Molecular genetic analysis of chloroplast gene promoters dependent on SIG2, a nucleus-encoded sigma factor for the plastid-encoded RNA polymerase, in Arabidopsis thaliana

Abstract

Most photosynthesis-related genes in mature chloroplasts are transcribed by a eubacterial-type RNA polymerase (PEP) whose core subunits are encoded by the plastid genome. It has been shown previously that six putative nuclear genes (SIG1 to SIG6) encode promoter-specificity factors for PEP in Arabidopsis thaliana, and we isolated a T-DNA insertion line of SIG2 (sig2-1 mutant) that manifests aberrant chloroplast development. With the use of S1 nuclease protection and primer extension analyses, we have now characterized the SIG2-dependent chloroplast promoters in A.thaliana. The amounts of transcripts derived from one of the multiple psbD promoters (psbD -256) and from the promoters of two tRNA genes (trnE-UUC and trnV-UAC) were markedly and specifically decreased in the sig2-1 mutant. The abundance of these transcripts was restored to wild-type levels by introduction into the mutant of a SIG2 transgene. The recombinant SIG2 protein mixed with Escherichia coli core RNA polymerase could bind to a DNA fragment that contains the SIG2-dependent psbD -256, trnE-UUC or trnV-UAC promoter. Sequences similar to those of the -35 and -10 promoter elements of E.coli were identified in the regions of the SIG2-dependent chloroplast genes upstream of the transcription initiation sites.

Figure 1

Pale-green phenotype of the sig2-1 mutant of A.thaliana during seedling growth. The wild-type (Wassilewskija, WT), sig2-1 mutant (MT), and sig2-1 mutant complemented with the entire genomic region of SIG2 (gB) were grown under continuous light at 23°C either for 1 week on an MS-Gelrite plate (A) or for 3 weeks on Jiffy 7 (B).

Figure 2

Transcript mapping of photosynthesis-related genes in A.thaliana. Transcripts of psbD (A) or of psbA and rbcL (B) were analyzed by the S1 nuclease protection assay with the indicated amounts of total RNA from wild-type (WT) and sig2-1 mutant (MT) leaves. The nucleotide positions of the 5′ ends of transcripts relative to the ATG translation initiation site were determined by primer extension analysis and are indicated on the left.

Figure 3

Transcript mapping of plastid tRNA genes in A.thaliana. Transcripts of trnE-UUC (A), trnV-UAC (B) or trnY-GUA, trnD-GUC, trnM-CAU and trnfM-CAU (C) were analyzed by the S1 nuclease protection assay with the indicated amounts of total RNA from wild-type (WT) and sig2-1 mutant (MT) leaves. The nucleotide positions of the 5′ ends of transcripts relative to those of the mature tRNAs are indicated on the left.

Figure 4

High-resolution mapping of the 5′ ends of SIG2-dependent (A–C) and SIG2-independent (D) transcripts. The psbD –256 (A), trnE-UUC (B), trnV-UAC (C) and rbcL (D) transcripts were analyzed by primer extension analysis with 25 µg of total RNA from wild-type (WT) leaves. The cDNA sequences (A, C, G and T) obtained with the same primers are also shown. Arrows indicate the positions of the 5′ ends of each transcript that correspond to the bands shown in Figures 2 and 3.

Figure 5

Ribonuclease protection assay for the in vitro capped primary transcript from psbD –256. Fifty micrograms of total RNA from wild-type leaves was capped by guanylyltransferase and [α-³²P]GTP, followed by RNase protection assay using in vitro transcribed antisence RNA for the psbD gene as a probe. Note that the probe protects a 37-nt fragment for the transcript from psbD –256. Lane M shows RNA marker of the sizes indicated at the right.

Figure 6

Gel-shift DNA binding assay with the recombinant SIG2 protein. The SIG2 protein was incubated with a ³²P-labeled DNA fragment containing psbD –256 (left), trnE-UUC (middle), trnV-UAC (right) promoter region in the absence (lane 2) or presence (lane 3) of E.coli core RNA polymerase. Labeled DNA alone (lane 1), and a DNA probe with core RNA polymerase (lane C) were also shown as controls. Competition experiments were carried out as in lane 3 with a 100-fold excess of unlabeled each promoter fragment (lane 4).

Figure 7

Alignment of nucleotide sequences of the regions upstream of the transcription initiation sites of SIG2-independent (A) and SIG2-dependent (B) Arabidopsis gene promoters. The arrows indicate the position corresponding to the 5′ end of each major transcript. The E.coli-like –35 and –10 promoter elements are boxed, and the underlined regions refer to conserved promoter elements specific to the three SIG2-dependent promoters. The consensus sequence motifs for the –35-like and –10-like elements of the SIG2-dependent promoters are also indicated in (B); upper case and lower case letters denote nucleotides that are completely or partially conserved among the three promoters.

Figure 8

In vivo complementation of SIG2-dependent transcriptional defects. The psbD (A) as well as trnE-UUC and trnV-UAC (B) transcripts were analyzed by S1 nuclease protection assays with the indicated amounts of total RNA from wild-type (WT), sig2-1 mutant (MT) and gB transgenic leaves. The nucleotide positions of the 5′ ends of transcripts relative to the ATG translation initiation site or to the 5′ end of the mature tRNA are indicated on the left.