Plant Science Letters, 32 (1983) 1--8 1 Elsevier Scientific Publishers Ireland Ltd.

PREPARATION OF ENZYMATICALLY CLEANED LEAF EPIDERMAL STRIPS OF NICOTIANA GLA UCA

STEPHEN HUDSON, JEFF TRAIL, DWAYNE SIMMONS, LARRY BALDWIN, REBECCA MYERS, BARRY TOM, JUDY MOHR and GARY TALLMAN*

Natural Science Division, Pepperdine University, Malibu, CA 90265 (U.S.A.)

(Received March 18th, 1983) (Revision received May 2rid 1983) (Accepted May 4th, 1983)

SUMMARY

A technique for enzymatically cleaning leaf epidermal strips of Nicotiana glauca so that guard cells remain the only intact and viable cells is described. The method relies on differential sensitivities of epidermal, mesophyll, and guard cell walls to digestion with a mixture of Cellulysin and hemicellulase. Guard cells isolated by this procedure remain joined at their ends as duplexes, retain continuous cell walls, and are free of epidermal and mesophyll cell wall fragments and cytoplasmic debris. Yields range from 90--95% of original cells. All recovered cells concentrate neutral red and exclude trypan blue. In addition, duplexes isolated from either adaxial or abaxial surfaces show significant increases in width anct~perture over dark controls when illumi- nated in solutions containing 30 mM KC1. Yields approach those necessary for standard methods of biochemical analysis. A brief comparison with existing techniques for guard cell isolation is included.

Key words: Guard cells - Stomata -- Isolation

INTRODUCTION

A number of techniques exist for the study of stomatal physiology and metabolism at the cellular level including the use of epidermal strips [ 1 ], 'rolled' epidermal strips [ 2], epidermal strips treated at low pH[ 3], sonicated epidermal strips [4], freeze-dried dissected guard cell duplexes [5], and guard cell protoplasts [6]. While each of these is suitable for certain types of

*To whom reprint requests should be sent. Abbreviations: MMS, modified salts of Murashige and Skoog medium.

0304-4211/83/$03.00 © 1983 Elsevier Scientific Publishers Ireland Ltd. Printed and Published in Ireland

analysis, none provides guard cells with continuous cell walls joined at the ends (guard cell duplexes) free of epidermal and mesophyll contamination in quantities sufficient for many standard methods of biochemical analysis.

We report a rapid method for the isolation of guard cell duplexes from leaf epidermis of N. glauca which relies on the differential sensitivities of epidermal, mesophyll, and guard cell walls to digestion with Cellulysin and hemicellulase. The method yields duplexes free of epidermal or mesophyll cell contamination. Quantities recovered approach those necessary for standard methods of biochemical analysis. We demonstrate that cells isolated by this method are viable and capable of transporting potassium to produce turgot when illuminated.

MATERIALS AND METHODS

Materials Chemicals and enzymes were purchased from Sigma Chemical Co., Inc.,

St. Louis, MO with the exception of Cellulysin purchased from Calbiochem Inc., La Jolla, CA.

Isolation of guard cell duplexes Epidermal strips were removed by a technique similar to that of Weyers

and Travis [7] from either adaxial or abaxial surfaces of healthy leaves ( 6 - 8 × 10--12 cm) taken from N. glauca plants growing on the Pepperdine University campus at Malibu, CA. Strips were peeled into Murashige and Skoog salts modified by replacing potassium salts with sodium salts at pH 6.5 (MMS) [8]. Major veins were removed with a razor blade and the strips were cut into sections of approx. 1 cm 2 in MMS on a non-stick surface. Peels were digested by incubation at 20--22°C for 2.5--5 h in a Dubnoff shaking water bath at 150 excursions/min in 20 ml of a solution containing 20 mg/ml Cellulysin, 20 mg/ml hemicellulase, 10 mg/ml polyvinylpyrrolidone 40 in MMS at pH 5.5. Incubation was carried out in a 250-ml beaker and all solutions were filtered through a 0.45 ~m Nalgene filter. Following incu- bation peels were transferred with forceps to 20 ml of MMS at pH 6.5 and washed by gentle vortexing. After 2 identical washes, peels were transferred to the same solution in preparation for viability analysis.

Analysis of contamination and viability Guard cell duplexes were photographed using an Olympus Vanox micro-

scope on Kodak panatomic-X film. Dark-field fluorescence microscopy (Zeiss microscope, LP 520 barrier filter, BG 12 exciting filter, and 200W Hg-vapor lamp) was used to detect contaminatidn with cell wall fragments or mesophyll chloroplasts. In this system cell wall fragments showed green refraction and mesophyll chloroplasts red fluorescence.

In some experiments, cleaned strips were treated with zinc chlor-iodide, phloroglucinol, and Sudan IV [9] in order to assess the composition of the material in which duplexes remained embedded.

Thirty ceils on 3 separate strips with 3 replicates were visually scored for intactness, ability to concentrate neutral red [ 9 ], and ability to exclude t rypan blue [10].

Responses to light and dark treatment in 30 mM KCI Strips cleaned by incubation for 2.5--3 h were used for analysis of guard

cell responses to light and dark treatment. Cleaned strips were transferred to MMS containing 30 mM KC1 at pH 6.8 and incubated for 2 h at ambient temperature in darkness or in light provided by a General Electric 600W Quartzline lamp at a photosynthet ic pho ton flux density of 1700 pmol m -2 sec -1 filtered through water. Photosynthet ical ly active radiation was measured with a Li-Cor LI-185 quantum/radiometer /photometer equipped with an LI-190S quantum sensor. Lengths, widths, and pore apertures of 20 duplexes on 2 separate cleaned strips from 3 separate leaves were measured under a green interference filter at the beginning and end of the light and dark incubation periods. Identical measurements were made on uncleaned, untreated strips. Measurements were performed using an American Optical microscope equipped with a TV camera and moni tor calibrated with a stage micrometer. All dimensions were measured at the sharpest focus in the depth of field when the cuticle of the cleaned strip faced the objective. Lengths were measured from end wall to end wall, widths as the widest points from the dorsal wall of 1 guard cell to the dorsal wall of the other, and apertures as the widest point from the ventral wail of 1 cell to the ventral wall of the opposing cell.

Strips from some experiments were stained for potassium with sodium cobaltnitri te stain as described by Allaway and Hsiao [2]. Stomatal densities were estimated by flattening strips from the adaxial or abaxial surfaces of ten leaves under the coverslip of a hemacytometer grid.

RESULTS AND DISCUSSION

Widths (means and 99% confidence intervals) o f closed duplexes on uncleaned strips (Fig. 1A) averaged 23.8 -+ 0.66 ~m for the adaxial surface compared to 24.8 -+ 0.74 pm for the abaxial surface. Lengths averaged 33.8 + 0.70 ~zm and 34.2 -+ 0.96 pm for adaxial and abaxial surfaces respectively. Stomatal densities averaged 1.24 × 104/cm 2 for the abaxial surface and 1.11 X 104/cm 2 for the adaxial surface.

After 2.5--3 h of digestion guard cells were the only remaining intact cells in epidermal strips (Fig. 1B). Epidermal ceils plasmolyzed during digestion and the resulting protoplasts were dest royed by agitation.' The remaining cytoplasmic debris washed into the incubation medium. Fluorescence microscopy revealed strips to be free of mesophyil chloroplasts (not shown) and guard cell chloroplasts showed red fluorescence. Strips cleaned for 2.5--3 h are probably more appropriate for physiological than biochemical analyses, since epidermal cytoplasmic contaminants not detectable by microscopic examination could be harbored in the remnants of epidermal

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cell walls. Responses of guard cell duplexes on 2.5--3 h strips to light and potassium chloride are summarized in Table I. In solutions containing 30 mM KC1, illuminated adaxial and abaxial guard cell duplexes increased signi- ficantly in width and aperture over dark controls, while most changes in length were marginally significant (Fig. 2A and B). Maximum widths and apertures achieved were significantly different for adaxial and abaxial duplexes so treated. Differences in responses of stomata from adaxial and abaxial surfaces have been reported [11]. Guard cells did develop some turgot in potassium-containing solutions in the dark reflected as increases in widths, lengths, and apertures of duplexes on both surfaces. Maximum apertures achieved in the dark were not significantly different for adaxial and abaxial surfaces. In some experiments, strips were transferred to dark- ness after 2 h of illumination in potassium-containing media. In such experi- ments apertures returned to those of the dark controls after 2 h of dark incubation, suggesting that, duplexes were capable of controlling turgor and tha t maintenance of maximum turgor was light-dependent. Cells treated with light in potassium-containing salts contained more potassium on the average at the end of the illumination period than dark controls as judged by sodium cobaltnitrite staining (Fig. 2C and D).

After 5 h of incubation remnants of epidermal cell walls were completely digested (Fig. 1C) but guard cell walls remained continuous and cell or- ganelles appeared intact in the light microscope. The material to which guard cell duplexes remained attached was judged to be primarily cutin after staining with zinc chlor-iodide, phloroglucinol, and Sudan IV. Between 90 and 95% of duplexes in cleaned strips concentrated neutral red and excluded trypan blue. Damaged cells were mainly confined to edges of strips penetrated

T A B L E I

C H A N G E S IN N I C O T I A N A G L A U C A G U A R D C E L L D U P L E X D I M E N S I O N S IN T H E L I G H T A N D D A R K A F T E R I N C U B A T I O N F O R 2 H IN P O T A S S I U M - F R E E S A L T S O F M U R A S H I G E A N D

S K O O G M E D I U M A N D T H E S A M E S A L T S C O N T A I N I N G 3 0 m M KC1.

Va lues a re m e a n s a n d s t a n d a r d e r r o r s f o r 1 2 0 d u p l e x e s ( 2 0 m e a s u r e m e n t s o n 2 s t r ips f r o m e a c h o f 3

s e p a r a t e leaves) .

S a m p l e W i d t h (~tm)

---KCI 3 0 m M KC1

L e n g t h (~m) A p e r t u r e (~zm)

- - K C I 3 0 m M KCI - - K C I 3 0 m M KCI

A b a x i a l D a r k 3 3 . 9 ( 0 . 2 3 ) 3 5 . 7 ( 0 . 3 3 ) b 3 2 . 4 ( 0 . 2 1 ) 3 1 . 5 ( 0 . 2 0 ) b 5 . 4 ( 0 . 1 6 ) 7 . 0 ( 0 . 2 4 ) b

L i g h t 3 3 . 0 ( 0 . 2 4 ) a 4 1 . 3 ( 0 . 3 4 ) a ' b 3 1 . 5 ( 0 . 2 3 ) a 3 2 . 4 ( 0 . 2 3 ) a ' b 5 . 1 ( 0 . 1 6 ) 1 2 . 7 ( 0 . 2 9 ) a ' b

Adax ia! D a r k 3 4 . 3 ( 0 . 2 1 ) 3 6 . 5 ( 0 . 3 9 ) b . 3 3 . 6 ( 0 . 2 3 ) c 3 2 . 7 ( 0 . 2 5 ) b ' c 4 . 3 ( 0 . 1 3 ) c 6 . 8 ( 0 . 1 7 ) b , L i g h t 3 3 . 8 ( 0 . 2 1 ) 3 8 . 2 ( 0 . 2 5 ) a ' ° ' c 3 3 . 4 ( 0 . 1 9 ) c 3 3 . 0 ( 0 . 2 9 ) 4 . 5 ( 0 . 1 2 ) c 8 . 7 ( 0 . 1 3 ) a ' ° ' c

a s i g n i f i c a n t a t 0 . 0 1 level when compared to d a r k c o n t r o l u s ing t - r a t io b s i g n i f i c a n t a t 0 . 01 level when compared to p o t a s s i u m - f r e e c o n t r o l u s ing t - r a t io Csignificant a t 0 .01 level when compared t o a b a x i a l e q u i v a l e n t us ing t - ra t io

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by the razor blade during trimming. It is significant that strips cleaned for 5 h were free of epidermal and mesophyU cell wall and membrane fragments that might interfere with subsequent biochemical measurement. Outlaw [ 12 ] has estimated that as little as 3--5% contaminat ion of Vi.cia guard cell pro- toplast preparations with mesophyll protoplasts can produce a mixture in which as much as 40--50% of chlorophyll is contr ibuted by the contami- hating cells. We calculate that 100 cm 2 of epidermis from the abaxial surface of a Nicotiana leaf would yield 2.4 × 106 cells. Because of ease of removal o f the epidermis, such amounts are readily obtainable.

Unlike protoplasting procedures that require 8--16 h [6] , the method is rapid and minimizes cell exposure to cellulolytic enzymes as well as phenols and flavanoids released from digested epidermal cells. The procedure also avoids potential ly harmful physical methods like 'rolling' [2] , and sonication [4] as well as extremes of pH [3] and temperature. The latter may be of particular importance, since recent studies suggest that temperatures com- monly used to produce guard cell protoplasts (28--30°C) may damage chloroplasts [13 ]. 'In contrast to intact peels, 'rolled' strips, and peels treated at low pH, adhering epidermal cells and their contents are washed away from the duplexes during incubation.

We have successfully used this method, with slight modifications, for the isolation of guard cell duplexes from Pelargonium. We prefer Nicotiana, however, since trichomes on Pelargonium are killed but no t removed by this technique. We suggest that strips.cleaned for 5 h may be particularly useful for studies of guard cell enzymology [14], chloroplast fluorescence [15], and oxygen exchange [13] that require preparations of high yield and viability with no contamination.

ACKNOWLEDGEMENTS

We thank Stephen Davis, Eduardo Zeiger, Ken Perrin and the staff of the Natural Science Division for their helpful comments and technical assistance.

REFERENCES

1 C.K. PaUaghy, Planta, 101 (1971) 287. 2 W.G. Allaway and T.C. Hsiao, Aust. J. Biol. Sci., 26 (1973) 309. 3 G.R. Squire and T.A. Mansfield, New Phytol., 71 (1972) 1033. 4 T. Ogawa, D. Grantz, J. Boyer and Govindjee, Plant Physiol., 69 (1982) 1140. 5 W.H. Outlaw, Jr. and O.H. Lowry, Proc. Natl. Acad. Sci., U.S.A., 74 (1977) 4434. 6 E. Zeiger and P.K. Hepler, Plant Physiol., 58 {1976) 492. 7 J.D.B. Weyers and A.J. Travis, J. Exp. Bot., 32 (1981) 837. 8 T. Murashige and F. Skoog, Physiol. Plant., 15 (1962) 473. 9 D.A. Johansen, Plant Microtechnique, McGraw-Hill, New York, 1940.

10 H.J. Phillips, Dye exclusion tests for cell viability, in P. Kruse, Jr. and M. Patterson, Jr. (Eds.), Tissue Culture: Methods and Applications, Academic Press, New York, 1973, p. 406.

11 M.A. Pemadasa, J. Exp. Bot., 33 (1982) 92. 12 W.H. Outlaw, Jr., B.C. Mayne, V.E. Zenger and J. Manchester, Plant Physiol., 67

(1981) 12. 13 K. Shimazaki, K. Gotow and N. Kondo, Plant Cell Physiol., 23 (1982) 871. 14 H. Schnabl, C.H. Bornman and H. Ziegler, Planta, 143 (1978) 33. 15 E. Zeiger, P. Atmond and A. Melis, Plant Physiol., 67 (1980) 17.