INTRODUCTION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plastids of higher plants have evolved from a cyanobacterium-like ancestor. The 120- to 160-kb circular genome encodes about 120 genes that are subunits of PSI and PSII, Rubisco, ATPase, cytochrome b₆/f, and NDH complex and some genes required for the organelle's maintenance (Ohyama et al., 1986; Shinozaki et al., 1986). Most of the 2,000 to 3,500 genes required for plastid function are encoded by the nucleus (Abdallah et al., 2000; Arabidopsis Genome Initiative, 2000; Emanuelsson et al., 2000). Some of these nuclear genes regulate plastid functions.

Nuclear genes controlling plastid gene expression have been identified in mutant screens and by biochemical approaches. Most of the genes identified in mutant screens affect posttranscriptional processes, indicating the importance of posttranscriptional regulation in plastid gene expression. In Chlamydomonas reinhardtii, there is a large number of nuclear genes dedicated to regulating the expression of a single plastid gene (for review, see Barkan and Goldschmidt-Clermont, 2000). The genes cloned thus far are involved in mRNA translation, turnover (Boudreau et al., 2000; Vaistij et al., 2000), and trans-splicing (Perron et al., 1999; Rivier et al., 2001). In higher plants, biochemical approaches lead to the identification of nuclear genes that encode general components of the plastid RNA metabolism and are involved in mRNA processing and turnover (Schuster and Gruissem, 1991; Hayes et al., 1996; Yang et al., 1996). Furthermore, genetic screens in maize (Zea mays) and Arabidopsis lead to the identification of nuclear genes regulating the expression of one or more plastid genes at the posttranscriptional level (Brutnell et al., 1999; Fisk et al., 1999; Jenkins and Barkan, 2001; Meurer et al., 1998; Till et al., 2001; for review see; Barkan and Goldschmidt-Clermont, 2000). We report here on a plastid clpP-specific mRNA maturation factor discovered through expression of a clpP segment in a chimeric context.

To compare the translation efficiency of chimeric mRNAs, we tested accumulation of neomycin phosphotransferase (NPTII) from neo reporter genes in tobacco (Nicotiana tabacum) chloroplasts. The neo transgenes were transcribed from the Prrn promoter that normally drives transcription of the plastid ribosomal RNA (rrn) operon (Vera and Sugiura, 1995; Allison et al., 1996; Sriraman et al., 1998). In earlier studies, the Prrn promoter was fused with the 5'-untranslated region (UTR) and sequences downstream of the AUG of the plastid atpB and rbcL genes (Kuroda and Maliga, 2001b) and of the T7 phage gene 10 (Kuroda and Maliga, 2001a). Here, we report on tobacco plants expressing NPTII in chloroplasts from the Prrn promoter with the clpP 5'-UTR. The plastid clpP gene encodes the ClpP1 protein (Adam et al., 2001). Unlike in the earlier studies, tobacco plants carrying the neo transgene have a chlorotic phenotype. The mutant phenotype was linked to reduced (approximately 2 times) levels of the plastid ClpP1 protease subunit and overexpression of clpP 5'-UTR in the chimeric transcript. In the transgenic plants, intron-containing clpP transcripts accumulate at the expense of monocistronic mRNAs, suggesting competition for an mRNA maturation factor. Because maturation of 11 other intron-containing mRNAs was unaffected in mutant leaves, it appears that the factor is specific for clpP.

	RESULTS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Expression of the clpP 5'-UTR in a Chimeric mRNA Causes a Pigment-Deficient Phenotype

We have prepared two chimeric Prrn constructs, one with the clpP 5'-UTR (pHK33) and one with the atpB 5'-UTR (pHK31; Fig. 1A) driving the expression of NPTII encoded in the neo gene. The tobacco clpP gene is transcribed from the PclpP-53 and PclpP-173 nuclear-encoded plastid RNA polymerase and the PClpP-95 plastid-encoded plastid RNA polymerase promoters (Hajdukiewicz et al., 1997). Prrn was fused with the 5'-UTR of the PclpP-53 promoter. The pHK31 atpB fusion is different from the earlier atpB constructs (Kuroda and Maliga, 2001b) because it does not contain sequences downstream of the AUG. In vectors pHK31 and pHK33, the reporter gene is linked to a selectable spectinomycin resistance (aadA) gene (Fig. 1B). Transforming DNA was introduced into chloroplasts by the biolistic process; uniform transformation of plastid genomes was confirmed by DNA gel-blot analysis (data not shown). We obtained multiple independently transformed lines after transformation with both plasmids. Plants representing independently transformed lines with the same plasmid have identical phenotypes. In the paper, we have included data for one line of each, Nt-pHK31-1C and Nt-pHK33-2A, transformed with plasmids pHK31 and pHK33, respectively. These plants will be referred to as Nt-pHK31 and Nt-pHK33 plants.

View larger version (32K): [in this window] [in a new window]   Figure 1.   Vectors for insertion of chimeric neo genes into the tobacco plastid genome. A, DNA sequence of the Prrn with the atpB and clpP 5'-UTRs. The Prrn promoter sequence is underlined. The transcription initiation site is marked by a horizontal arrow. The translational initiation codon (ATG) is in bold. B, Targeting region of the pPRV111B vector derivatives. Shown are the relative positions of selectable spectinomycin resistance (aadA) and neo passenger genes, flanked by plastid DNA encoding rrn16, trnV, and rps12/7 (Shinozaki et al., 1986). The neo gene is expressed from the Prrn promoter; the rbcL 3'-UTR (TrbcL) stabilizes the mRNA. Wavy lines represent neo and neo-aadA transcripts. Abbreviation of restriction sites: E, EcoRI; B, BglII; H, HindIII; N, NheI; S, SacI; and X, XbaI. Restriction sites removed during plasmid construction in parenthesis. A map of the promoter regions is shown below.

Nt-pHK33 plants, in which the neo gene is expressed from Prrn with the clpP 5'-UTR (PrrnLclpP), have a distinct mutant phenotype. The young, fast-growing leaves of greenhouse-grown plants are lighter green in color (chlorotic), whereas the older leaves are normal green (Fig. 2A). The young leaves accumulate approximately 0.26%, whereas mature leaves only approximately 0.14% of the total soluble leaf protein (TSP) as NPTII (Fig. 3A). The cotyledons of seedlings germinated in sterile culture are also pale green (Fig. 2B). The chlorotic phenotype of Nt-pHK33 plants is not due to NPTII accumulation per se because the Nt-pHK31 leaves contain higher concentrations of NPTII, approximately 0.8% in young and approximately 1.1% in mature leaves (Fig. 3A), in the absence of any recognizable mutant phenotype (Fig. 2). NPTII accumulated to even higher levels, 11% and 23% of TSP, in plants transformed with other constructs, confirming that NPTII accumulation is not detrimental to plants (Kuroda and Maliga, 2001a, 2001b).

View larger version (110K): [in this window] [in a new window]   Figure 2.   Overexpression of neo gene containing clpP 5'-UTR causes pigment deficiency. A, Mutant phenotype of young leaves of greenhouse plants. Shown are Nt-pHK33 (PrrnLclpP::neo::TrbcL gene), Nt-pHK31 (PrrnLatpB::neo::TrbcL gene), and wild-type, nontransformed (Nt-wt) tobacco plants. B, Pigment deficiency of seedling cotyledons of Nt-pHK33 plants.

View larger version (33K): [in this window] [in a new window]   Figure 3.   Accumulation of NPTII and ClpP1 in transgenic tobacco leaves. Lanes for plant lines are designated with transforming plasmid; Wt, wild-type tobacco sample. Twenty micrograms of TSP was loaded per lane. A, NPTII as percent TSP. NPTII dilution series is shown for reference. Values are an average of four experiments. Y and M refer to samples from young and mature leaves, respectively. B, Amount of ClpP1 protease subunit relative to wild-type plants (100%). Calculation was based on a dilution series of the wild-type extract. Values are an average of three experiments.

Reduced ClpP1 Accumulation in Pigment-Deficient Leaves

Because pigment deficiency was observed only in plants expressing neo from Prrn with the clpP 5'-UTR, we measured the accumulation of the ClpP1 protein in the Nt-pHK33 plants. Samples were taken from the young, pigment-deficient leaves and the normal green, mature leaves of the same plants. Immunoblot analysis revealed that the young Nt-pHK33 leaf sample contained about one-half as much ClpP1 protein as the wild-type and transgenic Nt-pHK31 (neo expressed from Prrn with atpB 5'-UTR) plants (Fig. 3B). Although the ClpP1 level in the mature Nt-pHK33 leaves was lower than in the wild-type leaves, the mature Nt-pHK33 leaves were normal green. Thus, pigment deficiency in young leaves could be linked to reduced ClpP1 levels.

Reduced Steady-State Levels of Monocistronic clpP mRNA in the Pigment-Deficient Transgenic Leaves

RNA gel-blot analysis was carried out to determine if changes in the relative clpP transcript abundance could be linked to reduced ClpP1 protein levels and thus to the mutant phenotype. In tobacco leaves, the clpP gene is transcribed from two major promoters, with transcription initiation sites 95 and 53 nucleotides upstream of the translation initiation codon (Hajdukiewicz et al., 1997; Fig. 4). Downstream of the clpP gene are rps12 exon 1 (rps12 Ex1) see for example and rpl20. Because the clpP (5'-UTR; Ex1, Ex2, Ex3, In1, and In2), rps12 (Ex1), and rpl20 probes detected a 4-kb pre-mRNA (transcript a in Fig. 4; Fig. 5), the clpP-rps12 Ex1-rpl20 region seems to be cotranscribed as previously described (Sugita and Sugiura, 1996). In an earlier study, a transcript 5' end was mapped between clpP Ex3 and rps12 Ex1 at nucleotide position 72,386 (GenBank accession no. Z00044; Hildebrand et al., 1988). At present, it is not known whether this 5' end is a processed 5' end or a primary transcript. The transcript analysis is complicated by trans-splicing of rps12 Ex1 and rps12 Ex2, the latter being the first gene segment in a complex transcription unit (rps12 Ex2-ndhB region, 5.1-kb transcript l in Figs. 4 and 5; Koller et al., 1987; Zaita et al., 1987; Hildebrand et al., 1988). Given the large number of similar size transcripts derived from the two operons and from trans-splicing, a complete transcript analysis is beyond the scope of this paper. Transcripts that were identified are marked by letters in Figures 4 and 5.

View larger version (21K): [in this window] [in a new window]   Figure 4.   The clpP and rps12 Ex2 operon maps. The clpP gene in leaves is transcribed from a plastid-encoded plastid RNA polymerase (95, black circle) and a nuclear-encoded plastid RNA polymerase (53, white circle) promoter. The clpP and rps12 Ex2 operon are located in the large single-copy and repeated regions of the plastid genome, respectively (insert). Primary transcripts (a and l) and some of the processed (c, d, e, and i, j, k) and trans-spliced (b and g) products are depicted between the operon maps.

View larger version (88K): [in this window] [in a new window]   Figure 5.   RNA gel-blot analysis of clpP-containing transcripts. A, Blots probed for exons and introns of clpP and rps12/7 operons. Probes are listed above gels. For map position, see Figure 4. Leaf samples were tested from Nt-pHK33, Nt-pHK31, and wild-type (Wt) control plants. Y and M refer to samples from young and mature leaves, respectively. B, Individual transcripts are marked for young leaves of Nt-pHK33 and control Nt-pHK31 plants for the blots shown in Figure 5A. For size and map position of transcripts, consult Figure 4.

Probing with the clpP gene segments revealed that the pigment-deficient young Nt-pHK33 leaves contain reduced amounts (25%) of the fully spliced 0.8-kb monocistronic clpP mRNA (transcript e, Figs. 4 and 5). In addition, accumulation of an aberrant clpP pre-mRNA species lacking Ex1, designated transcript h, was identified in the mutant Nt-pHK33 leaves (transcripts d and h are similar in size and are not separated in Fig. 5B) but not in wild-type and Nt-pHK31 plants. Transcript h could be a splicing intermediate consisting of clpP In1, Ex2, and Ex3. This would suggest that reduced clpP level is due to reduced levels of the mature 0.8-kb transcript caused by competition between the clpP 5'-UTR expressed as part of the chimeric neo mRNA (16-fold wild-type level; Fig. 5A, clpP 5'-UTR probe) and the endogenous clpP pre-mRNA for an mRNA maturation factor.

RNA gel-blot analysis was carried out to test mRNA maturation for additional, intron-containing genes, using probes for rps12, ndhB (Fig. 5), atpF, petB, petD, ycf3, rpoC1, rps16, rpl2, rpl16, and ndhA (Fig. 6). If there were changes, the levels of processed mRNAs were higher (rpoC1, rps16, rpl16) rather than lower as compared with wild-type and Nt-pHK31 plants. Thus, it appears that 16-fold overexpression of the clpP 5'-UTR in a chimeric context affects maturation of only the clpP mRNA.

View larger version (69K): [in this window] [in a new window]   Figure 6.   Probing for transcript patterns of intron-containing plastid genes in samples of Nt-pHK33, Nt-pHK31, and wild-type (Wt) controls. Probes are listed above gels. Black and white triangles represent monocistronic and polycistronic transcripts, respectively. A short (S) and long (L) exposure are shown for the blot probed with rpoC1 Ex1. Y and M refer to samples from young and mature leaves, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

We report here that a 16-fold overexpression of clpP 5'-UTR in a chimeric context reduces the accumulation of the plastid-encoded ClpP1 subunit by 2-fold, the likely reason for the mutant phenotype. The mutant phenotype is most severe in fast-growing seedlings or in young leaves, and normalizes in older leaves (Fig. 2). The plastids in young Nt-pHK33 leaves contain 4 times less monocistronic clpP mRNA (0.8-kb transcript e in Figs. 4 and 5) than in wild-type leaves. Steady-state levels of other mRNAs containing the processed clpP coding region, such as transcript b, have also been reduced (Figs. 4 and 5). Thus, reduced levels of ClpP1 appear to be due to reduced levels of processed clpP mRNA.

There is one aberrant transcript that gives some insight into the function affected in the Nt-pHK33 plants. Wild-type and Nt-pHK31 plants accumulate transcript c, which lacks In2 but contains In1. This RNA species is reduced in Nt-pHK33 plants that instead accumulate transcript h, an RNA species lacking clpP Ex1 (Figs. 4 and 5). Thus, we speculate that overexpression of the clpP 5'-UTR interferes with splicing of In1 and causes degradation of Ex1, implicating the lack of a factor involved in clpP mRNA maturation.

The clpP gene In1 has been classified as a group II subgroup IIB1 intron (Michel et al., 1989). RNA gel-blot analysis was carried out for the other mRNAs in subgroup IIB1 (petB, petD, rps16, rpoC1, and ycf3 In2) and for mRNAs with introns that belong to other subgroups (atpF, ndhA, ndhB, rps12, rpl2, rpl16, and ycf3 In1; Michel et al., 1989). We found no obvious major reduction in the steady-state levels of processed mRNAs for any of these genes, suggesting that overexpression of clpP 5'-UTR does not affect the maturation of transcripts other than that of clpP. The gene whose function is affected by overexpression of the clpP 5'-UTR remains to be identified.

The plastid-encoded ClpP1 protein is part of a 350-kD ClpP1 protease complex with 10 different isoforms in Arabidopsis (Peltier et al., 2001). The plastid-encoded subunit appears to be essential for viability because attempts to delete it from the plastid genome have failed in both C. reinhardtii (Huang et al., 1994) and tobacco (Shikanai et al., 2001). Although deletion of clpP gene copies was incomplete, reduction of the plastid clpP gene copy number affected both plastid ultrastructure and leaf development. Our results support an essential role for the ClpP1 protein. A mutant phenotype caused by a 2-fold reduction in ClpP1 levels indicates that the plastid-encoded subunit (clpP gene product) is important in young leaves for normal chloroplast development. In contrast to tobacco, reduced ClpP1 accumulation (25%-40%) in the chloroplasts of the unicellular alga C. reinhardtii had no affect on the rate of growth (Majeran et al., 2000). The transplastomic Nt-pHK33 plants described here will be useful to study ClpP1 function in chloroplasts.

	MATERIALS AND METHODS

TOP ABSTRACT INTRODUCTION RESULTS DISCUSSION MATERIALS AND METHODS LITERATURE CITED

Plasmid Construction

The chimeric Prrn/5'-UTR sequences are contained within SacI-NheI fragments. PrrnLatpB (Prrn promoter with atpB 5'-UTR) is contained in plasmid pHK11 (a pUC118 plasmid derivative). PrrnLclpP (Prrn promoter with clpP 5'-UTR) is found in plasmid pHK13 (pUC118 derivative). The promoter fragments were constructed by PCR. The DNA sequence is shown in Figure 1A. Construction details are available upon request. The chimeric Prrn/5'-UTR derivatives were translationally fused with the neo coding region via an engineered NheI site. The engineered neo gene derives from plasmid pSC1, and was obtained by inserting the NheI restriction site (GCTAGC) between the ATG and the first codon (ATT) of the neo coding region (Chaudhuri and Maliga, 1996). The neo genes have the plastid rbcL gene 3'-UTR (TrbcL) to stabilize the mRNAs (Staub and Maliga, 1994). Plastid vectors pHK31 and pHK33 were obtained by cloning the neo gene from plasmids pHK11 and pHK13 as a SacI-HindIII fragment into plastid vector pPRV111B (Zoubenko et al., 1994). The map of the targeting region of the plastid transformation vectors is shown in Figure 1B.

Plastid Transformation and Regeneration of Transgenic Plants

Transforming DNA was introduced into tobacco (Nicotiana tabacum cv Petit Havana) leaves on the surface of tungsten particles (1 µm) using the PDS1000He biolistic gun (DuPont, Wilmington, DE). Transplastomic plants were selected on RMOP medium containing 500 mg L¹ spectinomycin dihydrochloride. A uniform population of transformed plastid genome copies in the regenerated shoots was confirmed by DNA gel-blot analysis (Svab and Maliga, 1993). The transgenic plants were rooted on Murashige and Skoog medium (Murashige and Skoog, 1962) containing 3% (w/v) Suc and 0.6% (w/v) agar, and then transferred to the greenhouse. Seedlings were obtained in sterile culture by germinating surface-sterilized seeds on the same medium.

RNA Gel-Blot Analysis

Total cellular RNA was prepared from the leaves of plants grown in the greenhouse (Stiekema et al., 1988). RNA (4 µg lane¹) was separated on 1.0% (w/v) agarose/formaldehyde gel, and then transferred onto Hybond N membranes (Amersham, Piscataway, NJ) using the PosiBlot Transfer apparatus (Stratagene, La Jolla, CA). Hybridization to the probe was carried out in Rapid Hybridization buffer (Amersham) overnight at 50°C with the clpP 5'-UTR probe or at 65°C with other probes. The template for probing neo was a gel-purified NheI-XbaI fragment excised from plasmid pHK30. Single-stranded ³²P-labeled plastid gene probes were prepared by primer extension with the Klenow fragment using one-oligonucleotide (-³²P) dATP and a double-stranded DNA template prepared by PCR. The following regions of the tobacco plastid genome (GenBank accession no. Z00044; Shinozaki et al., 1986; Wakasugi et al., 1998) were included in the probes: clpP 5'-UTR, 74560-74502; clpP Ex1, 74507-74437; clpP In1, 74220-73921; clpP Ex2, 73595-73338; clpP In2, 73200-72910; clpP Ex3, 72700-72473; rps12 Ex1, 72334-72221; rps12 Ex2, 100854-100623; rpl20, 71261-71033; atpF Ex2, 12463-12220; ycf3 Ex2, 45402-45175; petB Ex2, 78221-78470; petD Ex2, 79803-80005; rps16 Ex2, 5306-5097; rpl16 Ex2, 84036-83724; rpl2 Ex1, 88218-87931; rpoC1 Ex1, 24280-24057; ndhA Ex1, 123870-123607; and ndhB Ex1, 143365-143595. RNA hybridization signals were quantified using a PhosphorImager (Molecular Dynamics, Sunnyvale, CA).

SDS-PAGE and Immunoblot Analysis

SDS-PAGE and immunoblot analysis were carried out as described previously (Kuroda and Maliga, 2001b). Antibody to detect the tobacco ClpP1 protein was kindly provided by Dr. Zach Adam (Hebrew University, Jerusalem). ClpP1 was detected with the SuperSignal West Dura Extended Duration Substrate (Pierce, Rockford, IL) and NPTII was detected with the ECL Western Blotting Detection System (Amersham). The NPTII antibody was purchased from 5Prime  3Prime, Inc. (Boulder, CO).