Phage-type RNA polymerase RPOTmp transcribes the rrn operon from the PC promoter at early developmental stages in Arabidopsis

Abstract

The plastid genome of higher plants is transcribed by two different types of RNA polymerases named nucleus encoded RNA polymerase (NEP) and plastid encoded RNA polymerase. Plastid encoded RNA polymerase is a multimeric enzyme comparable to eubacterial RNA polymerases. NEP enzymes represent a small family of monomeric phage-type RNA polymerases. Dicotyledonous plants harbor three different phage-type enzymes, named RPOTm, RPOTp, and RPOTmp. RPOTm is exclusively targeted to mitochondria, RPOTp is exclusively targeted to plastids, and RPOTmp is targeted to plastids as well as to mitochondria. In this article, we have made use of RPOTp and RPOTmp T-DNA insertion mutants to answer the question of whether both plastid-located phage-type RNA polymerases have overlapping or specific functions in plastid transcription. To this aim, we have analyzed accD and rpoB messenger RNAs (mRNA; transcribed from type I NEP promoters), clpP mRNA (transcribed from the -59 type II NEP promoter), and the 16S rRNA (transcribed from the exceptional PC NEP promoter) by primer extension. Results suggest that RPOTp represents the principal RNA polymerase for transcribing NEP-controlled mRNA genes during early plant development, while RPOTmp transcribes specifically the rrn operon from the PC promoter during seed imbibition.

Figure 1.

Characterization of two different RPOTmp T-DNA insertion mutants. A, Schematic presentation of the T-DNA insertions and the locations of the primers that have been used for PCR analyses. B, Selection of two different homozygous lines, 842 and 115, after the second backcross. The presence or absence of wild-type DNA was verified by PCR using primers 1 and 3 (lanes 1–3) and the presence and orientation of the T-DNA insertion was analyzed by PCR using primers LB and 1 (lanes 4 and 5) or LB and either 2 or 3 (lanes 6 and 7). C, Wild-type plants and the homozygous RPOTmp T-DNA insertion mutant (line 115) grown under a 16-h-light/8-h-dark cycle on agar plates for 1 week and then transferred to soil for 2 weeks. [See online article for color version of this figure.]

Figure 2.

Selection and characterization of a homozygous line for rpoTp. A, Schematic presentation of the T-DNA insertions and the location of the primers that have been used for PCR analyses. B, Characterization of a homozygous line after the third backcross. The absence of wild-type DNA was verified by PCR using primers 4 and 5 (lanes 1 and 2) and the presence and orientation of the T-DNA insertion was analyzed by PCR using primers LB and 5 (lines 3 and 4) or primers LB and 4 (lines 5 and 6). C, The absence of RPOTp (line 2) or RPOTmp (line 3) transcripts in the two T-DNA insertion mutants has been verified by RT-PCR. Transcripts for RPOTm and APT have been analyzed as controls. D, Wild-type and homozygous RPOTp T-DNA insertion mutants grown in soil under a 16-h-light/8-h-dark cycle for 3 weeks. Scale bar = 1 cm. [See online article for color version of this figure.]

Figure 3.

Phenotypes of wild-type and mutant plants 2 to 4 d after germination. Physiological stages of plantlets that have been used for microarray and primer extension experiments are boxed. The line indicates 5 mm.

Figure 4.

Characterization of different NEP- or PEP-specific precursor RNAs. Total RNA was prepared from 2-d-old wild-type (lane 1) and 3- to 4-d-old rpoTp (lane 2) or rpoTmp (lane 3) Arabidopsis plantlets and analyzed by primer extension. A, Analyses of RNAs of two exclusively NEP-transcribed genes, e.g. accD and rpoB. The insert on the right shows 5′-RACE of the −538 rpoB transcript with (+) or without (−) TAP treatment of mRNAs. The sequence of the 5′ end and the beginning of the linker is shown below. B, Analyses of RNAs of two exclusively PEP-transcribed genes, rbcL and psbA. C, Analyses of RNAs of two genes, clpP and rrn16S, that are transcribed from nonconsensus-type NEP promoters. The insert on the right shows the PC-initiated transcript after longer exposure. [See online article for color version of this figure.]

Figure 5.

Characterization of the 16S PC promoter. 16S precursor RNAs have been analyzed by primer extension using total RNA prepared from different plant materials. A, Dry seeds (lane 1), seeds after imbibition (lane 2), and leaves from mature Arabidopsis plants (lane 3). B, Dry seeds (lanes 1 and 4), seeds after vernalization (lanes 2 and 5), and plantlets 2 d after germination (lanes 3 and 6) of wild-type (lanes 1–3) and rpoTmp plantlets (lanes 4–6). C, Dry seeds (lanes 1 and 4), seeds after vernalization (lanes 2 and 5), and plantlets 2 d after germination (lanes 3 and 6) of rpoTp (lanes 1–3) and wild-type plantlets (lanes 4–6). D, Seeds of the tt2-1 mutant have been germinated and grown in the absence (−) or presence (+) of Tagetin and total RNA has been prepared 1 d after germination. 16S precursor RNAs have been analyzed by primer extension. The insert on the left shows a longer exposure of the PC-initiated rrn transcript.