The unfolded protein response (UPR) is a central regulatory pathway that ensures the proper function of the endoplasmic reticulum (ER) through efficient protein folding and quality control. In Arabidopsis, bZIP60 mRNA is activated by an IRE1-mediated unconventional splicing that excises a 23-nucleotide intron, resulting in the spliced form (bZIP60s mRNA) that encodes the active bZIP60 transcription factor lacking a transmembrane domain. In this study, we investigated the functional role of the spliced form-specific C-terminal extension, hereafter referred to as ORF2. Transient expression assays in Arabidopsis mesophyll protoplasts demonstrated that full-length bZIP60s potently activates the BiP3 promoter compared to a truncated variant lacking ORF2. Fusion of ORF2 to transcription factors unrelated to the UPR did not enhance their transcriptional potency, underscoring its specialized role in the context of bZIP60s. Furthermore, mutation in a conserved nuclear localization signal within ORF2 decreased promoter activation by bZIP60s. Fusion of ORF2 to GFP enhanced the nuclear localization of GFP. Our results suggest that ORF2 is critical for the full transcriptional activity of bZIP60s to ensure an efficient UPR.

Catechins includ galloylated catechins and non-galloylated catechins, among which galloylated catechins exhibit stronger antioxidant, anti-inflammatory and anti-cancer activities. Section Chrysantha Chang, the only group of yellow Camellia with rich catechins in their flowers, is a common health drink in southern China. To date, few studies have examined galloylated catechins biosynthesis in flowers of this group. To enrich the genetic information of the galloylated catechins biosynthesis, the ONT sequencing platform was used to perform full-length transcriptome sequencing of C. perpetua flowers and 7,972,574 transcripts was identified, including 42,883 simple sequence repeats (SSRs), 41,961 coding sequences (CDSs) and 2,602 long non-coding RNAs (lncRNAs). 36,516 transcripts were successfully annotated, and 147 critical enzyme-encoding genes were identified as involved in the galloylated catechins biosynthesis pathway, including 17 CpSCPL1A genes. Bioinformatics analysis revealed that each CpSCPL1A protein consisted of 427–506 amino acids, and all CpSCPL1A proteins were divided into 5 groups with conserved motifs 1, 4, 5, 6 and 8. Based on the correlation analysis between the gene expression of 17 CpSCPL1A genes and the content of galloylated catechins, 11 candidate CpSCPL1A genes were identified to be involved in the biosynthesis of 4 types of galloylated catechins in C. perpetua flowers. The results enrich the transcriptome data for C. perpetua and provide valuable insights into the importance of the CpSCPL1A gene family members in the galloylated catechins biosynthesis.

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.

Cassava is a globally important food source. Given the increasing frequency of climate change-induced drought, enhancing the drought resilience of cassava is paramount. Chemical priming can bolster tolerance to stress factors. We previously determined that pretreatment with low concentrations of ethanol enhances abiotic stress tolerance in Arabidopsis, tomato, and cassava. Nevertheless, the efficacy of ethanol treatment in complex natural settings remains to be fully explored. In this study, we assessed the impact of ethanol treatment on cassava under varying light photon flux densities (PFDs) and drought conditions. We observed that drought tolerance was enhanced by ethanol pretreatment at high (∼400 µmol photons m⁻² s⁻¹) and medium (∼60 µmol photons m⁻² s⁻¹) light PFDs but not under low light PFD (∼4 µmol photons m⁻² s⁻¹). Ethanol pretreatment under high and medium light PFDs promoted stomatal closure and drought avoidance, thereby preserving higher water content in plant tissues. Furthermore, ethanol pretreatment under these PFDs upregulated expressions of genes associated with ABA signaling and heat shock proteins (HSPs) relative to water pretreatment. In addition, starch accumulation in leaves was observed under all light PFDs with ethanol pretreatment. We hypothesize that ethanol pretreatment at light PFDs exceeding 60 µmol photons m⁻² s⁻¹ facilitates ethanol-mediated drought avoidance in cassava by activating at least three pathways: 1) ABA signaling, 2) protein folding-related response via triggering of the HSP/chaperone network, and 3) alterations in sugar and starch metabolism. Our findings support the application of optimal light PFDs to enhance the benefits of ethanol-induced drought avoidance in cassava.

Gentians are important ornamental plants, and gentian cultivars have been actively bred for decades. However, limited genetic resources are currently available for breeding; therefore, artificial mutagenesis has been applied to generate mutants. In this study, we developed a simple and efficient regeneration-mediated method for ion beam mutagenesis in the Japanese gentian hybrid cultivar ‘Albireo’ (Gentiana scabra × G. triflora). Carbon and neon ion species were tested. Effect of ion beam irradiation on callus formation from leaves was initially evaluated. Tissue culture was then continued, adventitious shoots were induced from calli, and many regenerated plants were obtained. These plants were cultivated until flowering, and two cultivated lines exhibiting a double-flowered phenotype were identified from leaves exposed to 9 and 12 Gy of neon ion beam irradiation among approximately 200 individuals. We analyzed one line derived from irradiation with 9 Gy of neon ions, named Ne9Gy#34, in detail. The agamous gene (AG1), previously identified as the gene responsible for the double-flower phenotype in gentians, was not amplified in the G. scabra allele by genomic polymerase chain reaction. Moreover, next-generation sequencing also indicated that the reads were mapped to the genomic region of the G. triflora AG1 but not to that of G. scabra, suggesting that the deletion of G. scabra AG1 led to the double-flowered phenotype. Ne9Gy#34 also exhibited increased flower size, suggesting additional mutations in genes other than AG1. In summary, the developed regeneration-mediated method represents a promising approach for inducing gentian mutagenesis and efficiently producing novel traits in this plant.

Hedysari Radix, a significant Chinese herbal medicine from Northwest China’s arid region, is renowned for its unique tonic effects in traditional Chinese medicine practices. This plant, a member of the Leguminosae family, forms a symbiotic relationship with nitrogen-fixing rhizobia. However, the Hedysarum polybotrys-rhizobium symbiotic system remains underexplored. The root nodule structure of H. polybotrys was examined using an optical microscope (OM). This examination revealed that its root nodules consist of meristematic zone, infection zone, nitrogen fixation zone, and senescence zone, arranged from top to bottom. This structure suggests that the root nodules of H. polybotrys belong to the indeterminate nodule category. In the fields of transmission electron microscopy (TEM) and fields emission scanning electron microscopy (FESEM), significant differences were observed between infected and un-infected cells. Rhizobium, identified via 16S rRNA technology and classified as the genus Mesorhizobium through phylogenetic analysis. Reinoculation of rhizobium into H. polybotrys seedlings resulted in nodule formation on the roots. Notably, inoculated plants exhibited a considerable increase in nodule number, leaf count, leaf length, aboveground height, aboveground fresh weight, root length, and root diameter compared to uninoculated controls, demonstrating that rhizobium inoculation enhances plant growth.

Nuclear staining using fluorescent dyes is crucial for cytological studies in plants. However, few fluorescent dyes are suitable for live-cell imaging of the nucleus. Here, we demonstrate that dimidium bromide (DimBr), a commercially available fluorescent dye, can be used to stain the nucleus (nucleolus and nucleoplasm) in living plant cells. DimBr emits peak fluorescence at 600 nm at an excitation wavelength of 525 nm, making it well suited for use with green fluorescent protein. DimBr staining can be used in various plant species and allows time-lapse observation of the nucleus. Therefore, DimBr can be used to visualize the nucleus in living plant cells, making it a valuable tool for plant cell biology.

The agroinfiltration technique using sprouts as a host is one of the most cost-effective, efficient, and rapid methods for producing recombinant proteins. We previously reported that radish sprouts were the best host for this purpose. To find suitable alternative sprouts comparable to radish sprouts, we investigated rye sprouts using a wheat dwarf virus (a geminivirus) DNA-containing expression vector. Various rye cultivars were tested, and Raitaro and Ryokuhiyo sprouts exhibited the highest enhanced green fluorescent protein (EGFP) productivity. When agroinfiltrated after a 5-d cultivation period, including 1 day of seed imbibition, approximately 1.8 mg of EGFP was produced per gram fresh weight of leaf in areas exhibiting EGFP fluorescence. This yield is comparable to that of mature leaves from Nicotiana benthamiana and radish sprouts. However, only a limited number of leaves produced the protein, and production was confined to areas near the leaf tips. Elevated production levels were observed in the guard cells of stomata and at wounded sites via microneedling, suggesting that the limiting factors for protein production may involve the entry of Agrobacterium into the leaves and/or the subsequent transfer of T-DNA into the plant cells.

Variability in environmental conditions and farming practices often leads to discrepancies between experimental results and outcomes in farmers’ fields. This gap poses a challenge for understanding the effects of agricultural inputs and methods under real-world conditions, particularly in fruit cultivation systems, where large-scale experimental data are limited. In this study, we applied a cohort study approach leveraging data from farmers’ fields to investigate the effects of pesticide and fertilizer application methods on fruit quality and soil properties in mandarin orange orchards. Biases arising from differences in covariates among cultivation methods were controlled using the inverse probability weighting (IPW) based on propensity scores. Consequently, compared to local-scale analysis between adjacent fields, the nationwide cohort analysis detected a greater number of significant effects of cultivation methods by utilizing its larger sample size. Through this analysis, we found important insights into the effects of pesticide and fertilizer application methods on plant pathogens, nutritional quality, and soil properties in sustainable cultivation systems of mandarin orange. This study demonstrates that cohort analyses using real-world data have great potential to advance agricultural biotechnology by providing effective feedback from farmers’ fields and bridging the gap between scientific research and real-world agriculture.

Quantitative trait loci involved in reducing high temperature injury during grain filling were investigated using fifty-four lines of the chromosome segment substitution lines (CSSLs) of the indica cultivar Kasalath in the japonica cultivar Nipponbare background. The ratio of chalky seeds was examined when they were ripened under the high-temperature conditions. Among these lines, SL13 seeds showed an obvious trait of alleviating injury. In SL13, a part of the chromosome 3 was substituted with the Kasalath genome, which contained a locus of this trait locus. Fine mapping of this locus using the progenies obtained by crossing SL13 with Nipponbare narrowed it down to a region of 3.36–3.81 Mb on chromosome 3. This region included the OsSUT1 gene encoding sucrose transporter 1. The Nipponbare OsSUT1 contained 19-nucleotide insertion in the promoter region, suggesting that this diversity might affect the transcription level of this gene. The progeny plants of SL13 containing the Kasalath OsSUT1, which had a significantly higher expression level of this gene, obviously reduced the high-temperature injury. Transformants carrying the Kasalath OsSUT1 gene showed significant alleviation in the high-temperature injury during grain filling. Our results indicate that OsSUT1 is a major factor consisting of the locus involved in reducing the high temperature injury in SL13. These results suggest that the enhanced function of OsSUT1 provides sufficient carbon assimilates to immature seeds even under high temperature conditions, leading to normal seed formation.

Commercial tomatoes have been increasingly criticized for their declining aroma, driving a search for tomato cultivars with more robust aroma profiles. Volatile organic compounds (VOCs) in tomato fruits play a crucial role in determining their aroma potential. This study investigates tomato cultivars with desirable aroma profiles by examining the levels of free- and glycoside-derived VOCs across 13 cultivars over a three-year period using non-targeted VOC profiling. The analysis detected 41 free VOCs and 35 VOCs released from the glycoside-derived precursors (glycoside-derived VOCs). Principal component analysis of the annotated free and glycoside-derived VOCs revealed that year-to-year differences were more pronounced than cultivar-to-cultivar variations. Among the annotated VOCs, 18 compounds were classified as unique free VOCs, while 12 were unique glycoside-derived VOCs. In 2020, the cultivar Livingstone’s stone exhibited significantly higher levels of several key aroma compounds compared to other cultivars and the control, Ailsa Craig. These compounds are known for their low odor thresholds and positive contributions to the overall aroma. These findings suggested that the growth year may substantially influence VOC production in tomato fruits, particularly for specific cultivars or VOCs, likely due to variations in climatic conditions. Consequently, optimizing the release of glycoside-derived VOCs offers a promising strategy to enhance tomato fruit aroma. This approach would be most effective by focusing on key VOCs from specific cultivars, while also accounting for the significant influence of growing year.

Suspension-cultured cells of a temperate bamboo species (Phyllostachys nigra) accumulate substantial amounts of hydroxycinnamic acid derivatives and lignin under culture conditions that promote xylogenesis. In our previous study, we found a metabolite specifically produced in bamboo cells cultured under lignification-inducing conditions in a medium containing N⁶-benzyladenine (BA), but the chemical structure was not elucidated. In this study, we purified and identified this compound as BA N⁹-β-D-glucopyranoside (BA-9G). Despite the presence of three nitrogen positions (N-3, N-7, and N-9) that may be glucosylated in the adenine moiety of BA, bamboo cells specifically produced BA-9G (i.e., without other glucoside types) when cells were cultured in the presence of BA. This finding suggests that bamboo cells possess a regio-specific N-glucosyltransferase for catalyzing cytokinin glucoside formation. The biological activity of BA-9G as a cytokinin was compared with that of BA on the basis of adventitious shoot formation on internodal segments of ipecac (Carapichea ipecacuanha) plants grown under in vitro conditions. The activity of BA-9G was more moderate than that of BA, but BA-9G was less cytotoxic than BA at a high concentration, suggesting that BA-9G may be useful as a plant growth regulator. The development of a viable system for the regio-specific bioproduction of BA-9G in bamboo cells may increase the availability of this highly expensive and rare cytokinin derivative.

Papaya (Carica papaya L.), a tropical plant belonging to the Caricaceae family, is widely cultivated in tropical and subtropical countries. Young leaves grow from the stem tips, petioles elongate, and leaf color changes from light green to dark green during development. Papaya leaves are used as therapeutic agents in folk medicine and potential functional food materials; however, the specific associations between the leaf development stage and functional components of papaya remain unknown. Therefore, in this study, we aimed to conduct a non-targeted analysis of the four developmental stages of papaya leaves via liquid chromatography coupled with quadrupole-time-of-flight mass spectrometry. Specifically, we focused on carpaine derivatives and γ-aminobutyric acid that have attracted attention in the Japanese functional food industry. Carpaine derivatives were abundant in young leaves; however, their levels decreased with increasing leaf maturity. In contrast, γ-aminobutyric acid levels increased with increasing leaf maturity. Multivariate analyses revealed that the metabolites changed more significantly during the transition to the dark green phase than during the transition from the early yellow green to bright green phase. Additionally, proteolytic activity was evaluated using casein as a substrate. Proteolytic activity decreased with increasing leaf maturity. In conclusion, our findings suggest that leaves at different developmental stages should be selected based on their functional components and intended application.

Bioproduction of high-value natural compounds in cultured plant cells represents a favorable strategy compatible with the framework of green sustainable chemistry. Raspberry ketone, a phenylpropanoid-derived compound utilized as a food additive and cosmetics constituent, is a distinctive aromatic component that is accumulated in the ripe fruit of raspberry (Rubus idaeus). Its natural abundance is extremely limited. Consequently, it is imperative to develop methodologies for efficient bioproduction of raspberry ketone. In the present study, we examined the effect of expressing the raspberry ketone biosynthetic genes RpBAS and RiRZS1 in bamboo (Phyllostachys nigra) cells, which had been demonstrated previously to be an appropriate host for production of phenylpropanoid-derived compounds, on the bioconversion of precursor compounds into raspberry ketone. The maximal production yield of raspberry ketone, primarily accumulated in the glycosylated form, reached 293 µg g⁻¹ fresh weight when the RpBAS-RiRZS1-transgenic bamboo cells were cultured in medium supplemented with 4-hydroxybenzalacetone, the immediate precursor of raspberry ketone. These findings underscore the potential utility of bamboo cells for the bioproduction of raspberry ketone from accessible precursors.

A plant-based expression system provides a cost-effective, scalable, and safe alternative to traditional cell culture platforms. In this study, recombinant human interleukin-15 (IL-15) was transiently expressed in Nicotiana benthamiana plants using agroinfiltration. IL-15 is a cytokine with significant potential in cell engineering, immunotherapy, and cancer therapy. A codon-optimized IL-15 gene was cloned into a binary vector designed for plant expression and introduced into Rhizobium radiobacter (formerly Agrobacterium tumefaciens). The R. radiobacter for human IL-15 expression was infiltrated into N. benthamiana leaves. Following purification, receptor-binding assays confirmed that the plant-derived IL-15 could bind to the IL-15 receptor comparably to its mammalian-produced counterpart. This first report of IL-15 expression in plants highlights the promise of plant-based systems for biopharmaceutical production and lays the groundwork for further development of IL-15 for applications in cell engineering, clinical therapies, and the cultured meat industry.

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.