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1. High‐resolution map of plastid‐encoded RNA polymerase binding patterns demonstrates a major role of transcription in chloroplast gene expression

   https://doi.org/10.1111/tpj.15882  

   Palomar, V. Miguel ; Jaksich, Sarah ; Fujii, Sho ; Kuciński, Jan ; Wierzbicki, Andrzej T. ( July 2022 , The Plant Journal)

   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 matureArabidopsis thalianachloroplasts, 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/.

    

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2. Transcriptome and phenotyping analyses support a role for chloroplast sigma factor 2 in red‐light‐dependent regulation of growth, stress, and photosynthesis

   https://doi.org/10.1002/pld3.43  

   Oh, Sookyung ; Strand, Deserah D. ; Kramer, David M. ; Chen, Jin ; Montgomery, Beronda L. ( February 2018 , Plant Direct)

   Sigma factor (SIG) proteins contribute to promoter specificity of the plastid‐encodedRNApolymerase during chloroplast genome transcription. All six members of theSIGfamily, that is,SIG1–SIG6, are nuclear‐encoded proteins targeted to chloroplasts. Sigma factor 2 (SIG2) is a phytochrome‐regulated protein important for stoichiometric control of the expression of plastid‐ and nuclear‐encoded genes that impact plastid development and plant growth and development. AmongSIGfactors,SIG2 is required not only for transcription of chloroplast genes (i.e., anterograde signaling), but also impacts nuclear‐encoded, photosynthesis‐related, and light signaling‐related genes (i.e., retrograde signaling) in response to plastid functional status. AlthoughSIG2 is involved in photomorphogenesis in Arabidopsis, the molecular bases for its role in light signaling that impacts photomorphogenesis and aspects of photosynthesis have only recently begun to be investigated. Previously, we reported thatSIG2 is necessary for phytochrome‐mediated photomorphogenesis specifically under red (R) and far‐red light, thereby suggesting a link between phytochromes and nuclear‐encodedSIG2 in light signaling. To explore transcriptional roles ofSIG2 in R‐dependent growth and development, we performedRNAsequencing analysis to compare gene expression insig2‐2mutant and Col‐0 wild‐type seedlings at two developmental stages (1‐ and 7‐day). We identified a subset of misregulated genes involved in growth, hormonal cross talk, stress responses, and photosynthesis. To investigate the functional relevance of these gene expression analyses, we performed several comparative phenotyping tests. In these analyses, strongsig2mutants showed insensitivity to bioactiveGA₃, high intracellular levels of hydrogen peroxide (H₂O₂) indicative of a stress response, and specific defects in photosynthesis, including elevated levels of cyclic electron flow (CEF) and nonphotochemical quenching (NPQ). We demonstrated thatSIG2 regulates a broader range of physiological responses at the molecular level than previously reported, with specific roles in red‐light‐mediated photomorphogenesis.

    

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3. A phosphorylation‐deficient ribosomal protein eS6 is largely functional in Arabidopsis thaliana , rescuing mutant defects from global translation and gene expression to photosynthesis and growth

   https://doi.org/10.1002/pld3.566  

   Dasgupta, Anwesha ; Urquidi Camacho, Ricardo A. ; Enganti, Ramya ; Cho, Sung Ki ; Tucker, Lindsey L. ; Torreverde, John S. ; Abraham, Paul E. ; von Arnim, Albrecht G. ( January 2024 , Plant Direct)

   The eukaryote‐specific ribosomal protein of the small subunit eS6 is phosphorylated through the target of rapamycin (TOR) kinase pathway. Although this phosphorylation event responds dynamically to environmental conditions and has been studied for over 50 years, its biochemical and physiological significance remains controversial and poorly understood. Here, we report data fromArabidopsis thaliana, which indicate that plants expressing only a phospho‐deficient isoform of eS6 grow essentially normally under laboratory conditions. The eS6z (RPS6A) paralog of eS6 functionally rescued a double mutant in bothrps6aandrps6bgenes when expressed at approximately twice the wild‐type dosage. A mutant isoform of eS6z lacking the major six phosphorylatable serine and threonine residues in its carboxyl‐terminal tail also rescued the lethality, rosette growth, and polyribosome loading of the double mutant. This isoform also complemented many mutant phenotypes ofrps6that were newly characterized here, including photosynthetic efficiency, and most of the gene expression defects that were measured by transcriptomics and proteomics. However, compared with plants rescued with a phospho‐enabled version of eS6z, the phospho‐deficient seedlings retained a mild pointed‐leaf phenotype, root growth was reduced, and certain cell cycle‐related mRNAs and ribosome biogenesis proteins were misexpressed. The residual defects of the phospho‐deficient seedlings could be understood as an incomplete rescue of therps6mutant defects. There was little or no evidence for gain‐of‐function defects. As previously published, the phospho‐deficient eS6z also rescued therps6aandrps6bsingle mutants; however, phosphorylation of the eS6y (RPS6B) paralog remained lower than predicted, further underscoring that plants can tolerate phospho‐deficiency of eS6 well. Our data also yield new insights into how plants cope with mutations in essential, duplicated ribosomal protein isoforms.

    

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4. Sexual specific functions of Tub1 beta‐tubulins require stage‐specific RNA processing and expression in Fusarium graminearum

   https://doi.org/10.1111/1462-2920.14441  

   Chen, Daipeng ; Wu, Chunlan ; Hao, Chaofeng ; Huang, Panpan ; Liu, Huiquan ; Bian, Zhuyun ; Xu, Jin‐Rong ( October 2018 , Environmental Microbiology)

   The wheat head blight fungusFusarium graminearumhas two highly similar beta‐tubulin genes with overlapping functions during vegetative growth but onlyTUB1is important for sexual reproduction. To better understand their functional divergence during ascosporogenesis, in this study we characterized the sequence elements important for stage‐specific functions ofTUB1.Deletion ofTUB1blocked the late but not initial stages of perithecium formation. Perithecia formed bytub1mutant had limited ascogenous hyphae and failed to develop asci. Silencing ofTUB1by MSUD also resulted in defects in ascospore formation. Interestingly, the 3′‐UTR ofTUB1was dispensable for growth but essential for its function during sexual reproduction. RIP mutations that specifically affected Tub1 functions during sexual reproduction also were identified in two ascospore progeny. Furthermore, site‐directed mutagenesis showed that whereas the non‐editable mutations at three A‐to‐I RNA editing sites had no effects, the N347D (not T362D or I368V) edited mutation affected ascospore development. In addition, the F167Y, but not E198K or F200Y, mutation inTUB1conferred tolerance to carbendazim and caused a minor defect in sexual reproduction. Taken together, our data indicateTUB1plays an essential role in ascosporogenesis and sexual‐specific functions ofTUB1require stage‐specific RNA processing and Tub1 expression.

    

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5. The dicot homolog of maize PPR103 carries a C-terminal DYW domain and may have a role in C-to-U editing of some chloroplast RNA transcripts

   https://doi.org/10.1007/s11103-024-01424-1  

   McCray, Tyra N. ; Azim, Mohammad F. ; Burch-Smith, Tessa M. ( March 2024 , Plant Molecular Biology)

   In plants, cytidine-to-uridine (C-to-U) editing is a crucial step in processing mitochondria- and chloroplast-encoded transcripts. This editing requires nuclear-encoded proteins including members of the pentatricopeptide (PPR) family, especially PLS-type proteins carrying the DYW domain.IPI1/emb175/PPR103is a nuclear gene encoding a PLS-type PPR protein essential for survival inArabidopsis thalianaand maize. Arabidopsis IPI1 was identified as likely interacting with ISE2, a chloroplast-localized RNA helicase associated with C-to-U RNA editing in Arabidopsis and maize. Notably, while the Arabidopsis andNicotianaIPI1 orthologs possess complete DYW motifs at their C-termini, the maize homolog, ZmPPR103, lacks this triplet of residues which are essential for editing. In this study we examined the function of IPI1 in chloroplast RNA processing inN. benthamianato gain insight into the importance of the DYW domain to the function of the EMB175/PPR103/ IPI1 proteins. Structural predictions suggest that evolutionary loss of residues identified as critical for catalyzing C-to-U editing in other members of this class of proteins, were likely to lead to reduced or absent editing activity in theNicotianaand Arabidopsis IPI1 orthologs. Virus-induced gene silencing ofNbIPI1led to defects in chloroplast ribosomal RNA processing and changes to stability ofrpl16transcripts, revealing conserved function with its maize ortholog.NbIPI1-silenced plants also had defective C-to-U RNA editing in several chloroplast transcripts, a contrast from the finding that maize PPR103 had no role in editing. The results indicate that in addition to its role in transcript stability, NbIPI1 may contribute to C-to-U editing inN. benthamianachloroplasts.

    

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