-Tubulin Is Essential for Microtubule Organization and Development in Arabidopsis

Supplemental Data

Files in this Data Supplement:

• Supplemental Table 1 - Mitotic defects in mutant pollen
• Supplemental Table 2 - Mitotic defects in tubg1-1 tubg2-2 roots
• Supplemental Figure 1 - Pollen viability from F1 TUBG1/tubg1-1; TUBG2/tubg2-1 plants
  Top panels : Alexander staining of wild-type (left) and F1 (right) anthers. A purple cytoplasmic coloration indicates viability, whereas a green one denotes dead pollen. No sign of pollen degeneracy was observed in pollen of F1 plants, which are expected to segregate 25% double mutant pollen (in which about 2/3 is not genetically transmitted).
  Middle panels : the same coloration was used for mature anthers at dehiscence anthers (wild-type: left; F1: right). At this later stage either, no indication for pollen death is visible on F1 plants.
  Lower panels : In vitro germination of pollen collected from wild-type (left) and F1 (right plants. Arabidopsis germination tests were as described (Derksen et al., 2002). From observation and measurements of ∼400 wild-type and ∼850 F1 pollen tubes, no particular germination defect nor morphological alterations were noted in the pollen from F1 plants.
• Supplemental Figure 2 - Microtubule organization during male gametophyte development
  Overlay images of anti-α-tubulin immunolocalization (in green) and DAPI staining (in blue). (A-F) Meiotic division of the sporogenous cell. At metaphase I (A) and anaphase I (B), the spindle poles are broad and spindles are weakly stained with the anti-a-tubulin antibody. (C) Telophase I of meiosis. Metaphase II (D) and telophase II (E and F) of meiosis. The spindle poles in metaphase II are focused (D) and the phragmoplast at telophase II (E and F) build walls between microspores. (G) Tetrade of microspores. (H) Individual cells of the tetrade are released as free microspores. (I-J) Microspore mitosis I. As highlighted by the spindle (I) and phragmoplast (J) position, the first mitotic division of the microspore is asymmetric and produces a large vegetative cell and a smaller sperm cell enclosed in the vegetative cell. At telophase (J) the phragmoplast encircles the sperm nuclei always pushed against the wall. (K) In binucleate pollen grains, the sperm nuclei is surrounded by dense teardrop-shaped microtubule arrays. (L-M) Pollen mitosis II. Metaphase (L) and anaphase (M) of the second mitotic division of the sperm cell that produces two gametes. (N and O) A dense microtubule array encircled the two sperm cells in trinucleate pollen grains. Scale bars = 8μm in all panels except 4μm in panel I.
• Supplemental Figure 3 - Time-lapse observations of dividing root tip cells of tubg1-1 tubg2-2 double mutant
  (A-B) At the end of G2 phase, the preprophase band is formed and condensed, whereas microtubules accumulate on the nuclear surface at the poles of division. In C, the spindle is formed and later evolves in an abnormally bent spindle (D). (F-G) Abnormal phragmoplast with two poorly organized sets of opposing microtubules. (H) Karyokinesis has however been achieved and two nuclei are present, one small and one large. (I) The new cell wall separating the two daughter cells is visible. Microtubule arrays are revealed by the GFP-MBD fusion protein and imaged by confocal microscopy. All images are stacks of multiple images taken 0.6μm apart. Bars = 10 μm in all images. Time is indicated in minutes on each panel.
• Supplemental Figure 4 - Time-lapse observations of dividing cells in tubg1-1 tubg2-2 double mutant root tip
  Two adjacent dividing cells (1 and 2 in (A)) undergoing different progression in mitosis. The cell #1 was in preprophase in(A), and entered mitosis first. This cell displayed abnormal spindle structures during 130 min (B-I), before forming a rather normal phragmoplast (J, K), and finally separating into two daughter cells (L). Cell #2 although entering M phase after cell #1 as evidenced by the presence of the preprophase band in A-C, terminates mitosis earlier. In this cell, an abnormally bent spindle (E) is formed, evolving after 40 minutes towards a rather normal phragmoplast (F-H). Microtubule arrays are revealed by the GFP-MBD fusion protein and imaged by confocal microscopy. All images are stacks of multiple images taken 0.6μm apart. Bars = 10 μm in all images. Time is indicated in minutes on each panel.