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

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Abstract
Exudate from the cut stems of Cucurbita pepo gels when exposed to the atmosphere for 1 or 2 minutes. Gelling was prevented when exudate was collected into a buffer containing 2-mercaptoethanol In the absence of gelling a soluble fraction and a structural fraction were obtained from these samples by centrifugation and filtration, which is evidence that solids in the exudate do not arise from the gelling reaction. The supernatants and the filtrates gelled when 2-mercaptoethanol was removed. Since 2-mercaptoethanol and dithiothreitol are both —SH reducing agents, and prevented gelling equally, it is likely that gel formation involves the oxidation of—SH groups to disulphide bonds. The soluble fraction was separated into 11 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 from the structural fraction were run on the same G200 Sephadex column and both revealed three protein peaks. The proteins from the solids were eluted from the column earlier indicating they are of higher molecular weight than the soluble proteins. Gel electrophoresis revealed 17 protein bands from the soluble fraction in addition to the basic, gelling protein, and by the same method only six bands were produced from a sample of partially dispersed solids At least four of these bands were not represented in the soluble fraction. The results indicate that structures in the exudate do not arise by the aggregation of soluble proteins. Gel formed from soluble protein is structureless in the light microscope, and has no organized fine structure in the electron microscope In the light microscope the structural fraction contains fibrils and particles which are sometimes associated in strands up to 10 μm in diameter. In the electron microscope strands of microscopic dimensions consist either of groups of parallel membrane-like structures or of parallel fine filaments which are less than 300 Å in diameter. The term P-protein (phloem protein) has been widely used to describe filaments such as these, and we believe they can now be described more accurately as PF protein. The term PS can be used to describe soluble proteins and the term PS/G to identify the protein of this group which gels Since membranes were found with PF protein in the structural fraction of exudate, it is interpreted here as a constituent of cytoplasm exuded from sieve elements. Although the protein which gels may not be the sole constituent of the slime plug, we suggest that gelling is the primary factor in the formation of the plugs. The possibility that PS/G protein interferes with the fixation of the protoplasts of sieve elements is discussed.

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