Light-Regulated Hypocotyl Elongation InvolvesProteasome-Dependent Degradation of the MicrotubuleRegulatory Protein WDL3 in ArabidopsisC W OA

Xiaomin Liu,1 Tao Qin,1 Qianqian Ma, Jingbo Sun, Ziqiang Liu, Ming Yuan, and Tonglin Mao2

State Key Laboratory of Plant Physiology and Biochemistry, Department of Plant Sciences, College of Biological Sciences, ChinaAgricultural University, Beijing 100193, China

ORCID ID: 0000-0002-8418-2870 (TM).

Light significantly inhibits hypocotyl cell elongation, and dark-grown seedlings exhibit elongated, etiolated hypocotyls.Microtubule regulatory proteins function as positive or negative regulators that mediate hypocotyl cell elongation by alteringmicrotubule organization. However, it remains unclear how plants coordinate these regulators to promote hypocotyl growthin darkness and inhibit growth in the light. Here, we demonstrate that WAVE-DAMPENED 2–LIKE3 (WDL3), a microtubuleregulatory protein of the WVD2/WDL family from Arabidopsis thaliana, functions in hypocotyl cell elongation and is regulatedby a ubiquitin-26S proteasome–dependent pathway in response to light.WDL3 RNA interference Arabidopsis seedlings grownin the light had much longer hypocotyls than controls. Moreover, WDL3 overexpression resulted in overall shortening ofhypocotyl cells and stabilization of cortical microtubules in the light. Cortical microtubule reorganization occurred slowly incells from WDL3 RNA interference transgenic lines but was accelerated in cells from WDL3-overexpressing seedlingssubjected to light treatment. More importantly, WDL3 protein was abundant in the light but was degraded through the 26Sproteasome pathway in the dark. Overexpression of WDL3 inhibited etiolated hypocotyl growth in regulatory particle non-ATPase subunit-1a mutant (rpn1a-4) plants but not in wild-type seedlings. Therefore, a ubiquitin-26S proteasome–dependentmechanism regulates the levels of WDL3 in response to light to modulate hypocotyl cell elongation.

INTRODUCTION

Arabidopsis thaliana seedlings exhibit different developmentalpatterns, depending on the ambient light. Seedlings perceivelight signals via multiple photoreceptors and transduce thesesignals to activate downstream regulators, resulting in preciseregulation of photomorphogenic developmental processes,such as termination of hypocotyl elongation, rapid root growthto anchor young plants in the soil, and opening of the cotyledon(Chen et al., 2004; Monte et al., 2007; Whitelam and Halliday,2007). By contrast, seedlings grown in darkness become etio-lated, which is associated with the presence of a rapidly elon-gating hypocotyl, small unopened cotyledons on an apicalhook, and a short primary root (Fankhauser and Chory, 1997;Jaillais and Vert, 2012). Numerous studies have revealed thatexternal and internal cues mediate the antagonistic effects of lightand darkness on hypocotyl elongation, including plant photo-receptors, phytohormones, calcium, and transcription factors(Wang et al., 2002; Folta et al., 2003; Castillon et al., 2007; Josseet al., 2008; Tsuchida-Mayama et al., 2010; Luo et al., 2010). For

example, the phytohormone ethylene has been shown to promotehypocotyl elongation in the light and suppress elongation in thedark, largely due to concomitant activation of two contrastingpathways (Shinkle and Jones, 1988; Ecker, 1995; Smalle et al.,1997; Zhong et al., 2012). Although an increasing number ofupstream effectors have been identified in these signaling path-ways, how plants coordinate the downstream negative andpositive regulators of hypocotyl elongation in darkness and lightremains an unanswered question.Genetic and physiological studies have demonstrated that

cortical microtubules play a crucial role in the regulation of cellelongation and expansion through orienting cellulose fibrils andcellulose fibril arrays to build the cell wall (Buschmann andLloyd, 2008; Lloyd and Chan, 2008; Sedbrook and Kaloriti,2008; Lloyd, 2011). The function of cortical microtubules is in-timately linked to their organization, which can be altered bydevelopmental and environmental cues (Dixit and Cyr, 2004).Previous studies have shown that the orientation of corticalmicrotubules varies with the status of hypocotyl growth (Leet al., 2005; Crowell et al., 2011). In rapidly elongating hypocotylcells, the parallel array of cortical microtubules is predominantlytransversely oriented to the hypocotyl longitudinal growth axis.By contrast, cortical microtubules are predominantly found inthe oblique or longitudinal direction once the accelerative phaseof cell elongation slows (Dixit and Cyr, 2004; Le et al., 2005;Li et al., 2011; Lloyd, 2011). Notably, reorganization of corticalmicrotubules from a transverse orientation into an oblique andlongitudinal array in hypocotyl cells occurs in response to light,which also inhibits hypocotyl growth (Ueda and Matsuyama,2000; Le et al., 2005; Sambade et al., 2012). However, the

1 These authors contributed equally to this work.2 Address correspondence to maotonglin@cau.edu.cn.The author responsible for distribution of materials integral to the findingspresented in this article in accordance with the policy described in theInstructions for Authors (www.plantcell.org) is: Tonglin Mao (maotonglin@cau.edu.cn).C Some figures in this article are displayed in color online but in black andwhite in the print edition.W Online version contains Web-only data.OAOpen Access articles can be viewed online without a subscription.www.plantcell.org/cgi/doi/10.1105/tpc.113.112789

The Plant Cell, Vol. 25: 1740–1755, May 2013, www.plantcell.org ã 2013 American Society of Plant Biologists. All rights reserved.

molecular mechanisms underlying this process are largelyunclear.

Microtubule regulatory proteins regulate the organization anddynamics of microtubules (Kaloriti et al., 2007; Buschmann andLloyd, 2008; Sedbrook and Kaloriti, 2008). An increasing numberof microtubule regulatory proteins have been reported to be in-volved in the regulation of hypocotyl elongation through alterationof microtubule organization and dynamics. For example, decreasedexpression of Arabidopsis MICROTUBULE-DESTABILIZING PRO-TEIN40 (MDP40) results in a shorter etiolated hypocotyl phenotype,demonstrating that MDP40 functions as a positive regulator ofhypocotyl elongation (Wang et al., 2012). By contrast, over-expression ofMDP25 dramatically inhibits hypocotyl elongation inresponse to changes in cytosolic calcium levels, suggesting thatMDP25 functions as a negative regulator of hypocotyl elongationin Arabidopsis (Li et al., 2011). However, how plants coordinatethese downstream positive and negative regulators to mediatethe different hypocotyl growth states in response to light is largelyunknown.

It is well known that many fundamental cellular processes areregulated by the ubiquitin-26S proteasome system, which con-trols the degradation rates of numerous proteins in plants (Smalleand Vierstra, 2004; Vierstra, 2009). One such process involvesthe CONSTITUTIVE PHOTOMORPHOGENIC/DE-ETIOLATED/FUSCA (COP/DET/FUS) pathway, which serves to repress pho-tomorphogenesis in the dark by playing a central role in the in-tegration of far-red, red, and blue light signaling (Kwok et al.,1996). The well-known E3 ligase COP1 partitions between thenucleus and cytosol in response to darkness and light, re-spectively. COP1 targets positive regulators of photomorphogen-esis, such as HYPOCOTYL5, LONG AFTER FAR-RED LIGHT1,and PHYTOCHROME A, for ubiquitin-mediated degradation bythe 26S proteasome in order to repress photomorphogenic de-velopmental processes, including hypocotyl elongation (Denget al., 1992; Osterlund et al., 2000; Saijo et al., 2003; Duek et al.,2004). Lines carrying mutations in components of the ubiquitin-26S proteasome system, such as cop1 and rpn1a-4, exhibit de-fective etiolated hypocotyl growth (Wang et al., 2009; Chang et al.,2011). Therefore, investigation of a potential role for the 26S pro-teasome pathway in regulation of microtubules during hypocotylgrowth is of great interest.

WDL3 belongs to the microtubule regulatory protein WAVE-DAMPENED2 (WVD2)/WVD2-LIKE (WDL) family, which containstargeting proteins for Xenopus kinesin-like protein2 in Arabi-dopsis (Yuen et al., 2003; Perrin et al., 2007). The conservedpentapeptide KLEEK motif was identified in an amino acid se-quence from the WVD2/WDL family, which was also found inMAP70-5, a member of the microtubule regulatory proteinMAP70 family (Yuen et al., 2003; Korolev et al., 2007). Consti-tutive expression ofWVD2 results in short, thick stems and rootsand inverted handedness of twisting hypocotyls and roots (Yuenet al., 2003). While MAP70-5 is involved in tracheary elementdevelopment, decreased MAP70-5 resulted in reduced in-florescence stem length and diameter (Korolev et al., 2007;Pesquet et al., 2010), suggesting diverse physiological roles ofKLEEK-containing proteins in plant growth and cell morpho-genesis. In this study, we functionally characterized WDL3,which plays an important role in the regulation of hypocotyl cell

elongation by altering the stability of cortical microtubules. Moreimportantly, we also found that WDL3 levels are regulated by anubiquitin-26S proteasome–dependent pathway in response tolight. Our study reveals a mechanism by which the ubiquitin-26Sproteasome–dependent pathway regulates microtubule organi-zation in response to light in Arabidopsis.

RESULTS

WDL3 Functions as a Negative Regulator of HypocotylCell Elongation

There are seven WVD2-like genes in the Arabidopsis genome,from WDL1 to WDL7 (Yuen et al., 2003). WDL3 expression issignificantly altered in brassinosteroid phytohormone mutantsbased on published microarray data (Sun et al., 2010), sug-gesting a potential role of WDL3 in cell elongation. To analyzethe function of WDL3 in Arabidopsis, WDL3 loss-of-function andWDL3–green fluorescent protein (GFP)–overexpressing seed-lings were generated. Since knockdown or knockout T-DNAinsertion lines of WDL3 are unavailable, we generated RNA in-terference (RNAi) lines to analyze the function of WDL3 in Arab-idopsis. Of the fifty-five WDL3 RNAi transgenic lines that wereobtained, 38 had longer hypocotyls after growth in the light. Inaddition, of 34 WDL3-GFP–overexpressing lines obtained, 20exhibited shorter hypocotyls after growth in the light. Line 2 ofthe WDL3 RNAi transgenic lines and line 7 of the WDL3-GFPtransgenic lines, which exhibited typical phenotypes, were se-lected for further analysis. The level of WDL3 transcription wasconsiderably enhanced in the overexpressing line and wasdramatically reduced in the RNAi line (Figure 1A). In addition, thetransgenic seedlings of plants carrying WDL3 without the GFP tagand WDL3-GFP exhibited similar phenotypes (see SupplementalFigures 1A to 1C online), demonstrating that the GFP tag did notinterfere with WDL3 function.Observation of 1-week-old seedlings from the WDL3-

overexpressing line revealed that the hypocotyl length wasconsiderably reduced. By contrast, the hypocotyls were muchlonger in the WDL3 RNAi Arabidopsis than in wild-type plantswhen the seedlings were grown in the light (Figure 1B). Statis-tical analysis using paired Student’s t tests indicated that thesedifferences in hypocotyl length were significant (Figure 1C).Scanning electronic microscopy revealed no major differencesin the cell profiles of the hypocotyls among the wild-type,WDL3-overexpressing, and WDL3 RNAi seedlings (Figure 1D).The cell numbers in individual hypocotyl-epidermal cell filesin the different seedlings were similar (;20 to 22). The aver-age widths of hypocotyl epidermal cells in wild-type, WDL3-overexpressing, andWDL3 RNAi seedlings were 27.886 5.13 mm,28.39 6 4.92 mm, and 31.66 6 5.71 mm, respectively (n > 500).Statistical analysis using the paired Student’s t test indicatedthat this difference was not significant. However, the length ofhypocotyl cells was decreased in WDL3-overexpressing seed-lings and significantly increased in WDL3 RNAi Arabidopsis,particularly in the middle region (Figure 1E), suggesting thatWDL3 is involved in hypocotyl cell elongation. We also mea-sured the elongation rates of hypocotyls at different times and

Proteasome-Mediated Degradation of WDL3 1741

positions along hypocotyls from plants grown in the light. Therewas little growth of hypocotyl cells in the wild-type (Figure 1F;Sambade et al., 2012), WDL3-overexpressing, and WDL3 RNAiArabidopsis at days 2 and 3, but the cells were greatly elongatedby day 4, especially in the middle region. In comparison to wild-type plants, the length of WDL3 RNAi hypocotyl cells was sig-nificantly increased, and the length of WDL3-overexpressinghypocotyl cells was relatively decreased at days 4 and 5 (Figure1F). These results suggest that WDL3 plays a role in light-inhibitedhypocotyl elongation.

To confirm that the hypocotyl phenotype in the WDL3 RNAiline was linked to WDL3 expression levels, we randomly se-lected four additional RNAi lines for RT-PCR analysis. Theseresults showed that the WDL3 expression levels correspondedwith the long-hypocotyl phenotype of plants grown in the light(see Supplemental Figures 2A to 2C online). Additionally, a sec-ond WDL3 RNAi (RNAi-1) construct using another WDL3 cDNAsequence was generated. Seventeen WDL3 RNAi-1 lines ex-hibited a longer hypocotyl phenotype, and three independentWDL3 RNAi-1 lines were selected for further analyses. These

Figure 1. WDL3 Negatively Regulates Hypocotyl Cell Elongation in the Light.

(A) RT-PCR analysis of WDL3 transcript levels in seedlings from the wild-type (WT) Columbia ecotype, WDL3-GFP transgenic (OE), and WDL3 RNAiArabidopsis lines (RNAi). The 18S rRNA was used as a control.(B) and (C) WDL3 RNAi seedlings have longer hypocotyls, whereas the hypocotyls are shorter in WDL3-GFP transgenic Arabidopsis (t test, **P < 0.01)grown on half-strength MS in the light for 7 d. The graph shows the average hypocotyl length measured from a minimum of 30 seedlings. Error barsrepresent the mean 6 SD.(D) Scanning electron microscopy images of hypocotyl epidermal cells from the wild-type, WDL3-GFP transgenic, and WDL3 RNAi Arabidopsis lines.The pink outlines show the profiles of epidermal cells in the middle of hypocotyls. Bar = 100 mm.(E) Length of hypocotyl cells of the wild-type, WDL3-GFP transgenic, andWDL3 RNAi Arabidopsis lines grown in the light for 7 d. t test, *P < 0.05, **P <0.01. Error bars represent the mean 6 SD.(F) Hypocotyl cell length of light-grown wild-type, WDL3-GFP transgenic, andWDL3 RNAi Arabidopsis seedlings was measured from days 2 to 5. The xaxis shows cell number from the bottoms of the hypocotyls, and the y axis shows cell length in micrometers.

1742 The Plant Cell

results showed that the transcription levels of WDL3 were dra-matically reduced, and the hypocotyl length in 7-d-old light-grownseedlings from the WDL3 RNAi-1 lines was much longer (seeSupplemental Figures 3A to 3C online). These results confirm thatthe longer hypocotyl phenotype of WDL3 RNAi lines grown in thelight is dependent upon the expression level of WDL3. BecauseWDL3 belongs to the WVD2/WDL family in Arabidopsis, we de-tected expression of other genes in WDL3 RNAi and RNAi-1 lines.Quantitative real-time PCR analysis showed that WVD2, WDL1,WDL2, WDL4, WDL5, WDL6, and WDL7 expression in theWDL3 RNAi and RNAi-1 lines was similar to the wildtype (seeSupplemental Figure 4 online), demonstrating that decreasedWDL3 expression did not affect transcription levels of other genesin the WVD2/WDL family. Together, those results demonstrate thatWDL3 plays a negative regulatory role in hypocotyl cell elongationin the light.

Organization and Stability of Cortical MicrotubulesAre Affected in the Epidermal Cells of WDL3Transgenic Seedlings

Because WDL3 belongs to the microtubule regulatory proteinWVD2/WDL family (Yuen et al., 2003; Perrin et al., 2007), wetested whether increasing or decreasing WDL3 expression af-fected the organization of cortical microtubules in hypocotylcells. Because the transverse, oblique, and longitudinal orien-tation patterns of cortical microtubules are related to the elon-gation rate of hypocotyl epidermal cells, hypocotyls are frequently

used to explore the mechanisms that underlie cell elongation andcortical microtubule organization (Le et al., 2005; Crowell et al.,2011; Motose et al., 2011). Therefore, we analyzed cortical mi-crotubules in epidermal cells from the hypocotyl middle region ofwild-type, WDL3-overexpressing, and WDL3 RNAi Arabidopsisseedlings carrying tubulin tagged with GFP. Cells were examinedat days 4 and 5 of exposure to light. The parallel arrays of corticalmicrotubules were generally transversely and obliquely orientedrelative to the longitudinal hypocotyl growth axis in epidermalcells from wild-type hypocotyls (Figures 2A and 2D). In compar-ison, random, oblique, or longitudinal cortical microtubules wereobserved in most of the WDL3-overexpressing hypocotyl cells,while dominantly transverse cortical microtubule arrays weredetected in the WDL3 RNAi Arabidopsis cells at day 4 (Figures2B to 2D). Cortical microtubules were mostly oriented in a lon-gitudinal manner in the wild-type and WDL3-overexpressinghypocotyl cells at day 5 (Figures 2E, 2F, and 2H), while obliqueand transverse cortical microtubules were observed in most ofthe WDL3 RNAi Arabidopsis at day 5 (Figures 2G and 2H). Thealtered microtubule arrays observed in WDL3 RNAi seedlingswere consistent with the significant increase in hypocotyl cellelongation caused by reduced expression of WDL3.In order to further characterize the effect of WDL3 on cortical

microtubules, the microtubule-disrupting drug oryzalin wasapplied to epidermal hypocotyl cells from wild-type, WDL3-overexpressing, and WDL3 RNAi seedlings. To quantify the effectof oryzalin on the stability of cortical microtubules, we estimatedthe density of cortical microtubules in hypocotyl epidermal cells.

Figure 2. The Cortical Microtubule Array Is Greatly Altered in Hypocotyl Epidermal Cells of WDL3 Transgenic Seedlings.

(A) to (C) and (E) to (G) Cortical microtubules in epidermal cells from the middle region of the hypocotyl of wild-type (WT), WDL3-GFP transgenic (OE),and WDL3 RNAi (RNAi) seedlings with a GFP-tubulin background were observed by confocal microscopy after growth in the light for 4 or 5 d. Bar in(G) = 10 mm.(D) and (H) Frequency of different microtubule orientation patterns in light-grown hypocotyl epidermal cells from wild-type, WDL3-GFP transgenic, andWDL3 RNAi seedlings (n > 150 cells).[See online article for color version of this figure.]

Proteasome-Mediated Degradation of WDL3 1743

A paired Student’s t test was used to identify significant dif-ferences. Our results revealed that cortical microtubules weresimilar in density before oryzalin treatment in the epidermal cellsof wild-type, WDL3-overexpressing, and WDL3 RNAi seedlings(Figure 3A). However, the number of cortical microtubules inepidermal cells of wild-type, WDL3-overexpressing, and WDL3RNAi seedlings was significantly different after drug treatment(Figure 3F). Microtubules were disrupted in epidermal cells fromthe WDL3 RNAi line after treatment with 5 mM oryzalin for 5 min(Figures 3B and 3F), whereas microtubules in wild-type andWDL3-overexpressing cells were largely unaffected. Increases

in the concentration and duration of oryzalin treatment re-sulted in disruption of the majority of cortical microtubules inboth wild-type and WDL3 RNAi Arabidopsis cells. However,cortical microtubules remained relatively unaffected in WDL3-overexpressing cells at doses of up to 10 µM oryzalin (Figures 3C,3D, and 3F). In addition, although cortical microtubules mostlyrecovered in cells from wild-type and WDL3-overexpressingArabidopsis hypocotyls after oryzalin washout, many cortical mi-crotubules remained disrupted in cells from WDL3 RNAi Arab-idopsis hypocotyls (Figures 3E and 3F). Thus, microtubules weremore sensitive to oryzalin treatment in WDL3 RNAi cells and lesssensitive when expression of WDL3 was increased. These resultsconfirm that WDL3 functions as a microtubule stabilizer. Micro-tubule dynamics were analyzed using confocal time-lapse imag-ing. Microtubules with clearly visible leading plus ends (identifiedby growth rates) were selected for measurement in wild-type,WDL3-overexpressing, and WDL3 RNAi cells (see SupplementalTable 1 online). The results show that microtubule dynamics werealtered in WDL3-overexpressing and RNAi hypocotyl epidermalcells. The catastrophe frequency (frequency of transitions fromgrowth or pause to shortening) of individual microtubules wassignificantly lower in WDL3-overexpressing cells (0.006 s21 forcatastrophe) and higher in WDL3 RNAi cells (0.042 s21 for ca-tastrophe) compared with wild-type cells (0.027 s21 for catas-trophe), suggesting that individual microtubules are more proneto growth when expression of WDL3 is increased but are morelikely to shrink in the absence of WDL3.

WDL3 Levels Are Downregulated by 26SProteasome–Dependent Degradation in the Dark

Unlike plants grown in the light, the lengths of etiolated hypo-cotyls from wild-type, WDL3-overexpressing, and WDL3 RNAiseedlings were not obviously different (see SupplementalFigures 5A and 5B online), suggesting that WDL3 function isinhibited in the dark. To better understand this phenomenon,levels of the WDL3 transcript were assessed to determinewhether WDL3 was expressed in the absence of light. A con-struct was made in which the b-glucuronidase (GUS) reportergene was placed under control of the ;1.7-kb WDL3 promoter.This construct (PWDL3:GUS) was introduced into wild-typeplants using Agrobacterium tumefaciens–mediated trans-formation. Thirty-seven independent transgenic lines werestained for GUS activity. GUS staining revealed that WDL3 wasexpressed in a majority of the tissues and organs of Arabi-dopsis (see Supplemental Figures 6A to 6H online), includingthe hypocotyls of seedlings grown in the light or in the dark(Figure 4A). To verify this result, RNA was purified from the hy-pocotyls of light- and dark-grown wild-type seedlings. RT-PCRrevealed that the WDL3 transcript was abundant in both light-grown and etiolated hypocotyls (Figure 4B), demonstrating thattranscription of WDL3 does not appear to be regulated by thepresence or absence of light. Next, WDL3 protein levels wereanalyzed using WDL3-GFP transgenic seedlings. Confocal mi-croscopy showed that WDL3-GFP filaments were clearly detectedin the hypocotyl epidermal cells of light-grown seedlings but notin etiolated hypocotyl epidermal cells of WDL3-GFP transgeniclines (Figure 4C). Protein gel blots using an anti-GFP antibody

Figure 3. Cortical Microtubules are Hypersensitive to Oryzalin Treat-ment in WDL3 RNAi Cells but More Resistant in WDL3 Transgenic Cells.

(A) to (D) Cortical microtubules were observed in epidermal cells from light-grown hypocotyls in wild-type (WT), WDL3 transgenic (OE), and WDL3 RNAi(RNAi) seedlings after treatment with 0 mM oryzalin (A), 5 mM oryzalin for5 min (B), 10 mM oryzalin for 10 min (C), and 10 mM oryzalin for 30 min (D).(E) After the treatment in (D), oryzalin was washed off, and cortical mi-crotubules were observed after 2 h. Bar = 10 mm.(F) Quantification of cortical microtubules in hypocotyl epidermal cells ofwild-type, WDL3 transgenic, and WDL3 RNAi seedlings using ImageJsoftware (n > 28 cells for each sample). The y axis represents the numberof cortical microtubules that crossed a fixed line (;10 mm) perpendicularto the orientation of the majority of cortical microtubules in the cell.Student’s t tests were performed to compare the numbers of corticalmicrotubules in hypocotyl epidermal cells from WDL3 transgenic andWDL3 RNAi lines with that of wild-type plants under the same conditions.**P < 0.01 and *P < 0.05. Error bars represent the mean 6 SD.[See online article for color version of this figure.]

1744 The Plant Cell

confirmed this result (Figure 4D), demonstrating that WDL3-GFPlevels weremore abundant in the light and less abundant in the dark.

To analyze the potential molecular mechanism of WDL3degradation in the dark, seedlings were treated with MG132and MG115, two 26S proteasome inhibitors (Lee and Goldberg,1998; Planchais et al., 2000). Confocal microscopy revealedthat WDL3-GFP fluorescence was recovered in etiolated hy-pocotyls from WDL3-GFP transgenic seedlings treated withMG132 and MG115 (Figures 5A to 5C). By contrast, mocktreatment with ethanol (Figure 5D) or with other general pro-tease inhibitors (leupeptin and phenylmethylsulfonyl fluoride)had little or no effect on the degradation of WDL3-GFP (Figures5E and 5F). Thus, the degradation of WDL3 is prevented bytreatment with proteasome-specific inhibitors, suggesting thatthe proteasome is largely responsible for the degradation ofWDL3 in the absence of light.

Next, we examined WDL3 protein levels during dark–lighttransitions. WDL3-GFP transgenic seedlings were grown in

darkness for 6 d and then transferred to continuous light for 4, 8,12, 16, 20, and 24 h. Protein gel blots of protein extracts fromthese seedlings revealed that the abundance of WDL3 was sig-nificantly increased after 4 h and 8 h of light. Seedlings grown incontinuous light were included as a positive control, and the WDL3levels in these plants were significantly higher than that of plantssubjected to the dark (Figure 5G). Treatment with cycloheximide,

Figure 4. WDL3 Expression is regulated by Light at the Protein Level.

(A) Histochemical staining of GUS in PWDL3:GUS:TWDL3 transgenic seed-lings grown in the light and the dark.(B) RT-PCR analysis of WDL3 transcript levels in light-grown andetiolated hypocotyls of wild-type seedlings, with 18S rRNA used asa control.(C) Confocal images of epidermal cells from light-grown and etiolatedhypocotyls in WDL3-GFP transgenic plants. Bar = 10 mm.(D) Protein gel blots showed that the WDL3-GFP levels were moreabundant in the light (lane 1) but less abundant in the dark (lane 2). Lane 3contains lysate from wild-type (WT) cells as a control.[See online article for color version of this figure.]

Figure 5. WDL3 Degradation Is Mediated by the 26S ProteasomePathway.

(A) to (F) Confocal images of epidermal cells from etiolated hypocotyls ofWDL3-GFP transgenic plants treated with different inhibitors. (A) iswithout inhibitor; (B) to (F) are proteasome inhibitors MG115 (40 mM) andMG132 (40 mM), mock buffer, leupeptin (40 mM), or phenylmethylsulfonylfluoride PMSF (4 mM). Bar = 10 mm.(G) Protein gel blot of WDL3-GFP seedlings grown in the dark for 6 d,followed by transfer to light for 0 (lane 2), 4 (lane 3), 8 (lane 4), 12 (lane 5),16 (lane 6), 20 (lane 7), and 24 h (lane 8). Seedlings grown in the lightwere included as a positive control (lane 9). WT, the wild type.(H) Protein gel blot of WDL3-GFP seedlings treated with 200 mMcycloheximide (+CHX) or buffer only (2CHX). Seedlings were grown incontinuous light (L) for 6 d before treatment and then either kept in thelight or transferred to darkness (D). Protein extracts were isolated at 0,12, and 24 h after the addition of cycloheximide. Nonspecific bandsserved as loading controls.[See online article for color version of this figure.]

Proteasome-Mediated Degradation of WDL3 1745

an inhibitor of cytoplasmic protein synthesis, was then used todetermine whether protein degradation or protein synthesis rep-resents the key regulatory step that modulates differential abun-dance of WDL3 in light and darkness. Six-day-old WDL3-GFPtransgenic seedlings grown in continuous light were transferred todarkness and cultured in the presence of 200 mM cycloheximide.The cultures were then treated in darkness for 0, 12, and 24 h,and the levels of WDL3-GFP were measured. Samples maintainedin the light served as controls. In the absence of cycloheximide,the amount of WDL3 was decreased when seedlings were trans-ferred to darkness. The amount of WDL3 decreased within 24 hunder both light and dark conditions in the presence of cyclo-heximide; however, the rate of decrease was significantly greaterat 12 h after transfer to darkness (Figure 5H). Because cyclohex-imide effectively blocks new protein synthesis, the reduced levelsof WDL3 must therefore be due to the degradation of preexistingprotein. Together, these results suggest that WDL3 is degraded bya 26S proteasome–dependent pathway in the absence of light.

Because our pharmacological results suggested that degrada-tion of the WDL3 protein was mediated by the 26S proteasome,

we next tested whether overexpression of WDL3 could inhibitetiolated hypocotyl growth in a 26S proteasome mutant back-ground. The RPN1 protein, encoded by RPN1a, is a non-ATPasesubunit of the regulatory particles of the 26S proteasome complexin Arabidopsis and plays a crucial role in the functioning of the 26Sproteasome (Rosenzweig et al., 2008). Therefore, we crossed theWDL3-GFP transgenic plant with rpn1a-4, a null mutant of RPN1a(Wang et al., 2009). Fifteen resulting WDL3-GFP;rpn1a-4 lines ex-hibited a shorter etiolated hypocotyl phenotype, and line 2, whichexhibited the typical phenotype, was selected for further analyses.Confocal images revealed that WDL3-GFP was present in fila-mentous structures in etiolated hypocotyl epidermal cells fromhomozygous WDL3-GFP;rpn1a-4 mutant seedlings but not WDL3transgenic wild-type seedlings (Figures 6A and 6B). These fila-mentous structures containing WDL3-GFP could be disrupted bytreatment with oryzalin (Figure 6C) and recovered after oryzalinwashout (Figure 6D), demonstrating that WDL3 is also capable ofbinding cortical microtubules in the dark. Observation of 5-d-oldetiolated seedlings from rpn1a-4 and WDL3-GFP;rpn1a-4 lines re-vealed that overexpression of WDL3-GFP substantially decreased

Figure 6. Overexpression of WDL3 Suppresses Etiolated Hypocotyl Growth in the 26S Proteasome Mutant rpn1a-4.

(A) and (B) Confocal images of epidermal cells from etiolated hypocotyls in WDL3-GFP (OE) transgenic wild-type and WDL3-GFP;rpn1a-4 mutants.(C) The filamentous pattern of WDL3-GFP was disrupted when cells were treated with oryzalin.(D) The filamentous WDL3-GFP structures were partially recovered after oryzalin washout. Bar = 10 mm.(E) The WDL3-GFP transgenic etiolated rpn1a-4 mutant exhibits shorter hypocotyls after growth on half-strength MS in the dark for 5 d. The graphshows the average hypocotyl length measured from at least 38 seedlings under dark growth conditions.(F) Scanning electron microscopy images of hypocotyl epidermal cells from rpn1a-4 and WDL3-GFP transgenic etiolated rpn1a-4 mutants in the darkfor 3 d. The outlines show the profiles of epidermal cells in the middle of hypocotyls. Bar = 100 mm.(G) Length of hypocotyl cells from rpn1a-4 and WDL3-GFP transgenic etiolated rpn1a-4 mutants grown in the dark for 3 d. t test, *P < 0.05, and **P <0.01. Error bars represent the mean 6 SD.[See online article for color version of this figure.]

1746 The Plant Cell

the etiolated hypocotyl length of the rpn1a-4 mutant (Figure 6E).Scanning electronic microscopy revealed no major differences inthe cell profiles of hypocotyls between rpn1a-4 and WDL3-GFP;rpn1a-4 seedlings (Figure 6F). The cell numbers in individual hypo-cotyl-epidermal cell files in different seedlings were similar (;20 to22). The average widths of hypocotyl epidermal cells in rpn1a-4 andWDL3-GFP;rpn1a-4 seedlings were 21.48 6 3.52 mm and 22.31 63.81 mm (n > 300), respectively. Statistical analysis using pairedStudent’s t test indicated that this difference was not significant.However, the length of hypocotyl cells was significantly de-creased in WDL3-GFP transgenic rpn1a-4 mutants (Figure 6G),which is consistent with a negative role of WDL3 in hypocotylcell elongation.

Alteration of WDL3 Expression Levels Affects CorticalMicrotubule Reorganization in Response to Light

Since light induces cortical microtubule reorganization from trans-verse arrays into oblique and longitudinal patterns in hypocotyl

cells after exposure of etiolated plants to light (Ueda andMatsuyama, 2000; Le et al., 2005), we investigated the effects ofWDL3 on this transition in elongating epidermal cells. Wild-type,WDL3-overexpressing, andWDL3 RNAi seedlings were grown for 3d in the dark on half-strength Murashige and Skoog (MS) medium,followed by exposure to white light for 0, 30, or 60 min. First, weassessed WDL3-GFP protein levels after transfer from darkness tolight. Protein gel blots showed that WDL3-GFP levels were greatlyincreased after treatment with light for 30 and 60 min (Figure 7A).Confocal microscopy revealed that, after 3 d of growth in thedark, parallel arrays of cortical microtubules were transversely ori-ented relative to the hypocotyl growth axis in epidermal cells fromthe etiolated hypocotyl upper regions from wild-type, WDL3-overexpressing, and WDL3 RNAi seedlings (Figures 7B, 7E, 7H,and 7K). After treatment with light for 30 min, transverse andoblique cortical microtubule arrays were dominantly observed inhypocotyl cells of the wild-type andWDL3 RNAi seedlings (Figures7C, 7I, and 7K) but not in the WDL3-overexpressing Arabidopsiscells. The overexpressing cells predominately exhibited longitudinal

Figure 7. Light-Induced Reorganization of Cortical Microtubules Occurs More Quickly in WDL3 Transgenic Arabidopsis Cells and More Slowly inWDL3RNAi Arabidopsis Cells.

(A) Protein gel blot of WDL3-GFP seedlings grown in the dark for 3 d, then transferred to light for 0 (lane 2), 30 (lane 3), and 60 min (lane 4). Wild-type(WT) seedlings grown in the light served as a control (lane 1). A nonspecific band appearing on the blot serves as a loading control.(B) to (J) Etiolated hypocotyls from wild-type ([B] to [D]), WDL3 transgenic ([E] to [G]), and WDL3 RNAi ([H] to [J]) seedlings with a GFP-tubulinbackground were grown on half-strength MS medium until day 3 and then transferred to the light for 0, 30, and 60 min. Cortical microtubules wereobserved in epidermal cells from the upper regions of the hypocotyls. (B), (E), and (H) are without light treatment; (C), (F), and (I) are treated with light for30 min; (D), (G), and (J) are treated with light for 60 min. Bar in (J) = 10 mm.(K) Frequency of microtubule orientation patterns in the epidermal cells from the upper regions of the hypocotyls of the wild-type, WDL3 transgenic, andWDL3 RNAi lines (n > 104 cells).[See online article for color version of this figure.]

Proteasome-Mediated Degradation of WDL3 1747

microtubule arrays (Figures 7F and 7K). Increasing the duration oftreatment induced the formation of longitudinal cortical microtubulearrays in the wild-type andWDL3-overexpressing cells (Figures 7D,7G, and 7K) but not in the WDL3 RNAi line (Figures 7J and 7K).These results indicate that cortical microtubule reorganization washindered in WDL3 RNAi cells but accelerated in WDL3-overexpressing cells in response to light treatment, demon-strating that WDL3 is necessary for light-regulated corticalmicrotubule reorganization.

Recombinant WDL3 Binds to, Bundles, and StabilizesMicrotubules in Vitro

Given that WDL3 is required for cortical microtubule stabilityin hypocotyls and normal hypocotyl cell elongation, we in-vestigated the molecular basis for WDL3 regulation of micro-tubules in vitro. We cloned the full-length CODING SEQUENCE(CDS) into the pGEX-4T vector and transformed the plasmid intoEscherichia coli. The resulting glutathione S-transferase (GST)–tagged WDL3 fusion protein was then purified using a glutathi-one sepharose 4B resin column. SDS-PAGE revealed thatGST-WDL3 was present in two major bands, the lower of whichwas predicted to be a degradation product based on immuno-blots using an anti-GST antibody (see Supplemental Figures 7Aand 7B online). In order to determine whether WDL3 binds tomicrotubules, a cosedimentation assay was used to analyzebinding of WDL3 to paclitaxel-stabilized microtubules. GST-WDL3 (4 mM) was incubated with preformed 5 mM paclitaxel-stabilized microtubules at room temperature for 20 min, followedby centrifugation. SDS-PAGE analysis revealed that GST-WDL3,but not GST alone, bound and cosedimented with the micro-tubules (Figure 8A). Then, the subcellular localization of WDL3 wasinvestigated. A construct expressing WDL3 fused with a C-terminalGFP tag under control of the 35S promoter was generated andtransiently introduced into Arabidopsis cells. Confocal microscopyrevealed that WDL3-GFP formed filamentous structures in pave-ment cells (Figure 8B). The filaments were mostly intact in thepresence of latrunculin A (LatA), a reagent that depolymerizes actinfilaments (Figure 8C), but were disrupted by treatment with themicrotubule-disrupting reagent oryzalin (Figure 8D). After washoutof oryzalin, the filamentous WDL3-GFP structures recovered(Figure 8E). To confirm this result, WDL3-GFP and MBD-mCherry(mCherry-tagged microtubule binding domain of MAP4) weretransiently coexpressed in Arabidopsis pavement cells. Confocalmicroscopy showed that the green fluorescent signal of WDL3-GFP overlapped with the red fluorescent signal of MBD-mCherry,confirming that WDL3 localized to microtubules (Figures 8F to 8H).Colocalization was analyzed by plotting the signal intensities ofWDL3-GFP and MBD-mCherry using ImageJ software (Figures 8Hand 8I). Filamentous structures that could be disrupted by oryzalin,but not LatA, were also observed in hypocotyl epidermal cellsof Arabidopsis seedlings stably expressing WDL3-GFP (seeSupplemental Figures 8A to 8C online). By contrast, actin filamentsdecorated with stably expressed fABD2-GFP (GFP tagged thesecond actin binding domain of Arabidopsis fimbrin) were dis-rupted by treatment with LatA (see Supplemental Figures 8D and8E online). These data demonstrate that WDL3 colocalizes withmicrotubules in vitro and in cells.

Effects of WDL3 on microtubules were investigated by con-focal and electron microscopy. Paclitaxel-stabilized microtubulespolymerized from rhodamine-labeled tubulins exhibited singlefilament patterns in the absence of WDL3, as observed usingconfocal microscopy (Figure 9A) and negative-staining electronmicroscopy (Figure 9E). However, addition of GST-WDL3 orWDL3 (without the GST tag) resulted in formation of large micro-tubule bundles (Figures 9B, 9C, 9F, and 9G). To strip WDL3 fromthe microtubules, 200 mM NaCl was added to the reaction. Fol-lowing the addition of NaCl, WDL3-induced microtubule bundlesdispersed into single filaments (Figure 9D). To further confirmmicrotubule bundling by WDL3, a low-speed cosedimentation

Figure 8. WDL3 Directly Binds to Microtubules.

(A) GST-WDL3 was cosedimented with paclitaxel-stabilized micro-tubules. GST-WDL3 was most abundant in the supernatant (S) in theabsence of microtubules (MTs) but cosedimented with microtubules intothe pellets (P). WDL3 colocalizes with cortical microtubules in cells.(B) WDL3-GFP was transiently expressed in Arabidopsis pavement cells,where it formed filamentous structures.(C) and (D) The filamentous pattern of WDL3-GFP was essentially un-affected when treated with 100 nM LatA for 30 min but was disruptedwhen the cells were treated with 10 mM oryzalin for 30 min.(E) The filamentous WDL3-GFP structures were partially recovered afteroryzalin washout.(F) to (H) Analysis of colocalization of transiently expressed WDL3-GFPand MBD-mCherry. Bar in (H) = 10 mm.(I) Plot of a line scan drawn in (H) showing a strong correlation betweenspatial localization of WDL3-GFP and MBD-mCherry.

1748 The Plant Cell

assay was performed. Arabidopsis microtubule-bundling proteinMAP65-1 was used as a control (Mao et al., 2005). Tubulin (20 mΜ)was assembled at 35°C for 30 min in the presence or absenceof 8 mΜ WDL3 or 6 mM MAP65-1 and centrifuged at 5900g.Microtubules alone did not sediment following low speed

centrifugation (Figure 9H, lanes 1 and 2). However, the ma-jority of microtubules appeared in the low-speed pellets whenincubated with WDL3 or the MAP65-1 fusion protein (Figures 9H,lanes 3 to 6, and 9I). These data indicate that WDL3 plays a role inmicrotubule bundling.

Figure 9. WDL3 Bundles and Stabilizes Microtubules in Vitro.

(A) to (H)WDL3 induces formation of microtubule (MT) bundles in vitro. Fluorescent images are shown in (A) to (D), and electron micrographs are shownin (E) to (G). Bar in (G) = 500 nm.(A) and (E) Microtubules polymerized in the absence of WDL3.(B) and (F) Microtubule bundles induced by GST-WDL3.(C) and (G) Microtubule bundles induced by WDL3.(D) Microtubule bundles induced by WDL3 were sensitive to NaCl. Single microtubules were observed after treatment with 200 mM NaCl.(H) Low-speed cosedimentation of WDL3 with microtubules. Tubulin (20 µM) was assembled with or without 8 µM WDL3 or 6 mM MAP65-1 fusion proteins for30 min and centrifuged at low speed. Lane 1, microtubule pellet; lane 2, microtubule supernatant; lane 3, microtubule pellet in the presence of WDL3; lane 4,microtubule supernatant in the presence ofWDL3; lane 5, microtubule pellet in the presence of MAP65-1; lane 6, microtubule supernatant in the presence of MAP65-1.(I) Statistical analysis for (H). The resulting gels were scanned to determine the distribution of tubules in the pellets in the presence of 0, 8 µM WDL3, or6 mM MAP65-1, respectively. Error bars represent 6 SD (n = 3). WDL3 stabilized microtubules against cold and dilution induced depolymerization.(J) to (R) Images of microtubules polymerized from rhodamine-labeled tubulin (20 µM) incubated in the presence or absence of 3 µMWDL3 or MAP65-1protein for 30 min. Samples from (J) to (L) were subjected to 10°C for 30 min ([M] to [O]) or diluted with a solution containing WDL3 or MAP65-1 in 503PEM buffer ([P] to [R]). Bar in (D) and (R) = 10 mm.

Proteasome-Mediated Degradation of WDL3 1749

Next, we tested whether WDL3 could protect microtubulesagainst cold-induced and dilution-induced depolymerization.MAP65-1, which has been shown to stabilize microtubulesunder these conditions (Mao et al., 2005), was used as a con-trol. Rhodamine-labeled tubulin (20 mM) was incubated in thepresence and absence of WDL3 (3 mM) or MAP65-1 (3 mM) toallow tubulin polymerization (Figures 9J to 9L). The solutionswere then incubated at 10°C for 30 min (Figures 9M to 9O) ordiluted with 503 prewarmed buffer and incubated at 35°C for60 min (Figures 9P to 9R) prior to fixation. After fixation, thesamples were observed by confocal microscopy. The resultsshowed that microtubule filaments in the absence of WDL3were fully disassembled after cold and dilution treatments(Figures 9M and 9P). However, many microtubules persisted inthe presence of WDL3 (Figures 9N and 9Q) and MAP65-1(Figures 9O and 9R) after the cold and dilution treatments.These results indicate that WDL3 is capable of stabilizingmicrotubules against low-temperature and dilution induceddepolymerization.

DISCUSSION

In this study, we demonstrate that the ubiquitin-26S proteasomedegradation system is involved in modulation of the microtubuleregulatory protein WDL3. Together, these proteins comprisea regulatory mechanism that mediates the antagonistic effectsof light and darkness on hypocotyl cell elongation.

Stabilization or Destabilization of Cortical MicrotubulesLeads to Altered Hypocotyl Growth

The orientation of cortical microtubules is widely accepted tobe closely associated with hypocotyl cell elongation (Le et al.,2005). Microtubule regulatory proteins affect microtubule sta-bility in order to remodel microtubule arrays in plant cells.However, it remains unclear how microtubule stabilizers anddestabilizers differentially contribute to the status of hypocotylgrowth. Genetic and pharmacological evidence has shown thatdestabilization of cortical microtubules results in inhibition ofhypocotyl cell elongation (Le et al., 2005; Li et al., 2011). Forexample, overexpression of MAP18 and MDP25 results ina disordered cortical microtubule orientation and destabilizesmicrotubules in hypocotyl epidermal cells, inhibiting hypocotylelongation (Wang et al., 2007; Li et al., 2011). Arabidopsisdouble mutants for microtubule-stabilizing protein MAP65-1 andMAP65-2 also exhibit defective hypocotyl growth (Smertenkoet al., 2004; Mao et al., 2005; Gaillard et al., 2008; Li et al., 2009;Lucas et al., 2011). However, our findings support the possibilitythat overstabilization of cortical microtubules also alters mi-crotubule organization and consequently inhibits hypocotylcell elongation. Together, these studies suggest that alterationof cortical microtubule stability, either by overstabilization ordestabilization, can induce abnormal hypocotyl cell elongation.

Although WDL3 exhibited microtubule-bundling activity invitro, large microtubule bundles were not observed in WDL3-overexpressing seedlings. This phenomenon could be due to (1)relatively low expression of WDL3 or (2) relatively weaker WDL3

microtubule-bundling activity, as suggested by the low-speedcosedimentation assay compared with MAP65-1 (Figures 9Hand 9I). Overexpression of WDL3 results in the formation ofshort hypocotyls by stabilizing cortical microtubules; however,the molecular mechanisms underlying this regulatory pathwayare complicated. It remains unclear whether the stabilizing ac-tivity of WDL3 is transient or prolonged in nature with respectto inhibition of hypocotyl growth. Analysis of transcription dem-onstrated that stabilizers and destabilizers, such as MAP18 andWDL3, are expressed in hypocotyls from seedlings grown in thelight (Wang et al., 2007; this study). Thus, how plants specificallycoordinate these regulators in the same cell in order to favor in-hibition of hypocotyl elongation in the light will be a subject forfuture studies.

Multiple Layers of Regulation Are Involved inMicrotubule-Mediated Hypocotyl Elongation inResponse to Light

Previous studies and our results indicate that organization ofcortical microtubules is altered when etiolated hypocotyls aretreated with light (Figure 7; Le et al., 2005). In addition, corticalmicrotubules in the hypocotyl epidermal cells of light-grownseedlings are more stable than those of seedlings grown indarkness (see Supplemental Figure 9 online; Sambade et al.,2012). Thus, precise regulation of reorganization and stabiliza-tion of cortical microtubules is required for hypocotyl growth inresponse to light. Importantly, these processes can be regulatedby specific microtubule regulatory proteins.Many microtubule regulatory proteins play positive or nega-

tive roles in hypocotyl cell elongation by altering the stability andorganization of cortical microtubules. Plants must properly co-ordinate multiple pathways to modulate the activity of these

Figure 10. Model for WDL3 Function in the Regulation of CorticalMicrotubules during Light-Mediated Hypocotyl Cell Elongation.

In the dark, the effect of WDL3 on inhibition of hypocotyl elongation isdecreased due to degradation by the 26S proteasome pathway. In thelight, WDL3 alters the stability of cortical microtubules and reorganizesthe cortical microtubules, resulting in modulation of hypocotyl cell elon-gation in response to light.

1750 The Plant Cell

regulators in order to favor different hypocotyl elongation statesin the light and the dark. One such mechanism that has beenshown to play a role in influencing the activity of microtubuleregulators to modulate hypocotyl growth in response to light isCa2+-dependent signaling. Cytosolic calcium levels are regu-lated by light in hypocotyl cells. Furthermore, Ca2+ regulates theactivity of MDP25 on cortical microtubules, and overexpressionof MDP25 has been shown to inhibit hypocotyl elongation bydestabilizing cortical microtubules (Johnson et al., 1995; Foltaet al., 2003; Li et al., 2011). Thus, MDP25 regulates hypocotylelongation in a Ca2+-dependent manner in response to light. Asecond mechanism that influences the activity of microtubuleregulatory proteins during hypocotyl elongation involves tran-script regulation. For example, SPIRAL1 (SPR1) transcripts weredetected in the cells of etiolated hypocotyls, but not light-grownhypocotyl cells, and the hypocotyls of spr1 mutant plants dis-play helical growth defects when grown in the dark (Nakajimaet al., 2004, 2006). Therefore, light affects SPR1 expression,which modulates SPR1-mediated regulation of hypocotyl growthin response to light. A third mechanism that may be involved inmediating microtubule regulatory protein function during hypo-cotyl elongation is posttranslational regulation. The Arabidopsisdouble map65-1 map65-2 mutant exhibited defective hypocotylgrowth, suggesting a role for these proteins in the regulation ofmicrotubule-dependent hypocotyl elongation (Lucas et al., 2011).Furthermore, Nt-MAP65-1, the tobacco (Nicotiana tabacum)homolog of MAP65-1 and MAP65-2, is phosphorylated and itsmicrotubule-bundling activity is regulated by kinases of thetobacco mitogen-activated protein kinase cascade, specificallyNRK1/NTF6 (Hussey et al., 2002; Sasabe et al., 2006). In addi-tion, phosphatidic acid, a product of phospholipase D, directlybinds MAP65-1 and promotes its microtubule-polymerizing andbundling activities to stabilize cortical microtubules (Zhang et al.,2012). Thus, phosphorylation and phosphatidic acid–mediatedposttranslational regulation may play key roles in MAP65-mediatedhypocotyl growth.

Our study has revealed that the proteasome-dependent pro-tein degradation pathway is also important for posttranslationalregulation of the abundance of microtubule regulatory proteinsin order to modulate hypocotyl growth in light and darkness.Plants may regulate the levels of proteins that regulate hypo-cotyl elongation in order to alter the organization and stabiliza-tion of cortical microtubules to facilitate hypocotyl growth in thedark or inhibit hypocotyl elongation in the light. In addition tothese pathways, future studies should explore whether otherposttranslational modifications are involved in modulating thefunctions of microtubule regulatory proteins, and thereby hy-pocotyl cell elongation, in response to the antagonistic effects oflight and darkness.

The Ubiquitin-26S Proteasome System Is Involved in theRegulation of Hypocotyl Elongation in Response to Light

The ubiquitin-26S proteasome system regulates the accumula-tion of many upstream positive and negative regulators to pro-mote optimal Arabidopsis hypocotyl growth in the dark and thelight (Osterlund et al., 2000; Catalá et al., 2011; Chang et al.,2011). A few studies have shown that the 26S proteasomal

degradation pathway is involved in regulating plant microtubulesin response to environmental and developmental cues. Arabi-dopsis SPR1, a microtubule-stabilizing protein, is degraded bythe 26S proteasome in order to promote microtubule disas-sembly during acclimation of plant cells to salt stress (Wanget al., 2011), suggesting that proteasome-dependent deg-radation of microtubule regulatory proteins is also requiredfor microtubule-based functions.Previous studies of rpn10-1 and rpn1a-4 mutants have shown

that abnormal functioning of the 26S proteasome system inhibitsetiolated hypocotyl growth (Wang et al., 2009). In addition, corticalmicrotubules are more stable in the rpn10-1 mutant background(Wang et al., 2011), suggesting that 26S proteasome–regulatedhypocotyl growth requires destabilization of cortical microtubules.Promotion of microtubule destabilization may be achievedthrough controlled degradation of microtubule stabilizers, such asWDL3. In this study, although we failed to generate an anti-WDL3antibody capable of detecting endogenous levels of the nativeWDL3 protein, we showed that genetic and pharmacologicalinactivation of the 26S proteasome in Arabidopsis leads to theaccumulation of WDL3 in the dark. Furthermore, inhibition ofetiolated hypocotyl elongation was observed in the 26S protea-some mutant rpn1a-4. These data suggest that WDL3 plays a rolein 26S proteasome–mediated hypocotyl growth in response tolight. In addition, a previous study showed that constitutiveexpression of WVD2 resulted in defective growth of etiolatedhypocotyls, demonstrating that the WVD2 protein functions inthe dark (Yuen et al., 2003). Filament structures of three othermembers of the WVD2/WDL family (WDL4-GFP, WDL5-mCherry,and WDL6-mCherry) were detected in the light- and dark-grownhypocotyl cells of transgenic Arabidopsis (see SupplementalFigures 10A to 10C online). This evidence suggests that a spe-cific function of WDL3 involves 26S proteasome–mediated hy-pocotyl cell elongation in response to light.Characterization of WDL3 provides strong evidence for a role

for the ubiquitin-26S proteasome degradation system in theregulation of microtubules in plant cells in order to coordinateantagonistic effects on hypocotyl growth in light and darkness.Based on these observations, we propose the following modeldescribing the function of WDL3 in light-inhibited hypocotyl cellelongation (Figure 10). In the dark, we propose that the hypo-cotyl-elongating inhibiting activity of WDL3 is inhibited due todegradation of WDL3 by the ubiquitin-26S proteasome system.By contrast, WDL3 is stabilized in the light, resulting in WDL3-mediated stabilization of cortical microtubules, which promoteslongitudinal orientation of the microtubules and inhibits hypo-cotyl cell elongation.

METHODS

Plant Materials and Growth Conditions

All Arabidopsis thaliana plants and materials used in this study were in theColumbia-0 ecotype background. Seeds were sterilized and placed onhalf-strength MS medium (Sigma-Aldrich) containing 0.8% agar and 1%Suc. For hypocotyl measurement, plates were placed at 22°C in the lightfor 5 or 7 d after stratification at 4°C for 3 d. Mutant rpn1a-4 plants and35S:Tubulin6A-GFP transgenic plants have previously been described(Wang et al., 2007; Wang et al., 2011).

Proteasome-Mediated Degradation of WDL3 1751

Isolation of WDL3 cDNA Clones from Arabidopsis

The full-length WDL3 cDNA sequence was amplified by RT-PCR. Primersused to amplifyWDL3were 59-GGATCCATGGATATCTGTATGGACAAGG-39 and 59-GTCGACTTAGCAGGCAAAAGCAGTCTC-39. GST-tagged fu-sion proteins were expressed and purified according to the manufacturer’sprotocols. Protein concentration was determined using a Bio-Rad proteinassay kit. Protein samples were analyzed by SDS-PAGE.

Microtubule Cosedimentation Assay

Porcine brain tubulinswere purified using a previously publishedmethod byCastoldi and Popov (2003). These purified tubulins were used for sedi-mentation assays. Tubulin assembly and cosedimentation of microtubuleswith the fusion proteins were performed as described by Mao et al. (2005)and Li et al. (2011).The purified proteins were centrifuged at 150,000g at4°C for 20 min before use. Prepolymerized, paclitaxel-stabilized micro-tubules (5 mM) were incubated with 4 mM WDL3 fusion proteins in PEMbuffer (1 mM MgCl2, 1 mM EGTA, and 100 mM PIPES-KOH, pH 6.9) plus20 mM paclitaxel, at room temperature for 20 min. After centrifugation at100,000g for 20min, the supernatant andpelletswere subjected toSDS-PAGE.

Low-Temperature and Dilution Assays

Purified tubulin was conjugated to 5-(and 6-)carboxytetramethylrhodaminesuccinimidyl ester (NHS)-rhodamine, as previously reported (Hyman, 1991).NHS-rhodamine–labeled tubulin underwent an additional roundof assembly/disassembly with 30% (v/v) glycerin prior to storage in liquid nitrogen.

GST-WDL3 protein or GST-MAP65-1 protein (3 mM) was added to20 mM rhodamine-labeled tubulin in PEM buffer containing 1 mM GTP.After tubulin assembly at 35°C for 40 min, the temperature was imme-diately decreased to 10°C and maintained for 30 min for low-temperatureexperiments. For dilution treatments, the assembled tubulin samplesdescribed abovewere dilutedwith 503 prewarmed PEMbuffer containingWDL3 or MAP65-1 and incubated for 60 min at 35°C prior to fixation.Samples were fixed with 1% glutaraldehyde for observation by confocalmicroscopy.

Analysis of WDL3 Promoter: GUS Activity

A DNA fragment of the WDL3 promoter containing 1689 bp upstreamof the translation start site was amplified by PCR. The primers usedfor amplification were 59-GGATCCGGGACCGACCCACTTA-39 and 59-GAATTCTTTTCTTGTGACAGACC-39. The sequencewas then cloned intothe pCAMBIA1391 vector (Invitrogen). The resulting construct was thentransformed into Arabidopsis plants using Agrobacterium tumefaciens(strain GV3101) by the Arabidopsis floral dip method (Clough and Bent,1998). Thirty-seven independent transgenic lines were obtained, and thehomozygous seedlings were used for histochemical localization of GUSactivity in hypocotyl cells. The GUS staining procedure was performed aspreviously described by Wang et al. (2007).

Light Treatment

Three-day-old etiolated wild-type, WDL3-overexpressing, or WDL3 RNAiArabidopsis with a GFP-tubulin background grown on half-strength MSmedium were treated with light (33 mmol m22 s21) for 30 and 60 min, andcortical microtubules were observed using confocal microscopy.

Generation of WDL3 Overexpression and RNAi Arabidopsis Lines

To prepare stable WDL3 RNAi Arabidopsis lines, a WDL3 RNAi vectorwas generated by amplifying 306- or 360-bp WDL3 coding se-quences in the sense and antisense orientations and inserting them

into the pFGC5941 vector. Primers used for amplification of WDL3RNAi sequences were 59-ATTTAAATCTTCGTTCCGATCCACTG-39and 59-GGCGCGCCCGCCTCGAAGCTCCTTTTG-39; and 59-CGG-ATCCATTTAAATCTTCGTTCCGATCCACTG-39 and 59-TCTAGAGG-CGCGCCCGCCTCGAAGCTCCTTTTG-39. The following primers wereused for amplification of WDL3 RNAi-1 sequences: 59-CCATGGATGGA-TATCTGTATGGACAAG-39 and 59-CTCGAGTGAAGCACGTTTCTCAGTTG-39;and 59-GGATCCCCATGGATGGATATCTGTATGGACAAG-39 and 59-TCTAG-ACTCGAGTGAAGCACGTTTCTCAGTTG-39.

To stably express WDL3-GFP, the full-length WDL3 cDNA was am-plified by PCR and subcloned into the pBI221 vector (Invitrogen), whichexpressed WDL3 under control of the 35S promoter and a nopalinesynthase terminator. GFP was amplified and inserted at the C terminus ofWDL3. The cDNAs encoding WDL3 and GFP were then amplified andsubcloned into the pCAMBIA1300 expression vector and transformedinto wild-type (Columbia ecotype) Arabidopsis plants with Agrobacterium(strain GV3101). The transgenic homozygous Arabidopsis lines from theT3 generation were used.

RT-PCR and Immunoblot Analysis

RT-PCR and quantitative real-time PCR analysis was performed to assessWDL3 and otherWVD2/WDL transcript levels inWDL3 RNAi, RNAi-1, andoverexpressing seedlings. Total RNA was isolated using TRIzol reagent(Invitrogen) followed by treatment with RNase-free DNase I (Takara) at37°C for 30min to degrade genomic DNA. The treated RNA samples (2 mgeach) were used as templates for first-strand cDNA synthesis (Takara).Real-time PCR was performed using Applied Biosystems 7500 real-timePCR system with SYBR Premix Ex Taq (Takara). Relative expressionlevels were calculated as described (Huang et al., 2010). All experimentswere repeated at least three times. Primerswere described in SupplementalTable 2 online. The 18S rRNA was also amplified as a loading controlusing the following primers: 59-CGGCTACCACATCCAAGGAA-39 and59-GCTGGAATTACCGCGCGGCT-39.

Protein extracts were prepared from wild-type and WDL3-GFP trans-genic seedlings. Blots were probed with an anti-GFP antibody (Roche) ata dilution of 1:5000 in TBST (50 mM Tris, 150 mMNaCl, and 0.05% Tween20, pH 7.5) and alkaline phosphatase–conjugated goat anti-mouse IgGsecondary antibody (Sigma-Aldrich) at a dilution of 1:10,000. Actin ornonspecific bands on the blot were used as loading controls.

Measurement of Individual Microtubule Dynamics

To analyze the dynamics of individual microtubules, hypocotyl cells of4-d-old seedlings from the wild-type, WDL3-overexpressing, and WDL3RNAi lines with GFP-tubulin backgrounds were used. Time series images300 s in length (with 5-s intervals) were obtained under spinning discconfocal microscopy. Measurements were performed using ImageJ toolsas described by DeBolt et al. (2007). Microtubules with clearly visibleleading plus ends and at least 10 times the number of phase transitionswere selected for measurements in the wild-type (n = 36 microtubulesfrom nine seedlings), WDL3-overexpressing (n = 36 microtubules from20 seedlings), and WDL3 RNAi cells (n = 40 microtubules from 12seedlings). Rescue and catastrophe event frequencies were measuredand analyzed according to Kirik et al. (2012). All of the data were pro-cessed using Excel software (Microsoft Office 2003).

Ballistics-Mediated Transient Expression in Leaf Epidermal Cells

Subcellular localization of WDL3-GFP and cortical microtubules wasvisualized using transiently expressed 35S:WDL3-GFP and 35S:MBD-mCherry constructs expressed in Arabidopsis (Columbia ecotype) leafepidermal cells. These experiments were performed as previously

1752 The Plant Cell

described by Fu et al. (2002).We used 1mg of 35S:WDL3-GFP and 1mg of35S:MBD-mCherry DNA for particle bombardment using a PDS-1000/Hesystem (Bio-Rad). Six to 8 h after bombardment, GFP and mCherrysignals were detected using a Zeiss LSM 510META confocal microscope.Filamentous structures containingWDL3-GFP in leaf epidermal cells werevisualized after treatment with 10 mM oryzalin for 10 min.

Quantification of Cortical Microtubules

The procedure was performed as previously described by Li et al. (2011).Briefly, we used ImageJ software (http://rsb.info.nih.gov/ij/) to quantify thedensity of cortical microtubules in the cell. A vertical line was drawnperpendicular to the majority of the cortical microtubules, and the densityof cortical microtubules crossing the line was quantified. Measurementswere performed in triplicate for each cell, and a minimum of 24 cells pertreatment were used. The values were recorded, and significant differ-ences were analyzed using a paired Student’s t test.

Accession Numbers

Sequence data described in this article can be found in the ArabidopsisGenome Initiative under accession numbers At3g23090 (WDL3) andAt2g20580 (RPN1a).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. The GFP Tag Does Not Disturb thePhysiological Functions of WDL3.

Supplemental Figure 2. Expression of WDL3 Is Associated with theHypocotyl Phenotype in WDL3 RNAi Lines.

Supplemental Figure 3. Decreased Expression of WDL3 in WDL3RNAi-1 Arabidopsis Enhances Hypocotyl Growth.

Supplemental Figure 4. Expression of WVD2/WDL Is Unaffected inWDL3 RNAi and RNAi-1 Lines.

Supplemental Figure 5. Etiolated Hypocotyl Length Is Similar in Wild-Type, WDL3 Transgenic, and WDL3 RNAi Seedlings.

Supplemental Figure 6.WDL3 Expression in Arabidopsis Tissues andOrgans.

Supplemental Figure 7. Expression and Purification of the GST-Tagged WDL3 Fusion Protein.

Supplemental Figure 8. WDL3 Decorates Cortical Microtubules inWDL3-GFP Transgenic Arabidopsis.

Supplemental Figure 9. Light and Dark Treatments Affect Sensitivityof Cortical Microtubules to Oryzalin.

Supplemental Figure 10. WDL4/5/6 Expression Is Not Regulated byLight at the Protein Level.

Supplemental Table 1. Microtubule Dynamic Parameters in Wild-Type, WDL3-Overexpressing, and WDL3 RNAi Lines.

Supplemental Table 2. Primers Used to Detect Transcripts in theWVD2/WDL Family.

ACKNOWLEDGMENTS

We thank Jan A. Smallea (University of Kentucky) for generouslyproviding the Arabidopsis rpn1a-4 mutant seeds. This research wassupported by grants from the National Basic Research Program of China

(2012CB114200 to M.Y.), the Natural Science Foundation of China(31222007 and 31070258 to T.M.), and the Program for New CenturyExcellent Talents in University (NCET-12-0523 to T.M.).

AUTHOR CONTRIBUTIONS

T.M. designed the project. X.L.,T.Q., Q.M., J.S., and Z.L. performedspecific experiments and analyzed the data. T.M. wrote the article. M.Y.and T.M. revised and edited the article.

Received April 16, 2013; revised April 16, 2013; accepted April 19, 2013;published May 7, 2013.

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Proteasome-Mediated Degradation of WDL3 1755

DOI 10.1105/tpc.113.112789; originally published online May 7, 2013; 2013;25;1740-1755Plant Cell

Xiaomin Liu, Tao Qin, Qianqian Ma, Jingbo Sun, Ziqiang Liu, Ming Yuan and Tonglin MaoArabidopsisMicrotubule Regulatory Protein WDL3 in

Light-Regulated Hypocotyl Elongation Involves Proteasome-Dependent Degradation of the

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