Vicilin Protein Research Articles

Structural Aspects of Protein Accumulation in Developing Pea Cotyledons. III. Immunocytochemical Localization of Legumin and Vicilin Using Antibodies Shown to Be Specific by the Enzyme-Linked Immunosorbent Assay (ELISA)

The site of sequestration of the storage proteins legumin and vicilin during development of cotyledons from pea (Pisum sativum L.) has been determined using improved immunocytochemical techniques. Antibodies to legumin and vicilin were made monospecific by affinity chromatography. They were allowed to react on sections of glycol methacrylate-embedded cotyledon tissue and detected by indirect immunocytochemical localization using rhodamine-labelled antibodies. The enzyme-linked immunosorbent assay (ELISA) technique was adapted to verify antibody specificity at a sensitivity up to 300 times greater than that of immunodiffusion. Legumin and vicilin 4 are localized in small peripheral deposits within large vacuoles as early as day 8 after flowering. As the vacuoles fragment during development the storage proteins continue to be localized in the vacuolar deposits until, at day 16, they entirely fill vacuoles, now termed protein bodies. Thereafter, the protein bodies become more densely packed and retain a similar form from day 22 to maturity. Wherever the same vacuolar deposit of protein body could be observed in adjacent sections, antilegumin and antivicilin 4 labelled both deposits, clearly indicating that both storage proteins are sequestered into the same area of protein.

Immunofluorescent localization of pea storage proteins in glycol methacrylate embedded tissue.

Improved immunofluorescent techniques have been developed for the high resolution light microscopic localization of intracellular antigens in plant tissue. Thin sections of pea cotyledon tissue which had been fixed in paraformaldehyde and embedded in glycol methacrylate were reacted with mono-specific antibodies to the storage proteins legumin and vicilin. These antibodies were raised in sheep, purified by affinity chromatography and tested by immunoelectrophoresis and immunodiffusion. Using the indirect technique, rhodamine-labeled antibodies permitted specific fluorescent localization of the legumin and vicilin to small (ca. 1 micrometer) cytoplasmic organelles in near mature tissue. Subsequent histochemical staining verified the proteinaceous nature of these organelles. Parameters affecting staining specificity and background fluorescence are discussed.

Growth of Immature Pea Cotyledons in Culture

Detached immature pea (Pisum sativum) cotyledons grow for several days in liquid medium and synthesize considerable quantities of chlorophyll, starch, DNA, RNA and protein (including the storage proteins vicilin and legumin). Seeds developing in detached pods also synthesize both major storage proteins.