Light-Dependent Ion Translocation in Spinach Chloroplasts - NCBI

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1 X 108 by means of a Coulter Counter and checked by a hemocytometer. 3 Abbreviations: ..... To test this possibility, the follow- ilng experimenital (lesign w\Tas.

Light-Dependent Ion Translocation in Spinach Chloroplasts", Park S. Nobel and Lester Packer Department of Physiology, University of California, Berkeley, California

Introduction The absorption of light by chloroplasts is the first step for several energy-requiring systems such as ATP synthesis and CO, fixation. Recently. it has been found that light can alter the structure and volume of the chloroplast (7, 15, 17, 26). Therefore the question was raised whether light could affect ion movements in isolated chloroplasts under conditions where structural changes occur. This action of light on ion uptake by chloroplasts has received little attention, even though ion movements in algae and higher plants have been known for some time. Hoagland and Davis in 1923 were among the earliest in demonstrating a role of light in the movement of ions in plant cells by showing light-induced increases of chloride and bromide in Nitella cells (10). These observations were extended in 1934 by Jacques and Osterhout, who found that light increased the rate

of K entry into Valonia rnacrophvtsa (16). Later, Arens reported Ca movements through Elodea leaves by a process dependent on photosynthesis (1), and Ingold found that chloride, K and phosphate were accumulated by such leaves in the light (14). Although subsequent investigations showed that lightdependent uptake of ions is a general process in plants (4, 19), still little is known about the mechanism of this process in chlorophyll-containing tis-

Lehninger, who reported that for each pair of electrons traversing a phosphorvlating site in the respiratory chain, 1.0 molecules of phosphate and 1.67 of Ca were accumulated (29). During Ca and phosphate accumulation, other ions also undergo concentrationi changes; e.g., the intramitochondrial K content falls and Na rises, although only 2 to 3 % as much monovalent ion is moved as Ca (5). It now appears that ion uptake by plant mitochondria occurs under similar conditions as ion uptake by animal mitochondria (12, 22). By an approach initially based on mitochondrial investigations for the uptake of Ca and phosphate, it has now been discovered that the basic conditions of electron flow, Mg and ATP support the uptake of these ions by isolated spinach chloroplasts. [Preliminary reports of this research have recentlv appeared (24. 25)]. This ioln tranislocationi process is associated with the light-triggered Mig-activated hydrolysis of ATP in chloroplasts (11, 21, 28). The ability of spinach chloroplasts to take up other ions has been surveyed and it has been found that Na is also taken up by an eniergy-depenldenit process.

Methods

Certain features of the problem outlined above for chloroplasts are relevant to the properties of mitochondria. Mitochondria are known to regulate their structure and also to translocate ions by an energy-dependent mechanism. In 1961, Ca uptake by mitochondria was reported by Vasington and Murphy (32) and by DeLuca and Engstrom (6). Ca uptake in rat kidney mitochondria was dependent on respiration and required ATP, Mg, and phosphate (33). Ca binding was prevented by uncouplers of oxidative phosphorylation and electron-transport inhibitors. Brierley, Murer, and Green found that ADP inhibited divalent cation uptake, possibly by competing with the ion for a high-energy intermediate (3). The close association of ion uptake with phosphorylation in mitochondria was shown by Rossi and

Spinach was purchased commercially and stored at 40 until it was used. Leaves were rinsed with water, their midribs removed, and approximately 100 g of leaf material was added to 200 ml of 175 mm NaCl, 50 mm Tris-HCl (pH 7.9) in a Waring Blendor and homogenized for 10 seconds. The homogenate was filtered through 4 layers of cheesecloth and the filtrate was centrifuged for 1 minute at 200 X g. The supernatant fraction was removed and centrifuged for 10 minutes at 200 X g to form the chloroplast-containing pellet. This method of isolation was found to give a high percentage of whole chloroplasts as determined by the height of the chloroplast peak at 55 /3 in a Coulter Counter. The amount of chloroplasts used in experiments was based on chlorophyll content ( 34). The number of chloroplasts/mg chlorophyll was determined to be 15 + 1 X 108 by means of a Coulter Counter and checked by a hemocytometer.

'Received December 11, 1964. This research was supported by the National Science Foundation (GB-1550).

3- (3, 4-dichlorophenyl) -1, 1-dimethylurea; DNP, dinitrophenol; GTP, guanosine triphosphate; ITP, inosine triphosphate; PCMB, p-chloromercuribenzoate; PMS, phenazine methosulfate; UTP, uridine triphosphate.

sues.

3 2

633

Abbreviations: CTP, cytosine triphosphate; DCMU,

0)34

:PLA NT PIIYSIOLOGY

Except where otherwise noted, the composition of the reaction mixture was that for light-triggered chloroplast adenosine triphosphatase and included 17-5 iiM N aCl, 50 nmi Tris-HCl (pH /7.9). 5 nM MIgCL.., 10 tim PMS8, 3 ni,I ATP, either 5 mm re(luce(l lipoic acid or 1 nilaI mercaptopropionic acid, and 200 1ug of chlorophyll/nlI, plus other additions including radioisotopes (10 l c of beta emitters or 0.5 tsc of ganimia emitters,/5 ml) as indicated. Aliquots (5 mil) of this reactioni mixture w7ere incubated in celntrifuge tubes at 25 Those in the light received 25,000 lux (which proved optimum for these experinlieits) froni a 150 w reflector flood lamp; the (lark onies wvere wrapped in alumintuiiu foil. For Ca4 studies, 10 i CaCl. (i.e., 50 m15noles/5 nil) lplus I10 c of Ca' as chloride per 5 ml was used. After inicubatioln for 10 miniutes the tubes were centrifuged at 27.000 X g for 10 niinutes to form a firm pellet. The supernatant fluids were next renioved, the i)ellets wvere resuspeiided, and these fractions were counted in a scintillation counter. The fraction of the radioactive ion taken up was determined by dividing the pellet cpmi byr tlle total cpm in the initial reactioln iixture. Except in figures 1 through 3, the pellet cpm was corrected for 100 % trapped supernatanlt i.e., the pellet was wA-eighed, a density of 1.000 g/ml wvas assumiied, aiid the cpmll in ani equivalenit amiiount of supernatant fluid was subtracted fromii the piellet cpiii. Pellet volumie averaged 0.1 50 ml in the light andl 0.080 in the (lark, therefore the correctioni for trapped sulernatalnt iiiaterial teli(ls to lower the appareiit uptake iiiore in the light thall the (lark. The fraction of the radioactive ioil taken tl -as converted into niiimoles by mutltiply-inlg by up the aiiiounit of ion present initially (iusually 50 ni15 niloles) The light-induced ion uptake xwas (lefined as the aniouit taken ui) in the light imiinlus thiat in the dark. The amounts of Ca accunmulated were determiined by atomlic absorption spectroscol)v alid Na 1y flamlie photonietrv with the assistance of Drs. T-. Sanui and C. M. Johnson. Phosphate was (leteriiiiiied spectrophotometrically ('13). O., evolution was mieasured polarographicall1 wvith a Clark electrode.

Results Basic Coniditionis for Ca Uptakc. The chief factors influencing light-iniduced Ca uptake into chloroplasts are showni in table I. This experimenit was selected to shoxN the requiremiients for Mg. ATP, reduced lipoic acid, and PMS for maximal Ca uptake into chlioroplasts. Nolie of these factors has ai appreciable effect in the dark. In other experiments, the omllissioli of one of the componenits of the basic reaction niixtuire lead to the folloxxing- decrease in lightin(lulc(d Cal upxtak: 7() % for- ATP, 33 %0 for re-73 foi- PA\ IS. Al \vu as the (lltce(l hipoic ac(id. anl(l 13 nliost imiportant conliponeint of the reactioni mixtuire

Table I. Factors Influtenicing Ca4, UJptakc i1to Chloroplast Fractio,t The complete reaction mixture includes: 175 mNi NaCl, 50 mnA Tris-HCl (pH 7.9), 5 mm MgCl*, 3 nim ATP, 5 mna reduced lipoic acid, 10 1ur PMS, 50 mntmoles CaCl., plus 10 uc of Ca47/5 ml, and 200 ug chlorophyll/ml.

Ca uptake

mn,unioles/mg clhloropllyll per 10 min Complete systenii -MgCl., (5 maI) - ATP (3 mam) -Reduced lipolc acid (5 mm) -PMS (10 pr)

Liglht 9.87 1.08 3.49

Dark

1.53 1.00 1.69

7.18

1.57

3.44

1.44

contributing to a light: lark difference in Ca uptake in that it was absolutely required. The reliability of these results is good as in 10 experimenits under the conditions in table I, the Ca uptake was 10.82 -+ 1.19 (SD) nmMnmoles/nig chlorophyll per 10 minutes in the light and 1.66 + 0.41 (SD) in the dark, i.e., a light: dark ratio of 6.5. The above conditions vere also foundel necessary for an uptake of P1 that acconilpaliies Ca uptake (cf. refereiice 24 aiid Ca: P, stoichoinietry given below). These conditionls supporting miiaximumii Ca alnd Pi uptake are precisely those whiclh have been fotulnd optimutim for MIg-activ-ate(l lighlt-triggeredI hydrolysis of ATP in spinlach chloroplasts (21). The pH optiniia o f pllotophosphorylation and(l a(leiosilie triplhosphatase are similar in spiniaclh chloroplasts. Heence, a study w-as iiiade on the depeidenice of Ca uptake on pH. Coiiceintrationis of ATP auid cliloroplasts xx ere redluced in these experimlenits to mininiiize pH changes frolmi ATP hydrolysis as well as the buffering effect of the chlorop!asts. Figuire 1 shows a strikinig pH depeiidenice of Ca uptake in the light wvith maxiniium uptake near 7.9. The dark results are also initeresting as the uiptake decreases by a factor of 2 wheni the pH is iiicreased froni 6.9 to 9.0 Ini order to extend the findiuigs described above. experiments were undertaken to determine wvhat changes could be made in the electron flow and ATP hydrolysis re(quirements of light-induced Ca uptake. On varying the concentration of PMS, it was found that the Ca uptake was highest between 5 to 20 /0oi, ith a peak at 10 piu. Although omission of PMS iiihibited Ca uptake, establishing colnditions for noncyclic flowr using 30 um NADP (plus 30 ,ug of ferredoxin/5 ml) supported Ca uptake 80 % as well as the usual P'MS system. Ca45 uptake in the NADI' system was found to be 80 % inihibited by 10 ai\ DCATIU. A noncyclic systeni with 0.2 m3a ferricvaiii(le supported light-indulced Ca ulptake 60 % as xWell as the PMlS systemii. 'l'ie factors affecting tCa uptake tund(ler VI' P livd(lrolx sis conditioins xxwere first stti(lie(l lbv xarviny

NOBEL AND PACKER-ION TRANTSLOCATION IN SIMINACTI CHLOROPLASTS

which inhibits adenosine triphosphatase in chloroplasts (27), inhibited Ca uptake both in the presence and absence of ATP. Ammonium chloride and quinacrine abolished Ca uptake. Some conditions whiclh vould disrupt chloroplast structure were examined for their effect on Ca uptake. It was completely inhilited by digitonin and 300 ,g/ml triton. Much lower concentrations of triton (2-30 4g/ml) gave a small enhancement of Ca uptake with a maximum at 20 ug/ml. M\Iechanical disruption of chloroplasts in a Nossal cell disintegrator for 10 seconds does not abolish light-activated adenosine triphosphatase (27), but light-induced Ca uptake was not detectable. Al-

12

z *-

10

0o

o.o

UIhIT _

>-

2v

8

CL

90 °

oC -X E 3

KDARK

*~--

E IV o -C E tJ E2 uw

l-

7.0

8.S

8.0

7.5

9.0

so,

pH

E 1:

;;

:E

'

CL

U0

o

,9

0-Ac ki

ED 'E

vE

,

RK

0

5

3

1

7.5

10

ATP, mM

FIG. 1. pH Dependence of Ca uptak e. Chloroplasts isolated as in Methods, but witlhout Tris-HCf. Usual conditions for Ca45 uptake were n and the pH pH of of lows: 1 mM ATP, 100 ,ug chlorophyll/ml, and wvere

indicated. FIG. 2. Influence of ATP on Ca uptakce in the chloroplast fraction.

50

mM

Tris-HCl

as

the concentration of ATP. Its optirnal concentration for light-induced Ca uptake was ab yout 3 mM (fig 2). Although the uptake of Ca in thie dark did not _11r, A ml%11 * _ _ .1 _ __ a slight enhancing effect of ATP at vary greatly, low concentrations was observed, i.e., ATP seemed to support a small amount of Ca uptake in the absence of light. Other nucleoside triphosphates at a concentration of 3 mm were then substituted for ATP. GTP was less than 40 % as effective as ATP, while ITP, CTP, and UTP had no appreciable effect on1 the Ca uptake. Although the requirement for nuticleoside triphosphates was quite specific, substitutioIns coti(i be made for the Mg and thiol (see below) requiirements. Ca uptake was maximal in the range of 5 to 10 milM MgCl2, while the dark was little affected. The requirement of Mg could be completely replaced by manganese; in fact, in the presence of 5 mM MnCl., the uptake was 15 % higher. Various other compounds were testedl for their effect on Ca uptake (table II). Valinomycin, DNP, and ouabain were without effect. However, ADP, I.

91

lowering the NaCl from 175

mm

to

44

mm

in the

usual reaction mixture abolished the light-induced Ca uptake. Uptake in the light varies greatly with the incuin about 30 minbation reaching a utes (fig 3). The optimum temperature range for Ca uptake was 25 to 300 for an incubation time of 15 minutes. Using data at 17 and 270, a Q10 for Ca tuptake was calculated to be 1.9. Amounts of Ca Accumulated. It was important to determine whether the uptake of Ca45 represented accumulation. As the concentration of the ion in

nmaximum

time,

O

0X-

oLo 15

6335

the reaction mixture is

increased,

the absolute amount

of the ion removed by the chloroplasts increased. while the percentage of the total Ca lost from the supernatant solution decreased. In figure 4, the light-induced change in these quantities is plotted as a

As expected, the of Ca removal occurred for lower

function of Ca concentration.

greater percentage

concentrations of Ca, hence most experiments were 10 /M Ca. The maximum amount of Ca taken up occurred at approximately 1 nmM CaCi2. These results show that Ca accumulation by chloroplasts, rather than exchange, is being measured

perfornmed using in

the uptake experiments. Atomic

absorption

definite measure of For these studies, the reaction mixaccumulation. ture (as described in MIethods) was used except

spectroscopy provided

a more

Table II. Inihibition of Ca45 Uptake

Light-induced uptake

Addition

None

Vahlilomycin DNP

Ouabain

0.05 ug/mtnl 0.5 mit 1 3

mlu

100 100 99 1()1

62 mii 1 muN '22 50 uAN Quinacrine m5- 2 N4C i 119 20 Triton ug/nml 0 300 gg/ml Triton 0 1 mg/ml Digitonin * ATP was not present in the reaction mixture in this experiment or its control.

ADP ADP*

636

PLANT PHIYSIOLO(Y

tul)es was inicubated in the li-ht an(d dark for 10 minutes in the usual way. A secoln(d pair without ATP

°

inicubated in the same mlanner. After 10 minthe light was turned off anid ATP immediately a(l(led to the second pair. The reaction was conitillue(l 10 miiinutes after placinig all tubes in the (larlk. The tube which lhad receixe(l the ATlP after the liglht was turned off took up 70 % as muclh ATPdependenit Ca a.s the one that had .\TP present throughout. This showed that light is niot requiredl while the Ca uptake is proceeding but rather it ftunictioned as a trigger analogous to its activation of adenosine triphosphatase (11, 21). The dark decay of the light-triggered Ca uptake was examine(d by adding ATP at various time intervals after the terminiation of the period required for light-triggering. 'Fhe amotunt of Ca uptake was reduced to half if the timiie of ATP addition was 3 miiintutes after the ein(l of the light period. A special featture of light-triggered azlenlosinie

>.

tril)hosphatase

was

utes,

Incubation

time, minutes

80

E

-

E

30

-

60

0

E E

40

° 20

Q 9 ° D ,

is

the ilnvolvenment of thiol grouips.

loni uptake is substantially elevated by tlhiols andl the optimumlli concenitration of each comllpotun1d teste(d and its effect on Ca tuptake is presenlted in table III.

aE E 4

E 20

o 10 _

0.001

0 E

C(1$5 Uptakc Table III. Actiont of Sulfhydryl Groups Reaction mixture as in Methods for Ca45 except thiol compounds were omitted. on

Addition

10

0.1

0.01

D °

CaCI2 concentration, mM

Fic. 3. Time course of Ca uptake. F(;. 4. Ca loss from me(diumiii an(l uptake

by

chloro-

plasts.

that the concenitration of Ca was 100 scM. Ca accumulation is higher under these conditionis (cf. fig 4). These experiments were done in duplicate and the results were averaged for 3 different preparations. The endogenous Ca contenit was 0.25 tcmole/ mg. After a 30 minute incubation period, analysis of the chloroplast samples showed that the Ca content was 20 % higher in the dark and 80 % higher in the light. Therefore, the light-in(luce(d increase wvas 0.15 numole Ca/mg chlorophyll per 30 miinutes. Frolmi the ntumber of chloroplasts (see Mlethods), it was calculated that 6 X 107 Ca atom.s were accutmulated per chloroplast. Using a volumiiie of 55 u,u per 1.8 imir increase chloroplast, this corresponds to in Ca if chloroplast volumie chalnges are igniore(l anid if it is assumiiie(l thalt Ca is unliformily (listril)ute(l. [fig/t-T1 iyycrmy al(d '1'11)iol ACti7'atioii. Since a

Conlditionis

established

for

Ca

uptake

were

those

for

light-triggered a(lenosiile triphosphatase, it was of interest to inivestigate whether the uptake could be triggered by light or whether continuous illumination To test this possibility, the followwas niecessary. ilng experimenital (lesign w\Tas iise(l. Onle pair of

None Reduced lipoic acid Cysteine

Mercaptopropiollic acid Mercaptoethanol Oxidized lipoic acid Cystine PCMB AgCl

Conc

Light-induced uptake

mM\

%

5 50 10 5

0.5 1 0.001

100 141 120 146 133 -3 64 0

0

Compounds containing S-S linkages such as oxidized lipoic acid or cystine inhibited Ca uptake. Also, it was not surprising to find that the sulfhydryl poisots, PCMB and AgCl, completely inhibited lightinduced Ca uptake. In order to check how the thiol compound was involved in the ionl uptake process, the following experiment was performed. Chloroplasts were incubated for 10 miniutes in the light or dark anid then ATP was added just as the light was ture(ed off together with either oxidized lipoic acid All tulbes were then kept another 10 or I)CMB. minutes in the (lark. Unider these conditions, PCMH, (1 imM) gave 9J5 % anlel oxidized lipoic acid (5 mM) 85 % inhibition of Ca uptake. These results indicate that sulfhydryl groups were important in the lighttriggered process occurring in the dark. Ca: Phosphate Stoichiometry. The stoichiometry of Ca and plhosplhate uptake was stul(lied by per-

NOBEL AND PACKER-ION TRANSLOCATION IN SIPINACH CHILOROPLASTS

63

forming the reactions in the dark following a preillumination period. ATP was omitted in these experiments, since phosphate formation accompanying its hydrolysis would interfere with the determinations. Ten uM Ca and/or phosphate was added after the 10-minute preincubation (light or dark), and before a 10-minute all-dark period as follows (table IV): 1 pair of tubes received labeled phosphate plus cold Ca, while another pair received labeled phosphate

Table V. Specificity of Ion Uptake by Chloroplasts Reaction conditions as in Methods, with the following modifications: in the Na experiments, isolation and incubation were carried out in media where all Na salts were replaced by K salts; in chloride studies, nitrates replaced chlorides; for C14-ATP, the reaction mixture contained 0.1 mNi CaCl, and was studied both by the usual procedure as well as by depositing the chloroplasts on a millipore filter by negative pressure and then counting on planchets in a thin window gas flow counter.

Table IV. Relation between Ca45 and P13' Uptake Reaction conditions as in Methods for Ca45 uptake, except ATP was absent. Also, Ca and/or Pi (10 AM) were added after a 10-minute preincubation (light or dark) and before a 10-minute dark incubation.

Light-induced uptake Ca45 0.15 ,umole/mg per 30 min* 0.002-5 Mn54 None 0.005 0.005 None Fe59 Zn65 None 0.005 > Ca45 0.002-10 Na22 K42 0.002-10 None 0.01 None Rb86 None 5-36 Cl36 10-4 X Ca45 BrS2 0.002-0.01 10-4 X Ca45 0.002-0.01 I131 _ Ca45 0.002-2 P3204 None 0.002-0.01 S3504 0.1 None C14-ATP None C14-D-glucose 0.01 * Initial concentration - 100 pi.

Additions + Ca Pi32 p 32

Ca45 + Pi Ca45

Ca uptake m,amoles/mg chlorophyll Preincubation Dark Light 4.73 2.72 2.69 2.44 3.87 1.64 2.11 1.45

only; by subtracting the light-inlduced phosphate uptake in the second pair from the first, the light-stimulated Ca-dependent phosphate uptake was obtained. Similarly, other pairs of tubes contained labeled Ca plus cold phosphate or labeled Ca with no phosphate: from these tubes, the light-stimulated phosphatedependent Ca uptake was determined. In this way, it was found in a series of 6 experiments that 0.9 ± 0.2 (SD) moles of Ca were taken up per mole of phosphate. Ca uptake can proceed in the absence of added phosphate either as P1 or ATP (cf. fig 2, table I, IV). To clarify the extent of Ca uptake in the absence of added Pi (and vice versa), further experinients were performed under the following conditions: K salts replaced Na salts in the medium (see below), ATP was omitted, the incubation period was lengthened to 30 minutes, and 10 MM Ca45C12 was added. In 3 such experiments there was an uptake of 5.7, 5.6, and 4.5 m,umoles of Ca/mg chlorophyll in the light and 2.5, 2.5 and 2.6 for the corresponding values in the dark. To test for Pi uptake under these conditions 10 UM pj32 replaced Ca45. In 2 experiments, no light-dark difference in Pi uptake was observed (1.8 mamoles Pi/mg chlorophyll in both cases). When ATP is present, its hydrolysis provides sufficient phosphate for Ca uptake, viz., 1.7 ii1M and 0.4 mm in 30 minutes in the light aiid (lark, respectively. This is significant because the addition of Pi results in an increased Ca uptake in both the light and dark. For example, 1 mM Pi added to the usual reaction mixture results in an additional uptake of about 1.7 m,umoles of Ca/mg chlorophyll per 10 minutes both in the light and in the dark.

mm

Studies on the stoichiometry of electron transport and Ca uptake indicate that the uptake process is inefficient. Systems with NADP (plus 30 Mg of ferredoxin/5 ml) or ferricyanide have been used, and the electron transport calculated from 0o evolution. It was calculated that about 5 Ca atoms were taken up per 100 pairs of electrons flowing, although optimum conditions of light intensity, chloroplast concentration, and medium tonicity have not been developed for simultaneously examining ion uptakes and Hill reaction activity. Specificity of Ion Uptake. A survey of the uptake of other ions (table V) was made under the optimum conditions established for Ca and phosphate uptake. As controls for these experiments, ATP, which is required for Ca uptake, and a neutral molecule, glucose, were investigated. No lightdependent uptake of either ATP or glucose was observed. The uptake in the light and dark was also not significantly different for Mn54, Fe59, Zn65, and S3504. A small light dependent uptake of I131 and Br82 was observed, but it was less than 0.01 % as much as the Ca uptake and(l could be due to iodination of a protein or other such1 reaction. The uptake of I- and Cl- was also stu(iie(d ill a mediumil in which the chloride in the isolation miiediunli and reaction mixtuire was replaced by nitrate. \While no IT31 or C136 uptake was observed in these chloroplasts, onily about 20 % as much Ca as usual was taken up in the NO5medium. Of the monovalent cations tested, an appreciable uptake of Na was found while K42 and Rb88 were not taken up. In fact, after correcting for trapped supernatant, the K decreased in the light

638PLANT PHYSIOLOGY' 638S Table VI. Factors Influtencintg Na2 Uptake Reaction conditions as in Methods, except K salts in place of Na salts in all media and Na22 replaced Ca45.

Light-induced uptake 10') 100 39 34

Coml)lete systemii -

MgCl, (5 mmI )

-PMS (10 -

p/Ax)

ATP (3 mM)

-Mercaptopropionic

acid (1

unim)

+-NH4Cl (5 mar)

+ CaCl., (5 mm) Ouabain (1 mat)

76 23 74 81

about 0.2 mMmole/mg chlorophyll per 10 miniiutes anid increased by about the same amilouniit in the dark. A study of the cofactors for Na tuptake showed that maniy of the same cofactors as for Ca uptake were requiired (table V,I). Na uptake was light(lependent, required ATP, PIS, and(I a sulfhydrylcontainiing compound, and was inhibited b1 NH4Cl. Na uptake did( not depend on M\g. This is an importanit difference froml Ca uptake, which was red(uced 100 % by onmissioni of Mg (cf. table I). Another difference is that additioni of Pi (10 uM5 mM) did not increase Na uptake in the light or dark. Ca reduces Na uptake; for example, adding 5 nmi CaCl9 decreases Na"2 uptake 33 % from a mediumu containing 10 /Am Na'. Conversely, the presence of 5 mm NaCl decreases the uptake of 10 jkr Ca++ by 42 % in a medium containing K salts. The uptake of Na as a function of its concentration in the medium was similar to the concentration dependence of Ca uptake (cf. fig 4). except that more Na was taken up. In experiments witlh 1 ima NaCl, analysis of Na by flame photometry showed nio increase of Na occurred in the chloroplast pellet in the dark, but an accumulation of 0.51 jumole of Na/mg chlorophyll with a 30-minute inicubation period was obtained in the light. This is about a 5-fold increase over the endogenous Na present in chloroplasts. Ca uptake was studied in the KCl medium (tused for Na studies) and was founldc to be only 20 % less. This indicates that the light-dependent Na tranisport is not a restult of tlle inhibition of Ca uptake.

by

Discussion Spiniach chloroplasts in vitro have been shown

take up Ca, phosphate an(l Na tunider coniditionis that support light-activated ATP hydrolysis. The optimum requirements for this adenosinie triphosp1hatase at pH 8 are light, Mg, PMS, anid a thiol coinpound; ADP and NH,Cl inhibit the reactioni (27). Marchant and Packer (21i) and Hoch and Martin (11) found that the role of light is indirect, as considerable enhancement of the adenosine triphosphatase activity was obtained in the dark followving a light to

preincubation. Ion uptake can also occur in the dark after a light preincubation and both ATl' hydrolysis (2, 21) and ion uptake decay in the d(ark. Furthermore, manganese can replace AM1g for both adeniosine triphosphatase an1d ion1 utptake, atInd the effect of triton is similar in both systemiis. 'l'his parallelism betwN-een ion uptake and adlenosinie triphosphatase has earlier been observed for structural changes (measured by light-scattering) and adenosine triphosphatase (27). In fact, no conditioni has been found in wvhich adenosine triphosphatase, light-scattering changes, and ion1 uptake differ. Hence. structural changes, which reflect water movemenits, anid ion translocations nmay be controlled by the samie process. A possible reason why conditions for ATP hydrolysis were optimumiii for Ca uptake could be that phate is produced, since the addition of PI. inicreases Ca uptake in both the light anld dark. llowever. there is always sonme light-dependenit Ca uptake in the absence of added Pi or ATP. In the experiments reported above, this usually amloutnits to 2 to 3 mnitnoles Ca/mug chlorophyll, or about 25 %°/ of the Ca uptake obtained at the optimal ATf) concentration. In mitochondria, energy is necessary for cation uptake, either from substrate or from ATP. Both substrate- (light) and ATP-supported catioln uptake is indicated for chloroplasts. Light results in Ca uptake by chloroplasts in the absence of ATP. but in its presence, light leads to more Ca uptake. analogous to mitochondria. Other similarities for ioIn uiptake in these 2 organelles are: 1) uncouplers ot phosphorylation abolish ion uptake; 2) requiiremlenlts for ATP and the relative ineffectiveeness of other nucleoside triphosphates; 3) maximal Ca uptake at 3 mm ATP, possibly due to ATP chelation of Ca or Mg; and 4) addition of Pi is unnecessary for the uptake of low concentrations of Ca (6, 33). The conditions for establishing ion uptake are consistent with suggestions that a high energy intermediate may be involved in ion translocation in chloroplasts similar to its proposed role in mitochondria (3). Perhaps, the inhibition of Ca uptake by ADP may be due to a competition of the nucleotide \ith the ion1 for a hiigh energy intermediate. The stoichiometry and amounits of Ca uptake are different in chloroplasts than in mitochoIndria. The time course is similar (29, 33) but the amotints are less (31). Since the localization of Ca is unknown in the chloroplasts, the sign]ificanice of these differences requires clarification. Also the efficiency of Ca upt)take when calculated onl the basis of pairs of electrons traversilng a phosphorylating site is much less for chloroplasts thaln for mitochondria. The stoichliometry of light-stimiiulated interdependent Ca and phosphate uptake gave a Ca: phosphate ratio of 0.9 + 0.2, which agrees with some mitochondrial studies (12), but not all (3, 5, 29). Interdependent light-induced

'phos-

NOBEL AND PACKER-ION TRANSLOCATION IN SPINACH CHLOROPLASTS

Ca and phosphate uptake has been found in Hvdrodictyvon and Sphaero plea by Frank (9) who suggested that the insoluble deposits found in the algae were hydroxyapatite. In chloroplasts, light-induced Na uptake is greater than Ca. Although this is not the case for mitochondria, a rise in intrainitochond.drial Na has been reported during the process of Ca accumulation (5). The interdependence of Na and Ca uptake is also seen in other biological systems (18), and it has been suggested that they compete for transport sites. Na uptake differs from Ca uptake in that it does not depend on the addition of Mg or Pi. Hence, the release of Pi by ATP hydrolysis does not seem to be involved in Na accumulation, indicating that another, as yet unspecified. mechanisn exists for the action of ATP on cation uptake. The ion translocations found in this research reflect the net uptake of ions; unidirectional fluxes have not been determined. Thus, the absence of significant light-induced uptake of K+, Rb+, AIn", Zn++, Fe+++, S04=, I-, Br-, and Cl- might result from an efflux of these ions (either in the light or in the dark) exceeding their influx. For example. Saltman, Forte, and Forte observed the efflux of Na, Br, and Rb from Nitella chloroplasts (30). Dilley believes that the efflux of K from spinach chloroplasts is light-dependent (8), which is consistent with our finding that K is not taken up by a light-triggered process. An understanding of the reversible light-stimulated uptake of H+ ions (or release of OH-) described by Neumann and Jagendorf for spinach chloroplasts would appear to be important for a more complete comprehension of ion movements (23). Also, MacRobbie has found an elevated chloride concentration in the chloroplast fraction of Nitella indicating active transport of this ion (20). The existence of a light-dependent mechanism for the regulation of ions suggests a role for chloroplasts in ion uptake systems in plant cells. Ion translocation may be important for maintaining an ionic .balance in the chloroplast or for sequestering certain ions from the cell. It is suggested that light may play an important role in controlling the ionic relationships of plant cells by regulating the movement of water and ions in chloroplasts.

maximum at pH 8. No difference was found in chemical or light requirements for adenosine triphosphatase, previously reported structural changes measured by light-scattering or ion uptake, possibly indicating a common mechanism of action. A number of analogies between ion uptake in chloroplasts and mitochondria have been established: both substrate and ATP support ion uptake; calcium uptake is maximum at 3 mM ATP and other nucleoside triphosphates are relatively ineffective; uptake reaches a maximum after about 30 minutes; and both are inhibited by ADP and uncouplers of phosphorylation. In chloroplasts the stoichiometry of light-induced calcium: phosphate uptake is 0.9 ± 0.2 and the net calcium accumulation is 0.15 ,umole/mg chlorophyll per 30 minutes. Sodium accumulation in chloroplasts (from a potassium-containing medium) was 3.3 times larger under the same conditions. Light does not result in an appreciable uptake of K42, Rb86, M\ln54, Zn65, Fe59, S3504, I131, Br82 and C136 in chloroplasts. It is concluded that spinach chloroplasts contain an energy-dependent light-stimulated mechanism for the regulation of their ionic composition.

Literature Cited ARENS, K. 1936. Physiologisch polarisierter Massenaustausch und Photosynthese bei submersen Wasserpflanzen. II. Die Ca(HC03) 2 -Assimilation. Jahrb. Wiss. Botan. 83: 513 -60. 2. BENNUN, A. AND M. AVRON. 1964. Light-dependent and light-triggered adenosine triphosphatases in 1.

chloroplasts. Biochim. Biophys. Acta 79: 646-48.

3. BRIERLEY, G. P., E. MURER, AND D. E. GREEN.

1963. Participation of an intermediate of oxidative phosphorylation in ion accumulation by mito-

chondria. Science 140: 60-62.

4. BRIGGs, G. E., A. B. HOPE, AND R. N. ROBERTSON. 1961. Electrolytes and plant cells. F. A. Davis, Philadelphia. p 168-74. 5. CARAFOLI, E., C. S. RosSI, AND A. L. LEHNINGER. 1964. Cation and anion balance during active ac-

6. 7.

Summary A light-triggered process that brings about ioln translocatioin in spinach chloroplasts in vitro is described. Calcium, phosphate, aln(l so(liumil were showni to be taken ul) using radioactive isotopes, under Simiconditions favoring the hydrolysis of ATP. lar to light-triggered chloroplast adenosine triphosphatase, calcium uptake could occur in the dark following a light preincubation, was stimulated by compounds containing sulfhydryl groups, and was

639

8. 9.

10.

cumulaticn of Ca++ and Mg++ by isolated mitochondria. J. Biol. Chem. 239: 3055-61. DELucA, H. F. AND G. W. ENGSTROM. 1961. Calcium uptake by rat kidney mitochondria. Proc. Natl. Acad. Sci. U. S. 47: 1744-50. DILLEY, R. A. AND L. P. VERNON. 1964. Lightinduced conformational changes of chloroplasts produced by high-energy intermediates of photoplhosphorylation. Biochem. Biophys. Res. Commuin. 15: 473-78. DILLEY, R. A. 1964. Light-inducedl potassium efflux from spinach chloroplasts. Biochem. Biophys. Res. Commun. 17: 716-22. FRANK, E. 1962. Vergleichende Untersuchungen zum Calcium-, Kalium- und Phosphathaushalt von Grunalgen. I. Calcium, Phosphat und Kalium bei Hydrodictyon und Spaeroplea in Abhiingigkeit von der Belichtung. Flora 152: 139-56. HOAGLAND, D. R. AND A. R. DAVIS. 1923. Further experiments on the absorption of ions by

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PLANT PHYSIOLOGY

p)lants, including observations oin the effect of light. J. Gen. Physiol. 6: 47-62. Hocii, G. ANI) I. MARTIN. 1963. Photo-potentiation of adenosine triphosphate hydrolysis. Biochem. Biophys. Res. Commun. 12: 223-28. HODGES, T. K. AND J. B. HA\NSON. 1965. Calciuim accumulatioin by maize mitochondria. Plant Phbisiol. 40: 101-09. HoRwi-mI, B. N. 1952. D)etermination of iniorganiic serum )pllosphate by means of stainious chloride. J. Biol. Chem. 199: 537-41. INGOLI), C. T. 1936. The effect of light on the absorption of salts by Elodea canadensis. New Phytol. 35: 132-41.

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Shrinkage of xwhole chloroplasts upon illumination. Biochim. Biophys. Acta 66: 319-27. 16. J1CQI-E.s, A. G. ANO) W. J. VT. OsTERHoUT. 1934. The accumulationI of electrolytes. VI. The effect of external pH. J. Gen. Pbysiol. 17: 727-50. 17. JAGENDORF, A. T. AN1D G. HIND). 1963. Studies on the mechanism of photoplhosphorylation. In :Photosynttletic Mechanisms of Green Plants, Natl. Acad. Sci., Washingtoni, D. C. p 599-610.

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