Coffee seeds contain 11S storage proteins

A legumin-like seed protein was purified from the endosperm of coffee (Coff ea avabica L. cv. Colombia). In contrast to legumes, n here efficient stora ge globulin extraction requires buffered saline solutions well above the ac idic pK(1) of the globulins, coffee legumin is readily extracted with acidi c aqueous buffers. The coffee legumin migrates like other 11S storage globu lins in sucrose gradients. Subunits of coffee legumin have an apparent mole cular mass of about 55 kDa after one-dimensional SDS-polyacrylamide gel ele ctrophoresis in the absence of a reducing agent, In the presence of 2-merca ptoethanol, two polypeptides appear that have apparent molecular masses of 33 and 24 kDa. Two full-length cDNAs were generated from mRNA of developing seeds that were more than 98% homologous. They had open reading frames of 1458 and 1467 bp. Each encoded legumin precursors of 486 and 489 amino acid s, respectively (M-r = 54136 and 54818). Examination of a 5' promoter regio n from a coffee legumin gene revealed a putative legumin-box. Genomic DNA f rom C. arabica was digested with six different restriction endonucleases, A fter separation of the fragments by electrophoresis, single discrete fragme nts on DIVA blots hybridized strongly to a cDNA probe for the acidic chain. Other fragments that hybridized weakly with this probe were visible after hybridization at very low stringency, DNA from other species and commercial ly important cultivars that comprise the genus Coffea produced similar resu lts. Immunocytochemical studies revealed that some legumin was detected in the cytoplasm in mature coffee seeds, but that the majority of it was in la rge storage vacuoles that accounted for most of the cell volume.