Résumé
α-
d-Galactosidase (
α-Gal; EC 3.2.1.22) is one of three principal enzymes involved in the modification or degradation of plant cell wall galactomannans. In the present paper it is shown that
α-galactosidase activities in field-grown coffee beans are variable amongst cultivars of the two species investigated (
Coffea arabica and
C. canephora var. Robusta). Higher activities were found in Arabica cultivars. Using beans from greenhouse-cultivated
C. arabica as a model, we showed that
α-Gal activity was undetectable in the bean perispem tissue, but increased gradually during the endosperm development, to reach a peak at approximately 30 weeks after flowering (WAF) which coincided with the hardening of the endosperm.
α-Gal-specific transcripts detected at 22 and 27 WAF accompanied the peak of
α-Gal activity, but were reduced to be undetectable in mature beans at 30 WAF, while
α-Gal activity still persisted. Two isoforms were distinguished in 2-DE profiles of crude protein extracts by N-terminal sequencing analysis. Analysis of two-dimensional gel electrophoresis profiles demonstrated that both isoforms accumulated in a linear fashion throughout grain maturation.
α-Gal activity was also observed to increase to high levels during in vitro germination of coffee beans suggesting an important function of this enzyme in this process.
α-Gal cDNA sequences from Arabica and Robusta were sequenced and their deduced proteins appeared to be very similar, differing by only eight amino acids. Southern-blot analysis suggests that the enzyme was encoded by at least two genes in
C. arabica that could explain the existence of the two isoforms identified in 2-DE profiles.