A comparative analysis of the structure and function of VP1/ABI3 in moss and seed plants

The phytohormone abscisic acid (ABA) plays a role in the development and physiology of plants by regulating a variety of processes including seed development and responses to abiotic stress. The transcriptional regulator VIVIPAROUS1 (VP1)/ABSCISIC ACID INSENSITIVE 3 (ABI3) is involved in ABA signaling in seed development and is composed of four, highly conserved, functional domains, which have been characterized only in seed plants. Three ABI3-like genes (PpABI3) were isolated from the moss Physcomitrella patens and I undertook a comparative approach to understand the role of the B1 and B2 functional domains in the ABA response. I confirmed the activity of VP1 and ABI3 in the ABA response using the wheat Em promoter in cells of P. patens protonema and barley aleurone. I found that PpABI3A functions in the ABA response of protonemal cells using an ABA-responsive promoter from P. patens (PpLEA1). PpABI3A can also activate the Em promoter in aleurone, but is unable to fully complement the abi3-6 mutant phenotypes in Arabidopsis. I show that this is most likely due to a weak interaction between the B1 domain of PpABI3A and the bZIP transcription factor ABI5. I further demonstrate that the interaction of VP1/ABI3 and ABI5 requires a specific leucine in the B1 domain, corresponding to the abi3-8 mutation. However, altering only this site in B1 of PpABI3A is not sufficient for functional interaction with ABI5. Substituting the B1 domain from VP1 for the B1 in Pp ABI3A gave only partial recovery of activity, indicating that other domains are required. I isolated two ABI5-like genes from P. patens and show that PpABI5A fully interacts with VPI in protonema and aleurone. However, PpABI5A interacts with PpABI3A only in protonema and not aleurone cells. I show that the B2 domain is required for nuclear localization of VP1. I further defined a nuclear localization signal (NLS) by comparing alignments of B2 domains and confirmed it by site-directed mutagenesis. A single amino acid change in this NLS (i.e. the abi3-7 mutation) prevents activation of Em promoter but not nuclear localization, showing that the B2 domain is bi-functional, regulating both nuclear localization and transcriptional activation.