Plants require larger quantities of nitrogen (N) than of other soil nutrients, making N availability critical for plant growth and crop yield. However, levels of N sources available in soils vary across different regions of the world, but are often insufficient to support optimum plant growth. In addition, N availability fluctuates both temporally and spatially. Therefore, plants must sense and respond to environmental variations in N levels or to N shortages. Nitrate is both the primary source of N for plants and a critical signaling molecule that regulates N assimilation and many other physiological processes in plants. Recent studies have unraveled the mechanism of nitrate signaling and placed it at the center of the regulatory pathways governing plant responses to N availability. This review briefly summarizes the recent advances that revealed the mechanisms controlling plant responses to N availability. It focuses particularly on the mechanism of nitrate signaling, before addressing the new questions that have emerged from recent findings. Finally, it discusses how recent insights into the mechanisms regulating plant responses to N availability can be utilized in the field to improve nitrogen use efficiency in crops.

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.

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.

Ehretia asperula Zoll. et Mor. (“Xạ đen”) is a valuable medicinal plant widely used in traditional Vietnamese medicine due to its rich content of bioactive compounds. This study investigated the phytochemical composition, antioxidant activity, and genetic diversity of samples collected from three regions in Vietnam. ITS marker and methods for quantifying secondary metabolites were used to assess the genetic diversity and chemical composition of E. asperula across different collection sites. Although ITS1 sequence analysis showed no significant genetic variation among accessions, there were notable differences in secondary metabolite content. Plants from Hoa Binh province contained the highest levels of total phenolic, flavonoids, and rosmarinic acid, followed by those from Dong Nai and Vinh Phuc provinces. Among the tested solvents, an ethanol-water (30 : 70, v/v) proved most effective for extracting the targeted compounds. In addition, a micropropagation protocol was successfully established using nodal explants from the plants collected from Hoa Binh province. Optimal surface sterilization was achieved with 0.1% HgCl₂ for 16 min. Shoots were most efficiently regenerated from nodal explants on MS medium supplemented with 3.0 mg l⁻¹ kinetin. Shoot proliferation was most effective on MS medium supplemented with 1.5 mg l⁻¹ 2-isopentenyladenine, while root induction reached 100% efficiency on MS medium containing 1.0 mg l⁻¹ indole-3-butyric acid. These findings highlight the potential of E. asperula as a sustainable natural antioxidant source and support its continued use in traditional medicine.

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.

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.

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.

Cytoplasmic male sterility (CMS) is driven by the incompatibility between a mitochondrial gene and the nuclear RESTORER OF FERTILITY (Rf) gene. Previously, we identified orf288 as the cryptic CMS-causing gene in the mitochondrial genome of japonica rice (Oryza sativa) Taichung 65 (T65) through the analysis of CMS lines of O. glaberrima carrying the cytoplasm of japonica rice; orf288 RNA was greatly reduced in the presence of a T65-derived nuclear Rf gene, which was linked to a region harboring rf4 encoding a pentatricopeptide repeat (PPR) protein and other Rf-like PPR genes. In this study, we knocked out Rf-like PPR genes in T65 to identify the Rf-like PPR gene regulating the reduction of orf288 RNA and pollen fertility. Among the CRISPR-Cas9-mediated mutations in PPR461 (Os10g0495100), PPR782/rf4 (Os10g0495200), and PPR794 (Os10g0497300), mutations in PPR782/rf4 (Os10g0495200) allowed the expression of orf288; however, the orf288 RNA level was lower than that in the O. glaberrima CMS line and did not affect pollen fertility. Our results indicate that PPR782/rf4, a non-functional allele of the RF4 gene responsible for fertility restoration in wild-abortive (WA)-type CMS, is functional and suppresses the accumulation of orf288 RNA in the japonica rice.

Oxalate is a simple dicarboxylate that accumulates primarily in terrestrial parts of plants. Inhibition experiments of a previous study focusing on “new leaves” of Rumex obtusifolius revealed that the isocitrate pathway, one of three oxalate pathways, is the primary contributor to oxalate accumulation. However, that experiment was conducted in the dark. In the present study, we conducted inhibitor experiments to evaluate the contribution of the isocitrate pathway to oxalate accumulation in the light. Oxalate accumulation was not inhibited by itaconate in the light, but uptake of itaconate in leaves of R. obtusifolius was greater in the light than in the dark. Multivariate statistical analyses revealed that itaconate only slightly affected the metabolite content under light conditions, but itaconate significantly affected the contents of oxalate and related compounds in the dark. These data suggest that the oxalate synthesis pathways that contribute to oxalate accumulation differ between day and night.

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.

Plants produce dimerized phenolic compounds as secondary metabolites. Hordatine A (HA), a dehydrodimer of p-coumaroylagmatine (pCA), is an antifungal compound that accumulates substantially in young barley (Hordeum vulgare) seedlings. The first committed step of the HA biosynthetic pathway is the formation of pCA via condensation of p-coumaroyl-CoA and agmatine, which is catalyzed by agmatine coumaroyltransferase (ACT). Although two ACT-encoding genes (HvACT-2HL1/2) on the long arm of barley chromosome 2H (2HL) have been identified, our previous study suggested the presence of another ACT locus on the short arm of barley chromosome 2H (2HS). In this study, an analysis of dissection lines of wheat (Triticum aestivum) carrying aberrant barley 2H chromosomes detected pCA in wheat lines carrying the distal region of 2HS. This chromosomal region, which includes genes encoding the laccase catalyzing the last committed step of the HA biosynthetic pathway, was revealed to also contain a putative ACT gene (HvACT-2HS1), with the encoded amino acid sequence similar to that of HvACT-2HL1 (46% sequence identity). Changes in HvACT-2HS1 transcript levels were in accordance with those in the pCA-forming enzymatic activity and the pCA level in barley seedlings. Additionally, recombinant HvACT-2HS1 heterologously expressed in Escherichia coli had pCA-forming enzymatic activity, with high specificity for agmatine as the acyl acceptor. Moreover, a phylogenetic analysis indicated that HvACT-2HS1 is not a paralog of HvACT-2HL1/2. These results suggest that HvACT-2HS1 and HvACT-2HL1/2, which originated from different ancestral genes, jointly mediate the formation of pCA for HA biosynthesis in barley.

For fruit trees, inefficient transformation and prolonged juvenile phase lasting for years to decades remain two major bottlenecks for breeding and functional gene analysis. Recently, fipexide (FPX) has been identified as a novel chemical that enhances callus induction, regeneration, and Agrobacterium-mediated transformation. In this study, we aimed to optimize FPX treatment for improving shoot regeneration rate and transformation efficiency in European pear (Pyrus communis), highbush blueberry (Vaccinium corymbosum), and persimmon (Diospyros kaki). Explants were cultured on media supplemented with various concentrations of FPX for different durations. In European pear ‘La France’, treatment with 3 µM FPX for one week significantly enhanced regeneration compared with the conventional phytohormone condition, whereas higher concentrations (≥10 µM) and extended exposure inhibited regeneration. Similar patterns were observed in European pear ‘Bartlett’, highbush blueberry ‘O’Neal’ and persimmon ‘Jiro’. Furthermore, 10 µM FPX enhanced Agrobacterium-mediated transient GFP expression in all 3 species. We also attempted to introduce Arabidopsis FLOWERING LOCUS T (FT) into persimmon to shorten juvenile phase and induce precocious flowering. Three transgenic ‘Jiro’ lines with AtFT overexpression were successfully obtained using 10 µM FPX. Collectively, our findings demonstrate that FPX is a potent enhancer of regeneration and transformation in multiple fruit trees species, offering a promising strategy for accelerating breeding programs and gene function analysis in recalcitrant woody species.

Tomatoes are extremely important plants that are cultivated worldwide, with various varieties grown in different regions. The traits required can vary depending on the region and intended use. Parthenocarpy, a trait that confers numerous advantages, reduces the labor required for pollination and minimizes the incidence of poor fruit set owing to temperature fluctuations. Mutations in SlIAA9 induce parthenocarpy in tomatoes, and the introduction of this trait into processed varieties via genome editing suggests its potential to markedly shorten the breeding timeline. Genome editing has gained considerable attention as a breeding technique because it enables precise mutations in specific genes. However, only a few recent studies have reported examples of genome editing in Japanese tomato varieties for processing. In this study, we employed a genome-editing technique targeting SlIAA9 to induce parthenocarpy in the Japanese tomato variety Natsunokoma for processing purposes, thereby reducing the labor required for pollination. The null-segregant Sliaa9 mutant exhibited enhanced parthenocarpy and fruit set. These results suggest that improvements in fruit-bearing and parthenocarpic traits enhance the quality of tomato varieties that are mainly used for processing.

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.

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.

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.