Plant Biotechnology
Online ISSN : 1347-6114
Print ISSN : 1342-4580
ISSN-L : 1342-4580
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Original Papers
  • Zhongrui Duan, Kohji Ito, Motoki Tominaga
    2020 Volume 37 Issue 3 Pages 253-259
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: June 16, 2020
    JOURNALS FREE ACCESS

    Camelina sativa is a Brassicaceae oilseed plant used as a biotechnology platform for biofuel and healthy vegetable oil. As Camelina is closely related to the model plant Arabidopsis, the genetic tools of Arabidopsis are considered useful when applied to Camelina. Myosin XI-2 is one of the major motive forces driving cytoplasmic streaming in Arabidopsis. In our previous study, high-speed chimeric myosin XI-2, a myosin XI-2 artificially modified by genetically exchanging the motor domain of Arabidopsis myosin XI-2 with the faster Chara myosin XI, was shown to accelerate cytoplasmic streaming and promote plant growth in Arabidopsis. Here, we heterologously transformed this high-speed Chara-Arabidopsis chimeric myosin XI-2 gene in Camelina. The transgenic plants exhibited not only enhancement of leaf development and main stem elongation but also early flowering and seed setting, indicating that the high-speed chimeric myosin XI-2 can improve plant growth in Camelina. Interestingly, total seed yield was significantly increased in the transgenic plants as the total seed number increased. Our results suggest that the high-speed myosin XI system might also be effective to improve the growth of other closely related plant species.

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  • Mahjabin Kabir, Naruke Haruki, UmaMaheswari Rajagopalan, Mikihisa Umeh ...
    2020 Volume 37 Issue 3 Pages 261-271
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 31, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Statistical interferometric technique (SIT) is a highly sensitive, high speed non-contact, and non-destructive optical technique developed by our group capable of measuring instantaeoues sub-nanometer displacements. SIT applied to plant leaf elongation revealed nanometric intrinsic fluctuaitons (NIF) that are robust and sensitive to variations in the environment making NIF as a measure of healthiness of the plants. In this study, exogenous plant hormones, auxin (2,4-dichlorophenoxyacetic acid-2,4-D), and gibberellic acid (GA3), along with an auxin transport inhibitor 2,3,5-triiodobenzoic acid-TIBA, that affect plant growth were used to investigate their effects on NIF. Rice (Oriza sativa) seedlings were used, and their roots were exposed to 1, 2, and 4 µM 2,4-D, and the auxin transport inhibitor, TIBA, of 10, and 20 µM for 22 h and GA3 solution of different concentrations of 10, 40, and 100 µM for 5 h. Results showed significant increment in NIF for 1 µM and reduction for 4 µM 2,4-D while applicaiton of both 10, and 20 µM TIBA led to reduction in NIF. On the other hand, significant increment in NIF for 40 µM, and a significant reduction at a higher concentration of 100 µM for 5 hours of GA3 were also observed in comparison to those of control. Our results indicate that NIF as revealed by SIT could show both the positive and negative effects depending on the concentration of exogenous hormones, and transport inhibitors. Results suggest that SIT could be a valuable tool being sensitive enough to speedily assess the effects of plant growth hormones.

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  • Chiaki Hori, Xiang Yu, Jenny C. Mortimer, Ryosuke Sano, Tomoko Matsumo ...
    2020 Volume 37 Issue 3 Pages 273-283
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: August 14, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Growth of biomass for lignocellulosic biofuels and biomaterials may take place on land unsuitable for foods, meaning the biomass plants are exposed to increased abiotic stresses. Thus, the understanding how this affects biomass composition and quality is important for downstream bioprocessing. Here, we analyzed the effect of drought and salt stress on cell wall biosynthesis in young shoots and xylem tissues of Populus trichocarpa using transcriptomic and biochemical methods. Following exposure to abiotic stress, stem tissues reduced vessel sizes, and young shoots increased xylem formation. Compositional analyses revealed a reduction in the total amount of cell wall polysaccharides. In contrast, the total lignin amount was unchanged, while the ratio of S/G lignin was significantly decreased in young shoots. Consistent with these observations, transcriptome analyses show that the expression of a subset of cell wall-related genes is tightly regulated by drought and salt stresses. In particular, the expression of a part of genes encoding key enzymes for S-lignin biosynthesis, caffeic acid O-methyltransferase and ferulate 5-hydroxylase, was decreased, suggesting the lower S/G ratio could be partly attributed to the down-regulation of these genes. Together, our data identifies a transcriptional abiotic stress response strategy in poplar, which results in adaptive changes to the plant cell wall.

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  • Chihiro Suketomo, Tomohiko Kazama, Kinya Toriyama
    2020 Volume 37 Issue 3 Pages 285-292
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: September 17, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Cytoplasmic male sterility (CMS) is a trait that produces nonfunctional pollen caused by the interaction between mitochondrial and nuclear genes. In Chinese-wild (CW) type CMS, CWA, in rice (Oryza sativa L.), its mitochondria enhance the expression of the nuclear gene RETROGRADE-REGULATED MALE STERILITY (RMS), which causes pollen abortion. Fertility is recovered when its expression decreases in a restorer line, CWR. The expression of RMS is controlled by the single nucleotide polymorphism (SNP) located in the promoter region 2,286 bp upstream of the start codon of RMS. However, another gene, PPR2, which encodes pentatricopeptide repeat-domain containing protein, is predicted in the reverse strand of this region and a premature stop codon is created in CWR by the SNP. To prove RMS is directly involved in restoring fertility of CW-CMS, we introduced mutations into RMS and PPR2 using CRISPR/Cas9. Fertility was recovered in the genome-edited CMS plants with reduced expression of RMS and unaltered expression of PPR2, when the mutation was introduced in the promoter regions of RMS within or outside the coding sequence (CDS) of PPR2. Fertility restoration was not obtained when the mutation was introduced within the CDS of RMS. Our results demonstrated that PPR2 is not responsible for fertility restoration, and fertility was recovered by reduced expression of RMS, providing us with a new artificial fertility restorer line for agronomical use.

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  • Aki Yamada, Miku Kondo-Kaneko, Kan’ichiro Ishiuchi, Toshiaki Makino, K ...
    2020 Volume 37 Issue 3 Pages 293-299
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Anthraquinones are widely distributed in various organisms and known as bioactive ingredients. Some of the anthraquinones accumulate as glycosides in higher plants. Plant secondary product glycosyltransferases (PSPGs) are the well-characterized enzymes producing plant secondary metabolite glycosides. However, PSPGs involved in the formation of anthraquinone glycosides remains unclear. The rhizome of Rheum palmatum contains anthraquinones as laxative agents, some of which are accumulated as glucosides. We isolated a glucosyltransferase, R. palmatum UDP-glycosyltransferase (RpUGT) 1 from the rhizome of R. palmatum, and characterized functionally. RpUGT1 glucosylated emodin yielding emodin-6-O-glucoside, and it also glucosylated rhapontigenin, a compound belonging to stilbenes, yielding rhaponticin. The expression patterns of RpUGT1 and the accumulation of the metabolites revealed that RpUGT1 contributes to the production of these glucosides in R. palmatum. These results may provide important information for the substrate recognition of the PSPGs for anthraquinones and stilbenes.

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  • Kai Uchida, Toshio Aoki, Hideyuki Suzuki, Tomoyoshi Akashi
    2020 Volume 37 Issue 3 Pages 301-310
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Most leguminous plants produce (−)-type enantiomers of pterocarpans as the phytoalexin, but pea (Pisum sativum L.) produces the opposite stereoisomer of pterocarpan, (+)-pisatin. Biosynthesis of (−)-pterocarpan skeleton is completely characterized at the molecular level, and pterocarpan synthase (PTS), a dirigent (DIR) domain-containing protein, participates in the last dehydration reaction. Similarly, isoflav-3-ene, a precursor of (+)-pisatin, is likely to be biosynthesized by the DIR-mediated dehydration reaction; however the biosynthesis is still unknown. In the present study, we screened PTS homologs based on RNA-sequence data from (+)-pisatin-producing pea seedlings and demonstrated that one of the candidates encodes isoflav-3-ene synthase (I3S). Real-time PCR analysis revealed that transcripts of I3S, in addition to other genes involved in the (+)-pisatin pathway, transiently accumulated in pea upon elicitation prior to the maximum accumulation of (+)-pisatin. I3S orthologs were also found in soybean and Lotus japonicus that are not known to accumulate (+)-pterocarpan, and the catalytic function of gene products was verified to be I3S by the in vitro enzyme assay. Incubation of the crude extract of elicited soybean cells with isoflav-3-ene yielded coumestrol, suggesting that isoflav-3-ene is a precursor of coumestrol biosynthesis in soybean.

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  • Phookaew Pawittra, Takaomi Suzuki, Harunori Kawabe, Arika Takebayashi, ...
    2020 Volume 37 Issue 3 Pages 311-318
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: August 14, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    The plant-specific NAC transcription factor VASCULAR-RELATED NAC-DOMAIN 7 (VND7) functions in xylem vessel cell differentiation in Arabidopsis thaliana. To identify novel factors regulating xylem vessel cell differentiation, we previously performed ethyl methanesulfonate mutagenesis of a transgenic 35S::VND7-VP16-GR line in which VND7 activity can be induced post-translationally by glucocorticoid treatment. We successfully isolated mutants that fail to form ectopic xylem vessel cells named seiv (suppressor of ectopic vessel cell differentiation induced by VND7) mutants. Here, we isolated eight novel dominant seiv mutants: seiv2 to seiv9. In these seiv mutants, ectopic xylem vessel cell differentiation was inhibited in aboveground but not underground tissues. Specifically, the shoot apices of the mutants, containing shoot apical meristems and leaf primordia, completely lacked ectopic xylem vessel cells. In these mutants, the VND7-induced upregulation of downstream genes was reduced, especially in shoots compared to roots. However, endogenous xylem vessel cell formation was not affected in the seiv mutants. Therefore, the seiv mutations identified in this study have repressive effects on cell differentiation in shoot meristematic regions, resulting in inhibited ectopic xylem vessel cell differentiation.

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  • Kosuke Shimizu, Atsuhiko Kushida, Ryota Akiyama, Hyoung Jae Lee, Yuya ...
    2020 Volume 37 Issue 3 Pages 319-325
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: September 11, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Cyst nematodes (Globodera spp. and Heterodera spp.) are highly evolved sedentary endoparasites that are considered as harmful pests worldwide. The hatching of the dormant eggs of cyst nematodes occurs in response to hatching factors (HFs), which are compounds that are secreted from the roots of host plants. Solanoeclepin A (SEA), a triterpene compound, has been isolated as HF for potato cyst nematode (PCN) eggs, whereas other compounds, such as steroidal glycoalkaloids (SGAs), are also known to show weak hatching stimulation (HS) activity. However, the structures of both compounds are different and the HF-mediated hatching mechanism is still largely unknown. In the present study, we observed specific hatching of PCN eggs stimulated by the hairy root culture media of potato and tomato, revealing the biosynthesis and secretion of HFs. SGAs, such as α-solanine, α-chaconine, and α-tomatine, showed significant HS activity, despite being remarkably less activities than that of SEA. Then, we evaluated the contribution of SGAs on the HS activities of the hairy root culture media. The estimated SGAs content in the hairy root culture media were low and nonconcordant with the HS activity of those, suggesting that the HS activity of SGAs did not contribute much. The analysis of structure–activity relationship revealed that the structural requirements of the HS activity of SGAs are dependent on the sugar moieties attached at the C3-hydoroxyl group and the alkaloid property of their aglycones. The stereochemistry in the EF rings of their aglycone also affected the strength of the HS activity.

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  • Kumpei Shiragaki, Rie Nakamura, Shigeki Nomura, Hai He, Tetsuya Yamada ...
    2020 Volume 37 Issue 3 Pages 327-333
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: August 26, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Hybrid lethality observed in hybrid seedlings between Nicotiana suaveolens and N. tabacum is characterized by browning, initially of the hypocotyls and eventually of entire seedlings. We investigated the mechanism underlying this browning of tissues. A phenylalanine ammonia-lyase (PAL) gene codes an enzyme involved in a pathway producing phenolic compounds related to the browning of plant tissues. The expression of PAL rapidly increased with the induction of hybrid lethality. Phenolic compounds were observed to be accumulated in whole parts of hybrid seedlings. Treatment of hybrid seedlings with L-2-aminooxy-3-phenylpropionic acid (AOPP), an inhibitor for PAL, suppressed browning and decreased the phenolic content of hybrid seedlings. Although programmed cell death (PCD) was involved in hybrid lethality, AOPP treatment also suppressed cell death and enhanced the growth of hybrid seedlings. These results indicated that PAL is involved in hybrid lethality, and phenolic compounds could be the cause of hybrid lethality-associated tissue browning.

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Short Communication
  • Anung Wahyudi, Chikako Fukazawa, Reiko Motohashi
    2020 Volume 37 Issue 3 Pages 335-341
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS

    Lipocalins are very important proteins for stress resistance in plants. To better understand the function of tomato lipocalins, we observed responses to oxidative stress using over-expressed SlTIL1, SlTIL2, SlCHL, and silenced-plants. Significant differences in reactive oxygen species accumulation (oxidative damage) were observed in all tested plants under heat stress. Plants with over-expressed SlTIL1, SlTIL2, and SlCHL showed less oxidative damage compared with wild-type plants under heat stress. The expression of SlSODs was induced in over-expressed SlTIL1, SlTIL2, and SlCHL plants under normal and heat stress conditions. Furthermore, silenced PDS, SlTILs, and SlCHL plants showed slightly increasing oxidative damage under heat stress alongside with lower SlSODs under normal and stress conditions. These results suggest that SlTIL1, SlTIL2, and SlCHL were involved in antioxidant defense by eliminating ROS in tomato plants.

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Notes
  • Hidehiko Sunohara, Shingo Kaida, Shinichiro Sawa
    2020 Volume 37 Issue 3 Pages 343-347
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS

    The root-knot nematode (RKN) Meloidogyne incognita is one of the most economically damaging plant-parasitic nematodes. Molecular studies of the plant–RKN interaction have been vigorously carried out in dicotyledonous model plants, while the host range of M. incognita is wide including monocotyledonous plants. As M. incognita causes quality and yield losses in rice (Oryza sativa L.) cultivated in both upland and irrigated systems, we developed a method to examine the plant–RKN interaction in this model monocotyledonous crop plant. Here, we show that a transparent paper pouch could be used to evaluate nematode infection rates in rice with similar results to that of the traditional soil method. The system using a transparent paper pouch can be used to observe the spatial and temporal distribution of developing galls and can save the space of growth chamber.

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  • Mika Ohmagari, Yusuke Kono, Rumi Tominaga
    2020 Volume 37 Issue 3 Pages 349-352
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS

    Phosphate (Pi) starvation affects root hair formation to increase the absorptive surface area of the roots. CAPRICE (CPC) and its homolog genes, including TRIPTYCHON (TRY), ENHANCER OF TRY AND CPC1 (ETC1), ETC2, and ETC3, positively regulate root hair formation in a partially redundant manner. In particular, ETC1 responds to Pi deficiency. To clarify role sharing among the CPC homolog genes under Pi-deficient condition, we analyzed the expression of five CPC homolog genes under Pi-deficient condition, using the real-time polymerase chain reaction analysis. Pi starvation enhanced the expression of not only ETC1, but also ETC3. Furthermore, ETC3, which is rarely expressed in the roots, was induced by Pi deficiency. The expression levels of CPC, TRY, and ETC2 in response to Pi deficiency were not significantly different from those under the control conditions. These results suggest that CPC homologs can be divided into two groups, genes that respond to Pi deficiency (ETC1 and ETC3) and those that do not (CPC, TRY, and ETC2).

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  • Deepchandi Lekamge, Shin-ichi Yamamoto, Shuuichi Morohashi, Toshikazu ...
    2020 Volume 37 Issue 3 Pages 353-357
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: August 14, 2020
    JOURNALS FREE ACCESS

    Natural seed germination is difficult to achieve in numerous plant species of wide economic importance. The germination of Polygonatum macranthum seeds takes as long as one and a half years under natural conditions. In addition, propagation by rhizome is also extremely slow in this species. Therefore, the natural propagation of P. macranthum through seeds or rhizome is not efficient. In this study, an efficient in vitro propagation system for P. macranthum from immature seeds with seed coat was developed, using a new surface sterilization protocol that utilized a low concentration of hypochlorite. In vitro germination was achieved at a rate of 30% within 9 weeks after inoculation on 1/2 MS medium. Shoot explants from seedlings were successfully cultured on 1/2 MS medium. Supplementation of the 1/2 MS medium with cytokinin 6-benzylaminopurine (BAP) facilitated efficient propagation by microrhizome. An efficient propagation rate of 1.3 microrhizomes per shoot in an 8-week culture period could be achieved by using a concentration of 1 mg l−1 BAP. During 4 weeks of acclimatization, 88% of shoots were rooted and started to grow into juvenile plants. After about 16 weeks in the field, 13% of the acclimatized plants showed viable growth and healthy regenerating shoots. The cultivation system demonstrated in this study can be used to propagate P. macranthum.

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  • Maruya Suzuki, Ryota Numazaki, Tomomi Nakagawa, Naoto Shibuya, Hanae K ...
    2020 Volume 37 Issue 3 Pages 359-362
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS

    Receptor complex formation at the cell surface is a key step to initiate downstream signaling but the contribution of this process for the regulation of the direction of downstream responses is not well understood. In the plant-microbe interactions, while CERK1, an Arabidopsis LysM-RLK, mediates chitin-induced immune responses, NFR1, a Lotus homolog of CERK1, regulates the symbiotic process with rhizobial bacteria through the recognition of Nod factors. Concerning the mechanistic insight of the regulation of such apparently opposite biological responses by the structurally related RLKs, Nakagawa et al. previously showed that the addition of YAQ sequence, conserved in NFR1 and other symbiotic LysM-RLKs, to the kinase domain of CERK1 switched downstream responses from defense to symbiosis using a set of chimeric receptors, NFR1-CERK1s. These results indicated that such a subtle difference in the cytoplasmic domain of LysM-RLKs could determine the direction of host responses from defense to symbiosis. On the other hand, it is still not understood how such structural differences in the cytoplasmic domains determine the direction of host responses. We here analyzed the interaction between chimeric NFR1s and NFR5, a partner receptor of NFR1, by co-immunoprecipitation (Co-IP) of these proteins transiently expressed in Nicotiana benthamiana. These results indicated that the cytoplasmic interaction between the LysM-RLKs is important for the symbiotic receptor complex formation and the YAQ containing region of NFR1 contributes to trigger symbiotic signaling through the successful formation of NFR1/NFR5 complex.

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  • Akinori Kiba, Kotoko Fukui, Maki Mitani, Ivan Galis, Yuko Hojo, Tomono ...
    2020 Volume 37 Issue 3 Pages 363-367
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: July 30, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Phosphatidic acid plays an important role in plant immune responses against phytopathogenic bacteria in Nicotiana benthamiana. Here we focused on phosphoinositide dependent protein kinases (PDKs) as a candidate required for phosphatidic acid signaling. Based on Arabidopsis PDK sequences, we identified four putative PDK orthologs in N. benthamiana genome. To address the role of PDKs in plant defense responses, we created all four NbPDKs-silenced plants by virus-induced gene silencing. the NbPDKs-silenced plants showed a moderately reduced growth phenotype. Induction of hypersensitive cell death was compromised in the NbPDKs-silenced plants challenged with Ralstonia solanacearum. The hypersensitive cell death induced by bacterial effectors was also reduced in the NbPDKs-silenced plants. the NbPDKs-silenced plants showed decreased production of salicylic acid, jasmonic acid and jasmonoyl-L-isoleucine, as well as hydrogen peroxide after inoculation with R. solanacearum. These results suggest that NbPDKs might have an important role in the regulation of the hypersensitive cell death via plant hormone signaling and oxidative burst.

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  • Daiki Nishioka, Hiroharu Banno
    2020 Volume 37 Issue 3 Pages 369-372
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: August 14, 2020
    JOURNALS FREE ACCESS

    Ligation-independent cloning (LIC), such as Gibson Assembly, tends to produce clones without an insert, depending on the sequences present at the ends of linearized vectors. We used a nicking enzyme-mediated LIC (NE-LIC) method to construct a cDNA library in a binary vector pER8. Prior to constructing the cDNA library, pilot experiments were carried out, in which the GUS coding sequence was cloned into pER8 using NE-LIC. Approximately 12% of input vector DNAs were converted to plasmids carrying a GUS insert, and no plasmids without an insert were detected, indicating that this strategy is highly effective for cloning with the binary vector pER8. Therefore, NE-LIC was adopted to construct a cDNA library in pER8, by using cDNA that was PCR-amplified from a library constructed in another vector. As a result, a cDNA library in pER8 was successfully constructed. During library construction, it is important to exclude plasmids without an insert, since contamination from plasmids without inserts decreases the efficiency of screening. Therefore, NE-LIC is useful for the construction of cDNA libraries.

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  • Ryutaro Aida, Katsutomo Sasaki, Satoshi Yoshioka, Naonobu Noda
    2020 Volume 37 Issue 3 Pages 373-375
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: September 17, 2020
    JOURNALS FREE ACCESS

    The shoot apical meristem (SAM) is typically divided into three cell layers: the outermost epidermal layer (L1), the subepidermal layer (L2) and the inner corpus region (L3). Structures within the cell layers are normally maintained throughout development; however, through vegetative propagation of a periclinal chimeric chrysanthemum expressing a fluorescent protein gene only in the L1 layer, we collected twelve independent shoots that had partially mosaic fluorescent inner cells (L2, L3) in addition to fluorescent epidermal cells (L1). Furthermore, the elongated tissues of nine shoots out of the twelve had no internal fluorescent cells, i.e., they had the original L1 chimerism. Observations of the fluorescence distribution suggested that the change in chimerism occurred at the nodes, indicating previously unnoticed cell layer dynamics occurring at the nodes.

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  • Yasuhide Hiraga, Takeshi Ara, Yoshiki Nagashima, Norimoto Shimada, Noz ...
    2020 Volume 37 Issue 3 Pages 377-381
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: August 14, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    The model land plant Physcomitrella patens synthesizes flavonoids which may act as protectant of ultraviolet-B radiation. We aimed to uncover its flavonoid profile, for which metabolome analysis using liquid chromatography coupled with Ion trap/Orbitrap mass spectrometry was performed. From the 80% methanol extracts, 661 valid peaks were detected. Prediction of the elemental compositions within a mass accuracy of 2 ppm indicated that 217 peaks had single elemental composition. A compound database search revealed 47 peaks to be annotated as secondary metabolites based on the compound database search. Comprehensive substituent search by ShiftedIonsFinder showed there were 13 peaks of potential flavonoid derivatives. Interestingly, a peak having m/z 287.0551, corresponding to that of luteolin, was detected, even though flavone synthase has never been identified in P. patens. Using P. patens labeled with stable isotopes (13C-, 15N-, 18O-, and 34S), we confirmed the elemental composition of the peak as C15H10O6. By a comparison of MS/MS spectra with that of authentic standard, the peak was identified as luteolin or related flavone isomers. This is the first report of luteolin or related flavones synthesis and the possibility of the existence of an unknown enzyme with flavone synthase activity in P. patens.

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  • Takeshi Ara, Kunihiro Suda, Masayuki Amagai, Kiyoshi Namai, Hideyuki S ...
    2020 Volume 37 Issue 3 Pages 383-387
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: September 17, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Metabolome analysis of flavored vegetables, green spring onion (Allium fistulosum), Chinese chive (A. tuberosum), and their interspecies hybrid Negi-Nira chive, was conducted using liquid chromatography-Fourier transform ion cyclotron resonance-mass spectrometry, with ca. 2 ppm mass accuracy. Ion peaks in the chromatograms of four biological replicates of the vegetable leaves were processed using the alignment software PowerGet for metabolite comparison, from which we obtained the potential chemical formulae. In total, 860 ion peaks were reproducibly detected; of these, 506, 525, and 336 peaks were found in the hybrid, A. tuberosum, and A. fistulosum, respectively. There were 130 peaks specific to the hybrid; from these, 31 metabolites were annotated by searching compound databases. The sulfur-containing compounds and flavonoids were further analyzed using bioinformatics, to examine the sulfur metabolism of Allium volatiles and the flavonoid pathways in these species. In conclusion, our metabolome analysis of this interspecies hybrid and its parents provides a unique opportunity to elucidate their metabolic background.

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  • Takao Koeduka, Hideyuki Suzuki, Goro Taguchi, Kenji Matsui
    2020 Volume 37 Issue 3 Pages 389-392
    Published: September 25, 2020
    Released: September 25, 2020
    [Advance publication] Released: September 05, 2020
    JOURNALS FREE ACCESS
    Supplementary material

    Methyl jasmonate and jasmonic acid play important roles as signaling molecules in regulating plant development and stress-related responses. Previous studies have shown that jasmonic acid carboxyl methyltransferase (JMT), which belongs to the SABATH methyltransferase gene family, catalyzes the transfer of methyl groups from S-adenosyl-L-methionine to the carboxyl groups of jasmonic acid. In the present study, we used RNA-seq analysis to identify a putative JMT gene, EujJMT, in wasabi (Eutrema japonicum). The EujJMT proteins showed the highest similarity (89% identity) to JMT proteins of Brassica rapa. Functional characterization of a recombinant EujJMT protein expressed in Escherichia coli showed the highest level of activity with jasmonic acid among the different carboxylic acids tested. The apparent Km value of EujJMT using jasmonic acid as substrate was 62.6 µM, which is comparable to the values of known JMTs. Phylogenetic analysis suggested that EujJMT shares a common ancestor with the JMTs of Arabidopsis and Brassica species and that the strict substrate specificity toward jasmonic acid is conserved among Brassicaceae JMTs.

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