The cytosol must flow: physiological signals coordinate transport through plasmodesmata

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)