Differential accumulation of four phaseolin glycoforms in transgenic tobacco.
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.
Bustos
M M
MM
Department of Biology, Texas A & M University, College Station 77843-3258.
Kalkan
F A
FA
VandenBosch
K A
KA
Hall
T C
TC
D000595:Amino Acid Sequence; D001483:Base Sequence; D007887:Fabaceae / Q000235:genetics; D006031:Glycosylation; D016253:Microscopy, Immunoelectron; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009693:Nucleic Acid Hybridization; D010940:Plant Proteins / Q000378:metabolism*; D010946:Plants, Medicinal; D010947:Plants, Toxic; D011134:Polysaccharides / Q000378:metabolism*; D012333:RNA, Messenger / Q000032:analysis; D011994:Recombinant Proteins / Q000378:metabolism*; D012639:Seeds / Q000378:metabolism; D014026:Nicotiana / Q000235:genetics* / Q000378:metabolism; D014170:Transformation, Genetic / Q000502:physiology
D010940:Plant Proteins; D011134:Polysaccharides; D012333:RNA, Messenger; D011994:Recombinant Proteins; C048826:phaseolin protein, Phaseolus vulgaris