How do tubulin mutations result in the organ-twisting phenotype in Arabidopsis?

• Project/Area Number: 26840095
• Research Category: Grant-in-Aid for Young Scientists (B)
• Allocation Type: Multi-year Fund
• Research Field: Plant molecular biology/Plant physiology
• Research Institution: Nara Institute of Science and Technology
• Principal Investigator: Hotta Takashi 奈良先端科学技術大学院大学, バイオサイエンス研究科, 助教 (50644457)
• Project Period (FY): 2014-04-01 – 2016-03-31
• Project Status: Completed (Fiscal Year 2015)
• Budget Amount: ¥4,160,000 (Direct Cost: ¥3,200,000、Indirect Cost: ¥960,000); Fiscal Year 2015: ¥1,950,000 (Direct Cost: ¥1,500,000、Indirect Cost: ¥450,000); Fiscal Year 2014: ¥2,210,000 (Direct Cost: ¥1,700,000、Indirect Cost: ¥510,000)
• Keywords: 植物細胞骨格 / 微小管 / チューブリン

Outline of Final Research Achievements

Microtubules (MTs) are polymerized from tubulin dimers and play an essential role in the regulation of polarized cell elongation in plants. Tubulin mutations alter the orientation of MTs by unknown mechanisms, which results in organ-twisting phenotype in Arabidopsis. To unveil the molecular mechanisms, it would be important to reconstitute MTs in vitro using mutant version of tubulin. To this end, I established an easy method to purify particular tubulin isotypes from Arabidopsis culture cells. Engineered tubulin with internal 6xHis-tag was purified with tubulin purification column (TOG) and subsequent Ni column. Purified tubulin was polymerized under microscope and polymerization dynamics were measured. It was found that plant MTs are highly dynamic and unstable compared to those polymerized from pig brain tubulin. It is now feasible to purify mutated version of Arabidopsis tubulin and analyze the polymerization dynamics and ultrastructure of the MTs containing mutant tubulin.

Research Products

• [Journal Article] Affinity purification and characterization of functional tubulin from cell suspension cultures of Arabidopsis and tobacco (2016)
  • Author(s): Hotta,T., Fujita,S., Uchimura,S., Noguchi,M., Demura, T. Muto,E., Hashimoto, T.
  • Journal Title: Plant Physiol. Volume: 170 Issue: 3 Pages: 1189-1205
  • DOI: 10.1104/pp.15.01173
  • Peer Reviewed
• [Presentation] 精製植物チューブリンを用いた微小管のin vitroダイナミクス解析 (2016)
  • Author(s): 堀田 崇, 内村 誠一, 野口 真大, 出村 拓, 武藤 悦子, 橋本 隆
  • Organizer: 第57回日本植物生理学会年会（盛岡年会）
  • Place of Presentation: 岩手大学 上田キャンパス （岩手県盛岡市）
  • Year and Date: 2016-03-18
• [Presentation] TOGカラムを用いた植物培養細胞からのチューブリン精製 (2015)
  • Author(s): 堀田 崇、橋本 隆
  • Organizer: 第56回日本植物生理学会年会
  • Place of Presentation: 東京農業大学世田谷キャンパス（東京都）
  • Year and Date: 2015-03-16 – 2015-03-18