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Plant Physiology and Biochemistry 63 (2013) 227e235

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Plant Physiology and Biochemistry

journal homepage: www.elsevier .com/locate/plaphy

Research article

Expression of a Medicago falcata small GTPase gene, MfARL1 enhancedtolerance to salt stress in Arabidopsis thaliana

Tian-Zuo Wang, Xiu-Zhi Xia, Min-Gui Zhao, Qiu-Ying Tian, Wen-Hao Zhang*

State Key Laboratory of Vegetation and Environmental Change, Institute of Botany, The Chinese Academy of Sciences, Beijing 100093, PR China

a r t i c l e i n f o

Article history:Received 30 October 2012Accepted 17 December 2012Available online 26 December 2012

Keywords:MfARL1Medicago falcataArabidopsis thalianaSalt stressSmall GTPasesNaþ accumulation

Abbreviations: ABA, abscisic acid; Arf, ADP-riboCaMV, cauliflower mosaic virus; CAT, catalase; GAPGEF, guanine nucleotide exchange factors; GFP, greenglucuronidase; MDA, malondialdehyde; MS, Murasreading frame; POD, peroxidase; RACE, rapid-amplreactive oxygen species; RT-qRCR, real-time quantidismutase; SSH, suppression subtractive hybridization* Corresponding author. Tel.: þ86 10 62836697; fax

E-mail addresses: tzwang@ibcas.ac.cn (T.-Z. W(X.-Z. Xia), zhaomingui@ibcas.ac.cn (M.-G. Zha(Q.-Y. Tian), whzhang@ibcas.ac.cn (W.-H. Zhang).

0981-9428/$ e see front matter � 2013 Elsevier Mashttp://dx.doi.org/10.1016/j.plaphy.2012.12.004

a b s t r a c t

To understand the role of small GTPases in response to abiotic stress, we isolated a gene encoding a smallGTPase, designated MfARL1, from a subtracted cDNA library in Medicago falcata, a native legume speciesin semi-arid grassland in northern China. The function ofMfARL1 in response to salt stress was studied byexpressing MfARL1 in Arabidopsis. Wild-type (WT) and transgenic plants constitutively expressingMfARL1 showed comparable phenotype when grown under control conditions. Germination of seedsexpressing MfARL1 was less suppressed by salt stress than that of WT seeds. Transgenic seedlings hadhigher survival rate than WT seedlings under salt stress, suggesting that expression of MfARL1 conferstolerance to salt stress. The physiological and molecular mechanisms underlying these phenomena wereelucidated. Salt stress led to a significant decrease in chlorophyll contents in WT plants, but not intransgenic plants. Transgenic plants accumulated less amounts of H2O2 and malondialdehyde than theirWT counterparts under salt stress, which can be accounted for by the higher catalase activities, loweractivities of superoxide dismutase, and peroxidase in transgenic plants than in WT plants. Transgenicplants displayed lower Naþ/Kþ ratio due to less accumulation of Naþ than wild-type under salt stressconditions. The lower Naþ/Kþ ratio may result from less accumulation of Naþ due to reduced expressionof AtHKT1 that encodes Naþ transporter in transgenic plants under salt stress. These findings demon-strate that MfARL1 encodes a novel stress-responsive small GTPase that is involved in tolerance to saltstress.

� 2013 Elsevier Masson SAS. All rights reserved.

1. Introduction

Being sessile organism, plants are frequently exposed tochanging environment that results in holdback of growth anddevelopment. Plants have evolved various mechanisms to avoidand/or alleviate the adverse effects of abiotic stress. Upon exposureof plants to a stressed environment, numerous molecular andphysiological processes are altered [1]. Environmental signals areoften sensed by plants and transduced to activate downstream

sylation factor; Arl, Arf-like;, GTPase-activating proteins;fluorescent protein; GUS, b-

hige and Skoog; ORF, openification of cDNA end; ROS,tative PCR; SOD, superoxide; WT, wild-type.: þ86 10 62592430.ang), xiaxiuzhi@ibcas.ac.cno), tianqiuying@ibcas.ac.cn

son SAS. All rights reserved.

targets through intricate signaling network, in which transcriptionfactors and signal-transducing GTPases play key roles. Small GTPaseproteins aremonomeric G proteins that are related to the subunit ofheterotrimeric G proteins with molecular masses of 20e40 kDa.Similar to heterotrimeric G proteins, small GTP-binding proteinscan cycle between GTP and GDP-bound states [2]. Once stimulatedby upstream signals, the GDP-bound inactive form can be con-verted into the GTP-bound active form by guanine nucleotideexchange factors (GEF), thus activating downstream targets.Conversely, GTPase-activating proteins (GAP) can catalyze the GTPform to the GDP form [3].

Members of small GTPase share several common structuralfeatures, including four guanine nucleotide-binding domains andan effector-binding domain [2]. However, small GTPases alsoexhibit a remarkable diversity in both structure and function. Theyare further divided into four main subfamilies in plants: Rab, Rop,Ran and Arf [4]. A diverse function of plant small GTPases has beenreported in the literature. For instance, the Rab and Arf GTPasefamilies have been suggested to regulate distinct steps ofmembrane trafficking [5,6]. Ran GTPases are involved in mediationof transport of proteins and RNA across the nuclear envelope [7].

Fig. 1. Phylogenetic tree analysis of small G proteins. Phylogenetic analysis was per-formed using MEGA 5 software. Accession numbers are as follows: AtRabG3e(Q9XI98.1), OsRab5a (CAC19792.1), OsRab7 (AAO67728.1), McRab5b (CAA06922.1),SsRab2 (AAD30658.1), AtRop1 (AEE78776.1), AtRop2 (Q38919.1), AtRop5 (AEE86595.1),OsRacB (AAT84075.1), TaRan1 (AAM08320.1), OsRan2 (BAB82438.1), AtARLB1(AAA87882.1), AtSARA1a (AEE28409.1), TITAN5 (AAM22961.1), MtARF1 (CAI29265.1),MfARL1 (JX292786).

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235228

Rop GTPases regulate several physiological processes, ranging frompollen growth and root hair development to abscisic acid (ABA)response [8]. These findings highlight that small GTPasesmay act asimportant molecular switches associated with plant signaling.

In recent years, the involvements of small GTPases in responseof plants to abiotic stresses have been investigated. Several genes inthe Rab GTPase family have been shown to be responsive to abioticstress, including response of SsRab2 to water stress [9], OsRab7 tocold stress [10] and AtRabG3e to salt/osmotic stress [11]. Amongthem, OsRab7 is differentially regulated by several environmentalstimuli including cold, salt, dehydration and phytohormone ABA[10]. In addition, OsRab7 is localized to the vacuolar membrane,suggesting that OsRab7 may be implicated in a vesicular transportto the vacuole in plant cells [10]. In Arabidopsis thaliana, transgenicplants of overexpressing AtRabG3e exhibited accelerated endocy-tosis in roots, leaves and protoplasts. In addition, the transgenicplants also showed increased tolerance to salt stress due to reducedaccumulation of reactive oxygen species [11]. These results implythat vesicle trafficking may play an important role in plant adap-tation to stress. Furthermore, expression of AtRop2 renders seedgermination less sensitive to ABA [8], suggesting that Rop GTPasesnegatively regulate ABA signaling. A recent study demonstratedthat overexpression of OsRAN2 in rice confers tolerance to coldstress by regulating cell cycle [12]. In contrast to cold stress, over-expression OsRAN2 in rice renders transgenic plants hypersensitiveto salinity and osmotic stress [13].

ADP-ribosylation factor (Arf) is an important regulator ofmembrane-trafficking pathways, and was initially identified due totheir ability to stimulate the ADP-ribosyltransferase activity ofcholera toxin A [14]. The production of three types of vesicle coatproteins (COPI, COPII and clathrin) is related to Arf GTPases [15].The protein sequences of Arf-like (Arl) GTPases are highly similar tothat of Arf GTPases. Knockout of an Arl GTPase, TITAN, leads todramatic alterations in mitosis and cell cycle control during seeddevelopment in Arabidopsis [16]. However, there has been noreport to evaluate the role of Arf GTPases in response and adapta-tion of plants to abiotic stresses.

Medicago falcata is a native legume species that widely occurs inthe areas of Russia, Mongolia, and northern China. M. falcata isdistinguished by its outstanding capacity to tolerate abiotic stress,and has been widely used as a general source of germplasm forbreeding forage of alfalfa. Several recent studies have investigatedthe physiological and molecular mechanisms by which M. falcatatolerates to cold [17,18], drought stress [19] and phosphorus defi-ciency [20,21].

In this study, we isolated an Arl GTPase gene, designatedMfARL1from M. falcata, a native legume species exhibiting great toleranceto abiotic stress in Inner Mongolia in northern China using themethod of suppression subtractive hybridization. We furtherfunctionally characterized MfARL1 by expressing this gene in Ara-bidopsis. Our results demonstrated that expression of MfARL1 inArabidopsis led to an enhanced tolerance to salt stress due to lessaccumulation of Naþ and H2O2 and MDA.

2. Results

2.1. Isolation and sequence analysis of MfARL1 cDNA

On the basis of the segment sequence of MfARL1, RACE wasperformed to obtain the full-length cDNA. The assembled resultsshowed that MfARL1 had a 618 bp open reading frame encodinga protein of 205 amino acid residues, with a calculated molecularmass of about 23 kDa. Sequence comparison revealed that theputative protein was highly homologous to the small GTP-bindingproteins (Fig. 1, Supplemental Fig. S1), and accordingly was

designated MfARL1. The sequence data have been deposited inGenBank (accession No. JX292786).

MfARL1 protein contains 6 conserved functional domains.Similar domains have been found in many reported small GTP-binding proteins (Supplemental Fig. S1). Domains IeIV areinvolved in GTP-binding, and domain E is an effector region whichcan be recognized by GTPase-activation proteins (GAPs), and isessential for regulation of GTPases. Domain P, the C-terminal motif,is a prenylation site and important for attachment of small GTP-binding proteins to membrane [2,3].

Phylogenetic trees based on the full-length amino acidsequences of MfARL1 proteins were constructed using the MEGA 5software (Fig. 1). The resulting trees contained four families, Rab,Ran, Rop and Arf. According to the phylogenetic trees, MfARL1protein had the highest similarity with AtARLB1, a small GTP-binding protein of Arf family in A. thalianawith unknown function.

2.2. Expression pattern and subcellular localization of MfARL1

The MfARL1 was isolated from the salt stress suppressionsubtractive hybridization library. To validate the response ofMfARL1 to various abiotic stresses, RT-qRCR was performed. Asshown in Fig. 2A, transcripts of MfARL1 were found to be accu-mulated after 2 h of exposure to salt stress, and peaked after 5 h ofsalt stress. Expression of MfARL1 was also up-regulated by treat-ments with low temperature (4 �C) and osmotic stress (20%PEG6000) (Fig. 2B). Transcripts of MfARL1 were detected in roots,stems, leaves, flowers and pods under non-stressed conditions,with the expression being greatest in leaves, followed by roots andstems, lowest in flowers (Fig. 2C).

To examine the subcellular localization of MfARL1, an openreading frame of MfARL1 was fused to the C-terminus of the GFPreporter gene of pEGAD [22]. The recombinant constructs of theMfARL1-GFP fusion gene and GFP alone were introduced intotobacco leaf epidermal cells by Agrobacterium injection. The resultsshowed that the MfARL1eGFP fusion protein was specificallylocalized in the cell membrane, whereas GFP alone showed ubiq-uitous distribution in the whole cell (Fig. 3).

Fig. 2. Real-time quantitative PCR analysis of the expression of MfARL1 in shoot tissue of M. falcata in response to 200 mM NaCl for varying periods (A), PEG-induced drought stress(20% PEG6000) and 4 �C cold for 5 and 10 h (B). The basal expression levels of MfARL1 in various unstressed tissues (C). Data are means� SE of three biological replicates.

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235 229

2.3. Expression of MfARL1 enhanced tolerance to salt stress

To study the function of MfARL1, an overexpressing construct,under the control of a Cauliflower mosaic virus (CaMV) 35Spromoter, was transformed into A. thaliana (Col-0) and five trans-genic lines were obtained. The transgenic lines were confirmed byhygromycin selection, b-Glucuronidase (GUS) staining, and RT-qPCR. Compared with the untransformed WT Arabidopsis, theabundance of MfARL1 transcript was much higher in the over-expressing lines (Supplemental Fig. S2). Three independent trans-genic lines (Line 1, 2 and 3) were used for further physiologicalstudies throughout this paper. Given that the expression of MfARL1was induced by salt stress in M. falcata, we investigated the role ofMfARL1 played in response to salt stress by comparing the perfor-mance of transgenic plants expressing MfARL1 and wild-typeseedlings under salt stress.

The effect of salt stress on germination of WT and transgenicseeds expressing MfARL1 was investigated. Exposure of both WTand transgenic seeds to NaCl reduced their germination rate, andno difference in seed germination between WT and the transgenicseeds in the absence of NaCl in the incubation medium wasobserved. However, germination of MfARL1-expressing transgenicseeds was less suppressed by NaCl than that of WT seeds (Fig. 4A).For instance, seed germination rate of the threeMfARL1-expressinglines was 92.2%, 67.8 and 75.6% when incubated in 1/2 MS medium

Fig. 3. Subcellular localization of MfARL1. Green fluorescent protein (A, B, C) and GFP fusion win the materials and methods. Images were taken in the dark field for green fluorescence (Aimages (C, F) are of (A, B) and (D, E), respectively.

supplemented with 100 mMNaCl, while germination rate for wild-type seeds was 50.0% under the same conditions, suggesting thatexpressing MfARL1 confers transgenic seeds more tolerant to saltstress during seed germination.

In addition to seed germination, the effect of NaCl on seedlinggrowth was also examined by exposure of four-week-old seedlingsto 1/2 MS medium supplemented with and without 200 mM NaClfor 7 days. The MfARL1-expressing plants and WT plants exhibitedcomparable phenotype in the absence of NaCl, while the transgenicplants appeared healthier thanWT in the presence of 200 mMNaCl(Fig. 4B).

Moreover, the MfARL1-expressing seedlings exhibited highersurvival rate under salt stress when grown in the agar plate. Forexample, the survival rate of the threeMfARL1-expressing lines was30.8%, 21.8 and 39.2% after treatment with 150 mM NaCl for 14days, respectively, while the survival rate forWT seedlings was only10.0% when challenged by the same salt stress (Fig. 5).

2.4. Expression of MfARL1 conferred transgenic plants higherchlorophyll content, less H2O2 and MDA under salt stress

A decrease in chlorophyll content is often observed when plantssuffer from salt stress [23]. There were significant decreases incontents of both chlorophyll a and chlorophyll b ofWT plants whenchallenged with NaCl (Fig. 6A and B). In contrast, no significant

ith MfARL1 (D, E, F) were transiently expressed in tobacco epidermal cells as described, D), while the outline of the cell (B, E) was photographed in a bright field. The merged

Fig. 4. Effect of salt stress on seed germination and phenotypes of transgenic and WT plants. (A) Germination rate of WT and transgenic seeds on 1/2 MS medium with or without100 mM NaCl after 48 h of imibition. (B) Phenotypes of seedlings after irrigated by 1/2 MS medium supplemented with and without 200 mM NaCl for 7 days. Data are means� SE ofthree biological replicates. Asterisks represent statistically significant differences between WT and transgenic lines. *P� 0.05, **P� 0.01.

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235230

changes in both chlorophyll a and chlorophyll b contents wereobserved for the three transgenic lines expressing MfARL1 whentreated with the same NaCl concentration (Fig. 6A and B).

To investigate whether the transgenic plants differ from WT intheir sensitivity to oxidative stress associated with salt stress, H2O2and MDA contents in WT and transgenic plants grown in theabsence and presence of NaCl were measured. As shown in Fig. 7,

Fig. 5. The survival state of seedlings in the absence (A) or presence (B) of NaCl, andthe survival rate under salt stress (C), after two-day-old seedlings of WT and transgenicplants were transfer to 1/2 MS agar plate supplemented with and without 150 mMNaCl for 14 days. Data are means� SE of three biological replicates. Asterisks representstatistically significant differences between WT and transgenic lines. *P� 0.05,**P� 0.01.

there were significant increases in H2O2 contents in both WT andtransgenic plants upon exposure to solution containing NaCl.However, the salt stress-induced accumulation of H2O2 in the threetransgenic lines was significantly less than in WT plants. A similardifferential increase in MDA content in WT and the transgenicplants was also observed when the seedlings were treated withNaCl. For instance, MDA content in WT and the transgenic plantswas comparable in the absence of NaCl in the incubation solution,and MDA content was increased by 319%, 181%, 66% and 73% in WTand the three transgenic lines after exposure to solution containingNaCl, respectively (Fig. 7B).

We further explored the physiological mechanisms by whichthe transgenic lines accumulate less H2O2 andMDA thanWT plantsunder salt stress by measuring the activities of antioxidantenzymes. The CAT activity of transgenic plants showed little changein response to NaCl treatment, while wild-type plants exhibitedsignificant decreases in CAT activity by the same treatment(Fig. 8A). In contrast to CAT activity, SOD activity in WT and thetransgenic plants were increased by salt stress with the increasebeing significantly higher in WT than in the transgenic plants(Fig. 8B). A marked increase in activity of POD was also observed inboth WT and the transgenic plants with the increase in WT beingapprox. 2.3 times greater than in the transgenic Line 3 (Fig. 8C).

2.5. Naþ content of transgenic plants under salt stress

Naþ is toxic to plant cells when accumulated in excess amountsunder salt stress, leading to damage to plants. To further elucidatethe physiological mechanisms underlying the enhanced toleranceof the MfARL1-expressing plants, the effects of salt stress on Naþ

and Kþ concentrations in shoots of WT and the transgenic plantswere investigated. No differences in both Naþ and Kþ concentra-tions in shoots of WT and the transgenic plants were found whenthey were grown in the control medium without NaCl (Fig. 9A andB). There were marked increases in Naþ concentrations in both WTand the transgenic plants when they were exposed to solutioncontaining 200 mM NaCl. However, Naþ concentration in the threetransgenic lines was significantly lower than that inWT (Fig. 9A). Incontrast to Naþ, Kþ concentrations in both WT and the transgenicplants were equally reduced when NaCl was present in the growthmedium (Fig. 9B). Accordingly, an increase in Naþ/Kþ ratio in bothWT and the transgenic lines was observed under salt stress. Forinstance, Naþ/Kþ ratio in WT plants was increased from 0.06 to2.39, while Naþ/Kþ ration in the transgenic Line 3 was increased to1.45 when these plants were treated with NaCl (Fig. 9C). Further-more, the effects of salt stress on the expression of AtHKT1 genethat encodes a Naþ influx transporter in both WT and the trans-genic lines were examined at transcriptional levels by real-timequantitative. As shown in Fig. 9D, transcripts of AtHKT1 in thetransgenic plants were significantly less than in WT under

Fig. 6. The contents of chlorophyll a (A) and chlorophyll b (B) of transgenic plants and WT in the absence and presence of NaCl. Four-week-old seedlings irrigated with or without200 mM NaCl for 5 days were used in the experiment. Data are means� SE of three biological replicates. Asterisks represent statistically significant differences between WT andtransgenic lines. *P� 0.05, **P� 0.01.

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235 231

conditions of salt stress. This result suggests that MfARL1 maydown-regulate the expression of AtHKT1, thus contributing toenhanced tolerance of MfARL1-expressing lines due to less accu-mulation of Naþ in shoots of the transgenic plants.

3. Discussion

There is increasing evidence demonstrating that small GTPaseproteins are involved in mediation of numerous physiologicalprocesses, ranging from pollen growth and root hair developmentto response to abiotic stress [8,12]. Signal transduction pathwaymediated by GTP-binding protein-coupled receptor is involved inmany physiological processes in plants [24]. Small GTP-bindingprotein, acting as molecular switches, can be “activated” by GTPand “inactivated” by the hydrolysis of GTP to GDP [4]. Among theGTPases, a few studies have demonstrated that Arf GTPases areinvolved in the regulation of membrane trafficking [5,6]. In recentyears, several small GTPases have been reported to be involved inresponse of plants to abiotic stresses. For example, overexpressionof AtRabG3e in A. thaliana leads to less sensitive to salt stress thanWT plants. Further studies revealed that Naþwas accumulated intothe vacuolar in the transgenic plants, thus minimizing toxic effectof Naþ in the cytosol and facilitating water uptake due to effectiveosmoregulation [11]. In rice, it has been shown that OsRAN2 playsdifferent roles when plants exposed to cold, salinity and osmoticstress [12]. The expression of OsRAN2 is up-regulated by cold stress,and overexpression of OsRAN2 in rice confers tolerance to coldstress [12]. However, there has been no report to evaluate the roleof Arf GTPases in response and adaptation of plants to abioticstresses. In the present study, we isolated a novel Arl gene, designed

Fig. 7. Effect of salt stress on contents of H2O2 (A) and malondialdehyde (B) of transgenic plawere tested in the experiment. Data are means� SE of three biological replicates. Asterisks r**P� 0.01.

MfARL1, that encodes a small GTPase protein from a legume pastureplant native to the semi-arid grassland in northern China, froma SSH cDNA library associated with salt stress in M. falcata. MfARL1have high similarity to AtARLB1 in terms of their sequences, but thefunction of AtARLB1 remains to be characterized. Our resultsshowed that expression of MfARL1 in Arabidopsis seedlingsconferred the transgenic plants more tolerant to salt stress. To thebest knowledge of authors, this is the first report showing theinvolvement of a plant Arl GTPase protein in response to salt stress.We further demonstrated that the enhanced tolerance of transgenicplant expressing MfARL1 to salt stress could be accounted for byreduced accumulation of Naþ, and H2O2 as well as MDA under saltstress.

Photosynthetic activity is suppressed by salt stress, and thereduction in photosynthetic activity can be accounted for by thedecline in chlorophyll content [23]. In the present study, we foundthat WT plants and MfARL1-expressing plants differed in theirchlorophyll contents in response to salt stress. For example, nochanges in both chlorophyll a and chlorophyll b contents ofMfARL1-expressing transgenic plants were observed when theywere exposed to solution containing NaCl (Fig. 6). In contrast,a significant decrease in photosynthetic activity and chlorophyllcontents was found inWT plants when challenged by the same saltstress (Fig. 6). The higher chlorophyll content in the MfARL1-expressing transgenic plants would allow plants to maintainphotosynthetic rate, thus facilitating normal growth of plantsunder salt stress.

Plants suffering from salt stress often display symptoms ofoxidative damage as indicated by marked accumulation of reactiveoxygen species such as H2O2 [25]. MDA has beenwidely recognized

nts and WT. Four-week-old seedlings irrigated with or without 200 mM NaCl for 5 daysepresent statistically significant differences between WT and transgenic lines. *P� 0.05,

Fig. 8. Effect of salt stress on activity of antioxidant enzymes, including catalase (A),superoxide dismutase (B), and peroxidase (C). Four-week-old seedlings irrigated withor without 200 mM NaCl for 5 days were tested in the experiment. Data are mean-s� SE of three biological replicates. Asterisks represent statistically significant differ-ences between WT and transgenic lines. *P� 0.05, **P� 0.01.

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235232

as a parameter for lipid peroxidation [26]. As shown in Fig. 7, theamounts of H2O2 and MDA in the transgenic plants were signifi-cantly less than in WT plants when challenged by salt stress, sug-gesting the transgenic plants were more tolerant to oxidative stressunder salt stress. A similar less accumulation of H2O2 in thetransgenic Arabidopsis overexpressing AtRabG3ewas also observed[11]. The less accumulation of H2O2 under salt stress may resultfrom an enhanced capacity for scavenging ROS. Under salt stress,the activity of CAT inWT plants wasmarkedly suppressed, while no

effect of salt stress on the activity of CAT was observed in thetransgenic plants (Fig. 8A). The higher CAT activity in the transgenicplants would effectively catalyze H2O2 to nontoxic H2O and O2, thusminimizing oxidative stress associated with accumulation of H2O2under salt stress (Fig. 8A). Moreover, the higher H2O2 contents inWT plants under salt stress may activate the activities of SOD andPOD, thus leading to the observed the higher activities of SOD andPOD in WT plants under salt stress (Fig. 8B and C).

To elucidate the mechanism underpinning the enhanced toler-ance of MfARL1-expressing plants to salt stress, we also measuredNaþ contents in shoots of both WT and transgenic plants in theabsence and presence of 200 mMNaCl in the medium. It was foundthat expression of MfARL1 led to reductions in Naþ contents inshoots under salt stress (Fig. 9A). In contrast to Naþ, there was nodifference in Kþ concentrations in bothWTand transgenic plants inthe absence or presence of NaCl in the growth medium (Fig. 9B).Consequently, the transgenic plants exhibited a lower Naþ/Kþ ratiothan WT under salt stress (Fig. 9C). The lower Naþ/Kþ ratio isbeneficial for plants to maintain physiological processes under saltstress, thus contributing to the enhanced tolerance of the MfARL1-expressing plants to salt stress. In plants, influx of Naþ into thecytosol and efflux of Naþ from the cytosol to apoplast and vacuolesare mediated by several transporters, including SOS1 [27], HKT1[28] and NHX1 [29]. Mutations in the AtHKT1 gene suppressed sos3mutant phenotypes, and analysis of ion contents in the sos3hkt1mutant demonstrated that AtHKT1 is involved in mediation of Naþ

influx into plant cells [28]. In addition, mutations that disruptedAtHKT1 function could also suppress sensitivity of sos1 and sos2 tosalt stress [30]. These results indicate that suppressing theexpression of AtHKT1 can enhance the ability of salt tolerance inArabidopsis. In the present study, we found that the expressionlevels of AtHKT1 in the transgenic plants were significantly less thanin WT under conditions of salt stress (Fig. 9D). The less expressionof AtHKT1 in MfARL1-expressing may prevent Naþ influx into plantcells, thus conferring plants more tolerance to salt stress by alle-viating toxic effect of Naþ on plant cells. The reduced accumulationof Naþ in the transgenic plants under salt stress is in contrast to thetransgenic Arabidopsis plants overexpressing AtRabG3e in whichNaþ is transported into the vacuoles, thus the overall Naþ content inthe transgenic plants is not reduced under salt stress [11].

In summary, expression of MfARL1 conferred the enhancedtolerance of the transgenic Arabidopsis seedlings to salt stress bymaintaining relatively higher chlorophyll contents, suppressedaccumulation of H2O2 and MDA, and reduced Naþ influx into plantcells via down-regulation of AtHKT1 expression. Therefore, ourfindings highlight that MfARL1, encoding a small GTPase protein,may act as an important regulator involved in response to saltstress.

4. Materials and methods

4.1. Plant material and treatments

Seeds of M. falcata L. cv Humeng were soaked in concentratedsulfuric acid for approximately 8 min, and then thoroughly rinsedwith tap water. After chilled at 4 �C for 2 d, the seeds were grown ina pot (diameter 10 cm) filled with vermiculite: peat soil (2:1) undercontrolled conditions (26 �C day/20 �C night,14-h photoperiod, and50% relative humidity) as described by Wang et al. [31].

Four-week-old seedlings were treated with varying abioticstresses. Salt and osmotic stresses were achieved by exposing 4-week-old seedlings to 1/2MSmedium supplementedwith 200 mMNaCl and 20% PEG6000 for different periods, respectively. For coldtreatments, the seedlings were exposed to 4 �C for 5 h and 10 h.Shoots were sampled for isolation of RNA and quantitative PCR.

Fig. 9. Effect of 200 mM NaCl on Naþ content (A), Kþ content (B), Naþ/Kþ ratio (C) and expression of AtHKT1 (D) in shoots of WT and transgenic plants. Four-week-old seedlingsirrigated with and without 200 mM NaCl for 5 days were used in the experiment. Data are means� SE with three replicates. Asterisks represent statistically significant differencesbetween WT and transgenic plants in response to salt stress between WT and transgenic lines. *P� 0.05, **P� 0.01.

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235 233

4.2. Cloning the full-length sequence of MfARL1 cDNA

To identify gene fragments in response to abiotic stresses,suppression subtractive hybridization (SSH) was used to constructa cDNA library. SSH was carried out using a PCR-Select cDNASubtraction Kit (Clontech) according to the manufacturer’sinstruction and the methods of Diatchenko et al. [32]. Briefly,shoots of M. falcata treated with solution containing 200 mM NaCland 0 mM NaCl (control) for 5 h were collected for RNA isolation,and then RNA was reverse-transcribed to cDNA. After digestion,two rounds of hybridization were performed to separate saltresponsive cDNA. After enriched by PCR, these cDNAwas ligated topGEM-T Easy vector (Promega), and then transformed intoEscherichia coli. The selected positive clones were sequenced usingABI 3730xl sequencer.

A positive clone was identified from the subtracted cDNA libraryof M. falcata. On the basis of the sequence of the clone, rapid-amplification of cDNA end (RACE) was performed to get the full-length cDNA using GeneRacer� RLM-RACE Kit (Invitrogen). Toobtain 50 end, amplify the first-strand cDNA using a reverse gene-specific primer (50-CCC TCC AGT CCA AGC ACT TGA TGG-30) andGeneRacer� 50 primer (homologous to the GeneRacer� RNAOligo). Each reaction contained 4.5 mL of 10 mM GeneRacer�50

promer, 1.5 mL of 10 mM reverse gene-specific primer, 1 mL RTtemplate, 5 mL of 10�Pfx amplification buffer, 1.5 mL dNTP (10 mMeach), 0.5 mL Platinum Pfx DNA polymerase (2.5 U/mL) and 1 mLMgSO4 (50 mM) in a final volume of 50 mL. The thermal cycle usedwas 94 �C for 2 min, 5 cycles of 94 �C for 30 s, 72 �C for 2 min, 5cycles of 94 �C for 30 s, 70 �C for 2 min, 22 cycles of 94 �C for 30 s,65 �C for 30 s, 68 �C for 2 min, and 68 �C for 10 min. A forwardgene-specific primer (50-ACT GGA GGG TTT TCT GCT TCT GCC-30)and GeneRacer� 30 primer were used to obtain 30 end of this geneby the same method. The complete full-length sequence was thenassembled by results of 50-RACE and 30-RACE using the DNAMANsoftware.

4.3. RNA isolation and real-time quantitative PCR

Total RNA was isolated using RNAiso Plus reagent (TaKaRa) andtreated with RNase-free DNase I (Promega). The total RNA wasreverse-transcribed into first-strand cDNA with PrimeScript� RTreagent Kit (TaKaRa).

Real-time quantitative PCR (RT-qPCR) was performed using ABIStepone Plus instrument. Gene-specific primers used for RT-qPCRwere designed using software Primer Premier 5, and were asfollows: forMfARL1 (50-AAG TGC TTG GAC TGGAGGGTT T-30 and 50-CAC CTA ATC GGA CTC CTT TCA C-30), for AtHKT1 (50-CAT CTG GCTCCT AAT CCC T-30 and 50-ACC ATA CTC GTC ACG CTT T-30), MtActingene (accession No. BT141409) and AtActin11 (accession No.NM_112046) were used as internal control with primers (50-ACGAGC GTT TCA GAT G-30 and 50-ACC TCC GAT CCA GAC A-30) and (50-TGT TCT TTC CCT CTA CGC T-30 and 50-CCT TAC GAT TTC ACG CTC T-30). The primer sequence of MtActin has been used previously[18,33], and AtActin11 was always used as a reference gene tonormalize the expression [34,35]. Each reaction contained 10.0 mLof SYBR Green Master Mix reagent (TOYOBO), 0.8 mL cDNA samples,and 1.2 mL of 10 mMgene-specific primers in a final volume of 20 mL.The thermal cycle used was 95 �C for 2 min, 40 cycles of 95 �C for30 s, 55 �C for 30 s, and 72 �C for 30 s. The relative expression levelwas analyzed by the comparative CT method using the MicrosoftExcel 2010 as described by Livak and Schmittgen [36].

4.4. Subcellular localization of MfARL1

The open reading frame (ORF) of MfARL1 was ligated to the C-terminal of green fluorescent protein (GFP) in pEGAD [22]. Thisconstruct was transformed to Agrobacterium tumefaciensGV3101 byelectroporation. The Agrobacterium was infiltrated to leaves ofNicotiana tabacum as described by Sparkes et al. [37]. After 48 h ofculture, GFP fluorescence in transformed tobacco epidermal cellswas observed under a Zeiss LSM 510 confocal microscope.

T.-Z. Wang et al. / Plant Physiology and Biochemistry 63 (2013) 227e235234

4.5. Transformation and regeneration of Arabidopsis

The ORF of MfARL1 was amplified with the primers 50-GGC GGATCC ATG TTT TCG TTA TTT TAT G-30 (BamHI site underlined) and 50-CTT GAG CTC CTAGGC AGGACC TGGACC C-30 (SacI site underlined).The EcoRI/BamHI e digested product was inserted the downstreamof cauliflower mosaic virus 35S (CaMV 35S) promoter of pSN1301[38]. After pSN1301: MfARL1 was transformed to A. tumefaciensEHA105 by electroporation, transformation of A. thaliana (Col-0)was performed using the A. tumefaciens e mediated floral dipmethod as described by Zhang et al. [39]. Several independent linesof the T3 generation were randomly chosen for further physiolog-ical studies.

4.6. Determination of tolerance to salt stress

To determinate seed germination rate in the absence andpresence of NaCl in the medium, sterile seeds were pointed to 1/2MS plate (0.8% agar) supplemented with and without 100 mMNaClat 25 �C. There were 40 seeds in each plate and the seeds wereconsidered to be germinated at the emergence of the plumule andscored. Seed germination was recorded after 48 h of incubation.

The effect of NaCl on seedling growth was also examined byirrigation of four-week-old seedlings using 1/2 MS medium sup-plemented with and without 200 mM NaCl for 7 days.

To determinate the survival rate of wild-type and transgenicplants, 40 two-day-old seedlings each replicate were transfer to 1/2MS agar plate supplemented with and without 150 mM NaCl for 14days. Seedlings that survived the salt treatments can be distin-guished from the dead plants, and the survival rate was determinedby counting the survived seedlings.

4.7. Determination of chlorophyll, H2O2 and malondialdehyde(MDA)

Four-week-old seedlings that were irrigated with or without200 mMNaCl for 5 days harvested for determination of chlorophyll,H2O2 and MDA contents. Chlorophyll was extracted and deter-mined following protocols used by Arnon [40]. Hydrogen peroxidewas measured as described by Alexieva et al. [41]. MDA content inleaves was determined following the protocol described by Krameret al. [42].

4.8. Determination of antioxidant enzyme activity

Seedlings irrigated with 200 mM NaCl for 5 days were used fordetermination of antioxidant enzymes. Leaves were sampled fordetermination of antioxidant enzyme activity. Catalase (CAT)activity was assayed using the method by Aebi [43]. Superoxidedismutase (SOD) activity was measured spectrophotometricallybased on inhibition in the photochemical reduction of nitrobluetetrazolium described by Giannopotitis and Ries [44]. Peroxidase(POD) was determined through measuring the oxidation of guaia-col [45].

4.9. Determination of Naþ and Kþ concentration

Four-week-old seedlings of transgenic and WT plants wereirrigated with 1/2 MS solution containing 200 mM NaCl for 5 days,washed with ultrapure water for five times, fixed at 105 �C for10 min and baked at 80 �C for 24 h to constant weight. As much as50 mg of dry material was weighed and placed in a digestion tube,and 5 mL of nitric acid and 1 mL of hydrogen peroxide were addedfor digestion. The digested fluid volume was finalized to 100 mLand ion content was measured by ICP-AES (Thermo).

4.10. Statistical analyses

All data were analyzed by analysis of variance using SPSS17.0statistics program. Statistical differences are referred to as signifi-cant when P� 0.05.

Acknowledgments

This work was supported by State Key Laboratory of Vegetationand Environmental Change (80006F2018).

Appendix A. Supplementary data

Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.plaphy.2012.12.004.

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