Planta
Planta (1982)155:146-153
9 Springer-Verlag 1982
Stomatal responses of Argenteum - a mutant of Pisum sativum L. with readily detachable leaf epidermis P.C. Jewer, L.D. Inco11, and J. Shaw Department of Plant Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Abstract. Epidermis is easily detached from both adaxial and abaxial surfaces of leaf four of the Argenteum mutant of Pisum sativum L. The isolated epidermis has stomata with large, easily-measured pores. Hairs and glands are absent. The density of stomata is high and contamination by mesophyll cells is low. In the light and in CO2-free air, stomata in isolated adaxial epidermis of Argenteum mutant opened maximally after 4 h incubation at 25 ~ C. The response of stomata to light was dependent on the concentration of KC1 in the incubation medium and was maximal at 50 tool m - 3 KC1. Stomata did not respond to exogenous kinetin, but apertures were reduced by incubation of epidermis on solutions containing between 10 - s and 10 -1 m o l m -3 abscisic acid (ABA). The responses of stomata of Argenteum mutant to light, exogenous KC1, ABA and kinetin were comparable with those described previously for stomata in isolated epidermis of Commelina communis. A method for preparing viable protoplasts of guard cells from isolated epidermis of Argenteum mutant is described. The responses of guard cell protoplasts to light, exogenous KC1, ABA and kinetin were similar to those of stomata in isolated epidermis except that the increase in volume of the protoplasts in response to light was maximal at a lower concentration of KC1 (10 mol m -3) and that protoplasts responded more rapidly to light than stomata in isolated epidermis. The protoplasts did not respond to exogenous kinetin, but when incubated for 1 h in the light and in CO2free air on a solution containing 10-3 mol m - 3 ABA, they decreased in volume by 30%. The advantages of using epidermis from Argenteum mutant for experiments on stomatal movements are discussed. Key words: Abscisic acid Cytokinin Mutant (barley) - Pisum - Potassium and stomata protoplast Stomata (epidermis). Abbreviations: ABA-abscisic acid; MES=2-(N-morpholino)ethanesulfonic acid
0032-0935/82/0155/0146/$01.60
Introduction Contemporary studies of stomatal physiology rely extensively on epidermis which has been removed from leaves (see reviews of Hsiao 1976; Raschke 1975, 1979). Although isolated epidermis was used in studies of stomatal opening around 1940 (Small and Maxwell 1939; Small et al. 1942), it was not until the late 1960s that doubts about the value of the technique were finally dispelled and the technique was generally accepted (Fujino 1967; Fischer 1968; Willmer and Mansfield 1969). However, the number of species with readily detachable epidermis is very small. Consequently, most stomatal research has been done on Commelina, Vicia and Allium, which all yield large sheets of epidermis free from contamination by mesophyll cells. Raschke (1979) and Weyers and Travis (1981) have advocated caution in generalising about stomatal physiology from data from a few species. Unfortunately new sources of material yielding clean epidermis are conspicuously absent in the literature. Indeed, the C4 grass AnthephOra pubescens Nees (Incoll and Whitelam 1977; Jewer and Incoll 1980) remains the only new source of leaf epidermis to be described in the last ten years. Thus the recent report of a new mutant of pea, Argenteum, which is characterised by a grey-green silvery cast of its leaves and stipules (Marx 1978), aroused our interest because it had been shown that the silvery cast was a consequence of extensive intercellular air spaces between the epidermis and palisade parenchyma (Hoch et al. 1980). Hoch et al. demonstrated that both adaxial and abaxial epidermes were easily detached, it frequently being possible to remove an entire epidermis in a single sheet. Moreover, examination by scanning electron microscopy of the exposed inner surface of isolated epidermis of Argenteum mutant showed that remnants of the palisade cells or cell walls were observed only occasionally (Hoch et al. 1980).
P.C. Jewer et ai. : Stomatal responses of a mutant of pea
While these properties suggested that epidermis of Argenteum mutant might be useful for experiments on stomatal movement, it was necessary to rigorously test the material by defining the responses of its stomata to various exogenous stimuli and by comparing these responses with those of stomata in isolated epidermis of the species which have been used commonly in stomatal research. We describe the characteristics of isolated epidermis of Argenteum mutant and discuss the suitability of this material for experiments on stomatal physiology by comparing these characteristics with criteria set out previously by Willmer and Mansfield (1969) and Weyers and Travis (1981). We have investigated the optimal conditions for opening the stomata in isolated epidermis of Argenteum mutant by defining their responses to light and exogenous KC1. We have also described their responses to the plant growth regulators, abscisic acid (ABA) and kinetin. Recent work has shown that protoplasts of guard cells provide a simple and effective system for studying the responses of stomata (Zeiger and Hepler 1976, 1977; Schnabl et al. 1978; Weyers, personal communication). Viable guard cell protoplasts of Vicia (Schnabl et al. 1978), Allium (Zeiger and Hepler 1976; Schnabl et al. 1978) and Commelina (Weyers, personal communication) have been prepared which respond to light, exogenous K +, ABA and fusicoccin by changing in volume, that is, they respond to the same stimuli that affect stomatal movements in isolated epidermis of these species (Raschke 1979). We therefore defined a method for preparing guard cell protoplasts from isolated epidermis of Argenteum mutant. The responses of these protoplasts to light, exogenous KC1 and to ABA and kinetin are compared with the responses of stomata on isolated epidermis of the same material. Material and methods Plant material. Plants of Argenteum mutant were raised from seed in a standard peat-based compost (J. Arthur Bowers, Lincoln, U.K.) in trays (0.5 m x 0.35 m) in a heated greenhouse. After the first visible leaf had emerged, plants were transferred to a constantenvironment growth cabinet (Model CM94 PG, Fisons, Loughborough, U.K.). The plants were iIIuminated by a bank of warm-white fluorescent and incandescent lamps for a 16h photoperiod (0800~400 h) at a photon flux density of 300 gmol m-Z s - 1 (Model LI 190SR Quantum Sensor, LICOR, Lincoln, USA). Temperatures in the light and dark were 25.0+ 1.5~ and 20.0_+ 1.0~ respectively and plants were kept in moving air (u < 1 m s - 1) with a maximum vapour-pressure deficit of 0.34 kPa (Type W V U psychrometer, Delta-T Devices, Cambridge, U.K.). An automatic watering system irrigated the plants with deionised water.
Preparation and incubation of epidermal strips. Leaflets were selected from the third visible leaf (Leaf four and node five) of 18- to 22-day-old plants. The leaflets were cut transversely along the midrib with a razor blade. Leaf margins were removed before stripping
147 the epidermis because otherwise fragments of mesophyll occasionally remained attached along the margins. Epidermes were carefully peeled from each half of the lamina in turn by pulling vertically with forceps. We avoided putting pressure on the leaf throughout the stripping procedure. Staining with Evans blue (60 mg m-3) and neutral red (50 mg m-3) showed that all cells remained vital except those at the cut edges of the strips; these cells were never measured in experiments. Incident fluorescence microscopy (Model IV FI, epiftuorescence microscope, Carl Zeiss, Oberkochen, FRG) of isolated epidermis showed much lower contamination by mesophyll cells of adaxial compared with abaxial epidermis, therefore adaxial epidermis was used in all subsequent experimental work. Pieces of epidermis, approx. 10 m m x 5 mm, were floated, cuticle uppermost, on 15 cm 3 of solution in transparent polystyrene boxes (55 cm 3 capacity). The boxes were partially immersed in a controlled-temperature water bath at 25.0_+0.8~ C and illuminated from above by a 400 W metal-halide lamp (Type HQI-T, Wotan, London, U.K.) which gave a photon flux density inside the boxes of 1000_+20 gmol m -2 s -1. A wide-band hot mirror (Type 6022001, OCLI, Dunfermline, U.K.) which reflected radiation of wavelength above 700 rim, was placed below the lamp to reduce the sensible heat load on the boxes. CO2-free air, saturated with water vapour and having a CO2 concentration of less than 1 cm 3 m - 3 (Type 225 Mk 2 infra-red gas analyser, A D C Hoddesdon, U.K.), was passed over the solutions at 0.85+0.01 cm 3 s- 1 per box. For incubation of epidermal pieces in the dark, the polystyrene boxes were covered with two layers of self-adhesive, aluminised polyester film.
Preparation and incubation of guard cell protoplasts. The method was modified from that of Schnabl et al. (1978). Preliminary experiments in a Sykes-Moore culture chamber (Zeiger and Hepler 1976) on the time course of digestion of guard cell walls by cellulase, defined the timing of the various stages in the following procedure. Pieces of epidermis, approx. 10 mm • 5 ram, from the adaxial surface of leaf four, were floated on a solution of 250 m o l m -3 mannitol containing 1 mol m - 3 CaC12 ; routinely, 70-80 pieces were prepared per 5 cm 3 of solution. A solution of 4% (w/v) cellulase (Onozuka R-10, Yakult Honsha Co., Ltd., Nishinomiya, Japan) in 250 m o l m 3 mannitol containing 1 tool m -3 CaClz was centrifuged (21000g for 20 rain) to remove cellular debris. Epidermal pieces were transferred to this solution and incubated with shaking in a centrifuge tube for l h at 30 ~ C. After removing epidermal cell protoplasts and cell wall debris by centrifugation (300g for 10 min), the epidermal pieces were transferred to a solution of 4% (w/v) cellulase in 4 0 0 m o l t o -3 mannitol containing 1 tool m -3 CaC12 and incubated with shaking at 30~ for 2 h. Guard cell protoplasts were collected by centrifugation (300g for 10 rain), either immediately or after storing the mixture at 4 ~ C overnight. The protoplasts were resuspeuded in 400 tool m - 3 mannitol containing 1 tool m -3 CaC12. Viability of the protoplasts was assessed by their ability to accumulate the vital stain, neutral red, or fluorescein from a solution of the fluorescent stain, fluorescein diacetate (Larkin 1976) or to exclude the mortal stain, Evans blue. In all cases the stains were dissolved in 400 m o l m 3 mannitol. Recovered protoplasts were counted at x 400 magnification with a haemocytometer. Solutions containing the protoplasts were incubated in small glass dishes (10 mm diameter) which were placed in the polystyrene boxes. All other conditions for incubation were as described for epidermal strips.
Experimental design and analysis. The design and analysis of experiments was similar to that described previously (Jewer and Incoll 1980), except for the following modifications to experiments with guard cell protoplasts. After incubation, 20 protoplasts in each
148
P.C. Jewer et al. : Stomatal responses of a mutant of pea
Table 1. Characteristics of isolated epidermis from leaf four of Argenteum mutant of
P. sativum L.
Epidermis
Stomatal density (pores mm -2)
Stomatal index a
Orientation of stomata
Number of subsidiary cells
Contamination by mesophylI cells (cells mm -2)
Adaxial Abaxial
158 180
20 23
Random Random
0 0
2.5 14.0
No. of stomata per unit area . x 100 a Stomatal index = No. of stomata per unit area + No. of epidermal cells per unit area Table 2. Dimensions (gm) of open and closed stomata on isolated epidermis from leaf four of Argenteum mutant of P. sativum L.
Epidermis
Adaxial Abaxial
Dimensions of stomatal guard cells (gm)
Length of stomatal pore (gm)
Stomata open
Stomata open
Stomata closed
13.2_+0.3 14.1_+0.1
14.4_+0.4 17.5_+0.2
Stomata closed
Length
Breadth
Length
Breadth
28.7+0.4 a 30.3_+0.3
9.6• 9.1_+0.2
21.6_+0.3 29.5_+0.2
8.8_+0.1 6.6_+0.3
a Dimensions are given-+the standard error
of five, 10 mm 3 drops of the suspension were selected at random and their diameters measured at x 400 magnification with an eyepiece graticule. Volumes of protoplasts were calculated from these data by assuming that they were perfect spheres. All experiments were repeated at least once and the replicate values were used in the analyses.
Solutions for incubations. In experiments with epidermal pieces, solutions of KC1, ABA and kinetin were prepared in 10 mol m -3 2-(N-morpholino)ethanesulphonic acid (MES) buffer (Sigma, Poole, U.K.), pH 6.15, with buffer alone as the control. In experiments with protoplasts, solutions of KC1 were prepared in 400 tool m- 3 mannitol containing 1 mol m- 3 CaC12. Before each experiment solutions were degassed as described previously (Jewer and Incoll 1980).
Results
Characteristics of isolated epidermis. E p i d e r m i s det a c h e d easily f r o m b o t h surfaces o f b o t h leaflets o f leaf f o u r o f Argenteum m u t a n t . I s o l a t e d e p i d e r m i s was easy to h a n d l e a n d u p to 300 m m 2 (that is, approx. six, 10 m m x 5 m m pieces) were easily o b t a i n e d f r o m one f u l l y - e x p a n d e d leaflet. The e p i d e r m e s readily f l o a t e d a n d a l t h o u g h they s o m e t i m e s f o l d e d w h e n p l a c e d on solutions, folds were easily r e m o v e d with a c a m e l - h a i r brush. T h e high density o f s t o m a t a in b o t h surfaces (Table 1), e n a b l e d s t o m a t a to be r a p i d l y l o c a t e d for m e a s u r e m e n t . R e p r o d u c i b l e m e a s u r e m e n t s o f s t o m a t a l a p e r t u r e were p o s s i b l e b y light m i c r o s c o p y b e c a u s e the ventral walls o f g u a r d cells were visible within a n a r r o w d e p t h o f field. This suggested the absence o f either an a n t e c h a m b e r a b o v e the s t o m a t a l pore, o r o f t h i c k e n e d ridges a l o n g the ventral walls, p r o p e r t i e s which f r e q u e n t l y i m p e d e
m e a s u r e m e n t o f a p e r t u r e in species such as Kalanchoe daigremontiana (Weyers a n d Travis 1981). F u r t h e r m o r e , g u a r d cells were large e n o u g h ( T a b l e 2) to be easily a n d a c c u r a t e l y m e a s u r e d a t a m a g n i f i c a t i o n o f x 400 o r x 1000. A p a r t f r o m t h e m a r k e d difference in c o n t a m i n a t i o n b y m e s o p h y l l cells a n d in the h a n dling characteristics, all o t h e r m o r p h o l o g i c a l p r o p e r ties o f i s o l a t e d e p i d e r m i s o f Argenteum m u t a n t were similar to t h o s e o f a n o n - m u t a n t p e a (Pisum sativum cv. H u r s t G r e e n Shaft) which were d e s c r i b e d b y W e y e r s a n d T r a v i s (1981).
Experiments with isolated adaxial epidermis. S t o m a t a on isolated e p i d e r m i s o p e n e d m a x i m a l l y after 4 h inc u b a t i o n in the light a n d in CO2-free air at 2 5 ~ (Fig. 1). A n i n c u b a t i o n p e r i o d of 4 h was thus used in all s u b s e q u e n t e x p e r i m e n t s with i s o l a t e d epidermis. In n e i t h e r o f the two replicate e x p e r i m e n t s d i d stom a t a close c o m p l e t e l y in CO2-free air in the d a r k , t h a t is, at time zero (Fig. 1). T h e a d d i t i o n o f KC1 to the i n c u b a t i o n m e d i u m h a d a m a r k e d effect on final a p e r t u r e in the light a n d d a r k . In the light, a p e r t u r e s increased linearly when e p i d e r m i s was i n c u b a t e d on c o n c e n t r a t i o n s o f KC1 between 0 a n d 50 m o l m - 3, b u t d i d n o t c h a n g e significantly at higher c o n c e n t r a t i o n s (Fig. 2a). In the d a r k , a p e r t u r e s increased p r o g r e s s i v e l y a n d a l m o s t linearly, with increasing e x o g e n o u s c o n c e n t r a t i o n o f KC1. T h e r e were no significant differences ( P < 0 . 0 5 ) between a p e r t u r e s in the light a n d d a r k when epidermis was i n c u b a t e d on 0 a n d 2 0 0 m o l m - 3 KC1 (Fig. 2a). T h e difference b e t w e e n s t o m a t a l a p e r t u r e s in the light a n d d a r k , t h a t is, the response o f s t o m a t a
P.C. Jewer et al. : Stomatal responses of a mutant of pea
E:a,.
149
10
10
if8
,i., ~-o~ ~
~
8
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\
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/
2l, 2 m2
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2 3 ~ 5 Durotion in light I h
I
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6
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Fig, 1. The apertures (gm) of stomata in isolated epidermal strips of Argenteum mutant of P. sativum L. incubated in the light and
in CO2-free air on 10molm 3 MES buffer containing 50 tool m-3 KC1. Strips were incubated at 25~ and apertures were measured at half-hourly intervals. Each point represents the mean of 400 measurements. Vertical bar shows least significant difference between any two treatment means using Duncan's multiple range test at P= 0.05 10
I
I
Fig. 3. The apertures (gin) of stomata of Argenteum mutant of P. sativum L. in isolated epidermal strips when incubated on 10 mol m 3 MES buffer+50 m o l m -3 KCl containing various concentrations of ABA, for 4 h at 25 ~ C in the light and in CO2-free air. Each value is the mean of 600 measurements. Vertical bar represents least significant difference at P = 0.05
r 2
Experiments with isolated guard cell protoplasts. Be-
::k
o/~
~
//s
8 6
~ ~. Ear
c "1:3 Q/ ~
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apertures in each replicate solution were not significantly different (P < 0.05). The presence of kinetin in the incubation medium over the range of concentration 10- s-10-1 mol m - 3 had no significant effect ( P < 0.05) on final aperture in the light and in C Q - f r e e air (Table 3).
E
2
I
10-5 10-~ 10-3 10-2 10-I Eoncentrution of ABA I tool m-3
I
I
I
50 100 150 200 Eoncenfrafion of KEI I tool m-3
0
b
1
1
I
I
]
50 100 150 200 Concentration of KCl I mot m-3
Fig. 2a, b. The effect of concentration of KC1 on response of stomata of Argenteum mutant of P. sativum L. to light. Epidermal strips were incubated on 10 tool m 3 MES buffer containing various concentrations of KCI at 25 ~ C in CO2-free air. a Apertures measured after 4 h in the light (o) or dark (e). b Differences between stomatal apertures in the light and dark after 4 h, that is, the response to light, Each value is the mean of 400 measurements. Vertical bar represents least significant difference at P = 0.05
to light, was maximal when epidermis was incubated on 50 mol m -3 KC1 (Fig. 2b). Although incubation on 10- 5 mol m - 3 ABA had no significant effect ( P < 0.05) on stomatal opening, apertures decreased with increasing ABA concentrations between 10 -5 and 10 - 1 m o l t o -3, so that at 10-1 tool m - 3 ABA, apertures were only 8 % of the control (Fig. 3). Errors associated with the preparation of the ABA solutions by serial dilution were not significant; in both experiments solutions were prepared from different stock solutions and mean
tween 70 and 80% of guard cell protoplasts were considered viable because they accumulated neutral red and fluoresced brightly after incubation with fluorescein diacetate. Of the 20-30% that accumulated Evans blue stain and were thus deemed non-viable, the majority had ruptured plasmalemmas. The isolation procedure yielded 240 protoplasts per m m 2 of epidermis, that is, for a mean stomatal density of 158 m m -2 (Table 1), 75% of the guard cell protoplasts were recovered. When the protoplasts were stabilised in 400 tool m - 3 mannitol solution containing 1 tool m - 3 CaC12 they became spherical. Isolated guard cell protoplasts of Argenteum mutant showed marked changes in volume following incubation for 1 h in various concentrations of KC1 in the light and dark. In solutions containing 010 tool m -3 KC1, volumes of protoplasts in the light increased linearly to a m a x i m u m in 10 mol m - 3 KC1 (Fig. 4a). However, volume was significantly reduced (P < 0.05) by incubation in the higher concentrations of 25 and 5 0 m o l t o -3 KC1 (Fig. 4a). In the dark, protoplasts swelled with increasing concentrations of KC1 to a m a x i m u m volume in solutions containing 50 mol m - 3 KC1 (Fig. 4a). The response of isolated protoplasts to light was maximal at a concentration
150
P.C. Jewer et al. : Stomatal responses of a m u t a n t of pea
Table
3. Apertures (jam) of stomata of Argenteum m u t a n t of P. sativum L. in isolated epidermal strips when incubated on various
concentrations of kinetin in 10 tool m 3 MES buffer+ 50 mol m -3 KC1 for 4h at 25~ in light and in CO2-free air. Each value is the mean of 400 measurements Concentration of kinetin (mol m- 3)
Aperture (gm)
0
l0 -5
10 -4
l0 -3
l0 -2
10 -1
7.89" (100) b
8.09 a (103)
7.96 a (101)
8.20 a (104)
8.25" (104)
7.95 ~ (101)
A. priori comparisons were made between the control a n d five treatments using the least significant difference. Values followed by the same letter are not significantly different at P = 0 . 0 5 b N u m b e r s in brackets are percentages of the control value
;0001/\
000_
20~ r
L.J
~/+50 ~I0007""
o 300 ~o~
o s00 -
zoo
0
L
i '
0 a
m [ [
'
10 20 30 I+0 50 Concenfrafion _of KC[ I mol rff-1
0
~
0i
"
0 b
10 20 30 /+0 50 Concenfrcffion of KC[ I tool m-3
I
I
I
I
I
0
30
60
90
120
Duration in light
/ min
Fig. 4a, b. The effect of concentration of KC1 on response of guard cell protoplasts of Argenteum m u t a n t of P. sativum L. to light. Protoplasts were incubated in 400 tool m -3 m a n n i t o l + 1 mol m -3 CaC12 containing various concentrations of KC1 at 25 ~ C in CO2-free air. a Volumes measured after 1 h in the ligJat (9 or dark (e). b Differences between volumes of protoplasts in the light and dark after 1 h, that is, the response to light. Each value is the mean of 400 measurements. Differences between light and dark treatments were significant at all KCI concentrations except 0 and 50 tool m - 3 , as determined by the M a n n - W h i t n e y
Fig. 5. The volumes (jam3) of isolated guard cell protoplasts of
test for non-parametric data (P=0.05)
the Mann-Whitney test for non-parametric data (P= 0.05)
of 10 mol m -3 KC1 (Fig. 4b). At the lowest and highest concentrations of KC1, 0 and 50 tool m -2, there was no significant response to light. Guard cell protoplasts incubated in a solution of 400 mol m - 3 mannitol containing 1 mol m - 3 CaC12 and 10 mol m -3 KC1, in the light and CO2-free air, reached a maximum volume after 1 h incubation (Fig. 5). The addition of 10 .3 m o l m -3 kinetin to a similar solution had no significant effect (P
Planta (1982)155:146-153
9 Springer-Verlag 1982
Stomatal responses of Argenteum - a mutant of Pisum sativum L. with readily detachable leaf epidermis P.C. Jewer, L.D. Inco11, and J. Shaw Department of Plant Sciences, University of Leeds, Leeds LS2 9JT, U.K.
Abstract. Epidermis is easily detached from both adaxial and abaxial surfaces of leaf four of the Argenteum mutant of Pisum sativum L. The isolated epidermis has stomata with large, easily-measured pores. Hairs and glands are absent. The density of stomata is high and contamination by mesophyll cells is low. In the light and in CO2-free air, stomata in isolated adaxial epidermis of Argenteum mutant opened maximally after 4 h incubation at 25 ~ C. The response of stomata to light was dependent on the concentration of KC1 in the incubation medium and was maximal at 50 tool m - 3 KC1. Stomata did not respond to exogenous kinetin, but apertures were reduced by incubation of epidermis on solutions containing between 10 - s and 10 -1 m o l m -3 abscisic acid (ABA). The responses of stomata of Argenteum mutant to light, exogenous KC1, ABA and kinetin were comparable with those described previously for stomata in isolated epidermis of Commelina communis. A method for preparing viable protoplasts of guard cells from isolated epidermis of Argenteum mutant is described. The responses of guard cell protoplasts to light, exogenous KC1, ABA and kinetin were similar to those of stomata in isolated epidermis except that the increase in volume of the protoplasts in response to light was maximal at a lower concentration of KC1 (10 mol m -3) and that protoplasts responded more rapidly to light than stomata in isolated epidermis. The protoplasts did not respond to exogenous kinetin, but when incubated for 1 h in the light and in CO2free air on a solution containing 10-3 mol m - 3 ABA, they decreased in volume by 30%. The advantages of using epidermis from Argenteum mutant for experiments on stomatal movements are discussed. Key words: Abscisic acid Cytokinin Mutant (barley) - Pisum - Potassium and stomata protoplast Stomata (epidermis). Abbreviations: ABA-abscisic acid; MES=2-(N-morpholino)ethanesulfonic acid
0032-0935/82/0155/0146/$01.60
Introduction Contemporary studies of stomatal physiology rely extensively on epidermis which has been removed from leaves (see reviews of Hsiao 1976; Raschke 1975, 1979). Although isolated epidermis was used in studies of stomatal opening around 1940 (Small and Maxwell 1939; Small et al. 1942), it was not until the late 1960s that doubts about the value of the technique were finally dispelled and the technique was generally accepted (Fujino 1967; Fischer 1968; Willmer and Mansfield 1969). However, the number of species with readily detachable epidermis is very small. Consequently, most stomatal research has been done on Commelina, Vicia and Allium, which all yield large sheets of epidermis free from contamination by mesophyll cells. Raschke (1979) and Weyers and Travis (1981) have advocated caution in generalising about stomatal physiology from data from a few species. Unfortunately new sources of material yielding clean epidermis are conspicuously absent in the literature. Indeed, the C4 grass AnthephOra pubescens Nees (Incoll and Whitelam 1977; Jewer and Incoll 1980) remains the only new source of leaf epidermis to be described in the last ten years. Thus the recent report of a new mutant of pea, Argenteum, which is characterised by a grey-green silvery cast of its leaves and stipules (Marx 1978), aroused our interest because it had been shown that the silvery cast was a consequence of extensive intercellular air spaces between the epidermis and palisade parenchyma (Hoch et al. 1980). Hoch et al. demonstrated that both adaxial and abaxial epidermes were easily detached, it frequently being possible to remove an entire epidermis in a single sheet. Moreover, examination by scanning electron microscopy of the exposed inner surface of isolated epidermis of Argenteum mutant showed that remnants of the palisade cells or cell walls were observed only occasionally (Hoch et al. 1980).
P.C. Jewer et ai. : Stomatal responses of a mutant of pea
While these properties suggested that epidermis of Argenteum mutant might be useful for experiments on stomatal movement, it was necessary to rigorously test the material by defining the responses of its stomata to various exogenous stimuli and by comparing these responses with those of stomata in isolated epidermis of the species which have been used commonly in stomatal research. We describe the characteristics of isolated epidermis of Argenteum mutant and discuss the suitability of this material for experiments on stomatal physiology by comparing these characteristics with criteria set out previously by Willmer and Mansfield (1969) and Weyers and Travis (1981). We have investigated the optimal conditions for opening the stomata in isolated epidermis of Argenteum mutant by defining their responses to light and exogenous KC1. We have also described their responses to the plant growth regulators, abscisic acid (ABA) and kinetin. Recent work has shown that protoplasts of guard cells provide a simple and effective system for studying the responses of stomata (Zeiger and Hepler 1976, 1977; Schnabl et al. 1978; Weyers, personal communication). Viable guard cell protoplasts of Vicia (Schnabl et al. 1978), Allium (Zeiger and Hepler 1976; Schnabl et al. 1978) and Commelina (Weyers, personal communication) have been prepared which respond to light, exogenous K +, ABA and fusicoccin by changing in volume, that is, they respond to the same stimuli that affect stomatal movements in isolated epidermis of these species (Raschke 1979). We therefore defined a method for preparing guard cell protoplasts from isolated epidermis of Argenteum mutant. The responses of these protoplasts to light, exogenous KC1 and to ABA and kinetin are compared with the responses of stomata on isolated epidermis of the same material. Material and methods Plant material. Plants of Argenteum mutant were raised from seed in a standard peat-based compost (J. Arthur Bowers, Lincoln, U.K.) in trays (0.5 m x 0.35 m) in a heated greenhouse. After the first visible leaf had emerged, plants were transferred to a constantenvironment growth cabinet (Model CM94 PG, Fisons, Loughborough, U.K.). The plants were iIIuminated by a bank of warm-white fluorescent and incandescent lamps for a 16h photoperiod (0800~400 h) at a photon flux density of 300 gmol m-Z s - 1 (Model LI 190SR Quantum Sensor, LICOR, Lincoln, USA). Temperatures in the light and dark were 25.0+ 1.5~ and 20.0_+ 1.0~ respectively and plants were kept in moving air (u < 1 m s - 1) with a maximum vapour-pressure deficit of 0.34 kPa (Type W V U psychrometer, Delta-T Devices, Cambridge, U.K.). An automatic watering system irrigated the plants with deionised water.
Preparation and incubation of epidermal strips. Leaflets were selected from the third visible leaf (Leaf four and node five) of 18- to 22-day-old plants. The leaflets were cut transversely along the midrib with a razor blade. Leaf margins were removed before stripping
147 the epidermis because otherwise fragments of mesophyll occasionally remained attached along the margins. Epidermes were carefully peeled from each half of the lamina in turn by pulling vertically with forceps. We avoided putting pressure on the leaf throughout the stripping procedure. Staining with Evans blue (60 mg m-3) and neutral red (50 mg m-3) showed that all cells remained vital except those at the cut edges of the strips; these cells were never measured in experiments. Incident fluorescence microscopy (Model IV FI, epiftuorescence microscope, Carl Zeiss, Oberkochen, FRG) of isolated epidermis showed much lower contamination by mesophyll cells of adaxial compared with abaxial epidermis, therefore adaxial epidermis was used in all subsequent experimental work. Pieces of epidermis, approx. 10 m m x 5 mm, were floated, cuticle uppermost, on 15 cm 3 of solution in transparent polystyrene boxes (55 cm 3 capacity). The boxes were partially immersed in a controlled-temperature water bath at 25.0_+0.8~ C and illuminated from above by a 400 W metal-halide lamp (Type HQI-T, Wotan, London, U.K.) which gave a photon flux density inside the boxes of 1000_+20 gmol m -2 s -1. A wide-band hot mirror (Type 6022001, OCLI, Dunfermline, U.K.) which reflected radiation of wavelength above 700 rim, was placed below the lamp to reduce the sensible heat load on the boxes. CO2-free air, saturated with water vapour and having a CO2 concentration of less than 1 cm 3 m - 3 (Type 225 Mk 2 infra-red gas analyser, A D C Hoddesdon, U.K.), was passed over the solutions at 0.85+0.01 cm 3 s- 1 per box. For incubation of epidermal pieces in the dark, the polystyrene boxes were covered with two layers of self-adhesive, aluminised polyester film.
Preparation and incubation of guard cell protoplasts. The method was modified from that of Schnabl et al. (1978). Preliminary experiments in a Sykes-Moore culture chamber (Zeiger and Hepler 1976) on the time course of digestion of guard cell walls by cellulase, defined the timing of the various stages in the following procedure. Pieces of epidermis, approx. 10 mm • 5 ram, from the adaxial surface of leaf four, were floated on a solution of 250 m o l m -3 mannitol containing 1 mol m - 3 CaC12 ; routinely, 70-80 pieces were prepared per 5 cm 3 of solution. A solution of 4% (w/v) cellulase (Onozuka R-10, Yakult Honsha Co., Ltd., Nishinomiya, Japan) in 250 m o l m 3 mannitol containing 1 tool m -3 CaClz was centrifuged (21000g for 20 rain) to remove cellular debris. Epidermal pieces were transferred to this solution and incubated with shaking in a centrifuge tube for l h at 30 ~ C. After removing epidermal cell protoplasts and cell wall debris by centrifugation (300g for 10 min), the epidermal pieces were transferred to a solution of 4% (w/v) cellulase in 4 0 0 m o l t o -3 mannitol containing 1 tool m -3 CaC12 and incubated with shaking at 30~ for 2 h. Guard cell protoplasts were collected by centrifugation (300g for 10 rain), either immediately or after storing the mixture at 4 ~ C overnight. The protoplasts were resuspeuded in 400 tool m - 3 mannitol containing 1 tool m -3 CaC12. Viability of the protoplasts was assessed by their ability to accumulate the vital stain, neutral red, or fluorescein from a solution of the fluorescent stain, fluorescein diacetate (Larkin 1976) or to exclude the mortal stain, Evans blue. In all cases the stains were dissolved in 400 m o l m 3 mannitol. Recovered protoplasts were counted at x 400 magnification with a haemocytometer. Solutions containing the protoplasts were incubated in small glass dishes (10 mm diameter) which were placed in the polystyrene boxes. All other conditions for incubation were as described for epidermal strips.
Experimental design and analysis. The design and analysis of experiments was similar to that described previously (Jewer and Incoll 1980), except for the following modifications to experiments with guard cell protoplasts. After incubation, 20 protoplasts in each
148
P.C. Jewer et al. : Stomatal responses of a mutant of pea
Table 1. Characteristics of isolated epidermis from leaf four of Argenteum mutant of
P. sativum L.
Epidermis
Stomatal density (pores mm -2)
Stomatal index a
Orientation of stomata
Number of subsidiary cells
Contamination by mesophylI cells (cells mm -2)
Adaxial Abaxial
158 180
20 23
Random Random
0 0
2.5 14.0
No. of stomata per unit area . x 100 a Stomatal index = No. of stomata per unit area + No. of epidermal cells per unit area Table 2. Dimensions (gm) of open and closed stomata on isolated epidermis from leaf four of Argenteum mutant of P. sativum L.
Epidermis
Adaxial Abaxial
Dimensions of stomatal guard cells (gm)
Length of stomatal pore (gm)
Stomata open
Stomata open
Stomata closed
13.2_+0.3 14.1_+0.1
14.4_+0.4 17.5_+0.2
Stomata closed
Length
Breadth
Length
Breadth
28.7+0.4 a 30.3_+0.3
9.6• 9.1_+0.2
21.6_+0.3 29.5_+0.2
8.8_+0.1 6.6_+0.3
a Dimensions are given-+the standard error
of five, 10 mm 3 drops of the suspension were selected at random and their diameters measured at x 400 magnification with an eyepiece graticule. Volumes of protoplasts were calculated from these data by assuming that they were perfect spheres. All experiments were repeated at least once and the replicate values were used in the analyses.
Solutions for incubations. In experiments with epidermal pieces, solutions of KC1, ABA and kinetin were prepared in 10 mol m -3 2-(N-morpholino)ethanesulphonic acid (MES) buffer (Sigma, Poole, U.K.), pH 6.15, with buffer alone as the control. In experiments with protoplasts, solutions of KC1 were prepared in 400 tool m- 3 mannitol containing 1 mol m- 3 CaC12. Before each experiment solutions were degassed as described previously (Jewer and Incoll 1980).
Results
Characteristics of isolated epidermis. E p i d e r m i s det a c h e d easily f r o m b o t h surfaces o f b o t h leaflets o f leaf f o u r o f Argenteum m u t a n t . I s o l a t e d e p i d e r m i s was easy to h a n d l e a n d u p to 300 m m 2 (that is, approx. six, 10 m m x 5 m m pieces) were easily o b t a i n e d f r o m one f u l l y - e x p a n d e d leaflet. The e p i d e r m e s readily f l o a t e d a n d a l t h o u g h they s o m e t i m e s f o l d e d w h e n p l a c e d on solutions, folds were easily r e m o v e d with a c a m e l - h a i r brush. T h e high density o f s t o m a t a in b o t h surfaces (Table 1), e n a b l e d s t o m a t a to be r a p i d l y l o c a t e d for m e a s u r e m e n t . R e p r o d u c i b l e m e a s u r e m e n t s o f s t o m a t a l a p e r t u r e were p o s s i b l e b y light m i c r o s c o p y b e c a u s e the ventral walls o f g u a r d cells were visible within a n a r r o w d e p t h o f field. This suggested the absence o f either an a n t e c h a m b e r a b o v e the s t o m a t a l pore, o r o f t h i c k e n e d ridges a l o n g the ventral walls, p r o p e r t i e s which f r e q u e n t l y i m p e d e
m e a s u r e m e n t o f a p e r t u r e in species such as Kalanchoe daigremontiana (Weyers a n d Travis 1981). F u r t h e r m o r e , g u a r d cells were large e n o u g h ( T a b l e 2) to be easily a n d a c c u r a t e l y m e a s u r e d a t a m a g n i f i c a t i o n o f x 400 o r x 1000. A p a r t f r o m t h e m a r k e d difference in c o n t a m i n a t i o n b y m e s o p h y l l cells a n d in the h a n dling characteristics, all o t h e r m o r p h o l o g i c a l p r o p e r ties o f i s o l a t e d e p i d e r m i s o f Argenteum m u t a n t were similar to t h o s e o f a n o n - m u t a n t p e a (Pisum sativum cv. H u r s t G r e e n Shaft) which were d e s c r i b e d b y W e y e r s a n d T r a v i s (1981).
Experiments with isolated adaxial epidermis. S t o m a t a on isolated e p i d e r m i s o p e n e d m a x i m a l l y after 4 h inc u b a t i o n in the light a n d in CO2-free air at 2 5 ~ (Fig. 1). A n i n c u b a t i o n p e r i o d of 4 h was thus used in all s u b s e q u e n t e x p e r i m e n t s with i s o l a t e d epidermis. In n e i t h e r o f the two replicate e x p e r i m e n t s d i d stom a t a close c o m p l e t e l y in CO2-free air in the d a r k , t h a t is, at time zero (Fig. 1). T h e a d d i t i o n o f KC1 to the i n c u b a t i o n m e d i u m h a d a m a r k e d effect on final a p e r t u r e in the light a n d d a r k . In the light, a p e r t u r e s increased linearly when e p i d e r m i s was i n c u b a t e d on c o n c e n t r a t i o n s o f KC1 between 0 a n d 50 m o l m - 3, b u t d i d n o t c h a n g e significantly at higher c o n c e n t r a t i o n s (Fig. 2a). In the d a r k , a p e r t u r e s increased p r o g r e s s i v e l y a n d a l m o s t linearly, with increasing e x o g e n o u s c o n c e n t r a t i o n o f KC1. T h e r e were no significant differences ( P < 0 . 0 5 ) between a p e r t u r e s in the light a n d d a r k when epidermis was i n c u b a t e d on 0 a n d 2 0 0 m o l m - 3 KC1 (Fig. 2a). T h e difference b e t w e e n s t o m a t a l a p e r t u r e s in the light a n d d a r k , t h a t is, the response o f s t o m a t a
P.C. Jewer et al. : Stomatal responses of a mutant of pea
E:a,.
149
10
10
if8
,i., ~-o~ ~
~
8
~ T
\
/e../o/ele~~
/
2l, 2 m2
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0
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2 3 ~ 5 Durotion in light I h
I
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6
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Fig, 1. The apertures (gm) of stomata in isolated epidermal strips of Argenteum mutant of P. sativum L. incubated in the light and
in CO2-free air on 10molm 3 MES buffer containing 50 tool m-3 KC1. Strips were incubated at 25~ and apertures were measured at half-hourly intervals. Each point represents the mean of 400 measurements. Vertical bar shows least significant difference between any two treatment means using Duncan's multiple range test at P= 0.05 10
I
I
Fig. 3. The apertures (gin) of stomata of Argenteum mutant of P. sativum L. in isolated epidermal strips when incubated on 10 mol m 3 MES buffer+50 m o l m -3 KCl containing various concentrations of ABA, for 4 h at 25 ~ C in the light and in CO2-free air. Each value is the mean of 600 measurements. Vertical bar represents least significant difference at P = 0.05
r 2
Experiments with isolated guard cell protoplasts. Be-
::k
o/~
~
//s
8 6
~ ~. Ear
c "1:3 Q/ ~
t
0
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0 a
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apertures in each replicate solution were not significantly different (P < 0.05). The presence of kinetin in the incubation medium over the range of concentration 10- s-10-1 mol m - 3 had no significant effect ( P < 0.05) on final aperture in the light and in C Q - f r e e air (Table 3).
E
2
I
10-5 10-~ 10-3 10-2 10-I Eoncentrution of ABA I tool m-3
I
I
I
50 100 150 200 Eoncenfrafion of KEI I tool m-3
0
b
1
1
I
I
]
50 100 150 200 Concentration of KCl I mot m-3
Fig. 2a, b. The effect of concentration of KC1 on response of stomata of Argenteum mutant of P. sativum L. to light. Epidermal strips were incubated on 10 tool m 3 MES buffer containing various concentrations of KCI at 25 ~ C in CO2-free air. a Apertures measured after 4 h in the light (o) or dark (e). b Differences between stomatal apertures in the light and dark after 4 h, that is, the response to light, Each value is the mean of 400 measurements. Vertical bar represents least significant difference at P = 0.05
to light, was maximal when epidermis was incubated on 50 mol m -3 KC1 (Fig. 2b). Although incubation on 10- 5 mol m - 3 ABA had no significant effect ( P < 0.05) on stomatal opening, apertures decreased with increasing ABA concentrations between 10 -5 and 10 - 1 m o l t o -3, so that at 10-1 tool m - 3 ABA, apertures were only 8 % of the control (Fig. 3). Errors associated with the preparation of the ABA solutions by serial dilution were not significant; in both experiments solutions were prepared from different stock solutions and mean
tween 70 and 80% of guard cell protoplasts were considered viable because they accumulated neutral red and fluoresced brightly after incubation with fluorescein diacetate. Of the 20-30% that accumulated Evans blue stain and were thus deemed non-viable, the majority had ruptured plasmalemmas. The isolation procedure yielded 240 protoplasts per m m 2 of epidermis, that is, for a mean stomatal density of 158 m m -2 (Table 1), 75% of the guard cell protoplasts were recovered. When the protoplasts were stabilised in 400 tool m - 3 mannitol solution containing 1 tool m - 3 CaC12 they became spherical. Isolated guard cell protoplasts of Argenteum mutant showed marked changes in volume following incubation for 1 h in various concentrations of KC1 in the light and dark. In solutions containing 010 tool m -3 KC1, volumes of protoplasts in the light increased linearly to a m a x i m u m in 10 mol m - 3 KC1 (Fig. 4a). However, volume was significantly reduced (P < 0.05) by incubation in the higher concentrations of 25 and 5 0 m o l t o -3 KC1 (Fig. 4a). In the dark, protoplasts swelled with increasing concentrations of KC1 to a m a x i m u m volume in solutions containing 50 mol m - 3 KC1 (Fig. 4a). The response of isolated protoplasts to light was maximal at a concentration
150
P.C. Jewer et al. : Stomatal responses of a m u t a n t of pea
Table
3. Apertures (jam) of stomata of Argenteum m u t a n t of P. sativum L. in isolated epidermal strips when incubated on various
concentrations of kinetin in 10 tool m 3 MES buffer+ 50 mol m -3 KC1 for 4h at 25~ in light and in CO2-free air. Each value is the mean of 400 measurements Concentration of kinetin (mol m- 3)
Aperture (gm)
0
l0 -5
10 -4
l0 -3
l0 -2
10 -1
7.89" (100) b
8.09 a (103)
7.96 a (101)
8.20 a (104)
8.25" (104)
7.95 ~ (101)
A. priori comparisons were made between the control a n d five treatments using the least significant difference. Values followed by the same letter are not significantly different at P = 0 . 0 5 b N u m b e r s in brackets are percentages of the control value
;0001/\
000_
20~ r
L.J
~/+50 ~I0007""
o 300 ~o~
o s00 -
zoo
0
L
i '
0 a
m [ [
'
10 20 30 I+0 50 Concenfrafion _of KC[ I mol rff-1
0
~
0i
"
0 b
10 20 30 /+0 50 Concenfrcffion of KC[ I tool m-3
I
I
I
I
I
0
30
60
90
120
Duration in light
/ min
Fig. 4a, b. The effect of concentration of KC1 on response of guard cell protoplasts of Argenteum m u t a n t of P. sativum L. to light. Protoplasts were incubated in 400 tool m -3 m a n n i t o l + 1 mol m -3 CaC12 containing various concentrations of KC1 at 25 ~ C in CO2-free air. a Volumes measured after 1 h in the ligJat (9 or dark (e). b Differences between volumes of protoplasts in the light and dark after 1 h, that is, the response to light. Each value is the mean of 400 measurements. Differences between light and dark treatments were significant at all KCI concentrations except 0 and 50 tool m - 3 , as determined by the M a n n - W h i t n e y
Fig. 5. The volumes (jam3) of isolated guard cell protoplasts of
test for non-parametric data (P=0.05)
the Mann-Whitney test for non-parametric data (P= 0.05)
of 10 mol m -3 KC1 (Fig. 4b). At the lowest and highest concentrations of KC1, 0 and 50 tool m -2, there was no significant response to light. Guard cell protoplasts incubated in a solution of 400 mol m - 3 mannitol containing 1 mol m - 3 CaC12 and 10 mol m -3 KC1, in the light and CO2-free air, reached a maximum volume after 1 h incubation (Fig. 5). The addition of 10 .3 m o l m -3 kinetin to a similar solution had no significant effect (P
