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Nucleic Acids Res. Jan 2007; 35(2): 455–464.
Published online Dec 14, 2006. doi:  10.1093/nar/gkl1067
PMCID: PMC1802608

Nucleus-encoded plastid sigma factor SIG3 transcribes specifically the psbN gene in plastids

Abstract

We have investigated the function of one of the six plastid sigma-like transcription factors, sigma 3 (SIG3), by analysing two different Arabidopsis T-DNA insertion lines having disrupted SIG3 genes. Hybridization of wild-type and sig3 plant RNA to a plastid specific microarray revealed a strong reduction of the plastid psbN mRNA. The microarray result has been confirmed by northern blot analysis. The SIG3-specific promoter region has been localized on the DNA by primer extension and mRNA capping experiments. Results suggest tight regulation of psbN gene expression by a SIG3-PEP holoenzyme. The psbN gene is localized on the opposite strand of the psbB operon, between the psbT and psbH genes, and the SIG3-dependent psbN transcription produces antisense RNA to the psbT–psbH intergenic region. We show that this antisense RNA is not limited to the intergenic region, i.e. it does not terminate at the end of the psbN gene but extends as antisense transcript to cover the whole psbT coding region. Thus, by specific transcription initiation at the psbN gene promoter, SIG3-PEP holoenzyme could also influence the expression of the psbB operon by producing psbT antisense RNA.

INTRODUCTION

Plastids are semiautonomous plant organelles harbouring their own transcription system that originate from a cyanobacteria- and proteobacteria-like endosymbiote. The cyanobacteria-like ancestor of chloroplasts has contributed with a eubacteria-type RNA polymerase and the proteobacteria-like ancestor of mitochondria with a phage-type RNA polymerase to the transcriptional apparatus of higher plants (13). Transcription regulation of the plastid genome of higher plants is rather complex. In dicotyledon plants, two different phage type RNA polymerases (NEPs, nucleus encoded RNA polymerases, RPOTp and RPOTmp) and one eubacteria-type RNA polymerase (PEP, plastid encoded RNA polymerase) participate in the transcription of the ~120 genes that are encoded on the plastid genome. The PEP core enzymes is composed of four different subunits, α, β, β′ and β″, which are encoded on the plastid genome in two different transcription units. Genes coding the β, β′ and β″ subunits are arranged as operon analogous to the rif operon of Escherichia coli. The α subunit is encoded in a S10 or spe-like operon together with genes coding for ribosomal proteins [(4), reviewed in (5)].

The activity of the PEP core enzyme (α2, β, β′, β″) is regulated by sigma-like transcription factors (SLFs), which have at first been characterized by in vitro transcription assays (68). The first cDNA sequences coding plant nucleus-encoded SLFs have been described much later (915) and finally six different sigma factors, SIG1–SIG6, have been described for Arabidopsis thaliana (16). The mRNAs of these SLFs are translated in the cytoplasm and corresponding proteins are subsequently imported as precursor proteins into the plastids. The functions of all these sigma factors are not yet completely elucidated. Besides of specificity in the recognition of different promoter regions, SLFs are also differentially expressed during plant development and plastid differentiation (17,18). Transcription of most of the sigma factor coding genes is under light control, but tissue/organ specific expression and regulation by circadian rhythm have also been described previously (11,13,19). In addition, regulation of PEP activity by phosphorylation either of SLFs or RNA polymerase subunits has been described (20,21).

In general, it seems that SLFs have overlapping as well as specific functions (15,22,23). Although overlapping functions have been demonstrated by in vitro transcription assays that are performed without competition by other sigma factors, the specific functions are more easily detected by analyses that reflect in vivo competition conditions, i.e. by characterization of specific sigma knock-out plants. Most of the results concerning the specific functions of plant sigma factors have been obtained by analyses of Arabidopsis T-DNA insertion mutants. From these results it can be concluded that a SIG2-PEP holoenzyme transcribes specifically some of the tRNA genes (24) and the psaJ gene (25). SIG5 has been shown to play an important role in the recognition of the blue-light dependent promoter of the psbD gene (2628) and might in addition have specific functions during embryogenesis (29). SIG6 plays a more general role during early plastid differentiation and plant development (30), and SIG4 is of specific importance for ndhF gene transcription (31). The functions of SIG1 and SIG3 in vivo have not yet been described.

From results obtained by in vitro transcription assays, it is suggested that the activity of SIG1 might be regulated by its interaction with additional protein(s) (32) and that the activity of SIG3 might be regulated by proteolytic cleavage (15,22). In the present paper, we have analysed the plastid gene expression pattern of an Arabidopsis SIG3 T-DNA insertion mutant in order to characterize the function of SIG3 in plastid gene transcription.

MATERIALS AND METHODS

Isolation of SIG3 T-DNA insertion lines

Two different A.thaliana (ecotype Columbia, Co) SIG3 T-DNA insertion lines have been obtained from the SALK collection (SALK_009166 and SALK_081321, named sig3-2 and sig3-4, respectively). The T-DNA is inserted at the border of intron 1 and exon 2 in line sig3-2 and within exon 4 in line sig3-4. None of the two lines was kanamycin resistant. The heterozygous SIG3 insertion lines were at first backcrossed with wild-type (WT, Co.) plants two times in order to eliminate any other T-DNA insertion or mutations. Every generation resulting from self-pollination was analysed by PCR for the presence of the T-DNA insertion in the SIG3 gene. Resulting homozygotes were isolated for both lines. The sequences of the primers that have been used for the characterization of the T-DNA lines are as follows:

1: 5′-GATGATACTGGTTGTGCCGCC-3′; 2: 5′-AACGGCAAGCACAAAGAGACG-3′; 3: 5′-TGCCAAAAGGTTCTTTGCCAG-3′; 4: 5′-GCGTGGACCGCTTGCTGCAACT-3′; 5: 5′-TTCAATTCGTTCCCCATTCCC-3′. PCRs have been performed as described previously (31).

Plant material and RNA isolation

Surface-sterilized Arabidopsis seeds were spread on MS agar plates, kept for 72 h at 4°C in darkness and then transferred into a growth chamber and grown for 6 days at 23°C under 16/8 h light/dark cycle at 110 μmol of photons m−2 s−1. Total RNA was prepared from seedling as described in (23).

DNA microarray preparation

The A.thaliana plastid DNA microarray was constructed by spotting 60mer synthetic oligonucleotides that corresponded to sequences of 80 protein genes on nitrocellulose membranes. Oligonucleotides have been chosen within 200 nt sequences downstream of the ATG translation initiation codons. The spotting procedure was performed by Eurogentec (Belgium). Each DNA sample was spotted two times on a nitrocellulose membrane.

cDNA synthesis and array analyses

Total RNA was treated twice with DNase I (2 U/μg RNA) in order to remove traces of DNA. An aliquot of 4 μg of each RNA preparation have been labelled for microarray hybridization. RNA was reverse transcribed using specific primers corresponding to the 80 protein coding genes that we wanted to analyse on the microarray. Primers are localized as near as possible to the 3′ end of the 60mers that have been spotted onto the filters. The reaction was performed as described (31) in the presence of 100 μCi of [α-32P]dATP (Amersham Bioscience) using Superscript II reverse transcriptase (Invitrogene). Samples were treated with RNase H at 37°C for 15 min and non-incorporated deoxyribonucleotides were removed by passage through Sephadex G50. An aliquot of each of the synthesized cDNAs was analyzed on a 6% denaturing polyacrylamide gel in order to verify the quality of the synthesized cDNA. Hybridization was performed under the same conditions as indicated for northern experiments, however, hybridization time was extended to 3 days. After 3 weeks exposure to Fujifilm Imaging Plates, the plates were analyzed using a Phosphoimager (Fujifilm FLA-8000) and the accompanying software. Background substraction was performed by application of the R Project for Statistical Computing (33) (http://www.R-project.org). Before calculating mean values and standard deviations, results from all independent experiments were normalized taking the highest-labelled experiment as reference.

Primer extension

Using isolated total DNA from Arabidopsis as template, the clpP, atpH, psbN and psbT promoter regions have been PCR amplified and cloned into pCRR2.1-TOPOR (Invitrogen) with the following primers: 5′-CCAATATGCAATGGGGG-3′ and 5′-GTATCTCCTTCTCCAGG-3′ (clpP), 5′-GGATAGGAACTACTATC-3′ and 5′-GTCCAATAGAAGCAAGC-3′ (atpH, −44), 5′-GTGAGTCTATGGAAGGTC-3′ and 5′-CGCTAAGATTAATCCAGCC-3′ (atpH, −413), 5′-CATCCAGTAGAACAGAAG-3′ and 5′-CTCTTAGTTGTTGAGAGG-3′ (psbN) and 5′-AGGTTGGTTTACGTTTG-3′ and 5′-TCATTTTTTAGTTGAAATTTT-3′ (psbT). Primer extension experiments have been performed as described (31) using 10 μg of total RNA. The following primers have been used for primer extension and to establish the accompanying sequence ladders: 5′-GTATCTCCTTCTCCAGG-3′ (clpP), 5′-GTCCAATAGAAGCAAGC-3′ (atpH), 5′-CAAAGGCAGTATATAGAGC-3′ (psbN), 5′-GAAATTTTAGGTGGTTC-3′ (psbT) and 5′-ATGGAAGCATTGGTTTATAC-3′ (psbN/psbT-antisense).

Capping

In vitro capping reactions were performed in a final volume of 30 μl, using 15 μg of total RNA and 10 U of guanylyltransferase (Ambion), in the presence of 100 μCi of [α-32P]GTP (3000 Ci/mmol) and 20 U of RNase inhibitor. The reactions were incubated at 37°C for 1 h. RNAs were purified with phenol/chloroform extractions and precipitated with 3 vol of ethanol. Transcripts were then hybridized to 5 ng of complementary riboprobe and subjected to ribonuclease protection assay, using the RPA IIITM Ribonuclease Protection Assay Kit (Ambion), according to the manufacturer's protocol.

5′-RACE

The discrimination between transcription start sites and processing sites of precursor RNAs was done by RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE Kit; Ambion) without and with previous TAP treatment of RNAs. Reactions were performed according to the supplier's protocol but without removal of free 5′-phosphates by calf intestine alkaline phosphatase. PCR products were analysed on agarose gels after two successive PCR amplifications, the first using two outer primers and the second using two inner primers. Primers are as follows: psbT-as outer: 5′-ATGGAAGCATTGGTTTATACATTTC-3′, psbT-as inner: 5′-CGGGAACCACCTAAAATTTCAAC-3′, psbN outer: 5′-CTCTTAGTTGTTGAGAGG-3′ and psbN inner: 5′-CCCAAAGGCAGTATATAGAC-3′. The inner and outer Adapter primers are those of the RLM-RACE Kit.

Northern

For northern blot hybridization, PCR fragments have been 32P-labelled by random priming (psbH, psbN, psbB and rbcL) or riboprobes have been generated by T7 RNA polymerase (psbT). The gene-specific PCR fragments have been obtained with the following primers: 5′-GGCTACACAAACTGTTGAAG-3′ and 5′-CTAATTCACTGAAATTCCATCC-3′ (psbH), 5′-GGAAACAGCAACCCTAGTCGCC-3′ and 5′-CCCCGTGTTCCTCGAATGGATC-3′ (psbN), 5′-GGTCCTGGAATATGGGTATCC-3′ and 5′-GCCCCGAATTCCACTTGAGC-3′ (psbB), 5′-AACCAAGGATACTGATATCTTGGC-3′ and 5′-ATCGTCCTTTGTAACGATCAAGGC-3′ (rbcL). Prehybridization (1 h at 65°C) and hybridization (24 h at 65°C) were performed in 0.5 M NaHPO4, pH 7. 2, 1 mM EDTA, 7% SDS and 1% BSA. After hybridization filters were washed in 40 mM NaHPO4, pH 7. 2, 1 mM EDTA and 7% SDS at room temperature for 10 min followed by washing at 65°C for 5 min.

The psbT PCR fragment has been cloned into pCR2.1-TOPO vector (Invitrogen), the construct was linearized by BamHI and transcribed using T7 RNA polymerase in order to obtain a psbT antisense riboprobe. Hybridization of the psbT antisense RNA was performed overnight in 50% formamide, 5× SSC, 5× Denhardt and 1% SDS at 60°C. After hybridization, filters were washed twice in 0.2× SSC containing 0.1% SDS at 42°C for 10 min.

RESULTS

Isolation of SIG3 T-DNA insertion lines

Two different A.thaliana SIG3 T-DNA insertion lines have been obtained from the SALK collection (named here sig3-2 for SALK_009166 and sig3-4 for SALK_081321). The positions of the T-DNA insertions in the SIG3 gene are indicated in Figure 1A. Homozygous plants were selected for each of the two lines by PCR analyses using one primer pair that amplifies the border between T-DNA and the SIG3 gene (primers 2 and 5 for sig3-2 and primers 3 and 5 for sig3-4) and one primer pair that amplifies part of the SIG3 gene (primers 1 and 2 for sig3-2 and primers 3 and 4 for sig3-4). Figure 1B shows the PCR analyses obtained after cleaning of the mutants by two successive backcrosses (see Materials and Methods). Results reveal three homozygous plants for sig3-2 (Figure 1B, lanes 1/7, 3/9 and 4/10) and one homozygous plant for sig3-4 (Figure 1B, lanes 2/8). These plants have been propagated and their descendents have been analysed in the following experiments. Figure 1C shows the visible phenotypes of WTand mutant plants (sig3-4) when grown at 23°C under 16/8 h light/dark cycle at 110 μmol of photons m−2 s−1, i.e. visible phenotypes are not different. We have also compared different light conditions, varying from 50 to 200 μmol of photons m−2 s−1, using light/dark cycle or continuous illumination. Up to now, we could not detect visible differences between phenotypes of WT and sig3 (sig3-2 and sig3-4) plants (data not shown). If not otherwise indicated, in all following experiments sig3-4 plants have been analysed.

Figure 1
Characterization of two different SIG3 T-DNA insertion mutants. (A) Schematic representation of the T-DNA insertions and the location of the primers that have been used for PCR analyses. (B) Selection of homozygous lines for sig3-2 and sig3-4 after the ...

Analyses of plastid gene expression in the SIG3 T-DNA insertion mutants

In order to characterize the function of SIG3 in plastid gene expression, we performed an overall transcript profiling by microarray hybridization. Transcript levels of 6-day-old SIG3 insertion mutants corresponding to 80 ORFs of the A.thaliana plastid genome (see Materials and Methods) were compared to that of WT plants. Three independent experiments have been made. Normalized average values (sig3/WT) and standard deviations are summarized in Table 1 (Supplementary Table S1 is at available at NAR online). If the commonly used threshold value of 0.66 (1/1.5) is applied (34), only two mRNAs, psbN and atpH, are significantly reduced in the sig3 plants thus representing valuable candidates for specific transcription by a SIG3-PEP holoenzyme. These two mRNAs have been analysed in more detail by primer extension in order to determine the promoter region for specific SIG3 dependent transcription (Figure 2A and B).

Table 1
Transcript analysis of plastid genes in sig3 compared with WT plants
Figure 2
Analyses of psbN, atpH and clpP precursor RNAs. Total RNA was prepared from 6-day-old Arabidopsis WT and sig3 (Δ3) plantlets, RNA was either reverse transcribed and cDNAs were separated on 6% denaturing polyacrylamide gels (psbN, atpH and clp ...

The determination of 5′ ends of precursor RNAs by primer extension shows that psbN mRNA is transcribed from a single PEP promoter (Figure 2A, lanes 5 and 6). Transcription initiation at position −32 was confirmed by in vitro capping of psbN mRNA (Figure 2A, lanes 7–9). The upstream DNA sequence of the transcription start site harbours −35 and −10 sequences reminiscent of prokaryotic-type promoter elements (Figure 2D, upper lane). The psbN transcript is strongly reduced in the sig3 plants, thus indicating that the psbN transcription is under specific control of SIG3. This experiment was made with the two different sig3 mutants, sig3-2 and sig3-4, and in both cases we obtained the same result (data not shown). This shows that the lack of psbN mRNA in the two mutants is indeed caused by the lack of sigma factor 3 and not by an additional, undetected, mutation.

The atpH gene yields two different transcripts. One of them ends up at position −44 from the ATG translation initiation codon (Figure 2B, lanes 1 and 2). This mRNA should result from transcription by NEP, as its upstream sequence does not reveal recognizable prokaryotic-type promoter elements. The exact position of the longer mRNA in Figure 2B could not be determined since the accompanying sequence ladder could not be read up to this point. Therefore, we have cloned the DNA sequence upstream of the −44 RNA and repeated the primer extension analysis using a primer that is close enough to determine the 5′-end of the long atpH transcript (Figure 2B, lanes 11 and 12). The upstream DNA sequence of the long transcript starting at position −413 is reported in Figure 2D, lower lane. This long RNA should result from specific transcription by SIG3-PEP holoenzyme because this RNA is completely absent in sig3 plants (Figure 2B, lane 12). Altogether, these results show that the atpH gene is under two-promoter/two-RNA polymerase control, and one of the two promoters is recognized specifically by a SIG3-PEP holoenzyme. As control for an mRNA whose expression does not change, i.e. it is not under control of SIG3, we have analysed also clpP mRNAs by primer extension. The predominant −53 precursor RNA that is produced by NEP (35) is present in equal amount in WT and sig3 plants (Figure 2C, lanes 4 and 5).

The lack of psbN mRNA does not change the processing of the psbB operon

The psbN mRNA represents naturally occurring antisense RNA to the psbB polycistronic mRNA (36). Therefore, we wanted to know whether the strong reduction of the psbN mRNA in the sig3 plants influences the processing of the polycistronic transcript of the psbB operon which consists of psbB, psbT, psbH, petB and petD mRNAs (37,38). The organization of the psbB operon is schematically represented in Figure 3A.

Figure 3
Analysis of the expression of the psbN gene and of the psbB operon by northern experiments. (A) Schematic representation of the psbB operon. (B) Gel blot hybridization of WT and sig3 (Δ3) RNAs. Total RNA (10 μg) of 6-day-old plantlets ...

At first we have analysed the intermediary RNA species that result from processing of the long co-transcript by northern analyses (Figure 3B). By using probes that correspond to the psbB, psbT and psbH genes northern analysis do not show remarkable differences between WT and sig3 RNAs (Figure 3B). The rbcL mRNA is analysed because the microarray analyses for this gene had a high average value and a high standard deviation (see Table 1). The rbcL probe had been spotted in one of the corners of the nitrocellulose membrane that had been utilized to handle the membrane. Background hybridization was very high in some experiments and the microarray results are not conclusive. Therefore, we have analysed the rbcL transcripts by northern hybridizations. As shown in Figure 3B, the rbcL mRNA level does not change significantly in sig3 plants. The psbN mRNA has been analysed in order to verify the microarray result. The result confirms that psbN transcription is indeed very much reduced in the sig3 mutant.

The psbT sense and antisense mRNAs were analysed by primer extension, a method that is more sensitive than northern analysis (Figure 4). The localizations of the primers that have been used in these experiments is schematically indicated in Figure 4A. Primer extension analysis using primer (1), i.e. analysing psbT-antisense RNA, yields two different RNAs (~330 and ~140 bases) that are both absent in sig3 plants (Figure 4B, left-hand side). From this result we can conclude that both of these RNAs are under the control of SIG3. The shorter of the two transcripts results from processing and the longer one is a primary transcript, as shown by 5′-RACE without or after treatment of mRNAs with tobacco acid pyrophosphatase [TAP, (39)] (Figure 4B, left-hand side, lanes 7 and 8). This means that the longer RNA corresponds to a psbN/psbT-antisense co-transcript, and the shorter one (i.e. the psbT-antisense RNA) results from processing of the long psbN/psbT-antisense co-transcript. The shorter RNA ends up in the intergenic region, between psbT and psbN. The exact cleavage site is shown in Figure 4C (upper lane). The processing event separates the psbT-antisense RNA from the psbN mRNA. Altogether, this experiment shows that transcription from the psbN promoter produces antisense RNA that covers the whole psbT reading frame and whose production is dependent on sigma factor 3.

Figure 4
Transcript patterns of the psbT sense and antisense transcripts. (A) Schematic representation of part of the psbB operon. Primers that have been used for primer extension analyses are indicated by horizontal, closed, arrows and by numbers. RNAs that have ...

Primer extension using primer (2) reveals two different RNAs having their 5′ ends located either in the intergenic region of the psbB and the psbT genes (several transcripts ~154 bases) or within the coding region of the psbB gene (~345 bases). Both RNAs are present in WT as well as in sig3 plants (Figure 4B, right-hand side, lanes 5 and 6). The ~154 bases transcripts diverge between the last 6 nt at the 5′ end. These RNAs could be processing intermediates of the larger RNA. But the sequence upstream of these mRNAs reveals two possible NEP promoter consensus sequences, underlined in Figure 4C, that could also be responsible for transcription initiation of RNAs starting at different, closely situated, sites.

DISCUSSION

In the present paper, we have analysed plastid gene expression in two different SIG3 T-DNA insertion mutants that had been obtained from the SALK collection. Plastid mRNAs have been characterized by three different methods. At first, RNA levels of all plastid mRNA coding genes have been monitored by hybridization on a plastid specific microarray and in a second step, selected mRNAs have been analysed in more detail either by northern or by primer extension experiments. In each analysis, RNAs prepared from sig3 mutants have been compared to RNAs obtained from WT plants.

Microarray analysis revealed only two significantly reduced mRNAs in sig3 plants, i.e. psbN and atpH (Table 1). These two mRNAs have been further characterized by primer extension, a method that allows localization of the 5′ ends of the corresponding precursor RNAs (Figure 2A and B). This analysis shows that the psbN gene is under control of a single promoter that is specifically recognized by SIG3-PEP holoenzyme. The promoter sequence of the SIG3-specific PEP promoter of psbN is shown in Figure 3 (upper line). The −10 sequence is remarkably rich in adenine, while the −35 element harbours the consensus TTG triplet. On the other hand, the atpH gene is transcribed from two different promoters. The presence of two different promoters explains why the atpH mRNA is less reduced in sig3 mutants than the psbN mRNA. Only the minor, longer, atpH RNA (−413) is under control of SIG3-PEP holoenzyme.

The comparison of the two different SIG3-specific promoter sequences reveals similarities in an extended −35 region, in the region between the −10 and −35 consensus elements and immediately upstream of the transcription start site. The −10 consensus element is less conserved (Figure 2D). The question of what makes the specificity of SIG3 to these promoters cannot be answered by simple sequence comparison and represents a challenge for future work. The same holds true for other sigma factor/promoter interactions such as SIG2/psaJ (25), SIG2/trnE-V-M and Q (24), SIG4/ndhF (31) and SIG5/psbD BLRD (27). At a first glance, the specificity of SIG3 reported here for the psbN gene promoter seems to be in contradiction with previous publications showing recognition of the psbA and rbcL promoters by SIG3 (15,23). However, these experiments have been made using in vitro transcription conditions without sigma competition. Similar results showing sigma specificity in vivo under competitive conditions and apparently less specificity under non-competitive conditions are also reported for SIG2 with respect to the psbA promoter (15,23,25,27). In addition to redundant action of sigma factors under non-competitive conditions we can also not exclude that additional SIG3-specific promoters exist in the multiple promoter regions that precede many of the plastid transcription units. Some of these promoters might be more easily revealed in other developmental or environmental situations than used in our studies. On the other hand, the absence of one sigma factor might be compensated by overexpression of other sigma factors that might initiate from another PEP promoter within the same promoter region thus compensating the lack of one sigma factor for the overall transcription of a given gene. In such case, no change will be detectable by microarray analysis.

Under our experimental conditions, there is only one gene on the plastid genome whose expression is remarkably reduced when SIG3/PEP is lacking. This is psbN. The function of the PsbN protein is still unknown. On the basis of antibody experiments it had been shown that PsbN is localized on the thylakoid membranes (40). However, the suggestion that PsbN represents one of the small proteins of photosystem II (PSII) has been recently contradicted by systematic sequencing of all PSII centre proteins (41). We have grown sig3 plants under various light conditions (50–200 μmol of photons m−2 s−1, 8 h/16 h dark/light cycle or continuous light), but we did not observe changes in the visible phenotype when compared with WT plants. The question of why evolution has conserved one sigma factor to regulate specifically the transcription of the psbN gene cannot be answered at the moment. The psbN gene expression might serve a regulatory function for the expression of the psbB operon that is located on the opposite DNA strand (see Figure 3A). The expression of the psbB operon has been extensively studied in Euglena gracilis (42), Chlamydomonas reinhardtii (43,44) and Arabidopsis (4547). In fact, two different 5′ leader segments have been described in Arabidopsis for processed psbH transcripts, one of them overlapping with the 5′ leader of the psbN transcript (45). The psbN mRNA might therefore influence the processing of the psbH mRNA. To verify this hypothesis we have analysed the expression pattern of the psbB operon in WT and sig3 plants by northern hybridization and by primer extension (Figures 3B and and4B).4B). Using these two methods, we could not detect changes in the processing pattern of the psbB operon in sig3 plants. Equally, western blot analyses of protein extracts prepared from WT and sig3 plants using commercially available antibodies against PsbB and PsbH proteins do not reveal remarkable differences (data not shown). Unfortunately, antibodies against the PsbT and PsbN proteins are not yet available in order to test also these two proteins.

Primer extension using a primer that starts at the ATG of the psbT gene to detect psbT antisense transcripts (primer 1 in Figure 4A and B) shows that the psbN mRNA extends at it 3′ end at least up to the beginning of psbT thus covering the entire psbT gene as antisense RNA. Both mRNAs, psbN and psbT, could therefore be regulated by antisense RNA. Noteworthy, by specific transcription of the psbN gene, SIG3 could also regulate the expression of the psbB operon by producing psbT antisense RNA.

Primer extension analysis of the psbT sense RNAs revealed a yet unknown psbT transcript whose 5′ end is located within the coding region of psbB (~345 bases, Figure 4A and B). It would be interesting to investigate whether this RNA results from transcription initiation or from processing. Initiation of the psbT mRNA within the psbB coding region would mean that under certain conditions both genes, psbB and psbT, need to be transcribed separately. On the other hand, processing of the polycistronic mRNA within the psbB coding region means inactivation of the psbB mRNA. Thus, our results indicate an additional, so far unknown, mode of regulation of the psbB operon, concerning especially the two proximal genes, psbB and psbT. The shorter psbT sense transcript (~154 bases, Figure 4A and B) ends up within the intergenic region between the psbB and psbT genes. The 5′ ends of these transcripts extend over six bases that are located downstream of two different potential NEP promoters. Thus, although cleavage of a dicistronic psbB-psbT mRNA is expected as part of processing of the polycistronic psbB precursor RNA we cannot exclude that the shorter psbT mRNA is produced by transcription initiation. It has recently been shown that antisense transcripts stabilize polyadenylated mRNA in chloroplasts (48). Therefore, it would be likely to assume that the absence of psbN/psbT-antisense RNA in sig3 plants destabilized the psbT sense transcript. However, the quantity of the ~154 bases psbT sense transcripts in sig3 plants is not significantly different from WT plants. Thus, regulation by antisense stabilization is not applicable at least not under the growth conditions that have been routinely used in the present work (i.e. 110 μmol of photons m−2 s−1). Experiments are actually in progress to analyse quantitative changes in psbN and psbT sense and antisense RNAs during plant growth and development and under specific stress conditions. We hope that these experiments will provide an idea under which conditions psbT antisense RNA regulation might be important in plastids. The psbT protein has been shown to play a role in dimerization of PSII (49) and to be required for efficient repair of photodamaged PSII reaction centre (50). Environmental conditions that lead to photodamage of PSII might therefore be connected to a specific induction of PsbT protein expression, regulated by transcription from independent promoter(s) and/or by antisense RNA. It would be interesting to test such a hypothesis in the future. The sig3 plants described here represent a powerful means to investigate on such type of regulation of the psbB operon, but also to elucidate the function of the psbN protein.

SUPPLEMENTARY DATA

Supplementary Data are available at NAR Online.

Acknowledgments

We are grateful to S. Lemaille for his help in adaptation of the R computing software (http://www.R.project.org.) for our special purpose, to H. Pesey and E. Lambert for helpful technical assistance and to J.-P. Alcaraz for excellent sequencing service. Part of the work was financed by the European Community (FP6-2002-LifeSciHealth, PLASTOMICS, Proposal No. 503238). Funding to pay the Open Access publication charges for this article was provided by the European Community.

Conflict of interest statement. None declared.

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