Mutations affecting spatial and temporal regulation of a beta‐phaseolin gene encoding the major storage protein of bean (Phaseolus vulgaris) were analyzed by stable and transient transformation approaches. The results substantiate the value of transient assays for rapid determination of the functionality of cis‐acting sequences and the importance of stable transformation to identify tissue‐specific determinants. Spatial information is specified primarily by two upstream activating sequences (UAS). UAS1 (−295 to −109) was sufficient for seed‐specific expression from both homologous and heterologous (CaMV 35S) promoters. In situ localization of GUS expression in tobacco embryos demonstrated that UAS1 activity was restricted to the cotyledons and shoot meristem. A second positive domain, UAS2 (−468 to −391), extended gene activity to the hypocotyl. Temporal control of GUS expression was found to involve two negative regulatory sequences, NRS1 (−391 to −295) and NRS2 (−518 to −418), as well as the positive domain UAS1. The deletion of either negative element caused premature onset of GUS expression. These findings indicate combinatorial interactions between multiple sequence motifs specifying spatial information, and provide the first example of the involvement of negative elements in the temporal control of gene expression in higher plants.

A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.

A 0.8-kilobase fragment from the 5'-flanking region of a French bean beta-phaseolin gene yielded strong, temporally regulated, and embryo-specific expression of beta-glucuronidase (GUS) in transgenic tobacco plants, paralleling that found for the seed protein phaseolin [Sengupta-Gopalan, C., Reichert, N.A., Barker, R.F., Hall. T.C., and Kemp, J.D. (1985) Proc. Natl. Acad. Sci. USA 82, 3320-3324]. Gel retardation and footprinting assays using nuclear extracts from immature bean cotyledons revealed strong binding of nuclear proteins to an upstream region (-628 to -682) that contains two inverted A/T-rich motifs. Fusion of a 103-base pair fragment or a 55-base pair synthetic oligonucleotide containing these motifs to a minimal 35S promoter/GUS cassette yielded strong GUS expression in several tissues. A different pattern of GUS expression was obtained in immature embryos and germinating seedlings from the nominally constitutive, full-length, 35S promoter. Whereas GUS expression under the control of the 0.8-kilobase beta-phaseolin regulatory region is limited to immature embryos, expression from constructs containing the A/T-rich motifs is strongest in roots. These data, combined with S1 mapping, provide direct evidence that a plant upstream A/T-rich sequence that binds nuclear proteins can activate transcription in vivo. They also indicate that additional regulatory elements in the beta-phaseolin 5'-flanking region are required for embryo-specific gene expression.

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.

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