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Isolation and characterization of galactinol synthases from hybrid poplar.

Unda F, Canam T, Preston L, Mansfield SD - J. Exp. Bot. (2011)

Bottom Line: Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII 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.Interestingly, spatial and temporal expression analyses revealed that Pa×gGolSII transcript levels varied seasonally, while Pa×gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme.This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Wood Science, The University of British Columbia, Vancouver, BC, Canada.

ABSTRACT
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 alba×grandidentata). 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 (Pa×gGolSI and Pa×gGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII 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. Pa×gGolSI inherently possessed a broader pH and temperature range when compared with Pa×gGolSII. Interestingly, spatial and temporal expression analyses revealed that Pa×gGolSII transcript levels varied seasonally, while Pa×gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.

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Enzymatic activity of the recombinant Pa×gGolSI (homologue of PtGolS2) and Pa×gGolSII (homologue of PtGolS3) in response to pH at 37.5 °C. Relative expression (%) was determined colorimetrically (Ribeiro et al., 2000). Error bars correspond to the standard error of the mean.
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fig4: Enzymatic activity of the recombinant Pa×gGolSI (homologue of PtGolS2) and Pa×gGolSII (homologue of PtGolS3) in response to pH at 37.5 °C. Relative expression (%) was determined colorimetrically (Ribeiro et al., 2000). Error bars correspond to the standard error of the mean.

Mentions: GolSI and GolSII from hybrid poplar were cloned and expressed in the methylotrophic yeast Pichia pastoris GS115. Both isoforms were able to catalyse the formation of galactinol from UDP-galactose and myo-inositol. GolSI displayed a greater pH stability, ranging from 5.5 to 9.0 with an optimum of 7, and losing only a small percentage of its original activity at the extreme pH (Fig. 4). In contrast, GolSII displayed a very narrow pH tolerance, with activity decreasing considerably when the pH deviated from the 7.5 optimum (Fig. 4). A similar pattern was apparent when the enzyme activity was measured at different temperatures: GolSI was more stable at different temperatures than GolSII, showing maximal activity at 45 and 37 °C, respectively (Fig. 5).


Isolation and characterization of galactinol synthases from hybrid poplar.

Unda F, Canam T, Preston L, Mansfield SD - J. Exp. Bot. (2011)

Enzymatic activity of the recombinant Pa×gGolSI (homologue of PtGolS2) and Pa×gGolSII (homologue of PtGolS3) in response to pH at 37.5 °C. Relative expression (%) was determined colorimetrically (Ribeiro et al., 2000). Error bars correspond to the standard error of the mean.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC3295395&req=5

fig4: Enzymatic activity of the recombinant Pa×gGolSI (homologue of PtGolS2) and Pa×gGolSII (homologue of PtGolS3) in response to pH at 37.5 °C. Relative expression (%) was determined colorimetrically (Ribeiro et al., 2000). Error bars correspond to the standard error of the mean.
Mentions: GolSI and GolSII from hybrid poplar were cloned and expressed in the methylotrophic yeast Pichia pastoris GS115. Both isoforms were able to catalyse the formation of galactinol from UDP-galactose and myo-inositol. GolSI displayed a greater pH stability, ranging from 5.5 to 9.0 with an optimum of 7, and losing only a small percentage of its original activity at the extreme pH (Fig. 4). In contrast, GolSII displayed a very narrow pH tolerance, with activity decreasing considerably when the pH deviated from the 7.5 optimum (Fig. 4). A similar pattern was apparent when the enzyme activity was measured at different temperatures: GolSI was more stable at different temperatures than GolSII, showing maximal activity at 45 and 37 °C, respectively (Fig. 5).

Bottom Line: Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII 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.Interestingly, spatial and temporal expression analyses revealed that Pa×gGolSII transcript levels varied seasonally, while Pa×gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme.This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.

View Article: PubMed Central - PubMed

Affiliation: Department of Wood Science, The University of British Columbia, Vancouver, BC, Canada.

ABSTRACT
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 alba×grandidentata). 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 (Pa×gGolSI and Pa×gGolSII) showed characteristics of GolS proteins from other species, including a serine phosphorylation site and the ASAAP pentapeptide hydrophobic domain. Kinetic analyses of recombinant Pa×gGolSI and Pa×gGolSII 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. Pa×gGolSI inherently possessed a broader pH and temperature range when compared with Pa×gGolSII. Interestingly, spatial and temporal expression analyses revealed that Pa×gGolSII transcript levels varied seasonally, while Pa×gGolSI did not, implying temperature-regulated transcriptional control of this gene in addition to the observed thermosensitivity of the respective enzyme. This evidence suggested that Pa×gGolSI may be involved in basic metabolic activities such as storage, while Pa×gGolSII is probably involved in seasonal mobilization of carbohydrates.

Show MeSH
Related in: MedlinePlus