Complete chloroplast DNA sequence of the moss Physcomitrella patens: evidence for the loss and relocation of rpoA from the chloroplast to the nucleus

Abstract

The complete chloroplast DNA sequence (122 890 bp) of the moss Physcomitrella patens has been determined. The genome contains 83 protein, 31 tRNA and four rRNA genes, and a pseudogene. Four protein genes (rpoA, cysA, cysT and ccsA) found in the liverwort Marchantia polymorpha and the hornwort Anthoceros formosae are absent from P.patens. The overall structure of P.patens chloroplast DNA (cpDNA) differs substantially from that of liverwort and hornwort. Compared with its close relatives, a 71 kb region from petD to rpoB of P.patens is inverted. To investigate whether this large inversion and the loss of rpoA usually occur in moss plants, we analyzed amplified cpDNA fragments from four moss species. Our data indicate that the large inversion occurs only in P.patens, whereas the loss of the rpoA gene occurs in all mosses. Moreover, we have isolated and characterized the nuclear rpoA gene encoding the alpha subunit of RNA polymerase (RNAP) from P.patens and examined its subcellular localization. When fused to green fluorescent protein, RpoA was observed in the chloroplasts of live moss protonemata cells. This indicates that chloroplast RNAP is encoded separately by chloroplast and nuclear genomes in the moss. These data provide new insights into the regulation and evolution of chloroplast transcription.

Figure 1

Gene map of the moss P.patens chloroplast genome. IR sequences (IR_A and IR_B) are separated by the SSC and LSC regions. Genes on the inside of the map are transcribed clockwise and genes on the outside are transcribed counter-clockwise. Genes with related functions are shown in the same color. Asterisks denote intron-containing genes or split genes. Arrows indicate positions of the endpoint of large inversion relative to liverwort M.polymorpha and hornwort A.formosae cpDNA.

Figure 2

(A) Representation of the strategy used to detect the orientation of the 71 kb inversion. The P.patens (top), M.polymorpha and A.formosae (bottom) genomes are shown in the region of the 71 kb inversion. The positions of primers P1–P4, located adjacent to the inversion endpoints, are indicated. (B) DNA regions amplified with either the P1 and P2 pair or the P1 and P3 pair are illustrated, with the length (bp) of the intergenic spacer.

Figure 3

Schematic diagram showing the domain structure of the α subunit of E.coli RNAP, P.patens nuclear PpRpoA and M.polymorpha chloroplast RpoA. αNTD and αCTD denote α subunit N-terminal and C-terminal domains, respectively. The gray boxes denote regions conserved in sequence between α homologs of prokaryotic, archaebacterial, chloroplast and eukaryotic RNAPs.

Figure 4

Localization of fusion protein encoded by the PpRpoA-gfp construct to chloroplasts. The PpRpoA-gfp was introduced into the P.patens protoplasts of protonemata. The localization of GFP and chloroplast pigments (chlorophyll) in transformed cells was detected by fluorescent microscopy. Merged images are shown on the right. Bars are 10 µm.