Antisense down-regulation of the strawberry β-galactosidase gene FaβGal4 increases cell wall galactose levels and reduces fruit softening

Strawberry softening is characterized by an increase in the solubilization and depolymerization of pectins from cell walls. Galactose release from pectin side chains by β-galactosidase enzymes has been proposed as one reason for the increase in soluble pectins. A putative β-galactosidase gene, FaβGal4, has been identified using a custom-made oligonucleotide-based strawberry microarray platform. FaβGal4 was expressed mainly in the receptacle during fruit ripening, and was positively regulated by abscisic acid and negatively regulated by auxins. To ascertain the role of FaβGal4 in strawberry softening, transgenic plants containing an antisense sequence of this gene under the control of the CaMV35S promoter were generated. Phenotypic analyses were carried out in transgenic plants during three consecutive growing seasons, using non-transformed plants as control. Two out of nine independent transgenic lines yielded fruits that were 30% firmer than control at the ripe stage. FaβGal4 mRNA levels were reduced by 70% in ripe fruits from these selected transgenic lines, but they also showed significant silencing of FaβGal1, although the genes did not share significant similarity. These two transgenic lines also showed an increase in pectin covalently bound to the cell wall, extracted using Na2CO3. The amount of galactose in cell walls from transgenic fruits was 30% higher than in control; notably, the galactose increase was larger in the 1 M KOH fraction, which is enriched in hemicellulose. These results suggest that FaβGal4 participates in the solubilization of covalently bound pectins during ripening, reducing strawberry fruit firmness.