Impaired function of the phage-type RNA polymerase RpoTp in transcription of chloroplast genes is compensated by a second phage-type RNA polymerase

Abstract

Although chloroplast genomes are small, the transcriptional machinery is very complex in plastids of higher plants. Plastidial genes of higher plants are transcribed by plastid-encoded (PEP) and nuclear-encoded RNA polymerases (NEP). The nuclear genome of Arabidopsis contains two candidate genes for NEP, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. We have analyzed the use of PEP and NEP promoters in transgenic Arabidopsis lines with altered RpoTp activities and in Arabidopsis RpoTp insertion mutants lacking functional RpoTp. Low or lacking RpoTp activity resulted in an albino phenotype of the seedlings, which normalized later in development. Differences in promoter usage between wild type and plants with altered RpoTp activity were also most obvious early in development. Nearly all NEP promoters were used in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene, however, was not used in mutant seedlings lacking RpoTp activity. Our data provide evidence for NEP being represented by two phage-type RNA polymerases (RpoTp and RpoTmp) that have overlapping as well as gene-specific functions in the transcription of plastidial genes.

Figure 1.

Overexpression and partially silencing the native AthRpoTp gene by insertion of ectopic AthRpoTp gene copies leads to different phenotypes. (a) Single copy plants (Tp1x) are not distinguishable from wild-type plants. Multiple copy plants (Tp5x) germinate into white seedlings. The white seedlings will eventually become green reaching wild-type levels after two weeks (b). (c) In northern blot analyses, 50 μg total RNA of Tp1x (lane 1), Tp5x (lane 2) and wild-type plants (lane 3) was separated in 1% agarose-formaldehyde gels. Ethidium bromide stained gel image is shown as a loading control (bottom panel). The RNA blot was hybridized with a single-stranded full-length AtRpoTp antisense DNA probe. Positions of rRNAs are given on the margin. AtRpoTp RNA accumulates to highest amounts in overexpressor plants with only one AtRpoTp transgene (Tp1x, lane 1). However, in multiple copy plants AtRpoTp RNA levels (Tp5x, lane 2) are reduced to lower than wild-type RNA levels (wt, lane 3).

Figure 2.

Northern blot analysis of psbA (a) and atpB (b) transcript levels in wild type and partially silenced Tp5x plants. Five microgram of total RNA of 6-day-old cotyledons of wild type (lanes 1) and Tp5x (lanes 3), and of 3-week-old leaves of wild type (lanes 2) and Tp5x (lanes 4) plants was separated in 1% agarose-formaldehyde gels. The RNA blot was hybridized to the indicated plastid gene sequence (upper panel). The same blot hybridized with an 18S rRNA probe is shown as a loading control (18S, bottom panel). Transcript sizes (kb) are given on the margin.

Figure 3.

Partial silencing of AthRpoTp in multiple copy plants negatively affects transcription of the type-I rpoB NEP promoter. Less abundant rpoB transcript levels most likely lead to white seedlings due to delayed PEP synthesis. However, the type-II clpP-58 promoter is not affected. Primer extension data are shown for the psbA (a), rpoB (b) and clpP genes (c). Mapped NEP type-I (filled circle), type-II (filled square) and PEP (open circle) promoters are identified by their distance between the transcription initiation site and the translation initiation codon in nucleotides (30). For reference, the same end-labeled primer was used to generate a DNA sequence ladder.

Figure 4.

Mapping of rpoB (a), psbA (b), atpB (c) and clpP (d) transcription sites in wild type and in rpoTp mutant seedlings. RNA isolated from 2-day-old wild type (lane 1), 4-day-old RpoTp T-DNA insertion lines sca3-3 (lane 2) and sca3-2 (lane 3), 3-week-old sca3-2 leaves (lane 4), 4-day-old plants with single (Tp1x, lane 5) and multiple (Tp5x, lane 6) AthRpoTp transgene copies were analyzed by primer extension. The lower panels show products generated with a second, cytoplasmatic 18S ribosomal RNA primer in the same primer extension reactions as shown above (18S). Mapped NEP type-I (filled circle), type-II (filled square) and PEP (open circle) promoters are identified by their distance between the transcription initiation site and the translation initiation codon in nucleotides.

Figure 5.

Mapping of rrn16 (a and b) and ycf1 (c and d) transcription sites in wild type and in rpoTp mutant seedlings. RNA isolated from 2-day-old wild type (lane 1), 4-day-old RpoTp T-DNA insertion lines sca3-3 (lane 2) and sca3-2 (lane 3), 3-week-old sca3-2 leaves (lane 4), 4-day-old plants with single (Tp1x, lane 5) and multiple (Tp5x, lane 6) AthRpoTp transgene copies were analyzed by primer extension (a and c). To control for loading and recovery, the primer extension reactions shown above contained a second primer for the cytoplasmatic 18S ribosomal RNA. The generated products are shown in the lower panels (18S). A physical map of the rrn16 region (b) and the ycf1 region (d) showing promoter usage in wild type (top) and in the sca3 mutant (bottom) is shown below. Primary transcripts from NEP and PEP promoters are marked by filled circles and open circles and labeled with their distance between the transcription initiation site and the translation initiation codon in nucleotides.