Despite the importance of pentatricopeptide repeat (PPR) proteins in organellar RNA metabolism and plant development, the functions of many PPR proteins remain unknown. Here, we determined the role ...of a mitochondrial PPR protein (At1g52620) comprising 19 PPR motifs, thus named PPR19, in Arabidopsis thaliana.
The ppr19 mutant displayed abnormal seed development, reduced seed yield, delayed seed germination, and retarded growth, indicating that PPR19 is indispensable for normal growth and development of Arabidopsis thaliana. Splicing pattern analysis of mitochondrial genes revealed that PPR19 specifically binds to the specific sequence in the 3′-terminus of the NADH dehydrogenase 1 (nad1) transcript and stabilizes transcripts containing the second and third exons of nad1. Loss of these transcripts in ppr19 leads to multiple secondary effects on accumulation and splicing of other nad1 transcripts, from which we can infer the order in which cis- and trans-spliced nad1 transcripts are normally processed.
Improper splicing of nad1 transcripts leads to the absence of mitochondrial complex I and alteration of the nuclear transcriptome, notably influencing the alternative splicing of a variety of nuclear genes.
Our results indicate that the mitochondrial PPR19 is an essential component in the splicing of nad1 transcripts, which is crucial for mitochondrial function and plant development.
Cyanobacteriochromes (CBCRs) are linear tetrapyrrole bilin‐binding photoreceptors of cyanobacteria that exhibit high spectral diversity, gaining attention in optogenetics and bioimaging applications. ...Several engineering studies on CBCRs were attempted, especially for designing near‐infrared (NIR) fluorescent proteins with longer fluorescence wavelengths. However, despite continuous efforts, a key component regulating fluorescence emission property in CBCRs is still poorly understood. As a model system, we focused on red/green CBCR Slr1393g3, from the unicellular cyanobacterium Synechocystis sp. PCC 6803 to engineer Pr to get far‐red light‐emitting property. Energy profiling and pairwise structural comparison of Slr1393g3 variants effectively reveal the mutations that are critical to the fluorescence changes. H497 seems to play a key role in stabilizing the chromophore environment, especially the α3 helix, while H495, T499, and Q502 are potential key residues determining fluorescence emission peak wavelength. We also found that mutations of α2 and α4 helical regions are closely related to the chromophore binding stability and likely affect fluorescence properties. Taken together, our computational analysis suggests that the fluorescence of Slr1393g3 is mainly controlled by the stabilization of the chromophore binding pocket. The predicted key residues potentially regulating the fluorescence emission property of a red/green CBCR will be advantageous for designing improved NIR fluorescent protein when combined with in vitro molecular evolution approaches.
•The epistatic interactions between Arabidopsis HY5 and PAP1 are unknown.•HY5 mutation reduced the PAP1 transcript level under far-red and blue light conditions.•Chip-qPCR and promoter assays ...indicated that HY5 binds directly to the PAP1 promoter.•PAP1, along with other anthocyanin structural genes, is a novel HY5 target gene.
Several positive transcription factors regulate Arabidopsis anthocyanin biosynthesis. HY5, a component of light-signaling pathways, and PAP1, an R2R3-MYB transcription factor, share common regulatory targets on anthocyanin biosynthesis genes. The epistatic interactions between the two transcription factors are currently unknown. To address this problem, we analyzed crosses between hy5 and pap1 mutants (hy5pap1) or pap1D overexpressors (hy5pap1D), performed chromatin immunoprecipitation-qPCR, and determined the PAP1 promoter region through deletion analysis. The results show that HY5 regulates PAP1 expression via direct binding to G- and ACE-boxes in the promoter region, which suggests bifurcate regulation of anthocyanin biosynthesis by HY5 via transcriptional activation of PAP1.
Serotonin N‐acetyltransferase (SNAT) catalyzes conversion of serotonin into N‐acetylserotonin, which is a direct precursor for melatonin biosynthesis in all organisms. Molecular cloning of plant SNAT ...from rice led to a screening for SNAT homolog genes in other species. We identified a cyanobacterium SNAT‐like gene (cSNAT) that showed 56% amino acid homology with the rice SNAT. To confirm whether cSNAT encoded SNAT enzyme activity, we expressed cSNAT DNA in Escherichia coli and purified the cSNAT protein as a C‐terminal His‐tagged form. The purified cSNAT protein exhibited SNAT enzyme activities, transferring the acetyl group into either serotonin or tryptamine substrates. The optimum temperature was 55°C, but it was still highly active at 70°C, suggesting that cSNAT is a thermotolerant enzyme. The Km and Vmax were 823 μm and 1.6 nmol/min/mg protein, respectively. The cSNAT gene is highly conserved in all cyanobacterial taxa and seems to be an origin of SNAT in higher plants. The thermotolerance of cSNAT suggests that melatonin plays a role in the response to high‐temperature stress. Further analysis of this role of melatonin in higher plants is needed.
Phytochrome B (phyB) is a plant photoreceptor that forms a membraneless organelle called a photobody. However, its constituents are not fully known. Here, we isolated phyB photobodies from ...Arabidopsis leaves using fluorescence-activated particle sorting and analyzed their components. We found that a photobody comprises ~1,500 phyB dimers along with other proteins that could be classified into two groups: The first includes proteins that directly interact with phyB and localize to the photobody when expressed in protoplasts, while the second includes proteins that interact with the first group proteins and require co-expression of a first-group protein to localize to the photobody. As an example of the second group, TOPLESS interacts with PHOTOPERIODIC CONTROL OF HYPOCOTYL 1 (PCH1) and localizes to the photobody when co-expressed with PCH1. Together, our results support that phyB photobodies include not only phyB and its primary interacting proteins but also its secondary interacting proteins.
Summary
Nannochloropsis species, unicellular industrial oleaginous microalgae, are model organisms for microalgal systems and synthetic biology. To facilitate community‐based annotation and mining of ...the rapidly accumulating functional genomics resources, we have initiated an international consortium and present a comprehensive multi‐omics resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single‐cell.cn). Via the Tripal toolkit, it features user‐friendly interfaces hosting genomic resources with gene annotations and transcriptomic and proteomic data for six Nannochloropsis species, including two updated genomes of Nannochloropsis oceanica IMET1 and Nannochloropsis salina CCMP1776. Toolboxes for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis, a genome browser, etc. are also included. In addition, functional validation of genes is indicated based on phenotypes of mutants and relevant bibliography. Furthermore, epigenomic resources are also incorporated, especially for sequencing of small RNAs including microRNAs and circular RNAs. Such comprehensive and integrated landscapes of Nannochloropsis genomics and epigenomics will promote and accelerate community efforts in systems and synthetic biology of these industrially important microalgae.
Significance Statement
Nannochloropsis species have emerged as model organisms for microalgal systems and synthetic biology, but a centralized portal to store, mine and disseminate the rapidly expanding multi‐omics datasets is lacking. Here, we initiate an international consortium and present a comprehensive resource database named Nannochloropsis Design and Synthesis (NanDeSyn; http://nandesyn.single‐cell.cn). NanDeSyn hosts genomic and epigenomic resources such as gene annotation, transcriptome, proteome, small RNA and mutant information, and provides tools for search, Blast, synteny view, enrichment analysis, metabolic pathway analysis and a genome browser.
Abstract
Although the importance of chloroplast RNA splicing and ribosome maturation (CRM) domain-containing proteins has been established for chloroplast RNA metabolism and plant development, the ...functional role of CRM proteins in mitochondria remains largely unknown. Here, we investigated the role of a mitochondria-targeted CRM protein (At3g27550), named CFM9, in Arabidopsis thaliana. Confocal analysis revealed that CFM9 is localized in mitochondria. The cfm9 mutant exhibited delayed seed germination, retarded growth and shorter height compared with the wild type under normal conditions. The growth-defect phenotypes were more manifested upon high salinity, dehydration or ABA application. Complementation lines expressing CFM9 in the mutant background fully recovered the wild-type phenotypes. Notably, the mutant had abnormal mitochondria, increased hydrogen peroxide and reduced respiration activity, implying that CFM9 is indispensable for normal mitochondrial function. More important, the splicing of many intron-containing genes in mitochondria was defective in the mutant, suggesting that CFM9 plays a crucial role in the splicing of mitochondrial introns. Collectively, our results provide clear evidence emphasizing that CFM9 is an essential factor in the splicing of mitochondrial introns, which is crucial for mitochondrial biogenesis and function and the growth and development of Arabidopsis.
Summary
Panax ginseng C. A. Meyer, reputed as the king of medicinal herbs, has slow growth, long generation time, low seed production and complicated genome structure that hamper its study. Here, we ...unveil the genomic architecture of tetraploid P. ginseng by de novo genome assembly, representing 2.98 Gbp with 59 352 annotated genes. Resequencing data indicated that diploid Panax species diverged in association with global warming in Southern Asia, and two North American species evolved via two intercontinental migrations. Two whole genome duplications (WGD) occurred in the family Araliaceae (including Panax) after divergence with the Apiaceae, the more recent one contributing to the ability of P. ginseng to overwinter, enabling it to spread broadly through the Northern Hemisphere. Functional and evolutionary analyses suggest that production of pharmacologically important dammarane‐type ginsenosides originated in Panax and are produced largely in shoot tissues and transported to roots; that newly evolved P. ginseng fatty acid desaturases increase freezing tolerance; and that unprecedented retention of chlorophyll a/b binding protein genes enables efficient photosynthesis under low light. A genome‐scale metabolic network provides a holistic view of Panax ginsenoside biosynthesis. This study provides valuable resources for improving medicinal values of ginseng either through genomics‐assisted breeding or metabolic engineering.
Summary
Pumilio RNA‐binding proteins are largely involved in mRNA degradation and translation repression. However, a few evolutionarily divergent Pumilios are also responsible for proper pre‐rRNA ...processing in human and yeast. Here, we describe an essential Arabidopsis nucleolar Pumilio, APUM24, that is expressed in tissues undergoing rapid proliferation and cell division. A T‐DNA insertion for APUM24 did not affect the male and female gametogenesis, but instead resulted in a negative female gametophytic effect on zygotic cell division immediately after fertilization. Additionally, the mutant embryos displayed defects in cell patterning from pro‐embryo through globular stages. The mutant embryos were marked by altered auxin maxima, which were substantiated by the mislocalization of PIN1 and PIN7 transporters in the defective embryos. Homozygous apum24 callus accumulates rRNA processing intermediates, including uridylated and adenylated 5.8S and 25S rRNA precursors. An RNA–protein interaction assay showed that the histidine‐tagged recombinant APUM24 binds RNAin vitro with no apparent specificity. Overall, our results demonstrated that APUM24 is required for rRNA processing and early embryogenesis in Arabidopsis.
Significance Statement
Pumilio RNA‐binding proteins possess multiple tandem repeat Puf domains, and participate in RNA degradation and/or translation repression. Twentyfive Pumilios are found in Arabidopsis, however their functions are largely unknown. Here, we show that nucleolar Pumilio APUM24 is implicated in the processing of 27SB and 5.8S precursor rRNAs. The apum24 mutant embryo is lethal before globular stage, emphasizing the essential role of APUM24 during developmental process in Arabidopsis.
• In higher plants, the plastidic glucose translocator (pGlcT) is assumed to play a role in the export of starch degradation products, but this has not yet been studied in detail. • To elucidate the ...role of pGlcT in the leaves of Arabidopsis thaliana, we generated single and double mutants lacking three plastidic sugar transporters, pGlcT, the triose-phosphate/phosphate translocator (TPT), and the maltose transporter (MEX1), and analyzed their growth phenotypes, photosynthetic properties and metabolite contents. • In contrast to the pglct-1 and pglct-2 single mutants lacking a visible growth phenotype, the double mutants pglct-1/mex1 and tpt-2/mex1 displayed markedly inhibited plant growth. Notably, pglct-1/mex1 exhibited more severe growth retardation than that seen for the other mutants. In parallel, the most severe reductions in sucrose content and starch turnover were observed in the pglct-1/mex1 mutant. The concurrent loss of pGlcT and MEX1 also resulted in severely reduced photosynthetic activities and extreme chloroplast abnormalities. • These findings suggest that pGlcT, together with MEX1, contributes significantly to the export of starch degradation products from chloroplasts in A. thaliana leaves, and that this starch-mediated pathway for photoassimilate export via pGlcT and MEX1 is essential for the growth and development of A. thaliana.
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