Abstract
Abscisic acid (ABA) regulates seed maturation, germination, and adaptation of vegetative tissues to environmental stresses. The mechanisms of ABA action and the specificity conferred by signaling components in overlapping pathways are not completely understood. The ABI5 gene (ABA insensitive 5) of Arabidopsis encodes a basic leucine zipper factor required for ABA response in the seed and vegetative tissues. Using transient gene expression in rice protoplasts, we provide evidence for the functional interactions of ABI5 with ABA signaling effectors VP1 (viviparous 1) and ABI1 (ABA insensitive 1). Co-transformation experiments with ABI5 cDNA constructs resulted in specific transactivation of the ABA-inducible wheat Em, Arabidopsis AtEm6, bean beta-Phaseolin, and barley HVA1 and HVA22 promoters. Furthermore, ABI5 interacted synergistically with ABA and co-expressed VP1, indicating that ABI5 is involved in ABA-regulated transcription mediated by VP1. ABI5-mediated transactivation was inhibited by overexpression of abi1-1, the dominant-negative allele of the protein phosphatase ABI1, and by 1-butanol, a competitive inhibitor of phospholipase D involved in ABA signaling. Lanthanum, a trivalent ion that acts as an agonist of ABA signaling, potentiated ABI5 transactivation. These results demonstrate that ABI5 is a key target of a conserved ABA signaling pathway in plants.
Highlights
Abscisic acid (ABA) regulates seed maturation, germination, and adaptation of vegetative tissues to environmental stresses
To test the role of ABI5 in ABA signaling in rice protoplasts and further examine the conservation of ABA signaling machinery among species, we measured the effect of overexpressed ABI5 cDNA, driven by the Ubi promoter, on various ABA-inducible promoters
We have demonstrated synergistic interactions of ABA with ABI5 and VP1, alone and in combination, in transient gene expression of both monocot and dicot ABA-inducible promoters in rice protoplasts
Summary
Plant Materials—Embryonic rice suspension cultures (Oryza sativa L. cv IR-54) were kindly provided by Dr W. Aliquots of transformed protoplast samples were treated with or without ABA and pharmacological agents for 16 h in the dark in a final volume of 0.8 ml of Krens solution. Prior to use, required dilutions of ABA, lanthanum chloride, and 1-butanol were made in Krens solution, and control samples received the same volumes of solvents as in ABA and pharmacological treatments. A construct (pDH359) containing the maize Ubi promoter [52] driving 1.4-kb ABI5 cDNA was created by digesting pDH349 (Ubi::VP1-Myc) with EcoRI and filling in the linearized product with Klenow fragment before digesting with BamHI to release the VP1-Myc fragment. Fold induction (ABA treatment and/or ABI5 transactivation) were calculated relative to control (zero ABA added, equal to unity) in paired samples co-transformed with either UbiϻABI5 cDNA effector construct or Ubiϻexpression vector alone and treated as described under “Experimental Procedures.”. The values are the averages Ϯ S.E. of four replicate transformations
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