Differential accumulation of four phaseolin glycoforms in transgenic tobacco

Abstract

An intron-less phaseolin gene was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical beta-phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (beta wti-) and mutant phaseolin glycoforms (beta dgly1, beta dgly2 and beta dgly1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the beta dgly1 and beta dgly2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin beta dgly1,2 gene. Additionally, the profile of 25-29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.

Heat‐induced synthesis and tunicamycin‐sensitive secretion of the putative storage glycoprotein conglutin γ from mature lupin seeds

“…Covalently linked carbohydrate may stabilize or control the conformation of a glycoprotein, its assembly and solubility [33], thus triggering the correct folding and/or oligomerization of the secretion-competent form of the protein [34-371. Indeed lack of glycosylation has proved to alter the accumulation and stability of a modified storage protein [38]. We cannot exclude that this might also be the case for conglutin y ; indeed a decrease of solubility during chemical and enzymatic deglycosylation of purified conglutin y has been observed (Duranti, M. and Gius, C., unpublished results).…”

Protein quality control along the route to the plant vacuole.

“…The introduction of a peptide enriched in methionine residues in the structure of phaseolin does not make the protein incompetent for transport, but the mutated phaseolin is much more unstable than is the wild-type counterpart once it reaches the storage vacuoles of transgenic tobacco seeds, suggesting that its conformation has been altered (Hoffman et al, 1988;Pueyo et al, 1995). In addition, fully unglycosylated phaseolin is considerably less stable than is glycosylated phaseolin when expressed in transgenic tobacco seeds, and again, instability is due to degradation in protein storage vacuoles (Bustos et al, 1991). Trimerization of phaseolin is unaffected by the lack of glycans and is only partially affected by the introduction of the methionine-rich peptide (Hoffman et al, 1988).…”

The Bean Seed Storage Protein [beta]-Phaseolin Is Synthesized, Processed, and Accumulated in the Vacuolar Type-II Protein Bodies of Transgenic Rice Endosperm

“…The exact chemical nature of oligosaccharide side chains in these /3-phaseolin glycoforms from rice endosperms remains to be elucidated. Even though the precise role that glycosylation plays in the mature phaseolin is not known, removal of glycosylation sites from phaseolin or phytohemagglutinin did not interfere with correct targeting of the nonglycosylated proteins into the vacuolar protein bodies, although it reduced protein accumulation in the protein bodies (Voelker et al, 1989;Busto et al, 1991).…”