The role of endo-β-1,4-glucanase in strawberry fruit development

Woolley, Lindsey C (2001). The role of endo-β-1,4-glucanase in strawberry fruit development. PhD thesis The Open University.



The ripening of strawberry fruit is characterized by changes in the composition and structure of the cell wall polysaccharides leading to textural changes and loss of firmness of the fruit. An endo-β-1,4-glucanase (EGase) 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 dqdecyl sulphate- polyaciylamide gel electrophoresis. A 25 amino acid N-terminal sequence showed strong homology with the proteins encoded by recently identified EGase genes from different strawberry cultivars and from Arabidopsis, pepper and tomato. The enzyme specifically cleaved the β-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 to 7.0 and 1.3 mg ml-1 respectively. A cDNA of the corresponding ripening-enhanced, fruit-specific gene, cel1, was isolated from a ripe fruit cDNA library. This was used to down-regulate cel1 expression in transgenic strawberry plants in order to assess the potential role(s) played by Cel1 during strawberry fruit ripening. In several transgenic lines, Cel1 mRNA was suppressed to undetectable levels in ripe fruit. However, EGase activity and firmness of these fruit were indistinguishable from control fruit. A second strawberry EGase gene, cel2, is also expressed in ripening fruit and this presence has prevented specific down- regulation of cel1 from revealing its role in fruit softening. Southern analysis of cel1 and cel2 revealed the presence of related sequences in the strawberry genome indicating a small multigene family, consistent with the isolation of two different EGase cDNAs from strawberry.

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