kinetics energetics light-enhanced potassium … each sample contained app)roximately 170 mg of leaf...

Plknt Physiol. (1968) 43, 394-400

Kinetics and Energetics of Light-enhanced PotassiumAbsorption by Corn Leaf Tissue

D. W. RainsKearney Foundation of Soil Science, University of California, Davis, California 95616

Received Cactober 10, 1967.

Abstract. The effect of illumination on the absorption of K+ by leaf tissue of Zea mayswas investigated. The rate of K+ absorption was enhanced by exposure of slices of corn leaftissue to light, even of relatively low intensities. Potassium was transported inward, withvirtually no efflux of previously accumulated K+. The evidence indicates that the transportmechanism for absorption of K+ is the same in the light as in the dark, but that the source

of energy for absorption of K+ is different in the light from 'that in the dark. Various anti-metabolites were used to establish that the energy utilized for active ion transport in the lightcame partly from ATP supplied by cyclic photophosphorylation. Expenditure of ATP was

required in the dark too, but this ATP was formed by oxidative phosphorylation. Establishingthe ultimate source of energy for active ion uptake by higher plants might be facilitated bydemonstration of an ion-transport process that is not linked directly with the transfer ofelectrons in the mitochondrial cytochrome chain.

The principal organ,s which accumutlate ioIns in

higher plan'ts are ro,ots, hut leaf cells also acctumu-late nutrient ions. These ioIns are absorbed froma dilute soltutioin suirrouindinig these cel,ls. This solui-tion is suppilieed by the xylem (17).

Investigation,s o.f celluilar ioIn aibsorption by leaftissuie ,of terrestrial plants have not been feasibleuinti,l recenitly. The ioInic environments of leaf cellsare variaible because of differences in leaf tempera-tutres, iinequa.l ionl distribution in lleaf tisssue duie tothe lenlgth of the diffusion path that the ionIs musttraverse, and change,s in transpirattion rates. How-ever, these variables have been eliminated bv a

techhniqtue idevedloped by Smith and Epsteini (35).Their stutdlies with narrowx leaf slices demon,stratedthat the absorption of ionIs proceeded through thecut e(iges and not the sturface of ithe leaf lamina.'his woulld seem 'to precluide the involvement ofstomates in the aibsorption of ions when tusiing theleaf szlice te,chnique. By the uise of thin slices ofleaves, leaf cellts were exposed to the external solul-tioIn wi,th nlo diffusion limitations on1 ioIn mlovenlemnt.

This techn,ique made possiblle comprehensive studiesof the kinietics of ion absorption in leaf tissue ofcotri, Zea 7n1(1wvs (36), an,d the mangrove, Azicenniainarina (31). Those studies form a b+asis for fuir-ther invet-gations of the effect of light on ionabsorption I) green tissule. Some earlier work on

'Abbreviations: DCMU, 3- (3,4 dichlorophenyl) -1. 1-dimethylurea; FCCP, carbonyl cyanide p-trifluoronieth-oxy-phenylhydrazone; m'Cl-CCP, carboniyl cyanide in-

chlorophenylhydrazone; Fe-EDTA, ferrous sulfate inequal parts of NH,-ethvlened(iamiiiuetetraacetic acid;ATP, adenosine triphosphate.

394

the effecit of lighit on ioln tranlsport by green tissulehas been review ed in a i-eceilt paper (29).

CGreen tissuie contains 2 possible sources ofenergy for metabolic activities sulclh as iOI trans'port.One soturce is oxi(hative phosphorylation. Respi,ra-tory substrates are uitilized in the miltochondria toform high-energy phosphate compounds. Thesecomlp)ounds are then metabolized to transi) )rt ioInsagaiinst concentrationi gradients. The links betweenicelluflar ion trailsport and respiratory processes haveleen reviewed by Epstein (5) andi(i Suitcliffe (38).The other site of energy production ini green tissuieis the chioroplasts;. In chloroplasts ATNP is pro-(lilced by photosynithetic reactions (1,40).

Understanding- of the energetics invOlve(l in thea,bs);orption of ions in the light has growN-n in thelas,t few years from (letailed studies of the operationof chloroplasts, particulllarly their participation inion trainsport (2, 14, 27, 28, 33, 37). Papers byArnon et al. (1) anld Vernon and Avron (40) givecomprehensive discussions of the energetics of thesephoto.synthetic processes.

AlacRobbie (22, 23) investigated the effect oflight oln the absorption of K+ ainid Cl- b)y the fre,h-water ailga NVitel1a translucens. She concluded thatK+ absor,ption uitilized einergy from ATP formed bycyclic photophosphorylation whereias Cl- transportwas linked to electron transfer duiring the operationof noncydclic photophosphorylation.

The dependence of ailion absorption OIn electr-oIntransfer is not a general case. Smith (34) demoln-strated that light-enhanced P,13- absorption byNitella is duile to the expendituire of ATP formedby cyclic photophosphorylation. Ailso, Jeschke (15)has sho,wn that absorption of Cl- by Elodca( in thelight is nolt in-hibited when noncyclic electronl flo,wis eliminated 1)v DC\U,.'

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RAINS-LIGHT-ENHANCED POTASSIUM ABSORPTION BY CORN LEAF

Cyclic electron flow has been demonstrated inblue-green allgae. When DCMU was added, pig-ment turnover was observed even though 02 evolu-tion was eiliminiated. The cyclic electron flow was

enhanced when FCCP was aidded, indicating coulpledATP formlaition t(39).

The evidence discutssed above would indicate thatthe absorption of cations in the light is not a passiveprocess controlled by eilecitron-mediaited anion trans-port. In general, ca.tion and anion transport re-

quires the expenditure of energy in the form ofATP.

The preseinit investigation was made to studythe ef'fect of light on cellular K+ absorption by corn

leaf tissue and to determine the sou'rce lof energy

for adtive K+ tranlsport in the light.Some prelliminary experimen(ts on liigih't-enhanced

K+ absorption by corn leaf tli,ssue h'ave been re-

porte(l elsewhere (29).

Materials and Methods

Corn (Zctu mia(yS 'DeKalib 805') was germinatedin the '(ldark and then grown in nitrient solution,s inthe greenihouse for approximately 2 weeks. Thenatrient soklutionIs contained one-filfth the normal

concentration of nutirien'ts supplied in Johnson'ssolnition (16) excep-t that K+ was present a,t only0.3 mm, about one-4twentieth the con,centraition nor-

mally added. Iron was supplied as Fe-EDTA at a

concentration of 0.2 mm.At the eind of 2 weeks the leaves were excised

from ithe ipliainits and brought into the lajboratory.Leaf slices were prepareld by tlhe meithod of Smithand Epstein (35) except for minor differences intechnique.

Ten pieces of corn l-eaves approximately 10 mm

wide aind 30 to 40 m.m long were placed between2 blocks of stvrofoam of about -the size of the leaf.This width of leaf represen'ted 'the width o,f an

entire leaf blade of a 2-week-old plant. The blocksand leaves were then placed i,n a hand microtomefor slicing. Each sample yidl;ded 60 slices, 400wide. The slices were wrapped in cheesecl6th, andthe ends of the dllth were gathered and tied withthread (8).

The bags containing t'he samples were suspendedfor 1 hour in a solution contaiining 0.5 mm CaSO4at 30°. This pretreatment was done in the darkwith samples to be subjeckted to a dark treatment,anid in the light with 'sam(ples to be exposed to light.The pH of all solutions was 5.7 + 0.2 pH tunits.

At the end of the pretreatment period the sam-

ples were placed in a K+ solution radioactivelylabeled with 86Rsb and containiing CaSO4 at a con-

centra,tion of 0.5 mm. Experimental solutions ofKOl were la-beled wiith 86Rb to the extent of ap-

proximatiely 0.02 /,c/pmole K. Tihis specific activitywas heild constant throughout any 1 experiment.The use of 8,Rb as a laibel for K+ in investigationis

of ion absorption by leaf tisstue has 'been shown tobe a valid procedure (31). Also experiments re-ported by Smtiith -and Epstein (36) indiciated thatK+ and Rb+ are transported by the same site in cornleaf slices and there is mutual competition betweenK+ and Rb+ in the absoiption 'process. The validityof tusing 86Rb to label K+ was tesIted in my experi-ments. In 2 differenit expveriments K+ soluitionswere labeled with 42K or 86RRb. An experimentsimilar to the one reported in figuire 2 in the section-on resulits was carried oult and there was no dif-ference in the exchangeable fraction wlheither it wasdeterm,ined by tusing 86Rb or 42K. In another ex-periment u,sing these 2 radioisotcopes t,o lalbel K+solutions there was no significant quantitative orqualitative difference in K+ absorption in it-he lightor dark. The possibility of isotopic exchange wasfurt'her testeid in the experiment described below.Samples were prepared as dcecr-.bod in this sectionexcept each sample contained app)roximately 170 mgof leaf tissuie. Triplicate samples were exposed to3 different treatments. The first set of 3 sampleswas blotted, weighed, asqhe,d anld the K+ content wasdetermnined by uisling flame spectrophotometry. An-other set of samples was exposed for 4 hours to aso'ltu,tfon containing K- at a concen,tration of 0.2 mM,radioactively labeled with 80R'b, anld CaSO, at aconcentra'tion of 0.5 mm. The third sot of sampleswas exposed to a solution containing 0.5 mM CaSO4for 4 hours. At tihe end of this pei4iod the saimpleswere rinsed for 30 mninu'tes in 0.5 'mm CaSO4 toremove any freely exchangeable K+. Tihe samipleswere bllotted, weighed, a§hed and the K+ conten't ofthe leaf tisstue was determined by flame spectro-photometry. Ailso the K+ absorbed was calctulatedon the basi.s of 86Rb taken uip by the itisstue. Theamoulnt of K+ 'absorbed from the sohlution containingK+ wa,s determined chemically by the difference inthe K+ content of the leaf t,issule at the beginningand end of the 4-hour period and amouinted to6.5 1umoles per gram of fresh weigh't. The K+absoribed as deitermined by the uptake of 86Rib w-as6.1 jumoles per gram of fresh weight. The valuespresented are averages of the replicates. Therewas no significant loss of K+ from the tissue ex-posed for 4 hours to the CaS04 solution. Theseobservations gave confidence in the use of 86>Rb tolabel K+ solutions. It is mtlchl more convenient totuse this longer-lived isotope and iit was thereforeu,sed for alil the experiments re-ported iin this paper.Calcitum w.as indluded in all experimenital 'sollutionsbecatuse of its essentiality for uinimipaired ion trans-port (4, 13).

The samples were exposed to the radioactivesolutiions for various periods. Then they wereplaced for 30 mintuites at room temiper;aiture (21°)in a desoirbing solution containing 1 mM KOl and0.5 mM CaSO4. 'Mlhe desorption removed any freelydiffusible or exchiangeable radioacotive ions (8).At the end of the desorbing period thie samples wererinsed in dist-illed water and thung u1p to drain.

395



PLANT PHYSIOLOGY

The cheesecloth was then rewere blotted to remove excesssample was weighed. Tihe weiiglexperiments ranged from 50 to i1 experiment the samples did ncby more th,an 2 mg.

T:he weighed samples were trchets and ashed for 1 hour at 5Cwetted, spread with a drop of 1(

and dried under iheat lamnps.The samples were counlted 3 t

window GM gas-flow couinter,least 2560 counts each time. Thepared wiith a standard, and th(absorbed were calculated oIn the 1tfresh weight per hioulr.

Results

The ra,te of K+ absorptionfunction of l,ight initensity asGeneral Electric Light Meter, Tof cheesecloth was placed over thso as to du,plicate the lighit internwere actualily exposed to inside thThe resuilts are presented in figtnitration of K+ was 0.1 m\r, andCa2+ was 0.5 m-\. The absorptilminuites. TIhe rate of K+ absorptiover that of the dairk control, i.ep,mole g-1 hrF', when the lightcreased to 500 lutimen m-2. ThiFimaxi,m,al respontse, reached ait Iiless than 5000 lulmen m -.

Since sttudies with algae indi,ction caused an efflux iof variotusexperiment was designed to tes-t 1of K+. The ileaf tissue was expothe liight or dark to a radioactive

2.SF

-J

w 20

o 1.5

0

4 1

I

0 5,500 11,000

LUMEN M-2

FIG. 1. Effect of inicreasinig ligrate of K+ absorptioni. PotassiumCaSO4: 0.5 mmr. pH 5.7 anid tempeition period 60 minutes. All treatmcircles represent the memins of 2 val,short horizonital lines. Horizontal linthe distance between them would haless than diameter of the circle.

'moved, the sliceswater, and each

iits in the various70 mg, buit in anyvt vary in weight

ansferred to plan-)0O. The ash wasdetergenit soluition,

times with a thin-for a tootail of atcounits were corm-

e amounts of K+)asis of a gram of

was studied as ameasured with aype 213. A piece, re,eofat,r wind,ra.w

41

T

hIA-J0

a 1

hI.E

L ightI.

Dark. * 0

1

2 -

. I I0 10 20 30 40 50 60

TIME, MINUTE

FIG. 2. Potassium retained by corn leaf tissue as afunction of time. Potassium: 0.1 ni,m\i Ca: 0.5 mNi dur-ing absorption period. Potassium: 1.0 niI, Ca: 0.5 m-rduring desorption period. Points on zero time ordinaterepresent values for samples rinsed in water only. Otherconditions and conventions as for figure 1 except sam-ples were not replicated.

silty tlhe leaf slices containing 0.1 mM K+ anid 0.5 mM Ca2-. Theie dheeSsedllot.h balg. values represented by open and closed circles on thele 1. Thecsoncen- zero time ordinate in figure 2 were obtained fromconcentration of samples that were rin,sed in distitlled water on'ly and

i,on period was 60 then analyzed for radioactivity. The other samplesion increased 50 % were placed in ithe light or dark for various periods'., from 1.4 to 2.2 in a nonradioactive solution containling 1.0 mM KCIuinltensiitv was in- and 0.5 m-r CaSO4. The resullts indicate little ors is 80 % of the no loss of previou!sl1y accumulated K` in the lightight intensities of or dark.

To study the efifect of increasing K- concenitra-

zate that ililuminia- tion on the rate of K+ absorption in the llight an(dcaitiouns (3, 33), an dark, the K+ concentration was varied from 0.01 m-mthe possible efflux to 0.2 mMr, while Ca2+ wa.s held constant at 0.5 mM.)sed for 1 hour in The tisstues were exposed for 60 minutes to the4y labeled solution solutioii indicalted above. The cuirves in figutre 3

indicate saturation-type kinetics, with the rate ofK+ absorption greater in the lighit than in the dark.The kineitics were anaJlyzed essentially as describedby Epstein et al. (7). The apparent MIichaelisconstant (Kim), an alpproximate measure of theaffinlity of a site for an ioIn (6, 7, 30), was deter-mine(l for the dark and light treatments. TheKm's were similar, 0.035 mM\ in the light and0.030 m-m in the dark. The maximal velocity( V\, ax ), the theoretical maximal rate of absorptionat non-limiting substrate concentrations, varied bymore than 60 %. Tihe maximal rates of K- ahbsorp-tion were 3.40 Pmole g-' hr-1 in the light anl(l 2.08

22,000 27,500 unmole g-1 hr'I in the dark.The data indicate a light-enhanced K+ ab,sorp-

h.tintensity on the tioIl. The next qtuestion concern,s the source of:0.1 mAI, Ca2+ as energy for this accelera.ted rate of uiptake in the

rature 300. Absorp - ..

tents are replicated; light. With this in mind, experiments were carriedLues indicated by the ouit writh variou"s antimetabolites. The resiults aretes not drawNn where presented in figture 4.wve been equal to or The concentration of K+ was 0.1 mM, an(l that

of Ca2 -was 0.5 mM. At the end of the 60-nmilnute

I

396

3



397RAINS-LIGHT-ENHANCED POTASSIUM ABSORPTION BY CORN LEAF

K,mM

FIG. 3. Rate of K+ absorption as a function of theconcentratioui of K+ in the light and dark. Potassium:0.01 to 0.2 maE, Ca: 0.5 mm. Other conditions andconventions as for figure 1.

absorption period the samples were placed in solu-tions conitaining nonradioactive K+ as describedearlier. The concentraitions of inhibitors added tothe solutions in these experiments were chosen soas to give maximal inhibition without irreversibledamage. These concentrations were such that afterthe 'sample was removed from the solution con-taining the inhibitor and rinsed free of the inhibitor,a ra'te o'f K+ absorptiion was restored which waasequivallent to that of bhe con'trol.

Sodium cyanide was added at a concen-tration of0.01 mM. This inshibitor depressed K+ absorptionin the dark but had little effedt on uptake in thelight.

The iunicotupler of oxidative phosphory4lation,DNP, at a concentration of 0.01 mai inhibited K+absorpt'ion in the dark to one-sixth of the rate in

4

31

2

LIGHT

Di

.1-sOU

ARK0.

190sU

a2

0a0

0

W..

0

o

z

0

I- I- &

CL0.

91-la

W I

0 2

oILza

a

tfhe absence of DNP. In the fight, inhibition dueto DNP was cons!idera(bly less, to abotitt half theuninhibited rate.

Sod,ium azide at 0.01 mm had approximaitely thesame effect a's DNP, but was somewhait less effec-Five as an inhiibitor in the dark.

In the dark ithe effect of mCl-GCP at a con-centration of 0.001 mm was similtar to thatt of DNPand NaN3. In the light, however, mCl-CCP wastwice as effective at inhib,iting K+ absorption aswas NaN3 or DNP.

Dich'lorouhenyl-1,1-dimet'hyl'urea is an inhibitorof photosynthettic 'reactions, particularly those in-volved in 02 evolution (1, 15, 23, 39, 40). Figure 5

4 LIGHT

to DARKw-J0

w 0. 2 as a0 z

00

figre 0teIodtosadcnetinasfrig

0 of a +

dar i th pegsne 001 NP 0.00 mUm - _ 0

0 -~~

FIG. 5. Effect of antimetaboilites on theraste of Kabsorption in the light and dark. Potassium: 01 mm,Ca: 0.5 mm, inhibitor concentrations as indicated onfigure. OthSer conditions and conventions as for fig-ure 1.

givmes the results of an exiperiment in whiohabesorp-tion of Khwas 'studied as a funbtion of light anddark in the presence of 0.01 mm DNP, 0.001 mmDCMU, and DNP + DCMU at the same concen-trations. Dinietrophenol inhibitedKo absorption to-the same extent as shown in figure 4. There wasno effect of DCMU on K abbsorption. The 2inhibitors comlbined h-ad the same effecot as DNPalone. Since green tissue evolves 0.in the light,0. cainot be removed from the leafceMU environ-ment slimply by maintaining externiall anaerobic con-ditions. Tbhere is the possibiity that 02 evollvedmight be igghtly coupled to the respiratory chain,alliltowing electrons to flow and oxidative ph-osphory-lation to prioceeid.

An experiment was carried out to determineWxhether the cytoch,rome isystem i,s closelly linkedwith io-n absorptio.n. The results 'are presented infiguire 6. The con'cen'trations were 0.1 mm K',0.5 mm Ca21, and 0.001 mm DCMU. Nitrogen,which h'ad been washed by a 0.2 m soluttion ofKOH t'o remove any CO2, was buibbled throutgh t'hesolutions. The absorption periods were 30 mintutes.Thirty-minuite per!iods were used 'because leaf tissue

FIG. 4. Effect of antimetabolites on the rate of K+absorption in the light and dark. Potassium: 0.1 mMCa: 0.5 nmi, inhibitor concentrations as indicated onfigure. Other conditions and conventions as for figure 1.

-II0 L-----i

I



398

3

-i

0

a

010

co

.4x

LIGHT

DARK

"I

a

0

0o

20 04

44

0

z

0O

(0

0

0

44z

d9

FIG. 6. Effect of anaerobiosis and DCMU on therate of K+ absorption in the light and dark. Potassium:0.1 mm, Ca: 0.5 ma\. Absorption period: 30 minutes,rate calculated for 1 hour. Other conditionis and con-

ventions as for figure 1.

exposed to anaerobic conditions for loniger was

irreversibly injured. Absorption of K+ was elimi-nated a4lmost completely When anaerobic conditionswere maintained in the dark. The presence ofDCMU made little differen'ce. In the light therewas considerable K+ absorption when anaerobic con-

ditions were mainitaine!d. This was true 'even whenO, evoiluition by photosynthetic processels was pre-

sumably eliminated by the addition of DCMU.

Discussion

The absorption 'of K+ by corn leaf tissuie isenh'an'ced w,hen the tisssue is illutminated. The light-enhan'ced rate is established after a 10-minute lightexpossu,re of tilssuie previously exposed to a 60-minuteda,rk perio,d (29). The light-enhanced K+ absorp-tion is obtained ait rel,ativelly low light intensities,and light siaturation is reached ait a lower lightintensity for the 'ion-transpo'rt process than forphotosynthesis (20). The light-response cuirve isvery similar in sha,pe to the response curve observedby v'an Lookeiren Cam'pagne (21) for Cl- absorptionby Valliseria.

The light-enhanced ef flux of ions demonstratedby some investigators (2, 3, 33) was not observedunder these experimentail conditions. The 86Ri'hlabeiled K+ ionis, once they were accurmulated, re-

mained in tihe tisstue even in the presence of a largeexcess olf nonradioactive K+. T'he amount of K+in corn leaf tissue culttured by the procedulre de-scribed in the section on Materials anid Meth,ods isapproximately 30 ,umo,les K+ per gram of freshweight. If it is assumed that a gram of freshtisstue is nearly equivalent to 1 mil then the concen-

PHYSIOLOGY

tration of K+ is approximately 30 mmi. The "'Ribtaken up by the planit tissue shou'ld be isotopicallydiluited with the K+ in the tissue. Ten samples witha tota.l weight of appromimately 0.5 g (albout 0.5 mlin volume) are suspended in 2000 Ma of a 1.0 m-mK+ solu"tion during the desorption period. Thismeans that thiis 0.5 md volume of solution, con-taining 15 jumolles of K+ la,beled with 86Rb is ex-po-sed to a soilttion containAing 2000 utmoles of un-lalbeled K+. If appreciaible exchange took place86Rb in the tissue should be diluted by the betterthan 1%0-fold excess of K+ in the external solution.As can be seen by the data shown in figure 2 thereis no measurable movement otiit o'f tihe tissute. Thesame type of experiment was carried oult with 42Kas a label for K+ and the same restults as shown infigure 2 wvere obtained. Previously absorbed labelis not exclhanged with non-labeled ions. The initialK- content of the leaf slices was not reduced evenafuter 4 hours of exposure to a sohlution containingCaSO, at a concentration of 0.5 mM. This wouldseem to reduce the possibility of exchange in thepreseiice or absence of K+. On the contrary, thedata woutlld seem to indicate that there is somebarrier to free movement of ions and unrestrictedisotopic exchange does not take place in this sys`tem.

An investigation of the mechanisms controdlingstomatal aperture was condlucted by Fischer (9).He demonstrated that DNP and mCl-CCP, at con-centrations 'used in mv experiments, had the sameeffec,t oIn stomatal closing in the lIight and in thedark. Th'is is quite different from results reportedin this study. These inhibitors resulited in qutitedifferent amounts of inhibiition of ion absorptionin the light and dairk. The present findings,together with those of Fischer (9), support theearlier condclutsion that stoma'tes are not a controllingfactior in ion absorption by narrow leaf slices (35).

The demonstration of more than 1 ion-transportmechanism in higher plants (6, 7, 30, 31) sutggeststhat an additional mechanism might become opera-tional in the light. Since an analysis of the kineticconstants showed that the Km',s in the light anddark were virtually identical, it is likely that themeclhanism for K+ absorption was the same in thelight and tthie dark. Similar experiments 'on K+ andRb+ absorption by corn leaf tissue were carried ottby Smith anid Epstein (36). They demonstratedthat K+ and Rb+ compete in a common ion absorp-tion process for which Na+ has little affinjity. Inthe present experiments, the rate of K+ absorptionwas considerably greater in the light than in thedark, indicating a faster tuirnover of the mechanism.The greaiter rate of turnover of a K+-abisorbingmechanism in the l'ight is possibly due to a morereadily available supply of high-energy sulbstances*rather than to the activation of a seconid mechanismof K+ transport.

The source for this energy could be ATP. Theresults illustrated in the last 3 figures (fig 4, 5, 6)lend support to the idea that K+ absorption in the

1 1 1ol



RAINS-LIGHT-ENHANCED POTASSIUM ABSORPTION BY CORN LEAF

light is closely linked to ATP formed by cyclicphotophoslphorylation (22, 23, 29). Cyanide and un-oouplers of oxidative phosphorylation, DNP, mC4-CCP, or NaN3 (1, 11, 12, 18, 19, 32), manifestedtheir greatest effect in the dark when a largepercenitage of ATP is probably formed by respira-tory processes. A'bsorption in the dark was lessthan 20 % of thiatt of the cTontroil when DNP,mCl-cCP, or NaN3 was present. When DNP waisadded in the liighit the absorption was 60 % of thatof the controil. With m,Cl-CCP present in the lightthe rate of a:bsorption was onlly 30 % of that of thecontrol. A:ltthoulgh DNP at concentraitions used haslittle or nio effecot on photophosphorylation (1, 26,40), mCl-CCP is a potent uincouplpleer of oxidativeand photophosphorylation (11,34,40). In the lightthe decrease in K+ uptake with mOl-CCP presentcould possilbly be attributed to a lower level ofavailable ATP, resulting from a decreased rate ofphotophosphorylation

The possibility that enhanced ion transport inthe li.ght couilld be tlhe resulit of increased electronf1low due to 'photoresspiration' does not appear likelyin corni tisstue (10, 24, 25). It seemed advisable,however, to consider the po!ssib lity. Electrontransfer along the cytolchrome chain can be pre-vented by removal of O., (the terminail electronacceptor. However, in green tisstue, O., is evolvedduring photosynthesis, and this 02 might be tightlycoupled ito the cytochrome system, allowing elec-trons to flow. Respiration is sevierely restricted bymaintaining external anaerobic conditions and in-hibiting 0° evolution by tihe ad'dition of DCMU(39). This can be accoimiplished witiho-ut interferingwith cyclic photophosphorylaition (1, 14, 22, 23, 34,39, 40). The system was mad,e anaer,obic by buib-bling CO-free nitrogen throutgh the system, 02evolution anid oxidative phosiphorylaition were as-stumed (to be inhibited, iand a considerable amount.of K+ was absorbed in the light. The possibilitythat DCMU might n'ot be completeily inhibiting 02evoluhtion in this system was not entirely eliminated.However, the major source of energy for activeK-F absorption in the light under these confditionswas assutmed to be cyclic photophosphorylation.This process cotu'ld suppily ATP required in theenergetic operation of the active ion-transportsystem. This concluision is in agreement with workreported by Weigl (41). He concluded that ATPcould be formed tinder anaerobic conditions whengreen tissue of Linmnophila gratioloides was exposedto ligh(t. This ATP formed by photophosphoryla-tion supplied energy for active ion transport. Hed'id not, however, eliminate photolsynthetic evolttionof 02 and some of the ATP formed cotlld be theresult of oxidaltive phosphorvlation. In a morerecent paper (42) Wei,gl stuld ed the absorption ofanions by Elodea and found that energy for activeion transport was available in the light even thoughanaerobiosis was maiintained and DCMU waspresent to prevent O2 evoluttion.

I't is not likely that the uptake of K+ is dependentupon the absorption oif Gl-. In experiments re-ported by Smitth and Epstein (36) thhe absorption ofK+ by corn leaf slices was independent of t;heaccompanying anion, whether it was the moreslloWly a'bsorbed S042- or the more rapidly absorbedCl' anion. In unpublished data obtained in thislab-oratory similar resilits were produce-d in the lightand dark treatmenets.

Inhibitors were used in this study on the assump-tion that th'e effect of these compounds would bespecific and thelir mode of action would parallelthat reported in the volluminoous literature on tihesubjecit. The effect of these inh'ibitors is notlimited to studies on cell particulates (chloroplastsand mitochondria). Algal eellts have been used(22, 23, 34) and virtuallly identical resullts were re-ported when these inhibitors were emnployed inlight-dark treatments. Also similar resuilts havebeen repor'ted for tissues of aquatic angiosperms,tissues more highily organized than those o'f ailgae(21,41,42).

Differential penetration of these inh(ibitors ac-cording to the lighit treatment is not inidicated bythis sttudy. Anaerobic conditions gave virtualily thesame differenitia)l inhibition in thie light and darkas did the chemical inhibitors. Iit is not likely thatN, penetrates at different rates in the ligh't anddark (cf. figs 5 and 6). The argument that thepenetrati'on of inhibitors varies with the light treat-ment would also apply to the ex'periments withchilloroplasts, mito,chondria and algae unider variouslight regimes. All of these enutities are also sur-rounided by limiting membranes. A study of 'theliterature concerned with these particulates does notsupport the hypothesis of light-dependenit differen-tial penetration of commonly used inhibitoirs.

The pres-ent demonstration of an ion-bransportsystem whioh is coupled to a process oif ATP for-maltion by a systeim other than respiratory electronflow may prove to be an important tool in deter-mining !sources o'f energy available for the accumu-lation of ions by higher plants.

Acknowledgments

The author is indebted to Dr. Emanuel Epstein formany helpful suggestions and to Dr. C. R. Stockingwho kindly supplied dichlorophenyl-1,1-dimethylurea. Ialso thank Mr. E. Ramsey of the Ramsey Seed Com-pany, California Bank Building, 1155 E. 14th Street,San Leandro, California, for donating the DeKalb 805corn seed.

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PLANT PHYSIOLOGY

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kinetics energetics light-enhanced potassium … each sample contained app)roximately 170 mg of leaf...

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