Two-dimensional Gel Electrophoretic Analysis of the Changes in Soluble Proteins During Development of Primary Oat (Avena sativa L.) Leaves

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Abstract
Two-dimensional polyacrylamide gel electrophoresis was used to follow the pattern of soluble proteins in primary oat ( Avena sativa L.) leaves from 7 to 37 d of plant development. Phenol-soluble protein samples were separated into over 400 spots by isoelectric focusing between pH 5.0 and 8.0 in the first dimension and 12.5% SDS slab gel electrophoresis in the second dimension, as visualized by silver staining. By immunoblotting the two subunits of ribulose- bisphosphate carboxylase were identified. 122 protein spots were further characterized by their presence in isolated chloroplasts. Upon comparison, three classes of proteins could be distinguished: spots originally present but disappearing during development below the limit of detection (disappearing spots); spots originally absent which become apparent and remain throughout leaf development (appearing spots); spots originally present, which do not undergo substantial changes during development (persisting spots). Major changes occurred between 7 and 12 d after sowing, when the primary leaves rapidly expanded to their final length. During this period, 39 mostly low-molecular-weight polypeptides disappeared and 34 mostly highmolecular weight spots became apparent. Of these, 16 and 13, respectively, were associated with chloroplasts. During the subsequent loss of protein and chlorophyll, indicative of senescence, the number of spots decreased gradually without new polypeptides becoming apparent, but 75% of the total number was still detectable in yellow, 37-d-old leaves. About half of the disappearing spots were associated with leaf expansion. No polypeptides were detected that were present exclusively in the later stages of leaf development: maturity, senescence, and final collapse.

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