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A potato α‐glucosidase gene encodes a glycoprotein‐processing α‐glucosidase II‐like activity. Demonstration of enzyme activity and effects of down‐regulation in transgenic plants
Author(s) -
Taylor* Mark A.,
Ross Heather A.,
McRae Diane,
Stewart Derek,
Roberts Ian,
Duncan George,
Wright Frank,
Millam Steve,
Davies Howard V.
Publication year - 2000
Publication title -
the plant journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.058
H-Index - 269
eISSN - 1365-313X
pISSN - 0960-7412
DOI - 10.1046/j.1365-313x.2000.00873.x
Subject(s) - endoplasmic reticulum , glucosidases , sucrose , biology , biochemistry , gene expression , fructose , transgene , gene , starch , enzyme , microbiology and biotechnology
Summary In order to elucidate more fully the function of a potato gene ( MAL1 ) encoding α‐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 α‐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 α‐glucosidase II of mammalian and yeast origin than to other plant α‐glucosidases. Using [ 14 C‐Glc]‐labelled Glc 2 Man 9 GlcNAc 2 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.