Salinity effects on the stomatal behaviour of grapevine

New Phytol. (1990), 116, 499-503 Salinity effects on the stomatal behaviour of grapevine BY W. J. S. DOWNTON, B. R. LOVEYS AND W. J. R. GRANT CSIRO Division of Horticulture, GPO Box 350, Adelaide, 5001, Australia {Received 25 April 1990 ; accepted 26 July 1990) SUMMARY An investigation of the time-course of inhibition of photosynthesis in salt-stressed grapevine {Vitis vinifera L.) leaves revealed two types of stomatal behaviour. Up to tissue concentrations of 165 mm chloride the inhibition was due to a uniform decrease in stomatal conductance, as indicated from autoradiograms of "CO^ fixation and no change in the relationship of assimilation to calculated intercellular partial pressure of COg {A-C,) compared with control plants. The occurrence of non-stomatal inhibition of photosynthesis at higher levels of leaf chloride, suggested by a decline in the slope of the calculated A-C. relationship, was associated with non-uniform "CO^ uptake over the leaf surface similar to that previously observed for ABA-treated and water-stressed grapevine leaves where non-stomatal inhibition of photosynthesis was shown to be an artifact arising from non-uniform stomatal behaviour. These observations also provide an explanation for the stimulation of photorespiration during salt stress. Key words: Chloride, photosynthesis, salt stress, stomata, Vitis vinifera. INTRODUCTION Previous time-course studies on salt-stressed grapevines have documented an initial stomatal inhibition of photosynthetic gas exchange followed by the onset of non-stomatal inhibition at higher levels of leaf chloride (Walker et a/., 1981; Walker & Downton, 1982). These effects have been associated with increased photorespiration as evidenced by greatly enhanced photosynthesis when oxygen is lowered from 21 to 2% O^ (Walker et al., 1981; Walker & Downton, 1982) and stimulated labelling of photorespiratory pathway intermediates in leaves exposed to ^^COg (Downton, 1977). Recently it has become evident that conclusions drawn from gas exchange data can be considerably in error, particularly during stress where stomata may not behave uniformly (Downton, Loveys & Grant, 19886; Sharkey & Seemann, 1989), thereby generating false evidence for direct (non-stomatal) effects of stress on photosynthesis. In view of the possibility that this may also occur during salt stress we have re-examined the time-course of salt-induced inhibition of photosynthesis in grapevine and document two types of stomatal behaviour depending on the extent to which chloride has accumulated within the leaf. MATERIALS AND METHODS Grapevines {Vitis vinifera L. cv. Sultana syn. Thompson Seedless) were grown as rooted cuttings (Downton & Loveys, 1978) in a soil mix consisting of 10% peatmoss:90% coarse sand during the period October-January (Southern Hemisphere). Plants were grown in a temperature-controlled glasshouse (mean maximum 28±2 C, mean minimum 17 ± 2 C) covered with 50 % shadecloth which reduced maximum photon flux density at mid-day to about 900 jimol photons m~^ s~^ (PAR). Following budburst, plants were trimmed to one shoot and regularly watered with a commercial water soluble fertilizer (Aquasol, Hortico Australia Pty Ltd; 0-12 gt^) which was supplemented with 50 mg 1"^ Ca(NO3)2.4H2O and 6 mg T^ FeEDTA. The Aquasol solution contained approx. 0-5 mm chloride. Salt treatment was applied to half of the vines following an establishment period of 7 wk by which time shoots were approx. 06 m long with about 10 leaves. Salt treatment consisted of the addition of 75 mm NaCl to the basic nutrient solution. Midshoot leaves which had just attained maximal photosynthetic rates (30-40 d old) were studied over the next 6-5 wk period. Photosynthetic rates were measured at intervals

500 W. J. S. Dotmton, B. R. Loveys and W. J. R. Grant during the course of the experiment. The relationship of assimilation {A) to intercellular partial pressure of COg (Q) was determined at decreasing partial pressure of COg external to the leaf (C^) from 340 to 80ytfbar COg using an open gas-exchange system (Downton, Grant & Robinson, 1985) and results calculated according to von Caemmerer & Farquhar (1981). Cuvette conditions were maintained at 26 + 2 C, with a leaf-to-air water vapour difference of 1-4 to 1-8 kpa. The photon irradiance incident on the leaves was 800 /*mol photons m"^ s~^ (PAR). The uniformity of assimilation was assessed by exposing leaves to ^^COg for 20 s in the gas exchange cuvette under the conditions described above (0-18 MBq ^^COg; Ca - 300/tbar). The leaf was rapidly frozen between two sheets of aluminium cooled to 60 C and the adaxial surface placed directly against film (Beta-max, Amersham) to produce autoradiograms. Leaf tissue was oven-dried at 70 C and powdered in a mortar. Chloride was measured by silver ion titration with a Buchler-Cotlove chloridometer (Nuclear Chicago, NJ, USA) and results expressed on a tissue water basis. 50 150 250 Intercellular CO2 (/ibar) Figure 1. Response of photosynthesis to the partial pressure of intercellular COg in salt-stressed grapevine leaves. Control leaf: curve A (48 mm Cl~). Salt-treated leaves: point B (162 mm Cr), curve C (217 mm Cl"), curve D (293 mm C1-), curve E (300 mm Cl"), curve F (317 mm cn. RESULTS AND DISCUSSION Plants receiving salt treatment continued to accumulate chloride into leaves over the course of the experiment (Table 1) and necrosis was evident on the margins of some of the leaves by day 46. These salt 'burn' symptoms occurred at tissue levels of chloride of about 350 mm as observed previously (Downton & Millhouse, 1983). Basal levels of chloride in leaves of untreated plants, which ranged between 35 and 50 mm and originated from the cutting itself and from the low level of chloride in the nutrient solution, did not change with time. These control plants maintained photosynthetic rates of 14-5 ±0-7/^mol COg m~^ s"^, stomatal conductances of 0*35 ±0-03 mol m~^ s~^ and an intercellular COg partial pressure of 262 ±5 /ihar (n = 9), whereas the salt-treated plants showed a steady decline in photosynthesis and stomatal conductance with time (Table 1). Intercellular partial pressure of COg fell from 260 to 200 /*bar following salt treatment and remained at that level for most of the experiment. The relationship of A vs. Q was also calculated to determine the extent of stomatal and non-stomatal limitations to photosynthesis. Q is routinely calculated from gas exchange data to determine the photosynthetic behaviour of leaves in the absence of stomatal limitations (von Caemmerer & Farquhar, 1981) and a decrease in slope of the A-C^ relationship with treatment is taken as evidence for direct (nonstomatal) inhibition of photosynthesis. Line A in Figure 1 shows a typical A-C^^ curve for the untreated leaves. Plants receiving salt-treatment for 14 d also followed this same A-C^ relationship, except that Q was much reduced (point B on line A; Table 1). Beyond 14 d, there was a progressive decline in the slope of the A C^ relationship as tissue levels of chloride increased beyond 165 mm, indicative of increasing non-stomatal inhibition of photosynthesis. Table 1. Photosynthesis (A), stomatal conductance (g), intercellular partial pressure of CO^ (Q) and chloride concentration in leaves of salt-treated grapevines. Mean + SE for 3 replicate plants Days of treatment A (jimol COg m"^! 3-^) g (mol m~^ s-^) c. (/ibar COg) ci- (mm) 0 14 21 39 46 12-7 + 0-2 10-1+0-7 8-5 + 1-0 3-9 + 0-5 l-3±0-l 0-29 + 0-014 0-13 + 0-013 0-09 + 0-011 0-03+0-003 0-01 ±0-001 256 + 3 199 + 8 189+15 143 + 12 196±8 41+2 165 + 5 212 + 8 277 + 11 305 ±10

Effects of salinity on stomata of grapevine 501 Figure 2. Autoradiograms of salt-stressed grapevine leaves exposed to the same leaves whose A-C^ curves are presented in Figure 1. Bar, 5 mm These results accord well with those of Walker et al (1981) who observed stomatal inhibition of photosynthesis in salt-treated Sultana vines up to tissue levels of chloride of about 150 mm, followed by 35 for 20 s. Autoradiograms are for increased mesophyll resistances at higher chloride levels. However, it is also now well documented that evidence for non-stomatal inhibition of photosynanp 116

502 W. y. S. Downton, B. R. Loveys and W. J. R. Grant thesis derived from A-Ci curves can be artifactual if stomata do not behave uniformly (Laisk, 1983; Farquhar et al., 1987; Downton, Loveys & Grant, 19SSa,b: Terashima et al, 1988; Sharkey & Seemann, 1989). To test this possibility, leaves were exposed to ^^COg for a short period of time and autoradiograms prepared in order to visualize spatial variation in stomatal conductance to COg as previously described (Downton et al, 1988a, fe). Figure 2 shows autoradiograms of the same leaves whose A-Ci curves are presented in Figure 1. Figure 2 fl is an example of a control leaf which shows uniform uptake of COg over the leaf surface. This uniformity was evident prior to commencement of salt-treatment and for control leaves during the experiment. Figure 2 b shows a leaf which had received 14 d of salt treatment. As expected from its position on the A-C^^ curve (Fig. 1), ^^COg uptake by leaf B was uniform, but the intensity of labelling was less due to reduced stomatal conductance. Figure 2{c-f) depicts autoradiograms of leaves containing progressively higher concentrations of chloride and showing decreasing slopes of A-C^. It is evident that these changes are associated with increasingly patchy CO2 fixation. Using a combination of chlorophyll fluorescence and gas exchange techniques we earlier demonstrated that non-uniform stomatal closure could completely account for the inhibition of photosynthesis in grapevine leaves treated with ABA or experiencing water stress (Downton et al, 1988 a, b). Although we did not utilize chlorophyll fluorescence quenching methodology in this study, it seems likely that at least part of the apparent non-stomatal inhibition of photosynthesis evident at the higher levels of tissue chloride arises from the non-uniform stomatal conductance to CO2 evident in the autoradiograms. This is supported by observations that cessation of salt treatment to Sultana vines results in a complete recovery of photosynthesis in leaves with high mesophyll resistances and containing about 200 mm chloride (Walker et al, 1981). Furthermore, although high salt treatment of Plantago maritima leads to a reduced A-C^ relationship at atmospheric levels of CO2, there is no difference from low-salt plants in oxygen evolution when measured at high partial pressures of CO2 in a leaf disc electrode (Flanagan & Jefferies, 1989). This is a similar response to that observed by Robinson, Grant & Loveys (1988) where high partial pressures of CO2 were found to largely overcome low stomatal conductances induced by ABA-treatment. We have previously observed increases in leaf ABA levels in salt-treated sultana vines, which remained elevated for some weeks (Downton & Loveys, 1981) and this may be associated with the non-uniform COg uptake evident in the autoradiograms. The present study also clarifles earlier observations on changes in photosynthetic carbon metabolism in salt-stressed grapevines. Increased salt concentrations in leaves were found to be associated with decreased **C-labelIing of sugar monophosphates, 3- phosphoglyceric acid and sucrose and increased labelling of ribulose,l-5-bisphosphate, glycolic acid, glycine and serine (Downton, 1977). This increased labelling of the photorespiratory pathway with salt stress, which resembles the effect of decreased partial pressure of CO2 on photosynthesis originally described by Wilson & Calvin (1955), can now be interpreted simply in terms of stomatal inhibition of photosynthesis and CO2 depletion in regions of low stomatal conductance, rather than to unknown direct effects on salinity or carbon metabolism itself. These changes in stomatal behaviour accompanying salt stress also account for the larger than expected low oxygen enhancement of photosynthesis in leaves with seemingly high mesophyll resistances (Walker et al, 1981). It can be concluded that for woody perennial crop plants such as grapevine with highly compartmentalized leaves, salt and water stress, which seemingly cause direct inhibition of photosynthesis, do so mainly by altering stomatal behaviour which restricts access of CO2 into the leaf. This implies that these environmental stress factors have no immediate effects on the photosynthetic process itself a viewpoint that is gaining increased support (Flanagan & Jefferies, 1989; Robinson, Downton & Loveys, 1989; Sharkey & Seemann, 1989). The important implication of this flnding for vineyard and orchard management in the irrigation areas is that management decisions can be taken to reverse stomatal inhibition of photosynthesis, readily detectable by porometry as a decrease in leaf conductance, before the duration and extent of the stress secondarily results in longer term damage to the photosynthetic apparatus which may not be readily reversible. REFERENCES DOWNTON, W. J. S. (1977). Photosynthesis in salt-stressed grapevines. Australian Journal of Plant Physiology 4, 183-192. DOWNTON, W. J. S., GRANT, W. J. R. & ROBINSON, S. P. (1985). Photosynthetic and stomatal responses of spinach leaves to salt stress. Plant Physiology, 78, 85-88. DOWNTON, W. J. S. & LOVEYS, B. R. (1978). Compositional changes during grape berry development in relationship to abscisic acid and salinity. Australian Journal of Plant Physiology 5,415-423. DOWNTON, W. J. S. & LOVEYS, B. R. (1981). Abscisic acid content and osmotic relations of salt-stressed grapevine leaves. Australian Journal of Plant Physiology 8, 443-452. DOWNTON, W. J. S., LOVEYS, B. R. & GRANT, W. J. R. (1988o). Stomatal closure fully accounts for the inhibition of photosynthesis by abscisic acid. New Phytologist 108, 263-266. DOWNTON, W. J. S., LOVEYS, B. R. & GRANT, W. J. R. (19886). Non-uniform stomatal closure induced by water stress causes putative non-stomatal inhibition of photosynthesis. New Phytologist 110, 503-509. DOWNTON, W. J. S. & MILLHOUSE, J. (1983). Turgor maintenance during salt stress prevents loss of variable fluorescence in grapevine leaves. Plant Science Letters 31, 1-7.

Effects of salinity on stomata of grapevine FARQUHAR, G. D., HUBICK, K. T., TERASHIMA, I., CONDON, A. G. & RICHARDS, R. A. (1987). Genetic variation in the relationship between photosynthetic CO2 assimilation rate and stomatal conductance to water loss. In: Progress in Photosynthesis Research, vol. 4 (Ed. by J. Biggins), pp. 209-212. Martinus Nijhoff, Dordrecht. FLANAGAN, L. B. & JEFFERIES, R. L. (1989). Photosynthetic and stomatal responses of the halophyte Piantago maritima L. to fluctuations in salinity. Plant, Cell and Environment 12 559-568. LAISK, A. (1983). Calculation of leaf photosynthetic parameters considering the statistical distribution of stomatal apertures. Journal of Experimental Botany 34, 1627-1635. ROBINSON, S. P., DOWNTON, W. J. S. & LOVEYS, B. R. (1989). Stress effects on photosynthesis. In: Plant Water Relations and Growth Under Stress, Proceedings of the Yamada Conference XXII, pp. 109-116. Yamada Science Foundation and Myu K.K., Tokyo. ROBINSON, S. P., GRANT, W. J. R. & LOVEYS, B. R. (1988). Stomatal limitation of photosynthesis in abscisic acid-treated and in water-stressed leaves measured at elevated Australian Journal of Plant Physiology 15, 495-503. 503 SHARKEY, T. D. & SEEMANN, J. R. (1989). Mild water stress effects on carbon-reduction-cycle intermediates, ribulose bisphosphate carboxylase activity, and spatial homogeneity of photosynthesis in intact leaves. Plant PhysiologyOT, 1060-1065. TERASHIMA, I., WONG, S. C, OSMOND, C. B. & FARQUHAR, G. D. (1988). Characterisation of non-uniform photosjmthesis induced by abscisic acid in leaves having different mesophyll anatomies. Plant and CeU Physiology 29, 385-394. VON CAEMMERER, S. & FARQUHAR, G. D. (1981). Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153, 376-387. WALKER, R. R. & DOWNTON, W. J. S. (1982). Photosynthesis in salt stressed woody perennials. In: Biosaline Research : A Look To The Future (Ed. by A. San Pietro), pp. 549-554. Plenum Publishing Corp., New York. Walker, R. R., Torokfalvy, E., Scott, N. S. & Kriedemann, P. E. (1981). An analysis of photosynthetic response to salt treatment in Vitis vinifera. Australian Journal of Plant Physiology 8, 359-374. WILSON, A. J. & CALVIN, M. (1955). The photosynthetic cycle. CO2 dependent transients. Journal of American Chemical Society 77, 5948-5957. 35-2