Monoclonal antibody against a cell wall marker protein for embryogenic potential of Dactylis glomerata L. suspension cultures

Abstract

We identified and isolated a monoclonal antibody (MAb 3G2) raised against extracellular proteins from microcluster cells of orchard grass (Dactylis glomerata L.) embryogenic suspension culture. MAb 3G2 recognized with high specificity an antigen ionically bound within the primary cell wall and in the culture medium of microcluster cells. Two-dimensional polyacrylamide gel analysis and blotting of proteins on PVDF membrane showed that MAb 3G2 detected a single polypeptide of apparent molecular mass of 48 kDa and an isoelectric point (pI) of 5.2, designated EP48. A transient expression during somatic embryogenesis was observed for EP48. Indirect immunofluorescence showed that this protein highly accumulated in the cell walls of some single cells, microclusters and partly in proembryogenic masses (PEMs), but not in globular embryos of the embryogenic cell line and microclusters from the non-embryogenic cell line. Signal intensity varied between individual cells of the same population and in successive stages of somatic embryo development. Screening of several D. glomerata L. embryogenic and non-embryogenic cell lines with MAb 3G2 indicated the presence of ECP48 in only embryogenic suspension cultures at early stages of embryo development long before morphological changes have taken place and thus it could serve as an early marker for embryogenic potential in D. glomerata L. suspension cultures.

Identification, Molecular Cloning, and Recombinant Gene Expression of an Extracellular A-Amylase fromDactylis GlomerataL. Embryogenic Suspension Cultures

“…The membrane was immunostained with monoclonal antibody MAb 3G2 essentially as previously described (44).…”

“…In a previous study, an extracellular protein EP48 was found to be a useful early marker for embryogenic potential in D. glomerata L. suspension cultures (44). To identify the EP48, extracellular proteins secreted by microclusters into the medium of D. glomerata L. embryogenic suspension culture were separated using 2-DE and stained with Colloidal CBB G-250 (Fig.…”

“…It is found in the cell wall and the medium of single cells and dividing microclusters during their developmental transition to proembryogenic masses and is not detected in differentiating embryos. In dividing cells it is located predominantly at the region of cell-to-cell adhesion (44). Based on this data, it seems more likely that the secreted DgAmy1 may act on some unknown carbohydrate in the cell wall liberating signals necessary for the development of microclusters into PEMs or it may locally modify the cell wall and thus ensure close cell-to-cell contact between the developing embryogenic structures.…”

“…Both, an acidic esterase with a putative role in providing a close cell-to-cell contact of different morphological structures during somatic embryogenesis and an endochitinase-like protein secreted only into the medium of Dactylis glomerata L. embryogenic suspension cultures have been proposed as early embryogenic markers (45,46). Recently, a monoclonal antibody MAb3G2 against a cell wall protein (EP48) secreted into the medium by the earliest morphological structures (microclusters) during somatic embryogenesis was selected to monitor the expression of embryogenic potential in D. glomerata L. suspension cultures long before any morphological changes have occurred (44). EP48 was transiently expressed during early embryo development up to the globular stage.…”

“…Orchardgrass α-amylase is found both in the medium and the cell wall of embryogenic microclusters (44 . Taken together, these differences between the structure of αAmy3, AmyI-1 and DgAmy1 make unlikely the localization of DgAmy1 in the amyloplasts of microcluster cells and hence its participation in starch degradation.…”