Plant Physiol. (1989) 90, 1435-14390032-0889/89/90/1 435/05/$01 .00/0

Received for publication November 1, 1988and in revised form March 20, 1989

Syringomycin, a Bacterial Phytotoxin, Closes Stomata1

Keith A. Mott* and Jon Y. TakemotoBiology Department UMC 5305, Utah State University, Logan, Utah 84322

ABSTRACT

The effects of the bacterial phytotoxin, syringomycin, on sto-mata were investigated using detached leaves of Xanthium stru-marium and isolated epidermes of Vicia faba. Syringomycin isknown to cause K+ efflux in fungal and higher plant cells. Dosesof syringomycin as low as 0.3 unit per square centimeter (about0.88 pmole per square centimeter) resulted in measurable sto-matal closure when applied through the transpiration stream ofdetached leaves; higher doses produced larger reductions instomatal conductance. Stomatal apertures of isolated epidermeswere also reduced by low concentrations (3.2 units per milliliter;10-8 molar) of syringomycin. The effects of syringomycin weresimilar to those of ABA. Both compounds closed stomata at asimilar rate and at similar concentrations. In addition, neithercompound significantly affected the relationship between photo-synthesis and intercellular CO2 based on data taken after sto-matal conductance had stabilized following the treatment. It ispossible that synngomycin and ABA activate the same K+ exportsystem in guard cells, and syringomycin may be a valuable toolfor studying the molecular basis of ABA effects on guard cells.

Syringomycin is a small (1224 kD) peptide-containing phy-totoxin produced by the bacterium Pseudomonas syringaepv. syringae van Hall that affects ion transport across theplasmalemma of plant and fungal cells. Perhaps the largest ofthese effects is a rapid K+ efflux that occurs when cells insuspension are exposed to low concentrations of this com-pound (14). Syringomycin also stimulates a plasmalemmaATPase (1) but stops acidification of the medium by cells(14), which has led to speculation that it stimulates a H+/K+antiport system (14). These effects may be mediated by pro-tein phosphorylation because syringomycin stimulates phos-phorylation of several plasma membrane proteins, one ofwhich corresponds in mol wt (100,000) to the proton pumpATPase (1). Phosphorylation of membrane proteins in re-sponse to syringomycin is inhibited by the calcium chelatorEGTA, suggesting that Ca2+ is involved in the process, andrecently Takemoto et al. (16) have shown that syringomycincauses Ca2+ influx into slices of red beet storage tissue.Many of the processes affected by syringomycin are similar

to those involved in the turgor-driven opening and closing ofstomata. It is well-established that turgor changes in guardcells are caused by changes in cellular osmotic pressure andthat the major osmoticum in this process is K+. Changes in

'Supported by National Science Foundation grants DMB 8515578and DMB 8704077 and the Utah Agricultural Experiment Station,journal paper No. 3718.

1435

intercellular K+ concentration of guard cells are effected bytransport of K+ across the plasma membrane, and transportinto the cell has been linked to a proton pump ATPase (12).The fungal toxin fusicoccin, which stimulates proton expul-sion from cells (3, 10), has been shown to open stomata byincreasing the rate of K+ transport into guard cells from theextracellular spaces (18). Although little is known about theprocesses involved in K+ efflux from guard cells, Ca2" de-creases stomatal aperture in isolated epidermes (9), and theability ofABA or darkness to close stomata is greatly reducedin the absence of Ca2+ (6, 15).

Because of the similarities between the processes that areaffected by syringomycin and those involved in turgor regu-lation of guard cells, the effects of syringomycin on stomatalconductance and stomatal aperture were tested. Small dosesof syringomycin (in the pm cm-2 range) were applied todetached leaves by feeding low concentrations (-I0-' M) ofpurified syringomycin through the transpiration stream. Theeffects of this procedure on stomatal conductance were deter-mined by monitoring plant gas exchange parameters before,during, and after the treatment. The effect of syringomycinon stomatal aperture was assessed by measuring stomatalapertures of isolated epidermes in the presence of variousconcentrations of syringomycin.

MATERIALS AND METHODS

Syringomycin Purification

Syringomycin was purified as described by Bidwai et al. (2).Because individual preparations varied in activity, syringo-mycin quantities are reported in activity units. Activity wasassayed using the yeast Rhodotorula pilmanae grown on po-tato-dextrose agar plates, and one unit of activity was definedas the amount of syringomycin that completely inhibited thegrowth of the yeast using a 10 uL drop (20). For mostpreparations, 32,000 units were approximately equal to 1,umol of purified syringomycin.

Plant Material

Xanthium strumarium L. (for intact leaf studies) was grownin greenhouses, and Viciafaba (for isolated epidermes studies)was grown in controlled environment growth chambers. ForV. faba, light intensity was maintained at approximately 350jgmol (m2s)-' at the top of the plant, photoperiod was 15 h,and air temperatures were 28 and 20°C during the light anddark periods, respectively. All plants were grown in 1 gallonnursery pots and watered as necessary with one-fourthstrength modified Hoagland solution.

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MOTT AND TAKEMOTO

Gas Exchange

Leaves for gas exchange (X. strumarium) were selected foruniformity of age and appearance. The entire leaf and petiolewere immersed in water, and the petiole was cut with a razor

blade. The leaf was removed from the water with the petiolesubmerged in a small flask of water, blotted dry, and insertedinto a gas exchange chamber, which clamped onto the leaf,forming separate chambers above and below it. Separatemixing and analysis systems were used to control and monitorthe two surfaces of the leaf, as described by Mott (1 1). Lightintensity was maintained at 360 .mol(m2s)-', which was

below saturating for photosynthesis. Leaf temperature wasmaintained at 25°C by controlling the chamber temperatureand the temperature of the air entering the chamber, andwater vapor pressure ofthe air in the chamber was maintainedat 15 mbar by controlling the humidity ofthe air entering thechamber.When photosynthesis and stomatal conductance had

reached steady state, syringomycin, fusicoccin, or ABA wasapplied to the leaf by quickly removing the petiole fromwater, drying the shaft (but not the cut end), and placing it inthe appropriate solution. This procedure was performed whilethe leaf was in the gas exchange chamber and took less than2 s. After the petiole had been in the solution for a predeter-mined length of time, it was returned to distilled water by thesame procedure. Gas exchange parameters were measuredcontinuously during the period of time that syringomycin,ABA, or fusicoccin was applied and for several hoursthereafter.

Portions of the leaf blade that were not in the gas exchangechamber were not transpiring because they were clampedbetween two pieces of opaque, closed cell foam. Therefore,the amount of syringomycin or ABA applied to the leaf couldbe calculated using the transpiration rate of the enclosed leafarea, the concentration ofthe solution, and the length oftimethat the petiole was in the solution. Both the concentrationof the solution and the duration of feeding were varied toachieve specific doses.

Isolated Epidermes

Two leaflets from a V. faba plant were removed from thesame node and submerged in distilled water for 5 to 10 min.The lower epidermis was then peeled from the leaf, and pieceswere placed in covered Petri dishes containing 5 mM Hepes(pH 6.5), 10 mm KCl, 100 AM CaCl2, and various concentra-tions of syringomycin. A 1000 W metal halide lamp withinfrared and neutral density filters provided 350 1Amol(m2s)-'to the epidermal pieces.

After a 2 h incubation period, epidermes were wet-mountedusing incubation buffer and examined under a light micro-scope. There was virtually no contamination of epidermalpieces with mesophyll cells when viewed through the micro-scope. To facilitate measurement of stomatal aperture, fourphotomicrographs ofeach epidermis, each containing approx-imately 10 stomata, were taken. Pictures were focused on a

sharply defined ridge at the inside edge of the guard cells. Theresultant slides were projected, and stomatal apertures were

measured and averaged.

RESULTS

The application of syringomycin, ABA, or fusicoccin todetached leaves involved removing the petiole from distilledwater and placing it in a solution of one of these compounds.The effect of this procedure on stomatal conductance wastested by transferring the petiole from distilled water backinto distilled water while monitoring gas exchange of theblade. The procedure had no effect on stomatal conductance(data not shown).

In contrast, transpiration rate and calculated stomatal con-ductance decreased rapidly shortly after removing the petiolefrom distilled water and immersing it in a solution of syrin-gomycin or ABA. This was accompanied by an increase inleaftemperature (which was compensated for and maintainedat 25°C) and a decrease in photosynthetic rate, as would beexpected as stomata closed. A typical result for syringomycinis shown in Figure 1. In this case, 3.5 units cm-2 (- 105 pmcm-2) of syringomycin were applied by feeding 320,000 unitsmL-' (-10I- M) for 16.5 min. Following the initial rapiddecrease, conductance usually oscillated once or twice beforestablizing at a lower value, although stomatal cycling wasoccasionally observed. Stomatal conductance remained con-stant at the lower value in all experiments (for as long as 4 h)unless subjected to further manipulation. This stable con-ductance value, which was achieved 1 to 2 h after the intialdecrease, was used to quantify the response of stomatal con-ductance to the imposed dose ofsyringomycin. Figure 2 showsthe results of a similar experiment with a 200 pm cm-2 doseof ABA produced by feeding 10-6 M ABA for 7.75 min. Aswith syringomycin, conductance stabilized at a lower valueapproximately 1 h after application and did not recover, evenafter several hours. In all experiments, the conductance re-sponses of the upper and lower leaf surfaces were parallel, andstomata remained sensitive to light and CO2 (data not shown).

Fusicoccin reversed stomatal closure caused by syringo-mycin or ABA. Continuous feeding of 10-4 M fusicoccin aftersyringomycin or ABA treatment produced a slow increase instomatal conductance and eventually resulted in higher sto-

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time (min)Figure 1. Effect of syringomycin and fusicoccin on stomatal con-ductance. At the time indicated, the petiole of the leaf was removedfrom water and immersed in 320,000 units mL-1 (-10-5 M) syringo-mycin for 16.5 min. The petiole was then returned to water until itwas immersed in 1 0-4 M fusicoccin at the time indicated.

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1 436 Plant Physiol. Vol. 90,1989

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SYRINGOMYCIN CLOSES STOMATA

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0.400

0.300

0.200

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Figure 2. Effect of ABA and fusicoccin on stomatal conductance. Atthe time indicated, the petiole of the leaf was removed from waterand immersed in 10-5 M ABA for 7.75 min. The petiole was thenreturned to water until it was immersed in 10-4 M fusicoccin at thetime indicated.

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Figure 3. Dose-response curve for syringomycin and stomatal con-

ductance. Panel a shows the reduction in conductance caused byvarious doses of syringomycin. Panel b expresses the data as percentreduction in conductance. The lines show second order polynomialregressions of the data.

matal conductances than were observed before treatment withsyringomycin or ABA (Figs. 1 and 2).The relationship between the reduction in stomatal con-

ductance and the size of the syringomycin dose is shown inFigure 3a. There was some variability in the steady stateconductances before syringomycin treatment, which mayhave been due to seasonal variations in light intensity in thegreenhouse. Plants with high initial conductances showed

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Figure 4. Effect of syringomycin and ABA on mesophyll photosyn-thetic capacity. The response of photosynthesis to Ci was determinedbefore and after 1.4 units cm-2 (40 pmole cm-2) syringomycin (panela) or 200 pmol cm-2 ABA (panel b).

larger effects of syringomycin than did those with low initialconductances. For example, a leafwith an initial conductanceof 0.81 mole m-2 s-' showed a decrease in conductance of0.47 mole m2 s' when subjected to a 2.8 units cm2 dose of

syringomycin. But for a leaf with an initial conductance of0.50 mole m-2 s-', a dose of 3.3 units cm-2 produced a

decrease of only 0.29 mole m-2 s-'. Both treatments causeda reduction in stomatal conductance of approximately 60%from the initial value, however, and Figure 3b shows the datafrom Figure 3a expressed as a percent reduction in conduct-ance. In both analyses, higher doses of syringomycin resultedin greater stomatal closure, but the pattern was more consist-ent when the stomatal response to syringomycin was ex-

pressed as a percentage of the initial conductance (Fig. 3b).Polynomial (second order) regression of the data shown inFigure 3b demonstrated a positive relationship (r2 = 0.67)between the dose of syringomycin and percent reduction instomatal conductance. Regression ofthe data shown in Figure3a failed to demonstrate a consistently positive relationshipand showed a lower coefficient of determination (r2 = 0.42).The responses of photosynthesis to C,2 before and after

providing 1.4 units cm-2 (40 pmol cm-2) of syringomycin toa leafare shown in Figure 4a. Although this treatment reducedconductance by 30%, the relationship between photosynthesisand C, was not affected. The responses of photosynthesis toCi before and after 200 pmol cm-2 of ABA was applied toleaves are shown in Figure 4b, and, as with syringomycin, therelationships were similar. The photosynthesis versus Ci re-

2 Abbreviation: Ci, intercellular CO2 concentration.

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1 437

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MOTT AND TAKEMOTO

sponses shown in Figure 4 were obtained after conductancehad stabilized following the syringomycin or ABA treatments.The relationship between photosynthesis and Ci was highlyvariable while conductance was changing in response to eithercompound.To determine if guard cells responded directly to syringo-

mycin, stomatal apertures were measured for isolated epi-dermes of Vicia faba at various concentrations of syringo-mycin (Fig. 5). Mean aperture was reduced significantly (P <0.001, using a t-test for unpaired samples with unequal vari-ances) by the lowest concentration of syringomycin tested,3.2 units mL-' (- 10-8 M). Linear regression of the data inFigure 5 revealed a negative slope that was significantly dif-ferent from zero (P < 0.001), indicating that smaller apertureswere correlated with higher doses of syringomycin. Frequencyhistograms showed that the distribution of stomatal aperturesaround the mean approximated a normal distribution in allcases.

DISCUSSION

ABA or syringomycin was applied to a detached leaf byimmersing the cut petiole in a solution ofthat compound andallowing the transpiration stream to carry it to the leaf blade.In contrast with many studies employing a similar method-ology, these compounds were not applied continuously.Rather, ABA or syringomycin was introduced into the tran-spiration stream for a specific period of time, after which thepetiole was put back into water. This procedure allowed a

specific amount of either compound to be drawn into thepetiole, and because portions of the leaf blade not in the gasexchange chamber were not transpiring, water loss by theportion of the leaf blade in the chamber was equal to wateruptake by the petiole. Assuming that ABA and syringomycin

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Syringomycin concentration(units/ml)

Figure 5. Effect of syringomycin on stomatal aperture of isolatedepidermes. Abaxial epidermes were peeled from V. faba leaflets andfloated on 5 mm Hepes (pH 6.5), 10 mm KCI, 100 AM CaC12, andvarying concentrations of syringomycin. Error bars indicate 95%confidence levels. Linear regression showed a negative slope thatwas significantly different from zero (P < 0.001). Mean aperture atthe lowest concentration of syringomycin (3.2 units mL1) was signif-icantly different from that with no syringomycin (P < 0.001) as

determined with a t-test for unpaired samples with unequal distribu-tions.

followed the transpiration stream with minimal diffusionalspreading, the amount of syringomycin or ABA applied tothe leaf area in the chamber was accurately controlled byvarying either the concentration of the compound or theduration of feeding.Cummins et al. (5) reported that the closing response of

stomata caused by applying ABA through the transpirationstream could be reversed if the ABA solution was replacedwith water. This reversal did not occur in the experimentsreported here. Instead, a dose of either ABA or syringomycinproduced a rapid decline in stomatal conductance, whichoften oscillated once or twice before stabilizing at a lowervalue. A new steady state conductance was usually achievedwithin 1 or 2 h of the initial decline in conductance, and thislower conductance was stable unless subjected to furthermanipulation.The data presented in this study indicate that syringomycin

acts on guard cells to close stomata. The reduction in stomatalconductance by syringomycin was dose-dependent in isolatedepidermes and intact leaves, and measurable stomatal re-

sponses occurred at syringomycin doses as low as 0.3 unitcm2 (-0.88 pmole cm-2) for intact leaves and 3.2 units ml-'(_ 10-8 M) for isolated epidermes. The effect of syringomycinwas reversible by fusicoccin, which opens stomata by increas-ing the rate of proton expulsion by guard cells (10), therebystimulating K+ uptake. Fusicoccin also reversed stomatalclosure induced by ABA, consistent with previous studies(18). In this study, continuously feeding fusicoccin after treat-ment with ABA or syringomycin produced conductanceshigher than those observed before treatment, which suggeststhat syringomycin affects guard cells by changing membranetransport processes and not by a general breakdown of mem-brane integrity.The fact that syringomycin did not affect the photosynthetic

capacity of the mesophyll cells supports the hypothesis thatsyringomycin affects membrane transport processes of guardcells. Ifsyringomycin affected guard cells by disrupting plasmamembranes, mesophyll as well as guard cells would have beenaffected, which would have decreased mesophyll photosyn-thetic capacity. Several studies have shown a reduction inphotosynthesis at a constant C1 (4, 13) caused by ABA,suggesting a direct effect ofABA on mesophyll photosyntheticcapacity. However, these effects may have been the result ofheterogeneous stomatal closure in response to ABA (17), andABA effects on mesophyll photosynthetic capacity have notbeen fully explained. The data reported in this study indicatethat syringomycin had no measurable effect on photosyn-thetic capacity after stomatal conductance had stabilized atthe lower value. Since heterogenous stomatal closure causes

parallel decreases in stomatal conductance and mesophyllcapacity, the data presented suggest that stomatal closure wasnot heterogenous after conductance had stabilized at the lowervalue. This conclusion is supported by the data from isolatedepidermes of Vicia faba. Syringomycin did not alter thevariation around the mean nor the pattern of variation ofstomatal closure. Had syringomycin reduced conductance byselectively closing some stomata while leaving others unaf-fected, a bimodal pattern of variation with a larger variancewould have been expected.

n=43n=52 n=52I~~~~~~~~~~~~~~~~~~TS n=42

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SYRINGOMYCIN CLOSES STOMATA

The discrepancy between the results reported here, whichshow no effect ofABA on photosynthetic capacity, and thoseshowing a reduction in the photosynthesis versus Ci relation-ship (4, 13) may be due to differences in the length of timebetween ABA application and the measurement of mesophyllcapacity. It should be noted, however, that not all previousstudies have shown a reduction in photosynthetic capacityfollowing treatment with ABA (e.g. 7).There were many similarities between stomatal responses

to ABA and syringomycin in this study. Both had effects in a

similar time frame and were reversible by fusicoccin (Figs. 1

and 2). The sensitivity of stomatal conductance to syringo-mycin was comparable to that ofABA, which has been shownto produce stomatal responses at doses of only a few fmolmm-2 (12) or at concentrations as low as 10-8 M. Stomataremained sensitive to CO2 after exposure to syringomycin or

ABA, and neither compound significantly changed the pho-tosynthetic capacity of the mesophyll cells.

It seems likely that both syringomycin and ABA initiate a

series of events that stimulate K+ export. While the eventsleading to K+ efflux may differ for syringomycin and ABA,the K+ pump is probably identical. Both syringomycin andABA have been shown to stop medium acidification by cellsin addition to promoting K+ efflux, and the effects of bothcompounds are Ca2` dependent and occur within seconds ofexposure (12, 14). Closing of stomata by ABA or darkness isenergy-dependent (8, 19) and can be blocked by uncouplerssuch as CCCP. While Karlsson and Schwartz (8) attribute thisphenomenon to the fact that either ATP or a membranepotential is required to open K+ channels in the plasma-lemma, it is also consistent with a H+/K+ antiport system,which has been proposed for syringomycin-induced K+ efflux.

In addition to guard cells, syringomycin affects yeast cellsand other plant cells, on which ABA has no apparent effect,even at high concentrations (KA Mott, JY Takemoto, unpub-lished observation). This is consistent with the idea that theturgor regulation system of guard cells is merely an amplifi-cation of the system used by other plant cells and fungi (9).Because syringomycin affects cells that are more easily isolatedand manipulated than guard cells, its molecular mechanismcan be more readily investigated than can the action ofABAon guard cells. If all or part of the mechanisms for ABA andsyringomycin are identical, then syringomycin may be valu-able in studying the molecular basis for ABA effects on guardcells. We are currently investigating this possibility.

ACKNOWLEDGMENTS

We thank Carl Bachman and Rand Hooper for excellent technicalassistance and Susan Durham for statistical advice.

LITERATURE CITED

1. Bidwai AP, Takemoto JY (1987) Bacterial phytotoxin, syringo-mycin, induces a protein kinase-mediated phosphorylation ofred beet plasma membrane polypeptides. Proc Natl Acad SciUSA 84: 6755-6759

2. Bidwai AP, Zhang L, Bachman RC, Takemoto JY (1987) Mech-anism of action of Pseudomonas syringae phytotoxin, syrin-gomycin. Plant Physiol 83: 39-43

3. Blum W, Key G, Weiler EW (1988) ATPase activity in plasma-lemma-rich vesicles isolated by aqueous two-phase partitioningfrom Vicia faba mesophyll and epidermis: Characterizationand influence of abscisic acid and fusicoccin. Physiol Plant 72:279-287

4. Cornie G, Miginiac E (1983) Nonstomatal inhibition of net CO2uptake by (±) abscisic acid in Pharbitis nil. Plant Physiol 73:529-533

5. Cummins WR, Kende H, Raschke K (1971) Specificity andreversibility of the rapid stomatal response to abscisic acid.Planta 99: 347-351

6. de Silva DLR, Hetherington AM, Mansfield TA (1985) Syner-gism between calcium ions and abscisic acid in preventingstomatal opening. New Phytol 100: 473-482

7. Dubbe DR, Farquhar GD, Raschke K (1978) Effect of abscisicacid on the gain of the feedback loop involving carbon dioxideand stomata. Plant Physiol 62: 406-417

8. Karlsson PE, Schwartz A (1988) Characterization of the effectsof metabolic inhibitors, ATPase inhibitors and potassium-channel blocker on stomatal opening and closing in isolatedepidermis of Commelina communis L. Plant Cell Environ 11:165-172

9. MacRobbie EAC (1987) Ionic relationsofguard cells. In E Zeiger,GD Farquhar, IR Cowan, eds, Stomatal Function. StanfordUniversity Press, Stanford, CA, pp 125-162

10. Manre E (1979) Fusicoccin: A tool in plant physiology. AnnuRev Plant Physiol 30: 273-288

11. Mott KA (1988) Do stomata respond to CO2 concentrationsother than intercellular? Plant Physiol 86: 200-203

12. Raschke K (1987) Action of abscisic acid on guard cells. In EZeiger, GD Farquhar, IR Cowan, eds. Stomatal Function.Stanford University Press, Stanford, CA, pp 253-280

13. Raschke K, Hedrich R (1985) Simultaneous and independenteffects of abscisic acid on stomata and the photosyntheticapparatus in whole leaves. Planta 115: 47-57

14. Reidl HH, Takemoto JY (1987) Mechanism ofaction ofbacterialphytotoxin, syringomycin. Simultaneous measurement ofearlyresponses in yeast and maize. Biochim Biophys Acta 898: 59-69

15. Schwartz A, Ilan N, Grantz DA (1988) Calcium effects onstomatal movement in Commelina communis L. Plant Physiol87: 583-587

16. Takemoto JY, Giannini JL, Vassey T, Briskin DP (1989) Syrin-gomycin effects on plasma membrane Ca2" transport. In AGraniti, RD Durbin, A Ballio, eds, Phytotoxins and PlantPathogenesis. Springer-Verlag, New York, pp 167-175

17. Terashima I, Wong S-C, Osmond CB, Farquhar GD (1988)Characterization of non-uniform photosynthesis induced byabscisic acid in leaves having different mesophyll anatomies.Plant Cell Physiol 29: 385-394

18. Turner NC, Graniti A (1969) Fusicoccin: A fungal toxin thatopens stomata. Nature 223: 1070-1071

19. Weyers JDB, Paterson NW, Fitzsimons PJ, Dudley JM (1982)Metabolic inhibitors block ABA-induced stomatal closure. JExp Bot 33: 1270-1278

20. Zhang L, Takemoto JY (1987) Effects of Pseudomonas syringaephytotoxin, syringomycin, on plasma membrane functions ofRhodotorula pilimanae. Phytopathology 77: 297-303

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