搜索结果：Plant biotechnology (Tokyo, Japan)

Next generation long-read sequencing is a powerful approach to generate de novo genome assemblies, however it requires high-quality and high molecular weight (HMW) DNA to reach near-chromosome level assemblies. Some plants are reported as recalcitrant to high-quality HMW DNA extraction due to high levels of secondary metabolites. Streptocarpus schliebenii (Gesneriaceae) is one of those highly recalcitrant plants and its DNA extraction failed repeatedly with previously published protocols. Percoll™ is a silica-based colloid coated with polyvinylpyrrolidone and utilized for various aspects in phase separation including plants' nuclei isolation for Hi-C library and DNA extraction to generate bacterial artificial chromosome clones. In this study, we developed a HMW DNA extraction protocol for long-read sequencing that included a Percoll™ gradient step. To establish a stable protocol, we examined and modified buffers and steps of several previous Percoll™ gradient protocols. Instead of the previously used agarose plug method for Percoll™ DNA extraction, CTAB lysis followed by Qiagen Genomic-Tips was employed. Three Streptocarpus species generated optimal quality and HMW DNA. The method was further tested in 12 species across a wide range of plant lineages. The results were species specific. While HMW DNA was obtained from seven species, HMW and high-quality DNA were obtained from four species, i.e. Iris pseudacorus, Pulmonaria affinis, Corytoplectus speciosus, and Ilex aquifolium. This indicates the wide applicability of this protocol for plants. This protocol provides a useful resource for those who are working on de novo plant genome projects of recalcitrant material to obtain optimal DNA for long-read sequencing.

Modern biotechnological approaches in technology for cassava propagation depend on somatic embryogenic calli (SEC) induction and somatic embryo (SE) regeneration techniques. Consequently, it is essential to develop SEC induction and SE regeneration for cassava genotypes. By assessing the effects of different auxins and explant types, this study aims to develop efficient techniques for inducing cassava SEC and SE. Callus induction medium (CIM) supplemented with 10 mg l-1 2,4-dichlorophenoxyacetic acid (2,4-D) or 12 mg l-1 Picloram was used to cultivate five different types of cassava explants. Subsequently, the induced calli were maintained on CIM until the formation of SEC, which were sub-cultured on the same CIM to promote proliferation and induce SE formation. These SEs subsequently germinated and developed into shoots, which were rooted to produced complete plantlets . These findings indicate that culturing-induced axillary bud explants on CIM supplemented with 12 mg l-1 Picloram is an effective method for inducing SEC in cassava. Histological analyses and cryogenic electron microscopy observations confirmed the development of SECs and SEs on CIM. Our finding, in which up to 202.3 SEs were regenerated from 10 induced axillary bud explants on CIM supplemented with 12 mg l-1 Picloram in the Menti genotype, highlights the effects of auxin and explant types on SEC and SE induction. Although further research is required, the methods developed in this study will contribute to the successful breeding and micropropagation of cassava.

In animals, genes of the cysteine aspartate-specific protease (caspase) family play a crucial role in inducing cell death. Genes homologous to animal caspase genes have not been found in plants, and to date, there are no examples of the ectopic expression of animal caspase genes inducing cell death in plants. In this study, we investigated whether cell death could be induced by expressing the human caspase-3 gene in plants. Wild-type caspase-3 (V266), inactive type (V266H), and constitutively active type (V266E) genes were expressed in leaves of Nicotiana benthamiana and/or Nicotiana tabacum using two types of Agrobacterium-infiltration-type virus vectors and one type of mechanical inoculation-type virus vector. In all cases, cell death symptoms were induced by V266 and V266E but not V266H. Similar results were obtained even when the caspase-3 gene was split into coding regions for subunit 1 and subunit 2 and co-expressed. As for virus infection, expression of V266 and V266E suppressed the infection of the tomato mosaic virus (ToMV). This suppression effect was particularly pronounced when the constitutively active type V266E was expressed using a virus vector of ToMV. With V266E, cell-to-cell movement of the virus was inhibited and long-distance movement did not occur. Furthermore, the expression of V266 and V266E tended to increase the mRNA levels of defense-related genes in N. benthamiana. These results suggest that the expression of the human caspase-3 gene induces cell death in plants, affects plant gene expression, and exerts an inhibitory effect against ToMV infection.

Phage therapy is being used to combat pathogenic bacterial infections that threaten plant, animal, and human health. However, its application remains limited by high host specificity and the emergence of bacterial resistance. In this study, we addressed the key issues in phage therapy using rice bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) strain N1 and its lytic phage NP1. Strain N1 acquired resistance to the phage NP1 through mutations and downregulation of lipopolysaccharide (LPS) biosynthesis genes. A directed evolution assay using phage NP1 and the resistant strain N1R resulted in the development of phage E12-2, which overcame bacterial resistance, expanded its host range and improved bacterial suppression by targeting alternative LPS binding sites. Moreover, genome analysis identified two amino acid substitutions (V303L and G317V) in its tail fiber protein. Additionally, phage E12-2 improved disease control efficiency by 51 % compared to the wild-type phage NP1 and induced plant immunity in a plant disease model. These findings enhance our understanding of how bacteria-phage evolution shapes the dynamics of phage therapy in plants.

The SARS-CoV-2 papain-like protease (PLpro) is an essential viral enzyme that promotes viral polyprotein processing while simultaneously suppressing the host innate immune response, which makes it a primary target for developing antiviral drugs. The present study employs a comprehensive approach integrating untargeted metabolomic profiling, in silico molecular docking and dynamics simulations, Molecular Mechanics Generalized Born Surface Area (MM-GBSA) energetic assessments, and biochemical enzyme assays. This integrated method aims to discover natural PLpro inhibitors from two ethnomedicinal plants, Lippia javanica and Acorus calamus, which have long been utilized in African traditional medicine to treat respiratory diseases. Comprehensive metabolite profiling using untargeted Ultra-Performance Liquid Chromatography-Tandem Mass Spectrometry (UPLC-MS/MS) and Global Natural Products Social (GNPS) molecular networking revealed flavonoid glucuronides and phenylpropanoid derivatives as the major constituents in both plant species. In situ histochemical staining further offered spatial validation of phenolic- and lignin-associated tissues, supporting the phenolic-dominated molecular families detected by GNPS molecular networking. In silico evaluation of six selected compounds demonstrated spontaneous and thermodynamically favorable binding to PLpro, with ΔG_bind values ranging from -5.63 to -6.43 kcal/mol. Catechin-7-glucoside emerged as the lead compound, establishing multiple hydrogen bond networks with Asp164, Gln269, Tyr264, and Asn267, supplemented by hydrophobic engagement with Pro247 and Pro248, and π-π stacking with the blocking loop 2 (BL2 loop). Molecular dynamics simulations confirmed the stability of the protein-ligand complexes. Biochemical enzyme assays confirmed concentration-dependent inhibition of PLpro proteolytic and deubiquitinating activity by both crude plant extracts and isolated bioactive compounds. However, S-adenosyl-methionine showed comparatively high PLpro proteolytic activity (IC50 5.872 µM) compared to catechin-7-glucoside, with an IC50 of 7.493 µM, exhibiting efficacy similar to the reference inhibitor GRL0617. Both the extracts of L. javanica and A. calamus have shown significant inhibitory activity while maintaining cell viability in Human embryonic kidney 293T cell (HEK293T) culture models, indicating a favorable safety profile of the tested concentrations. Based on these results, catechin-based polyphenols and phenylpropanoid derivatives appear as promising lead compounds for the development of PLpro inhibitors. To progress toward therapeutic use, further work is necessary in pharmacokinetics, structural optimization, and antiviral validation in cell models.

Despite widespread concern over global biodiversity loss, the balance between gains and losses within local plant communities remains contentious, largely due to a scarcity of integrative, long-term and large-scale analyses across different habitats and multiple facets of biodiversity. Here, we analyse 57,390 vegetation-plot time series of vascular plants across Europe to quantify the average and habitat-specific trends in taxonomic, functional, phylogenetic, and gamma diversity, alongside with changes in threatened Red List, non-native, and specialist versus generalist species. We find that, over the last 100 years, plant communities gained on average 0.7% in vegetation cover and 0.2% in species number per year, associated with gains in functional and phylogenetic diversity, non-native, Red List, and generalist species. Diversity changes are most pronounced in mire and wetland communities. Differences among habitat types and habitat-change trajectory (stable, successional, disturbed), together with the most recent observation year, explain 2.1%-36.6% of the variation in diversity trends. Habitat-specific gamma diversity showed no general trends and only increased in stable grasslands and successional sparsely vegetated habitats. By integrating habitat types and change trajectories, we reconcile some of the conflicting narratives on local biodiversity change in favour of a more nuanced understanding of the observed variation in local biodiversity change.

Controlling the digestibility of cellulosic biomass is important for its efficient use. We generated intragenic rice plants showing enhanced saccharification yield of rice straw. The rice cytokinin biosynthesis gene, LONELY GUY, under the control of the rice senescence-inducible STAY GREEN promoter, was introduced into the rice genome via particle bombardment. The rice-derived herbicide resistance gene ALS(G95A) was used as a selection marker gene. Regenerated intragenic rice plants with no foreign sequences showed enhanced saccharification yields from the leaves at harvest, whereas no significant differences were observed at the heading stage. Because the saccharification yields of rice straw are reduced after senescence, which is suppressed by cytokinin, we propose that the enhanced saccharification yields of intragenic rice plants are caused by the delay in senescence of the rice leaves due to the expression of the introduced cytokinin biosynthesis gene upon senescence.

Coccidiosis is a highly pathogenic disease caused by a protozoan parasite, severely impacting the global livestock economy. Due to the emergence of drug resistance to current anticoccidial agents, developing novel anticoccidial compounds, such as plant secondary metabolites, is necessary, as previously studied. The objective of the present study is to deepen the understanding of the anticoccidial activity of these natural compounds and to investigate their potential as anticoccidial drugs. Here, we examined the anticoccidial activity of 27 natural compounds, containing 28 secondary metabolites, derived from 8 alpine plants, using the mouse Eimeria species, Eimeria krijgsmanni. E. krijgsmanni sporozoites were exposed to the compounds at a final concentration of 100 μM. After incubation for 24 h, the viability of sporozoites was assessed using trypan blue staining. Twelve compounds, containing 14 secondary metabolites, reduced the sporozoite viability by 4.5%-69.5% and seven compounds were newly recognized to have anticoccidial activity against E. krijgsmanni. The compounds containing β-sitosterol showed the most potent effect. Although further studies are needed to analyze the in vivo efficacy, these anticoccidial compounds have potential as effective anticoccidial drugs.

This study demonstrated that covalently localized zwitterionic moieties in zwitterionic polypeptides (ZIPs) effectively disrupt hydrogen bonds in cellulosic substrates, including filter paper and plant cell wall materials, without significant cytotoxicity. ZIPs with varying densities of zwitterionic side chains were synthesized via the postpolymerization modification of histidine-containing oligopeptides. The newly developed ZIPs predominantly comprised repeating units with zwitterionically converted side chains. Such ZIPs can cleave multiple hydrogen bonds by anchoring the zwitterionic structure at specific sites, thereby partially dissociating the polysaccharide chains in the cell wall. They are especially effective in dissolving amorphous cellulose, even at low concentrations in aqueous solutions. Importantly, this effect was achieved with minimal cellular toxicity, harnessing the advantages of ionic liquid-like properties while mitigating their high-toxicity limitations. This biofriendly approach to cell wall denaturation highlights a novel method for controlling hydrogen bond networks in polysaccharides and cell walls. These findings indicate a new approach for reducing biomass recalcitrance and developing next-generation biobased materials and fuels derived from plant cell walls.

Evolutionary transitions in land plant fertilization from zooidogamy to siphonogamy were characterized by transformations of male reproductive cells. Basal land plants such as bryophytes and pteridophytes have motile sperm, whereas most seed plants have nonmotile sperm, delivered by a pollen tube. Despite being seed plants, gymnosperm cycads and ginkgo uniquely form highly multiflagellated and large motile sperm within pollen tubes. However, the evolutionary state of these male reproductive cells remains unknown. We clarified the gene expression profiles of Cycas revoluta pollen tubes and motile sperm swimming toward female reproductive cells. Male cycad cells expressed fewer genes associated with transcription, translation, and related processes, which is consistent across land plants. We compared the distinctive orthologous groups (OGs) of the genes specifically expressed in sperm and pollen tubes with those in other plants. Cycad pollen tubes shared several OGs with angiosperms but possessed significantly fewer gene copies and lacked cell wall remodeling and plasma membrane-localized receptor genes that contribute to rapid and guided growth. The growth mechanism of cycad pollen tubes might be largely different from angiosperm pollen tubes. In contrast, despite their morphological uniqueness, cycad sperm shared representative OGs with angiosperm sperm cells to the same extent as egg cells. In addition, a sperm-specific histone variant may contribute to transcriptional regulation via chromatin condensation like other male gametes. As an extant gymnosperm that retains zooidogamy with pollen tubes, the cycad represents a molecular intermediate state in the transition from zooidogamy to siphonogamy, providing insight into the evolution of land plant fertilization.

For the functional analysis of Dianthus and carnation endogenous genes, we investigated a viral vector derived from the apple latent spherical virus (ALSV) as a tool for reverse genetic analysis. ALSV can infect the aerial parts, such as leaves and flower organs, of Dianthus and carnation plants, without causing viral symptoms. Partial sequences of the chalcone synthase (CHS), 1-aminocyclopropane-1-carboxylate (ACC) synthase (ACS), and ACC oxidase (ACO) genes were cloned into the ALSV vector and then used to infect the plant. Plants infected with ALSV vectors carrying these genes exhibited knockdown phenotypes typical of CHS, ACS, and ACO. Plants infected with the ALSV vector carrying CHS showed white flower petals, whereas those infected with the ALSV vector carrying ACS and ACO generated long-lived flowers. Thus, ALSV vectors can promote virus-induced gene silencing (VIGS) in the petals and gynoecium. ALSV infects plants without viral symptoms and effectively induces VIGS in several flower organs; thus, the ALSV vector is a valuable tool for determining the functions of genes of interest in Dianthus and carnation plants.

In plants, the functional characterization of essential genes is often hindered by the lethality associated with complete loss-of-function alleles. Here, we present a genome editing-based strategy to generate viable hypomorphic alleles through selective removal of the translation start codon. Using Arabidopsis thaliana NON-SMC ELEMENT 1, which encodes a conserved component of the Structural Maintenance of Chromosomes (SMC) 5/6 complex involved in DNA repair and genome stability, as a model, we generated CRISPR-Cas9-edited alleles lacking the start codon. These homozygous mutants exhibited severe developmental defects, including stunted growth and failure to form true leaves. Moreover, they displayed molecular hallmarks of genome instability, such as increased DNA fragmentation, upregulation of DNA repair and cell cycle checkpoint genes, and root meristem cell death. Complementation assays using wild-type and mutated NSE1 genomic constructs confirmed that these alleles retained partial gene function. Overall, the use of start codon removal as an editing strategy is a robust and broadly applicable approach for generating hypomorphic alleles without relying on transcript-level manipulations, such as RNAi. This work therefore provides a practical demonstration of a novel editing strategy for dissecting the functions of essential genes that are otherwise genetically intractable. Consequently, this approach expands the functional genomics toolkit and opens new avenues for basic plant biology and advanced biotechnological applications.

In the present study, an efficient regeneration protocol via somatic embryogenesis and organogenesis has been developed for cassava var. Vamas 1 utilizing leaf and node explants, respectively. Leaves were inoculated on Murashige and Skoog (MS) medium containing different concentrations (4, 8, and 12 mg l-1) of Picloram or 2,4-dichlorophenoxyacetic acid (2,4-D) with 6 mg l-1 1-naphthaleneacetic acid (NAA). The maximum callus formation (100%) was recorded in medium containing 4 mg l-1 Picloram or 8 mg l-1 2,4-D. However, the callus fresh weight (0.11 g) was higher in presence of 4 mg l-1 Picloram with 2.72 scoring of callus proliferation after 3 weeks. After subculture, 12 mg l-1 Picloram with 6 mg l-1 NAA proved optimum medium that formed maximum 10.25±3.49 embryos (44.00±0.04% response) under dark conditions after 6 weeks. The green cotyledons were produced after 2 weeks of light incubation on 0.2 mg l-1 6-benzyladenin (BA). which further formed shoots within 5 weeks. Simultaneously, nodal explants were placed in MS media augmented with BA (2, 4, 8, and 10 mg l-1) individually and in combinations with 0.02 mg l-1 NAA. Results revealed that maximum 4.13±0.56 shoots/explant were formed with 11.07±2.79 number of leaves and 3.61±0.17 cm shoot length at 2 mg l-1 BA. These shoots induced 7.33±0.58 number of roots after 2 weeks in basal MS medium. At last, the plantlets derived via both the pathways were transferred to soil : rice husk (1 : 1 w/w), and they were successfuly acclimatized with 80% survival in greenhouse. Since the cassava plant regeneration is genotype-dependent, the developed protocol can be applied for mass-propagation of this recently released Indonesian superior variety Vamas 1. This will generate large number of plantlets for the farmers and also the protocol will be utilized for genetic improvement studies.

Calcium (Ca) availability is vital for optimal plant growth and immune signaling, yet the underlying mechanisms remain elusive. Here, we reveal that Arabidopsis vacuolar H⁺-pyrophosphatase (AVP1)-regulated cytosolic inorganic pyrophosphate (PPi) homeostasis governs leaf growth by maintaining cellulose synthesis to suppress autoimmune activation upon Ca deficiency. Ca deficiency reduces the AVP1 abundance, while AVP1 eliminates excess cytosolic PPi, which impairs guanosine triphosphate-dependent microtubule assembly and reduces cellulose synthase 3-mediated cellulose synthesis. This cell-wall disruption activates isochorismate synthase 1-mediated salicylic acid production, triggering autoimmune responses and inhibiting new leaf growth. Enhancing PPi hydrolysis genetically improves plant growth tolerance to low Ca availability (low-Ca). The link between Ca-dependent PPi metabolic regulation, autoimmunity, and leaf growth is conserved in tomato, highlighting the broad relevance of AVP1 and PPi homeostasis in plant resilience. Our findings offer potential strategies for improving crop tolerance to nutrient-limited environments.

Plant temperature-induced lipocalins (TILs) have been shown to be responsive to heat stress. The expression of TIL in wheat and Arabidopsis is induced by heat shock treatment and cold acclimation, but other responses and functions of lipocalins remain unknown. In this study, we focused on the response of lipocalins to phytohormones in tomato, as cis-element analysis revealed the presence of multiple phytohormone-responsive elements involving ethylene, abscisic acid (ABA), and jasmonic acid (JA). The expression levels of SlTIL1 and SlTIL2 increased after ABA treatment in young leaves, and tomato plants exhibited enhanced drought stress tolerance 24 h after ABA application. In addition, SlTIL1 expression increased in tomato fruits at the yellow stage following ABA treatment from the orange stage, thereby accelerating fruit ripening. We compared wild-type plants with overexpressing lines of SlTIL1, SlTIL2, and SlCHL and found that both ethylene gas production and expression of the ethylene synthesis gene SlACS2 were elevated from the yellow stage in SlTIL1-OX and SlTIL2-OX lines compared to wild-type. We suggest that ethylene and ABA treatments induce reactive oxygen species (ROS) in tomatoes, to which lipocalins respond by not only contributing to ROS accumulation scavenging but also promoting leaf senescence and fruit ripening.

Plants continuously respond to changes in nutrient availability by adjusting their growth and development. However, the role of RNA metabolism in this process is unclear. We performed seedling growth assays using Arabidopsis thaliana mutants defective in RNA metabolism. When grown on full-strength Murashige and Skoog (MS) medium containing 1% sucrose, all mutants had shorter primary roots than the wild type, suggesting the importance of RNA metabolic regulation for seedling growth under nutrient-rich conditions. Primary root growth exhibited distinct responses to the presence of sucrose in the following mutants: ccr4a ccr4b, with defects in the deadenylases CARBON CATABOLITE REPRESSOR4a (CCR4a) and CCR4b, which function in poly(A) tail degradation; mtr4-2, with a mutation in the exosome co-factor mRNA TRANSPORT4 (MTR4), which is required for 3'-5' RNA degradation; and rid1-1, with a defect in the RNA helicase ROOT INITIATION DEFECTIVE1 (RID1), which functions in pre-mRNA splicing. Whereas the ccr4a ccr4b seedlings did not exhibit sucrose-dependent changes in root growth, the mtr4-2 and rid1-1 seedlings exhibited more pronounced growth inhibition in response to a lack of sucrose and reduced MS salt and vitamin concentrations, respectively, compared to the wild type. When grown on 0.1×MS medium without sucrose, upf3-1 seedlings, which lack functional UP-FRAMESHIFT3 (UPF3), a component of nonsense-mediated mRNA decay (NMD), were larger than the wild type, suggesting the importance of NMD in regulating seedling growth under nutrient-limited conditions. Therefore, different RNA metabolic pathways play distinct roles in the nutrient-dependent regulation of plant growth, adjusting plant fitness to different environments.

In the midst of a worldwide outbreak of lepidopteran chewing pests such as the fall armyworm (FAW; Spodoptera frugiperda), innovative strategies for pest control are needed. Recent findings have linked the resistance of rice (Oryza sativa) to lepidopteran pests to diterpenoid phytoalexins (DPs), antimicrobial compounds produced in response to disease and abiotic stress conditions. In this study, we explored the involvement of DP biosynthesis-related genes in the response and resistance of rice plants to lepidopteran pests. In interactions between rice and FAW or the oriental armyworm (Mythimna separata), feeding damage from larvae induced the expression of DITERPENOID PHYTOALEXIN FACTOR (DPF), which encodes a key transcription factor in DP biosynthesis, as well as DP biosynthetic genes. Transcriptional analysis suggested that DPF promotes DP biosynthesis in response to chewing by lepidopteran herbivores and other stresses. When lepidopteran larvae were reared on the leaves of DPF-overexpressing transgenic rice plants, which persistently accumulate high DP levels, FAW larvae exhibited poor growth within days. Overexpression of DPF also suppressed the growth of larvae from the oriental armyworm, although the suppression was more moderate. These results demonstrate that DPF overexpression enhances plant resistance to lepidopteran pests, highlighting the potential of DPF as a tool for biotechnological pest control.

Sugar content is among the most important agronomic traits in tomatoes. High-sugar tomatoes are usually produced by applying water stress, which results in a significant reduction in fruit yield. We developed a hydroponic method involving the use of coral sand as a solid medium and optimized a nutrient management protocol to produce high-sugar tomatoes while largely maintaining yield. In this study, we analyzed the mechanism by which coral sand increases sugar content. Transcriptome analysis revealed that the expression of phosphorus deficiency-inducible genes increased in the leaves of tomato plants grown on coral sand. The phosphate concentration in both the nutrient solution and leaves decreased when the plants were grown on the coral medium, suggesting that the response to mild low-phosphate conditions may be associated with the increase in sugar content. In addition, under mild low phosphate in hydroponic culture, the sugar content in the fruit increased, even though the fruit yield decreased. Interestingly, comparisons of gene expression levels under these conditions with those from previously reported mild drought experiments showed that homologs of genes that are induced by coral sand were also upregulated in response to mild drought. Collectively, our findings reveal a previously unrecognized ability of hydroponic cultivation using coral sand to create mild low-phosphate conditions that trigger sugar accumulation pathways associated with mild drought. This study not only provides a practical strategy for producing high-sugar tomatoes using a coral sand cultivation system with minimal impact on yield, but also suggests a role for low-phosphate responses in regulating sugar accumulation in fruits.

Transferrin is one of the major soluble serum proteins and is responsible for iron transport. Industrially, it is significant as a component of mammalian cell culture media, where a safe and stable supply is necessary. However, because transferrin is a glycoprotein containing 19 disulfide bonds, it is difficult to produce as a recombinant protein in bacteria, and at present it is mainly sourced from animals. In glycoprotein production, stability of the N-glycan profile is crucial, as glycans play important roles in diverse biological processes and influence the efficacy of glycoproteins. In this study, we aimed to produce recombinant human transferrin (rhTF) with stable N-glycan profiles. We generated transgenic rice calli expressing human TF (hTF) as a secretory glycosylated protein. rhTF was successfully produced as a soluble protein in the liquid culture medium of transgenic rice calli and subsequently purified. We confirmed that rhTF contained two plant-specific N-glycans and that these profiles were consistent across production batches. Purified rhTFs promoted the proliferation of cultured animal cells and human iPS cells, similar to serum-derived transferrin. Our results demonstrate new possibilities for producing recombinant glycoproteins with stable N-glycan profiles using a plant cell culture-based secretory protein expression system.