Insights into stereoselective ring formation in canonical strigolactone: Identification of a dirigent domain-containing enzyme catalyzing orobanchol synthesis.

Strigolactones (SLs) are plant apocarotenoids with diverse roles and structures. Canonical SLs, widespread and characterized by structural variations in their tricyclic lactone (ABC-ring), are classified into two types based on C-ring configurations. The steric C-ring configuration emerges during the BC-ring closure, downstream of the biosynthetic intermediate, carlactonoic acid (CLA). Most plants produce either type of canonical SLs stereoselectively, e.g., tomato (Solanum lycopersicum) yields orobanchol with an α-oriented C-ring. The mechanisms driving SL structural diversification are partially understood, with limited insight into functional implications. Furthermore, the exact molecular mechanism for the stereoselective BC-ring closure reaction is yet to be known. We identified an enzyme, the stereoselective BC-ring-forming factor (SRF), from the dirigent protein (DIR) family, specifically the DIR-f subfamily, whose biochemical function had not been characterized, making it a key enzyme in stereoselective canonical SL biosynthesis with the α-oriented C-ring. We first confirm the precise catalytic function of the tomato cytochrome P450 SlCYP722C, previously shown to be involved in orobanchol biosynthesis [T. Wakabayashi et al., Sci. Adv. 5, eaax9067 (2019)], to convert CLA to 18-oxocarlactonoic acid. We then show that SRF catalyzes the stereoselective BC-ring closure reaction of 18-oxocarlactonoic acid, forming orobanchol. Our methodology combines experimental and computational techniques, including SRF structure prediction and conducting molecular dynamics simulations, suggesting a catalytic mechanism based on the conrotatory 4π-electrocyclic reaction for the stereoselective BC-ring formation in orobanchol. This study sheds light on the molecular basis of how plants produce SLs with specific stereochemistry in a controlled manner.

Discontinuous Translocation of a Luciferase Protein beyond Graft Junction in Tobacco.

Transgrafting, a grafting technique that uses both genetically modified (GM) and non-GM plants, is a novel plant breeding technology that can be used to improve the efficiency of crop cultivation without introducing foreign genes into the edible parts of non-GM plants. This technique can facilitate the acquisition of disease resistance and/or increased yield. However, the translocation of low-molecular-weight compounds, ribonucleic acid (RNA), and proteins through graft junctions raises a potential safety risk for food crops. Here, we used a transgenic tobacco plant expressing a firefly luciferase gene (LUC) to examine the translocation of the LUC protein beyond the graft junction in grafted plants. We observed the bi-directional translocation of LUC proteins in transgrafted tobacco plants, i.e., from the rootstock to scion and vice versa. Transcriptomic analysis revealed that transcripts of the LUC gene were undetectable in non-GM plant bodies, indicating that the LUC protein itself was translocated. Moreover, the movement of the LUC protein is an episodic (i.e., non-continuous) event, since non-GM samples showing high LUC activity were flanked by non-GM samples showing no apparent LUC activity. Translocation from the GM to non-GM part depends on the characteristics of GM plant bodies; here, the enhanced translocation of the LUC protein into the non-GM scion was observed when LUC-expressing rootstocks with hairy roots were used. Moreover, the quantity of translocated LUC protein was far below the level that is generally required to induce an allergenic response. Finally, since the LUC protein levels of plants used for transgrafting are moderate and the LUC protein itself is relatively unstable, further investigation is necessary regarding whether the newly expressed protein in GM plants is highly stable, easily translocated, and/or highly expressed.

The structure-activity relationship of aryloxyacetylthioureas for the inhibition of Orobanche minor radicle elongation.

Orobanchaceae root parasitic weeds cause significant damage to agriculture and become threats to global food security. Integrated pest management is a key concept in modern agriculture and requires chemicals with various modes of action. Planteose accumulates as a storage carbohydrate in the dry seeds of root parasitic weeds. In Orobanche minor seeds, planteose is hydrolyzed by an α-galactosidase, OmAGAL2, during germination. It was found that the OmAGAL2 inhibitor, PI-28, suppressed the radicle elongation of germinating O. minor seeds. This inhibitory activity against O. minor radicle elongation was evaluated for a series of aryloxyacetylthioureas synthesized based on the structure of PI-28. Compounds with a 3-Cl or 4-Cl substituent on the benzene ring in the phenoxy moiety in PI-28 exhibited more potent activity than the parent PI-28. This is the first report on the effect of aryloxyacetylthioureas on a root parasitic weed and will contribute to the development of control reagents for root parasitic weeds.

Multi-omics Analyses of Non-GM Tomato Scion Engrafted on GM Rootstocks.

Grafting has been widely applied in agricultural production in order to utilize agriculturally valuable traits. The use of genetically modified (GM) plants for grafting with non-GM crops will soon be implemented to generate chimeric plants (transgrafting)*, and the non-GM edible portions thus obtained could fall outside of the current legal regulations. A number of metabolites and macromolecules are reciprocally exchanged between scion and rootstock, affecting the crop properties as food. Accordingly, the potential risks associated with grafting, particularly those related to transgrafting with GM plants, should be carefully evaluated based on scientific evidence. In this study, we prepared a hetero-transgraft line composed of non-GM tomato scion and GM-tobacco rootstock expressing firefly luciferase. We also prepared a homograft line (both rootstock and scion are from non-GM tomato) and a heterograft line (non-GM tobacco rootstock and non-GM tomato scion). The non-GM tomato fruits were harvested from these grafted lines and subjected to comprehensive characterization by multi-omics analysis. Proteomic analysis detected tobacco-derived proteins from both heterograft and hetero-transgraft lines, suggesting protein transfer from the tobacco rootstock to the tomato fruits. No allergenicity information is available for these two tobacco-derived proteins. The transcript levels of the genes encoding two allergenic tomato intrinsic proteins (Sola l 4.0101 and Sola l 4.0201) decreased in the heterograft and hetero-transgraft lines. Several differences were observed in the metabolic profiles, including α-tomatine and nicotine. The accumulation of tobacco-derived nicotine in the tomato fruits of both heterograft and hetero-transgraft lines indicated that the transfer of unfavorable metabolites from rootstock to scion should be assessed as a food safety concern. Further investigations are needed to clarify whether variable environmental conditions and growth periods may influence the qualities of the non-GM edible parts produced by such transgrafted plants.

Omics Profiles of Non-GM Tubers from Transgrafted Potato with a GM Scion.

"Transgrafting" is a grafting procedure whereby a transgenic plant body is grafted to a non-transgenic plant body. It is a novel plant breeding technology that allows non-transgenic plants to obtain benefits usually conferred to transgenic plants. Many plants regulate flowering by perceiving the day-length cycle via expression of FLOWERING LOCUS T (FT) in the leaves. The resulting FT protein is translocated to the shoot apical meristem via the phloem. In potato plants, FT is involved in the promotion of tuber formation. Here we investigated the effects of a genetically modified (GM) scion on the edible parts of the non-GM rootstock by using potato plants transformed with StSP6A, a novel potato homolog of the FT gene. Scions prepared from GM or control (wild-type) potato plants were grafted to non-GM potato rootstocks; these were designated as TN and NN plants, respectively. After tuber harvest, we observed no significant differences in potato yield between TN and NN plants. Transcriptomic analysis revealed that only one gene-with unknown function-was differentially expressed between TN and NN plants. Subsequent proteomic analysis indicated that several members of protease inhibitor families, known as anti-nutritional factors in potato, were slightly more abundant in TN plants. Metabolomic analysis revealed a slight increase in metabolite abundance in NN plants, but we observed no difference in the accumulation of steroid glycoalkaloids, toxic metabolites found in potato. Finally, we found that TN and NN plants did not differ in nutrient composition. Taken together, these results indicate that FT expression in scions had a limited effect on the metabolism of non-transgenic potato tubers.

Omics Profiles of Non-transgenic Scion Grafted on Transgenic RdDM Rootstock.

Grafting of commercial varieties onto transgenic stress-tolerant rootstocks is attractive approach, because fruit from the non-transgenic plant body does not contain foreign genes. RNA silencing can modulate gene expression and protect host plants from viruses and insects, and small RNAs (sRNAs), key molecules of RNA silencing, can move systemically. Here, to evaluate the safety of foods obtained from sRNA-recipient plant bodies, we investigated the effects of rootstock-derived sRNAs involved in mediating RNA-directed DNA methylation (RdDM) on non-transgenic scions. We used tobacco rootstocks showing RdDM against the cauliflower mosaic virus (CaMV) 35S promoter. When scions harboring CaMV 35S promoter sequence were grafted onto RdDM-inducing rootstocks, we found that RdDM-inducing sRNAs were only weakly transported from the rootstocks to the scion, and we observed a low level of DNA methylation of the CaMV 35S promoter in the scion. Next, wild-type (WT) tobacco scions were grafted onto RdDM-inducing rootstocks (designated NT) or WT rootstocks (designated NN), and scion leaves were subjected to multi-omics analyses. Our transcriptomic analysis detected 55 differentially expressed genes between the NT and NN samples. A principal component analysis of proteome profiles showed no significant differences. In the positive and negative modes of LC-ESI-MS and GC-EI-MS analyses, we found a large overlap between the metabolomic clusters of the NT and NN samples. In contrast, the negative mode of a LC-ESI-MS analysis showed separation of clusters of NT and NN metabolites, and we detected 6 peak groups that significantly differed. In conclusion, we found that grafting onto RdDM-inducing rootstocks caused a low-level transmission of sRNAs, resulting in limited DNA methylation in the scion. However, the causal relationships between sRNA transmission and the very slight changes in the transcriptomic and metabolomic profiles of the scions remains unclear. The safety assessment points for grafting with RdDM rootstocks are discussed.

Transcriptome and metabolome analyses revealed that narrowband 280 and 310 nm UV-B induce distinctive responses in Arabidopsis.

In plants, the UV-B photoreceptor UV RESISTANCE LOCUS8 (UVR8) perceives UV-B and induces UV-B responses. UVR8 absorbs a range of UV-B (260-335 nm). However, the responsiveness of plants to each UV-B wavelength has not been intensively studied so far. Here, we performed transcriptome and metabolome analyses of Arabidopsis using UV light emitting diodes (LEDs) with peak wavelengths of 280 and 310 nm to investigate the differences in the wavelength-specific UV-B responses. Irradiation with both UV-LEDs induced gene expression of the transcription factor ELONGATED HYPOCOTYL 5 (HY5), which has a central role in the UVR8 signaling pathway. However, the overall transcriptomic and metabolic responses to 280 and 310 nm UV-LED irradiation were different. Most of the known UV-B-responsive genes, such as defense-related genes, responded only to 280 nm UV-LED irradiation. Lipids, polyamines and organic acids were the metabolites most affected by 280 nm UV-LED irradiation, whereas the effect of 310 nm UV-LED irradiation on the metabolome was considerably less. Enzymatic genes involved in the phenylpropanoid pathway upstream in anthocyanin biosynthesis were up-regulated only by 280 nm UV-LED irradiation. These results revealed that the responsivenesses of Arabidopsis to 280 and 310 nm UV-B were significantly different, suggesting that UV-B signaling is mediated by more complex pathways than the current model.

Effect of high-dose 290 nm UV-B on resveratrol content in grape skins.

UV-C irradiation increases resveratrol content in grape skins, but it reaches a maximum at a certain UV-C dose. In contrast, UV-B has a weak resveratrol-enhancing effect at low doses, but it has not been investigated at high doses. In this study, we investigated the effect of high-dose UV-B on resveratrol contents in grape skins. Irradiation of Muscat Bailey A with 290 nm UV-B LED at 22 500 and 225 000 µmol m-2 increased the resveratrol contents in the grape skins by 2.1- and 9.0-fold, respectively, without significant increases in other phenolic compounds. The effect was also confirmed for 2 other cultivars: Shine Muscat and Delaware. Transcriptome analysis of the grape skins of Muscat Bailey A immediately after irradiation with UV-B at 225 000 µmol m-2 showed that genes related to biotic and abiotic stresses were upregulated. Hence, it was suggested that high-dose UV-B irradiation induces a stress response and specifically activates resveratrol biosynthesis.

Involvement of α-galactosidase OmAGAL2 in planteose hydrolysis during seed germination of Orobanche minor.

Root parasitic weeds of the Orobanchaceae, such as witchweeds (Striga spp.) and broomrapes (Orobanche and Phelipanche spp.), cause serious losses in agriculture worldwide, and efforts have been made to control these parasitic weeds. Understanding the characteristic physiological processes in the life cycle of root parasitic weeds is particularly important to identify specific targets for growth modulators. In our previous study, planteose metabolism was revealed to be activated soon after the perception of strigolactones in germinating seeds of O. minor. Nojirimycin inhibited planteose metabolism and impeded seed germination of O. minor, indicating a possible target for root parasitic weed control. In the present study, we investigated the distribution of planteose in dry seeds of O. minor by matrix-assisted laser desorption/ionization-mass spectrometry imaging. Planteose was detected in tissues surrounding-but not within-the embryo, supporting its suggested role as a storage carbohydrate. Biochemical assays and molecular characterization of an α-galactosidase family member, OmAGAL2, indicated that the enzyme is involved in planteose hydrolysis in the apoplast around the embryo after the perception of strigolactones, to provide the embryo with essential hexoses for germination. These results indicate that OmAGAL2 is a potential molecular target for root parasitic weed control.

Exploration and characterization of chemical stimulators to maximize the wax ester production by Euglena gracilis.

Euglena gracilis, a phototrophic protist, is a valuable biomass producer that is often employed in sustainable development efforts. E. gracilis accumulates wax esters as byproducts during anaerobic ATP production via the reductive tricarboxylic acid cycle, utilizing the storage carbohydrate ß-1,3-glucan paramylon as the carbon source. Here, we report a library screening for chemical stimulators that accelerate both wax ester production and paramylon consumption. Among the 115 compounds tested, we identified nine compounds that increased wax ester production by more than 2.0-fold relative to the solvent control. In the presence of these nine compounds, the paramylon content decreased compared with the control experiment, and the residual paramylon content varied between 7% and 26% of the initial level. The most active compound, 1,4-diaminoanthracene-9,10-dione (OATQ008), stimulated wax ester production up to 2.7-fold within 24 h, and 93% of the cellular paramylon was consumed. In terms of the structural features of the chemical stimulators, we discuss the potential target sites to stimulate wax ester production in mitochondria under anaerobic conditions.

Effect of Transgenic Rootstock Grafting on the Omics Profiles in Tomato.

Grafting of non-transgenic scion onto genetically modified (GM) rootstocks provides superior agronomic traits in the GM rootstock, and excellent fruits can be produced for consumption. In such grafted plants, the scion does not contain any foreign genes, but the fruit itself is likely to be influenced directly or indirectly by the foreign genes in the rootstock. Before market release of such fruit products, the effects of grafting onto GM rootstocks should be determined from the perspective of safety use. Here, we evaluated the effects of a transgene encoding ß-glucuronidase (GUS) on the grafted tomato fruits as a model case. An edible tomato cultivar, Stella Mini Tomato, was grafted onto GM Micro-Tom tomato plants that had been transformed with the GUS gene. The grafted plants showed no difference in their fruit development rate and fresh weight regardless of the presence or absence of the GUS gene in the rootstock. The fruit samples were subjected to transcriptome (NGS-illumina), proteome (shotgun LC-MS/MS), metabolome (LC-ESI-MS and GC-EI-MS), and general food ingredient analyses. In addition, differentially detected items were identified between the grafted plants onto rootstocks with or without transgenes (more than two-fold). The transcriptome analysis detected approximately 18,500 expressed genes on average, and only 6 genes were identified as differentially expressed. Principal component analysis of 2,442 peaks for peptides in proteome profiles showed no significant differences. In the LC-ESI-MS and GC-EI-MS analyses, a total of 93 peak groups and 114 peak groups were identified, respectively, and only 2 peak groups showed more than two-fold differences. The general food ingredient analysis showed no significant differences in the fruits of Stella scions between GM and non-GM Micro-Tom rootstocks. These multiple omics data showed that grafting on the rootstock harboring the GUS transgene did not induce any genetic or metabolic variation in the scion.

Fertile Arabidopsis cyp704b1 mutant, defective in sporopollenin biosynthesis, has a normal pollen coat and lipidic organelles in the tapetum.

The exine acts as a protectant of the pollen from environmental stresses, and the pollen coat plays an important role in the attachment and recognition of the pollen to the stigma. The pollen coat is made of lipidic organelles in the tapetum. The pollen coat is necessary for fertility, as pollen coat-less mutants, such as those deficient in sterol biosynthesis, show severe male sterility. In contrast, the exine is made of sporopollenin precursors that are biosynthesized in the tapetum. Some mutants involved in sporopollenin biosynthesis lose the exine but show the fertile phenotype. One of these mutants, cyp704b1, was reported to lose not only the exine but also the pollen coat. To identify the cause of the fertile phenotype of the cyp704b1 mutant, the detailed structures of the tapetum tissue and pollen surface in the mutant were analyzed. As a result, the cyp704b1 mutant completely lost the normal exine but had high-electron-density granules localized where the exine should be present. Furthermore, normal lipidic organelles in the tapetum and pollen coat embedded between high-electron-density granules on the pollen surface were observed, unlike in a previous report, and the pollen coat was attached to the stigma. Therefore, the pollen coat is necessary for fertility, and the structure that functions like the exine, such as high-electron-density granules, on the pollen surface may play important roles in retaining the pollen coat in the cyp704b1 mutant.

Germination stimulatory activity of bacterial butenolide hormones from Streptomyces albus J1074 on seeds of the root parasitic weed Orobanche minor.

Damage caused by Orobanchaceae root parasitic weeds is a substantial agricultural problem for global food security. Many studies have been conducted to establish practical methods of control, but efforts are still required for successful management. Seed germination of root parasitic weeds requires host-derived germination stimulants including strigolactones (SLs). Studies on SLs have revealed that a butenolide ring is the essential moiety for SL activity as a germination stimulant. Interestingly, recent studies have revealed that butenolide hormones regulate the biosynthesis of secondary metabolites and mediate communication in actinomycete bacteria. Because of the structural similarity between SLs and the bacterial butenolides, we evaluated the germination stimulatory activity of butenolides isolated from Streptomyces albus J1074 on root parasitic weeds. These butenolides were found to specifically induce seed germination of Orobanche minor. Our findings contribute to understanding the molecular mechanisms of germination stimulant perception and to the development of a method for their biological control.

Identification of a Flavin Monooxygenase-Like Flavonoid 8-Hydroxylase with Gossypetin Synthase Activity from Lotus japonicus.

Lotus japonicus is a model legume that accumulates 8-hydroxyflavonol derivatives, such as gossypetin (8-hydroxyquercetin) 3-O-glycoside, which confer the yellow color to its petals. An enzyme, flavonoid 8-hydroxylase (F8H; LjF8H), is assumed to be involved in the biosynthesis, but the specific gene is yet to be identified. The LjF8H cDNA was isolated as a flavin adenine dinucleotide (FAD)-binding monooxygenase-like protein using flower buds and flower-specific EST data of L. japonicus. LjF8H is a single copy gene on chromosome III consisting of six exons. The conserved FAD- and NAD(P)H-dependent oxidase motifs were found in LjF8H. Phylogenetic analysis suggested that LjF8H is a member of the flavin monooxygenase group but distinctly different from other known flavonoid oxygenases. Analysis of recombinant yeast microsome expressing LjF8H revealed that the enzyme catalyzed the 8-hydroxylation of quercetin. Other flavonoids, such as naringenin, eriodictyol, apigenin, luteolin, taxifolin and kaempferol, also acted as substrates of LjF8H. This broad substrate acceptance was unlike known F8Hs in other plants. Interestingly, flavanone and flavanonol, which have saturated C-C bond at positions 2 and 3 of the flavonoid C-ring, produced 6-hyroxylflavonoids as a by-product of the enzymatic reaction. Furthermore, LjF8H only accepted the 2S-isomer of naringenin, suggesting that the conformational state of the substrates might affect product specificity. The overexpression of LjF8H in Arabidopsis thaliana and Petunia hybrida synthesized gossypetin and 8-hydroxykaempferol, respectively, indicating that LjF8H was functional in plant cells. In conclusion, this study represents the first instance of cloning and identification of F8Hs responsible for gossypetin biosynthesis.

The effect of nojirimycin on the transcriptome of germinating Orobanche minor seeds.

Orobanchaceae root parasitic weeds cause serious agricultural damage worldwide. Although numerous studies have been conducted to establish an effective control strategy for the growth and spread of root parasitic weeds, no practical method has been developed so far. Previously, metabolomic analyses were conducted on germinating seeds of a broomrape, Orobanche minor, to find novel targets for its selective control. Interestingly, planteose metabolism was identified as a possible target, and nojirimycin (NJ) selectively inhibited the germination of O. minor by intercepting planteose metabolism, although its precise mode of action was unclear. Here, transcriptome analysis by RNA-Seq was conducted to obtain molecular insight into the effects of NJ on germinating O. minor seeds. Differential gene expression analysis results suggest that NJ alters sugar metabolism and/or signaling, which is required to promote seed germination. This finding will contribute to understanding the effect of NJ and establishing a novel strategy for parasitic weed control.

Rhizotaxis Modulation in Arabidopsis Is Induced by Diffusible Compounds Produced during the Cocultivation of Arabidopsis and the Endophytic Fungus Serendipita indica.

Rhizotaxis is established under changing environmental conditions via periodic priming of lateral root (LR) initiation at the root tips and adaptive LR formation along the primary root (PR). In contrast to the adaptable LR formation in response to nutrient availability, there is little information on root development during interactions with beneficial microbes. The Arabidopsis root system is characteristically modified upon colonization by the root endophytic fungus Serendipita indica, accompanied by a marked stimulation of LR formation and the inhibition of PR growth. This root system modification has been attributed to endophyte-derived indole-3-acetic acid (IAA). However, it has yet to be clearly explained how fungal IAA affects the intrinsic LR formation process. In this study, we show that diffusible compounds (chemical signals) other than IAA are present in the coculture medium of Arabidopsis and S. indica and induce auxin-responsive DR5::GUS expression in specific sections within the pericycle layer. The DR5::GUS expression was independent of polar auxin transport and the major IAA biosynthetic pathways, implicating unidentified mechanisms responsible for the auxin response and LR formation. Detailed metabolite analysis revealed the presence of multiple compounds that induce local auxin responses and LR formation. We found that benzoic acid (BA) cooperatively acted with exogenous IAA to generate a local auxin response in the pericycle layer, suggesting that BA is one of the chemical signals involved in adaptable LR formation. Identification and characterization of the chemical signals will contribute to a greater understanding of the molecular mechanisms underlying adaptable root development and to unconventional technologies for sustainable agriculture.

Identification of novel cytochrome P450 monooxygenases from actinomycetes capable of intermolecular oxidative C-C coupling reactions.

The cross-coupling reaction is one of the most important chemical reactions in the modern organic chemistry. Biocatalysts capable of catalyzing C-C coupling reactions are desired in the chemical industry for sustainable development. Cytochrome P450 monooxygenases (P450s) have received considerable attention as biocatalysts capable of catalyzing such reactions. Here, we focused on actinomycete P450s, which have high homology with CYP158A2, involved in the oxidative C-C coupling reaction for flaviolin dimerization in Streptomyces coelicolor A3(2). The screening of a chemical library composed of 426 aromatic compounds identified several combinations of P450s and their potential substrates. The type-I difference spectrum indicated that the identified substrates bind to the active sites of a P450, named StVI from Streptomyces violaceusniger. A redshift of the absorption maximum of the reaction products, together with LC-MS analysis suggested the presence of extended conjugate systems in the products through direct C-C coupling between two aromatic rings. The results demonstrated that actinomycete P450s have great potential to be utilized as biocatalysts for oxidative C-C coupling reactions and to facilitate the synthesis of diverse coupling products.

Wax Ester Synthase/Diacylglycerol Acyltransferase Isoenzymes Play a Pivotal Role in Wax Ester Biosynthesis in Euglena gracilis.

Wax ester fermentation is a unique energy gaining pathway for a unicellular phytoflagellated protozoan, Euglena gracilis, to survive under anaerobiosis. Wax esters produced in E. gracilis are composed of saturated fatty acids and alcohols, which are the major constituents of myristic acid and myristyl alcohol. Thus, wax esters can be promising alternative biofuels. Here, we report the identification and characterization of wax ester synthase/diacylglycerol acyltrasferase (WSD) isoenzymes as the terminal enzymes of wax ester production in E. gracilis. Among six possible Euglena WSD orthologs predicted by BLASTX search, gene expression analysis and in vivo evaluation for enzyme activity with yeast expressing individual recombinant WSDs indicated that two of them (EgWSD2 and EgWSD5) predominantly function as wax ester synthase. Furthermore, experiments with gene silencing demonstrated a pivotal role of both EgWSD2 and EgWSD5 in wax ester synthesis, as evidenced by remarkably reduced wax ester contents in EgWSD2/5-double knockdown E. gracilis cells treated with anaerobic conditions. Interestingly, the decreased ability to produce wax ester did not affect adaptation of E. gracilis to anaerobiosis. Lipid profile analysis suggested allocation of metabolites to other compounds including triacylglycerol instead of wax esters.

Seed Metabolome Analysis of a Transgenic Rice Line Expressing Cholera Toxin B-subunit.

Plant-based human vaccines have been actively developed in recent years, and rice (Oryza sativa L.) is one of the best candidate crops for their production and delivery. By expressing a modified cholera toxin B (CTB) subunit, we previously developed MucoRice-CTB, a rice-based vaccine against cholera, which is caused by infection of the intestine with the bacteria Vibrio cholerae. MucoRice-CTB lines have been extensively characterized by whole-genome sequencing and proteome analyses to evaluate the mutation profiles and proteome status, respectively. Here, we report non-targeted metabolomic profiling of the MucoRice-CTB transgenic rice line 51A (MR-CTB51A), MucoRice-RNAi (MR-RNAi), and their non-transgenic parent line by using gas chromatography-time-of-flight mass spectrometry. The levels of several amino acids, organic acids, carbohydrates, lipids, and secondary metabolites were significantly increased in MR-CTB51A compared with the non-transgenic parent line. These metabolomics results complement essential information obtained by genome sequencing and proteomics approaches, thereby contributing to comprehensive understanding of the properties of MucoRice-CTB as a plant-based vaccine.

Critical Involvement of Environmental Carbon Dioxide Fixation to Drive Wax Ester Fermentation in Euglena.

Accumulation profiles of wax esters in Euglena gracilis Z were studied under several environmental conditions. The highest amount of total wax esters accumulated under hypoxia in the dark, and C28 (myristyl-myristate, C14:0-C14:0) was prevalent among all conditions investigated. The wax ester production was almost completely suppressed under anoxia in the light, and supplying exogenous inorganic carbon sources restored wax ester fermentation, indicating the need for external carbon sources for the wax ester fermentation. 13C-labeling experiments revealed specific isotopic enrichment in the odd-numbered fatty acids derived from wax esters, indicating that the exogenously-supplied CO2 was incorporated into wax esters via the propionyl-CoA pathway through the reverse tricarboxylic acid (TCA) cycle. The addition of 3-mercaptopicolinic acid, a phosphoenolpyruvate carboxykinase (PEPCK) inhibitor, significantly affected the incorporation of 13C into citrate and malate as the biosynthetic intermediates of the odd-numbered fatty acids, suggesting the involvement of PEPCK reaction to drive wax ester fermentation. Additionally, the 13C-enrichment pattern of succinate suggested that the CO2 assimilation might proceed through alternative pathways in addition to the PEPCK reaction. The current results indicate that the mechanisms of anoxic CO2 assimilation are an important target to reinforce wax ester fermentation in Euglena.