Cloning, functional analysis, and subcellular localization of two isoforms of NADH:cytochrome b5 reductase from developing seeds of tung (Vernicia fordii)

Two genes and the corresponding cDNAs for NADH:cytochrome b5 reductase (VfCBR1A and VfCBR1B) from tung (Vernicia fordii) were cloned and characterized. Both tung genes are expressed at similar levels in various organs throughout the plant, but differ substantially in their genomic architecture. Phylogenetic comparisons of many cloned and putative CBR genes from plants and yeast revealed two general classes of sequences. The separation of the classes likely reflects differences in the subcellular targeting of the two types of proteins. Immunofluorescence analyses of tobacco BY-2 cells containing transiently expressed tung CBR1A, CBR1B, or Arabidopsis thaliana CBR revealed definitive targeting of the proteins to the endoplasmic reticulum while a previously uncharacterized Arabidopsis CBR protein was targeted specifically to mitochondria. After overexpression in Saccharomyces cerevisiae, VfCBR1A was enzymatically active, and like its Arabidopsis ortholog, displayed strict specificity for NADH as the reductant. The subcellular localization and biochemical properties of the tung enzymes are consistent with a potential role in fatty acid desaturation and conjugation.