Discovery of novel membraneless organelles in Arabidopsis thaliana
Keywords: Biology.

Membraneless organelles are cellular compartments that form through liquid-liquid phase separation of macromolecules. This process has recently emerged as a universal mechanism to explain how cells compartmentalize biochemical processes and create or-der within a seemingly chaotic and dilute milieu. While the first membraneless organelle, the nucleolus, was discovered in the 19th century, it was only within the last decade that the nucleolus and other organelles such as Cajal bodies, speckles, paraspeckles, stress granules, P-bodies, and pyrenoids were recharacterized as phase separated liquid-like compartments. In animals, phase separation research has gained a lot of interest due to its role in neurodegenerative diseases, but in plants research in this field has lagged be-hind and fewer phase separated organelles have been described. In Chapter 1, I summarize the current knowledge of phase separation in plants and ar-gue that biases in plant biology research have left phase separation processes unex-plored. I show that Arabidopsis thaliana's uncharacterized proteome is enriched in bio-chemical features that are frequently found in phase separating proteins, and that we can therefore expect more phase separation events to be discovered in plants. I also hypothe-size that multiple uncharacterized structures that have been reported in the literature using terms such as "puncta", "foci" and "bodies" may actually be the products of phase sepa-ration and that, therefore, a second look into them is warranted. In Chapter 2, I explored Arabidopsis thaliana's uncharacterized seed proteome and discovered a new membraneless organelle that is involved regulating seed germination. While, traditionally, research into seed germination has mostly focused on hormones (e.g. abscisic acid and gibberellic acid) as well as on descriptive studies of seed mechan-ics, I posited that exploring the unknown seed proteome would reveal unexplored and novel mechanisms that would expand our understanding of how seeds make the critical decision of germinating. My search led to the discovery of a novel prion-like protein, that I named FLOE1, which regulates seed germination through a liquid-liquid phase separation mechanism. Through a close collaboration across multiple disciplines that I initiated, my collaborators and I used genetic, biochemical, live imaging, electron mi-croscopy, structural modeling, physiological, and comparative genomic approaches to provide evidence that FLOE1 functions as a water sensor in desiccated seeds. Addition-ally, a series of mutants with altered levels and variant forms of FLOE1 revealed the first biologically relevant function in vivo for a protein liquid-to-solid switch in a multicellular organism. In Chapter 3, I describe novel Arabidopsis components of another type of membrane-less organelles, stress granules (SGs), which are cytoplasmic assemblies of ribonucleo-proteins that emerge upon exposure to various stresses. First, I characterize seven Ara-bidopsis homologs of G3BP1/2, which are core components of human SGs. I show that while they have conserved properties in Arabidopsis SGs, they also exhibit plant-specific features. Second, by exploring the physical interaction network around AtG3BPs, I discovered that a mostly uncharacterized plant-specific family of transcrip-tional regulators—the GIP family—is also involved in SG biology. Finally, co-immunoprecipitation coupled to mass spectrometry of GIP1 and AtG3BPL3 led to the discovery of a new type of membraneless organelles that I named "rhamnosomes", which contains all four enzymes involved in the biosynthesis of rhamnose. In Chapter 4, I propose future directions to study the molecular mechanism of FLOE1 as well as to explore the role of FLOE1 homologs in both Arabidopsis thaliana and oth-er plants. I also comment on the impact of my research and its potential applications in agriculture and healthcare