Search

Families of papain- and legumain-like cysteine proteinases (CPR) were found in Vicia seeds. cDNAs and antibodies were used to follow organ specificity and the developmental course of CPR-specific mRNAs and polypeptides. Four papain-like cysteine proteinases (CPR1, CPR2, proteinase A and CPR4) from vetch seeds (Vicia sativa L.) were analysed. CPR2 and its mRNA were already found in dry embryonic axes. CPR1 was only detected there during early germination. Both CPR1 and CPR2 strongly increased later during germination. In cotyledons, both CPR1 and CPR2 were only observed one to two days later than in the axis. Proteinase A was not found in axes. In cotyledons it could only be detected several days after seeds had germinated. CPR4 mRNA and polypeptide were already present in embryonic axes and cotyledons during seed maturation and decreased in both organs during germination. Purified CPR1, CPR2 and proteinase A exhibited partially different patterns of globulin degradation products in vitro. Although the cDNA-deduced amino acid sequence of the precursor of proteinase A has an N-terminal signal peptide, the enzyme was not found in vacuoles whereas the other papain-like CPRs showed vacuolar localization. Four different legumain-like cysteine proteinases (VsPB2, proteinase B, VnPB1 and VnPB2) of Vicia species were analysed. Proteinase B and VnPB1 mRNAs were detected in cotyledons and seedling organs after seeds had germinated. Proteinase B degraded globulins isolated from mature vetch seeds in vitro. VsPB2 and proteinase B are localized to protein bodies of maturing seeds and seedlings, respectively, of V. sativa. Like VsPB2 from V sativa, also VnPB2 of V. narbonensis corresponds to vacuolar processing enzymes (betaVPE). Based on these results different functions in molecular maturation and mobilization of storage proteins could be attributed to the various members of the CPR families.

A single-chain Fv antibody (scFv) gene, which has previously been used to immunomodulate abscisic acid (ABA) activity in transgenic tobacco to create a 'wilty' phenotype, was put under control of the seed-specific USP promoter from Vicia faba and used to transform tobacco. Transformants were phenotypically similar to wild-type plants apart from their seeds. Anti-ABA scFv embryo development differed markedly from wild-type embryo development. Seeds which accumulated similar levels of a scFv that binds to oxazolone, a hapten absent from plants, developed like wild-type embryos. Anti-ABA scFv embryos developed green cotyledons containing chloroplasts and accumulated photosynthetic pigments but produced less seed storage protein and oil bodies. Anti-ABA scFv seeds germinated precociously if removed from seed capsules during development but were incapable of germination after drying. Total ABA levels were higher than in wild-type seeds but calculated free ABA levels were near-zero until 21 days after pollination. We show for the first time seed-specific immunomodulation and the resulting switch from the seed maturation programme to a germination programme. We conclude that the immunomodulation of hormones can alter the development programme of target organs, allowing the study of the directly blocked endogenous molecules and manipulation of the system concerned.

cDNA and genomic clones encoding narbonin, a 2S globulin from the seed of narbon bean (Vicia narbonensis L.), were obtained using the polymerase chain reaction (PCR) and sequenced. The full-length cDNA as well as genomic clones contain a single open reading frame (ORF) of 873 bp that encodes a protein with 291 amino acids comprising the mature narbonin polypeptide (M(r) ca. 33 100) and an initiation methionine. The deduced amino acid sequence lacks a transient N-terminal signal peptide. The genomic clones do not contain any intron. No homology was found to nucleic acid and protein sequences so far registered in sequence data libraries. The biosynthesis of narbonin during embryogenesis is developmentally-regulated and its pattern of synthesis closely resembles that of typical seed storage globulins. However, during seed germination narbonin was degraded very slowly, indicating that it may have other function than storage protein. Southern analysis suggests the existence of a small narbonin gene family. Narbonin genes were also found in four different species of the genus Vicia as well as in other legumes such as Canavalia ensiformis and Glycine max. In Escherichia coli a recombinant narbonin was produced which yielded crystals like those prepared from narbonin purified from seeds.

Proteinase B, an asparagine-specific endopeptidase, has been purified from germinating vetch (Vicia sativa L.) seeds. The final preparation consists of two enzymically active proteins with molecular masses of approximately 39 kDa and 37 kDa. Synthetic substrates were used to confirm cleavage specificity of the proteinase B preparation. As expected, the enzyme cleaves the substrates at the C-terminal side of Asn residues. The octapeptide ETRNGVEE was digested most efficiently. When Gly was replaced by Ile or Glu, cleavage took place with lower efficiency. Polyclonal antibodies displayed both proteins in cotyledon extracts of germinated vetch seeds. In addition, a strong cross-reacting protein band was found in cotyledon extracts of developing seeds, indicating the presence of a very similar enzyme during seed development. cDNA clones encoding proteinase B precursor have been obtained on the basis of the N-terminal amino acid sequence DDDFEGTRWAILLAGS, by means of the polymerase chain reaction. The cDNA clones contain an open reading frame of 1479 bp encoding a polypeptide of 493 amino acids. The precursor displayed 59% sequence identity to the cDNA-derived amino acid sequence of a vacuolar Asn-specific enzyme from the developing castor beam endosperm which is thought to catalyze the post-translational processing of pro-proteins into the mature forms. Proteinase B is synthesized de novo during seed germination. The results of Southern-blot analyses suggested that there are at least two genes for proteinase B.