Plant Physiol. (1987) 85, 239-2420032-0889/87/85/0239/04/$0 1.00/0

Light Induced Polarity of Redox Reactions in Leaves ofElodea canadensis Michx.'

Received for publication October 29, 1986 and in revised form May 26, 1987

J. THEO M. ELZENGA* AND HIDDE B. A. PRINSUniversity ofGroningen, Department ofPlant Physiology, P. 0. Box 14, 9750AA Haren, The Netherlands

ABSTRACT

This paper reports that extraceliular reductase activity in leaves ofElodeca densis, hitherto never associated with polar processes thoughtto be involved in bicarbonate utilization, also shows a very markedpolarity in light. The effect of ferricyanide, applied to the lower side ofilluminated leaves, was a depolarization of the membrane electricalpotential of up to 110 millivolts, while no depolarization was inducedwhen ferricyanide was applied to the upper side. In the dark ferricyanideinduced a depolarization when applied to either the upper or to the lowerside of the leaf. Staining with tetrazolium salts, specific indicators forreductase activity, resulted in the formation of a precipitate on the lowerside of the leaf when illuminated and on both sides in the dark. Theprecipitate was only located along the plasmalemma.

The transport properties of the leaves of submerged aquaticweeds like Elodea canadensis, Elodea nuttalli, Egeria densa, andPotamogeton lucens show a marked polarity. The solution incontact with the morphological lower side of the leaf becomesacid upon illumination, while the pH increases on the upperside. At the same time, the upper side becomes electricallynegative compared with the lower side and there is a net transportof cations from the lower to the upper side (5, 20). It has beenproposed that these processes have a function in photosyntheticutilization of bicarbonate, namely that the proton gradient overthe plasmamembrane on the lower side could energize a carrierfacilitated HCO3 influx ( 11) or the release ofprotons might shiftthe bicarbonate-CO2 equilibrium to the CO2 side and so raisethe CO2 concentration in the apoplast and unstirred layer andincrease the diffusion of CO2 into the cell (21). Central to bothproposed mechanisms is the operation of a light dependentproton extrusion pump located at the plasmalemma of the cellsin the lower epidermis. A similar function has been proposed forthe acid and alkaline bands in Chara (10). There are also ana-tomical differences between the upper and lower sides. On thelower side the epidermal cells are often transfer cells (3). Transfercells are characterized by ingrowths of cell wall material whichincreases the surface area ofthe plasmalemma (18). Furthermore,they contain numerous mitochondria and chloroplasts. In sub-merged aquatic plants these cells are thought to play a role insolute exchange (4). The role of a proton pumping ATP-ase inthe establishment of a proton electrochemical gradient is gener-ally accepted (25). There are, however indications that electrontransport may also occur in plasmamembranes and have a

'Supported by the Foundation for Fundamental Biological Research(BION) which is subsidized by the Netherlands Organization for theAdvancement of Pure Research (ZWO). This is an Ecotrans publication.

function in generating the electrochemical proton gradient (17)and in the uptake of nutrients (8, 14, 16). Novak and Ivankina(17) concluded from their experiments with E. canadensis thata redox system functions in the plasmalemma which transportsprotons out of the cell and utilizes intracellular oxygen as thefinal electron acceptor. This charge separation has a hyperpolar-izing effect on the AE.2 Another redox system has been describedby Sijmons et al. (24). In that system, extracellular Fe3+ is thenatural electron acceptor and it functions in roots of iron defi-cient bean plants to make the iron available for uptake. When aredox system functions in the plasmalemma, the reduction ofanexternal electron acceptor can induce a depolarization ofthe AE.Ivankina et al. (6) found that the reduction of externa ferricya-nide induces a depolarization of the AE of Elodea leaf cells. Asimilar depolarization could be demonstrated in root cells ofirondeficient bean plants after application of ferricyanide or DCPIP(24).The aim ofthe present study was to investigate the relationship

between the redox processes described by Novak and Ivankina(17) and the above mentioned polarity. The effect of an electronacceptor, applied to one side of the leaf only, on the AE in lightand in darkness was, therefore, determined. Furthermore, thelocalization ofplasmalemma bound redox reactions was studiedwith both light and EM techniques, using tetrazolium salts.

MATERIALS AND METHODSElodea canadensis Michx. was grown inside in concrete tanks

on a clay substrate covered with 2 cm ofwashed sand. The tankswere filled with demineralized water. This resulted in low nu-trient levels in the water and a pH between 7.5 and 8.2. The lightregime was 12 h light/ 12 h dark. The light intensity used was220 tmol.m-2.s-'.

Electric Potential Measurements. The experimental solutionused contained a 4 mol m-3 CaCl2 and 1.5 mol m-3 K+(Cl- orHCO3 ). Using an appropriate ratio between KCI an KHCO3the pH was brought to 7.2. An equivalent amount ofK3Fe(CN)6(0.5 mol m-3) instead of the KCl/KHCO3 mixture was used fortesting the effect of ferricyanide. Freshly excised Elodea leaveswere used. The AE of the epidermal cells was measured usingconventional microelectrodes of the Ag-AgCl type, filled with 3.103 mol m-3 KCI, brought to a pH of 2 with HCI. The resistanceof the intracellular electrodes was about 10 Mohm and the tippotential never exceeded 10 mV. Readings were not correctedfor this tip potential. Measurements were performed with bothsides ofthe leafin contact with the experimental solution or withonly the upper or lower side exposed. It was possible to expose

2 Abbreviations: AE, membrane electrical potential; GA, glutaralde-hyde; BSPT, 2-(2'-benzothiazolyl)-5-styryl-3-(4'-phtalhydrazidyl)-tetra-zolium chloride; DCPIP, dichlorophenolindophenol; NBT, 2,2'-di-p-nitro-phenyl-3,3'-(3,3'-dimethoxy-4,4'-biphenylene ditetrazolium chlo-ride (nitro blue tetrazolium).

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ELZENGA AND PRINS

only one side of the leaf to the medium by pressing the leaf in alump of synthetic soft plastic material, Therostat.A K4Fe(CN)6was used in control experiments. The light intensity during thelight period was 460 zmol/m2 .s-'.

Histochemistry. For localization of reductase activity in theEM, BSPT, a tetrazolium salt which gi-ves an osmiophilic difor-mazan when reduced (23) was used and for light microscopyNBT. When reduced, NBT forms a blue precipitate, which isvisible in light microscopy. Young shoots, about 2 cm long wereexcised and incubated in a solution containing 4 mol m-3 CaCl2,1.5 mol m-3 KCI, and 1 mol m-3 NaCl. The pH of the solutionwas brought to 7 with KHCO3. After 1 h at 25°C the shoots weretransferred to a medium with the same composition plus 0.5 molm-3 NBT or 0.2 molm-3 BSPT. In this medium the shoots werekept either in the dark or in the light. The light intensity was 275umol m-2 s-', for 20 min at 25C. As a control the NBTreduction in a strongly buffered solution was examined. Thesimultaneous measurements of the pH on both sides of the leafwere performed as previously described (19). First the pH was

measured in the medium without the buffer. In the light the pHon the upper side was raised to pH 9 and on the lower side waslowered to pH 4.5. This agrees with results previously described(20). Subsequently the medium was replaced by a solution withthe same composition but supplemented with 50 mol m-3 Tris/Tes buffer (pH 9). In this medium no changes in pH at the leafsurface could be measured upon dark/light transitions. The pHmeasured was 9 on both upper and lower sides. NBT was addedto this medium to a final concentration of 0.5molm-3 and theleaf was illuminated for a further 20 min.

Electron Microscopy. The leaves were cut, with a razorblade,into strips1 mm wide and fixed in 2.5% GA in 0.1 103 molm-3cacodylate buffer (pH 7.0) plus 0.2molm-3 MgCl2 and 0.2 molm-3 CaCl2 for 45 min. Subsequently the material was washedtwice in demineralized water and postfixed in 1%OSO4 over-night. The strips were then dehydrated in a graded ethanol/propylene-oxide series and embedded in Epon resin. Serie coupes(gold) were sectioned on a LKB 2088 Ultrotome and examinedin a Philips 201 or 300 transmission electron microscope.

Light Microscopy. The material was treated as for EM, withthe exception that the postfixation withOS04 was omitted. Thesections were cut with a microtome.

RESULTSWhen the leaves were placed in a solution containing 0.5 mol

m3 NBT the leaf stained dark blue within 20 min. This bluecolor is caused by a precipitate of water insoluble diformazanformed after reduction of NBT. Figure 1, A and B, shows thatalthough a precipitate was formed both in the dark and in light,there was a marked difference in the location of the precipitateunder both conditions. In the light a precipitate was only visibleon the lower side of the leaf, while in the dark a precipitate wasformed on both the upper and lower side. In the dark theprecipitate on both sides was less dense than that on the lowerside of illuminated leaves. Thus, the reductase activity parallelsthe other reactions that are polar in the light but not in the dark.It is evident from Figure 2A that the precipitate was located onlyat the plasmalemma. Under the culturing conditions applied,the cells on the lower side developed the cell-wall invaginationstypical for transfer cells (3). The diformazan precipitate at theplasmamembrane was clearly visible and it followed the invagi-nations formed by protuberances of cell wall material. Thisindicates that the reduction of tetrazolium is the result of plas-malemma associated reductase activity. At the plasmalemma ofcells on the upper side of the leaf no precipitate was visible (Fig.2B).

Reduction of an external electron acceptor can induce a de-polarization of the AE (8, 17, 24). In our material ferricyanide

also caused a rapid depolarization of up to 110 mV. Figure 3shows the results of an experiment in which both sides of theleaf were exposed and the leaf was illuminated. The depolarizingeffect of ferricyanide on the AE can be explained by assumingthat where, in a steady state situation the charge flows over themembrane are balanced, the addition of ferricyanide could causean extra flow of electrons over the membrane which is initiallyout of phase with the balancing flows. This then causes a changein the AE. As the change measured is a depolarization theprimary process is the transfer of electrons. After the ferricyanidewas removed from the experimental solution, the AE hyperpo-larized again to approximately its original value. In the series ofexperiments shown here the AE was -225.5 (±3.6 SE, n = 4) mVin the light and -189 (±2.4 SE, n = 4) mV in the dark. Addingferrocyanide instead of ferricyanide, had no effect on the AE. Inorder to expose only one side of the leaf to the solution the leafwas carefully pressed with one side into a piece of synthetic softplastic material. Figure 4, A and B, shows typical results of theseexperiments. The effect of exposing the lower side to the solutionand adding ferricyanide while the leaf was kept in the dark isshown in Figure 4A. Here ferricyanide also induced a depolari-zation of AE (mean depolarization 79 [±2.5 SE, n = 3] mV). Adepolarization was also observed when ferricyanide was admin-istered in the light (Fig. 4A) (mean depolarization 57 [±6.0 SE, n= 3] mV). However, when the upper side was exposed, ferricya-nide still induced a depolarization when applied in the dark(mean depolarization 34 [±4.7 SE, n =4] mV), but when the leafwas illuminated there was no longer any effect (Fig. 4B) (meandepolarization 2.9 [±2.1 SE, n = 5] mV). In some experimentsthe depolarization had a transient character as it was followedby a repolarization. In other experiments the potential remainedat the depolarized level for more than 40 min.The results of the experiments with diformazan precipitation

and the changes of AE induced by ferricyanide indicate thatreductase activity can be demonstrated on the plasmalemma onboth sides of the leaf in the dark but that, when the leaf isilluminated, the reductase activity is no longer apparent on theupper side. The influence of buffering at a high pH on thediformazan reduction was investigated as a control for the pos-sibility that the high pH at the upper side of the leaf duringillumination was inhibiting the reductase activity. The leaf wasincubated in a medium which also contained 50 mol m-3 Tris/Tes (pH 9). This buffer proved effective in eliminating the pHchanges at the leaf surface which were normally measured uponillumination. In this medium the leaf was still stained blue in 20min with the precipitate located mainly at the lower surface ofthe leaf (results not shown).

DISCUSSION

The staining of the plasmalemma with tetrazolium salts andthe depolarization induced by ferricyanide but not by ferrocya-nide indicates trans-plasmamembrane reductase activity in Elo-dea leaves. Previously Novak and Ivankina (17) and Kurkovaand Verkhovskaya (7), also using tetrazolium salts and ferricya-nide, arrived at the same conclusion. In this study we found amarked difference in reductase activity between the upper andlower sides ofthe leafwhen illuminated: a difference that parallelsthe polarity of the leaf with respect to acidification, polar trans-port of cations, a trans-leaf potential difference and the differencein anatomy of the epidermal cells. Transfer cells, which containnumerous mitochondria and chloroplasts and which are thoughtto play a role in short distance transport of solutes, are onlyfound in the lower epidermis. This complex of characteristicssuggests that transport processes predominantly take place onthe lower side of the leaf. The generation of a proton gradient isa prerequisite for the proposed mechanisms of bicarbonate uti-lization and for the transport of other nutrients. This gradient is

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POLARITY OF REDOX REACTIONS IN ELODEA LEAVES

A B10 urM

i

CC

4

FIG. 1. A, Micrograph of a cross-section of an E. canadensis leaf incubated in 4 mol m-3 CaCk2, 1.5 mol m-3 KCI, I mol m-3 NaCl, and 0.5 molm-3 NBT and illuminated for 20 min with a light intensity of 275 Amol/m-2. s-' at 25°C. Arrows indicate the diformazan precipitate. C: chloroplast.B, See (A), except that the incubation with NBT was performed in the dark.

k - A

1 urnim ~B

w.seH\'. * ''pm

FIG. 2. A, Electron micrograph of a section of an epidermis cell atthe lower leaf surface. The material was incubated for 20 min at 25°Cwith a light intensity of 275 Amol.m-2.s-' in a medium similar to thatof Figure 1, except that the NBT was replaced by 0.2 mol m-3 BSPT.Note the invaginations of cell wall material typical for transfer cells andthe precipitate at the plasmalemma. B, Electron micrograph of a sectionof an epidermis cell at the upper leaf surface. See (A). Note the absenceof the precipitate on the plasmalemma (here slightly plasmolysed). PM:plasmalemma, C: chloroplast.

generated mainly on the acidified lower side of the leaf. Sincethe reductase activity in the light is only found on the lower side,it is apparently connected with the transport processeIs. There isa great deal of evidence that the plasmalemma-bound protonpump is fueled by ATP (25). Electron transfer, however, mayalso be involved either indirectly by regulating the ATP-aseactivity (22) or directly by generating H+ transport across themembrane (8). MacRobbie (12) found that both cation andchloride fluxes were stimulated by light, but that only the cationfluxes were stimulated in far red light or in the presence ofDCMU, and concluded that Cl- transport depends on the pro-duction of a reduced component. Loppert (9) and Mimura et al.(13) found that the rate of the ATP-ase driven pump was inde-pendent of the ATP level and concluded that another regulatingfactor was involved. Ivankina et al. (6) found that reduction offerricyanide, and thus transplasmamembrane reductase activity,was also accompanied by proton extrusion. The fact that wefound light-dependent acidification as well as reductase activity

&E,mV0,

-150O

-200

-250

0 30 60 90 min

FIG. 3. Effect of ferricyanide on the AE of E. canadensis cells. Themedium contained 4 mol m-3 CaC12, and 1.5 mol m-3 KCI or KHCO3(pH 7.2). The light intensity was 465 smolm2 s-'. At the point indi-cated by the arrow labeled ferricyanide the KCI/KCO3 was replaced by0.5 mol m-3 K3Fe(CN)6 and at the point labeled ferrocyanide by 0.5 molm-3 K4Fe(CN)6. The whole leaf was in contact with the experimentalsolution.

predominantly on the lower side, does not necessarily mean thata redox system is driving the proton extrusion. The redox systemcould work in parallel with a proton pumping ATP-ase as sug-gested by Lin (8) in corn protoplasts or the electron transportsystem might have a regulating function on the ATP-ase activity(2, 22).The difference in reductase activity between the upper and

lower sides in the light cannot be attributed to a difference indistribution of the redox system, since both sides exhibit activityin the dark. The observation of reductase activity in a solutionbuffered at pH 9 indicates that the absence of reductase activityin the light at the upper side ofthe leafis not caused by inhibitionof the reductase activity by the high pH encountered there. It is,however, possible that the OH- ions excreted at the upper side

CONTROL

I

FERROCYANIDEFERRICYANIDE

241

_100

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ELZENGA AND PRINS

AE,mV

&E, mV

-15

_2C

-250L0 30 60 90 min

FIG. 4. A, Effect of ferricyanide, applied to the lower side of the leaf

only, on the AE of E. canadensis cells. See Figure 3, except that the leaf

is pressed with the upper side in soft plastic material leaving only the

lower side exposed to the experimental solution. Light (465 umol/m-2.s') and dark periods are indicated by the bar at the top. B, Effect of

ferricyanide, applied to the upper side of the leaf only, on the AE of E.

canadensis cells. See Figure 4A, except that only the upper side of the

leaf is exposed to the experimental solution.

inhibit the electron transport over the membrane by lowering

the surface potential (1) and that the protons released at the

lowsr side, by raising the surface potential (an effect that is not

influenced by pH buffering), facilitate the transport of electrons,

and so create the polarity in redox reactions. The polarity which

was found cannot, however, be explained by the high surface

potential on the lower side facilitating the approach of the

negative ferricyanide ions to the membrane and the low potentialon the upper side repelling the ferricyanide ions, since the same

polarity is found with tetrazolium salts which are positivelycharged. Novak and Ivankina (16, 17), Ivankina et al. (6), and

Kurkova and Verkhovskaya (7), using the same Elodea species,do not mention a polarity in reductase activity. Recently we

found that the occurrence of the light induced polar changes in

pH on upper and lower sides of the leaf were dependent on a

low carbon concentration in the growing and experimental me-

diums (results to be published).It is possible that polarity was

not induced under the conditions used by Novak et al.

The ferricyanide induced depolarization of the AE had a

transient character in some experiments while in other experi-

ments the depolarization lasted for over 40 min. This differenceis probably due to the difference in energy charge that can existbetween the leaves used in the different experiments. Novackyet al. (15) and Ullrich-Eberius et al. (26) described how pretreat-ment of the material can influence the rate of AE recovery.

In conclusion, the reductase activity parallels the polar proc-esses, pH difference, electrical potential difference, and net cationfluxes between the upper and lower side, which are normallyassociated with bicarbonate utilization. The reductase activitycould be polar as a result of the change in surface potentialcaused by the extrusion of protons on the lower side. The otherpossibility is that the redox reactions do not follow the polaritybut have a regulating function in the membrane processes gen-erating the polarity. This latter hypothesis is the subject of furtherresearch.

LIERATURE CMTD1. BoRST PAuwELs GWFH 1981 Ion transport in yeast. Biochim Biophys Acta

650: 88-1272. CRAIG TA, FL CRANE 1980 Hormonal control of a transplasmamembrane

electron transport system in plant cells. Proc Indiana Acad Sci 91: 150-1543. FALK H, P SITTE 1963 Zellfeinbau bei Plasmolyse 1. Der Feinbau der Elodea-Blattzellen. Protoplasma 57: 290-3034. GUNNING BES, JS PATE 1969 'Transfer cells'. Plant cells with wall ingrowths,

specialized in relation to short distance transport of solutes-their occur-rence, structure and development. Protoplasma 68: 107-133

5. HELDER RJ, J BOERmA 1973 Exchange and polar transport of rubidium ionsacross the leaves of Potamogeton. Acta Bot Neerl 22: 686-693

6. IVANKINA NG, VA NOVAK, AI MIKLANSHEVICH 1984 Redox reactions andactive H`-transport in the plasmalemma of Elodea leaf cells. In WJ Cram,K Janacek, R Rybova, K Sigler, eds, Membrane Transport in Plants.Academia, Prague, pp 404-405

7. KURKOVA EB, ML VERKHOVSKAYA 1984 Redox components in the plasma-lemma of plant cells. Soviet Plant Physiol (English transl Fiziol Rast) 31:386-393

8. LIN W1984FurthercharacterizationofthetransportpropertyofplasmalemmaNADH oxidation systemin isolated corn root protoplasts. Plant Physiol 74:219-222

9. LOPPERT H 1981 Energy coupling for membrane hyperpolarization in Lemna:evidence against an ATP-fueled electrogenicpump as the exclusive mecha-nism. Planta 151: 293-297

10. LucAs WJ 1976 Plasmalemma transport of HCO3- andOH- in Chara coral-lina: non-antiporter systems.J Exp Bot 27: 19-31

11. LucAs WJ 1983 Photosyntheticassimilation of exogenous HCO3- by aquaticplants Annu Rev Plant Physiol 34: 71-104

12. MACROBBIE EAC 1971 Fluxes and compartmentation in plant cells. Annu RevPlant Physiol 22: 75-96

13. MIMURA T, T SHIMMEN, M TAZAWA 1984 Adenine-nucleotide levels andmetabolism-dependent membrane potential in cells of Nittelopsis obtusaGroves. Planta 162: 77-83

14. MIsRA PC, TA CRAIG, FL CRANE 1984 A link between transport and plasma-lemma redox system(s) in carrot cells. Bioenerg Biomembr 16: 143-152

15. NOVACKY A, CA ULLRICH-EBERIUS, ULUTTGE 1978 Membrane potentialchanges during transport of hexoses in Lemna gibba GI. Planta 138: 263-270

16. NOVAK VA, NG IVANKINA 1978 Light induced absorbtion of ions by cells offreshwater plants. Soviet Plant Physiol (English tansl Fiziol Rast) 25: 248-253

17. NOVAK VA, NG IVANKINA 1983 Influence ofnitro blue tetrazolium on themembrane potential and ion transport in water thyme. Soviet Plant Physiol(Eng transl Fliziol Rast) 30: 845-853

18. PATE JS, BES GUNNING 1972 Transfer cells. Annu Rev Plant Physiol 23: 173-196

19. PRINS HBA,JFH SNEL, RJ HELDER, PE ZANSTRA 1980 Photosynthetic HCO3utilization andOH- excretion in aquaticangiosperms.Light induced changesat the leaf surface. Plant Physiol 66: 818-822

20. PINs HBA,JF SNEL, PE ZANSTRA 1982 The mechanism of photosyntheticbicarbonate utilization. InJJ Symoens, SS Hooper, P Compere, eds Studieson Aquatic Vascular Plants, Royal Botany Society of Belgium, Brussels, pp120-126

21. PRmNs HBA, JFH SNEL, PE ZANsTRA, RI HELDER 1982 Themecsm ofbicarbonate assimilation by the polar leaves of Potamogeton and Elodea.CO2 concentrations at the leaf surface. Plant Cell Environ 5:207-214

22. RUBINSTEIN B, Al STERN, RG STouT 1984 Redox activity at the surface of oatroot cells. Plant Physiol 76: 386-391

23. SEXTON R, JL HALL 1978 Enzyme cytochemistry. InJL Hall, ed, ElectronMicroscopy and Cytochemistry of Plant Cells, Elsevier, Amsterdam, pp 63-147

24. SIJMoNs PC, FC LANFERMEYER, AH DE BOER, HBAPRINS, HF BIENFAIT 1984Depolarization of cell membrane potental duringtransplasmamembraneelectrontransfer to extracellular electron acceptors in iron deficient roots ofPhaseolus vulgaris L. Plant Physiol 76: 943-946

25. SPANSWICK RM 1981 Electrogenic pumps. Annu Rev Plant Physiol 32: 267-289

26. ULLRIcH-EaERius CI, A NOVACKY, E BALL 1983 Effect of cyanide in dark andlight on the membranepotential and ATP level of young and maturegreentissues of higher plants. Plant Physiol 72: 7-15

CONTROLCONTROL I

~~~~tO- FERRICYANIDE FERRI- LIgHT

CYANIDE ONFERRICYANIDE

q ~~~~~~~~~CONTROL

B

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