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Zhang, Chang

Plant Biology

2015

PhD

Abscisic acid (ABA) is a plant hormone that regulates various developmental processes and environmental stress responses. ABA modulates growth of both primary roots and lateral roots, helping to shape root architecture. The lateral root organ defective (latd) mutants, disrupted in the MtLATD/NIP gene, encoding a nitrate transporter, have severe root growth defects that can be rescued by applying ABA. However, the way in which ABA stimulates latd root growth is unclear, and the downstream components of MtLATD/NIP and ABA signaling are completely unknown. To answer these questions, this dissertation focuses on two major potential downstream regulators: Reactive Oxygen Species (ROS) and transcription factors (TFs). ROS are important signaling molecules required in ABA-induced stomatal closure under drought or osmotic stresses, but their role in ABA regulation of root development is unclear. I found that latd mutant roots have increased ROS levels, and the expression level of several MtRboh genes, which encode major ROS-producing enzymes, the NADPH oxidases, is also increased. ABA decreases the amount of ROS in latd roots and also reduces expression of MtRbohC, in particular. In addition, I observed that latd mutant roots have cell elongation defects, which can also be rescued by exogenous ABA. I demonstrated that pharmaceutically decreasing ROS levels using an NADPH oxidase inhibitor, or reducing the expression of MtRbohC using RNA interference can increase cell elongation and stimulate lateral root elongation in latd roots. These findings have revealed a mechanism by which ABA restores root growth in latd mutant roots via regulating ROS levels, and identified MtRbohC as an important downstream target of ABA signaling mediated by MtLATD/NIP. TFs act as regulatory nodes controlling the transcription of gene clusters and playing a crucial role in plant growth and development. Using a high-throughput TF profiling approach, I have identified 20 TFs that exhibit altered expression levels in latd mutant roots as compared to wild type, 60% of which can be restored to normal levels by ABA. My analysis also revealed that ABA regulates the expression of a different set of TFs in latd roots, suggesting that MtLATD/NIP is crucial for ABA regulation of TF expression. Moreover, ABA changes the TFs regulated by MtLATD/NIP almost completely, indicating a tight control of ABA on TFs regulated by MtLATD/NIP. Surprisingly, I found that the expression of NODULATION SIGNALING PATHWAY 2 (MtNSP2), a GRAS family TF required for nodulation, is regulated by MtLATD/NIP, ABA and nitrate in non-symbiotic roots. In symbiotic roots, MtLATD/NIP is required for the transcriptional signaling pathway downstream of MtNSP2 in the epidermis as well as induction of MtNSP2 expression by cytokinin and subsequent activation of its downstream targets in the cortex. These findings indicate that MtLATD/NIP functions in nodulation signal transduction upstream of MtNSP2, and mediates crosstalk with cytokinin. Together, these two approaches have begun to characterize a signaling pathway downstream of ABA and MtLATD/NIP that involves ROS, MtNSP2, and a core group of TFs in the regulation of root development and nodulation in M. truncatula.