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First published online August 11, 2006; 10.1104/pp.106.085878 Plant Physiology 142:642-650 (2006) © 2006 American Society of Plant Biologists OPEN ACCESS ARTICLE
Dual Temporal Role of Plastid Sigma Factor 6 in Arabidopsis Development1,[OA]Plant Cell Physiology, University of Bochum, D–44780 Bochum, Germany
Plants contain nuclear-coded sigma factors for initiation of chloroplast transcription. The in vivo function of individual members of the sigma gene family has become increasingly accessible by knockout and complementation strategies. Here we have investigated plastid gene expression in an Arabidopsis (Arabidopsis thaliana) mutant with a defective gene for sigma factor 6. RNA gel-blot hybridization and real-time reverse transcription polymerase chain reaction together indicate that this factor has a dual developmental role, with both early and persistent (long-term) activities. The early role is evident from the sharp decrease of certain plastid transcripts only in young mutant seedlings. The second (persistent) role is reflected by the up- and down-regulation of other transcripts at the time of primary leaf formation and subsequent vegetative development. We conclude that sigma 6 does not represent a general factor, but seems to have specialized roles in developmental stage- and gene-specific plastid transcription. The possibility that plastid DNA copy number might be responsible for the altered transcript patterns in mutant versus wild type was excluded by the results of DNA gel-blot hybridization. Retransformation of the knockout line with the full-length sigma 6 cDNA further established a causal relationship between the functional sigma gene and the resulting phenotype.
Chloroplasts and other plastid types contain their own genetic system consisting of DNA and a full set of proteins for gene expression. Transcription, the first step leading to primary RNA molecules, involves at least two different RNA polymerases. Plastid-encoded polymerase (PEP) is the multisubunit bacterial-type enzyme, whereas nucleus-encoded polymerase (NEP) is of the single-subunit type shared with phage T3/T7 and mitochondrial enzymes (Hedtke et al., 1997  ; Maliga, 1998; Cahoon and Stern, 2001). The promoters recognized by each polymerase differ, with –35/–10 elements in the case of PEP and GAA/YRTA motifs in the case of NEP (Liere and Maliga, 2001; Shiina et al., 2005). PEP has major roles in functional chloroplasts, which is reflected by the large number of regulatory proteins surrounding the catalytic core (Pfannschmidt et al., 2000; Loschelder et al., 2004; Suzuki et al., 2005; Pfalz et al., 2006), including those that associate transiently, such as the sigma factors (Tiller and Link, 1993a, 1993b).
Sigma factors are the principal regulators of transcription initiation in bacteria (Borukhov and Nudler, 2003
Whereas basic sigma functions have been tested to a large extent using heterologous in vitro systems with authentic plastid or bacterially expressed sigma proteins and Escherichia coli RNA polymerase (Hakimi et al., 2000
Characterization of the Arabidopsis Sig6-2 Mutant Allele
We have chosen the knockout mutant line sig6-2 that had been generated in the GABI-Kat program at the Max-Planck-Institute fuer Zuechtungsforschung, Cologne (242G06; Rosso et al., 2003
Using reverse transcription (RT)-PCR and gene-specific primers (see "Materials and Methods"), transcripts from individual members of the Arabidopsis sigma gene family were assessed. As shown in Figure 1B, the signals for Sig1 to Sig6 were all clearly visible with wild-type RNA, whereas the Sig6 transcript was absent with mutant RNA. The phenotype of sig6-2 differs from that of the wild type in a developmental stage-specific way. Homozygous mutant seedlings develop normally shaped cotyledons, but with increasing chlorophyll deficiency. Younger stages until approximately 4 d after sowing have pale-green cotyledons, which then become yellowish and finally white during the next 6 to 8 d (Fig. 1C). In contrast, the primary leaves and subsequent rosette leaves are seemingly unaffected. Except for the remainder of the white cotyledons, mutant plants are green and have a normal morphological appearance (Fig. 1C). They tend, however, to be slightly smaller in size than wild type of the same age, which could be related to delayed germination and/or seedling development.
Transcript levels of representative chloroplast genes at different times in development were assessed by northern-blot hybridization. RNA samples from cotyledons (seedlings 4–10 d after sowing) or rosette leaves (plants 21 and 28 d after sowing) were fractionated and hybridized with gene-specific RNA probes (Fig. 2
) as described in "Materials and Methods." The results were grouped according to the observed expression mode in the mutant, taking into account the classification of plastid genes based on their transcription by PEP versus NEP (Hajdukiewicz et al., 1997
The 1.3-kb psbA transcript (Fig. 2A, top row) is a prominent band in all wild-type lanes from 4 to 28 d after sowing. In the mutant, this transcript is dramatically down-regulated at 4 d (i.e. the youngest seedling stage analyzed), and is then rapidly restored to wild-type levels. A similar time course was noticeable both for the 1.9-kb large subunit of Rubisco (rbcL) transcript (second row) and the 0.6-kb precursor of the intron-containing trnV(UAC) gene (third row), again with a decrease only in the 4-d mutant sample. In addition, the trnV probe revealed a high-molecular (3.0 kb) signal in the mutant, but not in the wild type. Unlike all other transcripts in Figure 2A, this RNA species was not detectable before the 8-d stage.
In contrast to the genes in Figure 2A, those in Figure 2B did not give rise to transcripts with an early decrease at the 4-d mutant stage. The dicistronic (Sugita and Sugiura, 1996 Finally, as shown in Figure 2C (first row), the 2.5-kb transcript of the accD gene was present in the mutant in amounts that were equal to (21 and 28 d) or higher (4–10 d), but never lower, than those in the wild type. A similar pattern was also observed for the 1.2-kb clpP transcript (second row). To assess steady-state transcript levels of selected monocistronic genes more rigorously, quantitative real-time PCR experiments were carried out (Fig. 3 ). Again, psbA and rbcL were found to give decreased transcript levels in 4-d-old, but not 10-d-old, sig6-2 seedlings (Fig. 3, A and B). For psbA (Fig. 3A), the down-regulation compared to the wild type exceeded a factor of 4 at the 4-d stage and was less than 0.5 at 10 d, whereas for rbcL (Fig. 3B) the factors were greater than 2 (4 d) versus less than 0.5 (10 d). In contrast, real-time RT-PCR showed greater than 3-fold up-regulation for the clpP transcript at day 4 and 2-fold at day 10 (Fig. 3C). This pattern is in agreement with that observed for accD and clpP in the northern-blot experiments (Fig. 2C). Because of their multiple overlapping transcripts, the polycistronic transcription units studied in Figure 2B (atpB-E, ndhC-K-J) were not investigated by real-time quantification.
Plastid-Nuclear DNA Ratio Is Not Responsible for Altered Expression Patterns in Sig6-2 As changes in copy number of plastid DNA might contribute to the altered RNA patterns in the mutant, we tested this possibility by Southern hybridization with both plastid and nuclear probes. Total DNA was prepared from wild type and mutant (sig6-2), either at the 4-d seedling stage (Fig. 4A ) or from 28-d plants (Fig. 4B). After digestion of equal amounts of DNA with HindIII, followed by gel fractionation and hybridization, a single signal at 7.5 kb was generated with the plastid psbA probe (Fig. 4B, left), and a 9.0-kb band with the nuclear 18S rDNA probe (Fig. 4B, right). The wild-type and sig6-2 lanes always revealed bands of equal intensity, suggesting that DNA copy number was not responsible for the different psbA transcript levels in mutant versus wild type that were observed at 4 d, but not 28 d (Fig. 2).
Rescue of SIG6 Gene Function by Complementation
To further confirm that insertional inactivation of the AtSig6 gene in the sig6-2 mutant is directly responsible for its phenotype, complementation experiments using full-length cDNA were carried out. Following RT and amplification, the cloned AtSig6 cDNA was fused to the cauliflower mosaic virus 35S promoter of the binary vector pBINAR (Höfgen and Willmitzer, 1990
We first examined the integration of the pBINAR T-DNA into the sig6-2 mutant line using genomic Southern-blot analyses (Fig. 5A). The sulf probe (Fig. 5A, left) established the absence of the primary T-DNA from the wild type, and its presence in both the sig6-2 knockout and the complemented mutant line. The neomycin phosphotransferase II (nptII) probe (Fig. 5A, middle) specifically detected the T-DNA insertion resulting from the secondary transformation, with a signal visible only in the complemented mutant. Using the sig6 probe (Fig. 5A, right), a single 5.3-kb band was generated in the wild type, whereas a 3.5-kb band was noticeable in both the knockout and retransformed plants. The latter also showed two additional bands at approximately 6.0 and 4.5 kb. As these two bands were consistently observed under a variety of experimental conditions, they probably indicate the presence of an additional EcoRI site adjacent to the insertion rather than partial digestion (data not shown). In any case, none of them is visible in the wild-type and knockout lanes, suggesting that they mark a single secondary insertion at a unique site. We next analyzed the gene expression patterns of the complemented line in comparison with those from the wild type and the sig6-2 knockout. As shown in Figure 5B (top left), RT-PCR amplification from total RNA of 4-d seedlings established that the AtSig6 transcripts are absent in the knockout, but are present in both wild-type and complemented lines, and similar results were obtained with RNA from 10-d seedlings (data not shown). Using RNA gel-blot hybridization with a psbA probe (Fig. 5B, left), the intensity of the 1.3-kb transcript was decreased in the knockout compared with the wild type (see also Fig. 2A), but was restored to at least wild-type levels in the complemented line. These quantitative differences in signal strength among lines were much more pronounced for the 4-d (left) than for the 10-d seedlings (right). The hybridization results with the atpB and atpE probes (Fig. 5B, right) again showed the typical transcript patterns for wild type and knockout (compare Fig. 2B), and the complete restoration of the retransformed mutant to the wild-type situation. Whereas the 2.0-kb (and 0.7-kb) transcripts are visible in all lanes, the 2.6-kb transcript is present only in wild-type and complemented lines, and the 4.8-kb band only in the knockout mutant line. Together, these data provide evidence that the retransformed line has acquired wild-type properties with regard to AtSig6-dependent plastid gene expression. This notion is further supported by the visible phenotype, which is indistinguishable from wild type (data not shown).
In this work, we have characterized a new AtSig6 mutant allele, sig6-2, both at the DNA and RNA level, as well as by complementation with intact cDNA. This reverse-genetics strategy established a causal link between the introduced gene and the visual and molecular phenotype of the rescued transformants, both of which resembled that of the wild type (Fig. 5). PCR and Southern-blot analysis together established the gene-specific (single) T-DNA insertion both in the knockout mutant and in the complemented line (Fig. 1). Using the same techniques, evidence was obtained that plastid DNA copy number does not seem to be a significant factor responsible for distinct plastid RNA patterns of wild type versus mutant (Fig. 4).
RT-PCR and northern-blot analysis together suggested that gene expression patterns in both wild-type and complemented lines are similar, if not identical, and those in the knockout mutant are clearly different (Figs. 2, 3, and 5). The plastid RNA patterns were thus of diagnostic value, in both the comparison of different Arabidopsis (wild type, mutant, and complemented) lines and different developmental stages of one single line. Furthermore, the expression patterns helped integrate the picture obtained for genes of different classes. For instance, as is evident from Figure 2, both the class I genes (psbA and rbcL) and the split trnV(UAC) gene gave rise to transcripts of similar expression mode. We feel that the term expression mode can be particularly useful if multiple transcripts are considered, as is the case for the genes presented in Figure 2B (atpB/E, trnV, ndhC; Sato et al., 1999
Unlike most mutants described for other Arabidopsis sigma factors (for review, see Shiina et al., 2005
The transcript patterns (Figs. 2 and 3) of sig6-2 were in agreement with those obtained for sig6-1 in at least some cases. This is evident for transcripts of the class I genes psbA and rbcL, each of which showed a sharp decrease in steady-state concentration at day 4, but not at day 8, in sig6-1 (Ishizaki et al., 2005
A notable difference, however, is evident from the trnV(UAC) transcript pattern (Fig. 2A, third row), consisting of two RNA species with different time courses during development. The smaller (0.6-kb) band shows the early decrease (4 d), as was seen for the class I transcripts psbA and rbcL (first and second row). The large 3.0-kb signal is visible only in the mutant, and only later throughout day 8 to 28. Neither effect was previously described for trnV in sig6-1 (Ishizaki et al., 2005
This view is strengthened by the data obtained with the polycistronic ndhC transcription unit (Fig. 2B, third row), which also results in two RNAs of different time courses. The smaller (1.8-kb) species appears to be present in relatively constant amounts without a decrease at the 4-d seedling stage. The mutant-specific 3.0-kb RNA is first visible at day 8 and then remains at a constant level (i.e. both its size and time course match those of the large trnV transcript; Fig. 2A, row 3). As trnV and ndhC are immediately adjacent (Sato et al., 1999 The atpB-E operon (Fig. 2B, first and second row) gives rise to several transcripts, none of which shows an early decrease comparable to that of the class I RNAs (Fig. 2A). (1) The major 2.0-kb (atpB-E) and the 0.7-kb (monocistronic atpE) RNAs were both present in roughly constant amounts throughout development. (2) The 2.6-kb RNA species was visible both in wild type and sig6-2 at 4 d, but was absent in the mutant at all subsequent stages. (3) The mutant-specific 4.8-kb species accumulated transiently between 4 and 10 d and then completely disappeared (Fig. 2B, first and second row).
Together, the data presented in Figure 2 indicate an unexpected complexity of SIG6-dependent responses in Arabidopsis development. The model depicted in Figure 6
suggests a dual role consisting of both an early and persistent (long-term) activity of the factor. An early decrease was seen for the transcripts of class I genes (Fig. 2A), but also for the 0.6-kb trnV transcript (expression mode I). The opposite effect (i.e. the early increase of the accD and clpP transcripts; Fig. 2C) may be functionally related, although it could be due to efficient NEP transcription (Allison et al., 1996
The transient 4.8-kb RNA of the atpB/E region (Fig. 2) may be a consequence of the fact that both the early and persistent (long-term) functions of SIG6 are absent in the mutant. If not generated by an alternative sigma factor and PEP, this mutant-specific RNA could be the result of NEP-dependent transcription. A similar mechanism (i.e. formation of a large [polycistronic] transcript by usage of a NEP promoter in the absence of SIG6), could explain the 3.0-kb trnV (and ndhC) transcript. Furthermore, it was previously established that trnV is a PEP-dependent gene preferentially transcribed in the presence of SIG2 (Kanamaru et al., 2001 ; Hanaoka et al., 2003; Privat et al., 2003). The early decrease of the 0.6-kb RNA at day 4 (Fig. 2A) suggests that SIG6, in addition to SIG2, may have a temporally restricted role in the transcription of this tRNA gene.
A question that emerges relates to the mechanisms involved in the functional overlap of plastid sigma factors throughout development or only at certain times (Kanamaru and Tanaka, 2004
In addition to phosphorylation and redox control, a number of other mechanisms could be envisaged for time- and promoter-specific usage of individual plastid sigma factors, including proteolytic cleavage (Hakimi et al., 2000
Plant Material, Growth Conditions, and Developmental Stages
The sig6-2 mutant of Arabidopsis (Arabidopsis thaliana ecotype Columbia) was identified in a collection of T-DNA insertion lines of the GABI-Kat project at the Max-Planck-Institute fuer Zuechtungsforschung (Rosso et al., 2003
For PCR analysis of the AtSig6 mutant, total DNA samples were isolated from rosette leaves of either wild type or progeny of the GABI-Kat line (T3 or later) by using the plant mini kit (Qiagen). The primer pair for the sulfonamide resistance gene of the T-DNA plasmid pAC161 (Rosso et al., 2003
Total RNA (2 µg) from 6-d-old Arabidopsis seedlings was mixed with random primers (10 pM; Promega), incubated at 70°C for 10 min, and chilled on ice for 1 min. After addition of 6 µL avian myeloblastosis virus-reverse transcriptase buffer (Promega), 1 µL RNasin (40 units/µL; Promega), 3 µL dNTPs (0.25 mM each), and 3 µL avian myeloblastosis virus-reverse transcriptase (10 units/µL) to a final volume of 30 µL, the reaction was incubated at 37°C for 90 min. Following heating to 95°C for 10 min, the mixture was chilled on ice for 1 min. One microliter of RNase A (10 µg/µL; Sigma) was then added and incubation continued at 37°C for 15 min. The cDNAs corresponding to each Arabidopsis sigma factor were amplified using Taq DNA polymerase (Promega). Primers were RTSIG1-1 (5'-TTTTCTGCATGGTGGTTTGA-3') and RTSIG1-2 (5'-ACCGCTCTCTATGGCTCTGA-3') for Sig1; RTSIG2-2 (5'-GAAAGAGGCACGAAAGCAAC-3') and RTSIG2-3 (5'-CCAACGAATCCCATTACCAC-3') for Sig2; RTSIG3-1 (5'-GAAAGCAAGGAGGTCGAGTG-3') and RTSIG3-2 (5'-TCCATCGTTGTGTCTGGTGT-3') for Sig3; RTSIG4-1 (5'-ACGACGATTCCCACTACAGC-3') and RTSIG4-2(5'-CTCGAAAGCTTCAGCGACTT-3') for Sig4; RTSIG5-2 (5'-TCCTCCTCGTGAGCAAGTTT-3') and RTSIG5-3 (5'-CATACCCGCTTGACAAAGGT-3') for Sig5; and RTSIG6-1 (5'-GCGTCGGTTCTCTCACAGGAGCCA-3') and RTSIG6-2 (5'-CTAGACAAGCAAATCAGCATA-3') for Sig6. PCR reactions consisted of an initial heating step at 95°C for 2 min, followed by 35 cycles each at 95°C for 30 s, 57.5°C to 60°C for 30 s, and 72°C for 2 min.
Cotyledon or rosette leaf samples (100 mg) were frozen in liquid nitrogen and ground to powder. RNA was isolated by the acid guanidinium-phenol-chloroform method (Chomczynski and Sacchi, 1987
Real-time one-step RT-PCR was carried out using the QuantiTect SYBR Green RT-PCR kit (Qiagen). The 50-µL PCR reaction contained gene-specific primer sets (0.5 µM each) to yield amplicons of 150 to 200 bp, QuantiTect SYBR Green plus RT Mix (Qiagen), and 0.02- to 20-ng template RNA. Primers were QpsbA-1 (5'-TTTCCGGTGCCATTATTCCT-3'), QpsbA-2 (5'-TCATAAGGACCGCCGTTGTA-3'), QrbcL-1 (5'-TCGGTGGAGGAACTTTAGGC-3'), QrbcL-2 (5'-TGCAAGATCACGTCCCTCAT-3'), QclpP-1 (5'-ATTCCATGAGCTTGGGCTTC-3'), and QclpP-2 (5'-ACTTCGCGAAACCATCACAA-3'). Experiments were carried out in an Opticon 2 DNA engine (MJ Research) using cycling conditions as follows: 50°C for 30 min, 95°C for 15 min, 94°C for 15 s, 50°C for 30 s, 72°C for 30 s, followed by 40 cycles at 94°C for 15 s and 55°C or 60°C for 1 min. To check for absence of dimer formation, the primers were subjected to melting curve analysis with incremental steps from 60°C to 95°C every 0.3°C for 3 s. Amplicon size for each primer pair was verified by gel electrophoresis and each reaction was carried out in triplicate. Primer pair efficiency was calculated using LinRegPCR (Ramakers et al., 2003
Full-length AtSIG6 cDNA, including the transit peptide, was PCR amplified from wild-type RNA using the primer pair SigF1 (5'-ATGGAAGCTACGAGGAACTTGG-3') and SigF2 (5'-CTAGACAAGCAAATCAGCATA-3') and cloned into the EcoRV site of vector pBSKS(–) (Stratagene). The insertion of the resulting intermediate plasmid was controlled by sequencing, cut out, and ligated into the BamHI and SalI sites downstream from the cauliflower mosaic virus 35S promoter of the binary vector pBINAR (Höfgen and Willmitzer, 1990
Genomic DNA for Southern-blot analysis was prepared from cotyledons and rosette leaves by using the cetyltrimethylammonium bromide procedure (Doyle and Doyle, 1987
We gratefully acknowledge the generous supply of the sig6-2 mutant line by Professor B. Weisshaar, University of Bielefeld, and the GABI-Kat team at the Max-Planck-Institute fuer Zuechtungsforschung, Cologne. We would also like to thank Dr. M. Nowrousian and Dr. I. Kubigsteltig at the Biology Department of the University of Bochum for their guidance and helpful discussions of the real-time RT-PCR and transgenic work, respectively. Received June 27, 2006; accepted August 1, 2006; published August 11, 2006.
1 This work was supported by the Deutsche Forschungsgemeinschaft (SFB 480).  The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Gerhard Link (gerhard.link{at}ruhr-uni-bochum.de).
[OA] Open Access articles can be viewed online without a subscription. www.plantphysiol.org/cgi/doi/10.1104/pp.106.085878 * Corresponding author; e-mail gerhard.link{at}ruhr-uni-bochum.de; fax 49–234–3214–188.
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ABSTRACT
RESULTS
-mercaptoethanol. After addition of 150 µL 2 M potassium acetate, pH 4.0, RNA was extracted with phenol-chloroform-isoamylalcohol (25:24:1) and precipitated with ethanol. Total RNA (1 µg/lane) was separated on a 1.2% (w/v) agarose-formaldehyde gel, blotted to positively charged nylon membrane (Roche), and hybridized with digoxygenin (DIG)-labeled RNA probes according to the Roche manual. Probes were generated by RT-PCR amplification and cloning of corresponding cDNAs into pGEM-T Easy (Promega), followed by in vitro transcription using phage RNA polymerases. The following primer sets were used: NorpsbA1 (5'-TTACCCAATCTGGGAAGCTG-3'), NorpsbA2 (5'-GCCTCAACAGCAGCTAGGTC-3'), RT7 (5'-GACAACTGTGTGGACCCATG-3'), RT8 (5'-TTCACCTGTTTCAGCCTCTG-3'), NorAccD-1 (5'-TCGCAATTTCATATCGGATG-3'), NorAccD-2 (5'-CTTCTTGCATTCGTGCTCCT-3'), atpB1nor (5'-GGGGAACCCGTTGATAATTT-3'), atpB2nor (5'-AACGCTCAATTTTTCGTGCT-3'), NorAtpEa (5'-GACTCCGAATCGAATTGTTTG-3'), NorAtpEb (5'-GTGTCCGAGCTCGTCTGAG-3'), NorndhC-1 (5'-TGCTATTCCTGTTTTGGCATT-3'), NorndhC-2 (5'-CCATTCCAATGCTCCTTTTC-3'), NortrnV-1 (5'-CTCGAACCGTAGACCTGCTC-3'), and NortrnV-2 (5'-GAGTCCATCACGCAATCAAA-3'). 
