Subcellular Localization and Function Analysis of 1, 4-β-glucanase from Lilium Longiflorum Pollen
Pollen germination and pollen tube elongation are crucial processes of double fertilization in higher plants. Pollen tubes are representative tip-growing cells, it is an ideal model for the study of cell tip-growth. The molecular structure and network of polysaccharide components in pollen grain and tube walls are modified by appropriate enzymes, which results in the alternation of the cell wall structure. The rheological property of the cell wall is important regulation factor regarding to pollen germination and pollen tube growth. In this paper, subcellular localization, enzymatic kinetics and biological function of LlpCel1, an endo-1,4-β-glucanase of pollen from Lilium longiflorum Thunb., was studied intensively.LlpCel1 was expressed in Escherichia coli, purified, and assayed for its hydrolytic activity in vitro. 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 optimum activity is 6.0 and the value of Km calculated from CMC was 5.0 mg/ml. Ca~(2+) is important for LlpCel1 enzyme hydrolytic activity. Adding EDTA resulted in the total loss of the enzymatic activity, and this effect could be restored by the addition of Ca~(2+). Other ions, like Mg~(2+) and Zn~(2+), could partially compensate enzyme activity, but had much less effect compare to Ca~(2+). The activity of LlpCel1 was, in certain extent, decreased by nojirimycin, an inhibitor of glucosidase.Polyclonal antiserum with high specificity was raised against recombinant LlpCel1. Western blotting analysis showed that LlpCel1 protein was specifically expressed in pollen grain, rehydrated pollen and germinating pollen, but not detectable in elongating pollen tube. A significant increase in the level of LlpCel1 protein was observed during pollen germination stage. The immunofluorescence labeling study also indicated the presence of LlpCel1 at the rehydrated pollen and beginning of germination, but not in the elongating pollen tube. This is well consistent with the above result of Western blotting analysis that the expression of LlpCel1 is prominently up-regulated at the pollen germination stage and distinctly down-regulated during pollen tube elongating stage. In addition, from the immunofluorescence labeling images we can see that the distribution of fluorescence signals were localized in cell wall and associated with germination apertures. Therefore, we proposed that the physiological function of LlpCel1 protein might be hydrolyze the cell wall of pollen aperture region.When incubation LlpCel1 with pollen, the germination percentage and the length of pollen tube were increased; this effect got higher increased when treated with cold-stored pollen. The expression level of LlpCel1 could be influenced by the concentration of Ca~(2+) in the medium which can also influence the pollen germination and tube growth。The optimum [Ca~(2+)] for pollen germination is 0.03% (w/v). But for the LlpCel1, when [Ca~(2+)] was more than 0.03%, the expression level of LlpCel1 was still get increased. We proposed that LlpCel1 is regulated by Ca~(2+) and influence the pollen germination.Taken together, these results showed that LlpCel1 is specifically expressed in pollen grain wall and pollen aperture region, and could play an important role in the regulation of lily pollen germination and the initiation of pollen tube growth.