α-l-Arabinofuranosidase from strawberry fruit: Cloning of three cDNAs, characterization of their expression and analysis of enzymatic activity in cultivars with contrasting firmness

Abstract

Softening of fleshy fruits during ripening is associated to catabolism of cell wall components. In strawberry, pectin degradation, as well as loss of neutral sugars (mainly arabinose), increases during ripening, and probably contributes to fruit softening. In this work, we report the activity of a-L-arabinofuranosidase (a-L-arafase) and the expression of related genes in strawberry. Activity of a-L-arafase was measured during ripening of cultivars with contrasting firmness. An important increment in the specific activity of a-L-arafase was detected during ripening in both cultivars. However, in the softest one (Toyonoka) the specific activities were higher than in the firmest (Camarosa). A combination of semiquantitative reverse transcriptase-PCR (RT-PCR) with degenerate primers and a screening of a cDNA library allowed the isolation and cloning of three cDNAs encoding putative a-L-arafases (FaAra1, FaAra2 and FaAra3). The deduced proteins revealed that FaAras belong to the glycoside hydrolase family 51 and not to glycoside hydrolase family 3. Expression studies, carried out by means of Northern-blot and semiquantitative RT-PCR, revealed that FaAras were predominantly expressed in fruit tissue and detected over the entire ripening process. Due to similarity of FaAras sequences, Northern-blot analysis probably grouped the expression of the three genes. The expression was high at small green stage, decreased at white stage and increased thereafter. The increment of the expression from white to 50% red stage was more evident in the softest cultivar (Toyonoka). Semi quantitative RT-PCR analysis allowed determining the expression of individual FaAras. The expression of the three genes was detected in all developmental and ripening stages. However, differences in expression levels could be detected between cultivars. In the softest cultivar, the expression of the three FaAras was higher at 50% and 75% red stages, and in the case of FaAra3 a higher expression was found also at 100% red stage. Overall, specific activity of a-L-arafase was higher in the softest cultivar; such activity reflects the expression of at least three putative FaAra genes.