Arabidopsis AUGMIN subunit8 is a microtubule plus-end binding protein that promotes microtubule reorientation in hypocotyls

Abstract

In plant cells, cortical microtubules provide tracks for cellulose-synthesizing enzymes and regulate cell division, growth, and morphogenesis. The role of microtubules in these essential cellular processes depends on the spatial arrangement of the microtubules. Cortical microtubules are reoriented in response to changes in cell growth status and cell shape. Therefore, an understanding of the mechanism that underlies the change in microtubule orientation will provide insight into plant cell growth and morphogenesis. This study demonstrated that AUGMIN subunit8 (AUG8) in Arabidopsis thaliana is a novel microtubule plus-end binding protein that participates in the reorientation of microtubules in hypocotyls when cell elongation slows down. AUG8 bound to the plus ends of microtubules and promoted tubulin polymerization in vitro. In vivo, AUG8 was recruited to the microtubule branch site immediately before nascent microtubules branched out. It specifically associated with the plus ends of growing cortical microtubules and regulated microtubule dynamics, which facilitated microtubule reorientation when microtubules changed their growth trajectory or encountered obstacle microtubules during microtubule reorientation. This study thus reveals a novel mechanism underlying microtubule reorientation that is critical for modulating cell elongation in Arabidopsis.

Figure 1.

AUG8 Participates in Hypocotyl Cell Elongation. (A) Five-day-old dark-grown seedlings of the wild type (Col), aug8, AUG8-OX-1, AUG8-OX-2, and two complementary lines that express AUG8_pro:AUG8-GFP in an aug8 background (COM-1 and COM-2), showing their etiolated hypocotyls. (B) Five-day-old dark-grown aug8 seedlings had longer hypocotyls (13.4 ± 1.4 mm, n = 32, P < 0.05, Student’s t test), and AUG8-OX-1 (8.6 ± 0.6 mm, n = 30) and AUG8-OX-2 (9.2 ± 0.5 mm, n = 32) seedlings had shorter hypocotyls (Student’s t test, P < 0.05) than the wild type (10.8 ± 0.8 mm, n = 31). The lengths of hypocotyls of COM-1 (11.4 ± 0.7 mm, n = 31) and COM-2 (11.3 ± 0.4 mm, n = 35) seedlings were similar to those of the wild type (P > 0.05, Student’s t test). (C) and (D) Scanning electronic microscopy images of wild-type, aug8, AUG8-OX-1, and COM-1 etiolated hypocotyls. Hypocotyl epidermal cells of aug8 exhibited twisted cell files, whereas no such twisted cell files were present in the wild type or AUG8-OX-1. The epidermal cells of aug8 hypocotyls were longer than those of the wild type, whereas AUG8-OX-1 epidermal cells (429 ± 20 µm, n = 100) were much shorter than those of the wild type (575 ± 28 µm, n = 100; P < 0.05, Student’s t test). No obvious difference was found between the epidermal cells of COM-1 (588 ± 35 µm, n = 100) and the wild type (P > 0.05, Student’s t test). Bars = 5 mm in (A) and 10 µm in (C). The data are expressed as mean ± sd. [See online article for color version of this figure.]

Figure 2.

Microtubule Organization in Hypocotyl Cells. (A) Cortical microtubules were visualized in cells of the upper part of aug8, AUG8-OX-1, and wild-type etiolated hypocotyls expressing GFP-tubulin. Bar = 5 µm. (B) Cartoon defining the angle of orientation (θ). Transverse: 90° ≥ θ > 67.5° or −90° ≤ θ < −67.5°. Oblique: 67.5° ≥ θ > 22.5° or −22.5° ≥ θ > −67.5°. Longitudinal: 22.5° ≥ θ > −22.5°. This cartoon was modified from a previously reported definition (Crowell et al., 2011). (C) Microtubules displayed a transverse orientation in 90% of aug8 hypocotyl epidermal cells (n = 122) and 59% of wild-type hypocotyl epidermal cells (n = 114) but primarily displayed random orientations in 78% of AUG8-OX-1 hypocotyl epidermal cells (n = 129). (D) Quantification of microtubules in aug8 and wild-type cells (n > 30 cells of each line). To quantify the microtubule (MT) density in the cell, a fixed line (10 µm) was drawn perpendicularly to the orientation of most cortical microtubules, and the number of microtubules across this line was counted. Five measurements were performed per cell. The data are expressed as mean ± sd.

Figure 3.

AUG8 Binds to Microtubules and Promotes Tubulin Polymerization. (A) Recombinant His-AUG8 cosedimented with taxol-stabilized microtubules prepolymerized from 5 µM tubulin. After high-speed centrifugation, the pellets were analyzed by SDS-PAGE. The amount of His-AUG8 in the pellets increased when higher concentrations of His-AUG8 protein were added. His-NtMAP65-1c was used as a positive control and BSA as a negative control. (B) Densitometry analysis of the results in (A) showed that the binding of recombinant His-AUG8 to microtubules was saturated at a stoichiometry of 0.25 M His-AUG8 per mole of tubulin dimers. (C) The time course of tubulin polymerization from 20 μM tubulin in the presence of various concentrations of His-AUG8 was monitored turbidimetrically by absorbance at 350 nm. BSA was used as a negative control. (D) Amount of tubulin in the pellets after microtubule polymerization. The increase in the amount of tubulin in the pellets corresponded to the increase in the amount of His-AUG8 protein added. Tubulin polymerized with 20 μM taxol was used as a positive control. (E) Immunofluorescence images showing the specific binding of His-AUG8 to the ends of microtubules. AUG8 (green) formed a dot-like structure at one end of a microtubule (red). The right panel shows enlarged images of microtubules with His-AUG8 binding to their ends. (F) and (G) No such association was observed in samples in which the protein was denatured by boiling (F) or when the samples were stained with secondary antibody alone (G). Bars = 5 µm in (E) to (G).

Figure 4.

AUG8 Specifically Binds to the Plus End of Growing Microtubules in Vivo. (A) The colocalization of AUG8-GFP and mCherry-EB1 in a hypocotyl epidermal cell indicates that AUG8 was associated with the plus ends of microtubules. (B) A time series of a hypocotyl epidermal cell expressing mCherry-TUA5 and AUG8-GFP. Closed arrowheads indicate the AUG8-GFP signal (green) associated with the growing end of a microtubule (red). AUG8-GFP disappeared from the microtubule end during shrinkage but reappeared when microtubule growth resumed. Open arrowheads indicate the plus end of a microtubule that started shrinking at 6 s. Bars = 5 µm in (A) and (B). (C) Kymographs of a microtubule are indicated by the closed arrowhead in (B), showing that the AUG8-GFP signal tracked the microtubule growing end.

Figure 5.

Histochemical GUS Staining of AUG8_pro:GUS Transgenic Plants. (A) GUS staining of a 10-d-old transgenic Arabidopsis plant harboring an AUG8_pro:GUS construct. (B) Seedlings of AUG8_pro:GUS were grown in the dark for 3, 4, 5, and 6 d. GUS staining showed that AUG8 expression was promoted when the elongation of hypocotyls decreased. Bar = 10 mm. (C) Quantitative real-time PCR analysis of AUG8 in hypocotyls of 6-d-old dark- or light-grown wild-type seedlings. The expression of AUG8 was higher in hypocotyls of light-grown seedlings than in those of dark-grown seedlings. (D) Quantitative real-time PCR analysis of AUG8 in different parts of hypocotyls. The expression of AUG8 gradually increased from the basal to apical regions of hypocotyls. The difference between different parts was significant (P < 0.001, Student’s t test).

Figure 6.

AUG8 Promotes Light-Induced Microtubule Reorientation. The cells in the upper part of etiolated hypocotyls of 3-d-old wild-type, AUG8-OX-1, and aug8 seedlings were observed. Microtubules were visualized using transgenically expressed GFP-tagged tubulin. The cortical microtubules in wild-type cells shifted their orientation from transverse to longitudinal 40 min after exposure to light, with randomly oriented microtubules appearing at ∼30 min. In AUG8-OX-1 cells, the reorientation of cortical microtubules was completed 48 ± 8 min (n = 8) after exposure to light, which was not significantly different from our observations in the wild type (43 ± 4 min, n = 10, P > 0.05, Student’s t test). Randomly oriented microtubules in AUG8-OX-1 cells were also observed at ∼30 min (middle row of images). In aug8 cells, transverse cortical microtubules were still dominantly observed 30 min after exposure to light. Randomly oriented microtubules occurred at ∼40 min and were persistent up to 50 min after exposure to light. Further observations revealed that complete microtubule reorientation in aug8 took an average of 67 ± 4 min (n = 10), which was significantly slower than in the wild type (P < 0.05, Student’s t test). The data are expressed as mean ± sd. Bar = 5 µm.

Figure 7.

AUG8-Associated Microtubules Display a Shift in Orientation Prior to That of Total Microtubules. The cells in the upper part of etiolated hypocotyls of seedlings that expressed AUG8-GFP and mCherry-TUA5 were used for observation and measurements. A distribution histogram of the observed microtubule (MT) angles relative to the longitudinal cell axis is presented in groups of 10° bins. Microtubules that were perpendicular to the longitudinal cell axis (i.e., transverse microtubules) were assigned to the 90° angle. Measurements were taken from the same group of cells before and after light exposure. No significant differences in the distribution of microtubule angles were found between AUG8-associated microtubules and total microtubules before light exposure (A) and after the completion of microtubule reorientation (D). However, during the process of microtubule reorientation, more AUG8-associated microtubules established orientations that were close to longitudinal alignments as opposed to transverse alignments compared with the distribution of orientation angles of total microtubules ([B] and [C]). Images in the right panel are representative photographs that correspond to the charts on the left. Bar = 5 μm.