Cytokinin treatment of leaf discs failed to enhance recovery of stomatal aperture, although it enhanced stomatal recovery of identically treated epidermal tissue.
PHYSIOLOGIA PLANTARUM 75: 399-404, Copenhagen 1989
Is proline involved in stomata regulation of Commelina communis plants recovering from salinity stress? Aviva Klein and Chanan ltai
Klein, A. and ltai, C. 1989. Is proline involved in stomata regulation of Commelina communis plants recovering from salinity stress? - Physiol. Plant. 75: 399-404. Plants of Commelina communis L. were grown in culture solution to which NaCl was added for 48 h. The solutions were then replaced with normal medium, so that the plants could recover from the stress. The water potential increased almost to that of the controls during 4 h of recovery, but stomatal resistance stayed high. Cytokinin treatment of leaf discs failed to enhance recovery of stomatal aperture, although it enhanced stomatal recovery of identically treated epidermal tissue. Proline levels in leaves correlated well with stomatal resistance. Incubation of epidermal tissue in D-proline inhibited stomatal opening. NaCI and benzyladenine interacted with the effect of proline, and the effect of abscisic acid and was additive to that of proline. Key words - Commelina communis, proline, salinity stress, stomata. A. Klein and C. ltai (corresponding author), Dept of Biology, Ben-Gurion Univ., Beer-Sheva 84105, Israel.
Introduction
After a period of exposure to water or salinity stress the water potential returns to normal much faster than transpiration recovers (Allaway and Mansfield 1970). ltai and Benzioni (1976) proposed that the lag in transpirational recovery is due to a below normal ratio of cytokinin to abscisic acid (ABA). However, attempts to overcome the after-effect by application of cytokinin proved only partially successful (Bengtson et al 1979), so that an additional factor may be involved. Since proline levels during and after stress are elevated, and since proline is involved in the recovery process from stress i^Blum and Ebercon 1976, ltai and Paleg 1982) and directly affects stomatal movement (Rajagopal 1981, Raghavendra and Reddy 1987), we investigated its involvement in the after-effect response of transpiration.
Materials and methods Plant material
Commelina communis L. seeds were germinated in wet vermiculite for 2 weeks. The seedlings were then transferred to aerated half-strength Hoagland solution (Hoagland and Arnon 1950). The plants were kept in a growth room in which relative humidity was not regulated, with a photon flux density at the plant level of 2(K) l^mol m - s ' (400-7CK) nm) emitted by fluorescent tubes (40 W preheatrapid start F400W CW, Philips, NY, USA) supplemented with 25 W Tungsten lamps. The plants were grown at 20/4 h day/night cycles, and 20 ± 5°C for 4—5 weeks. Relative humidity was not controlled in the room and it varied between 40 and 92%. To impose the stress, NaCl was added at different concentrations (as indicated) for 48 h. For recovery to take place, solutions were changed back to the regular ones.
Abbreviations - ABA, abscisic acid; MES, (2-N-morpholino)ethanesulfonic acid.
Received 25 August, 1988; revised 29 September, 1988; in final state 10 November, 1988 Phvsiol. Plant. 75. 1989
399
Epidermal peeling 5 -
Leaves were vacuum infiltrated with pectinase (from Aspergillus niger, obtained from Sigma) and incubated for 5 min. They were thoroughly rinsed with HiO, and the abaxial epidermis was peeled off.
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Measurement and analytical methods
Stomatal aperture was measured under the microscope, with a calibrated ocular microscope disc. A strict timetable was employed during the experiments, based on intervals of 2 min, which was the time needed for the measurement of one strip (20 stomata). The epidermal strips were kept under the microscope in liquid paraffin to diminish post-experimental changes. In each treatment 80 stomata from 4 different strips were measured. Each experiment was repeated as indicated in the legends. The aperture of stomata on non-peeled leaf discs was determined with the aid of silicon rubber impressions (Willmer 1983). Water potential was determined by a pressure bomb (A. R. L, Kfar Charouv, Israel). Stomatal resistance was determined using a Li 700 porometer (LI-COR Inc., Lincoln, NE, USA) on the abaxial surface of one appropriate leaf on each of 6 plants per treatment. Proline of leaf tissue, from which the main veins had been removed, was determined by a spectrophotometric analysis at 515 nm of the ninhydrin (2,2-dihydroxy-l,3-indanedione) reaction according to Singh et al. (1972). Each sample contained at least 0.7 g fresh weight.
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