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Chloroplast transcription, untangling the Gordian Knot

2015; Wiley; Volume: 206; Issue: 3 Linguagem: Inglês

10.1111/nph.13388

1469-8137

Peter Kindgren, Åsa Strand,

Plant nutrient uptake and metabolism

'The key question now is to address the order in which the subunits assemble into the multimeric PEP-complex. How do the building blocks fit together?' The promoters used by PEP are similar to the Escherichia coli σ70 promoters and consist of −35 and −10 consensus elements. However, in contrast to the bacterial RNA polymerase, the activity of the PEP transcription machinery depends on additional nuclear encoded components and these additional proteins do not bear any resemblance to bacterial proteins. Considerable effort over a number of years has been devoted to isolating the plastid RNA polymerase complex to identify its associated proteins (reviewed in Pfalz & Pfannschmidt, 2013). Biochemical analyses revealed that the core rpo subunits of PEP are present in both the insoluble RNA polymerase preparation called transcriptionally active chromosome (TAC), and the soluble RNA polymerase preparation (sRNAP) (Pfalz & Pfannschmidt, 2013). Gel filtration and mass spectrometry analysis from different plant species including spinach, tobacco, mustard and Arabidopsis (reviewed in Yu et al., 2014) concluded that the TAC complex contains 43 nuclear-encoded proteins. Ten of those 43 proteins were confirmed to be tightly associated with the PEP core and were therefore named polymerase-associated proteins (PAPs) (Steiner et al., 2011) (Fig. 1). Analysis of mutants of the PAP proteins demonstrated that the loss of these proteins results in albino or pale green phenotypes and severely impaired chloroplast development. The remaining proteins identified in the TAC complexes most likely represent more loosely associated components of the transcriptional machinery (Pfalz et al., 2006), possibly with regulatory functions (Kindgren et al., 2012). The phenotypes of those mutants also display more subtle phenotypes (Pfalz & Pfannschmidt, 2013). Two TAC components, pTAC7 and MurE-like were not identified as true PAPs but based on the phenotype of the respective T-DNA lines in Arabidopsis these two proteins were also proposed to be PAPs (Pfalz & Pfannschmidt, 2013). The PAPs can be divided into different groups dependent on their potential function, such as DNA/RNA metabolism, redox regulation or protection, or completely unknown (Fig. 1) (Yu et al., 2014). The proteins in the first group bind DNA, RNA or both DNA and RNA and are believed to be involved in promoter recognition and transcription initiation, elongation and possibly these proteins link plastid transcription to translation. The second group consist of putative regulatory proteins and the third group are proteins involved in protecting the PEP against reactive oxygen species (ROS). Two proteins, associated with PEP, pTAC6 and AtMurE, have so far unknown functions. Interactions between the different PAP proteins have been identified (Fig. 1) (Gao et al., 2011; Yu et al., 2013). Although pTAC3 was found to be associated with the rpo subunits by immunoprecipitation analysis (Yagi et al., 2012) direct interaction between PAPs and the PEP core subunits has so far not been confirmed. Identifying the PAPs required for functional PEP-mediated transcription represents a major breakthrough towards our understanding of the transcriptional machinery in the chloroplast. However, we still know very little about the specific roles of the individual PAPs and the order in which the subunits assemble into the multimeric PEP-complex. To investigate the specific roles of the PAPs Pfalz et al. (2015) performed detailed analysis of the Zmptac12 mutant alleles in maize and determined the localization of ZmpTAC12 in the cell. In contrast to Arabidopsis, maize has two isoforms of ZmpTAC12, but like Arabidopsis ZmpTAC12 showed dual localization to plastids and the nucleus, and within the plastids, both isoforms are present in the PEP-complex. Similar to other pTAC proteins ZmpTAC12 exhibits both ssRNA and ssDNA-binding activity (Fig. 1). To address a possible role of specific PAP subunits in the assembly of the multimeric PEP-complex, Pfalz et al. (2015) very elegantly took advantage of maize mutants lacking the different PEP-complex subunits pTAC12, pTAC2, pTAC10, and MurE. PEP-complex assembly was explored by blue native PAGE analysis and sucrose-gradient sedimentation of total leaf extracts. All the PAP components investigated were clearly shown to be required for accumulation of a fully assembled PEP-complex. This result strongly suggests that the PAPs act as critical building blocks in the assembly process of a functional active PEP complex (Pfalz et al., 2015, Fig. 5). Assembly of the PEP-complex occurs early during chloroplast development, and Zea mays is an excellent model to study the process of chloroplast development because the leaf contains a natural developmental gradient; with the youngest cells, containing pro-plastids, at the base and the oldest cells, containing mature chloroplasts, at the tip (Baker & Leech, 1977). The ZmpTAC12 protein demonstrated a clear development dependent accumulation with reduced concentrations at the leaf tip compared to the base supporting an important role of pTAC12 for PEP assembly during early chloroplast development (Pfalz et al., 2015, Fig. 1e). pTAC12 is clearly dually localized to both the chloroplasts and nuclei, as shown both in Arabidopsis (Chen et al., 2010) and in maize (Pfalz et al., 2015, Fig. 2a). In Arabidopsis pTAC12 was recently identified as a novel signaling component in the phytochrome response referred to as HEMERA (HMR) (Chen et al., 2010). In the nucleus, photoactivated phytochrome was shown to directly bind HMR/pTAC12 and promote photomorphogenesis through degradation of PIFs (Galvao et al., 2012). Curiously, the nuclear pTAC12 forms in maize and Arabidopsis have similar molecular mass as the plastidic proteins (Pfalz et al., 2015, Fig. 2a; Chen et al., 2010). Whirly1 is another protein associated to PEP that has been shown to be dually located where the protein also has the same size in chloroplasts and the nucleus. This suggests that a translocation towards the nucleus occurs after processing of the transit peptide in the plastids. Indeed, movement of Whirly1 from the chloroplast to the nucleus was demonstrated by inserting a HA-Whirly1 fusion protein into the plastid genome of tobacco and although the tagged protein was synthesized in the plastids, it was detected in the nucleus of the transplastomic tobacco line (Isemer et al., 2012). Detailed investigations of the timing of the localization of the pTAC12 during early light response and chloroplast development could reveal the exciting prospect that pTAC12 might be involved in the coordination of photosynthetic gene expression in the nucleus and in the chloroplast in response to light. Pfalz et al. (2015) have clearly demonstrated that the PAPs pTAC12, pTAC2, pTAC10 and MurE are required for the assembly of a functional PEP complex. During embryo development in Arabidopsis very high expression levels were observed for the genes encoding pTACs (Kremnev & Strand, 2014) suggesting that PEP requires the PAPs already during embryo development, supporting a structural role for the PAPs. The work by Pfalz and others propose that a structural establishment of a functional PEP represents a developmental checkpoint in the establishment of photosynthesis and for proper chloroplast development. If disturbed, it leads to abortion of proper chloroplast biogenesis as confirmed by the severe phenotype of the mutants lacking the PAPs. The key question now for the future is to address the order in which the subunits assemble into the multimeric PEP-complex. How do the building blocks fit together? We know that some subunits interact with each other but do they form structural or functional modules? To untangle this knot we need a system where chloroplast development and the assembly of the PEP complex can easily be followed, such as the maize leaf or greening cell cultures. From an evolutionary perspective it is intriguing that PEP has deviated so far from its bacterial origin. How is it that the nucleus now exerts such control over the transcriptional activities in the chloroplast? The answer could be that it is beneficial to coordinate the early photoreceptor mediated light response in the nucleus with the establishment of the photosynthetic machinery in the chloroplast.