Nucleus-encoded RNA Polymerase Research Articles

Accumulation of the RNA polymerase subunit RpoB depends on RNA editing by OsPPR16 and affects chloroplast development during early leaf development in rice.

Summary Plastid‐encoded genes are coordinately transcribed by the nucleus‐encoded RNA polymerase (NEP) and the plastid‐encoded RNA polymerase (PEP). Resulting primary transcripts are frequently subject to RNA editing by cytidine‐to‐uridine conversions at specific sites. The physiological role of many editing events is largely unknown.Here, we have used the CRISPR/Cas9 technique in rice to knock out a member of the PLS‐DYW subfamily of pentatricopeptide repeat (PPR) proteins.We found that OsPPR16 is responsible for a single editing event at position 545 in the chloroplast rpoB messenger RNA (mRNA), resulting in an amino acid change from serine to leucine in the β‐subunit of the PEP. In striking contrast to loss‐of‐function mutations of the putative orthologue in Arabidopsis, which were reported to have no visible phenotype, knockout of OsPPR16 leads to impaired accumulation of RpoB, reduced expression of PEP‐dependent genes, and a pale phenotype during early plant development. Thus, by editing the rpoB mRNA, OsPPR16 is required for faithful plastid transcription, which in turn is required for Chl synthesis and efficient chloroplast development.Our results provide new insights into the interconnection of the finely tuned regulatory mechanisms that operate at the transcriptional and post‐transcriptional levels of plastid gene expression.

FRUCTOKINASE-LIKE PROTEIN 1 interacts with TRXz to regulate chloroplast development in rice.

Chloroplast genes are transcribed by the plastid-encoded RNA polymerase (PEP) or nucleus-encoded RNA polymerase. FRUCTOKINASE-LIKE PROTEINS (FLNs) are phosphofructokinase-B (PfkB)-type carbohydrate kinases that act as part of the PEP complex; however, the molecular mechanisms underlying FLN activity in rice remain elusive. Previously, we identified and characterized a heat-stress sensitive albino (hsa1) mutant in rice. Map-based cloning revealed that HSA1 encodes a putative OsFLN2. Here, we further demonstrated that knockdown or knockout of the OsFLN1, a close homolog of HSA1/OsFLN2, considerably inhibits chloroplast biogenesis and the fln1 knockout mutants, created by clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associate protein 9, exhibit severe albino phenotype and seedling lethality. Moreover, OsFLN1 localizes to the chloroplast. Yeast two-hybrid, pull-down and bimolecular fluorescence complementation experiments revealed that OsFLN1 and HSA1/OsFLN2 interact with THIOREDOXINZ (OsTRXz) to regulate chloroplast development. In agreement with this, knockout of OsTRXz resulted in a similar albino and seedling lethality phenotype to that of the fln1 mutants. Quantitative reverse transcription polymerase chain reaction and immunoblot analysis revealed that the transcription and translation of PEP-dependent genes were strongly inhibited in fln1 and trxz mutants, indicating that loss of OsFLN1, HSA1/OsFLN2, or OsTRXz function perturbs the stability of the transcriptionally active chromosome complex and PEP activity. These results show that OsFLN1 and HSA1/OsFLN2 contribute to chloroplast biogenesis and plant growth.

EMB2738, which encodes a putative plastid-targeted GTP-binding protein, is essential for embryogenesis and chloroplast development in higher plants.

In higher plants, chloroplasts carry out many important functions, and normal chloroplast development is required for embryogenesis. Numerous chloroplast-targeted proteins involved in embryogenesis have been identified. Nevertheless, their functions remain unclear. In this study, a chloroplast-localized protein, EMB2738, was reported to be involved in Arabidopsis embryogenesis. EMB2738 knockout led to defective embryos, and the embryo development in emb2738 was interrupted after the globular stage. Complementation experiments identified the AT3G12080 locus as EMB2738. Cellular observation indicated that severely impaired chloroplast development was observed in these aborted embryos. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) analysis showed that chloroplast-encoded photosynthetic genes, which are transcribed by plastid-encoded RNA polymerase (PEP), are predominantly decreased in defective embryogenesis, compared with those in the wild-type. In contrast, genes encoding PEP core subunits, which are transcribed by nucleus-encoded RNA polymerase (NEP), were increased. These results suggested that the knockout of EMB2738 strongly blocked chloroplast-encoded photosynthesis gene expression in embryos. Silencing of the EMB2738 orthologue in tobacco through a virus-induced genome silencing technique resulted in an albinism phenotype, vacuolated chloroplasts and decreased PEP-dependent plastid transcription. These results suggested that NtEMB2738 might be involved in plastid gene expression. Nevertheless, genetic analysis showed that the NtEMB2738 coding sequence could not fully rescue the defective embryogenesis of the emb2738 mutant, which suggested functional divergence between NtEMB2738 and EMB2738 in embryogenesis. Taken together, these results indicated that both EMB2738 and NtEMB2738 are involved in the expression of plastid genes in higher plants, and there is a functional divergence between NtEMB2738 and EMB2738 in embryogenesis.

A Major Role for the Plastid-Encoded RNA Polymerase Complex in the Expression of Plastid Transfer RNAs

Chloroplast transcription in land plants relies on collaboration between a plastid-encoded RNA polymerase (PEP) of cyanobacterial ancestry and a nucleus-encoded RNA polymerase of phage ancestry. PEP associates with additional proteins that are unrelated to bacterial transcription factors, many of which have been shown to be important for PEP activity in Arabidopsis (Arabidopsis thaliana). However, the biochemical roles of these PEP-associated proteins are not known. We describe phenotypes conditioned by transposon insertions in genes encoding the maize (Zea mays) orthologs of five such proteins: ZmPTAC2, ZmMurE, ZmPTAC10, ZmPTAC12, and ZmPRIN2. These mutants have similar ivory/virescent pigmentation and similar reductions in plastid ribosomes and photosynthetic complexes. RNA gel-blot and microarray hybridizations revealed numerous changes in plastid transcript populations, many of which resemble those reported for the orthologous mutants in Arabidopsis. However, unanticipated reductions in the abundance of numerous transfer RNAs (tRNAs) dominated the microarray data and were validated on RNA gel blots. The magnitude of the deficiencies for several tRNAs was similar to that of the most severely affected messenger RNAs, with the loss of trnL-UAA being particularly severe. These findings suggest that PEP and its associated proteins are critical for the robust transcription of numerous plastid tRNAs and that this function is essential for the prodigious translation of plastid-encoded proteins that is required during the installation of the photosynthetic apparatus.

Differential regulation of Arabidopsis plastid gene expression and RNA editing in non-photosynthetic tissues

RNA editing is one of the post-transcriptional processes that commonly occur in plant plastids and mitochondria. In Arabidopsis, 34 C-to-U RNA editing events, affecting transcripts of 18 plastid genes, have been identified. Here, we examined the editing and expression of these transcripts in different organs, and in green and non-green seedlings (etiolated, cia5-2, ispF and ispG albino mutants, lincomycin-, and norflurazon-treated). The editing efficiency of Arabidopsis plastid transcripts varies from site to site, and may be specifically regulated in different tissues. Steady state levels of plastid transcripts are low or undetectable in etiolated seedlings, but most editing sites are edited with efficiencies similar to those observed in green seedlings. By contrast, the editing of some sites is completely lost or significantly reduced in other non-green tissues; for instance, the editing of ndhB-149, ndhB-1255, and ndhD-2 is completely lost in roots and in lincomycin-treated seedlings. The editing of ndhD-2 is also completely lost in albino mutants and norflurazon-treated seedlings. However, matK-640 is completely edited, and accD-794, atpF-92, psbE-214, psbF-77, psbZ-50, and rps14-50 are completely or highly edited in both green and non-green tissues. In addition, the expression of nucleus-encoded RNA polymerase dependent transcripts is specifically induced by lincomycin, and the splicing of ndhB transcripts is significantly reduced in the albino mutants and inhibitor-treated seedlings. Our results indicate that plastid gene expression, and the splicing and editing of plastid transcripts are specifically and differentially regulated in various types of non-green tissues.

Editing of accD and ndhF chloroplast transcripts is partially affected in the Arabidopsis vanilla cream1 mutant

The vanilla cream1 (vac1) albino mutant is defective in a gene encoding a chloroplast-localized pentatricopeptide repeat protein of the DYW subgroup. However, the carboxyl-terminal DYW motif is truncated in VAC1. To identify vac1-specific phenotypes, we compared 34 chloroplast RNA editing sites and approximately 90 chloroplast gene expression patterns among wild type, vac1 and another albino mutant ispH, which is defective in the plastid isoprenoid biosynthesis pathway. We found that the editing of accD and ndhF transcripts is partially affected in vac1. In addition, steady-state levels of chloroplast rRNAs are significantly decreased in vac1. The expression of plastid-encoded RNA polymerase transcribed genes is down-regulated, whereas the expression of nucleus-encoded RNA polymerase transcribed genes is up-regulated in vac1. Although the development and function of mutant chloroplasts are severely impaired, steady-state mRNA levels of nucleus-encoded photosynthetic genes are not affected or are only slightly decreased in vac1. The ZAT10 gene encodes a transcription factor and its expression is down-regulated by norflurazon treatment in wild type. This norflurazon effect was not observed in vac1. These results suggest that the VAC1 protein may be involved in plastid-to-nucleus retrograde signaling in addition to its role in chloroplast RNA editing and gene expression. A defect in a key biosynthetic pathway can have many indirect effects on chloroplast gene expression as is seen in the ispH mutant. Similarly, the vac1 mutant has pleiotropic molecular phenotypes and most of which may be indirect effects.

Phage-Type RNA Polymerase RPOTmp Transcribes the rrn Operon from the PC Promoter at Early Developmental Stages in Arabidopsis

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.

Hybrid transcription system for controlled plastid transgene expression

Plastid transformation technologies have developed rapidly over the last few years, reflecting their value in the study of the principal mechanisms of plastid gene expression and commercial interest in using plastids as bioreactors. Application of this technology is still limited by the difficulty of obtaining regulated, selective expression of plastid transgenes. The plastid genome is transcribed by two different types of RNA polymerase. One of them is of the eubacterial type of polymerase, and its subunits are encoded in the plastid genome [plastid-encoded RNA polymerase (PEP)]. The other one is of the phage type and nucleus-encoded [nucleus-encoded RNA polymerase (NEP)]. To obtain selective transgene expression, we have made use of the similarities and differences between the eubacterial and the plastid eubacterial type transcription systems. We created a hybrid transcription system in which the transgene is placed under the control of a eubacterial promoter which does not exist in the plastid genome and which is not recognized by the plastid endogenous transcriptional machinery. Selective transcription of the transgene is achieved by the supply of a chimeric transcription factor that interacts with PEP and directs it specifically to the foreign eubacterial-type transgene promoter. This hybrid transcription system could be used for biotechnological and fundamental research applications as well as in the characterization of the evolutionary differences between the eubacterial and the plastid eubacterial-type transcription systems.

Plastid RNA Polymerases

Plastids have a very interesting transcription apparatus that gives us an opportunity to investigate mono- and multisubunut RNA polymerase interaction under conditions of complex biogenesis of the organelles and the necessity to coordinate the expression of genes located in different cell compartments. The last decade has been a breakthrough in the study of chloroplast RNA polymerases. The most important advances are the discovery of nucleus-encoded RNA polymerase and nuclear gene family for sigma subunits of plastid-encoded RNA polymerase, the obtaining of knockout-mutants on the subunits of plastid-encoded RNA polymerase, and the revelation of this RNA polymerase reorganization during plastid biogenesis. The hypothesis concerning labor division between two plastid RNA polymerases has been proposed. The review focuses on the analysis of modern information about organization, function and evolution of plastid RNA polymerases.

Transcription in chloroplasts and mitochondria: A tale of two polymerases

D espite their eubacterial origin’, chloroplasts and mitochondria employ different strategies for expressing the genetic information they contain (Fig. 1). Chloroplast genomes encode a multicomponent, Eubacteria-like (aJ@‘) core RNA polymerasez. In contrast, mitochondria use a singlepolypeptide, bacteriophage T3/T7like RNA polymerase encoded in the nucleus’. Recently, clues to the evolution of these distinct transcriptional modes have surfaced. Although the plastid-encoded RNA polymerase (PEP)“ has an essential role in transcription in the chloroplast, a second, nucleusencoded, RNA polymerase (NEP)4 has been implicated in chloroplast gene expression (reviewed in Ref. 5). Early work suggested that the NEP (which must be imported into the chloroplast) might be a singlepolypeptide, phage-like RNA polymerase6. Distinct promoter classes for PEP and NEP have been identified in chloroplast DNA (Refs 4,7), with each polymerase functioning to transcribe a subset of chloroplast genes. A candidate NEP gene has now emerged with the recent report8 of two different but related phage-type RNA polymerase genes in the nuclear genome of the dicotyledonous angiosperm Arabidopsis thaliana. These genes, RpoY and RpoZ, encode proteins that share substantial sequence similarity with both fungal (Saccharomyces cerevisiae and Neuvospora cvassa) mitochondrial and bacteriophage (T3, T7 and SP6) RNA polymerases. In experiments in which the recombinant proteins (RPOY and RPOZ, respectively) were incubated with isolated organelles in vitro, RPOY was found to be specifically targeted to and imported into mitochondria, whereas RPOZ was taken up by chloroplasts but not mitochondria. These data strongly suggest that RPOY represents the mitochondrial RNA polymerase, whereas RPOZ represents the previously described nucleus-encoded, chloroplast-localized enzyme (NEP). Further biochemical analysis will be necessary to show definitively that RPOZ functions in chloroplasts; however, the signs are good that this is indeed the case. The degree of sequence conservation between RPOY and RPOZ is not exceptionally high (63% nucleotide identity, 55% amino acid identity); nevertheless, phylogenetic analysis shows that these genes are more closely related to one another than to the homologous fungal mitochondrial RNA polymerase genes*. Moreover, RpoY and RpoZ share the same organization of 19 exons (16 of which are identical in length), with all 18 intron insertion sites precisely conserved with respect to the amino acid sequences. This implies that the two genes are the products of a relatively recent gene duplication (Fig. Id-e). Phage-type (putative mitochondrial) RNA polymerase genes are widely distributed within the eukaryotic lineage9, having been found in eukaryotes that both do and do not contain chloroplasts (Fig. 2). Thus, it is reasonable to postulate that a gene encoding the mitochondrial RNA polymerase was duplicated at some