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SUMMARY Plastids contain their own genomes, which are transcribed by two types of RNA polymerases. One of those enzymes is a bacterial‐type, multi‐subunit polymerase encoded by the plastid genome. The plastid‐encoded RNA polymerase (PEP) is required for efficient expression of genes encoding proteins involved in photosynthesis. Despite the importance of PEP, its DNA binding locations have not been studied on the genome‐wide scale at high resolution. We established a highly specific approach to detect the genome‐wide pattern of PEP binding to chloroplast DNA using plastid chromatin immunoprecipitation–sequencing (ptChIP‐seq). We found that in mature
Arabidopsis thaliana chloroplasts, PEP has a complex DNA binding pattern with preferential association at genes encoding rRNA, tRNA, and a subset of photosynthetic proteins. Sigma factors SIG2 and SIG6 strongly impact PEP binding to a subset of tRNA genes and have more moderate effects on PEP binding throughout the rest of the genome. PEP binding is commonly enriched on gene promoters, around transcription start sites. Finally, the levels of PEP binding to DNA are correlated with levels of RNA accumulation, which demonstrates the impact of PEP on chloroplast gene expression. Presented data are available through a publicly available Plastid Genome Visualization Tool (Plavisto) athttps://plavisto.mcdb.lsa.umich.edu/ . -
Summary A network of peptidases governs proteostasis in plant chloroplasts and mitochondria. This study reveals strong genetic and functional interactions in Arabidopsis between the chloroplast stromal CLP chaperone‐protease system and the PREP1,2 peptidases, which are dually localized to chloroplast stroma and the mitochondrial matrix.
Higher order mutants defective in CLP or PREP proteins were generated and analyzed by quantitative proteomics and N‐terminal proteomics (terminal amine isotopic labeling of substrates (TAILS)).
Strong synergistic interactions were observed between the CLP protease system (
clpr1‐2 ,clpr2‐1 ,clpc1‐1 ,clpt1 ,clpt2) and both PREP homologs (prep1 ,prep2 ) resulting in embryo lethality or growth and developmental phenotypes. Synergistic interactions were observed even when only one of the PREP proteins was lacking, suggesting that PREP1 and PREP2 have divergent substrates. Proteome phenotypes were driven by the loss of CLP protease capacity, with little impact from the PREP peptidases. Chloroplast N‐terminal proteomess howed that many nuclear encoded chloroplast proteins have alternatively processed N‐termini inprep1prep2 ,clpt1clpt2 andprep1prep2clpt1clpt2 .Loss of chloroplast protease capacity interferes with stromal processing peptidase (SPP) activity due to folding stress and low levels of accumulated cleaved cTP fragments. PREP1,2 proteolysis of cleaved cTPs is complemented by unknown proteases. A model for CLP and PREP activity within a hierarchical chloroplast proteolysis network is proposed.
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Prasinophytes form a paraphyletic assemblage of early diverging green algae, which have the potential to reveal the traits of the last common ancestor of the main two green lineages: (i) chlorophyte algae and (ii) streptophyte algae. Understanding the genetic composition of prasinophyte algae is fundamental to understanding the diversification and evolutionary processes that may have occurred in both green lineages. In this study, we sequenced the chloroplast genome of
Pyramimonas parkeae NIES 254 and compared it with that ofP. parkeae CCMP 726, the only other fully sequencedP. parkeae chloroplast genome. The results revealed thatP. parkeae chloroplast genomes are surprisingly variable. The chloroplast genome ofNIES 254 was larger than that ofCCMP 726 by 3,204 bp, theNIES 254 large single copy was 288 bp longer, the small single copy was 5,088 bp longer, and theIR was 1,086 bp shorter than that ofCCMP 726. Similarity values of the two strains were almost zero in four large hot spot regions. Finally, the strains differed in copy number for three protein‐coding genes:ycf20 ,psaC , andndhE . Phylogenetic analyses using 16S and 18SrDNA andrbcL sequences resolved a clade consisting of these twoP. parkeae strains and a clade consisting of these plus otherPyramimonas isolates. These results are consistent with past studies indicating that prasinophyte chloroplast genomes display a higher level of variation than is commonly found among land plants. Consequently, prasinophyte chloroplast genomes may be less useful for inferring the early history of Viridiplantae than has been the case for land plant diversification. -
Summary Plant small
RNA s (sRNA s) modulate key physiological mechanisms through post‐transcriptional and transcriptional silencing of gene expression. SmallRNA s fall into two major categories: those are reliant onRNA ‐dependentRNA polymerases ( s) for biogenesis and those that are not. KnownRDR /RDR 12 /6 ‐dependentsRNA s include phased and repeat‐associated short interferingRNA s, while known /RDR 12 /6 ‐independentsRNA s are primarily microRNA s (miRNA ) and other hairpin‐derivedsRNA s. In this study we produced and analyzedsRNA ‐seq libraries fromrdr1 /rdr2 /rdr6 triple mutant plants. We found 58 previously annotated miRNA loci that were reliant on , ‐RDR 12 , or ‐6 function, casting doubt on their classification. We also found 38 /RDR 12 /6‐independentsRNA loci that are not s or otherwise hairpin‐derived, and did not fit into other known paradigms forMIRNA sRNA biogenesis. These 38sRNA ‐producing loci have as‐yet‐undescribed biogenesis mechanisms, and are frequently located in the vicinity of protein‐coding genes. Altogether, our analysis suggests that these 38 loci represent one or more undescribed types ofsRNA inArabidopsis thaliana .