Cloning and characterization of indole synthase (INS) and a putative tryptophan synthase α-subunit (TSA) genes from Polygonum tinctorium

• Published in: Plant cell reports 2016-12, Vol.35 (12), p.2449-2459
• Main Authors: Jin, Zhehao, Kim, Jin-Hee, Park, Sang Un, Kim, Soo-Un
• Format: Article
• Language: English
• Subjects: 5' Untranslated Regions - genetics; Amino Acid Sequence; Base Sequence; Biomedical and Life Sciences; Biotechnology; Cell Biology; Cloning, Molecular; Escherichia coli; Gene Expression Regulation, Plant - drug effects; Genes, Plant; Genetic Complementation Test; Green Fluorescent Proteins - metabolism; Indoles - chemistry; Indoles - metabolism; Life Sciences; Organ Specificity - drug effects; Organ Specificity - genetics; Original Article; Phylogeny; Plant Biochemistry; Plant Proteins - chemistry; Plant Proteins - genetics; Plant Proteins - metabolism; Plant Sciences; Polygonum - drug effects; Polygonum - enzymology; Polygonum - genetics; Protein Subunits - chemistry; Protein Subunits - genetics; Protein Subunits - metabolism; Protein Transport - drug effects; RNA, Messenger - genetics; RNA, Messenger - metabolism; Sequence Alignment; Subcellular Fractions - metabolism; Thiadiazoles - pharmacology; Tryptophan Synthase - chemistry; Tryptophan Synthase - genetics; Tryptophan Synthase - metabolism
• ISSN: 0721-7714; 1432-203X
• DOI: 10.1007/s00299-016-2046-3

Key message Two cDNAs for indole-3-glycerol phosphate lyase homolog were cloned from Polygonum tinctorium. One encoded cytosolic indole synthase possibly in indigoid synthesis, whereas the other encoded a putative tryptophan synthase α-subunit. Indigo is an old natural blue dye produced by plants such as Polygonum tinctorium . Key step in plant indigoid biosynthesis is production of indole by indole-3-glycerol phosphate lyase (IGL). Two tryptophan synthase α-subunit (TSA) homologs, PtIGL -short and -long, were isolated by RACE PCR from P. tinctorium . The genome of the plant contained two genes coding for IGL. The short and the long forms, respectively, encoded 273 and 316 amino acid residue-long proteins. The short form complemented E. coli Δ tnaA Δ trpA mutant on tryptophan-depleted agar plate signifying production of free indole, and thus was named indole synthase gene ( PtINS ). The long form, either intact or without the transit peptide sequence, did not complement the mutant and was tentatively named PtTSA . PtTSA was delivered into chloroplast as predicted by 42-residue-long targeting sequence, whereas PtINS was localized in cytosol. Genomic structure analysis suggested that a TSA duplicate acquired splicing sites during the course of evolution toward PtINS so that the targeting sequence-containing pre-mRNA segment was deleted as an intron. PtINS had about two to fivefolds higher transcript level than that of PtTSA , and treatment of 2,1,3-benzothiadiazole caused the relative transcript level of PtINS over PtTSA was significantly enhanced in the plant. The results indicate participation of PtINS in indigoid production.