Plant Physiol. (1989) 89, 602-6090032-0889/89/89/0602/08/$.00/0

Received for publication January 26, 1988and in revised form June 18, 1988

Expression of the Major Light-Harvesting Chlorophyll a/b-Protein and Its Import into Thylakoids of Mesophyll and

Bundle Sheath Chloroplasts of Maize1

Alexander Vainstein*2, Paulo Ferreira, Camille C. Peterson, Judith A. Verbeke, and J. Philip ThornberDepartment of Biology, University of California, Los Angeles, California 90024 (A.V., P.F., C.C.P., J.P.T.), and

Department of Biological Sciences, University of Illinois at Chicago, Chicago, Illinois 60680 (J.A.V.)

ABSTRACT

Distribution of the major light-harvesting chlorophyll a/b-pro-tein (LHCII) and its mRNA within bundle sheath and mesophyllcells of maize (Zea mays L.) was studied using in situ immuno-localization and hybridization, respectively. In situ hybridizationwith specific LHCII RNA probes from maize and Lemna gibbadefinitively shows the presence of high levels of mRNA for LHCIIin both bundle sheath cells and mesophyll cells. In situ immuno-localization studies, using an LHCII monoclonal antibody, dem-onstrate the presence of LHCII polypeptides in chloroplasts ofboth cell types. The polypeptide composition of LHCII and theamount of LHCII in bundle sheath cells are different from thosein mesophyll cells. Both mesophyll and bundle sheath chloro-plasts can take up, import and process the in vitro transcribedand translated LHCII precursor protein from L. gibba. Althoughbundle sheath chloroplasts incorporate LHCII into the pigmentedlight-harvesting complex, the efficiency is lower than that inmesophyll chloroplasts.

Leaves ofC4 plants contain two distinct types of photosyn-thetic cells, mesophyll (M3) and bundle sheath (BS), that arequite distinctly organized both structurally and functionally.Since the discovery of C4-dicarboxylic acid photosynthesis,much effort has been directed to determining the sequence ofreactions in that pathway (3, 11, 20, 26, 28, 29). Accumulateddata agree upon the existence in C4 plants of an active PSI inboth cell types and of PSII in M cells; however, there isdisagreement about the existence of PSII activity in BS cellsof maize and even about the presence of some of the constit-uents of PSII in BS cells (2, 3, 10, 20, 26, 28, 29). Particularlycontroversial is the occurrence in BS cells of the light-har-vesting complex of PSII (LHCII), which contains the majorprotein component in the thylakoid membranes of higher

'Research was supported by grants from the Binational Agricul-tural Research and Development Fund to A. V., from the NationalScience Foundation (NSF) and the United States Department ofAgriculture to J. P. T., and from NSF to J. A. V.

2 Present address: The Hebrew University of Jerusalem, Faculty ofAgriculture, Department of Horticulture,, P.O. Box 12, Rehovot 76-100, Israel.

3 Abbreviations: M, mesophyll; BS, bundle sheath; LHCII, themajor light-harvesting Chl a/b-protein of PSII; M, mesophyll; pre-

LHCII, LHCII precursor; FITC, fluorescein isothiocyanate.

602

plants (30). Some reports show the presence and some theabsence of LHCII transcripts and of LHCII polypeptides inmaize BS cells (3, 26, 28). In all of these studies, BS cells werefirst separated from M cells and then examined for the pres-ence ofmRNA for LHCII, or for the polypeptide itself.The apoproteins of LHCII are encoded by a nuclear gene

family and synthesized on cytoplasmic ribosomes as a water-soluble, higher mol wt precursor form(s), preLHCII. Duringits insertion into the thylakoid membranes it is processed toits mature, water-insoluble form, and photosynthetic pig-ments are added to it (7, 14, 24, 25, 30). The sequence ofevents that leads to its incorporation into the LHCII complexis, however, largely unknown. Uptake experiments in whichin vitro synthesized preLHCII is incubated with a suspensionof intact chloroplasts show that the efficiency with which thepreLHCII is processed to its mature size and incorporatedinto the pigmented LHCII complex depends upon the stageof plastid development (5). Import ofthe precursor by plastidsis energy dependent and requires a putative receptor in thechloroplast envelope (9, 13, 16). A stromal factor(s), probablyproteinaceous, is required for insertion into the thylakoidmembrane (5, 8).

In the present study we show qualitatively, using in situhybridization and in situ immunolocalization techniqueswhich avoid fractionation of the two cell types, that mRNAfor LHCII and the LHCII gene translation product(s) accu-mulate to a detectable level in both maize BS and M cells.The polypeptide composition of LHCII in M thylakoids dif-fers, however, from that in the BS membranes. Both cell typespossess the machinery for uptake and incorporation of in vitrosynthesized preLHCII into the pigmented LHCII complex.

MATERIALS AND METHODS

Plant Material

Zea mays W273 seeds (Wisconsin certified) were grown invermiculite in the greenhouse for 2 weeks (approximately 12h light, 12 h dark periods). Small pieces of secondary andtertiary leaves were used for in situ hybridization.

Hybridization in situ

Fresh leaf material for in situ hybridization was preparedaccording to the method of Barker et al. (1). Leaf tissue wascut into 2 to 3 mm pieces in a small pool of 1.5% glutaral-

PSII PROTEINS IN BUNDLE SHEATH AND MESOPHYLL CELLS

dehyde in 0.05 M sodium cacodylate buffer (pH 7.0). Thetissue was fixed for 3 h at room temperature with occasionalagitation, washed in buffer, dehydrated in a graded ethanolseries, and embedded in paraplast plus. Transverse and lon-gitudinal sections (8-10 ,m thick) were mounted on subbedslides. The slides were deparaffinized in xylene and the ma-terial serially rehydrated in ethanol followed by distilled water.Slides were coated with 1% BSA (to block any positivecharges), washed with water and incubated with proteinase Kto make cytoplasmic RNAs accessible to the hybridizationprobe. Slides were dipped into 0.1 M triethanolamine (pH8.0), followed by washing with 0.25% v/v acetic anhydride in0.1 M triethanolamine to acylate any remaining positivecharges. Slides were then washed with 2 x SSC (0.3 M NaCIand 0.03 M Na-citrate), dehydrated in an ethanol series andfinally air dried.

Hybridization probes were prepared using an SP6 and T7transcription vector system (Promega Biotec). Recombinantplasmids containing a 1.3 kb fragment of a maize LHCII gene(gift of Dr. W. Taylor, University of California, Berkeley) anda 1.1 kb Lemna gibba LHCII gene (AB30) (gift of Dr. E.Tobin, University of California, Los Angeles) (19) were line-arized and transcription reactions were carried out as de-scribed by Barker et al (1). Following hybridization and post-hybridization washes, slides were dipped in Kodak NB-2photographic emulsion in darkness, dried and stored in light-tight boxes for 3 to 6 days at 4°C. Subsequently, the emulsionwas developed with Kodak developer, D-19. Following themounting of cover slips with Permount, slides were viewedthrough an Olympus microscope and photographed. Thesystem was initially optimized for maize leaf material byhybridization of probes to ribulose bisphosphate carboxylase,the mRNA of which is known to be located in the bundlesheath cells (21, 23).

In Situ Immunofluorescent LabelingFresh leaf material for indirect immunofluorescent anti-

body labeling was prepared according to the method of Hat-tersley et al. (15). Leaf tissue was cut into pieces (3 x 3 mm),embedded and frozen in OTC compound (Tissue Tek II)under CO2. Transverse sections (6-8 ,um) were cut in acryostat, transferred to slides and fixed in 70% ethanol for 2h at room temperature, followed by washing in 0.1 M K-phosphate buffer. Sections were then incubated for 40 min atroom temperature with an LHCII monoclonal antibody(MLH 1, a gift of S. Darr [10]), or with 0.1 M K-phosphatebuffer and preimmune serum (control). After rinsing threetimes with K-phosphate buffer, the sections were incubatedwith FHTC-labeled goat-anti mouse antisera (1:40 dilution inPBS) for 40 min and subsequently rinsed as above. Sectionswere examined on a Nikon fluorescence microscope andphotographed. Optimal conditions were established by prob-ing with an antibody to ribulose bisphosphate carboxylasepolypeptides which was known to be located in BS cells (23).

Preparation of BS and M Plastids and ThylakoidMembranes for Uptake Experiments

Separation of BS from M chloroplasts was done accordingto the procedure of Chollet and Ogren (6), with some modi-

fications. Secondary and tertiary leaves, chilled on ice, werecut with a razor blade into 5 mm transverse segments. Theywere ground for 3 s in a razor-blade blender ( 18) in a suspen-sion buffer containing 0.6 M sorbitol, 10 mM Hepes-KOH(pH 7.5), and 2 mM CaCl2. The suspension was filteredthrough two layers of Miracloth and an 80 ,um nylon mesh.The filtrate (M plastids) was pelleted by centrifugation in aSorvall GSA rotor by accelerating to 3000 rpm and applyingthe brakes when that speed was reached. The pellet was gentlyresuspended and an estimation made of its Chl and proteincontent (5, 19). An appropriate amount of plastids was thentransferred to a 15 ml Corex tube and centrifuged in a SorvallSS34 rotor by accelerating to 500 rpm and then immediatelybraking. The pellet was resuspended in suspension buffer andused for uptake experiments.To obtain BS cells, the residue from the grinding and

filtration steps described above was resuspended and blendedfor one additional minute in a razor-blade blender (18), andthen filtered through Miracloth. This process was repeatedfour times. The final homogenate was filtered through 20 and35 mesh sieves and the filtrate blended in a Polytron Disin-tegrator (Brinkmann, Switzerland) for 30 s. The suspensionwas then filtered through an 80 ,um nylon net. This step wasrepeated three times. The final residue was blended for 5 s ina razor-blade blender. Following filtration through two layersof Miracloth, the BS chloroplasts were pelleted (Sorvall GSA,3000 rpm, up and down) and gently resuspended to anappropriate Chl concentration. Chloroplasts prepared in thismanner were used in uptake experiments (see next paragraph)after the purity of the preparation had been monitored bylight microscopy. BS cells had a Chl a/b ratio of 5.5-6.0(compared with 2.0-2.2 in M cells), and the specific activityofNADP-malate dehydrogenase in BS cells was 0.07% ofthatin the M chloroplast. The specific activity of the "malicenzyme" in the M chloroplast was 0.01% of that in the BScells (17). Note that the amount of the enzyme activitymeasured in cells that should have had no activity is scarcelydifferent from the zero control level; i.e., there is virtually nocross-contamination of the M and BS preparations.

Thylakoid membranes were obtained from plastids andused for polypeptide composition analysis as described inThornber et al. (31). Western blot analysis of thylakoid poly-peptides using a monoclonal antibody that reacts specificallywith LHCII apoproteins (10) for immunodecoration was per-formed, using alkaline phosphatase and 5-bromo-4-chloro-3-indoyl phosphate to locate the antibody-antigen complex (32).

Transcription, Translation and in Vitro Uptake of Precursorto LHCII

The in vitro expression of L. gibba AB30 gene was per-formed as described by Kohorn et al. (19). The uptake mix-ture, in a final volume of 300 ,l, consisted of 100 ,l plastidsuspension, 125 ,lA [35S]methionine-labeled translation prod-uct, 5 mM methionine and 8 mM ATP, in suspension buffer.Plastids were incubated for 1 h at room temperature withoccasional, gentle shaking. Afterward 68 ,ug/ml thermolysinand 8 mm CaCl2 were added for 30 min at 4°C to digest anyprecursor molecules associated with the outer envelope. Fol-lowing protease treatment, intact plastids were recovered by

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Plant Physiol. Vol. 89,1989

gradient centrifugation through Ficoll-polyethylene glycol(19). Intact plastids were then washed and thylakoids preparedand examined by PAGE (19). The procedures for fractiona-tion of the pigment-protein complexes by non-denaturing gelelectrophoresis, for polypeptide analysis of materials by fullydenaturing SDS-PAGE on 10 to 15% linear gradient poly-acrylamide gel, and for fluorography are described in Thorn-beret al. (31).

Chemicals

[35S]Methionine, [35S]UTP, SP6, and T7 polymerases were

from Amersham, Arlington Heights, IL. Paraplast-plus was

purchased from Fisher. Deriphat- 160 was from Henkel Corp,Hawthorne, CA. Acrylamide and photographic emulsion werefrom Eastman Kodak Co, Rochester, NY. All other chemicalswere obtained from Sigma Chemical Co, St. Louis, MO.

RESULTS

Presence of the LHCII mRNA and Its Translation Productin BS Cells

The distribution of LHCII at the transcriptional level be-tween M and BS cells was studied by in situ hybridization.Two different, specific probes, one homologous and one

heterologous to the maize cells, were derived from: (a) a maizeLHCII gene, and (b) the L. gibba AB30 gene, respectively.The hybridization of the AB30 probe with total maize RNAisolated from unfractionated leaf tissue, is shown in Figure1E. A similar specificity for a single size RNA was obtainedwith the maize probe (data not shown).The presence ofmRNAs for LHCII in BS cells, in addition

to M cells, is shown in Figure 1. The silver grains in Figure 1

represent the duplex RNA molecules formed between the T7promoter-generated "antisense" LHCII mRNA and theLHCII mRNA that was fixed in the leaf. There is no notice-able difference between the homologous or the heterologousprobe. In the control (Fig. lD), the SP6 promoter-generated"sense," mRNA was used as a probe. The scattered silvergrains in Figure 1 D, represent nonspecific binding, consideredto be background.

Because BS cells are cylindrical, maximal BS cell surfacearea is revealed in longitudinal sections. Therefore, to estab-lish more definitively the controversial (3, 26, 28) presence ofmRNAs for LHCII in BS cells in maize leaves, longitudinalsections were probed with the homologous and the heterolo-gous probes (Fig. 2). The BS cells do indeed contain substan-tial amounts of the LHCII mRNA.The presence of LHCII protein in maize M and BS cells

was examined using an immunofluorescent technique appliedto sections of leaf tissue. We emphasize again the importanceof this approach in avoiding problems encountered withseparation of the two cell types. The monoclonal antibody(MLH 1), which cross-reacts with the major apoproteins ofLHCII (10), was confirmed to be absolutely specific by thedata in Figure 3E, in which thylakoid membranes from un-

fractionated leaf tissue was electrophoresed and immunodec-orated with MLH 1. The band revealed is of 28 kD andcorresponds to the major LHCII polypeptide (cJf 31). The

immunofluorescent labeling of M and BS chloroplasts of asection incubated first with MLH1 and then with FITC-conjugated goat-antimouse antibody is shown in Figure 3B.Using buffer (or preimmune serum, data not shown) as acontrol instead of MLH1, the fluorescence is shown to bespecific to MLH 1 antibodies, and not to nonspecific bindingof the antimouse antibody or to autofluorescence of the tissueitself (Fig. 3D).

LHCII Composition in BS and M Cells

The polypeptide composition of the LHCII complex wasfollowed during cell development by harvesting maize leavesafter different periods of greening (Fig. 4). The M cells wereseparated from BS cells and the thylakoids were preparedfrom each cell type (see "Materials and Methods"), prior topolypeptide analysis using SDS-PAGE. The pattern of LHCIIpolypeptides was studied by Western blot analysis using theLHCII monoclonal antibody (MLH1). M cells are alwayshighly enriched, even after only 4 h greening, in the lowestmol wt form of LHCII that reacts with this antibody, ascompared to the BS cells (Fig. 4). Lanes M'G and B'G arefrom a separate experiment in which light-grown tissue wasexamined and they are included to emphasize the differencebetween the two forms of LHCII (see arrows) resolved by thisgel electrophoresis system.

LHCII Assembly into the Green Complex in BSChloroplasts

Previously it has been shown by us and others that isolatedchloroplasts of some higher plants can take up, process andinsert the LHCII precursor polypeptide into their thylakoidmembranes (5, 14, 19, 30). To find out whether BS and Mchloroplasts of maize each possess this mechanism for uptakeand insertion of cytoplasmic proteins into thylakoid mem-branes, we separated M and BS chloroplasts (see "Materialsand Methods"). Isolated chloroplasts were incubated with thein vitro synthesized, radiolabeled preLHCII of L. gibba. Thy-lakoids from each type of intact chloroplast were then isolatedand the fate of heterologous preLHCII was analyzed by SDS-PAGE followed by fluorography. Labeled polypeptides thatcorrespond to both the mature and the precursor forms ofLHCII are seen in the thylakoid membranes of both M andBS cells (Fig. 5). Neither the precursor nor the mature poly-peptides could be detected in the stromal fraction ofM or BSchloroplasts (data not shown). To determine whether thelabeled LHCII polypeptides had been assembled into themajor light-harvesting complex in M and BS cells, we sepa-rated the Chl-protein complex from each cell type (31). Thethylakoids obtained from intact chloroplasts of M and BScells after uptake of the radiolabeled LHCII precursor weresolubilized in a mixture of octyl glucoside, nonyl glucoside,and SDS (20:10:3) and electrophoresed for 40 min through a6.5% polyacrylamide gel (Fig. 6). The green band of anoligomeric form of LHCII (apparent size, 65 kD) was cut outand fully denaturing SDS-PAGE used to resolve its polypep-tides. Note that any unassembled preLHCII (apparent size,32 kD) in the plastid would not be contained in the excisedoligomeric LHCII band because of the large difference in size.

604 VAINSTEIN ET AL.

PSII PROTEINS IN BUNDLE SHEATH AND MESOPHYLL CELLS

E

1.2 kb

Figure 1. Distribution of LHCII mRNA in maize leaf sections using in situ hybridization. Transverse sections (10 Mim) of maize leaves (2 weeksold) were hybridized to homologous probes prepared from maize LHCII gene antisense (A, B) and sense strands (D). A section hybridized to an"antisense" probe prepared from L. gibba LHCII gene (AB30) (19) is shown in section C. Section A was observed in light-field microscopy whileB, C, and D were in dark-field microscopy. Section E shows the specificity of the L. gibba AB30 probe as analyzed by a Northern blot. RNA wasprepared from unfractionated maize leaves, electrophoresed, blotted and hybridized to nick-translated AB30 (3) (F = M cells; -- = BS cells).

Figure 2. In situ hybridization usinglongitudinal sections of a maize leaf.Two-week-old tissue was longitudi-nally sectioned (10 fm) and hybridizedwith maize LHCII "antisense" probe(A, B) or with L. gibba LHCII [Anti-sense" probe (C). Sections were ob-served in dark-field (A) and light-field(B, C) microscopy (-* = BS cells).

Fluorography of this gel (Fig. 7) shows the presence of themature and precursor forms of the LHCII apoproteins in thegreen complex ofM and, at a lower level, ofBS cells (comparethe intensity per mg protein in lanes 2 and 3 in Fig. 7). Noradioactivity was associated with other green complexes re-solved by the first dimension gel electrophoresis (data notshown).

DISCUSSION

Dimorphism of chloroplasts in C4 plants has attractedmuch attention since its discovery (3, 11, 20, 26, 28, 29).Several laboratories have published results correlating thestructural dimorphism with the absence, or greatly diminished

amount, of PSII activity in BS cells, and it became dogma tomany researchers to equate agranal chloroplasts of BS cellswith a lack of PSII activity (2, 3, 20, 26, 28). To determinewhich components of PSII were actually missing from agranalBS chloroplasts, various laboratories used the same strategy,namely enzymatic or mechanical separation, to fractionate Mcells from BS cells. Some analyses of thylakoids in theseseparated tissues showed the presence, and some the absence,ofthe most abundant component of PSII, i.e., the major light-harvesting complex II. Furthermore, when these tissues wereinvestigated for presence of the LHCII mRNA, no single,definitive result was obtained. One can explain the opposingobservations in a number of ways: (a) The necessity of sepa-rating the cells prior to their analysis leads to contamination

605

Plant Physiol. Vol. 89, 1989v* t - 'v v >es'Figure3. In situ immunolocaliza-3> & @. Sis\ tS.i5'*-agbltion of LHCII polypeptides. lmmu-

nofluorescent labeling of LHCIIwas performed on transverse sec-

i'As<o tions of 2-week-old maize leaves

15jjma ;flX !r :using an LHCII monoclonal anti-

-' ~~~~~~~~~~~body(MLH1) followed by labeling9* c,e.|* , S.S twith FITC-conjugated immuno-30%. "NO> t ;' C<L, ¢ t .,>aS'\^ globulin and observation by (A)

Jim A light or by (B) fluorescence micros-**-*44. * .. t- copy. In the control sections, 0.1

M K-phosphate buffer or preim-mune serum (not shown) replacedMLH1, and the sections were ob-served by (C) light and (D) fluores-cence microscopy. The specificityof the MLH1 antibody as revealedby Westem blot analysis of unfrac-tionated maize leaf thylakoids sub-jected to a 10% SDS-PAGE (see"Materials and Methods") is shownin (E) (-. = BS chloroplasts, * =M chloroplasts).

nnD ~~~~~M;BvIVIDo ~~~GG

G 36 16 8 4 0 0 4 16 36 G (h)

Figure 4. LHCII polypeptide pattem of maize M and BS cells during greening. M and BS (B) thylakoids were prepared from 2-week-old etiolatedleaves after different lengths of time of greening (G = light-grown tissue). Polypeptides (25 gg thylakoid protein) were separated using 10% to15% SDS-PAGE gradient and probed with a monoclonal (MLH1) antibody (10, 32). Arrows point to the highest and lowest mol wt immunostainedapoproteins of LHCII. Lanes M'G and B'G were obtained in a separate experiment using fully greened tissue and photographed at a highermagnification after immunodecoration.

of one cell type by the other. (b) The thorough washingrequired during the multiple steps in the separation procedurecauses some BS components to be washed away or degraded;alternatively, the resultant chloroplast population could befrom a class that is enriched in or is lacking some compo-

nent(s). (c) Different types of plants were used.

To bypass these criticisms of C4 studies, we applied in situhybridization techniques to look qualitatively for the presenceof the mRNA for LHCII in thin sections of intact maizeleaves, thereby avoiding complications inherent in the tissuefractionation approach. In situ hybridization studies of thedistribution of stromal enzymes between M and BS cells has

606 VAINSTEIN ET AL.

PSII PROTEINS IN BUNDLE SHEATH AND MESOPHYLL CELLS

1 2 3k D

__-

k D

3 2

28

3 2

Figure 5. Uptake and processing of in vitro transcribed and trans-lated L. gibba LHCII precursor. M and BS chloroplasts were sepa-

rated and the uptake of the L. gibba [35S]methionine-labeled preLHCII(lane 1) by the chloroplasts was performed. After the uptake incuba-tion period, chloroplasts were treated with thermolysin (68 Mg/lI) for30 min at 40C. A higher concentration of thermolysin (110 Mig/MlI) didnot affect the results (data not shown). Approximately 1 to 2% of theradiolabeled precursor that had been incubated with the chloroplastswas still associated with M thylakoids after thermolysin treatment.Fluorography of M (30 Ag protein) (lane 2) and BS (80 MAg protein)(lane 3) thylakoid membranes fractionated on fully denatunng SDS-PAGE was performed.

Figure 7. Processing and incorporation of the precursor of LHCIIinto the LHCII complex of maize M and BS thylakoids. The pigmentedoligomeric LHCII complex from M (40 Mg protein) and BS (120 Mg

protein) plastids was isolated (cf. Fig. 6), and each band was reelec-trophoresed under fully denaturing SDS-PAGE conditions; thereafter,fluorography were performed. Lane 1 was loaded with the L. gibbaprecursor of LHCII apoprotein(s), lane 2 with the LHCII complexisolated from M thylakoids, and lane 3 with the LHCII complex fromB thylakoids.

1 2

,, LHC II

-FP

Figure 6. Fractionation of M and BS thylakoid membranes into Chl-protein complexes on nondenatunng gel electrophoresis. M (lane 1)and BS (lane 2) thylakoids (15 Mg Chl/lane) were loaded on a 6.5%acrylamide gel and electrophoresis was performed for 40 min at RT.After electrophoresis the gel was photographed without staining.Note that a greater percentage of the total Chi is associated with PSIin the BS than in the M thylakoids, whereas the latter contain a

greater percentage of their Chi in LHCII (CCI, core pigment-proteincomplex of PSI; FP, free pigment).

been successfully used (21, 23); however, thylakoid polypep-tide distribution has not been examined previously. We foundLHCII mRNA present in both M and BS cells. Since BS cellshave a cylindrical structure, we examined longitudinal (Fig.2) as well as transverse sections in order to see the maximalcell surface area. Conversely, we examined only transversesections ofM cells, which are basically isodiametric with theirsurface area the same in both planes. As can be seen in Figure

2 the BS cells are packed with grains that represent theformation of duplex RNA molecules and hence the presenceofLHCII mRNA. It should be noted that in situ hybridizationstudies do not permit unequivocal quantitative data to beobtained.To look for the presence of LHCII protein we used an

equivalent approach, namely sections of maize leaves were

immunodecorated with monoclonal antibodies to LHCII. Wehave conclusively shown that BS plastids contain the LHCIIpolypeptide(s) (Fig. 3), albeit in apparently lesser amountsthan found in M plastids as judged by the fluorescence inten-sity per unit area (compare the fluorescence intensity of Mand BS chloroplasts). The possibility of a less concentratedcytoplasm or of fluorescence quenching cannot be disre-garded, however, as an explanation of lower level of fluores-cence in BS plastids.These findings make it less surprising that BS as well as M

cells possess the machinery for uptake, processing and incor-poration of LHCII into the pigmented complex; however,these processes have never been shown previously to occur inBS cells. To examine this situation we applied the more

commonly used strategy on C4 plants, i.e., physical separationof the M and BS chloroplasts. To minimize the disadvantagesof this necessary approach, we examined the purity of theisolated tissues by light microscopy, determined the Chl a/bratio and the relevant enzymatic activities, and discardedpreparations which did not meet the known characteristics ofeach class of cells (see "Materials and Methods"). Incubationof [35S]preLHCII with BS chloroplasts resulted in the appear-

1 2 3

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Plant Physiol. Vol. 89,1989

ance of a processed form of LHCII in the thylakoids (Fig. 5).This processed form was precipitated by specific LHCII anti-bodies (data not shown) and then shown to be contained inthe pigmented LHCII complex (Fig. 7). Thylakoids obtainedfrom chloroplasts incubated with [35S]preLHCII were appliedto a non-denaturing gel electrophoretic system (31) whichseparated the pigmented complex of LHCII from the rest ofthe photosynthetic components. Fluorography of a fully de-naturing gel analysis of this green complex (Figs. 5 and 7)revealed that it contained a processed form of the LHCIIproteins. The multiple bands seen below the processed form(28 kD) may be due to degradation occurring during theexperiment, although naturally occurring post-translationalmodifications may also occur. Although the machinery forthe introduction of preLHCII into the thylakoid LHCII com-plex was present in BS cells, its activity was less than that inM cells, based on the spectrophotometrically determined au-toradiographic intensity per mg protein of plastids (cJf Figs. 5and 7). Studies are underway to determine which step in theuptake process is limiting. The ability of BS cells to take upand process the preLHCII cannot be attributed to contami-nation of our isolated BS cells by M cells. There are so few Mcells in our BS cell fraction, as judged by microscopy, enzy-matic activities and Chl a/b ratios (cf 3, 26, 28) that if onlythe M cells had the chloroplasts that can take up preLHCII,then the level of uptake would have been below the limits ofour detection.

Considerable advances have been made recently in theidentification of multiple apoproteins of the LHCII complexof different species (4, 10, 12, 19, 22, 24, 25, 27, 30, 31). Wehave used the monoclonal antibodies characterized by Darret al. (10) to study the identity and biogenesis of LHCIIpolypeptides in maize by examining LHCII's subunit com-position in thylakoids ofM and BS cells separated from leavesat different stages of greening. Western blot analyses of thy-lakoid proteins (Fig. 4) reveal that BS cells are deficient in thesmallest apoprotein of LHCII that cross-reacts with the anti-body. Alternatively, no difference could be detected in thepolypeptide composition of LHCI in the two cell types, butsubunit VI of the core complex of PSI does differ duringcell development between the two types (manuscript inpreparation).

In conclusion, photosynthetically active plastids frommaize M and BS cells differ slightly but interestingly not onlyin the polypeptide composition of PSI and PSII but also inthe efficiency with which they can translocate and incorporateLHCII into the thylakoid membrane. Hence, the lower levelsof LHCII in BS thylakoids observed by us and several othergroups (3, 27, 28) could be due not only to transcriptional/post-transcriptional control, but also to a less active transportsystem for LHCII in BS cells than in M cells.

Acknowledgments

We would like to thank Gary Drews for his assistance with the insitu hybridization studies; Drs. R. Goldberg, J. Cascarano, and C.Bulinski for their help; Drs. W. Taylor and Sylvia Darr for generouslyproviding a maize LHCII gene and monoclonal antibodies, respec-tively; and Dr. Elaine Tobin for providing the AB30 gene of L. gibbaand for helpful discussion throughout the work.

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