Abscisic Acid (ABA) Transport, Sensitivity and Receptor Engineering in Arabidopsis
Abscisic acid (ABA) is a phytohormone that regulates developmental processes and response to abiotic and biotic stresses. It mediates seed dormancy, inhibits seed germination and lateral root formation and controls seedling development. It also limits water loss in times of low water availability through stomatal closure. Core ABA signaling components consist of recently identified PYR/PYL/RCAR receptors that directly regulate clade A PP2C phosphatases. Inhibition of PP2C activity releases SnRK2 kinase inhibition, which enables phosphorylation of downstream targets.
In Chapter 1, I identified four ABA transporters, which were identified by their ability to promote ABA uptake into S. cerevisiae cells, as indicated by measuring PYR1-PP2C interactions. For each protein identified, I cloned selected homologues and measured their ability to promote ABA transport in to S. cerevisiae by measuring 3H labeled ABA uptake. I also determined Kms for the two strongest ABA transporters identified (NRT1.2 and SUC7) and examined whether other substrates altered their ABA transport. Seed germination and root assay data showed that knockout mutants for a subset of the identified genes possessed altered sensitivity to ABA compared with wild type, which suggests that the genes AT1G69850, AT1G66570, AT1G09380 and AT4G22790 may function as ABA transporters in Arabidopsis. Collectively, the data suggest that there are likely many ABA transporters in Arabidopsis that function redundantly or in different tissues.
In Chapter 2, I provided evidence for the existence and characteristics of ABA desensitization, measured at the transcriptional level. RNA-Seq analysis demonstrates that a large (~10%) subset of ABA responsive genes are less responsive to ABA for a period of hours after initial ABA exposure. I also present data describing the desensitization response based on transcriptomic and metabolomic profiles, which demonstrates the desensitization is not only restricted to several specific genes, but is genome wide and can be measured both in metabolic and transcriptional responses.
In Chapter 3 I characterize engineered variants of the ABA receptor PYR1 that possess nanomolar sensitivity to the agrichemical mandipropamid. These were developed by others in my thesis lab to create a system for orthogonal control of ABA responses. My analyses of transcriptomic data show that the engineered receptor can induce an ABA-like response in transgenic Arabidopsis in response to mandipropamid and provide support for the functioning of the engineered receptor in vivo.