A de novo gene originating from the mitochondria controls floral transition in Arabidopsis thaliana.

De novo genes created in the plant mitochondrial genome have frequently been transferred into the nuclear genome via intergenomic gene transfer events. Therefore, plant mitochondria might be a source of de novo genes in the nuclear genome. However, the functions of de novo genes originating from mitochondria and the evolutionary fate remain unclear. Here, we revealed that an Arabidopsis thaliana specific small coding gene derived from the mitochondrial genome regulates floral transition. We previously identified 49 candidate de novo genes that induce abnormal morphological changes on overexpression. We focused on a candidate gene derived from the mitochondrial genome (sORF2146) that encodes 66 amino acids. Comparative genomic analyses indicated that the mitochondrial sORF2146 emerged in the Brassica lineage as a de novo gene. The nuclear sORF2146 emerged following an intergenomic gene transfer event in the A. thaliana after the divergence between Arabidopsis and Capsella. Although the nuclear and mitochondrial sORF2146 sequences are the same in A. thaliana, only the nuclear sORF2146 is transcribed. The nuclear sORF2146 product is localized in mitochondria, which may be associated with the pseudogenization of the mitochondrial sORF2146. To functionally characterize the nuclear sORF2146, we performed a transcriptomic analysis of transgenic plants overexpressing the nuclear sORF2146. Flowering transition-related genes were highly regulated in the transgenic plants. Subsequent phenotypic analyses demonstrated that the overexpression and knockdown of sORF2146 in transgenic plants resulted in delayed and early flowering, respectively. These findings suggest that a lineage-specific de novo gene derived from mitochondria has an important regulatory effect on floral transition.

Engineered Mutants of a Marine Photosynthetic Purple Nonsulfur Bacterium with Increased Volumetric Productivity of Polyhydroxyalkanoate Bioplastics.

Polyhydroxyalkanoates (PHAs) are green and sustainable bioplastics that could replace petrochemical synthetic plastics without posing environmental threats to living organisms. In addition, sustainable PHA production could be achieved using marine photosynthetic purple nonsulfur bacteria (PNSBs) that utilize natural seawater, sunlight, carbon dioxide gas, and nitrogen gas for growth. However, PHA production using marine photosynthetic PNSBs has not been economically feasible yet due to its high cost and low productivity. In this work, strain improvement, using genome-wide mutagenesis coupled with high-throughput screening via fluorescence-activated cell sorting, we were able to create Rhodovulum sulfidophilum mutants with enhanced volumetric PHA productivity, with an up to 1.7-fold increase. The best selected mutants (E6 and E6M4) reached the stationary growth phase 1 day faster and accumulated the maximum PHA content 2 days faster than the wild type. Maximizing volumetric PHA productivity before the stationary growth phase is indeed an additional advantage for R. sulfidophilum as a growth-associated PHA producer.

Draft Whole-Genome Sequence of Bacillus paramycoides LB_RP2, a Putative Polyhydroxyalkanoate-Producing Bacterium Isolated from an Amazonian Blackwater River.

Bacteria of the genus Bacillus have been investigated due to the ability that many species have of accumulating polyhydroxyalkanoates (PHA) via a wide variety of raw materials as their carbon source. Herein, we report the draft whole-genome sequence of the putative PHA-accumulating strain Bacillus paramycoides LB_RP2, isolated from an Amazonian river.

Peptide-Mediated Gene Transfer into Marine Purple Photosynthetic Bacteria.

Use of photosynthetic organisms is one of the sustainable ways to produce high-value products. Marine purple photosynthetic bacteria are one of the research focuses as microbial production hosts. Genetic transformation is indispensable as a biotechnology technique. However, only conjugation has been determined to be an applicable method for the transformation of marine purple photosynthetic bacteria so far. In this study, for the first time, a dual peptide-based transformation method combining cell penetrating peptide (CPP), cationic peptide and Tat-derived peptide (dTat-Sar-EED) (containing D-amino acids of Tat and endosomal escape domain (EED) connected by sarcosine linkers) successfully delivered plasmid DNA into Rhodovulum sulfidophilum, a marine purple photosynthetic bacterium. The plasmid delivery efficiency was greatly improved by dTat-Sar-EED. The concentrations of dTat-Sar-EED, cell growth stage and recovery duration affected the efficiency of plasmid DNA delivery. The delivery was inhibited at 4 °C and by A22, which is an inhibitor of the actin homolog MreB. This suggests that the plasmid DNA delivery occurred via MreB-mediated energy dependent process. Additionally, this peptide-mediated delivery method was also applicable for E. coli cells. Thus, a wide range of bacteria could be genetically transformed by using this novel peptide-based transformation method.

A marine photosynthetic microbial cell factory as a platform for spider silk production.

Photosynthetic microorganisms such as cyanobacteria, purple bacteria and microalgae have attracted great interest as promising platforms for economical and sustainable production of bioenergy, biochemicals, and biopolymers. Here, we demonstrate heterotrophic production of spider dragline silk proteins, major ampullate spidroins (MaSp), in a marine photosynthetic purple bacterium, Rhodovulum sulfidophilum, under both photoheterotrophic and photoautotrophic growth conditions. Spider silk is a biodegradable and biocompatible material with remarkable mechanical properties. R. sulfidophilum grow by utilizing abundant and renewable nonfood bioresources such as seawater, sunlight, and gaseous CO2 and N2, thus making this photosynthetic microbial cell factory a promising green and sustainable production platform for proteins and biopolymers, including spider silks.

Effect of small coding genes on the circadian rhythms under elevated CO2 conditions in plants.

Increase in atmospheric carbon dioxide (CO2) has a significant effect on plant growth and development. To explore the elevated-CO2 response, we generated transcriptional profiles over a time course (2 h-14 days) of exposure to elevated CO2 in Arabidopsis thaliana. Genes related to photosynthesis were down-regulated and circadian rhythm-related genes were abnormally regulated in the early to middle phase of elevated CO2 exposure. To understand the novel mechanism of elevated CO2 signaling, we focused on 42 unknown small coding genes that showed differential expression patterns under elevated CO2 conditions. Four transgenic plants overexpressing the small coding gene exhibited a growth-defective phenotype under elevated CO2 but not under current CO2. Transcriptome analysis showed that circadian rhythm-related genes were commonly regulated in four transgenic plants. These circadian rhythm-related genes were transcribed in the dark when CO2 concentrations in the leaf was high. Taken together, our identified four small coding genes are likely to participate in elevated CO2 signaling to the circadian rhythm.

Carotenoids in the eyespot apparatus are required for triggering phototaxis in Euglena gracilis.

Carotenoids are the most universal and most widespread pigments in nature. They have played pivotal roles in the evolution of photosensing mechanisms in microbes and of vision in animals. Several groups of phytoflagellates developed a photoreceptive organelle called the eyespot apparatus (EA) consisting of two separable components: the eyespot, a cluster of carotenoid-rich globules that acts as a reflector device, and actual photoreceptors for photobehaviors. Unlike other algal eyespots, the eyespot of Euglenophyta lacks reflective properties and is generally considered to act as a shading device for the photoreceptor (paraflagellar body, PFB) for major photomovements. However, the function of the eyespot of Euglenophyta has not yet been fully proven. Here, we report that the blocking carotenoid biosynthesis in Euglena gracilis by suppressing the phytoene synthase gene (crtB) caused a defect in eyespot function resulting in a loss of phototaxis. Raman spectroscopy and transmission electron microscopy suggested that EgcrtB-suppressed cells formed eyespot globules but had a defect in the accumulation of carotenoids in those packets. Motion analysis revealed the loss of phototaxis in EgcrtB-suppressed cells: a defect in the initiation of turning movements immediately after a change in light direction, rather than a defect in the termination of cell turning at the appropriate position due to a loss of the shading effect on the PFB. This study revealed that carotenoids are essential for light perception by the EA for the initiation of phototactic movement by E. gracilis, suggesting one possible photosensory role of carotenoids in the EA for the phototaxis.

Method for the facile transformation of marine purple photosynthetic bacteria using chemically competent cells.

Marine purple photosynthetic bacteria are ideal organisms for the production of useful materials at reduced costs and contributing to a sustainable society because they can utilize sunlight, seawater, and components of air, including carbon dioxide and nitrogen gases, for their growth. However, conjugation is the only applicable method for the transformation of marine purple photosynthetic bacteria so far. Here, we examined a calcium chloride-mediated method for the transformation of marine purple photosynthetic bacteria. Plasmid DNAs containing the kanamycin resistance gene were successfully transferred into chemically competent cells of two strains of marine purple photosynthetic bacteria (Rhodovulum sulfidophilum and Roseospira marina). Heat shock treatment increased the transformation efficiency in R. sulfidophilum, whereas the addition of cell-penetrating peptide did not improve it. We also found that prolonged incubation in agar plates containing kanamycin led to spontaneous mutation of the 16S rRNA, resulting in kanamycin resistance in R. marina. Thus, we developed an efficient and facile transformation method using chemically competent cells of marine purple photosynthetic bacteria with calcium chloride.

Marine Purple Photosynthetic Bacteria as Sustainable Microbial Production Hosts.

Photosynthetic microorganisms can serve as the ideal hosts for the sustainable production of high-value compounds. Purple photosynthetic bacteria are typical anoxygenic photosynthetic microorganisms and are expected to be one of the suitable microorganisms for industrial production. Purple photosynthetic bacteria are reported to produce polyhydroxyalkanoate (PHA), extracellular nucleic acids and hydrogen gas. We characterized PHA production as a model compound in purple photosynthetic bacteria, especially focused on marine strains. PHA is a family of biopolyesters synthesized by a variety of microorganisms as carbon and energy storage materials. PHA have recently attracted attention as an alternative to conventional petroleum-based plastics. Production of extracellular nucleic acids have been studied in Rhodovulum sulfidophilum, a marine purple non-sulfur bacterium. Several types of artificial RNAs have been successfully produced in R. sulfidophilum. Purple photosynthetic bacteria produce hydrogen via nitrogenase, and genetic engineering strategies have been investigated to enhance the hydrogen production. This mini review describes the microbial production of these high-value compounds using purple photosynthetic bacteria as the host microorganism.

Acetate-Inducing Metabolic States Enhance Polyhydroxyalkanoate Production in Marine Purple Non-sulfur Bacteria Under Aerobic Conditions.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that a variety of microorganisms accumulate as carbon and energy storage molecules under starvation conditions in the presence of excess carbon. Anoxygenic photosynthetic bacteria exhibit a variety of growth styles and high PHA production activity. Here, we characterized PHA production by four marine purple non-sulfur bacteria strains (Rhodovulum sulfidophilum, Rhodovulum euryhalinum, Rhodovulum imhoffii, and Rhodovulum visakhapatnamense) under different growth conditions. Unlike the well-studied PHA-producing bacteria, nutrient limitation is not appropriate for PHA production in marine purple non-sulfur bacteria. We found that marine purple non-sulfur bacteria did not accumulate PHA under aerobic conditions in the presence of malate and pyruvate. Interestingly, PHA accumulation was observed upon the addition of acetate under aerobic conditions but was not observed upon the addition of reductants, suggesting that an acetate-dependent pathway is involved in PHA accumulation. Gene expression analysis revealed that the expression of isocitrate dehydrogenase in the tricarboxylic acid (TCA) cycle decreased under aerobic conditions and increased with the addition of acetate, indicating that TCA cycle activity is involved in PHA production under aerobic conditions. We also found that expression of PdhRrs, which belongs to the GntR family of transcription regulators, in Rhodovulum sulfidophilum was upregulated upon the addition of acetate. Taken together, the results show that the changes in the metabolic state upon the addition of acetate, possibly regulated by PdhR, are important for PHA production under aerobic conditions in marine purple non-sulfur bacteria.

Transcriptome Analysis and Identification of a Transcriptional Regulatory Network in the Response to H2O2.

Hydrogen peroxide (H2O2) is a common signal molecule initiating transcriptional responses to all the known biotic and abiotic stresses of land plants. However, the degree of involvement of H2O2 in these stress responses has not yet been well studied. Here we identify time-dependent transcriptome profiles stimulated by H2O2 application in Arabidopsis (Arabidopsis thaliana) seedlings. Promoter prediction based on transcriptome data suggests strong crosstalk among high light, heat, and wounding stress responses in terms of environmental stresses and between the abscisic acid (ABA) and salicylic acid (SA) responses in terms of phytohormone signaling. Quantitative analysis revealed that ABA accumulation is induced by H2O2 but SA is not, suggesting that the implied crosstalk with ABA is achieved through ABA accumulation while the crosstalk with SA is different. We identified potential direct regulatory pairs between regulator transcription factor (TF) proteins and their regulated TF genes based on the time-course transcriptome analysis for the H2O2 response, in vivo regulation of the regulated TF by the regulator TF identified by expression analysis of mutants and overexpressors, and in vitro binding of the regulator TF protein to the target TF promoter. These analyses enabled the establishment of part of the transcriptional regulatory network for the H2O2 response composed of 15 regulatory pairs of TFs, including five pairs previously reported. This regulatory network is suggested to be involved in a wide range of biotic and abiotic stress responses in Arabidopsis.

Optimal iron concentrations for growth-associated polyhydroxyalkanoate biosynthesis in the marine photosynthetic purple bacterium Rhodovulum sulfidophilum under photoheterotrophic condition.

Polyhydroxyalkanoates (PHAs) are a group of natural biopolyesters that resemble petroleum-derived plastics in terms of physical properties but are less harmful biologically to the environment and humans. Most of the current PHA producers are heterotrophs, which require expensive feeding materials and thus contribute to the high price of PHAs. Marine photosynthetic bacteria are promising alternative microbial cell factories for cost-effective, carbon neutral and sustainable production of PHAs. In this study, Rhodovulum sulfidophilum, a marine photosynthetic purple nonsulfur bacterium with a high metabolic versatility, was evaluated for cell growth and PHA production under the influence of various media components found in previous studies. We evaluated iron, using ferric citrate, as another essential factor for cell growth and efficient PHA production and confirmed that PHA production in R. sulfidophilum was growth-associated under microaerobic and photoheterotrophic conditions. In fact, a subtle amount of iron (1 to 2 µM) was sufficient to promote rapid cell growth and biomass accumulation, as well as a high PHA volumetric productivity during the logarithmic phase. However, an excess amount of iron did not enhance the growth rate or PHA productivity. Thus, we successfully confirmed that an optimum concentration of iron, an essential nutrient, promotes cell growth in R. sulfidophilum and also enhances PHA utilization.

AtPep3 is a hormone-like peptide that plays a role in the salinity stress tolerance of plants.

Peptides encoded by small coding genes play an important role in plant development, acting in a similar manner as phytohormones. Few hormone-like peptides, however, have been shown to play a role in abiotic stress tolerance. In the current study, 17 Arabidopsis genes coding for small peptides were found to be up-regulated in response to salinity stress. To identify peptides leading salinity stress tolerance, we generated transgenic Arabidopsis plants overexpressing these small coding genes and assessed survivability and root growth under salinity stress conditions. Results indicated that 4 of the 17 overexpressed genes increased salinity stress tolerance. Further studies focused on AtPROPEP3, which was the most highly up-regulated gene under salinity stress. Treatment of plants with synthetic peptides encoded by AtPROPEP3 revealed that a C-terminal peptide fragment (AtPep3) inhibited the salt-induced bleaching of chlorophyll in seedlings. Conversely, knockdown AtPROPEP3 transgenic plants exhibited a hypersensitive phenotype under salinity stress, which was complemented by the AtPep3 peptide. This functional AtPep3 peptide region overlaps with an AtPep3 elicitor peptide that is related to the immune response of plants. Functional analyses with a receptor mutant of AtPep3 revealed that AtPep3 was recognized by the PEPR1 receptor and that it functions to increase salinity stress tolerance in plants. Collectively, these data indicate that AtPep3 plays a significant role in both salinity stress tolerance and immune response in Arabidopsis.

Class I Polyhydroxyalkanoate Synthase from the Purple Photosynthetic Bacterium Rhodovulum sulfidophilum Predominantly Exists as a Functional Dimer in the Absence of a Substrate.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that accumulate as carbon and energy storage compounds in a variety of micro-organisms. The marine purple photosynthetic bacterium Rhodovulum sulfidophilum is capable of synthesizing PHA. In this study, we cloned a gene encoding a class I PHA synthase from R. sulfidophilum (phaCRs ) and synthesized PhaCRs using a cell-free protein expression system. The specific activity of PhaCRs increased linearly as the (R)-3-hydroxybutyryl-coenzyme A (3HB-CoA) concentration increased and never reached a plateau, even at 3.75 mM 3HB-CoA, suggesting that PhaCRs was not saturated because of low substrate affinity. Size exclusion chromatography and native polyacrylamide gel electrophoresis analyses revealed that PhaCRs exists predominantly as an active dimer even in the absence of 3HB-CoA, unlike previously characterized PhaCs. The linear relationship between the PhaCRs activity and 3HB-CoA concentrations could result from a low substrate affinity as well as the absence of a rate-limiting step during PHA polymerization because of the existence of predominantly active dimers.

Sucrose supplementation suppressed the growth inhibition in polyhydroxyalkanoate-producing plants.

Polyhydroxyalkanoate (PHA) is a thermoplastic polymer with several advantageous properties, including biomass origin, biocompatibility, and biodegradability. PHA is synthesized in transgenic plants harboring 3 enzymatic genes: phaA, phaB, and phaC (collectively referred to as phaABC). PHA-producing plants exhibit severe growth inhibition that leads to extremely low PHA accumulation when these enzymes are localized in the cytosol. This growth inhibition could be attributed to the deleterious effects of the PHA biosynthetic pathway on endogenous essential metabolites or to PHA cytotoxicity itself. We performed precise morphological observations of phaABC-overexpressing Arabidopsis (ABC-ox), which displayed typical growth inhibition. On growth medium without sucrose, ABC-ox exhibited a pale green phenotype, dwarfism, including small cotyledons and true leaves, and short roots. ABC-ox partially recovered from this growth inhibition when the growth medium was supplemented with 1% sucrose. This recovery was reversed after ABC-ox grown on 1% sucrose medium was transferred to soil. ABC-ox grown on 1% sucrose medium not only demonstrated recovery from growth inhibition but were also the only examined plants with PHA accumulation, suggesting that growth inhibition was not caused by PHA cytotoxicity but rather by a lack of essential metabolites.

A Screening Method for the Isolation of Polyhydroxyalkanoate-Producing Purple Non-sulfur Photosynthetic Bacteria from Natural Seawater.

Polyhydroxyalkanoates (PHAs) are a family of biopolyesters accumulated by a variety of microorganisms as carbon and energy storage under starvation conditions. We focused on marine purple non-sulfur photosynthetic bacteria as host microorganisms for PHA production and developed a method for their isolation from natural seawater. To identify novel PHA-producing marine purple non-sulfur photosynthetic bacteria, natural seawaters were cultured in nutrient-rich medium for purple non-sulfur photosynthetic bacteria, and twelve pink- or red-pigmented colonies were picked up. Gas chromatography mass spectrometry analysis revealed that four isolates synthesized PHA at levels ranging from 0.5 to 24.4 wt% of cell dry weight. The 16S ribosomal RNA sequence analysis revealed that one isolate (HM2) showed 100% identity to marine purple non-sulfur photosynthetic bacteria. In conclusion, we have demonstrated in this study that PHA-producing marine purple non-sulfur photosynthetic bacteria can be isolated from natural seawater under nutrient-rich conditions.

Synthesis of High-Molecular-Weight Polyhydroxyalkanoates by Marine Photosynthetic Purple Bacteria.

Polyhydroxyalkanoate (PHA) is a biopolyester/bioplastic that is produced by a variety of microorganisms to store carbon and increase reducing redox potential. Photosynthetic bacteria convert carbon dioxide into organic compounds using light energy and are known to accumulate PHA. We analyzed PHAs synthesized by 3 purple sulfur bacteria and 9 purple non-sulfur bacteria strains. These 12 purple bacteria were cultured in nitrogen-limited medium containing acetate and/or sodium bicarbonate as carbon sources. PHA production in the purple sulfur bacteria was induced by nitrogen-limited conditions. Purple non-sulfur bacteria accumulated PHA even under normal growth conditions, and PHA production in 3 strains was enhanced by nitrogen-limited conditions. Gel permeation chromatography analysis revealed that 5 photosynthetic purple bacteria synthesized high-molecular-weight PHAs, which are useful for industrial applications. Quantitative reverse transcription polymerase chain reaction analysis revealed that mRNA levels of phaC and PhaZ genes were low under nitrogen-limited conditions, resulting in production of high-molecular-weight PHAs. We conclude that all 12 tested strains are able to synthesize PHA to some degree, and we identify 5 photosynthetic purple bacteria that accumulate high-molecular-weight PHA molecules. Furthermore, the photosynthetic purple bacteria synthesized PHA when they were cultured in seawater supplemented with acetate. The photosynthetic purple bacteria strains characterized in this study should be useful as host microorganisms for large-scale PHA production utilizing abundant marine resources and carbon dioxide.

Possible Involvement of MYB44-Mediated Stomatal Regulation in Systemic Resistance Induced by Penicillium simplicissimum GP17-2 in Arabidopsis.

The plant growth-promoting fungus (PGPF), Penicillium simplicissimum GP17-2 (GP17-2), induces systemic resistance against Pseudomonas syringae pv. tomato DC3000 (Pst) in Arabidopsis thaliana. The molecular mechanisms underlying induced systemic resistance (ISR) by GP17-2 were investigated in the present study. Microscopic observations revealed that stomatal reopening by Pst was restricted by elicitation with the culture filtrate (CF) from GP17-2. A gene expression analysis of MYB44, which enhances abscisic acid signaling and consequently closes stomata, revealed that the gene was activated by CF. CF-elicited myb44 mutant plants failed to restrict stomatal reopening and showed lower resistance to Pst than wild-type plants. These results indicate that stomatal resistance by GP17-2 is mediated by the gene activation of MYB44. We herein revealed that the MYB44-mediated prevention of penetration through the stomata is one of the components responsible for GP17-2-elicited ISR.

BPG3 is a novel chloroplast protein that involves the greening of leaves and related to brassinosteroid signaling.

Brassinosteroids are plant steroid hormones that regulate plant organs and chloroplast development. The detailed molecular mechanism for plant development by BR signaling is yet to be revealed, and many points regarding the relationship between BR signaling and chloroplast development remain unknown. We identify here the dominant mutant Brz-insensitive-pale green3-1D (bpg3-1D) from the Arabidopsis FOX lines that show reduced sensitivity to the chlorophyll accumulation promoted by the BR biosynthesis inhibitor, Brassinazole (Brz), in the light. BPG3 encodes a novel chloroplast protein that is evolutionally conserved in bacteria, algae, and higher plants. The expression of BPG3 was induced by light and Brz. The inhibition of electron transport in photosystem II of the chloroplasts was detected in bpg3-1D. These results suggest that BPG3 played an important role in regulating photosynthesis in the chloroplast under BR signaling.

Substantial expression of novel small open reading frames in Oryza sativa.

In our previous integrated study combining informatics and molecular biology analyses, we revealed that Arabidopsis small open reading frames (sORFs) predicted by computational analysis have biological functions in morphogenesis. Here, we report that sequences homologous to Arabidopsis sORFs are abundant in intergenic regions of the rice genome. These sequences represent a subset of non-protein-coding DNA, and some are transcribed into mRNA. These results indicate that many sORFs associated with morphogenesis are hidden in the genomes of crop species.