Salt Stress–Induced Disassembly of Arabidopsis Cortical Microtubule

Salt Stress–Induced Disassembly of Arabidopsis Cortical Microtubule Arrays Involves26S Proteasome–Dependent Degradation of SPIRAL1C W

Songhu Wang,a Jasmina Kurepa,a Takashi Hashimoto,b and Jan A.Smalle a,1

a Plant Physiology,Biochemistry,Molecular Biology Program,Department of Plant and Soil Sciences,University of Kentucky, Lexington,Kentucky40546

b Graduate School of Biological Sciences,Nara Institute of Science and Technology,Ikoma,Nara630-0192,Japan

The dynamic instability of cortical microtubules(MTs)(i.e.,their ability to rapidly alternate between phases of growth and shrinkage)plays an essential role in plant growth and development.In addition,recent studies have revealed a pivotal role for dynamic instability in the response to salt stress conditions.The salt stress response includes a rapid depolymerization of MTs followed by the formation of a new MT network that is believed to be better suited for surviving high salinity. Although this initial depolymerization response is essential for the adaptation to salt stress,the underlying molecular mechanism has remained largely unknown.Here,we show that the MT-associated protein SPIRAL1(SPR1)plays a key role in salt stress–induced MT disassembly.SPR1,a microtubule stabilizing protein,is degraded by the26S proteasome,and its degradation rate is accelerated in response to high salinity.We show that accelerated SPR1degradation is required for a fast MT disassembly response to salt stress and for salt stress tolerance.

INTRODUCTION

The ubiquitin/26S proteasome system(UPS)regulates many fundamental cellular processes by controlling the degradation rates of numerous proteins(Hershko and Ciechanover,1998; Vierstra,2009).For the majority of UPS substrates,the concerted action of E1,E2,and E3enzymes leads to the covalent attach-ment of a multiubiquitin chain to the protein destined for degra-dation.The polyubiquitinated target protein is then degraded by the26S proteasome(Glickman,2000),an evolutionarily conserved multicatalytic protease that contains an enclosed proteolytically active core particle and one or two regulatory particles(RPs).The main roles of the RPs are in substrate rec-ognition,which is performed by RP non-ATPase subunits(RPNs), and in the unfolding and translocation of substrates to the core particle by RP triple A ATPase subunits(RPTs)(Smalle and Vierstra,2004;Kurepa and Smalle,2008).

In Arabidopsis thaliana,like in other eukaryotes,proteasome mutants and proteasome activity inhibitors are used to uncover the identity of UPS-regulated pathways and UPS target poteins (Kurepa and Smalle,2008).Loss of function of RP subunits RPN1a and RPN10,for example,revealed that the26S protea-some is essential for cell division and expansion,the modulation of responses to hormones and proteostatic drugs,and game-tophyte development(Smalle et al.,2003;Kurepa et al.,2008, 2009a,2009b,2010;Wang et al.,2009).These studies also revealed that the stress responses of rpn1a and rpn10mutant plants are altered:compared with the wild type,proteasome mutants are more tolerant of oxidative stress and less tolerant of protein misfolding stresses such as heat shock and salt stress (Smalle et al.,2003;Kurepa et al.,2008;Wang et al.,2009). The most frequently used proteasome inhibitor is MG132,a reversible,cell-permeable peptidyl aldehyde that inhibits the proteasome-speci?c chymotrypsin-like protease b5(Lee and Goldberg,1998).Studies using MG132have shown that the UPS is involved in the regulation of plant cell microtubule(MT) networks(Yanagawa et al.,2002;Oka et al.,2004;Sheng et al., 2006;S.Wang et al.,2011).MTs are polymers of a/b-tubulin heterodimers,which are incorporated into MTs directionally so that a-tubulin is exposed at the so-called lagging(2)and b-tubulin at the leading(+)ends.MTs play important roles in numerous cellular processes,including cell division and directional cell expansion(Nogales,2001;Sedbrook and Kaloriti,2008).

The polymerization-depolymerization dynamics of MTs are essential for their functionality in cellular processes,and they include two types of GTP hydrolysis-dependent dynamic behav-iors:dynamic instability and treadmilling.Treadmilling is the net growth of MTs at their(+)ends and shortening at their(2)ends, and dynamic instability is the switching between episodes of growth(polymer assembly)and shortening(polymer disassembly) at the(+)ends.The transition from growing to shortening is a dynamic instability parameter called catastrophe,and transition from shortening to growing is known as rescue.Growth,shrink-age,catastrophe,and rescue rates depend on MT-associated

1Address correspondence to jsmalle@https://www.360docs.net/doc/1e10795561.html,.

The author responsible for distribution of materials integral to the

?ndings presented in this article in accordance with the policy described

in the Instructions for Authors(https://www.360docs.net/doc/1e10795561.html,)is:Jan A.Smalle

(jsmalle@https://www.360docs.net/doc/1e10795561.html,).

C Some?gures in this article are displayed in color online but in black

and white in the print edition.

W Online version contains Web-only data.

The Plant Cell,Vol.23:3412–3427,September2011,https://www.360docs.net/doc/1e10795561.html,?2011American Society of Plant Biologists.All rights reserved.

(+)-end-tracking proteins(+TIPs)(Hirokawa,1994;Mandelkow and Mandelkow,1995;Lloyd and Hussey,2001;Schuyler and Pellman,2001;Sedbrook,2004;Hamada,2007;Sedbrook and Kaloriti,2008;Lyle et al.,2009a,2009b).

A large number of+TIPs have been identi?ed,and some of them are found in widely diverged species,suggesting that they regulate a basic,evolutionarily conserved component of the dynamic instability process(Bisgrove et al.,2004).For example,Arabidop-sis has functional homologs of end binding protein1(EB1)and cytoplasmic linker-associated protein1(CLASP1),which were ?rst identi?ed in human cells(Chan et al.,2003;Mathur et al.,2003; Galjart,2005;Vaughan,2005;Ambrose et al.,2007;Kirik et al., 2007;Bisgrove et al.,2008;Komaki et al.,2010).Other+TIPs,such as SPIRAL1(SPR1),are found only in plants(Nakajima et al.,2004, 2006;Sedbrook et al.,2004).Because plants overexpressing SPR1have increased tolerance to MT-destabilizing drugs,and spr1mutants have morphological defects consistent with altered MT stability,it has been concluded that this protein,similar to EB1 and CLASP1,is important for the regulation of dynamic instability (Nakajima et al.,2004,2006;Sedbrook et al.,2004;Abe and Hashimoto,2005;Ishida et al.,2007).

Similar to other eukaryotes,the structure of the plant MT cy-toskeleton is modulated not only by various developmental cues, but also in response to environmental signals and stress condi-tions(Smertenko et al.,1997;Himmelspach et al.,1999;Mathur and Chua,2000;Wang and Nick,2001;Abdrakhamanova et al., 2003;Van Bruaene et al.,2004;C.Wang et al.,2007,2011; Hamant et al.,2008).For example,short-term salt stress pro-motes the reorientation of MTs in maize(Zea mays)roots from transverse to parallel relative to the longitudinal axis and in tobacco(Nicotiana tabacum)BY-2cells from a structured to a seemingly random organization(Blanca?or and Hasenstein, 1995;Dhonukshe et al.,2003).Long-term salt stress also affects cortical MT organization in Arabidopsis and suppresses the right-handed helical growth of spr1mutants(Shoji et al.,2006). Furthermore,prolonged exposure to salt stress conditions was shown to trigger a biphasic response starting with a massive MT depolymerization phase followed by the formation of new MT networks that are thought to be better suited for surviving high salinity(Wang et al.,2007).Because treatments with a MT-destabilizing drug improved survival and growth under salt stress conditions and treatments with a MT-stabilizing drug caused salt stress hypersensitivity,the initial MT depolymerization response is believed to be essential for maintaining salt stress tolerance (Wang et al.,2007).

The speci?c mechanism that mediates the initial,salt stress–induced MT destabilization is currently not well understood. Because all tested proteasome mutants are hypersensitive to salt stress,we hypothesized that the MT reorganization needed for salt stress tolerance might be impaired in these lines due to an altered dynamic instability of MTs.Since the dynamic instability is regulated by the action of MAPs and in particular+TIPs,we tested if one of these proteins is conditionally degraded by the proteasome to allow MT restructuring required for an optimal salt stress response.Here,we show that in Arabidopsis,salt stress accelerates the26S proteasome–dependent degradation of SPR1and that this facilitates MT disassembly and promotes

Figure1.26S Proteasome Mutants Have Increased Tolerance to MT-Destabilizing Drugs.

(A)and(B)Effect of propyzamide(A)and oryzalin(B)on primary root elongation in the wild type(Col-0)and proteasome mutants rpn1a-4, rpt2a-2,rpn10-1,and rpn12a-1.Seedlings grown on MS/2medium for 5d were transferred to media containing the denoted doses of propy-zamide or oryzalin.The increase in root length was measured after3d of treatment.The root length of untreated seedlings of each genotype was set at100%,and mean values6SD(n$25)are shown as percentages of the root length of the respective controls.The difference in root length between Col-0and rpn1a-4,rpn10-1,or rpn12a-1was statistically sig-ni?cant for all tested doses(n$25,P<0.0001;two-way ANOVA fol-lowed by Bonferroni multiple comparisons post-test).For clarity,only the statistical signi?cance for Col-0versus rpt2a-2is marked in the graphs (*P<0.05).

(C)Effects of4m M propyzamide on primary root morphogenesis. The experimental conditions and timeline were the same as in(A). Bar=250m m.

Proteasomal Degradation of SPR13413

RESULTS

26S Proteasome Mutants Have Increased Tolerance to

MT-Destabilizing Drugs

To assay the stability of cortical MTs in26S proteasome mutants, we?rst tested the responses of rpn1a-4,rpt2a-2,rpn10-1,and rpn12a-1mutants to the MT-destabilizing drugs oryzalin,which binds to a-tubulin,and propyzamide,which binds to b-tubulin (Nakamura et al.,2004;Lyons-Abbott et al.,2010).We showed previously that the total26S proteasome activity in the four tested proteasome mutants is affected to different levels,with rpt2a-2carrying the weakest and rpn10-1the strongest defect in 26S proteasome function(Kurepa et al.,2008).We determined the effects of MT-destabilizing drugs by measuring their inhibi-tion of root elongation and their promotion of root tip swelling, two plant growth responses that were shown to be caused by MT disassembly(Baskin et al.,1994).

All proteasome mutants were more tolerant to both propyza-mide and oryzalin(Figures1A and1B).The strongest mutant, rpn10-1,was the most tolerant,whereas the weakest mutant, rpt2a-2,showed a statistically signi?cant increase in propyza-mide and oryzalin tolerance only at a single dose(6m M for propyzamide,P<0.05;100nM for oryzalin,P<0.05).In addi-tion,the propyzamide tolerance of the double mutant rpn1a-4 rpn10-1was further increased compared with the respective single mutants(see Supplemental Figure1online).

We also analyzed the morphology of propyzamide-treated roots.Earlier reports have shown that seedlings grown on media supplemented with MT-destabilizing drugs contain swollen pri-mary roots(Furutani et al.,2000).After a3-d-long treatment with 4m M propyzamide,root swelling was obvious in the Columbia-0 (Col-0)plants,whereas the diameter of primary roots of all proteasome mutants did not increase(Figure1C;see Supple-mental Figure1C online).

Changes in rpn10-1MT Dynamics

To analyze the stability of MTs in proteasome mutants further,we compared the dynamics of individual MTs in Col-0and rpn10-1 backgrounds(Figure2).Previous studies have shown that green ?uorescent protein(GFP)fusions with a-(GFP-TUA6)or b-tubulin (GFP-TUB6)are incorporated into MTs and can be used to visualize MT dynamics in vivo(Ueda et al.,1999;Nakamura et al., 2004;Abe and Hashimoto,2005).However,whereas GFP-TUB6–labeled MTs have wild-type properties,the incorporation of GFP-TUA6changes MT dynamics and induces right-handed helical growth(Nakamura et al.,2004;Abe and Hashimoto, 2005).Thus,we chose to introduce the35S:GFP-TUB6trans-gene into the rpn10-1mutant and analyzed MT dynamics using confocal time-lapse imaging.

Kymographs of individual MT(+)-ends and representative life history plots showed that in rpn10-1,the MT growth phase was longer and the shrinkage phase was shorter than in the wild type (Figure2).Indeed,most of the parameters of MT dynamic instability were altered in the rpn10-1background(Table1). The average growth rate of the leading ends and the frequency of while the frequency of rescue events in rpn10-1was increased. Thus,individual MTs in rpn10-1were less dynamic and more prone to polymerization.In contrast with the(+)-end,there was no signi?cant difference in(2)-end dynamics between rpn10-1 and the wild type(Table1),implying that it is the process of dynamic instability and not treadmilling that was affected by the inhibition of proteasome activity.These results combined with the increased tolerance of proteasome mutants to MT-destabilizing drugs indicated that26S proteasome–dependent proteolysis plays an important role in the regulation of MT dy-namic instability.

SPR1Mediates the Tolerance of Proteasome Mutants

to Propyzamide

Previous studies showed that overexpression of the plant-speci?c+TIP SPR1leads to increased propyzamide

tolerance

Figure2.MT Plus-End Dynamics Are Altered in the rpn10-1Mutant.

(A)Kymograph of a160-s-long recording showing(+)-end dynamics of individual MTs.Four-day-old seedlings expressing35S:GFP-TUB6in the Col-0and rpn10-1backgrounds were used for time-lapse confocal imaging.Epidermal cells of upper hypocotyl regions were photographed every4s.Bar=5m m.

(B)The life history plots(length versus time)of three GFP-TUB6–labeled MTs in Col-0and three GFP-TUB6–labeled MTs in the rpn10-1back-ground.MT length was measured from confocal micrographs using Image J.

3414The Plant Cell

(Nakajima et al.,2004).Since proteasome mutants are also more tolerant of MT-destabilizing drugs(Figure1)and have altered MT dynamics,(Figure2),we tested whether SPR1is a26S protea-some target and whether its stabilization in proteasome mutants can explain the observed phenotypes.

To assay SPR1stability,we used cycloheximide(CHX)to inhibit de novo protein synthesis.After a12-h-long CHX treat-ment,the SPR1level was reduced to;30%of the control.This decrease was blocked by the proteasome inhibitor MG132, suggesting that SPR1is an unstable protein that is targeted for 26S proteasome–dependent proteolysis(Figure3A).Indeed,the SPR1level was increased in26S proteasome mutants rpn1a-4 and rpn10-1and was even more abundant in the rpn1a-4 rpn10-1double mutant(Figure3B).The SPR1mRNA abundance remained unchanged in the proteasome mutant backgrounds, con?rming that the increase in SPR1protein level was caused by a posttranscriptional mechanism(see Supplemental Figure2 online).Finally,CHX chase immunoblotting analyses(Yewdell et al.,2011)showed that the SPR1degradation rate was de-creased in proteasome mutant backgrounds(Figures3C and 3D),con?rming a role for the UPS in regulating SPR1abundance. To test if SPR1accumulation is a cause for the increased tolerance of proteasome mutants to MT-destabilizing drugs,we crossed the spr1-3mutation into the rpn1a-4and rpn10-1mutant backgrounds(Figure4;see Supplemental Figure3online). Analyses of the root tip morphology in propyzamide-treated double mutants showed that the reduced root tip swelling in proteasome mutants was suppressed by the spr1-3mutation (Figure4A).To quantify the effect of the spr1-3mutation,we measured the width of the primary root in the elongation zone (Figure4B).As expected,the propyzamide treatment did not promote any substantial root swelling in the proteasome mu-tants.The spr1-3mutation caused a mild increase in root width compared with the wild type(344.565m m and372.967m m for Col-0and spr1-3,respectively;n=25,P<0.001).By contrast, the root width of both rpn1a-4spr1-3and rpn10-1spr1-3double mutants was strongly increased compared with the respective single proteasome mutants(n=25,P<0.001),although the increases in swelling did not reach the wild-type level(P<0.01for Col-0).We concluded that the removal of SPR1suppressed the propyzamide tolerance of proteasome mutants but did not fully revert this phenotype to the wild-type level,suggesting the existence of other MT-stabilizing proteins that are also degraded by the26S proteasome.

The root elongation assay con?rmed that SPR1contributes to the propyzamide tolerance of proteasome mutants(Figure4C). The rpn1a-4spr1-3and rpn10-1spr1-3double mutants dis-played an increase in right-handed helical growth(see Sup-plemental Figure3online).For example,the right-handed root skewing of spr1-3was enhanced in both the rpn1a-4and rpn10-1mutant backgrounds.This was the most striking in rpn1a-4spr1-3seedlings that had upward growing roots(see Supplemental Figure3C online).However,low propyzamide doses fully suppressed the enhanced root skewing phenotype in both double mutants(see Supplemental Figure4online),allow-ing us to compare the propyzamide tolerance levels of all lines by the root elongation assay.Similar to the root tip swelling response,the root elongation assay showed that spr1-3sup-presses the propyzamide tolerance of both the rpn1a-4and rpn10-1mutants(Figure4C).

To analyze directly the effects of spr1-3on MT stability,we attempted to cross the35S:GFP-TUB6transgene into the spr1-3 and rpn10-1spr1-3mutants.We were unable to isolate spr1-3 or rpn10-1spr1-3mutants expressing GFP-TUB6in spite of screening more than1000F2seedlings,and we hypothesized that the35S:GFP-TUB6transgene is located in the proximity of the SPR1locus.However,analysis of the35S:GFP-TUB6locus revealed that the T-DNA is not linked to SPR1(data not shown). The reason GFP-TUB6expression and the spr1-3mutation are incompatible is currently unknown.As an alternative,we intro-duced the35S:GFP-TUA6transgene into the spr1-3and rpn10-1 spr1-3mutants.To assay the MT stability in these lines,we treated4-d-old seedlings with a high dose of propyzamide to ensure a fast and complete MT disruption(20m M propyzamide for1h)and analyzed epidermal cells of the upper hypocotyl regions by confocal microscopy(Figure4D).In rpn10-1,the GFP-TUA6–labeled cortical MTs remained partially intact,while MTs in the wild-type and rpn10-1spr1-3cells were disrupted,and the

Table1.Dynamic Instability Parameters in Col-0and rpn10-1

Dynamic Parameters (+)-Ends(à)-Ends

Col-0rpn10-1Col-0rpn10-1

Growth rate(m m/min) 5.4464.61 4.0462.73* 1.9161.15 1.7261.03

Shrinkage rate(m m/min)10.368.898.9168.44 3.5364.16 3.8264.17 Catastrophe(events/second)0.0410.030*0.2120.196

Rescue(events/second)0.0960.141*0.0470.051

Time spent on growth67.2%76.6%9.3%10.7%

Time spent on pause9.6%11.2%48.4%48.7%

Time spent on shrinkage20.4%12.3%42.4%40.6%

Dynamicity(m m/min) 5.9161.46 4.1962.41* 1.6761.92 1.9961.28

MT dynamic instability parameters were quanti?ed from confocal micrographs.Velocities were calculated from38leading and22lagging ends for 35S:GFP-TUB6in Col-0and45leading and32lagging ends for35S:GFP-TUB6in the rpn10-1background.Total measurements include1111and 1763velocities(4-s intervals)for GFP-TUB6in Col-0and rpn10-1,respectively.Dynamic parameters are expressed as mean6SD.Statistical signi?cance was calculated using Student’s t test comparing the mutant and wild-type values(*P<0.05).

Proteasomal Degradation of SPR13415

Collectively,these experiments suggested that the stabilization of SPR1in proteasome mutants is a cause for both their in-creased tolerance to MT-destabilizing drugs and altered MT dynamicity.

Salt Stress Promotes Proteolysis of SPR1

Based on earlier reports of the salt stress hypersensivitiy of26S proteasome mutants(Smalle et al.,2003;Wang et al.,2009)and the fact that salt stress tolerance requires MT disassembly function increases salt stress sensitivity by stabilizing SPR1, which in turn leads to increased MT stability.To test this,we?rst determined if salt stress leads to a conditional26S proteasome–dependent degradation of SPR1,which could facilitate the re-arrangement of cortical MTs needed for salt tolerance. Prolonged treatment(16h)of4-d-old Col-0seedlings with150 mM NaCl caused a decrease in SPR1level,and this decrease was blocked by MG132(Figure5A).CHX-chase immunoblotting analyses showed that a16-h-long NaCl treatment led to an ;80%reduction of the SPR1level in Col-0,whereas the SPR1 level in rpn1a-4and rpn10-1mutants was reduced by only;30

and;20%,respectively(Figures5B and5C).These results

indicated that the salt stress–induced decrease in SPR1level was a result of proteasome-dependent degradation.

During treatments with a higher concentration of salt(200mM), degradation of SPR1was detectable already after3h of incu-bation(see Supplemental Figures5A to5C online).To test if high salt concentrations promote SPR1degradation via a global increase in26S proteasome–dependent proteolysis,we moni-tored total proteasome activity by analyzing the abundance of polyubiquitinated proteins and by measuring proteasomal che-motryptic activity(see Supplemental Figures5D and5E online). After5h of exposure to200mM NaCl,we observed a signi?cant decrease in the SPR1protein level,no change in the overall levels of ubiquitinated proteins,and a mild but signi?cant decrease in proteasome activity.We concluded that the accelerated proteasome-dependent SPR1degradation during salt stress is not caused by a general increase in proteasome activity but is more likely the result of a speci?c destabilization mechanism that is activated by salt stress.Finally,we con?rmed that the SPR1 depletion in response to salt is indeed the result of a posttrans-criptional mechanism by observing that the SPR1mRNA level did not change in response to salt stress treatments(see Sup-plemental Figure5F online).

Previous studies have shown that cortical MT arrays depoly-merize in response to salt stress(Wang et al.,2007).Since the molecular mechanisms that govern this process were unknown, it remained possible that the salt stress–induced degradation of SPR1re?ects not a direct effect on SPR1,but an indirect effect caused by the MT disassembly.For example,the MT disassem-bly might increase the amount of free SPR1that could be more susceptible to degradation than the tubulin-bound version.To distinguish between these possibilities,we monitored the SPR1 level in Col-0plants treated with propyzamide.In Col-0seedlings treated for3h with10m M propyzamide,the cortical MTs were depolymerized(see Supplemental Figure6online),but the SPR1 level remained the same as in the untreated control(Figure5D). The propyzamide treatment also did not in?uence the salt stress–induced degradation of SPR1(Figure5D).

In addition to ionic stress,high salinity is also known to cause osmotic stress(Zhu,2001).To test if osmotic stress in?uences the stability of SPR1,we treated Col-0seedlings with high doses of the osmolyte mannitol(Figure5E).The mannitol treatments did not affect SPR1stability,suggesting that the signal triggering SPR1destabilization was speci?cally ionic stress(Figure5F).We concluded that salt stress,but not osmotic stress or MT disas-sembly per se,promotes the26S proteasome–dependent deg-

Figure3.SPR1Is a26S Proteasome Target.

(A)Immunoblotting analyses using anti-SPR1and anti-GS antisera. Seven-day-old seedlings were treated with100m M MG132and/or200 m M CHX for16h and used for the extraction of total protein.The anti-GS sera recognizes both the chloroplastic(45kD)and cytosolic(40kD)GS isoforms.

(B)SPR1levels in8-d-old Col-0,rpn1a-4,rpn10-1,and rpn1a-4rpn10-1 seedlings.Anti-RPN1and anti-RPN10sera were used to con?rm the genotype of proteasome mutants.

(C)Representative CHX-chase immunoblots.The stability of SPR1was tested on total protein extract of10-d-old wild-type and mutant seed-lings treated with200m M CHX for the indicated time periods.

(D)Quanti?cation of SPR1stability in CHX-treated Col-0,rpn1a-4,and rpn10-1plants.Immunoblots,representatives of which are shown in(C), were used to quantify signal intensities.The average signal intensity of the zero time point sample for each line was set to100%,and the mean values6SD(n=3)are shown as percentages of the respective control. The asterisks represent the statistical signi?cance of the difference be-tween degradation rates in Col-0and both proteasome mutants(****P< 0.0001;ANOVA followed by Bonferroni multiple comparisons post-test). [See online article for color version of this?gure.]

3416The Plant Cell

SPR1Stabilization Causes Salt Stress Hypersensitivity

To test if SPR1stabilization causes the salt hypersensitivity of26S proteasome mutants(Smalle et al.,2003;Wang et al.,2009),we ?rst analyzed if the increased MT stability is responsible for the changes in salt tolerance(Figure6A),then determined the salt tolerance levels of rpn1a-4spr1-3and rpn10-1spr1-3mutants (Figures6B to6D),and?nally compared the effects of salt stress on the MT arrays of rpn10-1and rpn10-1spr1-3cells(Figure7). To test the relationship between MT stability and salt tolerance, we analyzed the effects of combined salt stress and propyzamide treatments on the root elongation of rpn1a-4and rpn10-1mutants (Figure6A).In the absence of propyzamide,root length of wild-type plants grown on100mM NaCl was;70%of the control,whereas the root length of proteasome mutants was reduced to ;50%.As expected,low doses of propyzamide counteracted the salt-induced inhibition of root elongation in all lines.However, whereas1m M propyzamide led to an8%increase in root length in the wild type(statistically nonsigni?cant,P>0.05,n$15;analysis of variance[ANOVA]with Bonferroni post-test),the increase in root length in the proteasome mutants was;25%(P<0.001,n$15). Thus,since low doses of an MT-destabilizing drug reverted the salt hypersensitivity of proteasome mutants to wild-type levels,we concluded that the increased MT stability in proteasome mutants is indeed responsible for their salt hypersensitivity.

This conclusion,together with our observation that spr1-3 suppressed the increased MT stability in rpn1a-4and rpn10-1 mutants(Figure4),prompted us to test if the spr1-3mutation

also

Figure4.spr1-3Suppresses the Propyzamide Tolerance of Proteasome Mutants.

(A)Effects of4m M propyzamide on primary root morphogenesis in Col-0,rpn1a-4,spr1-3,and the double mutant rpn1a-4spr1-3.Five-day-old seedlings were transferred to fresh MS/2medium or MS/2medium with propyzamide.The seedlings were grown on vertically positioned plates for3d and then photographed.Bar=1mm.

(B)Effects of4m M propyzamide on root width.Seedlings were grown and treated as in(A).The width of primary roots at the elongation zone was measured from micrographs using ImageJ.The signi?cance was analyzed using ANOVA followed by Bonferroni multiple comparisons post-test. Crosses represent the signi?cance of the differences between the untreated Col-0and untreated mutant lines,and asterisks mark the signi?cance between the treated Col-0and treated mutant lines(**P<0.01;???and***P<0.001;and????and****P<0.0001).

(C)Root lengths of5-d-old seedlings grown on MS/2media with4m M propyzamide(****P<0.0001;signi?cance for Col-0versus mutants).The statistical analyses and data presentation are as in(B).

(D)In vivo analyses of the MT-destabilizing effect of20m M propyzamide in the Col-0wild type and in the rpn10-1and rpn10-1spr1-3mutants.The35S: GFP-TUA6transgene was crossed into the rpn10-1and rpn10-1spr1-3mutant backgrounds,and the GFP-TUA6–labeled cortical MTs were analyzed by confocal microscopy.Hypocotyl epidermal cells of4-d-old seedlings treated for1h are shown.Bar=20m m.

Proteasomal Degradation of SPR13417

suppresses the salt hypersensitivity of proteasome mutants (Figures 6B and 6D).Salt stress induces a range of morphological alternations in both the aerial parts of the plant and the roots.For example,NaCl treatments lead to reductions in root length and lateral root number,a decrease in leaf and petiole size,and a delay in leaf emergence (Burssens et al.,2000).Furthermore,prolonged or intense salt stress treatments cause leaf chloro-sis,bleaching,and necrosis.All adverse effects of salt stress,including leaf necrosis and the inhibition of root and rosette growth,were apparent at lower doses in rpn10-1compared with the wild type and were suppressed by the spr1-3mutation (Figure 6B).The fresh weight of rpn1a-4spr1-3and rpn10-1spr1-3plants grown for 2weeks on salt-containing media was signi?cantly increased compared with the single rpn1a-4and rpn10-1mutants but did not reach the wild-type levels (Figure 6C).The effects of NaCl on root elongation were measured after 3d of treatment (Figure 6D).On the control media,spr1-3sup-pressed root elongation both in the wild type and in proteasome mutants,probably because it caused the right-hand skewing of roots.On NaCl-containing media,root elongation of rpn1a-4spr1-3and rpn10-1spr1-3plants was increased compared with the respective single 26S proteasome mutants but again did not reach the length of the wild-type roots treated with the same dose of NaCl (Figure 6D).Thus,the spr1-3mutation did indeed suppress the salt hypersensitivity of proteasome mutants,but this suppression was partial.

Next,we performed a time-course analysis of the impact of salt stress on GFP-TUA6–labeled MT arrays in wild-type,spr1-3,rpn10-1,and rpn10-1spr1-3plants (Figure 7).In the wild type,MT disassembly was nearly complete after a 4-h-long exposure to 200mM NaCl (Figure 7A).By contrast,the cortical MT network in rpn10-1cells remained largely intact up until 6h into the treatment.The spr1-3mutation suppressed the delayed MT depolymerization in the rpn10-1mutant,but similarly to the effects of spr1-3on whole-seedling salt tolerance,it did not fully revert the MT disassembly rate back to the wild-type level.Analyses of the number of MTs per unit length con?rmed both the delayed salt response in rpn10-1and the partial suppression of this delay by the spr1-3mutation (Figure 7B).We concluded that the SPR1stabilization in proteasome mutants causes salt hy-persensitivity by slowing down the salt stress–induced depoly-merization of MTs.However,since the proteasome mutant salt stress responses did not completely revert back to the wild-type level when the spr1-3mutation was introduced,we also con-cluded that SPR1is not the only proteasome target that inhibits salt stress–induced MT

depolymerization.

Figure 5.Salt Stress Promotes 26S Proteasome–Dependent Proteolysis of SPR1.

(A)Immunoblotting analyses of SPR1in Col-0seedlings treated with NaCl and MG132.Four-day-old seedlings were treated for 16h with 150mM NaCl alone or 150mM NaCl and 100m M MG132.

(B)CHX-chase immunoblot.Ten-day-old seedlings were transferred to liquid MS/2media containing 200m M CHX and 200mM NaCl.Seedlings were treated for the indicated times.

(C)Quanti?cation of immunoblots represented by those in (B).The signals of the untreated samples were set at 100%.Data are shown as mean values 6SD (n =3)as percentages of the respective controls.The asterisks represent the statistical signi?cance of the difference between the degradation rates in Col-0versus rpn10-1and rpn1a-4(****P <0.0001;ANOVA followed by Bonferroni multiple comparisons post-test).

(D)and (E)Immunoblotting analyses of SPR1in Col-0seedlings treated with the denoted doses of NaCl and (D)propyzamide or mannitol (E).Four-day-old seedlings were transferred to water containing the test compounds,treated for 3h,and used for the isolation of total proteins.3418The Plant Cell

The fast,salt-induced MT depolymerization response in cells of the wild type and rpn10-1spr1-3double mutant was followed by the start of new MT formation(8-h time point;Figure7).By contrast,after8h of treatment,the MT disassembly was just completed and no distinct MTs could be detected in the rpn10-1 seedlings.This con?rms an earlier report stating that fast MT disassembly in response to salt stress is required for the timely formation of new MT networks potentially better suited to toler-ate high salt concentrations(Wang et al.,2007).

Additional proof that increased SPR1abundance is a cause for salt stress hypersensitivity was obtained by analyzing transgenic plants that overexpress SPR1.In theory,plants that overexpress SPR1should respond to salt stress similarly to proteasome mutants in which the SPR1level is increased because of a reduced degradation rate.Previous work already showed that SPR1overexpression indeed leads to increased tolerance to propyzamide in shoot organs(Nakajima et al.,2004).Here,we show that increased tolerance to propyzamide can also be observed in the root-swelling assay and is indeed associated with salt stress hypersensitivity(see Supplemental Figure7 online).Collectively,these results con?rm the importance of SPR1removal for maintaining salt stress tolerance.MG132Promotes MT Stability and Salt

Stress Hypersensitivity

Finally,to independently test the role of SPR1proteolysis in the salt stress–induced restructuring of cortical MT arrays,we ana-lyzed the combined effects of salt,propyzamide,and MG132on Col-0and spr1-3seedlings(Figure8).

Similar to the genetic suppression of proteasome activity, pretreatment of Col-0seedlings with MG132inhibited the propyzamide-induced swelling of root tips(Figure8A).Although MG132also inhibited the swelling of spr1-3roots,this effect was not as strong as in the wild type(Figure8A).MG132also counteracted the inhibitory effect of propyzamide on root elon-gation(Figure8B).Treatment with either MG132or propyzamide inhibited root elongation in Col-0and spr1-3seedlings.However, while MG132reduced the root lengths to;85%of the respec-tive controls,the propyzamide treatment caused a more severe growth inhibition(to;30%of the control values).Roots of Col-0 plants treated with both MG132and propyzamide were;50% longer than when treated with propyzamide alone,indicating that the proteasome inhibitor counteracts the effect of the MT destabilizing drug.Furthermore,this MG132effect was

also

Figure6.spr1-3Suppresses the Salt Hypersensitivity of26S Proteasome Mutants.

(A)Salt hypersensitivity of proteasome mutants was suppressed by propyzamide treatments.Five-day-old seedlings grown on MS/2medium were transferred to fresh MS/2plates or MS/2plates with100mM NaCl and the indicated doses of propyzamide.Plates were positioned vertically,and the root lengths were measured after3d.Data represent relative root length(n$20)with SD.The average root length of untreated Col-0seedlings was set at100%.

(B)Five-day-old seedlings grown on MS/2medium were transferred to fresh MS/2medium or MS/2containing100mM NaCl.Test plates were positioned vertically,and plants were grown for2weeks.The arrows highlight necrotic spots in the rpn10-1mutant grown on100mM NaCl.

(C)and(D)The salt tolerance levels were quanti?ed by comparing the fresh weight after a2-week-long treatment(C)and root lengths after a3-d-long treatment(D).Data are shown as mean values6SD of three independent experiments(n$15for each).The asterisks represent the signi?cances of the differences between the double mutants and the respective single proteasome mutants and were calculated using ANOVA followed by Bonferroni

Proteasomal Degradation of SPR13419

partially attenuated in the spr1-3mutant,implying a role for SPR1 in the MG132-induced MT stabilization(Figure8B).

To test if MG132also increases salt sensitivity,we analyzed the response of GFP-TUB6–labeled MT arrays and the growth of Col-0and spr1-3on medium supplemented with200mM NaCl (Figures8C to8E).Indeed,MG132suppressed the salt-induced depolymerization of cortical MTs(Figure8C)and decreased the ever,compared with the wild type,MG132was less effective in suppressing leaf expansion(Figure8D)and root elongation of salt-treated spr1-3seedlings(Figure8E),con?rming that the salt hypersensitivity caused by proteasome inhibition requires SPR1 function.Thus,we?nd that the increased MT stability and salt stress hypersensitivity of proteasome mutants can be phe-nocopied by treating wild-type plants with the proteasome inhibitor MG132.

DISCUSSION

Posttranscriptional and,in particular,proteasome-dependent regulation of tubulin/MT system dynamics have been described in considerably greater detail in animals than in plants.MT dynamics depend on the availability of tubulin heterodimers and on the activities of MAPs,and recent studies have shown that proteasome-dependent protein degradation determines the sta-bility of both MT dynamics determinants.For example,the role of the proteasome and tubulin-speci?c chaperones in the degra-dation of tubulins released by MT depolymerization has been documented in animals(Bhamidipati et al.,2000;Ren et al., 2003;Bartolini et al.,2005;Voloshin et al.,2010).Depolymeriza-tion of MTs also leads to the proteasome-dependent degrada-tion of tubulins in plants,but the molecular players that prime tubulin for proteolysis and that deliver tubulin heterodimers(or monomer-chaperone complexes)to the proteasome are still unidenti?ed(S.Wang et al.,2011).

In animals,the proteasome has also been shown to regulate MT dynamics by in?uencing the stability of MAPs(David et al., 2002;Petrucelli et al.,2004;Peth et al.,2007;Poruchynsky et al., 2008;Ban et al.,2009).Our study extends this observation to plants and reveals that proteasome-dependent stability control is essential for the restructuring of MT arrays and the?ne-tuning of MT dynamics.We show that genetic and pharmacological inactivation of26S proteasome activity in Arabidopsis leads to an increase in MT stability and,consequently,to an improved tolerance of MT destabilizing drugs.The increased MT stability in proteasome mutants was largely the result of stabilization of the plant-speci?c+TIP SPR1.Thus,the accumulation of SPR1 resulting from either overexpression of the SPR1transgene (Nakajima et al.,2004)or a reduced degradation rate(this study) is suf?cient to promote increased MT stability in plant cells. Although the increased stability of SPR1in26S proteasome mutants implies that this MAP is targeted for degradation in a ubiquitin-dependent manner,we were unable to detect any candidate ubiquitinated SPR1forms with higher molecular weights.Similar results were described for several other known proteasome targets(Dill et al.,2001;Lopez-Molina et al.,2001; Xie et al.,2002;Smalle et al.,2003;Gagne et al.,2004),sug-gesting that the detection of ubiquitin conjugates can be tech-nically challenging.Proteomic studies have shown that only a very small fraction of any given26S proteasome target protein exists in its ubiquitinated form(Kaiser and Tagwerker,2005). Furthermore,it can be envisioned that even upon partial stabi-lization in proteasome mutant backgrounds,the ubiquitinated SPR1forms remain undetectable due to the actions of ubiquitin

Figure7.SPR1Stabilization in rpn10-1Delays the Salt-Induced Disas-sembly of Cortical MT Arrays.

(A)Visualization of GFP-TUA6–labeled cortical MTs in upper hypocotyl epidermal cells from4-d-old Col-0,rpn10-1,and rpn10-1spr1-3seed-lings treated with200mM NaCl for the indicated times.Bar=10m m.

(B)The density of cortical MTs per unit length in upper hypocotyl cells was determined by counting the MTs crossing the longitudinal axis of a cell.For each line and treatment,a minimum of32hypocotyl cells from?ve separate seedlings was photographed and used for measurements.The asterisks represent the signi?cance of the difference between Col-0and the mutants treated for the same time and were calculated using ANOVA followed by Bonferroni multiple comparisons post-test(****P<0.0001). [See online article for color version of this?gure.]

3420The Plant Cell

ubiquitinated proteins back to their unmodi?ed forms (Smalle and Vierstra,2004;Kaiser and Tagwerker,2005).

While our data show that SPR1stabilization plays a pivotal role in the altered MT dynamics of proteasome mutants,they also suggest the involvement of other MAPs.For example,whereas the increased rescue frequencies and decreased frequencies of catastrophe (Figure 2,Table 1)that re?ect an overall increase in MT stability in rpn10-1cells are in agreement with the predicted effects of increased SPR1action,the slower growth of MTs (Table 1)cannot be explained by an increase in SPR1activity and suggests that loss of proteasome function also affects one or more proteins that control the MT polymerization rate.Another example is presented in Figure 4:The spr1-3mutation did not fully suppress the increased propyzamide tolerance of protea-some mutants,suggesting the stabilization of one or more additional proteins that promote MT stability.The involvement of MT-stabilizing factors other then SPR1could also explain the observation that the right skewing of spr1-3roots was enhanced in the rpn10-1spr1-3and rpn1a-4spr1-3double mutants (see Supplemental Figure 3online).The spr1mutants are unusual in that they combine a decrease in MT stability with the right skewing of roots even though this developmental phenotype is typically the result of MT stabilization (Sedbrook and Kaloriti,2008).Because spr1-3partially reversed the increased MT stability of proteasome mutants (Figure 4),we expected that the right skewing of spr1-3roots would also be reversed.However,since the right skewing was enhanced in the double mutants,we propose that this phenotype re?ects the function of other MT-stabilizing MAPs whose activity is enhanced by their stabilization in the proteasome mutant backgrounds.

Our observation that MT stability depends on the SPR1degradation rate suggested that the activity of this protein is controlled posttranslationally and implied the existence of de-velopmental or environmental cues that in?uence MT dynamics by regulating SPR1stability.Indeed,we found that SPR1pro-teolysis is enhanced under salt stress conditions (Figure 5)and that SPR1stabilization leads to salt stress hypersensitivity in 26S proteasome mutants (Figure 6).Furthermore,we showed that the increased salt stress sensitivity of proteasome mutants is caused by a slower rate of MT disassembly,which is a direct result of the SPR1stabilization.These results are in agreement with the results of an earlier study that described that suppression of MT disassembly by the MT-stabilizing drug paclitaxel caused salt stress hypersensitivity (Wang et al.,

2007).

Figure 8.MG132Stabilizes MTs and Increases the Sensitivity to Salt Stress.

(A)and (B)Four-day-old Col-0and spr1-3seedlings were transferred to MS/2medium containing 4m M propyzamide,40m M MG132,or 4m M propyzamide and 40m M MG132.Plants were grown vertically for three days before the root tips were photographed ([A];bar =0.5mm),and the root lengths were measured (B).In (B),data represent an average root length with SD (n =26),and the asterisks represent the signi?cances of the differences between the wild type and spr1-3(****P <0.0001;ANOVA followed by Bonferroni multiple comparisons post-test).

(C)Visualization of GFP-TUB6–labeled cortical MTs in upper hypocotyl epidermal cells.Four-day-old seedlings were pretreated with 100m M MG132or DMSO in liquid MS/2medium for 6h,and then 20m M propyzamide or 200mM NaCl was added.Propyzamide treatments were done for 1h and the NaCl treatments for 3h prior to microscopy.Bars =10m m.

(D)and (E)Four-day-old Col-0and spr1-3seedlings were transferred to MS/2medium containing 100mM NaCl,50m M MG132,or both.Seedlings were photographed,and the root lengths were measured after 3d of treatment.In (E),data represent an average root length with SD (n $25),and the asterisks represent the signi?cances of the differences between the wild type and spr1-3(****P <0.0001,***P <0.001,and **P <0.01;ANOVA followed Proteasomal Degradation of SPR13421

Arabidopsis26S proteasome mutants are also characterized by a decrease in growth rate that re?ects a reduced rate of mitosis (Kurepa et al.,2009b).Since MT depolymerization is also known to inhibit mitosis,an alternative explanation for the salt stress hyper-sensitivity of26S proteasome mutants is that the reduced mitotic rates make them more susceptible to the growth inhibitory effects of salt stress.However,this explanation is unlikely because the spr1-3mutation suppressed the salt hypersensitivity but not the reduced growth rate of proteasome mutants(Figure6).Further-more,the SPR1overexpression lines were also salt hypersensitive but did not display any decrease in growth rate(see Supplemental Figure7online),thus con?rming that the increased SPR1abun-dance in26S proteasome mutants is indeed the most likely cause for their salt stress hypersensitivity.

Collectively,our data reveal an important role for proteasome-dependent regulation of SPR1in the survival of plants challenged by high salinity.Figure9outlines a model that summarizes the role of SPR1in the MT disassembly response to salt stress.According to this model,SPR1is a moderately stable protein under normal growth conditions and is localized predominantly at the growing ends of MTs where it inhibits their disassembly. Upon salt stress,26S proteasome–dependent degradation of SPR1is accelerated,and the MT depolymerization needed for the survival of plant cells under salt stress is facilitated.By contrast,salt stress–induced SPR1degradation is attenuated in proteasome mutants,thus slowing down MT disassembly and causing salt stress hypersensitivity.Since the spr1-3mutant was not more tolerant to salt stress than the wild type,we can also conclude that this destabilization mechanism is strong and fast enough to suppress SPR1activity to a level where it does not interfere with the salt-induced MT disassembly process.Ac-cordingly,we propose that SPR1destabilization does not initiate the salt-induced MT disassembly,but its removal is required to allow the timely completion of this process.

The rapid MT depolymerization response to salt stress is thought to facilitate the formation of a new MT network that

allows

Figure9.Model Summarizing the Role of SPR1Proteolysis in the Salt Stress Tolerance of Wild-Type and Proteasome Mutant Cells.

SPR1,a(+)-end MAP,is degraded by the26S proteasome(26SP).In26S proteasome mutants,the degradation rate is reduced and the MTs are more stable due to SPR1accumulation.Upon salt stress perception,a still unknown mechanism leads to the increased proteasome-dependent degradation of SPR1that facilitates MT depolymerization.In proteasome mutants,salt stress–induced degradation of SPR1is reduced,which slows down MT depolymerization and causes salt stress hypersensitivity.This schematic is simpli?ed for the purpose of clarity:no other(+)-end MAPs but SPR1are 3422The Plant Cell

cells to better withstand the damaging impacts of high salt concentrations.Indeed,in plant cells exposed to prolonged salt stress,the initial massive MT depolymerization was followed by the formation of new MT networks(Wang et al.,2007).It has been established that salt stress leads to a reduction and reorientation of cell expansion and that these growth alterations are important for adapting to and surviving high salinity(Munns and Tester, 2008).As major determinants of the direction and rate of cell expansion,MT networks would indeed have to be rapidly reor-ganized to promote and facilitate such changes in growth.The new MT network has a more random MT organization compared with cells of unstressed plants(Wang et al.,2007).This corre-sponds well with the need for a reduced growth rate,as rapid cell elongation tends to require a transverse orientation of MTs to the direction of growth(Chan et al.,2011;Crowell et al.,2011). Because MT depolymerization is known to increase calcium channel activity(Thion et al.,1998),the initial MT disassembly response is also thought to be important for increasing the cytosolic calcium concentration,which is a major requirement for the adaptation to salt stress(Wang et al.,2007;Mahajan et al., 2008).The calcium burst was also shown to be essential for the formation of new MTs after prolonged salt stress exposure, suggesting that the initial MT depolymerization response not only allows the development of new MT networks but also establishes optimal conditions for MT synthesis(Wang et al.,2007).

While our study highlights the importance of proteasome-dependent proteolysis in the regulation of MT dynamics during salt stress,it also raises a number of questions.The?rst question relates to the stress-speci?c effects on MTs.We have shown that osmotic stress does not cause MT disassembly and does not promote SPR1destabilization.Other stresses,such as cold, heat,and treatments with nanoparticles(Smertenko et al.,1997; S.Wang et al.,2011),induce changes in plant MT networks,and future studies need to address whether this is also mediated via the stability control of MAPs.Whereas SPR1is currently the only known26S proteasome target among the plant MAPs,the partial suppression of26S proteasome mutant MT phenotypes by spr1-3suggests the existence of other proteins with SPR1-like activities that are also stabilized when proteasome function is impaired.Some obvious candidates to consider are the family of SPR1-like proteins,which were shown to have functions similar to SPR1(Nakajima et al.,2006),and the evolutionarily conserved, MT-stabilizing protein EB1,which is known to be targeted for proteasome-dependent proteolysis in human cells(Peth et al., 2007)and was reported to interact with SPR1potentially to regulate directional plant cell expansion(Kaloriti et al.,2007). The second question relates to the mechanism by which the perception of the salt stress signal leads to proteasome-dependent degradation of SPR1.Signal-induced site-speci?c phosphory-lation often initiates the ubiquitin-dependent targeting of a pro-tein to the26S proteasome(Chen et al.,1995;Matsuzaki et al., 2003;Smalle and Vierstra,2004).Phosphorylation is also a general regulation mechanism that reduces the binding af?nity of MAPs for MTs and thus leads to MT destabilization(Drewes et al.,1998;Matenia and Mandelkow,2009;Beck et al.,2010). On the other hand,it has been reported that a mitogen-activated protein kinase(MAPK)cascade plays a critical role in the salt salt stress–induced proteasome-dependent control of MT de-polymerization could involve a salt stress–activated MAPK cas-cade that leads to the phosphorylation of SPR1,followed by its interaction with a speci?c ubiquitin ligase and degradation by the 26S proteasome.This sequence of events would require that a salt stress–responsive MAPK localizes close to SPR1or is relocated to SPR1upon stress(i.e.,constitutively or conditionally associated with MTs).Recent studies in Arabidopsis identi?ed a number of MAPKs(MAK18and MAK4)involved in stress signal-ing that are associated with MTs or are involved in the regulation of MT dynamics(Walia et al.,2009;Beck et al.,2010).On the other hand,the SPR1protein contains nine putative phosphor-ylation sites(as predicted by NetPhos2.0),and one of them(Thr-76)is a part of MAPK consensus sequence PXS/TP or S/TP, suggesting that the potential for MAPK-dependent SPR1regu-lation is indeed an interesting topic for future research. METHODS

Plant Materials and Growth Conditions

Arabidopsis thaliana plants were grown on plates containing half-strength Murashige and Skoog medium with1%Suc(MS/2)as described previ-ously(Smalle et al.,2003).Plants were grown in a controlled environment chamber at228C with continuous light(140m mol photons m22s21).The proteasome mutants rpn1a-4,rpt2a-2,rpn10-1,and rpn12a-1(all in Col-0 background,and all carrying the kanamycin resistance gene)have been described(Kurepa et al.,2008;Wang et al.,2009).The spr1-3mutant and GFP-TUA6and GFP-TUB6overexpression lines(all in Col-0background) have also been described(Nakajima et al.,2004;Abe and Hashimoto, 2005).For the generation of double mutants,putative homozygous dou-ble mutants were selected based on their phenotypes,and their geno-types were con?rmed by immunoblotting analyses.

Treatments

Oryzalin and propyzamide were purchased from Sigma-Aldrich and MG132from Enzo Life Sciences.All drugs were made as10003stocks. MG132,CHX,and propyzamide were dissolved in DMSO and oryzalin in 100%ethanol.All control experiments included a13dose of the solvent. For all root elongation and root tip assays,plants grown on vertically positioned MS/2plates for5d were transferred to drug-or mock-supplemented media.Test plates were positioned vertically,and the root length was marked daily.After3d of treatments,plants were photo-graphed.Photomicrographs of representative root tips were taken with an Olympus SZX12microscope equipped with a DP12camera.For stress tolerance assays,4-or5-d-old seedlings were transferred to test plates with NaCl or mannitol and were grown vertically.For all morphometric analyses,the relevant parameter was measured from digital images using ImageJ(https://www.360docs.net/doc/1e10795561.html,/ij/).Unless speci?ed otherwise,data are presented as mean values,and the error bars represent standard devi-ation.Statistical signi?cance was determined by ANOVA tests followed by post hoc Bonferroni multiple comparison test.Post hoc statistical signi?cance is indicated in the?gures by asterisks or crosses.For all experiments,descriptive statistics,plotting,and the hypothesis testing were done using Prism5.0d software(GraphPad Software).

Confocal Microscopy Analysis of MTs and Scoring

MT distributions were analyzed in35S:GFP-TUA6and35S:GFP-TUB6

Proteasomal Degradation of SPR13423

Olympus Fluoview FV1000confocal laser scanning microscope equipped with an argon ion laser for the excitation of GFP(excitation488nm and barrier500to550nm)essentially as described(S.Wang et al.,2011).For all experiments,4-d-old seedlings were used,and epidermal cells of upper hypocotyl regions were analyzed.Except for the MT dynamics measurements,seedlings were mounted,incubated,and observed in water or an aqueous solution of the tested drugs.For the measurement of individual MT dynamics,seedlings were mounted in liquid MS/2medium, and time-lapse imaging was performed at1%laser power and4-s in-tervals for3to6min.Images were processed and analyzed using ImageJ as previously described(Buschmann and Lloyd,2008).The dynamics and the frequency of catastrophe and rescue were calculated as described (Dhonukshe and Gadella,2003;Abe and Hashimoto,2005).To quantify MT network density,MTs crossing the middle long axis of upper hypo-cotyl cells were counted.The results were expressed as MT number per unit distance as described previously(Ishida et al.,2007). Immunoblotting Analyses

For all analyses,plants were weighed and transferred to1.7-mL tubes for treatment.After treatment,plants were blotted,frozen in liquid N2,and ground in three volumes of23Laemmli sample buffer.Total proteins were separated by SDS-PAGE(14%acrylamide separating gel for SPR1 analyses,8%for RPN1analyses,and10%for all other analyses). Proteins were transferred to nitrocellulose membranes(Hybond C-Extra; GE)and probed as previously described(S.Wang et al.,2011).Antibodies against RPN1and SPR1were described(Nakajima et al.,2004;Wang et al.,2009).To test if the signal intensity for SPR1in all experiments falls within the linear dynamic range of the assay,immunoblots with a dilution series of total protein extracts were probed with anti-SPR1(dilution 1:1000),developed using chemiluminescent substrate(Thermo Scienti?c Pierce ECL Plus substrate)and analyzed using the linear best?t analyses (see Supplemental Figure8online).The anti-RPN10sera were obtained from Enzo Life Sciences.The anti-glutamine synthase(GS)serum was purchased from Agrisera AB.GS was assayed as a control.This is a stable protein,and its steady state levels are not affected by inhibition of proteasome activity(Kurepa et al.,2010).Horseradish peroxidase–conjugated and alkaline phosphatase–conjugated secondary antibodies were purchased from Santa Cruz Biotechnology.

Transcript Analyses

Total RNA was isolated using TRIzol reagent(Invitrogen).RNA was treated with TURBO DNase(Ambion),and the RNA concentrations were determined spectrophotometricaly(NanoDrop2000;Thermo Scienti?c). One microgram of RNA per sample was used for the synthesis of the?rst-strand cDNA(iScript reverse transcription supermix;Bio-Rad).

For the quanti?cation of SPR1transcripts,real-time RT-PCR was performed using the StepOne real-time PCR system(Applied Biosys-tems)and the DyNAmo Flash SYBR Green qPCR kit(Finnzymes)in total reaction volume of20m L.The SPR1primers used were59-AGCCTGCA-GAGCTTAACAAG-39and59-TGAACTTTGGTCGAAGGACG-39,and the primers for reference genes ACT2,ACT8,EF-1a,and GADPH were as described(Czechowski et al.,2005).Selection of the reference gene(s) best for the normalization in each experiment was calculated using geNorm(Vandesompele et al.,2002).

Analyses of Ubiquitin Conjugates and Proteasome Activity

Immunoblotting analysis of ubiquitin conjugates was done as described (Kurepa et al.,2008).The antipolyubiquitinated protein serum was from Enzo Life Sciences.For proteasome activity assays,samples were ground in1.25volumes of extraction buffer as described(Kurepa et al.,(Bio-Rad),and total proteasome activity was measured using the Suc-LLVY-AMC assay as described(Kurepa et al.,2008).

Generation of Transgenic Plants Overexpressing SPR1

To generate the overexpression construct,SPR1cDNA was ampli?ed using attB PCR primers attB1SPR159-GGGGACAAGTTTGTACAAAAA-AGCAGGCTTAATGGGTCGTGGAAACAGC-39and attB2SPR159-GGG-GACCACTTTGTACAAGAAAGCTGGGTCTTACTTGCCACCAGTGAAGA-39. The cDNA was introduced into pDONR221via BP reaction and then to pEarlyGate100(Earley et al.,2006)by LR recombinase(Invitrogen). Transgenic plants were selected on MS/2plates containing10m M L-phosphinothricin(Gold Biotechology),and homozygous T3plants were used for the analyses.

Accession Numbers

Sequence data from this article can be found in The Arabidopsis Information Resource(https://www.360docs.net/doc/1e10795561.html,/)under the following accession numbers:RPN1a(At2g20580),RPT2a(At4g29040),RPN10 (At4g38630),RPN12a(At1g64520),SPR1(At2g03680),TUA6(At4g14960), and TUB6(At5g12250).Arabidopsis T-DNA insertion mutants and trans-genic lines and their identi?cation numbers are SALK_027970(rpn1a-4), SALK_005596(rpt2a-2),35S:GFP-TUA6(CS6551),and35S:GFP-TUB6 (CS6550).

Supplemental Data

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

Supplemental Figure1.Visible Phenotypes and Propyzamide Re-sponses of the Double Mutant rpn1a-4rpn10-1.

Supplemental Figure2.The Steady State SPR1Transcript Levels in Proteasome Mutants Are the Same as in the Wild Type.

Supplemental Figure3.Right-Handed Helical Growth Is Enhanced in rpn1a-4spr1-3and rpn10-1spr1-3Mutants.

Supplemental Figure4.Propyzamide Treatment Reverts Ampli?ed Root Skewing in the rpn1a-4spr1-3Mutant.

Supplemental Figure5.Effects of NaCl Treatments on the SPR1 Transcript Level,SPR1Protein Abundance,Accumulation of Ubiq-uitinated Proteins,and Proteasome Activity.

Supplemental Figure6.Treatment with10m M Propyzamide Causes Depolymerization of Cortical MTs.

Supplemental Figure7.Overexpression of SPR1Increases MT Stability and Leads to Salt Stress Hypersensitivity.

Supplemental Figure8.Linear Relationship between SPR1Abun-dance and Signal Intensity on Immunoblots Developed Using Chemi-luminescent Substrate.

ACKNOWLEDGMENTS

This work was supported in part by the Kentucky Science and Engi-neering Foundation(Grant148-502-06-189)and the Kentucky Tobacco Research and Development Center.We thank the ABRC for providing seeds of the GFP-TUA6and GFP-TUB6transgenic lines.

AUTHOR CONTRIBUTIONS

The research was designed by S.W.under guidance from J.A.S.The

3424The Plant Cell

New analytical tools were contributed by T.H.The data were analyzed and the article was written by S.W.,J.K.,and J.A.S.

Received August1,2011;revised August30,2011;accepted September 12,2011;published September27,2011.

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Supplemental Figure 1. Visible Phenotypes and Propyzamide Responses of the Double Mutant rpn1a-4 rpn10-1.

(A) Rosette phenotypes of in vitro-grown three-week-old Col-0, rpn1a-4, rpn10-1 and rpn1a-4 rpn10-1 plants. Rosettes of the single proteasome mutants are smaller than those of the the wild type, and this phenotype is further enhanced in the double mutant. Arrowheads point to the first leaves in the double mutant that accumulate more anthocyanins than single mutants or the wild type. The immunoblot showing the absence of both RPN1a and RPN10 subunits in the double mutant is presented in Figure 3B.

(B) The effect of propyzamide on primary root elongation was measured after three days of treatment. Root elongation is shown in relative values due to the large differences in root lengths of the untreated controls. For all lines, the average root length of the untreated control was assigned the value of 100%, and mean values ± SD (n≥25) are shown as percentages of the respective controls. The double mutant had an increased propyzamide tolerance compared to single mutants (p < 0.0001, Bonferroni multiple comparisons post-test), and its root length was significantly increased compared to the untreated double mutant plants (p < 0.001). Statistical significances of the root length of the mutants vs. Col-0 are shown on the graph (*, p < 0.05; **, p < 0.01; ***, p < 0.001; ****, p < 0.0001).

(C) Effect of propyzamide (propyz.) on the diameter of primary roots. Roots were photographed after three days of treatment. Bar = 250 μm.

S P R 1 l e v e l s

Col-0 rpn1a-4

rpn10-1 1.2

1.0 0.8 0.6 0.4 0.2 0

Supplemental Figure 2. The Steady-State SPR1 Transcript Levels in Proteasome Mutants are the Same as in the Wild Type.

Real-time RT-PCR analyses of total RNA isolated from 8-day-old seedlings. Relative SPR1 mRNA levels are expressed as a ratio of SPR1

expression normalized against expression of ACT2

(At3g18780). Test was done in triplicate.

Supplemental Figure 3. Right-Handed Helical Growth is Enhanced in rpn1a-4 spr1-3 and rpn10-1 spr1-3 Mutants.

(A) To confirm that spr1-3 rpn1a-4 and spr1-3 rpn10-1 are homozygous for both mutations, total protein extracts from 10-day-old seedlings were used for immunoblotting analyses with anti-RPN1, anti-RPN10 and anti-SPR1 sera. Immunoblot showing GS isoforms is added as a loading control.

(B) Increased epidermal cell file skewing and increased diameter of etiolated hypocotyls in double mutants. All lines were grown on MS/2 plates in the dark for five days. Bar = 1 mm.

(C) Root phenotypes of ten-day-old Col-0, rpn10-1, spr1-3, rpn10-1 spr1-3 and rpn1a-4 spr1-3 seedlings. Seedlings were grown on vertically positioned plates.

(D) The epidermal cell files in the root elongation zone of plants shown in (C).

(E) Right-handed skewing of true leaves in plants grown on soil for three weeks.

Supplemental Figure 4.Propyzamide Treatment Reverts Amplified Root Skewing in the rpn1a-4 spr1-3 Mutant.

Seedlings were grown for five days on MS/2 plates, and than transferred to fresh MS/2 media or media containing 2 μM propyzamide. The root length was then marked. The photographs were taken after three days of treatment.