The E3 ligase MREL57 modulates microtubule stability and stomatal closure in response to ABA

Abstract

Regulation of stomatal movement is critical for plant adaptation to environmental stresses. The microtubule cytoskeleton undergoes disassembly, which is critical for stomatal closure in response to abscisic acid (ABA). However, the mechanism underlying this regulation largely remains unclear. Here we show that a ubiquitin-26S proteasome (UPS)-dependent pathway mediates microtubule disassembly and is required for ABA-induced stomatal closure. Moreover, we identify and characterize the ubiquitin E3 ligase MREL57 (MICROTUBULE-RELATED E3 LIGASE57) and the microtubule-stabilizing protein WDL7 (WAVE-DAMPENED2-LIKE7) in Arabidopsis and show that the MREL57-WDL7 module regulates microtubule disassembly to mediate stomatal closure in response to drought stress and ABA treatment. MREL57 interacts with, ubiquitinates and degrades WDL7, and this effect is clearly enhanced by ABA. ABA-induced stomatal closure and microtubule disassembly are significantly suppressed in mrel57 mutants, and these phenotypes can be restored when WDL7 expression is decreased. Our results unravel UPS-dependent mechanisms and the role of an MREL57-WDL7 module in microtubule disassembly and stomatal closure in response to drought stress and ABA.

Fig. 1. Cortical microtubules in 26S proteasome mutant plants are insensitive to ABA-induced disassembly.

a Cortical microtubules in guard cells from WT and rpn1a-4 seedlings treated with 5 μM oryzalin for the indicated times. Scale bar = 10 μm. b The graph shows the densities of microtubules in a. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. c Cortical microtubules in guard cells from WT and rpn1a-4 seedlings treated with 10 μM ABA for 40 min. Scale bar = 10 μm. d The graph shows the densities of microtubules in c. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. e Detached rosette leaves from WT, rpn1a-4, and rpn10-1 were incubated in opening buffer for 2 h and then treated with 10 μM ABA plus 0 or 5 μM oryzalin for 2 h. The box and whiskers plots represent minimum and maximum values. The line in the box indicates the median value and the boundaries demonstrate the 25th percentile (upper) and the 75th percentile (lower). Different letters represent significant differences at p < 0.01 (one-way ANOVA). The experiment was repeated three times as different biological replicates with a minimum of 100 stomatal pores.

Fig. 2. WDL7 is degraded during ABA-induced stomatal closure.

a Detached rosette leaves from YFP-tubulin were incubated in opening buffer for 2 h and then treated with 10 μM ABA plus 0 or 20 μM Taxol for 40 min. Scale bar = 10 μm. The graphs show the relative fluorescence of YFP-tubulin and quantitative analysis of microtubule bundling (skewness). Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. Different letters represent significant differences at p < 0.01 (one-way ANOVA). b, c Detached rosette leaves from WDL7-GFP transgenic seedlings were incubated in opening buffer for 2 h and then treated with 10 μM ABA, 10 μM ABA plus 20 μM Taxol, 10 μM ABA plus 20 μM Taxol and 50 μM MG132 for 40 min. Confocal images of the guard cells and pavement cells were taken. Scale bar = 10 μm. The graph shows the relative fluorescence of WDL7-GFP in b and c. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. Different letters represent significant differences at p < 0.01 (one-way ANOVA). d Ten-day-old WT seedlings were treated with mock buffer, 10 μM ABA, or 10 μM ABA plus 50 μM MG132 for 1 h and then total proteins were extracted. Purified MBP-WDL7-FLAG was incubated with equal amount of total proteins for the indicated times. MBP-WDL7-FLAG was detected with anti-FLAG antibody. Actin was used as a control. e Quantitative analysis of protein levels in d. The WDL7 protein level at 0 h was set to 1 as a reference for calculating the relative protein levels at the various time points. Data represent the mean ± SD for three independent experiments. Two-tailed Student’s t test, *p < 0.05. f Ten-day-old WDL7-GFP transgenic seedlings were treated with mock buffer, 10 μM ABA, or 10 μM ABA plus 50 μM MG132 for indicated times and then total proteins were extracted from the leaves. WDL7-GFP was detected with anti-GFP antibody. Actin was used as a control. g Quantitative analysis of protein levels in f. The protein level of WDL7-GFP treated with mock buffer was set to 1 as a reference for calculating relative protein levels of various time points. Data represent the mean ± SD for three independent experiments. Two-tailed Student’s t test, *p < 0.05.

Fig. 3. Overexpression of WDL7 impairs the stomatal closure in response to ABA.

a Detached rosette leaves from Pro_WDL7:GUS transgenic seedlings were incubated in opening buffer for 2 h and then treated with or without 10 μM ABA for 2 h. The epidermal strips were peeled from the rosette leaves and GUS staining was performed. Scale bar = 10 μm. b Quantitative real-time PCR analysis of WDL7 and MREL57 RNA levels in leaves after various treatment durations using 10 μM ABA. UBQ11 was used as a reference gene and RD29A was a positive control. Gene expression levels in seedlings treated with mock buffer were set to 1. The data represent the mean ± SD for three independent experiments. Significant differences from corresponding untreated seedlings are indicated by different letters (p < 0.01), as determined by one-way ANOVA. c WDL7 expression was detected by quantitative real-time PCR in wdl7-1 mutant and two WDL7-overexpressing lines (OE#1 and OE#2). The data represent the mean ± SD for three independent experiments. d Fresh weights of the detached leaves of seedlings from WT, wdl7-1, wdl7-2, OE#1, and OE#2 were measured at the indicated times. The experiment was repeated three times with independent treatments. Error bars indicate standard deviation. e Drought phenotypes of WT, wdl7-1, wdl7-2, OE#1, and OE#2 seedlings in soil. Three-week-old seedlings were subjected to drought stress by withholding water for two weeks before being photographed. Scale bar = 2 cm. Values represent mean ± SD for three independent experiments. Two-tailed Student’s t test, **p < 0.01. f Infrared thermography of seedlings from WT, wdl7-1, wdl7-2, OE#1, and OE#2. Images of 4-week-old plants in soil were taken using an infrared camera. The graph shows the leaf temperature measured using infrared camera software. Data represent mean ± standard deviation (SD) values. Different letters represent significant differences at p < 0.01 (one-way ANOVA). The experiment was repeated three times with independent treatments. g Detached rosette leaves of seedlings from WT, wdl7-1, wdl7-2, OE#1, and OE#2 were incubated in opening buffer for 2 h and then treated with 10 μM ABA plus 0 or 50 μM MG132 for another 2 h. The box and whiskers plots represent minimum and maximum values. The line in the box indicates the median value and the boundaries demonstrate the 25th percentile (upper) and the 75th percentile (lower). Different letters represent significant differences at p < 0.01 (one-way ANOVA). The experiment was repeated three times as different biological replicates with a minimum of 100 stomatal pores.

Fig. 4. WDL7 binds to and stabilizes microtubules in vitro and in vivo.

a WDL7-GFP colocalizes with cortical microtubules detected in guard cells. Detached rosette leaves of seedlings from WDL7-GFP transgenic plants on an mCherry-tubulin background were incubated in opening buffer for 2 h. The experiment repeated three times with similar results. Scale bar = 10 μm. b Plot of a line scan drawn in a showing a strong correlation between the spatial localization of WDL7-GFP and cortical microtubules. c His-WDL7-GFP colocalizes with microtubules polymerized from rhodamine-labeled tubulin in vitro. The experiment repeated three times with similar results. Scale bar = 10 μm. d WDL7 induced the formation of microtubule bundles in vitro. Images show microtubules polymerized from rhodamine-labeled tubulin incubated with 3 μM His-WDL7-GFP, GST-WDL5-His, or His-GFP protein for 30 min. The experiment repeated three times with similar results. Scale bar = 5 μm. e WDL7 stabilizes microtubules against cold and dilution-induced depolymerization. The experiment repeated three times with similar results. Scale bar = 10 μm. f Cortical microtubules in guard cells from WT and WDL7-GFP transgenic seedlings (WDL7ox) on an mCherry-tubulin background treated with 5 μM oryzalin for 10 and 20 min. Scale bar = 10 μm. The graph shows the densities of microtubules in f. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. g Cortical microtubules in guard cells from WT and WDL7-GFP transgenic seedlings (WDL7ox) on an mCherry-tubulin background treated with 0 and 10 μM ABA for 40 min. Scale bar = 10 μm. The graph shows the densities of microtubules in g. Data represent mean ± standard deviation (SD) values for three independent experiments with a minimum of 15 cells each. Different letters represent significant differences at p < 0.01 (one-way ANOVA).

Fig. 5. The E3 ligase MREL57 interacts with and ubiquitinates WDL7.

a Schematic representation of MREL57 truncated proteins. b WDL7 interacts with MREL57 and MREL57 N in a yeast two-hybrid assay. c A semi-in vivo pull-down assay showed an interaction between MREL57 and WDL7. MBP-WDL7-FLAG and MBP were marked with asterisks, respectively. The experiment repeated three times with similar results. d Split-luciferase complementation assay to analyze the interaction between MREL57 and WDL7 in N. benthamiana leaves. MG132 at 50 μM was injected into the leaf tissues 12 h before imaging. The pseudo-color bar shows the range of luminescence. Scale bar = 1 cm. e ABA enhances the interaction between MREL57 and WDL7. MYC-MREL57^C486A-nluc and cluc-WDL7-FLAG were transformed into N. benthamiana leaves. After a 2-day incubation, the leaves were treated with mock buffer or 100 μM ABA for 2 h. Scale bar = 1 cm. f WDL7 and MREL57 protein levels were detected by western blotting using anti-FLAG and anti-MYC antibodies. Actin was used as a control. g Luminescence values were determined using a spectrophotometer. The experiment was repeated three times. Data represent mean ± standard deviation (SD) values. Two-tailed Student’s t test, **p < 0.01. h MREL57 ubiquitinates WDL7 in vitro. Recombinant proteins were purified from E. coli and then incubated at 30 °C for 3 h. The ubiquitination of WDL7 was detected with anti-Ub and anti-FLAG antibodies. The experiment repeated three times with similar results. i MREL57 ubiquitinates WDL7 in vivo. WDL7-GFP and WDL7-GFP/mrel57-1 transgenic seedlings were treated with 0 or 10 μM of ABA for 1 h in the presence of 50 μM MG132. Total proteins were extracted and incubated with anti-GFP mAb-magnetic agarose. Anti-Ub was used to detect the polyubiquitination of WDL7. j Quantitative analysis of the signal intensity in i. Data represent the mean ± SD for three independent experiments. Different letters represent significant differences at p < 0.01 (one-way ANOVA).

Fig. 6. MREL57 destabilizes microtubules and positively regulates stomatal closure in response to ABA.

a Detached rosette leaves from Pro_MREL57:GUS transgenic seedlings were incubated in opening buffer for 2 h and then treated with or without 10 μM ABA for 2 h. The epidermal strips were peeled from the rosette leaves and GUS staining was performed. The experiment repeated three times with similar results. Scale bar = 10 μm. b MREL57 expression was detected by quantitative real-time RT-PCR in WT, mrel57-1, and mrel57-2 mutant seedlings. The experiment was repeated three times as different biological replicates. Error bars indicate standard deviation. c Fresh weights of detached leaves of seedlings from WT, mrel57-1, and mrel57-2 were measured every 30 min for a total of 3 h. The experiment was repeated three times with independent treatments. d Drought phenotypes in the wildtype, mrel57-1, and mrel57-2 in soil. Three-week-old seedlings were subjected to drought stress by withholding water for 2 weeks before being photographed. Values represent mean ± SD for three independent experiments. Two-tailed Student’s t test. **p < 0.01. e Infrared thermography of seedlings from WT, mrel57-1, and mrel57-2. Leaf temperature was measured using infrared camera software. The experiment was repeated three times with independent treatments. Error bars indicate standard deviation. Data represent mean ± standard deviation (SD) values. Different letters represent significant differences at p < 0.01 (one-way ANOVA). f Detached rosette leaves from WT, mrel57-1, and mrel57-2 were incubated in opening buffer for 2 h and then treated with 10 μM ABA with or without oryzalin. The box and whiskers plots represent minimum and maximum values. The line in the box indicates the median value and the boundaries demonstrate the 25th percentile (upper) and the 75th percentile (lower). Different letters represent significant differences at p < 0.01 (one-way ANOVA). The experiment was repeated three times as different biological replicates with a minimum of 100 stomatal pores. g Cortical microtubules in guard cells from WT and mrel57-1 seedlings treated with 5 μM oryzalin for the indicated times. Scale bar = 10 μm. The graph shows the densities of microtubules. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. h Cortical microtubules in guard cells from WT, mrel57-1 mutant, and mrel57-1wdl7-1 double mutant seedlings treated with 10 μM ABA for 40 min. Scale bar = 10 μm. The graph shows the densities of microtubules. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each.

Fig. 7. Degradation of WDL7 is required for MREL57-mediated stomatal closure in response to ABA.

a Detached rosette leaves of seedlings from YFP-tubulin, WDL7-GFP, and WDL7-GFP/mrel57-1 were incubated in opening buffer for 2 h and then treated with 10 μM ABA plus 20 μM Taxol for 40 min. Confocal images of the guard cells and pavement cells were taken. Scale bar = 10 μm. b The graphs show the relative fluorescence in a. Data represent mean ± standard deviation (SD) values from three independent experiments with a minimum of 15 cells each. Different letters represent significant differences at p < 0.01 (one-way ANOVA). c Degradation of WDL7 was clearly decreased in mrel57-1 mutant in a cell-free degradation assay. Purified MBP-WDL7-FLAG was incubated with equal amounts of total proteins from 10-day-old WT and mrel57-1 seedlings. MBP-WDL7-FLAG was detected with anti-FLAG antibody. Actin was used as a control. d Quantitative analysis of protein levels in c. The WDL7 protein level at 0 h was set to 1 as a reference for calculating the relative protein levels at the various time points. Data represent the mean ± SD for three independent experiments. Two-tailed Student’s t test, *p < 0.05. e Ten-day-old WDL7-GFP and WDL7-GFP/mrel57-1 transgenic seedlings were treated with 100 μM CHX, 100 μM CHX plus ABA, or 100 μM CHX plus ABA and MG132 for the indicated times and then total protein was extracted from the leaves. WDL7-GFP was detected with an anti-GFP antibody. Actin was used as a control. f Quantitative analysis of the protein levels in e. The WDL7 protein level in the leaves from mock buffer-treated seedlings was set to 1 as a reference for calculating the relative protein levels at the various time points. Data represent the mean ± SD for three independent experiments. Two-tailed Student’s t test, *p < 0.05, **p < 0.01. g, Fresh weights of detached leaves of seedlings from WT, mrel57-1, wdl7-1, mrel57-1wdl7-1#1, and mrel57-1wdl7-1#2 were measured every 30 min over a total of 3 h. The experiment was repeated three times with independent treatments. Error bars indicate standard deviation. h Infrared thermography of seedlings from WT, mrel57-1, wdl7-1, mrel57-1wdl7-1#1, and mrel57-1wdl7-1#2. Leaf temperature was measured using infrared camera software. Data represent mean ± standard deviation (SD) values. i Detached rosette leaves of seedlings from WT, mrel57-1, wdl7-1, mrel57-1wdl7-1#1, and mrel57-1wdl7-1#2 were incubated in opening buffer for 2 h and then treated with 10 μM ABA for 2 h. The box and whiskers plots represent minimum and maximum values. The line in the box indicates the median value and the boundaries demonstrate the 25th percentile (upper) and the 75th percentile (lower). Different letters represent significant differences at p < 0.01 (one-way ANOVA). The experiment was repeated three times as different biological replicates with a minimum of 100 stomatal pores.

Fig. 8. A hypothetical model for MREL57-WDL7 module function in stomatal closure in response to ABA.

Working model for the role of WDL7 proteolysis in ABA-induced stomatal closure and microtubule disassembly in WT and mrel57 mutant guard cells. WDL7 is a microtubule-stabilizing protein. In WT, the E3 ligase MREL57 ubiquitinates and degrades WDL7 in response to ABA, which facilitates microtubule disassembly and stomatal closure. In mrel57 mutant, the degradation of WDL7 is blocked. WDL7 binds to and stabilizes microtubules, resulting in stomatal closure insensitivity to ABA in mrel57 mutant.