Expression of the phloem lectin is developmentally linked to vascular differentiation in cucurbits

Abstract

The conducting elements of phloem in angiosperms are a complex of two cell types, sieve elements and companion cells, that form a single developmental and functional unit. During ontogeny of the sieve element/companion cell complex, specific proteins accumulate forming unique structures within sieve elements. Synthesis of these proteins coincides with vascular development and was studied in Cucurbita seedlings by following accumulation of the phloem lectin (PP2) and its mRNA by RNA blot analysis, enzyme-linked immunosorbent assay, immunocytochemistry and in situ hybridization. Genes encoding PP2 were developmentally regulated during vascular differentiation in hypocotyls of Cucurbita maxima Duch. Accumulation of PP2 mRNA and protein paralleled one another during hypocotyl elongation, after which mRNA levels decreased, while the protein appeared to be stable. Both PP2 and its mRNA were initially detected during metaphloem differentiation. However, PP2 mRNA was detected in companion cells of both bundle and extrafascicular phloem, but never in differentiating sieve elements. At later stages of development, PP2 mRNA was most often observed in extrafascicular phloem. In developing stems of Cucurbita moschata L., PP2 was immunolocalized in companion cells but not to filamentous phloem protein (P-protein) bodies that characterize immature sieve elements of bundle phloem. In contrast, PP2 was immunolocalized to persistent P-protein bodies in sieve elements of the extrafascicular phloem. Immunolocalization of PP2 in mature wound sieve elements was similar to that in bundle phloem. It appears that PP2 is synthesized in companion cells, then transported into differentiated sieve elements where it is a component of P-protein filaments in bundle phloem and persistent P-protein bodies in extrafascicular phloem. This differential accumulation in bundle and extrafascicular elements may result from different functional roles of the two types of phloem.

Phloem sap proteins from Cucurbita maxima and Ricinus communis have the capacity to traffic cell to cell through plasmodesmata

“…Immunolocalization and in situ studies performed on wheat (13), cucurbit (20,29), rice (30), and tobacco, potato, and tomato (28) have established that, when expressed in vivo, the sieve tube proteins are confined to the companion cell-sieve tube complex. Thus, our present findings reveal two important facets associated with plasmodesmal function: (i) the plasmodesmal supramolecular structure does not constitute, in itself, the only discrimination site for cell-to-cell transport of macromolecules; and (ii) additional cell-specific factors (cytoplasmic or plasmodesmal) are likely involved in the regulation of macromolecular trafficking across specific cellular boundaries (e.g., the companion cell-sieve tube member interface) (11).…”

Diversity of the Superfamily of Phloem Lectins (Phloem Protein 2) in Angiosperms

“…In addition, PP2 has the capacity to interact with mesophyll plasmodesmata to increase the size exclusion limit and traffic cell-to-cell (Balachandran et al, 1997). This property reflects the apparent intercellular movement of PP2 within the sieve element-companion cell complex: PP2 mRNA was detected only in companion cells, although the protein accumulates in the sieve elements (Bostwick et al, 1992;Dannenhoffer et al, 1997). Additional experiments demonstrated that soluble, unpolymerized PP2 subunits translocate within sieve elements from source to sink tissues, and cycle between sieve elements and companion cells (Golecki et al, 1999).…”

Translocation of Structural P Proteins in the Phloem

“…The phloem filament protein and phloem lectin have been localized immunocytochemically to both sieve elements and companion cells (Smith et al, 1987;Clark et al, 1997;Dannenhoffer et al, 1997). However, in situ hybridization experiments in hypocotyls of Cucurbita maxima seedlings established that PP1 and PP2 mRNAs accumulate only in companion cells in both immature and differentiated sieve element-companion cell complexes (Bostwick et al, 1992;Clark et al, 1997;Dannenhoffer et al, 1997). Thus, PP1 and PP2 apparently are synthesized in companion cells and subsequently transported into sieve elements via pore-plasmodesma contacts.…”

“…Analysis of soluble phloem filaments present in phloem exudates of cucurbits indicated that PP1 monomers and PP2 dimers were covalently cross-linked via disulfide bonds, forming high molecular weight polymers Northcote, 1983a, 1983b). The phloem filament protein and phloem lectin have been localized immunocytochemically to both sieve elements and companion cells (Smith et al, 1987;Clark et al, 1997;Dannenhoffer et al, 1997). However, in situ hybridization experiments in hypocotyls of Cucurbita maxima seedlings established that PP1 and PP2 mRNAs accumulate only in companion cells in both immature and differentiated sieve element-companion cell complexes (Bostwick et al, 1992;Clark et al, 1997;Dannenhoffer et al, 1997).…”