ELSEVIER Plant Science 109 (1995) 119-127

Guard cell protoplasts contain activity

S. Madhavan*a, Gautam SarathaTb, Bong Ho Lee’, Randall S. Pegdenb

‘Department of Biochemistry, N258, The Beadle Center, University of Nebraska-Lincoln, Lincoln, NE 68588-0664. USA bProtein Core Facility. Center for Biotechnology, The &adle Center, University of Nebraska, Lincoln, NE 68588-0664, USA ‘Depurtment of Industrial Chemistry. The National University of Taejon. Samsung 2-dong, Dong-gu, Taejon. 300-I 72. South Korea

Received 24 February 1995; revision received 17 May 1995; accepted 17 May 1995

Abstract

Acetylcholinesterase activity has been detected in extracts of guard cell protoplasts from Viciufabu L. and Nicotiunu gluucu Graham. Guard cell protoplast homogenates from V. fubu exhibited 16.4 and 6.7-fold greater specific activities for acetylthiocholine hydrolysis compared to homogenates of mesophyll cell protoplasts or whole leaves, respectively. Extracts of N. gluucu guard cell protoplasts also displayed highest specific activity for acetylthiocholine hydrolysis. Guard cell protoplast extracts from both species displayed a distinct substrate preference for acetylthiocholine. In con- trast, no substrate specificity for ester hydrolysis was observed in extracts of mesophyll cell protoplasts or whole leaves. In both species, specific reversible inhibitors of mammalian acetylcholinesterase, BW284c51 and neo- , inhibited 40-m? of guard cell protoplast acetylcholinesterase activity. Exogenously applied (1 mM) induced an 80% closure of stomata in abaxial epidermal peels of V. fubu leaves within 5 min, while only a lo-15% stomata1 closure was induced by either or propionylcholine. BW284c51, and eserine also induced varying degrees of stomata1 closure in epidermal peels of V. j&u. Results from these studies demonstrate that guard cells have acetycholinesterase activity and suggest that acetylcholine might have a physiological role in stomata1 movement.

Keywork Acetylcholinesterase; Guard cell protoplasts; Mesophyll cell protoplasts; Stomata1 aperture; Viciu fiba; Nicotiunu gluucu

1. IlltroductIon Abbreviations: ACh, acetytholine; AChE, acetylcholin- esterase; ATCh, acetylthiocholine; BuCh, butyrylcholine; Acetvlcholine (ACh). _ is a well known neuro- BuTCh, butyrylthiocholine; ChE, ; GCP, guard transmitter that mediates signal transmission, cell protoplasts;GCP-AChE, guard cell protoplast acetylcho- causing the opening of certain cation-selective linesterase; MCP, mesophyll cell protoplasts; PrCh, pro- pionylcholine, PrTCh, propionylthiocholine. channels in nerve synapses of higher animals [ 11.

l Corresponding author, Tel.: (402)472 2939; Fax: (402)472 However, its presence has also been reported in the 7842. non-nervous tissue of multicellular animals, pro-

0168-94521956.09.50 0 1995 Elsevier Science Ireland Ltd. All rights reserved SSDI 0168-9452(95)04164-P 120 S. Ma&van et al. /Plant Science 109 (1995) 119-127 tozoa, bacteria and several plant species [2]. 2.2. Plant material Acetylcholinesterase, AChE, (E.C. 3.1.1.7), can Broad bean (Vicia faba L.), and tree regulate ACh levels by hydrolyzing AChE to ace- (Nicotiana glauca Graham) plants were grown in a tate and choline. Acetylcholinesterase has been greenhouse under natural light conditions at detected in many plant species [2,3]. Recently, day/night temperatures of 22”C/17”C and at Momonoki [4] reported the presence of AChE at 60-70% relative humidity. the stele-cortex interface in Zea mays seedlings and suggested that the ACh-receptor-esterase sys- 2.3. Leaf extracts tem might operate to transmit a chemical stimulus Only young and fully expanded leaves of 3-5 across the plasmodesmatal junctions between week-old K faba plants and 8-10 week-old N. plant cells, thereby propagating membrane depol- gluucu plants, were used. Whole leaves were de- arization. veined and homogenized in 20 mM sodium phos- Though it still lacks a clearly defined role in phate buffer, pH 7.5, containing 4% (w/v) plants, ACh has been suggested to be a second (NH4)$04, 5 mM EDTA and 1% (w/v) insoluble messenger in various cellular processes such as PVP at 4°C. Extracts were filtered through 4 layers phytochrome action ($61, auxin-mediated salt ac- of cheesecloth and centrifuged at 27 000 x g for cumulation [7], leaf movements [8], regulation of 20 min. The supernatant fractions from these ex- the ATPNADPH ratio in chloroplasts and ion tracts were used as the source of enzyme. permeability of the chloroplast thylakoid mem- branes [9]. A majority of these studies suggest that 2.4. Protoplast isolation the primary mechanism of action of ACh in plant Guard cell protoplasts were isolated following cells is in the regulation of membrane permeability the procedures of Shimazaki et al. [l l] from epi- to ions. dermal strips that were carefully peeled from the Mechanisms that control ion fluxes are highly abaxial surfaces of leaves of 3 week-old K faba developed in guard cells. Ion fluxes across the plants. A 20 pm nylon net was used at the final plasma membrane of these cells not only regulate stage to ensure that GCP were free of contamina- gas exchange but also control water status and car- tion from other epidermal and mesophyll pro- bon assimilation in plants [lo]. toplasts. GCP from N. glauca were isolated by the In this study we present data supporting the lo- procedures of Cupples et al. [12]. Mesophyll cell calization of a true AChE, in guard cell pro- protoplasts (MCP) were isolated following the toplasts (GCP) of two higher plant species. This procedures of Spalding et al. [13] from leaves free finding suggests that guard cells could have a com- of abaxial epidermis. plete ACh system which perhaps might be involv- ed in their physiology. 2.5. Determination of acetylcholinesterase activity and inhibition studies 2. Materials and methods Acetylcholinesterase activity in plant extracts was determined by the method of Ellman et al. [ 141 2. I. Chemicals at a final concentration of 1 mM ATCh, PrTCh, Cellulase Onozuka R-10 was obtained from or BuTCh as substrate. All reactions were per- Yakult Honsha Co. Ltd., Tokyo, Japan and formed in 20 mM sodium phosphate buffer, pH Cellulysin from Calbiochem, San Diego, CA. 7.5, at 35°C. Specific reversible inhibitors of mam- Neostigmine bromide (3-[(dimethylamino)car- malian AChE, BW284c51 (the bis phenyl ketone), bonylloxy-N,N,N-trimethylbenzammonium bro- eserine and neostigmine were also studied for their mide), eserine (), BW284c5 1 [ 1,5- inhibition of GCP-AChE. For assays involving in- bis(Callyldimethy1 ammonium phenyl) pentan-3- hibitors, an aliquot of GCP extract was prein- one dibroriride] and all other chemicals were from cubated with the specific inhibitor at a final Sigma, St. Louis, MO. concentration of 1 mM for 5 min. The reaction S. Modhavan et-al. /Plant Science 109 (199s) 119-127 121 was started with the addition of ATCh to give a 3. Results final concentration of 1 mM ATCh in the reaction mixture. Separate experiments were performed 3.1. GCP-AChE activity and its substrate specificity with different concentrations of the inhibitors and Acetylcholinesterase activities detected in crude each experiment was repeated at least 3 times. The extracts of GCP, MCP and whole leaves of V.faba effect of incubation time on inhibition of GCP- are shown in Fig. 1A. Crude extracts of GCP AChE by eserine (1 mM) was performed over a displayed an activity of 100.3 f 3.4 nmol min-’ period of O-60 min using procedures described mg-’ protein, which was 16.4 and 6.7-fold greater above. than those observed in the MCP and whole leaf ex- tracts, respectively. The greater specific activity for 2.6. Measurement of stomata1 responses in epider- ATCh hydrolysis suggested that the GCP enzyme ma1 peels might display a distinct substrate preference. Fully expanded, young leaves of 3 week-old V. When the GCP cholinesterase was assayed with 3 faba plants were harvested 3-4 h into the light thiocholinester substrates, ATCh hydrolysis was period. Abaxial epidermal strips from these leaves approximately lO-fold faster than hydrolyses of were floated cuticle uppermost on 0.1 mM CaClz either PrTCh or BuTCh. In contrast, the crude ex- medium containing 0.2% ascorbic acid, cut into tracts of both MCP and the whole leaf did not pieces about 0.5 cm* and then transferred to 10 show any apparent substrate specificity. V. faba mM MES buffer, pH 6.2 containing 1 mM EDTA. GCP-AChE displayed a substrate saturation of 1.O The epidermal peels were pretreated in light mM for ATCh (not shown). (PPFD = 200 pm01 m -* s-‘) for 2 h. After this For N. glauca, a 2.5-fold increase in the specific preincubation, epidermal strips were floated at activity of AChE was observed in GCP extracts as room temperature, in 10 mM MES, pH 6.2 con- compared to those for whole leaf extracts (Fig. taining 1 mM CaC12, with or without 10 mM KC1 1B). The enzyme catalyzed hydrolysis of the 3 and containing 1 mM concentrations of ACh, thiocholine esters in N. glauca GCP extracts BuCh and PrCh. Additionally, epidermal strips generally exhibited a similar trend to that observed were also floated on solutions containing with V.faba GCP extracts (Fig. 1B). Again no ap- BW284c51, eserine and neostigmine. At different parent substrate specificity for thiocholine esters time intervals the epidermal strips were mounted was observed in the leaf extracts of N. glauca. in the same medium in which they were treated and photographed (Kodak Ektachrome, 400 ASA) 3.2. Effects of AChE inhibitors on GCP-AChE ac- using a Nikon Labophot-2 microscope at a fixed tivity magnification. Inhibition of both V. faba and N. glauca GCP- The stomata1 aperture width for at least 50 AChE by specific reversible inhibitors of mamma- stomata were measured by projecting the slides lian AChE, BW284c51 and neostigmine, were onto a screen from a fixed distance. Results are studied. BW284c51 and neostigmine rapidly inac- presented as the mean percentage * S.E. of tivated the enzyme present in V.faba GCP extracts stomata1 opening as compared to those of the (Table 1). The 1e.s (50% inhibition) values as respective controls. In all experiments utilizing epi- determined graphically from Job plots (not shown; dermal strips, strips floated on 10 mM MES, pII [ 161) for BW284c51 and neostigmine were 0.04 6.2 containing 10 mM KC1 and kept illuminated mM and 0.1 mM. Nicotiana glauca GCP-AChE served as controls. Stomata in these strips was was inhibited similarly by BW284c51 and neo- found to be maximally open. stigmine (Table 1). Inhibition of GCP-AChE ac- tivity by neostigmine, in N. glauca, was 20?! lower 2.7. Protein determination than in I’. f&a while iahibition by BW284c51 ap- Protein concentration in the various tissue ex- peared to be of an identical magnitude to that of tracts was determined by the method of Bradford P. f&a. In contrast to the GCP-AChE, the en- [ 151 using BSA as a standard. zymes from the whole leaf extracts of V. faba 122 S. Ma&van et al. /Plant Science 109 (1995) 119-127

Vicia faba A q GCP

ATCh PrTCh BuTCh

Nicotiana glauca n GCP B 0 Leaf I

il’: ATCh PrTCh BuTCh Choline ester substrate

Fig. I (A) Comparison of acetylcholinesterase activity in GCP, MCP and leaf tissue extracts of V. fiba and their specificity towards different chbline ester substrates. AChE activity was assayed in a final volume of 1ml, at 35°C in 20 mM Pi buffer, pH 7.5, containing 1 mM ATCh, or PrTCh or BuTCh and 0.5 mM 5,5 ‘-dithio bis (2-nitro benzoic acid). Reactions were initiated with the addition of 50-100 ~1 of tissue extract and the rate of substrate hydrolysis was followed spectrophotometrically at 412 nm. One unit of activity is defined as the amount of enzyme required to liberate 1 nmol of thiocholine from the respective thiocholine substrate per minute under the conditions of the assay. (B) Comparison of AChE activity in GCP and leaf tissue extracts of N. glauca. Tissue extracts were analyzed as described in (A). S. ~Ua&avan et al. / Plan1 Science 109 (1595) 119-127 123

Table 1 displayed only a 15-23% inhibition of their activ- Effect of specific inhibitors of animal acetylcholinesterase on ity when treated with these inhibitors. Guard cell GCP-AChE activity protoplast acetylcholinesterase activity was also inhibited by eserine. However, the results indicate Inhibitor GCP-AChE activitys, nmol min-’ mg-t protein that the inhibition of GCP-AChE by eserine was slower than that of the other two inhibitors V. faba N. glauca studied (not shown). Eserine inactivation of GCP- AChE activity required almost a 60 min prein- No inhibitor 81.4 l 7.4 24.3 zt 1.8 cubation as compared to l-2 min for other in- BW284cS1,l mM 9.3 f 1.1 4.2 zt 0.8 hibitors. Neostigmine, 1 mM 17.4 l 2.1 10.1 f 1.9

*AChE activity was assayed in a final volume of 1 ml at 3.3. Effect of choline esters on stomata1 movement 35°C. in 20 mM Pi buffer, pH 7.5 containing 1 mM ATCh, 0.5 A comparative study on the effects of K+ ions, mM 5,5’-dithio bis (Znitrobenxoic acid), 50-100 ~1 GCP ex- ACh, PrCh and BuCh on stomata1 movement was tract and 1 mM BW284c51 or neostigmine. Guard cell pro- made with detached abaxial epidermal peels from toplast extract was preincubated with the inhibitor for 5 min K faba leaves (Fig. 2). As controls, abaxial epider- prior to the addition of I mM ATCh to start the reaction. The rate of substrate hydrolysis was followed at 412 nm. ma1 peels from fully developed leaves of 3-4 week old V. faba plants were floated on 10 mM MES

_

ACh PrCh BuCh

Fig. 2. Comparison of stomata1 responses to exogenous application of choline esters. Abaxial epidermai peels from V. @a leaves that were floated cuticle uppermost on IO mM MES buffer, pH 6.2 and pretreated with light (PPFD, 200 pmol mm2 s-‘) served as controls. Epidermal peek that were pretreated with light (PPFD, 200 pmol m -2 s-r) in buffered medium containing 10 mM K+ were used to determine the maximal extent of stomatal opening. Epidermal peels were also floated on medium containing 1 mM of the respective choline esters for either 5 or 30 mitt at room temperature. At indicated times peels were photographed at a fixed magnifica- tion to obtain data from at least 50 stomata for each treatment. Data are normalixed with respect to the control and represent the mean f S.E. of at least 3 independent experiments. 124 S. IUA~UWA~ et ~1. /Plant Science 109 11995) 119-127

120

?

0 -! L! ,,,I 1 10 100 1000 Log ACh Cone, pM

Fig. 3. Dose response of ACh on stomata1 movement in the abaxial leaf epidermal peels of V. faba. Control epidermal peels were floated on buffered medium with or without IO mM KCI. Peels were also incubated in buffered medium with or without IO mM KCI containing ACh at the indicated concentrations. Peels were photographed for the measurement of stomata1 apertures after 30 min incubation. Data represent the mean f S.E. of 3 independent experiments.

buffer, pH 6.2, containing 1 mM CaClz and abaxial epidermal peels of V. faba were studied pretreated with light (PPFD, 200 pmol m-’ s-r). (Fig. 3). Control peels were floated on buffered Stomata in these peels were open. Addition of 10 medium with or without 10 mM KC1 and stomata1 mM KC1 to the medium in which the peels were apertures determined at zero time and at 30 min. floated induced a further 10% increase in stomata1 Almost 20% decrease in stomata1 aperture was ef- opening within 5 min. The extent of stomata1 fected by 10 PM ACh in the bathing solution. At opening in these peels was similar to those an exogenous concentration of 40 PM, ACh ef- pretreated with KCl, indicating that maximal fected a 50% stomata1 closure. As observed earlier, stomata1 opening was achieved and that the 1 mM ACh induced the greatest closure of stomata were responding in a physiologically com- stomates. Addition or omission of K+ ions (10 petent manner. Exogenous application of 1 mM mM) to or from the incubation media did not ap- ACh to these peels induced an 80% closure of pear to influence the effects of ACh on stomata in stomata within 5 min (Fig. 2) as compared to a 10 these peels. and 15% stomata1 closure induced by the addition of either 1 mM BCh or 1 mM PrCh. 3.5. Effects of AChE inhibitors on stomata1 movement 3.4. Stomata1 movement and dose response for ACh Opening and closing responses of stomata to an The effects of increasing concentrations of ACh exogenous application of AChE inhibitors, BW- on the induction of stomata1 closure in detached, 284c51 (1 mM), neostigmine (1 mM) and eserine S. M&van et al. /Plant Science 109 (1995) 119-127 125

BW284c51 Neostigmine Eserine Acetylcholinesterase Inhibitor

Fig. 4. Effect of AChE inhibitors, BW284c51, neostigmine and eserine on stomatal movement, in V.f&a abaxial leaf epidermal peels. All inhibitors were added to the MES buffer medium (with CaCI, and KCI) at a final concentration of I mM. Other conditions were as described in Fig. 2. Results represent the mean l S.E. of determinations from 3 experiments.

(1 mM) were also studied with the abaxial epider- tained from inhibitor studies further support this mal peels of I’. fuba leaves (Fig. 4). In contrast to conclusion. Inhibitors, BW284c5 1, eserine and control epidermal peels floated on medium con- neostigmine, which are known to inhibit AChE taining 10 mM KCl, BW284c51 and neostigmine from animal sources, also inhibited GCP-AChE induced a 90% decrease in stomata1 aperture activity. The 1s.s value for neostigmine towards within the first 5 min of treatment which did not GCP-AChE reported here is higher than that change over a 30 min time period. Eserine induced reported for a purified pea root AChE (I0.S 0.6 a smaller but a noticeable effect on stomata1 move- PM) by the same compound [ 181. It is conceivable ment (Fig. 4). that the purified guard cell enzyme might display greater sensitivity to this inhibitor. In contrast to 4. DisarssIon the inhibitors BW284c51 and neostigmine on the inhibition of GCP-AChE, eserine, displayed a Guard cell protoplasts isolated from V.fuba and relatively slow inhibition of GCP-AChE. This N. glaucu leaves are enriched in AChE activity re- effect by eserine is consistent with literature lative to the MCP and whole leaf extracts. The reports on purified pea root AChE [ 18,191. These greatest specificity for ATCh, rather than the long- findings suggest that the enzyme activity detected er propionyl and butyryl esters would suggest that in GCP extracts is a true plant AChE. the GCP enzyme is a true AChE 13,171,in contrast The presence of AChE and indeed all the com- to the more non-specific esterases apparently pres- ponents of the ACh pathway, namely, ACh, cho- ent in the MCP and whole leaf extracts. Data ob- line acetyltransferase, and putative receptors have 126 S. Madman et al. /Plant Science 109 (1995) 119-127

directly or indirectly been shown to occur in plant Guard cells constitute about 2% or less of the cells and plant protoplasts (see [2]). A cyto- epidermal cell population [23], and constitute an chemical detection of acetylcholinesterase activity even smaller percentage of the total leaf cells. has also been reported in stomata of Murchantia Thus, the enrichment of ChE activity in guard cells pofymorpha L. [20]. A suggested role for the over other cell types of the leaf may indicate a system in plants has been in influenc- physiological significance for ACh and AChE in ing inositol and cellular ion fluxes these cells. Detection of a true AChE in GCP ex- [2,3], both of these mechanisms have been im- tracts and the consistency of the effects of ex- plicated in the signal transduction pathway involv- ogenous ACh and AChE inhibitors on stomata1 ed in stomata1 movements [21,22]. movements suggest that the cholinergic system Data from the exogenous application of ACh, could represent another potential signal-trans- PrCh and BuCh on stomata1 movement, indicate duction pathway in guard cells. that the response to these choline esters parallels the apparent substrate specificity of the GCP- Acknowledgments AChE. Acetylcholine causes maximal stomata1 We thank Dr. Gary Tallman, Natural Science closing, PrCh induces some closure after 30 min Division, Pepperdine University, Malibu, CA For and BuCh is minimally effective. It is also possible the generous gift of N. gluucu seeds. This research that chain length of choline esters could impact on was supported in part by a grant from the Center their uptake and transport in stomata, thereby in- for Biotechnology, University of Nebraska- fluencing stomata1 movement, independent of a Lincoln and NIH grant GM 43043. This paper is cholinergic system. published as Paper No. 11147, Journal Series, That ACh is capable of inducing a relatively Nebraska Agricultural Research Division. rapid (< 5 min) closing of stomata is intriguing. The concentration required for causing a 50% re- References sponse was approximately 40 PM, although an effect was observed at 10 PM exogenous ACh. In- 111Y.S. Momonoki and T. Momonoki, Changes in acetyl- duction of stomata1 closure at these low pmolar choline levels following leaf wilting and leaf recovery by concentrations could be consistent with a heat stress in plant cultivars. Jpn. J. Crop Sci., 60 (1991) physiological role of ACh. However, a definition 283-290. 121A. Tretyn and R.E. Kendrick, Acetylcholine in plants: of its role is dependent on elucidating the existence presence, metabolism and mechanism of action. Bot. of ACh pathway in guard cells. Acetylcholineste- Rev., 57 (1991) 33-73. rase inhibitors also promoted closing of stomata. 131 E. Hartmann and R. Gupta, Acetylcholine as signalling Whereas both neostigmine and BW284c51 exerted system in plants, in: W.F. Boss and D.J. Morre (Eds.), an effect which was as pronounced as that of ACh Second Messengers in Plant Growth and Development, Alan R. Liss Inc., New York, 1989, pp. 257-287. application, eserine caused only a 50% stomata1 141 Y.S. Momonoki, Occurrence of acetylcholine-hydroly- closure. Whether this is due to inefficient uptake, zing activity at the stele-cortex interface. Plant Physiol., or other factors remains to be elucidated. How- 99 (1992) 130-133. ever, the data clearly demonstrate two important 151 M.J. Jaffe, Phytochrome mediated bioelectric potentials points, (1) exogenous application of AChE in mung seedlings. Science, 162 (1968) 1016-1017. substrates induces changes in stomata1 movement, 161 M.J. Jaffe, Evidence for the regulation of phytochrome mediated process in bean roots by the neurohumor, with the greatest effect seen in the presence of ACh acetylcholine. Plant Physiol., 46 (1970) 768-777. and (2) conditions that could increase the cellular 171 T.A. Bennet-Clark, Salt accumulation and mode of ac- concentrations of ACh appear to initiate stomata1 tion of auxin: a preliminary hypothesis, in: R.L. Wain closure. Similar complementary actions of ACh and F. Wightman (Eds.), The Chemistry and Mode of and AChE inhibitors have been reported in other Action of Plant Growth Substances, Butterworths, Lon- don, 1956, pp. 284-291. plants [2]. However an unequivocal existence of PI H. Toriyama and M.J. Jaffe, Migration of calcium and the complete choline@ pathway in plants re- its role in the regulation of seismonasty in the motor cells mains to be demonstrated. of Mimosu pudica L. Plant Phyiol., 49 (1972) 72-81. S. Maahvan et al. /Plant Science 109 (1995) 119-127 127

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