Quinoa, a pseudocereal and leafy vegetable native to South America, is highly nutritious and can grow in harsh environments. One of the most prominent morphological features of quinoa is that the above-ground portion is covered with a layer of epidermal bladder cells (EBCs), and the role of EBCs in quinoa's high stress tolerance is of interest. Recent studies have shown that two WD40-repeat proteins, Reduced number of EBC (REBC) and REBC-like1, are required for EBC formation and that EBCs contribute defense mechanisms against biotic stress rather than abiotic stress. However, the role of EBCs in drought stress tolerance remains controversial due to the pleiotropic effects of these genes, including their impact on plant growth. Here, we show that REBC and REBC-like1 mediate water retention in detached quinoa leaves. Using a virus-induced gene silencing (VIGS) system, we found that downregulation of both REBC and REBC-like1 had no apparent effect on plant growth, but reduced the number of EBCs in both lowland and highland quinoa lines. Further, downregulation of both genes increased water loss in detached leaves of quinoa plants, supporting the notion that EBCs mediate water retention in quinoa leaves. Interestingly, we found higher EBC density in the southern highland lines grown in drier areas. Thus, we demonstrate that the effective use of VIGS in the analysis of genes with pleiotropic effects allows analyses that were difficult to perform using mutants alone, and that unlike mutants, functional genomics studies of quinoa can be easily performed in various lines using VIGS.

Rational metabolic-flow switching is an effective strategy that we previously proposed to produce exogenous high-value natural products in cultured plant cells through redirecting a highly active inherent metabolic pathway to a pathway producing related exogenous compounds. In previous proof-of-concept studies, we demonstrated that bamboo (Phyllostachys nigra; Pn) cells are a suitable host for production of phenylpropanoid-derived compounds, in particular those derived from feruloyl- and p-coumaroyl-CoAs. To expand the utility of Pn cells for production of exogenous metabolites via the rational metabolic-flow switching strategy, it is important to evaluate the metabolic potential of Pn cells under diverse culture conditions. In this study, we examined highly active metabolic pathway (s) in Pn suspension cells cultured under light. The Pn suspension cells strongly accumulated two light-induced compounds. These compounds were isolated and identified as 3-O-caffeoylquinic acid (neochlorogenic acid) and its regioisomer 5-O-caffeoylquinic acid (chlorogenic acid). Through optimization of the culture conditions, production titers of 3-O-caffeoylquinic acid and 5-O-caffeoylquinic acid in the Pn suspension cells reached 121 and 77.9 mg l⁻¹, respectively. These findings indicate that Pn cells are a suitable host for bioproduction of exogenous metabolites, in particular those derived from caffeoyl-CoA via the rational metabolic-flow switching strategy.

Plant viruses cause significant damage to global crop protection, since they can reduce plant quality and quantity, and the estimated annual cost of virus-induced damage is approximately $30 billion. Tomato mosaic virus (ToMV), a member of the Tobamovirus genus, presents a major threat to tomatoes and other solanaceous plants. Agricultural chemicals, including plant growth regulators, are commonly used to control the spread of pathogens, but these can be ineffective against viruses. In this study, we aimed to develop an antiviral agent using micronutrients such as zinc, iron, and copper. The plant virus disease control effects of these micronutrients was evaluated by applying zinc gluconate (ZnGluc), iron gluconate (FeGluc), and copper gluconate, (CuGluc) solutions to Nicotiana benthamiana plants that were subsequently inoculated with ToMV. Our results showed that ZnGluc exhibited the highest disease control activity and did not cause phytotoxic effects. Further analysis via quantitative real-time polymerase chain reaction analysis confirmed these findings. In addition, a mixture of ZnGluc and proanthocyanidins sourced from Alpinia zerumbet extracts exerted a synergistic disease control effect. Overall, we provide the first evidence that micronutrients, especially ZnGluc, exhibit significant disease control activity against ToMV, and thereby suggest that these treatments have potential as an agricultural chemical.

Japanese cedar or sugi (Cryptomeria japonica D. Don) is among the most important plantation conifers in Japan, occupying 12% of the total land area in the country. We have successfully established a CRISPR/Cas9-based genome editing system in C. japonica. However, in practical use, we encountered problems of low efficiency when generating biallelic mutations, i.e., target gene knockouts. As part of our efforts to improve efficiency, we codon-optimized the Cas9 gene, evaluated by the genome editing efficiency of CjChl I, a gene encoding a chlorophyll biosynthesis enzyme. As a result, our codon-optimized SpCas9, named ^CjSpCas9, performed the highest genome editing efficiency of two targets (t4, t1+t2). Specifically, the biallelic disruption efficiency of the CjChl I with ^CjSpCas9 was 1.8-fold higher than that of the SpCas9 gene optimized for Arabidopsis thaliana (^AtSpCas9) and 2.0-fold higher than that of the SpCas9 gene optimized for Orysa sativa (^OsSpCas9) for t4, respectively. For t1+t2, the efficiency was 4.9-fold higher than that of ^AtSpCas9 and 1.4-fold higher than that of ^OsSpCas9, respectively. Our western blotting analysis proved that the Cas9 protein accumulation increased upon codon frequency optimization. We concluded that the observed efficiency improvement was due to the increased Cas9 protein quantity. The efficient genome editing system we report here would accelerate molecular breeding in conifers.

Coronaridine, a monoterpenoid indole alkaloid, is present in Tabernanthe iboga and the related species Tabernaemontana divaricata. Recent exhaustive analysis revealed its presence in Catharanthus roseus, though specific details remain unknown. We conducted a detailed analysis of coronaridine in C. roseus, detecting it in seedlings post-germination up to 8 weeks after sowing, with peak abundance at 3–4 weeks. Gradual decrease occurred from the flowering stage, and it was absent during seed formation. The accumulation varied dramatically with the plant's growth phase. LC–MS/MS analysis confirmed (−) coronaridine, consistent with T. iboga. Additionally, cultivating at 35 °C increased coronaridine accumulation over 10-fold. These findings hold potential for enhancing the stable production of iboga alkaloids for pharmaceutical use.

Methylglyoxal synthase (MGS), which converts dihydroxyacetone phosphate to methylglyoxal (MG), is found in only prokaryotes. Synechocystis sp. PCC 6803 possesses the gene sll0036, which encodes MGS. To clarify the biological function of MGS, we constructed a gene-disruption strain of Synechocystis sp. PCC 6803. Expression analysis showed that MG metabolic genes (sll0036, sll0067, and slr1167) were upregulated under photoautotrophic conditions compared to mixotrophic conditions. The sll0036-deficient strain (Δ0036) exhibited a higher growth rate than the wild-type (WT) strain under mixotrophic conditions, whereas no significant difference was observed under photoautotrophic conditions. When cells were cultured in a medium supplemented with sorbitol or mannitol instead of glucose, the growth enhancement observed in the Δ0036 strain disappeared. This suggests that the difference in growth between Δ0036 and WT is influenced by glucose-related metabolism rather than osmotic stress. MG contents were found to be decreased in the Δ0036 strain compared to WT under mixotrophic conditions. This suggests that the reduction of MG level might activate the cell proliferation of Synechocystis sp. PCC 6803 under mixotrophic conditions.

KODA, a type of oxylipin, has stimulatory effects on plant growth under limiting conditions of stress, such as promoting flowering, rooting, and resistance to pathogens, for use in agriculture. KODA is released from Lemna paucicostata under drought, heat, and osmotic pressure, and is produced from α-linolenic acid by a two-step enzymatic reaction with 9-lipoxygenase and allene oxide synthase. In this paper, we report the outstanding KODA productivity of L. paucicostata, SH strain screened from 56 Lemna species. We constructed co-expression vectors for 9-lipoxygenase gene (LpLOX) and allene oxide synthase gene (LpAOS) isolated from the SH strain to be transformed into E. coli. The productivity (per fresh weight) using E. coli is 25.3 mg KODA /g as compared to 0.366 mg /g from L. paucicostata, SH strain, which requires a longer culture time, light irradiation and larger space for culture.

α-Ketol octadecadienoic acid (KODA), an oxylipin, is generated from linolenic acid by 9-specific lipoxygenase, while jasmonic acid is ultimately synthesized from the same linolenic acid by 13-specific lipoxygenase. KODA has a unique action different from jasmonic acid, such as promotion of flower formation, activation of rooting, increase of shoot germinating in spring, and breaking endodormancy. We report here that KODA promotes the systemic growth in juvenile Populus alba cultured in vitro probably through the activation of immature tissue.

Two newly growing shoots emerging from axillary buds of Populus alba shoots cultured in vitro, were cut off. One was immersed in 10 μM KODA for 3 min while the other in water as a control. The growth of the plants developing from the shoots was observed one month later. KODA strongly promoted the growth of the primary roots and the aerial parts, in which leaves were mainly contributed. Measurement of the length of each internode revealed that KODA significantly acted on the elongation zone in the stem; clearly extending the length of the second and the third position of internode. The total node number was not significantly different from that in the control. Accordingly, KODA had little effect on the height of the whole shoot. Combined with the previous research of KODA, these findings suggest that KODA application systemically promotes the growth of Populus alba cultured in vitro by improving the growth of immature tissues of all organs including roots, stem, and leaves.

Plant hormones like salicylic acid (SA) and jasmonic acid (JA) play crucial roles in regulating defense gene expression systems. SA mainly regulates defense against biotrophic pathogens, while JA mediates defense against necrotrophic pathogens. Compounds called plant activators including probenazole, acibenzolar-s-methyl and 2,6-dichloroisonicotinic acid (INA) activate plant immune systems, providing protection against pathogens. Unlike conventional pesticides that directly target pathogens, plant activators boost the host's defense mechanisms, potentially reducing the likelihood of drug resistance development. Various high-throughput screening systems (HTS) have been developed with the aim of searching for plant activators. Transgenic Arabidopsis lines expressing luciferase under the control of defense gene promoters allow us to monitor the activity of defense-related gene in vivo. To investigate the influence of nutrients on the HTS system, we conducted luciferase assays using Arabidopsis seedlings and observed the suppression of defense gene expression in response to the treatment of plant activators. Reverse transcription quantitative polymerase chain reaction (RT-qPCR) was employed to monitor the expression levels of endogenous genes in response to nutrient-rich conditions and confirmed the suppression effect of defense gene expression as observed in the luciferase reporter assays. The findings highlight the importance of considering nutrient effects when evaluating plant activators and screening for compounds that induce defense gene expression under nutrient-rich conditions.

Wheat, the second most produced cereal globally, is primarily cultivated in cooler regions. Unexpected freezing temperatures can severely impact wheat production. Wheat and other temperate plants have a cold acclimation mechanism that enhances freezing tolerance, but reduces growth under low, non-freezing temperatures. During cold acclimation, plants break down storage polysaccharides like starch and fructan to accumulate soluble sugars such as glucose and fructose. These soluble sugars aid freezing tolerance through osmotic adjustments, membrane stabilization, and freezing point depression. However, plant cell walls, composed of insoluble polysaccharides, are the first line of defense against extracellular freezing. We analyzed the contributions of soluble sugars, storage polysaccharides, and cell wall polysaccharides to freezing tolerance and growth under cold acclimation in wheat. The study involved two Japanese winter cultivars (Yumechikara and Norin-61) and one Japanese spring cultivar (Haruyokoi). While Yumechikara's showed poor growth after four weeks of cold acclimation, it exhibited higher freezing tolerance than the other cultivars. Our analysis revealed that Yumechikara accumulated higher levels of glucose, fructose, starch, and fructan than Norin-61 and Haruyokoi, whereas no significant differences in cell wall composition among the cultivars were observed. Gene expression patterns related to soluble sugar metabolism supported these findings. Additionally, the distribution of sugar changes between leaves (source) and crown (sink) correlated with the relationship between growth and freezing tolerance. These results suggest that freezing tolerance in wheat involves a balance between sugar accumulation and growth regulation during cold acclimation.

Chinese chestnut (Castanea mollissima Blume) is distinguished by its remarkable nut quality and robustness against disease and environmental stressor. However, its somatic embryogenesis process is notably slow, presenting a significant bottleneck in its cultivation. This study focuses on the WUSCHEL (WUS)-related homeobox 2 gene (WOX2), a member of WOX transcription factors gene family, known for its critical role in the somatic embryo development of Arabidopsis. We have identified and explored the function of a WOX2 homolog in Chinese chestnut, termed CmWOX2, in the context of somatic embryogenesis. Our analysis revealed seven WUS gene family members in the species, with CmWOX2 being uniquely upregulated in callus. Our experiments demonstrated that suppression of CmWOX2 expression diminishes somatic embryo production, whereas its overexpression enlarges the embryonic callus diameter. Notably, CmWOX2 expression levels are threefold higher in varieties with high embryogenic competence, such as ‘Jingshuhong’ and ‘Huaihuang’, compared to those with lower competence, including ‘Jiujiazhong’ and ‘Shandonghongli’. These findings underscored the pivotal role of CmWOX2 in the initial stages of Chinese chestnut somatic embryogenesis, highlighting its potential as a target for enhancing somatic embryogenesis in this species.

Chili presents challenges for Agrobacterium-mediated transfection due to its highly recalcitrant nature. One way to overcome this challenge is by using PEG-mediated transfection of protoplasts, which enhances the likelihood of successfully introducing transgenes into the cells. The tape sandwich method for isolating chili leaf protoplasts was optimized by adjusting enzyme concentrations and incubation duration, resulting in a high yield of 1.3×10⁶ cells ml⁻¹ per 0.1 g of leaves. The efficiency of transfecting GFP-encoding plasmids and Cas9 protein using PEG molecules of different sizes was also examined. The highest plasmid transfection efficiency was achieved with 5 µg of plasmid in 50 µl⁻¹, with an average efficiency of 48.71%. For Cas9 protein transfection, the most effective treatment involved using 1000 µg of protein in 100 µl⁻¹, mediated by 40% PEG 4000, resulting in an average efficiency of 2.94% due to protein aggregation. Nevertheless, this optimized protocol reduces the time required for chili protoplast isolation and enhances plasmid transfection efficiency by nearly 50%.

We present a near-complete genome assembly of tomato (Solanum lycopersicum) cultivar Micro-Tom, which has been recognized as a model cultivar for fruit research. The genome DNA of Micro-Tom, provided by the National BioResource Project (NBRP) Tomato of Japan, was sequenced to obtain 72 Gb of high-fidelity long reads. These reads were assembled into 140 contigs, spanning 832.8 Mb, with an N50 length of 39.6 Mb. The contigs were aligned against the tomato reference genome sequence SL4.0 to establish a chromosome-level assembly. The genome assembly of Micro-Tom contained 98.5% complete BUSCOs and a total of 31,429 genes. Comparative genome structure analysis revealed that Micro-Tom possesses a cluster of ribosomal DNA genes spanning a 15 Mb stretch at the short arm of chromosome 2. This region was not found in the genome assemblies of previously sequenced tomato cultivars, possibly because of the inability of previous technologies to sequence such repetitive DNA. In conclusion, the near-complete genome assembly of Micro-Tom reported in this study would advance the genomics and genetics research on tomato and facilitate the breeding of improved tomato cultivars.

The development of cell-free protein synthesis technology has made it possible to easily and quickly synthesize recombinant proteins. Among cell-free protein synthesis systems, wheat germ cell-free protein synthesis using eukaryotic ribosomes is an efficient approach to synthesize proteins with diverse and complex structures and functions. However, to date, cell-free protein synthesis systems, including wheat germ cell-free systems, have not been widely used in plant research, and little is known about their applications. Here, I first introduce a basic overview of the cell-free protein synthesis system of wheat germ. Next, I will focus on our previous research examples on plants and present the applications in which the wheat germ cell-free system is used. We provide protein expression and protein function screening methods at the semi-genomic level and also introduce new approaches to enhance study of chemical biology by adapting the cell-free system of wheat germ. With this review, I would like to highlight the potential of the wheat germ cell-free system and position it as a widely used tool for the previously difficult task of recombinant protein preparation and functional analysis.

Recently a cytidine deaminase-based method for highly efficient C-to-T targeted base editing was developed and has been used with CRISPR-mediated systems. It is a powerful method for genome engineering, although it is prone to off-target effects and has a limited targeting scope. Transcription activator-like effector (TALE)-based tools which allow longer recognition sequences than do CRISPR/Cas9 systems, can also be used for targeted C-to-T base editing. Here, we describe a method that efficiently achieved targeted C-to-T substitutions in Arabidopsis nuclear genes using cytidine deaminase fused to a TALE DNA-binding domain. We used a single pair of TALEs with a novel TALE-repeat unit that can recognize all four DNA bases, especially to allow for variations in the third base of codons in homologous genes. This targeting strategy makes it possible to simultaneously base edit almost identical sites in multiple isoforms of a gene while suppressing off-target substitutions.

Transgrafting, a technique involving the use of genetically modified (GM) plants as grafting partners with non-genetically modified (non-GM) crops, presents non-GM edible harvests from transgrafted crops, often considered as non-GM products. However, the classification of the non-GM portions from transgrafted crops as non-GM foods remains uncertain, therefore it is critical to investigate the potential translocation of substances from GM portions to non-GM edible portions in transgrafted plants. In this study, we explored the translocation of exogenous proteins (luciferase and green fluorescent protein) in model transgrafted plants consisting of GM plant rootstocks and non-GM tomato scions. Our results suggest that exogenous proteins accumulated in the stem tissues of non-GM tomato scions in all cases investigated. The levels and patterns of exogenous protein accumulation in the non-GM tomato stem tissues varied among the individual transgrafted plants and rootstock plant species used. However, exogenous proteins were not detected in the fruits, the edible part of the tomato, and in mature leaves in non-GM tomato scions under the current experimental conditions. Our results provide basic knowledge for understanding exogenous protein translocation in transgrafted plants.