production of guard cell protoplasts fromonion and tobacco1 · the onion epidermal peels, guard...

Plant Physiol. (1976) 58, 492-498

Production of Guard Cell Protoplasts from Onion and Tobacco1Received for publication April 22, 1976 and in revised form June 21, 1976

EDUARDO ZEIGER AND PETER K. HEPLERDepartment of Biological Sciences, Stanford University, Stanford, California 94305

ABSTRACT

Guard cell protoplasts (GCP) from young cotyledons of onion andtobacco were isolated in culture microchambers where optimal isolatingand culture conditions could be determined in situ. The digestion coursewas quantified by following under polarized light the loss of.retardationof the birefringent cellulose of the guard cells. The assay showed thatdriselase has a 5-fold higher cellulytic activity than cellulysin. Driselaseis, however, harmful to the GCP. Calcofluor staining was less adequatefor establishing digestion courses because it increases sharply after ex-posing guard cells to cellulysin.

Osmotic conditions were crucial for GCP survival. Onion guard cellsfragment in the presence of strong plasmolyticum (>0.45 M) indicatingcytoplasmic connnections between neighboring guard cells and/or cyto-plasmic attachments to the wail. Tobacco guard cells plasmolyzed with0.7 M mannitol revealed several areas of strong attachment to the wallwhich resulted in severe damage to the cells. Healthy tobacco GCP areobtained by an initial digestion with 4% (w/v) cellulysin in 0.23 Mmannitol for 2 to 3 hours followed by an increase in the osmoticum to 0.7M to stabilize the forming protoplasts.

Onion GCP were obtained by digesting paradermal slices with 4% (w/v) cellulysin in 0.23 M mannitol. Protoplasts can be osmotically releasedby replacing the enzyme solution with 0.23 M mannitol at early stages ofdigestion. They are also available after prolonged digestion (6-12hours). Paradermal slices also yield mesophyll and epidermal cell proto-plasts but they can be selectively washed away if a pure preparation ofGCP is desired. Onion GCP have been kept alive in a simple culturesolution for up to 10 days.

Plant cell protoplasts are valuable experimental objects fordevelopmental and physiological investigations. Most of thestudies thus far have dealt with protoplasts isolated from tissueculture cells or the relatively undifferentiated leaf mesophyll (1,6). Protoplasts of differentiated, highly specialized cells, on theother hand, have gone unexamined in spite of their potential forapproaching basic questions on cell determination, genetic con-trol of differentiation, and the cellular expression of specializedphysiological functions.

In this study we report on the production and use of proto-plasts from the guard cells of the stomatal complex in onion andtobacco. Guard cells are of great interest because of their dis-tinctive morphogenetic and physiological properties. They havea well defined developmental pathway (15) and a prominentspecialized shape. During differentiation, a central wall thicken-ing is formed, from which cellulose microfibrils fan outward in aradial pattern that provides the reinforcement necessary to de-termine the cell shape. The deposition of the wall and especiallythe cellulose microfibrils is anticipated and presumably con-trolled by a precisely oriented radial array of cortical cytoplasmic

I This work was supported by Grant BMS-74-15245 from the Na-tional Science Foundation.

microtubules (10). The physiological properties of the guardcells are also striking (14).

In spite of a great deal of available information on stomataldevelopment and function, the cellular control of the guard cellactivities remains unknown. Experimental work with guard cellprotoplasts offers a novel approach that could provide newinsights into those long standing questions.

This work reports on the isolation and culture of guard cellprotoplasts using microchambers. The chambers permit observa-tion of the entire sequence of wall degradation, protoplastrelease, and the response of the protoplasts to different osmoticand culture conditions. The use of the chambers has also led tothe development of a novel method involving polarized lightmicroscopy for the quantitative, in situ monitoring of the cellu-lose digestion within the wall.

MATERIALS AND METHODS

Plant Material. The species used were Allium cepa cv. Cimahybrid (Keystone Co., Hollister, Cal.) and Nicotiana tabacumvar. Mammoth. The seeds were sown in pots with Vermiculiteand grown in a greenhouse with a temperature range of 20 to 30C. Tap water was supplied every 24 or 48 hr.

Preparations and Microchamber Assembly. Onion prepara-tions were obtained by dissecting 4- to 15-day-old cotyledons.Epidermal peels were made by tearing a small portion of tissue 2to 3 cm below the hook, with a pair of fine forceps and thenpulling upward. The resulting epidermal peel was cut at theattached end and mounted. Paradermal sections were done witha scalpel inserted slightly below the epidermis. A 2 to 3 cmparadermal cut was made parallel to the long axis of the cotyle-don; two transverse cuts within the area of the paradermalincision released a slice that was then picked up with forceps andmounted. Tobacco epidermal peels were obtained from theabaxial surface of 10- to 20-day-old cotyledons by tearing offepidermal strips with fine forceps. The peels, 2 to 5 mm long,were collected in a drop of 0.23 M mannitol solution, unfolded asmuch as possible, and mounted.Most of the digestion and protoplast formation studies were

done in a Sykes Moore culture chamber (Bellco Glass Inc.,Vineland, N. J.), using a 1.5 mm thick o-ring which provides0.65 ml of medium volume. To allow Kohler illumination in aReichert Zetopan microscope, the chamber was trimmed onboth top and bottom to reduce its thickness and the observationswere made with-the bottom part of the chamber facing theobjective of the scope.The onion peels or sections were mounted in round, No. 1

thickness coverslips in 1 drop of 0.23 M mannitol, with thecuticle against the glass. Some gentle pressure was applied to theends of the strips so that they lay straight and flat against thecoverslip surface. Excess liquid was removed with Kimwipes andboth ends were adhered to the glass with a hot mixture of 40%Vaseline, 40% anhydrous lanolin, and 20% (w/v) paraffin,melting point 55 to 57 C (5). The strips were then bathed againwith 0.23 M mannitol solution and subjected to a vacuum for 30to 90 sec. The coverslip was placed in the bottom of the cham-

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GUARD CELL PROTOPLASTS

ber, an o-ring and a second coverslip were introduced on top ofit, and the chamber was sealed (Fig. 1). The enzyme solutionwas injected through the o-ring with a syringe holding a 25-gauge needle. A 23-gauge needle was inserted on the oppositeside to release the air. One air bubble was usually left in.Solutions were changed by perfusion through the needles. Theentrance needle was connected to a Hamilton 4-way valve withTygon tubing. The valve was connected to 1 to 4 reservoirscontaining the washing or nutrient solutions. A tube inserted tothe exit needle carried the liquid out of the chamber. Theperfusion was done by gravity; the degree of opening of the valveand the height of the reservoir regulated the flow. The procedureallowed one to perfuse the chamber while it was being observedunder the microscope; flow rates of 1 to 2 cc/min were gentleenough to prevent the washing away of the released protoplasts.When a new solution was introduced, 30 to 50 cc were perfusedthrough the chamber, to ensure adequate replacement of theprevious one.Enzyme Solutions. Cellulysin (Calbiochem) and driselase

(Kyowa Hakko Kogyo Co., Tokyo, Japan) were dissolved in0.23 to 0.40 M mannitol, adjusted to pH 5 with HCI, and filteredthrough Nalge filter units with a 0.20 ,um membrane.Measurements of Retardation in Polarized Light. Retardation

of the cellulose component of the cell wall was measured on aReichert Zetopan polarizing microscope equipped with a Zeiss40X oil immersion strain-free epiachromat objective and a Zeiss1/30 X Brace-Kohler compensator. Retardation (r) is defined inthe following equation: F = (n, - n2) t where n, and n2 are thetwo refractive indices of the birefringent cellulose microfibril andt is the thickness of the object being examined.

Cell Wall Staining. Calcofluor white ST (American CyanamidCo.), 0.1N% (w/v), in 0.23 to 0.4 M mannitol was used to stainthe cell walls (13). The stain was removed after 5 min. The cellswere washed twice with a mannitol solution and observed in aReichert Zetopan with a high pressure mercury lamp, an E2excitation filter with a maximum transmission at 355 nm, and aSpl barrier filter with a 50% cut-off at 460 nm.

Culture Medium. Washed onion protoplasts were cultured in asimple medium containing (mg/ml): glucose, 18; sucrose, 20;ribose, 0.5; xylose, 0.5; CaCl2 - 2H2O, 0.5; MgSO4 * 7H2O, 0.25;KH2PO4, 0.1; mannitol, 18.5; gentamicin, 0.05. The pH wasadjusted to 6 with 1 N HCI, the calculated osmolarity was 280milliosmols.

RESULTS

Cytological and Osmotic Properties of Epidermal Tissue. Inthe onion epidermal peels, guard cells remain with their wallsintact (Figs. 2 and 3), although 30 to 50% of them showcytoplasmic damage. Most of the epidermal cells are destroyedin the process of tearing. In a 0.15 M mannitol solution, intactguard cells display active cytoplasmic streaming that persists fora few hours. Paradermal slices provide intact epidermal andguard cells, except for the cells in the edge of the preparation

which are damaged during the dissection. In 0.15 M mannitol,both epidermal and guard cells show vigorous streaming thatcontinues up to 24 to 48 hr.Tobacco epidermal peels also have mostly destroyed epider-

mal cells. Many guard cells are dead, as judged by their inabilityto respond to osmotic changes. However, each peel usually hassome guard cells with intact cytoplasm (Fig. 4). No streamingcould be seen on those cells which display, instead, saltatorymotion of the organelles.Mannitol is used as a plasmolyticum to preserve protoplast

integrity. Developing onion guard cells are highly resistant toplasmolysis and remain turgid for several minutes in the pres-ence of mannitol in concentrations as high as 0.7 M; in contrast,epidermal or mesophyll cells plasmolyze rapidly in 0.2 M manni-tol. Fully mature guard cells, on the other hand, have lowerturgor values; 0.15 M mannitol is hypotonic, 0.23 M is in theisotonic range, and more concentrated solutions are hypertonic.When mature onion guard cells are abruptly subjected to os-motic pressures higher than 0.45 M mannitol, the cytoplasmoften constricts and breaks into two protoplast spheres (Fig. 6).In situ observations show that, upon introduction of the osmoti-cum, the cell contracts along the longitudinal walls and themiddle portion of the cytoplasm becomes progressively thinner,finally leading to the breakage. Both parts show streaming afterthe rupture. The observations suggest that there may be rela-tively strong connections between both guard cell cytoplasms ora strong membrane-cell wall attachment at the distal ends of thecells which prevents uniform plasmolysis. If the cells are plasmo-lyzed gradually the fission is seldom seen.The response of tobacco guard cells to 0.7 M mannitol solu-

tions is even more suggestive of a strong plasmalemma-cell wallattachment. Upon plasmolysis, some portions of the cytoplasmremain strongly attached to the wall while the rest of the cyto-plasm contracts markedly (Fig. 5). The strands of cytoplasmbetween the attached areas and the bulk of the contracted cellare subject to a high tension that very likely results in membranedamage. This conclusion is supported by the fact that no intactguard cell protoplasts could be obtained from peels digested inhigh osmoticum.

Birefringence in Intact Cells. Examination of guard cell wallsin polarized light (Fig. 7) reveals that the cellulose fibers fanoutward from the central pore (refs. 10 and 16; Palevitz andHepler, in preparation). That feature allowed us to make meas-urements of the amount of ordered cellulose present. In prac-tice, retardation measurements are made at two places: (a) onthe fan shaped fibers on the periclinal face of the guard cell wallmidway between the pore and the outer lateral wall (radialbirefringence), and (b) on the anticlinal face of the outer lateralwall itself (edge birefringence). Edge birefringence is observedin virtually all kinds of plant cells and depends upon the fact thatthe polarizing microscope integrates over the entire thickness ofthe cell. Thus, when a wall is observed parallel to its plane, i.e.edge on, cellulose microfibrils that may even run predominatelyalong the optical axis, if tilted slightly will have a projection in

FIG. 1. Illustration of the microchamber assembly. From left: chamber, o-ring, coverslip (25 mm in diameter) with a slice held in place withValap, chamber seal, and assembled chamber. A second coverslip is not shown.

493Plant Physiol. Vol. 58, 1976

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ZEIGER AND HEPLER

FIG. 2 AND 3. Scanning electron micrographs of a stomatal complex in an epidermal peel from an onion cotyledon. 2: top view showing the cuticleand the stomatal pore surrounded by a thick ridge. 1500 x. 3: underside view of the epidermal peel showing the pore delimited by a pair of guardcells. Note that most of the epidermal cells around the guard cells are damaged. 1900 X.

FIG. 4 AND 5. Light micrographs of a stomatal complex in an epidermal peel from a young tobacco cotyledon. 4: turgid guard cells show intactcytoplasm with numerous chloroplasts. 800 x. 5: in guard cells plasmolyzed in 0.7 M mannitol, the cytoplasm has pulled away from the wall (0). Thebulk of the cells volume is seen in both sides of the central pore. Cytoplasmic strands connect the plasmolyzed main portions to points of strongattachment on the walls. 900 x.

FIG. 6. Onion guard cells in epidermal peels abruptly plasmolyzed in 0.45 M mannitol. The bottom guard cell of each complex has undergonecytoplasmic fission, producing two separated cell fragments. 900 X.

494 Plant Physiol. Vol. 58, 1976



Plant Physiol. Vol. 58, 1976

io ., ., t

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rw -4i

r Ar "'

FIG. 7 AND 8. Radial and edge birefringence in onion guard cells seen under polarized light. 7: mature guard cells showing both radial and edgebirefringence. The four light spots in the center of the cells are produced by the radial cellulose microfibrils fanning outward from the stomatal pore.Edge birefringence is seen as four light areas on the periphery of the cells. 850 x. 8: very young onion guard cells showing edge birefringence. Thesecells have not yet acquired a significant amount of radially oriented cellulose microfibrils, hence they do not display radial birefringence. 550 x.

FIG. 9. Intact (left: 800 x) and fully digested (right: 600 x) guard cells in the onion epidermis. Only the ridge surrounding the pore remainsundigested after a 10 hr digestion with 4% (w/v) cellulysin. The particles in the background belong to the cuticle.

FIG. 10 AND 11. Fluorescence micrographs of onion epidermal peels treated with 0.1 % (w/v) Calcofluor. 200 x. 10: control peels treated withCalcofluor for 5 min. Both the epidermal and guard cell walls show the characteristic fluorescence, but the guard cells fluoresce poorly, relative totheir cellulose content. 1 1: epidermal peels digested in 4% (w/v) cellulysin for 1 hr and treated with Calcofluor for 5 min. The epidermal cell wallshave been completely digested. In contrast, the walls of the guard cells have only been partially digested and they now fluoresce intensely.

GUARD CELL PROTOPLASTS 495



496 ZEIGER AND HEPLER

the plane normal to the optical axis. Thus, in guard cells edgebirefringence indicates that the order is parallel to the plane ofthe membrane.

Fully mature guard cells on epidermal peels mounted onordinary slides show the highest values reaching 170 (Fig. 7).Younger guard cells found in progressively more basal portionsof the cotyledon have decreasing values that approach zero (Fig.8) at the stage where the radial reinforcement of the walls hadjust started. Measurements of cells on paradermal slices, or onthe thicker culture chambers give lower initial values, due toincreasing optical scattering, especially by subjacent mesophyllcells. Fully mature tobacco guard cells have retardation valuesfor radial birefringence comparable to those found in matureonion guard cells. Retardation values for edge birefringence, onthe other hand, are lower; onion guard cells in epidermal peelsmeasure only 70 A.

Loss of Birefringence after Enzymic Digestion. In onion guardcells, total loss of birefringence is obtained after digestion withboth cellulysin (Fig. 9) and driselase. Each enzyme, however,gave characteristic digestion time courses (Tables I and II).Retardation diminishes slowly with cellulysin even at the ratherhigh concentration of 4% (w/v). In addition, actual decreases inretardation are always preceded by a lag period. Driselase, onthe other hand, is five times more potent than cellulysin. Withepidermal peels, retardation values decrease at the onset of thedigestion without a lag period. Higher concentrations of drise-lase give higher relative velocities of digestion, suggesting aconcentration dependence, but the relationship is not linear.Tobacco guard cells in epidermal peels digested with 4% (w/v)

cellulysin show reductions in retardation at rates comparable tothose obtained with onion guard cells (Table III).

Staining with Calcofluor white ST, a rather specific fluorescingstain for cellulose (9), provided an independent method forfollowing the loss of cellulose with digestion time. Two unex-pected results are found. First, onion guard cells in epidermalpeels stain poorly with Calcofluor, as compared with neighbor-ing epidermal cells (Fig. 10). Staining periods of up to 12 hr didnot increase the intensity of the fluorescence. Second, there is adramatic increase in the fluorescence of guard cell walls afterexposure to 4% (w/v) cellulysin (Fig. 1 1). In the presence of theenzyme, stained walls maintained the control levels of fluores-cence for about 30 min, followed by an increase that peakedaround 90 min. The fluorescence decreases thereafter and ismostly gone at the end of the digestion.

Protoplast Release. Onion guard cell protoplasts are obtainedby digesting paradermal slices with 4% (wlv) cellulysin in 0.23 Mmannitol, pH 5. While cells treated with 0.23 M mannitol showlittle or no plasmolysis, the addition of 4% cellulysin has a clearosmotic contribution and the cells plasmolyze markedly. Theprotoplasts can be made by osmotic release or by prolongedenzymic digestion. Osmotically released protoplasts are ob-

Table I. Loss of Retardation in the Walls of Onion Guard CellsDigested with 4% (wlv) Cellulysin

Digestion Relative

Preparation time Retardation velocity

min R R/min

epidermal peel 0 122 ...40 12188 113 0.15118 92 0.70153 25 1.91

paradermal slice 0 121 ...115 121 ...155 112 0.22180 94 0.72215 79 0.43

Plant Physiol. Vol. 58, 1976

Table II. Loss of Retardation in the Walls of Onion Guard CellsDigested with Driselase

Enzyme Digestion Relative

Preparation concentration time Retardation velocity

7. (w/v) min R X/min

epidermal peel 0.5 0 131 ...13 119 1.0032 83 1.9038 63 3.3048 47 1.60

epidermal peel 1 0 16219 85 4.0034 47 2.5049 18 1.90

epidermal peel 2 0 155 ...

18 142 0.7233 58 5.6043 23 3.50

paradermal slice 2 0 124 ...30 12460 104 0.6680 61 2.1695 29 2.16

Table III. Loss of Retardation in the Walls of Tobacco Guard CellsDigested with 4% (w/v) Cellulysin

Digestion Relative

Preparation time Retardation velocity

min gi/min

epidermal peel 0 13545 135 ...80 130 0.14112 122 0.25140 110 0.43180 76 0.85202 43 1.50235 13 0.91

tained from preparations which have been digested with 4% (w/v) cellulysin for 2 to 6 hr, with the guard cells exhibiting retarda-tion levels of less than 100 A. The enzyme mixture is removedand the microchamber is perfused with 20 to 50 cc of 0.23 Mmannitol. The protoplasts resume vigorous streaming and swell,becoming tightly appressed to the walls. Upon restoration of theturgor pressure, the guard cell walls deform and acquire a morerounded shape. Protoplasts then protrude through the lateralwall opposite the pore and emerge to the outside. After release,they become spherical and continue to stream. If left in 0.23 Mmannitol, the protoplasts are stable for a few hours, but theyswell steadily and finally burst. Upon release of the protoplasts,retardation levels in the guard cell walls drop abruptly to zero.Guard cell protoplasts can also be obtained by prolonged

digestion of paradermal slices. After 6 to 10 hr, retardation is nolonger measurable and the protoplasts become spherical (Fig.13). Many remain in the vicinity of the pore, presumablytrapped in the remaining weakened matrix of the wall. Othersmove away from the pore but keep adhering to the epidermalcuticle. The wall material that in many cases is left surroundingthe pore after prolonged digestion with cellulysin is Calcofluor-negative and no longer birefringent. Thus it is most likely non-cellulosic but its precise nature remains unknown.

Besides guard cell protoplasts, digested paradermal slices alsoproduce mesophyll and epidermal cell protoplasts. The threeclasses are morphologically distinct when observed under themicroscope. Guard cell protoplasts are small (18-25 ,um indiameter) and have a dense, granular cytoplasm, whereas epi-dermal cell protoplasts are large (averaging 45 gm in diameter)and are highly vacuolated. Mesophyll cell protoplasts have largenumbers of green chloroplasts. If it is desirable to make prepara-



GUARD CELL PROTOPLASTS

FIG. 12 AND 13. Guard cell protoplasts of tobacco (Fig. 12) and onion (Fig. 13). The undigested pore ridge is seen in both preparations. Largechloroplasts and a central vacuole are prominent in the tobacco protoplasts. Onion guard cell protoplasts lack visible chloroplasts and their cytoplasmis denser and more granular. 900 x.

tions free of mesophyll and epidermal cell protoplasts, these twocell types, which form into protoplasts more quickly than guardcells, can be readily washed away at an earlier stage of digestion.An air bubble is introduced in the chamber and is displaced a fewtimes across the mounted slices, causing the dispersal of theformed protoplasts into the medium. The solution is then re-moved with a syringe and replaced with fresh enzyme. Thedigestion is then allowed to proceed until the formation of theguard cell protoplast is complete.Tobacco guard cell protoplasts are obtained from epidermal

peels torn from young cotyledons. Unfortunately, the morefavorable paradermal slices could not be made in that material.The usual plasmolyticum utilized for tobacco mesophyll proto-plast formation, 0.7 M mannitol, is harmful in the production ofguard cell protoplasts. Favorable results are obtained by collect-ing the peels in 0.23 M mannitol and then transferring them to4% (w/v) cellulysin in 0.23 M mannitol, pH 5. Protoplastsemerge within 2 to 3 hr (Fig. 12), but burst rapidly unlessstabilized with 0.7 M mannitol. Alternatively, peels can betreated with 4% (w/v) cellulysin in 0.23 M mannitol for 2 hr,followed by a 2- to 4-hr period with 4% (w/v) cellulysin in 0.7 Mmannitol. After protoplast release, the preparation is washedwith 0.7 M mannitol. Protoplasts thus obtained are stable for 24to 48 hr.

Driselase, although very effective in cellulose digestion is notuseful for protoplast production because of its toxicity, even atlow (0.5% w/v) concentrations. Guard cells showed cytoplasmicdamage within minutes after being exposed to the enzyme andstreaming was never resumed upon its removal. Charcoal-ad-sorbed driselase (4) also failed to produce healthy guard cellprotoplasts.

Protoplast Culture. Thus far attempts to culture guard cellprotoplasts have been made only with onion. Protoplasts cul-tured in microchambers in 0.3 to 0.4 M mannitol solution remainalive and actively streaming for periods up to 24 hr. Their lifespan could be extended up to 10 days, with a simple mediumcontaining a mixture of sugars and a few major salts. Protoplastsresumed streaming but did not form a detectable wall. Attemptsto use more complex media were unsuccessful. Gamborg's B5(7) is toxic, and the protoplasts died within a few minutes.

Linsmaier and Skoog (12) medium is more favorable; proto-plasts remained alive for 3 to 4 days, and on two instances therehave been indications of cell wall formation, which however didnot prevent the subsequent death of the cells.

DISCUSSION

Our results show that protoplasts of onion and tobacco guardcells can be made and that they retain their viability, especially inthe case of onion, for several days. The cytoplasm of guard cellsappears to be more tightly adhered to the cell wall than othercells which have been traditionally used as a source of proto-plasts. Thus, when guard cells are strongly plasmolyzed, theplasmalemma develops lesions leading to cell death. This isparticularly the case with tobacco and might explain previouslyreported failures in obtaining guard cell protoplasts from thatspecies (3, 14). Gentle plasmolysis allows a more gradual disso-ciation of the plasmalemma-cell wall association and has provencrucial for the production of viable guard cell protoplasts.Microchambers have assisted us greatly in isolating guard cell

protoplasts by allowing continuous in situ observations of thecourse of the digestion, of the release of the protoplasts, and oftheir subsequent response to culturing. On the other hand, onlya small number of cells (100-200/slice) can be handled in themicrochamber and that limitation prevents the design of manyexperiments where large quantities of protoplasts are needed.This technique therefore complements, but by no means re-places, the large scale isolation of guard cell protoplasts.The measurement of the loss of retardation of the cell wall

seen under polarized light permits us to quantify the course ofthe digestion. The method opens the way for a better under-standing of the response of the cell wall to the digestion andoptimal digestion times. It also provides a convenient standardassay to test different cellulolytic enzymes. In undisturbed cells,the progressive loss of retardation implies the degradation of theanisotropic cellulose microfibril. On the other hand, the fast,sharp decreases observed after the osmotic release of the proto-plasts can best be explained by a sudden loss of order in theweakened wall. Loss of retardation can be monitored withoutany disturbance to the cells and in that regard, this technique is

497Plant Physiol. Vol. 58, 1976



498 ZEIGER Al

more favorable than Calcofluor staining. The latter has theadditional disadvantage of an increase of intensity preceding theactual decline in fluorescence.The comparison of the time courses of loss of retardation using

cellulysin and driselase shows that the latter can digest the cellwall about five times faster than the former. In addition, drise-lase-treated preparations fail to show any lag period beforeactual loss of retardation, possibly reflecting a higher activity ofnoncellulolytic components, which digest elements of the matrixand thus expose the cellulose microfibrils to the cellulolyticenzymes. On the other hand driselase is toxic to the guard cellprotoplasts and for that reason, in experiments where healthyprotoplasts were desired, cellulysin has been exclusively used.There have been several reports where driselase was successfullyused to isolate viable protoplasts (2, 8, 1 1). so it is unclear if thetoxicity that we observe reflects a characteristic sensitivity of theguard cell protoplasts or is due to a particularly toxic batch ofenzyme, since great variability has been observed between dif-ferent batches of driselase (Eriksson, personal communication).

It is apparent to us that the availability of protoplasts fromguard cells and stomatal cell initials will allow more direct ap-proaches to questions in stomatal development and function.Since they are freed from neighboring tissues, culture experi-ments should reveal more clearly their specific capabilities todivide and redifferentiate. Protoplasts should also be very usefulto investigate the physiological properties of the guard cells atthe cellular level. Initial experiments have already proven thatthe onion guard cell protoplasts retain their ability to respond tolight (Zeiger and Hepler, in preparation). It is most likely thatother physiological responses such as their sensitivity to abscisicacid or the nature of their ion exchange could also be effectivelystudied using guard cell protoplasts.

N]D HEPLER Plant Physiol. Vol. 58, 1976

Acknowledgment- We thank N. Burstein for making the scanning micrographs.

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