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. 2018 Mar;176(3):2071-2081.
doi: 10.1104/pp.17.01124. Epub 2018 Feb 5.

Ethylene Signaling Modulates Cortical Microtubule Reassembly in Response to Salt Stress

Liru Dou  1 , KaiKai He  1 , Takumi Higaki  2 , Xiangfeng Wang  1 , Tonglin Mao  3
Affiliations

Ethylene Signaling Modulates Cortical Microtubule Reassembly in Response to Salt Stress

Liru Dou et al. Plant Physiol. 2018 Mar.

Abstract

Regulation of cortical microtubule reorganization is essential for plant cell survival under high salinity conditions. In response to salt stress, microtubules undergo rapid depolymerization followed by reassembly to form a new microtubule network that promotes cell survival; however, the upstream regulatory mechanisms for this recovery response are largely unknown. In this study, we demonstrate that ethylene signaling facilitates salt stress-induced reassembly of cortical microtubules in Arabidopsis (Arabidopsis thaliana). Microtubule depolymerization was not affected under salt stress following the suppression of ethylene signaling with Ag+ or in ethylene-insensitive mutants, whereas microtubule reassembly was significantly inhibited. ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, was shown to play a central role in microtubule reassembly under salt stress. In addition, we performed functional characterization of the microtubule-stabilizing protein WAVE-DAMPENED2-LIKE5 (WDL5), which was found to promote ethylene-associated microtubule reassembly and plant salt stress tolerance. These findings indicate that ethylene signaling regulates microtubule reassembly by up-regulating WDL5 expression in response to salt stress, thereby implicating ethylene signaling in salt-stress tolerance in plants.

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Figures

Figure 1.
Figure 1.
Blockage of ethylene signaling results in abnormal cortical microtubule reassembly in response to salt stress. A, Cortical microtubules in cotyledon pavement cells from wild-type (YFP-tubulin) seedlings treated with 125 mm NaCl for the indicated times. B, Cortical microtubules in the cotyledon pavement cells from wild-type (YFP-tubulin) seedlings treated with 125 mm NaCl with 20 μm Ag+ for the indicated times. C, Cortical microtubules in the cotyledon pavement cells from mutant ein2-5 (YFP-tubulin) seedlings treated with 125 mm NaCl for the indicated times. Scale bar = 20 μm. D, Density of cortical microtubules in A through C, quantified using ImageJ software. Data represent the mean ± sd of 3 independent experiments with a minimum of 10 cells from 3 seedlings assessed in each experiment. Student’s t test, **P < 0.01.
Figure 2.
Figure 2.
EIN3 is essential for microtubule reassembly in response to salt stress. A, Cortical microtubules in the cotyledon pavement cells from wild-type (YFP-tubulin) and ein3eil1 (YFP-tubulin) seedlings treated with 125 mm NaCl for the indicated times. Scale bar = 20 μm. B, Density of cortical microtubules in A quantified using ImageJ software. Data represent the mean ± sd of 3 independent experiments with a minimum of 10 cells from 3 seedlings assessed in each experiment. Student’s t test, **P < 0.01. C, ein3eil1 seedlings expressing 35S:EIN3-GFP were treated with NaCl for 0, 4, or 30 h, and EIN3-GFP fluorescence was examined in hypocotyl epidermal cells. ACC treatment was used as a control. Scale bar = 20 μm. D, Immunoblot analysis of EIN3-GFP accumulation in ein3eil1 seedlings expressing 35S:EIN3-GFP treated with NaCl for 0, 4, or 30 h. Wild-type seedlings served as a comparison. Actin was used as a loading control. E, Cortical microtubules in the cotyledon pavement cells of wild-type (YFP-tubulin) and ein3eil1 (YFP-tubulin) seedlings treated with 125 mm NaCl for 12 h and then transferred onto medium containing 125 mm NaCl with 0 or 10 μm ACC and grown for a further 12 h. Scale bar = 20 μm. F, Density of cortical microtubules in E quantified using ImageJ software. Data represent the mean ± sd of 3 independent experiments with a minimum of 10 cells from 3 seedlings assessed in each experiment. Student’s t test, **P < 0.01.
Figure 3.
Figure 3.
Salt stress activates WDL5 expression via ethylene signaling. A, Histochemical GUS staining of seedlings expressing ProWDL5:GUS treated with NaCl for 0, 4, or 30 h. B, Histochemical GUS staining of seedlings expressing ProWDL5:GUS treated with NaCl with Ag+ for 0, 4, or 30 h. C, WDL5 expression was determined by RT-qPCR using RNA isolated from cotyledons of wild-type or ein3eil11 seedlings that had been treated with NaCl for 0, 4, or 30 h. Error bars represent ± sd (n = 3).
Figure 4.
Figure 4.
WDL5 is a positive regulator of microtubule reassembly in response to salt stress. A, Cortical microtubules in the cotyledon pavement cells from wild-type (YFP-tubulin) seedlings treated with 125 mm NaCl for the indicated times. B, Cortical microtubules in the cotyledon pavement cells from mutant wdl5-1 (YFP-tubulin) seedlings treated with 125 mm NaCl for the indicated times. C, Cortical microtubules in cotyledon pavement cells from WDL5-overexpressing (YFP-tubulin, OE) seedlings treated with 125 mm NaCl for the indicated times. Scale bar = 20 μm. D, Density of cortical microtubules in A to C quantified using ImageJ software. Data are the mean ± sd of 3 independent experiments with a minimum of 10 cells from 3 seedlings assessed in each experiment. Student’s t test, **P < 0.01. E, Wild-type (Col-0 ecotype), wdl5-1, wdl5-1 expressing ProWDL5: WDL5 (Com), and WDL5-overexpressing (OE) seedlings were grown for 5 d and then transferred to plates containing 200 mm NaCl and grown for a further 3 d. F, The survival rates of wild-type (Col-0 ecotype), wdl5-1, wdl5-1 expressing ProWDL5: WDL5 (Com), and WDL5-overexpressing (OE) seedlings. Data represent the mean ± sd of 3 independent experiments with a minimum of 80 seedlings assessed in each experiment. Student’s t test, **P < 0.01.
Figure 5.
Figure 5.
Knockout of WDL5 partially suppresses ethylene-promoted microtubule reassembly in response to salt stress. A, Cortical microtubules in cotyledon pavement cells from wild-type (YFP-tubulin) and mutant wdl5-1 (YFP-tubulin) seedlings treated with 125 mm NaCl for 12 h and then transferred onto medium containing 125 mm NaCl with 0 or 10 μm ACC for a further 12 h growth. Scale bar = 20 μm. B, Density of cortical microtubules quantified using ImageJ software. Data represent mean ± sd of 3 independent experiments with a minimum of 10 cells from 3 seedlings assessed in each experiment. C, Wild-type (Col-0 ecotype), ACC-pretreated wild-type, wdl5-1, and ACC-pretreated wdl5-1 seedlings were grown for 5 d and then transferred to plates containing 200 mm NaCl and grown for a further 3 d. D, The graph shows the survival rate of seedlings. Data shown are the mean ± sd of 3 independent experiments with a minimum of 80 seedlings assessed in each experiment. E, RT-qPCR analysis of WDL5 transcripts in wild-type, ein3eil1, and 2 ein3eil1 lines expressing pSuper:WDL5-Myc (OE#1/ein3eil1 and OE#2/ein3eil1). F, Wild-type, ein3eil1, OE#1/ein3eil1, and OE#2/ein3eil1 seedlings were grown for 5 d and then transferred to plates containing 200 mm NaCl and grown for a further 3 d. G, The graph shows the survival rate of seedlings. Data represent the mean ± sd of 3 independent experiments with a minimum of 80 seedlings assessed in each experiment. Student’s t test, **P < 0.01.
Figure 6.
Figure 6.
Ethylene signaling participates in microtubule reassembly under salt stress: a working model. Cortical microtubules initially depolymerize and then recover themselves under salt stress. PHS1-mediated phosphorylation of α-tubulin promotes microtubule depolymerization under salt stress. SPR1 is degraded by the proteasome-mediated protein degradation pathway, which facilitates salt-induced rapid depolymerization of cortical microtubules. Ethylene signaling plays a positive role in microtubule reassembly under salt stress. WDL5, as a downstream effector of ethylene signaling, promotes microtubule reassembly and salt tolerance in plants. In addition, PA binding to MAP65-1 and relocalization of RIC1 favor microtubule reassembly under salt stress. Scale bars = 20 μm.
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