Planta 9 by Springer-Verlag 1978

Planta 143, 33-39 (1978)

Studies on Isolated Starch-containing (Vicia faba) and Starch-deficient (Allium cepa) Guard Cell Protoplasts H. Schnabl, Chris H. Bornman, and H. Ziegler Institut ffir Botanikund Mikrobiologieder TechnischenUniversitfitM~nchen, Arcisstral3e21, D-8000 M/inchen2, Federal Republic of Germany

Guard cell protoplasts from starch-containing Vicia faba and starch-deficient Alliurn cepa stomata were isolated, stabilized and recovered with an efficiency-in relation to the potential y i e l d - o f approx. 62% and 77%, respectively. In vitro, guard cell protoplasts (GCP) respond to abscisic acid and fusicoccin by respectively contracting and swelling, that is, decreasing or increasing in diameter by about 15% and more in comparison to the control. This in vitro response correlates with, but is more than 4 times as rapid as, the in vivo response of the stomata. Among the advantages presented by working with isolated GCPs are: greater sensitivity in response; freedom from influences of cuticular ridges, cell walls, subsidiary cells, and epidermal cells; and direct and parallel comparisons of starch-containing and starch-deficient GCP systems. Abstract.

Abscisic acid - A l l i u m cell protoplasts - Vicia.

Key words: Guard

-

Fusicoccin

-

Introduction

Numerous reports and several reviews on the isolation and culture of plant and fungal cell protplasts have appeared in recent years (e.g., Cocking, 1972; Courts, 1974; Uchimiya and Murashige, 1974; Raveh and Galun, 1975; Vasil, 1976; Zelcer and Galun, 1976; Gamborg, 1976; Bajaj, 1977; Kinnersley et al., 1978). In the majority of cases, protoplasts were isolated from leaf mesophyll. However, in 1976 Zeiger and Hepler isolated and maintained in culture for 10 days protoplasts from a highly specialized type of cell, Abbreviations." ABA=abscisic acid; FC=fusicoccin; ECP, MCP, and GCP=epidermal, mesophyll, and guard cell protoplasts, respectively; PPV=packed protoplast volume

namely, the stomatal guard cells of Allium and Nicoliana. Accordingly, there is a paucity of information on the physiologic responses of guard cell protoplasts. Zeiger et al. (1977a, b) and Moody and Zeiger (1978), demonstrating a negative membrane potential difference in the guard cells of Alliurn, proposed that ion uptake during stomatal opening is mediated by a proton-motive force that drives a passive K + influx across the guard cell membrane. In this paper we report firstly on the differential separation of epidermal, mesophyllous, and guard cell protoplasts from starch-containing Vicia and starchdeficient Alliurn and secondly, compare the responses of isolated guard cell protoplasts with those of intact stomata to abscisic acid and the fungal toxin, fusicoccin. ABA and FC were selected because of their respective effects on stomatal closure and inhibition of K § uptake in guard cells (Mittelheuser and Van Steveninck, 1969, 1971; Horton, 1970; Squire and Mansfield, 1972), and on stomatal opening and correlated K § accumulation in guard cells (Turner and Graniti, 1969; Turner, 1973). Rollo etal. (1977) reported that FC promotes inter alia proton extrusion and K § uptake by isolated mesophyll protoplasts in Nicotiana and Spinacia. We want to show that isolated GCPs present a simple and effective system from which extrapolations of stomatal behavior might be made. Since the respective GCPs of Vicia and Alliurn are starch containing and starch deficient (Fujino and Jinno, 1972; Allaway and Setterfield, 1972; Schnabl and Ziegler, 1977), a unique model is further available by which those ion uptake and H § extrusion phenomena that play a regulatory role in stomatal opening and closure, can be studied in parallel situations. A question raised was whether opening and closure of stomata could be directly equated with measurable physiologic responses such as, for example, swelling and shrinking of isolated GCPs. 0032-0935/78/0143/0033/$01.40

34

Materials and Methods

H. Schnabl et al. : Studies on G u a r d Cell Protoplasts Table 1. Flow chart summarizing the isolation procedure of Allium and Vicia guard cell protoplasts

Bulbs of Allium cepa L. (cv. Stuttgarter Riesen) and seeds of Vicia faba (Lagomarsino Seeds, Inc. Sacramento, California) were germinated and grown in peat moss in a growth chamber at 22_+ 2 ~ C (day) and 1 8 + 2 ~ (night) with a 12-h photoperiod (15,000 lx). Allium was watered twice and Vicia thrice weekly with distilled water,

Incubate at 30~ intact brushed, epidermal peels in:

Allium

Vicia

4% cellulysin in 0.25 M mannitol 30 min

4% cellulysin in 0.25 M mannitol, 30 rain

II Epidermal Peels The upper 20 30 m m of the Allium leaves (pilot work had shown the opening and closing responses of the stomata to decrease progressively from apex to base) and the 2nd to 4th fully expanded leaves of 3-week-old Vicia plants, were cut before c o m m e n c e m e n t of the light period and placed in distilled water. Strips of epidermis (abaxial in Vicia) were peeled, floated on either distilled water or 0.25 M mannitol, and brushed gently with a dissecting needle on the innerside to dislodge most of the mesophyll parenchyma. The strips were then cut into approx. 10 x 5 m m pieces with microscissors and incubated from 15-180 min in a CO2-free air stream on either 10 - 4 M FC (in 10 - 2 M KCI; Raschke, 1977) or 10 - s M A B A ; temperature was 22_+1~ and light intensity 15,000Ix. Stomatal aperture was measured with an eyepiece micrometer. F o u r strips were used per treatment and ten stomata per strip were measured. Experiments were repeated three times.

Isolation of Guard Cell Protoplasts ( GCP) To emphasize differences between Allium and Vicia in the molarity of the osmotic stabilizer during incubation as well as during other procedures, the isolation procedure, based on the experience of 37 experiments, is summarized in Table 1. The cell wall was lysed with a 4% (wt/vol) solution of cellulysin (Calbiochem) in various molar concentrations of mannitol; to stabilize G C P membranes, the solutions in the final stages were made I m M with respect to CaC1 z. The pH of all solutions was 5.0. Incubation was carried out on a gyratory shaker in a water bath at 30 ~ C. Packed protoplast volumes and G C P counts were determined as follows: F r o m 50 (Allium) to 80 (Vicia) 1 0 x 5 m m pieces of stripped and brushed epidermal peels were used per 5 ml incubation m e d i u m for a final yield of 2.4-3.0 x 10 s stomata. Washed pellets of isolated and stabilized GCPs were resuspended in 5 ml 0.7 M mannitol in 1 m M CaC12 (Allium) or 0.5 M mannitol in 0,1 m M CaC12 (Vicia), transferred to hematocrit tubes, and centrifuged at 120 g for 10 min. The PPV was measured in the capillary tube. Cell n u m b e r was determined by resuspending the protoplasts in the hematocrit tube with the aid of a fine-tipped Hamilton syringe and by vortex mixing. An aliquot was removed and diluted so that 1 ml of suspension would contain approx. 0.1 m m a of packed protoplasts. Duplicate counts of intact GCPs were made in 0.1 m m 3 suspension at a magnification of 440 x using a Levy cell-counting chamber. Average PPV and n u m b e r of cells were determined from three separate experiments with Allium and five in the case of Vieia, representing a total of 7400 and 20,700 m m 2 of epidermal peel, respectively. The presence of cell walls and cell wall debris was qualitatively determined by staining with H 2 S O 4 - Z n C 1 2 (Jensen, 1962).

Incubation of Isolated Guard Cell Protoplasts Isolated and washed Allium and Vicia GCPs were incubated over a period of 30 rain in solutions of 0.7 M and 0.5 M mannitol, respectively. These solutions contained either 1 0 - 4 M FC (in

4% cellulysin in 0.4 M mannitol, 30 min

I1 wash (1 x ) with 0.4 M mannitol, centrifuge at 120 x g, 7 min

wash (1 x ) with 0.4 M mannitol, centrifuge at 120 x g, 7 rain

wash (1 x ) with 0.4 M mannitol

wash (1 x ) with 0.4 M mannitol

4% cellulysin in 0.7 M mannitol plus l m M CaC12 2 h ; centrifuge 100 x g , 5rain

4% cellulysin in 0.4 M mannitol plus 1raM CaClz 3 h; centrifuge 100 x g , 5min

wash (2 x ) with 0.7 M mannitol i 1 m M CaClz, centrifuge at 100 x g, 5 min

wash (2 x ) with 0.5 M mannitol in I 1 m M CaC12, I centrifuge at 100 x g , 5rain

Discard pellet containing burst ECPs, MCPs, and cell wall debris W a s h supernatant containing epidermal peels with intact stomata

Discard supernatant containing empty epidermal < peels and wall fragments of guard cells W a s h pellet consisting of GCPs Discard supernatant containing washed-out enzyme':

n]

I I

Take pellet up in:

0.7 M mannitol in 1 m M CaC12

[•

0.5 M mannitol in] 1 m M CaC12

I

Isolated, stabilized guard cell protoplasts /

10 - 2 M KCI) or 10 5 M A B A and were 1 m M with respect to CaC12. Incubation was at 2 2 ~ and in the presence of 15,000 lx. Mannitol only served as control. Diameters of fully stabilized GCPs were measured at intervals of 5 and 10 min with an eyepiece micrometer at a magnification of 480 x under phase contrast. The

H. Schnabl et al. : Studies on G u a r d Cell Protoplasts diameters of ten protoplasts were measured in each case and the experiments were repeated three times.

35 Table 2. N u m b e r s of guard cells and guard cell protoplasts, range and mean diameter, volume, packed protoplast volume, ratio of intact to damaged GCPs, and efficiency of isolation procedure

Results

Table 2 is a summary of pertinent data concerning the size and number of GCPs as well as the efficiency of isolation, while Figures 1-10 illustrate features of the epidermis, stomata, and GCPs of Allium and Vicia. Figures 11 and 12 are time-course responses of in vivo guard cells and in vitro GCPs to ABA and FC, respectively. I. Isolation Procedure

An optimal incubation time of 30 rain (Vicia) and 60 rain (Allium) is required for the lysis of epidermal and mesophyll cell walls. In the case of Allium it is essential that after 30 rain the osmotic concentration of the enzyme solution be increased from 0.25 to 0.4 M in order to prevent subsequent damage to the GCPs. Following digestion of their walls, the protoplasts of the epidermal cells and those of the few remaining mesophyll cells that escaped the brushing procedure are mostly broken or burst, and are removed in the first centrifugation. Except for a few epidermal cells at the most, the peel with its intact stomata is clean (Fig. 1, Allium) and remains suspended in the supernatant during centrifugation. Those ECPs that remain attached to the washed epidermal peels following the first incubation, are usually broken. In Allium, surviving MCPs are recognized by their numerous chloroplasts and ECPs by their enormous longitudinal dimensions (Fig. 2), ranging up to ca. 170 gin, their larger nuclei, and apparent inability to stabilize. In Vicia the epidermal peel strips cleanly, leaving very few mesophyll cells attached; and the epidermal cells are digested more quickly and completely than is the case in Alliurn. GCPs of Allium begin to extrude after ca. 1.5 h in the fresh 0.7 M digesting medium. Figure 3 shows a dumbbell-shaped GCP shortly before extrusion. After 2 h the remains of the digested stomata are shown on the washed peels (Fig. 4) only by their cuticularized edges (Fig. 3 and 4). The guard cell walls of Vicia are much more tenacious that those of Allium and require enzymatic lysis of at least 3 h duration. Protoplasts may sometimes be found clumped (Fig. 5) in groups of up to hundreds, but we take this as an indication that their walls may not be completely digested. GCPs are usually released in a kidney-shaped form (Fig. 6), although, with the relatively high molarity of 0.7 M in the A llium procedure, many stabilize within the guard cells. When stabiliz-

Allium

Vicia 120

a) No. of G C P r a m - 2

240

b) Range and mean diameter of GCPs, ~tm

11.5 15.5 19 13-16.9-20.5

c) Average volume of GCPs, gm 3

1950

2525

d) Potential yield of GCPs from epidermal areas averaging 2465 m m 2 (Allium) and 4136 m m 2 (Vicia)

5.916 x 105

4.963 • l0 s

e) PPV of GCPs based on same areas as in (d), g m 3

9.83 x l0 s

9.90 x 108

f) No. of GCPs determined from PPV, (e)/(c)

5.04 x 105

3.92 x 105

g) Actual n u m b e r of intact GCPs counted (Levy cell chamber)

3.68 x l0 s

3.81 x 105

h) Efficiency of isolation procedure, % (g)/(d) • 100

62

77

i) Ratio of intact to damaged GCPs - epidermal peels floated on water during pretreatment

2.5 : 1

2.8 : l

- e p i d e r m a l peels floated on 0.25 M mannitol during pretreatment

2.7:l

3.l:1

ing, the GCPs go through an intermediate spherical to oblong stage (Fig. 7). Depending upon conditions, the stabilized GCPs of both Allium (Fig. 8) and Vicia (Fig. 9) have diameters that vary from 11.5 to 20.5 lain, despite the nearly twice greater dimension of the latter's guard cells. When the molarity of the medium in which the GCPs are finally suspended, is gradually reduced, it is possible to observe the coalescence and extrusion of the GCP-vacuoles from the burst and broken protoplasts (Fig. 10). Nearly doubling their volumes, some of these vacuoles attain diameters of up to 30 ~tm (as compared with 3 5 lain in the case of small vacuoles in in vivo guard cells), demonstrating the resilience of the tonoplast in comparison to that of the plasmalemma. Vicia GCPs are easily distinguished from those of Allium by the presence in them of welldeveloped chloroplasts (ca. 10 per protoplast). Allium GCPs contain no starch grains.

2. Potential and Actual Harvest of GCPs Allium and Vicia have approx. 120 and 60 stomata mm-2, respectively. Careful counts showed that the

36

H. Schnabl et al. : Studies on G u a r d Cell Protoplasts

H. Schnabl et al.: Studies on Guard Cell Protoplasts

ratio of intact to damaged GCPs in our isolation procedure was ca. 2.5:1 (Allium) to 2.8:1 (Vicia). However, if instead of on distilled water the epidermal peels are floated on 0.25 M mannitol during the sometimes lengthy preparatory stages, these ratios can be somewhat improved upon (Table 2). A 5-ml incubation sample containing, say 50 epidermal strips with a total area of 2500 mm 2, should therefore potentially yield 2.25 x 105 intact Vicia GCPs. Based on actual cell counts, the efficiency of our isolation procedure was ca. 62% for Allium and 77% for Vicia, and there is no doubt that with more refined techniques it can be improved. Based on PPV [compare (d) and (f) in Table 2], the loss of GCPs as a percentage of the potential yield is ca. 15% and 21% with regard to AlIiurn and Vicia, respectively. However, this exceptionally low loss in the case of Allium finds reflection in a higher PPV and is no doubt due to the inclusion of cell wall debris.

37

Vicia faba Alliurn cepa

?-

cI

-

O ,~

( 3

Studies on isolated starch-containing (Vicia faba) and starch-deficient (Allium cepa) guard cell protoplasts.

Guard cell protoplasts from starch-containing Vicia faba and starch-deficient Allium cepa stomata were isolated, stabilized and recovered with an effi...
1MB Sizes 0 Downloads 0 Views