Available online at www.sciencedirect.com

Journal of Genetics and Genomics 38 (2011) 307e313www.jgenetgenomics.org

The alkaline tolerance in Arabidopsis requires stabilizing microfilamentpartially through inactivation of PKS5 kinase

Juntao Liu a,b, Yan Guo a,*

a State Key Laboratory of Plant Physiology and Biochemistry, College of Biological Sciences, China Agricultural University, Beijing 100094, ChinabNational Institute of Biological Sciences, Beijing, No. 7 Science Park Road, Zhongguancun Life Science Park, Beijing 102206, China

Received 19 April 2011; revised 18 May 2011; accepted 19 May 2011

Abstract

High soil pH is harmful to plant growth and development. The organization and dynamics of microfilament (MF) cytoskeleton play importantroles in the plant anti-alkaline process. In the previous study, we determined that alkaline stress induces a signal that triggers MF dynamics-dependent root growth. In this study we identified that PKS5 kinase involves in this regulatory process to facilitate the signal to reach thedownstream target MF. Under pH 8.3 treatment, the depolymerization of MF was faster in pks5-4 (PKS5 kinase constitutively activated) thanthat in wild-type plants. The inhibition of wild-type, pks5-1, and pks5-4 root growth by pH 8.3 was correlated to their MF depolymerization rate.When the plants were treated with phalloidin to stabilize MF, the high pH sensitive phenotype of pks5-4 can be partially rescued. When theplants were treated with a kinase inhibitor Staurosporine, the MF depolymerization rate in pks5-4 was similar as that in wild-type under pH 8.3treatment and the sensitivity of root growth was also rescued. However, when the plants were treated with LaCl3, a calcium channel blocker, theroot growth sensitivity of pks5-4 under pH 8.3 was rescued but MF depolymerization was even faster than that of plants without LaCl3 treatment.These results suggest that the PKS5 involves in external high pH signal mediated MF depolymerization, and that may be independent of calciumsignal.

Keywords: Arabidopsis; Alkaline stress; Microfilament; Kinase activity

1. Introduction

Soil alkalinity is a severe stress in agriculture worldwideand a principal cause for yield and quality reduction in crops.Environmental pH is a highly variable factor that affects rootgrowth and plant development during plant life-cycle. Whenthe external pH is up to 7.5, a passive influx of OH� ions wasobserved, and the cytoplasmic pH increased according to theincrease of external pH (Gout et al., 1992), suggesting thatexternal pH can directly influence the cytoplasmic pH. At pH8.1, primary root growth is obviously suppressed (Yang et al.,2010). Plant root cells can modify apoplastic pH in response toextracellular signals (Lager et al., 2010). Extracellular alka-linization is a specific cellular response to heat shock stress

* Corresponding author. Tel/fax: þ86 10 6273 2882.

E-mail address: guoyan@cau.edu.cn (Y. Guo).

1673-8527/$ - see front matter Copyright � 2011, Institute of Genetics and Develop

Published by Elsevier Limited and Science Press. All rights reserved.

doi:10.1016/j.jgg.2011.05.006

(Chaidee and Pfeiffer, 2006). Environmental stresses oftenresult in cytoplasmic pH change, for example, increasingextracellular NaCl concentration can cause transient alkalin-ization of the cytoplasm in brown alga embryos (Gibbon andKropf, 1993). The pH of the nutrient solution used for fababean and maize culture was lower than initial pH of 6.0 afterincubating for a while (Zhou et al., 2009), suggesting thatplant cells continuously pump proton out of the plasmamembrane (PM).

The organization and dynamics of cytoskeleton play animportant role in plant response to diverse abiotic stresses(Wang et al., 2011). Cortical microtubule reorganization isrequired for salt tolerance (Wang et al., 2007). The microfil-ament (MF) cytoskeleton has diverse functions under stressesin both plants and animals. Bundled MF functions in intra-cellular mechanical stress distribution and signaling (Wang,2010). Heat shock stress induces depolymerization of MF(Malerba et al., 2010). Disrupting MF integrity is an early

mental Biology, Chinese Academy of Sciences, and Genetics Society of China.

308 J. Liu, Y. Guo / Journal of Genetics and Genomics 38 (2011) 307e313

intracellular signal for loss of tensile stress-induced modula-tion and may contribute to the pathogenesis of vasculardiseases in smooth muscle cells (Zheng et al., 2010). MFpolymerization is correlated with the ability of cerebellargranule neurons to decrease calcium influx through L-typevoltage-operated calcium channels in cultured condition(Tiago et al., 2011). Disruption of the MF organization islinked to alterations of the cytosolic calcium concentration(Tiago et al., 2011). MF assembly and bundle formation areassociated to plant NaCl and osmotic stress responses (Wanget al., 2010).

It is reported that MF disrupted rapidly at pH 8.0 or abovein pea and rice root (Andersland and Parthasarathy, 1993).Extracellular acidic stress can induce the heat shock proteinHLJ1 to bind to beta-actin in lung cancer cells (Chen et al.,2010). We previously found that MF depolymerizes in rootunder alkaline stress in Arabidopsis (Zhou et al., 2010).However, the components between the pH signal and the MFdynamics are unclear. In this study, we determined that PKS5kinase plays a critical role in this regulatory process. PKS5was identified as a negative regulator of intracellular pHhomeostasis in response to alkaline pH (Fuglsang et al., 2007;Yang et al., 2010). Knock-out mutant pks5-1 showeda decreased sensitivity to high external pH treatment, whilepks5-4 (the PKS5 kinase constitutively activated) showed anincreased sensitivity to this treatment, and its MF depoly-merization was faster than that in wild-type (WT). When pks5-

Fig. 1. MF in pks5-4 depolymerizes more quickly than that in WT and pks5-1 un

seedlings in root elongation zone. A and D: WT, Col-0; B and E: pks5-1; C a

pictures in D, E and F were taken 6 h after pH 8.3 treatment. In D, E and F, values

�SE of three experiments (n> 30 cells per experiment). Scale bars¼ 10 mm.

4 is treated with a kinase inhibitor Staurosporine, the high pHsensitive phenotype of pks5-4 is partially rescued and that iscorrelated to the slow MF depolymerization. These resultssuggest that protein kinase PKS5 involves in the MF dynamicsin alkaline tolerance.

2. Materials and methods

2.1. Plant materials

The 35S::GFP-fABD2-GFP line (Wang et al., 2008) wascrossed to pks5-1 and pks5-4. Seedlings were germinated andgrown erectly on MS media (pH 5.8) at 23�C under continuouslight. For calculation of MF disassembly rate, four-day-oldseedlings were treated with liquid MS media with or withoutdrugs at indicated times. For phenotype test, four-day-oldseedlings were transferred to different pH MS media with orwithout drugs. The root length was measured at 14 days aftertransferred by IMAG-J.

2.2. Confocal laser scanning microscopy

For MF organization observation, procedures weredescribed by Zhou et al. (2010).

Four-day-old seedlings were treated with liquid MS media(pH 8.3) with or without different drugs for indicated times.Fluorescence images of the MF organization in root mature

der pH 8.3 treatment. All the pictures show MF organization of four-day-old

nd F: pks5-4. Pictures in A, B and C were taken before pH 8.3 treatment;

below the images represent the percentage of cells in which MF was disrupted,

309J. Liu, Y. Guo / Journal of Genetics and Genomics 38 (2011) 307e313

and elongation zones were observed at 6 h or indicated timesand captured with a Zeiss LSM 5 Meta Confocal. The sampleswere excited at 488 nm using a kryptoneargon laser line anddetected using a 505e530 nm filter. More than thirty root cellsof each seedling were observed, and the cells with disruptedMF organization were recorded. The disassembly rate of MFin cells was calculated. Statistics for each assay, at least 100cells of three seedlings were observed.

3. Results

3.1. MF in the mutant pks5-4 depolymerizes morequickly than that in WT and pks5-1 under alkalinetreatment

MF dynamics is important to root growth under alkalinestress (Zhou et al., 2010). PKS5 protein kinase is a criticalregulator in plant response to alkaline stress (Fuglsang et al.,2007; Yang et al., 2010). In order to test whether PKS5involves in pH signal and MF depolymerization under alkalinestress, 35S::GFP-fABD2-GFP plasmid was transformed intoWT and pks5 mutant plants to label MF cytoskeleton (Wanget al., 2008). T3 homozygous lines were used in this study.We found that in mutant pks5-4 (point mutation that causesPKS5 kinase constitutively activated) MF depolymerized more

Fig. 2. Phenotypes of pks5-1 and pks5-4 under high pH stress. A and B: five-day

transferred to MS medium at pH 5.8 or at pH 8.3 with 75 mmol/L NaCl. Pictures

elongation on pH 8.3 with 75 mmol/L NaCl was measured 14 days after transfer; th

normalized to WT (plant number> 15). A Student’s t test was used to determine sta

1 and pks5-4 treated by pH 8.3 buffer were calculated at 2, 4 and 6 h, respectivel

quickly than that in WT (Fig. 1), while in the T-DNA insertionknock-out mutant pks5-1, MF disassembly rate was slightlylower than that in WT. After 6-h treatment under pH 8.3, MFdisassembly rate was 42.2% in WT, 36.7% in pks5-1 and77.8% in pks5-4 (Fig. 1). The status of MF depolymerizationunder pH 8.3 treatment in WT and two PKS5 mutants wascorrelated to the PKS5 kinase activity. The higher kinaseactivity PKS5 had, the quicker MF depolymerization andslower root growth were observed in their correspondingplants. The mutant pks5-1 was less sensitive and the mutantpks5-4 was more sensitive to high external pH than that of WT(Fig. 2AeC). This is consistent with our pervious finding(Yang et al., 2010). Our results suggest that PKS5 kinase playsa role in regulating high pH mediated MF depolymerization.

3.2. Stabilization of MF partially rescues the high pHsensitive phenotype of pks5-4

MF dynamics is important for plant stress responses.Decrease of MF stability inhibits the primary root growth inArabidopsis under alkaline stress (Zhou et al., 2010). To test iffast MF disassembly is a reason for the root growth inhibitionof pks5-4 under pH 8.3, we use 1.5 mmol/L phalloidin to treatthe seedlings of WT, pks5-1 and pks5-4 on the pH 8.3 MSmedia. The MF disassembly rate in pks5-4 is obviously lower

-old WT, pks5-1 and pks5-4 seedlings grown on MS medium at pH 5.8 were

were taken 7 days (A) or 14 days (B) after transfer. C: relative primary root

e elongation of WTwas used as standard and was set to 1, and the others were

tistical significance (* means P< 0.05). D: MF disassembly rates of WT, pks5-

y. Values are means� SE of three experiments (n> 30 cells per experiment).

310 J. Liu, Y. Guo / Journal of Genetics and Genomics 38 (2011) 307e313

than that when only treated with pH 8.3. However, it was stillhigher than that of WT and pks5-1 (Fig. 3). Interestingly, theinhibition of primary root growth of pks5-4 by pH 8.3 waspartially rescued by the presence of 1.5 mmol/L phalloidin(Fig. 3). Our results suggest that the root growth inhibition ofpks5-4 by alkaline stress is partially due to the fast MFdepolymerization.

3.3. Deactivation of PKS5 kinase is required forstabilization of MF under alkaline stress

The different types of mutation in PKS5 cause oppositephenotypes under alkaline stress (Yang et al., 2010), sug-gesting that maintaining PKS5 kinase activity in a certain levelis important for Arabidopsis alkaline tolerance. To determineif regulation of PKS5 kinase activity is critical for PKS5-mediated MF depolymerization, the WT, pks5-1 and pks5-4were treated with a kinase inhibitor, Staurosporine. Staur-osporine is an ATP-competitive serine/threonine kinaseinhibitor because it binds to the ATP-binding site of manykinases with high affinity (Karaman et al., 2008). It has beenshown that Staurosporine suppressed MF depolymerization inguard cells (Hwang and Lee, 2001). When WT, pks5-1 andpks5-4 seedlings were treated with Staurosporine, no signifi-cant difference was observed on the root growth and MF

Fig. 3. Stabilizing MF partially rescue the high pH sensitive phenotype of pks5-4. A:

were transferred to MS medium at pH 8.3 with 1.5 mmol/L phalloidin. Pictures were

1.5 mmol/L phalloidin was measured 14 days after transfer; the elongation of WT

(plant number> 15). C: primary root elongation of pks5-4 on pH 8.3 with or wi

14 days after transfer (plant number> 15). D: MF disassembly rates of WT, pks5-1

6 h were calculated. Values are means� SE of three experiments (n> 30 cells pe

(* means P< 0.05); significant differences (P� 0.05) in D are indicated by differ

depolymerization under pH 8.3 treatment in WT and pks5-1.However, in pks5-4, the MF disassembly rate decreased toeven slightly lower than that of WT and pks5-1, and theprimary root growth was rescued to the level of WT and pks5-1 (Fig. 4). Our results suggest that regulation of PKS5 kinaseactivity is important for MF dynamics in plant alkaline stressresponse.

3.4. HD-ATPase activity and MF dynamics areseparately regulated by PKS5 kinase under alkalinestress

Activation of PKS5 kinase requires the involvement ofcalcium signal. PKS5 plays an important role in regulation ofthe cytoplasmic pH homeostasis. We previously showed thatcalcium sensor SCaBP1 (SOS3 like Calcium BindingProtein 1) interacts with and activates PKS5, and this complexin turn suppresses plasma membrane (PM) Hþ-ATPase AHA2by directly phosphorylating its C-terminal Ser931 (Fuglsanget al., 2007). To test if calcium signal also involves in theMF depolymerization upon alkaline stress, we use calciumchannel inhibitor LaCl3 to treat the seedlings of WT, pks5-1and pks5-4. LaCl3 slightly accelerated the MF depolymeriza-tion in all three seedlings compared to pH 8.3 treatment only,and the MF depolymerized still faster in pks5-4 than that in

five-day-old WT, pks5-1 and pks5-4 seedlings grown on MS medium at pH 5.8

taken 14 days after transfer. B: relative primary root elongation on pH 8.3 with

was used as standard and was set to 1, and the others were normalized to WT

thout 1.5 mmol/L phalloidin relative to the corresponding WT was measured

and pks5-4 treated by pH 8.3 buffer with or without 1.5 mmol/L phalloidin for

r experiment); a Student’s t test was used to determine statistical significance

ent lowercase letters.

Fig. 4. Activation of PKS5 kinase is required for Arabidopsis MF depoly-

merization under alkaline stress. A: five-day-old WT, pks5-1 and pks5-4

seedlings grown on MS medium at pH 5.8 were transferred to MS medium at

pH 8.3 with 20 mmol/L Staurosporine. Pictures were taken 14 days after

transfer. B: relative primary root elongation on pH 8.3 with 20 mmol/L

Staurosporine was measured 14 days after transfer; the elongation of WT was

used as standard and was set to 1, and the others were normalized to WT (plant

number> 15). C: MF disassembly rates of WT, pks5-1 and pks5-4 were

measured after seedlings treated by pH 8.3 buffer with or without 20 mmol/L

Staurosporine for 6 h were calculated. Values are means� SE of three

experiments (n> 30 cells per experiment); a Student’s t test was used to

determine statistical significance; significant differences (P� 0.05) are indi-

cated by different lowercase letters.

311J. Liu, Y. Guo / Journal of Genetics and Genomics 38 (2011) 307e313

WTand pks5-1 (Fig. 5B). However, the primary root growth ofthe two mutants and WT had no significant difference, andboth pks5-1 and pks5-4 were rescued to the WT level(Fig. 5A). This result is consistent with our previous finding

that calcium signal involves in plant alkaline stress response(Fuglsang et al., 2007). The calcium signal might be repressedby LaCl3, which in turn inhibits the PKS5 kinase activity andreleases the PM Hþ-ATPase activity to rescue the pks5-4 highpH sensitive phenotype. The LaCl3 treatment had differenteffects on the root growth and MF depolymerization of WT,pks5-1 and pks5-4 plants, suggesting that the regulation of thePM Hþ-ATPase activity and MF dynamics by PKS5 kinaseunder alkaline stress involves in two independent pathways.

4. Discussion

4.1. Alteration of pH can function as a signal

External pH can act as a signal to launch the secretion ofeffectors in Salmonella enterica. Under pH 7.2 condition, theeffectors are secreted into the host cell quickly, but this doesnot happen at pH 6.0 (Yu et al., 2010). Transient alteration ofthe cytoplasmic pH is essential in many signal cascades thatactivate environmental responses or developmental processesin plant cells. The influx of Hþ from apoplast is often inhibitedin the hypersensitive response (Roos et al., 2006). Extracel-lular protons are involved in temperature sensing and signalingin Chenopodium cells, probably via a Kþ/Hþ antiportermediated pathway (Chaidee and Pfeiffer, 2006). Transientcytosolic pH regulation can be achieved through three ways:Hþ binding by buffering groups; Hþ transport out of thecytosol; and Hþ transport into the vacuole (Bethmann andSchonknecht, 2009). Temporary inactivation of the PM Hþ-ATPases could cause transient alkalization (Vodeneev et al.,2010). Arabidopsis AtCHX23 regulates the cytosol pHpossibly by maintaining a high pH level in the chloroplaststroma (Song et al., 2004).

The purpose of our study is to identify componentsinvolved in the early biological events of plant response toalkaline stress. The high external pH triggers a signal thatresults in MF depolymerization, and that associates with theinhibition of the primary root growth. Plants use this mecha-nism to avoid the alkaline stress. In this study, we determinethat PKS5 kinase plays a vital role in this process and theregulation of its kinase activity is critical for the MF dynamicsunder alkaline stress. When the external pH changed, PKS5kinase activity is repressed and that may in turn releaseunknown downstream target(s). The signal is transferred toMF and stabilizes MF. This signal transduction is depended onPKS5 kinase activity.

4.2. Calcium signal involves in PKS5-mediated plantalkaline stress response through two independentpathways

It is likely that the calcium signal activates SCaBP1-PKS5complex at normal condition to repress the PM Hþ-ATPaseactivity at a low level (Fuglsang et al., 2007). Upon alkalinestress, J3 protein interacts with and represses PKS5 kinaseactivity to release PM Hþ-ATPase (Yang et al., 2010). In bothplants and fungi, cells use the trans-membrane proton gradient

Fig. 5. Calcium signal is important for plant alkaline stress response but may not be important for PKS5-mediated MF depolymerization. A: relative primary root

elongation on pH 8.3 with 200 mmol/L LaCl3 was measured 14 days after transfer; the elongation of WTwas used as standard and was set to 1, and the others were

normalized to WT (plant number> 15). B:MF disassembly rates of WT, pks5-1 and pks5-4 were measured after seedlings treated by pH 8.3 buffer with or without

200 mmol/L LaCl3 for 6 h were calculated. Values are means� SE of three experiments (n> 30 cells per experiment); a Student’s t test was used to determine

statistical significance; significant differences (P� 0.05) are indicated by different lowercase letters.

312 J. Liu, Y. Guo / Journal of Genetics and Genomics 38 (2011) 307e313

to drive ion transport across the PM. These proton gradientsare settled by PM Hþ-ATPases (Palmgren, 2001). When theexternal environment becomes alkalinized, the proton gradientwould be demolished, which would alter the ion homeostasisin the cell. Changes of external pH stimulate the cytosoliccalcium ion current oscillation in Type-I hair cells (Malayevand Nelson, 1995; Almanza et al., 2008). In plant, highexternal pH increases cytoplasmic calcium concentration(Fuglsang et al., 2007) and triggers a signal that led to MFreorganization (Zhou et al., 2010). In cerebellar granuleneurons, SIN-1 inhibits L-type voltage-operated calciumchannels to decrease cytosolic calcium concentration anddisrupt the MF organization in cultured condition (Tiago et al.,2011). It is not well understood that the MF dynamics is theupstream or downstream event in plant stress responses. TheMF reorganization is important for calcium signal release inearly plant stress response. In this study, we found thatcalcium is involved in plant alkaline stress response throughPKS5 kinase but possibly is not required for the PKS5-mediated MF depolymerization process. When WT, pks5-1and pks5-4 were treated with LaCl3, the disassembly rate ofMF in these plants was slightly faster than in pH 8.3 only,suggesting that calcium may not play an important role in MFreorganization and that is independent of PKS5. However theLaCl3 treatment improved the primary root growth of theseplants at pH 8.3, and the growth of pks5-4 and WT was nearlyat a similar level, suggesting that other calcium signal plays animportant role in alkaline stress through regulating PKS5activity. Possibly calcium participates in this process ina much more complex manner.

4.3. The pH sensor in plant

Many studies have shown that severe pH change in cell orenvironment is harmful for plant. Bacteria perceive externalpH changes and trigger downstream response for survive(Kenjale et al., 2005; Torruellas et al., 2005). However, there isno evidence if pH sensor(s) exists in plant. Zhou et al. (2010)reported that alkaline stress may trigger a signal that leads theMF dynamics and in turn regulates root growth. In this studyour results indicate that protein kinase PKS5 plays a vital role

in this process. The more activated PKS5 form causes thefaster MF depolymerization and lower PM Hþ-ATPasesactivity. Our hypothesis is when the external pH changes,a signal may be generated in cells and PKS5 perceives thissignal and transfers it to either PM Hþ-ATPases or MF. Thissignal transduction is depended on PKS5 kinase activity.However, it is not known how PM Hþ-ATPase activity and MFstability are synergistically regulated by PKS5 under salt-alkaline stress.

5. Accession numbers

Sequence data from this article can be found in the Ara-bidopsis Genome Initiative under the following accessionnumber: PKS5, At2g30360.

Acknowledgements

This work was supported by the grant of China NationalFunds for Distinguished Young Scientists (No. 31025003) toY. Guo.

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