Module‐specific regulation of the β‐phaseolin promoter during embryogenesis

Abstract

SummaryThe phas promoter displays stringent spatial regulation, being very highly expressed during embryogenesis and completely silent during all phases of vegetative development in bean, Phaseolus vulgaris. This pattern is maintained in transgenic tobacco and, as shown here, Arabidopsis. Dimethyl sulphate in vivo footprinting analyses revealed that over 20 cis-elements within the proximal 295 bp of the phas promoter are protected by factor binding in seed tissues whereas none are bound in leaves. The hypothesis that this complex profile represents a summation of several module (cotyledon, hypocotyl, and radicle)-specific factor-DNA interactions has been explored by the incorporation of site-directed substitution mutations into 10 locations within the À295phas promoter. Only 2.6% of À295phas promoter activity remained after mutation of the G-box; the CCAAAT box, the E-box and the RY elements were also found to mediate high levels of expression in embryos. Whereas the CACA element has dual positive and negative regulatory roles, the vicilin box was identified as a strong negative regulatory element. The proximal (À70 to À64) RY motif was found to bestow expression in the hypocotyl while all the RY elements contribute to expression in cotyledons but not to vascular tissue expression during embryogenesis. RY elements at positions À277 to À271, À260 to À254, and À237 to À231 were found to orchestrate radicle-specific repression. The G-box appears to be the functional abscisic acid responsive element and the E-site may be a coupling element. The results substantiate the concept that autarkical cis-element functions generate modular patterning during embryogenesis. They also reflect the existence of both redundancy and hierarchy in cis-element interactions. Importantly, the virtually identical expression patterns observed for the two distantly related plants studied argue strongly for the generality of function for the observed factor-element interactions.

Sequence and Spacing of TATA Box Elements Are Critical for Accurate Initiation from the β-Phaseolin Promoter

“…2, histochemical staining patterns for embryos harboring mTATA1-gus (B) or mTATA2-gus (C) were similar to that obtained for embryos bearing the Ϫ295phas-gus control (A). The absence of GUS expression in the radicle is consistent with earlier findings that expression in this region is mediated by distal regulatory elements upstream of bp Ϫ295 (14). As with Ϫ295phas-gus control plants, no GUS expression could be detected in leaves of the stably transformed Arabidopsis plants harboring the TATA mutants (data not shown).…”

“…Plasmid Constructions-Promoter fragments bearing mutations in the TATA region were digested with HindIII and NcoI, and ligated to HindIII/NcoI-digested vector Ϫ295phas-gus-3Ј/pUC (14). Subsequently, the constructs were digested with HindIII and EcoRI to release fragments containing the mutant Ϫ295phas promoter, reporter gene and the 3Ј region.…”

“…The phas gene is under strict developmental and tissue-specific control (14,15), primarily at the level of transcription initiation (16). The AT rich proximal promoter region of phas contains a complex array of transcription initiation regulatory elements within 45 bp of the transcription start site.…”

Using Genomics to Study Legume Seed Development

“…Research in several laboratories with legume seed protein genes, such as b-conglycinin, glycinin, Kunitz trypsin inhibitor, and lectin, showed that their mRNA accumulation patterns are regulated temporally and spatially (Goldberg et al, 1981a(Goldberg et al, , 1983Meinke et al, 1981;Rerie et al, 1992) and controlled by both transcriptional and posttranscriptional processes (Evans et al, 1984;Beach et al, 1985;Chappell and Chrispeels, 1986;Walling et al, 1986). Subsequent work determined that cisregulatory sequences flanking legume seed protein genes could confer embryo-specific expression patterns in heterologous plants, such as tobacco (Nicotiana tabacum) and petunia (Petunia hybrida; Chen et al, 1986;Okamuro et al, 1986;Jofuku et al, 1987;Higgins et al, 1988;Naito et al, 1988;Baumlein et al, 1992;Wohlfarth et al, 1998;Chandrasekharan et al, 2003).…”

Repression of theLEAFY COTYLEDON 1/B3Regulatory Network in Plant Embryo Development byVP1/ABSCISIC ACID INSENSITIVE 3-LIKEB3 Genes

“…Activation of downstream genes is mediated by specific binding of the B3 domain (Suzuki et al, 1997;Kroj et al, 2003;Carranco et al, 2004;Monke et al, 2004;Braybrook et al, 2006) to the Sph/RY cis-element (Hattori et al, 1992;Kao et al, 1996;Ezcurra et al, 1999;Reidt et al, 2000;Chandrasekharan et al, 2003;Nag et al, 2005). In addition, ABI3 mediates ABA-regulated gene expression in the seed through interaction with specific basic Leu-zipper transcription factors that bind ABA response elements (Finkelstein et al, 2002).…”