著者

Ryota Akiyama Naoyuki Umemoto Masaharu Mizutani

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.185-191, 2023-09-25 (Released:2023-09-25)

参考文献数

38

被引用文献数

5

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Steroidal glycoalkaloids (SGAs) are specialized metabolites found in members of Solanum species, and are also known as toxic substances in Solanum food crops such as tomato (Solanum lycopersicum), potato (Solanum tuberosum), and eggplant (Solanum melongena). SGA biosynthesis can be divided into two main parts: formation of steroidal aglycones, which are derived from cholesterol, and glycosylation at the C-3 hydroxy group. This review focuses on recent studies that shed light on the complete process of the aglycone formation in SGA biosynthesis and structural diversification of SGAs by duplicated dioxygenases, as well as the development of non-toxic potatoes through genome editing using these findings.

著者

Masahiro Nishihara Akiko Hirabuchi Fumina Goto Aiko Watanabe Chiharu Yoshida Rie Washiashi Masashi Odashima Keiichirou Nemoto

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.40, no.3, pp.229-236, 2023-09-25 (Released:2023-09-25)

参考文献数

39

被引用文献数

3

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Japanese cultivated gentians are highly valued ornamental flowers in Japan, but the flower shape is mostly limited to the single-flower type, unlike other flowers such as roses and carnations. To overcome this limitation, we used the CRISPR/Cas9 genome editing system to increase double-flowered genetic resources in gentians. Our approach targeted an AGAMOUS (AG) floral homeotic gene (AG1), which is responsible for the natural mutation that causes double flowers in gentians. We designed two targets in exon 1 of AG1 for genome editing and found that 9 of 12 herbicide-resistant shoots had biallelic mutations in the target regions of AG1. These nine lines all produced double flowers, with stamens converted into petaloid organs, similar to the natural mutant. We also analyzed the off-target effects of AG2, which is homologous to AG1, and found that such effects occurred in gentian genome editing but with low frequency. Furthermore, we successfully produced transgene-free genome-edited plants (null segregants) by crossing with wild-type pollen. F1 seedlings were subjected to PCR analysis to determine whether foreign DNA sequences, two partial regions of the CaMV35S promoter and Cas9 gene, were present in the genome. As a result, foreign genes were segregated at a 1 : 1 ratio, indicating successful null segregant production. Using PCR analysis, we confirmed that four representative null segregants did not contain transfer DNA. In summary, our study demonstrates that the CRISPR/Cas9 system can efficiently produce double-flowered gentians, and null segregants can also be obtained. These genome-edited plants are valuable genetic resources for future gentian breeding programs.

著者

Kinuyo Yoneya Junji Takabayashi

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.31, no.5, pp.409-416, 2014-12-25 (Released:2015-02-27)

参考文献数

55

被引用文献数

5 42

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When exposed to herbivore-infested plant volatiles or volatiles from artificially damaged plants, intact plants enhance their defense against herbivores. This phenomenon is called plant-plant communication. Here, we outline studies on plant-plant communication from both ecological and plant physiological perspectives. Regarding the ecological perspective, we give an overview of studies showing that plant–plant communication affect direct and indirect defense levels of exposed plants, and herbivore performance on exposed plants. Cases of kin selection in plant–plant communications and intra-plant communication via airborne signals are also summarized. Regarding the plant physiological perspective, we give an overview of studies that showed specific responses of receiver plants to a volatile molecular species, to different configurations of a volatile molecular species and to blends of volatiles. Furthermore, we review the signaling pathways involved, priming, sensitivity, and how plants receive volatile compounds in plant–plant communications.

著者

Masaharu Kyo Momoko Hagiya Madoka Tada Akemi Matsura

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.22.1124a, (Released:2023-02-09)

参考文献数

9

---

A binary vector carrying two WUSCHEL-related homeobox (WOX) genes, WOX2 and WOX8, under the control of a chemical-inducible expression system, worked in the transformation in N. paniculata, a recalcitrant species of Nicotiana. The resulting transformants exhibited improved culture performance in regeneration from leaf segments and suspended cells. Multicellular masses generated from freely suspended cells showed a specific cell division pattern similar to that of somatic embryo, likely owing to the function of the two WOX genes.

著者

Ryota Inoue Naoto Nakamura Chie Matsumoto Hisabumi Takase Jiro Sekiya Rafael Prieto

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.39, no.4, pp.381-389, 2022-12-25 (Released:2022-12-25)

参考文献数

30

被引用文献数

1

---

Glutathione (GSH, γ-L-glutamyl-L-cysteinyl-glycine) has been implicated in a multitude of cellular functions, such as protection of cells against oxidative stress, detoxification of xenobiotics via degradation of GSH S-conjugates, and disease resistance. Glutathione also serves as a precursor of phytochelatins, and thereby plays an essential role in heavy metal detoxification. The Arabidopsis genome encodes three functional γ-glutamyltransferase genes (AtGGT1, AtGGT2, AtGGT4) and two phytochelatin synthase genes (AtPCS1, AtPCS2). The function of plant GGT has not yet been clearly defined, although it is thought to be involved in GSH and GSH S-conjugate catabolism. On the other hand, besides its role in heavy metal detoxification, PCS has also been involved in GSH S-conjugate catabolism. Herein we describe the HPLC characterization of GSH and GSH S-conjugate catabolism in Arabidopsis mutants deficient in GSH biosynthesis (pad2-1/gsh1), atggt and atpcs1 T-DNA insertion mutants, atggt pad2-1, atggt atpcs1 double mutants, and the atggt1 atggt4 atpcs1 triple mutant. The results of our HPLC analysis confirm that AtGGT and AtPCS play important roles in two different pathways related with GSH and GSH S-conjugate (GS-bimane) catabolism in Arabidopsis.

著者

Hyungjun Park Yosuke Narasako Tomoko Abe Hisato Kunitake Tomonari Hirano

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.39, no.3, pp.311-316, 2022-09-25 (Released:2022-09-25)

参考文献数

41

被引用文献数

1

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Sweet potato is a major root crop with nutritious tuberous roots. The mechanism of tuberous root development has not yet been adequately elucidated. Genetic resources are required to develop the molecular understanding of sweet potato. Heavy-ion beams were applied to hexaploid sweet potato for an increase in genetic variation, after which the comprehensive effects of heavy-ion beam irradiation were investigated. In vitro cultured shoots with an axillary bud of ‘Beniharuka’ were irradiated with Ar-ions at a dose of 1–5 Gy and C-ions at a dose of 5–20 Gy, and three irradiated lines were separated from each irradiated shoot. The shoot regeneration was inhibited at high doses of each ion irradiation. Ar-ion irradiation had an especially high biological effect on shoot regeneration. A total of 335 lines were obtained, consisting of 104 and 231 lines derived from Ar- and C-ion irradiation, respectively. The change in the DNA content of the lines was analyzed by flow cytometry to evaluate the irradiation-induced damage to the DNA. The two lines demonstrated significant differences in the DNA content and changes at the chromosome level. The screening for the morphological mutants was conducted in the field. Some irradiated lines showed inhibited or no tuberous root phenotype as mutant candidates. Additionally, the high-yield mutant candidates were dominated by Ar-ion irradiation. It was indicated that heavy-ion beam mutagenesis is effective in broadening the range of the phenotypes corresponding to tuberous root formation in hexaploid sweet potato.

著者

Emi Yumoto Naohisa Yanagihara Masashi Asahina

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.39, no.2, pp.199-204, 2022-06-25 (Released:2022-06-25)

参考文献数

18

被引用文献数

1

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L-3,4-dihydroxyphenylalanine (L-DOPA) is one of the important secondary metabolites of plants and has been used for various purposes, such as in clinical treatment for Parkinson’s disease and dopamine-responsive dystonia. In plants, L-DOPA is a precursor of many alkaloids, catecholamines, and melanin; the L-DOPA synthesis pathway is similar to that in mammals. L-DOPA acts as an allelochemical, has an important role in several biological processes, such as stress response and metabolism, in plants. L-DOPA is widely used in the clinical treatment as well as a dietary supplement or psychotropic drug, understanding of biosynthesis of L-DOPA in plant could lead to a stable supply of L-DOPA. This paper describes an improved method for simple and rapid quantification of L-DOPA content using liquid chromatography-tandem mass spectrometry. The standard quantitative methods for L-DOPA require multiple purification steps or relatively large amounts of plant material. In our improved method, quantification of L-DOPA was possible with extract of one–two pieces of cotyledon without any partitioning or column for purification. The endogenous L-DOPA (approximately 4,000 µg g−1 FW (fresh weight)) could be detected from the one pieces of cotyledon of the faba bean sprout using this method. This method was also effective for samples with low endogenous amounts of L-DOPA such as broccoli, Japanese white radish, pea, and red cabbage sprouts. Therefore, this improved method will allow to measurement of L-DOPA content easily and accurately from a small amount of plant tissue and contribute to understanding biosynthesis, catabolism, and transport of L-DOPA.

著者

Masato Nakamura Mamoru Nozaki Yuji Iwata Nozomu Koizumi Yasushi Sato

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.39, no.2, pp.129-138, 2022-06-25 (Released:2022-06-25)

参考文献数

48

被引用文献数

2

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Endoplasmic reticulum (ER) stress activates unfolded protein responses (UPRs), such as promoting protein folding under the control of specific gene expression. Our previous study showed that ER stress induced by ER stress inducers such as tunicamycin (Tm), an inhibitor of N-linked glycan synthesis, causes ectopic lignin deposition in Arabidopsis roots, but the relationship between UPR and ectopic lignin deposition remains unclear. The receptor-like kinase THESEUS1 (THE1) has been shown to sense cell wall damage (CWD) induced in Arabidopsis by cellulose synthase inhibitors such as isoxaben (ISO) and to activate ectopic lignin deposition. In this study, we assessed the involvement of THE1 in ectopic lignin deposition caused by the ER stress inducer Tm. The loss-of-function mutation of THE1, the1-3, suppressed Tm-induced root growth inhibition and ectopic lignin deposition, revealing that THE1 is involved in root growth defects and ectopic lignin deposition caused by ER stress. Similarly, ISO treatment induced ectopic lignin deposition as well as the expression of the UPR marker genes binding protein 3 (BiP3) and ER-localized DnaJ 3b (ERdj3b). Conversely, in the the1-3 mutant, ISO-induced ectopic lignin deposition and the expression of BiP3 and ERdj3b were suppressed. These results showed that THE1 is involved in not only root growth inhibition and ectopic lignin deposition caused by ER stress but also CWD-induced UPR.

著者

Ryo Nakabayashi Kei Hashimoto Tetsuya Mori Kiminori Toyooka Hiroshi Sudo Kazuki Saito

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.3, pp.311-315, 2021-09-25 (Released:2021-09-25)

参考文献数

14

被引用文献数

5

---

Spatial metabolomics uses imaging mass spectrometry (IMS) to localize metabolites within tissue section. Here, we performed matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resonance-IMS (MALDI-FTICR-IMS) to identify the localization of asparaptine A, a naturally occurring inhibitor of angiotensin-converting enzyme, in green spears of asparagus (Asparagus officinalis). Spatial metabolome data were acquired in an untargeted manner. Segmentation analysis using the data characterized tissue-type-dependent and independent distribution patterns in cross-sections of asparagus spears. Moreover, asparaptine A accumulated at high levels in developing lateral shoot tissues. Quantification of asparaptine A in lateral shoots using liquid chromatography-tandem mass spectrometry (LC-MS/MS) validated the IMS analysis. These results provide valuable information for understanding the function of asparaptine A in asparagus, and identify the lateral shoot as a potential region of interest for multiomics studies to examine gene-to-metabolite associations in the asparaptine A biosynthesis.

著者

Eri Kamon Chihiro Noda Takumi Higaki Taku Demura Misato Ohtani

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.21.0519a, (Released:2021-09-18)

参考文献数

52

被引用文献数

4

---

Secondary cell walls (SCWs) accumulate in specific cell types of vascular plants, notably xylem vessel cells. Previous work has shown that calcium ions (Ca2+) participate in xylem vessel cell differentiation, but whether they function in SCW deposition remains unclear. In this study, we examined the role of Ca2+ in SCW deposition during xylem vessel cell differentiation using Arabidopsis thaliana suspension-cultured cells carrying the VND7-inducible system, in which VND7 activity can be post-translationally upregulated to induce transdifferentiation into protoxylem-type vessel cells. We observed that extracellular Ca2+ concentration was a crucial determinant of differentiation, although it did not have consistent effects on the transcription of VND7-downstream genes as a whole. Increasing the Ca2+ concentration reduced differentiation but the cells could generate the spiral patterning of SCWs. Exposure to a calcium-channel inhibitor partly restored differentiation but resulted in abnormal branched and net-like SCW patterning. These data suggest that Ca2+ signaling participates in xylem vessel cell differentiation via post-transcriptional regulation of VND7-downstream events, such as patterning of SCW deposition.

著者

Na Yuan Chihiro Furumizu Baolong Zhang Shinichiro Sawa

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.137-143, 2021-03-25 (Released:2021-03-25)

参考文献数

37

被引用文献数

5

---

In plant-pathogen interactions, pathogens employ secreted molecules, known as effectors to overcome physical barriers, modulate plant immunity, and facilitate colonization. Among these diverse effectors, some are found to mimic the plant peptides, to target host’s peptide receptors, and intervene in the peptide-regulated defense pathways and/or plant development. To better understand how pathogens have co-evolved with their plant hosts in order to improve disease management, we explored the presence of plant peptide mimics in microbes by bioinformatic analysis. In total, 36 novel peptide mimics belong to five plant peptide families were detected in bacterial and fungal kingdoms. Among them, phytosulfokine homologues were widely distributed in 22 phytopathogens and one bacterium, thereby constituted the largest proportion of the identified mimics. The putative functional peptide region is well conserved between plant and microbes, while the existence of a putative signal peptide varies between species. Our findings will increase understanding of plant-pathogen interactions, and provide new ideas for future studies of pathogenic mechanisms and disease management.

著者

Takeshi Ara Nozomu Sakurai Hideyuki Suzuki Koh Aoki Kazuki Saito Daisuke Shibata

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.38, no.1, pp.167-171, 2021-03-25 (Released:2021-03-25)

参考文献数

15

被引用文献数

5

---

Depository of low-molecular-weight compounds or metabolites detected in various organisms in a non-targeted manner is indispensable for metabolomics research. Due to the diverse chemical compounds, various mass spectrometry (MS) setups with state-of-the-art technologies have been used. Over the past two decades, we have analyzed various biological samples by using gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, or capillary electrophoresis-mass spectrometry, and archived the datasets in the depository MassBase (http://webs2.kazusa.or.jp/massbase/). As the format of MS datasets depends on the MS setup used, we converted each raw binary dataset of the mass chromatogram to text file format, and thereafter, information of the chromatograph peak was extracted in the text file from the converted file. In total, the depository comprises 46,493 datasets, of which 38,750 belong to the plant species and 7,743 are authentic or mixed chemicals as well as other sources (microorganisms, animals, and foods), as on August 1, 2020. All files in the depository can be downloaded in bulk from the website. Mass chromatograms of 90 plant species obtained by LC-Fourier transform ion cyclotron resonance MS or Orbitrap MS, which detect the ionized molecules with high accuracy allowing speculation of chemical compositions, were converted to text files by the software PowerGet, and the chemical annotation of each peak was added. The processed datasets were deposited in the annotation database KomicMarket2 (http://webs2.kazusa.or.jp/km2/). The archives provide fundamental resources for comparative metabolomics and functional genomics, which may result in deeper understanding of living organisms.

著者

Akifumi Sugiyama Kazufumi Yazaki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.31, no.5, pp.431-443, 2014-12-25 (Released:2015-02-27)

参考文献数

120

被引用文献数

21 60

---

Flavonoids, one of the most-described group of plant “specialized metabolites”, consist of more than 10,000 structurally diverse compounds. Most flavonoids accumulate in plant vacuoles as glycosides, with some released by the roots into rhizospheres. These flavonoids are involved in biological communications with rhizobia, arbuscular mycorrhizal fungi, plant growth promoting rhizobacteria, pathogens, nematodes, and other plant species. Both aglycones and glycosides of flavonoids are found in root exudates and in soils. This review describes researches on the mechanisms of flavonoid secretion and the fate of flavonoids released into rhizospheres. This review also discusses the direction of future research that may elucidate the specific roles of flavonoids in biological communications in rhizospheres, enabling the utilization of flavonoid activities and functions in agricultural practice.

著者

Somnuk Bunsupa Kana Komastsu Ryo Nakabayashi Kazuki Saito Mami Yamazaki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.31, no.5, pp.511-518, 2014-12-25 (Released:2015-02-27)

参考文献数

31

被引用文献数

8 16

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Anabasine is an alkaloid found in a small number of Nicotiana species. The components of the anabasine biosynthetic pathway have yet to be identified. Here, we report the reinvestigation of biosynthetic pathways of anabasine and related tobacco alkaloids in genetically engineered cells. Hairy roots of N. tabacum harboring a lysine/ornithine decarboxylase gene from Lupinus angustifolius (La-L/ODC) were fed with labeled [ε-15N]- or [α-15N]-L-lysine. Relative to the unfed control, feeding of labeled 15N-L-lysine greatly enhanced anabasine levels 13.5-fold in La-L/ODC-expressing line compared to 5.3-fold in the control line, suggesting that both LDC activity and substrate supplied are important factors for the efficient production of anabasine. GUS-expressing line showed preferential incorporation of [ε-15N]-L-lysine into anabasine, indicating the main biosynthetic pathway of Δ1-piperideine intermediate in tobacco is asymmetrically processes. In contrast, the expression of La-L/ODC showed the symmetric labeling of 15N atom into anabasine, implying the occurrence of free cadaverine, which is produced by La-L/ODC enzyme, during the biosynthesis of Δ1-piperideine intermediate. No considerable incorporation of 15N into other tobacco alkaloids such as, nicotine, anatabine, and anatalline, was detected. Detailed analysis using ultra-high resolution mass spectrometry indicated that two 15N atoms were incorporated into anabasine in La-L/ODC-expressing lines after feeding [ε-15N]- or [α-15N]-L-lysine. Our results not only provide information insight into the biosynthesis of anabasine but also suggest an alternative route for the production of anabasine by genetic engineering.

著者

Shuhei Yasumoto Satoru Sawai Hyoung Jae Lee Masaharu Mizutani Kazuki Saito Naoyuki Umemoto Toshiya Muranaka

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.37, no.2, pp.205-211, 2020-06-25 (Released:2020-06-25)

参考文献数

24

被引用文献数

22

---

Genome editing using site-specific nucleases, such as transcription activator-like effector nucleases (TALENs) and clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9 (CRISPR-Cas9), is a powerful technology for crop breeding. For plant genome editing, the genome-editing reagents are usually expressed in plant cells from stably integrated transgenes within the genome. This requires crossing processes to remove foreign nucleotides from the genome to generate null segregants. However, in highly heterozygous plants such as potato, the progeny lines have different agronomic traits from the parent cultivar and do not necessarily become elite lines. Agrobacteria can transfer exogenous genes on T-DNA into plant cells. This has been used both to transform plants stably and to express the genes transiently in plant cells. Here, we infected potato, with Agrobacterium tumefaciens harboring TALEN-expression vector targeting sterol side chain reductase 2 (SSR2) gene and regenerated shoots without selection. We obtained regenerated lines with disrupted-SSR2 gene and without transgene of the TALEN gene, revealing that their disruption should be caused by transient gene expression. The strategy using transient gene expression by Agrobacterium that we call Agrobacterial mutagenesis, developed here should accelerate the use of genome-editing technology to modify heterozygous plant genomes.

著者

Tian Tian Tan Taku Demura Misato Ohtani

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.36, no.1, pp.1-6, 2019-03-25 (Released:2019-03-31)

参考文献数

45

被引用文献数

10

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Xylem is an essential conductive tissue in vascular plants, and secondary cell wall polymers found in xylem vessel elements, such as cellulose, hemicellulose, and lignin, are promising sustainable bioresources. Thus, understanding the molecular mechanisms underlying xylem vessel element differentiation is an important step towards increasing woody biomass and crop yields. Establishing in vitro induction systems, in which vessel element differentiation is induced by phytohormonal stimuli or by overexpression of specific transcription factors, has been vital to this research. In this review, we present an overview of these in vitro induction systems, and describe two recently developed in vitro induction systems, VISUAL (Vascular cell Induction culture System Using Arabidopsis Leaves) and the KDB system. Furthermore, we discuss the potentials and limitations of each of these new in vitro induction systems for advancing our understanding of the molecular mechanisms driving xylem vessel element differentiation.

著者

Ngoc-Ha Thi Tran Taichi Oguchi Etsuko Matsunaga Akiyoshi Kawaoka Kazuo N. Watanabe Akira Kikuchi

出版者

Japanese Society for Plant Cell and Molecular Biology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.18.0831a, (Released:2018-12-14)

参考文献数

33

被引用文献数

7

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Under the Japanese biosafety regulatory framework for transgenic plants, data for assessing a transgenic plant’s impact on biodiversity must be submitted in order to obtain approval for a confined field trial. We recently reported the development of four novel transgenic Eucalyptus camaldulensis clones expressing the bacterial choline oxidase A (codA) gene, i.e., codAH-1, codAH-2, codAN-1, and codAN-2, and evaluated their abiotic tolerance by semiconfined screen house trial cultivation. Here we evaluated the impacts of the transgenic E. camaldulensis clones on productivities of harmful substances from those clones to affect soil microorganisms and/or other plants in the environment. A comparison of the assessment data between the transgenic trees and non-transgenic comparators showed no significant difference in potential impacts on biodiversity. The results contribute to sound-science evidence ensuring substantial equivalence between transgenic and non-transgenic E. camaldulensis.

著者

Yuta Fujii Yutaka Kodama

出版者

日本植物細胞分子生物学会

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

vol.32, no.1, pp.81-87, 2015-03-25 (Released:2015-04-04)

参考文献数

31

被引用文献数

3 22

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Green fluorescent protein (GFP) was discovered from the jellyfish Aequorea victoria, and several improvements have been carried out to change its physicochemical properties. The resulting improved GFP variants have been used as reporter proteins for bioimaging techniques in various research fields including plant science. Almost all GFP variants were developed using Escherichia coli to improve fluorescence properties in mammalian cells, but the impact in other organisms such as plant cells remains to be determined. In this study, we performed comparative analysis of four improved GFP variants, GFP-S65T, eGFP, frGFP and sfGFP, with reference to the fluorescence intensity in Arabidopsis protoplasts, and found that sfGFP is the brightest. Using non-fluorescent fragments from the GFP variants, we also conducted bimolecular fluorescence complementation (BiFC) assays to find appropriate fragment pairs of GFP-based BiFC for visualization of protein–protein interactions in living plant cells. Our observations revealed that the brightest is the sfGFP-based BiFC. Further, as an evaluation method for the sfGFP-based BiFC, a BiFC competition assay was successfully completed for the first time in planta. The present study provides useful information for selection and improvement of the GFP molecule and its application to BiFC technology in plants.

著者

Hiroaki Kusano Ami Takeuchi Hiroaki Shimada

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.23.0611a, (Released:2023-09-07)

参考文献数

29

被引用文献数

3

---

Potato (Solanum tuberosum L.) has a tetraploid genome. To make a mutant lacking a specific gene function, it is necessary to introduce mutations into all four gene alleles. To achieve this goal, we developed a powerful genome editing tool, CRISPR/dMac3-Cas9, which installed the translation enhancer dMac3 that greatly increased the translation of the downstream open reading frame. The CRISPR/dMac3-Cas9 system employing three guide RNAs (gRNAs) greatly elevated the frequency of the generation rate of mutation. This system enabled to create the 4-allele mutants of granule-bound starch synthase (GBSS) and starch branching enzyme (SBE). These mutants indicated functionally defective features, suggesting that we succeeded in efficient genome editing of the potato tetraploid genome. Here, we show the effect of the number of gRNAs for efficient mutagenesis of the target gene using the mutants of the GBSS1 gene. CRISPR/dMac3-Cas9 employing three gRNA genes achieved a higher mutation efficiency than the CRISPR/dMac3-Cas9 with two gRNAs, suggesting being influenced by the dose effect of the number of gRNAs at the target region. The alleles of the SBE3 gene contained SNPs that caused sequence differences in the gRNAs but these gRNAs functioned efficiently. However, many rearrangement events and large deletions were induced. These results support the importance of accurate binding of gRNA to the target sequence, which may lead to a hint to avoid the unexpected mutation on the off-target sites.

著者

Ayako Nishizawa-Yokoi Seiichi Toki

出版者

Japanese Society for Plant Biotechnology

雑誌

Plant Biotechnology (ISSN:13424580)

巻号頁・発行日

pp.23.0525a, (Released:2023-08-28)

参考文献数

52

---

Transposons are mobile genetic elements that can move to a different position within a genome or between genomes. They have long been used as a tool for genetic engineering, including transgenesis, insertional mutagenesis, and marker excision, in a variety of organisms. The piggyBac transposon derived from the cabbage looper moth is one of the most promising transposon tools ever identified because piggyBac has the advantage that it can transpose without leaving a footprint at the excised site. Applying the piggyBac transposon to precise genome editing in plants, we have demonstrated efficient and precise piggyBac transposon excision from a transgene locus integrated into the rice genome. Furthermore, introduction of only desired point mutations into the target gene can be achieved by a combination of precise gene modification via homologous recombination-mediated gene targeting with subsequent marker excision from target loci using piggyBac transposition in rice. In addition, we have designed a piggyBac-mediated transgenesis system for the temporary expression of sequence-specific nucleases to eliminate the transgene from the host genome without leaving unnecessary sequences after the successful induction of targeted mutagenesis via sequence-specific nucleases for use in vegetatively propagated plants. In this review, we summarize our previous works and the future prospects of genetic engineering with piggyBac transposon.