Salt Stress–Induced Disassembly of Arabidopsis CorticalMicrotubule Arrays Involves 26S Proteasome–DependentDegradation of SPIRAL1 C W

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

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

Lexington, Kentucky 40546bGraduate School of Biological Sciences, Nara Institute of Science and Technology, Ikoma, Nara 630-0192, 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 the 26S proteasome, and its

degradation rate is accelerated in response to high salinity. We show that accelerated SPR1 degradation 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 themajority of UPS substrates, the concerted

action of E1, E2, and E3 enzymes 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 the 26S 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 performedbyRPnon-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 the 26S 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 rpn10 mutant

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-specific chymotrypsin-like protease b5 (Lee and

Goldberg, 1998). Studies using MG132 have 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

thata-tubulin is exposed at the so-called lagging (2) andb-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

proteins (MAPs) and, in particular, a subclass of MAPs called

1Address correspondence to jsmalle@uky.edu.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Jan A. Smalle(jsmalle@uky.edu).CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.111.089920

The Plant Cell, Vol. 23: 3412–3427, September 2011, www.plantcell.org ã 2011 American 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 havebeen identified, and someof them

are found inwidelydivergedspecies, 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 protein 1 (EB1) and

cytoplasmic linker-associated protein 1 (CLASP1), which were

first identified in human cells (Chan et al., 2003;Mathur et al., 2003;

Galjart, 2005; Vaughan, 2005; Ambrose et al., 2007; Kirik et al.,

2007; Bisgroveet al., 2008;Komaki et al., 2010).Other +TIPs, such

asSPIRAL1 (SPR1), are found only in plants (Nakajima et al., 2004,

2006; Sedbrook et al., 2004). Because plants overexpressing

SPR1 have increased tolerance to MT-destabilizing drugs, and

spr1 mutants 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 ismodulated 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-2 cells from a structured to a

seemingly random organization (Blancaflor 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 spr1 mutants (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 aMT-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 specific 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 tolerancemight 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 allowMT restructuring required for an optimal salt

stress response. Here, we show that in Arabidopsis, salt stress

accelerates the 26S proteasome–dependent degradation of

SPR1 and that this facilitates MT disassembly and promotes

salt stress tolerance.

Figure 1. 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/2 medium for

5 d were transferred to media containing the denoted doses of propy-

zamide or oryzalin. The increase in root length was measured after 3 d of

treatment. The root length of untreated seedlings of each genotype was

set at 100%, andmean values6 SD (n$ 25) are shown as percentages of

the root length of the respective controls. The difference in root length

between Col-0 and rpn1a-4, rpn10-1, or rpn12a-1 was statistically sig-

nificant 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 significance for Col-0 versus rpt2a-2 is marked in the graphs

(*P < 0.05).

(C) Effects of 4 mM propyzamide on primary root morphogenesis.

The experimental conditions and timeline were the same as in (A).

Bar = 250 mm.

Proteasomal Degradation of SPR1 3413

RESULTS

26S ProteasomeMutants Have Increased Tolerance to

MT-Destabilizing Drugs

To assay the stability of cortical MTs in 26S proteasomemutants,

we first tested the responses of rpn1a-4, rpt2a-2, rpn10-1, and

rpn12a-1 mutants 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 total 26S proteasome activity in the four

tested proteasome mutants is affected to different levels, with

rpt2a-2 carrying the weakest and rpn10-1 the 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 byMT

disassembly (Baskin et al., 1994).

All proteasome mutants were more tolerant to both propyza-

mide and oryzalin (Figures 1A and 1B). The strongest mutant,

rpn10-1, was the most tolerant, whereas the weakest mutant,

rpt2a-2, showed a statistically significant increase in propyza-

mide and oryzalin tolerance only at a single dose (6 mM for

propyzamide, P < 0.05; 100 nM for oryzalin, P < 0.05). In addi-

tion, the propyzamide tolerance of the double mutant rpn1a-4

rpn10-1 was further increased compared with the respective

single mutants (see Supplemental Figure 1 online).

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 a 3-d-long treatment with

4 mMpropyzamide, root swelling was obvious in the Columbia-0

(Col-0) plants, whereas the diameter of primary roots of all

proteasome mutants did not increase (Figure 1C; see Supple-

mental Figure 1C online).

Changes in rpn10-1MT Dynamics

To analyze the stability ofMTs in proteasomemutants further, we

compared the dynamics of individual MTs in Col-0 and rpn10-1

backgrounds (Figure 2). Previous studies have shown that green

fluorescent protein (GFP) fusions with a- (GFP-TUA6) or b-tubulin

(GFP-TUB6) are incorporated into MTs and can be used to

visualizeMT 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-TUA6 changes MT dynamics and induces right-handed

helical growth (Nakamura et al., 2004; Abe and Hashimoto,

2005). Thus, we chose to introduce the 35S:GFP-TUB6 trans-

gene into the rpn10-1 mutant 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

(Figure 2). Indeed, most of the parameters of MT dynamic

instability were altered in the rpn10-1 background (Table 1).

The average growth rate of the leading ends and the frequency of

catastrophe in rpn10-1 were significantly lower than in Col-0,

while the frequency of rescue events in rpn10-1 was increased.

Thus, individual MTs in rpn10-1 were less dynamic and more

prone to polymerization. In contrast with the (+)-end, there was

no significant difference in (2)-end dynamics between rpn10-1

and the wild type (Table 1), 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 that 26S proteasome–dependent

proteolysis plays an important role in the regulation of MT dy-

namic instability.

SPR1 Mediates the Tolerance of Proteasome Mutants

to Propyzamide

Previous studies showed that overexpression of the plant-

specific +TIP SPR1 leads to increased propyzamide tolerance

Figure 2. MT Plus-End Dynamics Are Altered in the rpn10-1 Mutant.

(A) Kymograph of a 160-s-long recording showing (+)-end dynamics of

individual MTs. Four-day-old seedlings expressing 35S:GFP-TUB6 in the

Col-0 and rpn10-1 backgrounds were used for time-lapse confocal

imaging. Epidermal cells of upper hypocotyl regions were photographed

every 4 s. Bar = 5 mm.

(B) The life history plots (length versus time) of three GFP-TUB6–labeled

MTs in Col-0 and three GFP-TUB6–labeled MTs in the rpn10-1 back-

ground. MT length was measured from confocal micrographs using

Image J.

[See online article for color version of this figure.]

3414 The Plant Cell

(Nakajima et al., 2004). Since proteasomemutants are also more

tolerant of MT-destabilizing drugs (Figure 1) and have alteredMT

dynamics, (Figure 2), we tested whether SPR1 is a 26S protea-

some target and whether its stabilization in proteasome mutants

can explain the observed phenotypes.

To assay SPR1 stability, we used cycloheximide (CHX) to

inhibit de novo protein synthesis. After a 12-h-long CHX treat-

ment, the SPR1 level was reduced to;30% of the control. This

decrease was blocked by the proteasome inhibitor MG132,

suggesting that SPR1 is an unstable protein that is targeted for

26S proteasome–dependent proteolysis (Figure 3A). Indeed, the

SPR1 level was increased in 26S proteasome mutants rpn1a-4

and rpn10-1 and was even more abundant in the rpn1a-4

rpn10-1 doublemutant (Figure 3B). The SPR1mRNA abundance

remained unchanged in the proteasome mutant backgrounds,

confirming that the increase in SPR1 protein level was caused by

a posttranscriptional mechanism (see Supplemental Figure 2

online). Finally, CHX chase immunoblotting analyses (Yewdell

et al., 2011) showed that the SPR1 degradation rate was de-

creased in proteasome mutant backgrounds (Figures 3C and

3D), confirming a role for the UPS in regulating SPR1 abundance.

To test if SPR1 accumulation is a cause for the increased

tolerance of proteasome mutants to MT-destabilizing drugs, we

crossed the spr1-3mutation into the rpn1a-4 and rpn10-1mutant

backgrounds (Figure 4; see Supplemental Figure 3 online).

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 spr1-3 mutation

(Figure 4A). To quantify the effect of the spr1-3 mutation, we

measured the width of the primary root in the elongation zone

(Figure 4B). As expected, the propyzamide treatment did not

promote any substantial root swelling in the proteasome mu-

tants. The spr1-3 mutation caused a mild increase in root width

compared with the wild type (344.56 5mmand 372.96 7mm for

Col-0 and spr1-3, respectively; n = 25, P < 0.001). By contrast,

the root width of both rpn1a-4 spr1-3 and rpn10-1 spr1-3 double

mutants was strongly increased compared with the respective

single proteasome mutants (n = 25, P < 0.001), although the

increases in swelling did not reach thewild-type level (P < 0.01 for

rpn1a-4 spr1-3 and P < 0.001 for rpn10-1 spr1-3 compared with

Col-0). We concluded that the removal of SPR1 suppressed 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 the 26S proteasome.

The root elongation assay confirmed that SPR1 contributes to

the propyzamide tolerance of proteasome mutants (Figure 4C).

The rpn1a-4 spr1-3 and rpn10-1 spr1-3 double mutants dis-

played an increase in right-handed helical growth (see Sup-

plemental Figure 3 online). For example, the right-handed root

skewing of spr1-3 was enhanced in both the rpn1a-4 and

rpn10-1 mutant backgrounds. This was the most striking in

rpn1a-4 spr1-3 seedlings that had upward growing roots (see

Supplemental Figure 3C online). However, low propyzamide

doses fully suppressed the enhanced root skewing phenotype in

both double mutants (see Supplemental Figure 4 online), 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 spr1-3 sup-

presses the propyzamide tolerance of both the rpn1a-4 and

rpn10-1 mutants (Figure 4C).

To analyze directly the effects of spr1-3 on MT stability, we

attempted to cross the 35S:GFP-TUB6 transgene into the spr1-3

and rpn10-1 spr1-3 mutants. We were unable to isolate spr1-3

or rpn10-1 spr1-3 mutants expressing GFP-TUB6 in spite of

screening more than 1000 F2 seedlings, and we hypothesized

that the 35S:GFP-TUB6 transgene is located in the proximity of

the SPR1 locus. However, analysis of the 35S:GFP-TUB6 locus

revealed that the T-DNA is not linked to SPR1 (data not shown).

The reason GFP-TUB6 expression and the spr1-3 mutation are

incompatible is currently unknown. As an alternative, we intro-

duced the 35S:GFP-TUA6 transgene into the spr1-3 and rpn10-1

spr1-3 mutants. To assay the MT stability in these lines, we

treated 4-d-old seedlings with a high dose of propyzamide to

ensure a fast and complete MT disruption (20 mM propyzamide

for 1 h) and analyzed epidermal cells of the upper hypocotyl

regions by confocalmicroscopy (Figure 4D). In rpn10-1, theGFP-

TUA6–labeled cortical MTs remained partially intact, while MTs

in the wild-type and rpn10-1 spr1-3 cells were disrupted, and the

GFP signal was localized in randomly dispersed aggregates.

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

Dynamic Parameters

(+)-Ends (�)-Ends

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

Growth rate (mm/min) 5.44 6 4.61 4.04 6 2.73* 1.91 6 1.15 1.72 6 1.03

Shrinkage rate (mm/min) 10.3 6 8.89 8.91 6 8.44 3.53 6 4.16 3.82 6 4.17

Catastrophe (events/second) 0.041 0.030* 0.212 0.196

Rescue (events/second) 0.096 0.141* 0.047 0.051

Time spent on growth 67.2% 76.6% 9.3% 10.7%

Time spent on pause 9.6% 11.2% 48.4% 48.7%

Time spent on shrinkage 20.4% 12.3% 42.4% 40.6%

Dynamicity (mm/min) 5.91 6 1.46 4.19 6 2.41* 1.67 6 1.92 1.99 6 1.28

MT dynamic instability parameters were quantified from confocal micrographs. Velocities were calculated from 38 leading and 22 lagging ends for

35S:GFP-TUB6 in Col-0 and 45 leading and 32 lagging ends for 35S:GFP-TUB6 in the rpn10-1 background. Total measurements include 1111 and

1763 velocities (4-s intervals) for GFP-TUB6 in Col-0 and rpn10-1, respectively. Dynamic parameters are expressed as mean 6 SD. Statistical

significance was calculated using Student’s t test comparing the mutant and wild-type values (*P < 0.05).

Proteasomal Degradation of SPR1 3415

Collectively, these experiments suggested that the stabilization

of SPR1 in 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 of 26S

proteasome mutants (Smalle et al., 2003; Wang et al., 2009) and

the fact that salt stress tolerance requires MT disassembly

(Wang et al., 2007), we hypothesized that loss of proteasome

function increases salt stress sensitivity by stabilizing SPR1,

which in turn leads to increased MT stability. To test this, we first

determined if salt stress leads to a conditional 26S proteasome–

dependent degradation of SPR1, which could facilitate the re-

arrangement of cortical MTs needed for salt tolerance.

Prolonged treatment (16 h) of 4-d-old Col-0 seedlings with 150

mM NaCl caused a decrease in SPR1 level, and this decrease

was blocked by MG132 (Figure 5A). CHX-chase immunoblotting

analyses showed that a 16-h-long NaCl treatment led to an

;80% reduction of the SPR1 level in Col-0, whereas the SPR1

level in rpn1a-4 and rpn10-1 mutants was reduced by only;30

and ;20%, respectively (Figures 5B and 5C). These results

indicated that the salt stress–induced decrease in SPR1 level

was a result of proteasome-dependent degradation.

During treatmentswith a higher concentration of salt (200mM),

degradation of SPR1 was detectable already after 3 h of incu-

bation (see Supplemental Figures 5A to 5C online). To test if high

salt concentrations promote SPR1 degradation via a global

increase in 26S 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 Figures 5D and 5E online).

After 5 h of exposure to 200 mM NaCl, we observed a significant

decrease in the SPR1protein level, no change in the overall levels

of ubiquitinated proteins, and a mild but significant decrease

in proteasome activity. We concluded that the accelerated

proteasome-dependent SPR1 degradation during salt stress is

not caused by a general increase in proteasome activity but is

more likely the result of a specific destabilizationmechanism that

is activated by salt stress. Finally, we confirmed that the SPR1

depletion in response to salt is indeed the result of a posttrans-

criptional mechanism by observing that the SPR1 mRNA level

did not change in response to salt stress treatments (see Sup-

plemental Figure 5F 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

SPR1 reflects 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 SPR1 that could be more

susceptible to degradation than the tubulin-bound version. To

distinguish between these possibilities, we monitored the SPR1

level in Col-0 plants treatedwith propyzamide. In Col-0 seedlings

treated for 3 h with 10 mM propyzamide, the cortical MTs were

depolymerized (see Supplemental Figure 6 online), but the SPR1

level remained the same as in the untreated control (Figure 5D).

The propyzamide treatment also did not influence the salt stress–

induced degradation of SPR1 (Figure 5D).

In addition to ionic stress, high salinity is also known to cause

osmotic stress (Zhu, 2001). To test if osmotic stress influences

the stability of SPR1, we treated Col-0 seedlings with high doses

of the osmolytemannitol (Figure 5E). Themannitol treatments did

not affect SPR1 stability, suggesting that the signal triggering

SPR1 destabilization was specifically ionic stress (Figure 5F). We

concluded that salt stress, but not osmotic stress or MT disas-

sembly per se, promotes the 26S proteasome–dependent deg-

radation of SPR1.

Figure 3. SPR1 Is a 26S Proteasome Target.

(A) Immunoblotting analyses using anti-SPR1 and anti-GS antisera.

Seven-day-old seedlings were treated with 100 mM MG132 and/or 200

mM CHX for 16 h and used for the extraction of total protein. The anti-GS

sera recognizes both the chloroplastic (45 kD) and cytosolic (40 kD) GS

isoforms.

(B) SPR1 levels in 8-d-old Col-0, rpn1a-4, rpn10-1, and rpn1a-4 rpn10-1

seedlings. Anti-RPN1 and anti-RPN10 sera were used to confirm the

genotype of proteasome mutants.

(C) Representative CHX-chase immunoblots. The stability of SPR1 was

tested on total protein extract of 10-d-old wild-type and mutant seed-

lings treated with 200 mM CHX for the indicated time periods.

(D) Quantification of SPR1 stability in CHX-treated Col-0, rpn1a-4, and

rpn10-1 plants. 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 to 100%, and the mean

values 6 SD (n = 3) are shown as percentages of the respective control.

The asterisks represent the statistical significance of the difference be-

tween degradation rates in Col-0 and both proteasome mutants (****P <

0.0001; ANOVA followed by Bonferroni multiple comparisons post-test).

[See online article for color version of this figure.]

3416 The Plant Cell

SPR1 Stabilization Causes Salt Stress Hypersensitivity

To test if SPR1 stabilization causes the salt hypersensitivity of 26S

proteasome mutants (Smalle et al., 2003; Wang et al., 2009), we

first analyzed if the increased MT stability is responsible for the

changes in salt tolerance (Figure 6A), then determined the salt

tolerance levels of rpn1a-4 spr1-3 and rpn10-1 spr1-3 mutants

(Figures 6B to 6D), and finally compared the effects of salt stress

on the MT arrays of rpn10-1 and rpn10-1 spr1-3 cells (Figure 7).

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-4 and rpn10-1mutants

(Figure 6A). In the absence of propyzamide, root length of wild-

type plants grown on 100 mM NaCl was ;70% of the control,

whereas the root length of proteasome mutants was reduced to

;50%. As expected, lowdoses of propyzamide counteracted the

salt-induced inhibition of root elongation in all lines. However,

whereas 1mMpropyzamide led to an 8% increase in root length in

the wild type (statistically nonsignificant, P > 0.05, n$ 15; analysis

of variance [ANOVA]withBonferroni post-test), the increase in root

length in the proteasomemutants was;25% (P < 0.001, n$ 15).

Thus, since lowdosesof anMT-destabilizingdrug 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-4 and rpn10-1

mutants (Figure 4), prompted us to test if the spr1-3mutation also

Figure 4. spr1-3 Suppresses the Propyzamide Tolerance of Proteasome Mutants.

(A) Effects of 4 mM propyzamide on primary root morphogenesis in Col-0, rpn1a-4, spr1-3, and the double mutant rpn1a-4 spr1-3. Five-day-old

seedlings were transferred to fresh MS/2 medium or MS/2 medium with propyzamide. The seedlings were grown on vertically positioned plates for 3 d

and then photographed. Bar = 1 mm.

(B) Effects of 4 mM 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 significance was analyzed using ANOVA followed by Bonferroni multiple comparisons post-test.

Crosses represent the significance of the differences between the untreated Col-0 and untreated mutant lines, and asterisks mark the significance

between the treated Col-0 and treated mutant lines (**P < 0.01; ††† and ***P < 0.001; and †††† and ****P < 0.0001).

(C) Root lengths of 5-d-old seedlings grown on MS/2 media with 4 mM propyzamide (****P < 0.0001; significance for Col-0 versus mutants). The

statistical analyses and data presentation are as in (B).

(D) In vivo analyses of the MT-destabilizing effect of 20 mMpropyzamide in the Col-0 wild type and in the rpn10-1 and rpn10-1 spr1-3mutants. The 35S:

GFP-TUA6 transgene was crossed into the rpn10-1 and rpn10-1 spr1-3 mutant backgrounds, and the GFP-TUA6–labeled cortical MTs were analyzed

by confocal microscopy. Hypocotyl epidermal cells of 4-d-old seedlings treated for 1 h are shown. Bar = 20 mm.

[See online article for color version of this figure.]

Proteasomal Degradation of SPR1 3417

suppresses the salt hypersensitivity of proteasome mutants

(Figures 6B and 6D). Salt stress induces a range ofmorphological

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 rpn10-1 compared with

the wild type and were suppressed by the spr1-3 mutation

(Figure 6B). The fresh weight of rpn1a-4 spr1-3 and rpn10-1

spr1-3 plants grown for 2 weeks on salt-containing media was

significantly increased compared with the single rpn1a-4 and

rpn10-1 mutants but did not reach the wild-type levels (Figure

6C). The effects of NaCl on root elongation were measured after

3 d of treatment (Figure 6D). On the control media, spr1-3 sup-

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-4

spr1-3 and rpn10-1 spr1-3 plants 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 spr1-3 mutation 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 rpn10-1 spr1-3 plants (Figure 7). In the wild type,

MT disassembly was nearly complete after a 4-h-long exposure

to 200mMNaCl (Figure 7A). By contrast, the cortical MT network

in rpn10-1 cells remained largely intact up until 6 h into the

treatment. The spr1-3 mutation suppressed the delayed MT

depolymerization in the rpn10-1 mutant, but similarly to the

effects of spr1-3 on whole-seedling salt tolerance, it did not fully

revert the MT disassembly rate back to the wild-type level.

Analyses of the number ofMTs per unit length confirmed both the

delayed salt response in rpn10-1 and the partial suppression of

this delay by the spr1-3mutation (Figure 7B). We concluded that

the SPR1 stabilization 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 spr1-3 mutation was introduced, we also con-

cluded that SPR1 is 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 SPR1 in Col-0 seedlings treated with NaCl and MG132. Four-day-old seedlings were treated for 16 h with 150 mM NaCl

alone or 150 mM NaCl and 100 mM MG132.

(B) CHX-chase immunoblot. Ten-day-old seedlings were transferred to liquid MS/2 media containing 200 mM CHX and 200 mM NaCl. Seedlings were

treated for the indicated times.

(C) Quantification of immunoblots represented by those in (B). The signals of the untreated samples were set at 100%. Data are shown as mean values

6 SD (n = 3) as percentages of the respective controls. The asterisks represent the statistical significance of the difference between the degradation

rates in Col-0 versus rpn10-1 and rpn1a-4 (****P < 0.0001; ANOVA followed by Bonferroni multiple comparisons post-test).

(D) and (E) Immunoblotting analyses of SPR1 in Col-0 seedlings 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 3 h, and used for the isolation of total proteins.

[See online article for color version of this figure.]

3418 The Plant Cell

The fast, salt-induced MT depolymerization response in cells

of the wild type and rpn10-1 spr1-3 double mutant was followed

by the start of new MT formation (8-h time point; Figure 7). By

contrast, after 8 h of treatment, the MT disassembly was just

completed and no distinct MTs could be detected in the rpn10-1

seedlings. This confirms 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 SPR1 abundance is a cause for

salt stress hypersensitivity was obtained by analyzing transgenic

plants that overexpress SPR1. In theory, plants that overexpress

SPR1 should respond to salt stress similarly to proteasome

mutants in which the SPR1 level is increased because of a

reduced degradation rate. Previous work already showed that

SPR1 overexpression 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 Figure 7

online). Collectively, these results confirm the importance of

SPR1 removal for maintaining salt stress tolerance.

MG132 Promotes MT Stability and Salt

Stress Hypersensitivity

Finally, to independently test the role of SPR1 proteolysis in the

salt stress–induced restructuring of cortical MT arrays, we ana-

lyzed the combined effects of salt, propyzamide, and MG132 on

Col-0 and spr1-3 seedlings (Figure 8).

Similar to the genetic suppression of proteasome activity,

pretreatment of Col-0 seedlings with MG132 inhibited the

propyzamide-induced swelling of root tips (Figure 8A). Although

MG132 also inhibited the swelling of spr1-3 roots, this effect

was not as strong as in the wild type (Figure 8A). MG132 also

counteracted the inhibitory effect of propyzamide on root elon-

gation (Figure 8B). Treatment with either MG132 or propyzamide

inhibited root elongation in Col-0 and spr1-3 seedlings. However,

while MG132 reduced 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 MG132 and 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 MG132 effect was also

Figure 6. spr1-3 Suppresses the Salt Hypersensitivity of 26S Proteasome Mutants.

(A) Salt hypersensitivity of proteasome mutants was suppressed by propyzamide treatments. Five-day-old seedlings grown on MS/2 medium were

transferred to fresh MS/2 plates or MS/2 plates with 100 mM NaCl and the indicated doses of propyzamide. Plates were positioned vertically, and the

root lengths were measured after 3 d. Data represent relative root length (n$ 20) with SD. The average root length of untreated Col-0 seedlings was set

at 100%.

(B) Five-day-old seedlings grown on MS/2 medium were transferred to fresh MS/2 medium or MS/2 containing 100 mM NaCl. Test plates were

positioned vertically, and plants were grown for 2 weeks. The arrows highlight necrotic spots in the rpn10-1 mutant grown on 100 mM NaCl.

(C) and (D) The salt tolerance levels were quantified by comparing the fresh weight after a 2-week-long treatment (C) and root lengths after a 3-d-long

treatment (D). Data are shown as mean values6 SD of three independent experiments (n$ 15 for each). The asterisks represent the significances of the

differences between the double mutants and the respective single proteasome mutants and were calculated using ANOVA followed by Bonferroni

multiple comparisons post-test (****P < 0.0001, ***P < 0.001, and **P < 0.01).

Proteasomal Degradation of SPR1 3419

partially attenuated in the spr1-3mutant, implying a role for SPR1

in the MG132-induced MT stabilization (Figure 8B).

To test if MG132 also increases salt sensitivity, we analyzed

the response of GFP-TUB6–labeled MT arrays and the growth of

Col-0 and spr1-3 on medium supplemented with 200 mM NaCl

(Figures 8C to 8E). Indeed, MG132 suppressed the salt-induced

depolymerization of cortical MTs (Figure 8C) and decreased the

salt tolerance of wild-type seedlings (Figures 8D and 8E). How-

ever, compared with the wild type, MG132 was less effective in

suppressing leaf expansion (Figure 8D) and root elongation of

salt-treated spr1-3 seedlings (Figure 8E), confirming that the salt

hypersensitivity caused by proteasome inhibition requires SPR1

function. Thus, we find 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 bothMT dynamics determinants. For example, the role of

the proteasome and tubulin-specific 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

unidentified (S. Wang et al., 2011).

In animals, the proteasome has also been shown to regulate

MT dynamics by influencing 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 fine-tuning

of MT dynamics. We show that genetic and pharmacological

inactivation of 26S 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-specific +TIP SPR1. Thus, the accumulation of SPR1

resulting from either overexpression of the SPR1 transgene

(Nakajima et al., 2004) or a reduced degradation rate (this study)

is sufficient to promote increased MT stability in plant cells.

Although the increased stability of SPR1 in 26S proteasome

mutants implies that this MAP is targeted for degradation in

a ubiquitin-dependent manner, we were unable to detect any

candidate ubiquitinated SPR1 forms 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 given 26S 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

SPR1 forms remain undetectable due to the actions of ubiquitin

proteases that are known to revert a substantial fraction of

Figure 7. SPR1 Stabilization in rpn10-1 Delays the Salt-Induced Disas-

sembly of Cortical MT Arrays.

(A) Visualization of GFP-TUA6–labeled cortical MTs in upper hypocotyl

epidermal cells from 4-d-old Col-0, rpn10-1, and rpn10-1 spr1-3 seed-

lings treated with 200 mM NaCl for the indicated times. Bar = 10 mm.

(B) The density of cortical MTs per unit length in upper hypocotyl cells was

determined by counting theMTs crossing the longitudinal axis of a cell. For

each line and treatment, a minimum of 32 hypocotyl cells from five

separate seedlings was photographed and used for measurements. The

asterisks represent the significance of the difference between Col-0 and

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 figure.]

3420 The Plant Cell

ubiquitinated proteins back to their unmodified forms (Smalle

and Vierstra, 2004; Kaiser and Tagwerker, 2005).

While our data show that SPR1 stabilization 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 reflect an overall increase in

MT stability in rpn10-1 cells are in agreement with the predicted

effects of increased SPR1 action, the slower growth of MTs

(Table 1) cannot be explained by an increase in SPR1 activity 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 spr1-3 mutation 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 SPR1 could also explain the

observation that the right skewing of spr1-3 roots was enhanced

in the rpn10-1 spr1-3 and rpn1a-4 spr1-3 double mutants (see

Supplemental Figure 3 online). The spr1 mutants 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 spr1-3 partially reversed the increased MT

stability of proteasome mutants (Figure 4), we expected that

the right skewing of spr1-3 roots would also be reversed.

However, since the right skewing was enhanced in the double

mutants, we propose that this phenotype reflects 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 SPR1

degradation rate suggested that the activity of this protein is

controlled posttranslationally and implied the existence of de-

velopmental or environmental cues that influence MT dynamics

by regulating SPR1 stability. Indeed, we found that SPR1 pro-

teolysis is enhanced under salt stress conditions (Figure 5) and

that SPR1 stabilization leads to salt stress hypersensitivity in 26S

proteasomemutants (Figure 6). Furthermore, we showed that the

increased salt stress sensitivity of proteasomemutants is caused

by a slower rate of MT disassembly, which is a direct result of the

SPR1 stabilization. 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. MG132 Stabilizes MTs and Increases the Sensitivity to Salt Stress.

(A) and (B) Four-day-old Col-0 and spr1-3 seedlings were transferred to MS/2 medium containing 4 mM propyzamide, 40 mM MG132, or 4 mM

propyzamide and 40 mM MG132. Plants were grown vertically for three days before the root tips were photographed ([A]; bar = 0.5 mm), and the root

lengths were measured (B). In (B), data represent an average root length with SD (n = 26), and the asterisks represent the significances 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 100 mMMG132 or

DMSO in liquid MS/2 medium for 6 h, and then 20 mM propyzamide or 200 mM NaCl was added. Propyzamide treatments were done for 1 h and the

NaCl treatments for 3 h prior to microscopy. Bars = 10 mm.

(D) and (E) Four-day-old Col-0 and spr1-3 seedlings were transferred to MS/2 medium containing 100 mM NaCl, 50 mM MG132, or both. Seedlings

were photographed, and the root lengths were measured after 3 d of treatment. In (E), data represent an average root length with SD (n $ 25), and the

asterisks represent the significances of the differences between the wild type and spr1-3 (****P < 0.0001, ***P < 0.001, and **P < 0.01; ANOVA followed

by Bonferroni multiple comparisons post-test).

Proteasomal Degradation of SPR1 3421

Arabidopsis 26S proteasomemutants are also characterized by

a decrease in growth rate that reflects a reduced rate of mitosis

(Kurepa et al., 2009b). SinceMTdepolymerization is also known to

inhibit mitosis, an alternative explanation for the salt stress hyper-

sensitivity of 26S proteasome mutants is that the reduced mitotic

rates make themmore susceptible to the growth inhibitory effects

of salt stress. However, this explanation is unlikely because the

spr1-3 mutation suppressed the salt hypersensitivity but not the

reduced growth rate of proteasome mutants (Figure 6). Further-

more, the SPR1overexpression lineswere also salt hypersensitive

but did not display any decrease in growth rate (see Supplemental

Figure 7 online), thus confirming that the increased SPR1 abun-

dance in 26S proteasomemutants is indeed the most likely cause

for their salt stress hypersensitivity.

Collectively, our data reveal an important role for proteasome-

dependent regulation of SPR1 in the survival of plants challenged

by high salinity. Figure 9 outlines a model that summarizes the

role of SPR1 in the MT disassembly response to salt stress.

According to this model, SPR1 is 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

SPR1 is accelerated, and the MT depolymerization needed for

the survival of plant cells under salt stress is facilitated. By

contrast, salt stress–induced SPR1 degradation 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 SPR1 activity to a level where it does not

interfere with the salt-induced MT disassembly process. Ac-

cordingly, we propose that SPR1 destabilization 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 newMT network that allows

Figure 9. Model Summarizing the Role of SPR1 Proteolysis in the Salt Stress Tolerance of Wild-Type and Proteasome Mutant Cells.

SPR1, a (+)-end MAP, is degraded by the 26S proteasome (26SP). In 26S proteasome mutants, the degradation rate is reduced and the MTs are more

stable due to SPR1 accumulation. Upon salt stress perception, a still unknown mechanism leads to the increased proteasome-dependent degradation

of SPR1 that facilitates MT depolymerization. In proteasome mutants, salt stress–induced degradation of SPR1 is reduced, which slows down MT

depolymerization and causes salt stress hypersensitivity. This schematic is simplified for the purpose of clarity: no other (+)-end MAPs but SPR1 are

depicted, and all (�)-end components are omitted.

3422 The 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 newMT 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 calciumconcentration,which is amajor 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 newMTnetworks 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 first question

relates to the stress-specific effects on MTs. We have shown

that osmotic stress does not cause MT disassembly and does

not promote SPR1 destabilization. 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 SPR1 is currently the only

known 26S proteasome target among the plant MAPs, the partial

suppression of 26S proteasome mutant MT phenotypes by

spr1-3 suggests 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 SPR1 potentially to

regulate directional plant cell expansion (Kaloriti et al., 2007).

The second question relates to the mechanism by which the

perceptionof the salt stresssignal leads toproteasome-dependent

degradation of SPR1. Signal-induced site-specific phosphory-

lation often initiates the ubiquitin-dependent targeting of a pro-

tein to the 26S proteasome (Chen et al., 1995; Matsuzaki et al.,

2003; Smalle and Vierstra, 2004). Phosphorylation is also a

general regulationmechanism that reduces the binding affinity 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

stress response of Arabidopsis (Teige et al., 2004). Therefore,

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 specific ubiquitin ligase and degradation by the

26S proteasome. This sequence of events would require that

a salt stress–responsive MAPK localizes close to SPR1 or is

relocated to SPR1 upon stress (i.e., constitutively or conditionally

associated with MTs). Recent studies in Arabidopsis identified a

number of MAPKs (MAK18 and 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 SPR1 protein contains nine putative phosphor-

ylation sites (as predicted by NetPhos 2.0), 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 SPR1 regu-

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 with 1% Suc (MS/2) as described previ-

ously (Smalle et al., 2003). Plants were grown in a controlled environment

chamber at 228C with continuous light (140 mmol photons m22 s21). The

proteasomemutants 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-TUA6 andGFP-TUB6 overexpression lines (all in Col-0 background)

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 confirmed by immunoblotting analyses.

Treatments

Oryzalin and propyzamide were purchased from Sigma-Aldrich and

MG132 from Enzo Life Sciences. All drugs were made as 10003 stocks.

MG132, CHX, and propyzamide were dissolved in DMSO and oryzalin in

100% ethanol. All control experiments included a 13 dose of the solvent.

For all root elongation and root tip assays, plants grown on vertically

positioned MS/2 plates for 5 d were transferred to drug- or mock-

supplemented media. Test plates were positioned vertically, and the root

length was marked daily. After 3 d of treatments, plants were photo-

graphed. Photomicrographs of representative root tips were taken with

anOlympus SZX12microscope equippedwith aDP12 camera. For stress

tolerance assays, 4- or 5-d-old seedlings were transferred to test plates

with NaCl or mannitol and were grown vertically. For all morphometric

analyses, the relevant parameter wasmeasured fromdigital images using

ImageJ (http://rsb.info.nih.gov/ij/). Unless specified otherwise, data are

presented as mean values, and the error bars represent standard devi-

ation. Statistical significance was determined by ANOVA tests followed

by post hoc Bonferroni multiple comparison test. Post hoc statistical

significance is indicated in the figures by asterisks or crosses. For all

experiments, descriptive statistics, plotting, and the hypothesis testing

were done using Prism 5.0d software (GraphPad Software).

Confocal Microscopy Analysis of MTs and Scoring

MT distributions were analyzed in 35S:GFP-TUA6 and 35S:GFP-TUB6

lines in Col-0, rpn10-1, and rpn10-1 spr1-3 backgrounds using an

Proteasomal Degradation of SPR1 3423

Olympus Fluoview FV1000 confocal laser scanningmicroscope equipped

with an argon ion laser for the excitation of GFP (excitation 488 nm and

barrier 500 to 550 nm) 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 themeasurement of

individual MT dynamics, seedlings were mounted in liquid MS/2 medium,

and time-lapse imaging was performed at 1% laser power and 4-s in-

tervals for 3 to 6 min. 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 to 1.7-mL tubes for

treatment. After treatment, plants were blotted, frozen in liquid N2, and

ground in three volumes of 23 Laemmli sample buffer. Total proteins

were separated by SDS-PAGE (14% acrylamide separating gel for SPR1

analyses, 8% for RPN1 analyses, and 10% 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 RPN1 and SPR1 were described (Nakajima et al., 2004; Wang

et al., 2009). To test if the signal intensity for SPR1 in 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 Scientific

Pierce ECL Plus substrate) and analyzed using the linear best fit analyses

(see Supplemental Figure 8 online). The anti-RPN10 sera 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 (NanoDrop 2000; Thermo Scientific).

Onemicrogram of RNA per sample was used for the synthesis of the first-

strand cDNA (iScript reverse transcription supermix; Bio-Rad).

For the quantification of SPR1 transcripts, 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 of 20 mL. The SPR1 primers used were 59-AGCCTGCA-

GAGCTTAACAAG-39 and 59-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 in 1.25 volumes of extraction buffer as described (Kurepa et al.,

2008). Protein concentrations were measured using Bradford reagent

(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, SPR1 cDNA was amplified

using attB PCR primers attB1SPR1 59-GGGGACAAGTTTGTACAAAAA-

AGCAGGCTTAATGGGTCGTGGAAACAGC-39 and attB2SPR1 59-GGG-

GACCACTTTGTACAAGAAAGCTGGGTCTTACTTGCCACCAGTGAAGA-39.

The cDNA was introduced into pDONR221 via BP reaction and then to

pEarlyGate100 (Earley et al., 2006) by LR recombinase (Invitrogen).

Transgenic plants were selected on MS/2 plates containing 10 mM

L-phosphinothricin (Gold Biotechology), and homozygous T3 plants were

used for the analyses.

Accession Numbers

Sequence data from this article can be found in The Arabidopsis

Information Resource (http://www.Arabidopsis.org/) 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 identification numbers are SALK_027970 (rpn1a-4),

SALK_005596 (rpt2a-2), 35S:GFP-TUA6 (CS6551), and 35S:GFP-TUB6

(CS6550).

Supplemental Data

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

Supplemental Figure 1. Visible Phenotypes and Propyzamide Re-

sponses of the Double Mutant rpn1a-4 rpn10-1.

Supplemental Figure 2. The Steady State SPR1 Transcript Levels in

Proteasome Mutants Are the Same as in the Wild Type.

Supplemental Figure 3. Right-Handed Helical Growth Is Enhanced in

rpn1a-4 spr1-3 and rpn10-1 spr1-3 Mutants.

Supplemental Figure 4. Propyzamide Treatment Reverts Amplified

Root Skewing in the rpn1a-4 spr1-3 Mutant.

Supplemental Figure 5. Effects of NaCl Treatments on the SPR1

Transcript Level, SPR1 Protein Abundance, Accumulation of Ubiq-

uitinated Proteins, and Proteasome Activity.

Supplemental Figure 6. Treatment with 10 mM Propyzamide Causes

Depolymerization of Cortical MTs.

Supplemental Figure 7. Overexpression of SPR1 Increases MT

Stability and Leads to Salt Stress Hypersensitivity.

Supplemental Figure 8. Linear Relationship between SPR1 Abun-

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 (Grant 148-502-06-189) and the Kentucky Tobacco

Research and Development Center. We thank the ABRC for providing

seeds of the GFP-TUA6 and GFP-TUB6 transgenic lines.

AUTHOR CONTRIBUTIONS

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

research was performed by S.W. with assistance from J.K. and J.A.S.

3424 The 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 August 1, 2011; revised August 30, 2011; accepted September

12, 2011; published September 27, 2011.

REFERENCES

Abdrakhamanova, A., Wang, Q.Y., Khokhlova, L., and Nick, P.

(2003). Is microtubule disassembly a trigger for cold acclimation?

Plant Cell Physiol. 44: 676–686.

Abe, T., and Hashimoto, T. (2005). Altered microtubule dynamics by

expression of modified a-tubulin protein causes right-handed helical

growth in transgenic Arabidopsis plants. Plant J. 43: 191–204.

Ambrose, J.C., Shoji, T., Kotzer, A.M., Pighin, J.A., and Wasteneys,

G.O. (2007). The Arabidopsis CLASP gene encodes a microtubule-

associated protein involved in cell expansion and division. Plant Cell

19: 2763–2775.

Ban, R., Matsuzaki, H., Akashi, T., Sakashita, G., Taniguchi, H., Park,

S.Y., Tanaka, H., Furukawa, K., and Urano, T. (2009). Mitotic

regulation of the stability of microtubule plus-end tracking protein

EB3 by ubiquitin ligase SIAH-1 and Aurora mitotic kinases. J. Biol.

Chem. 284: 28367–28381.

Bartolini, F., Tian, G., Piehl, M., Cassimeris, L., Lewis, S.A., and

Cowan, N.J. (2005). Identification of a novel tubulin-destabilizing pro-

tein related to the chaperone cofactor E. J. Cell Sci. 118: 1197–1207.

Baskin, T.I., Wilson, J.E., Cork, A., and Williamson, R.E. (1994).

Morphology and microtubule organization in Arabidopsis roots ex-

posed to oryzalin or taxol. Plant Cell Physiol. 35: 935–942.

Beck, M., Komis, G., Muller, J., Menzel, D., and Samaj, J. (2010).

Arabidopsis homologs of nucleus- and phragmoplast-localized kinase

2 and 3 and mitogen-activated protein kinase 4 are essential for

microtubule organization. Plant Cell 22: 755–771.

Bhamidipati, A., Lewis, S.A., and Cowan, N.J. (2000). ADP ribosyla-

tion factor-like protein 2 (Arl2) regulates the interaction of tubulin-

folding cofactor D with native tubulin. J. Cell Biol. 149: 1087–1096.

Bisgrove, S.R., Hable, W.E., and Kropf, D.L. (2004). +TIPs and

microtubule regulation. The beginning of the plus end in plants. Plant

Physiol. 136: 3855–3863.

Bisgrove, S.R., Lee, Y.R., Liu, B., Peters, N.T., and Kropf, D.L. (2008).

The microtubule plus-end binding protein EB1 functions in root

responses to touch and gravity signals in Arabidopsis. Plant Cell 20:

396–410.

Blancaflor, E.B., and Hasenstein, K.H. (1995). Growth and microtubule

orientation of Zea mays roots subjected to osmotic stress. Int. J. Plant

Sci. 156: 774–783.

Burssens, S., Himanen, K., van de Cotte, B., Beeckman, T., Van

Montagu, M., Inze, D., and Verbruggen, N. (2000). Expression of cell

cycle regulatory genes and morphological alterations in response to

salt stress in Arabidopsis thaliana. Planta 211: 632–640.

Buschmann, H., and Lloyd, C.W. (2008). Arabidopsis mutants and the

network of microtubule-associated functions. Mol. Plant 1: 888–898.

Chan, J., Calder, G.M., Doonan, J.H., and Lloyd, C.W. (2003). EB1

reveals mobile microtubule nucleation sites in Arabidopsis. Nat. Cell

Biol. 5: 967–971.

Chan, J., Eder, M., Crowell, E.F., Hampson, J., Calder, G., and Lloyd,

C. (2011). Microtubules and CESA tracks at the inner epidermal wall

align independently of those on the outer wall of light-grown Arabi-

dopsis hypocotyls. J. Cell Sci. 124: 1088–1094.

Chen, Z., Hagler, J., Palombella, V.J., Melandri, F., Scherer, D.,

Ballard, D., and Maniatis, T. (1995). Signal-induced site-specific

phosphorylation targets I kappa B alpha to the ubiquitin-proteasome

pathway. Genes Dev. 9: 1586–1597.

Crowell, E.F., Timpano, H., Desprez, T., Franssen-Verheijen, T.,

Emons, A.M., Hofte, H., and Vernhettes, S. (2011). Differential regu-

lation of cellulose orientation at the inner and outer face of epidermal

cells in the Arabidopsis hypocotyl. Plant Cell 23: 2592–2605.

Czechowski, T., Stitt, M., Altmann, T., Udvardi, M.K., and Scheible,

W.R. (2005). Genome-wide identification and testing of superior

reference genes for transcript normalization in Arabidopsis. Plant

Physiol. 139: 5–17.

David, D.C., Layfield, R., Serpell, L., Narain, Y., Goedert, M., and

Spillantini, M.G. (2002). Proteasomal degradation of tau protein. J.

Neurochem. 83: 176–185.

Dhonukshe, P., and Gadella, T.W., Jr. (2003). Alteration of microtubule

dynamic instability during preprophase band formation revealed by

yellow fluorescent protein-CLIP170 microtubule plus-end labeling.

Plant Cell 15: 597–611.

Dhonukshe, P., Laxalt, A.M., Goedhart, J., Gadella, T.W., and

Munnik, T. (2003). Phospholipase d activation correlates with micro-

tubule reorganization in living plant cells. Plant Cell 15: 2666–2679.

Dill, A., Jung, H.S., and Sun, T.P. (2001). The DELLA motif is essential

for gibberellin-induced degradation of RGA. Proc. Natl. Acad. Sci.

USA 98: 14162–14167.

Drewes, G., Ebneth, A., and Mandelkow, E.M. (1998). MAPs, MARKs

and microtubule dynamics. Trends Biochem. Sci. 23: 307–311.

Earley, K.W., Haag, J.R., Pontes, O., Opper, K., Juehne, T., Song, K.,

and Pikaard, C.S. (2006). Gateway-compatible vectors for plant

functional genomics and proteomics. Plant J. 45: 616–629.

Furutani, I., Watanabe, Y., Prieto, R., Masukawa, M., Suzuki, K.,

Naoi, K., Thitamadee, S., Shikanai, T., and Hashimoto, T. (2000).

The SPIRAL genes are required for directional control of cell elonga-

tion in Arabidopsis thaliana. Development 127: 4443–4453.

Gagne, J.M., Smalle, J., Gingerich, D.J., Walker, J.M., Yoo, S.D.,

Yanagisawa, S., and Vierstra, R.D. (2004). Arabidopsis EIN3-binding

F-box 1 and 2 form ubiquitin-protein ligases that repress ethylene

action and promote growth by directing EIN3 degradation. Proc. Natl.

Acad. Sci. USA 101: 6803–6808.

Galjart, N. (2005). CLIPs and CLASPs and cellular dynamics. Nat. Rev.

Mol. Cell Biol. 6: 487–498.

Glickman, M.H. (2000). Getting in and out of the proteasome. Semin.

Cell Dev. Biol. 11: 149–158.

Hamada, T. (2007). Microtubule-associated proteins in higher plants.

J. Plant Res. 120: 79–98.

Hamant, O., Heisler, M.G., Jonsson, H., Krupinski, P., Uyttewaal, M.,

Bokov, P., Corson, F., Sahlin, P., Boudaoud, A., Meyerowitz, E.M.,

Couder, Y., and Traas, J. (2008). Developmental patterning by

mechanical signals in Arabidopsis. Science 322: 1650–1655.

Hershko, A., and Ciechanover, A. (1998). The ubiquitin system. Annu.

Rev. Biochem. 67: 425–479.

Himmelspach, R., Wymer, C.L., Lloyd, C.W., and Nick, P. (1999).

Gravity-induced reorientation of cortical microtubules observed in

vivo. Plant J. 18: 449–453.

Hirokawa, N. (1994). Microtubule organization and dynamics dependent

on microtubule-associated proteins. Curr. Opin. Cell Biol. 6: 74–81.

Ishida, T., Kaneko, Y., Iwano, M., and Hashimoto, T. (2007). Helical

microtubule arrays in a collection of twisting tubulin mutants of

Arabidopsis thaliana. Proc. Natl. Acad. Sci. USA 104: 8544–8549.

Kaloriti, K., Galva, C., Parupalli, C., Khalifa, N., Galvin, M., and

Sedbrook, J.C. (2007). Microtubule associated proteins in plants and

the processes They manage. J. Integr. Plant Biol. 48: 1164–1173.

Kaiser, P., and Tagwerker, C. (2005). Is this protein ubiquitinated?

Methods Enzymol. 399: 243–248.

Kirik, V., Herrmann, U., Parupalli, C., Sedbrook, J.C., Ehrhardt, D.W.,

Proteasomal Degradation of SPR1 3425

and Hulskamp, M. (2007). CLASP localizes in two discrete patterns

on cortical microtubules and is required for cell morphogenesis and

cell division in Arabidopsis. J. Cell Sci. 120: 4416–4425.

Komaki, S., Abe, T., Coutuer, S., Inze, D., Russinova, E., and

Hashimoto, T. (2010). Nuclear-localized subtype of end-binding

1 protein regulates spindle organization in Arabidopsis. J. Cell Sci.

123: 451–459.

Kurepa, J., Karangwa, C., Duke, L.S., and Smalle, J.A. (2010).

Arabidopsis sensitivity to protein synthesis inhibitors depends on

26S proteasome activity. Plant Cell Rep. 29: 249–259.

Kurepa, J., and Smalle, J.A. (2008). Structure, function and regulation

of plant proteasomes. Biochimie 90: 324–335.

Kurepa, J., Toh-E, A., and Smalle, J.A. (2008). 26S proteasome

regulatory particle mutants have increased oxidative stress tolerance.

Plant J. 53: 102–114.

Kurepa, J., Wang, S., Li, Y., and Smalle, J. (2009a). Proteasome

regulation, plant growth and stress tolerance. Plant Signal. Behav. 4:

924–927.

Kurepa, J., Wang, S., Li, Y., Zaitlin, D., Pierce, A.J., and Smalle, J.A.

(2009b). Loss of 26S proteasome function leads to increased cell size

and decreased cell number in Arabidopsis shoot organs. Plant

Physiol. 150: 178–189.

Lee, D.H., and Goldberg, A.L. (1998). Proteasome inhibitors: Valuable

new tools for cell biologists. Trends Cell Biol. 8: 397–403.

Lloyd, C., and Hussey, P. (2001). Microtubule-associated proteins in

plants—Why we need a MAP. Nat. Rev. Mol. Cell Biol. 2: 40–47.

Lopez-Molina, L., Mongrand, S., and Chua, N.H. (2001). A postgermi-

nation developmental arrest checkpoint is mediated by abscisic acid

and requires the ABI5 transcription factor in Arabidopsis. Proc. Natl.

Acad. Sci. USA 98: 4782–4787.

Lyle, K., Kumar, P., and Wittmann, T. (2009a). SnapShot: Microtubule

regulators I. Cell 136: 380.

Lyle, K., Kumar, P., and Wittmann, T. (2009b). SnapShot: Microtubule

regulators II. Cell 136: 566.

Lyons-Abbott, S., Sackett, D.L., Wloga, D., Gaertig, J., Morgan, R.E.,

Werbovetz, K.A., and Morrissette, N.S. (2010). a-Tubulin mutations

alter oryzalin affinity and microtubule assembly properties to confer

dinitroaniline resistance. Eukaryot. Cell 9: 1825–1834.

Mahajan, S., Pandey, G.K., and Tuteja, N. (2008). Calcium- and salt-

stress signaling in plants: shedding light on SOS pathway. Arch.

Biochem. Biophys. 471: 146–158.

Mandelkow, E., and Mandelkow, E.M. (1995). Microtubules and

microtubule-associated proteins. Curr. Opin. Cell Biol. 7: 72–81.

Matenia, D., and Mandelkow, E.M. (2009). The tau of MARK: A

polarized view of the cytoskeleton. Trends Biochem. Sci. 34: 332–342.

Mathur, J., and Chua, N.H. (2000). Microtubule stabilization leads to

growth reorientation in Arabidopsis trichomes. Plant Cell 12: 465–477.

Mathur, J., Mathur, N., Kernebeck, B., Srinivas, B.P., and Hulskamp,

M. (2003). A novel localization pattern for an EB1-like protein links

microtubule dynamics to endomembrane organization. Curr. Biol. 13:

1991–1997.

Matsuzaki, H., Daitoku, H., Hatta, M., Tanaka, K., and Fukamizu, A.

(2003). Insulin-induced phosphorylation of FKHR (Foxo1) targets to

proteasomal degradation. Proc. Natl. Acad. Sci. USA 100: 11285–11290.

Munns, R., and Tester, M. (2008). Mechanisms of salinity tolerance.

Annu. Rev. Plant Biol. 59: 651–681.

Nakajima, K., Furutani, I., Tachimoto, H., Matsubara, H., and

Hashimoto, T. (2004). SPIRAL1 encodes a plant-specific microtubule-

localized protein required for directional control of rapidly expanding

Arabidopsis cells. Plant Cell 16: 1178–1190.

Nakajima, K., Kawamura, T., and Hashimoto, T. (2006). Role of the

SPIRAL1 gene family in anisotropic growth of Arabidopsis thaliana.

Plant Cell Physiol. 47: 513–522.

Nakamura, M., Naoi, K., Shoji, T., and Hashimoto, T. (2004). Low

concentrations of propyzamide and oryzalin alter microtubule dynam-

ics in Arabidopsis epidermal cells. Plant Cell Physiol. 45: 1330–1334.

Nogales, E. (2001). Structural insight into microtubule function. Annu.

Rev. Biophys. Biomol. Struct. 30: 397–420.

Oka, M., Yanagawa, Y., Asada, T., Yoneda, A., Hasezawa, S., Sato,

T., and Nakagawa, H. (2004). Inhibition of proteasome by MG-132

treatment causes extra phragmoplast formation and cortical micro-

tubule disorganization during M/G1 transition in synchronized to-

bacco cells. Plant Cell Physiol. 45: 1623–1632.

Peth, A., Boettcher, J.P., and Dubiel, W. (2007). Ubiquitin-dependent

proteolysis of the microtubule end-binding protein 1, EB1, is con-

trolled by the COP9 signalosome: Possible consequences for micro-

tubule filament stability. J. Mol. Biol. 368: 550–563.

Petrucelli, L., et al. (2004). CHIP and Hsp70 regulate tau ubiquitination,

degradation and aggregation. Hum. Mol. Genet. 13: 703–714.

Poruchynsky, M.S., Sackett, D.L., Robey, R.W., Ward, Y., Annunziata,

C., and Fojo, T. (2008). Proteasome inhibitors increase tubulin poly-

merization and stabilization in tissue culture cells: A possible mech-

anism contributing to peripheral neuropathy and cellular toxicity

following proteasome inhibition. Cell Cycle 7: 940–949.

Ren, Y., Zhao, J., and Feng, J. (2003). Parkin binds to a/b tubulin and

increases their ubiquitination and degradation. J. Neurosci. 23: 3316–

3324.

Schuyler, S.C., and Pellman, D. (2001). Microtubule “plus-end-tracking

proteins”: The end is just the beginning. Cell 105: 421–424.

Sedbrook, J.C. (2004). MAPs in plant cells: Delineating microtubule

growth dynamics and organization. Curr. Opin. Plant Biol. 7: 632–640.

Sedbrook, J.C., Ehrhardt, D.W., Fisher, S.E., Scheible, W.R., and

Somerville, C.R. (2004). The Arabidopsis sku6/spiral1 gene encodes

a plus end-localized microtubule-interacting protein involved in direc-

tional cell expansion. Plant Cell 16: 1506–1520.

Sedbrook, J.C., and Kaloriti, D. (2008). Microtubules, MAPs and plant

directional cell expansion. Trends Plant Sci. 13: 303–310.

Sheng, X., Hu, Z., Lu, H., Wang, X., Baluska, F., Samaj, J., and Lin, J.

(2006). Roles of the ubiquitin/proteasome pathway in pollen tube

growth with emphasis on MG132-induced alterations in ultrastructure,

cytoskeleton, and cell wall components. Plant Physiol. 141: 1578–

1590.

Shoji, T., Suzuki, K., Abe, T., Kaneko, Y., Shi, H., Zhu, J.K., Rus, A.,

Hasegawa, P.M., and Hashimoto, T. (2006). Salt stress affects

cortical microtubule organization and helical growth in Arabidopsis.

Plant Cell Physiol. 47: 1158–1168.

Smalle, J., Kurepa, J., Yang, P., Emborg, T.J., Babiychuk, E.,

Kushnir, S., and Vierstra, R.D. (2003). The pleiotropic role of the

26S proteasome subunit RPN10 in Arabidopsis growth and develop-

ment supports a substrate-specific function in abscisic acid signaling.

Plant Cell 15: 965–980.

Smalle, J., and Vierstra, R.D. (2004). The ubiquitin 26S proteasome

proteolytic pathway. Annu. Rev. Plant Biol. 55: 555–590.

Smertenko, A., Draber, P., Viklicky, V., and Opatrny, Z. (1997). Heat

stress affects the organization of microtubules and cell division in

Nicotiana tabacum cells. Plant Cell Environ. 20: 1534–1542.

Teige, M., Scheikl, E., Eulgem, T., Doczi, R., Ichimura, K., Shinozaki,

K., Dangl, J.L., and Hirt, H. (2004). The MKK2 pathway mediates

cold and salt stress signaling in Arabidopsis. Mol. Cell 15: 141–152.

Thion, L., Mazars, C., Nacry, P., Bouchez, D., Moreau, M., Ranjeva, R.,

and Thuleau, P. (1998). Plasma membrane depolarization-activated

calcium channels, stimulated by microtubule-depolymerizing drugs in

wild-type Arabidopsis thaliana protoplasts, display constitutively large

activities and a longer half-life in ton 2 mutant cells affected in the

organization of cortical microtubules. Plant J. 13: 603–610.

Ueda, K., Matsuyama, T., and Hashimoto, T. (1999). Visualization of

3426 The Plant Cell

microtubules in living cells of transgenic Arabidopsis thaliana. Proto-

plasma 206: 201–206.

Van Bruaene, N., Joss, G., and Van Oostveldt, P. (2004). Reorgani-

zation and in vivo dynamics of microtubules during Arabidopsis root

hair development. Plant Physiol. 136: 3905–3919.

Vandesompele, J., De Preter, K., Pattyn, F., Poppe, B., Van Roy, N.,

De Paepe, A., and Speleman, F. (2002). Accurate normalization of

real-time quantitative RT-PCR data by geometric averaging of multi-

ple internal control genes. Genome Biol. 3: RESEARCH0034.

Vaughan, K.T. (2005). TIP maker and TIP marker; EB1 as a master

controller of microtubule plus ends. J. Cell Biol. 171: 197–200.

Vierstra, R.D. (2009). The ubiquitin-26S proteasome system at the

nexus of plant biology. Nat. Rev. Mol. Cell Biol. 10: 385–397.

Voloshin, O., Gocheva, Y., Gutnick, M., Movshovich, N., Bakhrat, A.,

Baranes-Bachar, K., Bar-Zvi, D., Parvari, R., Gheber, L., and

Raveh, D. (2010). Tubulin chaperone E binds microtubules and

proteasomes and protects against misfolded protein stress. Cell.

Mol. Life Sci. 67: 2025–2038.

Walia, A., Lee, J.S., Wasteneys, G., and Ellis, B. (2009). Arabidopsis

mitogen-activated protein kinase MPK18 mediates cortical microtu-

bule functions in plant cells. Plant J. 59: 565–575.

Wang, C., Li, J., and Yuan, M. (2007). Salt tolerance requires cortical

microtubule reorganization in Arabidopsis. Plant Cell Physiol. 48:

1534–1547.

Wang, C., Zhang, L.-J., and Huang, R.-D. (2011). Cytoskeleton and

plant salt stress tolerance. Plant Signal. Behav. 6: 29–31.

Wang, Q.Y., and Nick, P. (2001). Cold acclimation can induce micro-

tubular cold stability in a manner distinct from abscisic acid. Plant Cell

Physiol. 42: 999–1005.

Wang, S., Kurepa, J., and Smalle, J.A. (2009). The Arabidopsis 26S

proteasome subunit RPN1a is required for optimal plant growth and

stress responses. Plant Cell Physiol. 50: 1721–1725.

Wang, S., Kurepa, J., and Smalle, J.A. (2011). Ultra-small TiO(2)

nanoparticles disrupt microtubular networks in Arabidopsis thaliana.

Plant Cell Environ. 34: 811–820.

Xie, Q., Guo, H.S., Dallman, G., Fang, S., Weissman, A.M., and Chua,

N.H. (2002). SINAT5 promotes ubiquitin-related degradation of NAC1

to attenuate auxin signals. Nature 419: 167–170.

Yanagawa, Y., et al. (2002). Cell-cycle dependent dynamic change of

26S proteasome distribution in tobacco BY-2 cells. Plant Cell Physiol.

43: 604–613.

Yewdell, J.W., Lacsina, J.R., Rechsteiner, M.C., and Nicchitta, C.V.

(2011). Out with the old, in with the new? Comparing methods for

measuring protein degradation. Cell Biol. Int. 35: 457–462.

Zhu, J.K. (2001). Plant salt tolerance. Trends Plant Sci. 6: 66–71.

Proteasomal Degradation of SPR1 3427

DOI 10.1105/tpc.111.089920; originally published online September 27, 2011; 2011;23;3412-3427Plant Cell

Songhu Wang, Jasmina Kurepa, Takashi Hashimoto and Jan A. SmalleDependent Degradation of SPIRAL1−Proteasome

Cortical Microtubule Arrays Involves 26S ArabidopsisInduced Disassembly of −Salt Stress

 This information is current as of February 26, 2020

 

Supplemental Data /content/suppl/2011/09/15/tpc.111.089920.DC1.html

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