title: An Arabidopsis T-DNA insertion mutant for galactokinase (AtGALK, At3g06580) hyperaccumulates free galactose and is insensitive to exogenous galactose.
abstract: Galactokinase (GALK, EC 2.7.1.6) is a cytosolic enzyme with a wide occurrence across the taxonomic kingdoms. It catalyzes the phosphorylation of α-d-galactose (Gal) to α-d-Gal-1-P. The cytotoxicity of free (unphosphorylated) Gal is well documented in plants and causes marked defects. An Arabidopsis GALK (AtGALK, At3g06580) was previously identified, cloned and functionally characterized in Escherichia coli and was suggested to occur as a single copy gene in Arabidopsis. We identified an AtGALK T-DNA insertion mutant (atgalk) that (i) is AtGALK transcript deficient; (ii) displays no GALK activity in vegetative tissues; and (iii) accumulates Gal up to 6.8 mg g(-1) FW in vegetative tissues, in contrast to wild-type plants. By constitutively overexpressing the AtGALK cDNA, atgalk was functionally rescued. Three independent transformed lines showed restored AtGALK transcripts and GALK activity and had low leaf Gal concentrations comparable with those observed in wild-type plants. Surprisingly, in vitro grown atgalk plants were largely insensitive to the exogenous application of up to 100 mM free Gal, while wild-type plants exhibited sensitivity to low Gal concentrations (10 mM). Furthermore, atgalk seedlings retained the capacity for uptake of exogenously supplied Gal (100 mM), accumulating up to 57 mg g(-1) FW in leaves. Leaves from soil-grown atgalk plants that exhibited no growth or morphological defects were used to demonstrate that the accumulating Gal occurred exclusively in the vacuoles of mesophyll protoplasts. Collectively, these findings suggest a novel Gal detoxification pathway that targets free Gal to the vacuole and is active in the atgalk mutant background.
authors.affiliation: Institute of Plant Biology, Molecular Plant Physiology, University of Zürich, Zollikerstrasse 107, CH-8008 Zürich, Switzerland.
authors.forename: Aurélie
authors.initials: A
authors.lastname: Egert
authors.forename: Shaun
authors.initials: S
authors.lastname: Peters
authors.forename: Christelle
authors.initials: C
authors.lastname: Guyot
authors.forename: Bruno
authors.initials: B
authors.lastname: Stieger
authors.forename: Felix
authors.initials: F
authors.lastname: Keller
mesh_terms: D017360:Arabidopsis / Q000187:drug effects / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D029681:Arabidopsis Proteins / Q000235:genetics* / Q000378:metabolism; D004269:DNA, Bacterial / Q000235:genetics*; D005686:Galactokinase / Q000235:genetics* / Q000378:metabolism; D005690:Galactose / Q000378:metabolism* / Q000494:pharmacology*; D058503:Mesophyll Cells / Q000187:drug effects / Q000378:metabolism; D016254:Mutagenesis, Insertional / Q000187:drug effects / Q000235:genetics*; D009154:Mutation / Q000235:genetics; D009928:Organ Specificity / Q000187:drug effects; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism; D018517:Plant Roots / Q000187:drug effects / Q000378:metabolism; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D036226:Seedlings / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D014617:Vacuoles / Q000187:drug effects / Q000378:metabolism
chemical_list: D029681:Arabidopsis Proteins; D004269:DNA, Bacterial; D012333:RNA, Messenger; C036483:T-DNA; C574656:GALK protein, Arabidopsis; D005686:Galactokinase; D005690:Galactose