Guard Cell Protoplasts Contain Acetylcholinesterase Activity
Total Page：16
File Type：pdf, Size：1020Kb
ELSEVIER Plant Science 109 (1995) 119-127 Guard cell protoplasts contain acetylcholinesterase 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 choline 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- stigmine, inhibited 40-m? of guard cell protoplast acetylcholinesterase activity. Exogenously applied acetylcholine (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 butyrylcholine or propionylcholine. BW284c51, neostigmine 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, cholinesterase; 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 tobacco 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 (physostigmine), 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.