Fractionation and polypeptide analysis of phloem tissue of Pinus sabiniana Dougl.

Abstract

The polypeptide complement of phloem tissue and xylem tissue in trunks of Pinus sabiniana collected in spring was compared by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Two polypeptides at 24 and 25 kdalton represented over 20% of the total polypeptide complement of the phloem but were absent in xylem tissue. In an attempt to relate the 24- and 25-kdalton phloem polypeptides (PPP) to cellular constitutents, phloem tissue was fractionated by sequential differential and density-gradient centrifugation utilizing the PPP as biochemical markers. In borate buffer, the fractions containing PPP pelleted at less than 12,000 g and were subsequently enriched in sucrose gradients at densities greater than 1.22. However, the cytological entities containing the PPP were almost completely dissociated when phloem tissue was processed with a 2-amino-2-(hydroxymethyl)-1,3-propanediol (Tris) buffer containing mercaptoethanol, and the PPP were then found in the supernatant of material spun at 45,000 g for 3 h. In electron micrographs of PPP-enriched fractions processed with borate buffer an assortment of structures that are associated with mature sieve cells of pine were found, including filaments, cup-shaped arrays, polyhedral crystals, and paracrystalline bodies. Similar structures were not found in identically processed xylem tissue or in phloem tissue processed with the Tris buffer. It is suggested that the PPP represent a proteinaceous component common to these phloem structures.