The CRISPR/Cas9 system is now widely used for precise genome editing in many crop species, allowing targeted mutagenesis and precise base substitution. While CRISPR/Cas9-mediated genome editing is highly accurate, a limitation of this technology is the requirement for an appropriate protospacer adjacent motif (PAM) for target site recognition. Cas9 from Brevibacillus laterosporus (BlCas9) has been reported to recognize N₄CNDD PAMs with a pronounced preference for A at the 7th and 8th positions of the PAM, and is used for genome editing in maize and human cells. Recently, the enhanced BlCas9 (enBlCas9) variant was reported that includes two amino acid mutations in the PAM-interacting domain of BlCas9 and exhibits enhanced genome editing activity with an expanded target scope in vitro and in human cells. Here, we demonstrate the BlCas9- and enBlCas9-mediated genome editing in rice. Both BlCas9 and enBlCas9 can introduce targeted mutations in rice, and enBlCas9 can broaden the target scope with a non-A residue at the 7th and 8th bases of the PAM. Furthermore, enBlCas9 is applicable for precise base editing with extended target sites, such as C-to-T and A-to-G base conversions. In addition, we developed and validated a proximal CRISPR targeting method (proxy-CRISPR) in which nuclease-dead SpCas9 (SpdCas9) bound near the target sites can improve the genome editing activity of BlCas9 and enBlCas9. These enBlCas9-based genome editing technologies are expected to broaden the scope of efficient targeted mutation and precise base editing in rice.

B-box (BBX) transcription factors play critical roles in plant acclimation to environmental stresses, yet their involvement in cold stress responses remains incompletely understood. Following a genome-wide analysis of BBX gene expression profiles in Arabidopsis thaliana, we focused on AtBBX11, whose transcript levels increased by up to 80-fold in shoots after 24 h of cold treatment. Co-expression analysis revealed that AtBBX11 is associated with key cold-responsive genes and stress-related pathways, including responses to cold, abscisic acid (ABA), water deprivation, and hypoxia. Functional characterization using overexpression lines demonstrated that AtBBX11 enhances chilling tolerance, as transgenic plants exhibited significantly less reduction in shoot fresh weight compared to wild type under two chilling regimes. Transcriptome analysis under cold stress identified numerous differentially expressed genes, including selected transcription factors from several families, with NAC and bHLH being the most represented. Gene Ontology enrichment highlighted processes related to hypoxia response, phosphate starvation, and ABA signaling. Several transcription factors and hypoxia-responsive genes were further validated by Reverse Transcription-quantitative Polymerase Chain Reaction (RT-qPCR), confirming RNA-seq reliability. Collectively, these findings establish AtBBX11 as a cold-responsive regulator that may modulate multiple stress-related pathways, providing new insights into BBX-mediated mechanisms underlying plant cold tolerance.

In Orchidaceae, the protocorm, which forms after seed germination, serves as a crucial genetic resource for plant preservation and breeding. To establish a long-term conservation method for the protocorm, in this study, we developed cryopreservation methods. Protocorms of Bletilla striata, a terrestrial orchid species, were precultured for 3 days in a medium containing 0.3 M sucrose and then cryopreserved using vitrification. Although viable cell staining indicated the presence of many surviving cells in the protocorms, the regrowth rate was extremely low. We categorized the stained regions based on the presence or absence of staining in the shoot apical meristem (SAM). The staining rate in the SAM region was low after cryopreservation, which was considered the cause of the low regrowth rate. To enable cryopreservation of the protocorms, the V-Cryo-plate method was adopted, and the treatment times for the solutions used in dehydration were investigated. Treatment times of 45 min and 2 h for the loading and vitrification solutions were found to be appropriate, respectively. Furthermore, an investigation of the effect of adding plant growth regulators to the preculture medium showed that adding 0.1 µM 1-naphthalene acetic acid resulted in a high SAM survival rate of 88% and regrowth rate of 87%, comparable to that observed with survival cell staining. Therefore, survival in the SAM region is important for regrowth in cryopreserved protocorms. Confirmation of survival regions is an important indicator for bridging the gap between the survival and regrowth rates.

Some functions of flavonoid glycosides are determined by the glycan structure or enhanced by increasing the attached sugars. Therefore, engineering the glycan moiety can modify the functions of the flavonoid. We attempted to construct a biotransformation method for producing a flavonoid diglycoside, naringin (NRGI) as a model target, using engineered yeast. Naringenin (NRG) glycosylation is initiated by the regiospecific 7-O-glucosyltransferase (7-O-GlcT) to form naringenin-7-O-glucoside (N7G), followed by further rhamnosylation via the branching-type α1,2-rhamnosyltransferase (1,2RhaT), producing NRGI as the final product. In this study, we introduced three α1,2-rhamnosyl-glucoside synthesis-related enzymes—7-O-GlcT from Arabidopsis thaliana (AtGT-2), 1,2RhaT from Citrus maxima (Cm1,2RhaT), and UDP-Rha synthase from A. thaliana (AtRHM2)—into the fission yeast Schizosaccharomyces pombe. To improve the titer of NRGI, we examined the effects of biotransformation medium composition, initial cell concentration, and cell-permeabilizing reagents. Consequently, we successfully constructed a biotransformation method for producing a flavonoid diglycoside from the aglycone via sequential glycosylation using a single recombinant yeast. Using the optimized biotransformation method, we produced 23.1±1.1 mg l⁻¹ of NRGI, 8.0±0.4% molar conversion, from 136 mg l⁻¹ of NRG in 24 h cultivation. This study demonstrated the first example of flavonoid O-diglycoside production in engineered S. pombe via sequential glycosylation by uridine diphosphate sugar-dependent glycosyltransferases (UGTs) under optimized production conditions.

Kudzu (Pueraria montana var. lobata) produces several isoflavones that have beneficial effects in humans. These isoflavones accumulate in a tissue-specific manner, with 5,7,4′-trihydroxy-6-methoxyisoflavone (tectorigenin) and its derivatives accumulating mainly in flowers. Tectorigenin has various beneficial effects on humans and is in high demand, but its biosynthesis has not been elucidated, making large-scale production difficult. In this study, we elucidated the steps in tectorigenin biosynthesis in kudzu by identifying a novel isoflavone 6-hydroxylase and isoflavone O-methyltransferase through a transcriptome analysis and related enzyme assays. Furthermore, using yeast engineered to express genes encoding these enzymes, we successfully produced the 6-methoxyisoflavones tectorigenin, glycitein, and afrormosin from the inexpensive isoflavones genistein, daidzein, and formononetin, respectively. The tectorigenin production rate was 40 mg l⁻¹ after being cultured for 72 h. These results contribute to large-scale tectorigenin production and to tectorigenin-related research.

Modulation of plant vascular tissues is of interest for crop improvement because xylem and phloem are responsible for wood production and sugar transport across the plant, respectively. Previous studies have shown that DELLA and ERECTA (ER) regulate vascular tissue development, as their respective mutants exhibit enlarged xylem and disorganized vascular tissue. The tomato hawaiian skirt-1 (hws-1) mutant accumulates microRNAs and displays a strongly enlarged phloem together with slightly delayed xylem development in pedicels. In this study, della and er mutant lines were crossed with hws-1 to evaluate the effect of disrupted DELLA and ER function on the large-phloem phenotype of hws in the resulting double mutants. Measurement of miRNA164 levels, a microRNA that accumulates in hws, revealed that its abundance returned to wild-type levels in the double mutants, indicating that the effects of della and er mutations dominate over hws. Consistently, plant architecture and vascular phenotypes in the double mutants closely resembled those of the della and er single mutants, and the characteristic phloem enlargement of hws was absent. These results indicate that DELLA and ER functions are required for the manifestation of the hws large-phloem phenotype in tomato cv. Micro-Tom and that these genes are largely epistatic to hws for the majority of the morphological traits examined.

Apomixis is the reproduction mode in which only the mother’s genes are transmitted from generation to generation. If this trait can be put to practical use, it is expected to be a truly epoch-making breeding method, as seed production costs can be greatly reduced by fixing F₁ hybrids. This study aimed to isolate the aposporous apomixis gene and analyze how the gene(s) will be expressed from/in the aposporous guineagrass (Panicum maximum Jacq.). A new classification method using the ovary length as an index was developed to sample different developmental stages of ovaries and buds in obligate sexual plants and apomicts. A cDNA library was derived from the ovaries of aposporous accession N68/96-8-o-11 staged at the appearance of aposporous initial cells (AICs) to isolate AIC stage-specific genes. Using differential screening, four AIC stage-specific cDNA clones obtained from ten thousand of plaques by Northern blot hybridization showed the same start codon and sequences, ranging in lengths from 577 to 1182 bp. The characteristics and their homologies of the four cDNA clones are similar to Apomixis-specific gene-1 (ASG-1), indicating that they are the different cDNA clones of Apomixis-specific gene-1 homolog (ASG-1H). In situ expression analysis detected signals without distinguishing between ASG-1 mRNA and the ASG-1H on gene expression specifically in AIC, AIC-derived embryo sacs, and root tips and shoot apical meristems of aposporous accession. The finding and identification of ASG-1H expressed at the times of AIC appearance and AIC-derived embryo sac formation, may represent an initial step towards isolating an apospory gene.

β-Aminobutyric acid (BABA) is a nonproteinogenic amino acid that functions as a potent plant defense activator, conferring broad-spectrum resistance in many plants. Although its effects on biotrophic and necrotrophic pathogens are well known, the molecular basis of BABA-induced resistance (BABA-IR) against the hemibiotrophic fungus Colletotrichum higginsianum remains unclear. In this study, we investigated BABA’s effects on C. higginsianum-infected Arabidopsis thaliana. Foliar BABA application (1.25–10 mM) reduced lesion size and fungal biomass in a concentration-dependent manner, with 5–10 mM being most effective. Microscopy showed that BABA treatment prevented fungal invasion beyond the appressorium stage. Mutant analysis revealed that BABA-IR depends on salicylic acid (SA) biosynthesis (NahG and eds16-1) and the signaling components EDS1 and NDR1. In contrast, mutants insensitive to jasmonic acid and ethylene (jar1-1 and ein2-12, respectively) retained BABA-mediated protection. These findings indicate that BABA primes SA-dependent defenses against C. higginsianum while highlighting its potential for improving sustainable crop protection.

Identification of novel functional genes through mutant analysis is one of the most powerful approaches in plant science. In the model plant Arabidopsis thaliana, this approach typically combines whole-genome sequencing of the mutant with fine mapping using molecular polymorphic markers between two ecotypes to narrow down the genomic region containing the mutant allele. Among PCR-based mapping methods, insertion/deletion (InDel) markers allow for precise and rapid genotyping with relatively simple technology. In this report, we present a set of 49 new InDel markers distributed across the entire genome between Columbia (Col-0) and Landsberg erecta (Ler) accessions, specifically suitable for initial mapping experiments. These markers are designed to detect insertions in Col-0 or deletions in Ler with a length polymorphism greater than 100 bp and will help facilitate map-based gene cloning.

Selectable marker genes are essential for recovering rare transformants during plant genetic transformations. However, only a limited number of markers are routinely used in Arabidopsis thaliana, particularly for nuclear transformation by the floral dip method. Aminoglycoside-3″-adenylyltransferase (AadA) has been widely used in plastid transformation to confer resistance to Streptomycin and Spectinomycin; however, its applicability to nuclear transformation in Arabidopsis has not been systematically examined. Here, we evaluated an Arabidopsis-codon-optimized AadA1 (Streptomycin/Spectinomycin Resistance: SmR) gene derived from Escherichia coli and lacking any plastid-targeting sequence, as a selectable marker for Arabidopsis nuclear transformation. Dose–response analyses revealed consistent Streptomycin and Spectinomycin sensitivity profiles across the four accessions (Col, L. er, Ws, and C24). When introduced via Agrobacterium-mediated floral dip, SmR conferred robust resistance to 50 mg l⁻¹ Streptomycin or 10 mg l⁻¹ Spectinomycin, enabling clear visual discrimination between resistant transformants and bleached non-transformants. Segregation analyses of T₂ progeny revealed Mendelian 3 : 1 ratios, indicating successful transformation of the nuclear genome. Importantly, SmR transformants remained fully sensitive to Kanamycin and Hygromycin, demonstrating that SmR does not confer cross-resistance to these commonly used antibiotics. This compatibility enables the simultaneous use of Spectinomycin, Kanamycin, and Hygromycin for triple selection, allowing efficient isolation of triple transgenic plants. These results establish the non-targeted SmR as an efficient and cost-effective selectable marker for Arabidopsis nuclear transformation and expand the practical repertoire of plant selectable marker systems.