Proteins and Fine Structural Components in Exudate from Sieve Tubes in Cucurbits pepo Stems

Exudate from the cut stems of Cucurbita pepo gels when exposedto the atmosphere for 1 or 2 minutes. Gelling was preventedwhen exudate was collected into a buffer containing 2-mercaptoethanolIn the absence of gelling a soluble fraction and a structuralfraction were obtained from these samples by centrifugationand filtration, which is evidence that solids in the exudatedo not arise from the gelling reaction. The supernatants andthe filtrates gelled when 2-mercaptoethanol was removed. Since 2-mercaptoethanol and dithiothreitol are both —SHreducing agents, and prevented gelling equally, it is likelythat gel formation involves the oxidation of—SH groupsto disulphide bonds. The soluble fraction was separated into11 protein components by D.E.A E. chromatography but only one,a basic protein with a sedimentation coefficient S°_{20, W}of 4 32S, gelled when 2-mercaptoethanol was removed. The soluble fraction and a 2M potassium chloride extract fromthe structural fraction were run on the same G200 Sephadex columnand both revealed three protein peaks. The proteins from thesolids were eluted from the column earlier indicating they areof higher molecular weight than the soluble proteins. Gel electrophoresisrevealed 17 protein bands from the soluble fraction in additionto the basic, gelling protein, and by the same method only sixbands were produced from a sample of partially dispersed solidsAt least four of these bands were not represented in the solublefraction. The results indicate that structures in the exudatedo not arise by the aggregation of soluble proteins. Gel formed from soluble protein is structureless in the lightmicroscope, and has no organized fine structure in the electronmicroscope In the light microscope the structural fraction containsfibrils and particles which are sometimes associated in strandsup to 10 µm in diameter. In the electron microscope strandsof microscopic dimensions consist either of groups of parallelmembrane-like structures or of parallel fine filaments whichare less than 300 Å in diameter. The term P-protein (phloemprotein) has been widely used to describe filaments such asthese, and we believe they can now be described more accuratelyas PF protein. The term PS can be used to describe soluble proteinsand the term PS/G to identify the protein of this group whichgels Since membranes were found with PF protein in the structuralfraction of exudate, it is interpreted here as a constituentof cytoplasm exuded from sieve elements. Although the proteinwhich gels may not be the sole constituent of the slime plug,we suggest that gelling is the primary factor in the formationof the plugs. The possibility that PS/G protein interferes with the fixationof the protoplasts of sieve elements is discussed.