Molecular analysis of interacting abscisic acid signaling effectors in protoplasts

Abscisic acid (ABA) is a plant hormone that plays an important role in growth and development, regulation of seed maturation, germination, and adaptation to environmental stresses. ABA signaling pathways overlap with other signaling pathways such as hormone-, developmental-, sugar-, and stress-response pathways suggesting a complex network of interactions. Although physiochemlcal and molecular genetic approaches have provided fundamental insights into ABA signaling, our knowledge about transcriptional regulation of ABA signaling and components required for ABA signal transduction is far from complete.

I have taken a multidisciplinary approach involving biochemical, molecular, genetic and pharmacological techniques to characterize ABA signaling in protoplasts. I have developed and utilized transient gene expression in protoplasts to functionally analyze the interactions of ABA regulatory genes {ABA INSENSITIVE-1, -3 and-5-Like gene families). Using a pharmacological approach, I demonstrated that phospholipase D (PLD) functions in ABA-inducible gene expression in rice. Antagonism of ABA, VIVIPAR0US1 (VP1) and lanthanum synergy by 1-butanol, a specific inhibitor of PLD, was similar to the inhibition by co-expression of ABI1-1. These results have shown that ABA, VP1, lanthanum, PLD and ABM are all involved in ABA-regulated gene expression and are consistent with an integrated model whereby La^"" acts upstream of PLD. Using transient gene expression in rice and maize protoplasts, I have shown that overexpressed ABI5 transactivates several ABA-inducible promoters from both monocots and dicots and synergizes with ABA and co-expressed VP1. ABI5 also interacted with other known ABA signaling effectors such as ABU, lanthanum and PLD to regulate ABA-inducible gene expression demonstrating that ABI5 is a key target of a conserved ABA signaling pathway in plants. Based on my results, I propose that overexpression of ABI5-Like family members in combination with VP1 will confer value-added traits to transgenic plants including drought and salt tolerance, viability under stress and tissue-specific target gene expression. Reverse genetic analysis of ABF3, a member of the ABI5-Like family, demonstrated that ABF3 mutants are insensitive to ABA-inhibition of root growth confirming the role o1 ABF3 in vegetative ABA responses. Regulation of ABA signaling by post-transcriptional mRNA processing is discussed.