Preparation and applications of Arabidopsis thaliana guard cell protoplasts

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Research

Blackwell Science Ltd

Preparation and applications of

Arabidopsis thaliana

guard cell protoplasts

Sona Pandey, Xi-Qing Wang*, Sylvie A. Coursol* and Sarah M. Assmann

Biology Department, The Pennsylvania State University, 208 Mueller Laboratory, University Park, PA, USA, 16803

Summary

• Guard cells play an important role in the physiology and development of plants.The genetic resources available for


make it the most favorableplant species for the study of guard cell processes, but it is not easy to isolate highlypurified preparations of large numbers of guard cells from this species. Here, wedescribe methods for isolation of both guard cell and mesophyll cell protoplasts from

A. thaliana

and their use in the study of unique biochemical and cellular propertiesof these cell types.• Protocols developed for large- and small-scale preparation of guard cell proto-plasts and mesophyll cell protoplasts are described, followed by specific examples oftheir use in electrophysiological, biochemical and molecular approaches such aspatch clamping, enzyme assays, and reverse-transcription polymerase chain reaction.• The protocols described yield millions of highly purified, viable guard cell proto-plasts and mesophyll cell protoplasts from

A. thaliana.

These protoplasts have beenused successfully in the study of ion channel properties, assay of ABA activation inphospholipase D activity and comparisons of gene and protein expression levels.• These techniques make it possible to elucidate electrophysiological, biochemicaland molecular genetic pathways of guard cell function.

Key words:

abscisic acid, anion channel;


, gene expression,guard cell protoplast, K

+

channel, phospholipase D, stomata.

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Author for correspondence:

Sarah M. Assmann Tel: +1 814 863 9579 Fax: +1 814 865 9131

Email:

[email protected]

Received:

30 July 2001

Accepted:

30 October 2001

Introduction

Guard cells play a vital physiological role in regulating gaseousfluxes across the plant epidermis. For the plant scientist, guardcells also have become a model system for the study of signaltransduction pathways, as they report physiological responsesto environmental signals in a readily accessible manner (Blatt,2000; MacRobbie, 2000; Assmann & Wang, 2001; Ng

et al.

,2001; Schroeder

et al.

, 2001). However, it is an intrinsiccharacteristic of stomatal development and spacing (Palevitz,1981; Sack, 1987; Zhao & Sack, 1999) that guard cellscomprise a minor component of the epidermal tissue anda yet more minute fraction of the total cells in a leaf.Thus, researchers wishing to investigate unique cellular orbiochemical attributes of guard cells must first develop wayseither to assess these attributes on a single-cell basis within

the epidermal tissue (Roelfsema & Prins, 1997; Outlaw &Zhang, 2001) and/or to obtain preparations of purified guardcell protoplasts (GCPs).

The first methods for the small-scale isolation of GCPsfrom

Allium cepa

, and

Nicotiana tabacum

were developed aquarter of a century ago (Zeiger & Hepler, 1976). Methodsfor GCP isolation from

Vicia faba

(Schnabl

et al.

, 1978),

Paphiopedilum harrisianum

(Zeiger, 1981), the

Argenteum

mutant of

Pisum sativum

( Jewer

et al

., 1982),

Commelinacommunis

(Fitzsimons & Weyers, 1983),

Nicotiana glauca

(Cupples

et al.

, 1991),

Zea mays

(Fairley-Grenot & Assmann,1992), and

Beta vulgaris

(Hall

et al.

, 1996) have also beendeveloped (reviewed by Boorse & Tallman, 1999). In par-ticular, GCPs from

V. faba

have proven to be a vital tool forsingle-cell electrophysiological assays of plant ion channels/transporters and the signals that regulate them (reviewed byHedrich & Schroeder, 1989; Assmann, 1993; Ward

et al.

,1995; Schroeder

et al.

, 2001). Large-scale preparations of

*These two authors contributed equally.

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V. faba

GCPs, facilitated by the ease with which the epidermallayer can be mechanically stripped from the rest of the leafin this particular species (Kruse

et al.

, 1989), have enabledbiochemical and molecular studies of guard cell function.Biochemical assays of

V. faba

GCPs have been used to evaluateH

+

-ATPase activity (Becker

et al.

, 1993; Kinoshita

et al.

,1995; Kinoshita & Shimazaki, 1999b) and guard cell photo-synthetic capacity and carbon metabolism (reviewed byAssmann, 1993; Talbott & Zeiger, 1998; Goh

et al.

, 1999).Biochemical assays of

V. faba

GCPs have also identified phos-pholipase D (PLD) as an abscisic acid (ABA) signalingelement in guard cells ( Jacob

et al.

, 1999). cDNA librariesderived from

V. faba

GCPs have allowed identification ofcDNAs encoding guard cell H

+

-ATPases (Nakajima

et al.

,1995; Hentzen

et al.

, 1996), 14-3-3 proteins (Emi

et al.

,2001), cyclophilin (Kinoshita & Shimazaki, 1999a) and anABA-activated kinase (Li

et al.

, 2000).

Vicia faba

is advantageous in that it is a crop species, whichmeans that results derived from it may be directly applicableto agronomic improvement, not only of

V. faba

but also ofother important leguminous crops such as soybean. There arealso disadvantages, however, to the use of

V. faba

, includinglarge genome size, which precludes efficient application ofmany genetic techniques, and difficulty in achieving stabletransformation (although transient transformation of

V. faba

guard cells is feasible; Li

et al.

, 2000). With regard to the easeof molecular genetic manipulations,

A. thaliana

is emergingas a system of choice. The main disadvantage of

A. thaliana

isthat the small stature of the plant and the comparatively smallsize of the guard cells necessitate growth of a large number ofplants for any biochemical assessments of guard cell function.Moreover, the texture of the

A. thaliana

leaf is very differentfrom species such as

V. faba

and

C. communis

, which havebeen used for many studies involving guard cell physiology.Epidermal peels from

A. thaliana

invariably have significantamounts of mesophyll cells adhering to them, which limitstheir usefulness for most molecular biological and biochemicalstudies of guard cell function. Thus, it becomes necessaryto develop efficient protocols to isolate large quantities ofcomparatively pure guard cell protoplasts from this species.

In the present report, we describe methods for both small-and large-scale isolation of

A. thaliana

GCPs, and we also giveexamples of several applications that we have pursued usingprotoplasts from this species. Just as for the initial methods ofGCP isolation (Zeiger & Hepler, 1976; Zeiger, 1981), ourpurification protocols rely on the fact that the thickenedguard cell wall allows a series of sequential cell wall digestionsteps. Mesophyll and epidermal cell protoplasts are releasedearly in the digestion and can be washed away from theepidermal peels. The peels then retain guard cells as theonly intact cell type and can be further digested to releasethe GCPs. It is our hope that the detailed methodologiesprovided below will facilitate use of

A. thaliana

GCPs byplant biologists.

Methods

Plant growth

Plants of

A. thaliana

ecotype WS are grown in growthchambers in Metro-Mix 200 potting mixture (The Scotts Co.,Marysville, OH, USA) under an 8-h light/16-h dark regimeat a light intensity of 200 µmol m

−

2

s

−

1

and 22

°

C/18

°

C in lightand dark. They are bottom-watered with quarter-strengthHoagland’s solution for the first two weeks and thereaftertop-watered with the same solution twice weekly. Plants are4–6 weeks old when the leaves are harvested and have not yetinitiated flower bolts. Protoplasts are isolated from healthygreen leaves taken from the third level of the rosette. FinalGCP yield and contamination level are greatly affected by thechoice of leaves. Typically, leaves that are easier to peel by handalso give better results in the protocols described below.

Isolation of

A. thaliana

GCPs: same-day large-scale and small-scale protocols

These protocols were modified from protocols originallydeveloped for

V. faba

(Kruse

et al.

, 1989; Lee

et al.

, 1996;Miedema & Assmann, 1996; Assmann & Romano, 1999).Except for the last purification step and the amounts of leaftissue and solutions used, the large- and small-scale protocolsare identical. In the paragraphs that follow, large-scalequantities are given first followed by small-scale quantities inparentheses.

The protoplasting solutions are as follows. Basic solution:5 m

M

2-[N-morpholino] ethare sulfonic acid hydrate-Tris(Mes–Tris), pH 5.5, 0.5 m

M

CaCl

2

, 0.5 m

M

MgCl

2

, 10 µ

M

KH

2

PO

4

, 0.5 m

M

ascorbic acid (Sigma, St Louis, MO, USA),and 0.55

M

sorbitol. Enzyme solution 1: 0.7% cellulysin cel-lulase,

Trichoderma viride

(Calbiochem, La Jolla, CA, USA),0.1% (w/v) polyvingl pyrrolidone 40 000 (PVP-40), 0.25%(w/v) bovine serum albumin (BSA Fraction V, Sigma # A-4503,Sigma Chemical Co.) and 0.5 m

M

L

-ascorbic acid (final con-centrations), dissolved in 55% (v/v) basic solution and 45%(v/v) distilled water. Enzyme solution 2: 1.3% (w/v) OnozukaRS cellulase (Yakult Honsha, Tokyo, Japan), 0.0075% (w/v)Pectolyase Y-23 (Seishin Pharmaceutical, Tokyo, Japan), 0.25%(w/v) BSA, and 0.5 m

M

L

-ascorbic acid in basic solution. Forinformation on ordering wall-digesting enzymes, see Assmann& Romano (1999). Before use, the pH of enzyme solution 2is reduced to pH 3.5 for 5 min, using HCl according toMiedema & Assmann (1996). Most commercially availablewall-digesting enzymes are not highly purified and the lowpH treatment is thought to inactivate contaminating proteases(Fairley-Grenot & Assmann, 1992). The pH is then raised topH 5.5 using KOH. Finally, the enzyme mixture is vacuum-filtered through a MSI micronsep nitrocellulose disc filter(Osmonics Laboratory Products, Minnetonka, MN, USA) toremove any insoluble and/or precipitated material.


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After excision of the major veins with a razor blade, 80–100(8) young, fully expanded leaves are blended for 1 min andthen again for 30 s to 1 min (twice for 30 s) in 100 ml coldtap water, using a commercial Waring blender connected to aVariac set to 85 V. Total blending time is dependent on leafnumber and toughness: older leaves require the longer blend-ing period. In general, if blending time is shortened, there willbe more mesophyll cell contamination in the final GCP pre-paration. If blending time is too long, the peels become verysmall (ideal peel size is 0.4–200 mm

2

) and it is not easy to findand observe them during the digestion process; ultimately, theyield is reduced. The blended mixture is poured through a200-µm mesh (The Spectrum Companies, Laguna Hills, CA,USA) to remove broken mesophyll and epidermal cells. Ifany large green specks of mesophyll tissue remain, they areremoved with forceps. The epidermal peels retained (Fig. 1a)are rinsed thoroughly with deionized water until the foamproduced by blending has essentially gone. The peels, whichshould be pale green, are then transferred into a 250-ml(50 ml) Erlenmeyer flask containing 50 ml (10 ml) of enzymesolution 1. The purpose of the first digestion is to releaseremaining epidermal cells, whose protoplasts then burst as aresult of the low osmolality. The flask is placed in a shakingwater bath (American Scientific Products, Model YB-521) at27

°

C for 30 min, with the shaking speed set to 140 excursionsper min. As guard cells are responsive to light, digestion is per-formed in darkness (Dietrich

et al.

, 2001). After 30 min,150 ml (30 ml) basic solution is added, and the mixture isshaken for an additional 5 min under the same conditions.This step provides an intermediate osmolality betweenenzyme solution 1 and enzyme solution 2; sudden plasmolysiswould damages the guard cell membrane. The digested peels(Fig. 1b) are collected on 200 µm nylon mesh and rinsedgently with basic solution, then placed into 50 ml (10 ml) of

enzyme solution 2 in a 250-ml (50 ml) Erlenmeyer flask.Since this step releases guard cell protoplasts into solution, theosmolality is increased to maintain protoplast integrity. Theflask is shaken at 40 excursions per min in the dark at 20

°

Cfor 50–60 min, until most of the GCPs have rounded up(Fig. 1c). (The small-scale preparation is shaken at 60 excur-sions per min for 30 min. At this point the protoplasts shouldhave started to round up. The shaking speed is then reducedto 40 excursions per min and digestion is stopped at 40–45 min. By this time, only approximately 50% of the GCPshave rounded up but in our hands further digestion decreasesthe success rate for obtaining stable high-resistance sealsrequired for patch clamp analysis.) At the end of the digestionperiod the flask is swirled gently by hand for a few seconds;this may improve release of GCPs from the peels. The mixtureis filtered through 20 µm nylon mesh (The Spectrum Com-panies). The peels remaining on the mesh are washed with100–150 ml basic solution to release GCPs adhering to them.The filtrate, which contains the GCPs, is collected in four(one) 50-ml centrifuge tubes. The protoplasts are sub-sequently spun in a benchtop centrifuge for 5 min at 200

g

. Allbut 1–2 ml of supernatant is carefully removed from each loosepellet, and then the protoplast pellet is gently resuspended.The tubes are refilled with 25 ml (50 ml) basic solution and asecond, identical centrifugation is performed. After removalof the supernatant, each GCP pellet is gently resuspended,yielding a volume of 2–3 ml. For the small scale preparation,used for single cell assays such as patch clamping, the purityof this preparation is more than adequate: 70–90% GCPs(calculated on a cell number basis), with mesophyll cell pro-toplasts (MCPs) and chloroplasts as the major contaminants.

For large-scale isolations that will be used for molecular andbiochemical assays, an additional step is performed that sig-nificantly improves the purity of the GCP preparation. Basic

Fig. 1 Isolation of Arabidopsis thaliana guard cell protoplasts. (a) A. thaliana epidermal peels exhibit epidermal cell walls and intact, healthy guard cells after brief blending. (b) Intact and healthy guard cells are seen in the epidermal peels in the absence of epidermal cell walls after the first digestion and intermediate osmolality treatment. Scale bar in (a) is 20 µm and refers to (a) and (b). (c) A pair of guard cell protoplasts rounds up after 40 min of digestion in enzyme solution 2; bar, 10 µm. (d) Millions of very pure guard cell protoplasts can be obtained with the large-scale protocol. Scale bar in (d) is 20 µm and refers to panels (d) and (e). (e) Mesophyll cell protoplast preparation. (f) A. thaliana guard cell protoplast. Scale bar in (f) is 10 µm and refers to panels (f) and (g). (g) A. thaliana mesophyll cell protoplast. Note the difference in protoplast size between (f) and (g).



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solution is added to the combined protoplast pellets to givea final volume of 10 ml. Five milliliters of Histopaque(No. 1077, Sigma Chemical Co.) is carefully pipetted into thebottom of each of two 50-ml centrifuge tubes. Five millilitersof GCP suspension is then carefully layered on top. The GCPsuspension is introduced slowly down the side of the tubeusing a pipettor equipped with a 1-ml tip cut at the end toincrease the size of the opening and decrease the shear stresson the GCPs. Layering is a crucial step: if there is too muchmixing and two distinct layers are not formed, the yield andpurity of the GCPs will be substantially reduced. The tubes arecentrifuged for 15 min at 200

g

.

The fuzzy opaque layer ofguard cells is isolated from the interface of the two solutionswith a bent-tip Pasteur pipette or a pipettor equipped with a1-ml tip cut at the end, and expelled into a new 50-ml centrifugetube. The volume of the GCP suspension is doubled with basicsolution and the tube is centrifuged for 5 min at 200

g

. Thesupernatant is removed and the protoplast pellet is gentlyresuspended in 1 ml of basic solution. The protoplasts arekept on ice in the dark for at least 1 h before use. This stepappears to be important for recovery of protoplast membranestability and health following the centrifugation steps. Tenmicroliters of GCP suspension is taken and the number ofprotoplasts estimated with a hemocytometer. The typicalyield is of the order of 2.2–2.7

×

10

6

GCPs per 80–100leaves. The typical purity is 98–99.5% on a cell basis, withcontamination originating from MCPs (Fig. 1d).

Isolation of

A. thaliana

GCPs: overnight large-scale protocol

We have also developed an alternative large-scale isolationmethod that is less labor intensive and uses considerably smalleramounts of enzymes in an overnight digestion. This method hasbeen developed for 100 leaves and is modified from a methoddescribed by Pei

et al

. (1997). The protocol is similar to thelarge-scale protocol described in the previous section, with thefollowing changes. After blending, the peels are rinsed first withdeionized water and then with basic solution. The peels arethen transferred into a 250-ml flask containing 50 ml of basicsolution with 0.65% Onozuka RS cellulase, 0.35% MacerozymeR10 (Yakult Honsha Co, Ltd), 0.25% (w : v) BSA and 0.001%kanamycin, and placed in a slow-shaking water bath (40excursions per min) at 22

°

C for 12–13 h. At this point, mostof the protoplasts should be round and detached from theepidermal peels when viewed under the microscope. TheGCPs are then further processed as described in the previoussection. Typically, 4.7–5.2

×

10

6

GCPs, 97.5–99% pure areobtained, the contaminants being broken and intact MCPs.

Isolation and purification of

A. thaliana

MCPs

The method for isolating

A. thaliana

MCPs is modified fromthat described by Romano

et al.

(1998). Ten fully expanded

leaves are excised and immersed in distilled water in the darkbefore processing. The midrib and abaxial epidermis of eachleaf are removed with the aid of a razor blade and forceps andthe leaves are cut into 1 cm

2

pieces. The leaf pieces are thentransferred to 25 ml of enzyme solution (5 mM Mes, 0.65

M

sorbitol, 1 m

M

CaCl

2

, 0.4% (w : v) Macerozyme R-10, 1%(w : v) Cellulase R-10, 0.2% (w : v) BSA and 0.1% (w : v)PVP-40, pH 5.5). The leaf pieces are vacuum infiltrated withenzyme solution for 3 min. Broken cells and debris are removedby decanting the solution and the leaf tissue is transferred to25 ml of fresh enzyme solution. Digestion is performed at24

°

C for 45 min with slow shaking (30 excursions per min)in a water bath. The enzyme solution develops a greenish hue,indicating the release of protoplasts. The solution is filteredthrough 30 µm nylon mesh (The Spectrum Co.) into a 50-mlcentrifuge tube. The leaf pieces retained are rinsed with 25 mlof incubation medium (0.65

M

sorbitol, 1 m

M

CaCl

2

) and thefiltrate is also collected. The suspension is centrifuged at 150

g

for 4 min and the pellet obtained is resuspended in 50 ml ofincubation medium. The solution is centrifuged, as before,and the final pellet is gently resuspended in 1 ml of incubationmedium and kept on ice in the dark until use. This protocolyields approximately 1.2

×

10

7

MCPs at a purity > 99%, withvascular elements and cell debris as contaminants (Fig. 1e,g).

Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) of soluble and membrane proteins

Soluble and membrane protein fractions of GCP and MCPwere prepared according to Li & Assmann (1996). Briefly, theprotoplasts (isolated by overnight digestion protocol) werepelleted by centrifugation and frozen in liquid nitrogen. Allsubsequent steps were performed at 4

°

C. Protoplasts (2.5

×

10

6

)were homogenized in 50 µl buffer in prechilled microfugetubes. The buffer was 50 m

M

Tris-HCl, pH 7.5, 1 mM MgCl2,2 mM ethylenediaminetetraacetic acid (EDTA), 0.25 mM

ethyleneglycoltetraacetic acid (EGTA), 250 mM sucrosecontaining 2 mM dithiothreitol (DTT), 1 mM phenyl-methylsulfonyl fluoride (PMSF) and 10 µg ml–1 each of theprotease inhibitors leupeptin, pepstatin and aprotinin (SigmaChemical Co.). The homogenate was then centrifuged at10 000 g for 15 min and the resulting supernatant (crudeextract) was centrifuged for 45 min at 150 000 g. Thissupernatant was removed as the soluble fraction and the pelletwas resuspended in homogenization buffer and recentrifugedat 150 000 g for 45 min. The final pellet was used as themicrosomal membrane fraction. Both soluble and membranefractions were immediately analyzed by SDS-PAGE (Fig. 2).

Patch clamping

Protoplasts used for patch clamping are always isolated withthe small-scale protocol. Standard patch clamp protocols


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(Fig. 3) were employed as described by Pei et al. (1997) andWang et al. (2001). Solutions for K+ channel recording wereas described in Wang et al. (2001): 10 mM Mes-Tris, pH 6.0,10 mM K-Glutamate, 4 mM MgCl2, and 1 mM CaCl2,osmolality 540 mmol kg–1 (bath solution), and 10 mM N-2-hydroxyethylpiperazine-N ′-2-ethanesulfonic acid (HEPES)-Tris, pH 7.8, 80 mM K-glutamate, and 20 mM KCl,osmolality 560 mmol kg–1 (pipette solution). ATP (5 mM

from a 0.5 M Mg-ATP stock solution in 0.5 M Tris) was addeddaily to the pipette solution. Final osmolalities were obtainedby the addition of sorbitol. ABA (500 µm) was addedimmediately after achieving a stable whole-cell recording andcurrent responses were recorded 10 min later.

The pipette solution for anion channel recording was alsoas described in Wang et al. (2001): 150 mM CsCl, 2 mM

MgCl2, 6.7 mM EGTA, 3.35 mM CaCl2, 10 mM HEPES-Tris, pH 7.5, osmolality 540 mmol kg–1. Five mM Mg-ATPand 5 mM Tris GTP (final concentrations) were added justbefore the experiment. The bath solution for anion channelexperiments was: 10 mM Mes-Tris, pH 5.6, 30 mM CsCl, 2 mM

MgCl2, 5 mM CaCl2, osmolality 560 mmol kg–1. Protoplastswere pretreated at least 1.5 h with ABA (500 µm), and 50 µM

ABA was added to bath and pipette solutions, following Peiet al. (1997).

Assay of PLD activity

For assay of PLD activity, protoplasts were isolated by thesame-day large-scale protocol. Aliquots of the GCPsuspension (100 µl, approximately 0.9 × 105 protoplasts) wereincubated in 1.5 ml microfuge tubes at room temperaturefor 10 min, and then treated with 20 µM ± ABA (finalconcentration from a 1 mM ABA stock in 10 mM-hydroxyl-1,1-bis(hydroxymethyl)ethylamino-1-propane sulfonic acid(TAPS), pH 8.0, with HCl). After the appropriate incubationtime, each sample was processed as described by Ritchie &Gilroy (1998), with the following modifications to the PLDassays: a 40-µl sample was incubated at room temperature for10 min, with continuous shaking at 100 excursions per min,in 80 µl reaction mixture containing 40 mM Mes-NaOH,pH 6.5, 283 µM NBD-PtdCho (1-oleoyl-2-(-12-((7-nitro-2–1,3-benzoxadiazol-4-yl)amino)dodecanoyl])-sn-glycero-3-phosphocholine; R1, 18 : 1; R2, 12 : 0-N-NBD, Avanti Polar

Fig. 2 Sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS-PAGE) analysis of total proteins from Arabidopsis thaliana guard cell protoplasts vs mesophyll cell protoplasts. Total protein was isolated from guard cell protoplasts (GCPs) and mesophyll cell protoplasts (MCPs) and fractionated into soluble ( GCP sol and MCP sol) and membrane (GCP memb and MCP memb) fractions as described in Methods. Ten micrograms of each fraction was separated by SDS-PAGE. Arrows mark the small (14 kDa) and large (56 kDa) subunits of Rubisco. Lane Mr shows protein molecular mass markers (in kDa). Fig. 3 ABA regulation of ion channels in Arabidopsis thaliana guard

cell protoplasts. (a) Representative A. thaliana guard cell whole-cell K+ currents before and after ABA (50 µM) treatment. The currents were recorded from a voltage family stepping from –216 to +64 mV in +20 mV increments, with a holding potential of –76 mV. (b) Representative A. thaliana guard cell whole-cell slow anion currents with and without ABA (50 µM) treatment. The voltage protocol ranges from –145 mV to +35 mV in +30 mV increments, at a holding potential of 0 mV.

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Lipids, Inc., Alabaster, AL, USA), 50 mM CaCl2, and 0.25 mM

SDS. 1-Butanol (0.9%, v : v) was included in the assay topermit detection of the transphosphatidylation product,NBD-PBut (NBD-phosphatidylbutanol), as a metabolicallystable index of PLD activity (Liscovitch et al., 1993, Fig. 4a).The reaction was stopped by the addition of 300 µlchloroform/methanol (1 : 2, v : v). Chloroform (80 µl) and2 M KCl (80 µl) were added sequentially to generate a two-phase system. After vigorous vortexing, phases were separatedby centrifugation for 10 min at 10 000 g and the aqueousphase was removed. The chloroform extract was dried usinga speed vacuum concentrator and the residue was suspendedin 40 µl of chloroform before thin-layer chromatography onheat-activated precoated silica gel plates (VWR, SouthPlainfield, NJ, USA). To analyze the production of NBD-PtdBut, plates were developed in the organic phase of amixture of ethyl acetate/2,2,4-trimethylpentane/acetic acid/water (13 : 2 : 3 : 10, v : v : v : v), and were visualized withUV light (UV transilluminator FBTIV-88, Fisher Scientific,Pittsburgh, PA, USA). The spots corresponding to NBD-PtdBut were scraped from the plates, placed in 650 µlchloroform–methanol–H2O (5 : 5 : 1, v : v : v) in 1.5-mltubes, vortexed, and centrifuged for 10 min at 12 000 g. Thefluorescence of a 200-µl aliquot of the eluted lipid wasmeasured in a fluorescence spectrometer (excitation 460 nm,emission 535 nm) (HTS 7000, Perkin Elmer Instruments,Norwalk, CI, USA).

Isolation of RNA and use in reverse-transcription polymerase chain reaction (RT-PCR)

Both same-day and overnight large-scale protocols havebeen used for RT-PCR experiments. Total RNA was isolatedfrom GCPs and MCPs using TRIzol reagent (Gibco BRL,Rockville, MD, USA) according to the manufacturer’sprotocol. One milliliter of reagent was used per 5 × 106 GCPsand 5 × 105 MCPs, respectively. Typically, 12–15 µg RNAwas obtained from these amounts of protoplasts with anA260 : 280 ratio > 1.8.

Two micrograms of total RNA was used for first strandsynthesis using the Superscript preamplification system(Gibco-BRL) according to the manufacturer’s protocol. RNAwas primed with the oligo dT primers and the first strandsynthesis reaction allowed to continue for 50 min at 42°C.The reaction was terminated by incubation at 70°C for15 min and either used immediately or stored in small aliquotsat –20°C until further use.

Because the polymerase chain reaction (PCR) is anextremely sensitive technique, and the guard cell preparationdoes have some contamination from mesophyll cells (≈ 1–2%), it is important to assess whether PCR products obtainedoriginate from GCPs or from contaminating MCPs. Foraccurate conclusions regarding genes expressed specifically inguard cells or genes expressed in guard cells at a higher levelcompared with mesophyll cells, PCR conditions were

Fig. 4 Phospholipase D (PLD) activity is stimulated in Arabidopsis thaliana guard cell protoplasts treated with ABA. The PLD activity was measured in vitro using protein extracts of A. thaliana guard cell protoplasts that had been treated with ± ABA (20 µM) for various times. (a) PLD activity was assessed using its unique ability to transfer the phosphatidyl group of its substrate (a structural phospholipid, such as phosphatidylcholine, tagged with a fluorescent probe (NBD-PtdCho)) to a primary alcohol (in this case, 1-butanol) instead of water, forming phosphatidylalcohol (instead of phosphatidic acid (NBD-PtdOH)). The amount of phosphatidylbutanol (NBD-PtdBut) formed is a relative measure of PLD activity. (b) Fluorescence image of a thin-layer chromatography plate separating NBD-PtdBut. (c) PLD activity expressed as the percentage of product formation in the absence of ABA at t = 0 min (b) and (c) show data representative of one of three separate experiments.



Research 523

modified (Fig. 5). The reactions were either performed withserial dilution of the first strand synthesis reaction or withdifferent numbers of cycles. For serial dilution PCR, the firststrand reaction from both guard cells and mesophyll cells wasdiluted 1 : 10, 1 : 100, 1 : 1000, 1 : 10 000 and 1 : 100 000,and 1 µl of undiluted or diluted product was used in a 25-µlreaction mixture. Alternatively, the reaction was performed atone particular dilution, but stopped after different number ofcycles, e.g. 10, 15, 20, 25, 30, 35 and 40, assuming 1.8-foldamplification per cycle (Freeman et al., 1999).

If a gene has equal expression in both the cell types, it showsequivalent amplification at all the dilutions and all termina-tion times. If an amplification product disappears at a lowerdilution of the PCR products of one cell type compared withthe other, it is considered to have lower expression in the celltype from which the PCR product disappears first. Similarly,if a product appears at a certain number of cycles in one celltype and only after a greater number of cycles in the secondcell type, it is considered to have higher expression in the firstcell type. Standard PCR conditions are used depending on the

primers. For serial dilution, the products are typically ampli-fied for 30 cycles; for different numbers of cycles a 1 : 100dilution of first strand reaction is used. A control reaction isalways performed with actin primers to confirm equalamounts of starting cDNA.

Using different dilutions of cDNA to perform the compar-ative PCR assay requires good pipetting technique to acquirean accurate dilution series. Using different cycle numbersrequires that the researcher be present to remove the appro-priate tubes at the established cycle numbers. The differentcycle number approach may be more quantitative since intheory one can go down to the level of difference of one cycleand see the relative expression of the gene in the two samples.

Results and Discussion

We have described three methods for isolating guard cellprotoplasts from A. thaliana at small and large scales. OnceA. thaliana GCPs are produced, they can be used for dissectingspecific stomatal functions that can be investigated using

Fig. 5 Reverse-transcription polymerase chain reaction analysis of gene expression in Arabidopsis thaliana guard cell protoplasts vs mesophyll cell protoplasts. RNA isolated from purified guard cell protoplasts (GCPs) and mesophyll cell protoplasts (MCPs) was used for the first strand synthesis reaction. For estimation of relative abundance of transcripts, cDNA to be amplified was either serially diluted (left) or was amplified for varying numbers of cycles (right). (a) expression of a putative protein kinase gene (Accession No. AL031032). Kinase primer sequences were: forward primer 5′-CAAGGATATTGGCTCCGGTA-3′; reverse primer 5′-CCTGCAGGAGGAACAGTTG-3′. The kinase shows higher expression in guard cells as the transcript could be observed until 1 : 10 000 dilution in GCPs vs 1 : 1000 in MCPs and appears at the 25th cycle of amplification in GCPs vs the 30th cycle in MCP. (b) Expression of carbonic anhydrase (Accession No. X65541) at a higher level in MCPs than in GCPs. Carbonic anhydrase primer sequences were: forward primer 5′-GCTTCTCTTCGCCAACAAAT-3′; reverse primer 5′-ATTCAAGTCCCCAAAGCTCA-3′. (c) Amplification of the KAT1 gene under identical conditions as a control for predominantly guard cell-expressing gene. KAT1 (Accession No. X93022.1) primers were: forward primer 5′-TTTGTTCCACAGCACTTCAGCCACTA-3′; reverse primer 5′-AGTGTCGGAAGTCGGATTCGTAACATCTA-3′. (d) Amplification of actin transcripts under identical conditions as in (a) and (b) as a control for equal expression in both the cell types and to show amounts of initial cDNA template. Actin primer sequences were: forward primer 5′-GTTGGGATGAACCAGAAGGA-3′; reverse primer 5′-GAACCACCGATCCAGACACT-3′. Lane Mr represents molecular weight markers in kb.



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a diversity of assays, such as electrophysiology, biochemistryof signal transduction, and expression profiling of guard cell-expressed genes.

Figure 1a–c shows various stages in the production ofA. thaliana GCPs. Figure 1d,f illustrates the high quality ofthe protoplasts obtained; these are round, with intact, evenlydistributed chloroplasts. Figure 1d,e illustrates the purity offinal large-scale preparations of GCPs and MCPs. Fluoresceindicctate (FDA) staining of the protoplasts obtained by eitherof the large-scale protocols shows 95% GCP viability.Although all of the results reported here were obtained withthe WS ecotype, methods and yields of GCPs were alsocomparable for the Columbia (0) ecotype (data not shown).

We described two methods for the large-scale isolation ofA. thaliana GCPs. The overnight method gives a significantlyhigher yield of GCPs than the same-day large-scale prepara-tion. This protocol, yields approximately 5 million GCPs,providing approximately 30 µg of soluble and 10 µg of mem-brane protein; we use a minimum of 10 µg protein per lanefor Coomassie-stained SDS-PAGE protein gels (Fig. 2). It ispossible that the longer the GCPs are exposed to the enzymesolution the greater the impact on the physiology of theprotoplasts, although comparable results have been obtainedfor patch clamping studies using a rapid protoplastingprotocol (Wang et al., 2001) or an overnight protoplastingprotocol (Pei et al., 1997). Experimental determination of theoptimal protoplasting method is suggested for a given assay.

The GCPs isolated by the same-day small-scale methodyield ABA-regulated K+ and anion currents (Fig. 3) com-parable to those described by Pei et al. (1997), who used anovernight protoplasting protocol. A major advantage ofA. thaliana over other species is the relative ease with whichmutants can be identified by both forward and reverse geneticapproaches. Schroeder and colleagues have used GCPs fromA. thaliana mutants in ABA signaling such as abi1-1, abi2-1,era1, det3 and gca2 to assess resultant alterations in ionchannel activity (Pei et al., 1997, 1998, 2000; Allen et al., 1999,2000). Recently, we have also described alterations in ABAsignaling in G protein α subunit (gpa1) mutants (Wang et al.,2001) and Szyroki et al. (2001) have described K+ currentsin a kat1 mutant.

ABA regulation of ion channels is achieved through complexand interwoven signal transduction pathways (Leung &Giraudat, 1998; Munnik, 2001). In Fig. 4b,c we illustratethat, as previously shown for V. faba ( Jacob et al., 1999), ABAactivates PLD activity in A. thaliana guard cells isolated bysame-day large-scale method. It will be of interest to assaythe available A. thaliana ABA-related mutants to determinewhich, if any, are altered in ABA activation of PLD.

Despite the large-scale protocols described here, theamount of GCP isolated is still limiting for RNA gel blotanalysis of gene expression. Five million GCPs from the large-scale overnight approach typically yield only enough RNA fortwo or three lanes on a Northern blot. In contrast, sensitive

RT-PCR approaches can provide an assessment of transcriptlevels starting with much smaller quantities of RNA. Wedescribed two RT-PCR-based methods for assessing relativegene expression levels in GCPs vs MCPs. The two approachesshould and do yield qualitatively similar results, as illustratedin Fig. 5.

We had previously cloned the cDNA for an ABA-activatedprotein kinase (AAPK) from V. faba guard cells (Li et al.,2000). Based on sequence analysis, the A. thaliana genomecontains several possible AAPK orthologs. Figure 5a showsexpression levels for one of these. Expression is significantlyhigher in GCPs than in MCPs, although there is some expres-sion in the latter cell type, in contrast to V. faba where AAPKexpression appears to be limited to guard cells (Li et al., 2000).By contrast, a chloroplastic carbonic anhydrase (Raines et al.,1992), which facilitates CO2–HCO3

– conversion, shows sig-nificantly lower expression in GCPs than in MCPs, consistentwith the expectation from other plant species that GCPs havea limited photosynthetic capacity (Goh et al., 1999). A com-parable result was obtained in preliminary microarray analysisof GCP vs. MCP expressed genes, in which carbonic anhy-drase and several other photosynthesis-related genes wereobserved to be expressed at higher levels in MCPs than inGCPs (S. Pandey and S. M. Assmann, unpubl. data). TheRT-PCR of two characterized genes as ‘controls’ also validatesour results. Amplification of the KAT1 gene which encodes aninward K+ channel (as a guard cell-specific gene control) andactin genes (as a gene showing equal expression in all the celltypes) clearly shows a greater abundance of KAT1 transcriptin GCPs and approximately equal expression of actin genes inGCPs and MCPs. Such results demonstrate that modified,semiquantitative RT-PCR methods are an efficient method tocompare expression profiles of GCPs and MCPs. Becausesuch approaches are rapid, simple, use small quantities ofRNA and are cost effective, they can be used in an initial sur-vey of many genes. Once the candidate genes with interestingdifferences in the expression levels have been identified in thismanner, one can proceed to more quantitative but expensive(in terms of reagents or labor) techniques such as real timeRT-PCR (Bustin, 2000; Szyroki et al., 2001), RNA gel blotand/or microarrays to elucidate the absolute quantitative differ-ence in the expression levels.

Owing to the presence of mesophyll cells as contaminantsand the extreme sensitivity of RT-PCR (compared with non-amplification hybridization techniques such as Northernblots) it is always advisable to compare the expression profilein both the cell types. If a RT-PCR product is observed at alower cycle number or at a higher dilution in GCPs than inMCPs, then it is logical to conclude that the gene of interestis indeed expressed by the guard cells. One can also keep inmind that mesophyll contamination is a problem mostly with‘amplification’ techniques such as PCR and RT-PCR, wherevery low quantities can be amplified to a significant level,whereas for techniques such as Northern blot or SDS-PAGE,



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where no amplification is involved, 1% MCP contaminationdoes not contribute significantly to the results. Other contam-inants, such as broken cell debris, do not contribute to thecDNA, as confirmed by observation of SYTO (MolecularProbes, Eugene, OR, USA) staining of nucleic acids only inintact protoplasts (data not shown). Staining was performedaccording to the manufacturer’s protocol.

In addition to the assays described above, A. thaliana GCPshave been used in fluorescence assays of cellular processes.Schreiber and colleagues have applied fluorescence transientanalysis to assess guard cell photosynthesis (Goh et al., 1999).Since fluorescent indicators for ions, lipids, and other signal-ing molecules continue to proliferate (Gilroy, 1997), thepotential for application to A. thaliana GCPs appears great.Although their small size renders A. thaliana GCPs less likelythan GCPs from other species to be practical for assays of pro-toplast volume change in response to environmental signals,in all other respects, A. thaliana GCPs appear to be a highlyuseful addition to the plant physiologist’s toolkit.

Acknowledgements

Research in the authors’ laboratory on guard cell signaltransduction is supported by grants from the National ScienceFoundation (MCB 98-74438; MCB 00-86315) and the USDepartment of Agriculture (00-35100-9420; 01-35304-09916). Two anonymous reviewers are also thanked forcomments that improved the manuscript.

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Preparation and applications of Arabidopsis thaliana guard cell protoplasts

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