Biology subjects

Kurotani, K.-i.

Publications and source records attributed to Kurotani, K.-i..

9 recordsLinked to original sources

bioRxiv2026

A network analysis for the identification of gene modules in the transcriptome during Nicotiana benthamiana interfamily grafting

BackgroundNicotiana benthamiana has been found to exhibit grafting capability with phylogenetically distant plant species by accomplishing cell-cell adhesion as the first step in graft establishment. Morphological and physiological studies combined with time-course transcriptome analysis revealed that this grafting triggered various biological processes. Thus, further elucidation of accumulated datasets is required to describe the processes during grafting. ResultsIn this study, we performed a Bayesian network analysis to identify crucial gene modules in the transcriptome of Nicotiana benthamiana interfamily grafting. Our bioinformatics analyses of the transcriptome included threshold-based clustering, functional annotation, Bayesian network analysis, module analysis, and hub gene identification. We defined six distinct temporal gene expression patterns in the transcriptome data. Gene ontology enrichment was performed for each expression pattern, and results were summarized as Gene ontology supercluster treemaps. Bayesian gene networks were constructed using the SiGN-BN HC + BS program along with 120 N. benthamiana transcriptome data covering the whole life cycle. Gene modules were identified using two module detection algorithms: Molecular Complex Detection (MCODE) and GLay. Gene modules were characterized by identifying Gene Ontologies and hub genes as the most interconnected nodes in the gene network using the Cytohubba plugin. ConclusionThis study provides further knowledge and enhances our understanding of the molecular mechanisms underlying interfamily grafting.

systems biology↗

bioRxiv2023

Microfluidic device for simple diagnosis of plant growth condition by detecting miRNAs from filtered plant extracts

Plants are exposed to a variety of environmental stress and starvation of inorganic phosphorus can be a major constraint in crop production. In plants, in response to phosphate deficiency in soil, miR399, a type of microRNA (miRNA), is upregulated. By detecting miR399, the early diagnosis of phosphorus deficiency stress in plants can be accomplished. However, general miRNA detection methods require complicated experimental manipulations. Therefore, simple and rapid miRNA detection methods are required for early plant nutritional diagnosis. For the simple detection of miR399, microfluidic technology is suitable for point-of-care applications because of its ability to detect target molecules in small amounts in a short time and with simple manipulation. In this study, we developed a microfluidic device to detect miRNAs from filtered plant extracts for the easy diagnosis of plant growth conditions. To fabricate the microfluidic device, verification of the amine-terminated glass as the basis of the device and the DNA probe immobilization method on the glass was conducted. In this device, the target miRNAs were detected by fluorescence of sandwich hybridization in a microfluidic channel. For plant stress diagnostics using a microfluidic device, we developed a protocol for miRNA detection by validating the sample preparation buffer, filtering, and signal amplification. Using this system, endogenous sly-miR399 in tomatoes, which is expressed in response to phosphorus deficiency, was detected before the appearance of stress symptoms. This early diagnosis system of plant growth conditions has a potential to improve food production and sustainability through cultivation management.

plant biology↗

bioRxiv2023

Establishing a comprehensive web-based analysis platform for Nicotiana benthamiana genome and transcriptome

Nicotiana benthamiana has long served as a crucial plant material extensively used in plant physiology research, particularly in the field of plant pathology, because of its high susceptibility to plant viruses. Additionally, it serves as a production platform to test vaccines and other valuable substances. Among its approximately 3.1 Gb genome, 57,583 genes have been annotated within a 61 Mb region. We created a comprehensive and easy-to-use platform to use transcriptomes for modern annotation. These tools allow to visualize gene expression profiles, draw molecular evolutionary phylogenetic trees of gene families, perform functional enrichment analyses, and facilitate output downloads. To demonstrate their utility, we analyzed the gene expression profiles of enzymes within the nicotine biosynthesis pathway, a secondary metabolic pathway characteristic of the Nicotiana genus. Using the developed tool, expression profiles of the nicotine biosynthesis pathway genes were generated. The expression patterns of eight gene groups in the pathway were strongly expressed in the roots and weakly expressed in leaves and flowers of N. benthamiana. The results were consistent with the established gene expression profiles in Nicotiana tabacum and provided insights into gene family composition and expression trends. The compilation of this database tool can facilitate genetic analysis of N. benthamiana in the future. Significance statementA tool was developed to visualize gene expression profiles, draw molecular evolutionary phylogenetic trees of gene families, perform functional enrichment analyses, and facilitate output downloads of Nicotiana benthamiana. The database developed using the tool can evolve into a comprehensive all-in-one analysis platform by continuously incorporating transcriptome data released to date, newly released RNA-seq data, and annotations in the future.

plant biology↗

bioRxiv2022

Nicotiana benthamiana XYLEM CYSTEINE PROTEASE genes facilitate tracheary element formation in interfamily grafting

Grafting is a plant propagation technique widely used in agriculture. A recent discovery of the capability of interfamily grafting in Nicotiana has expanded the potential combinations of grafting. In this study, we showed that xylem connection is essential for the achievement of interfamily grafting and investigated the molecular basis of xylem formation at the graft junction. Transcriptome and gene network analyses revealed gene modules for tracheary element (TE) formation during grafting that include genes associated with xylem cell differentiation and immune response. The reliability of the drawn network was validated by examining the role of the Nicotiana benthamiana XYLEM CYSTEINE PROTEASE (NbXCP) genes in TE formation during interfamily grafting. Promoter activities of NbXCP1 and NbXCP2 genes were found in differentiating TE cells in the stem and callus tissues at the graft junction. Analysis of a Nbxcp1;Nbxcp2 loss-of-function mutant indicated that NbXCPs control the timing of de novo TE formation at the graft junction. Moreover, grafts of the NbXCP1 overexpressor increased the scion growth rate as well as the fruit size. Thus, we identified gene modules for TE formation at the graft boundary and demonstrated potential ways to enhance Nicotiana interfamily grafting.

plant biology↗

bioRxiv2021

Quantitative Analysis of Plasmodesmata Permeability using Cultured Tobacco BY-2 Cells Entrapped in Microfluidic Chips

Plasmodesmata are unique channel structures in plants that link the fluid cytoplasm between adjacent cells. Plants have evolved these microchannels to allow trafficking of nutritious substances as well as signaling molecules for intercellular communication. However, tracking the behavior of plasmodesmata in real time is difficult because they are located inside tissues. Hence, we developed a microfluidic device that traps cultured cells and fixes their positions to allow testing of plasmodesmata permeability. The device has 112 tandemly aligned trap zones in the flow channel. Cells of the tobacco line BY-2 were cultured for 7 days and filtered using a sieve and a cell strainer before use to isolate short cell clusters consisting of only a few cells. The isolated cells were introduced into the flow channel, resulting in entrapment of cell clusters at 25 out of 112 trap zones (22.3%). Plasmodesmata permeability was tested from 1 to 4 days after trapping the cells. During this period, the cell numbers increased through cell division. Fluorescence recovery after photobleaching experiments using a transgenic marker line expressing nuclear-localized H2B-GFP demonstrated that cell-to-cell movement of H2B-GFP protein occurred within 200 min of photobleaching. The transport of H2B-GFP protein was not observed when sodium chloride, a compound known to cause plasmodesmata closure, was present in the microfluid channel. Thus, this microfluidic device and one-dimensional plant cell samples allowed us to observe plasmodesmata behavior in real time under controllable conditions.

plant biology↗

bioRxiv2020

An in vitro grafting method to quantify mechanical forces of adhering tissues

AbstractsGrafting is an indispensable agricultural technology for propagating useful tree varieties and obtaining beneficial traits of two varieties/species--as stock and scion--at the same time. Recent studies of molecular events during grafting have revealed dynamic physiological and transcriptomic changes. Strategies focused on specific grafting steps are needed to further associate each physiological and molecular event with those steps. In this study, we developed a method to investigate the tissue adhesion event, an early grafting step, by improving an artificial in vitro grafting system in which two pieces of 1.5-mm thick Nicotiana benthamiana cut stem sections were combined and cultured on medium. We prepared a silicone sheet containing five special cutouts for adhesion of cut stem slices. We quantitatively measured the adhesive force at these grafting interfaces using a force gauge and found that graft adhesion started 2 days after grafting, with the adhesive force gradually increasing over time. After confirming the positive effect of auxin on grafting by this method, we tested the effect of cellulase treatment and observed significant enhancement of graft tissue adhesion. Compared with the addition of auxin or cellulase individually, the adhesive force was stronger when both auxin and cellulase were added simultaneously. The in vitro grafting method developed in this study is thus useful for examining the process of graft adhesion.

plant biology↗

bioRxiv2020

Host-parasite tissue adhesion by a secreted type of β-1,4-glucanase in the parasitic plant Phtheirospermum japonicum

Tissue adhesion between plant species occurs both naturally and artificially. Parasitic plants establish symbiotic relationship with host plants by adhering tissues at roots or stems. Plant grafting, on the other hand, is a widely used technique in agriculture to adhere tissues of two stems. While compatibility of tissue adhesion in plant grafting is often limited within close relatives, parasitic plants exhibit much wider compatibilities. For example, the Orobanchaceae parasitic plant Striga hermonthica is able to infect Poaceae crop plants, causing a serious agricultural loss. Here we found that the model Orobanchaceae parasite plant Phtheirospermum japonicum can be grafted on to interfamily species, such as Arabidopsis, a Brassicaceae plant. To understand molecular basis of tissue adhesion between distant plant species, we conducted comparative transcriptome analyses on both infection and grafting by P. japonicum on Arabidopsis. Through gene clustering, we identified genes upregulated during these tissue adhesion processes, which include cell proliferation- and cell wall modification-related genes. By comparing with a transcriptome dataset of interfamily grafting between Nicotiana and Arabidopsis, we identified 9 genes commonly induced in tissue adhesion between distant species. Among them, we showed a gene encoding secreted type of {beta}-1,4-glucanase plays an important role for plant parasitism. Our data provide insights into the molecular commonality between parasitism and grafting in plants. Significance StatementComprehensive sequential RNA-Seq datasets for parasitic infection of the root and grafting of the stem between P. japonicum and Arabidopsis revealed that molecular events of parasitism and grafting are substantially different and only share a part of events such as cell proliferation and cell wall modification. This study demonstrated that a secreted type of {beta}-1,4-glucanase gene expressed in cells located at the parasite-host interface as an important factor for parasitism in the Orobanchaceae.

plant biology↗

bioRxiv2020

Cell-cell adhesion in plant grafting is facilitated by β-1,4-glucanases

Plant grafting is conducted for vegetative propagation in plants, whereby a piece of living tissue is attached to another tissue through establishment of cell-cell adhesion. Plant grafting has a long history in agriculture and has been applied to improve crop traits for thousands of years1. Plant grafting has mostly relied on the natural ability of a plant for wound healing. However, the compatibility of cell-cell adhesion typically limits graft combinations to closely related species2-4, and the mechanism by which cell-cell adhesion of injured tissues is established is largely unknown. Here, we show that a subclade of {beta}-1,4-glucanases secreted into the extracellular region facilitates cell-cell adhesion near the graft interface. Nicotiana shows a propensity for cell-cell adhesion with a diverse range of angiosperms, including vegetables, fruit trees, and monocots, in which cell wall reconstruction was promoted in a similar manner to conventional intrafamily grafting5-7. Using transcriptomic approaches, we identified a specific clade of {beta}-1,4-glucanases that is upregulated during grafting in successful graft combinations but not in incompatible grafts and precedes graft adhesion in inter- and intrafamily grafts. Grafting was facilitated with an overexpressor of the {beta}-1,4-glucanase and, using Nicotiana stem as an interscion, we produced tomato fruits on rootstocks from other plant families. Our results demonstrate that the mechanism of cell-cell adhesion is partly conserved in plants and is a potential target to enhance plant grafting techniques.

plant biology↗

bioRxiv2020

Autophagy is induced during plant grafting for wound healing

O_LIGrafting is an important technique in agriculture to obtain several good traits such as high disease tolerance and high yield by exchanging root system. However, the underlined cellular processes to compensate the wound damage and repair tissues were largely unknown. C_LIO_LIWe analyzed two graft combinations: Nicotiana benthamiana (Nb) homograft as a compatible, wound repairing model and Nb heterograft with Arabidopsis thaliana (At) as an incompatible and more stressful model, which we recently identified as an exceptional maintainable interfamily grafting. C_LIO_LIIn both graft combinations, nutrient loss was observed in gene expression after grafting, where the level of nutrient loss was more sever in heterografts. Transmission electron micrographs of Nb/At heterografts suggested that microautophagy was induced in cells near the graft boundary. In At seedling micrografting, the fluorescence of autophagy protein marker GFP-ATG8 was highly observed at graft junction especially in cambial region. In At atg2 mutant homografts, growth after grafting decreased compared with wild-type homografts. Moreover, when NbATG5 knocked-down Nb scion was grafted to At stock, the successful rate of grafting was significantly decreased. C_LIO_LIAltogether, these results suggest that component of autophagy is induced during grafting and has a role in wound healing. C_LI

plant biology↗