A nuclear-encoded protein, mTERF6, mediates transcription termination of rpoA polycistron for plastid-encoded RNA polymerase-dependent chloroplast gene expression and chloroplast development

Abstract

The expression of plastid genes is regulated by two types of DNA-dependent RNA polymerases, plastid-encoded RNA polymerase (PEP) and nuclear-encoded RNA polymerase (NEP). The plastid rpoA polycistron encodes a series of essential chloroplast ribosome subunits and a core subunit of PEP. Despite the functional importance, little is known about the regulation of rpoA polycistron. In this work, we show that mTERF6 directly associates with a 3'-end sequence of rpoA polycistron in vitro and in vivo, and that absence of mTERF6 promotes read-through transcription at this site, indicating that mTERF6 acts as a factor required for termination of plastid genes' transcription in vivo. In addition, the transcriptions of some essential ribosome subunits encoded by rpoA polycistron and PEP-dependent plastid genes are reduced in the mterf6 knockout mutant. RpoA, a PEP core subunit, accumulates to about 50% that of the wild type in the mutant, where early chloroplast development is impaired. Overall, our functional analyses of mTERF6 provide evidence that it is more likely a factor required for transcription termination of rpoA polycistron, which is essential for chloroplast gene expression and chloroplast development.

Figure 1

Phenotype characterization and the T-DNA insertion sites identification of the mterf6 mutant. (A) Phenotype of the wild-type (WT; Columbia-0), mterf6-5 and mterf6-6 mutants, and mterf6-5 complemented plants grown on MS medium with or without 2% sucrose for 7 or 14 days. Bars = 0.1 cm. (B) Phenotype of the WT, mterf6-5 and mterf6-5 complemented plant grown in soil after being grown in MS medium with 2% sucrose for 2 weeks. Bar = 1 cm. (C) Positions of the two T-DNA insertions in the mterf6 mutants and the genomic fragment for complementation experiment. The black boxes, gray boxes and lines indicate the exons, untranslated regions and introns, respectively. The location of the primer pair (LP and RP) used for genotyping is shown by arrows. (D) MTERF6 expression in WT, mterf6-5 and mterf6-6 mutants and in the complementation line.

Figure 2

Tissue expression pattern of the MTERF6 gene. (A) The sketch map of MTERF6 and the position of the three specifically designed primers pairs (F1, R1; F2, R2; F3, R3). Black boxes show exons, gray boxes are UTRs, and black lines between boxes are introns. (B) The expression levels of the three transcripts in seedlings. (C,D) RT-PCR analysis (C) and quantitative real-time RT-PCR analysis (D) of MTERF6 transcripts in root, stem, leaf, and flower. Error bars indicate standard deviations for triplicates.

Figure 3

Characterization of the expression of three types of chloroplast genes and the RpoA protein accumulation in the WT and mterf6-5 mutant. (A) Quantitative real-time RT-PCR analysis of the three types of chloroplast genes in 7-day-old seedlings of WT and mterf6-5. Error bars indicate standard deviations for triplicates. (B) Western blot analysis of RpoA protein accumulation in 7-day-old seedlings of WT and mterf6-5. Coomassie brilliant blue R-250 (CBB) staining was a loading control. Each lane contains 40 μg total protein. Lane WT 1/8, WT1/4, WT 1/2 represents that the loading amount is 1/8, 1/4, 1/2 that of the WT, respectively. The arrow shows the position of the Rubisco large subunit (RbcL).

Figure 4

Quantitative real-time RT-PCR analysis of the read-through transcripts in the WT and mterf6-5. (A) An example of the ycf9 gene for the primer design. The primer sets of the inside (ycf9) and downstream (ycf9-1, ycf9-2, ycf9-3) are shown in the model. “Normal” represents supposed normal transcripts of ycf9, and “elongated” represents supposed elongated transcripts of ycf9. (B) Quantitative real-time RT-PCR analysis of the mRNA level of ycf9 in WT by primer sets. The primer pair of ycf9-3 was renamed “ycf9-T” (red) and selected for further study. Error bars indicate standard deviations for triplicates. (C) Selected primer sets and their locations in their corresponding genes. (D) Comparison of the expression of genes with the selected primer sets by quantitative real-time RT-PCR in the WT. Error bars indicate standard deviations for triplicates. (E) Comparison of the expression of genes with the selected primer pairs by quantitative real-time RT-PCR in the WT and mterf6-5. Error bars indicate standard deviations for triplicates.

Figure 5

Chromatin immunoprecipitation quantitative PCR (ChIP-qPCR) analysis of the DNA binding activity of mTERF6 in vivo. (A) Immunoprecipitation and immunoblot analysis of mTERF6. The samples consisting of the total leaf protein extract from pMTERF6::MTERF6.1-FLAG transgenic plants (mterf6-5 background) were immunoprecipitated with the FLAG antibody-conjugated protein G beads (IP+) or naked protein G beads (IP−) as a control. The immunoblot analysis was performed with the FLAG antibody. The lane “mTERF6.1-FLAG” is the input. (B) The position of the designed primer pairs for ChIP-qPCR analysis. (C) DNA immunoprecipitation analysis. The chloroplasts fixed with formaldehyde (FA) were extracted from the pMTERF6::MTERF6.1-FLAG transgenic plants, then all of the DNA was sonicated into short fragments under specific conditions and incubated with the FLAG antibody-conjugated protein G beads. With the interaction between the FLAG antibody and the mTERF6.1-FLAG protein, the associated DNA was immunoprecipitated and analyzed by quantitative real-time RT-PCR. Error bars indicate standard deviations for triplicates.

Figure 6

Electrophoretic mobility shift assay (EMSA) by recombinant mTERF6. (A) Scheme of the probes used in the EMSA assay. Black boxes, white boxes and straight lines represent the exons, introns and interval regions, respectively. The locations and sequences of probes A and B were 5′-end labeled with biotin. Probe A is a double-stranded DNA fragment produced by PCR amplification and purification. Probe B is a single strand DNA. (B) Expression and purification of MBP-mTERF6.1 by SDS-PAGE analysis. Lanes 1 and 2 are the total proteins from the pre-induction cultures and post-induction cultures (induced by 0.4 mM IPTG at 18 °C for 6 h), respectively; lane 3 is the soluble extract induced (by 0.4 mM IPTG at 18 °C) overnight; lane 4 is the purified recombinant MBP-mTERF6.1 protein. The arrow indicates the expected band (This is a cropped gel. Full-length gels are included in Original pictures of Supplementary Fig. 6B). (C) The binding of MBP-mTERF6.1 to probe A. “fp” represents free probe. (D) The binding of MBP-mTERF6.1 to probe B. (E) Comparison of the exact binding sites of rpoA (3′-end) and trnI.2 and the 3′-end DNA sequences of other genes (rbcL, ycf5 and petD). Red “*” indicates that the analyzed DNA sequences of ycf5 are inside its downstream adjacent gene.