Cytokinin Activity in Water - stressed Shoots ' Received

Water stress applied to the plant shoot through enhanced evaporative demands reduced cytokinin activity in extracts of xylem exudate and leaves. This reduction resembled the changes in cytokinin activity caused by water stress applied to the root. Cytokinin activity in detached wilting leaves decreased rapidly. Recovery took place after several hours in a humid chamber. Experiments with "4C-kinetin indicated that the mechanism of the inactivation and its reversal involve a chemical transformation of the cytokinin molecule. Plants may be water-stressed by limiting water availability to the root through drought, salinity, or osmotic media. Water stress can also be imposed by enhancement of transpiration demands. Plants respond similarly to various water stresses. Similarities lie in RNA and protein metabolism (13) and several developmental and morphogenic features (15). Evidence has been brought (5, 6) on the role of cytokinins in regulating plant response to water stress applied to the root. The work described here attempts to verify and extend the hypothesis that cytokinins play a role in the regulation of plant water stress responses, when transpiration demands are enhanced. MATERIALS AND METHODS Sixto 8-week old Nicotiana rustica plants were used which, at the beginning of the experimental period, had developed to almost the termination of the vegetative stage. Each plant was grown in 2 liters of half-strength Hoagland solution in a cooled greenhouse. Cytokinin activity in stem exudate was determined as described earlier (6). The leaf cytokinin extraction and separation method was modified from Heide and Skoog (4). About 100 g of mature leaves, with main veins removed, were frozen in liquid air. The leaves were blended in a Waring Blendor with 1 liter of 70% (v/v) ethanol and then boiled gently for 5 min at 85 C. The cooled extract was filtered, and the pH of the supernatant was adjusted to 8.8. After centrifugation (10,OOOg for 25 min), the precipitate was discarded, and 20 ml of saturated barium acetate were added to the supernatant. Centrifugation was done after an 18-hr period in darkness at 4 C. The precipitate was discarded, the volume of the supernatant was reduced to half, and the pH was adjusted to 2.5 with HCI. A cation exchange column was used for separation (180 ml, Amberlite IR-100 A.R. 20-50 mesh, 1The research was supported by the United States Department of Agriculture under Contract FG-IS-32. 2Present address: Volcani Institute for Agricultural Research, Beit Dagan, Israel. or Dowex 50W x 4 50-100 mesh). The column was rinsed with 1 liter of 70%to (v/v) ethanol and with H20. The active substance was eluted with 1 liter of 4 N NH40H. The eluate was concentrated to 300 ml under vacuum at 45 C, and the pH was adjusted to 8.0 and extracted twice on a shaker for 20 hr with 200 ml of n-butanol. The extracts were then combined and dried under vacuum. The residue was dissolved in 5 ml of water at pH 8. This solution was divided into equal portions and bioassayed by means of soybean tissue culture (9). The incorporation of '4C-L-leucine into trichloroacetic acidinsoluble leaf disc extracts was measured according to BenZioni et al. (2). Kinetin treatment was applied prior to incubation in "4C-L-leucine, by placing leaf discs for 45 min between Whatman No. 3 papers wet with kinetin solution. Leaf discs were exposed to two extreme relative humidities by placing them for 30 min in desiccators which contained either wet filter paper or CaCl2. Fresh weight change was recorded. Experiments with labeled kinetin were done as follows. The petiole of a fully expanded leaf was placed in a vial containing 2 ml of 0.5 4c kinetin-8-14C solution (Cal Atomic, Los Angeles, specific radioactivity 24.6 Ac/Mmole). The 2 ml were taken up by the leaf in 60 to 120 min. The leaf was cut in half, and the middle rib was removed. One half was treated as indicated in each experiment, and the other was put in a humid chamber for the duration of the treatment. On termination of treatment the halfleaf was frozen in liquid air and homogenized with 2 ml of cold ethanol. The homogenate was filtered off, and the residue was washed with 2 ml of cold water. To the combined filtrate 1 ml of chloroform was added. The nonpolar fraction contained no label. The polar fraction was separated and applied to Whatman No. 3 chromatography paper. The chromatogram was developed for 30 cm in an ascending-descending fashion in one of the two solvent mixtures: H20-acetic acid-n-butanol (1:1:4) or ethanolchloroform (9:1). The chromatogram was then dried and cut into 10 equal portions which were placed into vials containing dioxane-ethanol-toluene scintillation solution. The vials were then placed into a liquid scintillation counter (Packard TriCarb 3308) and assayed for "4C. RESULTS Plants were exposed for 30 min to an airstream which caused slight wilting of the leaves. The airstream was turned off and the shoots of both the treated and the control plants were sprinkled with water. After 10 min, leaves recovered their turgor, all the plants were topped, and the mature leaves were frozen in liquid air. A rubber hose was mounted on the stump, and exudate was collected for 6 hr. The amount of exudate did not differ between treatments. The data in Table I demonstrate a reduction in the activity of cytokinin in the leaves and in exudate when the stress was applied to the intact plant. This experiment was repeated three times. The range of the reduction was 39 to 60% in exudate and 22 to 59% in leaves. Table IIA demonstrates that such a reduction is also observed if the stress is applied to a detached leaf. In this experiment, 87 www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1971 American Society of Plant Biologists. All rights reserved. Plant Physiol. Vol. 47, 1971 leaves were assayed for cytokinin activity at short intervals, after being detached from the plants. Leaves were placed on the laboratory table with dorsal side up for the time between detachment and freezing. Fresh weight loss was determined prior to freezing. Cytokinin activity decreases as drying proceeds. These experiments were repeated eight times with several modifications. The reduction ranged between 70 and 89%70 after 10 min and 5 and 52% after drying for 30 min. Table IIB shows that cytokinin activity is partly reversible. In this experiment the leaves were placed in a humid chamber for different intervals after the drying period. Table III shows the results of 14C-leucine incorporation in discs obtained from leaves which were detached after the stress was applied. In a further experiment (Table IV) stress was applied by placing leaf discs for 30 min in containers usually used as desiccators. CaCl, was put into one container and water into the other. Tables III and IV show a reduction in incorporation due to atmospheric stress. This reduction is not reversible by kinetin treatment. The inability of kinetin to bring about recovery of '4C-leucine incorporation (Tables III and IV) does not agree with previous results for leaf discs where stress was applied to the roots through excess salinity or osmotica (5). This disagreeTable I. Cytokininl Activity in Leaves anid Exudaate of Tobacco Platits Stressed plants were exposed to air stream for 30 min. Callus WN'eight Kinetin Equivalent Activity mng pg/'g freslI wt Exudate Nonstressed 236.2 0.140 100 Stressed 107.5 0.055 39 Leaves Nonstressed 115.8 0.044 100 Stressed 87.7 0.026 59 Table II. Cytokinini Activity in Extracts of100 g ofDetached Leaves as Funiction of Dryintg antd Recovering Time in Humid Chamber Drying Recovery Time Callus Weight Kinetin Activity Time Equivalents hr mig pg/kg freshz wt m,/O A. 0 0 0.624 43.4 100 0.15 0 0.558 35.1 81 0.5 0 0.324 22.9 53 B. 0.5 0 0.254 15.9 36 0.5 18 0.505 31.7 73 Table III. Incorporationi of l4C-L-Leuicine i/ito Protein ofLeafDiscs Takeii from Stressed anid Nonistressed Plantts anzd Treated with Kinetin Solution Period of incorporation was 1 hr. H20 Kinetin, 1.0 pg/ml Percentage of Control cpmn/100 mg fresh wt Nonstressed 13,392 14, 868 111 Stressed 12,253 12,667 103 Table IV. Intcorporation of 14C-L-Leucine into Protein ofStressed anid Nonzstressed Leaf Discs Treated with Kinetint The period of incorporation was 1 hr. The concentration of kinetin was 1.0 ug/ml. Percentage Incorporation of Initial 'ramn Pretreatment Post-treatment cptn/disc %0 of control cpm/disc o of control 96 2146 100 4755 100 +I 2872 133 6251 131 75 1504 100 3044 100 + 1744 117 4132 135 ment may indicate that, when the leaves are stressed by drying as is done here, cytokinins may be inactivated. The experiments with labeled kinetin were intended to study the fate of kinetin during stress. Different distribution of the label is observed in stressed and nonstressed leaves (Fig. 1, A, D). Differences in distribution due to stress are eliminated after recovery of the leaves in a humid chamber for 7 hr (Fig. 1, E). The manner of application of the labeled kinetin did not modify the results (Fig. 1, B, C). DISCUSSION There are several reports in the literature which indicate that a measurable reduction in cytokinin activity in root exudate is observed when plant roots have undergone a period of water shortage (6) or water flooding (3), or the medium has been changed to impose excess salinity (5), excess osmotica (5), high or low root temperature (14), or a low pH (1). In these cases stress is applied to roots, and changes in shoot behavior are brought about through the effect of the stress on the root. In the experiments reported here an attempt is made to test the effect of direct drying of the leaves on cytokinin activity in exudate as well as in leaves. The results show that the level of cytokinin in both the exudate and the leaves is reduced by an air drying stress (Tables 1, II). This is of particular interest since the stress is very much shorter (30 min) than those commonly employed in root stress experiments (1 day to several days). It has been suggested that roots are the site of cytokinin synthesis in plants (11, 18) and that cytokinin biosynthesis in the shoot has so far been reported only in fruitlets (8). Furthermore, clear evidence exists that the shoot depends on root cytokinin (7, 11), and also that various shoot organs compete for cytokinins from the root (12). It is therefore feasible that biosynthesis in the root ceases at the moment the water tension in the leaf is enhanced. The signal to the site of synthesis may in this case be the changes in water potential which are transmitted through the plant. A different case is the reduction of cytokinin levels in detached leaves, where changes in biosynthesis in the root cannot account for the reduction. The short time of stress necessary to effect such a reduction, the inability of external kinetin to restore amino acid incorporation, and the ability to recover cytokinin level all suggest inactivation as a likely process. Such inactivation may be a result of either inhibitor accumulation during stress or actual transformation of the cytokinin molecule. Inhibitor accumulation may be responsible for lower cytokinin activity due to stress. Abscisic acid is one of those inhibitors (17) which accumulates during water stress (10, 19, 20). This could explain the data on leucine incorporation. However, several points contradict such an explanation for lower activity of cytokinins in stressed leaves. While cytokinin reduction is rapid, levels of abscisic acid increase only after 4 hr (20). Fur88 ITAI AND VAADIA www.plantphysiol.org on October 23, 2017 Published by Downloaded from Copyright © 1971 American Society of Plant Biologists. All rights reserved. CYTOKININ IN WATER-STRESSED SHOOTS