Imaging plant germline differentiation within Arabidopsis flowers by light sheet microscopy
- Masaryk University, Czech Republic
- VSB–Technical University of Ostrava, Czech Republic
- FEECS VSB – Technical University of Ostrava, Czech Republic
- Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Germany
- Max Planck Institute of Molecular Cell Biology and Genetics, Germany
Figures
Figure 1 with 1 supplement
Imaging Arabidopsis flower using LSFM.
(A) Workflow of sample preparation. (B) Maximum intensity projections (MIPs) of micrographs of HTA10:RFP flowers dissected from buds of the indicated sizes (upper panel, scale bar 200 µm). A …
Figure 1—figure supplement 1
Growth dynamics of a floral bud.
(A) Time lapse images of a single floral bud (marked with black) growing on a wild type plant. Scale bar 300 µm. (B) Quantification of the growth rate of Arabidopsis floral buds. The width of …
Figure 2 with 1 supplement
The 3D reconstruction of Arabidopsis flower from multiview imaging.
(A) MIPs of a 0.5 mm floral bud expressing ASY1:eYFP (green) and H2B:mRuby2 (magenta) viewed from eight different angles. Scale bar 200 µm. (B,C) Imaris MIP of 3D reconstructed flower. Longitudinal …
Figure 2—video 1
Animation of 3D reconstructed flower expressing ASY1:eYFP (green) and H2B:mRuby2 (magenta).
The movie shows rotation of the MIPs, cross-sections (partial MIPs) and surface renderings. Belongs to Figure 2B–D.
Figure 3 with 2 supplements
Time lapse imaging of a growing flower.
(A) MIPs of a flower (upper panel) expressing ASY1:eYFP (green) and H2B:mRuby2 (magenta) and one of its anther lobes (bottom panel) at indicated time points (scale bar 100 µm). (B) Detailed view of …
Figure 3—figure supplement 1
Development of a floral bud in the closed capillary.
(A) A 0.3 mm floral bud from the HTA10:RFP reporter line was embedded in media with low melting point agarose within the closed FEP/capillary system and imaged at time point 0 (MIP, upper panel; …
Figure 3—video 1
Time lapse imaging of floral bud development in 60 min intervals (ASY1:eYFP in green, H2B:mRuby2 in magenta).
Belongs to Figure 3A.
Figure 4 with 1 supplement
Spatiotemporal distribution of auxin response in flower.
(A) MIPs of DR5::N7-Venus signal of four different flower buds of different sizes. Scale bar 200 µm. (B) Time lapse imaging of a flower at developmental stage 12 expressing DR5::N7-Venus in 2 hr …
Figure 4—video 1
Time lapse imaging of a flower at developmental stage 12 expressing DR5::N7-Venus in 2 hr intervals.
Belongs to Figure 4B.
Figure 5 with 5 supplements
Time lapse imaging of subcellular processes within the flower.
(A) Chromosome segregation in meiosis I from diakinesis (0 m) to telophase I (64 m) visualized with the HTA10:RFP marker. Images were taken every 30 s, scale bar 10 µm. (B) Restitution mitosis in …
Figure 5—video 1
Time lapse imaging of chromosome segregation in PMCs from diakinesis through telophase II in 30 s intervals.
Chromatin is labeled by HTA10:RFP. Belongs to Figure 5A.
Figure 5—video 2
Time lapse imaging of restitution mitosis in tapetum cells in 60 s intervals.
Chromatin is labeled by HTA10:RFP. Belongs to Figure 5B.
Figure 5—video 3
Time lapse imaging of asymmetric pollen mitosis I in 5 min intervals.
Chromatin is labeled with H2A:RFP (magenta), 488 nm autofluorescence highlights the pollen wall (green). Belongs to Figure 5D.
Figure 5—video 4
Rapid movements of chromatin axes in zygotene in 5 s intervals.
Chromatin axes are visualized with ASY1:eYFP (green), somatic nuclei with H2B:mRuby2 (magenta). Belongs to Figure 5C.
Figure 5—video 5
Time lapse imaging of female meiosis in 10 min intervals.
MMC is marked with ASY1:eYFP (green), chromatin with HTA10:RFP (magenta). Belongs to Figure 5E.
Figure 6 with 2 supplements
Protein localization in meiotic S-phase.
(A) Time lapse imaging of PCNA:TagRFP during meiotic S-phase. Nuclear speckles are visible between 45 to 120 min. Images were taken every 15 min, scale bar 10 µm. (B) Time lapse imaging of …
Figure 6—video 1
Time lapse imaging of PCNA:TagRFP in PMCs in 15 min intervals.
Belongs to Figure 6A.
Figure 6—video 2
Time lapse imaging of PCNA:TagRFP (magenta) and ASY1:eYFP (green) in PMCs in 15 min intervals.
Belongs to Figure 6B.
Figure 7 with 3 supplements
Time lapse imaging of meiosis II in smg7-1 mutants.
Time point 0 min corresponds to prometaphase II when chromosomes start condensing. Middle panel depicts PMCs in smg7-1 PMCs arrested in aberrant anaphase II. Lower panel shows smg7-1 PMCs that …
Figure 7—video 1
Time lapse imaging of chromosome segregation in meiosis I and meiosis II in a wild type plant in 2 min intervals.
Chromatin is labeled with HTA10:RFP (magenta) and autofluorescence is in green. Belongs to Figure 7.
Figure 7—video 2
Time lapse imaging of meiosis II and irregular anaphase II in smg7-1 in 2 min intervals.
Chromatin is labeled with HTA10:RFP. Belongs to Figure 7.
Figure 7—video 3
Time lapse imaging of meiosis II with brief telophase II and irregular anaphase III in smg7-1 plant in 2 min intervals.
Chromatin is labeled with HTA10:RFP. Belongs to Figure 7.
Author response image 1
Comparison of different objectives and zooming options.
(A) Field of view when using objectives without zoom (0.36 zoom, upper panel) and with maximal zoom (2.5 zoom, lower panel). ASY1:YFP HTA10:RFP flowers were used in the experiment. (B) Comparison of …
Videos
Video 1
Preparation of a sample for imaging by LSFM.
Additional files
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Supplementary file 1
Supplementary material.
(A) Oligonucleotides used in the study. (B) Overview of image processing.
- https://cdn.elifesciences.org/articles/52546/elife-52546-supp1-v1.docx
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Transparent reporting form
- https://cdn.elifesciences.org/articles/52546/elife-52546-transrepform-v1.pdf
