Kinetic Properties of Guard-Cell Phosphoenolpyruvate Carboxylase

Biochem. Physiol. Pflanzen 186,317-325 (1990) Gustav Fischer Verlag lena

Kinetic Properties of Guard-Cell Phosphoenolpyruvate Carboxylase*) WILLIAM H. OUTLAW, Jr. Department of Biological Science (Unit I), Florida State University, Tallahassee, FL, U.S.A. Key Term Index: gas exchange, guard cell, stomata, phosphoenolpyruvate carboxylase

Summary The pivotal role of guard-cell phosphoenolpyruvate carboxylase (PEPC) in stomatal function is discussed. Appropriate tissue sources for this enzyme are suggested. The few kinetic data for guard-cell PEPC are compared with each other and to those of other PEPC's. Finally, an attempt is made to place these findings in the context of guard-cell cytoplasm. Directions for future research are indicated.

Introduction Phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31, orthophosphate: oxaloacetate carboxylase (phosphorylating) catalyzes the irreversible reaction phosphoenolpyruvate

+

HC0 3 -

~

oxaloacetate

+

Pi'

This enzyme is perhaps present at some level in all plant tissues. However, the specific activity varies widely, and high levels correlate with tissue- or cell-specific functions, of which there are many (LATZKO and KELLY 1983). For example, C 4 mesophyll cells are especially enriched with PEPC, which there has a role in the synthesis of a "COz-transport" species (malate or asparate). Another specific role of PEPC is in CAM; there, PEPC functions in the synthesis of a "COr storage" species (malic acid). In these two instances, diurnal reversible post-translational modifications synchronize the appropriate catalytic properties with the required in situ flux through the metabolic branchpoint catalyzed by PEPC. These examples can illustrate two points. First, distinctive allo- and isoenzymes with different properties have evolved to fulfill specialized roles in a developmentally specific and tissue-specific manner. Thus, the physiology of specific cells, like guard cells, cannot be understood from extrapolation of the properties of a foreign PEPC. Second, the enzymes must be studied in a physiologically relevant manner, as at least some PEPC's undergo temporal transitions (JIAO and CHOLLET 1989). For other general aspects of PEPC, I refer the reader to two good reviews (O'LEARY 1982; ANDREO et al. 1987); here, I will focus on guard-cell PEPC, which has been reviewed before (WILLMER 1983). The motor that drives stomatal movements is the strongly electrogenic H+ -extruding ATPase of the guard-cell plasmalemma. When this pump hyperpolarizes the membrane potential, voltage-sensitive potassium channels open (SCHROEDER et al. 1987), which results in stomatal opening as the internal potassium concentration increases nominally 300 mM (OUTLAW 1983). Thus, speaking broadly, on stomatal opening a pair of Vidafaba guard *)

Presented at the FESPP-Workshop "Stomata '89", held at Berlin, September 4 to 9, 1989. BPP 186 (1990) 5/6

317

cells extrudes ~ 3 pmol H+, which exceeds the expected pH buffering capacity. A small part of this proton extrusion is, in effect, compensated for by concomitant permeant anion (chloride) uptake, but most of the extruded protons are replaced by the protons that are released during the accumulation of carboxylates, prototypically divalent malate. The proposed pathway in guard cells - a pH-stat mechanism (e.g. SMITH and RAVEN 1979) - has been sketched earlier (OUTLAW 1982; SCHNABL 1983; WILLMER 1983; OUTLAW 1987). In outline, hexose (derived from starch) is oxidized glycolytically, and the reaction releases to solution one proton/triose-Po The "downstream" glycolytic intermediate, PEP, has several possible fates, one of which is carboxylation. For this latter, HC0 3 - is the cosubstrate; it is formed by the hydration of CO 2 , a reaction that releases a proton to solution. Obviously, PEP carboxylation is the pivotal step leading to malate accumulation. Albeit a weak acid, malate's pK's (~3.4, ~ 5.1) are well below vacuolar pH (for Commelina guard cells of open stomata, pH 5.6; PENNY and BOWLING 1975), indicating that malate accumulates there as the divalent salt. Thus, the accumulation of malate represents a proton debt; malate itself is the end-point. The accumulated malate is dissipated (spatially or metabolically) when stomata close. In essence, guard-cell PEPC functions at the metabolic branchpoint to direct the accumulation of potassium counterion. The high level of PEPC in guard cells is consistent with a specialized role there that corresponds to the large malate fluctuations these cells experience (see Fig. 5 of OUTLAW 1980). Before regulation of this crucial reaction can be explained, the kinetic properties of the enzyme must be described.

Sources of Guard-cell PEPC As they were by WILLMER (1983), comments concerning the choice of biological sample as a source of guard-cell PEPC will be made. Three types of samples have been used. (a) Ordinary epidermal peels have been important historically, and WILLMER (1983) has reviewed these investigations. However, because ordinary epidermal peels may comprise epidermal cells per se, trichomes, contaminating mesophyll cells, and guard-cells in some proportion depending on the plant, the growth conditions, and the method of harvesting the peel, they are not suitable for biochemical studies (WILLMER et al. 1987). (b) Guard-cell protoplasts (WEYERS et al. 1983) are perhaps the single most important tool available. In many ways (e.g., response to blue light), they behave like the guard cells from which they were derived; this conservation of property allows for physiologically relevant studies. Indeed, all reports, save one, that are reviewed here will be based on PEPC extracted from guard-cell protoplasts. (c) Finally, guard-cell PEPC can be extracted directly from guard-cell pairs that have been dissected from freeze-dried leaf (OUTLAW 1980; HAMPP and OUTLAW 1987). The amount of PEPC present in a single guard-cell pair (~3 pg) can easily be measured by recently developed methods (OUTLAW et al. 1985). WILLMER'S (1983) review of the microchemical approach was negative, but without sound reason.

Assay of PEPC The assay for PEPC has been revised over the last two years on two bases. First, the substrate was proposed to be PEP, Mg (MUKERJI 1977) (and not the free anion). This assertion was based on "smooth" hyperbolic kinetics when the velocity was the dependent 318

BPP 186 (1990) 5/6

variable against which limiting [PEP , MgJ was plotted. (The calculated [PEP, Mg)'s were achieved at various PEP and Mg2+ concentrations.) However , until recently (WEDDING et al. 1988), most PEPC kinetics studies were based on the assumption (O'LEARY 1982) that PEP(free) was the substrate. Further, common assay modifications cause inadvertent changes in PEP , Mg2+ and PEP' Mg concentration (OUTLAW 1990). For example, addition of malate, generally an inhibitor of PEPC, decreases [PEP· Mg2 +] (therefore increasing PEP(free) concentration) because malate itself complexes with Mg2+. Regardless whether the substrate should be PEP or PEP' Mg, formulation of assay cocktails should incorporate the knowledge that different species of the various components exist in solution. As few authors consider metal-ligand interactions , strict interpretation and comparison of kinetics results are problematical.

CD

®

HCO

NADH

PEP(-Mg)~,~ 'j" • OAt~f'®' ;::'0" Pi

MAL

CO 2

PYR

NAOH~ ®

t

NAD

LAC

>

CD ®

PEPC

MDH

®

Phosphatase

@)

Nonenzymic

®

LDH

Fig. 1. Reactions that occur in the assay cocktail for "raw-extract" phosphoenolpyruvate carboxylase (PEPC). (1), the reaction catalyzed by PEPC itself; (2), enzymic reduction of PEPC product OAA by analytical malic dehydrogenase; (3) , PEP conversion to pyruvate, which is catalyzed by tissue phosphatases and which results in an overestimate of PEPC when lactic dehydrogenase is present; (4), nonenzymic Mg2 + -stimulated decarboxylation of PEPC product OAA , which results in an underestimate of PEPC in malic-dehydrogenase-linked assays; (5), enzymic reduction of pyruvate, which is an indirect reaction product of PEPC , but which is also formed by hydrolysis of the PEPC substrate PEP . (Reprinted from TARCYNSKI and OUTLAW 1990).

Second, the assay depends on the formation of a stable C4 -acid product (commonly malate) from the unstable reaction product oxaloacetate. Before MEYER et al. (1988), the assumption had been that OAA reduction - in either the radiometric or the spectrophotometric assay - is stoichiometric with oxaloacetate formation (i.e., that the rate of reaction CD = reaction @, Fig . I). However, some oxaloacetate spontaneously decarboxylates to form pyruvate (reaction @, Fig. I). With purified PEPC, lactic dehydrogenase may be added (reaction @, Fig. I) so that the true PEPC activity is indicated as the simultaneous sum of pyruvate and oxaloacetate reduction (MEYER et al. 1988) . Unfortunately , endogenous phosphatases which also fom1 pyruvate from PEP (reaction @, Fig. 1) complicate the otherwise simple corrective measure of incorporating lactic dehydrogenase into the assay cocktail (SMITH et al. 1989; TARCZYNSKI and OUTLAW 1990). In summary, the apparent simplicity of assaying for PEPC belies the intricate series of interacting reactions in the assay cocktail; few investigators have considered these complications . BPP 186 (1990) 5/6

319

Guard-cell PEPC: Affinity with Substrate as a Function of pH, [Malate], and Physiological State Fig. 2A shows PEPC-PEp· Mg affinity at pH 8.3; I calculate the Km to be 0.07 ± 0.03 mM. [The value calculated by SCHNABL and KOTTMEIER (1984) was for PEP(free) (0.21 mM); the corresponding value I would have calculated (WILKINSON 1961) is 0.14 ± 0.07 mM. The reason for the difference is that they deleted the datum for 0.75 mM and retained the datum for 1.0 mM, whereas I did the opposite.] This Km was in the range of that (0.162 ± 0.02 mM) obtained by T ARCZYNSKI and OUTLAW (1990) at pH 8.5. These two investigations on Vida faba guard-cell PEPC used radically different approaches (protoplast extracts and quantitative histochemistry on microdissected samples, respectively). -I

PEP·Mg ,mM

-I

PEP, Mg- I, mM- 1

10

.

~

.~ ;;

20

l

40

A

B0 -; .~

Ci

f

f

7

7

>

~

>

o

80

10

o

0

40

Fig. 2. Double-reciprocal plots calculated from vl[PEP] plots published by (A) SCHNABL and their Fig. 4A "control") and (B) RASCHKE et al. (1988, their Fig. 10, extract from illuminated cells). [PEP, Mg 2+],s were calculated from the conditions in the original reports (see l OUTLAW 1990); it is sufficiently accurate for this graphical representation to plot [PEPr (X-axis) and [PEP, Mg2+r 1 (upper X-axis) as a single point. The circled datum points were not considered in the Km calculations, which were made according to the statistical protocol of WILKINSON (1961). (Points omitted in A were cases of substrate inhibition as interpreted by SCHNABL and KOTTMEIER (1984); points omitted in B were very low substrate concentrations that could not be precisely determined by the reviewer. The arrows indicate (app) Km- I . A: Viciafaba; assay, pH 8.3. B: Pisum sativum; assay, pH 7.0. KOTTMEIER (1984,

From these data, one generalizes that the Km(PEP' Mg) of Vida guard-cell PEPC at approximately pH 8.4 is 0.10 to 0.15 mM. Separate laboratories (KOTTMEIER and SCHNABL 1986; TARCZYNSKI and OUTLAW 1990) have found that this kinetic parameter increases at lower pH. For example, we (TARCZYNSKI and OUTLAW 1990) found that the Km(PEP'Mg) was ca. 4 X lower at pH 8.5 than atpH 7.0. Guard-cell PEPC Km has been obtained for two other species. RASCHKE et al. (1988) reported viS plots for Pisum sativum guard-cell PEPC, which I have calculated on the basis of Km(PEP'Mg) (Fig. 2B). At pH 7.0, the Km was 0.15 to 0.36 mM, depending on the preincubation conditions of the protoplasts. I calculate (WILKINSON 1961) the Km of Commelina guard-cell PEPC to be 0.3 to 0.6 mM at pH 7.2 (unpublished data of C. M. WILLMER). As briefly mentioned earlier, the CAM and C4 PEPC's are regulated by reversible posttranslational modification. These precedents in temporal regulation have been the impetus 320

BPP 186 (1990) 5/6

for several studies or the PEPC of guard cells, where the rate of malate accumulation increases dramatically upon stomatal opening. KOTTMEIER and SCHNABL (1986) used K+induced guard-cell protoplast swelling in darkness (SCHNABL, pers. comm.) as a physiological correlate of stomatal opening. They found that the app Km(PEP (total» decreased - II X if PEPC was extracted from maximally swollen protoplasts, but assay conditions (particularly pH) were not given. Part of this change in Km is possibly coincidental , as the authors were distinguishing a radius change that I calculate to be over the range from 0.16 to 1.17 !-lm. Under some conditions, RASCHKE et al. (1988) also detected a Km change that correlated with the physiological state of the extracted protoplasts. The small Km change (Table 1) for Pisum was detected as assay pH 7.0, but not at assay pH 8.0. Correlation of the Km shift with cell preincubation also depended on light as a preincubation variable (i.e., ± K + in the protoplast preincubation media did not produce a detectable Km difference (K. RASCHKE, pers. comm .). TARCZYNSKI and OUTLAW (1990) did not find a Km change that correlated with the rate of guard-cell malate accumulation ; C. M . WILLMER'S experiments (unpublished) with guard-cell protoplasts of Commelina did not reveal a Km shift either. Obviously, the question whether guard-cell PEPC is temporally regulated by reversible post-translational modifications remains unsolved. Table 1. KmIPEP .Mgj values (mM) calculated b y the reviewer from vl[PEP] plots of different investigators. Source for guard-cell PEPC

Assay pH

Vicia faba . protoplasts

8.3

0.07 ± 0 .03"

SCHNABL and KOTTMEIER (1984)

Vida faba . cells dissected from freeze-dried leaves

8.5 7.0

0.16 ± 0.02 0.71 ± 0 . 11

TARCZYNSKI and OUTLAW (1990)

Commelina communis. protoplasts

7 .2

0.61 ± 0 .20 WrLLMER , PETROPOULOU, (0.27 ± 0 . 18)b MAN ETAS (unpublished)

Pisum sativum. protoplasts

7.0

0.15 ± 0.01 (0.36 ± 0.02)

a b

Km

Reference

RASCHKE et al. (1988)

Km reported by SCHNABL and coworkers depends on the swelling state of the protoplasts. See text. Values in parentheses are Km ' s obtained with extracts of dark-adapted protoplasts .

An important development in temporal modulation, so far addressed only by SCHNABL'S group (MICHALKE and SCHNABL 1990) , isthat PEPC activity may oscillate with a periodicity of ca. 2 min. This discovery might be not only of intrinsic value in interpreting guard-cell physiology but possibly of usc in elucidating the mechanism by which PEPC is modulated (see above) . Like other PEPC's , guard-cell PEPC is inhibited by malate. When guard-cell protoplasts of Commelina were lysed directly in assay cocktail (WILLMER, PETROPOULOU and MANETAS , unpublished) , the malate concentration required for 50% inhibition was - 50 ftM (assay pH 7.2) . However, an extr act-initiated assay showed malate to be much less effective (halfinhibition at > 500 [lM). Because glycerol amendment to the extract resulted in much greater sensitivity to malate, it is tempting to speculate that the aggregation state of guard-cell PEPC BPP 186 (1990) 5/6

321

changes during extract storage. SCHNABL and KOTTMEIER (1984) also reported that malate inhibits (desalted) guard-cell PEPC (at 5 ruM malate, pH 8 .3, 82 % decrease in V max> and a 2.7 x increase in Km) . RASCHKE et al. (1988) reported that malate was a competitive inhibitor, being more effective at pH 8. The above authors did not consider malate· Mg interactions as did TARCZYNSKI and OUTLAW (1990); we found that malate(free) was a competitive inhibitor. At 1 ruM malate(free), the Km(PEP' Mg) was increased by almost 4-fold (to - 2.5 ruM) at pH 7.0 and 2-fold (to - 0.26 ruM) at pH 8.5; these results are thus in qualitative agreement with those of RASCHKE et al. (1988).

Comparison of Guard-cell PEPC with other PEPC's Beginning with the seminal paper of TING and OSMOND (1973), it has been widely reported that PEPC ' s of C 3 , C4 , and CAM photosynthetic tissue can be differentiated on the basis of kinetic properties. For example, with PEP(free) as the substrate, the Km of Flaveria cronquistii PEPC (C 3) was 1I17th that of the C4 F. trinervia (BAUWE and CHOLLET 1986) when assayed at pH 7.0. Likewise, the F. cronquistii PEPC Km(PEP(total)) was reported to be 1I9th that of F. trinervia (NAKAMOTO et a1. 1983). (Other intrageneric comparisons are reported by TING and OSMOND 1973, and HOLADAY and BLACK 1981.) It would, therefore, seem to be of interest to compare guard-cell PEPC (Table 1) with these other PEPC's. Unfortunately, such a comparison among various reports is not instructive, as the absolute values obtained under presumably similar conditions differ by as much as an order of magnitude ; the reason for the differences is not obvious. Double-reciprocal plots (Fig. 2) indicate that guard-cell PEPC obeys Michaelis-Menten kinetics, similar to C 3 PEPC (but see TING and OSMOND 1973), whereas the C 4 PEPC is usually reported to be allosteric (but see HOLADAY and BLACK 1981, for the C 4 PEPC from Panicum prionitis). Allosterism with C 4 PEPC's may depend on assay pH. SCHNABL and KOTTMEIER'S (1984) data indicate that guard-cell PEPC is inhibited at high substrate concentration. This phenomenon has not been observed in other guard-cell experimental systems . The important question of physiological activation is unresolved for guard-cell PEPC. A positive answer to this question would permit alliance to either C4 or CAM PEPC (which are temporally modulated). The limited evidence indicates that the predominant PEPC in C 3 leaves is unaffected by light (CHASTAIN and CHOLLET 1989).

Implications of Guard-cell PEPC Kinetic Properties for Stomatal Function The ultimate goal is to be able to base a partial explanation of guard-cell function on the kinetic properties of PEPC, determined in vitro. Obviously, simple assumptions (e.g., no discontinuities in cytosolic pH and metabolites) are necessary. At present, we have few of the requisite data in hand . The [pEP(totaI)J in guard cells has been estimated to be 0.27 mM (OUTLAW and KENNEDY 1978) compared with a model value of ca. 0 .0 2 ruM for a "typical" plant cell (BIELESKI 1973). (SCHNABL' S (1983) determination of 15 mM was not a definite one and needs to be investigated in more detail (SCHNABL, pers . comm.» If we take 0.5 mM as a reasonable value for cytosolic [Mg(free)J and pH 7.2 as typical (see Table 1 of LOUGHMAN et al. 1989) for the cytosol, we can caculate a PEP' Mg2+ concentration ranging from approximately 50 r-tM to approximately 2.5 ruM (using the assumption PEP is excluded from 322

BPP 186 (1990) 5/6

the vacuole). These values need to be refined, but even so, it appears that PEPC is potentially limited by substrate. Whole-cell malate concentration (e.g., OUTLAW and LOWRY 1977) is of little value in inferring an influence on PEPC, as malate is expected to be largely vacuolarly localized. To my knowledge, only MICHALKE and SCHNABL (1987; 1990) have attempted the subcellular localization of malate in guard cells. Their values will require independent confirmation; they show that the mitochondrial malate level is as high as 380 fmol . (guard-cell protoplast)-I (Fig. 2, ofMICHALKE and SCHNABL 1990). Consider that there are 23 mitochondrial guard-cell profile in Vicia (Table I of ALLA WAY and SETTERFIELD 1972) and that each mitochondrion is 0.63 [lm in diameter (measured from plate 7 of ALLAWAY and SETTERFIELD 1972). For a maximum (over) estimate of mitochondrial volume, one can regard each mitochondrion as a cylinder, H = 10 [lm (the thickness of the cell), and estimate that the mitochondrial volume is < 68 X 10- 18 m3 (~3.2 % of cell volume), which implies that the reported mitochondrial malate concentration is > 5.5 M. The only unambiguously determined cytoplasmic malate concentration for any plant cell of which I am aware is that published by CHANG and ROBERTS (1989), who obtained the values by NMR after incubation of tissue with H 13 C0 3 . Extrapolation from their findings (> 5 mM) makes clear that malate will attenuate PEPC, as also concluded by RASCHKE et al. (1988). The 4-fold decrease in efficiency (T ARCZYNSKI and OUTLAW 1990) with an increase in proton concentration implies that guard-cell cytosolic pH - which is unknown - may be an important factor in regulating carboxylate synthesis. Open Questions

This paper emphasizes that guard-cell PEPC is critically understudied and poses some questions that require answers: Is the assayed activity due to a cell-type-specific isoenzyme? Why do growth-lot-specific variations occur in PEPC kinetic parameters (see OUTLAW et al. 1979; SCHNABL 1985)? How does guard-cell PEPC respond to various effectors (e.g., glucose 6-P) that powerfully modulate other PEPC's? Does guard-cell PEPC undergo reversible regulatory phosphorylation? Are there aggregation isomers in vivo? Perhaps most neglected, what is the cytosolic environment of guard-cell PEPC (e.g., [Mg2+], [PEP, Mg], pH, etc.)? Until these questions are answered, it is my opinion that we will remain quite ignorant of the regulation of stomatal aperture, one of the most critical plant physiological functions. Acknowledgements WHO was supported by a grant from the U.S. DOE during the preparation of this article.

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BIELESKI, R. L.: Phosphate pools, phosphate transport, and phosphate availability. Annu. Rev. Plant Physiol. 24, 225-252 (1973). CHANG, K., and ROBERTS, 1. K. M.: Observation of cytoplasmic and vacuolar malate in maize root tips by 13C-NMR spectroscopy. Plant Physiol. 89, 197-203 (1989). CHASTAIN, C. J., and CHOLLET, R.: Interspecific variation in assimilation of 14C02 into C4 acids by leaves of C3 , C4, and C3 -C4 intermediate Flaveria species near the CO2 compensation concentration. Planta 179, 81-88 (1989). HAMPP, R., and OUTLAW, W. H. Jr.: Mikroanalytik in der pflanzlichen Biochemie. Naturwissen. 74, 431-438 (1987). HOLADAY, A. S., and BLACK, C. C.: Comparative characterization of phosphoenolpyruvate carboxylase in C3 , C4 , and C3 -C4 intermediate Panicum species. Plant Physiol. 67, 330-334 (1981). JIAO, J. -A., and CHOLLET, R.: Regulatory seryl-phosphorylation of C4 phosphoenolpyruvate carboxylase by a soluble protein kinase from maize leaves. Arch. Biochem. Biophys. 269, 526-535 (1989). KOTTMEIER, C. M., and SCHNABL, H.: The Km-value of phosphoenolpyruvate carboxylase as an indicator of the swelling state of guard cell protoplasts. Plant Sci. 43, 213-217 (1986). LATZKO, E., and KELLY, G. K.: The many faceted functions of phosphoenolpyruvate carboxylase in C3 plants. Physiol. Veg. 21, 805-815 (1983). LOUGHMAN, B. C., RATCLIFFE, R. G., and SOUTHON, T. E.: Observation on the cytoplasmic and vacuolar orthophosphate pools in leaf tissues using in vivo 31 P-NMR spectroscopy. FEBS Lett. 242, 279-284 (1989). MEYER, C. R., RUSTIN, P., and WEDDING, R. T.: A simple and accurate spectrophotometric assay for phosphoenolpyruvate carboxylase activity. Plant Physiol. 86, 325- 328 (1988). MICHALKE, B., and SCHNABL, H.: The status of adenine nucleotides and malate in chloroplasts, mitochondria and supernatant of guard cell protoplasts from Vida faba. J. Plant Physiol. 130, 243-253 (1987). MICHALKE, B., and SCHNABL, H.: Modulation of the activity of phosphoenolpyruvate carboxylase in K+ -induced swelling guard cell protoplasts of Vida faba L. after light and dark treatment. Planta 180, 188-193 (1990). MUKERJI, S. K.: Com leaf phosphoenolpyruvate carboxylases. The effect of divalent cations on activity. Arch. Biochem. Biophys. 182,352-359 (1977). NAKAMOTO, H., Ku, M. S. B., and EDWARDS, G. E.: Photosynthetic characteristics of C3-C4 intermediate Flaveria species. 11. Kinetic properties of phosphoenolpyruvate carboxylase from C3, C4 and C3-C4 intermediate species. Plant Cell Physiol. 24, 1387-1393 (1983). O'LEARY, M. H.: Phosphoenolpyruvate carboxylase: an enzymologist's view. Annu. Rev. Plant Physiol. 33, 297-315 (1982). OUTLAW, W. H. Jr.: A descriptive evaluation of quantitative histochemical techniques based on pyridine nucleotides. Annu. Rev. Plant Physiol. 31, 299-311 (1980). OUTLAW, W. H. Jr.: Carbon metabolism in guard cells. In: Cellular and Subcellular Localization in Plant Metabolism (Eds. CREASY, L. L., and HRAZDINA, G.) pp. 185-222. Plenum Press, New York 1982. OUTLAW, W. H. Jr.: Current concepts on the role of potassium in stomatal movements. Physiol. Plant. 59,302-311 (1983). OUTLAW, W. H. Jr.: An introduction to carbon metabolism in guard cells. In: Stomatal Function (Eds. ZEIGER, E., FARQUHAR, G. D., and COWAN, I. R.) pp. 115-123. Stanford Univ. Press, Stanford, CA 1987. OUTLAW, W. H. Jr.: Potential importance of metal-ligand interactions in enzyme assays demonstrated with the assay cocktail for phosphoenolpyruvate carboxylase. Plant Physiol. 92, 528-530 (1990). OUTLAW, W. H. Jr., and KENNEDY, J.: Enzymic and substrate basis for the anaplerotic step in guard cells. Plant Physiol. 62, 648-652 (1978). OUTLAW, W. H. Jr., and LOWRY, O. H.: Organic acid and potassium accumulation in guard cells during stomatal opening. Proc. Natl. Acad. Sci. (USA) 74, 4434-4438 (1977).

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OUTLAW, W. H . .Ir .. MANClIESrER, .I., and DICAMELU, C. A.: Histochemical approach to properties of Viciafaba guard cell phosphoenolpyruvate carboxylase. Plant Physiol. 64, 269-272 (1979). OUTLAW, W. H. Jr., SPRINGER. S. A., and TARCZYNSKI, M. C.: Histochemical technique: a general method for quantitative enzyme assays of single-cell "extracts" with a time resolution of seconds and a reading precision of femtomoles. Plant Physiol. 77, 659-666 (1985). PENNY, M. G., and BOWLING, D. J. F.: Direct determination of pH in the stomatal complex of Commelina. Planta 122,209-212 (1975). RASCHKE, K., HEDRICH. R., RECKMANN, U., and SCHROEDER, J. 1.: Exploring biophysical and biochemical components of the osmotic motor that drives stomatal movements. Bot. Acta 101,

283-294 (1988). SCHNABL, H.: The key role of phosphoenolpyruvate carboxylase during the volume changes of guard cell protoplasts. Physiol. Veg. 21, 955-962 (1983). SCHNABL, H.: Regulation of volume changes in guard cell protoplasts. In: The Physiological Properties of Plant Protoplasts (Ed. PILET, P. E.) pp. 162-170. Springer-Verlag, Heidelberg 1985. SCHNABL, H., and KOTTMEIER, H.: Determination of malate levels during the swelling of vacuoles isolated from guard-cell protoplasts. Planta 161, 27 - 31 (1984). SCHROEDER, J. I., RASCHKE, K., and NEHER, E.: Voltage dependence of K+ channels in guard-cell protoplasts. Proc. Natl. Acad. Sci. (USA) 84, 4108-4112 (1987). SMITH, F. A., and RAVEN, 1. A.: Intracellular pH and its regulation. Annu. Rev. Plant Physiol. 30,

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(1989).

TARCZYNSKI, M. c., and OUTLAW, W. H., Jr.: Partial characterization of guard-cell phosphoenolpyruvate carboxylase: kinetic datum collection in real time from single-cell activities. Arch. Biochem. Biophys. 280,153-158 (1990). TING, 1. P., and OSMOND, C. B.: Photosynthetic phosphoenolpyruvate carboxylases. Characteristics of the alloenzymes from leaves of C 3 and C 4 plants. Plant Physiol. 51, 439-447 (1973). WEDDING, R. T., RUSTIN, P., MEYER, C. R., and BLACK, M. K.: Kinetic studies of the form of substrate bound by phosphoenolpyruvate carboxylase. Plant Physiol. 88, 976-979 (1988). WEYERS, 1. D. B., FITZSIMONS, P. 1., MANSEY, G. M., and MARTIN, E. S.: Guard cell protoplasts aspects of work with an important new research tool. Physiol. Plant. 58, 331- 339 (1983). WILKINSON, G. N.: Statistical estimations in enzyme kinetics. Biochem. J. 80, 324-332 (1961). WILLMER, C. M.: Phosphoenolpyruvate carboxylase activity and stomatal operation. Physiol. Veg. 21,

943-953 (1983). WILLMER, C. M., JAMIESON, A., and BIRKENHEAD, K.: Leaf epidermal tissue is unsuitable to use for studying biochemical aspects of stomatal functioning. Plant Sci. 52, 105-110 (1987). Author's address: Dr. BILL OUTLAW, BlO, B-157, Florida State University, Tallahassee, FL 32306-3050, U.S.A.

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