||Abscisic acid is a plant hormone involved in seed development and abiotic stress adaptation. Numerous lines of evidence support the existence of multiple ABA signaling pathways, as well as overlapping yet separable ABA and stress response pathways in plants. Our long term objective is to use the Dc3-GUS reporter system in transgenic Ambidopsis to isolate genes affecting vegetative tissue-specific responses to ABA and drought stress. Dc3 is a member of the Late Embryogensis Abundant (LEA) class of genes expressed in developing seeds and stressed leaves and is thought to function in desiccation tolerance. Recombinant a ba (ABA-deficient), abil, and abi2 (ABA-_ insensitive) mutant lines were employed to validate the interactions of ABA as well as osmotic stress signaling with the carrot Dc3 promoter. Semi- quantitative histochemical analysis of GUS activities with the chromogenic substrate X-gluc provided evidence for synergy between ABA and drought stress responses, especially in roots. ABA and drought signaling pathways were shown to operate in root, vascular tissues and guard cells. ABIl and AB12 genes differentially affected guard cell expression of Dc3-GUS in response to ABA or mannitol, a non-penetrating solute that simulates drought stress. Quantitative GUS assays on leaves showed that ABIl acts primarily in drought-regulated Dc3 expression and AB12 acts primarily on ABA-regulated Dc3 expression. These results support the hypothesis that there are separate yet overlapping drought and ABA- response pathways, with ABIl and AB12 differentially involved in both. Northern blot analysis of the endogenous ABA and drought-regulated gene RD29A showed synergy between ABA and mannitol-induced transcript accumulation, consistent with the Dc3-GUS results. Furthermore, expression of RD29A as well as another stress- and ABA-inducible marker gene, COR47, was reduced in the abi2 mutants, consistent with ABI2 function in ABA and drought signaling. Taken together, these studies validate Dc3 as a suitable marker gene to genetically dissect ABA and stress signaling pathways in Arabidopsis. The molecular genetic characterization of ABA signaling mutants in Ara bid opsis may lead to strategies for genetic engineering of crops with improved responses to drought and other stresses and with more desirable seed traits.