies are required at molecular and cellular levels to unravel the molecular link between ABA and PIPs and to identify the localization of this interaction. However, the dialogue between the vascular parenchyma and the bundle sheath arises as a central component of the ABA-induced hydraulic control of stomata. In line with this, the junction between xylem and bundle sheath is thought to be a major bottleneck of leaf water transfer in Arabidopsis (Ache et al., 2010). The decrease in Kleaf lowers Ψleaf (or, more precisely, the water potential actually sensed by stomata), and gs in cascade according to a hydraulic mechanism, biophysical transduction of which is debated in the literature (Buckley, 2005; Peak & Mott, 2011). Finally, the decrease in gs lowers transpiration, which stabilizes Ψleaf.
Such a mechanism triggered by ABA mimics the response of plants subjected to high VPD (low humidity), labelled ‘regime B’ by Monteith (1995). In this regime, an increase in VPD leads to a dramatic stomatal closure in such a way that transpiration is reduced and hence Ψleaf can be maintained (or even increased). Interestingly, this regime has been proposed to result from a decline in Kleaf caused by xylem cavitation (Oren et al., 1999; Buckley & Mott, 2002; Dewar, 2002). A similar situation is induced in our experiments where Kleaf decreases upon ABA application. How stomata can respond to such a decrease in Kleaf remains controversial. We suggest that guard cells sense a local decrease in water potential before any measurable change at the bulk leaf level. Such fine-tuning can respond to even a slight decrease in Kleaf and avoid a catastrophic burst of cavitation.
Coordinating water supply and balance through stomatal sensitivity to vascular ABA: a determinant of species strategy to face drought stress?
The fact that ABA induces a decrease in Kleaf through changes in bundle sheath aquaporin is puzzling given that ABA generally increases root hydraulic conductivity (e.g. Ludewig et al., 1988; Zhang et al., 1995; Hose et al., 2000; Thompson et al., 2007), at least partly as a result of a transcriptional and post-translational regulation of aquaporins (Wan et al., 2004; Zhu et al., 2005; Parent et al., 2009). However, this makes sense at the molecular level because the aquaporin family comprises multiple isoforms with various degrees of functional specialization (Maurel et al., 2008). Furthermore, the physiological significance of this difference between roots and leaves in the response of water transport to ABA could be that it contributes to stabilize the water continuum in the plant, by increasing the inflow while decreasing the outflow as a response to an imbalance between water supply from the roots and water demand from the leaves. At the leaf level, controlling stomata by an ABA-induced hydraulic mechanism within vascular tissues could also be a crucial component of the coordination between water supply and water demand. Overall, positive correlations between Kleaf, gs, vein density and stomatal density (Brodribb & Jordan, 2011) converge to the conclusion that leaf water supply and demand are strongly coordinated in plants. Interestingly, some species have evolved bundle sheath extensions which improve the hydraulic coupling between the epidermis and the rest of the leaf and accelerate hydropassive stomatal movements (Buckley et al., 2011). Adding a signalling component upstream of stomata would improve the stability of such coordination under water stress. Hence, the hydraulic effect of ABA we have described here may have a preponderant role in elaborating species responses to drought. Under water stress, some species maintain leaf water potential (isohydric behaviour) while other favour stomatal conductance for the maintenance of CO2 assimilation (anisohydric behaviour). The isohydric behaviour is specifically a result of the apparent enhancement by low Ψleaf of the ABA effect on gs (Tardieu & Davies, 1992; Tardieu & Simonneau, 1998). This physiologically obscure interaction between ABA sensitivity and hydraulics could arise from the effect of ABA on Kleaf, Ψleaf and gs, as described in this study. On the other hand, aquaporin regulation has been suggested as a relevant molecular framework to distinguish between isohydric and anisohydric behaviours (Sade et al., 2009; Vandeleur et al., 2009). As a perspective, it is thus tempting to speculate that the vascular ABA-responsive component described here (putatively aquaporins) as well as Kleaf are much less sensitive to ABA in anisohydric species, providing a putative molecular basis and biophysical process for the difference between isohydric and anisohydric species.
We thank Nathalie Leonhardt for supplying seeds of ABA-insensitive mutants. Myriam Dauzat, Gaëlle Rolland, Alexis Bédiée and Remy Puppo are gratefully acknowledged for technical support during the experiments. This work was supported by the French Ministry of Research (PhD fellowship to F.P.), CNRS and PACA region (‘BDI’ fellowship to D.J.), the Fundação para a Ciência e Tecnologia, Portugal (postdoctoral fellowship ref. SFRH/BPD/34429/2006 to J.M.C.), the French Agence Nationale de la Recherche (LEAFFLUX project, BLAN 07-1-192876 to B.G. and F.M.), the European Commission through the FP6 AGRON-OMICS Integrated Project (grant no. LSHG–CT–2006–037704 to B.M.) and the Marie Curie FP5 Research Training Network program STRESSIMAGING (HNRT-CT_2002 00254 to B.G. and J.M.C.).