Degradation of ß-conglycinin ß-homotrimer by papain: independent occurrence of limited and extensive proteolysis.

Limited and extensive proteolysis occur when ß-conglycinin ß homo-trimer (ß(3)-conglycinin) from soybeans is attacked by papain. Slow limited proteolysis is restricted to cleavage of ß(3)-conglycinin polypeptides into subunit halves (N- and C-terminal domains) that are further slightly truncated. The kinetics of limited and extensive proteolyses analyzed separately indicates that the two processes occur independently from the very beginning of the reaction. In contrast, limited proteolysis of phaseolin from common beans has been found to be prerequisite for the onset of its extensive proteolysis. The dramatic distinction between the degradation patterns of ß(3)-conglycinin and phaseolin, homologous storage 7S globulins, suggests the existence of intrinsic differences in their structures. This hypothesis is supported by comparative analysis of the accessibilities to the solvent of amino acid residues in phaseolin and ß(3)-conglycinin structures, which indicated the relatively low packing density of the latter, resulting in enhanced susceptibility of it to extensive proteolysis.

Limited proteolysis regulates massive degradation of glycinin, storage 11S globulin from soybean seeds: an in vitro model.

The time course of glycinin hydrolysis by papain was followed using densitometry of SDS-PAGE patterns, quantification of the residual protein and determination of its molecular mass by gel filtration, and by other appropriate methods. The hydrolysis occurs in two steps. In the first step, a limited proteolysis was observed consisting of a gradual detachment of the α-chain C-terminal sequence region, leading to the formation of glycinin-P, a relatively stable proteolysis product retaining the primordial hexameric structure. Glycinin-P was found to be composed of the intact ß-chains covalently bound with the C-terminally truncated α-chains lacking the helix domain, strand J', and the C-terminal disordered region. Glycinin-P is further hydrolyzed in the second step exclusively by a one-by-one mechanism. Comparison of the kinetics of the limited and one-by-one proteolyses analyzed separately indicated that the decrease of protein concentration by 24-25% in the first step occurs almost exclusively due to the decrease of the molecular mass of the residual protein. Thus, the onset of the one-by-one proteolysis is delayed, suggesting a regulatory role of the preceding limited proteolysis in the subsequent massive degradation of glycinin. Probable structural alterations of glycinin generated by this limited proteolysis are discussed.

Identification and genetic analysis of the APOSPORY locus in Hypericum perforatum L.

The introduction of apomixis - seed formation without fertilization - into crop plants is a long-held goal of breeding research, since it would allow for the ready fixation of heterozygosity. The genetic basis of apomixis, whether of the aposporous or the diplosporous type, is still only poorly understood. Hypericum perforatum (St John's wort), a plant with a small genome and a short generation time, can be aposporous and/or parthenogenetic, and so represents an interesting model dicot for apomixis research. Here we describe a genetic analysis which first defined and then isolated a locus (designated HAPPY for Hypericum APOSPORY) associated with apospory. Amplified fragment length polymorphism (AFLP) profiling was used to generate a cleaved amplified polymorphic sequence (CAPS) marker for HAPPY which co-segregated with apospory but not with parthenogenesis, showing that these two components of apomixis are independently controlled. Apospory was inherited as a dominant simplex gene at the tetraploid level. Part of the HAPPY sequence is homologous to the Arabidopsis thaliana gene ARI7 encoding the ring finger protein ARIADNE7. This protein is predicted to be involved in various regulatory processes, including ubiquitin-mediated protein degradation. While the aposporous and sexual alleles of the HAPPY component HpARI were co-expressed in many parts of the plant, the gene product of the apomict's allele is truncated. Cloning HpARI represents the first step towards the full characterization of HAPPY and the elucidation of the molecular mechanisms underlying apomixis in H. perforatum.

Phylogenetic footprints in fern spore- and seed-specific gene promoters.

Spermatophyte seed-storage proteins have descended from a group of proteins involved in cellular desiccation/hydration processes. Conserved protein structures are found across all plant phyla and in the fungi and Archaea. We investigated whether conservation in the coding region sequence is paralleled by common gene regulatory processes. Seed- and spore-specific gene promoters of three phylogenetically diverse plants were analysed by transient and transgenic expression in Arabidopsis thaliana and tobacco. The transcription factors FUS3 and ABI3, which are central regulators of seed maturation processes, interact with cis-motifs of seed-specific promoters from distantly related plants. The promoter of a fern spore-specific gene encoding a seed-storage globulin-like protein exhibits strong seed-specific activity in both Arabidopsis and tobacco. The existence of phylogenetic footprints indicates good conservation of regulatory pathways controlling gene expression in fern spores and in gymnosperm and angiosperm seeds, reflecting the concerted evolution of coding and regulatory regions.

EFFECTOR OF TRANSCRIPTION2 is involved in xylem differentiation and includes a functional DNA single strand cutting domain.

EFFECTORS OF TRANSCRIPTION2 (ET) are plant-specific regulatory proteins characterized by the presence of two to five C-terminal DNA- and Zn-binding repeats, and a highly conserved cysteine pattern. We describe the structural characterization of the three member Arabidopsis thaliana ET gene family and reveal some allelic sequence polymorphisms. A mutation analysis showed that AtET2 affects the expression of various KNAT genes involved in the maintenance of the undifferentiated state of cambial meristem cells. It also plays a role in the regulation of GA5 (gibberellin 3-beta-dioxygenase) and the cell-cycle-related GASA4. A correlation was established between AtET2 expression and the cellular differentiation state. AtET-GFP fusion proteins shuttle between the cytoplasm and nucleus, with the AtET2 product prevented from entering the nucleus in non-differentiating cells. Within the nucleus, AtET2 probably acts via a single strand cutting domain. A more general regulatory role for ET factors is proposed, governing cell differentiation in cambial meristems, a crucial process for the development of plant vascular tissues.