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. 2015 Jul 1;169(1):325–337. doi: 10.1104/pp.15.00609

Ethylene Regulates the Arabidopsis Microtubule-Associated Protein WAVE-DAMPENED2-LIKE5 in Etiolated Hypocotyl Elongation1,[OPEN]

Jingbo Sun 1,2, Qianqian Ma 1,2, Tonglin Mao 1,*
PMCID: PMC4577400  PMID: 26134166

Ethylene affects the expression of a microtubule-associated protein and etiolated hypocotyl cell elongation in Arabidopsis.

Abstract

The phytohormone ethylene plays crucial roles in the negative regulation of plant etiolated hypocotyl elongation. The microtubule cytoskeleton also participates in hypocotyl cell growth. However, it remains unclear if ethylene signaling-mediated etiolated hypocotyl elongation involves the microtubule cytoskeleton. In this study, we functionally identified the previously uncharacterized microtubule-associated protein WAVE-DAMPENED2-LIKE5 (WDL5) as a microtubule-stabilizing protein that plays a positive role in ethylene-regulated etiolated hypocotyl cell elongation in Arabidopsis (Arabidopsis thaliana). ETHYLENE-INSENSITIVE3, a key transcription factor in the ethylene signaling pathway, directly targets and up-regulates WDL5. Etiolated hypocotyls from a WDL5 loss-of-function mutant (wdl5-1) were more insensitive to 1-aminocyclopropane-1-carboxylic acid treatment than the wild type. Decreasing WDL5 expression partially rescued the shorter etiolated hypocotyl phenotype in the ethylene overproduction mutant eto1-1. Reorganization of cortical microtubules in etiolated hypocotyl cells from the wdl5-1 mutant was less sensitive to 1-aminocyclopropane-1-carboxylic acid treatment. These findings indicate that WDL5 is an important participant in ethylene signaling inhibition of etiolated hypocotyl growth. This study reveals a mechanism involved in the ethylene regulation of microtubules through WDL5 to inhibit etiolated hypocotyl cell elongation.

Skotomorphogenesis occurs as buried seedlings fully elongate their hypocotyls upward in search of the soil surface. When elongated hypocotyls encounter mechanical obstacles during seedling extrusion from the soil, inhibition of rapid etiolated hypocotyl elongation is required to optimize the seedling’s ability to push through the soil without damaging its shoot meristem. Disturbing this physiological process significantly affects seedling emergence from the soil and survival (Zhong et al., 2014). The phytohormone ethylene plays a crucial role in the negative regulation of hypocotyl elongation in the dark. Ethylene functions through five membrane-bound receptors (ETHYLENE RESPONSE1 [ETR1], ETHYLENE RESPONSE SENSOR1 [ERS1], ETR2, ERS2, and ETHYLENE-INSENSITIVE4 [EIN4]) and a well-defined signal transduction pathway to activate the redundant nucleus-localized transcription factors EIN3 and ETHYLENE-INSENSITIVE3-LIKE1 (EIL1). EIN3 and EIL1 specifically bind to the promoters of ethylene-response target genes to activate or repress their expression, thereby modulating ethylene-related responses in plants (Boutrot et al., 2010; Zhang et al., 2011; Chang et al., 2013). The abundance of the EIN3 protein rapidly increases with ethylene treatment, but it is targeted by Skp1/Cullin1/F-boxEIN3-BINDING F-BOX PROTEIN1/2 (SCFEBF1/EBF2) complexes and degraded in the absence of ethylene (Guo and Ecker, 2003; Potuschak et al., 2003). One of the most widely documented ethylene responses in etiolated seedlings is the triple response, including a short, thickened hypocotyl when dark-grown Arabidopsis (Arabidopsis thaliana) seedlings are treated with ethylene or its biosynthetic precursor 1-aminocyclopropane-1-carboxylic acid (ACC; Bleecker et al., 1988; Ecker, 1995). Ethylene and ACC stimulate hypocotyl elongation in the light but suppress etiolated hypocotyl elongation in the dark, largely due to the concomitant activation of two contrasting pathways (Ecker, 1995; Zhong et al., 2012). Genetic evidence has shown that ethylene-overproduced or constitutive ethylene-response mutants generally display defective etiolated hypocotyl cell growth phenotypes. For example, the ethylene-overproducing mutant eto1-1 and the CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) mutant ctr1-1 have shorter etiolated hypocotyls than wild-type seedlings in the dark (Kieber et al., 1993). Treatment with ACC obviously inhibited the etiolated hypocotyl elongation of wild-type seedlings but not ein3eil1 seedlings, and overexpression of EIN3 significantly inhibited hypocotyl elongation in the dark (An et al., 2010), demonstrating that EIN3 and EIL1 are required for ethylene-inhibited hypocotyl elongation in the dark. Although ethylene has been implicated in the regulation of hypocotyl growth in the dark, the molecular mechanisms regarding the EIN3 regulation of downstream effectors that may directly participate in inhibiting etiolated hypocotyl elongation are largely unknown.

Cortical microtubules orient cellulose fibrils to control plant cell growth by building the mechanical properties of the cell wall (Lloyd and Chan, 2008; Lloyd, 2011; Bashline et al., 2014; Lei et al., 2014). Multiple approaches have demonstrated that regulation of the stabilization, organization, and dynamics of cortical microtubules is pivotal for hypocotyl cell growth. Etiolated Arabidopsis seedlings exhibit stunted hypocotyls when the microtubule-disrupting drug propyzamide is used to disturb cortical microtubules (Le et al., 2005). Mutation or overexpression of many microtubule-associated proteins (MAPs) also results in abnormal etiolated hypocotyl cell elongation by altering the stability and organization of cortical microtubules. For example, overexpression of the microtubule plus-end tracking protein SPIRAL1 promotes etiolated hypocotyl elongation by stabilizing cortical microtubules, whereas overexpression of MICROTUBULE-DESTABILIZING PROTEIN25 (MDP25) inhibits etiolated hypocotyl elongation by destabilizing cortical microtubules (Nakajima et al., 2004; Li et al., 2011; Galva et al., 2014).

Hypocotyl elongation is strongly influenced by developmental and environmental cues. Studies have detailed the mechanisms involved in hypocotyl cell elongation that are regulated by light, phytohormones, and transcription factors (Niwa et al., 2009; Luo et al., 2010; Fan et al., 2012). However, the role of microtubules in these physiological processes remains to be determined. A recent study showed that Arabidopsis MDP40 is involved in brassinosteroid (BR) signaling promotion of hypocotyl growth (Wang et al., 2012). Although ethylene has been reported to affect the organization of cortical microtubules in plant cells (Takahashi et al., 2003; Le et al., 2005; Soga et al., 2010; Polko et al., 2012), the molecular mechanisms regarding the effects of ethylene signaling on microtubule regulation in mediating hypocotyl elongation are largely unclear. The identification of MAPs involved in ethylene-mediated hypocotyl elongation will facilitate our understanding of the underlying mechanisms of ethylene-regulated cell growth.

WAVE-DAMPENED2-LIKE5 (WDL5) belongs to the MAP WAVE-DAMPENED2 (WVD2)/WAVE-DAMPENED2-LIKE (WDL) family in Arabidopsis (Yuen et al., 2003; Perrin et al., 2007). Seedlings with constitutive WVD2 expression exhibit short, thick stems and roots and inverted handedness of twisting hypocotyls and roots (Yuen et al., 2003). WDL3 is a negative regulator of hypocotyl elongation in the light and is degraded by the ubiquitin-26S proteasome-dependent pathway in the dark (Liu et al., 2013), suggesting diverse physiological roles of WVD2/WDL proteins in plant growth and plant cell morphogenesis. In this study, we demonstrate that ethylene regulates microtubules through WDL5, which is targeted by EIN3 and up-regulated by ethylene, to inhibit etiolated hypocotyl cell elongation. This study demonstrates that WDL5 is involved in ethylene-mediated etiolated hypocotyl cell elongation by altering the organization and stability of cortical microtubules.

RESULTS

WDL5 Is an Ethylene-Up-Regulated Gene

Given that WDL5 expression was shown to be regulated by ethylene in a microarray assay and its homolog WDL3 is involved in hypocotyl elongation in Arabidopsis (Zhong et al., 2009; Liu et al., 2013), we speculated that WDL5 may play a role in ethylene-regulated hypocotyl cell elongation. We first determined whether and how ethylene regulates WDL5 expression.

RNA was purified from etiolated seedlings of the ethylene overproduction mutant eto1-1 and the ethylene-insensitive mutant ein2-5, and quantitative real-time PCR analyses were performed. WDL5 expression was much higher in the eto1-1 mutant but lower in the ein2-5 mutant compared with the wild type (Fig. 1A). After etiolated wild-type seedlings were treated with 100 μm ACC, quantitative real-time PCR showed that WDL5 expression was induced by ACC treatment, with peak levels detected 6 h after treatment (Fig. 1B). These results indicate that ethylene up-regulates WDL5 expression.

Figure 1.

Figure 1.

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Ethylene up-regulates WDL5 expression. A, WDL5 expression was determined using quantitative real-time PCR with RNA purified from wild-type (WT), eto1-1, or ein2-5 etiolated seedlings. Wild-type gene expression levels were set to 1. The data represent means ± sd of three independent experiments. Significant differences from the corresponding wild type are indicated by asterisks (**, P < 0.01), as determined by Student’s t test. B, Quantitative real-time PCR analysis of WDL5 RNA levels in 4-d-old dark-grown seedlings after various treatment durations using 100 μm ACC or a mock buffer. UBIQUITIN11 (UBQ11) was used as a reference gene. Gene expression levels in untreated seedlings were set to 1. The data represent means ± sd of three independent experiments. Significant differences from corresponding untreated seedlings are indicated by asterisks (**, P < 0.01), as determined by Student’s t test.

WDL5 Functions as a Positive Regulator in Ethylene-Mediated Etiolated Hypocotyl Cell Elongation

WDL5 expression is significantly up-regulated by ethylene, suggesting a potential role of WDL5 in ethylene-regulated hypocotyl cell elongation. To determine the function of WDL5, the transfer DNA (T-DNA) insertion mutant wdl5-1 was obtained from The Arabidopsis Information Resource (CS436432). The homozygous wdl5-1 mutant contained a T-DNA insertion in the intron, and a full-length transcript was not detected by reverse transcription (RT)-PCR (Fig. 2, A and B). However, a partial transcript upstream of the T-DNA insertion site was identified (Supplemental Fig. S1). The wdl5-1 phenotype indicated that the function of WDL5 was abolished or dramatically affected in the mutant (Fig. 2, C and D). In addition, another WDL5 T-DNA insertion allele (wdl5-2-CS434701) with a T-DNA insertion site in the exon was detected, although a full-length WDL5 transcript was not detected. This mutant exhibited a phenotype similar to that of wdl5-1 (Supplemental Fig. S2, A–C).

Figure 2.

Figure 2.

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WDL5 is a positive regulator of ethylene-inhibited etiolated hypocotyl cell elongation. A, Physical structure of Arabidopsis WDL5. WDL5 contains eight exons and seven introns, which are represented by black boxes and lines, respectively. Positions of two T-DNA insertion mutants, wdl5-1 (T-DNA line CS436432, intron 7) and wdl5-2 (T-DNA line CS434701, exon 8), are noted by arrows above the diagram. B, RT-PCR analysis of WDL5 transcripts in the wild-type (WT) Columbia ecotype seedlings and wdl5-1 mutant, with UBQ as a control. C, The wdl5-1 mutant shows much longer etiolated hypocotyls when grown on MS medium for 5 d in the presence of 10 μm ACC. D, Relative hypocotyl length of seedlings grown on MS medium supplemented with 0, 0.25, 0.5, 1, 1.5, 2, 3, 5, 10, and 20 μm ACC in the dark for 5 d. Three independent experiments were performed with similar results, each with three biological repeats. More than 40 seedlings were measured in each replicate. **, P < 0.01, as determined by Student’s t test. The data represent means ± se. E, Scanning electron microscopy images of etiolated hypocotyl epidermal cells from wild-type and wdl5-1 seedlings when grown on MS medium for 5 d in the absence and presence of 10 μm ACC. Bar = 100 μm. F, Relative hypocotyl cell length of the wild type and wdl5-1 was measured and calculated from at least 500 cells under dark growth conditions. **, P < 0.01, as determined by Student’s t test. The data represent means ± se.

To analyze the role of WDL5 in ethylene-mediated etiolated hypocotyl elongation, wild-type and wdl5-1 seedlings were cultured on MS medium containing various concentrations of ACC, and etiolated hypocotyl lengths were measured. Observation of 5-d-old dark-grown wdl5-1 seedlings revealed that the hypocotyl length was longer than in wild-type plants without ACC treatment. This phenotype was complemented by PWDL5:WDL5 (Supplemental Fig. S3, A–C), indicating that the aberrant etiolated hypocotyl phenotype in wdl5-1 is associated with WDL5 expression levels. Hypocotyl length in 5-d-old etiolated seedlings from the wild type was dramatically reduced in the presence of ACC, while etiolated hypocotyls were much longer in wdl5-1 seedlings grown on the same medium (Fig. 2C). The effects of ACC on hypocotyl elongation were more pronounced in the wild type and decreased in the wdl5-1 mutant at all concentrations (Fig. 2D), indicating that wdl5-1 mutant seedlings are much less sensitive to ACC in etiolated hypocotyl elongation than the wild type. Thus, these observations demonstrate that WDL5 plays a positive role in ethylene-regulated etiolated hypocotyl elongation.

Scanning electronic microscopy revealed that the length of etiolated hypocotyl cells was longer in wdl5-1 than in wild-type seedlings in the presence of 10 μm ACC, particularly in the middle and top regions (Fig. 2E). Paired Student’s t test indicated that the difference in relative cell lengths between the wild type and the wdl5-1 mutant in response to ACC was significant (Fig. 2F). The number of cells in individual hypocotyl-epidermal cell files in wild-type and wdl5-1 seedlings was similar (approximately 20–22). These results suggest that WDL5 plays a positive role in the ethylene inhibition of etiolated hypocotyl cell elongation.

WDL5 Is an EIN3 Target Gene

The redundant transcription factors EIN3 and EIL1 play central roles in the ethylene regulation of plant growth and development. EIN3 and EIL1 bind to EIN3-binding sites (EBSs) on target gene promoters (Kosugi and Ohashi, 2000; Zhong et al., 2009; Shi et al., 2012). To determine if ethylene signaling directly regulates WDL5 expression, we analyzed the WDL5 promoter sequence. Bioinformatics analysis had revealed that the WDL5 promoter regions contain three putative EBSs (Zhong et al., 2009; located at −389 to −393, −1,111 to −1,115, and −1,205 to −1,209 upstream of the transcription start site; Fig. 3A). To determine whether the EIN3 protein binds to the WDL5 promoter, chromatin immunoprecipitation (ChIP) was performed. An EIN3-3×FLAG fusion protein was expressed using an estradiol-inducible promoter (Chen et al., 2009) and immunoprecipitated using an antibody recognizing the FLAG tag. Genomic DNA fragments that coimmunoprecipitated with EIN3-3×FLAG were analyzed using quantitative real-time PCR. Chromatin immunoprecipitated with the anti-FLAG antibody was enriched in fragments P1 (located from −332 to −510 upstream of the transcription start site) and P2 (containing two close EBSs; located from −1,086 to −1,247 upstream of the transcription start site) in the WDL5 promoter but not in a control in which DNA precipitated without the anti-FLAG antibody (Fig. 3B).

Figure 3.

Figure 3.

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WDL5 is an EIN3 target gene. A, Three distinctive EIN3-binding sites were predicted in the promoter region of the WDL5 gene. The numbers −389, −1,111, and −1,205 represent the starting nucleotide positions of the EBSs upstream of the transcription start site in the WDL5 promoter. Fragment P1 contained one EBS (−389 to −393) and was located from −332 to −510; fragment P2 contained two close EBSs (−1,111 to −1,115 and −1,205 to −1,209) and was located from −1,086 to −1,247; fragment P3 was located from −536 to −690 without putative EBSs upstream of the transcription start site in the WDL5 promoter. B, ChIP-quantitative RT-PCR assay of EIN3 binding to WDL5 promoters in vivo. Chromatin from dark-grown EIN3-3×FLAG transgenic seedlings was immunoprecipitated with an anti-FLAG antibody, and the amount of the indicated DNA in the immune complex was determined by quantitative RT-PCR. DNA precipitated without the addition of antibody (−Ab) was used as a negative control. At least three independent experiments were performed with similar results. Data are mean values of three replicates ± sd from one experiment. DMSO, Dimethyl sulfoxide. C and D, EMSA for EIN3 binding to WDL5 promoters. Each biotin-labeled DNA fragment was incubated with the GST-EIN3 protein. Competition for labeled promoter sequences was performed by adding an excess of unlabeled probes. A biotin-labeled DNA fragment (P3) that does not contain putative EBSs in the WDL5 promoter served as a negative control. The arrows indicate bands resulting from EIN3 binding to P1 (C) and P2 (D) fragments in the WDL5 promoter.

We further tested the direct binding of EIN3 to P1 and P2 of the WDL5 promoter with electrophoretic mobility shift assays (EMSAs) using 5-(and 6-)carboxytetramethylrhodamine succinimidyl ester (NHS)-biotin-labeled DNA fragments of the WDL5 promoter and a bacterially expressed truncated glutathione S-transferase (GST)-EIN3 protein (amino acids 141–352) containing the DNA-binding domain in vitro (Chen et al., 2009). The results showed that the GST-EIN3 fusion protein bound to P1 and P2, but not the −536 to −690 region (P3; without putative EBSs, upstream of the transcription start site), of the WDL5 promoter. Moreover, binding was abolished by the addition of increasing amounts of unlabeled P1 and P2 probes (Fig. 3, C and D), indicating that EIN3 can directly bind to the WDL5 promoter in vitro. These results demonstrate that WDL5 is an EIN3 target gene. A previous study showed that ethylene constantly activates a hypocotyl elongation-inhibiting pathway mediated by the APETALA2-type transcription factor ETHYLENE RESPONSE FACTOR1 (ERF1) in the dark (Zhong et al., 2012). We evaluated the WDL5 promoter sequence and found a typical ERF1-binding motif with a variant base site (AGCCGCT; Supplemental Fig. S4A, asterisk). A previous study demonstrated that this site is crucial for DNA binding of ERF1 (Fujimoto et al., 2000). EMSA in this study also showed that ERF1 did not bind to the WDL5 promoter due to this variant site (Supplemental Fig. S4B), demonstrating that ethylene regulates WDL5 expression through EIN3 but not ERF1.

Decreasing WDL5 Expression Partially Suppresses Short Etiolated Hypocotyls in the eto1-1 Mutant

Because the above-mentioned results show that WDL5 is an EIN3 target and an ethylene-up-regulated gene, we hypothesized that decreased WDL5 expression could suppress the short etiolated hypocotyl phenotype induced by the overproduction of ethylene. We crossed wdl5-1 with eto1-1 to create a wdl5-1eto1-1 double mutant. All 15 of the wdl5-1eto1-1 lines obtained exhibited the longer etiolated hypocotyl phenotype, and line 2 was selected for further analyses (Fig. 4B; data not shown).

Figure 4.

Figure 4.

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Decreasing WDL5 expression partially rescues shorter etiolated hypocotyls in the eto1 mutant. A, RT-PCR analysis of WDL5 transcripts in wild-type (WT), eto1-1, and wdl5-1eto1-1 seedlings. B, The wdl5-1eto1-1 double mutant shows longer etiolated hypocotyls than eto1-1 grown on MS medium in the dark for 5 d. C, Average hypocotyl lengths measured from at least 40 seedlings under dark growth conditions. **, P < 0.01, as determined by Student’s t test. The data represent means ± sd. D, Scanning electron microscopy images of etiolated hypocotyl epidermal cells of the wild type, eto1-1, and wdl5-1eto1-1. Bar = 100 μm. E, Length of etiolated hypocotyl cells from the wild type, eto1-1, and wdl5-1eto1-1 grown in the dark for 5 d. **, P < 0.01, as determined by Student’s t test. The data represent means ± sd.

RT-PCR showed that WDL5 transcription levels were considerably decreased in wdl5-1eto1-1 seedlings (Fig. 4A). Decreased WDL5 expression was correlated with a dramatic increase in the etiolated hypocotyl length of eto1-1 mutants in 5-d-old etiolated seedlings (Fig. 4, B and C). Scanning electronic microscopy revealed that etiolated hypocotyl cell length in eto1-1 mutants was increased when WDL5 expression was reduced (Fig. 4D). Hypocotyl cell length was significantly increased in wdl5-1eto1-1 mutants (Fig. 4E). This evidence demonstrates that WDL5 is a downstream factor in the ethylene signaling pathway and is associated with inhibited etiolated hypocotyl cell elongation in response to ethylene.

WDL5 Regulates Cortical Microtubule Organization and Stability in Response to Ethylene

Because cortical microtubule organization is associated with the growth status of etiolated hypocotyls (Le et al., 2005; Crowell et al., 2011) and WDL5 plays a positive role in ethylene-mediated etiolated hypocotyl elongation, we investigated the effects of WDL5 on the regulation of cortical microtubule organization in response to ethylene. Wild-type and wdl5-1 seedlings were grown for 4 d in the dark and treated with ACC. After 4 d, parallel arrays of cortical microtubules were mostly transversely oriented to the longitudinal hypocotyl growth axis in the upper regions of etiolated hypocotyls in the wild type and wdl5-1 mutants (Fig. 5, A and E). After treatment with 100 μm ACC for 40 min, most of the cells in wild-type hypocotyls had random, oblique, or longitudinal microtubule arrays (Fig. 5, B and I), while almost 40% of transverse cortical microtubules remained in the hypocotyl cells of wdl5-1 seedlings (Fig. 5, F and I). Increasing the duration of treatment induced predominantly longitudinal cortical microtubules in the wild-type cells but not in wdl5-1 cells (Fig. 5, C, G, and I), indicating that cortical microtubule reorganization was partially hindered in wdl5-1 cells in response to ACC treatment. Cortical microtubule arrays in wild-type and wdl5-1 cells did not differ after the cells were treated with mock buffer for 90 min (Fig. 5, D, H, and I). This demonstrates that the much longer etiolated hypocotyl phenotype in wdl5-1 cells is correlated with a defect in microtubule reorganization from transverse to longitudinal in response to ethylene.

Figure 5.

Figure 5.

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The organization of cortical microtubules in wdl5-1 cells is insensitive to treatment with ACC. A to H, Wild-type (WT; A–D) and wdl5-1 mutant (E–H) etiolated hypocotyls with a yellow fluorescent protein (YFP)-tubulin background were treated with mock buffer or 100 μm ACC. Cortical microtubules from the upper region of hypocotyl epidermal cells were observed without ACC treatment (A and E), treated with ACC for 40 min (B and F), treated with ACC for 90 min (C and G), or treated with a mock buffer for 90 min (D and H). Bar in H = 20 μm. I, Frequency of microtubule orientation patterns in the upper region of etiolated hypocotyl epidermal cells from the wild type and the wdl5-1 mutant (n > 120 cells).

To gain insight into the mechanism by which WDL5 mediates etiolated hypocotyl cell elongation through the regulation of cortical microtubule organization in response to ethylene, wild-type and wdl5-1 epidermal hypocotyl cells pretreated with ACC were treated with the microtubule-disrupting drug oryzalin. Epidermal cells in the top region were used to compare cortical microtubule stability. To quantify the effects of oryzalin on cortical microtubules following treatment with ACC, the number of cortical microtubules in each treatment group was determined. The density of cortical microtubules in wild-type epidermal cells was similar to the density in wdl5-1 cells before treatment (Fig. 6, A, D, G, and J). However, the relative microtubule numbers were significantly different after oryzalin treatment (Fig. 6M). In the absence of ACC pretreatment, cortical microtubules were mostly disrupted in wdl5-1 epidermal cells after treatment with 10 μm oryzalin for 3 min, while more microtubules were observed in wild-type cells (Fig. 6, B and E). Statistical analysis using paired Student’s t test indicated that this difference was significant (Fig. 6M, blue asterisks), suggesting that WDL5 functions as a microtubule stabilizer in vivo.

Figure 6.

Figure 6.

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Cortical microtubules are more sensitive to treatment with oryzalin in wdl5-1 cells in response to ACC. A to L, Cortical microtubules were observed in the upper region of etiolated hypocotyl epidermal cells from the wild type (WT; A–C and G–I) and the wdl5-1 mutant (D–F and J–L) with a YFP-tubulin background pretreated with mock buffer or 100 μm ACC after treatment with 0 μm oryzalin (A, D, G, and J) or 10 μm oryzalin for 3 min (B, E, H, and K) or 8 min (C, F, I, and L). Bar in L = 20 μm. M, Relative number of cortical microtubules in hypocotyl epidermal cells from the wild type and wdl5-1 using ImageJ software (n > 50 cells from each sample). Student’s t tests compared the number of cortical microtubules in hypocotyl epidermal cells of wdl5-1 with the number in the wild type under the same conditions: **, P < 0.01 and *, P < 0.05. The data represent means ± sd.

In addition, the percentage of remaining cortical microtubules increased in wild-type epidermal cells (from approximately 52% to approximately 68%) when pretreated with 100 μm ACC for 90 min and then treated with 10 μm oryzalin for 3 min (Fig. 6, B, H, and M, red asterisks) but increased slightly in wdl5-1 cells (from approximately 38% to approximately 43%; Fig. 6, E, K, and M). Increasing the duration of oryzalin treatment resulted in the disappearance of most of the cortical microtubules in mock buffer-pretreated and ACC-pretreated wdl5-1 cells. The percentage of remaining cortical microtubules was approximately 12% and approximately 18%, respectively (Fig. 6, F, L, and M). However, the percentage of cortical microtubules was increased significantly in ACC-pretreated wild-type cells compared with mock buffer-pretreated wild-type cells (from approximately 20% to approximately 56%; Fig. 6, C, I, and M, green asterisks). Statistical analysis using paired Student’s t test indicated that the differences were significant (Fig. 6M). These results demonstrate that WDL5 functions as a microtubule stabilizer in ethylene-inhibited etiolated hypocotyl elongation.

WDL5 Binds to and Stabilizes Microtubules in Vitro

Given that WDL5 is required for cortical microtubule stability in ethylene-mediated hypocotyl elongation, the molecular basis for WDL5 regulation of microtubules was investigated in vitro. A GST-WDL5-His fusion protein was purified from Escherichia coli, and a cosedimentation assay was performed to determine whether WDL5 directly binds to microtubules. GST-WDL5-His (4 μm) was incubated with preformed 5 μm paclitaxel-stabilized microtubules at room temperature for 20 min, followed by centrifugation. SDS-PAGE analysis revealed that GST-WDL5-His, but not GST alone, bound to and cosedimented with the microtubules (Fig. 7A). To investigate the localization pattern of WDL5 in vivo, a construct expressing WDL5 fused with a C-terminal GFP tag under the control of the 35S promoter was generated and transiently introduced into Arabidopsis pavement cells. Confocal microscopy observations showed that WDL5-GFP exhibited filamentous localization in the cells (Fig. 7B). The filamentous localization was disrupted by treatment with oryzalin (Fig. 7D) but was nearly intact in the presence of Latrunculin A (LatA), a reagent that depolymerizes actin filaments (Fig. 7C). To confirm this result, WDL5-GFP and MBD-mCherry were transiently coexpressed in Arabidopsis pavement cells. Confocal microscopy showed that the WDL5-GFP green fluorescent signal overlapped with the MBD-mCherry red fluorescent signal, confirming that WDL5 colocalized with microtubules (Fig. 7, E–G). Colocalization was analyzed by plotting WDL5-GFP and MBD-mCherry signal intensities using ImageJ software (Fig. 7, G and H). Colocalization of WDL5-mCherry with cortical microtubules was also observed in hypocotyl epidermal cells of Arabidopsis seedlings stably expressing WDL5-mCherry in a GFP-tubulin background (Supplemental Fig. S5). These data demonstrate that WDL5 colocalizes with microtubules in vitro and in cells.

Figure 7.

Figure 7.

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WDL5 directly binds to microtubules. A, GST-WDL5-His was cosedimented with paclitaxel-stabilized microtubules (MTs). GST-WDL5-His was most abundant in the supernatant (S) in the absence of microtubules but cosedimented with microtubules into pellets (P). WDL5 colocalizes with cortical microtubules in cells. B, WDL5-GFP was transiently expressed in Arabidopsis pavement cells, where it localized to filamentous structures. C and D, The filamentous pattern of WDL5-GFP was essentially unaffected when treated with 100 nm LatA for 30 min (C) but was disrupted when the cells were treated with 10 μm oryzalin for 30 min (D). E to G, Analysis of the colocalization of transiently expressed WDL5-GFP and MBD-mCherry. Bars = 10 μm (D), and 20 μm (G). H, Plot of the line scan drawn in G showing a strong correlation between the spatial localization of WDL5-GFP and MBD-mCherry.

To investigate the direct effect of WDL5 on microtubule stability, low-temperature and dilution treatments that disrupt microtubules were applied in vitro. MAP65-1, which stabilizes microtubules under these conditions (Mao et al., 2005), was used as a control. Rhodamine-labeled tubulin (20 μm) was incubated in the presence or absence of WDL5 (3 μm) or MAP65-1 (3 μm) to allow tubulin polymerization (Fig. 8, A–C). The solutions were then incubated at 10°C for 30 min (Fig. 8, D–F) or diluted with 50× prewarmed buffer and incubated at 35°C for 60 min (Fig. 8, G–I) prior to fixation. After fixation, samples were observed by confocal microscopy. Like WVD2 and WDL3 in the Arabidopsis WVD2/WDL protein family, WDL5 fusion proteins induced the formation of large microtubule bundles in vitro (Fig. 8B; Supplemental Fig. S6). Microtubule filaments in the absence of WDL5 were fully disassembled after low-temperature and dilution treatments (Fig. 8, D and G). However, many large microtubule bundles remained in the presence of WDL5 (Fig. 8, E and H) or MAP65-1 (Fig. 8, F and I) after the treatments. These results indicate that WDL5 is capable of stabilizing microtubules against low temperature- and dilution-induced depolymerization.

Figure 8.

Figure 8.

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WDL5 stabilizes microtubules in vitro. A to C, Images of microtubules polymerized from rhodamine-labeled tubulin (20 μm) incubated in the presence or absence of 3 μm GST-WDL5-His or GST-MAP65-1 protein for 30 min. D to I, Samples from A to C were subjected to 10°C for 30 min (D–F) or diluted with a solution containing WDL5 or MAP65-1 in 50× PEM buffer (see “Materials and Methods”; G–I). Bar in I = 20 μm. J, Model of WDL5 functions on cortical microtubules in ethylene-inhibited etiolated hypocotyl cell elongation. Ethylene activates the transcription factor EIN3/EIL1 by a well-defined signal transduction pathway; EIN3 directly regulates WDL5 expression; WDL5 alters the stability of and reorganizes cortical microtubules, which results in the inhibition of etiolated hypocotyl cell elongation. Arrows and bars represent positive and negative regulation, respectively.

DISCUSSION

Understanding how hormone signaling regulates cortical microtubules is essential in elucidating developmental mechanisms in plants. In this study, we demonstrate that the microtubule-stabilizing protein WDL5 participates in ethylene signaling-inhibited etiolated hypocotyl cell elongation.

The Hormone Signaling Pathway Directly Regulates MAPs in Hypocotyl Elongation

Many phytohormones play crucial roles in regulating hypocotyl elongation, such as GAs, auxin, BR, and ethylene. Microarray assays have shown that the transcriptional levels of many MAPs are regulated by hormones (Zhong et al., 2009; Sun et al., 2010). However, it is still unclear whether those proteins are involved in hormone signaling-mediated hypocotyl elongation. A previous study showed that Arabidopsis MDP40 promotes etiolated hypocotyl cell elongation via BR signaling (Wang et al., 2012). In this study, we showed that WDL5 participates in ethylene signaling-inhibited etiolated hypocotyl cell elongation. Although other hormones, such as GAs and auxin, are capable of altering cortical microtubule organization in growing cells (Nick et al., 1990; Shibaoka, 1993, 1994; Fujino et al., 1995; Vineyard et al., 2013), no MAPs have been identified that target and are regulated by their signaling pathways. Thus, investigating individual hormone signaling pathways through microtubules by directly targeting MAPs in hypocotyl elongation is important in understanding regulatory mechanisms.

Previous studies have shown that ethylene and BR signaling pathways play different roles in etiolated hypocotyl cells (Wang et al., 2002; An et al., 2010). Although BRs cross talk with ethylene in a broad spectrum of physiological and developmental processes (Choudhary et al., 2012), it is still unknown how plant cells coordinate opposite functions on microtubules within the same cell to promote or inhibit elongation. Future studies will be necessary to provide more experimental data to demonstrate whether similar regulatory mechanisms in microtubules are exploited by other environmental and developmental cues and how those pathways cross talk to mediate plant cell growth and morphogenesis via microtubules.

Microtubule-Stabilizing Proteins Are Involved in Ethylene-Regulated Etiolated Hypocotyl Cell Elongation

MAPs play positive and negative roles in hypocotyl cell elongation (Li et al., 2011; Liu et al., 2013). These proteins are considered to be microtubule stabilizers or destabilizers depending on their effect on stability (Heald and Nogales, 2002). In this study, cortical microtubule stability increased in etiolated hypocotyl epidermal cells from ACC-treated wild-type seedlings (Fig. 6). Coincidentally, expression of the microtubule stabilizer WDL5 was significantly increased by treatment with ACC (Fig. 1), and etiolated hypocotyl cell elongation in wdl5-1 was less sensitive to ACC treatment (Fig. 2). Additionally, 16 of the WDL5-overexpressing lines obtained exhibited the shorter etiolated hypocotyl phenotype (data not shown), and line 10 was selected for analysis. Observation of 5-d dark-grown seedlings from line 10 revealed that the etiolated hypocotyl length was considerably reduced (Supplemental Fig. S7, A–C). This evidence suggests that ethylene-inhibited etiolated hypocotyl cell growth may be required to increase the levels of negative regulators that function as microtubule stabilizers.

Our findings are in agreement with previous studies showing that microtubules are more stable in shorter etiolated hypocotyl cells from some mutants, such as the regulatory particle non-ATPase (RPN) subunit RPN10 partial loss-of-function mutant rpn10-1 and the BR-deficient mutant deetiolated2, than in the wild type (Wang et al., 2009, 2011, 2012). Increasing the expression of microtubule stabilizers, such as WDL3, also inhibits hypocotyl cell elongation (Liu et al., 2013). Destabilization of cortical microtubules and increased expression of microtubule destabilizers are necessary for the BR promotion of etiolated hypocotyl elongation (Wang et al., 2012). Thus, regulating the expression of microtubule stabilizers and destabilizers may play a crucial role in hormone-mediated hypocotyl cell elongation. However, the molecular mechanisms involved are complicated. For example, whether the microtubule-stabilizing or -destabilizing activity of those regulators is transient or prolonged in nature and the means through which they coordinate to maintain a dynamic cortical microtubule array in response to diverse hormone signaling are still unclear.

In addition, this study showed that decreased WDL5 expression only partially suppressed shorter etiolated hypocotyls in the eto1-1 mutant (Fig. 4). A possible explanation for this phenomenon is that other MAPs with WDL5-like activities are also involved in ethylene-inhibited etiolated hypocotyl elongation. In addition to MAP involvement, microarray assays have shown that expression levels of many negative regulators of plant cell elongation, such as RALF23, RALF31, and RALF33 from the Rapid Alkalinization Factor family, are obviously up-regulated by ethylene (Srivastava et al., 2009; Zhong et al., 2009; Morato do Canto et al., 2014), which may also be a cause of shorter etiolated hypocotyls in eto1-1. Thus, future studies will be necessary to functionally identify other MAPs and negative regulators of cell elongation involved in ethylene-mediated hypocotyl growth.

Ethylene stimulates hypocotyl elongation of Arabidopsis seedlings in the light. A previous study indicated that EIN3 and EIL1 are required for ethylene-promoting hypocotyl elongation in the light, mainly through the activation of the transcription factor PHYTOCHROME-INTERACTING FACTOR3 (PIF3; Zhong et al., 2012). We did not find the typical PIF3-binding motif (G box; Monte et al., 2004) in the WDL5 promoter sequence. Although WDL5 expression was found to be increased in light-grown EIN3-overexpressing seedlings and decreased in the ethylene-insensitive mutant ein2-5 compared with the wild type (Supplemental Fig. S8A), hypocotyl elongation in the wdl5-1 mutant was similar to that of wild-type seedlings in the absence or presence of ACC (Supplemental Fig. S8, B–D), suggesting that WDL5 may not be involved in ethylene-promoted hypocotyl elongation in the light. The potential physiological function of WDL5 in response to ethylene in the light should be further investigated.

The characterization of WDL5 provides strong evidence for the role of microtubules as a link between ethylene signaling and ethylene-mediated etiolated hypocotyl cell elongation. We propose the following model describing the function of WDL5 in ethylene-inhibited hypocotyl cell elongation in the dark (Fig. 8J): ethylene functions through a well-defined signal transduction pathway to activate EIN3/EIL1 transcription factors; EIN3 directly targets the WDL5 promoter to up-regulate WDL5 expression; and WDL5 acts on cortical microtubules with microtubule-stabilizing activity to maintain a longitudinal organization, which inhibits etiolated hypocotyl cell elongation.

MATERIALS AND METHODS

Plant Materials and Growth Conditions

All plant materials used in this study were from the Arabidopsis (Arabidopsis thaliana) Columbia ecotype background. The wdl5-1 (CS436432) and wdl5-2 (CS434701) mutants, ordered from the Arabidopsis Biological Resource Center, were from the Columbia ecotype background. PCR genotyping and sequencing results revealed that wdl5-1 and wdl5-2 are knockout mutants with a T-DNA insertion in the seventh intron and eighth exon of WDL5. Seeds were sterilized and placed on MS medium (Sigma-Aldrich) with 1% (w/v) agar and 3% (w/v) Suc. For hypocotyl measurement, plates were placed at 22°C in the light for 12 h after stratification at 4°C for 2 d and then transferred to the dark for 5 d. Mutants ein2-5 (Alonso et al., 1999) and eto1-1 (Kieber et al., 1993) and 35S:Tubulin5A-YFP transgenic plants (Kirik et al., 2012) were used in this study.

Isolation of WDL5 Complementary DNA Clones from Arabidopsis

The full-length WDL5 complementary DNA sequence was amplified using RT-PCR. The primers used to amplify WDL5 were 5′-TCTAGAATGGACCCTGAGAGTATCATGGC-3′ and 5′-GGTACCTTAATGCTCAACAGCAACCGC-3′. GST-WDL5-His-tagged fusion proteins were expressed and purified according to the manufacturer’s protocols. Protein concentration was determined using a Bio-Rad protein assay kit. Protein samples were analyzed by SDS-PAGE.

PWDL5:WDL5 Construction in Arabidopsis

To complement the hypocotyl phenotype of the wdl5-1 mutant, a fragment 2,138-bp upstream of the initiation codon (ATG) in WDL5 to the stop codon (TAA) was amplified and reconstructed into a pCAMBIA1300 vector. Complementary DNAs for WDL5 were amplified and reconstructed into the expression vector pCAMBIA1300 under the control of the WDL5 promoter and nopaline synthase terminator. Constructs were transformed into Arabidopsis plants by Agrobacterium tumefaciens (strain GV3101). Homozygous lines were used for subsequent analyses.

ACC Treatment

Four-day-old etiolated hypocotyls from wild-type and wdl5-1 plants with a 35S:Tubulin5A-YFP background grown on MS medium were used. Seedlings were treated with ACC at a concentration of 100 μm for 0, 40, and 90 min, and cortical microtubules were observed using confocal microscopy.

Microtubule Cosedimentation Assay

Porcine brain tubulins were purified using a previously published method by Castoldi and Popov (2003) and used for sedimentation assays. Tubulin assembly and cosedimentation of microtubules with GST-WDL5-His fusion proteins were performed as described by Mao et al. (2005) and Li et al. (2011). Purified proteins were centrifuged at 150,000g at 4°C for 20 min before use. Prepolymerized, paclitaxel-stabilized microtubules (5 μm) were incubated with 3 μm WDL5 fusion proteins in PEM buffer (1 mm MgCl2, 1 mm EGTA, and 100 mm PIPES-KOH, pH 6.9) plus 20 μm paclitaxel at room temperature for 20 min. After centrifugation at 100,000g for 20 min, the supernatant and pellets were subjected to SDS-PAGE.

Low-Temperature and Dilution Assays

Purified tubulin was conjugated to NHS-rhodamine as reported previously (Hyman, 1991). NHS-rhodamine-labeled tubulin underwent an additional round of assembly/disassembly with 30% (v/v) glycerin prior to storage in liquid nitrogen. GST-WDL5-His protein or GST-MAP65-1 protein (3 μm) was added to 20 μm rhodamine-labeled tubulin in PEM buffer containing 1 mm GTP. After tubulin assembly at 35°C for 40 min, the temperature was immediately decreased to 10°C and maintained for 30 min for low-temperature experiments. For dilution treatments, the assembled tubulin samples described were diluted with 50× prewarmed PEM buffer containing WDL5 or MAP65-1 and incubated for 60 min at 35°C prior to fixation. Samples were fixed with 1% (v/v) glutaraldehyde for observation by confocal microscopy.

PCR Analysis

RT-PCR and quantitative real-time PCR analyses were performed to assess WDL5 transcript levels in wild-type, wdl5-1, wdl5-2, eto1-1, and ein2-5 seedlings. Total RNA was isolated using TRIzol reagent (Invitrogen) from hypocotyls of 5-d-old seedlings grown in the dark. Three independent pairs of primers were used to determine the levels of full-length WDL5 transcripts (5′-ATGGACCCTGAGAGTATCATGGC-3′ and 5′-TTAATGCTCAACAGCAACCGC-3′), partial WDL5 transcripts located upstream of the T-DNA insertion site (5′-AAGTCAGAATGAGAATTCGGCAAAC-3′ and 5′-CATCGTCTGCTTTCGGACTATTAGA-3′), and partial WDL5 transcripts located downstream of the T-DNA insertion site (5′-CTTTTATCAAGAACCTCAGCCGCCT-3′ and 5′-TTAATGCTCAACAGCAACCGCTTCA-3′) in wdl5-1 and wdl5-2 mutants. UBQ was amplified as a loading control using the following primers: 5′-GACCATAACCCTTGAGGTTGAATC-3′ and 5′-AGAGAGAAAGAGAAGGATCGATC-3′.

For quantitative real-time PCR, an ABI 7500 real-time PCR system (Applied Biosystems) was used according to the manufacturer’s instructions. Primers used for subsequent detection of WDL5 expression were 5′-AAATGGTTCTGTTGCTCCTAATGTA-3′ and 5′-TTTGAGACTTTGGTTTCACCTTCT-3′. UBQ11 was used as an internal control (5′-GCAGATTTTCGTTAAAACC-3′ and 5′-CCAAAGTTCTGCCGTCC-3′). Three biological replicates and two to three technical replicates (for each biological replicate) were used for each treatment. Average and sd values were calculated from the biological replicates.

EMSA

EMSA was performed according to Zhang et al. (2012). Briefly, the recombinant GST-EIN3 truncated protein (amino acids 141–352) was purified from Escherichia coli according to the manufacturer’s instructions. Biotin-labeled DNA fragments were synthesized and used as probes, and biotin-unlabeled DNA fragments of the same sequences were used as competitors. Nucleotide sequences of the double-stranded oligonucleotides were as follows: for WDL5 P1, 5′-TTTTTTTGCCAACCACTTATGTCT-3′ and 5′-GTACATTGCGATTTTCAACCTTAAA-3′; for WDL5 P2, 5′-GATTTAATTCTTTTGGCCTACC-3′ and 5′-ATCAACAATATTTCAAAGTTGGAAT-3′; and for WDL5 P3, 5′-ACGAAAAGTTTATACCGTTT -3′ and 5′-GTCCAAATTAATACTTGTTATAAAA-3′. Primers were labeled using the Biotin 5′ End DNA Labeling Kit (Pierce). Standard reaction mixtures (20 μL) for EMSA contained 1 μg of purified proteins, 2 μL of biotin-labeled annealed oligonucleotides, 2 μL of 10× binding buffer (100 mm Tris, 500 mm KCl, and 10 mm dithiothreitol, pH 7.5), 1 μL of 50% (v/v) glycerol, 1 μL of 1% (v/v) Nonidet P-40, 1 μL of 1 m KCl, 1 μL of 100 mm MgCl2, 1 μL of 200 mm EDTA, 1 μL of 1 mg mL−1 poly(dI-dC), and 10 μL of ultrapure water. Reactions were incubated at room temperature (25°C) for 30 min and loaded onto a 6% (w/v) native polyacrylamide gel in TBE buffer (45 mm Tris, 45 mm boric acid, and 1 mm EDTA, pH 8.3). The gel was sandwiched and transferred to an N+ nylon membrane (Millipore) in 0.5× TBE buffer at 380 mA at 4°C for 60 min. Detection of biotin-labeled DNA by chemiluminescence was performed based on the instructions provided in the Light Shift Chemiluminescent EMSA Kit (Pierce).

ChIP

Five-day-old dark-grown seedlings were treated with 10 µm β-estradiol or dimethyl sulfoxide as a control under the same growth conditions for 4 h. ChIP was performed as described previously (Johnson et al., 2002) using an anti-FLAG monoclonal antibody (Sigma-Aldrich) for immunoprecipitation. Equal quantities of starting plant material and ChIP reagents were used for the PCR. Primers used to detect the EIN3 target WDL5 promoter were as follows: for WDL5 P1, 5′-TTTTTTTGCCAACCACTTATGTCT-3′ and 5′-GTACATTGCGATTTTCAACCTTAAA-3′; for WDL5 P2, 5′-GATTTAATTCTTTTGGCCTACC-3′ and 5′-ATCAACAATATTTCAAAGTTGGAAT-3′; with ACTIN2 as a control (5′-GGTAACATTGTGCTCAGTGGTGG-3′ and 5′-AACGACCTTAATCTTCATGCTGC-3′). ChIP experiments were performed independently three times.

Ballistics-Mediated Transient Expression in Leaf Epidermal Cells

Subcellular localization of WDL5-GFP and cortical microtubules was visualized using transiently expressed 35S:WDL5-GFP and 35S:MBD-mCherry constructs in Arabidopsis (Columbia ecotype) leaf epidermal cells. Experiments were performed as described previously by Fu et al. (2002). One microgram of 35S:WDL5-GFP and 1 μg of 35S:MBD-mCherry DNA were used for particle bombardment. Six to 8 h after bombardment, GFP and mCherry signals were detected using the Zeiss LSM 510 META confocal microscope. Filamentous structures containing WDL5-GFP in leaf epidermal cells were visualized after treatment with 10 μm oryzalin and 100 nm LatA for 30 min.

Quantification of Cortical Microtubules in the Cell

ImageJ software (http://rsb.info.nih.gov/ij/) was used to quantify the density of cortical microtubules in the cell. A vertical line that oriented to the majority of the cortical microtubules with a fixed length (approximately 10 μm) was drawn, and the density of cortical microtubules across the line was measured. Four repeated measurements were performed for each cell, and at least 36 cells from each treatment were used. The values were recorded, and significance was analyzed using the paired Student’s t test.

Sequence data for WDL5 can be found in the Arabidopsis Genome Initiative database under accession number At4g32330.

Supplemental Data

The following supplemental materials are available.

Supplementary Material

Supplemental Data
supp_169_1_325__index.html (1KB, html)

Acknowledgments

We thank Dr. Ming Yuan (China Agricultural University) for critical reading and comments on the article and Dr. Hongwei Guo (Peking University) and Dr. Shuhua Yang (China Agricultural University) for generously providing the ethylene-related Arabidopsis mutant seeds.

Glossary

ACC

1-aminocyclopropane-1-carboxylic acid

BR

brassinosteroid

MAP

microtubule-associated protein

T-DNA

transfer DNA

RT

reverse transcription

MS

Murashige and Skoog

EBS

ETHYLENE-INSENSITIVE3-binding site

ChIP

chromatin immunoprecipitation

EMSA

electrophoretic mobility shift assay

NHS

5-(and 6-)carboxytetramethylrhodamine succinimidyl ester

Footnotes

1

This work was supported by the National Basic Research Program of China (grant no. 2012CB114200 to T.M.), the Natural Science Foundation of China (grant nos. 31471272 and 31222007 to T.M.), and the Program for New Century Excellent Talents in University (grant no. NCET–12–0523 to T.M.).

T.M. conceived the original screening and research plans; T.M., J.S., and Q.M. supervised the experiments; J.S. and Q.M. performed most of the experiments; T.M., J.S., and Q.M. designed the experiments and analyzed the data; T.M. conceived the project and wrote the article with contributions of all the authors; T.M. supervised and complemented the writing.

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