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Nucleic Acids Research Pages 415-419

Nuclear encoding of a plastid [sigma] factor in rice and its tissue- and light-dependent expression
Introduction
Materials And Methods
   Screening of cDNA libraries
   DNA sequencing
   Computational analysis
   Southern blot hybridization
   RNA blot hybridization
Results
   Isolation of an O.sativa cDNA encoding a [sigma] factor-like protein
   Characteristics of the predicted Os-SigA protein
   Copy number of Os-sigA-related genes
   Expression of Os-sigA
Discussion
Acknowledgements
References


Nuclear encoding of a plastid [sigma] factor in rice and its tissue- and light-dependent expression

Nuclear encoding of a plastid [sigma] factor in rice and its tissue- and light-dependent expression Yuzuru Tozawa, Kan Tanaka1, Hideo Takahashi1 and Kyo Wakasa*

National Agriculture Research Center, 3-1-1 Kannondai, Tsukuba, Ibaraki 305, Japan and 1Institute of Molecular and Cellular Biosciences, The University of Tokyo, 1-1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan

Received October 30, 1997; Revised and Accepted November 7, 1997

DDBJ/EMBL/GenBank accession no. AB005290

ABSTRACT

A full-length cDNA encoding a putative [sigma] factor for a plastid RNA polymerase was isolated from the higher plant Oryza sativa. The nucleotide sequence of the corresponding nuclear gene, named Os-sigA (O.sativa sigma A), predicts a polypeptide of 519 amino acids that contains a putative plastid-targeting sequence in its N-terminal region. The predicted mature protein shows extensive sequence homology to bacterial [sigma] factors, encompassing the conserved regions 1.2, 2.1, 2.2, 2.3, 2.4, 3, 4.1 and 4.2 implicated in binding to -10 promoter elements, promoter melting and interaction with the core RNA polymerase enzyme. RNA blot analysis revealed that the abundance of Os-sigA transcripts was markedly greater in green shoots than in roots or in dark-grown etiolated shoots of rice seedlings. Furthermore, exposure of dark-grown etiolated seedlings to light resulted in a rapid increase in the amount of Os-sigA mRNA in the shoot. These observations suggest that regulation of expression of the nuclear gene for this putative plastid RNA polymerase [sigma] factor by light contributes to light-dependent transcriptional regulation of plastid genes.

INTRODUCTION

Plastids are intracellular semiautonomous organelles in plants that retain extensive similarities to prokaryotic organisms, including a circular double-stranded DNA genome, -10, -35 type consensus promoter elements and polycistronic gene clusters (1-7). It has been presumed that plastids of higher plants arose through endosymbiotic events in which a photosynthetic prokaryote entered a eukaryote host cell (8,9). Transcriptional regulation of plastid genes is important for plastid differentiation, such as chloroplast biogenesis during leaf formation. Biochemical analysis has identified at least two independent RNA polymerases (RNAPs) that appear to contribute to plastid gene expression (10,11): a nuclear encoded enzyme that has been purified as a single 110 kDa polypeptide from spinach chloroplasts (12) and a bacterial-type multisubunit enzyme. Sequence analysis has also indicated that the genomes of plastids from several plants encode counterparts of bacterial RNAP subunits, including [alpha] (RpoA), [beta] (RpoB) and [beta]' (RpoC1 and RpoC2) (13,14). Disruption of rpoB in the tobacco plastid genome resulted in impaired chloroplast development and reduced transcription of several plastid genes, suggesting that the bacterial-type RNAP is required for development of proplastids into chloroplasts (15).

In bacteria, accurate initiation of transcription requires not only the core RNAP enzyme ([alpha]2[beta][beta]') but also another subunit, [sigma] factor, that defines binding specificity to target promoter sequences. The use of different [sigma] factors with different promoter specificities thus contributes to regulation of gene expression (16,17). -10, -35 element-like sequences found in the 5' flanking regions of several plastid genes have suggested that [sigma] factor-like proteins might participate in regulation of plastid gene transcription by the bacterial-type RNAP. Indeed, purified plastid RNAP fractions from higher plants contain polypeptides that cross-react with antibodies to the principal [sigma] factor of cyanobacteria (18). Moreover, three [sigma]-like factors were identified biochemically in mustard and their promoter specificity was suggested to be altered by phosphorylation status (19,20). Although evidence indicates that plastid [sigma] factors are encoded by the nuclear genome, the corresponding genes have not been identified in higher plants.

Bacterial principal [sigma] factors, members of the [sigma]70 family, recognize -10, -35 elements and the amino acid sequences of their functional domains are highly conserved (21). This sequence conservation enabled isolation and characterization of previously unidentified [sigma] factors from many prokaryote species (22,23). Similarly, homology-based screening has revealed the existence of nuclear [sigma] factor genes in the unicellular eukaryote Cyanidium caldarium (24,25). These eukaryotic [sigma]-like factors possess transcriptional activity; one (SigA) was shown to be located in chloroplasts (24) and transcription of the gene encoding the other (RpoD) was shown to be induced by light (25).

A search of sequence databases with the amino acid sequence deduced from C.caldarium RK-1 sigA revealed an Arabidopsis thaliana expressed sequence tag (EST) that encoded a partial amino acid sequence with homology to a highly conserved region of members of the [sigma]70 family. This observation served as the basis for the present study, in which we describe the isolation and characterization of a [sigma] factor from the monocotyledon Oryza sativa (rice) and suggest its involvement in light-dependent expression of chloroplast genes.

MATERIALS AND METHODS

The nucleotide sequence of Os-sigA described in this paper has been deposited in GenBank under accession no. AB005290.

Screening of cDNA libraries

Standard recombinant DNA techniques were performed basically as described (26). PCR was performed with an A.thaliana cDNA library (kindly provided by K.Shinozaki-Yamaguchi) and oligonucleotide primers sigA1 (5'-TGGGAGCAGAAATGTCTGACCTTGTTCAG) and sigA2 (5'-AATGTAACTGTGATGAGTTTCTCCAG), based on the A.thaliana EST (GenBank accession no. T88387) with homology to [sigma] factors. The 50 µl reaction mixture contained 1* ExTaq buffer (Takara), 1 µg cDNA, 0.2 mM each deoxynucleoside triphosphate, 1 mM each primer and 2.5 U ExTaq DNA polymerase (Takara). Amplification was carried out in a Perkin Elmer DNA Thermal Cycler 480 with an initial denaturation step at 94°C for 90 s, followed by 35 cycles of denaturation at 94°C for 1 min, annealing at 55°C for 1 min and elongation at 72°C for 1 min. PCR products were cloned into the pCRII vector (Invitrogen) and sequenced. Amplified DNA fragments were also purified with a Nick spin column (Pharmacia) and used to prepare fluorescein isothiocyanate (FITC)-labeled probes with an ECL random prime labeling system (Amersham).

Total RNA was prepared as previously described (27) from 7-day-old O.sativa cv. Nipponbare plants that had been grown at 28°C under a 16 h light, 8 h dark cycle. Poly(A)+ RNA was purified from total RNA with an mRNA preparation kit (Pharmacia) and cDNA was synthesized with a Timesaver cDNA synthesis kit (Pharmacia) and cloned into [lambda]gt11 (Clontech) using Gigapack II Gold (Stratagene). The resulting cDNA library was expressed in Escherichia coli Y1088 and the ~150 000 plaques formed on each LB agar plate (235 * 235 mm; Nunc) supplemented with 10 mM MgSO4 were transferred to a Hybond-N+ membrane (220 * 220 mm; Amersham). Phage DNA was fixed on the membrane with alkali and plaque hybridization performed overnight at 60°C with FITC-labeled probe in a solution containing 5* SSC, 0.1% SDS, 5% (w/v) dextran sulfate and 5% ECL Liquid Block (Amersham). The membranes were washed sequentially twice (5 min each time) at room temperature with a solution containing 1* SSC, 0.1% SDS and once at 60°C for 15 min with 0.5* SSC, 0.1% SDS. Hybridization probe was detected with the ECL detection system and Hyperfilm (Amersham).

DNA sequencing

Cloned cDNAs were digested with appropriate restriction enzymes and the resulting fragments were subcloned into the Bluescript phagemid vector (Stratagene). Single-stranded DNA was recovered from each vector using helper phage VCS-M13 and was sequenced using FITC-labeled primers, an AutoRead sequencing kit (Pharmacia) and an ALF II DNA sequencer (Pharmacia).

Computational analysis

Database homology searches were performed with the BLAST program (28). Analysis of amino acid sequences was performed with ClustalW (29) software.

Southern blot hybridization

Genomic DNA was isolated from O.sativa cv. Nippponbare seedlings as described previously (30) and 15 µg were digested with various restriction enzymes, subjected to electrophoresis in a 1.0% agarose gel and transferred to a Hybond-N+ membrane by the capillary procedure with 20* SSC. The DNA fragments were fixed to the membrane by baking at 80°C for 2 h. Southern hybridization, washing and detection of hybridized probe were performed as described above for screening of cDNA libraries.

RNA blot hybridization

For detection of Os-sigA transcripts a digoxigenin-labeled antisense RNA probe was prepared with a DIG RNA labeling kit (Boehringer Mannheim). A 714 bp SacI-HindIII fragment of Os-sigA cDNA, corresponding to nt 817-1530, was subcloned into the pSPT18 vector and the antisense RNA probe was synthesized by in vitro transcription with SP6 RNA polymerase and digoxigenin-labeled UTP. A rice actin2 antisense RNA probe (31) was also synthesized as an internal marker. An EcoRI-PstI fragment, including 1 kb of the open reading frame, of actin2 cDNA was excised from the pRAC2 clone (kindly provided by Dr I.Mitsuhara) and subcloned into pSPT18 and a digoxigenin-labeled antisense RNA probe was synthesized. Poly(A)+ RNA corresponding to ~50 µg total RNA was purified using Oligotex-dT30<Super> (Nippon Roche), subjected to electrophoresis in a 1.2% agarose-formaldehyde gel and transferred to a Hybond-N+ membrane by the capillary method with 20* SSC. Transferred RNA was fixed to the membrane by baking at 80°C for 2 h. The membrane was incubated in a solution containing 5* SSC, 50% formamide, 0.02% SDS, 2% Blocking Reagent (Amersham) and 0.1% N-lauroylsarcosine and then subjected to hybridization for 16 h at 68°C in the same solution containing the RNA probe (100 ng/ml). After washing the membrane with 2* SSC, 0.1% SDS at room temperature for 10 min and then with 0.2* SSC, 0.1% SDS first at room temperature for 10 min and then twice at 65°C for 20 min, the hybridized probe was detected with a DIG Luminescent Detection Kit (Boehringer Mannheim) and Hyperfilm-ECL (Amersham). The amount of RNA loaded in each lane was verified by removing the Os-sigA probe and rehybridizing the membrane with the actin2 probe.

RESULTS

Isolation of an O.sativa cDNA encoding a [sigma] factor-like protein

A homology search revealed that, allowing for several frameshifts, an A.thaliana EST (GenBank accession no. T88387) encoded a partial amino acid sequence similar to that of SigA of C.caldarium RK-1 (24). We amplified the corresponding cDNA fragment from an A.thaliana cDNA library by PCR and determined its nucleotide sequence. The peptide encoded by the amplified cDNA was indeed homologous to conserved regions 2 and 3 of [sigma] factors (Fig. 1; 32). Screening of 450 000 plaques of an O.sativa cDNA library with a probe derived from this PCR product yielded eight positive clones and the complete 2038 bp nucleotide sequence of the longest one (clone pOSIG7) was determined. The gene from which this full-length cDNA was derived was named Os-sigA (O.sativa sigma factor A).


Figure 1. Comparison of the predicted amino acid sequences of two putative [sigma] factors from higher plants. The amino acid sequences of the predicted products of Os-sigA (GenBank accession no. AB005390) (this work) and At-sigA (GenBank accession no. D89993) (32) were aligned with the ClustalW program (29), with dashes representing gaps introduced to optimize alignment. Boxed sequences indicate potential cleavage sites (AVA-A and VDV-A) (32). Centered dots indicate residues identical in the two proteins. The underlined residues correspond to the sequence derived from the A.thaliana EST (GenBank accession no. T88387). Residue numbers are indicated on the right.

Characteristics of the predicted Os-SigA protein

The open reading frame of Os-sigA encodes a 519 amino acid protein. The entire amino acid sequences of Os-SigA and A.thaliana SigA are 50% identical (Fig. 1). The N-terminal region of Os-SigA, which shows especially high homology to At-SigA, is followed by a stretch of 13 glycine residues and a putative cleavage site (AVAA) that shows similarity to that of the plastid-targeting sequence LTVVAA (33,34).

The sequence was compared with those of [sigma] factors from eubacteria, cyanobacteria, red algae and A.thaliana (data not shown). The predicted amino acid sequence of Os-SigA showed substantial homology to conserved regions 1.2-4.2 of other [sigma] factors. The sequence known as the RpoD box (21,22), which overlaps regions 2.3 and 2.4 and is highly conserved among microbial principal [sigma] factors, was also especially well conserved in the [sigma] factor-like polypeptides of plants and C.caldarium; the consensus sequence of this region, taking into account all of the proteins, is G(F/Y)(R/K)STY(V/A)(Y/T)WWIRQ(G/S/A)(V/I) (S/T).

We constructed a phylogenetic tree to shed light on the evolutionary relationship among the various [sigma] factors (Fig. 2). Os-SigA and At-SigA were most closely related to each other, with the red alga SigA being the next closest relative of both. In turn, these plastid [sigma] factors are more closely related to [sigma] factors of cyanobacteria than to those of eubacteria. This observation is consistent with the endosymbiotic theory, the putative evolutionary relationship between plastids and cyanobacteria.


Figure 2. Phylogenetic tree of [sigma] factors from various species. The amino acid sequences of [sigma] factors from higher plants [O.sativa (Os) and A.thaliana (At)], a red alga [C.caldarium RK-1(Cy) (GenBank accession no. D83179)], cyanobacteria [Anabaena spp. PCC7120 (An) (GenBank accession no. M60036) and Synechococcus spp. PCC7942 (Sy) (GenBank accession no. D10973)] and eubacteria [E.coli (Ec) (GenBank accession no. J01687) and Bacillus subtilis (Bs) (GenBank accession no. X03897)] were aligned. The unrooted tree was constructed from the evolutionary distance data derived by the neighbor joining method (40). The bootstrap procedure sampled 1000 times with replacement by ClustalW (29). The number at each node represents the percentage of trees supporting the specific branching pattern in the bootstrap analysis. The bar indicates the distance corresponding to 4.8 changes per 100 amino acid positions.

Copy number of Os-sigA-related genes

Southern blot analysis of genomic DNA from O.sativa revealed that Os-sigA is a single-copy nuclear gene (Fig. 3). Whereas Southern analysis with a probe comprising the entire Os-sigA cDNA, which exhibits a high GC content in its 5'-region, generated a high background signal (data not shown), a probe corresponding to nt 767-1677 of the cDNA sequence (which encompasses regions 1.2-4.2 of the encoded protein) yielded specific signals (Fig. 3).


Figure 3. Southern analysis of Os-sigA. Genomic DNA (15 µg) from O.sativa was digested with HincII, PstI, SpeI, SphI, EcoRV, XhoI or SacI (lanes 1-7 respectively), fractionated by electrophoresis in a 1.0% agarose gel and subjected to Southern hybridization with a 911 bp FITC-labeled fragment, corresponding to nt 767-1677 of the cDNA. The positions of DNA size markers (HindIII digest of [lambda] DNA) are indicated in kilobase pairs.

Expression of Os-sigA

Expression of Os-sigA in rice plants was investigated by RNA blot analysis. To ensure specific hybridization with a low background signal we used an antisense RNA probe. Os-sigA transcripts were barely detectable by hybridization to total RNA, indicative of their low abundance (data not shown). We therefore repeated the analysis with poly(A)+ RNA (Fig. 4). The amount of 2.1 kb Os-sigA mRNA in the shoots of juvenile plants grown under a 16 h light, 8 h dark cycle was markedly greater than that in roots. In contrast, the amount of Os-sigA transcripts in the shoots of etiolated juvenile plants grown in complete darkness was markedly reduced and was similar to that in roots of plants grown under the light/dark cycle. However, exposure of such etiolated seedlings to light for 4 h resulted in a substantial increase in the abundance of Os-sigA transcripts.

DISCUSSION

With a homology screening approach we have identified a rice nuclear gene, Os-sigA, that encodes a [sigma] factor-like protein. This gene and genes that we have also identified in A.thaliana (32) appear to be the first [sigma] factor genes characterized in higher plants.

The recent determination of the crystal structure of a [sigma]70 protein from E.coli (35), together with the results of biochemical and genetic studies, has allowed identification of amino acid residues important in binding to the core RNAP enzyme, interaction with the -10 promoter element and promoter melting. These residues are located in the highly conserved regions 1.2, 2.1, 2.2, 2.3, 2.4, 3, 4.1 and 4.2 and are clustered together and closely associated in the tertiary structure (35). Sequence alignment reveals substantial homology among [sigma] factors from eubacteria, cyanobacteria, red algae and higher plants. Although the plant proteins have not yet been shown to function as [sigma] factors, their conserved structural features indicate that Os-SigA and At-SigA likely contribute to recognition of -10, -35-type plastid promoters as components of bacterial-type RNAP enzymes. However, in cyanobacteria and chloroplasts some promoters are devoid of distinct -35 sequences and, therefore, the importance of -35 elements has not been well established. Future analyses on -35 elements and the recognition domain of these [sigma] factors may shed light on the recognition mechanism in these systems.

We simultaneously isolated cDNAs encoding [sigma] factor homologs (SigA, SigB and SigC) from A.thaliana (32). Among them At-SigA showed the most extensive sequence similarity to Os-SigA. The N-terminal regions of both proteins are highly similar and appear to contain a plastid-targeting sequence. At-SigB and At-SigC also appear to contain plastid-targeting signals in their N-terminal regions (32). Localization of the red algal SigA protein to plastids (24) is consistent with the functionality of these putative plastid-targeting sequences in the plant proteins.

Southern blot analysis indicated that Os-sigA is a single-copy gene (Fig. 3). However, we also isolated two more rice partial cDNAs that appear to encode putative [sigma] factors resembling At-SigB (data not shown). The low sequence homology of these cDNAs with Os-sigA likely explains why the corresponding genes were not detected by Southern analysis with the Os-sigA probe. Our identification of multiple genes for putative [sigma] factors in rice and A.thaliana is consistent with biochemical identification of multiple [sigma]-like factors in mustard (19-20).


Figure 4. RNA blot analysis of Os-sigA expression. (A) Poly(A)+ RNA corresponding to ~50 µg total RNA from O.sativa seedlings grown under various conditions was subjected to RNA blot analyis with Os-sigA (upper panel) or actin2 gene (lower panel) probes. Lane 1, shoot of 7-day-old seedlings grown under a 16 h light, 8 h dark cycle, harvested in the middle of the light period; lane 2, shoot of 10-day-old etiolated seedlings grown under total darkness; lane 3, shoot of 10-day-old etiolated seedlings grown under total darkness but exposed to light for 4 h before harvesting; lane 4, root of seedlings corresponding to lane 1. (B) 5* longer exposure of the same membrane shown in (A) hybridized with the Os-sigA probe.

Expression of Os-sigA was markedly greater in the shoot than in the root of rice seedlings grown under a normal light/dark cycle (Fig. 4). Furthermore, whereas the abundance of Os-sigA transcripts was markedly reduced in the shoot of etiolated seedlings grown in the dark, it increased rapidly on exposure of such shoots to light (Fig. 4). Thus the pattern of Os-sigA expression appears to coincide with that of light-dependent chloroplast biogenesis.

Light induces marked changes in plant cells that are accompanied by coordinated transcription of genes in both the nucleus and plastids (36-38). Although biochemical studies have demonstrated a role for bacterial-type RNAP in transcription of several plastid genes, genes encoding subunits of this enzyme are transcribed by a different type of transcriptional apparatus in a light-independent manner (10). The light-dependent transcription of certain plastid genes that possess -10, -35 consensus promoter elements is blocked by tagetitoxin, an inhibitor of bacterial RNAP (11,39). Thus a role has been proposed for additional transcription factors that confer the property of light-dependent expression to plastid genes. Our data indicate that light-dependent transcription of Os-sigA may contribute to light-dependent expression of plastid genes during chloroplast biogenesis.

ACKNOWLEDGEMENTS

This work was supported by grants for Advanced Recombinant DNA Techniques to Y.T and K.W. from the Ministry of Agriculture, Forestry and Fisheries of Japan and by a Grant-in-Aid for Scientific Research in Priority Areas (no. 09274207) to K.T. from the Ministry of Education, Science and Culture of Japan.

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*To whom correspondence should be addressed. Tel/Fax: +81 298 38 8949; Email: kwakasa@narc.affrc.go.jp

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A RelA-SpoT homolog (Cr-RSH) identified in Chlamydomonas reinhardtii generates stringent factor in vivo and localizes to chloroplasts in vitro
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Y. Tozawa, H. Hasegawa, T. Terakawa, and K. Wakasa
Characterization of Rice Anthranilate Synthase {alpha}-Subunit Genes OASA1 and OASA2. Tryptophan Accumulation in Transgenic Rice Expressing a Feedback-Insensitive Mutant of OASA1
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K. Baba, T. Nakano, K. Yamagishi, and S. Yoshida
Involvement of a Nuclear-Encoded Basic Helix-Loop-Helix Protein in Transcription of the Light-Responsive Promoter of psbD
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S. D. Lahiri and L. A. Allison
Complementary Expression of Two Plastid-Localized sigma -Like Factors in Maize
Plant Physiology, July 1, 2000; 123(3): 883 - 894.
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S. Kapoor and M. Sugiura
Identification of Two Essential Sequence Elements in the Nonconsensus Type II PatpB-290 Plastid Promoter by Using Plastid Transcription Extracts from Cultured Tobacco BY-2 Cells
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S. Tan and R. F. Troxler
Characterization of two chloroplast RNA polymerase sigma factors from Zea mays: Photoregulation and differential expression
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M. Kim, K. E. Thum, D. T. Morishige, and J. E. Mullet
Detailed Architecture of the Barley Chloroplast psbD-psbC Blue Light-responsive Promoter
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Y. Nakahira, K. Baba, A. Yoneda, T. Shiina, and Y. Toyoshima
Circadian-Regulated Transcription of the psbD Light-Responsive Promoter in Wheat Chloroplasts
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T. Shiina, L. Allison, and P. Maliga
rbcL Transcript Levels in Tobacco Plastids Are Independent of Light: Reduced Dark Transcription Rate Is Compensated by Increased mRNA Stability
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