Functional and molecular characterization of fluoride exporter (FEX) from rice and its constitutive overexpression in Nicotiana benthamiana to promote fluoride tolerance.

Abstract

Early induction of OsFEX was insufficient for fluoride adaptation in IR-64. Overexpression of OsFEX in yeast and Nicotiana benthamiana enhanced fluoride tolerance. The present study delineates the regulation of fluoride exporter (FEX) in the fluoride-sensitive rice cultivar, IR-64 and its efficacy in generating high fluoride tolerance in transgenic Nicotiana benthamiana. Gene and protein expression profiling revealed that OsFEX exhibited early induction during fluoride stress in the vegetative and reproductive tissues of IR-64, although the expression was suppressed upon prolonged stress treatment. Analysis of OsFEX promoter in transgenic N. benthamiana, using β-glucuronidase reporter assay confirmed its early inducible nature, since the reporter expression and activity peaked at 12h of NaF stress, after which it was lowered. OsFEX expression was up regulated in the presence of gibberellic acid (GA) and melatonin, while it was suppressed by abscisic acid (ABA). Complementation of ΔFEX1ΔFEX2 yeast mutants with OsFEX enabled high fluoride tolerance, thus validating the functional efficiency of the transgene. Bioassay of transgenic N. benthamiana lines, expressing OsFEX either under its own promoter or under CaMV35S promoter, established that constitutive overexpression, rather than early induction of OsFEX was essential and crucial for generating fluoride tolerance in the transgenics. Overall, the suppression of OsFEX in the later growth phases of stressed IR-64 due to enhanced ABA conservation and lowered synthesis of GA, as supported by the application of the respective phytohormone biosynthetic inhibitors, such as sodium tungstate and paclobutrazol, accounted for the fluoride-hyperaccumulative nature of the rice cultivar.