Planta

Planta 144, 167-172 (1979)

9 by Springer-Verlag 1979

Uptake and Distribution of Abscisic Acid in Commelina Leaf Epidermis J.D.B. Weyers I and J.R. Hillman Botany Department, University of Glasgow, Glasgow G12 8QQ, U.K.

Abstract. Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a range of KC1 concentrations. Control apertures were greatest at high concentrations of the salt, and the effects of ABA, in terms of closure, were most pronounced below 1 0 0 m o l m - 3 KC1. Stomata opened on strips floated on buffer plus 5 0 m o l m -3 KC1 and closed within 10min when transferred to the same medium plus 0 . 1 m o l m -3 ABA. [2-14C]ABA was used to study uptake and distribution of the hormone by the epidermal strips. It was calculated that no more than 6 fmol ABA were present per stomatal complex at the time of closure, although uptake continued thereafter. Microautoradiography indicated that radioactivity from [2-14C]ABA accumulated in the stomatal complex at or near the guard cells within 20 min. T L C was used to examine the state of the label after 1 h incubation. Efflux of label from preincubated tissue appeared to occur in three phases (ta/2=7.2 s, 4.0 min, 35.2 min). Efflux was correlated with stomatal re-opening. The results confirm that ABA can accumulate in the epidermis of C. cornmunis. Key words: Abscisic acid - Commelina - Epidermal strip - Stomatal closure,

Introduction It is now well established that plant hormones are involved in natural higher-plant reactions to water stress (Vaadia, 1976), and the stomatal response to

ABA has received particular attention (Raschke, 1975; Mansfield et al., 1978; Jones, 1978). Water stress induces stomatal closure which may continue after the stress has been relieved (Meidner and Mansfield, 1968); it also results in a rapid rise in endogenous levels of ABA (Wright and Hiron, 1969; Kriedemann et al., 1972; Hiron and Wright, 1973). Moreover, the effects of applied synthetic ABA can mimic these responses (see Mansfield, 1976). Further corroborative evidence comes from the studies of Tal and co-workers on flacca, the 'wilty' mutant of tomato which is deficient in ABA (see Vaadia, 1976). Loveys (1977) showed that isolated leaf epidermis of Vicia was incapable of producing ABA in response to a stress but that levels of ABA were elevated in epidermis removed from stressed whole leaf tissue. It therefore appeared that transport of wilt-induced ABA occurred from the mesophyll tissue to the epidermis during water stress. ABA is known to alter the metabolic and ionic status of the guard cells (Mansfield and Jones, 1971 ; Horton and Moran, 1972) and it has been concluded that the hormone acts directly on guard cells (see Raschke, 1975). There is, however, little information about the uptake, distribution, or transport of ABA within epidermal tissue. We have recently shown (Itai et al., 1978) that the stomatal complex of Commelina is capable of accumulating label from [2-1~C]-ABA. In this paper we describe the characteristics of ABA uptake and efflux in more detail using a system in which the effects of the hormone on stomatal aperture were also measured.

Materials and Methods Plant Material

1 Present address: Department of Biological Sciences, Dundee University, Dundee, DD1 4HN, U.K. Abbreviation: ABA=Abscisic acid

Plants of C. communis L. were grown from seed (supplied by C. Willmer, Stirling University, U.K.) in standard compost (peat: sand:loam, 1 : 1:1, by volume, with John Innes Base) in 125 mm

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J.D.B. Weyers and J.R. Hillman: Uptake and Distribution of A B A in Commelina Epidermis

pots in a greenhouse. Supplementary lighting was given for 16 h daily (0300-i700 h) from Atlas ' K o l o r l u x ' 400 W M B F R / U highpressure mercury vapour lamps. Plants were used about 6 weeks after sowing and were transferred to the experimental room one day before use. Epidermis was peeled from the abaxial surface of the fully expanded leaf closest to the apex. Epidermal strips were removed at right angles to the lamina to optimise cell viability and minimise mesophyll contamination, using the method of Mansfield and Travis (personal communication).

Incubation Solutions Buffer solutions were based on 10 tool m -3 PIPES (B.D.H. Ltd., Poole, U.K.) adjusted to p H 6.8 with K O H . These contained a range of KC1 concentrations in the first experiment described, but in all other cases 50 tool m - 3 KC1 was employed. When used, (_+)-ABA (Sigma Chemical Co., Ltd., St. Louis, U.S.A.) or [2-14C] ( + ) A B A (225.7 GBq m o l - 1 ; The Radiochemical Centre, Amersham, U.K.) were supplied at 0.1 mol m -3.

Incubation Conditions and Aperture Measurement Strips of epidermis were cut to appropriate shapes and sizes and floated cuticle uppermost, on 20 • 10 . 6 m a buffer solution in glass petri dishes through which artificial air having a CO2 concentration of less than 1 v p m (BOC Ltd., London, U.K.) was bubbled continously at a rate of 1.67 x 10-6 m 3 s - ~ per dish. Experiments were always carried out at the same time of day: a 0.5 h preincubation was started at 11.30 h and experiments began at noon. The dishes were placed 300 m m below five 80 W Atlas white fluorescent tubes. The irradiance in the band 400 1000 rim, measured with an Optometer model 40X light measuring instrument fitted with a diffuser head (United Detector Technology Inc., Santa Monica, U.S.A.), was 16-19 W m - 2 . The temperature of the experimental room was maintained at 296 + 1.5 K. Stomatal apertures were measured by microscope with an eyepiece graticule to the nearest 0.75 ~tm. Generally 25 measurements from each of two strips were combined for each m e a n shown. When required, strip areas were measured by eyepiece graticule to the nearest 0.1 m m 2.

Radioassay For uptake studies and microautoradiography, strips taken from radioactive solutions were rinsed to remove free-space label in 6 x 15 s washes of l0 - 6 m 3 cold buffer without ABA. Radioactivity present in epidermal strips was estimated by liquid scintillation spectrometry after extraction for 12 h in 10 _6 m 3 methanol and allowance was made for quenching. The microautoradiographic procedure was a modification of that of Willmer et al., (1973). Rinsed epidermal tissue was applied, cuticle uppermost, to a subbed slide and immersed in liquid N z before freeze-drying for 12h. The emulsion side of a piece of K o d a k Pan-F film (18 DIN/50 ASA) was applied directly to the tissue and held in place with another slide which was then taped to the first, this operation being carried out in a dark room. The film was exposed for 10 weeks in the dark at 258 K before development with PQ Universal developer and H y p a m fixer (Ilford Ltd., llford, U.K.) using the manufacturer's recommended procedures. Controls for positive and negative chemography were always employed (Rogers, 1973). Efflux of radioactivity from 4 pieces of epidermal tissue incubated for 1 h in [2-1'~C]-ABA solution was studied by transferring the epidermis (total area 44.8 ram2), without rinsing, through a

series of buffer solutions containing non-radioactive ABA. Radioactivity present in the solutions was then estimated by liquid scintillation spectrometry. Stomatal aperture measurement during efflux studies was performed on tissue incubated with cold A B A for 1 h and transferred, after rinsing, to 20 x 10 . 6 m 3 buffer without ABA. This solution was changed after 1 h. Control strips received equivalent disturbance, The molecular state of the 14C label in methanol extracts of epidermis tissue incubated for 1 h in [2-14C]ABA solution was examined after T L C in four solvent systems (benzene : acetone: glacial acetic acid, 70 : 30 : 1 ; ethyl acetate: toluene: glacial acetic acid, 1 : 10 : 2; chloroform: methanol, 1 : 1 ; n-hexane: ethyl acetate, 1 : 1 ; all by volume) using liquid scintillation spectrometry.

Results and Discussion

The nature of the incubation medium is known to define to a great extent the reactions of guard cells in epidermal strips to various stimuli (Willmer and Mansfield, 1969). Thus, the ionic content of the medium has been shown to have a marked effect on stomatal opening (Fischer, 1968; Humble and Hsaio, 1969). The table shows that apertures attained by stomata on strips of Commelina epidermis increased with the KC1 concentration. When ABA was supplied, continously of after 3 h, its effects on closure were substantially modified by the KC1 concentration. Above 200 tool m - 3 KC1, ABA had little effect on aperture whilst it caused almost complete closure in both treatments at 25 and 50 mol m -3 KC1. Failure to obtain full closure of stomata on epidermal strips with ABA (e.g. Lancaster etal., 1977) may therefore be due to the use of excessive concentrations of KC1 in the medium, an observation also noted by Willmer et al. (1978). This phenomenon may have important implications for studies on the mode of action of ABA in stomata1 closure. Buffer containing 50 tool m - 3 KC1 was selected to investigate uptake and distribution of ABA and the kinetics of stomatal closure; this allowed adequate and reproducible opening whilst ensuring sensitivity in terms of closure. When epidermal strips were floated on this solution maximal apertures were noted after 2 h. A two-hour incubation to open stomata was therefore carried out before ABA treatment in all further experiments. Figure 1 shows that closure occurred within 10 rain on strips transferred to ABA solutions ; such a speed of reaction is compatible with results obtained with whole leaves, feeding ABA via the transpiration stream (Kriedemann et al., 1972). Stomatal re-opening was not observed within 3 h of continuous ABA application. The uptake of radioactivity by epidermal strips during a 1 h treatment substituting [2-14C]-ABA for 'cold' ABA is shown in Figure 2. Longer-term studies with [aH]-ABA indicated that there was little further

J.D.B. Weyers and J.R. Hillman: Uptake and Distribution of ABA in Commelina Epidermis

169

Table 1, Effect of ABA on stomata1 aperture at different KC1 concentrations. Series 1 A = 3 h in buffer control, B ~ 3 h in buffer plus 0.1 m o l t o -3 ABA. Series 2 C = 3 + 1 h in buffer control (equivalent disturbance), D = 3 h in b u f f e r + l h in buffer plus 0.1 m o l t o-3 ABA. Standard error values are given in parentheses, n = number of observations per treatment KCI concentration/ mol m - 3

Mean stomatal aperture/pro Series 1 n = 100

25 50 100 i50 200 300 a

Series 2 n = 50

A

B

B/A x 100

C

1.93 (0.21) 6.68 (0.24) 7.72 (0.33) i 1.6i (0.30) 14.73 (0.35) 16.99 (0.42)

0.03 (0.02) 0.20 (0.07) 3.75 (0.21) 9.38 (0.48) 14.57 (0.28) 15.78 (0.22)

1.55 2.99 48.58 80.79 98.91 92.98

2.20 8.31 13.70 12.93 13,14 15.35

(0.39) (0.39) (0.50) (0.44) (0.43) (0.75)

D

D/C x 100

0.03 (0,03) 0,60 (0.20) 6.67 (0.27) 10.63 (0,11) 11,90 (0.34) 15.25 ~ (1.06)

1.36 7.22 48.69 82.21 90.56 99.35

Based on 25 observations

10-

600

E

\ 0~

O .

30 TIME/rain

60

0

30

60

TIME/rain

Fig. 1. Effect of 0.1 mol m -3 ABA on stomatal aperture as a function of time. Epidermal strips were transferred after preincubation for 2 h on PIPES buffer with 50 m o l m -3 KC1 to either fresh buffer (9 or buffer plus 0.1 mol m -3 ABA (e). Bars represent twice standard error values

Fig. 2. Uptake of radioactivity from [2-14C]~ABA over a period of 1 h, Each point is the mean of six epidermal samples given a 90 s rinsing treatment to remove free-space label. Bars represent twice standard error values

net uptake during 1 to 3 h. Uptake continued after closure but calculations using the specific activity of the [2-14C]-ABA and an estimate of 50 stomata per mm 2 (based on observations) show that closure was complete with no more than 5.9 fmol ABA present per complex. This sensitivity estimate is greater than our previous calculation based on transpirationstream application (Itai et al., 1978), possibly'due to higher concentrations of ABA being available near the stomatal complex. The molecular state of the a4C-label after 1 h uptake was investigated by T L C in four solvent systems. In each case, a single peak containing over 90% of the total radioactivity was observed to be chromatographically similar to standard ABA. Microautora-

diography of tissue exposed to [2-t4C]-ABA (Fig. 3) confirmed that the radioactivity present was chiefly located within the stomatal complex. In this case, aggregation of radioactivity was noticeable after 20min exposure to [14C]-ABA. Controls for chemography and pressure artifacts were always negative. Sectioned freeze-dried epidermis material indicated that spatial artifacts were unlikely. Efflux of label from tissues incubated as above for 1 h in [I~C]-ABA solution was studied by sequentially transferring strips to fresh unlabelled solutions. A computer-assisted least-squares fitting process (Hipkins, 1978) was used to describe the decay in efflux rate as the sum of three first-order phases (Fig. 4): the first (ta/2=7.2 s) was assumed to be label

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J.D.B. Weyers and J.R. Hillman: Uptake and Distribution of ABA in Commelina Epidermis

Fig. 3a and b. Microautoradiographs of epidermal strips supplied with [2-14C]-ABA. a Appearance of freeze-dried tissue under light microscopy,b Correspondingdistribution of silver grains in the photographic emulsion. Rows: microautoradiographsfrom tissue incubated in [2-14C]-ABAfor 20min (top), 40rain (middle), and 60rain (bottom). m=contaminating mesophyll cell that has also taken up radioactivity. 90 x ; bar equivalent to I00 gm

associated with free space; the second and third (tl/2 =4.0 and 35.2min) to be protoplasmic in origin (cf. Baker and Hall, 1975). Attempts to fit the data to the sum of two first-order phases resulted in a higher sum of squared residuals compared with the three-phase fit. A rapid efflux of ABA has also been reported from Arena coleoptiles (Philipson, 1973) and lettuce fruits (McWha and Hillman, 1973). F r o m the

tl/z of the first phase, we have calculated that over 99% of label initially present in the free space was removed by the rinsing procedure used in uptake and autoradiographic studies. Only data from the second and third phases are dealt with in Figure 5, which shows that when apertures were measured in efftux studies, the stomata began to re-open 1 h after transfer from ABA solution, whilst stomata on strips con-

J.D.B. Weyers and J.R. Hillman: Uptake and Distribution of ABA in Commelina Epidermis

171

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Uptake and distribution of abscisic acid in Commelina leaf epidermis.

Closure of stomata by abscisic acid (ABA) was studied by floating leaf epidermal strips of Commelina communis L. in PIPES buffer (pH 6.8) containing a...
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