Partly dehydrated tobacco leaf tissue (Nicotiana rustica), stripped of the lower epidermis, was used to study the effect of kinetin on the rate of rehydration.
Plant Physiol. (1972) 49, 124-126
A Rapid Effect of Kinetin Tobacco Leaf Tissue
on
Rehydration of Received for publication July 7, 1971
AVINOAM LIvNE AND YOSEF GRAZIANI Division of Life Sciences, Negev Institute for Arid Zone Research, Beer-Sheva, Israel ABSTRACT
Partly dehydrated tobacco leaf tissue (Nicotiana rustica), stripped of the lower epidermis, was used to study the effect of kinetin on the rate of rehydration. Depending on the rate of rehydration in untreated tissue, kinetin either increased or decreased rehydration rates. The response to kinetin was very rapid and could be discerned in less than 2 minutes. On extensive dehydration, the tissue lost the capacity to respond to kinetin. Salinity stress, which decreases the endogenous level of cytokinins in the plant, conditions the leaf to stimulation of rehydration by kinetin. It is suggested that cytokinins may play a role in controlling water permeability of the leaf tissue.
in 0.2 ml of ethanol at 50 C and adding water to 5 ml. The stock solution was further diluted with unlabeled solution to 4 /c//imole. An equivalent amount of ethanol was added to the control. Direct radioactivity measurements of leaf discs by liquid scintillation counting were performed as described (5). Where equations are shown, the curves were computed according to the least square fit (2). RESULTS AND DISCUSSION
Tobacco leaf discs, stripped of the lower epidermis, lose water at a rate of 10 to 25 mg per g fresh weight per min. When the partly dried tissue is floated on water, the rate of rehydration is 20 to 40 mg per g fresh weight per min. Table I shows that preincubation of leaf discs with 50 [M kinetin for 5 min prior to the 33-min dehydration period greatly stimulated the rate of rehydration. Furthermore, Table I shows that the stimulation of rehydration by kinetin was apparent even without the pretreatment with kinetin. The time course of rehydration shows that the effect of kinetin was very Of the many chemical compounds which are known to rapid (Fig. 1). The curves of such measurements indicated that affect permeability (11), the plant hormones IAA (3) and kinetin exerted its effect in less than 2 min. The equations gibberellic acid (4) are particularly interesting. Application of given in Figure 1 fit the time course of rehydration of the a relatively low concentration of either hormone to plant cells discs and suggest a response even within seconds. This is increases water permeability, whereas a decrease in per- apparently the fastest response of a plant tissue to kinetin meability of the cells to water is observed at higher concentra- recorded in the literature. tions. No effect is apparent at intermediate concentrations. The stimulation of the rate of rehydration by kinetin Considering the known effect of kinetin and similar analogues depended on the degree of dehydration (Table II). The rehyon stomatal opening and transpiration (7, 9), it was of interest dration rates were similar if the discs were previously dehyto examine the effect of kinetin on water permeability of plant drated to either 350 or to 600 mg per g fresh weight. but if cells in general. excessive dehydration had taken place (600 mg per g fresh This communication reports a fast response of rehydrating weight), kinetin no longer affected rehydration. Possibly kinetin tobacco leaf tissue to kinetin. The response is apparently was not absorbed by the discs after excessive dehydration. To related to the endogenous level of cytokinins. An abstract of test this possibility we compared the rates of uptake of '4Cthis report has appeared (8). labeled kinetin (50 1-M) by discs which had lost 360 or 600 mg water per g fresh weight. When the discs lost water to the extent of 60% of their fresh weight, the rate of uptake of MATERIALS AND METHODS labeled kinetin was 20 to 40% higher than in discs which had The growth of the tobacco plants (Nicotiana rustica), the lost only 35% of their water content. Therefore. absorption preparation of leaf discs free of the lower epidermis and the of kinetin by the discs does not limit the stimulation of rehymeasurement of dehydration and rehydration were as described dration rate. An efflux experiment with labeled kinetin was performed to (5). Rates of hydration are expressed on the basis of the original fresh weight of the stripped tissue, prior to dehydra- test the possibility that a change of essential membrane struction. The data shown represent an average of experiments, tures, due to excessive drying, eliminates the stimulation of each performed in duplicate and, unless otherwise indicated, rehydration by kinetin. CaCl2 (0.5 mM) was included during the incubation of the tissue with labeled kinetin, since this repeated three times. Kinetin was obtained from Sigma Chemical Co., St. Louis incubation period was relatively long (30 min), but similar (Lot No. 469-0390), and from Calbiochem Ag, Luzern (Lot results were obtained without CaCl,. Figure 2 shows that No. 53515). Aqueous solutions of kinetin were prepared by when the discs had lost 300 mg water per g fresh weight, the autoclaving for 20 min. Kinetin-8-'4C was obtained from the rate of kinetin efflux was the same as from the control tissue. Radioactive Centre, Amersham, England. A stock solution of However, the effltix of kinetin was greatly increased when the labeled kinetin was prepared by dissolving a 50-ftc sample the tissue had lost 600 mg water per g fresh weight, indicating 124
125
REHYDRATION OF TOBACCO LEAF TISSUE
Plant Physiol. Vol. 49, 1972
changes of the membranes of the tissue
on
excessive drying
(5).
Figure 3 shows that kinetin not only stimulated but could also decrease the rehydration rate of tobacco leaf tissue. When the rehydration rate of the tissue was low, stimulation by kinetin was observed, while the reverse was true when the
20
15
Table 1. Effect of Kinetin on Rate of Rehydrationi of Partly Dehydrated Tissue Discs from tobacco leaves with the epidermis-free surface down were preincubated by being floated on either H20 or 50 LM kinetin for 5 min at 20 C. The discs were then allowed to lose 350 mg H20 per g fresh weight. Rehydration of the tissue at 20 C on either H20 or 50 MM kinetin was measured by frequent weighing at 2 to 3 min intervals. Rehydration rates are given as mg H20 taken up per g fresh weight per min during the initial 5 min of rehydration. Values of standard error are also given.
cq
10
5
10
Rate of Rehydration
Preincubation
H20
Kinetin mg/gfr uw*min
H20 Kinetin
25 I 43 ± 3
37 3 55 ± 4
20 TIME, minutes
30
FIG. 2. Efflux of "IC-kinetin from tobacco leaf discs. Discs were first incubated in the presence of 25 gM kinetin (4 Ac per ,umole) and 0.5 mm CaCls for 30 min, when the level of labeled kinetin in the tissue reached a plateau. The discs were then allowed to lose either 300 or 600 mg IIO per g fresh weight in evaporation. Control tissue with labeled kinetin was kept in a moist dish for 15 or 38 min, respectively, for the time required for dehydration. After dehydration, the discs were floated on water, sampled at the indicated intervals, blotted and measured directly for radioactivity.
300 +20
z
+
KINETIN
-
0
0~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~
"
200
0
CONTROL
410
LIU
z z
0
U
LLI c
0-
ll
ioo
I
7oX064_X171 Y= 45.2 X0 8 - X 1.73
+KINETIN Y= w Ir
1.
0
Z
'I E
CONTROL
OLLJX
L' L] E
0n
0
LL LUJ LL
6
8
10
12
14
y=-l.O49x+5285 r= 0.862\
z
TIME, minutes z
FIG. 1. Time course of rehydration of tobacco leaf discs. The discs were dehydrated to the extent of 350 mg per g fresh weight and rehydrated by being floated on 50 aM kinetin, or for control, on water.
20
20
30
40 REHYDRATION mg /g /min
50
FIG. 3. Kinetin-affected change in rehydration of tobacco leaf discs as a function of rate of rehydration in untreated tissue. Rehydration was measured as in Table I, but without preincubation. Kinetin concentration was 50 MuM.
Table II. Thze Dependency of the Stimulation of Leaf Tissue Rehydration by Kinetin on the Extent of Dehydration Discs from tobacco leaves were allowed to lose either 600 or 350 mg H20 per g fresh weight; rehydration then followed as described rate of rehydration was high. An inverse and linear relation in Table I. Rehydration rates are given as mg H20 taken up per g between the kinetin-affected change in rehydration and the fresh wt per min during the initial 5 min of rehydration. Values of rehydration rate was measured, with a regression coefficient standard error are also given. of 0.86 (Fig. 3). The slope of the curve (-1.05) indicates a feedback mechanism, so that kinetin changed the dehydration Rehydration rate from a range of 15-50 mg/g*min to a median value of Extent Dehydration about 33 mg/g-min. These results might be explained by the Medium Rate assumption that the level of endogenous cytokinins affects the rate of rehydration. The levels of these cytokinins apparently mg/gfr wt *rmin varied in our experiments. Daily and seasonal variation of the 4 31 Kinetin, 50,uM 1 600 mg/g fresh wt endogenous level of hormones in tobacco plants has been suggested by several workers at our Institute (ref. 10; Itai and 4 Kinetin, 50 2M44 3 350 mg/g fresh wt Richmond, personal communication). It is therefore anticipated that a treatment which decreases the endogenous
126
LIVNE AND GRAZIANI
Table III. Effect of Salinity Stress Applied to Tobacco Plants on Rehydration of Leaf Tissue For treatment A, the nutrient medium of the stress plants was supplemented with 50 mNs NaCl, followed by 100 mm NaCl after 24 hr. The measurements were taken 1 day later (3rd day). For treatment B, the nutrient medium of the plants was replaced on the 3rd day by a fresh one, without the supplementary NaCl, and analyzed 1 day later (4th day). Dehydration and rehydration of leaf discs, without preincubation, were as described in Table I. Kinetin concentration was 25,Mm. Rate of rehydration is given as mg H20 per g fresh weight per min. The number of experiments summarized are included in parentheses.
Rate
mg/g fr wt-min
LITERATURE CITED
Plant Medium
tobacco plants, under-the growth conditions and with the same facilities used in the present report. Table IIIA shows that salinity stress reduced the rate of rehydration by 23% and induced a 15% stimulation of the rehydration rate of kinetin. These differences in rates are small, yet significant at 95% level. Table IIIB shows that after 24 hr of recovery from the salinity stress, the original rate of rehydration was again observed. At the same time, the original level of cytokinins is restored (6). indicating that the level of endogenous cytokinins indeed may affect the rehydration rate. Acknowledgments-The authors are most grateful to Dr. Pieter J. C. Kuiper for reading the manuscript and for valuable suggestions. Constructive comments proffered by Dr. J. M. Daly and Mr. J. Schechter are appreciated. We wish to thank Mr. N. Zelingher for his help in mathematical analysis and Mrs. Judith Tal for capable technical assistance. Thanks are also extended to Mrs. Cynthia Bellon for editing the manuscript.
Rehydration Treatment
Plant Physiol. Vol. 49, 1972
Control
H20 Kinetin
35 4 1 29 :1= 2
Stress
H20 Kinetin
27 32
Control
H20 Kinetin
32 29
Post-stress
H20 Kinetin
36 26
A (3) 4 -+-
1 1
B (1)
cytokinin level could affect both the rate of rehydration and the response of the tissue to exogenous kinins. It has previously been shown (1, 6) that salinity decreases the supply of cytokinins from the roots to the shoot and produces symptoms of cytokinin deficiency in sunflower and
1. BEN-ZioNi, A., C. ITAI, AND Y. VAADIA. 1967. Water and salt stresses, kinetin and protein synthesis in tobacco leaves. Plant Physiol. 42: 361-365. 2. BROWNLEE, A. 1965. Statistical Theory and Methodology in Science and Engineering. John Wiley and Sons, New York. 3. HEINRICH, G. 1962. Fusarinsaiure in ihrer Wirkung auf die Wasserpermeabilitat des Protoplasmas. Protoplasma 55: 320-326. 4. HEINRICH, G. 1964. Huminsaure and Permeabilitat. Protoplasma 58: 402-425. 5. GRAZIAXI, Y. AND A. LIVN-E. 1972. Dehydration, water fluxes and permeability of tobacco leaf tissue. Plant Physiol. In press. 6. ITAI, C., A. RiCHxNO1), AND Y. VAADIA. 1968. The role of root cytokinins during water and salinity stress. Israel J. Bot. 17: 187-195. 7. LiVNE, A. AND Y. VAADIA. 1965. Stimulation of transpiration rate in barley leaves by kinetin and gibberellic acid. Physiol. Plant. 18: 658-664. 8. LIVNE, A. AND Y. GRAZIANI. 1971. Effect of kinetin on rehydration rate of tobacco leaf tissue. Israel J. Bot. 20: 332. 9. LIKE, H. H. AND T. E. FREEMAN. 1968. Stimulation of transpiration by cytokinins. Nature 217: 873-874. 10. ROTH-BEJERANO, N. AND S. H. Lips. 1970. Seasonal characteristics of the interaction between kinetin and gibberellic acid with regard to level of nitrate reductase in tobacco leaves. Israel J. Bot. 19: 30-36. 11. STADELMANN, E. J. 1969. Permeability of the plant cell. Annu. Rev. Plant Physiol. 20: 585-606.
