Proteome profiling of plant Cajal bodies uncovers kingdom-specific components.

Membraneless organelles (MLOs) in eukaryotic cells, such as the nuclear Cajal body (CB), have been notoriously difficult to biochemically isolate due to their dynamic and delicate nature. CBs, formed via liquid-liquid phase separation, mediate RNA processing and ribonucleoprotein assembly, essential for nuclear functions. In plants, this challenge has prevented any direct molecular characterization to date. Here, we report a successful isolation of plant CBs, achieved through a combination of biochemical purification and fluorescence-activated nuclei and particle sorting, enabling a previously unattainable biochemical access to this organelle. Mass spectrometry analysis defined a high-confidence set of plant CB-associated proteins, identifying 15 and 79 proteins uniquely localized and enriched in CBs, respectively, of which 18 are also labelled by CB markers in proximity labelling assays. While a partial overlap with known animal CB proteins exists, our data suggests a partially divergent composition of CB-associated proteins across kingdoms. This work breaks a major technical challenge, enabling detailed characterization of plant CBs and establishing a versatile platform adaptable for the isolation and molecular dissection of other MLOs.