Abstract
Two caleosin/peroxygenase isoforms from date palm, Phoenix dactylifera L., PdCLO2 and PdCLO4, were characterized with respect to their tissue expression, subcellular localization, and oxylipin pathway substrate specificities in developing seedlings. Both PdCLO2 and PdCLO4 had peroxygenase activities that peaked at the mid-stage (radicle length of 2.5 cm) of seedling growth and were associated with the lipid droplet (LD) and microsomal fractions. Recombinant PdCLO2 and PdCLO4 proteins heterologously expressed in yeast cells were localized in both LD and microsomal fractions. Each of the purified recombinant proteins exhibited peroxygenase activity but they were catalytically distinct with respect to their specificity and product formation from fatty acid epoxide and hydroxide substrates. We recently showed that date palm CLO genes were upregulated following exposure to the potent toxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) (Hanano et al., 2016), and we show here that transcripts of 9- and 13-lipoxygenase (LOX) genes were also induced by TCDD exposure. At the enzyme level, 9-LOX and 13-LOX activities were present in a range of seedling tissues and responded differently to TCDD exposure, as did the 9- and 13-fatty acid hydroperoxide reductase activities. This demonstrates that at least two branches of the oxylipin pathway are involved in responses to the environmental organic toxin, TCDD in date palm.
Highlights
Caleosins (Pfam reference PF05042) are a group of structurally related genes that appear to be ubiquitous in Streptophytes, including land plants, and in the Chlorophyte green algae; while similar genes are found in most, but not all, fungal clades (Naested et al, 2000; Partridge and Murphy, 2009; Hanano et al, 2015a)
These caleosin/peroxygenase proteins have been shown to be associated with bilayer membranes and/or lipid droplets (LDs) with some isoforms being able to bind to both types of lipid structure (Naested et al, 2000; Partridge and Murphy, 2009; Purkrtova et al, 2015)
We tested the subcellular distribution of these proteins in date palm seedlings where we found that both microsomes and lipid droplets (LDs) were able to catalyze the same co-oxidation reactions reported for other plant PXGs as follows
Summary
Caleosins (Pfam reference PF05042) are a group of structurally related genes that appear to be ubiquitous in Streptophytes, including land plants, and in the Chlorophyte green algae; while similar genes are found in most, but not all, fungal clades (Naested et al, 2000; Partridge and Murphy, 2009; Hanano et al, 2015a). PXG-LOX Pathways in Date Palm Seedlings conserved calcium-binding, EF-hand motif plus an invariant heme-binding histidine residue in the region proximal to the N terminus. This is followed by a relatively hydrophobic, potentially membrane-spanning, region plus a proline rich domain in the center of the protein. The lipid peroxygenase activity is associated with epoxy fatty acid biosynthesis as part of overall oxylipin metabolism in plants (Hanano et al, 2006; Blée et al, 2012) These caleosin/peroxygenase proteins have been shown to be associated with bilayer membranes and/or lipid droplets (LDs) with some isoforms being able to bind to both types of lipid structure (Naested et al, 2000; Partridge and Murphy, 2009; Purkrtova et al, 2015)
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