Ion-specific mechanisms of osmoregulation in bean mesophyll cells

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ionic mechanisms of osmoregulation in higher plant cells,. Introduction and the data are controversial at times. Curti et al. reported an increase in K+ uptake into.
Journal of Experimental Botany, Vol. 51, No. 348, pp. 1243–1253, July 2000

Ion-specific mechanisms of osmoregulation in bean mesophyll cells Sergey Shabala1,3, Olga Babourina2 and Ian Newman2 1 School of Agricultural Science, University of Tasmania, GPO Box 252–54, Hobart, Tas 7001, Australia 2 School of Mathematics and Physics, University of Tasmania, GPO Box 252–21, Hobart, Tas 7001, Australia Received 8 December, 1999; Accepted 2 March 2000

Abstract Transient kinetics of net H+, K+, Ca2+, and Cl− fluxes were measured non-invasively, using an ion-selective microelectrode technique, for bean (Vicia faba L.) leaf mesophyll in response to 150 mM mannitol treatment. In a parallel set of experiments, changes in the plasma membrane potential and the total proline content in leaves were monitored. Regardless of the ionic composition of the bath solution, hyperosmotic stress caused a significant increase in the K+ and Cl− uptake into mesophyll cells. At the same time, no significant proline changes were observed for at least 16 h after the onset of stress. Experiments with inhibitors suggested that potassium inward rectifier (KIR) channels, exhibiting mechanosensitive properties and acting as primary receptors of osmotic stress, are likely to be involved. Due to the coupling by membrane potential, changes in K+ and Cl− transport may modify activity of the plasma membrane H+-pump. Such coupling may also be responsible for the mannitol-induced oscillations (period of about 4 min) in net ion fluxes observed in 90% of plants. Calculations show that influx of K+ and Cl− observed in response to hyperosmotic treatment may provide an adequate osmotic adjustment in bean mesophyll, which suggests that the activity of the plasma membrane transporters for these ions should be targeted to improve osmotolerance, at least in this crop. Key words: Osmoregulation, plasma membrane, ion transporters, Vicia faba.

Introduction A large number of environmental stresses, including drought, salinity and low temperature, limit crop growth

and productivity by imposing osmotic stress on plants. To cope with the problem, plant cells must readjust their osmotic potential to prevent water losses. That can be achieved by either uptake of inorganic ions from the external solution, or by de novo synthesis of compatible solutes (amino acids, sugars, polyoles, quaternary amines) acting as osmolytes ( Wyn Jones and Pritchard, 1989; Delauney and Verma, 1993; Bohnert et al., 1995; Bohnert and Shen, 1999; Serrano et al., 1999). These two processes differ significantly in their time scales. Immediate changes in ion fluxes are believed to provide quick (within a few minutes) osmotic adjustment while a fine ‘tuning’ by means of biochemical synthesis of compatible solutes has a scale of hours and days ( Wyn Jones and Pritchard, 1989; Lew, 1996). Recent progress in molecular genetics has made biochemical mechanisms of osmolyte biosynthesis the primary target for genetic engineering of salt and drought tolerance in crops (see Yeo, 1998, for a review). However, the progress is disappointingly slow, and an increase in the osmotic tolerance in a field situation is only marginal (Bohnert and Shen, 1999). It appears that membrane transport processes play a more crucial role in plant osmoregulation than was believed so far. There is much evidence supporting this statement. Cerda et al. concluded that accumulation of inorganic ions was sufficient for osmotic adjustment in salt-stressed maize cultivars, and that no single organic solute appeared to be important in this process (Cerda et al., 1995). Similar conclusions were made by Huang and Redmann for cultivated barley genotypes (Huang and Redmann, 1995). In spite of these facts, little is known about the ionic mechanisms of osmoregulation in higher plant cells, and the data are controversial at times. Curti et al. reported an increase in K+ uptake into Arabidopsis cultured cells in response to hyperosmotic

3 To whom correspondence should be sent. Fax: +61 3 6226 2642. E-mail: [email protected] © Oxford University Press 2000

1244 Shabala et al. treatment (Curti et al., 1993). Mannitol-induced decrease in Cl− efflux was shown by Teodoro et al. for the same species ( Teodoro et al., 1998). However, Lew found a significant increase in outward K+ flux in Arabidopsis root hairs under the same conditions, with no significant changes in Cl− flux (Lew, 1998). The only uptake measured in his experiments was in the net Ca2+ flux; its magnitude, however, was too small for Ca2+ to be considered to act as an osmoticum. Lew concluded that observed changes in ion fluxes are likely to be a part of the initial signalling cascade, but not directly involved in the osmotic regulation (Lew, 1998). However, as his measurements were taken for only a single moment, 5 min after the onset of osmotic stress, some important features of the ion flux kinetics could be missed. Clearly, this question requires more thorough study. Could these differences be attributed to the different ion composition of the bath? Specific ionic mechanisms involved in osmotic stress perception are still elusive. Lew suggested that Arabidopsis root hair cells possess an osmo-sensing but not a turgorsensing mechanism (Lew, 1996); specific details of this process remain unknown. At least two mechanisms by which a plant can sense osmotic conditions have been suggested (Brownlee et al., 1999). First, the changes in cell volume could be sensed by mechanosensitive receptors on the PM (Cosgrove and Hedrich, 1991). Another option is that the intracellular osmosensing mechanisms may detect the degree of cytosol hydration (Brownlee et al., 1999, and references within). Teodoro et al. suggested that the primary targets in the osmosensory mechanism in cultured Arabidopsis cells were stretch-activated Cl− channels inactivated by hyperosmotic stress ( Teodoro et al., 1998). Is that the only mechanism present at the PM? In this paper, some of the above issues are addressed by non-invasive measurements of net H+, K+, Ca2+, and Cl− fluxes from bean leaf mesophyll in response to hyperosmotic treatment. In a parallel set of experiments, changes in the PM potential and the total proline content in leaves were monitored. It is concluded that mannitolinduced activation of the PM transporters is strongly dependent on the ionic composition of the external solution, and that the influx of K+ and Cl− observed in response to hyperosmotic treatment provides an adequate osmotic adjustment in bean mesophyll.

Materials and methods Plant material Broad beans ( Vicia faba L. cv. Coles Dwarf; Cresswell’s Seeds, New Norfolk, Australia) were grown from seed in 0.5 l plastic pots. The potting mixture included 70% composted pine bark, 20% coarse sand and 10% sphagnum peat (pH 6.0). A fertilizer mixture (1.8 kg m−3 Limil, 1.8 kg m−3 dolomite, 6.0 kg m−3

Osmocote Plus, and 0.5 kg m−3 ferrous sulphate) was added to each pot, and the plants were watered four times per week with tap water. Growth conditions were 16/8 h light/dark (model M1500-A lighting unit, Thorne, Moonah, Australia; total irradiance=150 W m−2 at the leaf level ) with temperature ranging from 20 °C (dark) to 28 °C ( light). Leaves from 20–30-d-old plants were used for measurements. Mesophyll tissue was isolated essentially as described previously (Shabala and Newman, 1999). Ion selective flux measurements Net fluxes of H+, K+, Cl−, and Ca2+ were measured noninvasively using ion-selective vibrating microelectrodes (the MIFETM technique; University of Tasmania, Hobart, Australia) generally as described in previous publications (Shabala et al., 1997; Shabala and Newman, 1999). Commercially available ionophore cocktails were used (Fluka catalogue numbers 95297 for hydrogen; 60031 for potassium; 24902 for chloride; 21048 for calcium). The electrodes were calibrated in sets of standards before and after use. Electrodes with a response of less than 50 mV/decade for monovalent ions (25 mV/decade for Ca2+) were discarded. Experimental protocol Isolated mesophyll segments were cut and floated peeled side (abaxial surface) down on the experimental solution (unbuffered 0.1 mM CaCl +1 mM KCl ) for 4–5 h essentially as described 2 previously (Shabala and Newman, 1999). Forty to 50 min prior to measurements the segment was mounted in a Perspex holder and placed in the measuring chamber under the dim green microscope light. Three types of measuring solutions were used: basic 0.1 mM CaCl +1 mM KCl; 0.1 mM CaCl ; and 0.1 mM 2 2 CaSO . 4 The chamber containing a mesophyll segment was placed on the microscope stage and the electrodes were positioned 50 mm above the leaf surface, with their tips separated by 2–3 mm and aligned parallel to the surface. Ion fluxes were measured in the steady state for 5 min and then the hyperosmotic treatment was given. To provide the required 150 mM mannitol concentration in the bath, 880 ml of 1 M mannitol stock was added into the 5 ml chamber. The solution was thoroughly mixed by sucking and expelling using a Pasteur pipette, and net ion fluxes were measured for another 60 min. The time required for stock addition, mixing, and establishing the diffusion gradients (unstirred layers) was about 2 min. This interval was later discarded from the analysis and appears as a gap in most figures. In special methodological experiments, a small amount of the bath solution was added into the chamber instead of the mannitol stock, and solution was then thoroughly mixed as described above. No significant changes were evident for any of the ion fluxes measured (H+, K+, and Ca2+; data not shown). It was concluded that some possible changes in aeration conditions had no significant effects on the measured ion flux kinetics. Membrane potential measurements Membrane potentials of the mesophyll cells were measured with glass microelectrodes (GC 150–10F, Clark Electromedical Instruments, Pangbourne, Berks, UK ) before and 40 min after the onset of hyperosmotic stress using the MIFE electrometer. Electrodes had a tip diameter