Plant cells are connected by nanoscopic channels in the cell wall called plasmodesmata (PD). PD allow neighboring cells to exchange cytosol, permitting small and large molecules (including water, ions, sugars, hormones, proteins, RNA, and viruses) to move from cell to cell. PD are absolutely essential for plant life: all land plants have PD, and no mutant lacking PD has ever been identified. Despite their importance to a range of biology, including hormonal signaling, transcription factor trafficking, and the movement of resources and nutrients, very little is currently known about how PD transport is regulated.

The Zambryski lab conducted a forward genetic screen for mutants severely defective in regulating PD transport during embryogenesis, and identified five mutants with increased PD transport (ise1–ise5) and one mutant with decreased PD transport (dse1) at the mid-torpedo stages of embryogenesis. After mapping and characterizing these mutants, I discovered that two major metabolic pathways regulate PD transport during development of plant embryos and shoots: chloroplast biogenesis and sugar signaling. Here, I use a combination of approaches from cell biology, transcriptomics, and physiology to unravel the complex pathways linking chloroplasts, sugars, and PD.

Upon discovering that two mutants defective in chloroplast biogenesis, ise1 and ise2, affect the expression of thousands of nuclear genes—including many that are not clearly related to chloroplast function—and prevent the restriction of PD transport during embryogenesis, I decided to pursue an open hypothesis that chloroplasts (and other plastid types) could transmit signals within the cell through thin, tubular projections called “stromules” (stroma-filled tubules). Previous work has shown that stromules physically associate with the nucleus, ER, mitochondria, and other subcellular locations, but there was no evidence that signals initiated within the plastid affected stromule activity. I demonstrate that stromules are induced during the day and that they respond to light-sensitive redox signaling pathways within the chloroplast. Furthermore, I show that stromules can form independently outside of their cellular context. In combination, these results support the hypothesis that stromules are potential routes for intracellular signal transduction (Chapter Two).

Previous work had suggested that PD transport is tightly regulated over developmental time scales and in response to abiotic and biotic stress. Our discovery that chloroplasts coordinate PD transport led us to explore whether intercellular trafficking changes over physiological time scales, particularly in response to light. I show that cytosol moves through PD much faster during the day than the night in leaves, and that this increase in PD transport during the day is light-dependent. Light is insufficient to induce high levels of PD transport at night, however, suggesting that PD transport is regulated by the circadian clock (Chapter Three).

PD transport decreases as leaves age, which promotes allocation of nutritional resources to younger, growing leaves in the shoot. Although well-studied, the mechanism of this transition from high to low PD transport remained unclear. I use the ise4 mutant as a starting point: ISE4 is a chaperone subunit required to activate a kinase, TOR, that is a central metabolic sensor in all eukaryotes. I show that TOR senses sugar availability to coordinate the restriction of PD transport during embryogenesis and during shoot development, and that TOR regulates plant growth and aging (Chapter Four).

This dissertation concerns the interrogation of plasmodesmatal regulation in the context of TARGET OF RAPAMYCIN (TOR) signaling. Plasmodesmata (PD) are nanoscopic channels between plant cells allowing for the exchange of diverse cytosolic molecules, with a rate of transportation that changes across developmental time. Meanwhile, TOR is a central metabolic kinase that coordinates multiple signaling pathways to influence growth and development. Previous work has connected TOR to PD in an antagonistic relationship, the precise underpinnings of which remain unclear. In the seven parts of this dissertation, I describe that relationship, build the tools necessary to investigate it, and perform experiments that uncover several previously unknown aspects of TOR signaling and PD regulation as they relate to the Reptin-Pontin-Tah1-Pih1 (R2TP) Complex.In Part I, I review our current knowledge of TOR signaling as it relates to PD, with special emphasis on an additional complex involved in sugar sensing known as the R2TP complex. To phosphorylate downstream targets, TOR must act in a complex known as TOR Complex 1 (TORC1). R2TP is essential for the dimerization of TORC1 in response to glucose and thus acts upstream of TOR signaling. Simultaneously, mutants in the R2TP-TOR signaling axis have been found to have a substantial impact on PD transport, indicating a direct link between glucose-stimulated TOR activity and PD regulation. In Part II, I detail the method I developed to quantify GFP movement as an indicator of PD transport in leaf epidermal tissue. In this method, individual cells are transformed with Agrobacterium tumefaciens to express GFP, which moves from the primary transformant into neighboring cells through PD. After two to three days, the amount of GFP movement is documented by confocal fluorescence microscopy and total movement is quantified in terms of the number of cells or rings of cells to which GFP has spread. These methods proved useful for experiments in modulating PD transport, as shown in Part V. In Part III, I outline the methods available for elucidating protein-protein interactions (PPIs) in several biological contexts for the purpose of determining which method is most appropriate for use in the TOR signaling network. I describe the benefits and pitfalls of methods such as yeast two-hybrid (Y2H), bimolecular fluorescence complementation (BiFC), fluorescence resonance energy transfer (FRET), co-immunoprecipitation (coIP), in silico programs, and finally proximity labeling. The ability of proximity labeling with TurboID to rapidly identify many potentially unknown interactors of diverse proteins leads me to choose that method for investigation of interactors in the R2TP-TOR signaling axis. In Part IV, I describe my creation of a new series of binary vectors for use in plants. While gateway plasmids have found much use in the plant community, my AVEC series of plasmids have a modular design allowing for a wide range of uses and customizability. I further test these vectors in the context of confocal microscopy, coIPs, proximity labeling, and the creation of transgenic lines, and determine that they are the optimal vehicle for delivering a TurboID-based proximity labeling system into plants for my future experiments in Part VI. In Part V, I probe the role of cytokinins in regulating PD transport, a function previously uncovered for TOR signaling. I provide evidence that cytokinins directly promote PD transport in leaves using genetic and pharmacological perturbations of the cytokinin signaling pathway in conjunction with the methods developed in Part II. I show that first that PD transport significantly increases when leaves are supplied with exogenous cytokinins or when a positive regulator of cytokinin responses is overexpressed. Following, I demonstrate that PD transport significantly decreases when cytokinin receptors are silenced or a negative regulator of cytokinin signaling is overexpressed. These results indicate that cytokinins contribute to dynamic changes in PD transport in plants, and in conjunction with transcriptomic data from R2TP mutants more fully flesh out our understanding of the connection between PD and the R2TP-TOR signaling axis. In Part VI, I demonstrate the utility of TurboID-based proximity labeling in probing the interactome of the R2TP complex in plants. Through transient expression in N. benthamiana and transgenic expression in Arabidopsis of Reptin, Pontin, and Spaghetti proteins fused to TurboID, I demonstrate that the R2TP complex in plants likely lacks a key protein component found in other eukaryotes. I therefore propose that the complex in plants be renamed to the R2T complex. Despite the lack of this key protein, results from mass spectrometry indicate that the R2T complex is able to fulfill many of its canonical activities and associate with several interacting partners known in other organisms, opening the door to future discoveries. Finally, in Part VII, I summarize the impact of these explorations on our knowledge of cytokinin signaling and the R2TP-TOR axis as they relate to PD regulation, and briefly propose future avenues of experimentation to further our knowledge of this complex system.