Critical role of cyclic electron transport around photosystem I in the maintenance of photosystem I activity.

In angiosperms, cyclic electron transport around photosystem I (PSI) is mediated by two pathways that depend on the PROTON GRADIENT REGULATION 5 (PGR5) protein and the chloroplast NADH dehydrogenase-like (NDH) complex, respectively. In the Arabidopsis double mutants defective in both pathways, plant growth and photosynthesis are impaired. The pgr5-1 mutant used in the original study is a missense allele and accumulates low levels of PGR5 protein. In this study, we generated two knockout (KO) alleles, designated as pgr5-5 and pgr5-6, using the CRISPR-Cas9 technology. Although both KO alleles showed a severe reduction in P700 similar to the pgr5-1 allele, NPQ induction was less severely impaired in the KO alleles than in the pgr5-1 allele. In the pgr5-1 allele, the second mutation affecting NPQ size was mapped to ~21 cM south of the pgr5-1 locus. Overexpression of the pgr5-1 allele, encoding the glycine130-to-serine change, complemented the pgr5-5 phenotype, suggesting that the pgr5-1 mutation destabilizes PGR5 but that the mutant protein retains partial functionality. Using two KO alleles, we created the double mutants with two chlororespiratory reduction (crr) mutants defective in the NDH complex. The growth of the double mutants was notably impaired. In the double mutant seedlings that survived on the medium containing sucrose, PSI activity evaluated by the P700 oxidation was severely impaired, whereas PSII activity was only mildly impaired. Cyclic electron transport around PSI is required to maintain PSI activity.

Glycosylated queuosines in tRNAs optimize translational rate and post-embryonic growth.

Transfer RNA (tRNA) modifications are critical for protein synthesis. Queuosine (Q), a 7-deaza-guanosine derivative, is present in tRNA anticodons. In vertebrate tRNAs for Tyr and Asp, Q is further glycosylated with galactose and mannose to generate galQ and manQ, respectively. However, biogenesis and physiological relevance of Q-glycosylation remain poorly understood. Here, we biochemically identified two RNA glycosylases, QTGAL and QTMAN, and successfully reconstituted Q-glycosylation of tRNAs using nucleotide diphosphate sugars. Ribosome profiling of knockout cells revealed that Q-glycosylation slowed down elongation at cognate codons, UAC and GAC (GAU), respectively. We also found that galactosylation of Q suppresses stop codon readthrough. Moreover, protein aggregates increased in cells lacking Q-glycosylation, indicating that Q-glycosylation contributes to proteostasis. Cryo-EM of human ribosome-tRNA complex revealed the molecular basis of codon recognition regulated by Q-glycosylations. Furthermore, zebrafish qtgal and qtman knockout lines displayed shortened body length, implying that Q-glycosylation is required for post-embryonic growth in vertebrates.

Ribosome slowdown triggers codon-mediated mRNA decay independently of ribosome quality control.

The control of mRNA stability plays a central role in regulating gene expression patterns. Recent studies have revealed that codon composition in the open reading frame determines mRNA stability in multiple organisms. Based on genome-wide correlation approaches, this previously unrecognized role for the genetic code is attributable to the kinetics of the codon-decoding process by the ribosome. However, complementary experimental analyses are required to clarify the codon effects on mRNA stability and the related cotranslational mRNA decay pathways, for example, those triggered by aberrant ribosome stalling. In the current study, we performed a set of reporter-based analyses to define codon-mediated mRNA decay and ribosome stall-dependent mRNA decay in zebrafish embryos. Our analysis showed that the effect of codons on mRNA stability stems from the decoding process, independent of the ribosome quality control factor Znf598 and stalling-dependent mRNA decay. We propose that codon-mediated mRNA decay is rather triggered by transiently slowed ribosomes engaging in a productive translation cycle in zebrafish embryos.

Evolutionary Model of Plastidial RNA Editing in Angiosperms Presumed from Genome-Wide Analysis of Amborella trichopoda.

Amborella trichopoda is placed close to the base of the angiosperm lineage (basal angiosperm). By genome-wide RNA sequencing, we identified 184C-to-U RNA editing sites in the plastid genome of Amborella. This number is much higher than that observed in other angiosperms including maize (44 sites), rice (39 sites) and grape (115 sites). Despite the high frequency of RNA editing, the biased distribution of RNA editing sites in the genome, target codon preference and nucleotide preference adjacent to the edited cytidine are similar to that in other angiosperms, suggesting a common editing machinery. Consistent with this idea, the Amborella nuclear genome encodes 2-3 times more of the E- and DYW-subclass members of pentatricopeptide repeat proteins responsible for RNA editing site recognition in plant organelles. Among 165 editing sites in plastid protein coding sequences in Amborella, 100 sites were conserved at least in one out of 38 species selected to represent key branching points of the angiosperm phylogenetic tree. We assume these 100 sites represent at least a subset of the sites in the plastid editotype of ancestral angiosperms. We then mapped the loss and gain of editing sites on the phylogenetic tree of angiosperms. Our results support the idea that the evolution of angiosperms has led to the loss of RNA editing sites in plastids.

Holliday junction resolvases mediate chloroplast nucleoid segregation.

Holliday junctions, four-stranded DNA structures formed during homologous recombination, are disentangled by resolvases that have been found in prokaryotes and eukaryotes but not in plant organelles. Here, we identify monokaryotic chloroplast 1 (MOC1) as a Holliday junction resolvase in chloroplasts by analyzing a green alga Chlamydomonas reinhardtii mutant defective in chloroplast nucleoid (DNA-protein complex) segregation. MOC1 is structurally similar to a bacterial Holliday junction resolvase, resistance to ultraviolet (Ruv) C, and genetically conserved among green plants. Reduced or no expression of MOC1 in Arabidopsis thaliana leads to growth defects and aberrant chloroplast nucleoid segregation. In vitro biochemical analysis and high-speed atomic force microscopic analysis revealed that A. thaliana MOC 1 (AtMOC1) binds and cleaves the core of Holliday junctions symmetrically. MOC1 may mediate chloroplast nucleoid segregation in green plants by resolving Holliday junctions.

C-Terminal Region of Sulfite Reductase Is Important to Localize to Chloroplast Nucleoids in Land Plants.

Chloroplast (cp) DNA is compacted into cpDNA-protein complexes, called cp nucleoids. An abundant and extensively studied component of cp nucleoids is the bifunctional protein sulfite reductase (SiR). The preconceived role of SiR as the core cp nucleoid protein, however, is becoming less likely because of the recent findings that SiRs do not associate with cp nucleoids in some plant species, such as Zea mays and Arabidopsis thaliana To address this discrepancy, we have performed a detailed phylogenetic analysis of SiRs, which shows that cp nucleoid-type SiRs share conserved C-terminally encoded peptides (CEPs). The CEPs are likely to form a bacterial ribbon-helix-helix DNA-binding motif, implying a potential role in attaching SiRs onto cp nucleoids. A proof-of-concept experiment was conducted by fusing the nonnucleoid-type SiR from A. thaliana (AtSiR) with the CEP from the cp nucleoid-type SiR of Phaseolus vulgaris The addition of the CEP drastically altered the intra-cp localization of AtSiR to cp nucleoids. Our analysis supports the possible functions of CEPs in determining the localization of SiRs to cp nucleoids and illuminates a possible evolutionary scenario for SiR as a cp nucleoid protein.