Developmentally regulated expression of the bean β-phaseolin gene in tobacco seed

Abstract

Recombinant phage X177.4 contains a gene for j3 phaseolin, a major storage glycoprotein of French bean seed. A 3.8-kilobase Bgl II-Bam}Il fragment containing the entire 1700-base-pair coding region, together with 863 base pairs of 5' and 1226 base pairs of 3' flanking sequence, was inserted into the A66 Ti plasmid of Agrobacterium tumefaciens and used to transform tobacco. The level of phaseolin in the seeds of plants regenerated from cloned tissue was 1000-fold higher than in other tissues. The molecular weight of the phaseolin RNA transcript in tobacco seeds was identical to that found in bean seeds. The phaseolin protein in tobacco seed was glycosylated and appeared to undergo removal of the signal peptide. However, a large proportion of the phaseolin was cleaved into discrete peptides. These same peptides were formed as phaseolin was degraded during tobacco seed germination. The phaseolin gene appeared to be inserted as a single copy, and the proportion of phaseolin per genome copy in tobacco seeds (up to 3% of the total embryo proteins) resembled that in the bean seeds (40% of total seed protein, expressed from about 14 copies per diploid genome). Furthermore, the transplanted gene was turned on during tobacco seed development, and its protein product, phaseolin, was localized in the embryonic tissues. Finally, the phaseolin gene was inherited as a Mendelian dominant trait in tobacco.The transfer of foreign genetic information to broad-leafed plants by means (4) and maintained on Murashige and Skoog medium (MS) without hormone supplement. The tissue was cloned by the feeder plate method (9) and then placed in liquid culture to induce shoot-stem elongation (10). The shoots were grafted (11) onto 6-to 8-week-old N. tabacum var. Xanthi plants and grown at 22°C with a 16-hr photoperiod. Flowers were self-pollinated, and the seeds were allowed to mature.Quantitative and Qualitative Protein Assays. Proteins were extracted and quantified as described (8). Phaseolin was quantitated in tissues by dot-immunobinding assay (12). Protein patterns were analyzed after fractionation on a 13% polyacrylamide gel (13) or by two-dimensional gel electrophoresis (14) followed by electrophoretic immunoblot analysis with polyclonal antiserum to phaseolin (15). Antigenantibody complexes were visualized by treating the filters with 251I-labeled Staphylococcus protein A, followed by autoradiography.For immunodetection of concanavalin A-bound proteins, tissue extracts were incubated with concanavalin ASepharose beads followed by elution of the bound fraction with 1% NaDodSO4. The bound and unbound fractions were subjected to NaDodSO4/polyacrylamide gel electrophoresis followed by immunoblot analysis as described earlier.Isolation of RNA and Blot-Hybridization Analysis. Total RNA was isolated from leaves as described (16). RNA from developing seeds was prepared by isolation of polysomes followed by phenol extraction of the polysome pellet (17 3320The publication costs of this article were defrayed in part by page charge pay...

Accumulation of 15-Kilodalton Zein in Novel Protein Bodies in Transgenic Tobacco

“…3B). As reported earlier (Sengupta-Gopalan et al, 1985), in addition to the authentic-size /3-phaseolin protein, the seeds also showed the accumulation of small molecular mass immunoreactive products, probably representing specific cleavage products. Whereas the leaf and stem showed very low levels of small molecular mass immunoreactive bands, the roots showed accumulation of a major immunoreactive band of 29 kD (Fig.…”

Lysine synthesis and catabolism are coordinately regulated during tobacco seed development.

“…DHPS activity in the homozygous transgenic plant AE26 was also first detected at 14 days after anthesis but continued to rise dramatically until maturity ( Fig. 2A), as expected from its regulation by the phaseolin promoter (17). Free lysine accumulation in seeds of the control plants correlated with the activity of DHPS, reaching a maximum at 18-22 days after anthesis and then declining to its lowest level in mature seeds (Fig.…”

Seed-Specific Gene Activation Mediated by the Cre/lox Site-Specific Recombination System

“…In transgenic tobacco seeds analyzed as bulk samples, phaseolin protein (Sengupta-Gopalan et al, 1985) and P-phaseolin promoter-expressed mRNA (Riggs et al, 1989) or marker gene activity (Bustos et al, 1989) were first detected at a midmaturation stage, Deletion analysis of this promoter in transgenic tobacco revealed a complex set of regulatory regions within the sequence between +20 and -795 (Bustos et al, 1991). Since our promoter fragment extends only to -410, it retains only a subset of these domains.…”