Tissue-specific expression and dynamic organization of SR splicing factors in Arabidopsis

Abstract

The organization of the pre-mRNA splicing machinery has been extensively studied in mammalian and yeast cells and far less is known in living plant cells and different cell types of an intact organism. Here, we report on the expression, organization, and dynamics of pre-mRNA splicing factors (SR33, SR1/atSRp34, and atSRp30) under control of their endogenous promoters in Arabidopsis. Distinct tissue-specific expression patterns were observed, and differences in the distribution of these proteins within nuclei of different cell types were identified. These factors localized in a cell type-dependent speckled pattern as well as being diffusely distributed throughout the nucleoplasm. Electron microscopic analysis has revealed that these speckles correspond to interchromatin granule clusters. Time-lapse microscopy revealed that speckles move within a constrained nuclear space, and their organization is altered during the cell cycle. Fluorescence recovery after photobleaching analysis revealed a rapid exchange rate of splicing factors in nuclear speckles. The dynamic organization of plant speckles is closely related to the transcriptional activity of the cells. The organization and dynamic behavior of speckles in Arabidopsis cell nuclei provides significant insight into understanding the functional compartmentalization of the nucleus and its relationship to chromatin organization within various cell types of a single organism.

Figure 1.

Expression and nuclear localization patterns of SR33-YFP, SR1/atSRp34-YFP, and atSRp30-YFP in Arabidopsis. Projections of a series of confocal optical sections of inflorescence stems, anthers, roots, and leaves are shown. (A, F, and K) In the basal section of primary root. (B, G, and L) In the tip section of primary root. (C, H, and M) In leaf. (D, I, and N) In anther (D′, enlarged view of a pollen grain, arrow highlights the vegetative nucleus and arrowheads highlight the nuclei of sperm cells; bar, 10 μm). (E, J, and O) In inflorescence stem. Different cell types are highlighted with arrows: Gu, guard cells; LEP, larger (C) and small (H) leaf epidermal pavement cells; RE, root epidermal cells; RH, root hairs; RM, root meristematic cells; Tri, trichomes. Bar, 100 μm.

Figure 2.

Localization of SR33-YFP, SR1/atSRp34-YFP, and atSRp30-YFP in different Arabidopsis cell types. Maximum projections of deconvolved optical sections are shown. The localization patterns of the three SR-YFP fusions are different among cell types with variable nuclear sizes and shapes. Bar, 5 μm.

Figure 3.

Immunoelectron microscopic localization of SR proteins in Arabidopsis. Root sections are immunolabeled with 3C5 antibody, which recognizes the SR family of pre-mRNA splicing factors in IGCs (arrows). Chr, chromatin; No, nucleolus. Bar, 200 nm.

Figure 4.

Time-lapse deconvolution microscopy of SR1/atSRp34-YFP in a living leaf epidermal pavement cell. The projections of deconvolved optical sections are shown at each time point. The arrows highlight the fusion and disassociation of two speckles, and the arrowheads highlight the splicing factors being released from speckles. The time is indicated in seconds. Bar, 5 μm.

Figure 5.

FRAP of SR proteins. (A) Leaf epidermal pavement cells expressing SR1/atSRp34-YFP were imaged before and during recovery from photobleaching. The white square frames represent the photobleached region. Bar, 5 μm. (B) Kinetics of recovery after bleaching of SR1/atSRp34-YFP. ^* indicates the photobleach point.

Figure 6.

Dynamics of SR1/atSRp34-YFP during mitosis in living root epidermal cells. (A) A cell (arrow) just before nuclear envelope breakdown. (B and C) In prophase, as the nuclear envelope breaks down, the splicing factors enter the cytoplasm. (D) In metaphase, splicing factors are diffusely distributed in the cytoplasm. (E–J) Splicing factors are reentering into daughter nuclei. Newly forming speckles are observed in telophase nuclei (arrows in F–I). Bar, 5 μm.

Figure 7.

Effect of transcription inhibition on the organization of SR proteins. The transgenic leaves were treated with 50 μg/ml DRB for 5 h at 22°C. In leaf epidermal pavement cells (B), after transcriptional inhibition, the splicing factors further accumulated in speckles with less labeling in the nucleoplasm. In trichomes (D), after transcriptional inhibition, the splicing factors organized into thousands of microspeckles. DRB (F) has no obvious effect on nuclear morphology and the diffuse distribution of a control protein YFP-tetR-NLS. In control samples, water treatment for 5 hat 22°C had no obvious effect on the distribution of SR-YFP or YFP-tetR-NLS (A, C, and E). Bar, 5 μm.