Plant Biotechnology最新文献

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.

The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system has been used for genome editing in various fruit trees, including apple (Malus × domestica). In previous studies, transfer DNA (T-DNA) expressing genome editing tools, Streptococcus pyogenes Cas9 (SpCas9) and single guide RNA (sgRNA), was stably integrated into the apple genome via Agrobacterium-mediated transformation. However, due to self-incompatibility, long generation period, and the high heterozygosity of apple, removing only the integrated T-DNA from the apple genome by crossbreeding while maintaining the introduced varietal trait is difficult. Therefore, an efficient SpCas9-sgRNA delivery system without transgene insertion is required for genome editing of apple. In this study, we used geminivirus-derived replicons (GVRs) for the transient expression of genome editing tools. Small DNA vectors were deconstructed by splitting the elements necessary for the production of GVRs from bean yellow dwarf virus into two vectors. Production of GVRs using these vectors was demonstrated in Arabidopsis and apple cells. Genome editing was improved by using the GVR-producing vectors with genome editing tools in Arabidopsis protoplasts. The use of the GVR-producing vectors for SpCas9 and sgRNA delivery into apple leaves improved the expression levels of SpCas9 and sgRNA, enabling the detection of targeted mutations introduced in the endogenous apple genome. These findings demonstrate the utility of GVRs in genome editing via transient gene expression in apple. It can be expected that our GVR-based genome editing technology has potential utility for transgene-free genome editing in apple.

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.

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%.

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.

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^-1 as compared to 0.366 mg g^-1 from L. paucicostata, SH strain, which requires a longer culture time, light irradiation and larger space for culture.

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.

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.

To efficiently produce useful compounds using biological cells, it is essential to optimally design all metabolic reactions and pathways, including not only the flow of carbon within the cell but also the production and consumption of energy and the balance of oxidation-reduction. Computational scientific methods are effective for the rational design of metabolic pathways and the optimization of metabolic fluxes. Based on this blueprint, it is crucial to accurately construct the cell, test and analyze whether it conforms to the design, and learn from the results to redesign the system in an effective cycle. This review introduces essential metabolic design techniques in synthetic biology and discusses the potential of using plant cells or plant genes effectively in synthetic biology for the production of useful compounds.

Synthetic biology, an interdisciplinary field at the intersection of engineering and biology, has garnered considerable attention for its potential applications in plant science. By exploiting engineering principles, synthetic biology enables the redesign and construction of biological systems to manipulate plant traits, metabolic pathways, and responses to environmental stressors. This review explores the evolution and current state of synthetic biology in plants, highlighting key achievements and emerging trends. Synthetic biology offers innovative solutions to longstanding challenges in agriculture and biotechnology for improvement of nutrition and photosynthetic efficiency, useful secondary metabolite production, engineering biosensors, and conferring stress tolerance. Recent advances, such as genome editing technologies, have facilitated precise manipulation of plant genomes, creating new possibilities for crop improvement and sustainable agriculture. Despite its transformative potential, ethical and biosafety considerations underscore the need for responsible deployment of synthetic biology tools in plant research and development. This review provides insights into the burgeoning field of plant synthetic biology, offering a glimpse into its future implications for food security, environmental sustainability, and human health.