Cloning, characterization and expression of a chloroplastic fructose-1,6-bisphosphatase from Porteresia coarctata conferring salt-tolerance in transgenic tobacco

A gene coding for the chloroplastic fructose 1,6-bisphosphatase ( PcCFR ) in Porteresia coarctata Tateoka (Roxb.), a halophytic wild rice, has been isolated along with its rice ( Oryza sativa ; var. indica ) homologue ( OsCFR ), cloned and sequenced. Comparison between the nucleotide and deduced amino acid sequences of these two revealed a difference in five amino acid residues, namely Glu 14 , Thr 24 , Ala 48 , Ala 163 and Arg 296 in OsCFR which have been found to be replaced by Ser 14 , Ile 24 , Ser 48 , Ser 163 and Lys 296 in PcCFR respectively. The purified recombinant PcCFR is found to retain its enzymatic activity in presence of up to 500 mM NaCl in vitro as opposed to OsCFR, which is inactivated even at lower salt concentration. The six in vitro point mutant proteins of PcCFR showed varied degree of sensitivity towards high salt, with the maximum OsCFR-like effect in the triple mutant S 14 A-S 48 A-S 163 A suggesting a possible concerted role of all three serine residues in the in vitro salt tolerance property of PcCFR protein. Transgenic tobacco plants with chloroplast targeted PcCFR and OsCFR gene(s) have been developed under constitutive expression of CaMV 35S promoter and NOS terminator. The PcCFR transgenics showed better plant growth during exposure to salt stress in comparison to either the OsCFR or the empty vector transformed plants. The PcCFR transgenics also revealed enhanced photosynthetic efficiency coupled with protection to both photodamage of PSII and chlorophyll degradation through better reactive oxygen species scavenging at higher concentration of NaCl during late salt-stress growth.