Abstract
Microtubules are highly dynamic cellular structures that are required for many biological processes. Cortical microtubules in plant play crucial roles during cell expansion. Its proper dynamics are required for plant growth and responses to environmental stimuli. Arabidopsis mutants, such as sav2/tub4 P287L, display a variety of growth defects, including short and twisting hypocotyls in dark and shade. Both microtubule organization and dynamics are altered in sav2. Here, we have identified a suppressor of sav2 (sus2), which surprisingly contains a missense mutation in another β tubulin gene, TUB6. The mutation results in a L246F substitution in TUB6. It locates at the interface of αβ-intradimer. This mutation partially suppressed the swirling microtubule arrangement in sav2 hypocotyl cells, leading to the partial rescue of sav2 phenotypes. As the mutant behaves as a semi-dominant mutation and the CFP-labeled tub6L246F can incorporate into microtubules, we propose that the incorporation of tub6L246F interferes with the normal function of microtubules. tub6 L246F single mutant is hypersensitive to drugs disrupting microtubule dynamics, such as colchicine, suggesting the mutation may affect microtubule dynamics. Moreover, we found the colchicine hypersensitivity of tub6L246F can be suppressed by tub4P287L, while tub6L246F interferes with the rescuing effect of EB1 on sav2. As P287 locates around M-loop, which is involved in interactions between microtubule protofilaments, we propose that altered interactions at αβ-intradimer interface may affect microtubule dynamics through M-loop mediated interactions between microtubule protofilaments.
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Acknowledgments
We thank Dr. Takashi Hashimoto for providing tub4 P287L seeds; Dr. Rong Yu and Tonglin Mao for providing 35S::GFP-TUA6 and 35S::EB1b-GFP seeds. This work was supported by the National Natural Science Foundation of China, 31171162, 31271298 to Y.T; Fundamental Research Funds for the Central Universities 2012121041 to Y.T.
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J. Yu and K. Dong have equally contributed to this study.
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10725_2016_186_MOESM1_ESM.tif
Sup Fig. 1 Phenotypic characterization of sus1 mutants. a Mutation sites on TUB4 amino acid in the sus1 intragenic suppressors. b Positions (shown in white) of sus1 mutations in the 3D structural model of the αβ-tubulin dimer described by Nogales et al. (1998; PDB accession number, 1TUB). c mTUB4 fragment (with P287L mutation only) under the control of its own promoter complemented sus1 in shade. Representative seedlings are shown. The scale bars representing 2 mm. d Quantitative measurements of hypocotyl length and width of Col-0, sav2 and sus1 mutants grown in continuous white light (Wc) and shade. Error bars represent standard error of mean (SEM) (n≥10); ***P < 0.001 by Student’s t-test; NS, not significant (TIF 25139 KB)
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Sup Fig. 2 cMT patterns in hypocotyl epidermal cells of shade treated Col-0, sav2, sus2 and tub6 L246F. Scale bar represent 20 μm. (TIF 24570 KB)
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Sup Fig. 3 sus2 behaves in a semi-dominant manner. Segregation of WT-like, intermediate and sav2-like seedlings in dark-grown sus2(♀)×sav2 (♂) and sav2(♀)×sus2 (♂) F1 and F2 population. Segregation ratio in F2 population fitted the expected 1:2:1(χ2 = 0.113 and 0.148 < χ2 0.05,2 = 5.991) (TIF 6441 KB)
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Sup Fig. 4 Computational modeling showing L246F interacting with GTP after mutation. GTP-interacting residues in the wild type (top panel) and in the simulated mutant protein structure (lower panel) are shown. Yellow arrow heads indicate the residue equivalent to residue 246 in Arabidopsis TUB6 (TIF 32981 KB)
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Sup Fig. 5 tub6L246F did not exert its suppression effect through increasing its expression. a Quantitative RT-PCR results showing relative expression level of TUB6 in Col-0, sav2, sus2, and tub6 L246F under Wc and shade. Five-d-old light grown seedlings were treated with Wc or simulated shade for 2 h. Error bars represent standard error of mean (n=3). b Protein level of α- and β-tubulin did not change in sav2 and sus2. Tubulin was detected by western using anti-α-tubulin and anti-β-tubulin antibody. Commassie blue (CBB) stain of total proteins are shown as loading control. (TIF 14207 KB)
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Yu, J., Dong, K., He, Q. et al. Studies on suppressors of sav2/shade avoidance 2 revealed altered interaction at the interface of αβ-tubulin intradimer affects microtubule dynamics. Plant Growth Regul 81, 71–79 (2017). https://doi.org/10.1007/s10725-016-0186-7
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DOI: https://doi.org/10.1007/s10725-016-0186-7