Sulfur-rich 2S proteins in Lecythidaceae and their methionine-enriched forms in transgenic plants

Abstract

The 28 seed proteins from three members of the Brazil nut family (Lecythidaceae), monkeypot, cannonball, and Brazil nut, were isolated, characterized, and compared in regard to physical and chemical properties including subunit structure, amino acid composition, immunoreactivity, N-terminal sequence, and processing. These 28 proteins contain about 24% sulfur amino acids, thus they are sulfur-rich proteins (8RPs). The 28 8RPs are major seed proteins in the three plant species and all consist of two subunits, 9 kD and 3 kD, linked by disulfide bonds. In addition, these 28 8RPs share a high degree of similarity in subunit structure, amino acid composition, immunoreactivity, and N-terminal sequence. However, the precursor processing patterns of these 28 proteins are distinctly different. In monkeypot and Brazil nut, the precursor is cleaved into mature subunit polypeptides in three steps, i.e. 18 kD → 15 kD → 12 kD → 9 + 3 kD, while in cannonball, only two steps are detected, i.e. 18 kD → 15 kD → 9 + 3 kD. A total of 9 cDNA clones encoding the 28 8RPs in monkeypot and cannonball have been isolated and characterized. DNA sequence analysis reveals that the 28 8RPs isolated from different members of the Brazil nut family share a high degree of homology in amino acid (>80%) and nucleotide (>90%) sequences. The methionine (Met) residues are clustered in two areas of the variable region (between the 6th and 7th Cys residues) of the larger subunit. The 2S SRPs are encoded by multigene families and can be classified into two subfamilies. Native monkeypot 2S SRP (MP2S) contains 16 Mol% Met. The variable region of the MP2S gene was modified through nucleotide sequence alternations to increase the protein's Met content. Eight Met-enriched MP2S genes were engineered to increase the Met varying amounts ranging from 18 to 24 Mol%. To test the effect of these modifications on the stability of the MP2S, chimeric genes containing coding sequences of three modified (19, 21, and 23% Met) and the wild type MP2S cDNA were transferred into tobacco plants via the Agrobacterium transformation system. Northern and Western blot analyses demonstrated that the genes for the modified MP2S were expressed in the transgenic tobacco seeds at levels comparable to that of the wild type MP2S gene. Both the wild type and modified MP2S proteins were correctly processed into mature subunits. These results suggest that the wild type as well as the modified MP2S genes are suitable candidates for use in protein quality improvement.