COMPARTMENTALIZATION IN THE COTYLEDONARY CELLS OF PHASEOLUS VULGARIS L. SEEDS: A DIFFERENTIAL SEDIMENTATION STUDY

Abstract

SummaryThe appearance and the yield of sedimentable organelles from the cotyledons of Phaseolus vulgaris L. were measured with respect to the influence of the method of homogenization, the choice of dry or imbibed seeds, and the composition and the concentration of the homogenization medium. Sucrose, (0.2–0.8 M), the addition of Mg2+ (2 × 10−4M) or EDTA (3 × 10−3M) or the replacement of sucrose with mannitol made little difference to the average size distribution or the amount recovered of the 21 000 gav× 10 min pelleted organelles, the main protein body‐containing fraction of the homogenate. Slicing the imbibed seeds with a razor blade or by a mechanical rotary cutter combined with a short extraction was the gentlest and the most efficient method for the production of organelles. Cell homogenates were fractionated on the basis of size by differential sedimentation into several subcellular fractions. These included a residue, starch particles (300 g× 10 min), a main protein body fraction (21 000 gav× 10 min), 38 000 gav× 60 min pellets, 78 000 gav× 240 min pellets and a final supernatant. The activity in these fractions of several catabolic enzymes was measured. Most of the activity of the acidic nucleases, amylases, the BAPA‐ase and a substantial part of the acidic phosphatase and the cathepsin A‐type enzyme(s) were found in the supernatant, while no specific DNA‐ase was found in these seeds. Protein bodies contained some of the trypsin inhibitors and most (if not all) of the lectins and Glycoprotein II. Most of the endopeptidase (azoproteinase) activity of the cotyledons appeared to be tightly bound to the cell walls or to other insoluble structural elements. Protein bodies and other intracellular organelles had very little autolytic activity and, thus, the main storage proteins of the seed were not degraded during the first 4 days of germination.