Differences in stomatal responses and root to shoot signalling between two grapevine varieties subjected to drought

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1 CSIRO PUBLISHING Functional Plant Biology, 21, 37, Differences in stomatal responses and root to shoot signalling between two grapevine varieties subjected to drought Alexandros Beis A and Angelos Patakas A,B A Laboratory of Plant Production, School of Natural Resources and Enterprises Management, University of Ioannina, G. Seferi 2, 3 1 Agrinio, Greece. B Corresponding author. apatakas@cc.uoi.gr Abstract. A comparative study on stomatal control between two grapevine varieties (Vitis vinifera L. cvs Sabatiano and Mavrodafni) differing in their ability for drought adaptation was conducted using 3-year-old own-rooted plants. The plants were subjected to prolonged drought stress by withholding irrigation water. The relationship between predawn water potential and maximum stomatal conductance indicated significant differences in stomatal sensitivity to drought between the two varieties. Stomatal closure occurred at higher values of predawn water potential in Sabatiano compared with Mavrodafni. No significant differences were found in plant hydraulic conductance and osmotic potential at full turgor (p 1 ) between the two varieties. Leaf and root ABA concentrations increased more rapidly in Mavrodafni compared with Sabatiano at the beginning of the drought period. Furthermore, Mavrodafni also exhibited significantly higher xylem ph values as well as higher stomatal sensitivity to ABA and ph increase compared with Sabatiano. Results suggest that these two grapevine varieties might have evolved different strategies in order to adapt under drought conditions. In particular, the greater ability for drought adaptation in Sabatiano might be attributed to the more efficient regulation of stomatal closure. In contrast, chemical signalling in Mavrodafni seems to be the main mechanism for drought adaptation. Additional keywords: abscisic acid, hydraulic conductance, isohydricity, stomatal conductance. Introduction Grapevine (Vitis vinifera L.) varieties are characterised by high heterogeneity as they are grown in the region from the cool temperate 5 N through the dry Mediterranean-type climates to the tropics (Schultz 23). This environmental variation is believed to influence the divergence among varieties in the plasticity of traits and also leads to large differences in genotypic components concerning their reaction to drought (Duan et al. 27). In general, varieties originating from more arid environments are considered to be more adaptive to drought compared with those with mesic origin, which are considered as more vulnerable (Schultz 23). It is believed that the above differences are closely related to different mechanisms used by each variety in order to withstand drought conditions. Moreover, predicted scenarios of climate change over the coming decades include global warming and shifts in the amount, seasonality and distribution of precipitation. The foreseen increase in both the intensity and length of the dry periods especially in the Mediterranean area is expected to result in extended and severe drought events (Rodrigues et al. 28). Therefore, evaluation of the different mechanisms of the way in which grapevine varieties adapt to drought is considered of major importance. Stomatal regulation has been proposed as a determining factor for the ability of grapevine varieties to withstand drought conditions (Schultz 23; Soar et al. 24). The regulation of stomatal closure under drought conditions is complex involving hydraulic, chemical and even electrical signals (Soar et al. 24; Pou et al. 28). It is generally accepted that ABA is one of the main components involved in the control of stomatal conductance as the soil dries (Wilkinson and Davies 22). However, previous studies on grapevines subjected to different water regimes indicated considerably variable results concerning the relation between stomatal conductance and ABA concentration. In particular, stomatal conductance was found to be closely related to xylem sap ABA concentration when measurements performed in the morning, whereas correlation was lacking during the course of the day (Correia et al. 1995). Moreover, xylem sap ABA during veraison did not seem to be involved in stomatal control in field-grown grapevines, whereas the opposite was evident at mid-ripening (Rodrigues et al. 28). Recently, Lovisolo et al. (22) showed that leaf ABA concentration exerts significant control on stomatal behaviour during water deficit in pot-grown grapevines. These findings, although diverging, do not necessarily contradict each other given that different methodologies were used in order to discriminate the possible role of ABA on stomatal regulation. In fact, in some of these studies, ABA concentration in the xylem sap was considered as a more accurate variable to identify ABA effects on stomatal conductance, whereas in the others the ABA concentration in leaves was used. There were also significant differences concerning the time of sampling as well as the degree of water stress intensity implied, which probably affect ABA concentration values making the interpretation of the results CSIRO /FP /1/2139

2 14 Functional Plant Biology A. Beis and A. Patakas even more difficult (Borel et al. 1997). Therefore, the possible role of ABA to drought adaptation in different grapevine varieties still remains more or less obscure. In contrast, hydraulic control was also included in models describing regulation of stomatal aperture in several publications (e.g. Lovisolo et al. 22; Soar et al. 24). Studies on two grapevine cultivars (Grenache and Syrah) attributed their contrasting stomatal sensitivity to drought to differences in hydraulic conductance (Schultz 23). It was also suggested that, in many cases, depending on the variety and/or experimental conditions, hydraulic limitations rather than chemical signalling might dominate stomatal control (Comstock 22; Rodrigues et al. 28). Thus, the hypothesis that there are differences in the mechanism of stomatal control between grapevine varieties that are related to their ability for drought adaptation, remains to be tested. Therefore, the aim of this study was to evaluate the relative contribution of either hydraulic factors or chemical signals in the regulation of stomatal behaviour in two grapevine varieties differing in their ability for drought adaptation. Materials and methods Plant material and treatments The experiment was conducted during two consecutive growing periods (26 to 27) at the experimental station of the University of Ioannina, located in Agrinio, western Greece (38 37 N, E). Two varieties of 3-year-old, own-rooted grapevine plants (Vitis vinifera L. cvs Sabatiano and Mavrodafni) were grown into 25-L pots under a permanent rain shelter. The pots were filled with a mixture of soil, sand, peat and vermiculite (3%, 1%, 3% and 3% v/v, respectively) and were wrapped in aluminium foil to minimise radiationinduced heating of the root system. The plants were pruned to a single-bud spur. After bud break, the single shoot of each plant was trained vertically, while laterals and clusters were removed immediately after formation. During the first 2 months, plants were uniformly irrigated to soil capacity on a daily basis. Thereafter, the plants of each variety were divided into two uniform groups consisting of 4 plants of similar leaf area. Two different water regimes were applied in each group over a 24-day period. In particular, the first group of each variety was irrigated to soil capacity every 3 days (well-watered treatment; WW), whereas the second group was deficit-irrigated receiving half of the water given to control plants at the same frequency (DI). After the 24-day drought period, stressed plants were fully irrigated to soil capacity. The same experiment was repeated three times in 26 and three times in 27. Water relations and gas exchange Soil moisture sensors (Echo EA-1, Decagon Devices, Pullman, WA, USA) were installed in three pots per treatment in order to continuously monitor the volumetric soil water content. Predawn leaf water potential (5 hours; Y PD ) and midday water potential (12 14 hours; Y MD ) were measured with a Scholander-type pressure chamber (SKPM 14/8; Skye Instruments, Powys, UK) every 3 days (nine times over the drought period corresponding to Days 1, 3, 6, 9, 12, 15, 18, 21 and 24, respectively), before irrigation, in six fully expanded leaves per treatment obtained from different plants. Osmotic potential at full turgor (p 1 ) was also determined on six leaves per treatment at the end of the drought cycle using the pressure volume technique (Patakas et al. 22). Maximum stomatal conductance (g s ), photosynthesis rate (P N ) and transpiration rate (E) were measured daily from 9 to 1 hours at saturating light intensity (PAR 11 mmol m 2 s 1 ) in six leaves per treatment using a portable gas exchange system (LCpro + ADC BioScientific, Herts, UK). The leaves that were used for gas exchange determinations were obtained from the same plants that were used for predawn leaf water potential measurements. Plant hydraulic conductivity (K l ) was calculated from the Ohm s law analogy for the soil plant atmosphere continuum (Lovisolo et al. 22; Pou et al. 28): E ¼ K 1 ðy soil Y leaf Þ where E, Y leaf and Y soil are transpiration rate, leaf water potential and soil water potential, respectively. Y PD was taken as a proxy for Y soil and Y MD was taken as Y leaf. ABA and ph determinations Xylem sap ph measurements were conducted at predawn on four of the six leaves per treatment that were previously used for Y PD determinations. Xylem sap was extracted from each leaf using a pressure chamber following methodology described by Beis et al. (27) where, after the balancing pressure was obtained, the cut surface of the leaf was blotted dry. Thereafter, the pressure was slowly increased in steps of.3 MPa min 1 until the first droplets of xylem sap appeared at the cut surface of the petioles. These droplets were discarded in order to avoid contamination from damaged tissues (Else et al. 1994, 1995; Wilkinson and Davies 1997). Sap collection from each leaf was fractionated into several successive extracts according to the pressure level finally applied: fraction (I), (II) and (III) corresponding to 1 MPa, 2 MPa and 2.5 MPa pressure, respectively. Xylem sap ph was measured using a microelectrode (HI 183, Hanna HI 926; HANNA instruments, Inc, Woonsocket, RI, USA) in the fraction III exudates sap. The sap of the other two fractions (I, II) was combined and used to determine xylem ABA. Concomitant measurements of leaf and root ABA concentration were performed on four leaves and root samples per treatment. Leaves, roots and xylem sap samples were all immediately frozen in liquid nitrogen and stored at 8 C until analysis for ABA concentration. ABA was extracted following the protocol described in Lovisolo et al. (22) with minor modifications. Frozen homogenised plant tissue was extracted for 48 h in 8% (v/v) methanol. Extracts were centrifuged twice at 1 g for 2 min and the supernatant was passed through a reversed phase C 18 -Sep-Pak cartridge (Waters, Milford, MA, USA) to remove lipids and pigments. Methanol was removed under vacuum and the aqueous residue was partitioned three times against ethyl acetate at ph 3.. Ethyl acetate of the combined organic fractions was removed under vacuum. The residue was re-suspended in Tris-buffered saline (15 mm NaCl, 1 mm MgCl 2 and 5 mm TRIS, ph 7.8) and subjected to an immunological ABA assay (Phytodetek Kit supplied by Agdia, Elkhart, IN, USA).

3 Drought signalling in grapevines Functional Plant Biology 141 Statistical analysis Except for the data in Fig. 3a, which are from measurements conducted in 26 7, all other data are from measurements conducted during 27. Statistical data analysis was performed according to a completely randomised ANOVA. Duncan s multiple range test (P <.5) were carried out to test the significance of differences between treatment means using SPSS ver. 15. for Windows (SPSS, Chicago, IL, USA). Analysis of covariance (ANCOVA) was also carried out to determine whether treatments (cvs Mavrodafni and Sabatiano under deficit irrigation) caused differences in the relationship between various x-covariates and y-independent variables (Figs 2 6). A change in the sensitivity of the y-independent variables to the x-covariates is given by a significant interaction term (x-covariate by treatment). Regressions analysis was calculated using Origin software (ver. 6.; Microcal Software, Inc., Northampton, MA, USA). Results Predawn leaf water potential (Y PD ) as well as soil water content decreased continuously in drought stressed treatments in both grapevine varieties reaching minimum values at the end of the drought period (Fig. 1). However, soil water content was higher in Sabatiano compared with Mavrodafni during the first days after starting the drought treatment (Fig. 1). Mavrodafni exhibited a steeper decline in Y PD in response to soil drying, which resulted in significantly lower values of Y PD compared with Sabatiano at the beginning of the drought period (Days 9 and 12) (Fig. 1). The time course of maximum stomatal conductance and photosynthetic rate also revealed significant differences between the two varieties. In particular, both g s and P N were lower in Mavrodafni deficit-irrigated plants compared with Sabatiano during the first days of the drought period (Days 6 12) (Fig. 1). Furthermore, midday leaf water potential values (Y MD ) in Mavrodafni were significantly lower compared with Sabatiano for the same values of predawn leaf water potential (Fig. 2). The relationship between Y PD and maximum stomatal conductance indicated significant differences in stomatal sensitivity between the two varieties (Fig. 3a). Mavrodafni exhibited higher values of stomatal conductance for the same values of Y PD in the beginning of the stress period (Y PD <.21 MPa) compared with Sabatiano. There were no significant differences in osmotic potential at full turgor (p 1 ) between the stressed plants of the two varieties, which measured MPa and MPa in Mavrodafni and Sabatiano, respectively. Plant hydraulic conductance (K l ) was significantly affected by changes in Y PD (Fig. 3b), but there were no differences in K l between the two varieties. Furthermore, as Y PD decreased, plant hydraulic conductance declined similarly in both varieties. Predawn leaf ABA concentration increased significantly in response to soil drying in both varieties (Table 1). Leaf ABA concentration was significantly higher in Mavrodafni deficitirrigated plants compared with Sabatiano at the beginning of the drought period (Days 6 12), whereas the opposite trend was evident at the end of the drought period (Table 1). Root ABA concentration was significantly higher in Mavrodafni deficitirrigated plants almost throughout the drought period. In Soil water content (% of volume) P N (µmol CO 2 m 2 s 1 ) g s (mol H 2 O m 2 s 1 ) Predawn leaf water potential (MPa) (b) (c) (a) (d) Drought period (Days) Fig. 1. Changes in (a) soil water content, (b) predawn leaf water potential (Y PD ), (c) stomatal conductance (g s ) and (d) photosynthetic rate (P N ) in wellwatered (WW) and deficit-irrigated (DI) plants of two grapevine varieties, Mavrodafni (MAV) and Sabatiano (SAB), over a 24-day drought period. Data shown refer to the three drought cycles conducted in 27. Each point represents the mean s.e. of 18 (6 leaf per cycle 3 drought cycles) replicates. contrast, no significant differences in xylem ABA concentration were observed between the two varieties at the beginning of the drought period (Table 1). Thereafter, stressed Mavrodafni plants exhibited higher xylem ABA concentration compared with Sabatiano. A negative correlation between leaf ABA concentration and stomatal conductance (Fig. 4) in both varieties was also observed indicating that ABA exerts significant control over the stomatal aperture during the drought period. However, stomatal sensitivity to leaf ABA concentration was

4 142 Functional Plant Biology A. Beis and A. Patakas Midday leaf water potential (MPa) Predawn leaf water potential (MPa) ψ P <.1 PD : F = 57.1, P <.1 Treatment : F =.26, P = P.621 =.621 Treatment ψ P =.44 PD : F = 4.89, P =.44 Fig. 2. Relationship between predawn leaf water potential (Y PD ) and midday water potential (Y MD ) in deficit-irrigated (DI) plants of two grapevine varieties, Mavrodafni (MAV) and Sabatiano (SAB). Data shown refer to the three drought cycles conducted in 27. Each point corresponds to measurements carried out on different sampling days (1, 3, 6, 9, 12, 15, 18, 21 and 24) of each drought cycle and represents the mean s.e. for both x and y axes of 18 (6 leaf per cycle 3 drought cycles) replicates. Regression lines were also fitted to each treatment (P <.5). F-values and P-values were determined by ANCOVA for each main effect (treatment and Y PD ) and their interaction is shown. K 1 (mmol MPa 1 m 2 s 1 ) g s (mol H 2 O m 2 s 1 ) (a) (b) ψ PD <.21 Mpa P <.1 ψ PD : F = 9.1, P <.1 Treatment : F =.27, P = P.64 =.64 Treatment ψ P =.12 PD : F = 6.42, P = Predawn leaf water potential (MPa) ψ PD : F = 72.61, P <.1 Treatment : F =.712, P =.412 Treatment ψ PD : F =.3, P =.955 significantly higher in Mavrodafni compared with Sabatiano (Fig. 4). Moreover, leaf apoplastic ph values increased in both varieties in response to drought intensity (Fig. 5). Mavrodafni exhibited significantly higher xylem ph values as well as higher ph values at a given Y PD compared with Sabatiano (Fig. 5). The relationship between stomatal conductance and xylem ph indicated a greater stomatal sensitivity to ph increase in Mavrodafni compared with Sabatiano (Fig. 6). Discussion The observed significant differences in stomatal sensitivity to Y PD (Fig. 3a) between Sabatiano and Mavrodafni provide evidence that the two varieties have evolved different strategies in response to drought. In particular, the tighter control on stomatal aperture in Sabatiano in the early stages of the drought period, despite the higher soil moisture levels, seems to be more efficient in order to regulate transpiration water loss and to maintain leaf hydration at near control levels for a longer time during the drought period (Figs 1, 2). The terms isohydric and anisohydric are used to divide plant species into two broad categories based on the extent to which tissue hydration is kept stable under fluctuating environmental conditions. Isohydry is generally attributed to strong stomatal control of the transpiration rate, which results in the similarity in midday leaf water potential (Y MD ) in droughted and well-watered plants (Franks et al. 27). In contrast, anisohydric plants typically Predawn leaf water potential (MPa) Fig. 3. Changes in (a) stomatal conductance (g s ) and (b) hydraulic conductance (K l ) in relation to predawn leaf water potential (Y PD ) in deficit-irrigated (DI) plants of two grapevine varieties, Mavrodafni and Sabatiano. Data in (a) represent the mean of the three drought cycles conducted in both 27 (54 points) and 26 (54 points) for each variety. Data in (b) refer to the three drought cycles conducted in 27. Each point corresponds to measurements carried out on different sampling days (1, 3, 6, 9, 12, 15, 18, 21 and 24) of each drought cycle and represents the mean s.e. for both x and y axes of 18 (6 leaf per cycle 3 drought cycles) replicates. F-values and P-values were determined by ANCOVA for each main effect (treatment and Y PD ) and their interaction is shown. exhibit less stomatal sensitivity to evaporative demand and soil moisture, thus allowing large fluctuations in Y MD. In our results, both varieties exhibited significant reductions in midday leaf water potential in response to drought (Fig. 2), clearly indicating anisohydric behaviour. The fact that the magnitude of Y MD reduction was significantly lower in Sabatiano implies that the two varieties may differ in the degree of anisohydricity, with Sabatiano displaying fewer anisohydric properties. However, interpretation of the differences in stomatal control between varieties is difficult given that several interconnecting chemical and hydraulic signals are involved. Stomata close under drought conditions in response to changes in soil to leaf chemical

5 Drought signalling in grapevines Functional Plant Biology 143 Table 1. Changes in leaf, root and xylem sap ABA concentration in relation to the drought period in two grapevine varieties, Mavrodafni and Sabatiano Data shown refer to the three drought cycles conducted in 27. Values are means s.e. of 12 replicates (4 ABA determinations per cycle 3 drought cycles). Statistically significant differences (P <.5) within the same row are indicated by different letters. DI, drought -irrigated; WW, well-watered Parameters Days of ABA concentration drought period Mavrodafni Sabatiano WW DI WW DI Bulk leaf ABA (nmol g 1 FW) ±.14a.643 ±.14a.821 ±.1a.821 ±.1a ±.19a ±.28b.74 ±.14a.857 ±.29a ±.5a ±.17c ±.15a 2.87 ±.27b ±.11a ±.61b ±.21a ±.17c ±.9a ±.14c ±.23b ±.41d Root ABA (nmol g 1 FW) 1.41 ±.6ab.42 ±.4b.335 ±.4ab.35 ±.1a ±.5a.565 ±.3b.324 ±.4a.318 ±.2a ±.7ab.816 ±.12c.354 ±.7a.614 ±.4b ±.5a.828 ±.7c.43 ±.4a.688 ±.1b ±.5a.843 ±.8b.385 ±.3a.762 ±.11b Xylem sap ABA (nmol ml 1 ) ±.3a.256 ±.2a.231 ±.1a.226 ±.1a ±.2a.47 ±.4b.282 ±.2a.445 ±.9b ±.4a 1.35 ±.1c.342 ±.3a.84 ±.11b ±.3a.844 ±.11c.247 ±.4a.587 ±.9b ±.2a ±.4c.257 ±.3a.732 ±.5b g s (mol H 2 O m 2 s 1 ) ABA : F = 445, P <.1 Treatment : F =.52, P =.472 Treatment ABA : F = 6.75, P =.11 Xylem sap ph ABA (nmol g 1 FW) Fig. 4. Relationship between leaf ABA concentration and stomatal conductance (g s ) in two grapevine varieties, Mavrodafni and Sabatiano, subjected to deficit irrigation (DI). Data shown refer to the three drought cycles conducted in 27. Regression lines were fitted to each treatment (P <.5). F-values and P-values were determined by ANCOVA for each main effect (treatment and ABA) and their interaction is shown. signals, leaf turgor and plant hydraulic conductivity (Pou et al. 28). In our results, the lack of differences in osmotic potential between the two varieties (Fig. 1) implies that the different stomatal behavior at the beginning of drought period could not be attributed to different ability for osmotic adjustment. As far as the hydraulic properties is concerned, no significant differences in ψ PD : F = 33.9, P <.1 6. Treatment : F = 24.8, P =.1 Treatment ψ PD : F = 6.99, P = Predawn leaf water potential (MPa) Fig. 5. Changes in xylem sap ph in relation to predawn leaf water potential (Y PD ) in two grapevine varieties, Mavrodafni (MAV) and Sabatiano (SAB), subjected to deficit irrigation (DI). Data shown refer to the three drought cycles conducted in 27. Each point corresponds to measurements carried out on different sampling days (1, 3, 6, 9, 12, 15, 18, 21 and 24) of each drought cycle and represents the mean s.e. for both x and y axes of four replicates. Regression lines were fitted to each treatment (P <.5). F-values and P-values were determined by ANCOVA for each main effect (treatment and Y PD ) and their interaction is shown. plant hydraulic conductance (K l ) in response to soil drying were found between the two varieties (Fig. 3b). However, K l measurement in the field is known to be subjected to errors

6 144 Functional Plant Biology A. Beis and A. Patakas g s (mol H 2 O m 2 s 1 ) ph : F = 62.7, P <.1 Treatment : F = 6.99, P =.11 Treatment ABA : F = 6.6, P = Xylem sap ph Fig. 6. Relationship between xylem sap ph and stomatal conductance (g s )in two grapevine varieties, Mavrodafni and Sabatiano, subjected to deficit irrigation (DI). Data shown refer to the three drought cycles conducted in 27. Each point corresponds to measurements carried out on different sampling days (1, 3, 6, 9, 12, 15, 18, 21 and 24) of each drought cycle and represents the mean s.e. for both x and y axes of four replicates. Regression lines were fitted to each treatment (P <.5). F-values and P-values were determined by ANCOVA for each main effect (treatment and xylem sap ph) and their interaction is shown. due to the fact that they are conducted under non-steady-state conditions (Hubbard et al. 21). Furthermore, Schultz (23) demonstrated, by comparing whole plant versus single organ hydraulics, that it is the differences in the water conducting capacity of stems and especially of petioles, rather than those in whole plant conductance, that accounts for differences in stomatal behaviour in grapevine varieties. If this is true, then the observed earlier reduction in stomatal conductance in Sabatiano might be attributed to hydraulic signals. Chemical signals might also be required to cause stomatal closure under drought conditions (Davies and Zhang 1991; Tardieu et al. 1996; Bacon et al. 1998; Dodd 25, 27;). The greater ability of Mavrodafni roots for ABA synthesis at the beginning of the drought period (Days 6 and 12) (Table 1) seems to be responsible for the greater ABA concentration in the leaves taking into consideration that no significant differences in hydraulic conductivity between the two varieties occurred (Fig. 3b). Lower leaf ABA concentration in Sabatiano suggests that the observed earlier reduction in g s during the drought period (Fig. 1) could not be attributed to ABA accumulation. However, the relation between leaf ABA concentration and stomatal conductance have been recently questioned due to the fact that part of the leaf ABA could be trapped inside the cytoplasm of mesophyll or epidermal cells away from the sites of action on the stomata according to the anion trap concept (Jia and Zhang 1999; Hartung et al. 22; Jia and Davies 27). Therefore, a discrete portion of the total leaf ABA located at the vicinity of the guard cells is considered to be more important in determining stomatal conductance (Jia and Zhang 1999). Thus, it could be assumed that differences in leaf ABA compartmentalisation between the two varieties might account for the earlier reduction in g s in Sabatiano plants. Leaf ABA compartmentalisation between apoplast and symplast is known to be mainly affected by changes in xylem ph (Slovik et al. 1995). In particular, an increase in ph is expected to promote accumulation of ABA near stomata thus enhancing stomatal closure even at lower values of leaf ABA concentration (Rodrigues et al. 28). However, Mavrodafni exhibited significant higher ph values compared with Sabatiano throughout the drought period (Fig. 5), which suggests that a higher ABA concentration would eventually reach the guard cells for the same values of total leaf ABA concentration. While these results are consistent with the higher stomatal sensitivity to changes in ABA observed in Mavrodafni (Fig. 4), they also imply that chemical signals might not be involved in the early reduction of stomatal conductance in Sabatiano plants at the beginning of the drought period. In contrast, it is worth noting that despite the relatively lower concentrations of ABA in both roots and xylem sap (Table 1) and the lack in differences in plant hydraulic conductivity between the two varieties, Sabatiano exhibited significant higher leaf ABA concentrations compared with Mavrodafni at the end of the drought period. This increase in ABA concentration in Sabatiano leaves might be attributed to either greater ability for local synthesis and/or lower rates of ABA degradation. ABA metabolism has been shown (Soar et al. 24) to be rapid inside leaf mesophyll cells (Ren et al. 27), with a halflife of less than 3 h reported for maize (Zea mays L.) (Jia and Zhang 1997) and less than 2 h in sunflower (Helianthus annuus L.) (Jia and Zhang 1999). Moreover, Daeter and Hartung (1995) demonstrated that the epidermal cells have a catabolic rate that is five-fold greater than that of the mesophyll cells. Assuming that there are no differences in metabolic enzyme activity between the two varieties, the ABA degradation rate is expected to be modulated by the leaf apoplastic ph values. (Slovik and Hartung 1992a, 1992b; Daeter et al. 1993; Davies et al. 22). In particular, even a small decrease in the ph of the apoplast can influence ABA diffusivity into leaf cells enhancing ABA catabolism and thus influencing total leaf ABA concentration (Wilkinson and Davies 22). In our results, Sabatiano exhibited significantly lower xylem ph values compared with Mavrodafni throughout the drought period (Fig. 5). Since low leaf ph values imply high rates of ABA catabolism, the higher leaf ABA concentration in Sabatiano could not be attributed to differences in the ABA degradation rate, which suggests that there is a greater ability for local ABA synthesis in Sabatiano leaves (Soar et al. 26). In conclusion, the present study provides evidence that the grapevine varieties Sabatiano and Mavrodafni have evolved different strategies in order to adapt under drought conditions. In particular, the greater ability for drought adaptation in Sabatiano might be attributed to the more efficient control on stomatal closure, which results in maintaining leaf hydration at near control levels despite the changes in soil water availability. The earlier reduction in stomatal conductance in Sabatiano in the

7 Drought signalling in grapevines Functional Plant Biology 145 absence of an increase in leaf ABA concentration provides evidence that other mechanisms, probably related to changes in plant hydraulic conductance, might be responsible for this stomatal behaviour. However, increases in both leaf ABA and ph under more severe drought stress conditions suggests that chemical signals might also be involved in stomatal control and thus stomatal closure at the end of the drought period results from the combined effects of both chemical and hydraulic signals. In contrast, the mechanism for drought adaptation in Mavrodafni is mainly based on chemical signals. In this variety, the higher ph values, which enhance leaf ABA concentration in the vicinity of guard cells, might counteract the limited hydraulic control on stomata, promoting stomatal closure. The higher sensitivity of stomatal conductance to leaf ABA concentration in Mavrodafni confirms the preponderant role of this mechanism in drought adaptation. Acknowledgements This research project (PENED) is co-financed by an E.U.-European Social Fund (8%) and the Greek Ministry of Development-GSRT (2%). References Bacon MA, Wilkinson S, Davies WJ (1998) ph-regulated leaf cell expansion in droughted plants is abscisic acid dependent. Plant Physiology 118, doi:1.114/pp Beis A, Zotos A, Patakas A (27) Changes in xylem ph induced by different levels of pressure in detached water stressed grapevines leaves. In Proceedings of the XVth international GESCO symposium, Porec, Croatia. (Ed. B Sladonja) pp (Institut za poljoprivredu i turizam: Porec) Borel CH, Simonneau TH, This D, Tardieu F (1997) Stomatal conductance and ABA concentration in the xylem sap of barley lines of contrasting genetic origins. 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