No abstract

The primary amino acid sequence of an abundant methionine-rich seed protein found in Brazil nut (Bertholletia excelsa H.B.K.) has been elucidated by protein sequencing and from the nucleotide sequence of cDNA clones. The 9 kDa subunit of this protein was found to contain 77 amino acids of which 14 were methionine (18%) and 6 were cysteine (8%). Over half of the methionine residues in this subunit are clustered in two regions of the polypeptide where they are interspersed with arginine residues. In one of these regions, methionine residues account for 5 out of 6 amino acids and four of these methionine residues are contiguous. The sequence data verifies that the Brazil nut sulfur-rich protein is synthesized as a precursor polypeptide that is considerably larger than either of the two subunits of the mature protein. Three proteolytic processing steps by which the encoded polypeptide is sequentially trimmed to the 9 kDa and 3 kDa subunit polypeptides have been correlated with the sequence information. In addition, we have found that the sulfur-rich protein from Brazil nut is homologous in its amino acid sequence to small water-soluble proteins found in two other oilseeds, castor bean (Ricinus communis) and rapeseed (Brassica napus). When the amino acid sequences of these three proteins are aligned to maximize homology, the arrangement of cysteine residues is conserved. However, the two subunits of the Brazil nut protein contain over 19% methionine whereas the homologous proteins from castor bean and rapeseed contain only 2.1% and 2.6% methionine, respectively.

An abundant seed protein, which is exceptionally rich in the sulfur-containing amino acids, methionine (18%) and cysteine (So/,), is synthesized in Brazil nut embryos about 9 months after flowering. This sulfur-rich protein consists of two low-molecular-mass polypeptide components, a 9-kDa polypeptide and a 3-kDa polypeptide. The two-subunit polypeptides associate through disulfide linkage(s) to form a 12-kDa protein molecule. We have demonstrated through in vitro translation studies, using RNA from 9-month-old embryos, that the sulfur-rich protein is synthesized as a larger precursor polypeptide of 18 kDa. In addition, data from in vivo labelling studies of 9-month-old Brazil nuts suggest that there are two intermediate precursors of the sulfur-rich protein, one of 15 kDa and another of 12 kDa. One of these precursors, the 12-kDa polypeptide, accumulates for a 2-month period in the developing embryos. From these data we infer that at least three stepwise cleavages are involved in the maturation of the sulfur-rich protein from its 18-kDa precursor.The seeds of Brazil nut (Bertholletia excelsa H.B.K.) contain 15-17% protein by fresh weight [l] and some 50% protein by dry weight of the defatted flour [2]. Brazil nuts are probably one of the richest food sources of the sulfurcontaining amino acids; the total seed protein is reported to contain about 8.3% methionine and cysteine by weight [3]. The total protein of Brazil nuts can be fractionated into three size classes of proteins, the 11 S, 7S, and 2s proteins [4]. The 2 S fraction, which is water-soluble and thus classified as albumin, comprises about 30% of the total protein and is exceptionally rich in the sulfur amino acids; about 30% methionine and cysteine [4]. From this fraction we have purified a sulfur-rich protein, which contains 18% methionine and 8% cysteine. We have found that this protein is composed of two polypeptide components with molecular masses of about 9 kDa and 3 kDa (unpublished work). In this paper we report on some of the structural properties of the sulfur-rich protein.In addition we report on the biosynthesis of the sulfurrich protein in developing Brazil nut embryos. The maturation of Brazil nut fruits is a lengthy process, usually taking over a year (12-14 months) [l]. Mature Brazil nut embryos consist mainly of hypocotyl [5], in which the sulfur-rich protein, other seed proteins and the oil reserve (75% by fresh weight) are localized. We demonstrate that the sulfur-rich protein is synthesized and begins to accumulate in Brazil nut seeds about 8 -9 months after flowering. In addition we have used both in vitro and in vivo labelling studies to show that the lowmolecular-mass sulfur-rich protein is synthesized initially as a larger precursor polypeptide, which undergoes stepwise cleavages to reach its mature form.

Phosphoenolpyruvate carboxylase (PEPCase; EC 4.1.1.31) from Flaveria trinervia Mohr (C4), F. floridana Johnston (C3-C4), and F. cronquistii Powell (C3) leaves were compared by electrotransfer blotting/enzyme-linked immunoassay (Western-blot analysis), mobility of the native enzyme in polyacrylamide gels and in isoelectric focusing (IEF) gels, peptide mapping, and in-vitro translation of RNA isolated from each plant. The PEPCases from the C3 and C3-C4 plants were very similar to each other in terms of electrophoretic mobilities on gels and isoenzyme patterns on IEF gels, and identical in peptide mapping. Quantitative differences were noted, however, in that the C3-C4 intermediate plant contained more PEPCase overall and that the relative activity of individual isoenzymes shifted between the C3 and C3-C4 intermediate PEPCases. The PEPCase from the C4 plant had a different isoenzyme pattern, a different peptide map, and was far more abundant than the other two enzymes. Western blot analysis demonstrated the cross-reactivity of PEPCases from all three Flaveria species with antibody raised against maize PEPCase. The results provide evidence, at the molecular level, that supports the view of C3-C4 intermediate species as C3-like plants with some C4-like photosynthetic characteristics, but there are differences from the C3 plant in the quantity and properties of the PEPCase from the C3-C4 intermediate plant.

We have cloned a full length cDNA for the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase from the C4 dicot Flaveria trinervia, deduced the complete nucleotide sequence of this cDNA and derived the amino acid sequence of the corresponding 1) Stiekema, W.