Isolation and characterization of galactinol synthases from hybrid poplar

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 2.4.1.123), which catalyses the first step in the biosynthesis of RFOs, were identified and cloned from hybrid poplar (Populus albaxgrandidentata). 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 (PaxgGolSI and PaxgGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant PaxgGolSI and PaxgGolSII resulted in K-m values for UPD-galactose of 0.80 and 0.65 mM and V-max values of 657.5 and 1245 nM min(-1), respectively. PaxgGolSI inherently possessed a broader pH and temperature range when compared with PaxgGolSII. Interestingly, spatial and temporal expression analyses revealed that PaxgGolSII transcript levels varied seasonally, while PaxgGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. This evidence suggested that PaxgGolSI may be involved in basic metabolic activities such as storage, while PaxgGolSII is probably involved in seasonal mobilization of carbohydrates.