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Article ; Online: Growth control of Marchantia polymorpha gemmae using nonthermal plasma irradiation.

Tsuboyama, Shoko / Okumura, Takamasa / Attri, Pankaj / Koga, Kazunori / Shiratani, Masaharu / Kuchitsu, Kazuyuki

2024 Volume 14, Issue 1, Page(s) 3172

Abstract: Several studies have documented that treatment by cold atmospheric pressure plasma (CAPP) on plants foster seed germination and growth in recent years. However, the molecular processes that underlie the action of CAPP on the seeds and plants remain ... ...

Abstract	Several studies have documented that treatment by cold atmospheric pressure plasma (CAPP) on plants foster seed germination and growth in recent years. However, the molecular processes that underlie the action of CAPP on the seeds and plants remain mostly enigmatic. We here introduce gemmae of Marchantia polymorpha, a basal liverwort, as a novel model plant material suitable for CAPP research. Treating the gemmae with CAPP for a constant time interval at low power resulted in consistent growth enhancement, while growth inhibition at higher power in a dose-dependent manner. These results distinctly demonstrate that CAPP irradiation can positively and negatively regulate plant growth depending on the plasma intensity of irradiation, offering a suitable experimental system for understanding the molecular mechanisms underlying the action of CAPP in plants.
MeSH term(s)	Marchantia ; Plant Development
Language	English
Publishing date	2024-02-07
Publishing country	England
Document type	Journal Article
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-024-53104-1
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Article ; Online: Enhanced Ca2+ binding to EF‐hands through phosphorylation of conserved serine residues activates MpRBOHB and chitin‐triggered ROS production

Hashimoto, Takafumi / Hashimoto, Kenji / Shindo, Hiroki / Tsuboyama, Shoko / Miyakawa, Takuya / Tanokura, Masaru / Kuchitsu, Kazuyuki

Physiologia Plantarum. 2023 , v. 175, no. 6 p.e14101-

2023

Abstract: NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca²⁺ ... ...

Abstract	NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca²⁺ binding and phosphorylation by various protein kinases, a mechanism linking Ca²⁺ binding and phosphorylation in the activity regulation remained elusive. Chitin‐triggered ROS production required cytosolic Ca²⁺ elevation and Ca²⁺ binding to MpRBOHB in a liverwort Marchantia polymorpha. Heterologous expression analysis of truncated variants revealed that a segment of the N‐terminal cytosolic region highly conserved among land plant RBOHs encompassing the two EF‐hand motifs is essential for the activation of MpRBOHB. Within the conserved regulatory domain, we have identified two Ser residues whose phosphorylation is critical for the activation in planta. Isothermal titration calorimetry analyses revealed that phosphorylation of the two Ser residues increased the Ca²⁺ binding affinity of MpRBOHB, while Ca²⁺ binding is indispensable for the activation, even if the two Ser residues are phosphorylated. Our findings shed light on a mechanism through which phosphorylation potentiates the Ca²⁺‐dependent activation of MpRBOHB, emphasizing the pivotal role of Ca²⁺ binding in mediating the Ca²⁺ and phosphorylation‐driven activation of MpRBOHB, which is likely to represent a fundamental mechanism conserved among land plant RBOHs.
Keywords	Marchantia polymorpha ; calcium ; calorimetry ; heterologous gene expression ; mosses and liverworts ; oxidoreductases ; phosphorylation ; plant development ; protein kinases ; serine ; signal transduction ; titration
Language	English
Dates of publication	2023-11
Publishing place	Blackwell Publishing Ltd
Document type	Article ; Online
Note	JOURNAL ARTICLE
ZDB-ID	2020837-6
ISSN	1399-3054 ; 0031-9317
ISSN (online)	1399-3054
ISSN	0031-9317
DOI	10.1111/ppl.14101
Database	NAL-Catalogue (AGRICOLA)

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Article: Novel in silico screening system for plant defense activators using deep learning-based prediction of reactive oxygen species accumulation.

Kogoshi, Masayuki / Nishio, Daiki / Kitahata, Nobutaka / Ohwada, Hayato / Kuchitsu, Kazuyuki / Mizuno, Hideyuki / Kurusu, Takamitsu

Plant methods

2023 Volume 19, Issue 1, Page(s) 142

Abstract: Background: Plant defense activators offer advantages over pesticides by avoiding the emergence of drug-resistant pathogens. However, only a limited number of compounds have been reported. Reactive oxygen species (ROS) act as not only antimicrobial ... ...

Abstract	Background: Plant defense activators offer advantages over pesticides by avoiding the emergence of drug-resistant pathogens. However, only a limited number of compounds have been reported. Reactive oxygen species (ROS) act as not only antimicrobial agents but also signaling molecules that trigger immune responses. They also affect various cellular processes, highlighting the potential ROS modulators as plant defense activators. Establishing a high-throughput screening system for ROS modulators holds great promise for identifying lead chemical compounds with novel modes of action (MoAs). Results: We established a novel in silico screening system for plant defense activators using deep learning-based predictions of ROS accumulation combined with the chemical properties of the compounds as explanatory variables. Our screening strategy comprised four phases: (1) development of a ROS inference system based on a deep neural network that combines ROS production data in plant cells and multidimensional chemical features of chemical compounds; (2) in silico extensive-scale screening of seven million commercially available compounds using the ROS inference model; (3) secondary screening by visualization of the chemical space of compounds using the generative topographic mapping; and (4) confirmation and validation of the identified compounds as potential ROS modulators within plant cells. We further characterized the effects of selected chemical compounds on plant cells using molecular biology methods, including pathogenic signal-triggered enzymatic ROS induction and programmed cell death as immune responses. Our results indicate that deep learning-based screening systems can rapidly and effectively identify potential immune signal-inducible ROS modulators with distinct chemical characteristics compared with the actual ROS measurement system in plant cells. Conclusions: We developed a model system capable of inferring a diverse range of ROS activity control agents that activate immune responses through the assimilation of chemical features of candidate pesticide compounds. By employing this system in the prescreening phase of actual ROS measurement in plant cells, we anticipate enhanced efficiency and reduced pesticide discovery costs. The in-silico screening methods for identifying plant ROS modulators hold the potential to facilitate the development of diverse plant defense activators with novel MoAs.
Language	English
Publishing date	2023-12-08
Publishing country	England
Document type	Journal Article
ZDB-ID	2203723-8
ISSN	1746-4811
ISSN	1746-4811
DOI	10.1186/s13007-023-01118-7
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Article ; Online: Rapid propagation of Ca2+ waves and electrical signals in a liverwort Marchantia polymorpha.

Watanabe, Kenshiro / Hashimoto, Kenji / Hasegawa, Kota / Shindo, Hiroki / Tsuruda, Yushin / Kupisz, Kamila / Koselski, Mateusz / Wasko, Piotr / Trebacz, Kazimierz / Kuchitsu, Kazuyuki

Plant & cell physiology

2024

Abstract: In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying ... ...

Abstract	In response to both biotic and abiotic stresses, vascular plants transmit long-distance Ca2+ and electrical signals from localized stress sites to distant tissues through their vasculature. Various models have been proposed for the mechanisms underlying the long-distance signaling, primarily centered around the presence of vascular bundles. We here demonstrate that the non-vascular liverwort Marchantia polymorpha possesses a mechanism for propagating Ca2+ waves and electrical signals in response to wounding. The propagation velocity of these signals was approximately 1-2 mm/s, equivalent to that observed in vascular plants. Both Ca2+ waves and electrical signals were inhibited by La3+ as well as tetraethylammonium chloride, suggesting crucial importance of both Ca2+ channel(s) and K+ channel(s) in wound-induced membrane depolarization as well as the subsequent long-distance signal propagation. Simultaneous recordings of Ca2+ and electrical signals indicated a tight coupling between the dynamics of these two signaling modalities. Furthermore, molecular genetic studies revealed that a GLUTAMATE RECEPTOR-LIKE (GLR) channel plays a central role in the propagation of both Ca2+ waves and electrical signals. Conversely, none of the three two-pore channels (TPCs) were implicated in either signal propagation. These findings shed light on the evolutionary conservation of rapid long-distance Ca2+ wave and electrical signal propagation involving GLRs in land plants, even in the absence of vascular tissue.
Language	English
Publishing date	2024-01-09
Publishing country	Japan
Document type	Journal Article
ZDB-ID	208907-5
ISSN	1471-9053 ; 0032-0781
ISSN (online)	1471-9053
ISSN	0032-0781
DOI	10.1093/pcp/pcad159
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Article ; Online: Quantitative Analysis for ROS-Producing Activity and Regulation of Plant NADPH Oxidases in HEK293T Cells.

Kimura, Sachie / Kaya, Hidetaka / Hashimoto, Kenji / Wrzaczek, Michael / Kuchitsu, Kazuyuki

Methods in molecular biology (Clifton, N.J.)

2022 Volume 2526, Page(s) 107–122

Abstract: Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling ... ...

Abstract	Reactive oxygen species (ROS) produced by plant NADPH oxidases, respiratory burst oxidase homologs (RBOHs), play key roles in biotic and abiotic stress responses and development in plants. While properly controlled amounts of ROS function as signaling molecules, excessive accumulation of ROS can cause undesirable side effects due to their ability to oxidize DNA, lipids, and proteins. To limit the damaging consequences of unrestricted ROS accumulation, RBOH activity is tightly controlled by post-translational modifications (PTMs) and protein-protein interactions. In order to analyze these elaborate regulatory mechanisms, it is crucial to quantitatively assess the ROS-producing activity of RBOHs. Given the high endogenous ROS generation in plants, however, it can be challenging in plant cells to measure ROS production derived from specific RBOHs and to analyze the contribution of regulatory events for their activation and inactivation. Here we describe human embryonic kidney 293T (HEK293T) cells as a heterologous expression system and a useful tool to quantitatively monitor ROS production by RBOHs. This system permits the reconstitution of regulatory events to dissect the effects of Ca
MeSH term(s)	Gene Expression Regulation, Plant ; HEK293 Cells ; Humans ; Kidney/metabolism ; NADPH Oxidases/metabolism ; Plants/metabolism ; Reactive Oxygen Species/metabolism
Chemical Substances	Reactive Oxygen Species ; NADPH Oxidases (EC 1.6.3.-)
Language	English
Publishing date	2022-07-13
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-0716-2469-2_8
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Article ; Online: Diversity and characteristics of plant immunity-activating bacteria from Brassicaceae plants.

Kaneko, Hiroki / Miyata, Fuma / Kurokawa, Mari / Hashimoto, Kenji / Kuchitsu, Kazuyuki / Furuya, Toshiki

BMC microbiology

2023 Volume 23, Issue 1, Page(s) 175

Abstract: Background: Microorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity-activating microorganisms ...

Abstract	Background: Microorganisms that activate plant immune responses are useful for application as biocontrol agents in agriculture to minimize crop losses. The present study was conducted to identify and characterize plant immunity-activating microorganisms in Brassicaceae plants. Results: A total of 25 bacterial strains were isolated from the interior of a Brassicaceae plant, Raphanus sativus var. hortensis. Ten different genera of bacteria were identified: Pseudomonas, Leclercia, Enterobacter, Xanthomonas, Rhizobium, Agrobacterium, Pantoea, Rhodococcus, Microbacterium, and Plantibacter. The isolated strains were analyzed using a method to detect plant immunity-activating microorganisms that involves incubation of the microorganism with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses. In this method, cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells serves as a marker of immune activation. Among the 25 strains examined, 6 strains markedly enhanced cryptogein-induced ROS production in BY-2 cells. These 6 strains colonized the interior of Arabidopsis plants, and Pseudomonas sp. RS3R-1 and Rhodococcus sp. RS1R-6 selectively enhanced plant resistance to the bacterial pathogens Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovorum subsp. carotovorum NBRC 14082, respectively. In addition, Pseudomonas sp. RS1P-1 effectively enhanced resistance to both pathogens. We also comprehensively investigated the localization (i.e., cellular or extracellular) of the plant immunity-activating components produced by the bacteria derived from R. sativus var. hortensis and the components produced by previously isolated bacteria derived from another Brassicaceae plant species, Brassica rapa var. perviridis. Most gram-negative strains enhanced cryptogein-induced ROS production in BY-2 cells via the presence of cells themselves rather than via extracellular components, whereas many gram-positive strains enhanced ROS production via extracellular components. Comparative genomic analyses supported the hypothesis that the structure of lipopolysaccharides in the outer cell envelope plays an important role in the ROS-enhancing activity of gram-negative Pseudomonas strains. Conclusions: The assay method described here based on elicitor-induced ROS production in cultured plant cells enabled the discovery of novel plant immunity-activating bacteria from R. sativus var. hortensis. The results in this study also suggest that components involved in the ROS-enhancing activity of the bacteria may differ depending largely on genus and species.
MeSH term(s)	Brassicaceae ; Reactive Oxygen Species ; Arabidopsis ; Pseudomonas syringae/genetics ; Plant Immunity ; Plant Diseases/microbiology
Chemical Substances	Reactive Oxygen Species
Language	English
Publishing date	2023-07-05
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2041505-9
ISSN	1471-2180 ; 1471-2180
ISSN (online)	1471-2180
ISSN	1471-2180
DOI	10.1186/s12866-023-02920-y
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Article ; Online: Enhanced Ca

Hashimoto, Takafumi / Hashimoto, Kenji / Shindo, Hiroki / Tsuboyama, Shoko / Miyakawa, Takuya / Tanokura, Masaru / Kuchitsu, Kazuyuki

Physiologia plantarum

2023 Volume 175, Issue 6, Page(s) e14101

Abstract	NADPH oxidases/RBOHs catalyze apoplastic ROS production and act as key signaling nodes, integrating multiple signal transduction pathways regulating plant development and stress responses. Although RBOHs have been suggested to be activated by Ca
MeSH term(s)	Phosphorylation ; Reactive Oxygen Species/metabolism ; Serine/metabolism ; Chitin/metabolism ; NADPH Oxidases/chemistry ; NADPH Oxidases/metabolism
Chemical Substances	Reactive Oxygen Species ; Serine (452VLY9402) ; Chitin (1398-61-4) ; NADPH Oxidases (EC 1.6.3.-)
Language	English
Publishing date	2023-12-26
Publishing country	Denmark
Document type	Journal Article
ZDB-ID	2020837-6
ISSN	1399-3054 ; 0031-9317
ISSN (online)	1399-3054
ISSN	0031-9317
DOI	10.1111/ppl.14101
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Article ; Online: Cell Cycle-Dependence of Autophagic Activity and Inhibition of Autophagosome Formation at M Phase in Tobacco BY-2 Cells.

Hanamata, Shigeru / Kurusu, Takamitsu / Kuchitsu, Kazuyuki

International journal of molecular sciences

2020 Volume 21, Issue 23

Abstract: Autophagy is ubiquitous in eukaryotic cells and plays an essential role in stress adaptation and development by recycling nutrients and maintaining cellular homeostasis. However, the dynamics and regulatory mechanisms of autophagosome formation during ... ...

Abstract	Autophagy is ubiquitous in eukaryotic cells and plays an essential role in stress adaptation and development by recycling nutrients and maintaining cellular homeostasis. However, the dynamics and regulatory mechanisms of autophagosome formation during the cell cycle in plant cells remain poorly elucidated. We here analyzed the number of autophagosomes during cell cycle progression in synchronized tobacco BY-2 cells expressing YFP-NtATG8a as a marker for the autophagosomes. Autophagosomes were abundant in the G2 and G1 phases of interphase, though they were much less abundant in the M and S phases. Autophagosomes drastically decreased during the G2/M transition, and the CDK inhibitor roscovitine inhibited the G2/M transition and the decrease in autophagosomes. Autophagosomes were rapidly increased by a proteasome inhibitor, MG-132. MG-132-induced autophagosome formation was also markedly lower in the M phases than during interphase. These results indicate that the activity of autophagosome formation is differently regulated at each cell cycle stage, which is strongly suppressed during mitosis.
MeSH term(s)	Autophagosomes/metabolism ; Autophagy ; Biomarkers ; Cell Cycle ; Cell Division ; Cell Line ; Fluorescent Antibody Technique ; Plant Cells ; Nicotiana/physiology
Chemical Substances	Biomarkers
Language	English
Publishing date	2020-12-01
Publishing country	Switzerland
Document type	Journal Article
ZDB-ID	2019364-6
ISSN	1422-0067 ; 1422-0067 ; 1661-6596
ISSN (online)	1422-0067
ISSN	1422-0067 ; 1661-6596
DOI	10.3390/ijms21239166
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Article ; Online: An efficient direct screening system for microorganisms that activate plant immune responses based on plant-microbe interactions using cultured plant cells.

Kurokawa, Mari / Nakano, Masataka / Kitahata, Nobutaka / Kuchitsu, Kazuyuki / Furuya, Toshiki

Scientific reports

2021 Volume 11, Issue 1, Page(s) 7396

Abstract: Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using ... ...

Abstract	Microorganisms that activate plant immune responses have attracted considerable attention as potential biocontrol agents in agriculture because they could reduce agrochemical use. However, conventional methods to screen for such microorganisms using whole plants and pathogens are generally laborious and time consuming. Here, we describe a general strategy using cultured plant cells to identify microorganisms that activate plant defense responses based on plant-microbe interactions. Microbial cells were incubated with tobacco BY-2 cells, followed by treatment with cryptogein, a proteinaceous elicitor of tobacco immune responses secreted by an oomycete. Cryptogein-induced production of reactive oxygen species (ROS) in BY-2 cells served as a marker to evaluate the potential of microorganisms to activate plant defense responses. Twenty-nine bacterial strains isolated from the interior of Brassica rapa var. perviridis plants were screened, and 8 strains that enhanced cryptogein-induced ROS production in BY-2 cells were selected. Following application of these strains to the root tip of Arabidopsis seedlings, two strains, Delftia sp. BR1R-2 and Arthrobacter sp. BR2S-6, were found to induce whole-plant resistance to bacterial pathogens (Pseudomonas syringae pv. tomato DC3000 and Pectobacterium carotovora subsp. carotovora NBRC 14082). Pathogen-induced expression of plant defense-related genes (PR-1, PR-5, and PDF1.2) was enhanced by the pretreatment with strain BR1R-2. This cell-cell interaction-based platform is readily applicable to large-scale screening for microorganisms that enhance plant defense responses under various environmental conditions.
MeSH term(s)	Agriculture ; Arabidopsis ; Arabidopsis Proteins/genetics ; Brassica rapa/microbiology ; DNA, Ribosomal/metabolism ; Endophytes ; Fungal Proteins/metabolism ; Gene Expression Regulation, Plant/drug effects ; Genes, Plant/drug effects ; Immune System ; Phylogeny ; Plant Cells/metabolism ; Plant Diseases/microbiology ; Plant Immunity/drug effects ; Plant Leaves/metabolism ; Plants/immunology ; Plants/microbiology ; Pseudomonas syringae/genetics ; RNA, Ribosomal, 16S/metabolism ; Reactive Oxygen Species ; Seedlings/metabolism ; Nicotiana/microbiology
Chemical Substances	Arabidopsis Proteins ; DNA, Ribosomal ; Fungal Proteins ; RNA, Ribosomal, 16S ; Reactive Oxygen Species ; cryptogein protein, Phytophthora cryptogea
Language	English
Publishing date	2021-04-01
Publishing country	England
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ZDB-ID	2615211-3
ISSN	2045-2322 ; 2045-2322
ISSN (online)	2045-2322
ISSN	2045-2322
DOI	10.1038/s41598-021-86560-0
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Article ; Online: "Fusion" in fertilization: interdisciplinary collaboration among plant and animal scientists.

Yamato, Katsuyuki T / Kuchitsu, Kazuyuki

Journal of plant research

2017 Volume 130, Issue 3, Page(s) 419–421

MeSH term(s)	Animals ; Arabidopsis/physiology ; Chlamydomonas reinhardtii/physiology ; Fertilization ; Interdisciplinary Communication ; Male ; Ovum ; Plants ; Plasmodium berghei/physiology ; Seeds ; Spermatozoa ; Zygote
Language	English
Publishing date	2017-04-10
Publishing country	Japan
Document type	Journal Article
ZDB-ID	2077362-6
ISSN	1618-0860 ; 0918-9440
ISSN (online)	1618-0860
ISSN	0918-9440
DOI	10.1007/s10265-017-0937-1
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