PAP1 and PAP7 are required for association of plastid-encoded RNA polymerase with DNA.

Plastid-encoded RNA polymerase (PEP) is a bacterial-type multisubunit RNA polymerase responsible for the majority of transcription in chloroplasts. PEP consists of four core subunits, which are orthologs of their cyanobacterial counterparts. In Arabidopsis thaliana, PEP is expected to interact with 14 PEP-associated proteins (PAPs), which serve as peripheral subunits of the RNA polymerase. The exact contributions of PAPs to PEP function are still poorly understood. We used ptChIP-seq to show that PAP1 (also known as pTAC3), a peripheral subunit of PEP, binds to the same genomic loci as RpoB, a core subunit of PEP. The pap1 mutant shows a complete loss of RpoB binding to DNA throughout the genome, indicating that PAP1 is necessary for RpoB binding to DNA. A similar loss of RpoB binding to DNA is observed in a mutant defective in PAP7 (also known as pTAC14), another peripheral PEP subunit. We propose that PAPs are required for the recruitment of core PEP subunits to DNA.

Membrane association of active genes organizes the chloroplast nucleoid structure.

DNA is organized into chromatin-like structures that support the maintenance and regulation of genomes. A unique and poorly understood form of DNA organization exists in chloroplasts, which are organelles of endosymbiotic origin responsible for photosynthesis. Chloroplast genomes, together with associated proteins, form membrane-less structures known as nucleoids. The internal arrangement of the nucleoid, molecular mechanisms of DNA organization, and connections between nucleoid structure and gene expression remain mostly unknown. We show that Arabidopsis thaliana chloroplast nucleoids have a unique sequence-specific organization driven by DNA binding to the thylakoid membranes. DNA associated with the membranes has high protein occupancy, has reduced DNA accessibility, and is highly transcribed. In contrast, genes with low levels of transcription are further away from the membranes, have lower protein occupancy, and have higher DNA accessibility. Membrane association of active genes relies on the pattern of transcription and proper chloroplast development. We propose a speculative model that transcription organizes the chloroplast nucleoid into a transcriptionally active membrane-associated core and a less active periphery.

N6 -Methyladenosine modification of mRNA contributes to the transition from 2D to 3D growth in the moss Physcomitrium patens.

Plants colonized the land approximately 470 million years ago, coinciding with the development of apical cells that divide in three planes. The molecular mechanisms that underly the development of the 3D growth pattern are poorly understood, mainly because 3D growth in seed plants starts during embryo development. In contrast, the transition from 2D to 3D growth in the moss Physcomitrium patens has been widely studied, and it involves a large turnover of the transcriptome to allow the establishment of stage-specific transcripts that facilitate this developmental transition. N6 -Methyladenosine (m6 A) is the most abundant, dynamic and conserved internal nucleotide modification present on eukaryotic mRNA and serves as a layer of post-transcriptional regulation directly affecting several cellular processes and developmental pathways in many organisms. In Arabidopsis, m6 A has been reported to be essential for organ growth and determination, embryo development and responses to environmental signals. In this study, we identified the main genes of the m6 A methyltransferase complex (MTC), MTA, MTB and FIP37, in P. patens and demonstrate that their inactivation leads to the loss of m6 A in mRNA, a delay in the formation of gametophore buds and defects in spore development. Genome-wide analysis revealed several transcripts affected in the Ppmta background. We demonstrate that the PpAPB1-PpAPB4 transcripts, encoding central factors orchestrating the transition from 2D to 3D growth in P. patens, are modified by m6 A, whereas in the Ppmta mutant the lack of the m6 A marker is associated with a corresponding decrease in transcript accumulation. Overall, we suggest that m6 A is essential to enable the proper accumulation of these and other bud-specific transcripts directing the turnover of stage-specific transcriptomes, and thus promoting the transition from protonema to gametophore buds in P. patens.

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

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) at https://plavisto.mcdb.lsa.umich.edu/.

Insights into the function of the phasiRNA-triggering miR1514 in response to stress in legumes.

We recently described the activity of miR1514a in response to water deficit in Phaseolus vulgaris. Pvu-miR1514a targets a NAC transcription factor mRNA for cleavage and subsequently triggers NAC-derived phasiRNA formation. Here we show that accumulation and activity of miR1514a are also conserved in the model legume Medicago truncatula. Consistently, we identified Mtr-miR1514a and detected its increased accumulation in response to stress conditions, targeting a NAC TF mRNA for cleavage and triggering phasiRNA production. In P. vulgaris, miR1514a inhibition in transgenic hairy roots was reported to increase NAC 700 mRNA levels and to affect expression patterns of several genes, including that of a Sec 14 homolog. We report here that in adult plant roots exposed to dehydration conditions, where miR1514a levels increased and NAC 700 mRNA decreased, there was a reduction of Sec 14 homolog mRNA levels, suggesting a direct transcriptional effect. The functions of miR1514a, NAC 700 and derived phasiRNAs have just begun to be elucidated in common bean; future understanding of their activities in this and other legumes species will advance our knowledge of microRNA functions in plants.