PEP associates with large regions of the chloroplast chromosome
At least three RNA polymerases are active in chloroplasts of dicotyledonous plants: PEP and two versions of NEP (the latter encoded by the nuclear genes RpoTp and RpoTmp). Attempts have been made to dissect the targets of these genes genetically: tests of RNA accumulation in polymerase-deficient mutants identified target genes of PEP and NEP (Hajdukiewicz et al., 1997; Kuhn et al., 2009). However, the results of such analyses must be treated with caution as knockouts of PEP as well as of the two nuclear-encoded polymerases have strong phenotypic effects, i.e. albinism in the case of PEP (Allison et al., 1996), and leaf deformations, bleaching and slower growth in the case of NEP (Baba et al., 2004; Hricova et al., 2006; Kuhn et al., 2009). Such massive secondary effects may interfere with transcription by the remaining RNA polymerase activities. For example, loss of chloroplast gene expression in general is known to generate so-called retrograde signals that lead to a massive reprogramming of nuclear gene expression (Woodson and Chory, 2008), including the NEP genes (Emanuel et al., 2004, 2006). Thus, modifying PEP activity will alter NEP activity as well (Zhelyazkova et al., 2012). Similarly, as chloroplast PEP genes are transcribed by NEP, it is difficult to obtain clean NEP phenotypes even in double knockouts of the two NEP genes RPOTmp and RPOTp. Finally, knockouts of the chloroplast- and mitochondrial-targeted RPOTmp protein have been shown to lead to compromised mitochondrial respiratory chain activity by obliterating complex I expression (Kuhn et al., 2009). As defective mitochondria are known to affect chloroplast gene expression (Leister, 2005; Van Aken and Whelan, 2012), this may also lead to problems identifying the true targets of the plastid RNA polymerases. These complex inter-relationships complicate the analysis of chloroplast transcription mutants.
A more direct approach to characterize the genes served by a specific polymerase involves ChIP experiments. Co-precipitation of DNA together with a particular RNA polymerase may provide strong evidence for a functional interaction, although it does not deliver proof of polymerase activity at the respective site. This technique has been widely used to characterize bacterial RNA polymerases (Wade et al., 2007) as well as eukaryotic polymerases, particularly RNA polymerase II (Sims et al., 2004). For the bacterial RNA polymerase, a large number of studies performed under various conditions and genetic backgrounds have uncovered regulatory roles for this enzyme (Wade et al., 2007).
For the chloroplast transcription machinery, first attempts to characterize bound DNA consisted of visualizing restriction-digested DNA isolated from TAC preparations. This demonstrated that the entire plastid chromosome is present in the TAC, but did not lead to a more refined view of transcribed DNA (Reiss and Link, 1985). Recently, immunoprecipitations of chloroplast RpoA using an antibody raised against almost the entire RpoA protein were performed, and bound DNA was detected by quantitative PCR for 14 probes (Yagi et al., 2012). Enrichment was found for probes detecting psbA, rbcL, psbD, the rrn operon, trnDY and psaA. Similarly, enrichment of psbA, psaA, rbcL and the rrn operon was found in immunoprecipitations of RpoB (Zhong et al., 2013). Likewise, ChIP/quantitative PCR of sigma factor 5 identified psbA, psbC/D, psbA/B and psbB/T as target regions (Noordally et al., 2013), and ChIP/quantitative PCR of sigma factor 1 identified rbcL, psbB/T, psaA/B, clpP and psbEFLJ as target regions (Hanaoka et al., 2012). This suggests that PEP is associated with several photosynthetic genes.
Our ChIP-on-chip approach confirms these targets and puts these genes in the context of the entire plastome, thus pinpointing the main targets of PEP. A technical benefit of our approach is use of the HA epitope tag, which enables highly specific immunoprecipitations that pull-down exclusively RpoA:HA; no cross-reactions were seen in protein gel-blot analysis of pelleted proteins or total protein extracts from wild-type plants. We also provide evidence that the precipitated PEP is intact, since the amount of precipitated RpoA protein appears to be similar to the other PEP subunits identified, which would reflect approximately the stoichiometry of PEP (A2:B:C1:C2). We conclude that most of the RpoA precipiated is probably within a PEP complex. Finally, as we are performing direct chemical labeling of DNA, our technique lacks any enzymatic reaction, unlike ChIP/quantitative PCR, which uses target amplification with the risk of introducing enzymatic biases.
The top target regions of PEP correspond to the rRNA operon and several photosynthetic genes. In fact, two of the genes identified as preferred targets for PEP were identified by all studies mentioned above: psbA and psaA/B. In addition, the psbC/D operon, rbcL and trnV–trnM are within the top five peaks in Figure 5(a). These results mirror the most abundant RNAs in tobacco, and therefore further confirm that PEP is the most important RNA polymerase for plastid transcription at least in green tissue (Legen et al., 2002; Zhelyazkova et al., 2012). The results also show that, in green tissue, the efficacy of PEP–DNA co-enrichment (Figure 5) reflects transcriptional activity in most cases (Legen et al., 2002).
Light-dependent PEP–DNA association
As we have investigated binding to DNA in the light and the dark, we are able to identify which are the main light-regulated genes at the transcriptional level. On a global scale, we determined how much of the chloroplast genetic information is subject to PEP-dependent light activation. We found that approximately 75% of the chloroplast chromosome is more strongly associated with PEP in the light than in the dark. Such global changes may be brought about by light-dependent activation of PEP mediated by changes in the phosphorylation state of core PEP subunits as well as sigma factors (Baginsky et al., 1997; Isono et al., 1997; Kanamaru et al., 1999; Tan and Troxler, 1999).
What are the main genes associated with PEP in the light? Light regulation of chloroplast genes at the level of transcription of individual genes has been known for a long time, including for the genes rbcL, psbA, rrn16 (DuBell and Mullet, 1995; Chun et al., 2001), psbD (Mochizuki et al., 2001; Thum et al., 2001) and psaB (DuBell and Mullet, 1995). Our analysis provides complementary and direct evidence that PEP is important for light regulation of these key genes in photosynthesis. In addition, our analysis reveals that genes for ribosomal RNA and tRNAs are among the main PEP targets in the light (including the rrn operon, the trnV (UAC) to trnM (CAU) region, the psbZ–trnG–trnfM region, and the trnR (ACG) trnN (GUU) orf75 region; see Table S2). This suggests that transcription of RNA components of the chloroplast translational apparatus is switched on in the light relative to the dark. Indeed, moderate increases in transcriptional activity of rRNA genes upon increasing light have been described in Sinapis alba (Baena-Gonzalez et al., 2001). The reason for light-induced PEP association with rRNA and tRNA genes in green tissue may be to replenish ribosome and tRNA pools that turnover between day and night. The recent finding that the PEP is in particular important for tRNA expression further supports this idea (Williams-Carrier et al., 2013). A time-resolved ChIP-on-chip analysis performed at the onset of daylight is necessary to clarify this issue.
However, these results are in contrast to those of studies on de-etiolation, which showed no increase in transcription or even a decrease in transcriptional activity for rDNA (Rodermel and Bogorad, 1985; Zhu et al., 1985; Deng and Gruissem, 1987; Klein and Mullet, 1990). Possibly, harvesting light-grown seedlings after a slightly extended dark period (as performed here) does not compare directly to the situation of etiolated tissue starting to green. In fact, several light regulatory mechanisms only occur in green, mature leaves (Aro et al., 1993), and action spectra as well as photoreceptors are different between etioplasts and chloroplasts (e.g. Fluhr et al., 1986; Chory et al., 1989; Quail, 1994). In addition, preparations of RNA polymerizing activities from chloroplasts and etioplasts have different protein compositions (Reiss and Link, 1985; Pfannschmidt and Link, 1994; Suck et al., 1996; Majeran et al., 2011). Thus, our data and data obtained from de-etiolation systems are not directly comparable.
Are there membrane anchors for PEP?
Two Spinacia oleracea NEP enzymes have been reported to be membrane-associated in mature chloroplasts in a non-DNA-mediated manner (Azevedo et al., 2006). Furthermore, in Arabidopsis, the NEP protein encoded by RpoTmp has been shown to be anchored to the thylakoid membrane by a RING-finger protein in response to light signals (Azevedo et al., 2008). No similar light-regulated attachment factor is known for PEP, despite the fact that membrane association has been known for a long time (Reiss and Link, 1985). PEP subunits have been detected in preparations of nucleoids and the TAC (Suck et al., 1996; Pfalz et al., 2006; Majeran et al., 2011), both of which are known to be localized to membranes. We quantified membrane association and demonstrate that more than 95% of PEP is membrane-bound and that this does not change in a light-dependent fashion. It will be interesting to investigate the mechanisms for PEP attachment to membranes in comparison to RING-finger protein-based attachment of NEP (RPOTmp; Azevedo et al., 2008).
We suspected that PEP may be linked to membranes via DNA, as chloroplast DNA is membrane-attached (Miyamura et al., 1986; Liu and Rose, 1992; Sato et al., 1993, 1999). However, digestion of DNA did not change the localization of PEP, at least in young tissue, suggesting that there is a permanent non-DNA anchor that retains PEP at the membrane irrespective of light conditions. The situation is further complicated by the finding that, in old tissue, PEP may be partially solubilized by digesting the DNA. Whether this is simply due to a change in transcriptional activity or is a property of the unknown membrane anchor remains to be seen. Candidates for attachment factors include the PEND protein and MFP1. PEND, a member of the bZIP protein family, is associated with the inner envelope membrane and is expressed at early stages of plastid development, i.e. at the same time as the nucleoids are found to co-localize with the plastid envelope (Sato et al., 1993, 1998). MFP1 is a coiled-coil protein with a C–terminal DNA-binding domain and is associated with nucleoids in vivo (Jeong et al., 2003). It will be interesting to test whether any of these general DNA-binding proteins directly interact with PEP.