Phytochemistry 76 (2012) 46–51

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Phytochemistry

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Nitric oxide exacerbates Al-induced inhibition of root elongation in rice beanby affecting cell wall and plasma membrane properties

Yuan Zhou, Xiao Yan Xu, Li Qian Chen, Jian Li Yang, Shao Jian Zheng ⇑Key Laboratory of Conservation Biology for Endangered Wildlife, Ministry of Education, College of Life Sciences, Zhejiang University, Hangzhou 310058, China

a r t i c l e i n f o

Article history:Received 12 November 2010Received in revised form 24 November 2011Available online 7 January 2012

Keywords:Rice beanAluminum toleranceNitric oxide (NO)Sodium nitroprusside (SNP)Plant cell wall

0031-9422/$ - see front matter � 2011 Elsevier Ltd. Adoi:10.1016/j.phytochem.2011.12.004

⇑ Corresponding author. Tel./fax: +86 571 8820643E-mail addresses: 10614051@zju.edu.cn (Y. Zh

(X.Y. Xu), wzmcclq1987@163.com (L.Q. Chen), yangsjzheng@zju.edu.cn (S.J. Zheng).

a b s t r a c t

Aluminum (Al) toxicity is one of the most widespread problems for crop production on acid soils, andnitric oxide (NO) is a key signaling molecule involved in the mediation of various biotic and abiotic stres-ses in plants. Here we found that exogenous application of the NO donor sodium nitroprusside (SNP)exacerbated the inhibition of Al-induced root growth in rice bean [Vigna umbellata (Thunb.) Ohwi &Ohashi ‘Jiangnan’, Fabaceae]. This was accompanied by an increased accumulation of Al in the root apex.However, Al treatments had no effect on endogenous NO concentrations in root apices. These results indi-cate that a change in NO concentration is not the cause of Al-induced root growth inhibition and theadverse effect of SNP on Al-induced root growth inhibition should result from increased Al accumulation.Al could significantly induce citrate efflux but SNP had no effects on citrate efflux either in the absence orpresence of Al. On the other hand, SNP pretreatment significantly increased Al-induced malondialdehydeaccumulation and Evans Blue staining, indicating an intensification of the disruption of plasma mem-brane integrity. Furthermore, SNP pretreatment also caused greater induction of pectin methylesteraseactivity by Al, which could be the cause of the increased Al accumulation. Taken together, it is concludedthat NO exacerbates Al-induced root growth inhibition by affecting cell wall and plasma membraneproperties.

� 2011 Elsevier Ltd. All rights reserved.

1. Introduction

Aluminum (Al) toxicity is a major factor limiting crop produc-tion on acid soils worldwide (Foy, 1988; Kochian, 1995). Al rapidlyinhibits root growth at micromolar concentrations and root apex isthe main target of Al toxicity (Ryan et al., 1993). A number of pos-sible mechanisms responsible for Al toxicity have been proposed.Thus, Al may interact with the cell wall, plasma membrane ormany intracellular components such as enzymes and other pro-teins resulting in the disruption of their functions (for a reviewsee Zheng and Yang, 2005; Ma, 2007). Al may also interact withsignal transduction pathways such as Ca2+-dependent signalingcascades (Rengel and Zhang, 2003). However, the primary causeof Al-induced root growth inhibition is still unclear.

Nitric oxide (NO) is a crucial gaseous signaling molecule in plants,and it plays significant roles in modulating physiological and bio-chemical functions (Crawford and Guo, 2005; Durner and Klessig,1999). It has also been suggested to be involved in responses to Alstress in plants. For instance, Wang and Yang (2005) reported thatexogenous NO ameliorated Al-induced inhibition of root growth in

ll rights reserved.

8.ou), xxy19832002@163.comjianli@zju.edu.cn (J.L. Yang),

Cassia tora by preventing Al-induced oxidative stress. The sameprotective effects of NO on Al-induced root growth inhibition hadalso been found in red kidney bean roots (Wang et al., 2010). Tianet al. (2007) demonstrated that Al-induced root growth inhibitionin Hibiscus moscheutos was associated with decrease of endogenousNO concentrations. On the other hand, accumulation of NO is toxic tocells and results in cell death. For example, infection of suspension-cultured soybean cells with the bacterial pathogen Pseudomonassyringae caused NO and reactive oxygen species increase whichresulting in hypersensitive response and programmed cell death(PCD). NO had also been implicated in Cd-induced PCD in Arabidop-sis suspension cultures (De Michele et al., 2009). These resultsindicate that NO mediates either negative or positive effects depen-dent upon concentrations of NO and plant species.

Many studies have focused the physiological functions of NO onoxidative stress. However, NO has been shown to taken part indifferent hormone signaling pathways and also acts in concert withwell-characterized second messengers. Pagnussat et al. (2002)showed NO accumulation in response to auxin treatment in cucum-ber explants during adventitious root formation. Studies also indi-cated that NO has profound effects on the regulation of ionchannels in plants (Ahern et al., 2002; Mannick and Schonhoff,2004). Moreover, it was demonstrated that artificially generatedNO had the ability to induce fast and transient extracellular Ca2+

uptake in tobacco cell cultures (Lamotte et al., 2004). Thus, Functions

1.5

2.0

gatio

n (c

m)

-SNP-Al-SNP+Al+SNP-Al+SNP+Al

Y. Zhou et al. / Phytochemistry 76 (2012) 46–51 47

of NO other than its antioxidative ability in response of plant to Al areof interest. In the present study, the effects of exogenous NO on rootgrowth responses to Al stress in rice bean were investigated. Theresults showed that exogenous NO exacerbates Al-induced rootgrowth inhibition which promoted exploration of a number ofpossible mechanisms with emphasis on the effects of NO on cell walland plasma membrane properties.

0 6 12 240.0

0.5

1.0

Treatment time (h)

Roo

t elo

n

Fig. 2. Effect of exogenous SNP (400 lM) on the Al-induced inhibition of rootgrowth in rice bean. Three-day-old seedlings were pretreated with or without SNPfor 12 h and then exposed to 0 or 25 lM Al for different times up to 24 h. Rootelongation was measured periodically. Data are means ± SD (n = 10).

2. Results

2.1. Effect of NO donor on Al-induced inhibition of root growth

Exposure of rice bean seedling roots to 25 lM Al for 24 h caused a35% inhibition of root elongation compared to no Al control (Fig. 1).The exogenous application of the nitric oxide donor, SNP, on rootgrowth was then examined. Pre-treatment of roots with SNP at vary-ing concentrations from 200 to 800 lM had no obvious effect on rootelongation in the absence of Al (Fig. 1A). However, when the seed-lings pretreated with SNP subjected to Al stress, dependent on theSNP concentrations, root elongation was further inhibited whencompared to that of no SNP pretreatment. As a consequence, the rel-ative root elongation showed progressively inhibition with an in-crease in SNP concentrations (Fig. 1B). Because SNP at 400 lM hada significant effect on Al-induced root growth inhibition, and a pre-vious study of exogenous application of SNP at this concentrationcaused an ameliorative effect on Al-induced root growth inhibitionin C. tora L. (Wang and Yang, 2005), this concentration was chosenfor the subsequent experiments to allow a direct comparison. Atime-course experiment showed that an adverse effect of SNP treat-ment could be observed after 24 h of Al stress, but root elongationwas inhibited greatly after only 6 h exposure to Al stress, indicatingthat the adverse effect of SNP on Al-induced inhibition of root elon-gation may be due to the enhancement of secondary effects of Alstress in rice bean.

In order to examine whether NO content was directly involvedin Al-induced root growth inhibition in rice bean, NO in root apiceswas labeled with a fluorescent probe 4-amino-5-methylamino-20,70-difluorofluorescein diacetate (DAF-FM DA) and visualized byepifluorescence microscopy. Al treatment had no effect on fluores-cence intensity. On the other hand, pretreatment with SNP resultedin about 38% increase of fluorescence intensity with or without Altreatment (Fig. 3). The results indicate that NO content of the rootapex is not the cause of Al-induced root growth inhibition.

0 200 400 600 8000.0

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SNP concentration (µM)

Roo

t elo

ngat

ion

(cm

)

-Al+Al

A B

a a a a a

a

bc c

ab

c

Fig. 1. Effect of the concentrations of the NO donor SNP on the Al-induced inhibition oconcentrations of SNP for 12 h and then exposed to 0 or 25 lM Al for 24 h. (A) root elongmeans ± SD (n = 10). Different letters above the bars represent significant difference am

2.2. Effect of NO donor on Al accumulation in root apices

In order to unravel the underlying basis of NO-exacerbated Al-induced root growth inhibition, the Al content in root apex wasmeasured (Fig. 4). There is a small amount of Al in root apex of ricebean in the absence of administered Al, possibly due to the impu-rity of the chemicals used. Al treatment resulted in significantaccumulation of Al in root apex, and SNP pretreatment furtherincreased the Al content by 60%, indicating that NO-aggravatedroot growth inhibition under Al stress might result from the in-creased accumulation of Al in the root apex.

2.3. Efflux of citrate from rice bean roots

To examine whether the increased accumulation of Al in rootapex after SNP pretreatment is due to dysfunction of the Al exclu-sion mechanism dependent on the Al-induced citrate secretion inrice bean, as our previous study demonstrated (Yang et al., 2006),the effect of SNP pretreatment on Al-induced citrate efflux fromroot apex was examined. While Al significantly induced citrateefflux, pretreatment with SNP had no effect on citrate efflux eitherin the presence or absence of Al (Fig. 5), indicating that the

0 200 400 600 8000

20

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SNP concentration (µM)

Rel

ativ

e ro

ot e

long

atio

n(+

Al/-A

l x10

0)

a

bcab

bcc

f root growth in rice bean. Three-day-old seedlings were pretreated with differentation and (B) relative root elongation (+/�Al at various SNP concentrations. Data areong different SNP concentrations treatments (P < 0.5).

a b

c d

-Al +Al -Al +Al0

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250

TreatmentsR

elat

ive in

tens

ity o

fN

O fl

uore

scen

ce (%

)

-SNP +SNP

A B

a ab b

Fig. 3. Effect of Al, exogenous SNP, and SNP pretreatment plus Al treatment onendogenous NO concentrations in rice bean root apex. (A) three-day-old seedlingswere pretreated with (c and d) or without (a and b) 400 lM SNP for 12 h and thenexposed to 0 (a and b) or 25 lM Al (c and d) for 24 h. Photographs of productionshown as green fluorescence in representative roots. (B), NO production expressedas relative fluorescence. Data are means ± SD (n = 10).

-Al +Al -Al +Al0.00

0.05

0.10

0.15

0.20

0.25

0.30

Treatments

Al c

onte

nt[µ

g (ro

ot ti

p)-1

]

-SNP +SNP

a

b

cc

Fig. 4. The Al contents of rice beans after Al treatment for 24 h. Seedlings were pre-treated with SNP at 400 lM for 12 h and then exposed to 25 lM Al for 24 h. Rootapices (1 cm) were excised and the Al contents were determined. Vertical barsrepresent the SD of the mean (n = 10). Different letters above the bars representsignificant difference among different treatments (P < 0.5).

-Al +Al -Al +Al0

500

1000

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3500

Treatments

Citr

ate

efflu

x[p

mol

(roo

t ape

x)-1

h-1

]

-SNP +SNP

a a

bb

Fig. 5. The effect of the NO donor SNP on the exudation of citrate from roots of ricebean. Seedlings were pre-treated with 0.4 mM SNP for 12 h and then exposed to25 lM Al for 24 h. Data are means ± SD (n = 3).

-Al +Al -Al +Al-SNP +SNP

B

-Al +Al -Al +Al0.00

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0.10

0.15

0.20

MD

A co

nten

t(µ

mol

g-1

pro

tein

)

-SNP +SNP

Aa

bc

c

Fig. 6. Evans blue staining (A) and malondialdehyde (MDA) concentration (B) in theroot tips of ricebean under different treatments. Three-day-old seedlings werepretreated with or without 400 lM SNP for 12 h and then exposed to 0 or 25 lM Alfor 24 h. Data are means ± SD (n = 3). (For interpretation of the references to color inthis figure legend, the reader is referred to the web version of this article.)

48 Y. Zhou et al. / Phytochemistry 76 (2012) 46–51

increased accumulation of Al in root apex by application of exoge-nous NO is not resulted from the reduced efflux of citrate from rootapex.

2.4. Effect of NO donor on Al-induced oxidative stress and lipidperoxidation

In the present study, Evans blue staining was used to indicatethe effect of exogenous NO on the integrity of plasma membrane.The roots of rice bean treated with Al alone were stained withEvans blue (Fig. 6A), whereas those pre-treated with SNP weremore intensively stained, indicating that application of exogenousNO aggravates Al-induced oxidative injury in rice bean roots.

Peroxidation of membrane lipids was further determined interms of malondialdehyde (MDA) accumulation. Al treatmentincreased accumulation of MDA significantly, and SNP pretreat-ment caused further increase of MDA content in the presence ofAl (Fig. 6B).

2.5. Effect of NO donor on cell wall compositions

The effect of SNP pretreatment on cell wall pectin content andits degree of methylation was examined in root apex of rice bean.As shown in Fig. 7A, the pectin content showed no differencesamong different treatments. On the other hand, Al treatmentresulted in a significant increase in pectin methylesterase (PME)activity in comparison with no Al treatment. Pretreatment withSNP alone had no effect on PME activity, whereas pretreatmentwith SNP plus Al treatment caused a further increase in PME activ-ity (Fig. 7B).

3. Discussion

Aluminum toxicity caused rapid inhibition of root growth, butthe underlying basis still remains unclear. NO has been implicatedin modulating numerous physiological processes such as growthand development, hypersensitive responses and responses to bioticand abiotic stresses (Chen et al., 2010; Graziano and Lamattina,2007; Wang and Yang, 2005; Wendehenne et al., 2004; Zhaoet al., 2007). Tian et al. (2007) found that Al-induced root growthinhibition is associated with a reduction of endogenous NO con-centrations. Therefore, it seems likely that NO is involved in Alstress response in plants. In this study, it was demonstrated thatexogenous application of NO exacerbated Al-induced root growthinhibition in rice bean (Figs. 1 and 2). This finding is contrary toa previous report on another plant species, C. tora L. (Wang and

-Al +Al -Al +Al0.0

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Treatments

PME

activ

ity(µ

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MeO

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rote

in)

-SNP +SNP

-Al +Al -Al +Al0

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Pect

in c

onte

nt[m

g G

aE g

-1 (c

ell w

all D

W)]

-SNP +SNP

A

B a

b

c c

aa

aa

Fig. 7. Effect of the NO donor SNP on the pectin content (A) and activities of pectinmethylesterase (PME) (B) in the cell wall of rice bean root apices. Seedlings werepre-treated with 0.4 mM SNP for 12 h and then exposed to 25 lM Al for 24 h. Thevalues are means ± SD (n = 3).

Y. Zhou et al. / Phytochemistry 76 (2012) 46–51 49

Yang, 2005). The discrepancy could be due to different plantspecies, because Al stress resulted in different effects on endoge-nous NO concentrations dependent on plant species. For instance,exposure of H. moscheutos to Al for 20 min caused a 34% reductionin endogenous NO concentration (Tian et al., 2007). However,exposure of red kidney bean roots to 50 lM Al for 24 h caused a58% increase in NO concentration (Wang et al., 2010). In the pres-ent study, exposure of rice bean roots to 25 lM Al for 24 h had noobvious effect on endogenous NO concentration (Fig. 3). Theseresults imply that NO signaling pathways associating with Alstress, if any, differ among different plant species. NO at certainconcentration would trigger the signal transduction pathways ofdefense mechanisms to Al toxicity like antioxidant enzymes in C.tora (Wang and Yang, 2005) while it exerts opposite effects in ricebean (Fig. 6).

Al accumulation in root apex is in many cases directly related toAl toxicity (Delhaize et al., 1993; Frantzios et al., 2001; Sivaguruand Horst, 1998; Wang et al., 2010). In this study, pretreatmentof rice bean roots with SNP resulted in a much greater accumula-tion of Al in root apex (Fig. 4), which could be the main cause ofaggravated root growth inhibition in the presence of Al. This resultalso suggested that disturbance of homeostasis of endogenous NOconcentration interfered with physiological processes functioningin protecting Al from entering roots.

Some plant species have adapted well to Al-toxic environments,suggesting that plant species possess Al resistance mechanisms (Ko-chian et al., 2004; Taylor, 1991). Based on whether they operateapoplastically or symplastically, these resistance mechanisms havebeen grouped into external exclusion and internal tolerance mecha-nisms, respectively. In most cases, stimulated secretion of organicacid anions protecting root tip from the entry of Al is among the wellestablished Al exclusion mechanisms (Kochian et al., 2004). Forexample, it has been demonstrated that Al-induced citrate secretionfrom rice bean roots underlies Al exclusion and citrate is the onlyorganic acid anion whose response is based on the presence of Al(Yang et al., 2006). Thus, whether enhanced accumulation of Al byNO results from decreased citrate efflux was examined. The resultshowed that the SNP pre-treated plants during the Al treatment se-creted as much citrate as those treated with Al alone (Fig. 5). Thissuggests that (1) exogenous NO-induced excessive Al accumulationis not through a process of decreasing citrate efflux from roots and(2) NO is not involved in the process of Al-induced citrate efflux inrice bean.

Oxidative stress is one consequences of Al toxicity, leading tolipid peroxidation of the plasma membrane in plants (Cakmak andHorst, 1991; Yamamoto et al., 2001). In C. tora, NO-alleviated, Al-in-duced inhibition of root elongation was accompanied by alleviationof oxidative stress, correlating with a decrease in Al accumulation inroot apexes (Wang and Yang, 2005). However, these results showedthat pre-exposure of the seedlings to the NO donor enhanced theAl-induced production of MDA, an index of lipid peroxidation andplasma membrane injury in roots (Fig. 6). It was also found that pre-treatment of rice bean roots with SNP resulted in much more accu-mulation of Cd and Cu in root apex (data not shown). Thisdiscrepancy may be due to differences in the metal concentrationand the different plant species tested. For example, Cd-induced celldeath is associated with a transient increase of intracellular NO con-centration in Arabidopsis suspension culture (De Michele et al.,2009). However, NO acts as an antioxidant in barley aleurone layersand delays programmed cell death (Beligni et al., 2002).

NO signaling in plant stress responses is usually related to itscross-talk with the reactive oxygen species (ROS) (Wu and Wu,2008). For example, it has been proposed that the defense responsein plants results from the simultaneous and balanced production ofNO and ROS (Bright et al., 2006; Zaninotto et al., 2006). An imbal-ance of the O2

��/NO ratio could favor oxidative conditions butcould also interfere with the signal transduction pathways of thedefense mechanism against stress (Delledonne et al., 2001). Laxaltet al. (2007) reported that NO triggers ROS production by means ofthe lipid signaling system. In plants, accumulation of ROS can alsoactivate the enzymes eliminating ROS such as catalase and perox-idase. Such evidence may suggest a role of NO in the up-regulationof the isoforms of peroxidase at the activity level, although themechanism involved is ambiguous. Therefore, NO exacerbation ofAl-induced lipid peroxidation in rice bean could be the result of abreak down of the NO equilibrium in plant cells.

Recently, accumulating evidence has indicated that cell wallproperties are associated with Al resistance and/or toxicity (Horstet al., 2010). As one of the cell wall components, pectin with its neg-ative charge has been proposed to be fundamental to interactionwith Al (Blamley et al., 1993). The binding of Al can change cell wallstructure, making it more rigid, reducing cell expansion andmechanical extensibility. It was reported that pretreatment of ricewith SNP increased root cell wall pectin and hemicellulose content,thus more Cd was retained in root cell walls and alleviatedCd-induced inhibition of root elongation (Xiong et al., 2009). Theseresults induced us to examine whether pretreatment of rice beanresulted in changes of cell wall properties. However, it was foundherein that pretreatment with SNP had no effect on pectincontent (Fig. 7), indicating that this is not involved in increased

50 Y. Zhou et al. / Phytochemistry 76 (2012) 46–51

accumulation of Al in rice bean. Not only pectin content but its de-gree of methylation contributes to the presence of negative charges.In the present study, pretreatment of rice bean with NO resulted insignificant increase of PME activity (Fig. 7). As a consequence, the re-moval of methyl groups from pectin by PME caused the exposure ofmore carboxyl groups which have a high affinity for Al. Thus, it islikely that the increased accumulation of Al is the result of anenhanced degree of cell wall pectin demethylation.

4. Conclusion

Nitric oxide (NO) has been implicated in the adaptation ofplants to a variety of abiotic stresses, including Al toxicity.However, the functions of NO could be positive or negativedepending on the plant species, stress strength and duration. Hereexperimental evidence is presented that application of SNP aggra-vates Al toxicity in rice bean roots by affecting cell wall and plasmamembrane properties, and thus, afford a novel insight into thedestructive role of NO in mediating Al-induced toxicity in plants.

5. Experimental

5.1. Plant material and growth condition

Seeds of rice bean [Vigna umbellata (Thunb.) Ohwi & Ohashi‘Jiangnan’] were germinated and cultured according to a previousreport (Yang et al., 2006). For Al and SNP treatments, 3-d-old seed-lings were pretreated with 0, 0.2, 0.4, 0.6, 0.8 mM SNP (blank solu-tion is 0.5 mM CaCl2, pH 4.5) for 12 h and then cultivated in0.5 mM CaCl2 (pH 4.5) plus 0 or 25 lM AlCl3 for 24 h. For a time-course experiment, 3-d-old seedlings were pretreated with orwithout 0.4 mM SNP (blank solution is 0.5 mM CaCl2, pH 4.5) for12 h and then exposed to a 0.5 mM CaCl2 (pH 4.5) solution plus 0or 25 lM AlCl3 for different times. Root lengths were measuredwith a ruler before and after treatments.

5.2. Determination of NO content in roots

The endogenous levels of NO in roots were determined using4-amino-5-methylamino-20,70-difluorofluorescein diacetate (DAF-FM DA) probes and epifluorescence microscopy. Root tips wereincubated with 5 lM DAF-FM DA in dark for 30 min, washed threetimes in PBS (pH 7.4) and analyzed microscopically. Photomicro-graphs recorded are representative of three different experimentsin which at least 10 seedlings were analyzed for each treatment.

5.3. Determination of Al content in root apexes

The collected 1 cm root apices (10 apices were pooled into onesample) were rinsed briefly with deionized H2O thrice before beingplaced in a 1.5 ml Eppendorf tube with 1 ml of 2 M HCl (Osawa andMatsumoto, 2001). The Al content in root tips was extracted andmeasured by Inductively Coupled Plasma (ICP) spectrometry.

5.4. Collection and analysis of root exudates

After treatments, root exudates were collected and purifiedaccording to Zheng et al. (2005). The concentration of citrate wasanalyzed enzymatically (Yang et al., 2006).

5.5. Histochemical analyses

Plasma membrane integrity in the root apex was monitored byimmersing the roots in Evans blue solution (Yamamoto et al.,2001). Stained roots were observed under a microscope andphotographed.

5.6. Determination of lipid peroxidation

Malondialdehyde (MDA) content was determined by the thio-barbituric acid reaction (Tewari et al., 2006).

5.7. Cell wall fractionation and measurement

Cell wall materials and pectin were extracted according toZhong and Läuchli (1993). Uronic acid content in the pectin frac-tion was assayed colorimetrically using galacturonic acid as thestandard (Correa-Aragunde et al., 2008).

5.8. PME activity assay

For extraction of PME, 1 cm root apices (20 apices for each sam-ple) were homogenized and suspended in 1 M NaCl solution(pH 6.0). Then the homogenate was centrifuged at 23,000g for10 min at 4 �C and the supernatant was collected as PME sample.The incubation contained 100 lL of 200 mM PBS containing0.64 mg mL�1 of pectin, 10 lL of alcohol oxidase (AO) at 0.001units lL�1 and 50 lL of PME sample. After incubated for 10 minat 30 �C samples were added 200 lL of a 0.5 N NaOH solution con-taining 5 mg ml�1 purpald were added. After incubation at 30 �Cfor 30 min, 550 lL of water were added to give a final volume of1.0 ml. The absorbance was measured at 550 nm.

5.9. Statistical analysis

Each experiment was repeated at least three times. Data wasanalyzed by one-way ANOVA and the means were compared byDuncan’s multiple range test. Different letters on the histogramsindicate that the means were statistically different at the P < 0.05level.

Acknowledgements

This work was financially supported by a fund from the NaturalScience Foundation of China (30830076, 31071849), ZhejiangScience and Technology Bureau (2008C12005-2), ChangjiangScholarship and the Fundamental Research Funds for CentralUniversities.

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