Nuclear-encoded Phage-type RNA Polymerase Research Articles

Albino seedling lethality 4; Chloroplast 30S Ribosomal Protein S1 is Required for Chloroplast Ribosome Biogenesis and Early Chloroplast Development in Rice

BackgroundRibosomes responsible for transcription and translation of plastid-encoded proteins in chloroplasts are essential for chloroplast development and plant growth. Although most ribosomal proteins in plastids have been identified, the molecular mechanisms regulating chloroplast biogenesis remain to be investigated.ResultsHere, we identified albinic seedling mutant albino seedling lethality 4 (asl4) caused by disruption of 30S ribosomal protein S1 that is targeted to the chloroplast. The mutant was defective in early chloroplast development and chlorophyll (Chl) biosynthesis. A 2855-bp deletion in the ASL4 allele was verified as responsible for the mutant phenotype by complementation tests. Expression analysis revealed that the ASL4 allele was highly expressed in leaf 4 sections and newly expanded leaves during early leaf development. Expression levels were increased by exposure to light following darkness. Some genes involved in chloroplast biogenesis were up-regulated and others down-regulated in asl4 mutant tissues compared to wild type. Plastid-encoded plastid RNA polymerase (PEP)-dependent photosynthesis genes and nuclear-encoded phage-type RNA polymerase (NEP)-dependent housekeeping genes were separately down-regulated and up-regulated, suggesting that plastid transcription was impaired in the mutant. Transcriptome and western blot analyses showed that levels of most plastid-encoded genes and proteins were reduced in the mutant. The decreased contents of chloroplast rRNAs and ribosomal proteins indicated that chloroplast ribosome biogenesis was impaired in the asl4 mutant.ConclusionsRice ASL4 encodes 30S ribosomal protein S1, which is targeted to the chloroplast. ASL4 is essential for chloroplast ribosome biogenesis and early chloroplast development. These data will facilitate efforts to further elucidate the molecular mechanism of chloroplast biogenesis.

Young Seedling Stripe1 encodes a chloroplast nucleoid-associated protein required for chloroplast development in rice seedlings.

Young Seedling Stripe1 (YSS1) was characterized as an important regulator of plastid-encoded plastid RNA polymerase (PEP) activity essential for chloroplast development at rice seedling stage. Chloroplast development is coordinately regulated by plastid- and nuclear-encoding genes. Although a few regulators have been reported to be involved in chloroplast development, new factors remain to be identified, given the complexity of this process. Here, we report the characterization of a temperature-sensitive young seedling stripe1 (yss1) rice mutant, which develops striated leaves at the seedling stage, particularly in leaf 3, but produces wild-type leaves in leaf 5 and onwards. The chlorotic leaves have decreased chlorophyll (Chls) accumulation and impaired chloroplast structure. Positional cloning combined with sequencing demonstrated that aberrant splicing of the 8th intron in YSS1 gene, due to a single nucleotide deletion around splicing donor site, leads to decreased expression of YSS1 and accumulation of an 8th intron-retained yss1 transcript. Furthermore, complementation test revealed that downregulation of YSS1 but not accumulation of yss1 transcript confers yss1 mutant phenotype. YSS1 encodes a chloroplast nucleoid-localized protein belonging to the DUF3727 superfamily. Expression analysis showed that YSS1 gene is more expressed in newly expanded leaves, and distinctly up-regulated as temperatures increase and by light stimulus. PEP- and nuclear-encoded phage-type RNA polymerase (NEP)-dependent genes are separately down-regulated and up-regulated in yss1 mutant, indicating that PEP activity may be impaired. Furthermore, levels of chloroplast proteins are mostly reduced in yss1 seedlings. Together, our findings identify YSS1 as a novel regulator of PEP activity essential for chloroplast development at rice seedling stage.

Effects of inefficient transcription termination of rbcL on the expression of accD in plastids of Arabidopsis thaliana.

The plastid accD gene encodes one subunit of a multimeric acetyl-CoA carboxylase that is required for fatty acid biosynthesis. In Arabidopsis thaliana, the accD gene is transcribed by the nuclear-encoded phage-type RNA polymerase, and the accumulation of accD transcripts is subjected to a dynamic pattern during chloroplast development. However, the mechanisms underlying the regulation of accD expression remain unknown. Here, we showed that the inefficient transcription termination of rbcL due to the absence of RHON1 impaired the developmental profile of accD, resulting in the constitutive expression of accD during chloroplast development. Moreover, the accumulation of accD transcripts accordingly resulted in an increase in accD protein levels, suggesting that transcript abundance is critical for accD gene production. Our study demonstrates that the interplay between accD and upstream rbcL regulates the expression of accD and highlights the significance of transcriptional regulation in plastid gene expression in higher plants.

Plastid encoded RNA polymerase activity and expression of photosynthesis genes required for embryo and seed development in Arabidopsis.

Chloroplast biogenesis and function is essential for proper plant embryo and seed development but the molecular mechanisms underlying the role of plastids during embryogenesis are poorly understood. Expression of plastid encoded genes is dependent on two different transcription machineries; a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. However, the division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear. We show here that PLASTID REDOX INSENSITIVE 2 (PRIN2) and CHLOROPLAST STEM-LOOP BINDING PROTEIN 41 kDa (CSP41b), two proteins identified in plastid nucleoid preparations, are essential for proper plant embryo development. Using Co-IP assays and native PAGE we have shown a direct physical interaction between PRIN2 and CSP41b. Moreover, PRIN2 and CSP41b form a distinct protein complex in vitro that binds DNA. The prin2.2 and csp41b-2 single mutants displayed pale phenotypes, abnormal chloroplasts with reduced transcript levels of photosynthesis genes and defects in embryo development. The respective csp41b-2prin2.2 homo/heterozygote double mutants produced abnormal white colored ovules and shrunken seeds. Thus, the csp41b-2prin2.2 double mutant is embryo lethal. In silico analysis of available array data showed that a large number of genes traditionally classified as PEP dependent genes are transcribed during early embryo development from the pre-globular stage to the mature-green-stage. Taken together, our results suggest that PEP activity and consequently the switch from NEP to PEP activity, is essential during embryo development and that the PRIN2-CSP41b DNA binding protein complex possibly is important for full PEP activity during this process.

The primary transcriptome of barley chloroplasts: numerous noncoding RNAs and the dominating role of the plastid-encoded RNA polymerase.

Gene expression in plastids of higher plants is dependent on two different transcription machineries, a plastid-encoded bacterial-type RNA polymerase (PEP) and a nuclear-encoded phage-type RNA polymerase (NEP), which recognize distinct types of promoters. The division of labor between PEP and NEP during plastid development and in mature chloroplasts is unclear due to a lack of comprehensive information on promoter usage. Here, we present a thorough investigation into the distribution of PEP and NEP promoters within the plastid genome of barley (Hordeum vulgare). Using a novel differential RNA sequencing approach, which discriminates between primary and processed transcripts, we obtained a genome-wide map of transcription start sites in plastids of mature first leaves. PEP-lacking plastids of the albostrians mutant allowed for the unambiguous identification of NEP promoters. We observed that the chloroplast genome contains many more promoters than genes. According to our data, most genes (including genes coding for photosynthesis proteins) have both PEP and NEP promoters. We also detected numerous transcription start sites within operons, indicating transcriptional uncoupling of genes in polycistronic gene clusters. Moreover, we mapped many transcription start sites in intergenic regions and opposite to annotated genes, demonstrating the existence of numerous noncoding RNA candidates.

Molecular Biology of the Plant Mitochondrion

Referee: Prof. C. J. Leaver, FRS, FRSE, Department of Plant Sciences, University of Oxford, South Parks Road, Oxford OX13RB UKThe comparison of the mitochondrial genomes in plants shows that the gene complement can slightly vary between individual species. The differentially evolving DNA arrangements and sequences offer unique time windows upon the phylogeny of embryophytes. Genes in plant mitochondria are most often transcribed from their own promoters, many of which conform to a conserved type, probably the point of entry for a nuclear-encoded phage-type RNA polymerase. Primary transcripts undergo various RNA processing steps during maturation, including RNA editing, cis- and trans-intron splicing and 5′- and 3′-trimming. RNA half-life regulation via 3′-mediated access for nucleases is an important factor in determining RNA availiability. This includes mRNA destabilization by the addition of non-encoded polyA tails. Present research interests focus on elucidating the regulatory and the metabolic networks integrating the organelle into the plant cell and we outline our visions of the future directions of research in plant mitochondria.