Plant Physiol. (1975) 55, 520-525

Photosynthetic Activi-ty and Membrane Polypeptide Compositionof Supergranal Chloroplasts from Plant Tissue CulturesContaining a Viruslike Particlet

Received for publication July 31, 1974 and in revised form October 10, 1974

DONNA D. SMITH2 AND RICHARD D. SJOLUNDDepartnment of Botany, University of Iowa, Iowa City, lowa 52242

ABSTRACT

Tissue culture cells of Streptanthus tortuosus (Kell.) var.orbiculatus (Greene) Hall (Cruciferae), having a viruslikeparticle in their nucleoli, the STV cell line, contain "super-granal" chloroplasts. Freeze-fracture studies of chloroplasts ofa control cell line, which lacks the viruslike particles, revealtwo complementary faces similar to those observed in spinachchloroplasts. Replicas of freeze-fractured STV supergranalchloroplasts, however, show that one membrane face (B) con-

tains widely spaced 80 A particles and the other face (C) isessentially smooth. Isolated STV supergranal chloroplasts lackphotosystem II activity as indicated by their inability to reducedichlorophenolindophenol and are unable to reduce NADP withelectrons from photosystem II or from ascorbate-reduced di-chlorophenolindophenol. However, partial photosystem I ac-tivity is indicated by the reduction of methyl viologen withelectrons from dichlorophenolindophenol-ascorbate. This sup-ports the concept that there is not a direct correspondencebetween grana formation and photosystem II activity. Elec-trophoresis shows that all of the major polypeptide bandspresent in the STV supergranal chloroplasts are also presentin the control chloroplast membranes. One band, molecularweight 33,000, is present in a greatly increased amount in theSTV supergranal chloroplast membranes and may be associatedwith grana stacking.

Tissue culture cells of Streptanthus tortuosuis (Kell.) var.orbiculatus (Greene) Hall (Cruciferae) which were shown bySjolund and Shih (25) to have a viruslike particle located intheir nucleoli, designated cell line STV, have been demon-strated by Sjolund and Smith (26) to contain supergranalchloroplasts. Previously reported freeze-fracture investigationshave shown these supergranal chloroplasts of the STV cellline to have a unique internal membrane substructure (26).Preliminary investigations of the photosynthetic activity of theSTV supergranal chloroplasts indicated that they exhibited nolight-dependent O, evolution or "4CO2 fixation (26). In the

This work was supported in part by National Science Founda-tion Grant GU2591.

2 Present address: Department of Biological Sciences, PurdueUniversity, West Lafayette, Ind. 47907.

present report we discuss the PS I3 and PS II activities ofisolated STV supergranal chloroplasts and the polypeptidecomposition of their thylakoid membranes.The work of Arntzen et al. (6) on digitonin-fractionated

spinach chloroplast membranes implicated the small C faceparticles and the large B face particles of freeze-fracturedmembranes as "markers" of PS I and PS II activities, respec-tively. Further studies by Sane et al. (24) and Goodchild andPark (11) have shown that the large B face particles occur onlyin partition regions of grana. The partition regions containboth size class particles while the stroma lamellae contain onlythe small, C face particles. In addition, they showed a func-tional difference between the two regions of the internal mem-brane structure. The partition regions of grana contain bothPS I and II activities while the stroma lamellae contain onlyPS I activity.Goodenough and Staehlin (13), however, have done freeze-

fracture studies of chloroplasts from mutant and wild typestrains of the green alga Chlamydomonas reinhardtii, and theyconcluded that a correlation exists between a greater density ofB-face particles and regions of membrane stacking, rather thana correlation between the particles and photosynthetic activity.The supergranal chloroplasts of the STV cell line which

contain only stacked chloroplast lamellae provide an oppor-tunity to investigate the relationship of the membrane sub-structure revealed with freeze-fracture to grana stacking andphotosynthetic function.

Recent studies of the polypeptide composition of the chloro-plast membranes of Chlamydomonas (18, 19) and higher plants(3, 19) have shown that membrane fractions enriched in PSI and II activities have different characteristic polypeptidecompositions and that agranal chloroplasts lack some of thepolypeptides associated with the fraction enriched in PS IIactivity. The STV cell line, which can survive by heterotrophicgrowth in tissue culture, represents in higher plants a uniqueopportunity to correlate internal membrane substructure asrevealed by freeze-fracturing, photosynthetic function, andmembrane polypeptide composition.We have found that the STV supergranal chloroplast lacks

PS II activity and is unable to reduce NADP. However, partialPS I activity is indicated by the reduction of methyl viologenwith electrons from DPIP-ascorbate. The polypeptide composi-tion of the STV supergranal chloroplasts is essentially the sameas control chloroplasts with the exception of an increasedamount of a polypeptide of mol wt 33,000.

'Abbreviations used: PS I: photosystem I; PS II: photosystemII; DPIP: 2 6-dichlorodiphenolindophenol; DPC: diphenylcarbo-hydrazide.

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SUPERGRANAL CHLOROPLASTS

MATERIALS AND METHODS

The callus tissues used in this investigation were grown ona liquid culture medium (21). The origin of the STV cell lineand the control cell line, 6045, has been previously reported(25, 26).

Tissue was prepared for electron microscopy by fixation inKarnovsky's fixative (16). embedded in Spurr's plastic (28),thin sectioned with a diamond knife, and examined with eithera Zeiss EM-9A or Hitachi HU-12 electron microscope.

Material for freeze-tracturing was fixed in 0.2% glutaralde-hyde and infiltrated in 25% glycerol for 12 hr. Freeze-fractur-ing, etching, and replication were performed using a Balzersfreeze-etch device (20).

Chloroplasts were isolated for use in photoreactions by theprocedure of Sane et al. (24). The grinding buffer contained0.05 M potassium phosphate (pH 7.4), 10 mm KCl, and 0.5 Msucrose. The resulting pellet of chloroplasts was resuspended in0.05 M potassium phosphate (pH 7.4) and 0.15 M KCl. Chloro-phyll determinations were performed by the method of Arnon(5).

Assays of the ability of isolated chloroplasts from the twocell lines to reduce NADP and DPIP were performed accord-ing to the procedure of Gorman and Levine (14). The reduc-tion of NADP was followed as an increase in absorption at340 nm using a Beckman Acta III recording spectrophotom-eter with an attached actinic light. The reaction cuvette con-tained in a 1 ml volume: chloroplasts containing Chl equalto approximately 10 jug, 10 mM KPO, (pH 7.0), 2.5 mMMgCl2, 20 mM KCI, 0.5 mM NADP, and an excess of NADPreductase and ferredoxin. Where ascorbate-reduced DPIP wasused as an artificial electron donor, 0.05 mm DPIP, 5 mmascrobate, and 0.01 mM DCMU were added. The reduction ofDPIP was followed as a decrease in absorption at 600 nm. Thereaction cuvette contained in a 1 ml volume: chloroplasts con-taining Chl equal to approximately 10 ,ig, 10 mm KPO, (pH7.0), 2.5 mM MgCl2, 20 mm KCl, and 0.1 mm DPIP. DPC,when used, was added to a concentration of 0.5 mm. Assaysof the ability of isolated chloroplasts to reduce methyl viologenwere done by monitoring the uptake of 02 using a Gilsonoxygraph equipped with a Clark electrode (Gilson MedicalElectronics, Inc., Middleton, Wis.). The reaction mixturecontained in a 1.7 ml volume: chloroplasts containing approxi-mately 75 jig Chl, 10 mM KPO4 (pH 7), 2.5 mM MgCl2, 20 mMKCI, and 0.1 mm methyl viologen. Where DPIP-ascorbate wasused as an artificial electron donor, 0.1 mm DPIP, 1 mMascorbate, and 0.01 mm DCMU were added.

Chloroplasts for determination of the polypeptide com-position of chloroplast membranes were isolated in the samemanner as those used in assays of light reaction activitiesexcept for the addition of 1 mM phenylmethylsulfonyl fluorideto the grinding buffer. The isolated chloroplasts were purifiedaccording to the procedure of Hoober (15). The membraneswere extracted with either 90% acetone or 95% ethanol andether. The membranes were then dissolved in the followingbuffer: 0.063 M tris-HCI (pH 6.8), 2% SDS, 10% glycerol, 5%mercaptoethanol, and 0.5 M urea. The solubilized membraneswere boiled for 4 min, and the samples were run on 15-cmsodium dodecyl sulfate acrylamide gels according to theelectrophoresis system of Laemmli (17) with the followingmodifications: the running gels were of 12% acrylamide; thefinal concentration of tris-HCl, (pH 8.8) was 0.19 M, a finalconcentration of 0.03% N, N, N', N'-tetramethylethelenedia-mine and 0.05% ammonium persulfate were used for polym-erization; plastic tubes were used instead of glass tubes; theelectrode buffer contained 0.05 M tris, 0.38 M glycine, and 0.1%sodium dodecyl sulfate at pH 8.3. The gels were run with

a constant voltage power supply at 50 v, 10 m amp, 120 pulses/sec, and 1 /if discharge for 3 hr or until the marker dye(bromophenol blue) had entered the running gel. The powerwas then increased to 100 v, 18 m amp for 5 to 7 hr or untilthe dye had reached the bottom of the gel. The gels were fixedin 12.5% trichloroacetic acid at 65 C for at least 3 hr, thenstained in a mixture of aqueous 0.4% Coomassie brilliant blueR-250-ethanol-glacial acetic acid (45:45:10) at 65 C for atleast 3 hr. Destaining was accomplished by several changes ofa destaining solution containing ethanol-glacial acetic acid-H20(25:10:65) at 65 C for at least 3 hr. The gels were thenscanned at 570 nm using a gel scanner attachment for a Beck-man Acta III spectrophotometer.

RESULTS

Chloroplast Structure in the STV and Control Cell Lines.Chloroplasts of the control cell line which lacks the viruslikeparticle are similar to those of most higher plants and containseveral small (average 0.5 jim) grana interconnected by stromalamellae (Fig. 1). Chloroplasts in the control tissue culturecells do contain starch deposits (26). In contrast, chloroplasts inthe STV cell line contain "supergrana" which frequently con-tain 30 or more thylakoids. These supergrana are not inter-rupted by interconnecting stroma lamellae and are usually aslong (average 4 ptm) as the entire chloroplast itself (Fig. 2). TheSTV supergranal chloroplasts never contain starch deposits.

Freeze-fracturing of cells of the control cell line reveals theinternal substructure of the chloroplast membranes to be essen-tially identical to that of spinach or other higher plants (Fig. 3).Two complementary faces are revealed; the B face-or thatdirected out from the thylakoid interior-is characterized bylarge, 175 A average diameter particles; the C face-or thatfacing the thylakoid interior-contains small, 120 A averagediameter particles. However, replicas of freeze-fractured super-granal chloroplasts from the STV cell line are completelydifferent (Figs. 4 and 5). Two complementary faces arerevealed, but the B face is characterized by small, widelyspaced 80 A average diameter particles and the C face is essen-tially smooth.

Light Reactions of Isolated Chloroplasts. A comparison ofthe ability of isolated chloroplasts from cell lines STV and6045 (control) to reduce NADP with electrons supplied fromwater is shown in Figure 6A. Chloroplasts from the control cellline reduce NADP at a rate of 50 ,umole/hr mg Chl, whereaschloroplasts from the STV cell line are totally unable toreduce NADP. Since NADP is the terminal electron acceptorin the light reaction, a lesion anywhere between H20 andNADP could cause the STV supergranal thylakoids to beunable to reduce NADP. The rates of the assays for light reac-tion activity in the control chloroplasts are low when comparedto spinach chloroplasts under identical conditions. This paperis the first report of activity in chloroplasts isolated from planttissue cultures, thus rates for comparison are not available. Therate of "4CO2 fixation by these tissue cultures as previouslyreported (26) is also low when compared to spinach orChlamydomonas. Plant tissue cultures are grown heterotrophi-cally and have low Chl content. Nevertheless, the ratesreported here are consistent, and are significantly differentfrom the STV chloroplasts. The STV cell line has as muchor greater Chl/g fresh weight as the control cell line.One reason that the STV chloroplasts are unable to reduce

NADP from water could be that it has a normally functioningPS I, but PS II is aberrant and unable to provide electrons tothe reaction centers of PS I. To test this possibility, electronswere supplied from an artificial electron donor, ascorbate-reduced DPIP in the presence of DCMU. Figure 6B is a com-

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SMITH AND SJOLUND

See f l e pag

See figuire legetids iiext, page

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523SUPERGRANAL CHLOROPLASTS

/STV

1 2 3 4 5TIME (min.)

czt

C-

(B) NADP Reductionfrom DPIP/Ascorbote

0.9

CZ 0.7

0o

IZ 0.6

/ STV --------

1 20 12 34 5

TIME (min.)

(C) DPIP Reduction

STV

Rates:Control = 81E1M/mg Chl/hr

STV=0

Control

0.5,

O 1 2 3 4 5TIME (min.)

FIG. 6. Comparison of the ability of isolated chloroplasts from the STV and control cell lines to reduce NADP and DPIP. A: NADP reduc-

tion with water as a source of electrons; B: NADP reduction with electrons supplied from ascorbate-reduced DPIP and electron flow from PS

II blocked with DCMU; C: DPIP reduction.

0.6

-CZ.13tl-lh

ZZLLAJ

(Z.l

0.5 -

0.4 -

0.3-

0.2 -

0.1-

0 0.5 1 1.5 5

TIME (min.)

FIG. 7. Ability of isolated STV supergranal chloroplasts to

reduce methyl viologen as followed by an uptake of 02- With

ascorbate-reduced DPIP as an electron donor ( ); with water

as an electron donor (--).

parison of the ability of chloroplasts from control and STV cell

lines to reduce NADP with electrons supplied from DPIP-

ascorbate. Chloroplasts from the control cell line reduced

NADP at the rate of 28 ,uM/hr-mg Chl, whereas chloroplastsfrom the STV cell line were unable to reduce NADP.

Although the inability of the STV supergranal chloroplastto reduce NADP with electrons supplied from DPIP-ascorbateindicates that it lacks a normally functioning PS I, it would

still be possible for it to have a normal PS II. To test this

possibility, the ability of isolated chloroplasts from the STV

cell line to reduce DPIP was determined. Figure 6C shows

that control chloroplasts reduce DPIP at a rate of 81 ,umole/hr- mg Chl. The STV chloroplasts failed to reduce DPIP even

in reactions run with very high concentrations of Chl over a

long time period. DPC has been shown to restore or enhance

the ability of spinach chloroplasts washed in tris buffer or

submitted to detergent treatment to reduce DPIP (29). Under

the conditions used here, DPC was demonstrated to enhancethe rate of DPIP reduction by spinach chloroplasts 3-fold.However, isolated STV chloroplasts remain unable to reduceDPIP even in the presence of 0.5 mM DPC. The STV

thylakoids, therefore, lack a functional PS II.

To examine the possibility that electron flow through the

reaction center of PS I is normal, but that a photosyntheticlesion exists between the reaction center of PS I and NADP,the ability of isolated chloroplasts from the STV cell line to

reduce methyl viologen was determined. Figure 7 shows that

STV supergranal chloroplasts are unable to reduce methylviologen with electrons supplied from water, which is con-

sistent with the conclusion, based upon their inability to reduceDPIP, that they lack a functional PS II. However, whenelectrons are supplied from the artificial electron donor DPIP-ascorbate, isolated STV chloroplasts are able to reduce methylviologen as indicated by an uptake of 02 at the rate of 19

,umoles/hr mg Chl. This result indicates that the STV chloro-plast thylakoids have functional PS I reaction centers, but are

unable to reduce NADP.To determine if some specific product induced by the

viruslike particle exists in the STV chloroplast pellet which iscapable of inhibiting the light reactions, STV chloroplastsand spinach chloroplasts were combined and assayed for thepresence of PS I and II activities. If such an inhibitory sub-stance were present, the rate of NADP or DPIP reduction ofthe spinach chloroplasts alone should be greater than therate of spinach chloroplasts combined with STV chloroplasts.No such inhibitory effect by the STV chloroplasts was found.

Polypeptide Composition of Chloroplast Membranes. The

polypeptide composition of the chloroplast membranes fromthe two cell lines was compared by SDS disc gel electrophoresisof solubilized membranes. Figure 8 shows that scans of gelsof the control and STV membranes from the cell lines exhibit1 1 bands, plus several smaller bands and shoulders. A small

FIG. 1. Thin sectioned chloroplasts from control cells. Arrows indicate grana. Bar equals 0.2 pm. X 45,000.

FIG. 2. Thin sectioned chloroplast from STV cell line showing supergranum (SG). Bar equals 0.2 ,um X 39,000.

FIG. 3. Freeze-fractured replica from control chloroplast. The exposed faces resemble those of freeze-fractured spinach chloroplasts. Large

(175 A) particles are seen on the B face and smaller (120 A) particles are seen on the C face. Bar equals 0.1I m. X 100,000.

FIGs. 4 and 5. Freeze-fracture replicas of STV supergranal chloroplasts showing the exposed complementary faces, B and C. The B face con-

tains 80 A particles and the C face is essentially smooth. Broad arrows indicate direction of metal evaporation in all fgures. Par ecuals 0.1 pJm.

X 65,000.

- DPIP/Ascorbote--- H20

-----------------_

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Plant Physiol. Vol. 55, 1975

top

Control-STV - - -

FIG. 8. Scans of SDS acrylamide gels of solubilized chloroplastmembranes from STV (--- ) and control ( ) cell lines. Broadarrow indicates the position of band 5. Small arrows indicate posi-tions of marker proteins. a: BSA (mol wt 67,000); b: ovalbumin(mol wt 45,000); c: myoglobin (mol wt 17,800).

front is present at the bottom of the gels. It is apparent thatthere is a close correspondence between the bands presentin the membranes of the STV supergrana and those in controlchloroplasts and that all of the bands present in the control gelsare present in the STV gels. The only difference that is evidentis the much greater amount of band number 5-mol wt 33,000daltons-in the STV membranes than in the control mem-

branes. The prominence of this band is a consistent and dis-tinguishing feature of the STV gels. In addition, when com-

pared to the control gels, the STV gels have a slightly increasedamount in the region of bands 8 through 11.

DISCUSSION

The bulk of the experimental evidence has indicated thatregions of appressed chloroplast lamellae, grana stacks, are

not necessary for PS II activity. Studies of greening chloro-plasts (2, 7, 8, 10) have shown that a correlation exists betweenthe time of the initiation of PS II activity and stacked thy-lakoids. However, when etiolated bean plants are exposed tofar red light the greening membranes accumulate primarilyChl a and little grana stacking takes place. Nevertheless, theseunstacked lamellae have PS II activity (22). The agranal bundlesheath cell chloroplasts of sorghum (27) and maize (1, 4) havePS II activity. Similarly, studies with Chlamydomonas (12, 13,23) have shown the presence of PS II in unstacked chloroplastlamellae.The data reported in this work with the supergranal chloro-

plast of the STV cell line, which has extensive regions ofappressed chloroplast membranes, support the concept thatthere is not a direct correspondence between grana formationand PS II activity. Although the thylakoids of the STVchloroplasts are arranged in supergrana, the STV chloroplastsconsistently failed to show any PS II activity as measured bytheir ability to reduce a Hill reaction electron acceptor, DPIP.

When considered with the instances cited above of agranalchloroplasts with PS II activity, the lack of activity in theSTV supergranal chloroplast shows that the phenomena (asyet undefined) which cause chloroplast membranes to beaggregated in a stacked structure are not directly linked tothe presence of an active PS II.The most striking characteristic of the freeze-fractured STV

supergranal chloroplast is the complete lack of large particlesin lamellae which are extensively aggregated into large grana.Thin sections of the STV supergrana (Fig. 2) clearly show thatthe thylakoid membranes are appressed to one another to formpartition regions. This result is the first reported instance ofstacked chloroplast lamellae which lack the large B faceparticles. This report indicates that in at least this higher plantthe large B face particles are not a result of the aggregation ofthylakoid membranes in grana stacks. It is not possible to

conclude, however, that the B face particles do not result fromsome specific interaction between membranes (which might beabnormal in the STV supergrana); we can only conclude thatthey do not result from the appression of one lamella onanother. The STV supergranal chloroplasts have limitedphotosynthetic function. They completely lack PS II activityand are unable to reduce NADP although they have functionalPS I reaction centers. Freeze-fractured STV supergrana lackthe 175 A B face particles and the 110 A C face particlescharacteristic of control chloroplasts. Instead, the B face con-tains 80 A particles and the C face is smooth (Figs. 4 and 5).The abnormal freeze-fracture substructure of the STV super-grana tends to support the conclusions of Arntzen et al. (6) thatthe two size classes of particles are markers of regions ofchloroplast membranes with normal photochemical activity.It is not possible to determine from the data presented herewhether the abnormal particle distributions of the faces ex-

posed in the freeze-fractured STV supergrana and its lack ofphotosynthetic function are due to an altered association oflipids and proteins within the membrane, to conformationalchanges of polypeptides, or to some change in the interactionbetween adjoining membranes in the partition regions.

If the particles seen on freeze-fractured membrane faces ofchloroplasts represent proteins or aggregates of several poly-peptides, membrane fractions with different particle sizes anddistributions should have different polypeptide compositions.This has been shown to be the case for spinach and Chlamnv-doomonas reinhardtii, wild type and ac-S mutant strains. Usingdigitonin-fractionated chloroplasts, Levine and co-workers (19)have demonstrated that a fraction enriched in PS II activitvhas a different polypeptide composition than a fraction en-riched in PS I activity. Similar results have been reported forpea chloroplasts (9).

However, analysis of the polypeptide composition of stackedand unstacked chloroplast lamellae of the ac-S mutant ofChlarnydomnonas showed that the unstacked membranes whichhave PS II activity, lack the group of polypeptides associatedwith fractions of membranes enriched in PS II activity (18).This analysis would lead the present authors to conclude thatif the unstacked membranes can have high PS II activity, butlack those polypeptides shown to be characteristic of mem-branes enriched in PS II activity, then the PS II group of poly-peptides alone must not be the molecular component whichmakes the chloroplast membranes competent to perform PS II.The polypeptide compositions of chloroplast membranes

from the STV and control cell lines are very similar, with a

close correspondence between the molecular weights of themajor bands. The STV supergranal chloroplast membranesdo not lack anv of the major polypeptides present in thecontrol cell chloroplast membranes. The only major difference

524 SMITH AND SJOLUND

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SUPERGRANAL (

evident is the much greater amount of one polypeptide, band5, mol wt 33,000. From these results, it can be concluded thatthe lack of photosynthetic capacity and abnormal freeze-frac-ture substructure of the STV supergranal chloroplast mem-branes is not due to a gross deficiency in one or several poly-peptides. This evidence, when considered with data from theac-S mutant showing a lack of correspondence between PS IIactivity and the polypeptides associated with membrane frag-ments enriched in PS II activity, suggests that the properties ofa chloroplast membrane which make it photosyntheticallycompetent are more complex than the simple presence of oneor several of its constituent polypeptides.The polypeptide of mol wt 33,000 present in the STV

supergranal chloroplast membranes in an increased amount,could correspond to a polypeptide associated with membranestacking. Levine and Duram (18) and Anderson and Levine (3)have shown that two proteins are reduced in content in agranalbundle sheath chloroplasts of C4 plants and also in algal andhigher plant mutants which have limited grana stacking. Theysuggest that these may be required to obtain stacked lamellae.The increased amount of the 33,000 mol wt protein in theSTV chloroplast membranes, which have greatly increasedregions of stacking compared to the control thylakoid mem-branes, may be an indication that it represents this type ofpolypeptide. The polypeptides found by Levine and Duram(18) and Anderson and Levine (3) to be deficient in chloro-plasts that lack grana stacks correspond to those of chloroplastmembrane fractions enriched in PS II activity. The molecularweight of band 5 (33,000 daltons in STV) and the molecularweights in the region of bands 8 to 11 which are present in aslightly higher concentration in STV are within the range ofmolecular weights of the polypeptides of PS II-enrichedspinach and Chlamydomonas fractions. This observation sug-gests that the differences in polypeptide concentrations betweenthe STV and control chloroplast membranes might be corre-lated with the extensive stacking of the STV supergranal chloro-plast.

LITERATURE CITED

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2HLOROPLASTS 525

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Plant Physiol. Vol. 55, 1975

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