NRS1, a nitrogen story, and the fuTORe of the Amino Sensing Pathway

This work addresses three pictures of nitrogen and amino acid sensing within a plant. Much of this work focuses on the role of the kinase TARGET OF RAPAMYCIN (TOR). TOR is the focus of many researchers interested in metabolic diseases and cancers since TOR dysregulation is a major cause of such diseases. The role of TOR in plants is relatively unexplored, however, and questions surrounding its upstream cues and downstream targets persist.TOR is a deeply conserved Ser/Thr protein kinase that integrates an array of metabolic processes to coordinate growth and development with nutritional status in eukaryotes. TOR is often dysregulated in many human diseases including diabetes and cancer, and as such, has been relatively well studied in mammalian systems. The TOR protein and complex are well-conserved across the eukaryotic kingdom; however, their role in plant growth and development is not as well understood. Recent work in this emerging field has shown that TOR plays a similar role in plants, serving as a metabolic rheostat modulating developmental processes in response to signals like light, sugars, and phosphorus abundance. In response, TOR regulates key pathways like nucleotide biosynthesis, ribosome biogenesis, and leaf initiation. In chapter one, as well as outlining the field of TOR in plants, I will also introduce the nuances and complexities of sensors, transducers, and transporters in the world of nitrogen sensing, detailing what it takes to be considered a “sensor” in plant cell biology. The section that follows will outline the developments in autonomous nitrogen pathways and the small body of knowledge of nitrogen sensing by TOR in plants, and explain important contextual background for nutrient dependent TOR regulation. Here, I introduce NRS1 in it’s starring role in this thesis. The Brunkard lab discovered a null 1 allele in a forward genetic screen for defective embryonic cell-cell (plasmodesmatal) trafficking that repeatedly identified mutants defective in TOR signaling (Brunkard et al 2020, Kim et al 2002), which led us to speculate that NRS1 could be required for TOR activity in plants. Jake Brunkard used RNA-Seq to define the NRS1 transcriptome (>850 differentially expressed genes using stringent cut-off parameters), which exhibits several signatures of TOR inactivation, including significant repression of ribosomal protein genes and induction of proteolytic and catabolism-related genes (Busche et al 2021, Brunkard et al 2020, Xiong et al 2013). NRS1 mutants and NRS1 knockdowns also show significantly less phosphorylation of S6K-T449 (orthologous to human S6K- T389), a residue that is uniquely phosphorylated by the TOR kinase in plants. In all, this makes NRS1 an attractive candidate for study in the TOR field./nIn chapter two, I tell a nitrogen story. I detail a series of experiments aimed at teasing out TOR’s role in sensing and responding to different exogenous forms of nitrogen. The first picture of my thesis engages the hypothesis that application of exogenous nitrogen has an impact on the TOR signaling pathway. Here, I will describe how nitrogenous nutrients stimulate TOR activity, with the hypothesis that TOR activity is promoted by the exogenous addition of nitrogenous nutrients when plants are grown under nitrogen limiting conditions. I established N-limiting growth conditions to determine the impact of nitrogenous nutrients on TOR activity, and tested nitrogen and amino acid addition in N- deprived plants to identify conditions that promote TOR activity. I found that the addition of nitrogenous nutrients and glucose to a nitrogenous nutrient limiting environment increases the phosphorylation of SK1, with glucose having less effect than the other nitrogen-rich substrates. The addition of asparagine, a nitrogen rich amino acid, has more impact than glucose, however, the addition of nitrate and ammonia to the substrate caused the most significant S6K1 phosphorylation, indicative of higher TOR activity. These findings suggest there must be some sensor that exists that modulates external nitrogen suppliance with TOR activity.In chapter three, I dive within plant metabolism, with the goal of elucidating the impact of genetically manipulating endogenous levels of specific amino acids on TOR activity. Unlike animals, plants synthesize all 20 of the amino acids, and have complex metabolic systems to produce and transport these amino acids throughout the plant. One of the limitations of only investigating the exogenous application of nitrogenous nutrient sources and the impact on TOR activity via western blot, is that it is not possible to know all the possible downstream effects of plant amino acid uptake and biosynthesis when adding exogenous amino acids and nitrogenous nutrients. The experiments in the previous chapter may guide our understanding of TOR activity as it responds to external output, but will not be sufficient to conclude that a particular metabolite is shifting TOR activity without looking at the plant’s endogenous metabolism. I cloned the suite of ASN genes present in A. thaliana (ASN1, ASN2, ASN3) and the L- ASPARAGINASES (Aspga1, Aspgb1). Arabidopsis thaliana plants were transformed to stably or transiently overexpress these genes. My hypothesis was that if asparagine is the primary amino acid involved in activating TOR in plants, then it is predicted that overexpressing L- asparaginase will decrease TOR activity, as measured by S6K1- Thr449 phosphorylation. In plants, these genes can either be stably overexpressed in transgenic plants or they can be transiently overexpressed in leaves by infiltration with Agrobacterium. However, the results showed that manipulating the individual asparaginases did not have a significant impact on TOR activity, turning my focus to NRS1. In chapter four, I put a singular focus on NRS1 and its paralogs. The known cytosolic NRS genes in Arabidopsis thaliana are NRS1, NRS2, and a recently evolved paralogue of NRS1, which is called NRS3. A few insertional mutants are available for NRS3, but none have been characterized or investigated for phenotypes, and the Brunkard Lab has shown that null NRS2 alleles exhibit no obvious phenotypes under normal growing conditions. Significantly, however, NRS1 mutants are lethal at the mid-torpedo stage of embryogenesis, which suggests that NRS1 has some nonredundant function that is not complemented by NRS2 or NRS3. Therefore, NRS1 is unique among aminoacyl tRNA transferases in plants because it is not strictly essential for cell survival, but it is required for plant development, even though there are two other NRS genes in Arabidopsis thaliana. In combination with a previous experiment, I agroinfiltrated plants with the three aminoacyl tRNA synthetase constructs. Genetic expression of the construct in the leaf occurs at 48 hours, and leaf samples were collected and TOR activity was assayed. As anticipated, overexpressing NRS1 has a significant impact on S6K1 phosphorylation. Finally, in chapter five, I embarked on a journey to tease out the molecular mechanisms of NRS1 and TOR activity. This chapter proposes to build on the discovery that NRS1 stimulates TOR activity by mechanistically defining how NRS1 activates TOR at a molecular level. I disrupted key functions of NRS1 as a tRNA synthetase and deleted the WHEP domain to establish the domains of NRS1 necessary to activate TOR. I cloned truncated versions of NRS1 to exclude the WHEP domain, i.e., NRS1ΔWHEP. This truncated NRS1ΔWHEP over-expression construct was compared against the Wt- NRS1 construct in western blot assays. The WHEP domain of WARS, for example, acts like a molecular “bridge”, interacting with both the phosphatidylinositol kinase-like kinase (PIKK) DNA-PKcs and the poly[ADP-ribose] (PAR) polymerase 1 (PARP1) to facilitate PARylation of DNA- PKcs and subsequent activation of the tumor suppressor p53 (Guo et al, 2010). Thus, there exists a model in humans where the WHEP domain mediates interaction between a tRNA synthetase and a protein kinase.

Advisor: Hake, Sarah
Brunkard, Jacob