The microtubule-associated protein WDL4 modulates auxin distribution to promote apical hook opening in Arabidopsis

Abstract

The unique apical hook in dicotyledonous plants protects the shoot apical meristem and cotyledons when seedlings emerge through the soil. Its formation involves differential cell growth under the coordinated control of plant hormones, especially ethylene and auxin. Microtubules are essential players in plant cell growth that are regulated by multiple microtubule-associated proteins (MAPs). However, the role and underlying mechanisms of MAP-microtubule modules in differential cell growth are poorly understood. In this study, we found that the previously uncharacterized Arabidopsis MAP WAVE-DAMPENED2-LIKE4 (WDL4) protein plays a positive role in apical hook opening. WDL4 exhibits a temporal expression pattern during hook development in dark-grown seedlings that is directly regulated by ethylene signaling. WDL4 mutants showed a delayed hook opening phenotype while overexpression of WDL4 resulted in enhanced hook opening. In particular, wdl4-1 mutants exhibited stronger auxin accumulation in the concave side of the apical hook. Furthermore, the regulation of the auxin maxima and trafficking of the auxin efflux carriers PIN-FORMED1 (PIN1) and PIN7 in the hook region is critical for WDL4-mediated hook opening. Together, our study demonstrates that WDL4 positively regulates apical hook opening by modulating auxin distribution, thus unraveling a mechanism for MAP-mediated differential plant cell growth.

Figure 1

WDL4 is required for apical hook opening. A, RT-PCR analysis of WDL4 transcript levels in the apical hook of 3-day-old etiolated seedlings of WT, wdl4-1 mutant, and two independent GFP-WDL4 OE transgenic lines. 18S rRNA was used as a control. B, Apical hook phenotypes of 3-day-old etiolated seedlings of WT, wdl4-1 mutant, and two independent OE lines. Scale bar = 0.5 mm. C, Quantification of the apical hook curvature angles, as shown in (B). Values represent mean ± sd (n > 30, ^**P < 0.01). D, Representative images of WDL4 expression pattern in the apical hook region from WDL4pro:GUS transgenic etiolated seedlings maintained in the dark for the indicated number of days. Scale bar = 0.2 mm. E, Kinematic analyses of apical hook development in etiolated seedlings of WT, wdl4-1 mutant, and OE lines. Compared to WT, the wdl4-1 mutant shows a defect at the hook opening stage.

Figure 2

Ethylene signaling negatively regulates WDL4 expression during hook development. A, RT-qPCR analysis of WDL4 transcript levels in 3-day-old etiolated WT seedlings grown on half-strength MS medium alone (mock) or treated with 100 µM ACC for 4 h. Values represent mean ± sd for three individual biological repeats (**P < 0.01). B, RT-qPCR analysis of WDL4 transcript levels in 3-day-old etiolated seedlings of WT, the eto1-1 mutant, the EIN3ox transgenic line, and the ein3 eil1 mutant. Values represent mean ± sd for three individual biological repeats (*P < 0.05, **P < 0.01). C, Representative images of GUS staining in the hook region of WDL4pro:GUS transgenic seedlings in medium (mock) or with 10  µM ACC treatment. Seedlings were stained and imaged after growth in the dark for 3 days. D, Representative images of GUS staining in the hook region of WDL4pro:GUS transgenic seedlings in medium without (control) or with the indicated chemicals, the ethylene receptor antagonist AgNO₃ and the ethylene biosynthesis inhibitor AVG. Seedlings were stained and imaged after growth in the dark for 1 or 2 days. E, Representative images of GUS staining in the hook region of WDL4pro:GUS transgenic ein3 eil1 seedlings. Two independent lines were checked after seedling growth in the dark for 1 or 2 days. Scale bar = 0.2 mm.

Figure 3

WDL4 is an EIN3 target gene. A, EMSA assay for EIN3 binding to the WDL4 promoter. Each biotin-labeled DNA fragment was incubated with recombinant GST-EIN3. Free GST was used as a control. Competition for the labeled promoter fragments was performed by adding an excess (30×) of unlabeled WT or mutated probes. Two putative EBSs (P1 and P2) in the WDL4 promoter were tested. White arrowhead indicates free probe and black arrowhead indicates bound complex. B, ChIP assay for EIN3 binding to the WDL4 promoter. Chromatin was immunoprecipitated from etiolated seedlings expressing EIN3-3 × FLAG under the β-estradiol-inducible promoter using an anti-FLAG antibody. Seedlings treated with mock buffer (half-strength MS medium with the same volume of DMSO used for β-estradiol treatment) were used as a control. The amount of indicated DNA in the immune complex was determined by qPCR. Values represent mean ± sd from three individual biological repeats. C, Transient expression of WDL4pro:GUS with or without EIN3 in N. benthamiana leaves. LUC was expressed from the Super promoter as an internal control. GUS and LUC activity was quantified, and the GUS/LUC ratio used to estimate the binding activity of EIN3 to the WDL4 promoter. At least three independent experiments were performed with similar results. D and E, Representative images and quantification of the hook curvature in the ein3 eil1 wdl4-1 triple mutant compared to that in WT and the ein3 eil1 double mutant. Two independent ein3 eil1 wdl4-1 triple mutant lines were used for analysis. Values represent mean  ± sd (n > 43 independent seedlings; ^**P < 0.01). Scale bar = 0.5 mm.

Figure 4

WDL5 driven by the WDL4 promoter does not rescue the delayed hook opening phenotype of wdl4-1 mutant. A, Representative images of microtubules incubated with WDL4 or WDL5 using in vitro assays under low temperature and dilution treatments. Scale bar = 10 μm. B and C, Representative images and quantification of hook curvature in wdl4-1 WDL4pro:GFP-WDL5 complementation lines. Three independent lines were used for analysis. Values in (C) represent mean ± sd (n > 46 independent seedlings; ^**P < 0.01). Scale bar = 0.5 mm. D and E, Light-triggered rapid hook opening process in WT, wdl4-1, wdl4-1 WDL4pro:WDL4-GFP complementation line (COM #1) and two independent wdl4-1 WDL4pro:GFP-WDL5 lines. Seedlings were germinated and grown vertically in the dark for 42 h, followed by short-term white light treatment as indicated. Representative photographs were taken, and hook curvature was quantified at different time points as labeled. Values in (E) represent mean ± sd (n > 5 independent experiments each containing >10 seedlings; ^**P < 0.01). Scale bar = 0.5 mm.

Figure 5

WDL4 mutation alters auxin maxima and impairs sensitivity to auxin efflux inhibitor during hook development. A and B, Representative images of DR5:GFP expression patterns and quantified fluorescence intensity in the hook region of dark-grown WT and wdl4-1 mutants at different time points. Values in (B) represent mean ± sd (n > 12 independent seedlings; ^**P < 0.01). Scale bar = 0.2 mm. C and D, Representative images of DR5:GFP expression patterns and quantified fluorescence intensity in the hook region of etiolated seedlings with induced overexpression of WDL4 (iWDL4ox). Seedlings were germinated and grown vertically in the dark for 48 h, and transferred to new plates with or without β-estradiol for another 24 h in the dark. Values in (D) represent mean ± sd (n > 12 independent seedlings; ^**P < 0.01). Scale bar = 0.2 mm. E and F, Representative images of DR5:GFP expression patterns and quantified fluorescence intensity during light-induced hook opening. WT and wdl4-1 seedlings were germinated and grown vertically in the dark for 42 h, and transferred to white light for another 3 h. Values in (F) represent mean ± sd (n > 15 independent seedlings; ^**P < 0.01). Scale bar = 0.2 mm. G and H, Representative images and quantified analysis of NPA effect on light-induced hook opening. WT, wdl4-1, and wdl4-1 WDL4pro:WDL4-GFP (COM #1) seedlings were germinated and grown vertically in the dark for 42 h, and transferred to white light for another 6 h with or without NPA treatment. Values in (F) represent mean ± sd (n > 17 independent seedlings; ^**P < 0.01). Scale bar = 0.5 mm.

Figure 6

WDL4 mutation attenuates the enhanced distribution of PIN1–GFP and PIN7–GFP at the PM during light-induced hook opening. A and B, Representative images shown as pseudocolors (red-green-blue palette) and relative fluorescence intensity of PM-localized PIN1–GFP in hook epidermal cells in WT and wdl4-1 seedlings expressing PIN1pro:PIN1-GFP. Seedlings were germinated and grown vertically in the dark for 42 h, and transferred to white light for another 2 h. Values represent mean ± sd (n > 14 independent seedlings; ^**P < 0.01). Scale bar = 20 μm. C, Relative expression of PIN1 in the apical hook of the seedlings used in A. One to two millimeter of the apical hook regions from seedlings were used for RNA extraction. Values represent mean ± SD for three individual biological repeats. D and E, Representative images shown as pseudocolors and relative fluorescence intensity of PM-localized PIN7–GFP in hook epidermal cells in WT and wdl4-1 seedlings expressing PIN7pro:PIN7–GFP. Seedlings were grown and treated the same way as in A. Values represent mean ± sd (n > 21 independent seedlings; ^**P < 0.01). Scale bar = 20 μm. F, Relative expression of PIN7 in the apical hook of the seedlings used in D. One to two millimeter of the apical hook regions from seedlings were used for RNA extraction. Values represent mean ± sd for three individual biological repeats.

Figure 7

Inactivation of WDL4 impairs trafficking of PIN1–GFP during light-induced hook opening. A and B, Representative images and quantified analysis of BFA effects on light-induced hook opening. WT, wdl4-1, and wdl4-1 WDL4pro:WDL4-GFP (COM #1) seedlings were germinated and grown vertically in the dark for 42 h, and transferred to white light for another 6 h with or without BFA treatment. Values in B represent mean ± sd (n > 36 independent seedlings; ^**P < 0.01). Scale bar = 0.5 mm. C, WT and wdl4-1 seedlings expressing PIN1pro:PIN1–GFP were germinated and grown vertically in the dark for 42 h, and transferred to white light with CHX pre-treatment (50 μM) for 30 min, followed by CHX + BFA (100 μM) for 1.5 h to induce the formation of BFA bodies. BFA was removed and seedlings were incubated in the presence of CHX and white light for another 30 or 60 min. The images of PIN1–GFP were taken at different time points. The red channel shows auto-fluorescence signals. Scale bar = 20 μm. D, Number of PIN1–GFP-labeled BFA bodies per cell and their relative size. Values represent mean ± sd (n > 14 independent seedlings; ^*P < 0.05, ^**P < 0.01).

Figure 8

A hypothetical model for WDL4 function in apical hook development. The formation of hook curvature in etiolated seedlings is achieved by growth inhibition along with asymmetric auxin accumulation at the concave side, while hook opening is achieved by growth promotion and a decrease in the auxin maxima. During the early stage of hook formation, the level of ethylene is high, and the key transcription factor EIN3 accumulates. EIN3 binds to the promoter region of WDL4 and suppresses WDL4 expression. During hook opening, when ethylene levels are relatively low, EIN3 is degraded, and the inhibition of WDL4 expression is released. WDL4 accumulates and promotes hook opening by influencing the auxin maxima and PIN protein distribution and trafficking. The question mark indicates the yet-unknown mechanism by which WDL4 regulates auxin distribution and the trafficking of auxin efflux carriers during hook opening.