Gamma-tubulin in basal land plants: characterization, localization, and implication in the evolution of acentriolar microtubule organizing centers

Abstract

Although seed plants have gamma-tubulin, a ubiquitous component of centrosomes associated with microtubule nucleation in algal and animal cells, they do not have discrete microtubule organizing centers (MTOCs) comparable to animal centrosomes, and the organization of microtubule arrays in plants has remained enigmatic. Spindle development in basal land plants has revealed a surprising variety of MTOCs that may represent milestones in the evolution of the typical diffuse acentrosomal plant spindle. We have isolated and characterized the gamma-tubulin gene from a liverwort, one of the extant basal land plants. Sequence similarity to the gamma-tubulin gene of higher plants suggests that the gamma-tubulin gene is highly conserved in land plants. The G9 antibody to fission yeast gamma-tubulin recognized a single band of 55 kD in immunoblots from bryophytes. Immunohistochemistry with the G9 antibody clearly documented the association of gamma-tubulin with various MTOC sites in basal land plants (e.g., discrete centrosomes with and without centrioles and the plastid surface in monoplastidic meiosis of bryophytes). Changes in the distribution of gamma-tubulin occur in a cell cycle-specific manner during monoplastidic meiosis in the liverwort Dumortiera hirsuta. gamma-Tubulin changes its localization from the plastid surface in prophase I to the spindle, from the spindle to phragmoplasts and the nuclear envelope in telophase I, and back to the plastid surfaces in prophase II. In vitro experiments show that gamma-tubulin is detectable on the surface of isolated plastids and nuclei of D. hirsuta, and microtubules can be repolymerized from the isolated plastids. gamma-Tubulin localization patterns on plastid and nuclear surfaces are not affected by the destruction of microtubules by oryzalin. We conclude that gamma-tubulin is a highly conserved protein associated with microtubule nucleation in basal land plants and that it has a cell cycle-dependent distribution essential for the orderly succession of microtubule arrays.

Figure 1.

Characterization of the γ-Tubulin Genes of Basal Land Plants. (A) Deduced phylogeny tree generated by the unweighted pair group method with arithmetic mean algorithm. The γ-tubulins used to generate the tree are listed in (B). (B) Amino acid sequence comparison of the C. japonicum and other plant γ-tubulins. The deduced amino acid sequence of the C. japonicum γ-tubulin is shown in the top row. Amino acid sequences of plant γ-tubulins were aligned, and amino acids identical to those of the C. japonicum γ-tubulin are indicated by dashes. Dots indicate gaps introduced for the alignment. The inferred recognition epitope of the G9 antibody is underlined. H. sap, Homo sapiens; X. lae, Xenopus laevis; A. tha (At), Arabidopsis thaliana; N. tab (Nt), Nicotiana tabacum; Z. may (Zm), Zea mays; C. jap (Cj), Conocephalum japonicum; P. pat (Pp), Physcomitrella patens; A. phy (Ap), Anemia phyllitidis; C. rei (Cr), Chlamydomonas reinhardtii; S. pom (Sp), Schizosaccharomyces pombe; A. nid, Aspergillus nidulans; S. cer, Saccharomyces cerevisiae.

Figure 2.

Immunoblot Analysis of G9 Antibody in Protein Extracts from Sporogenous Tissue of the Bryophytes C. japonicum and D. hirsuta. (A) C. japonicum. (B) D. hirsuta. Lane 1, Coomassie brilliant blue staining; lane 2, control immunoblot without primary antibodies; lanes 3 and 4, monoclonal antibodies against α- and β-tubulin, respectively; lane 5, G9 anti-γ-tubulin. The positions of molecular mass markers and their molecular masses (kD) are indicated at left.

Figure 3.

Triple Labeling for γ-Tubulin, α and β-Tubulin, and DNA Shows the Subcellular Localization of γ-Tubulin Homologs in Some Typical MTOC Sites in Bryophytes. (A) to (C) A QMS during meiotic prophase of the moss Entodon seductrix. Note that γ-tubulin homologs (B) are localized around the four daughter plastids (arrowheads in [B] and asterisks in [C]) from which microtubules emanate (A). (D) to (F) A prophase spindle with centrioles in the final spermatogenous mitotic division of the liverwort Makinoa crispate. Two distinctive fluorescent dots (E) at two distinctive MTOCs (D) are considered to be equivalent to centriolar centrosomes. (G) to (L) A prophase spindle with POs ([G] to [I]) and a metaphase spindle with broad poles ([J] to [L]) during archesporial mitosis of the liverwort Marchantia polymorpha. Note that although a prophase spindle arises from the discrete POs where γ-tubulin homologs locate (H), the polar distribution of γ-tubulin extending along proximal portions is seen in a metaphase spindle that has dispersed MTOCs (K). (M) to (P) A barrel-shaped metaphase spindle in meiosis I ([M] and [N]) and RMSs and phragmoplasts in meiotic telophase II ([O] and [P]) of the liverwort C. japonicum. Note that γ-tubulin homologs localized on the mature spindle microtubules, but distribution clearly was biased toward the broad spindle poles (N). RMSs emanating from telophase nuclei (asterisks in [O]) and phragmoplasts develop between nuclei. γ-Tubulin homologs localized at putative MTOCs, the nuclear surface, and phragmoplasts (P). Arrowheads in (P) show three newly forming cell plates that appear as dark, unstained lines. Microtubules ([A], [D], [G], [J], [M], and [O]), γ-tubulin homologs ([B], [E], [H], [K], [N], and [P]), and nuclei ([C], [F], [I], and [L]) were stained using anti-plant-tubulin, G9 anti-γ-tubulin, and 4′,6-diamidino-2-phenylindole (DAPI), respectively. Bars = 10 μm.

Figure 4.

Changes in the Subcellular Localization of γ-Tubulin Homologs during Plastid Division and Partitioning in Monoplastidic Meiosis of D. hirsuta (Premeiotic Interphase to Prophase I). (A) and (B) A premeiotic interphase cell with a single plastid (asterisk in [B]). Note that dots of γ-tubulin homologs are distributed randomly in the cytoplasm (B) with reticular arrays of microtubules (A). (C) and (D) An early prophase cell with two daughter plastids (asterisks in [D]). Note the γ-tubulin homologs localized around the two plastids. (E) to (H) A mid-prophase cell with four daughter plastids (asterisks in [F]). Note that the localization of γ-tubulin homologs around the four daughter plastids becomes more prominent. (G) and (H) show single optical sections of (E) and (F), respectively. (I) and (J) A mid-prophase cell with four daughter plastids (asterisks in [I]). The primary antibody (G9) is omitted (a similar stage is shown in [E]). Crosstalk and nonspecific signal of secondary antibody were not observed. Microtubules (green signal in [A], [C], [E], [G], and [I]), DNA (blue signal in [A], [C], [E], [G], and [I]), and γ-tubulin homologs (red signal in [B], [D], [F], [H], and [J]) were stained using anti-plant-tubulin, DAPI, and G9 anti-γ-tubulin, respectively. (A) and (B), (C) and (D), (E) and (F), (G) and (H), and (I) and (J) show the same cells with different color combinations. Bar = 10 μm.

Figure 5.

Changes in the Subcellular Localization of γ-Tubulin Homologs during the Progression of Meiosis I in Monoplastidic Cells of D. hirsuta (Prometaphase to Anaphase I). (A) and (B) A prometaphase cell with four daughter plastids (asterisks in [A]). Note that the γ-tubulin localizes distinctly near the spindle poles. (C) and (D) A cell in metaphase I. Note that γ-tubulin homologs are localized along spindle microtubule arrays but are absent from the kinetochore side. (E) and (F) A cell in anaphase I. γ-Tubulin homologs are localized at the polar surface of each daughter nucleus (arrowheads in [F]). (G) and (H) A cell in telophase I. γ-Tubulin homologs are localized both in phragmoplasts and on the nuclear surface. The position of the cell plate is shown by arrowheads. Microtubules (green signal in [A], [C], [E], and [G]), DNA (blue signal in [A], [C], [E], and [G]), and γ-tubulin homologs (red signal in [B], [D], [F], and [H]) were stained using anti-plant-tubulin, DAPI, and G9 anti-γ-tubulin, respectively. (A) and (B), (C) and (D), (E) and (F), and (G) and (H) show the same cells with different color combinations. Bar = 10 μm.

Figure 6.

Changes in the Subcellular Localization of γ-Tubulin Homologs during the Progression of Meiosis II in Monoplastidic Cells of D. hirsuta (Prophase II to Telophase II). (A) and (B) A cell in prophase II. Note the reappearance of the localization of γ-tubulin homologs around the plastids (asterisks in [B]). (C) and (D) A cell in metaphase II. The broad localization of γ-tubulin homologs in the polar region of spindles is identical to that in prophase I. (E) to (H) A cell in telophase II. Note that γ-tubulin homologs are localized at the nuclear surface and early phragmoplasts are localized between daughter nuclei. (G) and (H) show single optical sections of (E) and (F), respectively. Microtubules (green signal in [A], [C], [E], and [G]), DNA (blue signal in [A], [C], [E], and [G]), and γ-tubulin homologs (red signal in [B], [D], [F], and [H]) were stained using anti-plant-tubulin, DAPI, and G9 anti-γ-tubulin, respectively. (A) and (B), (C) and (D), (E) and (F), and (G) and (H) show the same cells with different color combinations. Bar = 10 μm.

Figure 7.

Localization of γ-Tubulin Homologs in Isolated Organelles, and Microtubule-Initiating Activity of Isolated Plastids from Sporocytes of D. hirsuta. (A) to (C) An isolated plastid. γ-Tubulin homologs are localized on the plastid surface. (D) to (F) An isolated nucleus. γ-Tubulin homologs are localized on the nuclear surface. DNA ([B] and [E]) and γ-tubulin homologs ([C] and [F]) were stained using DAPI and G9 anti-γ-tubulin. (A) and (D) show differential interference contrast views. (A) to (C) and (D) to (F) show the same cells. (G) and (H) Unfixed isolated plastid just after isolation. (I) and (J) Isolated plastid after incubation with rhodamine-labeled tubulin. Rhodamine-labeled tubulin nucleates from the plastid. (G) and (I) show differential interference contrast views of the same plastids in (H) and (J), respectively. (H) and (J) show views through a red filter to detect the rhodamine signal. Chlorophyll autofluorescence also is seen in these images. Bar = 10 μm.

Figure 8.

Effects of the Anti-Microtubule Drug Oryzalin on the Subcellular Localization of γ-Tubulin Homologs in Monoplastidic Cells of D. hirsuta. (A) and (B) A prophase I cell treated for 20 min with 20 μM oryzalin. Plastid-based microtubules are absent (A). Note that γ-tubulin homologs are detected around the plastids (B). (C) and (D) A metaphase I cell treated for 20 min with 20 μM oryzalin. γ-Tubulin homologs are localized along the remnant microtubule bundles. (E) and (F) A telophase II cell treated for 20 min with 20 μM oryzalin. Note that no γ-tubulin homologs are localized along the remnant microtubules of RMS. Oryzalin treatment did not affect the γ-tubulin homolog localization on the nuclear surface. (G) and (H) An early meiotic cell treated for 2 h with 20 μM oryzalin. Note that γ-tubulin homologs still remain around the plastids (asterisks in [H]). Microtubules (green signal in [A], [C], [E], and [G]), DNA (blue signal in [A], [C], [E], and [G]), and γ-tubulin homologs (red signal in [B], [D], [F], and [H]) were stained using anti-plant-tubulin, DAPI, and G9 anti-γ-tubulin, respectively. (A) and (B), (C) and (D), (E) and (F), and (G) and (H) show the same cells with different color combinations. Bar = 10 μm.

Figure 9.

Schemes of the Diversity of γ-Tubulin Localization in Prophase Spindles. (A) A cell with centrioles. In green algae and in spermatogenous cells of bryophytes, γ-tubulin localizes precisely at centriolar centrosomes. (B) A cell with POs. γ-Tubulin localizes distinctly at acentriolar POs of some liverworts. (C) The spindle during monoplastidic meiosis in some bryophytes arises from daughter plastids. γ-Tubulin localizes on the plastid surface. (D) In higher plants, γ-tubulin localizes indistinctly at polar regions of diffuse spindles. Microtubules, plastids, nuclei, and γ-tubulin are shown as dark green, lime green, blue, and red, respectively.

Figure 10.

Schemes of the MTOC Cycle during Monoplastidic Meiosis in Bryophytes. (A) Premeiotic interphase. (B) Early prophase. (C) Mid prophase. QMS and cytoplasmic lobing predict the polarity of the two meiotic divisions. (D) Late prophase. (E) Metaphase I. (F) Telophase I. (G) Early prophase II. (H) Early telophase II. Microtubules, plastids, nuclei, and γ-tubulin are shown as dark green, lime green, blue, and red, respectively.