The two RNA polymerases encoded by the nuclear and the plastid compartments transcribe distinct groups of genes in tobacco plastids

Abstract

deleting the rpoB gene encoding the essential β subunit 1 Present address: University of Nebraska, Lincoln, of the tobacco PEP, we established the existence of a N209 Beadle Center, Lincoln, NE 68588-0664, USA second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants (Allison et al., 1996). Corresponding authorDeletion of rpoB yielded photosynthetically defective, pigment-deficient plants. An examination of ΔrpoB plants The plastid genome in photosynthetic higher plants encodes subunits of an Escherichia coli-like RNA polyrevealed proplastid-like structures containing low levels of mRNAs for the photosynthetic genes rbcL, psbA and merase (PEP) which initiates transcription from E.coli σ 70 -type promoters. We have previously established psbD due to the lack of PEP promoter activity. In wildtype tobacco leaves, the ribosomal RNA operon (rrn) is the existence of a second nuclear-encoded plastid RNA polymerase (NEP) in photosynthetic higher plants. We transcribed by PEP. Interestingly, in the ΔrpoB plants the rrn mRNA accumulated close to wild-type levels due to report here that many plastid genes and operons have at least one promoter each for PEP and NEP (Class II transcription by NEP acting at a downstream non-σ 70 -type promoter. The rRNA operon is the first plastid transcription unit). However, a subset of plastid genes, including photosystem I and II genes, are transcribed transcription unit for which a promoter for both PEP and NEP was identified. from PEP promoters only (Class I genes), while in some instances (e.g. accD) genes are transcribed excluWe report here that the rRNA operon is not unique, but represents a class of plastid transcription units which have sively by NEP (Class III genes). Sequence alignment identified a 10 nucleotide NEP promoter consensus at least one promoter each for PEP and NEP. These genes or operons have a potential for expression by either of around the transcription initiation site. Distinct NEP and PEP promoters reported here provide a general the two plastid RNA polymerases. Furthermore, some genes are transcribed by only one of the two RNA mechanism for group-specific gene expression through recognition by the two RNA polymerases.polymerases. We propose that transcription by NEP and PEP, through recognition of distinct promoters, is a general Keywords: Nicotiana tabacum/nuclear-encoded plastid RNA polymerase (NEP)/plastid-encoded RNA mechanism of group-specific gene regulation during chloroplast development. A tentative NEP promoter conpolymerase (PEP)/plastid gene expression/rpoB deficient mutant sensus is derived by the alignment of the transcription initiation sites.

A Functional Component of the Transcriptionally Active Chromosome Complex, Arabidopsis pTAC14, Interacts with pTAC12/HEMERA and Regulates Plastid Gene Expression

“…Recent reports have revealed that mutations in several of the genes encoding TAC components cause deficiencies in PEP-dependent transcription (Pfalz et al, 2006;Garcia et al, 2008;Myouga et al, 2008). As shown in Figure 5, real-time RT-PCR and northern-blot hybridization showed that the expression pattern of plastid genes in ptac14-1 mutants resembles that observed in Drpo mutants (Serino and Maliga, 1998;Krause et al, 2000) and in mutants with lesions in PEP function (Hess et al, 1993;Allison et al, 1996;Hajdukiewicz et al, 1997;Chateigner-Boutin et al, 2008;Chi et al, 2008). These results suggest that pTAC14 is indispensable for the proper function of the PEP transcription machinery.…”

“…A few genes, mostly encoding components of the transcription/translation apparatus, are exclusively transcribed by NEP (class III genes), and nonphotosynthetic housekeeping genes are mostly transcribed by both PEP and NEP (class II genes). NEP promoters are more active in the youngest, nongreen tissues early in leaf development, while PEP increases its activity during the maturation of chloroplasts (Allison et al, 1996;Hajdukiewicz et al, 1997). The complexity of promoter specificity and chloroplast transcription initiated by PEP is further increased by multiple nucleus-encoded s factors, whose expression is spatially and temporally regulated by environmental cues (Hess and Börner, 1999;Allison, 2000;Shiina et al, 2005;Loschelder et al, 2006).…”

High efficiency plastid transformation in potato and regulation of transgene expression in leaves and tubers by alternative 5′ and 3′ regulatory sequences

“…These results support those obtained in expression profile analyses with total and polysomal plastid RNAs, which indicated clpP as one of the less downregulated genes in tubers compared to leaves (Valkov et al 2009). clpP has multiple PEP/NEP promoters (Hajdukiewicz et al 1997), but, in tubers, all native transcripts initiated from the strong -53 NEP promoter (Valkov et al 2009). However, based on GFP protein accumulation in leaves, tubers and petals, it is clear that the relative performance of rrn and clpP regulatory sequences, and their potential applicability in plastid transformation approaches, vary with plastid types.…”

“…These differences in transcript accumulation in tubers were mainly attributed to differential transcript stability (Brosch et al 2007;Valkov et al 2009). Although both the eubacterial-type (PEP) and phage-type (NEP) RNA polymerases were shown to be active in non-green plastids (Brosch et al 2007;Kahlau and Bock 2008;Valkov et al 2009), genes with relatively higher levels of transcripts in tubers (e.g., rrn, clpP, accD, ycf1, ycf2) contain either NEP or multiple PEP and NEP promoters (Hajdukiewicz et al 1997;Legen et al 2002;Valkov et al 2009), suggesting the involvement of NEP in tuber amyloplast gene expression. Indeed, recent data suggest that the two polymerases do not simply mediate gene classspecific transcription in different cells or plastid types, but differential processing, stability, and accumulation of the resulting transcripts and polypeptides (Legen et al 2002;Cahoon et al 2004;Zoschke et al 2007) are involved in regulating gene expression.…”

CIA2 Coordinately Up-Regulates Protein Import and Synthesis in Leaf Chloroplasts

“…The sequences of the cauliflower mosaic virus 35S promoter and NOS terminator from vector pBI121 (Invitrogen) and the coding sequence of CIA2 PCR amplified from Col cDNA were ligated into the binary vector pPZP221 (Hajdukiewicz et al, 1997) to create plasmid pCS146. The cia2 mutant was transformed with pCS146 using the floral dipping method (Clough and Bent, 1998), mediated by Agrobacterium tumefaciens strain GV3101.…”