A Transcriptomic Analysis of Tobacco Leaf with the Functional Loss of the Plastid rpoB Operon Caused by TALEN-Mediated Double-Strand Breakage

Abstract

At least two sets of RNA polymerase (RNAP), nucleus (NEP)- and plastid (PEP)-encoded polymerases, recognizing distinct promoters exist in the plastids of land plants. Most plastid genes are regulated by multiple promoters with different strengths in their response to developmental stages and environmental cues. Recently, we applied chloroplast-targeted transcription activator-like effector nuclease (cpTALEN) technology to site-specifically cause double-strand DNA breaks in the rpoB gene of tobacco, which encodes the β-subunit of PEP. The repair of damaged chloroplast DNA (cpDNA) through microhomology-mediated recombination caused the functional loss of the rpoB operon and resulted in the heterotrophic growth of an albino plant. We conducted a genome-wide analysis of the steady state of gene expression in the leaf tissue of PEP-deficient tobacco by RNA-Seq and compared it with that of wild-type plants. The expression of NEP genes was up-regulated in PEP-deficient tobacco; in particular, the level of RpoT3 transcripts encoding the specifically plastid-targeted NEP was significantly increased. Alongside most housekeeping genes, NEP also plays an important role in the regulation of gene expression involved in photosynthesis. In contrast, alongside the photosynthesis-related genes, PEP also plays an important role in the regulation of gene expression involved in some housekeeping functions. Furthermore, the mitochondrial DNA copy number and the level of most mitochondrial protein-coding transcripts were slightly increased in PEP-deficient tobacco. The disruption of PEP function not only affected plastid gene expression, but also nuclear and mitochondrial gene expression. This study demonstrated the intercompartmental retrograde signaling in the regulation of gene expression.

Yellow-Green Leaf 19 Encoding a Specific and Conservative Protein for Photosynthetic Organisms Affects Tetrapyrrole Biosynthesis, Photosynthesis, and Reactive Oxygen Species Metabolism in Rice

“…In the chloroplast, the large subunit is encoded by RBCL, and it catalyzes two competing reactions, CO 2 fixation in photosynthesis and the production of 2-phosphoglycolate in the photorespiratory pathway [71]. In rice and tobacco, deletion of the rpoA or rpoB encoding the subunits of plastid-encoded RNA polymerase resulted in photosynthetic incompetence [33,72]. In our study, plastid-encoded photosynthesis-related genes showed lower expression levels in the ygl19 mutant.…”

Integrative transcriptome and whole‐genome bisulfite sequencing analyses of a temperature‐sensitive albino tea plant cultivar

“…RPOB is a subunit of chloroplast DNA‐dependent RNA polymerase that affects chloroplast development. The lack of RPOB leads to pale and albino leaf phenotypes in rice and tobacco (Huang et al, 2020; Liu et al, 2022). Several DEGs related to impaired chloroplast complex activity were upregulated in albinism process of ZH2, including PsbP‐DOMAIN PROTEIN 1 (PPD1), PsbQ‐LIKE 2 (PQL2), ATS2, and STN8 (Liu et al, 2012; Schonberg et al, 2017; Suorsa et al, 2010; Yu et al, 2004).…”

Genome Editing of Plant Mitochondrial and Chloroplast Genomes

“…The DSB was repaired by homologous recombination (HR) including microhomology-mediated recombination, but not by non-homologous end-joining, which is predominantly observed in the nucleus. The following year, the same group reported analyses on the transcriptome of a heterotrophic albino mutant, in which rpoB1 (a gene of a subunit of the plastid-encoded RNA polymerase) was knocked out by the plastid-targeted TALENs ( Liu et al. 2022 ).…”