The raffinose family of oligosaccharides (RFOs) serve as transport carbohydrates in the phloem, storage compounds in sink tissues, and putative biological agents to combat both abiotic and biotic stress in several plant species. To investigate further the functional roles of this class of compounds in trees, two cDNAs encoding galactinol synthase (GolS, EC 18.104.22.168), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus alba3grandidentata). Phylogenetic analyses of the Populus GolS isoforms with other known GolS proteins suggested a putative role for these enzymes during biotic or abiotic stress in hybrid poplar. The predicted protein sequences of both isoforms (Pa3gGolSI and Pa3gGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant Pa3gGolSI and Pa3gGolSII resulted in Km values for UPD-galactose of 0.80 and 0.65 mM and Vmax values of 657.5 and 1245 nM min21, respectively. Pa3gGolSI inherently possessed a broader pH and temperature range when compared with Pa3gGolSII. Interestingly, spatial and temporal expression analyses revealed that Pa3gGolSII transcript levels varied seasonally, while Pa3gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermo-sensitivity of the respective enzyme. This evidence suggested that Pa3gGolSI may be involved in basic metabolic activities such as storage, while Pa3gGolSII is probably involved in seasonal mobilization of carbohydrates.
Unda, Faride; Canam, Thomas; Preston, Lindsay; and Mansfield, Shawn, "Isolation and characterization of galactinol synthases from hybrid poplar" (2012). Faculty Research & Creative Activity. 346.