Vicilin and Napin Storage-Protein Gene Promoters Are Responsive to Abscisic Acid in Developing Transgenic Tobacco Seed but Lose Sensitivity following Premature Desiccation

In transgenic tobacco (Nicotiana tabacum L.) seed, expression of chimeric [beta]-glucuronidase (GUS) genes containing the vicilin or napin storage-protein gene promoters is responsive to premature drying and declines upon rehydration (L. Jiang, W.L. Downing, C.L. Baszczynski, A.R. Kermode [1995] Plant Physiol 107: 1439-1449). Desiccation may cause changes in the content of or sensitivity to abscisic acid (ABA), partially or wholly removing the effects of this "modulator" of developmental gene expression. We studied the enhancement of GUS reporter enzyme activities in transgenic tobacco by exogenous ABA before and after drying. A racemic mixture of ABA at 10 [mu]M caused a 2- to 3-fold increase in GUS activity in developing transgenic seed expressing chimeric genes containing the vicilin or napin gene promoters. However, when these seeds were prematurely dried prior to their culture on ABA medium, enhancement of GUS activity was virtually abolished. Use of optically pure ABAs revealed that the enhancement in GUS activity in developing fresh seed was due largely to the natural (+) form of ABA. Chimeric constructs containing a viral 35S promoter did not respond to ABA whether or not premature drying was applied. Thus, vicilin and napin chimeric genes, initially sensitive to ABA, become relatively insensitive to the hormone following drying. A decline in ABA sensitivity may be an important factor in the cessation of storage-protein gene expression.     

Module-specific regulation of the beta-phaseolin promoter during embryogenesis

The 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 -295phas promoter. Only 2.6% of -295phas 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 (-70 to -64) 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 -277 to -271, -260 to -254, and -237 to -231 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.     

Cloning and expression of the pea gene encoding SBP65, a seed-specific biotinylated protein

Besides biotin-dependent carboxylases, which play key roles in basic metabolism, SBP65 (seed biotinylated protein of 65 kDa of apparent molecular mass), an atypical biotinylated protein, has been described in pea plants. This seed-specific protein is devoid of any carboxylase activity, and shares many physiological and molecular features with late embryogenesis-abundant (Lea) proteins. In a first step toward understanding the role of this peculiar protein, we have demonstrated the role of abscisic acid (ABA) and of the osmotic environment on its expression using northern blot analysis from immature embryos cultured in vitro and germinating mature seeds. Moreover, the cloning and characterization of its gene (referred to as sbp gene) allowed us to define various potential cis-acting elements within the promoter region to account for the observed strict seed-specific expression. The results described in this paper are consistent with a model in which ABA regulates, at least in part, expression of this gene. However, unlike most lea genes, ABA regulation of the sbp gene seems to occur in a very restricted fashion, being confined only to particular stages of embryo development. Such a strict spatial and temporal expression pattern is dependent on the osmotic environment of the developing embryos and on tissue-specific factors, presumably preventing biotin depletion in cells requiring this essential cofactor for basic metabolic activity.     

Abscisic acid-inducible nuclear proteins bind to bipartite promoter elements required for ABA response and embryo-regulated expression of the carrot<Emphasis Type="Italic"> Dc3</Emphasis> gene

The carrot (Daucus carota L.) lea-class gene Dc3 is expressed in developing seeds and in vegetative tissues subject to drought and treatment with exogenous abscisic acid (ABA). Cis regulatory elements involved in seed-specific expression and in response to ABA were identified in transgenic tobacco (Nicotiana tabacum L.) using -glucuronidase (GUS) reporter gene constructs containing a series of deletion and orientation mutants of the Dc3 promoter. These experiments demonstrated that the Dc3 promoter is comprised of a proximal promoter region (PPR) and a distal promoter region (DPR). TCGTGT motifs in the DPR in combination with the PPR comprise a novel, bipartite ABA module in the Dc3 gene promoter. The PPR contains cis-acting elements responsible for the developmental regulation of Dc3 expression in seeds. Five similar sequence motifs with the consensus ACACgtGCa were identified in the PPR. Both DPR and PPR interact with common nuclear proteins that are present in embryos and are inducible by ABA in vegetative tissues.