The EMBO Journal vol.5 no. 13 pp.3417 - 3422, 1986

Expression of the ribulose-1,5-bisphosphate carboxylase largesubunit gene and three small subunit genes in two cell types ofmaize leaves

Jenq-Yunn Sheen and Lawrence Bogorad

Department of Cellular and Developmental Biology, Harvard University,Cambridge, MA 02138, USA

Communicated by J.H.Weil

Transcripts of three distinct ribulose-1,5-bisphosphate carb-oxylase (RuBPC) small subunit (SS) genes account for - 90%of the mRNA for this protein in maize leaves. Transcriptsof two of them constitute >80% of the SS mRNA in 24-hgreening maize leaves. The third gene contribute - 10%.Transcripts of all three nuclear-encoded SS genes are detect-able in bundle sheath (BSC) and mesophyll cells (MC) of etio-lated maize leaves. The level of mRNA for each gene isdifferent in etioplasts of MC but all drop duringphotoregulated development of chloroplasts in MC and followa pattern of transitory rise and fall in BSC. The amounts ofLS and SS proteins continue to increase steadily well afterthe mRNA levels reach their peaks in BSC. The molar ratioofmRNA for chloroplast-encoded RuBPC large subunit (LS)to the nuclear genome encoded SS is about 10:1 although LSand SS proteins are present in about equimolar amounts.Key words: bundle sheath cells/cell-specific expression/light in-duction/mesophyll cells/multigene family

detected in MC of dark-grown seedlings declines and disappearsupon illumination.We show here that maize SS is encoded by a multigene fami-

ly. We have examined the expression of three SS genes in thetwo cell types of greening maize seedling leaves and have foundthat none is expressed solely in MC or in BSC. We also showin this paper that the expression of all three SS genes is similarto that of the LS gene in two cell types of etiolated, greeningand green maize leaves.

ResultsAnalysis of maize SS by two-dimensional gel electrophoresisTwo intensely labelled spots with Mr 13 000 were revealed bytwo-dimensional gel electrophoresis of SS immunoprecipitatedfrom extracts of plants supplied [35S]methionine. Some minorspots (2-4) were also detected (Figure 1). Earlier analyses oftobacco and pea SS indicated the existence of two major isoelec-tric variants and some minor ones. The variants of SS may repre-sent the products of separate genes as has been suggested forother plants (Chen et al., 1976; Gray et al., 1978; Ishiye et al.,1981).Identity and specificity of three SS cDNA clonesTwelve putative maize SS cDNA clones were obtained initiallyby differential screening of maize leaf cDNA libraries with size-

IntroductionRibulose-1,5-bisphosphate carboxylase (RuBPC) catalyzes thefirst step of CO2 fixation in the Calvin cycle in higher plants.The holoenzyme is a multimeric protein comprised of eight largesubunits (LS) and eight small subunits (SS) (Kung, 1977). TheLS is encoded in the chloroplast genome (Coen et al., 1977) andthe SS is encoded in the nuclear genome as a multigene familyin every plant species examined (Berry-Lowe et al., 1982; Broglieet al., 1983; Dunsmuir et al., 1983; Coruzzi et al., 1984; Deanet al., 1985a; Polans et al., 1985). Light has been shown tomodulate the accumulation of mRNAs (Coruzzi et al., 1984;Crossland et al., 1984; Nelson et al., 1984; Silverthorne andTobin, 1984; Berry et al., 1985; Morelli et al., 1985; Nagi etal., 1985; Inamino et al., 1985; Rodermel and Bogorad, 1985;Sheen and Bogorad, 1985; Timko et al., 1985; Tobin and Silver-thorne, 1985), and proteins (Nelson et al., 1984; Berry et al.,1985) of both LS and SS genes in various plant species.Maize is a C4 plant, LS and SS gene expression has been

demonstrated to be restricted at both the mRNA and protein levelsin bundle sheath cells (BSC) of green leaves (Huber et al., 1976;Matsumoto et al., 1977; Link et al., 1978; Broglie et al., 1984;Aoyagi and Nakamoto, 1985; Sheen and Bogorad, 1985; Schusteret al., 1985). In addition, we and Nelson et al. (1984) have foundsome LS and SS mRNAs and proteins in leaves of dark-grownmaize seedlings but the levels of mRNAs and proteins are greaterafter illumination. As described in a previously published paper(Sheen and Bogorad, 1985), LS mRNA is present in both BSCand MC of dark-grown maize seedlings. Light stimulates theaccumulation of LS mRNA in BSC but not MC. The LS mRNA

IRL Press Limited, Oxford, England

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Fig. 1. Maize SS analyzed by two-dimensional gel electrophoresis. SS wasimmunoprecipitated from extracts of maize seedlings supplied with[35S]methionine. The first dimension was the isoelectric focusing gel (IEF)and the second dimension was a SDS-polyacrylamide gel (12.5%acrylamide). Arrows indicate the locations of SS isoelectric variants.

3417

J.-Y.Sheen and L.Bogorad

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Fig. 2. Identity and specificity of three SS cDNA clones. (a) Hybrid selection, in vitro translation and immunoprecipitation results of SS1, ASS6 and ASS7were shown. In vitro translation products of poly(A)+ mRNA isolated from BSC (B) and MC (M) and the immunoprecipitated SS from (B) were run inparallel. (b) Cross-hybridization among three SS cDNA clones were with excised insert DNAs. (c) Southern blots of maize genomic DNA digested withBamHI(l), BgllI(2), EcoRI(3), HindlI(4), XbaI(5) and PstI(6) were first probed with two cDNA clones of a SS gene carrying a long insert -700 bp (SS7)and a short insert 200 bp (ASS7). (d) The short inserts of the other two SS cDNA clones (SS1, ASS6) and the long insert of SS7 were used to probemaize genomic blots. The maize genomic DNA was digested with BamHI.

enriched SS and BSC specific poly(A)+ mRNAs, however, onlythree of them, SS1, SS6 and SS7, were chosen for further analysisafter insert cross-hybridization experiments (data not shown)showed these to be distinctive. The cDNA clones SS6 and SS7,carrying inserts of 350 bp and 700 bp, respectively, were sub-jected to Bal31 deletion as described in Materials and methodsto generate short end-fragments to use as specific probes. Theidentities and specificities of these three SS cDNA clones - SS 1(220 bp), ASS6 (200 bp) and ASS7 (200 bp) were confirmedfirst by hybrid selection followed by in vitro translation and im-munoprecipitation with SS antibody, and, second, by insert cross-

hybridization, genomic Southern blot analysis, and Northern blotanalysis. These three cDNA clones hybrid-selected mRNAs that,when translated in vitro, gave polypeptides that co-migrated withSS precursors in SDS - polyacrylamide gel electrophoresis andcould be immunoprecipitated by SS antibody (Figure 2a). Theinserts of SS cDNA clones SS1, ASS6 and ASS7 show little or

no cross-hybridization to one another (Figure 2b). Furthermore,their inserts hybridize to one or other of the genomic bands thatare hybridized by SS7, a long-insert SS cDNA clone (Figure2c,d), and hybridize to RNAs of different abundance in Nor-thern blot analysis, (Figure 3). The genomic bands hybridizedby SS1 and ASS6 (Figure 2d) were located in the doublet revealedin Figure 2c (lane 1). Evidence from cross-hybridization and Nor-thern blot analyses supported the conclusion that they correspondto parts of two different genes. Therefore, we believe that wehave identified partial cDNA clones of three different SS genesthat are actively expressed in maize leaves. We designate thegenes rbcS-ml (clone SS1), rbcS-m2 (clone SS6) and rbcS-m3

(clone SS7). There are one or two more SS genes (estimated bygenomic blotting) in the maize genome but cDNAs for these havenot been obtained (Figure 2c).

Relative expression levels of three SS genes estimated by Nor-thern blot analysisTo estimate the relative expression levels of these three SS genes,we excised the cDNA-clone inserts, end-labelled them to equalspecific activity and used them to probe Northern blot strips con-taining equal amounts of poly(A)+ mRNA from 24-h greeningmaize leaves. As shown in Figure 3, SS1 hybridized to -45%of the total leaf SS mRNA, ASS7 to 35%, and ASS6 to 10%(examined by densitometer scanning). Thus, -90% of SSmRNA at its accumulation peak (in 24-h illuminated dark-grownmaize) was complementary to SS1, ASS6 and ASS7, i.e. the pro-ducts of rbcS-ml, m2 and m3. The uncloned SS genes may or

may not be expressed, as has been seen in petunia (Dean et al.,1985b).

Cell-specific expression of three SS genes in BSC andMC ojfmaizeleavesDot blot analyses revealed that transcripts of all three genes are

detectable in BSC and MC of dark-grown maize seedlings, butare overall about one-third as abundant in MC as in BSC.Moreover, the transcript ratio of three SS genes (rbcS-ml :m2:m3) is about 4:1:3 in etioplasts ofBSC and 2:1:1 in MC.Upon illumination, total SS mRNA increased - 3-fold (examin-ed by densitometer scanning) after 24 h of greening in BSC,whereas it dropped from the initial very low level in MC. rbcS-

3418

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Fig. 3. Relative levels of transcripts of three SS genes as revealed byNorthern blot analysis. One jg of poly(A)+ mRNA was fractionated on a

formaldehyde -agarose gel (1% agarose) and blotted onto Nytran. Three SScDNA clones with short inserts (SS1, ASS6, ASS7) were used as probes toestimate the relative level of RNA complementary to each SS gene probe as

compared with the total SS mRNA as revealed by a SS cDNA clone with a

long insert (SS7).

B M

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Fig. 4. Cell-specific differences in mRNAs of three SS genes estimated bydot blot analysis. Five Mg of total RNA isolated from BSC (B) and MC (M)of etiolated (0), greening (6, 12, 24, 48, 72, 96 h) and green (G) maizeleaves was spotted onto Nytran. Dot blot strips were probed with three SScDNA clones with short inserts (SS1, ASS6, ASS7). The signals for ASS6were obtained after exposing the film three times longer than for others.The control lane (C) was probed with an unknown cDNA clone that was

expressed equally in BSC and MC.

ml (SS1) and m3 (ASS7) transcripts were much more abundantthan those of m2 (ASS6) in leaves of all developmental stagesexamined, however, the influence of light on their expressionwas similar in BSC; in a quite different way, they respondedtogether in MC (Figure 4). The control lane was an unknowngene that is photoregulated and is expressed equally in the twocell types.

Fig. 5. Transcripts of LS and SS genes in BSC and MC assayed byNorthern blot analysis. Ten Atg and 30 ,g of total RNA samples as iiFigure 4 were fractionated on formaldehyde-agarose gels (1% agar(blotted onto nitrocellulose and probed with a RuBPC large subunit (Iclone (Rodermel and Bogorad, 1985; Sheen and Bogorad, 1985), a (

clone (Bedbrook et al., 1978) and an RuBPC small subunit clone (SEa long insert (SS7), respectively. (a) The four rRNA bands were see]u.v. light after staining with ethidium bromide. This shows that the aof RNA was approximately the same in each lane. (b) Northern blotanalyses for LS, QB and SS mRNAs are shown in parallel. The sign,SS mRNA were obtained by exposing the film 10 times longer thanmRNA. Bam8 and Bam9 indicated the quantitation of DNA templateQB and LS, respectively, in a Southern blot with DNA isolated fromset of samples used for quantitating the mRNAs. The DNA samplesdigested with BamHI fractionated on a 0.8% agarose gel, blotted ont,nitrocellulose and probed with LS and QB clones.

Expression of LS and SS genes in BSC and MCWith maize SS cDNA clone, SS7, carrying a long in!

700 bp, and thus presumed to have most of the conserved i

regions found in all maize SS genes [as discovered in ppetunia (Coruzzi et al., 1984; Dean et al., 1985b)], and theLS clone used in previous studies (Rodermel and Bogorad,Sheen and Bogorad, 1985), the accumulation patterns of ISS mRNAs in BSC and MC of etiolated, greening andleaves were analyzed by Northern blotting. As shown in:5, both mRNAs were present at - 20-30% of the 24-Ilevels in BSC of etiolated leaves (examined by densitometening). Upon illumination, both mRNAs increased to reachafter 24 h of greening then declined in BSC of green leI

steady-state levels slightly higher than in etiolated leave:LS mRNA was clearly detected in MC of etiolated leavdisappeared after 72 h of greening as described beforeand Bogorad, 1985). The mRNA of SS genes was barelytable in MC of etiolated leaves. About 10 times more LSS mRNA was estimated to be present in the total RN)of maize leaves as the film showing SS mRNA was expo

SS1 ASS6 &SS7 SS7M

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J.-Y.Sheen and L.Bogorad

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0 6 12 24487296G 0 6 12 24 487296 G hOn the other hand, the levels of LS and SS stayed about the samein MC throughout the course of greening to 96 h; these proteinswere not detectable in MC of green leaves.

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Discussion-6 6K 7hree distinct SS genes are actively expressed in maize leaves

As in C3 plants, the SS of the C4 plant maize is also encodedLS by a multigene family with at least 4-5 members. Four lines

_>-45K of evidence, including hybrid selection and immunoprecipitation,cross-hybridization, genomic Southern blotting and Northern blot-

-<31K ting with shortened cDNA clones, indicate that we have iden-tified cDNA clones of three distinct SS genes that are activelyexpressed in maize leaves. Two of them, rbcS-ml (complemen-

tx -21K tary to cDNA SSl) and rbcs-m3 (complementary to cDNAXs_ <14K \ASS7), account for - 80% of the SS mRNA detected in etiolated,

-SS greening and green maize leaves. The third gene, rbcS-m2(characterized through cDNA ASS6), contributes 10% of the

op SS mRNA detected in maize leaves. Two-dimensional gelanalysis of maize SS reveals two intensely labelled isoelectric

M224487296G h variants; these may be the products of two different highly ex-

pressed SS genes but there is no firm evidence at present thatsupports or refutes this possibility. In tobacco and pea, two ma-jor isoelectric variants are also revealed by isoelectric focusing_ _ < L S on two-dimensional gel and it has been suggested that each is

encoded by a different gene based on peptide mapping (Gay etal., 1978).

Fig. 6. Coordinate accumulation of LS and SS in BSC and MC of greeningmaize leaves. Total proteins were isolated from aliquots of the BSC (B) andMC (M) samples used for RNA isolation. The proteins loaded in each lanewere normalized with equal RNA content as for the same set of RNAsamples used in Figures 4 and 5. The proteins were either stained withCoomassie blue (a) or transferred onto nitrocellulose and probed with LSand SS antibodies (b).

times longer to obtain a comparable signal (Figure Sb) and threetimes more total RNA was used for Northern blotting of SSmRNA to compensate for differences in probe length when theLS and SS probes were of equal specific activity [the probe lengthof LS gene (2.2 kb) is three times longer than the SS gene(0.7 kb)].The patterns of accumulation of LS and SS proteins were

studied directly by Coomassie blue staining of the protein geland by Western blotting. As shown in Figure 6a, many proteinsaccumulated differently in BSC and MC of etiolated, greeningand green maize leaves. The two most dominant bands, with Mr52 000 and 13 000, in BSC were LS and SS, respectively. Thiswas further confirmed by Western blotting analysis with antiserato LS and SS (Figure 6b). LS and SS were present in BSC andMC of etiolated maize leaves at about equal molar ratios.However, the total amounts of LS and SS were 2- to 3-fold higherin BSC than in MC of etiolated leaves. Upon illumination, LSand SS started to accumulate progressively and reached a plateauafter 72 h of greening in BSC. The steady-state levels of LS andSS in BSC of green maize leaves were similar to the plateau level.

3420

Expression of LS and SS genes in BSC and MC of etiolated,greening and green maize leavesIt has been shown, in whole maize leaves, that LS and SS mRNAsincrease upon illumination of dark-grown maize seedlings (Nelsonet al., 1984; Rodermel and Bogorad, 1985; Zhu et al., 1985).After separating the two cell types of maize leaves, we have foundthat transcripts of three SS genes that are represented by slightlyto vastly different fractions of the total SS mRNA accumulatein the same pattern and in the same ratio to one another in BSCof etiolated, greening and green maize leaves. The pools ofRNAsfor the three SS genes we have studied here are present in theratio 4:1:3 in BSC at all stages of greening and 2: 1:1 in etiolatedMC for rbcS-ml :m2:m3. Thus, there is some degree of differen-tial expression of members of this gene family within each celltype as well as overall photoregulated suppression ofMC levelsof these genes in MC and stimulation in BSC. We estimate cross-contamination of BSC and MC preparations to be < 5% (Sheenand Bogorad, 1985). Although the differences in relative abun-dance of MCs of the three genes in etiolated BSC and MC areslight, the data argue against cross-contamination of the two celltype preparations being the sole basis for the resultsLS and SS mRNAs (Nelson et al., 1984; Rodermel and

Bogorad, 1985) and proteins (Nelson et al., 1984) are presentin etiolated maize leaves and the difference in BSC and MC ofetiolated leaves is only 2- to 3-fold at both mRNA and proteinlevels. On the other hand, these mRNAs and proteins are onlypresent in BSC of green, light-grown maize leaves (Huber et al.,1976; Link et al., 1978; Broglie et al., 1984; Aoyagi andNakamoto, 1985; Sheen and Bogorad, 1985; Schuster et al.,1985).We show here that the cell-specific expression of LS and SS

genes is enhanced dramatically during greening. The pools ofmRNAs of LS and SS genes rise and decline to steady-state levelsin BSC during 96 h of greening while they disappear from MC(Rodermel and Bogorad, 1985; Sheen and Bogorad, 1985). InBSC, levels of LS and SS proteins increase progressively follow-

a

Expression of RuBPC LS and SS genes in maize

ing the accumulation of their mRNAs and stay at high levelsdespite the drops in mRNAs. The proteins remain at low levelsin MC for up to 96 h of continuous illumination and become un-detectable in green leaves. The small amounts of LS and SSdetected in MC of greening leaves are probably residual stabletranslation products of mRNAs present in etiolated leaves forno LS and SS mRNAs are detected in MC after 72 h of greening.These data suggest that the localization of LS and SS in BSC

of green leaves is established through the BSC-specific accumula-tion of LS and SS mRNAs and proteins stimulated by light and/ora photoregulated MC-specific depression of the accumulation ofthese mRNAs during greening.We have found the molar ratio of LS to SS mRNA to be about

10: 1. To reach the final molar ratio of LS to SS protein at 1:1,more efficient transcription or mRNA with higher stability isneeded for SS genes as each cell has at least 100- to 2000-foldmore plastid-encoded LS gene than nuclear-encoded SS genes(Lamppa and Bendich, 1979; Scott and Possingham, 1980). Ad-ditionally, more efficient translation of SS mRNA is needed toobtain a final molar ratio of LS to SS of 1:1. Alternatively, 10times more LS could be made but then degraded if unassembledas has been suggested previously (Roy et al., 1982). Our valuefor the molar ratio of 10:1 for LS and SS transcripts does notagree with the 1:1 ratio reported previously (Nelson et al., 1984).We believe that the discrepancy comes from the RNA samplesused. Nelson et al. used poly(A)+ RNA for quantitating bothLS and SS mRNAs, however, only 10% of the LS mRNAco-purified with poly(A) + RNA using oligo(dT)-cellulose(J.-Y.Sheen and L.Bogorad, unpublished observation). Thus,their estimated 1:1 ratio is different from our data of 10:1 whentotal RNA samples are used.The changes in sizes of mRNA pools of three members of the

SS multigene family is coordinate in BSC of maize leaves dur-ing light-induced maturation of plastids; however, mRNAs ofthe three genes are present in different proportions in MC andBSC etioplasts. On the contrary, each of six members of theLHCP II multigene family (or families) in maize leaves isrepresented by a very different sized pool ofmRNA in BSC andMC and differs from the others in the degree of responsivenessto light (Sheen and Bogorad, 1986).

Materials and methodsPlant material and growthMaize seedlings (Zea mays: FR9crs x FR37, Illinois Foundation Seeds) weregrown in the darkroom before greening or in the greenhouse, as described bySheen and Bogorad (1985). All samples were isolated from the mature sectionsof second leaves (4- 10cm) as described previously (Sheen and Bogorad, 1985;Aoyagi and Bassham, 1986).Immunoprecipitation and two-dimensional gel electrophoresisMaize seedlings illuminated for 24 h were labelled with [35S]methionine (NEN)in situ by inserting cut leaf bases into the labelling solution. Total protein wasextracted by boiling in 50 mM Tris-HCI, pH 7.5, 2% SDS, 5% (3-mercaptoethanol, 6% sucrose. SS was immunoprecipitated by the method ofFranssen et al. (1982) and solubilized in the same extraction buffer describedabove. Two-dimensional, isoelectric focusing SDS gel electrophoresis was car-ried out with 0.5% (w/v) SDS present in the sample as described previously (O'Far-rell, 1975; Ames and Nakaiko, 1986). The Ampholine was pH 3.5-10 (LKB).Methods for cDNA cloning, colony hybridization and hybrid selectionThe first and second strands of cDNA were synthesized as described by Gublerand Hoffman (1983), except that the two reactions were carried out in the sametube sequentially by diluting the 10 Al of the first reaction to 100 JAI with a mix-ture for the second reaction. The double-stranded cDNAs were tailed with dGTPas described by Gubler and Hoffman (1983) and inserted into the SaI sites ofpUC13 plasmid. MC1061 competent cells were used for transformation (Maniatiset al., 1982). Differential screening with SS mRNA enriched by sucrose gra-dient fractionation and BSC-specific mRNA was done by the methods of Maas

(1983). Hybrid selection was done as described by Pames et al (1981) exceptthat 0.1-1 g of plasmid DNA was digested with BamHI for linearization beforethe alkaline denaturation. The in vitro translation products of hybrid-selectedmRNAs were immunoprecipitated with SS antibody to confirm the identity ofSS cDNA clones.Construction of specific probesSS cDNA clones containing inserts longer than 200 bp were subjected to Bal3ldeletion to generate short specific probes for each gene. For use in making dele-tions from both orientations of the inserts, 1 Ag of each clone was linearized byeither BamHI or EcoRI. Bal3l deletions were carried out in 100 1u of reactionbuffer (as described by the manufacturer) with 1 itg of linearized plasmid DNAand 1 U of Bal3l (BRL) at 30°C for 5-15 min to delete 150-450 bp. Thereaction was stopped by phenol extraction. T4 DNA polymerase was used toblunt the ends (Maniatis et al., 1982) and non-phosphorylated BamHI or EcoRIlinkers were ligated to them (Seth, 1984) to regenerate the deleted sites.DNA cross-hybridization and genomic Southern blot analysisInserts of different SS cDNA clones were excised by digestion with BamHI andEcoRI, fractionated on a 5 % polyacrylamide gel, and eluted in dialysis bags byelectrophoresis at 200 V for 15 min. Ten ng of each DNA insert sample wasdenatured in 10 u1 of 2.2 M formaldehyde, 1 mM EDTA at 80°C for 3 minbefore spotting onto Nytran (S & S). The prehybridization and hybridization wasdone in 4 x SSPE (1 x SSPE = 0.18 M NaCl, 10 mM NaH2PO4 H2O, 1 mMNa2EDTA, pH 7.4), 5 x Denhardt's (0.002% Ficoll, 0.002% BSA and 0.002%polyvinylpyrrolidone), 0.2% SDS, 100 ,g/ml calf thymus DNA at 65°C for 2 hand 16 h, respectively. The washing conditions were: 1 x SSC (0.15 M NaCl,0.015 M Na-citrate), 10 mM NaPPi, 0.2% SDS for 1 h at 68°C and then 0.1x SSC at 680C for 30 min. The filters were blotted dry and exposed to KodakX-ray film at -800C.

Total maize DNA was extracted from leaves of 2-week-old seedlings as describedby Bendich et al. (1981). Maize genomic DNA (10 ILg) was digested to comple-tion with restriction enzymes at a DNA concentration of 100 sg/ml and an en-zyme concentration of 5 U/4g for 16 h at 370C. Completely digested DNA sampleswere concentrated by phenol extraction, ethanol precipitation and were fractionatedelectrophoretically on an 0.8% agarose gel. After electrphoresis, gels were treatedwith 0.25 M HCI for 30 min, 0.5 M NaOH for 45 min and 1.5 NaCl, 1 MTris-HCI, pH 7.5 for 30 min before blotting onto Nytran (S & S) with 10 xSSC for 10 h. The hybridization and washing were the same as described above.Specific SS cDNA probes were made by nick-translating insert DNA with[a-32P]dATP and [a-32P]dCTP (Maniatis et al., 1982).Northern blot and dot blot analysesPoly(A)+ mRNA from 24-h greening leaves and total RNA from BSC and MCwere prepared as described previously (Sheen and Bogorad, 1985). For dot blotanalysis, 5 Ag of each total RNA sample was denatured in 5 mM Tris-HCI,pH 7.5 and 2.2 M formaldehyde at 800C for 3 min before spotting onto Nytran(S & S). Hybridization and washing were the same as for Northern blot analysisas described by Sheen and Bogorad (1985). The probes were insert DNAs end-labelled with [a-32P]dATP and [a-32P]dCTP to EcoRI and BamHI ends to equalspecific activity as described by Maniatis et al. (1982).Western blot analysisThe samples of BSC and MC protein were obtained by boiling with 50 mMTris-HCI, pH 7.5, 2% SDS, 6% sucrose, 5% 13-mercaptoethanol for 5 min.BSC and MC were prepared as described by Sheen and Bogorad (1985) exceptthat the starting material for preparation of BSC was the residue of enzymatical-ly digested leaves after release of MC. SDS-polyacrylamide gel (12.5%acrylamide) electrophoresis was used for protein fractionation with the Laemmlibuffer system (1970). A lower ratio of acrylamide to bis-acrylamide (30:0.4) wasused and the gel was run at a constant current of 20 mA at room temperatureto achieve maximal resolution. The method of Towbin et al. (1979) was usedfor blotting proteins onto nitrocellulose but 0.1% (w/v) SDS was included in thetransfer buffer. Bound antibodies were located by horse radish peroxidase con-jugated goat second antibody (Biorad) as described by the manufacturer.

AcknowledgementsWe thank S.Mosley and J.Martin-Perez for advice on two-dimensional gel pro-cedures, A.Cheung and L.D.Crossland for advice on Bal31 deletion experimentsand G.Link for preparing antibody against purified maize RuBSC SS. This workwas supported in part by a graduate fellowship from Harvard University to J.-Y.S. and a grant from the national Institute of General Medical Sciences. It wasalso supported in part by the Maria Moors Cabot Foundation for Botanical Researchof Harvard University.

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