Characterization of an endo-1,4-β-glucanase from Lilium longiflorum that functioned in pollen germination and pollen tube growth

Abstract

Endo-β-glucanases play vital roles in the regulation of pollen tube growth. Here, a previously identified endo-1,4-β-glucanase from Lilium longiflorum (lily), named LlpCel1, was expressed in Escherichia coli, purified, and further investigated for its physiological function. The recombinant LlpCel1 protein hydrolyzed carboxy-methylcellulose (CMC) and exhibited activity towards laminarin from Eisenia. arborea and 1,3:1,4-β-glucan of barley. The pH for the optimum activity was 6.0 and the value of Km calculated from CMC was 5.0 mg/mL. Adding EDTA resulted in the total loss of the enzymatic activity, and this effect could be restored by the addition of Ca2+. Western blotting analysis showed that LlpCel1 protein was present in pollen grains and rehydrated pollen grains, and the amount of the protein was increased during pollen germinating, but not in the pollen tube. Consistently, the immunofluorescence labeling study with the antibody against LlpCel1 also indicated the presence of LlpCel1 at the beginning of germination, but not in the elongating pollen tube. Furthermore, incubation of LlpCel1 with pollen at the beginning of pollen germination increased the germination percentage and the length of pollen tube. All of these results suggested that LlpCel1 could play an important role in the regulation of lily pollen germination and the initiation of pollen tube growth.