Subunit Structure and Interactions of the Phloem Proteins of Cucurbita maxima (Pumpkin)

Abstract

1. The two major proteins from the phloem exudate of Cucurbita maxima (pumpkin), PP1 and PP2, were stable in the absence of reducing agents after modification of their accessible cysteine residues with iodoacetamide. This permitted their purification without precautions to prevent oxidation.2. PP2, a lectin specific for oligomers of N-acetyl-D-glucosamine, was shown by sedimentation-equilibrium ultracentrifugation to be a dimer of M, of 48000. Neither dithiothreitol nor tri-(N-acetyl-D-glucosamine) altered this value. The constituent polypeptides were linked by two buried disulphide bridges. PP2 behaved aberrantly on gel-filtration on both Sephadex and Bio-Gel unless tri-(N-acetyl-D-glucosamine) was added to the elution buffer; the M , was then measured as 46000. Other proteins which bind oligomers of N-acetyl-D-glucosamine are also retarded on gel-filtration.3. Soluble phloem filaments were prepared by collection of exudate into deaerated buffer containing iodoacetamide but no reducing agent. Oxidative gellation of the filaments was prevented by rapid modification of their many accessible cysteine residues, and is assumed to have maintained the degree of polymerisation found in vivo.Those disulphide bridges which were present allowed the incorporation of approximatically 60 % of the PP1 and 80 % of the PP2 into polymeric material. It is concluded that PP1 and PP2 are both structural proteins present in the filaments observable in vivo.4. PP2 had an elongated binding-site for oligomers of N-acetyl-D-glucosamine. It is suggested that this lectin immobilises bacteria and fungi to the cross-linked filaments which seal wounded phloem sieve-tubes, and thus maintains sterility.The sieve-tube cells of phloem tissue conduct organic nutrients around plants by intracellular mass-flow of the translocated material [l]. Protein filaments known as P-protein are the major structures microscopically observable in the sieve-tube lumen, but their function and positioning are disputed [2-41. Both the stream of translocates and the P-proteins are contained in a fluid exudate collected from mature sieve-tubes of certain plant species [5 -81.Phloem exudate from Cucurbita (pumpkin) forms a gel on exposure to air due to cross-linking of the filaments, and this can be prevented or reversed by agents which reduce disulphide bridges [9,10]. Reduction gives two proteins: phloem protein 1 (PPl), which reconstitutes filaments on oxidation [lo-121, and phloem protein 2 (PP2), which also precipitates on oxidation but does not form filaments [12], and which is a lectin specific for oligomers of N-acetylglucosamine [13 -151. Several different M , measurements have been given for these proteins: 88000-136000 for PP1 [4,10, 11,161 and 20000-60000 for PP2 [4,11,14,16].We show here that PP2 is a dimer of M , of 48000 held together by buried disulphide bridges, and that filaments from Cucurbita phloem contain both PP1 and PP2, which are therefore both structural proteins.Abbreviations. (GlcNAc),, (GlcNAc), and (Glc NAc),, di-, tri-, tetra-~-(1+4)-(2...

Binding Properties of the N-Acetylglucosamine and High-Mannose N-Glycan PP2-A1 Phloem Lectin in Arabidopsis

“…PP2 lectins from cucurbits and the related protein Nictaba have been reported to be dimeric in planta (Read and Northcote, 1983a;Chen et al, 2002;Walz et al, 2004). The existence of a high-affinity site in PP2-A1 dimeric forms is consistent with these observations.…”

“…PP2s constitute an interesting family of phloem proteins. They were first identified as components of the socalled P-proteins in cucurbits and shown to act as dimeric chitin-binding lectins in these plants (Beyenbach et al, 1974;Sabnis and Hart, 1978;Allen, 1979;Read and Northcote, 1983a;Smith et al, 1987;Bostwick et al, 1992), leading to the suggestion of a role in defense mechanisms. In C. maxima, CbmPP2 presents several properties associated with NCAPs (Bostwick et al, 1994;Balachandran et al, 1997;Golecki et al, 1998Golecki et al, , 1999, suggesting a role in the trafficking of macromolecules.…”

“…The binding of PP2-A1 to other sugars (see below) probably renders the system even more complex, because these sugars may also have different affinities for PP2-A1 subunits and dimers. This raises the question of the conditions required for oligomerization in vivo and the role of PP2 subunits and aggregates in planta, as previously considered for the cucurbit PP1 and PP2 phloem proteins (Read and Northcote, 1983a;Leineweber et al, 2000).…”

“…In C. maxima, PP2 is produced in companion cells, but the protein accumulates in sieve elements and is transported across long distances (Golecki et al, 1998(Golecki et al, , 1999. Early studies suggested that the P-proteins of C. maxima exist in at least two structural states: large polymers immobilized in individual sieve elements, and small polymers or individual subunits translocated over long distances (Read and Northcote, 1983a). A phloem-specific pattern of expression has been reported for the PP2 genes in two other plant species: Apium graveolens (AgPP2-1), a member of the Apiaceae, and Arabidopsis (Arabidopsis thaliana; AtPP2-A1 and AtPP2-A2), a crucifer.…”

“…These proteins bind to chitin columns, demonstrating an affinity for oligomers of GlcNAc. These observations led Allen (1979) and Read and Northcote (1983a) to suggest a role in defenses against fungi or bacteria. Other lectins that bind to oligomers of GlcNAc have been identified in plants, and interactions with glycoproteins from plant pathogens, including fungi, have been demonstrated for some of these molecules (Peumans and Van Damme, 1995).…”