RESEARCH ARTICLE

Maize BMS cultured cell lines survive with massive plastid gene loss

Received: 28 March 2003 / Revised: 1 May 2003 /Accepted: 2 May 2003 / Published online: 13 June 2003� Springer-Verlag 2003

Abstract As part of developing an ex planta modelsystem for the study of maize plastid and mitochondrialgene expression, a series of established Black MexicanSweet (BMS) suspension cell lines was characterized.Although the initial assumption was that their organellebiochemistry would be similar enough to normal inplanta cells to facilitate future work, each of the threelines was found to have plastid DNA (ptDNA) differingfrom control maize plants, in one case lacking as muchas 70% of the genome. The other two BMS lines pos-sessed either near-wild-type ptDNA or displayed anintermediate state of gene loss, suggesting that theseclonal lines are rapidly evolving. Gene expression pro-files of BMS cells varied dramatically from those inmaize leaf chloroplasts, but resembled those of albinoplants lacking plastid ribosomes. In spite of lacking mostplastid gene expression and apparently mature rRNAs,BMS cells appear to import proteins from the cytoplasmin a normal manner. The regions retained in BMSptDNAs point to a set of tRNA genes universally pre-served among even highly reduced plastid genomes,whereas the other preserved regions may illuminatewhich plastid genes are truly indispensable for plant cellsurvival.

Keywords Plastid Æ Black Mexican Sweet ÆTissue culture Æ Maize

Introduction

Regulation of plastid and mitochondrial gene expres-sion by nucleus-encoded factors is of primary impor-tance in promoting organelle biogenesis and responseto environmental factors. Robust studies depend on theability to manipulate components of the system, usinggenetic, molecular, and biochemical approaches. Themost fruitful models to date are maize, Arabidopsis,and Chlamydomonas reinhardtii where, in each case,numerous nuclear mutants were isolated that affectchloroplast gene expression (for reviews, see Goldsch-midt-Clermont 1998; Barkan and Goldschmidt-Clermont 2000), or where mitochondrial dysfunctionarose in certain nuclear backgrounds (for a review, seeLeon et al. 1998). In addition, the ability to disruptplastid genes in tobacco played a critical role indefining gene function and associated cis elements (fora review, see Maliga 2002).

Each system has its advantages, although maize suf-fers from difficulties in nuclear transformation and has arelatively long generation time. An in vivo maize systemthat allows more rapid analysis than stable transfor-mation of plants would be useful for a number of studiesof nuclear–cytoplasmic interactions. For example,analysis of plastid-splicing and RNA stability factors inChlamydomonas was facilitated by the ability to epitope-tag and re-introduce the genes encoding them (e.g.Perron et al. 1999; Boudreau et al. 2000; Rivier et al.2001). In our own work, we isolated genes encoding thecatalytic subunits of both the maize plastid and mito-chondrial RNA polymerases (Chang et al. 1999) andwished to test whether they assemble into larger com-plexes, which might benefit from the same approach.Our first choice of a model was the Black Mexican Sweet(BMS) cell line (Chourey 1981; Sheridan 1982), due toits availability, prolific growth, ability to producetransformants easily, and proven use as a model forbiochemical studies (Forlani et al. 1994; Grotewold et al.1994, 1998).

Curr Genet (2003) 44: 104–113DOI 10.1007/s00294-003-0408-1

A. Bruce Cahoon Æ Katherine A. Cunningham

Thomas J. Bollenbach Æ David B. Stern

Communicated by F.-A. Wollman

A.B. Cahoon Æ K.A. Cunningham Æ T.J. BollenbachD.B. Stern (&)Boyce Thompson Institute for Plant Research,Tower Road, Ithaca, NY 14853, USAE-mail: ds28@cornell.eduTel.: +1-607-2541306Fax: +1-607-2556695

Present address: K.A. CunninghamDepartment of Biological Sciences,Stanford University, Stanford, California, USA

In spite of (and in some cases because of) their lack ofphotosynthetic capacity, plant cell culture lines provevery useful in the study of plastid gene expression. Riceand tobacco cell lines were essential in the dissection ofnuclear-encoded plastid RNA polymerase (NEP) speci-ficity and in the identification of NEP promoters (Veraand Sugiura 1995; Vera et al. 1996; Kapoor et al. 1997;Silhavy and Maliga 1998b; Liere and Maliga 1999).Also, albino mutants of maize (iojap) and barley(albostrians) were instrumental in the study of selectivetranscription by NEP (Han et al. 1993; Hess et al. 1993;Allison et al. 1996; Silhavy and Maliga 1998a; DeSantis-Maciossek et al. 1999), which shares duties with an eu-bacterial plastid-encoded polymerase (for a review, seeCahoon and Stern 2001). We hypothesized that BMScells would display a plastid gene expression patternsimilar to iojap and possess a highly active NEP ame-nable to biochemical manipulation in the absence ofplastid-encoded RNA polymerase (PEP).

As a cautionary note, however, we were aware thatphysiological alterations occur in tissue culture linesduring the process of becoming immortalized. Existingreports showed, for example, that long-term rice andbarley cell lines accrue large deletions in their plastidgenomes (Ellis and Day 1986; Harada et al. 1991;Kawata et al. 1995). Although similar data were notavailable for maize, this raised a concern that the BMSlines should be carefully characterized prior to their usefor the study of plastid gene expression. Here, wedetermine the gene content and sample plastid andmitochondrial gene expression and protein import inthree independently maintained BMS lines. We concludethat two lines, BMS-C and BMS-G, carry plastidgenomes with multiple deletions whereas a third,BMS-D, retains an essentially complete genome. Theseresults are discussed in terms of what might constitute anessential plastid gene complement and whether such celllines are suitable for certain types of functional assays.

Materials and methods

Maintenance of BMS cultures

BMS cells were maintained in liquid MS2D medium (Murashigeand Skoog 1962) with 2 mg 2,4-D/l. The medium was changedevery 4 days and the suspensions were subdivided each week. Thecells were grown in Erlenmeyer flasks at 25 �C in the dark on arotary shaker.

Preparation of total DNA and filter hybridization analysis

Total DNA was isolated from 20 ml packed volume of BMS cellsor 1 g of A188 leaf tissue, using a CTAB-based method (Dellaportaet al. 1983). For DNA gel blot analysis, 10 lg of total DNA wasdigested with 5 units of BamHI for 4 h, separated in 0.8% agarosegels, stained with ethidium bromide, and visualized with an Al-phaImager 2200 (Alpha Innotech Corp.). Transfer and UV-cross-linking were followed by prehybridization in aqueous hybridizationbuffer I (Ausubel et al. 1997). DNA probes were labeled with a32P-dCTP, using random hexamers. Membranes were probed overnight

and washed once for 20 min with 2· SSC and 2% SDS and oncefor 20 min with 0.2· SSC and 0.2%SDS. Washed blots wereexposed overnight to a PhosphorImager screen (MolecularDynamics).

For exonuclease digestion of total DNA, samples were incu-bated with 16 units of exonuclease III for 2 h, extracted twice withphenol:chloroform (1:1), incubated at 65 �C for 15 min, and thendigested with BamHI. Filter hybridization analysis was as describedabove.

Preparation of RNA and filter hybridization analysis

RNA was extracted from BMS cell suspensions in log phase growth(2500 cells/ml) and A188 seedling leaf tissue. Aliquots of BMS cellswere centrifuged at 200 g for 5 min and excess medium was re-moved. Cells were frozen in liquid nitrogen and ground with amortar and pestle. Total RNA was extracted using the standardprotocol for Tri reagent (Molecular Research Center). Total RNAsamples were mixed with an equal volume of 2· sample buffer [60%formamide, 20% formaldehyde, 1· MOPS buffer (0.2 M MOPS,10 mM EDTA, pH 8.0, 83 mM sodium acetate), 1% ethidiumbromide], heated to 65 �C for 15 min, and electrophoresed in 1%agarose/1· MOPS buffer/3% formaldehyde gels for 4 h at 80 V.The gels were blotted onto GeneScreen Nylon filters, using a cap-illary blotting apparatus and 5· SSC buffer, and UV-crosslinked.Probes, washing, and visualization were as described above forDNA hybridization analysis.

Primer extension

Primer extension was carried out on 15–20 lg of total RNA. RNAwas denatured at 95 �C for 5 min with 10 nmol of primer tRNE-C(5¢-aggcagcgggtattcgactt-3¢) and placed on ice. Then, 5 units ofavian myeloblastosis virus reverse transcriptase, 1· reaction buffer(Promega), and 40 lCi of a32P-dCTP were added and the reactionwas incubated at 42 �C for 15 min. Reaction products were sepa-rated in a 42% urea/20% polyacrylamide/1· Tris-borate EDTAsequencing gel and visualized using a PhosphorImager.

Dot blots

Clones of plastid genes in plasmid vectors (kindly provided by AliceBarkan, University of Oregon, and by Ruairidh Sawers and TomBrutnell, Boyce Thompson Institute) were blotted onto Nylonmembranes as paired dots (50 ng, 25 ng), using the BioDot appa-ratus (BioRad) according to the manufacturer¢s protocol. TotalBMS DNA was labeled with a32P-dCTP using random hexamers.The membranes were prehybridized, incubated with labeled probes,and washed as described above.

Biolistic transformation of BMS cells and subcellular localization

The N-terminal transit peptides of the mitochondrial T7-like RNApolymerase (RpoTm) and the small subunit of ribulose bisphos-phate carboxylase (RbcS) were fused to green fluorescent protein(GFP), using the BamHI site of the vector 35S-C4PPDK-sGFP.These and a cytosolic control lacking a transit peptide were con-structed as described by Chang et al. (1999). Suspended BMS cells(12 ml, approximately 106 cells) from a 4-day-old subculture weretransferred to a Buchner funnel containing a Whatman number 1filter. The cells were drawn onto the filter by gentle vacuum. Thefilters were then transferred to plates containing BMS agar and theexcess liquid was allowed to evaporate. Tungsten beads, previouslyloaded with 10 lg of transforming DNA, were bombarded onto thecells from a distance of 10 cm with a breaking pressure of5,500 KPa. The cells were then incubated in the dark at room

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temperature for 16 h, resuspended in 10 ml BMS medium, andincubated in the dark at room temperature in the presence of100 nM Mitotracker (Molecular Probes, Eugene, Ore.). Cells wereharvested by centrifugation at 100 g and resuspended in 1 ml BMSmedium. GFP fluorescence was observed by confocal laser scan-ning microscopy as described by Chang et al. (1999).

Results

The plastid genomes of some BMS cell lines carryvery large deletions

The scientific database is littered with accounts of tissueculture lines and cells that are anomalous, comparedwith their wild-type counterparts. One such problemspecifically affecting plastids is the loss of sometimesextensive portions of their genome (Ellis and Day 1986;Dunford and Walden 1991; Harada et al. 1991; Kawataet al. 1995), although stable cultures are also observed(e.g. Seyer et al. 1982). When we began our analysis ofBMS cells, we were led by preliminary experiments tosuspect that portions of the plastid genome might bemissing or rearranged. To screen a large number ofgenes simultaneously, we used a dot blot macroarray,arraying 50 maize plastid genes excluding tRNAs on aNylon membrane as paired dots. The 50 clones sampledthe entire plastid genome, based on its publishedsequence (Maier et al. 1995), and offered a cumulativebase for base coverage of 57%, with an average gapdistance between clones of 2.7 kb (gap range 0.5–9.9 kb). Total DNAs from a control, the inbred line

A188, and three BMS lines were radioactively labeledand hybridized to the dot blot arrays. As seen in Fig. 1,labeled total DNA from A188 (row A) and the BMS-Dline (row D) hybridized to all of the clones except anegative control, the pBluescript plasmid, and two verysmall genes, psbI and psbJ, probably due to their sizesand/or sequence divergence between the sequences usedand those of the maize lines tested.

Labeled total DNAs from BMS-C and BMS-G, incontrast, failed to hybridize to many of the plastid genes(Fig. 1, rows C, G), suggesting large regions of theirplastid genomes were missing. BMS-C retained appar-ently contiguous portions of the large single copy (LSC)and inverted repeat (IR) regions and four other non-contiguous regions distributed through the LSC and IR(see genome representation below each row). BMS-Ghad the most extensive deletions, appearing to retain asmaller but comparable contiguous portion of the LSCand the same region of the IR preserved in BMS-C.

To confirm and extend the dot blot results, a series ofgel blots were hybridized with single-gene probes (clpP,rps16, rps2, cemA, rpl16, rpoA, atpB, rbcL, psbD, rpl22,rpl32, ndhH, psbC, 23S, rpoB, rpl2, rpl20, 4.5S, rps12,rpoC2). Figure 2 shows representative results. Forexample, ndhH and rpl22 were predicted to be presentonly in A188 and BMS-D. As seen in Fig. 2, these geneshybridize in the predicted lines and show no unexpectedpolymorphisms. Two more examples of this analysis(clpP, rpoB) are mentioned by Cahoon et al. (2003).

Figure 2 also shows that, as expected, A188 andBMS-D cells carry two copies of rpl2 which straddle the

Fig. 1 Dot blot array analysisof the inbred line (A188) andBlack Mexican Sweet (BMS)plastid genomes. Clones of eachof the plastid genes shown inthis figure were blotted ontoNylon membranes as paireddots (50 ng, 25 ng). Theplasmid pBluescript (pBS) wasincluded as a negative control.Total A188 and BMS DNAswere labeled using randomhexamers and hybridized to themembrane. A188 (A) and threedifferent BMS cell lines (D, C,G) were examined. Markedbeneath each row are theapproximate extents of theinverted repeats (checkeredbars) and the large (black bars)and small (white bars) single-copy regions

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IR-LSC boundary: the 4.7-kb band is derived from IRA

and the 5.5-kb band from IRB. BMS-C and BMS-G hadsingle hybridizing fragments of 8 kb and 10 kb, respec-tively, suggesting that the IR exists as a single copy,albeit with some deletion and/or rearrangement. Thepreserved rpl2 copy in BMS-C and BMS-G is most likelyderived from IRA, because we were able to PCR-amplifya region spanning psbD and the rpl2 intergenic region inBMS-G (see Fig. 4; data not shown). A 23S rRNAprobe also hybridized with two IR copies in A188 andBMS-D. The 12.9-kb band in both lines is the IRA copy.The IRB copy reveals a polymorphism, perhaps due to adeletion or rearrangement in BMS-D. BMS-C andBMS-G each had a single hybridizing band, confirmingthe presence of a single IR region in these lines. Takentogether, the data in Fig. 2 suggest that there may beminor polymorphisms between the A188 and BMS-Dplastid genomes, and additionally demonstrate thatthere are substantial polymorphisms in the BMS-C andBMS-G lines, as would be expected from multipledeletion events.

Although plastid genomes are generally representedas closed circular molecules, it has been demonstratedthat they actually exist as populations of circular andlinear molecules (Ellis and Day 1986; Bendich and Smith1990; Collin and Ellis 1991; Backert et al. 1995; Kelch-ner and Wendel 1996; Nosek et al. 1998; Lilly et al.2001). With this in mind, we wanted to know whetherthe deletions in the BMS-C and BMS-G plastid genomesaffected their overall structures, for example shiftingthem to primarily linear forms. To test this, total DNAsfrom A188 and BMS cells were treated with exonucleaseIII, followed by deactivation of the enzyme and

subsequent digestion with BamHI. Exonuclease III has aprocessive 3¢-to-5¢ activity that attacks either protrudingor recessed 3¢ ends. We reasoned that, if A188 DNA wasa mixture of circular and linear molecules, but one ormore BMS plastid DNAs was chiefly linear, it would bemore sensitive to exonuclease III. BamHI digestion wasused to give discrete fragments that could be visualizedby filter hybridization.

As shown in Fig. 3, incubation of total A188 DNAwith exonuclease III resulted in a significant reduction ofthe BamHI fragment identified by a rpoB probe. Thissuggests that, under our isolation conditions, much ofA188 cpDNA is linear, probably due to random shear-ing during extraction and/or the in vivo conformation ofthe genome. Most important, however, is the presence ofexonuclease III-resistant molecules, which suggests thatthis methodology can be used to detect circular plastidgenomes. Using the same assay, we found that BMS-D,BMS-C, and BMS-G also have exonuclease III-resistantplastid genomes. This suggests that at least a portion ofBMS plastid DNAs exist in circular form and that thelarge deletions in BMS-C and BMS-G did not drasticallyalter the overall genome. As controls, a 1-kb ladder andintact plasmid DNA were also exonuclease-treated. Asexpected, the linear 1-kb ladder was highly susceptible toexonuclease digestion, whereas the circular plasmidDNA was not.

Figure 4 summarizes our genomic analyses of BMSand A188 ptDNAs. Each block on the outermost circlerepresents one gene in a complete, unadulterated maizechloroplast genome. White blocks are genes that weretested by either dot blot or DNA gel blot analysis,whereas black blocks are genes that were not directlytested but are assumed to be present, based upon thepresence of neighboring genes.

Based on our analysis, BMS-D may have minordeletions or rearrangements, but retains all the genespresent in A188; and thus it is unusually stable for atissue culture line. The middle gray circle representsthe BMS-C genome, which has multiple deletions in theLSC and short SC and has lost parts of both IRs. Thenet result of the deletions in the IRs is that only one copyof a given sequence remains. In other words, there is no

Fig. 2 DNA gel blot confirmation of dot blot results. Total A188and BMS DNAs (10 lg) were digested with BamHI, separated in a0.8% agarose gel, and blotted onto Nylon membranes. Probes forndhH, rpl22, rpl2, and rrn23 were radioactively labeled usingrandom hexamers. Molecular mass markers (shown at right) wereused to confirm the predicted fragment sizes for A188 and to deriveapproximate sizes for the BMS cell lines

Fig. 3 Analysis of cpDNA following exonuclease III treatment.Total A188 and BMS DNAs (10 lg) were digested (+) or not ())with 16 units of exonuclease III and then digested with BamHI, asdescribed in Materials and methods. A 1-kb DNA ladder andminiprep plasmid DNA were included as controls for sensitivityand resistance, respectively. rpoB was radioactively labeled usingrandom hexamers. The 1-kb ladder and plasmid controls are visibledue to non-specific binding of the labeled rpoB DNA

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longer a repeat. The BMS-G genome, shown on theinner circle, has multiple and catastrophic deletions,leaving only four apparent blocks of genes. Three ofthese regions, retained in both BMS-C and BMS-D,were named PGR-1, PGR-2, and PGR-3 (plastid gen-ome remnants; see Discussion).

Plastid gene expression is affected in all BMS cell lines

Considering the extent of the deletions in the BMS-Ggenome, we wondered whether any of the gene expres-sion machinery of this plastid was still operational andwhether any of the remaining genes were expressed. Totest this, we probed a dot blot array identical to thoseseen in Fig. 1 with radioactively labeled cDNA madefrom total BMS-G RNA. Unfortunately, the sensitivityof this assay was extremely limited and only highlyabundant rRNAs were detectable (data not shown). Atotal absence of transcription would have been anunprecedented result, suggesting the unlikely possibility(Race et al. 1999) that the plastid genome was dispens-able. We therefore used RNA gel blot analysis, a moresensitive technique, although one is realistically re-stricted to testing a smaller suite of genes.

Selected genes were tested for expression, as shown inFig. 5. Among the rRNAs, which are normally the mosthighly accumulating transcripts, 4.5S, 23S, and 16S wereundetectable in BMS-C and BMS-G lines. The smallamount of 4.5S and 23S hybridization seen in the BMS-D lanes shows a severe reduction or lack of transcript

processing. As examples of photosynthetic genes, psbDand petD were used. No RNA was detected in any BMSline for psbD, not an unexpected result for a gene with alight-regulated promoter (Christopher et al. 1992).In contrast, RNA for petD was detected in BMS-D.Indeed, petD shows residual transcription in tobaccoplants lacking PEP (Hajdukiewicz et al. 1997); and analtered mRNA pattern accumulates in a wide variety ofalbino or photosynthesis-deficient maize mutants(Barkan et al. 1986; Han et al. 1993).

The gene encoding a plastid protease subunit, clpP, hasbeen shown to be actively transcribed in tissue culturelines and plants that lack plastid ribosomes (Vera andSugiura 1995; Silhavy andMaliga 1998a; Zubko andDay2002). Since BMS-D apparently lacks ribosomes and,unlike the other two BMS lines, carries the clpP gene, wewondered whether it would be transcribed. Figure 5shows this is indeed the case and, since clpP is transcribedby the NEP (Silhavy and Maliga 1998b), the presence ofclpP mRNA in BMS-D suggests an active NEP.

Because clpP and petD genes are missing from theBMS-C and BMS-G cell lines, it was necessary to testfor the presence of other known NEP transcripts, suchas atpA, rpl2, and rps15. The atpA gene is retained in theBMS-D and BMS-C plastid genomes but not in BMS-G.In A188, we observed the same transcripts as describedby Stahl et al (1993). In BMS-D and BMS-C, however,the 4.9-kb species is missing, which retains the atpFintron. A band of this size was also missing whentobacco BY-2 cell RNA was probed with the atpF intron(Miyagi et al. 1998).

Fig. 4 Summary of plastidgenome analysis. Each block onthe outermost circle representsone gene in a complete maizechloroplast genome. Whiteblocks represent genes that weretested by either dot-blot orDNA gel blot analyses. Blackblocks mark genes that were notdirectly tested but are assumedto be present, based upon thepresence or absence ofneighboring genes. Genes<100 bp in length appear asblack lines, due the scale of themap. The two lines withseverely reduced genomes,BMS-C and BMS-G, arerepresented by the middle lightgray circle and the innermostcircle of genes, respectively. Theshort and long single-copyregions are denoted as SSC andLSC, respectively. The invertedrepeat regions are labeled asIRA and IRB. Three regions thatare conserved among the BMSgenomes and other publishedreduced plastid genomes(plastid genome remnants) arelabeled as PGR-1, PGR-2, andPGR-3

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rpl2 and rps15 encode ribosomal proteins, they arethe only two NEP-transcribed protein-coding genespresent in all three BMS cell lines, and their expressionpatterns have been characterized in albino maize leaves(Zubko and Day 2002). In A188 and BMS-D, rpl2mRNA is present in similar amounts. However, rps15mRNA is reduced in BMS-D but there is a relativelylarge amount of a 1.4-kb putative processing interme-diate. The BMS-C line shows a single 2.6-kb rpl2 tran-script, perhaps due to loss of the usual neighboringpromoters and genes. rps15 mRNA was undetectable inBMS-C. Surprisingly, rpl2 and rps15 mRNAs wereundetectable in BMS-G cells.

One gene expected to be expressed in all the lines wasthe tRNAGlu gene, trnE. tRNAGlu is required for tet-rapyrrole biosynthesis in plants (Schon et al. 1986;Grimm 1998) and in rice anther cell lines; and its pres-ervation in otherwise reduced plastid genomes is equatedwith its importance and assumed expression (Haradaet al. 1992). Earlier studies of trnE expression in non-photosynthetic plastids suggested that tRNAGlu wasgreatly reduced or perhaps even missing (Hess et al.

1992; Zubko and Day 2002). Similarly, our RNA gelblot analysis of trnE expression in BMS cells suggestedthat trnE was not expressed (data not shown). Becausethis result seemed improbable, we turned to the morequantitative and sensitive technique of primer extension,as shown in Fig. 6. These data clearly show that, in fact,trnE is expressed in the non-photosynthetic BMS celllines at levels comparable with wild-type photosynthetictissue, arguing that prior reports of low trnE expressionmay warrant reconsideration.

Mitochondrial transcript levels may be increasedin BMS cell lines

Considering the importance of mitochondrial function tocell viability, we expected that mitochondrial geneexpression would be robust in BMS cells. To test thisassumption, we checked the abundance of three mito-chondrial transcripts, as shown in Fig. 7. The same RNAgel blots used to check plastid transcription (Fig. 5) wereallowed to decay and were reprobed for atp6 (ATP syn-thase subunit VI), cob (cytochrome b), and cox1 (cyto-chrome c oxidase subunit I) transcripts. The transcriptswere present in the three BMS cell lines and were of theexpected sizes (Isaac et al. 1985; Wen and Chase 1999).However, there was a considerable difference in theabundance of transcripts between A188 and BMS mito-chondria, even taking into account the perhaps slightunderloading of A188 RNA (Fig. 5). For atp6, we esti-mate 8·, 3·, and 11· increases of the transcript in BMS-D, BMS-C, and BMS-G, respectively. The correspond-ing figures for cobwere 12·, 3·, and 9·, and for cox1 14·,3·, and 22·. These data suggest that BMS cells have anincreased mitochondrial transcription and/or a lowermRNA turnover rate, but we cannot exclude that part of

Fig. 5 RNA gel blot analysis of plastid genes. Total RNA (5 lg)was isolated from A188 leaf tissue (A) and log-phase BMS cells (D,C, G), separated in denaturing gels, and blotted onto Nylonmembranes. The gene-specific probes shown at the left of each panelwere radioactively labeled using random hexamers. rRNAs werevisualized by ethidium bromide staining. Known or deducedtranscript sizes, marked at right, were derived by comparison toan RNA ladder

Fig. 6 tRNAGlu primer extension analysis. Total RNAs from A188leaf tissue (A) and log-phase BMS cells (D, C, G) were denatured byheating and then quickly cooled prior to annealing an antisenseprimer to tRNAGlu. The trnE sense strand is underlined and theDNA primer is represented as an arrow to the right of the gel

image. Avian myeloblastosis virus reverse transcriptase and

a32P-dCTP were included, so that up to four radioactive nucleo-tides could be added. Bands denoted with an asterisk aredegradation products where nucleotides were lost from the 3¢ endof the primer

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the increase results from a higher number of mitochon-dria per cell, thus increasing overall mitochondrialtranscript abundance on a total RNA basis.

Photosynthetic proteins can be importedinto BMS plastids

Since one of our goals is to use BMS cells to expressepitope-tagged plastid- and/or mitochondrion-targetedproteins, we chose to test its feasibility by transfectingBMS cells with plasmids encoding GFP fused to knownplastid or mitochondrial transit peptides (Chang et al.1999). We chose the BMS-G line, since it had thesmallest genome and presumably the least gene expres-sion, with the assumption that this line represented themost defective plastid.

Transient expression in BMS-G cells was carried outas described in Materials and methods. Cells werestained with Mitotracker to locate mitochondria andGFP fluorescence was observed as early as 16 h afterbombardment, using confocal laser scanning micros-copy. As seen in Fig. 8, GFP fused to the transit peptideof the maize RpoTm localized to punctuate areas withincells. A merge of this image with the Mitotracker redchannel image indicates that the stain and the GFPlocalized to the same areas. Mitotracker also stainedgranules which appeared to coincide primarily with thevacuole. GFP fused to the transit peptide of the smallsubunit of RbcS localized to distinct oval organelles inGFP-expressing cells. When this image was overlaidwith the Mitotracker red channel, it was evident that theGFP and Mitotracker localized to different organelles,

consistent with a plastid localization and similar toresults obtained in tobacco tissue culture BY-2 cells(Mitsuhashi et al. 2000). The lower row of Fig. 8 dem-onstrates that GFP lacking transit peptide remains in thecytosol. Taken together, these experiments suggest thatboth mitochondrial and plastid protein import operatenormally in BMS cells.

Discussion

Suspension culture cells are very useful for the study ofplant cell biochemistry and physiology. Of particularinterest to us is their proven usefulness in the study ofplastid gene expression (Han et al. 1992, 1993; Hess et al.1993; Vera and Sugiura 1995; Allison et al. 1996; Veraet al. 1996; Kapoor et al. 1997). Our desire to find asuitable maize model for mechanistic studies of plastidgene expression led us to the BMS suspension culture(Chourey 1981; Sheridan 1982).

Here, we present detailed maps of severely reducedplastid genomes from an immortalized plant cell line anddemonstrate the inconsistencies of gene loss in threeBMS cell lines of different origins. We also provideevidence that transcription is limited but not completelylost in the lines and that the plastids are intact and stillcapable of importing proteins from the cytoplasm. Sincemitochondrial gene expression is rather robust, BMScells may be of even greater use for the study of proteinsencoded in the nucleus and imported into mitochondria.Plastid-targeted proteins whose functions depend on thepresence of certain plastid genes or mRNAs must,

Fig. 7 RNA gel blot analysis of mitochondrial genes. The RNA gelblots shown in Fig. 5 were allowed to decay and were then probedwith mitochondrial gene-specific probes as indicated

Fig. 8 Subcellular localization of green fluorescent protein (GFP)in BMS cells. Log phase BMS cells were transfected by particlebombardment with GFP linked to the mitochondrion-specific T7-like RNA polymerase (RpoTm) and the plastid-specific ribulosebisphosphate carboxylase (RbcS) transit peptides. At 16 h aftertransfection, cells were stained with Mitotracker dye and fluores-cence was visualized using confocal laser scanning microscopy. Theleft images show Mitotracker fluorescence, the right images showGFP fluorescence, and the middle images are the left and rightimages merged together

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however, be considered on a case-by-case basis. ForNEP-transcribed genes, BMS-D would appear to be themost suitable system.

Some BMS chloroplast genomes have large deletions

Using a combination of dot blot array and DNA gel blotanalysis, we determined that the BMS-G and BMS-Clines have lost up to 70% and 40% of their plastid ge-nomes, respectively. BMS-D appears to have a fullcomplement of genes, but does have polymorphisms,which suggests there are minor deletions or rearrange-ments. Plastid genome loss has been observed previouslyin anther and somatic cell-derived rice and barley cul-tures (Ellis and Day 1986; Harada et al. 1991; Kawataet al. 1995) from which albino plantlets can be derived.Where proteins required for chlorophyll-binding aredeleted (e.g. PSI or PSII reaction centers), the deletionsand/or a lack of plastid translation are likely to beresponsible, although a lack of photosynthesis in and ofitself does not necessarily cause a loss of pigmentation inplant cells. Reduced chloroplast genomes can also befound in the natural world, for example the non-pho-tosynthetic parasitic plant Epifagus virginiana, which hasa 70-kb genome (dePamphilis and Palmer 1990; Wolfeet al. 1992), and pathogenic apicomplexan species with35-kb plastid-like genomes (Wilson et al. 1996; Marechaland Cesbron-Delauw 2001).

A comparison of previously published reduced chlo-roplast genomes and the BMS lines indicates that atleast three of the regions retained in BMS-C and BMS-Gare conserved (PGR-1, PGR-2, PGR-3 in Fig. 4). Of thethree, PGR-1 appears to be universally conserved and isfound in the BMS lines, Epifagus, the apicoplexanPlasmodium faciparum, and 15 pollen and somaticallyderived rice cultures (Kawata et al. 1995; Wilson et al.1996; Marechal and Cesbron-Delauw 2001). Kawataet al. (1995) suggested this region may contain regionsessential for DNA replication and/or essential tRNAgenes; and the latter is consistent with the presence oftrnE. PGR-2 and PGR-3 are retained in the BMS cells,Epifagus, and P. falciparum, but not the rice lines(Harada et al. 1992). While PGR-2 and PGR-3 containrRNA or protein genes, one cannot postulate anyobvious function as coding regions, given the othermissing genes in the same functional categories and thefact that the 23S rRNA appears to be unprocessed(Fig. 5). Accumulation of rRNA precursors in maizechloroplasts is associated with defects in polysomeassembly (Barkan 1993), but is not necessarily associ-ated with albinism (Han et al. 1993).

Why have these cell lines lost much of their plastidgenomes? The losses seen with tissue culture lines hap-pen very quickly in comparison with gene migration onan evolutionary scale, following endosymbiosis, perhapsbecause the genes lost are functionally unimportant.Although the majority of the published data fail to rejectthis hypothesis, we present BMS-D as an exception,

because it has been in culture for close to 28 years(C. Donovan, personal communication) and hasretained its plastid genome. One of the distinguishingaspects of the BMS-C and BMS-G plastid genomes istheir lack of IRs. Palmer and Thompson (1982)hypothesized that loss of the IR allows an increasedfrequency of rearrangement, although this principle hasnot proved to be universal (Palmer et al. 1987). Perhapsin BMS-C and BMS-G, early damage to the IR regionsled to an eventual loss of much of the rest of the genome.It is worth noting that naturally occurring (and thusstable) reduced plastid genomes have intact IRs (Wolfeet al. 1992; Wilson et al. 1996).

In the process of becoming immortalized, these tissueculture cells were also likely to accumulate nuclearmutations. There is evidence for a nuclear mutation inDrosophila subobscura which results in an increase inmitochondrial DNA rearrangements and losses (Le Goffet al. 2002). It is certainly possible that a mutation in oneor more nuclear genes in the BMS-C and BMS-G linesled to the partial disintegration of their cpDNAs. Forexample in evening primrose, the nuclear plastomemutator locus controls correct replication of cpDNA(Chang et al. 1996).

BMS plastid gene expression

Our results using RNA gel blots and primer extensionfor tRNAGlu suggest that the majority of the remainingplastid genes in BMS-C and BMS-G do not give rise tostable transcripts and that BMS-D, although it has acomplete plastid genome, also does not transcribe themajority of its genome. Interestingly, even thoughrRNA genes are preserved in all the lines, they appar-ently are not expressed (or are improperly processed,such as 23S rRNA in BMS-D), suggesting that mRNAswhich are transcribed cannot be translated. The lack ofactive translation means that the PEP is not present andtherefore the few plastid RNAs in BMS cells must betranscribed by a NEP (Chang et al. 1999; Ikeda andGray 1999). In four cases (atpA, petD, rpl2, rps15),transcripts found in wild-type leaf chloroplasts aremissing in BMS. This could be due to improper RNAprocessing, suggesting that not all nuclear-encodedRNA-processing enzymes are present in these cells. It isalso possible that certain transcripts are absent becausethey initiate at PEP promoters.

The closest parallel to BMS in terms of plastid geneexpression in a monocot system is perhaps anther-derived barley plants. Dunford and Walden (1991)found that albino plants with large plastid DNA dele-tions failed to express 23S and 16S rRNAs and hadgreatly reduced transcript abundance among geneswhich were transcribed. Other examples can be found inthe studies of ribosome-deficient maize (iojap) and bar-ley (albostrians) lines (Walbot and Coe 1979; Hess et al.1993) Like the BMS cells described in this paper, albinoplants and cell lines apparently lack plastid ribosomes

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and also do not properly process the few transcripts thatthey do produce (Han et al. 1993; Hess et al. 1993, 1994;Zubko and Day 2002).

Directed rpoA knockout mutants in tobacco whichspecifically lack PEP (Allison et al. 1996; Legen et al.2002) have been used to delineate the roles of the twoplastid RNA polymerases in dicots. Data collected fromthis system suggest that the NEP transcribes only a fewplastid genes (clpP, rpoB, rpl2, rpl23, rps15) to greatabundance (Allison et al. 1996; Silhavy and Maliga1998a; Zubko and Day 2002) and yet transcribes allplastid genes to some degree (Legen et al. 2002). Theseresults suggest a level of complexity in dicot systems,which carry more than one NEP (Hedtke et al. 2000),that may not exist in monocots.

BMS plastids import proteins

Using GFP linked to plastid- and mitochondrion-spe-cific transit peptides, we demonstrated that BMS cellshave intact plastids and mitochondria that are able toimport proteins from the cytoplasm. Non-photosyn-thetic plastids are the site of starch metabolism, nitratereduction, fatty acid synthesis, and porphyrin synthesis(Emes and Neuhaus 1997). All of these processes requireproteins and cofactors to be imported in and out of theplastid. Our data suggest that, although the plastidgenome and its expression may be greatly reduced, theplastids in these cells are still fulfilling essential bio-chemical functions.

Acknowledgements The authors wish to thank Erich Grotewold ofthe Ohio State University, Prem Chourey of the USDA/Universityof Florida, and Chris Donovan of the University of Minnesota forsupplying the BMS-G, BMS-C, and BMS-D cell lines, respectively.We also thank Alice Barkan of the University of Oregon andRuairidh Sawers and Tom Brutnell of the Boyce ThompsonInstitute for providing the chloroplast DNA clones used in the dotblot analyses. Carol Bayles of the Cornell University MIF facilityprovided confocal microscope support. Finally, we thank membersof the Stern laboratory, the maize community, and G. HectorRodriguez for helpful discussions. A.B.C. is supported by UnitedStates Department of Agriculture–National Research Initiativepostdoctoral fellowship 2001-35301-10845. Work was also sup-ported by National Science Foundation award 0090658 to D.B.S.

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