Nuclear bodies and compartmentalization of pre-mRNA splicing factors in higher plants

We studied the fine structural organization of nuclear bodies in the root meristem during germination of maize and Arabidopsis thaliana using electron microscopy (EM). Cajal bodies (CBs) were observed in quiescent embryos as well as germinating cells in both species. The number and distribution of CBs were investigated. To characterize the nuclear splicing domains, immunofluorescence labelling with antibodies against splicing factors (U2B" and m3G-snRNAs) and in situ hybridisation (with U1/U6 antisense probes) were performed combined with confocal microscopy. Antibodies specific to the Arabidopsis SR splicing factor atRSp31 were produced. AtRSp31 was detected in quiescent nuclei and in germinating cells. This study revealed an unexpected nuclear speckled organization of atRSp31 in root epidermal cells where micro clusters of interchromatin granules were also observed by EM. Therefore, we examined the distribution of green fluorescent protein (GFP)-tagged atRSp31 in living cells after Agrobacterium-mediated transient expression. When expressed transiently, atRSp31-GFP exhibited a speckled distribution in leaf cells. Treatments with -amanitin, okadaic acid, staurosporine or heat-shock induced the speckles to reorganize. Furthermore, we have generated stable Arabidopsis transgenics expressing atRSp31-GFP. The distribution of the fusion protein was identical to the endogenous atRSp31. Three-dimensional time-lapse confocal microscopy showed that speckles were highly dynamic domains over time.