ABSTRACT
Here we describe methods for producing nuclei from Arabidopsis suspension cultures or root tips of Arabidopsis, wheat, or pea. These methods could be adapted for other species and cell types. The resulting nuclei can be further purified for use in biochemical or proteomic studies, or can be used for microscopy. We also describe how the nuclei can be used to obtain a preparation of nucleoli.
Subject(s)
Arabidopsis/chemistry , Cell Fractionation/methods , Cell Nucleolus/chemistry , Cell Nucleus/chemistry , Pisum sativum/chemistry , Triticum/chemistry , Antibodies/chemistry , Cell Fractionation/instrumentation , Cell Nucleolus/ultrastructure , Cell Nucleus/ultrastructure , Centrifugation, Density Gradient/instrumentation , Centrifugation, Density Gradient/methods , Culture Media/chemistry , Fluorescent Antibody Technique , Meristem/chemistry , Protoplasts/chemistry , Protoplasts/ultrastructure , Seeds/chemistry , Staining and Labeling/methodsABSTRACT
We describe a method for isolating nuclei from cultured Arabidopsis cells. The same method can be used to further isolate nucleoli. Cell walls are first digested to yield protoplasts, which are purified by flotation on a Percoll gradient. Mechanical homogenisation is used to release nuclei, or with more homogenisation, nucleoli. These fractions are most easily purified by gentle centrifugation. The method has been used for proteomic analysis of nucleoli, as well as for biochemical studies. We also describe a method for immunological labelling of isolated nuclei.
Subject(s)
Arabidopsis/metabolism , Cell Nucleolus/metabolism , Cell Nucleus/metabolism , Cytological Techniques , Biochemistry/methods , Centrifugation/methods , Chromatin/metabolism , Microscopy, Fluorescence/methods , Proteomics/methodsABSTRACT
The eukaryotic nucleolus is involved in ribosome biogenesis and a wide range of other RNA metabolism and cellular functions. An important step in the functional analysis of the nucleolus is to determine the complement of proteins of this nuclear compartment. Here, we describe the first proteomic analysis of plant (Arabidopsis thaliana) nucleoli, in which we have identified 217 proteins. This allows a direct comparison of the proteomes of an important nuclear structure between two widely divergent species: human and Arabidopsis. The comparison identified many common proteins, plant-specific proteins, proteins of unknown function found in both proteomes, and proteins that were nucleolar in plants but nonnucleolar in human. Seventy-two proteins were expressed as GFP fusions and 87% showed nucleolar or nucleolar-associated localization. In a striking and unexpected finding, we have identified six components of the postsplicing exon-junction complex (EJC) involved in mRNA export and nonsense-mediated decay (NMD)/mRNA surveillance. This association was confirmed by GFP-fusion protein localization. These results raise the possibility that in plants, nucleoli may have additional functions in mRNA export or surveillance.
