High-temperature Disruption of Guard Cells of Vicia faba

Abstract

Increased variability in stomatal aperture at high temperatures can be attributed, in part, to the differential sensitivity of guard cells to thermal damage. Individual stomata become increasingly open at higher temperatures until guard ceUls are lethaly damaged; at that temperature, apertures decrease. The extent of irreversible damage causing closure was estimated by K' uptake, neutral red accumulation, and visual scoring of chloroplasts. This study found that visual scoring of chloroplast disruption provided the best estimate of guard cell viability at high temperature. Removal of the damaged guard ceUs from the population sample resulted in a constant coefficient of variability for apertures over the temperature range 25 to 50 C. Stomatal apertures increase with increasing temperature up to some optimum before closure begins (2, 9, 10, 12, 14, 16). The temperature at which this reduction occurs is difficult to determine because of pronounced variability in aperture occurring at higher temperatures (8-10, 12, 16). Irreversible thermal damage to guard cell function was considered as a possible cause of this variability. The sensitivity of chloroplasts to thermal injury has been determined in other studies to limit the thermal resistance of mesophyll cells. Bjorkman (1) reported that the photosynthetic apparatus in chloroplasts was damaged by heat before other symptoms of injury were detected. Schreiber and Berry (11) determined heat damage to the photosynthetic apparatus ofseveral species utilizing the technique of Chl fluorescence. The lower limit of heat damage appeared to be between 41 and 49 C, depending on the plant species and the temperature acclimation of the plant. Previous studies with Viciafaba (9, 10) have shown aperture reduction to begin at temperatures above 40 C, which is consistent with Schrei-ber and Berry's observations of chloroplast thermal stability (11). Pallas (6) reported the disintegration of guard cell chloroplasts in Zebrina pendula, attributing the disintegration to pH damage of the chloroplast membranes. Pallas (6) also observed proto-plasmic streaming and staining with neutral red as indicators of guard cell viability; he found both methods unreliable. Stomata were found capable of opening without evidence of either proto-plasmic streaming or neutral red uptake. The purposes of the study presented here were (a) to determine a reliable method for detecting guard cell damage by observation ' of chloroplast structure; (b) to utilize this method to determine how guard cell chloroplast viability relates to variability in sto-matal aperture; and (c) to ascertain the temperature-aperture response at high temperatures for guard cells with visually intact chloroplasts. MATERIALS AND METHODS V. faba plants were grown from seed in a peat-Perlite mixture in a growth room. Epidermal strips were floated on 10 mm KCl in light at 25, 35, 40, 45, 47, and 50 C with C02-free air, saturated with H20 at the temperature of the experiment, passing over the strips. Details of the growth conditions and incubation periods are given in Rogers et al. (9, 10). At the end of the incubation period, strips were mounted on a glass slide on 10 mm KC1 solution and apertures were measured with a calibrated ocular micrometer at x490. Twenty to 40 apertures were measured on each of two strips for each incubation period and temperature with 3 to 12 replica-tions. Strips stained with sodium cobaltinitrite for K+ determination were treated as described by Rogers et al. (9, 10). Chloroplast integrity was determined concurrently with stoma-tal aperture. Chloroplasts within the guard cells bordering the stomatal pore were scored visually on a scale of 0 to 2. The chloroplasts that appeared green and round with the outer membrane smooth and distinct were scored "2," and the guard cells were considered healthy and viable (Fig. la). If the chloroplasts were colorless, irregular in shape or shrunken, with the outer membrane wrinkled or indistinct, they were scored "0" (Fig. lb). Chloroplasts intermediate between these two forms were scored "1." Sometimes the condition of the chloroplasts in the two guard cells bordering the pore were not identical in appearance. That pair would be scored with two numbers to indicate a "split-pair" condition. Figure lc shows a "1-2" split and Figure ld shows a "0-2" split. Guard cells having a split-pair condition were included in the total population but were excluded from the data set containing only guard cells with chloroplasts scoring "2." Additional strips were also treated with neutral red for comparison. Strips were placed on a 0.003% solution of neutral red stain for 30 min at room temperature. The strips were rinsed in distilled H20 and examined microscopically for neutral red accumulation in the guard cells. RESULTS To estimate the increase in variability in stomatal aperture at high temperatures, a large number of apertures were measured at 35, 40, and 45 C. Table I shows that variability, expressed as the CV5, increased from 43 to 81% as the temperature was raised from 5 Abbreviation: CV, coefficient of variability.