A potato alpha-glucosidase gene encodes a glycoprotein-processing alpha-glucosidase II-like activity. Demonstration of enzyme activity and effects of down-regulation in transgenic plants.
In order to elucidate more fully the function of a potato gene (MAL1) encoding alpha-glucosidase activity, transgenic plants in which MAL1 expression was down-regulated were generated using antisense technology. In transgenic lines severely down-regulated in the expression of MAL1, total alpha-glucosidase activity was not decreased in leaves and tubers, and the contents of starch, glucose, fructose and sucrose remained unchanged in tubers. Phylogenetic analysis indicated that the MAL1 gene product was more similar to the glycoprotein-processing alpha-glucosidase II of mammalian and yeast origin than to other plant alpha-glucosidases. Using [14C-Glc]-labelled Glc2Man9GlcNAc2 as a substrate, it was demonstrated that glucosidase II activity was markedly down-regulated in microsomes isolated from tubers of four independent antisense lines studied in detail, strongly suggesting that MAL1 encodes glucosidase II activity. In field trials (but not in the glasshouse), MAL1 down-regulation produced an extremely stunted phenotype - the leaves were curled and tuber yield was decreased by 90% compared to control values. Microscopic analysis of leaves revealed significant differences between the antisense and control samples. Plants with down-regulated glucosidase II activity showed a greater degree of plasmolysis, and an increase in the size of mesophyll intracellular spaces. Analysis of cell walls also indicated changes in structure as a result of MAL1 down-regulation. In leaves from four antisense lines, the steady-state transcript level corresponding to the endoplasmic reticulum chaperone, BiP, was enhanced. This is diagnostic of stress in the endoplasmic reticulum.
Taylor
M A
MA
Scottish Crop Research Institute, Invergowrie, Dundee DD2 5DA, UK. mtaylo@scri.sari.ac.uk
Ross
H A
HA
McRae
D
D
Stewart
D
D
Roberts
I
I
Duncan
G
G
Wright
F
F
Millam
S
S
Davies
H V
HV
D050260:Carbohydrate Metabolism; D002240:Carbohydrate Sequence; D016373:DNA, Antisense / Q000235:genetics; D015536:Down-Regulation; D017343:Genes, Plant; D006023:Glycoproteins / Q000378:metabolism; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry / Q000378:metabolism; D010641:Phenotype; D010802:Phylogeny; D010940:Plant Proteins / Q000378:metabolism; D030821:Plants, Genetically Modified; D011198:Solanum tuberosum / Q000201:enzymology* / Q000235:genetics*; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000235:genetics* / Q000378:metabolism*
D016373:DNA, Antisense; D006023:Glycoproteins; D009844:Oligosaccharides; D010940:Plant Proteins; D000520:alpha-Glucosidases