Shintaro Inoue, Takahito Watanabe, Taiki Hamaguchi, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji and Taro Mito : Combinatorial expression of ebony and tan generates body color variation from nymph through adult stages in the cricket, Gryllus bimaculatus., PLoS ONE, Vol.18, No.5, 2023.
(Summary)
Insect body colors and patterns change markedly during development in some species as they adapt to their surroundings. The contribution of melanin and sclerotin pigments, both of which are synthesized from dopamine, to cuticle tanning has been well studied. Nevertheless, little is known about how insects alter their body color patterns. To investigate this mechanism, the cricket Gryllus bimaculatus, whose body color patterns change during postembryonic development, was used as a model in this study. We focused on the ebony and tan genes, which encode enzymes that catalyze the synthesis and degradation, respectively, of the precursor of yellow sclerotin N-β-alanyl dopamine (NBAD). Expression of the G. bimaculatus (Gb) ebony and tan transcripts tended to be elevated just after hatching and the molting period. We found that dynamic alterations in the combined expression levels of Gb'ebony and Gb'tan correlated with the body color transition from the nymphal stages to the adult. The body color of Gb'ebony knockout mutants generated by CRISPR/Cas9 systemically darkened. Meanwhile, Gb'tan knockout mutants displayed a yellow color in certain areas and stages. The phenotypes of the Gb'ebony and Gb'tan mutants probably result from an over-production of melanin and yellow sclerotin NBAD, respectively. Overall, stage-specific body color patterns in the postembryonic stages of the cricket are governed by the combinatorial expression of Gb'ebony and Gb'tan. Our findings provide insights into the mechanism by which insects evolve adaptive body coloration at each developmental stage.
Shintaro Inoue, Rihito Morita and Yoshiko Minami : An indigo-producing plant, Polygonum tinctorium, possesses a flavin-containing monooxygenase capable of oxidizing indole., Biochemical and Biophysical Research Communications, Vol.534, No.1, 199-205, 2020.
(Summary)
Polygonum tinctorium (P. tinctorium) is an indigo plant that is cultivated for a specific metabolite that it produces i.e., indoxyl β-D-glucoside (indican). In this study, flavin-containing monooxygenase (PtFMO) from P. tinctorium was cloned. When recombinant PtFMO was expressed in E. coli in the presence of tryptophan, indigo production was observed. Furthermore, we measured the activity of PtFMO using the membrane fraction from E. coli and found that it could produce indigo using indole as a substrate. The co-expression of PtFMO with indoxyl β-D-glucoside synthase (PtIGS), which catalyzes the glucosylation of indoxyl, brought about the formation of indican in E. coli. The results showed that indican was synthesized by sequential reactions of PtFMO and PtIGS. In three-week-old P. tinctorium specimens, the first leaves demonstrated higher levels of PtFMO expression than the subsequent leaves. This result coincided with that of our prior study on PtIGS expression level. Our study provides evidence that PtFMO might contribute to indican biosynthesis.
Shintaro Inoue, Rihito Morita, Keiko Kuwata, Kazuo Ishii and Yoshiko Minami : Detection of candidate proteins in the indican biosynthetic pathway of Persicaria tinctoria (Polygonum tinctorium) using protein-protein interactions and transcriptome analyses., Phytochemistry, Vol.179, 112507, 2020.
(Summary)
Persicaria tinctoria (Polygonum tinctorium) synthesizes indican (indoxyl-β-D-glucoside) as a specialized metabolite. Indican is synthesized in the cytosol of leaf cells from indoxyl and UDP-glucose by the catalysis of indoxyl-β-D-glucoside synthase (PtIGS), then transported into vacuoles. As a portion of PtIGS is found on the microsomal membrane, we assume that it is present on the ER membrane as a large complex involving other indican metabolism-related proteins. Based on this hypothesis, the existence of such a complex was investigated using two separate approaches: a protein-protein interaction assay and transcriptome analysis. We first performed a co-immunoprecipitation using the anti-PtIGS antibody and a pull-down assay using recombinant PtIGS, then identified the candidate proteins through MS/MS analysis. Secondly, we performed a transcriptome analysis to examine the differential gene expression between the first and the second leaves. The expressions of candidate genes detected by protein-protein interaction analyses were collated with transcriptome data and validated by quantitative reverse transcription polymerase chain reaction, showing that the expression of sucrose synthase and cytochrome P450 genes decreased in the second leaves compared with the first leaves. Furthermore, we detected several additional proteins, such as heat shock and cytoskeletal proteins, suggesting that PtIGS may form a large complex, a metabolon.
Shintaro Inoue, Rihito Morita, Keiko Kuwata, Tadashi Kunieda, Haruko Ueda, Ikuko Hara-Nishimura and Yoshiko Minami : Tissue-specific and intracellular localization of indican synthase from Polygonum tinctorium., Plant Physiology and Biochemistry : PPB, Vol.132, 138-144, 2018.
(Summary)
The plant Polygonum tinctorium produces the secondary metabolite indican (indoxyl-β-D-glucoside), a precursor of the blue dye indigo. P. tinctorium synthesizes indican through the actions of the UDP-glucosyltransferase (UGT), indican synthase. Herein, we partially purified an indican synthase from the leaves and subsequently performed peptide mass fingerprinting analysis. Consequently, we identified a fragment that was homologous to a UDP-glucosyltransferase 72B (UGT72B) family member. We named it PtIgs (P. tinctoriumindoxyl-β-D-glucoside synthase) and obtained the full-length cDNA using rapid amplification of the cDNA ends. The primary structure of PtIGS, which PtIgs encoded, showed high identity with indican synthases (ItUGT1 and ItUGT2) from Indigofera tinctoria (Inoue et al., 2017). Moreover, in expression analyses of P. tinctorium, PtIGS mRNA was virtually found only in the leaves, was most highly expressed in the 1st leaves, and decreased with leaf age. Because PtIGS expression tended to reflect indican contents and synthesis activities, we concluded that PtIGS functions as an indican synthase in plant cells. To examine intracellular localization of PtIGS, crude leaf extracts were separated into cytosol and microsome fractions, and found PtIGS in the cytosol and in microsome fractions. Furthermore, microsomal PtIGS was soluble in the presence of detergents and urea and was strongly associated with membranes. Finally, we confirmed endoplasmic reticulum (ER) membrane localization of PtIGS using ultracentrifugation with a sucrose density gradient. These data suggest that PtIGS interacts with some kind of proteins on ER membranes to certainly carry out a delivery of substrate.
Shintaro Inoue, Toshiki Moriya, Rihito Morita, Keiko Kuwata, T Sanjog Thul, K Bijaya Sarangi and Yoshiko Minami : Characterization of UDP-glucosyltransferase from Indigofera tinctoria., Plant Physiology and Biochemistry : PPB, Vol.121, 226-233, 2017.
(Summary)
Indican is a secondary metabolite in Indigofera tinctoria; its synthesis from indoxyl and UDP-glucose is catalyzed by a UDP-glucosyltransferase (UGT). In this study, we partially purified UGT extracted from I. tinctoria leaves and analyzed the protein by peptide mass fingerprinting. We identified two fragments that were homologous to UGT after comparison with the transcriptomic data of I. tinctoria leaves. The fragments were named itUgt1 and itUgt2 and were amplified using rapid amplification of cDNA ends polymerase chain reaction to obtain full-length cDNAs. The resultant nucleotide sequences of itUgt1 and itUgt2 encoded peptides of 477 and 475 amino acids, respectively. The primary structure of itUGT1 was 89% identical to that of itUGT2 and contained an important plant secondary product glycosyltransferase (PSPG) box sequence and a UGT motif. The recombinant proteins expressed in Escherichia coli were found to possess high indican synthesis activity. Although the properties of the two proteins itUGT1 and itUGT2 were very similar, itUGT2 was more stable at high temperatures than itUGT1. Expression levels of itUGT mRNA and protein in plant tissues were examined by UGT assay, immunoblotting, and semi-quantitative reverse transcription polymerase chain reaction. So far, we presume that itUGT1, but not itUGT2, primarily catalyzes indican synthesis in I. tinctoria leaves.
Shintaro Inoue, Takahito Watanabe, Hamaguchi Taiki, Fujie Kai, Shimamura Ayane, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji and Taro Mito : Artificial modification of cricket body color: breeding for the next-generation of protein supply, International Conference of Non-Traditional Arthropod Model Systems, Aug. 2023.
2.
Fujie Kai, Shintaro Inoue, Hamaguchi Taiki, Yoshiyasu Ishimaru, Katsuyuki Miyawaki, Takeshi Nikawa, Akira Takahashi, Sumihare Noji, Takahito Watanabe and Taro Mito : The discovery of two paralogous dopamine-synthase genes in the two-spotted cricket Gryllus bimaculatus, International Conference of Non-Traditional Arthropod Model Systems, Aug. 2023.
Proceeding of Domestic Conference:
1.
INOUE Taketo, Noriko Saito-Tarashima, Shintaro Inoue, Sumihare Noji, Taro Mito and Noriaki Minakawa : フタホシコオロギを用いたsiRNAのin vivo活性評価系の検討, 日本薬学会第144年会, Mar. 2024.
Shintaro Inoue, HAMAGUCHI Taiki, Yoshiyasu Ishimaru, Taro Mito and Takahito Watanabe : フタホシコオロギの体色関連遺伝子のノックアウト系統作製および表現型解析, 第66回日本応用動物昆虫学会大会, Mar. 2022.
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Hamaguchi Taiki, Shintaro Inoue, Takahito Watanabe, Yoshiyasu Ishimaru and Taro Mito : フタホシコオロギにおけるクチクラ形成と色素合成に関わる遺伝子の機能解析, The 44th Annual Meeting of the Molecular Biology Society of Japan, Dec. 2021.