Plant Biotechnology最新文献

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.

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.

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.

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.

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.

The pulvinus is a unique motor organ found in leguminous plants. The motor cells surrounding the central vascular bundle of the pulvinus are divided into extensor and flexor halves. The asymmetric change in turgor pressure of the motor cells of the extensor/flexor halves is the driving force behind nyctinastic leaf movement. Omics analysis has recently revealed genes involved in pulvinar development and function, but the molecular mechanism orchestrating the pulvinar movement remains elusive. In this study, we investigated genes predominantly and highly expressed in the pulvinus to find out key genes involved in the regulation of nyctinastic movement. Gene expression in both the pulvinus and stem at dawn and dusk was examined using RNA sequencing analysis. As a result, several genes were identified that preferentially change in expression in the pulvinus at dawn. Among the genes, we first focused on genes that are more highly expressed in the pulvinus than in the stem and validated the results by reverse transcription-polymerase chain reaction (RT-PCR). We further focused on auxin-related genes, as auxin was found to be preferentially expressed in the pulvinus and has been reported to be involved in the regulation of nyctinastic leaf movement. Quantitative real-time PCR and in situ hybridization analyses revealed that at least two auxin-related genes, IAA19/FLS1, are dominantly expressed in the pulvinus. Thus, we provided a new dataset to identify genes involved in the regulation of nyctinastic leaf movement.

Recent developments have shown that the production of recombinant proteins in plants is more useful than in microbial, insect, or mammalian cell-based expression systems in terms of cost-effectiveness, scalability, safety, and sustainability. Furthermore, transient expression systems in plants may be superior to stable transgenic plants in terms of cost, yield, environmental impact, and regulation compliance. Recombinant proteins, such as enzymes, growth factors, scaffolds, and antibodies are in high demand for use in the food and chemical industries, and will be in even greater demand for diagnostic, therapeutic, and pharmaceutical applications that require high-quality proteins. In this review, we summarize the comparison of recombinant protein expression strategies in mammalian cells, microorganisms, insects, and plants. Furthermore, the efficacy of protein expression in plant cultivation environments, the optimal protein extraction, purification methods, and costs and risks are discussed. We should be aware that the production of recombinant proteins has not only scientific challenges, but also economic and political issues that must be overcome.

Plants display diverse resistance responses that are influenced by their age and the timing of pathogen exposure. In Arabidopsis thaliana (Arabidopsis), nonhost resistance (NHR) to Pyricularia oryzae varies with leaf age and the time of inoculation. While the circadian clock and photoperiod have been linked to the time-dependent regulation of NHR in Arabidopsis, the mechanism underlying leaf age-dependent NHR remains unclear. To identify key players in leaf age-dependent NHR to P. oryzae in Arabidopsis, we utilized rice-Full-length cDNA OvereXpressing (FOX) Arabidopsis lines and identified the rice eIF2β (eukaryotic translation initiation factor 2 beta subunit) variant (Os03g0333300-2). Overexpression of the rice eIF2β variant reduced NHR to P. oryzae and modulated host resistance (HR) to Colletotrichum higginsianum in Arabidopsis. The effect of Os03g0333300-2 expression on resistance is dependent on leaf age in Arabidopsis. These results suggest that overexpression of the rice eIF2β variant Os03g0333300-2 could contribute to defense responses in a leaf age-dependent manner in Arabidopsis. Our findings might suggest the involvement of the rice eIF2β variant in eIF2-dependent translation regulation of resistance response to pathogens in plants.

Rational metabolic-flow switching is an effective strategy that we previously proposed to produce exogenous high-value secondary metabolite(s) in cultured plant cells. Specifically, it involves redirecting a highly active inherent metabolic pathway to a pathway producing related exogenous compounds. The success of this strategy depends on the identification of at least one highly active metabolic pathway in host plant cells that can be redirected to produce a target compound following the introduction of exogenous biosynthetic gene(s) via genetic transformation. Active metabolic pathways may be predicted on the basis of the major metabolites that accumulate in cells. In previous proof-of-concept studies, we demonstrated that cultured cells of a temperate bamboo species (Phyllostachys nigra; Pn) are an appropriate host for producing phenylpropanoid-derived compounds. However, developing a series of host plant cells with a variety of metabolic properties is necessary to maximize the utility of rational metabolic-flow switching. In this study, we established cultured cells of two tropical bamboo species (Dendrocalamus giganteus and Dendrocalamus brandisii). By analyzing the metabolites that increased in abundance in response to phytohormone treatments, we determined that exogenous gibberellin A₃ (GA₃) substantially induced the accumulation of an unknown metabolite in D. giganteus (Dg) cells. This compound was isolated and identified as serotonin (5-hydroxytryptamine). After optimizing the culture conditions, the serotonin production titer in Dg suspension cells reached 360 mg l^-1. These findings indicate that Dg cells are potentially suitable for the bioproduction of exogenous tryptophan-derived indolic compounds via rational metabolic-flow switching.

DNA extraction with reliable purity and concentration is essential for most of the molecular genetics studies. Extracting DNA from young leaves in seedling stage is advantageous because it causes less damage to remaining plant which can be further used for phenotypic analysis. DNA extraction from seeds is even more advantageous in terms of saving time, labor, space, and cost for germination. Maize is one of the most important food and feed sources and provides great materials for genetic and breeding studies which are accompanied by genotyping and phenotyping. We present seed DNA extraction method which does not cause damage the seed's germination ability. DNA was extracted using cetyltrimethyl-ammonium bromide method or a commercial DNA extraction kit from the seed fragment, and the quantity and quality of the DNA were examined. Seed germination was tested for proportional seed cuts at 0, 10, 30, and 50% of the distal end of a seed, proportionally by weight. Extracting DNA from the distal seed fragments resulted in high-quality and sufficient amount of DNA. Germination rates were not significantly reduced when seed cuts were made at 10 or 30% of seed weight. DNA extraction from seeds cut can be an efficient way to obtain samples for genotyping and phenotyping. Moreover, it can be applied for high-throughput DNA extraction in maize and possibly to other smaller seeds.