Betalain pigments, primarily produced by the order Caryophyllales, are categorized into betacyanins (red/purple) and betaxanthins (yellow/orange). While the biosynthetic pathways of these pigments are well-studied, the genes responsible for betaxanthin biosynthesis in quinoa were previously unknown. This study identified three candidate genes, CqCYP76AD5v1, CqCYP76AD5v2, and CqCYP76AD130, as quinoa orthologs of beet CYP76AD5 and CYP76AD6. Agroinfiltration experiments in Nicotiana benthamiana revealed that CqCYP76AD5v1 exhibited L-DOPA synthesis activity, whereas CqCYP76AD130 did not. To enable large-scale production of betaxanthins, we developed a tobacco BY-2 cell line expressing CqCYP76AD5v1 and CqDODA1-1, with vulgaxanthin I identified as the predominant product. Furthermore, the betaxanthin mixture extracted from this line inhibited amyloid-β (Aβ) aggregation, a key factor associated with Alzheimer’s disease. These findings demonstrate the potential of betaxanthins derived from quinoa betaxanthin-biosynthesis genes for applications in health supplements and pharmaceuticals.

Arabinoxylan, a major hemicellulose in plant cell walls, particularly in grasses and cereals, plays a crucial role in structural integrity and biological functions, with diverse industrial applications such as food production and prebiotic development. Despite its significance, the molecular mechanism of arabinoxylan biosynthesis remains unclear. Here, we identified and characterized a xylan synthase catalytic subunit, Setaria viridis IRregular Xylem 10 (SvIRX10), from a new model plant for C₄-photosynthetic grasses, S. viridis A10.1. Bioinformatic analysis classified SvIRX10 as a glycosyltransferase 47 family member, conserved across various species. Recombinant SvIRX10 expressed in Expi293 cells exhibited xylan synthase activity for all tested xylotrimer (Xyl₃) acceptors with distinct fluorescent labels. The substrate conversion efficiency for 2-aminobenzoic acid-labeled Xyl₃ (Xyl₃-2AA) was highest, but those for other labeled Xyl₃ were lower. Nevertheless, the elongation efficiencies were comparable among tested acceptors when the xylan chains elongated enough. Structural prediction and docking simulations illustrated most frequently the productive conformations using Xyl₃-2AA and xylotetraose as ligands. The interactions between the two ligands and the active site were well-conserved, and all ligand units interacted with SvIRX10. These ligand conformations in the active site were similar, but those of other fluorescently labeled Xyl₃ differed except for the first xylosyl unit at the non-reducing end. Thus, SvIRX10 recognizes at least 4 xylosyl units in the xylan synthetic reaction. Together, these findings provide insights into the enzymatic mechanisms of SvIRX10 and the initiation of xylan elongation, offering potential applications for modifying plant cell walls in biomass utilization and functional food development.

Tip growth is vital for plant growth and development, yet the regulatory mechanisms governing this process remain incompletely understood. In this study, we identify Reagent F4, a novel small molecule that disrupts tip growth and polarized cell expansion in the moss, Physcomitrium patens protonemata. Through unbiased chemical screening, we found that Reagent F4 induces abnormal protonemal morphology, characterized by reduced cell elongation and stunted cell expansion. Our analyses revealed that F4 treatment triggers actin depolymerization and disrupts apical actin foci, which are critical for initiating and maintaining tip growth. Additionally, both acute and prolonged F4 exposure led to mislocalization of ROP GTPase, a key regulator of cell polarity. Transcriptomic analyses of F4 treated protonemata show significant downregulation of genes involved in lipid asymmetry, a process essential for polarized growth. These findings establish Reagent F4 as a valuable tool to investigate the molecular mechanisms governing tip growth in P. patens and highlight the potential role of lipid asymmetry in coordinating cytoskeletal organization and membrane polarity.

Uncaria plants, belonging to the Rubiaceae family, develop characteristic hooks at their leaf axils. In the Japanese Pharmacopoeia, the hooks from three Uncaria species, including U. rhynchophylla, are collectively defined as “Uncaria Hook” and are widely used as medicinal materials. The pharmacological properties of the diverse bioactive metabolites in U. rhynchophylla, particularly monoterpenoid indole alkaloids (MIAs), have been extensively studied. In this study, we aimed to establish sterile cultures of U. rhynchophylla as models for investigating MIA biosynthesis. LC-MS/MS-based untargeted metabolomic analysis revealed that the metabolomic profiles of stems from cultured plants showed strong similarity to those of medicinal parts from mature plants, specifically the hooks and stems. Furthermore, the analysis indicated that the contents of oxindole and indole alkaloids exhibited distinct variations depending on the plant part and developmental stage, both in sterile plant cultures and mature plants. Our findings demonstrate that U. rhynchophylla can be maintained under sterile conditions while stably producing MIAs. These cultured plants can serve as a model system not only for studying MIA biosynthetic pathways but also for ensuring quality control of Uncaria Hook in medicinal applications. This model system would contribute to the fundamental research by enhancing our understanding of the biosynthetic mechanisms and facilitating applications such as metabolic control of the contents of bioactive compounds in Uncaria Hook.

For ornamental plants, inflorescence architecture is one of the most important traits to determine their commercial values. However, molecular mechanisms of inflorescence architecture determination have not yet been fully elucidated. LEAFY (LFY), which encodes a plant-specific transcriptional factor, has been shown to play a key role in the switch from vegetative to reproductive phases. Recent studies have demonstrated that LFY is involved not only in floral meristem induction but also in inflorescence architecture determination. Tricyrtis spp., which belong to the family Liliaceae, show two different types of inflorescence architecture: T. hirta produces both apical and lateral flowers, whereas T. formosana produces only apical flowers. In the present study, we isolated LFY-homologous genes from T. hirta and T. formosana (designated as ThirLFY and TforLFY, respectively) and analyze their functions and expression patterns as a first step toward elucidation of molecular mechanisms of inflorescence architecture determination in Tricyrtis spp. Alignment analysis based on amino acid sequences showed that both ThirLFY and TforLFY have functional motifs of LFY, and only three amino acid differences are found between them. Transgenic Arabidopsis thaliana plants overexpressing ThirLFY or TforLFY showed early flowering and production of secondary inflorescences, and no functional differences were observed between ThirLFY and TforLFY. In situ hybridization analysis showed that ThirLFY was expressed in both apical and lateral buds of T. hirta, whereas TforLFY was only expressed in apical buds of T. formosana. Thus, two different types of inflorescence architecture in Tricyrtis spp. may be caused by different expression patterns of LFY-homologous genes.

Post-translational modification is critical for the bioactivity of small secreted-signaling peptides. The shoot apical meristem (SAM) activity that defines SAM size is controlled by the CLAVATA3 (CLV3) peptide ligand, which belongs to the CLV3/EMBRYO SURROUNDING REGIONRELATED (CLE) family, and its cognate receptor CLV1. The mature CLV3 peptide is post-translationally modified with tri-arabinose, increasing the binding affinity with CLV1. However, the mature form of most CLE peptides is unknown. Here we apply the synthetic CLE3 peptide with tri-arabinose to clv3 mutant to determine whether the CLE3 peptide can reduce the SAM size. We show that tri-arabinosylated CLE3 peptide exhibits stronger bioactivity in the SAM in a CLV1/BAM1-dependent manner. Our data emphasizes the importance of post-translational modification on peptide signaling, helping to characterize bona fide mature peptides.

Wheat (Triticum aestivum L.) consists of three genomes, and notable mutant phenotypes can be observed when all homoeologs are knocked out due to functional redundancy among the homoeologous gene copies. Therefore, high editing efficiency is required to rapidly obtain loss-of-function mutants in wheat. The endogenous tRNA processing system of CRISPR/Cas9 genome editing enables the expression of multiple single-guide RNA (sgRNAs) under the control of a single promoter, facilitating simultaneous multiple genome editing in an organism. Here, we evaluated the genome editing efficiency of multiple sgRNA expressions with the tRNA processing system. At first, using sgRNA of quantitative trait locus for seed dormancy 1, polycistronic tRNA–sgRNA vectors were introduced into immature embryos, and genome editing efficiency was evaluated in the transformed T₁ plants. In the use of three sgRNA modules, there was no difference in the efficiency of genome editing among the positions of the sgRNAs. We subsequently tested simultaneous genome editing of multiple homoeologous loci. Simultaneous expression of six sgRNAs per gene to target all homoeologous loci increased the editing efficiency of all homoeologous loci up to 100%. Our study indicates that the tRNA processing system is highly effective at simultaneous genome editing of homoeologous loci of wheat.

Bioluminescence monitoring techniques are widely used to study the gene expression dynamics in living plants. Monitoring the bioluminescence from a luciferase gene under the control of a circadian promoter is indispensable for examining plant circadian systems. The bioluminescence monitoring technique was successfully applied to physiological studies of circadian rhythms in duckweed plants. It has been reported that a luciferase gene under a constitutive promoter also exhibits a bioluminescent circadian rhythm in duckweed. However, the mechanisms underlying rhythm generation remain unknown. In this study, we performed a model-based analysis to evaluate the machinery that generates the bioluminescence rhythm. We hypothesized the rhythmic factor of three aspects regarding the bioluminescence intensities of luciferase in cells: luminescence efficiency, production rate, and degradation rate. Theoretically, if the latter two are involved in rhythm generation, the difference in luciferase stability affects the amplitude and phase relations of the bioluminescence rhythm. Luciferase stability is irrelevant to these rhythm properties if only the luminescence efficiency is involved. First, we simulated the bioluminescence rhythms of two luciferases with different stabilities associated with each of three rhythmic factors. Luciferase stability was set based on the reported values for Emerald-luciferase and Emerald-luciferase-PEST. We then experimentally examined the bioluminescence rhythms of reporters of these luciferases driven by the CAULIFLOWER MOSAIC VIRUS 35S promoter in the duckweed Lemna japonica. Their circadian properties matched those obtained from the simulation of the luminescence efficiency. This supports the view that cells in duckweed show circadian changes in physiological conditions associated with the luciferase enzyme reaction.

Paeonia lactiflora, the roots of which are used as a crude drug, is one of the most widely used and important medicinal plants. The long cultivation period and low proliferation rate of P. lactiflora makes it difficult to propagate large numbers of plants within a short period. We developed a bio-nursery system using plant tissue culture techniques to contribute to the supply of P. lactiflora seeds and seedlings in Japan. Here, we report on the improved tissue culture and acclimation conditions for a more stable and efficient bio-nursery system. We investigated the effect of culture conditions on shoot proliferation and the effect of calcium concentration during root induction and acclimation of cultured plantlets. The results demonstrated that the number of shoots increased under the 15/5°C diurnal temperature changing treatment [15°C, 12 h light (fluorescent light, 80–130 µmol m⁻² s⁻¹)/5°C, 12 h dark] compared to a constant temperature of 15°C. A higher calcium concentration (6 mM Ca²⁺) during root induction resulted in more vigorous growth after transplantation to the soil. In addition, it was found that planting in a closed greenhouse at a constant temperature of 20°C after cold treatment was suitable for acclimation of cultured plantlets. These findings are expected to contribute to the future seedling supply of P. lactiflora.

Histochemical detection of suberin lamellae has remarkably advanced our understanding of the roles of the apoplastic diffusion barrier in the root endodermis and the oxygen diffusion barrier in the root exodermis. Fluorol yellow 088 detects the aliphatic component of suberin and is one of the most reliable stains for detecting suberin lamellae in the endodermis and exodermis. Although fluorol yellow 088 staining can detect suberin lamellae in various plant roots by a simple procedure, conventional methods are time-consuming, as they need a long time to prepare the solution, stain, and wash the samples. Here, we propose a rapid method to minimize the time required to achieve suberin staining using root cross-sections. While conventional methods use polyethylene glycol-glycerol or lactic acid as the solvent, we found that fluorol yellow 088 readily dissolves into ethanol. This modification remarkably shortened the time required to prepare the solution, stain, and wash root cross-sections. Thus, our method will enhance the study of root anatomy and the histological development of plant roots.