A novel salt-tolerant L-myo-inositol-1-phosphate synthase from Porteresia coarctata (Roxb.) Tateoka, a halophytic wild rice: molecular cloning, bacterial overexpression, characterization, and functional introgression into tobacco-conferring salt tolerance phenotype.
l-myo-Inositol-1-phosphate synthase (EC 5.5.1.4, MIPS), an evolutionarily conserved enzyme protein, catalyzes the synthesis of inositol, which is implicated in a number of metabolic reactions in the biological kingdom. Here we report on the isolation of the gene (PINO1) for a novel salt-tolerant MIPS from the wild halophytic rice, Porteresia coarctata (Roxb.) Tateoka. Identity of the PINO1 gene was confirmed by functional complementation in a yeast inositol auxotrophic strain. Comparison of the nucleotide and deduced amino acid sequences of PINO1 with that of the homologous gene from Oryza sativa L. (RINO1) revealed distinct differences in a stretch of 37 amino acids, between amino acids 174 and 210. Purified bacterially expressed PINO1 protein demonstrated a salt-tolerant character in vitro compared with the salt-sensitive RINO1 protein as with those purified from the native source or an expressed salt-sensitive mutant PINO1 protein wherein amino acids 174-210 have been deleted. Analysis of the salt effect on oligomerization and tryptophan fluorescence of the RINO1 and PINO1 proteins revealed that the structure of PINO1 protein is stable toward salt environment. Furthermore, introgression of PINO1 rendered transgenic tobacco plants capable of growth in 200-300 mm NaCl with retention of approximately 40-80% of the photosynthetic competence with concomitant increased inositol production compared with unstressed control. MIPS protein isolated from PINO1 transgenics showed salt-tolerant property in vitro confirming functional expression in planta of the PINO1 gene. To our knowledge, this is the first report of a salt-tolerant MIPS from any source.
Majee
Manoj
M
Plant Molecular and Cellular Genetics, Bose Institute (Centenary Building), P-1/12, CIT Scheme VII M, Kolkata 700054, India.
Maitra
Susmita
S
Dastidar
Krishnarup Ghosh
KG
Pattnaik
Sitakanta
S
Chatterjee
Anirban
A
Hait
Nitai C
NC
Das
Kali Pada
KP
Majumder
Arun Lahiri
AL
D000595:Amino Acid Sequence; D000817:Anilino Naphthalenesulfonates / Q000494:pharmacology; D001483:Base Sequence; D015153:Blotting, Western; D002455:Cell Division; D002849:Chromatography, Gas; D003001:Cloning, Molecular; D004591:Electrophoresis, Polyacrylamide Gel; D017353:Gene Deletion; D005816:Genetic Complementation Test; D005822:Genetic Vectors; D007294:Inositol / Q000737:chemistry; D008969:Molecular Sequence Data; D009154:Mutation; D007296:Myo-Inositol-1-Phosphate Synthase / Q000737:chemistry / Q000494:pharmacology*; D012275:Oryza / Q000235:genetics*; D010641:Phenotype; D010788:Photosynthesis; D010944:Plants / Q000235:genetics; D030821:Plants, Genetically Modified; D006109:Poaceae / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry; D012441:Saccharomyces cerevisiae / Q000378:metabolism; D012492:Salts / Q000494:pharmacology*; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride / Q000494:pharmacology; D013050:Spectrometry, Fluorescence; D014026:Nicotiana / Q000235:genetics; D014364:Tryptophan / Q000737:chemistry
D000817:Anilino Naphthalenesulfonates; D011994:Recombinant Proteins; D012492:Salts; D012965:Sodium Chloride; D007294:Inositol; C035763:5,5'-bis(8-(phenylamino)-1-naphthalenesulfonate); D014364:Tryptophan; D007296:Myo-Inositol-1-Phosphate Synthase