Biology subjects

Osakabe, K.

Publications and source records attributed to Osakabe, K..

8 recordsLinked to original sources

bioRxiv2025

CRISPR/Cas9-mediated genome editing reveals the involvement of a polyphenol oxidase in the shikonin-specific biosynthesis in Lithospermum erythrorhizon

Shikonin, a 1,4-naphthoquinone derivative produced by several Boraginaceae species, exhibits unique pharmacological properties and is used as a natural dye. The regulatory factors of shikonin production have been demonstrated using a cell culture system of Lithospermum erythrorhizon. Among these factors, copper is known to be the strongest enhancer of shikonin production. Although shikonin biosynthesis has been studied for over 40 years, the steps of naphthalene ring formation are still unknown, as is the reason for the effect of copper. In this study, we explored candidate genes associated with shikonin production using a PCR-select subtraction experiment. Polyphenol oxidase (PPO), a dicopper-dependent oxidoreductase, was highlighted because it showed synchronous expression with shikonin production. Transcriptome analysis of hairy roots and cultured cells of this plant revealed that, of the five PPO genes expressed in L. erythrorhizon, only PPO1 showed a strong correlation with shikonin production. Next, we generated genome-edited hairy roots of LePPO1 using CRISPR/Cas9-mediated mutagenesis to analyze its impact on shikonin derivative and other specialized metabolite production. The results showed that shikonin content was markedly reduced in all LePPO1-ge lines. Interestingly, the content of deoxyshikonofuran, a hydroquinone derivative and shunt product that branches after GHQ-3''-OH in the shikonin biosynthetic pathway, remained unaffected in the LePPO1-ge lines. These findings suggest that LePPO1 participates in naphthalene ring formation and explain why a copper ion is crucial for shikonin biosynthesis.

biochemistry↗

bioRxiv2025

Stacked mutations disrupting syringyl and p-coumaroylated lignin biosynthesis in rice result in lignin dominated by guaiacyl units: insights into grass-specific lignin monomer biosynthesis and polymerization mechanisms

O_LIThe aromatic composition of lignin significantly impacts the usability of lignocellulosic biomass. In eudicots, transgenic and mutant lines with elevated guaiacyl (G) or syringyl (S) lignin units have been successfully generated by manipulating the expression level of CONIFERALDEHYDE 5-HYDROXYLASE (CAld5H). However, this bioengineering approach has proven less effective in grasses, implicating the potential existence of a grass-specific alternative pathway for S lignin biosynthesis. C_LIO_LIThrough characterization of genome-edited rice mutants, we demonstrated that S lignin in rice can be virtually eliminated by disrupting genes encoding CAld5H along with p- COUMAROYL-COENZYME A:MONOLIGNOL TRANSFERASE (PMT), a grass-specific enzyme essential for the biosynthesis of monolignol p-coumarate conjugates. In contrast, individual mutations in either CAld5H or PMT genes resulted in incomplete elimination of S lignin. These findings provide strong evidence that rice possesses a CAld5H-independent pathway leading to the grass-specific monolignol p-coumarate conjugates. C_LIO_LIIn-depth structural characterizations of G-dominated lignins from rice and Arabidopsis mutants, natural gymnosperm pine, and G-type synthetic lignin revealed pronounced effects of lineage-dependent cell wall environments on the linkage patterns and molecular weight distributions of the resulting lignin polymers. C_LIO_LIOverall, our findings highlight previously overlooked lineage-specific lignin monomer biosynthesis and polymerization patterns in grasses. C_LI

plant biology↗

bioRxiv2024

Essential Yet Dispensable: The Role of CINNAMATE 4-HYDROXYLASE in Rice Cell Wall Lignification

A comprehensive understanding of the intricate lignin biosynthesis in grasses could contribute to enhancing our ability to utilize grass biomass. CINNAMATE 4-HYDROXYLASE (C4H), in conjunction with PHENYLALANINE AMMONIA-LYASE (PAL), initiates the entry of phenylalanine into the cinnamate/monolignol pathway, leading to the production of diverse phenylpropanoids, including lignin monomers. Despite extensive research on C4H in eudicots, genetic studies of C4H in grasses remain considerably limited. Notably, the role of C4H in the presence of PHENYLALANINE/TYROSINE AMMONIA-LYASE (PTAL), a grass-specific ammonia-lyase that can bypass the conserved PAL-C4H pathway by recruiting tyrosine into the cinnamate/monolignol pathway, remains unclear. To address this gap, a set of genome-edited rice mutants harboring knockout mutations in rice C4H genes were generated and subjected to the analysis of growth phenotype and cell wall chemotype, alongside isotopic feeding and chemical inhibitor assays to test the contributions of the PAL-C4H and PTAL pathways. The phenotype and chemotype characterizations of C4H-knockout rice mutants demonstrated that class I (OsC4H1/CYP73A38) and class II (OsC4H2a/CYP73A39 and OsC4H2b/CYP73A40) C4Hs cooperatively contribute to lignin biosynthesis in rice. Nevertheless, the impacts of C4H-deficiency on plant development and lignin formation in rice appeared to be less prominent compared to those reported in eudicots. The 13C-labeled phenylalanine and tyrosine feeding experiments demonstrated that even with the phenylalanine-derived PAL-C4H pathway completely blocked, the C4H-knockout rice can still produce significant levels of lignin and maintain sound cell walls by utilizing the tyrosine-derived PTAL pathway. Overall, this study demonstrates the essential but dispensable role of C4H in grass cell wall lignification.

plant biology↗

bioRxiv2022

The trans-zeatin-type side-chain modification of cytokinins controls rice growth

Cytokinins (CKs), a class of phytohormones with vital roles in growth and development, occur naturally with various side-chain structures, including N6-({Delta}2-isopentenyl)adenine-, cis-zeatin- and trans-zeatin (tZ)-types. Recent studies in a model dicot plant Arabidopsis demonstrated that tZ-type CKs are biosynthesized via cytochrome P450 monooxygenase (P450) CYP735A, and have a specific function in shoot growth promotion. Although the function of some of these CKs has been demonstrated in a few dicotyledonous plant species, the significance of these variations and their biosynthetic mechanism and function in monocots and in plants with distinctive side-chain profiles than Arabidopsis, such as Oryza sativa (rice), remain elusive. In this study, we characterized CYP735A3 and CYP735A4 to investigate the role of tZ-type CKs in rice. Complementation test of the Arabidopsis CYP735A-deficient mutant and CK profiling of loss-of-function rice mutant, cyp735a3 cyp735a4, demonstrated that CYP735A3 and CYP735A4 encode P450s required for tZ-type side-chain modification in rice. CYP735As are expressed in both roots and shoots. The cyp735a3 cyp735a4 mutants exhibited growth retardation concomitant with reduction in CK activity in both roots and shoots, indicating that tZ-type CKs function in growth promotion of both organs. Expression analysis revealed that tZ-type CK biosynthesis is negatively regulated by auxin, abscisic acid, and cytokinin and positively by dual nitrogen nutrient signals, namely glutamine-related and nitrate-specific signals. These results suggest that the physiological role of tZ-type CKs in rice is different from that in Arabidopsis and they control growth of both roots and shoots in response to internal and environmental cues in rice.

plant biology↗

bioRxiv2021

The establishment of multiple knockout mutants of Colletotrichum orbiculare by CRISPR/Cas9 and Cre/loxP systems

Phytopathogenic fungi belonging to the Colletotrichum genus cause devastating damage for many plant species. Among them, Colletotrichum orbiculare is employed as a model fungus to analyze molecular aspects of plant-fungus interactions. Although gene disruption via homologous recombination (HR) was established for C. orbiculare, this approach is laborious due to its low efficiency. Here we developed methods to efficiently generate multiple knockout mutants of C. orbiculare. We first found that CRISPR/Cas9 system massively promoted gene-targeting efficiency. By transiently introducing a CRISPR/Cas9 vector, more than 90 % of obtained transformants were knockout mutants. Furthermore, we optimized a self-excision Cre/loxP marker recycling system for C. orbiculare because limited availability of desired selective markers hampers sequential gene disruption. In this system, integrated selective marker is removable from the genome via Cre recombinase driven by a xylose-inducible promoter, enabling reuse of the same selective marker for the next transformation. Using our CRISPR/Cas9 and Cre/loxP systems, we attempted to identify functional sugar transporters in C. orbiculare. Multiple disruptions of putative quinate transporter genes restrict fungal growth on media containing quinate as a sole carbon source, confirming their functionality as quinate transporters. Our analyses revealed that quinate acquisition is dispensable during fungal infection because this mutant displayed normal virulence to host plants. In addition, we successfully built mutations of 17 cellobiose transporter genes in a strain. From the data of knockout mutants established in this study, we inferred that repetitive rounds of gene disruption using CRISPR/Cas9 and Cre/loxP systems do not cause negative effects for fungal virulence and growth. Therefore, these systems will be powerful tools to perform systematic gene targeting approach for C. orbiculare.

plant biology↗

bioRxiv2020

Association between the extent of DNA methylation at the CpG sites of HIF3A and parameters of obesity in the general Japanese population

Obesity is a major public health problem worldwide owing to the substantial increase in risk of metabolic diseases. Hypoxia-inducible factors (HIFs) regulate transcriptional responses to hypoxic stress. DNA methylation in the CpG sites of intron 1 of HIF3A is associated with body mass index in the whole blood and adipose tissue. This study investigates the correlation between DNA methylation of HIF3A and parameters of obesity, including thickness of visceral (VAT) and subcutaneous adipose tissues, in the general Japanese population. Participants (220 men and 253 women) who underwent medical examination were enrolled in this cross-sectional study. We used pyrosequencing to quantify DNA methylation (CpG sites of cg16672562, cg22891070, and cg27146050) in HIF3A. DNA methylation of HIF3A was only different in women. Multiple regression analysis showed that DNA methylation level at cg27146050 was associated with thickness of VAT in women. DNA methylation level at cg27146050 also correlated with body mass index and percentage of body fat in women after excluding smokers and non-smokers who quit smoking with the last 5 years. DNA methylation in the CpG site (cg27146050) of HIF3A correlated with parameters of obesity in Japanese women.

biochemistry↗

bioRxiv2020

Genome editing in mammals and plants using CRISPR type I-D nuclease

Adoption of the CRISPR-Cas system has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I--the most abundant CRISPR system in bacteria--has been less developed. Type I systems in which Cas3 nuclease degrades the target DNA are known; in contrast, for the sub-type CRISPR type I-D (TiD), which lacks a typical Cas3 nuclease in its cascade, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d--a nuclease in TiD--is multi-functional in PAM recognition, stabilization and target DNA degradation. TiD can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. TiD off-target effects, which were dependent on the mismatch position in the protospacer of TiD, were also identified. Our findings suggest TiD as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.

molecular biology↗

bioRxiv2018

Efficient CRISPR/Cas9-based genome editing and its application to conditional genetic analysis in Marchantia polymorpha

Marchantia polymorpha is one of the model species of basal land plants. Although CRISPR/Cas9-based genome editing has already been demonstrated for this plant, the efficiency was too low to apply to functional analysis. In this study, we show the establishment of CRISPR/Cas9 genome editing vectors with high efficiency for both construction and genome editing. Codon optimization of Cas9 to Arabidopsis achieved over 70% genome editing efficiency at two loci tested. Systematic assessment revealed that guide sequences of 17 nt or shorter dramatically decreased this efficiency. We also demonstrated that a combinatorial use of this system and a floxed complementation construct enabled conditional analysis of a nearly essential gene. This study reports that simple, rapid, and efficient genome editing is feasible with the series of developed vectors.

plant biology↗