Nucleotide sequence of a pumpkin phloem lectin cDNA.

One of the characteristic events that occurs during phloem differentiation in dicotyledonous plants is the appearance of phloem protein within the sieve elements and companion cells of the phloem tissue (Esau and Cronshaw, 1967). Two very abundant phloem proteins, PPl and PP2, have been isolated from phloem exudates of pumpkin (Cucurbita maxima) (Read and Northcote, 1983). PPl is a 96-kD structural protein that forms polymeric filaments, and PP2 is a 48-kD lectin that is covalently linked to the PPl polymers (Sabnis and Hart, 1979; Read and Northcote, 1983). PP2 is composed of two subunits, a (M, 26,500) and @ (Mr 25,000), joined by disulfide linkages between Cys residues (Read and Northcote, 1983). It is unclear whether the two subunits are encoded by different genes or are modifications of a single gene product. cDNAs encoding phloem proteins were isolated by screening a pumpkin seedling cDNA library with a complex antiserum raised against pumpkin phloem exudate proteins (Table I). We subsequently demonstrated that two of these cDNAs, cPC13 (868 bp) and cPC20 (792 bp), encoded a functional PP2 subunit (Bostwick et al., 1992). Nucleotide sequence analysis of the cDNAs showed that the entire sequence of cPC20 was identical to nucleotides 46-837 of cPC13. Northern blot analysis demonstrated that the mRNA encoding a PP2 subunit migrates as a single species of approximately 1000 nucleotides (Sham and Northcote, 1987; Bostwick et al., 1992). Although cPC13 is not a full-length copy of the PP2 mRNA, we identified an ORF of 654 nucleotides that extends from nucleotide 31 to 684. The translation initiation sequence (5’-TGCAATGGA-3’) for the deduced polypeptide matched six of nine nucleotides from the consensus sequence for plant genes (5’-AACAATGGC-3’) (Lutcke et al., 1987). The ORF of cPC13 encoded a deduced polypeptide of 218 amino acids with a calculated molecular mass of 24,478 D. This coincides with the apparent molecular mass of 25,000 D for the p subunit of PP2 (Read and Northcote, 1983). Hydropathy analysis of the deduced polypeptide did not indicate a hydrophobic signal peptide or other hydrophobic domains, supporting previous observations (Allen, 1979) that PP2 is a cytosolic protein that is not glycosylated.