Plant Physiol. (1987) 85, 0850-08550032-0889/87/85/0850/05/$01 .00/0

Effects of Temperature on H+ Secretion and Uptake by ExcisedFlexor Cells during Dark-Induced Closure of Samanea Leaflets'

Received for publication March 23, 1987 and in revised form July 29, 1987

RUTH L. SATTER*, YUJIA XU, AND ANTHONY DEPASSDepartment ofCell and Molecular Biology, U-42, University ofConnecticut, Storrs, Connecticut 06268

ABSTRACrf MATERIALS AND METHODS

Previous studies reveal that dark-induced closure of Samanea leafletsis accompanied by H' secretion from flexor motor cells. We now reportthat flexor tissue excised in the light, incubated in a weakly bufferedbathing solution, and then darkened at different temperatures (18°C-30°C) acidified the medium (indicating net H' efflux) at all temperaturestested, but most rapidly at the highest temperature. However, pH changesreversed direction after 20 to 70 minutes; the lower the temperature, thelater pH reversal occurred, and the lower the pH at reversal and after 45minutes. These data provide a basis for the previously reported promotiveeffect of low temperature on dark-induced leaflet closure, assuming netH' and K' fluxes are opposite in direction. Net H' efflux at all temper-atures tested was greater when the impermeant molecule iminodiacetatereplaced small permeant anions in the bathing solution, suggesting thatH' uptake is coupled to anion uptake, probably via a Hf/anion symportsystem. When permeant anions were deficient, the amount of malate inthe tissue increased, presumably by new synthesis. Malate synthesiswould substitute for H /anion uptake in charge balance and in providingHW for cytoplasmic pH regulation.

Samanea saman is a nyctinastic legume with paired leaflets(pinnae and pinnules) that usually separate from each other(open) in the light and fold together (close) in the dark (22).Dark-induced closure is dependent upon loss of K+, Cl-, andwater from extensor cells and uptake of ions and water by flexorcells in the pulvinus (23). The K+ and Cl- fluxes, in turn, areaccompanied by oppositely directed H+ fluxes, i.e. H+ is takenup by extensor cells and is extruded from flexor cells (8).H+ efflux from flexor cells is dependent on 02 and inhibited

by KCN (8), consistent with an outwardly directed H+ pumpand leading one to predict increase in H+ efflux with increase intemperature. However, previous unpublished investigations inour laboratory revealed an unexpected response to temperature.Flexor tissue that was excised from a plant in the light, incubatedin a bathing solution, and then darkened for 45 min, acidifiedthe medium more at 18C than at 30C. In the present study, weexamined the effects of temperature on H+ fluxes in greaterdetail, monitoring pH of the medium continuously during thedark period, rather than only at its end. Our data provideevidence that two H+ transport systems operate over a broadtemperature range: a H+-secreting pump, as described in (8), anda co-transport system for coupled H+/anion uptake.

' Supported by National Science Foundation grant DMB 83-04613 toR. L. S.

Plant material. Samanea saman plants were grown from seedin a controlled chamber with a 16 h photoperiod (200 ,gmol m-2s-') at 260 C ± 1.50 C. Flexor tissue was as harvested fromterminal secondary pulvini of the third to eighth mature leaf,counting from the apex (8). Tissue from 12 pulvini (mean dryweight = 15.6 mg) was used for each experimental treatment.Whenever practical, we compared the effects of only two

treatments, using one member of each pair of 12 pulvini for onetreatment, and the other member of each pair for the othertreatment (Figs. 2-5; Tables I and IIB, and IDA2 and C1-treatments in Table IIA). The dry weight of flexor strips excisedfrom paired pulvini varied from one another by only 2 to 3%,whereas flexor strips excised from pulvini from different plantsvaried from one another by up to 33%. In Figure 1, where theeffects of four different treatments were compared, variabilitydue to differences in pulvinar age and physiology were minimizedby using pulvini from four plants, and including three flexorstrips from each plant and two strips from pulvini of each age(i.e. third through eighth leaf) in each group of 12 pulvini.Measurements of pH and H' flux. Flexor tissue was excised

at h 4 to 7 of the photoperiod, washed in bathing solution inwhite light for 10 min at room temperature, blotted dry, andthen transferred to 0.5 ml of fresh bathing solution and darkenedfor 40 to 80 min at 18, 22, 26, or 30C. All manipulationsconducted during the 'dark' period were performed using dimgreen 'safe-light' (8). The temperature was controlled to ±0.5°Cwith a circulating, constant temperature water bath. The bathingsolution contained 1.0 mM Mes, 1 mM Ca(NO3)2, 5 mm K2SO4,and 0.2 M mannitol (control solution), except for Figure 4 andTables I and II, where IDA, Cl-, or NO3 replaced the otheranions. The initial pH was adjusted to 5.5 with HCI (mostexperiments) or IDA (Fig. 4; Tables I and II). Mannitol wasincluded to lower the osmotic potential of the bathing solutionto a value close to (although more negative than) that measuredin vivo (6). Oxygen was bubbled through the medium continu-ously, except when replaced by N2 (Fig. 2). The pH ofthe bathingsolution was monitored continuously with a miniature pH elec-trode (model 410 Microelectrodes, Inc., Londonderry, NH) andrecorded on a chart recorder. In some experiments (Fig. 3; TablesI and II), the pH of the medium was rapidly titrated back to 5.5with KOH at the end of the experiment to determine net H+efflux. In other experiments, the pH was maintained close to 5.5during the first 20 min (Fig. 5A) or during the entire measure-ment period (Fig. 4) by titration with KOH or HG at regularintervals.

Inhibitors. Vanadate in the form of Na3VO4 was dissolved ina small amount of water and heated above 38C for 3 h, cooled,and then added to the medium (16) to a final concentration of

2Abbreviations: IDA, iminodiacetate; DCCD, N,N'-dicyclohexylcar-bodiimide; DES, diethylstilbestrol.

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EFFECTS OF TEMPERATURE ON H+ SECRETION AND UPTAKE IN SAMANEA

0.5 mm or 1.0 mm. DCCD and DES were each dissolved inabsolute ethanol and added to the medium to a final concentra-tion of 0.05 mm (DCCD) or 0.07 mm (DES). The concentrationof ethanol in the final solution was 1%. Controls contained thesame concentration of ethanol as the experimental solutions. Inall experiments with inhibitors except that shown in Figure 3C,the tissue was preincubated in the light in the inhibitor for 15min prior to darkening, to allow time for diffusion of the chem-ical to inner cells.

Determination of Malate Levels. Malate was extracted fromgroups of 12 flexor strips, following a modification ofthe methoddescribed in (26). The tissue was either (a) excised in the lightand then rapidly immersed in 5 ml of alkalinized, 80% ethanol(zero time controls), or (b) excised in the light, and then incu-bated in the dark or the light for 45 min (in a bathing solutioncontaining Cl- or IDA as the only anion) prior to immersion inethanol. The tissue was boiled in the ethanol for 20 min, ho-mogenized (glass on glass), and then centrifuged at 750g for 5min. The supernatant was recovered and the residue reextractedin 5 ml of alkalinized ethanol (these steps were repeated twice).The supernatant fractions were pooled and evaporated to drynessat 60°C under partial vacuum, and the residue was resuspendedin 1 ml of4.25% (v/v) hydrazine buffer (pH 9). The 1 ml sampleswere microfuged at 840g to remove insoluble materials.

Malate was assayed by modifications of the assays describedin Refs. 5 and 7. The assay depends upon the oxidation of malateto oxaloacetate accompanied by stoichiometric reduction ofNAD to NADH, determined spectrophotometrically. The pro-cedure was as follows: a 100 1l aliquot of each 1 ml sample wasincubated with 100 ,l of 80 mM f,-NAD, 1.25 ml 4.25% hydra-zine buffer (pH 9), and 10 ,u malate dehydrogenase at roomtemperature (25°C) for 70 min, using partial vacuum to speedup the removal of bubbles that formed during the incubation.The absorbance of each experimental solution at 340 nm wascompared to that of a solution that was similar, except it lackedmalate dehydrogenase. The amount of malate/12 flexor stripswas determined from a standard curve. Each value in Table IIrepresents the average of three determinations.

Presentation of Data. All experiments were performed at leastthree times, with similar results. Our pH tracings (obtained witha chart recorder and re-drawn for appropriate magnification)represent typical rather than averaged data, since pH is based ona logarithmic scale and thus averaged values are difficult tointerpret. Variability in the pH value and the time at which thepH changes reversed direction provide an indication of thevariability between replicate experiments: the pH varied from4.95 to 5.25, while the period of dark incubation varied from 18to 26 min (data from experiments conducted at 30°C, with acontrol bathing solution).

Sources of Reagents. DCCD and Na3VO4: Fisher; DES, IDA,NAD, and malate dehydrogenase: Sigma.

RESULTSChanges in pH of the Medium Bathing Flexor Tissue Incu-

bated at Different Temperatures. In our first experiments, wedetermined the kinetics of pH changes at 18, 22, 26, and 30°Cduring an 80 min dark period (Fig. 1). Changes in pH of themedium during the first 4 to 5 min of darkness (stage 0) werehighly variable, probably due to exchange of H+ between themedium and negative sites in the cell wall. During stage 1, flexortissue acidified the medium at a rate that increased with increasein temperature. The pH changes reversed direction after 17 to70 min of darkness, leading to the onset of stage 2 (alkaliniza-tion). The lower the temperature, the later stage 2 began. Fur-thermore, the pH at reversal and the pH after 70 min ofdarknesswere both lowest (i.e. acidification was maximal) at the lowesttemperature.

FIG. 1. Effects of temperature on the pH of a solution bathing flexortissue excised at h 5 of the photoperiod, temperature = 26°C, and thendarkened at the indicated temperature.

FIG. 2. Effect of anaerobiosis on the pH of a solution bathing flexortissue excised at h 5 ofthe photoperiod and then darkened at 18'C (upperpanel) or 30°C (lower panel).

Anaerobic conditions prevented acidification at both 18 and30°C (the latter confirming results reported in Ref. 8), but theseeffects were reversible when aerobic conditions were resumed(Fig. 2). These results indicate that aerobic metabolism, probablyinvolving ATP synthesis, was required for acidification over thetemperature range tested. After 02 was supplied, the pH changesat both temperatures assumed their usual time courses, i.e. aperiod of acidification that lasted about 70 min at 18°C andabout 20 min at 30°C, and was followed by alkalinization (30°Cdata in Fig. 2; 180 C data not shown). Imposition of anaerobicconditions from the end of stage 1 through stage 2 had no effecton pH patterns (data not shown), implying that H+fluxes duringstage 2 do not depend upon ATP synthesis.The H' Pump. We tested the effects of three different pump

inhibitors, since any one may have side effects. DCCD, a blockerof H+-ATPases (13), reduced acidification at both 18 and 30°Cat 0.05 mM (Fig. 3, A and B), implying that the H+pump makesa major contribution to medium acidification over the temper-ature range tested. In these experiments, the tissue was preincu-bated in the inhibitor for 15 min prior to darkening, to permitadequate time for uptake and diffusion of the chemical throughthe tissue. To determine whether the H+pump was also active

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Plant Physiol. Vol. 85, 1987

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FIG. 3. Effects of various inhibitors on the pH of solutions bathingflexor tissue excised at h 5 of the photoperiod and then darkened. Thetissue was preincubated in the inhibitor in the light for 15 min immedi-ately prior to darkening for all treatments except in panel C. The pH ofthe medium was back titrated to 5.5 with KOH at the end of eachexperiment to estimate the number of H+ equivalents released into themedium. (A-C): DCCD (0.05 mM), at (A) 18°C and (B-C) 30C, (B)with and (C) without preincubation. (D): DES'(0.07 mM), 30C. (E-F):NA3VO4, 26°C, at (E) 0.05 mm and (F) (1.0 mM). Inhibitor (- -);control (con) (-); addition of inhibitor (./). The number at the rightend ofeach tracing indicates nanoequivalents ofOH- (supplied as KOH)required for back titration to pH = 5.5.

during stage 2, in our next experiment we added DCCD to thebathing solution about 15 min prior to the end of stage 1 (i.e. atthe beginning of the dark period at 30C). The inhibitor wasineffective under these conditions (Fig. 3C), suggesting that pumpactivity ceases at the end of stage 1.DES, another H+ ATPase inhibitor (24), also reduced acidifi-

cation at 30C at 0.07 mM (Fig. 3D); effects were not tested at18C, since solubility was poor at this temperature. Na3VO4,inhibitor of plasma membrane-localized ATPases (2), reducedacidification at 26C (Fig. 3, E and F), although high concentra-tions (0.5-1.0 mM) were required, possibly due to limited uptakeof the chemical through the plasma membrane. We did not testeffects of Na3VO4 at lower temperatures, since the high concen-tration required at 26C raised questions about the inhibitor'smode of action.

Effects of Permeant and Nonpermeant Anions on H' Fluxes.The swelling of flexor cells during dark-induced leaflet closure isaccompanied by the uptake of K+, Cl-, and other anions (20). Ifanion uptake is coupled to H+ uptake, as proposed in Satter andGalston (21), the presence of permeable anions in the bathingsolution should promote H+ uptake. To test this possibility, weexamined H+ flux patterns when the bathing solution containedsmall permeable anions as compared to the large impermeantanion IDA. Since IDA increased the buffering capacity of thebathing solution substantially, we obtained data on H+ fluxesrather than pH changes.Net H+ efflux during a 45 min dark period was greater when

the bathing solution contained IDA rather than Cl- or NO3- asthe only anion (Table I), suggesting that the uptake of smallanions is accompanied by H+ uptake. To determine whether theavailability of small anions promotes H+ uptake during stage 1,stage 2, or both, we titrated the pH ofthe bathing solution to 5.5at regular intervals during the entire dark period, determiningthe amount ofOH- required for back titration when the solutioncontained IDA as compared to the permeable anions in thecontrol solution (Fig. 4). Net H+ efflux was higher in the IDAsolution during stage 1, but IDA had only a minimal effectduring stage 2, whether experiments were conducted at 18C,(stage 1 persists for about 70 min) or at 30C (stage 1 persists forabout 20 min).Malate Levels in Flexor Tissue Incubated in a Solution Con-

taining IDA as Compared to C1-. If the increase in net H+ effluxin the IDA-containing solution is indeed attributable to a defi-ciency of small anions, one might expect that malate levels inthe tissue would be higher when small anions are deficient, asoccurs in stomatal guard cells (26). Data in Table IIA supportthis prediction; malate levels were 30% higher when flexor tissuewas incubated in the dark in a solution containing IDA ratherthan Cl- or N03 as the only anion. Although the amount ofmalate in tissue that was darkened and incubated in the IDA

Table I. Effects ofSpecific Anions in the Bathing Solution on H+Effluxfrom the Tissue

Flexor tissue was excised at h 5 of the photoperiod and incubated inthe dark for 45 min at 26°C in a bathing solution containing only oneanion, initial pH = 5.5. Data represent neq OH- required for backtitration to 5.5 at the end ofthe experiment. Data from three experimentscomparing IDA and NO3- and three comparing IDA and Cl- are shown.The concentration of IDA (20 mM) was higher than that of NO3- andC1- (each 14 mM), since IDA was used for pH adjustment in addition toreplacing other anions in K+and Ca2" salts.

IDA Cl- IDA/Cl- IDA NO3- IDA/NO3-OH- neq ratio OH- neq ratio28 15 1.9 25 15 1.715 10 1.5 38 20 1.910 5 2.0 20 10 2.0

852 SATTER ET AL.

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EFFECTS OF TEMPERATURE ON H+ SECRETION AND UPTAKE IN SAMANEA

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FIG. 4. Number of nanoequivalents of OH- (cumulative) required tomaintain the pH of the bathing solution close to 5.5, as a function oftime the tissue was incubated in the dark, at 18C (upper panel) and 30°C (lower panel). KOH was used for titration where values of the ordinateincrease, and HCI was used where values decrease.

Table II. Amount ofMalate (Amol/12 Flexor Strips) in TissueIncubated in a Bathing Solation Containing IDA or Cl- as the Only

AnionThe tissue was excised in the light at h 6 of the photoperiod and

incubated in bathing solution for 45 min at 26C, either (A) in the dark,or (B) in the light. Zero time controls, from replicate tissue excised inthe light, were not incubated. (The pulvini used for part B were muchlarger than those used for part A, leading to higher values for the controlin B than in A.)

Experiment C* IDA Cl- Cl-/C IDA/C IDA/C1-malate (Mmol/12 flexor strips) ratiosAI 1.13 1.17 0.95 0.84 1.04 1.232 0.85 0.90 0.62 0.73 1.06 1.453 0.79 1.10 0.87 1.10 1.40 1.274 1.08 0.84 0.67 0.62 0.78 1.24

Experiment C* Cl- Cl-/Cmalate (jumol/12 flexor strips) ratioB1 1.94 1.50 0.772 1.83 1.48 0.80

* Zero time control.

solution for 45 min was not significantly higher than that ofcontrol tissue that was not darkened or incubated in bathingsolution, this may be a consequence of incubating excised tissuein a bathing solution, for the malate level decreased when flexor

tissue was incubated in the light for 45 min (Table IIB). Loss ofmalate from tissue incubated in the light might be due to leakagefrom the apoplast to the bathing solution.

Effect of Maintaining Constant pH during Phase 1. In ournext experiments, the pH was maintained close to 5.5 for thefirst 25 min of darkness by repeated back titration with KOH,or by incubating the tissue in a large volume of solution (Fig. 5).Note that alkalinization began at almost the same time as in thecontrols, even though the external pH did not attain its usuallow value.

DISCUSSION

During a previous investigation in our laboratory using anexperimental system similar to the one described herein, weconsidered several possible artifacts that might effect the relation-ship between changes in pH of the bathing solution and H+fluxes through Samanea motor cell membranes (8). We con-cluded that, at all times after stage 0, the possible release ofmalate or other weak organic anions was the only reaction ofthose we considered that would be expected to alter pH withoutaffecting H+ fluxes. Let us consider interconversions betweenmalate and malic acid:

COOH

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COOH

/oo

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FIG. 5. Effect of maintaining the pH close to 5.5 for 30 min on

subsequent changes in pH of a solution bathing flexor tissue excised at h5 of the photoperiod and then darkened. The pH was maintained closeto 5.5 during the first 30 min by back titration (indicated by arrows) withKOH at 3 to 4 min intervals (upper panel), or by incubating the tissuein a large (15 ml) volume of bathing solution (lower panel). The arrow

(lower panel) indicates the time when the tissue was transferred to a

small vial containing 0.5 ml of pH = 5.4 solution. The temperature was

26°C in both experiments.

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853

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Plant Physiol. Vol. 85, 1987

The doubly dissociated form would predominate in the cyto-plasm (pH close to 7.2), but an appreciable amount would be inthe singly dissociated form in the bathing solution, particularlyat low pH. This would lead to an underestimate ofH+ secretionthrough the pump during stage 1 and an overestimate of H+uptake during stage 2.H' Transport Systems. The direction and rate of net H+ flux

through flexor cell membranes depends upon the dynamic equi-librium between H+ secretion through the pump and H+ uptakethrough various transport systems. The pump requires aerobicconditions (Fig. 2; Ref. 8), presumably for ATP synthesis, isinhibited by DCCD and DES (Fig. 3, A, B and D), and functionsover a broad temperature range (1 8-30°C). H+ secretion throughthe pump is masked in part by H+ uptake through the H+/anionco-transporter, which also peaks during stage 1 (Fig. 4).H+ is taken up through a number of pathways: (a) a leakage

pathway that exists in all types of cells and is enhanced by injury(3), as occurs during excision; (b) a H+/sucrose co-transportsystem that functions in Samanea pulvinar cells at defined timesduring the circadian cycle (17) but would not have an appreciableeffect on H+ fluxes in the experiments reported here, sinceendogenously generated sucrose would be diluted by the bathingsolution; (c) a transport system that promotes the uptake of C1-or other small anions (10, 23) and alkalinizes the cellular exterior(Fig. 4; Table I); and (d) possibly, a symport system that co-transports H+ and K+ into the cell, as described recently forNeurospora (18), although we did not test this possibility. Wewill focus here on (c), the transporter that alkalinizes the mediumin the presence of small anions and functions during stage 1 (Fig.4), i.e. when pump activity is high.H+/anion co-transport (or OH-/anion counter-transport,

which is functionally similar) provides one possible explanationfor data in Figure 4 and Table I. This type of transporter wouldutilize energy stored in the transmembrane pH gradient to poweranion uptake. It has been studied most thoroughly in the giantalga Chara corallina, where electrophysiological data and ther-modynamic considerations led Sanders and Hansen (19) andSmith and Walker (25) to conclude that two H+ are co-trans-ported with one Cl-. Thus in Chara, H+/anion co-transportacidifies the cytoplasm, alkalinizes the apoplast, and depolarizesthe cell.HCO3- efflux coupled to anion uptake would also acidify the

cytoplasm and alkalinize the apoplast during anion uptake underour experimental conditions, if such a transporter were presentin flexor cell membranes. In red blood cell plasma membranes,HCO3- generated by respiration is transported out of the cell inexchange for Cl- via a HCO3 /Cl- countertransport system (14).HCO3- forms the major buffering system in the extracellularfluid. Interconversions between dissolved C02, H2CO3, HCO3-,and C022- are as follows:

CO2 (dissolved) + H20 = H2CO3 - H+pK=3.8

+ HCO3- - 2H+ + C032-pK= 10.2

Based on pH considerations, one would expect most of thedissolved CO2 released from Samanea motor cells during dark-induced closure to be in the HCO3- form, both in the cytoplasmand in the bathing solution. However, if flexor cell membranescontained a HCO3 /anion exchanger, as in red blood cells,continuous rapid bubbling of 02 in the bathing solution (asoccurs in our experiments) would displace CO2 and drive reac-tions in the bathing solution to the left, leading to low HCO3-.This, in turn, would increase the transmembrane HCO3: gradientand promote outward HCO3- transport through the HCO3 /anion exchanger, thereby alkalinizing the external solution.Thus, effects on pH of the bathing solution would be indistin-

guishable from those of H+/anion co-transport, using the meth-ods we have described. However, measurements of pH in theflexor apoplast that were made in situ without bubbling of 02(12), reveal dark-induced changes in pH similar to those de-scribed for stage 1 (Fig. 1), i.e. acidification ceases after 25 minat 26°C. Thus, conditions that would drive outward HCO3-transport are not required for the termination of stage 1. Fur-thermore, although inward HCO3- transport through the plasmamembrane has been proposed for the alga C. corallina (15),outward HCO3- transport coupled to inward anion transport hasnot been described in algae or higher plant cells, to the best ofour knowledge. Thus, H+/anion co-transport, rather thanHCO3/anion exchange, provides the most reasonable explana-tion for our data. Of course, it will be necessary to examine Cl-uptake in a more defined system (e.g. plasma membrane en-riched vesicles) before drawing definitive conclusions.

Role of Malate. Malate (1; A DePass, R L Satter, unpublisheddata), Cl- (23), and NO3- (1O) serve as counterions for K+ duringmovements of Samanea and Phaseolus pulvini. When a defi-ciency of small anions prevented anion uptake, malate levels inthe tissue increased (Table II), presumably by new synthesis.Increase in malate synthesis would serve two functions normallyserved by coupled H+/Cl- uptake: it would provide H+ ions forcytoplasmic pH regulation (4), and it would replace Cl- inbalancing the charge of K+.

Transition from Stage 1 to Stage 2: How is it Regulated? Weconsidered whether the change from acidification of the bathingsolution to alkalinization after a certain period in darkness hadphysiological significance, or whether it was merely a conse-quence of excising the tissue and submerging it in a bathingsolution. These experimental procedures would be expected topromote alkalinization, both by increasing inward H+ leakage,since it is enhanced by injury (3), and by leakage of malate fromthe apoplast to the bathing solution (Table IIB). However, theseexperimental procedures cannot provide the major explanationfor the end of stage 1, since: (a) extensor tissue that was excisedand submerged in a bathing solution acidified the mediumcontinuously for more than 75 min at 26 or 30°C (1 1); and (b)dark-induced acidification of the flexor apoplast was transitorywhen pH was measured in situ, even though alkalinization duringstage 2 was minor under these conditions (12).Coupled H+/anion transport is maximal during stage 1 rather

than stage 2 (Fig. 4), and thus cannot be responsible for thetransition from stage 1 to stage 2. Alternatively, if the pump wereturned off (or if its activity decreased sharply) after a certainperiod of darkness, this would signify the end of stage 1. Datarevealing that DCCD (Fig. 3C) and anaerobiosis (data not shown)have no effect on pH of the bathing solution during stage 2 areconsistent with this interpretation, as is the cessation of pumpactivity in situ (12), discussed above. Thus, it seems likely thatthe pump becomes inoperative after a dark period that rangesfrom 20 min at 30°C to 70 min at 18°C (Fig. 1), although otherprocesses such as leakage of H+ and/or malate might contributetoward alkalinization during stage 2.We considered two possible explanations for the cessation of

pump activity. (a) It might depend upon attainment of a lowexternal pH during stage 1, since external pH acts as a regulatorof H+ pump activity in other systems (9). Data in Figure 5 areat variance with this interpretation. Stage 2 began at close to itsusual time when external pH was maintained at its initial value(5.5) during the first 20 to 25 min of darkness at 30°C (Fig. 5).(b) It might depend upon an internal timing process that isinitiated by darkness. This interpretation is consistent with ourdata, assuming the timing process is temperature dependent (Fig.1) and requires aerobic conditions (Fig. 2). The nature of thetiming process remains to be determined.

Temperature Compensation of HU Fluxes. The H+ pump in

854 SATTER ET AL.

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EFFECTS OF TEMPERATURE ON H+ SE

flexor cells has a Q,o > 1.0, in accord with ion pumps in othercell types. Thus, one might expect a priori that acidification ofthe medium would be greater at 30C than at 18°C. However,the early termination of pump activity at high temperaturetogether with H+ uptake at all temperatures tested, leads to netH+ efflux that is relatively independent oftemperature at 20 minof darkness, and is promoted by low temperature during longerdark periods (Fig. 1). Thus H+ fluxes in flexor cells are temper-ature compensated during the early part of the dark period andbecome overly temperature compensated during longer darkperiods. Temperature compensation of H+ fluxes in flexor cellswould be expected to have important consequences for leafletmovement regulation. Leaflet closure at low temperature is onesuch consequence (assuming K+ fluxes are opposite to those ofH+), as discussed in Lee and Satter (1 1).

Acknowledgments-We thank Youngsook Lee and Drs. M. J. Morse and R. C.Crain for critical comments on the manuscript.

LITERATURE CITED

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2. BOWMAN BJ, CW SLAYMAN 1979 The effects of vanadate on the plasmamembrane ATPase of Neurospora crassa. J Biol Chem 254: 2928-2934

3. CHASTAIN CJ, JB HANSON 1982 Control of proton efflux from corn root tissueby an injury-sensing mechanism. Plant Sci Lett 24: 97-104

4. DAVIES DD 1986 The fine control of cytosolic pH. Physiol Plant 67: 702-7065. GOLDBERG ND, JV PASSONEAU 1974 L-Malate and fumarate. Fluorometric

determination. In HU Bergmeyer, ed, Methods of Enzymatic Analysis.Academic Press, New York, pp 1600-1603

6. GORTON H 1987 Water relations in pulvini from Samanea saman. I. Intactpulvini. Plant Physiol 83: 945-950

7. GUTMANN I, AW WAHLEFELD 1974 L(-)-Malate determination with malatedehydrogenase and NAD. In HU Bergmeyer, ed, Methods of EnzymaticAnalysis. Academic Press, New York, pp 1585-1589

8. IGLESIAS A, RL SArTER 1983 H+ fluxes in excised Samanea motor tissue. PlantPhysiol 72: 564-569

9. KAWAMURA G, T SHIMMEN, M TAZAWA 1980 Dependence of the membranepotential of Chara cells on external pH in the presence or absence of internal

,CRETION AND UPTAKE IN SAMANEA 855

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