Translocation of Structural P Proteins in the Phloem

Abstract

Phloem-specific proteins (P proteins) are particularly useful markers to investigate long-distance trafficking of macromolecules in plants. In this study, genus-specific molecular probes were used in combination with intergeneric grafts to reveal the presence of a pool of translocatable P protein subunits. Immunoblot analyses demonstrated that Cucurbita spp P proteins PP1 and PP2 are translocated from Cucurbita maxima stocks and accumulate in Cucumis sativus scions. Cucurbita maxima or Cucurbita ficifolia PP1 and PP2 mRNAs were not detected in Cucumis sativus scions by either RNA gel blot analysis or reverse transcription-polymerase chain reaction, indicating that the proteins, rather than transcripts, are translocated. Tissue prints of the Cucumis sativus scion, using antibodies raised against Cucurbita maxima PP1 or PP2, detected both proteins in the fascicular phloem of the stem at points distal to the graft union and in the petiole of a developing leaf, suggesting that the proteins move within the assimilate stream toward sink tissues. Cucurbita maxima PP1 was immunolocalized by light microscopy in sieve elements of the extrafascicular phloem of Cucumis sativus scions, whereas Cucurbita maxima PP2 was detected in both sieve elements and companion cells. INTRODUCTIONThe long-distance movement of macromolecules in vascular tissues can impact profoundly normal plant growth and development. The importance of long-distance signaling in response to wounding as well as systemic infections by plant pathogens, such as viruses, has been well documented (Narváez-Vásquez et al., 1995;Schaller and Ryan, 1995;Nelson and Van Bel, 1998). However, little is known about the mechanisms or effects of translocating the numerous proteins that are known to be expressed specifically within the phloem tissue. The phloem of most angiosperms contains proteinaceous structures, collectively called P proteins (phloem proteins), that accumulate in differentiating sieve elements and persist in translocating sieve elements. The P protein is deposited initially into ultrastructurally distinct polymorphous or crystalline bodies during sieve element differentiation (reviewed in Cronshaw, 1975;Cronshaw and Sabnis, 1990;Sabnis and Sabnis, 1995). P protein bodies either persist or more often disperse, forming a filamentous network in the parietal cytoplasm that is thought to be immobilized through interactions with the appressed endomembrane system (Smith et al., 1987). Disruption of sieve elements that occurs during wounding results in the accumulation of P protein filaments at the sieve plate, ostensibly blocking translocation by forming P protein plugs.P protein filaments in Cucurbita maxima (pumpkin) are composed of two very abundant proteins: phloem protein 1 (PP1), a 96-kD phloem filament protein, and phloem protein 2 (PP2), a 48-kD dimeric lectin that specifically binds poly( ␤ -1,4-N -acetylglucosamine) (Beyenbach et al., 1974;Sabnis and Hart, 1978;Allen, 1979;Read and Northcote, 1983b). Analysis of soluble phloem filaments present in phl...

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

“…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. However, it remains unclear whether PP2s from species other than cucurbits present such properties.…”

“…Alternatively, PP2-A1 may not be mobile in sieve elements, instead remaining bound to structural elements (Oparka and Cruz, 2000). Indeed, in C. maxima, it has been suggested that the phloem lectin attaches the P-proteins to the sieve element reticulum or plasma membrane (Smith et al, 1987), although there is also evidence to suggest that a fraction is translocated over long distances through graft unions (Golecki et al, 1998(Golecki et al, , 1999. We also cannot rule out the possibility that the method used to collect phloem sap, involving the use of EDTA, a chelator of Ca 2+ ions, affects the aggregation and mobility of PP2 proteins.…”

“…These observations suggest that PP2 might play a role in the cell-to-cell trafficking of macromolecules. 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).…”

Sieve element occlusion(SEO) genes encode structural phloem proteins involved in wound sealing of the phloem

“…This filament protein was immunolocalized in sieve element slime plugs and P-protein bodies, whereas the corresponding mRNA was shown to accumulate in companion cells (19). PP1 also was shown to be translocated within the extraordinary extrafascicular phloem system of Cucurbitaceae (20). However, the fact that PP1 is potentially unique to cucurbit species (21), whereas the typical P-protein structures in fascicular sieve elements are morphologically comparable in all dicotyledonous angiosperms, is still a matter of debate.…”

Long-distance movement of ArabidopsisFLOWERING LOCUS TRNA participates in systemic floral regulation

“…a cell-autonomous RNA specifically expressed in companion cells, 30 to test the specificity of RNA movement in our cleftgrafting experiments. The full-length cDNA of GAI and AtPP2A1 were driven by a CaMV35S promoter and introduced into Arabidopsis Columbia ecotype (Col, all transformants are in a Col background).…”