light-induced changes in the ultrastructure pea chloroplastsfor microdensitometer studies,...

Plant Physiol. (1972) 49, 535-541

Light-induced Changes in the Ultrastructure of Pea Chloroplastsin Vivo

RELATIONSHIP TO DEVELOPMENT AND PHOTOSYNTHESIS1

Received for publication August 16, 1971

MARCIA M. MILLER2 AND PARK S. NOBELDepartment of Botanical Sciences and Molecular Biology histitute, University of California, Los Angeles,California 90024

ABSTRACT

Light-induced structural changes of chloroplasts and theirlamellae were studied in leaves of Pisum sativum L., cv. BlueBantam, using electron microscopy. Upon illumination of 14-day-old plants with 2000 lux, the chloroplasts decreased inthickness by about 23% with an accompanying increase inelectron scattering by the stroma. Concomitantly, the averagethickness of granal lamellae (thylakoids) decreased from 195+ 4 angstroms in the dark to 152 ± 4 angstroms in the light,and this change was half-saturated at only 50 lux. Lamellarflattening at 50 lux and its reversal in the dark both had half-times of a minute or less. The thickness of a partition (a pairof apposed lamellar membranes) was 140 + 9 angstroms inboth the light and the dark, indicating that the observed light-induced change was in the volume enclosed within the thyla-koid. The effect of illumination could be inhibited by variousuncouplers of photophosphorylation but not by 3- (3, 4-di-chlorophenyl)-j,l-dimethylurea, suggesting that it dependedon ATP (or its precursor). In the presence of 0.5 micromolarnigericin, the thickness of the granal lamellae increased in thelight to 213 + 3 angstroms; this may reflect an uptake of K+into an osmotically responding space within the thylakoids.During development, the capacity of the chloroplasts to

flatten upon illumination increased in parallel with the amountof chlorophyll per gram of leaf and the number of lamellaeper chloroplast. In contrast, the capacity of the leaves to fixCO2 lagged nearly 2 days behind the development of chloro-phyll. C02 fixation developed in parallel with the stacking ofthe lamellae into grana, supporting the contention that suchorganization is related to the linkage of photosystem II to pho-tosystem I.

Effects of light on chloroplast shape in vivo have been ob-served in a variety of plants including algae and both primi-tive and advanced vascular plants (7, 8, 14, 17, 30, 43, 44).In general, chloroplasts flatten in the light and become more

'This investigation was supported in part by Public HealthService Research Grant GM 15183 from the National Instituteof General Medical Sciences. A preliminary report of this workhas been presented (Plant Physiol. 47: S32, 1971).

2 Predoctoral research fellow of the United States Public HealthService (GM 46035).

spherical in the dark. Such light-induced flattening of thechloroplasts is dependent upon certain specific photochemicalreactions. For instance, Izawa et al. (16) showed that the ac-tion spectrum for the structural changes in vitro correspondsclosely to the absorption spectrum of chloroplasts, except fora minor peak from 720 to 740 nm. A similar enhanced activityfor wavelengths beyond 680 nm was observed by Nobel (27)using chloroplasts that were illuminated in vivo and then rap-idly but gently isolated in order to retain the volume changesoccurring in the plant. Moreover, Heber (13) attributed ab-sorbance changes caused by far-red illumination of intactleaves to alterations in chloroplast shape. It thus appears thatthe chloroplast flattening can be caused by far-red light ab-sorbed by Photosystem I, suggesting that the energy source isATP or a high energy precursor to ATP. In support of thishypothesis, the light-induced flattening has been found to besuppressed by the uncouplers of photophosphorylation, nigeri-cin (27, 31, 40) and FCCP3 (13, 27), both in vivo (13, 27) andin vitro (31, 40).

Light-induced chloroplast flattening occurring in the plantcell is correlated with increased rates of photosynthesis in vivo(18, 30) and also with increased rates of photophosphorylation(18, 25) and CO2 fixation (29) by the isolated chloroplasts. Forinstance, Nobel (25, 29) has found that illuminating peaplants before chloroplast isolation causes an approximate dou-bling of the photophosphorylation and CO2 fixation rates invitro. Of special interest is the observation that the kineticsand light intensity responses of the metabolic changes are es-sentially identical to those for the reversible chloroplast flat-tening under the same conditions (25, 26). Thus, the structuralchange of the chloroplasts is accompanied by altered rates ofmetabolism, although it has not been possible to delineatecause and effect. One of the objectives of the present studyis to investigate the correlation between CO2 fixation and light-induced structural changes during chloroplast development.

Light may also affect chloroplasts in vivo at the ultrastruc-tural or lamellar level (4, 24, 32). Packer et al. (32) detectedno distinct change in granal lamellae upon illuminating spinachleaves. Murakami and Packer (24) indicated that the thick-ness of lamellae in algal thalli was little affected upon illumi-nation, unless they first infiltrated the thalli with a solutioncontaining sodium acet?te and ferricyanide or phenazine meth-osulfate. Under the latter rather "unphysiological" conditions.light caused a 28% decrease in the thickness of the lamellae,which is similar to the light-induced ultrastructural changes

3Abbreviation: FCCP: p-trifluoromethoxycarbonyl cyanidephenylhydrazone.

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Plant Physiol. Vol. 49, 1972

a. Major axis

Lamellar thicknessb.

Partition thickness 3

FIG. 1. Diagram of a mature pea chloroplast (a) and an ex-panded view of a granum (b). The major axis is the longest di-mension of the chloroplast in section, whereas the minor axis isthe shortest distance through the chloroplast bisecting the majoraxis. A lamella extends from the center of one partition (two ap-posed membranes within a granum) to the center of the adjacentpartition.

occurring in vitro (22, 23, 42). Consequently, there is un-certainty as to the actual effect of light on the structure andthickness of chloroplast lamellae in vivo. In the present ex-periments, the kinetics, light intensity dependence, and inhib-itor responses of light-induced changes in the lamellae of peachloroplasts in vivo are determined.

MATERIALS AND METHODS

Material. Seeds of Pisum sativum L., cv. Blue Bantam (W.Atlee Burpee Co., Riverside, Calif.) were soaked for 24 hrin the dark and then planted 2 cm below the surface in moistvermiculite. Plants were grown at 20 C and 50% relative hu-midity under a light intensity of 2000 lux provided by fluores-cent lights for 12 hr each day. After 14 days or other periodsas specified below, plants were cut at the surface of the vermic-ulite and then were placed with their stems in distilled waterfor 1 hr either in darkness or at a light intensity of 2000 lux(unless otherwise indicated). Under the same light conditions,the bottom (oldest) leaf was then detached and placed in aPetri dish containing the fixative for electron microscopy, andsmall pieces of the leaf near the midvein were cut out with arazor blade.

Fixation, Dehydration, Staining. Specimens were fixed at 20C in 4% (v/v) glutaraldehyde (EM Grade, Polysciences,Warrington, Pa.), 50 mm Na-KH2PO4, pH 7.9, for 1 hr fol-lowed by rinsing in 50 mm buffer for 24 hr; post fixation wascarried out in 1% (w/v) OsO4, 50 mM Na-KH2PO4, pH 7.9,for 1 hr. Fixed tissue was dehydrated in a graded series ofacetone solutions (10 min in each of 25, 50, 70, and 100%[v/v] acetone) and then embedded in an Epon-Araldite mix-ture (20:15 :5:1.2 by volume of dodecenyl succinic anhy-dride-Araldite 502-Epon 81 2-dibutyl phthalate, Ref. 21 asmodified by R. B. Addison). Tissue was stained with 0.5%(w/v) uranyl acetate for 24 hr in the 70% acetone used dur-ing the dehydration steps. Sections were prepared with glassknives on an LKB Ultrotome, poststained with 5% (w/v)Ba(MnO4)2 for 3 min, and then viewed with a Philips EM200 electron microscope.

Axes, Lamellae. Sections judged to be passing through thecenter of chloroplasts and in which the stacking of granallamellae was clearly visible were selected. and electron mi-

crographs were prepared. The major chloroplast axis wasdefined as the longest dimension in the chloroplasts, whilethe minor axis was the shortest distance through the chloro-plast bisecting this major axis (Fig. 1 [a]). The axial ratio is themajor axis divided by the minor axis. As an index of thenumber of lamellae per plastid, the number of lamellae tran-sected by the minor axis was determined. The number ofgranal lamellae per chloroplast was obtained by counting thenumber of lamellae which were formed into grana in the sec-tions of chloroplasts used in determining the axial ratios. Thestandard error of the mean of samples was calculated usingFortran programs.

Microdensitometry. For microdensitometer studies, nega-tives at 91,000 magnification were traced with a 20: 1 armratio on a Joyce, Loebl model MK III C double beam re-cording microdensitometer with a 30-,um slit (magnificationwas checked using a carbon grating with a spacing of 463nm). The thickness of lamellae (cf. Fig. 1[b]) was determinedfrom the distance between microdensitometer peaks obtainedupon tracing across grana (see also Fig. 2[b]). The thicknessof a partition (two apposed membranes within a granum)was estimated from the full width at the half maximal valuesfor a microdensitometer peak (the baseline was the densityof the surrounding stroma). Negatives at a magnification of46,000 were traced across a cell wall and then across thestroma present between the limiting membranes of the chloro-plast and the lamellar network. Assuming that the electronscattering of the cell wall was the same in both the light andthe dark samples, comparisons could be made between theelectron scattering of the stroma under different conditions.CO2 Fixation, Chlorophyll. To measure CO2 fixation, we

detached leaves from plants 4 to 14 days old and placed themin a chamber containing 0.03% CO2 (labeled with "4C), 21%02, and 79% N2. After equilibrating for 10 min, we illuminatedthe leaves for 10 min at 2000 lux with a tungsten lamp andthen immediately immersed them in liquid nitrogen. Theleaves were dried on planchets, and radioactivity was deter-mined with a Geiger-Muller counter (see ref. 30 for details).

For measurement of chlorophyll content, approximately0.1 g of leaves was weighed and then homogenized in 10 mlof 80% (v/v) acetone. After centrifugation and re-extractionof the leaves, the absorbance of the extracts was measuredat 645 and 663 nm using a Cary 14 recording spectrophotome-ter. The chlorophyll concentration was determined by themethod of MacKinney (19) with the use of the equation de-rived by Arnon (3).

Inhibitors, Uncouplers. Inhibitors and uncouplers were in-troduced by bathing cut stems in solutions containing 1 mMDCMU, 5 ptM FCCP, 0.5 /_M monensin, or 0.5 /tM nigericinin 20-ml beakers (27). A cut plant was placed in a particularsolution and then maintained in the dark or at a light intensityof 2000 lux for 4 hr to allow uptake, after which the bottomleaf was prepared for electron microscopy as described above.Rates of CO2 fixation measured in the manner described abovewere used to determine the effect of the inhibitor or the un-couplers in the same portion of the leaf as was employed inthe preparations for electron microscopy. Monensin and ni-gericin were generously provided by Dr. J. M. McQuire(Lily Research Laboratories, Indianapolis, Ind.) and FCCPwas provided by Dr. P. G. Heytler (E. I. du Pont de Nemoursand Co., Wilmington, Del.).

RESULTS

Striking changes in the thickness of chloroplast granal lamel-lae resulted from illumination of the plants. The spaces withingranal lamellae, which appear as light areas between the dark

536 MILLER AND NOBEL

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LIGHT AFFECTS CHLOROPLAST ULTRASTRUCTURE

., C

CENTER-TO -CENTERSPAC ING

b

f

d

hFIG. 2. Effect of light and nigericin on granal lamellae in chloroplasts of 14 day-old-pea plants. a: Granum in a plant which has been in the

dark for 4 hr prior to fixation; b: microdensitometer tracing across the granum in (a); c: granum in a plant which has been in the light tor 4hr prior to fixation; d: microdensitometer tracing across the granum in (c); e: granum from a plant which has been in the dark for 4 hr withthe cut stem in 0.5 Mm nigencin; f: microdensitometer tracing across the granum in (e); g: granum from a plant which has been in the light for4 hr with the cut stem in 0.5 ,M nigericin; h: microdensitometer tracing across the granum in (g).

lines in the electron micrographs, were about twice as large ingrana of plants in the dark (Fig. 2[a]) compared with the light(Fig. 2[c]). Microdensitometer tracings of grana (Fig. 2[b]and 2[d]) were used to measure this light-induced decreasein thylakoid thickness. Comparison of the center-to-centerspacings between tracing peaks showed that the thickness ofgranal lamellae in plants under a light intensity of 4000 luxwas 152 ± 4 A, a decrease of 22% from the 195 + 4 Athickness in the dark (Table I). This light-induced decrease wassaturated at a light intensity of only 200 lux and was half-saturated near 50 lux, where the center-to-center spacing ofthe tracing peaks was 172 + 3 A (Table I). After 1 min undera light intensity of 50 lux, the thWckness of the thylakoids was181 ± 4 A. This observation and other data in Table I indi-cate that the half-time for the light-induced decrease in granal

lamellar thickness is about 1 min at the extremely low lightintensity of 50 lux. At 4000 lux, the flattening was completein less than half a minute. Reversal of the flattening in thedark was also rapid and had a half-time of about 0.5 min forplants transferred from a light intensity of 2000 lux to dark-ness (Table I).As determined by measuring the full width of the half

maximal values of the microdensitometer tracings (see Fig.2[b]), the partition thickness was 140 + 9 A for grana fromplants either in the light or the dark. Therefore, the observed22% decrease in the thickness of granal lamellae in the lightwas attributed to a decrease in the space enclosed by thelamellar membranes (i.e., the volume within the thylakoid),with no detectable contribution coming from a decrease inthe thickness of the lamellar membranes themselves. The

Plant Physiol. Vol. 49, 1972 537

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Table 1. Effect of Illuminiationi atnd Time onz the Thickniessof Granal Lamellae in 14-Day-Old Pisum sativum

Plants were kept in the dark (or 2000 lux for the bottom fourtable entries) for 1 hr and then exposed to various light intensitiesfor the times indicated. The center-to-center spacings of thetracing peaks across granal lamellae were determined as illustratedin Figure 2(b). Data are presented as mean + SE.

LightIntensity

lIux

050

2002000

50505050500000

Time rNo. of Lamellae Center-to-CenterTime Measured Spacing

jnin i| A

60 145 195 460 122 172 ± 360 203 150 ± 260 85 152±40 359 192 30.2 25 190±40.5 70 186 ± 41.0 39 181 ± 3

60 122 172 ±430 33 150±30.5 50 173 33.0 65 186±4

30 61 194 ±+4

Uiicouiplers of Photosynithesisonl the Light-iliduced Decrease in the Thick,,ess

of Grantal LamellaeFour hours after placing plants cut below the first leaf node in

the solutions indicated, leaves were prepared for electron mi-croscopy under the same light conditions, viz., in the dark or at alight intensity of 2000 lux. For cut plants placed in the solutionsin the dark, the center-to-center spacings of the lamellar tracingswere not significantly different from the water control (191 + 3 A),and so only the lamellar thickness in the light is presented in thetable. To test whether the various compounds actually reachedthe leaves, we also assayed the CO2 fixation rates in leaves after4 hr in the bathing solutions; the light-dependent CO2 fixationrate of the water control was 20.0,umoles CO2 fixed per mg chloro-phyll in 10 min.

Solution BathingStem

Water1 mM DCMU5,M FCCP0.5 M51 Monensin0.5 Mm Nigericin

Relative C02 No. of Lamellae Center-to-CenterFixation Rate Mleasured Spacing

c

10012181112

.4

101 150 + 366 153 ± 3117 169 ± 398 190±4121 213 ± 3

stromal lamellae appeared to respond to light in the samemanner as did the granal lamellae, viz., they flattened 20% inresponse to light (2000 lux) with no apparent change in thethickness of the membranes of a stromal lamella.To study the energetic basis of the decrease in lamellar

thickness in the light, we performed experiments with un-

couplers and an inhibitor of photosynthesis (Table II). Theconcentrations listed in the table, e.g., 1 mm for the inhibitorDCMU, are those bathing the cut stems-the actual concen-trations in the leaf cells are not known. For determinationof the metabolic effect of the uncouplers and the inhibitor,rates of CO2 fixation in leaf tissues treated in the same wayas for the electron microscopy were measured. DCMU in-hibited CO2 fixation 88%; on the other hand, the thickness

of the lamellae in the light (153 + 3 A) and in the dark (188+ 3 A) in the presence of 1 mm DCMU was the same as inthe control samples (Table II). FCCP decreased CO2 fixationby 82% and also inhibited the light-induced decrease in la-mellar thickness. The light-induced change in the thicknessof the granal lamellae with 5 ptM FCCP was 22 A comparedwith the control where the thickness decreased 41 A in thelight (Table II). Another uncoupler, monensin, abolished thelight-induced decrease in the thickness of the granal lamellae.Nigericin, which is structurally similar to monensin and whichalso reduced CO2 fixation by about 90%, actually caused thethickness of the lamellae to increase to 213 + 3 A in thelight (Table II). This unusual effect is evident in Figure 2(e)to (h), where electron micrographs and microdensitometertracings demonstrate that the thickness of granal lamellae be-comes greater upon illumination in the presence of nigericin.There was no detectable change in the partition thickness inthe presence of the uncouplers. Thus the uncouplers reducethe light-induced decrease in the thickness of the lamellae byinhibiting the change in the space contained within the lamel-lae and not by affecting the thickness of the membranes.The stromal lamellae responded to the uncouplers in thesame way as did the granal lamellae.To investigate the possibility of light-induced changes in

the stroma, we made microdensitometer tracings across elec-tron micrographs of leaf cells. The cell walls provided a base-line for density, i.e., they were assumed to have the sameelectron scattering for plants in the light compared with thedark. Figure 3 shows representative microdensitometer trac-ings across the cell wall, plasmalemma, cytoplasm, limitingmembranes, and the stroma of chloroplasts. As can be seen,the electron scattering of the stroma in plants in the lightwas higher than in the dark. Such a light-induced increase inthe electron scattering of the stroma is consistent with theloss of water that accompanies the chloroplast flattening causedby illumination (7, 8, 14, 17, 30, 43, 44). In particular, themean axial ratio (major/minor axis) of 36 chloroplasts inilluminated plants prepared as usual using glutaraldehyde andosmium was 2.98 + 0.08. For 97 chloroplasts from plants inthe dark the mean axial ratio was 2.51 ± 0.06. When pre-pared with OsO alone, the axial ratios in the light (37 chloro-plasts) and in the dark (34 chloroplasts) were 2.15 ± 0.07and 1.71 + 0.05. respectively. Although the magnitudesof the axial ratios were different for the two fixatives, in bothcases the axial ratios of the chloroplasts increased about 20%

10

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c'STANCE TRACEDujm

FIG. 3. Microdensitometer tracings across electron micrographsof leaf cells of plants which have been in the light or the dark.Sections were stained with 5% (w/v) uranyl acetate for 30 min andthen with 1.7% (w/v) lead citrate for 5 min (36).

Table II. Effect of ani Inihibitor alid

Plasmolemma StromaChloroplast membranes light

Cell wall ; Kk

-N--


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in the light. (The lower values of the axial ratios in the OsO0preparations reflect the tendency of chloroplasts to becomemore spherical in this fixative.) In summary, the light-in-duced decreases in the thickness of the granal lamellae andthe increase in the electron scattering of the stroma is ac-companied by an increase in the axial ratio of the chloro-plasts, the latter reflecting a flattening of this organelle uponillumination.

For measurement of light-induced flattening during devel-opment, the major and the minor axes of chloroplasts in 6-,8-, 11-, and 14-day-old plants were determined in the lightand the dark (Fig. 4). Both axes increased in length as thechloroplasts developed. However, while the major axes hadnearly the same mean values in the light and the dark, de-creases in the minor axes in response to light became greaterwith age. In particular, when the plants were 6 days old, theminor axes of the chloroplasts in plants in the light or the darkwere essentially the same, while at 8 days the minor axis was0.30 Mum shorter in the light, and at 11 and 14 days this differ-ence increased to 0.53 and 0.56 ,um, respectively (Fig. 4).From the measurements of minor axes, it was calculated thatthe chloroplasts flattened 0, 16, 22, and 23% at 6, 8, 11, and14 days, respectively. Therefore, while the chloroplasts wereincreasing in size during development, they were also acquir-ing a capacity to flatten upon illumination.To relate the development of the capacity for light-induced

flattening to other processes in the chloroplasts, we alsostudied the amount of chlorophyll and the rate of CO2 fixa-tion in the developing plastids (Fig. 5). The increase in theamount of chlorophyll per g fresh weight of leaves coincidedwith the development of the capacity to flatten. In contrast,the capacity of the leaves to fix CO2 developed more slowlyand lagged about 2 days behind the formation of chlorophyll.For example, on the 8th day, the amount of chlorophyll wasapproximately 55% of that on the 14th day, while the CO2fixation rate was only about 7% of the rate on the 14th day(Fig. 5). By the 12th day, the chlorophyll content, the ca-

apr axes

4 /

3

z g

628 -i .)LLEAF A,E L AYK

FIG. 4. Effect of age and light conditions on axial lengths ofchloroplasts in developing pea leaves. Major and minor axes in thelight or the dark are presented with bars indicating the standarderror of the mean. Leaf tissue was fixed with 1% OsO4, 0.05 MNa-KH2PO4 (pH 7.9) for 1 hr followed by the usual dehydrationand embedding techniques.

FIG. 5. Effect of plant age on chlorophyll content, light-in-duced flattening, number of granal lamellae, and C02 fixation ofchloroplasts in pea leaves. Data are presented as percentage ofvalue observed on the 14th day, when the chlorophyll content was2.1 mg/g fresh weight of leaves, the flattening amounted to a 23%decrease in the minor axis in the light, there were 138 granallamellae/chloroplast in section, and the C02 fixation rate was19.5 jumoles C02 fixed/mg chlorophyll in 10 min (light minus darkrate).

pacity to undergo the light-induced flattening, and the CO2fixation rates of the chloroplasts had all reached values char-acteristic of the mature leaf.Next the chloroplast lamellae were examined at various

stages of development. An indication of the number of lamel-lae was obtained by counting the stromal and granal lamellaethat were transected by the minor axis of the chloroplast. Thisnumber was about three at 6 days, 16 by the 8th day, 20 onthe 11th day, and 25 after 14 days. The number of lamellaeincreased at a rate similar to both the development of chloro-plast flattening and the amount of chlorophyll per unit weightof leaf. As another index, the total number of granal lamellaeper chloroplast was counted. When the leaves were 6, 8, 9,11, and 14 days old, the number of granal lamellae per chloro-plast (in section) was 5, 30, 65, 127, and 138, respectively.As shown in Figure 5, the increase in granal lamellae occurredat the same rate as the development of the capacity of theintact leaves to fix CO2. Thus, the light-induced flatteningdeveloped at the same rate as the number of lamellae, whileCO2 fixation developed later, viz., at a time when the lamellaeaggregated into grana.

DISCUSSION

Two light-induced flattening processes may occur in chloro-plasts, one located at the limiting membranes and the otherin the lamellar system itself. The thickness of granal lamellaedecreased about 40 A or 22% upon illumination of pea plants,contrary to previous finding, in vivo with other species (24,32). The responses to light of both stromal and granal lamellaewere similar, in agreement with observations of Murakami

Plant Physiol. Vol. 49, 1972 539

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and Packer (22) on the light-induced changes in vitro. Thedecrease in the thickness of granal lamellae caused by il-lumination was saturated at only 200 lux. In contrast, thelight-induced flattening of the whole chloroplast in vivo,which results in a 23% decrease in thickness (cf. Fig. 4),saturates at 2000 lux (26). The kinetics of the two processesare also different. The thylakoid flattening in the light hasa half-time of about 1 min at 50 lux, while the reversal in thedark has a half-time of about 0.5 min. These responses aremuch faster than the light-induced flattening and its reversalfor whole chloroplasts, which have half-times of 3 to 5 min(26).The difference between the two light-induced flattening

processes is further supported by an estimate of the extent towhich flattening of the lamellae contributes to the over-allflattening of the chloroplasts. The number of lamellae tran-sected by the minor axis (about 25) multiplied by the 40 Adecrease in the light per lamella suggests that the change in thechloroplast thickness attributable to the lamellae is 1000 Aor 0.1 ,um, a value considerably less than the 0.56-,tm decreasein chloroplast thickness actually observed here. As other evi-dence that the ultrastructural change is not responsible forthe flattening of whole chloroplasts, Nobel (28) has calculatedthat upon illumination there is an efflux of 32% of the freewater in chloroplasts. (The observed increase in the electronscattering of the stroma in the chloroplasts of plants upon il-lumination correlates well with this efflux of water, since thechloroplast contents are then contained in a smaller volumeand would scatter electrons more.) Thus the mechanical as-pects of the light-induced flattening, including the light in-tensity for saturation and the kinetics, are different at thechloroplast level compared with the lamellar level.

Metabolic studies with uncouplers and an inhibitor suggestthat the light-induced decrease in lamellar thickness dependson photophosphorylation, similar to the over-all flattening ofchloroplasts caused by illumination (27). In particular, bath-ing the cut stems in FCCP inhibited the light-induced decreasein the thickness of granal lamellae by 46%, monensin elim-inated it, and nigericin actually caused a light-induced in-crease in thickness. In previous experiments with pea chloro-plasts in vivo (27), FCCP inhibited 80% of the light-inducedchloroplast flattening while nigericin eliminated the light ef-fect. Moreover, DCMU had little effect on either the light-induced chloroplast flattening (27) or the change in thylakoidthickness (although CO2 fixation was severely inhibited in itspresence), suggesting that neither process was dependent onnoncyclic electron flow. In short, ATP (or its precursor) is ap-parently responsible for both the light-induced decrease inthe lamellar thickness and the over-all chloroplast flatteningcaused by illuminating the plant.

With isolated chloroplasts, the uncoupling effect of nigeri-cin is generally dependent on the presence of K+ (31, 39, 40).Nobel (28) has estimated that the K+ concentration within peachloroplasts in vivo is about 100 mm. Nigericin may eliminatethe light-induced decrease in lamellar thickness by uncouplingphotophosphorylation in vivo using this potassium. The ob-served swelling of thylakoids in the light in the presence ofnigericin may be due to the accumulation of K+ within theosmotically responding space of the granal lamellae, similarto observations with isolated chloroplasts in the presence ofnigericin and 100 mm KCl (40).

In the present studies no major change was observed in thethickness of the lamellar membranes themselves upon illumi-nation. On the other hand, Murakami and Packer (22-24)observed a light-induced decrease of about 30 A per partition,but only in the presence of phenazine methosulfate plus so-dium acetate or phenylmercuric acetate. Such reagents may

exaggerate a normally occurring conformational change ofthe membrane proteins and thereby maKe it large enough to beresolved with the electron microscope. In this regard, a light-induced conformational change in lamellar membranes hasbeen postulated based on fluorescence measurements (33, 34).The capacity of chloroplasts to flatten in the light increases

in parallel with the amount of chlorophyll and total numberof lameilae in the developing plastids, but apparently thelight-induced flattening does not depend on grana formation(Fig. 5). Photosystem I activity in bean etioplasts develops inparallel with chlorophyll, while photosystem II reactions lagat least 5 hr behind (12). Also photochemical reduction offerricyanide (a test of noncyclic electron flow) by bean etio-plasts begins after 6 hr of illumination of the plants and cor-responds in time with the appearance of rudimentary grana asobserved by phase contrast microscopy (1). Since chloroplastflattening depends on ATP or its precursor as an energysource (27), it could be supported by cyclic photophosphoryla-tion (a photosystem I activity) before the advent of a func-tioning photosystem II during the development of the or-ganelles. In the present study, the capacity to fix CO., whichrequires the linking of photosystem I to photosystem II inorder to provide a pathway for noncyclic electron flow, lags2 days behind the development of chlorophyll. This lag inCO-fixing ability coincides with the delay in the developmentof grana within the chloroplasts (Fig. 5), further suggestingthat grana formation and photosystem II activity may be re-lated.

Although the relationship between grana structure and func-tion in higher plants is by no means fully understood, there isevidence that the stacking of lamellae into grana may be re-lated either to the presence of photosystem II activity (1, 2, 6,9-12, 15, 20, 35, 37, 38) or to the linking of photosystem IIwith photosystem I by an intermediate in the chain of electroncarriers (5, 41). For instance, the formation of the partitionregion within grana has been suggested to be an importantstep in the development of the CO2 fixation ability of barleyplastids (37). The localization of photosystem II in the granaof spinach chloroplasts has also been proposed on the basisof specific photochemical reactions observed in subchloroplastfractions prepared using the French press (38) or digitonindigestion (11, 35). The formation of grana apparently is nec-essary for the integrated functioning of photosystem I andphotosystem II and thus for CO2 fixation by the chloroplastsof higher plants. Other energy-requiring processes such as thelight-induced chloroplast and lamellar flattening can be sup-ported by photosystem I by itself and apparently are inde-pendent of grana formation.

Acknowledgment-Thorouah instruction concerning the use of the electronmicroscope by Professor John Fessler is gratefully acknowledged.

LITERATURE CITED

1. ANDERSON, J. 'M. AN-D N. K. BOARDNIANZ. 1964. Studies on the greening ofdark-grown bean plants. II. Development of photochemical activity. Aust.J. Biol. Sci. 17: 93-101.

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4. BARTELS, F. 1971. Strukturelle VerEnderungen der Chloroplasten-Thylakoidein Palisadenzellen von Peperomia metallica im Licht-Dunkelwechsel.Protoplasma 72: 27-41.

5. BiSHOP, D. G., K. S. ANDERSEN-. A'ND R. M. SMILLIE. 1971. Lamellar structureand composition in relation to photochemical activity. In: M. D. Hatch,C. B. Osmond, and R. 0. Slatyer, eds., Photosynthesis and Photorespira-tion. John Wiley, Interscience, New York. pp. 372-381.

6. BLACK, C. C., JR. AND H. H. TMOLLENHACER. 1971. Structure and distributionof chloroplasts and other organelles in leaves with various rates of photo-synthesis. Plant Pbsi-o. 47:-15;-3.


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7. B,-'SNNIN-G, E. 1942. Untersuchungen uiber den physiologischen Mechanismusder endogenen Tagesrhythmik bei Pflanzen. Z. Bot. 37: 433-486.

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17. KuSHIDA, H., M. ITOH, S. IZAWA, AND K. SHIBATA. 1963. Deformation ofchloroplasts on illumination in intact spinach leaves. Biochim. Biophys.Acta 79: 201-203.

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21. MOLLENHAUER, H. H. 1964. Plastic embedding mixtures for use in electronmicroscopy. Stain Technol. 39: 111-114.

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23. MURAKAMI, S. AND L. PACKER. 1970. Protonation and chloroplast membranestructure. J. Cell Biol. 47: 332-351.

24. IMURAXAMI, S. AND L. PACKER. 1970. Light-induced changes in the confor-mation and configuration of the thylakoid membranes of Ulva and Por-phyra in vivo. Plant Physiol. 45: 289-299.

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26. NOBEL, P. S. 1968. Light-induced chloroplast shrinkage in vivo detectableafter rapid isolation of chloroplasts from Pisum sativum. Plant Physiol.43: 781-787.

27. NOBEL, P. S. 1968. Energetic basis of the light-induced chloroplast shrink-age in vivo. Plant Cell Physiol. 9: 499-509.

28. NOBEL, P. S. 1969. Light-induced changes in the ionic content of chloroplastsin Pisum sativum. Biochim. Biophys. Acta 172: 134-143.

29. NOBEL, P. S. 1970. Increased CO2 fixation by Pisum sativum chloroplasts invitro reflecting a change in coupling caused by illuminating the plants.Plant Cell Physiol. 11: 467-474.

30. NOBEL, P. S., D. T. CHANG, C.-T. WANG, S. S. SMITH, AND D. E. BARCUS.1969. Initial ATP formation, NADP reduction, CO2 fixation and chloro-plast flattening upon illuminating pea leaves. Plant Physiol. 44: 655661.

31. PACKER, L. 1967. Effect of nigericin upon light-dependent monovalent cationtransport in chloroplasts. Biochem. Biophys. Res. Commu.n. 28: 1022-1027.

32. PACKER, L., A. C. BARNARD, AND D. W. DEAMER. 1967. Ultrastructural andphotometric evidence for light-induced changes in chloroplast structure invivo. Plant Physiol. 42: 283-293.

33. PAPAGEORGIOU, G. A-ND GOVINDJEE. 1968. Light-induced changes in the fluores-cense yield of chlorophyll a in vivo. I. Anacystis nidulans. Biophys. J. 8:1299-1315.

34. PAPAGEORGIOU, G. AND GOVINDJEE. 1968. Light-induced changes in thefluorescence yield of chlorophyll a in vivo. II. Chlorella pyrenoidosa. Bio-phys. J. 8: 1316-1328.

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41. SMILLIE, R. M., K. S. ANDERSEN, AND D. G. BISHOP. 1971. Plastocyanin-de-pendent photoreduction of NADP by agranal chloroplasts from maize.FEBS (Fed. Eur. Biochem. Soc.) Lett. 13: 318-320.

42. SUNQJIST, J. E. AND R. H. BURRIS. 1970. Light-dependent structural changesin the lamellar membranes of isolated spinach chloroplasts: measurementsby electron microscopy. Biochim. Biophys. Acta 223: 115-121.

43. VANDEN- DRIESSCHE, T. 1966. Circadian rhythms in Acetabularia: photosyn-thetic capacity and chloroplast shape. Exp. Cell Res. 42: 18-30.

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light-induced changes in the ultrastructure pea chloroplastsfor microdensitometer studies,...

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