CML24 is Involved in Root Mechanoresponses and Cortical Microtubule Orientation in Arabidopsis

Abstract

Mechanostimuli can influence plant root system architecture by causing alterations in the root tip growth direction and triggering lateral root initiation. However, how a plant root senses and translates mechanostimulation into appropriate growth and/or developmental responses remains largely unclear. The fast expression induction and transcript turnover of the Arabidopsis TCH genes by touch stimulation suggest that the TCH genes may function in mechano-related events. However, the physiological functions of the TCH genes in Arabidopsis mechanoresponses remain undetermined. Here we screened a suite of tch mutants by characterizing their root growth behaviors on hard-agar surfaces. Two calmodulin-like 24 (CML24 or TCH2) mutants, cml24-2 and cml24-4, exhibited reduced root length, biased skewing, and altered epidermal cell file rotation (CFR) phenotypes compared with wild type (Col-0). The mutant phenotypes were dependent on hard-agar surface contact and disappeared when seedlings were grown in liquid medium. Abnormal glass barrier responses of cml24 mutants further indicate touch response defects. Pharmacological tests revealed differential sensitivity of cml24 mutants to microtubule-targeted agents. Nonadditive effects of mutations in CML24 and transgenic expression of a functional microtubule label, MBD-GFP, on root skewing and CFR phenotypes suggest a potential microtubule-related role of CML24. In vivo visualization of microtubule structures with the MBD-GFP reporter revealed altered cortical microtubule orientation in the epidermal cells in cml24-4. Our observations indicate that CML24 has a role in Arabidopsis root mechanoresponses, possibly through the regulation of cortical microtubule orientation.

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Acknowledgments

This material is based upon work supported by the National Science Foundation under grant Nos. MCB 0817976 (JB) and MCB-0641288, by the National Aeronautics and Space Administration under grant No. NNX09AK80G (SG), and the National Natural Science of China under grant No. 10872223 (BW). We thank Elliot M. Meyerowitz for kindly providing the MBD-GFP seeds, and Sarah Swanson and William Deery for technical support in imaging. We are grateful to Yu-Chang Tsai and Won-Gyu Choi for helpful discussions and to Liz Eich for isolation and characterization of the cml12-2 mutant.

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Correspondence to Bochu Wang or Janet Braam.

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Supplementary material 1 (AVI 451 kb) Time-lapse movie of a wild-type root as in Figure 2a, showing a typical step-like growth response upon encountering a glass barrier. Time interval between fames is 10 minutes. Scale bars= 1mm

Supplementary material 2 (AVI 453 kb) Time-lapse movie of a cml24-2 root as in Figure 2f, showing an aberrant growth response upon encountering a glass barrier. Time interval between fames is 10 minutes. Scale bars= 1mm

Supplementary material 3 (AVI 426 kb) Time-lapse movie of a cml24-4 root as in Figure 2g, showing an aberrant growth response upon encountering a glass barrier. Time interval between fames is 10 minutes. Scale bars= 1mm

Supplementary material 4 (TIFF 4203 kb) Root waving of wild type, cam2-1, and cml12-2 grown on hard-agar surfaces. (a) Wild type Col-0 seedlings grown on hard-agar surfaces tilted 15o backward from vertical. No detectable biased root skewing or general root growth defect was found in (b) cam2-1 or (c) cml12-2 seedlings compared with (a) wild type in the same condition. Images were taken from above the agar surface. Scale bars: a-c = 10 mm

Supplementary material 5 (TIFF 5086 kb) Root cell file features of wild type, cml24-2, and cml24-4 grown on hard-agar surfaces supplemented with 1µM taxol. Representative root cell file features of (a) wild type, (b) cml24-2, and (c) cml24-4 when grown on 1µM taxol supplemented hard-agar surfaces tilted 15o backward from vertical. No swollen cells were found in (a) wild type or (c) cml24-4, whereas many swollen cells were detected in (b) cml24-2. Scale bars: a-c = 0.5mm

Supplementary material 6 (TIFF 2859 kb) Root waving and skewing of wild type, cml24-2, and wild-type (Col-0) YC3.6, YC3.6/cml24-2 grown on hard-agar surfaces. YC3.6 is a transgenic line producing a Ca2+ reporter (Monshausen and others 2009.) (a) Wild type Col-0, (b) cml24-2, (c) YC3.6, and (d) YC3.6/cml24-2 seedlings grown on hard-agar surfaces tilted 15o backward from vertical. No detectable rescue of the general growth defect was found in cml24-2 seedlings (b) expressing YC3.6 (d). Images were taken from above the agar surface. Scale bars: a-d = 10mm

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Wang, Y., Wang, B., Gilroy, S. et al. CML24 is Involved in Root Mechanoresponses and Cortical Microtubule Orientation in Arabidopsis . J Plant Growth Regul 30, 467–479 (2011). https://doi.org/10.1007/s00344-011-9209-9

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Keywords

  • Mechanoresponse
  • Root skewing
  • Barrier response
  • TCH genes
  • Microtubules
  • Arabidopsis