RNA polymerase subunits encoded by the plastid rpo genes are not shared with the nucleus-encoded plastid enzyme

Abstract

Plastid genes in photosynthetic higher plants are transcribed by at least two RNA polymerases. The plastid rpoA, rpoB, rpoC1, and rpoC2 genes encode subunits of the plastid-encoded plastid RNA polymerase (PEP), an Escherichia coli-like core enzyme. The second enzyme is referred to as the nucleus-encoded plastid RNA polymerase (NEP), since its subunits are assumed to be encoded in the nucleus. Promoters for NEP have been previously characterized in tobacco plants lacking PEP due to targeted deletion of rpoB (encoding the beta-subunit) from the plastid genome. To determine if NEP and PEP share any essential subunits, the rpoA, rpoC1, and rpoC2 genes encoding the PEP alpha-, beta'-, and beta"-subunits were removed by targeted gene deletion from the plastid genome. We report here that deletion of each of these genes yielded photosynthetically defective plants that lack PEP activity while maintaining transcription specificity from NEP promoters. Therefore, rpoA, rpoB, rpoC1, and rpoC2 encode PEP subunits that are not essential components of the NEP transcription machinery. Furthermore, our data indicate that no functional copy of rpoA, rpoB, rpoC1, or rpoC2 that could complement the deleted plastid rpo genes exists outside the plastids.

Figure 1

Targeted deletion of rpo genes from the plastid genome. A, Deletion of the rpoA gene. Homologous recombination events (hatched lines) between ptDNA sequences in vector pGS95 and the tobacco plastid genome yields a genome lacking rpoA. Probes for Southern blots in D are marked with thick horizontal lines. Map position of the probed restriction fragments with size in kilobases is shown below the maps. aadA, Chimeric spectinomycin resistance gene (Svab and Maliga, 1993); rpoA, rpoB, rpoC1, and rpoC2, the plastid genes encoding the α-, β-, β′-, and β"-subunits of PEP, respectively; atpI, petD, rps2, and rps11, plastid genes (Shinozaki et al., 1986). Restriction endonuclease cleavage sites: H, HincII; X, XbaI; Bg, BglII; Sc, ScaI; P, PstI; B, BamHI; Pp, Psp1406I; A, AccI; SI, SacI; SII, SacII; StI, StuI; E, EcoRV; Bs, BsrGI. Brackets indicate restriction sites eliminated during cloning. B, Deletion of the rpoC1 gene. Homologous recombination events (crossed lines) between ptDNA sequences in vector pGS97 and the tobacco plastid genome yields a genome lacking rpoC1. C, Deletion of the rpoC2 gene. Homologous recombination events (crossed lines) between ptDNA sequences in vector pGS99 and the tobacco plastid genome yields a genome lacking rpoC2. D, Southern probing demonstrates a uniform population of transformed plastid genomes. Total cellular DNA was isolated from the leaves of plants transformed with plasmids pGS95 (targeting rpoA), pGS97 (targeting rpoC1), and pGS99 (targeting rpoC2), and from wild-type green leaves (WT). Data are shown for two independently transformed lines (pGS95-2, pGS95-3), or two plants derived from the same transformation event (pGS97-2.2, pGS97-2.3 and pGS99-4.1, pGS99-4.4).

Figure 2

Isolation of homoplasmic ΔrpoA plants. A, Callus and shoots carrying a mixed population of wild-type and ΔrpoA plastid genomes are green. B, Chimeric leaves with white (transgenic) and green (wild-type) sectors. C, White, homoplasmic ΔrpoA plant with transgenomes only.

Figure 3

Accumulation of plastid mRNAs in wild-type and plastid rpo gene deletion derivatives. Data are shown for genes carrying only PEP promoters (rbcL), only NEP promoters (accD), or PEP and NEP promoters (clpP, 16S rDNA, atpB) in wild-type, ΔrpoA, ΔrpoB, ΔrpoC1, and ΔrpoC2 leaves. The excess of wild-type over Δrpo intensities (average of the four Δrpo lines) for each probe is given in parentheses. Gel blots were prepared with total leaf RNA (5 μg per lane) from wild-type plants, and in plants transformed with plasmids pGS95 (ΔrpoA), pGS97 (ΔrpoC1), and pGS99 (ΔrpoC2). Upper panels show blots probed for plastid genes. Lower panels show loading controls, obtained by probing the same filters for the cytoplasmic 25S rRNA. The blots were scanned with a phosphor imager (Molecular Dynamics, Sunnyvale, CA). Hybridization signals were quantified with Imagequant software (Molecular Dynamics) and normalized to the 25S rRNA signal.

Figure 4

Mapping of transcription-initiation sites in plastids of wild-type and rpo-deletion derivatives. Primer-extension data are shown for the rbcL, atpB, 16SrDNA, clpP, and accD genes. Mapped NEP (•) and PEP (○) promoters are identified by the distance between the transcription-initiation site and the translation-initiation codon (ATG) in nucleotides (Allison et al., 1996; Hajdukiewicz et al., 1997). Processing sites are also marked (✂). Schematic maps with transcription-initiation sites for NEP and PEP promoters are shown at the bottom of the figure.