Cytoskeletal inhibitors suppress the stomatal opening of
Vicia faba
L. induced by fusicoccin and IAA
Rong Feng Huang
a,c, Xue Chen Wang
b,c,*, Cheng Hou Lou
b,caBiotechnology Research Center,Chinese Academy of Agricultural Sciences,Beijing100081, People’s Republic of China bCollege of Biological Sciences,China Agricultural Uni6ersity,Beijing100094, People’s Republic of China cLaboratory of Plant Physiology and Biochemistry,Ministry of Agriculture,Beijing 100094, People’s Republic of China
Received 9 September 1999; received in revised form 22 November 1999; accepted 23 February 2000
Abstract
Stomatal movement is governed by osmotic potential, in which K+concentration plays the dominant role. Our previous work
has shown that both microtubules and microfilaments are involved in regulating stomatal movement. In the present investigation the relationships between cytoskeletal components and K+fluxes in stomatal opening were addressed by using fusicoccin (FC),
indoleacetic acid (IAA), and cytoskeletal inhibitors to treat both epidermal strips and protoplasts of guard cells. The results revealed that FC and IAA induced stomatal opening with or without KCl in the dark. Also FC or IAA induced guard cell protoplast swelling in the dark even without added KCl. However, the induction was partially suppressed when strips and protoplasts were pretreated with cytochalasin B (CB), an inhibitor of F-actin polymerization, or oryzalin, an inhibitor of plant microtubule polymerization. Thus our preliminary results indicate for the first time that microtubules and microfilaments can affect stomatal opening independently of K+fluxes. © 2000 Published by Elsevier Science Ireland Ltd. All rights reserved.
Keywords:Microtubules; Microfilaments; Fusicoccin (FC); Indoleacetic acid (IAA); Stomatal opening
www.elsevier.com/locate/plantsci
1. Introduction
Various environmental and internal signals, working through different signal transduction path-ways, control the process of stomatal movement. Changes of turgor pressure, caused by the osmotic potential of guard cells, trigger stomatal movement, and a central role of K+in stomatal movements is
undisputed in the current literature [1]. Stomatal aperture depends on the turgor differences between guard cells and the surrounding subsidiary or epi-dermal cells, as well as on the mechanical properties of the cell walls of guard cells. However, there is recent evidence indicating that microfilaments [2,3] and microtubules [4,5] are involved in both stomatal opening and closing. For example, the effects of actin antagonists on stomatal opening parallel their
effects on hyperpolarization-dependent inward K+
current, suggesting that activities of inward K+
rectified channels in guard cells are regulated by structural changes in actin filament [6].
Though K+has a central role in stomatal
open-ing, other factors must also be important. For instance, when there is no K+ in the incubation
medium the stomatal aperture can increase in re-sponse to blue light, without any K+accumulation
or starch loss [7]. And for this reason there has been discussion of the role of the dynamics of cortical microtubules and microfilaments in stomatal ment [2,8,9]. Thus, to understand stomatal move-ment it is very important to investigate the relationships between cytoskeletal components and K+fluxes in stomatal opening. In the present study
this point was addressed by using fusicoccin (FC) and indoleacetic acid (IAA) separately in order to induce stomatal movement [10 – 14] and proto-plast swelling in combinations with
cy-* Corresponding author: Tel.: +86-01-62892706 fax: + 86-01-62893450.
E-mail address:[email protected] (X.C. Wang)
toskeletal inhibitors to treat both epidermal strips and guard cell protoplasts.
2. Materials and methods
2.1. Plant materials
Plants of Vicia faba L. were grown in a green-house with 13 – 16 h of natural light per day at temperatures of 20 – 25°C. Plants were kept free from water stress at all stages of development. The two youngest fully expanded leaves were used from plants 3 – 4-weeks-old. Epidermal peels were in MAC buffer containing 10 mM 2-[N -mor-pholino] ethanesulfonic acid (Mes)-tris-(hydrox-ymethyl) aminomethane (Tris), pH 6.1, 1 mM AlCl3 and 0.1 mM CaCl2. And all experiments
were conducted in the dark at a temperature of 2592°C.
2.2. Isolation of guard cell protoplasts
Eight to ten leaflets were put in cold distilled water. The midveins and veins were removed using a razor blade, and the leaves were cut into small pieces. Then the leaf pieces were placed in 100 ml MBMC buffer (10 mM Mes-Tris, pH 6.1, 0.02% bovine serum albumin (BSA), 0.35 M mannitol, 0.1 mM CaCl2) in a small blending jar. Blender
speed-setting and blending time was adjusted ac-cording to the toughness of the leaves. The blended mixture was poured through a 200 mm
nylon mesh. Broken mesophyll and epidermal cells passed through the mesh. The pale whitish-green epidermal pieces were put in 10 ml enzyme solu-tion 1 (2% cellulase Onozuka R-10, 0.1% pec-tolyase Y-23, 2% BSA, 0.2 mM phenyl-methyl-sulfonyl-fluoride (PMSF) and 1 mM dithiothreitol (DTT) in MBMC buffer) in a 50 ml flask after rinsing thoroughly with cold distilled water. After shaking for at least 45 min at 78 – 85 rpm, the epidermal pieces were collected on 200 mm nylon mesh, and rinsed thoroughly with
MBMC buffer. Then the epidermal strips were transferred into 10 ml enzyme solution 2 (1% cellulase Onozuka R-10, 0.05% Pectolyase Y-23, 0.1 mM PMSF, 0.5 mM DTT in MBMC buffer) in a 50 ml flask with shaking at 35 – 45 rpm for 50 – 60 min. After guard cell protoplasts were sepa-rated from the guard cell walls, the sample was
filtered through 30 mm nylon mesh into a 50 ml
centrifuge tube. The protoplasts were centrifuged for 5 min at 200×g in MBMC buffer for three times, the supernatant was carefully pipetted out leaving a minimum volume at the bottom of the tube, and the pellet was resuspended with MBMC buffer.
2.3. Experimental procedures in6ol6ing
pharmacological treatments
The lower epidermal strips were peeled from detached leaves in the morning, and the mesophyll cells washed away with distilled water. Then the strips were incubated in 10 mM Mes-Tris (pH 6.1) in the dark to induce stomatal closing. After stom-ata were closed in the dark, the epidermal strips were transferred into FC or IAA solution to in-duce stomatal opening, with plus-30 mM KCl and minus-K+ separately.
A total of 20 mM Cytochalasin B (CB), an
inhibitor of F-actin polymerization [2,3,15], or 50
mM oryzalin, an inhibitor of plant microtubule
polymerization [16,17], were applied in this experi-ment to pre-treat closed stomata in the dark. Stomatal apertures did not change significantly within our applied concentrations for less than 60 min, which is consistent with the findings of Huang and Wang [3,5]. Then the epidermal strips were transferred into FC or IAA solution with or without cytoskeletal inhibitor.
For experiments with protoplasts, the freshly isolated protoplasts were suspended with MBMC buffer containing CB or oryzalin. Changes of vol-ume were less than 3% after incubation in CB, oryzalin, or MBMC buffer for 30 min in the dark. Then the protoplasts were treated with FC and IAA plus cytoskeletal inhibitor separately. The volume of protoplasts was calculated by measur-ing their diameters with a ruler in the eyepiece under the microscope.
In parallel with each experiment, controls were conducted with dimethyl sulfoxide in which CB and oryzalin were dissolved separately (the stock concentrations are 52 mM of CB, 100 mM of oryzalin). The final concentration of DMSO was less than 0.05% (v/v). No significant effect on stomatal movement was found at these concentrations.
the neutral red methods as described by Weyers and Meidner [18]. More than 90% stomata or protoplasts of guard cells had viability.
Measurements were made within 5 min of 50 stomata or protoplasts located at random, ten from each of the five strips or slides. Each experi-ment was conducted at least three times independently.
3. Results
3.1. Effect of FC, or IAA on stomatal opening
The effect of FC on stomatal opening was tested by treating epidermal strips for 90 min with differ-ent concdiffer-entration of FC. FC induced stomatal opening in both plus- and minus-KCl buffer (Fig. 1A). The apertures were significantly increased until the concentration of FC added in plus-KCl solution and minus-KCl solution reached 0.73 and 2.19 mM, respectively, but not further increased at
higher concentrations (Fig. 1A).
The time course of 1.46 mM FC on stomatal
opening in plus- and minus-K+ buffers is shown
in Fig. 1B. FC induced stomatal opening after 30 min. The maximum aperture was approached after 90 and 105 min after FC incubation in plus-KCl and minus-KCl, respectively (Fig. 1B).
IAA of different concentrations induced stom-atal opening in plus- and minus-KCl for 90 min (Fig. 2A). Stomatal aperture increased with IAA concentration. The maximum aperture at concen-tration of 0.2 mM IAA was 8.6 mm in plus-K+
and 7.6mm (Fig. 2A) in minus-K+buffer. For the
time course, the stomatal aperture was signifi-cantly increased in the first 90 min both in plus-KCl and minus-plus-KCl as 0.2 mM IAA was added (Fig. 2B). Thus the results indicated that FC, or IAA induced stomatal opening in the dark without requiring the addition of KCl.
3.2. Effect of CB or oryzalin on stomatal opening-induced by FC or IAA
When stomata were treated with 20mM CB for
50 min, the aperture induced by 1.46 mM FC was
only 58.8% of the control’s (Fig. 3 Treatment 3). If FC were added in CB solution after CB pretreat-ment, the aperture was 42.2% of control’s (Fig. 3 Treatment 4).
Also when stomata were pretreated with 50mM
oryzalin for 60 min, the aperture induced by 1.46
Fig. 1. Effect of FC on stomatal opening in plus-30 mM KCl (black circle) and minus-KCl (black triangle) in the dark. A: The closed stomata were incubated in different concentra-tions of FC for 90 min. B: Closed stomata incubated in 1.46
mM FC for different time to induce stomatal opening.
Epider-mal peels were in MAC buffer containing 10 mM Mes-Tris (pH 6.1) plus 1 mM AlCl3and 0.1 mM CaCl2. The stomatal
aperture was the average (9SE) of three separate experi-ments, counting at least 50 stomata for each experiment as described in Section 2.
Fig. 2. Effect of IAA on stomatal opening in plus-30 mM KCl (black circle), and minus-KCl (black triangle) in the dark. A: Closed stomata incubated in different concentrations of IAA for 90 min. B: Closed stomata incubated in 0.2 mM IAA for different time to induce stomatal opening. Epidermal peels were in MAC buffer containing 10 mM Mes-Tris (pH 6.1) plus 1 mM AlCl3 and 0.1 mM CaCl2. The stomatal
Fig. 3. Effect of 20mM CB or 50mM oryzalin pretreatment
on stomatal opening induced by 1.46mM FC for 90 min in
the dark. 1: control: Closed stomata were incubated in MAC buffer. 2: FC treatment, 3: FC treatment after 50 min pre-treatment of CB. 4: FC pre-treatment plus CB after prepre-treatment of CB. 5: FC treatment after 60 min pretreatment of oryzalin. 6: FC treatment plus oryzalin after pretreatment of oryzalin. Epidermal peels were in MAC buffer containing 10 mM Mes-Tris (pH 6.1) plus 1 mM AlCl3and 0.1 mM CaCl2. The
stomatal aperture was the average (9SE) of three separate experiments, counting at least 50 stomata for each experiment described as Section 2.
not change significantly. However, the protoplasts swelled if 0.73mM FC or 100mM IAA was added
to the MBMC buffer. The relative volume of guard cell protoplasts was increased by 28 – 35% within 50 – 60 min following the addition of FC or IAA (Fig. 5 and Fig. 6). After this period, 78.1 – 85.7% protoplasts were broken.
The volume of guard cell protoplasts did not change significantly if protoplasts were treated with 20 mM CB, or 50 mM oryzalin for 30 min in
minus-K+ buffer in the dark. The increase in
volume of the protoplasts induced by FC and IAA after CB and oryzalin pretreatment respectively was less than 3% (Figs. 5 and 6). This result on proto-plasts demonstrates that the induction is likely to reflect the inherent nature of the microtubule and microfilament system rather than some collateral effects of cell walls.
4. Discussion
Electrophysiological studies have demonstrated that there are two independent K+channels in the
Fig. 4. Effect of 20 mM CB or 50 mM oryzalin on stomatal
opening induced by 0.2 mM IAA in the dark for 90 min. 1: control: Closed stomata were incubated in MAC buffer. 2: IAA treatment. 3: IAA treatment after 50 min pretreatment of CB. 4: IAA treatment plus CB after pretreatment of CB. 5: IAA treatment after 60 min pretreatment of oryzalin. 6: IAA treatment plus oryzalin after pretreatment of oryzalin. Epidermal peels were in MAC buffer containing 10 mM Mes-Tris (pH 6.1) plus 1 mM AlCl3and 0.1 mM CaCl2. The
stomatal aperture was the average (9SE) of three separate experiments, counting at least 50 stomata for each experiment described as Section 2.
mM FC was 61.8% of control’s (Fig. 3 Treatment
5). If FC was added to the oryzalin solution after oryzalin pretreatment, the aperture was 44.1% of control’s (Fig. 3 Treatment 6).
Similarly, stomatal aperture induced by 0.2 mM IAA was suppressed when stomata were pretreated with either CB for 50 min or oryzalin for 60 min. Compared with the control the aperture was 59.3% for IAA treatment and 46.5% if IAA was added to the CB solution after CB pretreatment. Similar results were obtained with oryzalin (Fig. 4).
Thus our results demonstrate that pretreatment of CB or oryzalin suppressed stomatal opening induced by FC or by IAA in K+-free medium in the
dark. These suggest that microtubules and microfi-laments can affect stomatal opening independently of K+ fluxes.
3.3. Effect of CB, or oryzalin on the swelling of guard cell protoplasts induced by FC, or IAA
Fig. 5. Effect of 20mM CB or 50mM oryzalin on protoplast
swelling-induced by 0.73 mM FC in minus-K+ in the dark.
Protoplasts were treated with FC only (black circle). A: The protoplasts were incubated in FC plus CB after CB pretreat-ment for 30 min as indicated with arrow (black triangle). B: The protoplasts were incubated in FC plus oryzalin after oryzalin pretreatment for 30 min as indicated with arrow (black triangle). All the protoplasts were in MBMC buffer. The volume data were the average of three separate experi-ments, counting at least 50 protoplasts for each experiment. And the relative volume of guard cell protoplasts (GCPs) is the percentage of protoplast average volume (9SE) com-pared with control’s (100%).
not change significantly (data not shown). These results suggest that stomatal opening induced by FC or IAA is independent of the K+ fluxes.
Plant growth regulators affect plant development and movement through the dynamics of microtubules or microfilaments. For example IAA changes the orientation of cortical microtubule [21,22], and regulates the elasticity of the actin network [23]. FC has many effects similar to IAA, such as promoting proton translocation [24 – 27], but it is not clear whether FC also regulates the cytoskeletal network. In stomatal movements, there is some evidence that microtubules and micro-filaments are involved in stomatal opening and closing depending on K+[1 – 5,8,9] and the activities
of K+ channels [6]. In this paper our
experi-Fig. 6. Effect of 20 mM CB or 50mM oryzalin on protoplast
swelling-induced by 0.1 mM IAA in minus-K+in the dark.
Protoplasts were treated with IAA only (black circle). A: The protoplasts were incubated in IAA plus CB after CB pretreat-ment for 30 min as indicated with arrow (black triangle). B: The protoplasts were incubated in IAA plus oryzalin after oryzalin pretreatment for 30 min as indicated with arrow (black triangle). The protoplasts were in MBMC buffer. The volume data were the average of three separate experiments, counting at least 50 protoplasts for each experiment. And the relative volume of guard cell protoplasts (GCPs) is the per-centage of protoplast average volume (9SE) compared with those of control’s (100%).
guard cell plasma membrane, e.g. inward rectified K+ channels and outward rectified K+ channels
[1,19]. Al3+ was reported as an inhibitor of the
inward rectified K+ channel [20], suggesting the
central role played by K+ in stomatal movement.
However, when KCl is absent from the incubation medium, stomatal aperture increases in response to blue light without any K+ accumulation [7].
Further, in K+-free medium in the dark, our results
demonstrate that FC (Fig. 1) and IAA (Fig. 2) induce both stomatal opening and protoplast swelling (Figs. 5 and 6). The K+ concentration for
minus-K+buffer was less than 0.05 mM (measured
ments provided further evidence that stomatal opening and protoplast swelling induced by FC or IAA in the absence of KCl in the dark could be suppressed by CB or by oryzalin (Figs. 3 and 4). These results, taken together with those in the literature [2,8,9], suggest that microtubules and microfilaments might independently affect stomatal opening.
To further eliminate the effect of the cell wall in stomatal movement, we used guard cell proto-plasts, thereby investigating a direct role for cy-toskeletal components in stomatal opening. Our results show that protoplast swelling induced by FC or IAA was suppressed when protoplasts were pre-treated with CB or oryzalin (Figs. 5 and 6). This suggests that microtubules and mi-crofilaments trigger stomatal opening directly rather than acting indirectly on guard cell walls. In the light of this we hypothesize that the dynamics of both microtubules and microfila-ments directly modulate the swelling of guard cell protoplasts at the beginning of stomatal opening. These changes of guard cell volume are, ultimately, the result of water movement. Aqua-porins form water selective channels, allowing water to pass freely while excluding ions and metabolites [28 – 30]. It seems likely that water channels or aquaporins in the plasma membrane [31 – 33] control water movement in guard cells. The changes in the activity of microtubules and microfilaments could activate aquaporins which, depending on the existing osmotic potential, would drive water into guard cells, resulting in stomatal opening. Thus our results provide new clues for a fuller understanding of the mecha-nism of stomatal movement.
Acknowledgements
The present study was supported by National Natural Science Foundation of China, and The Major State Basic Research Program of PR China (G1999011704). We thank Dr DowElanco in Indianapolis, USA, for his kindly gift of oryzalin, Professor Clive W. Lloyd at John Innes Center, Norwich, UK and Dr Ming Yuan at China Agricultural University for their critical reading of the manuscript and suggestions for the discussion.
References
[1] S.M. Assmann, Signal transduction in guard cells, Annu. Rev. Cell Biol. 9 (1993) 345 – 375.
[2] M. Kim, P.K. Hepler, S.-O. Eun, Y. Lee, Actin filaments in mature guard cells are radially distributed and involved in stomatal movement, Plant Physiol. 109 (1995) 1077 – 1084.
[3] R.F. Huang, X.C. Wang, Effect of cytochalasin B on stomatal movement ofVicia fabaL., Acta PhytoPhysiol. Sinica. 22 (1996) 404 – 408.
[4] L. Couot-Gastelier, P. Louguet, Effet de la colchicine sur les mouvements des et l’utrastructure des cellules stoma-tiques deTradescantia6irginiana, Bull. Soc. Bot. Fr. Lett.
Bot. 139 (1992) 345 – 356.
[5] R.F. Huang, X.C. Wang, Role of microtubules in stomatal movement ofVicia fabaL, Acta Bot. Sinica. 39 (1997a) 253 – 258.
[6] J.U. Hwang, S. Suh, H. Yi, J. Kim, Y. Lee, Actin filaments modulate both stomatal opening and inward K+ -chan-nel activities in guard cells ofVicia fabaL, Plant Physiol. 115 (1997) 335 – 342.
[7] G. Tallman, E. Zeiger, Light quality and osmoregulation in Vicia faba guard cells: Evidence for involvement of three metabolic pathways, Plant Physiol. 88 (1988) 887 – 895.
[8] R.F. Huang, X.C. Wang, Effect of abscisic acid-induced changes of cortical microtubule orientation on stomatal movement ofVicia fabaL, Acta Bot. Sinica. 22 (1997b) 375 – 378.
[9] M. Fukuda, S. Hasezawa, N. Asai, N. Nakajima, N. Kondo, Dynamic organization of microtubules in guard cells ofVicia fabawith diurnal cycles, Plant Cell Physiol. 39 (1998) 80 – 86.
[10] B.E. Anderson, J.M. Ward, J.I. Schroeder, Evidence for an extracellular reception site for abscisic acid inCom
-melinaguard cells, Plant Physiol. 104 (1994) 1177 – 1184. [11] S.M. Assmann, A. Schwartz, Synerdistic effect of light and fusicoccin on stomatal opening. Epidermal peel and patch clamp experiments, Plant Physiol. 98 (1992) 1349 – 1355. [12] A. Vavasseur, G. Lasceve, A. Cousson, Guard cell re-sponses to potassium ferricyanide, Physiol. Plant. 93 (1995) 253 – 258.
[13] L.K. Levit, D.B. Stein, B. Rubinstein, Promotion of stomatal opening by indoleacetic acid and ethrel in epidermal strips ofVicia fabaL, Plant Physiol. 85 (1987) 318 – 321.
[14] P.J. Dunleavy, P.D. Ladley, Stomatal responses ofVicia fabato indoleacetic acid and abscisic acid, J. Exp. Bot. 46 (1995) 95 – 100.
[15] H. Hartwiga, T.P. Stossel, Cytochalasin B and structure of actin gels, J. Mol. Biol. 134 (1979) 539 – 546. [16] I.D. Hugdahl, L.C. Morejohn, Rapid and reversible
high-affinity binding of the dinitroaniline herbicide oryzalin to tubulin from Zea mays L, Plant Physiol. 102 (1993) 725 – 740.
[18] J.D.B.Weyers, H. Meidner, Methods in stomatal Re-search. Longman Scientific & Technical, 1990.
[19] M.R. Blatt, Potassium channel currents in intact stom-atal guard cells: rapid enhancement by abscisic acid, Planta 180 (1990) 445 – 455.
[20] H. Schnable, E. Zeiger, The influence of aluminum ions on movement of the stomata in Vicia faba epidermis strips, Z. Pflanzenphysiol. Bd. 74 (1975) 394 – 403. [21] R. Bergfeld, V. Speth, P. Schopfer, Reorientation of
microfibrils and microtubules at the outer epidermal wall of maize coleoptiles during auxin-mediated growth, Bot. Acta. 101 (1988) 57 – 67.
[22] H. Shibaoka, Plant hormone-induced changes in the orientation of cortical microtubules: Alteration in the cross-linking between microtubules and the plasma membrane, Annu. Rev. Plant Physiol. Plant Mol. Biol. 45 (1994) 527 – 544.
[23] S. Grabski, M. Schindler, Auxins and cytokinins as antipodal modulators of elasticity within the actin net-work of plant cells, Plant Physiol. 110 (1996) 965 – 970. [24] P. Aducci, A. Ballio, J.P. Blein, et al., Functional recon-stitution of a proton-translocating system responsive to fusicoccin, Proc. Natl. Acad. Sci. USA. 85 (1988) 7849 – 7851.
[25] P. Aducci, M. Marra, V. Fogliano, M.R. Fullone, Fusic-occin receptors: perception and transduction of the fu-sicoccin signal, J. Exp. Bot. 46 (1995) 1463 – 1478.
[26] H. Edelmann, Wall extensibility during hypocotyl growth: a hypothesis to explain elastic-induced wall loosening, Physiol. Plant. 95 (1995) 296 – 303.
[27] H. Edelmann, K. Kohler, Auxin increases elastic wall-properties in rye coleoptiles: implications for the mecha-nism of wall loosening, Physiol. Plant. 93 (1995) 85 – 92. [28] M.L. Chrispeels, P. Agre, Aquaporins: water channel proteins of plant and animal cells, Trends Biochem. Sci. 19 (1994) 421 – 425.
[29] T. Henzler, E. Steudle, Reversible closing of water chan-nels in Chara internodes provides evidence for a com-posite transport model of the plasma membrane, J. Exp. Bot. 46 (1995) 199 – 209.
[30] S. Yamda, M. Katsuhara, W.B. Kelly, C.B. Michalowski, H.J. Bohnert, A family of transcripts en-coding water channel proteins: tissue-specific expression in the common ice plant, Plant Cell 7 (1995) 1129 – 1142. [31] R.F. Huang, X.C. Wang, C.H. Lou, Effect of HgCl2on
stomatal opening regulated by cytoskeleton. J. China Agri. Uni. 2 (1997) 6, 40.
[32] X.C. Wang, M.J. Zhu, Y. Kang, J. Chen, Preparation of antibody against GST-RD28 and its localization in guard cell, Academic Periodical Abstracts of China 4 (1998) 1234 – 1235.
[33] M.J. Zhu, X.C. Wang, J. Chen, Advances in aquaporin research, Prog. Biochem. Biophys. 25 (1998) 508 – 512.
