Postdoctoral Researcher Carnegie Institution for Science SAN FRANCISCO, California
Rhamnose is a monosaccharide found throughout plants and bacteria. In plants, rhamnose is an essential component of rhamnogalacturonan I and II that are major building blocks of the cell wall. Rhamnose is synthesized in the cytoplasm as uridine diphosphate (UDP)-rhamnose from UDP-glucose through three sequential reactions performed by the trifunctional enzyme RHAMNOSE (RHM). Arabidopsis thaliana has three functionally redundant RHM enzymes (RHM1, RHM2, RHM3) that are expressed in different tissues. RHM1 is the most broadly expressed throughout the plant, and mutants in rhm1 have twisted petals and roots. RHM1 localizes to cytoplasmic biomolecular condensates in petal cells, but we do not yet know how RHM1 localizes to condensates and if this localization is important for the biochemical function and developmental role of RHM1. To understand if RHM1 localization to biomolecular condensates is important for petal development, we investigated when and how RHM1 localizes to condensates, and tested if RHM1 localizing to condensates was essential for its biochemical function. To determine whether the RHM1-containing condensates have been previously characterized, we co-localized RHM1 with published condensate marker proteins for stress granules in Nicotiana benthamiana and human U2OS cells. The RHM1-condensates did not co-localize with any of the markers, suggesting RHM1-condensates represent a new type of condensate. In Arabidopsis, RHM1-condensates formed in petals, leaves, and in the developing seed coat. RHM1 formed condensates by a hydrophobic interaction through the RHM1 homodimerization domain, and a single point mutation in this domain (RHM1A161K) was sufficient to prevent condensate formation. We tested if RHM1 condensates were essential for growth and development by generating stable Arabidopsis lines in the rhm1-2 background that expressed RHM1WT or RHM1A161K driven by the native RHM1 promoter. When compared to wild type and RHM1WT lines, rhm1-2 and RHM1A161K plants had misshapen petal cells and petals, smaller leaves, and had less seed mucilage. To test if RHM1 condensates are required for UDP-rhamnose synthesis, we expressed RHM1WT and RHM1A161K in a heterologous yeast expression system. RHM1WT formed RHM1 condensates that synthesized UDP-rhamnose while RHM1A161K did not. Overall, these experiments demonstrate RHM1 condensates are required for petal, leaf, and seed development in Arabidopsis and for UDP-rhamnose synthesis. Together, this study describes the formation and function of a biomolecular condensate formed by RHM1, which has not been previously characterized, and we termed these novel condensates “rhamnosomes”.