Purification and properties of an endo-beta-1,4-glucanase from strawberry and down-regulation of the corresponding gene, cel1.
An endo-beta-1,4-glucanase (EG) was purified from ripe strawberry (Fragaria x ananassa Duch.) fruit using cellulose affinity chromatography. The purified enzyme gave a single protein band of 54 kDa on sodium dodecyl sulphate-polyacrylamide gel electrophoresis. The N-terminal amino acid sequence of this protein showed strong homology with the proteins encoded by recently identified EG genes from different strawberry cultivars and from Arabidopsis, pepper and tomato. The enzyme specifically cleaved the beta-1,4-glucosyl linkages of xyloglucan but was unable to hydrolyze those of insoluble cellulose. The pH optimum and Km of the enzyme against the artificial substrate carboxymethylcellulose (CMC) were pH 5.0-7.0 and 1.3 mg ml-1, respectively. To assess the role of the Cell enzyme in fruit softening a cDNA of the corresponding fruit-specific and ripening-enhanced strawberry gene, cell, was used to down-regulate cell gene expression in transgenic strawberry plants. In several primary transformants, cell mRNA was strongly suppressed in ripe fruit. However, the EG activity and firmness of these fruit were indistinguishable from those of control fruit. The expression of a second gene, cel2, encoding a different strawberry EG was unaltered in the fruits of these transformants. The presence of the cel2 transcript in transgenic plants may have prevented the specific down-regulation of cell from revealing its role in fruit softening.
Woolley
L C
LC
Horticulture Research International, Wellesbourne, Warwick, CV35 9EF, UK.
James
D J
DJ
Manning
K
K
D015152:Blotting, Northern; D002480:Cellulase / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D043366:Cellulose 1,4-beta-Cellobiosidase; D018076:DNA, Complementary / Q000032:analysis; D015536:Down-Regulation; D005638:Fruit / Q000201:enzymology*; D018506:Gene Expression Regulation, Plant; D007527:Isoenzymes / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D030821:Plants, Genetically Modified; D027824:Rosaceae / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
D018076:DNA, Complementary; D007527:Isoenzymes; D002480:Cellulase; D043366:Cellulose 1,4-beta-Cellobiosidase