SummaryWe have identi®ed the barley gene and cDNA encoding the plastid phage-type RNA polymerase (RNAP), nuclear-encoded plastid RNAP (RpoTp), and the nearly full-length cDNA of the mitochondrial RNAP, nuclear-encoded mitochondrial RNAP (RpoTm). RpoTp spans more than 9000 nt, consists of 19 exons and 18 introns, gives rise to a 3632-nt mRNA and is localized to the long arm of chromosome 1 (7H). The length of the deduced polypeptide is 948 residues. The mRNA levels of RpoTp and RpoTm were determined in roots and primary leaf sections of 7-day-old barley seedlings of the albostrians mutant, which were either phenotypically normal and exhibited a gradient of chloroplast development, or contained ribosome-de®-cient undifferentiated plastids. Transcript levels of RpoTp and RpoTm in almost all sections reached higher concentrations in plastid ribosome-de®cient leaves than in the wild-type material, except in the most basal part of the leaf. These data indicate a role of plastid-to-nucleus signalling in the expression of the two RpoT genes. The mRNA levels of the plastid genes, b-subunit of plastid-encoded RNAP (rpoB), proteolytic subunit of the Clp protease (clpP) and ribosomal protein Rpl2 (rpl2), all known to be transcribed by the nuclearencoded RNAP (NEP), followed closely the pattern of RpoTp mRNA accumulation, strongly suggesting that RpoTp and NEP are identical. Transcripts of RpoTm and RpoTm-transcribed mitochondrial genes cytochrome oxidase subunit 2 (coxII) and ATPase subunit 9 (atp9) accumulated to the highest levels in the most basal parts of the leaf and declined considerably towards the leaf tip with a pronounced reduction in green versus white leaves. Our data revealed a marked in¯uence of the developmental stage of the plastid on the expression and activity of organellar phage-type RNAPs and their target genes. Thus, interorganellar cross-talk in the regulated expression of nuclear-encoded plastid and mitochondrial RNAP genes might be a key element governing the concerted expression of genes located within plastids, mitochondria and the nucleus of the plant cell.

Although chloroplast genomes are small, the transcriptional machinery is very complex in plastids of higher plants. Plastidial genes of higher plants are transcribed by plastid-encoded (PEP) and nuclear-encoded RNA polymerases (NEP). The nuclear genome of Arabidopsis contains two candidate genes for NEP, RpoTp and RpoTmp, both coding for phage-type RNA polymerases. We have analyzed the use of PEP and NEP promoters in transgenic Arabidopsis lines with altered RpoTp activities and in Arabidopsis RpoTp insertion mutants lacking functional RpoTp. Low or lacking RpoTp activity resulted in an albino phenotype of the seedlings, which normalized later in development. Differences in promoter usage between wild type and plants with altered RpoTp activity were also most obvious early in development. Nearly all NEP promoters were used in plants with low or lacking RpoTp activity, though certain promoters showed reduced or even increased usage. The strong NEP promoter of the essential ycf1 gene, however, was not used in mutant seedlings lacking RpoTp activity. Our data provide evidence for NEP being represented by two phage-type RNA polymerases (RpoTp and RpoTmp) that have overlapping as well as gene-specific functions in the transcription of plastidial genes.

Arabidopsis thaliana possesses three RpoT genes which encode three different phage-type RNA polymerases with yet unknown function in organelle transcription: RpoTm and RpoTp, imported into mitochondria and plastids, respectively, and RpoTmp, co-targeted into both organelles. Expression of the RpoT genes was analyzed by quantitative RT-PCR, histochemical beta-glucuronidase (GUS) assays and in situ hybridization. Transcripts of all three RpoT genes accumulated to very low amounts in all organs. Surprisingly, RT-PCR revealed their highest levels in flower tissues. RpoTm transcripts were the most abundant in all organs, except mature leaves, in which RpoTp transcripts showed the highest accumulation. In the developing seedling, RpoTm::GUS and RpoTmp::GUS expression precedes that of RpoTp::GUS, the latter showing up only 7 days after germination. The RpoTm and RpoTmp promoters expressed GUS mainly in meristematic and mitochondria-rich cells such as the distal part of the root and companion cells flanking the phloem, whereas RpoTp::GUS activity was found in green tissues as the parenchyme cells of young leaves, the primary cortex of the stem, and sepals of buds and young flowers. Sites of GUS expression coincided spatially with those of in situ hybridization. Our data demonstrate an overlapping expression pattern of RpoTm and RpoTmp, and a completely differing pattern of RpoTp expression. The results suggest that RpoTm and RpoTmp recognize different types of mitochondrial promoters. The plastid polymerase RpoTp might play a major role in green tissue, i.e. in chloroplast transcription, whilst the dual-targeted RpoTmp in plastids should function mainly in the transcription of genes in non-green types.

The barley plastome mutant CL2 (cytoplasmic line 2) carries a point mutation in the infA gene, a homologue of the bacterial gene for the conserved translation initiator factor 1 (IF1). The function of infA in plastids is not known. The mutation in CL2 leads to a temporal chlorophyll deficiency in the primary leaf blade that is normalised in the basal and middle parts during further development. We have compared the expression of selected nuclear and plastid genes in different parts of primary leaves of CL2 and wild-type and found no indication for an adverse effect of the mutation on plastidial transcription. We observed an enhanced expression of RpoTp (encoding the phage-type nuclear-encoded plastid RNA polymerase) suggested to be caused by retrograde plastid signalling. Decreased amounts of plastid rRNA in basal and top sections are in agreement with the idea that the mutation in infA leads to a time- and position-dependent defect of plastid translation that causes a delay in plastid development. The normalisation of the phenotype in the middle section of CL2 leaves correlates with wild-type levels of chloroplast 16S rRNA and RbcL and increased expression of plastid housekeeping genes. The normalisation was not observed in cells at the tip of CL2 leaves suggesting different ways of regulating chloroplast development in cells at the tip of primary barley leaves as compared with other leaf sections.