REGULAR ARTICLE

Effects of 1-aminocyclopropane-1-carboxylate (ACC)deaminase-overproducing Sinorhizobium meliloti on plantgrowth and copper tolerance of Medicago lupulina

Zhaoyu Kong & Bernard R. Glick & Jin Duan &

Shulan Ding & Jie Tian & Brendan J. McConkey &

Gehong Wei

Received: 9 December 2014 /Accepted: 1 March 2015 /Published online: 15 March 2015# Springer International Publishing Switzerland 2015

AbstractBackground and aims Rhizobia typically produce alower level of ACC deaminase compared with free-living plant growth-promoting bacteria. While the en-dogenous rhizobial ACC deaminase is important inlegume nodulation, it is not sufficient to protect hostplants against environmental stresses. The main goal ofthis study was to assess the effects of a geneticallyengineered Sinorhizobium meliloti strain overproducingACC deaminase, and its symbiotic performance inMedicago lupulina under copper stress conditions.Methods The engineered strain was transformed withan exogenous acdS gene by triparental conjugation. Aplant growth assay was conducted to assess its plant

growth promotion ability under copper stress condi-tions. The expressions of antioxidant genes in theseplants were analyzed using quantitative real-time PCR.Results Plants nodulated with the engineered strainshowed a greater dry weight, a decreased ethylene levelin roots, a higher total copper uptake but a lower level ofcopper translocation to aerial parts, as compared withthe plants nodulated with the wild-type strain undercopper stress conditions. These results were positivelycorrelated with higher expression of antioxidant genesin the roots of these plants exposed to severe copperstress.Conclusions The engineered strain could improve plantgrowth as well as copper tolerance of M. lupulina, andenhance the antioxidant defense system.

Keywords ACC deaminase . Symbiosis . Excesscopper . Antioxidant responses . Phytoremediation

Introduction

Copper (Cu) is an essential micronutrient for normalgrowth and development of plants, as it is directlyinvolved in a variety of metabolic pathways, includingrespiration, photosynthesis, protein synthesis, cell wallmetabolism and lignification, ethylene sensing and ox-idative stress protection. These properties make the cu-pric ion indispensable for the life of plants; however,they are also the reason why the copper ion could bestrongly toxic for plants when it is present in excess.Over the centuries, as a result of industrial production,

Plant Soil (2015) 391:383–398DOI 10.1007/s11104-015-2434-4

Responsible Editor: Katharina Pawlowski.

Electronic supplementary material The online version of thisarticle (doi:10.1007/s11104-015-2434-4) contains supplementarymaterial, which is available to authorized users.

Z. Kong : S. Ding :G. Wei (*)State Key Laboratory of Soil Erosion andDryland Farming onthe Loess Plateau, College of Life Sciences, Northwest A&FUniversity, Yangling, Shaanxi 712100, Chinae-mail: weigehong@nwsuaf.edu.cn

Z. Kong : B. R. Glick : J. Duan : B. J. McConkey (*)Department of Biology, University of Waterloo, 200University Avenue West, Waterloo, Ontario N2L 3G1,Canadae-mail: mcconkey@uwaterloo.ca

J. TianCollege of Horticulture, Nanjing Agricultural University,Nanjing 210095, China

sewage irrigation and the extensive use of chemicalfertilizers and pesticides, copper has contaminated nu-merous soils and waters, especially in industrial areas(Figueira et al. 2002; Lu et al. 2009; Srinivasa Gowdet al. 2010). One of the main constraints that limit plantgrowth in heavy metal-contaminated soils is nutrientdeficiency, especially nitrogen and phosphorus (Pajueloet al. 2011). Legumes are well known for their ability toform root nodules with compatible rhizobial strains,within which nitrogen fixation take place, therefore,the legume-Rhizobium symbiosis is of great importanceboth ecologically and agriculturally (Graham and Vance2003). Recently, concerns about environmental pollu-tion have stimulated interest in the use of the legume-Rhizobium symbiosis as a tool for bioremediation ofheavy metals (Pajuelo et al. 2011).

It is generally believed that copper ions can interactwith membrane proteins and lead to lipid peroxidationthrough the photosynthetic electron transport system.Moreover, high metal concentrations can cause oxida-tive stress in plants resulting in the overproduction ofreactive oxygen species (ROS). ROS are toxic, but alsocan participate in signaling events. To counter ROS,plant cells utilize at least two different antioxidant de-fense systems, which can alleviate oxidative damage byscavenging the ROS. The first defense system is in-volved directly with keeping active forms of oxygen ata low level, including superoxide dismutase (SOD),catalase (CAT) and peroxidase (POD), and the secondtype of system is involved in the regeneration of oxi-dized antioxidants, such as ascorbate peroxidase (APX)and glutathione reductase (GR) (Mittler 2002). An in-crease of oxidative stress and ROS accumulation hasbeen reported in plants grown in the presence of excessCu (Jouili and El Ferjani 2003; Tewari et al. 2006;Zhang et al. 2008). Furthermore, the antioxidant defenseresponse against Cu stress was found to be less effectivein white lupin nodules than those of soybean, whichresulted in greater oxidative stress, inhibition of nodu-lation processes and reduction in nitrogen fixation(Sánchez-Pardo et al. 2012).

Ethylene, a key regulator in many aspects of plantgrowth and development, is involved in plant responseto abiotic stresses including drought, salt, freezing, hightemperature, and toxic metals (Bari and Jones 2009;Czarny et al. 2006; Gamalero and Glick 2012; Glick2010). Ethylene was found to be involved in the inhib-itory action of excess Cu on the leaf and root growth ofdicotyledonous plants (Maksymiec and Krupa 2007).

Furthermore, ethylene is also induced by rhizobia dur-ing nodulation initiation and adversely affects the for-mation and functioning of nodules (Ding and Oldroyd2009; Nandwal et al. 2007). Rhizobia that express ACCdeaminase can promote nodulation of host legumesthrough conversion of the immediate ethylene precursorACC intoα-ketobutyrate and ammonia, thus decreasingethylene levels that could otherwise inhibit nodulation(Ma et al. 2003a, 2004; Uchiumi et al. 2004). However,rhizobia produce lower levels of ACC deaminase com-pared with free-living plant growth-promoting bacteria(Glick et al. 2007). Thus, while rhizobia have sufficientenzyme activity to lower ACC levels that occur duringnodulation, they are unable to sufficiently lower theACC levels that occur as a consequence of abiotic stresssuch as the presence of heavy metals. From this per-spective, the approach of reducing the deleterious eth-ylene levels in plants by increasing the activity of ACCdeaminase of symbiotic rhizobia may be an effectivestrategy to improve plant growth and resistance to heavymetal stress.

Positive effects of expression of an exogenous ACCdeaminase gene in different rhizobia genera, and theirabilities to nodulate legume host plants have previouslybeen demonstrated (Conforte et al. 2010;Ma et al. 2004;Nascimento et al. 2012). Moreover, it was recentlyshown that the expression of ACC deaminase gene inMesorhizobium spp. improved the growth of chickpeaplants under salt stress (Brígido et al. 2013). On theother hand, mutants of the ACC deaminase gene (acdS)and its regulatory gene (lrpL) of Rhizobiumleguminosarum bv. viciae 128C53K, neither of whichexpressed ACC deaminase, showed reduced nodulationefficiency compared to the native strain (Ma et al.2003a).

In order to further improve the performance of le-gume-Rhizobium symbiosis in the presence of excessCu, an ACC deaminase-overproducing S. meliloti strainwas constructed through transformation with an exoge-nous acdS gene that encodes a high level of ACCdeaminase in Pseudomonas putida UW4 (Ma et al.2003b). It was of considerable interest to: (1) study theeffect of Cu stress on the growth ofMedicago plants andthe symbiotic performance of the wild-type and theACC deaminase-overproducing strains under excessCu conditions; (2) evaluate the Cu uptake and transportbehavior in plants from roots to aerial parts in thepresence of bacterial ACC deaminase; (3) determinethe effects of ACC deaminase overproduction on the

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antioxidant defense responses in plants during Cu stress.The results of this investigation indicate that theengineered rhizobial strain that could express a higherlevel of ACC deaminase activity played a positive rolein improving adaption of legume host plants to excessCu stress.

Materials and methods

Bacterial growth conditions

The plasmids and bacterial strains used in this work arelisted in Table 1. The Cu-resistant bacterium S. melilotistrain CCNWSX0020 was isolated from the root nod-ules of M. lupulina plants growing in lead-zinc minetailings in China (Fan et al. 2010). This strain wasdeposited in the Agricultural Culture Collection of Chi-na and named ACCC19736. The complete genomesequence of S. meliloti CCNWSX0020 has been report-ed, including a number of predicted protein-codinggenes involved in Cu resistance (Li et al. 2012).P. putida UW4 was originally isolated from the rhizo-sphere of common reeds, based on its ability to utilizeACC as a sole source of nitrogen (Glick et al. 1995). TheP. putida UW4 strain, growing in TSB medium (DifoLaboratories, Detroit, MI, USA) at 30 °C, was used asthe co-inoculant with S. melilotiCCNWSX0020 in plantexperiments. S. meliloti strains were grown at 28 °Cwith shaking at 150 rpm in tryptone yeast extract (TY)medium (5 g tryptone, 3 g yeast extract, and 0.7 g

CaCl2·2H2O per liter; pH 7.2) or in modified M9 min-imal medium (Robertsen et al. 1981). The growth me-dium for the S. meliloti derivative strain was supple-mentedwith tetracycline (10μgmL−1). TheEscherichiacoli strains were grown in Luria-Bertani medium (Greenand Sambrook 2012) at 37 °C with shaking at 150 rpm.For E. coli strains carrying pRKACC, the medium wassupplemented with 15 μg mL−1 of tetracycline, whilefor E. coli strains containing pRK600, 25 μg mL−1 ofchloramphenicol was used.

Triparental conjugation

The plasmid pRKACC, carrying the P. putida UW4acdS gene, was used to transform the S. melilotiCCNWSX0020 strain by triparental conjugation as de-scribed by Nascimento et al. (2012). To confirm thesuccessful transformation of plasmid pRKACC toS. meliloti cells, the plasmid was extracted from theputatively transconjugant S. meliloti cells following themanufacturer’s instructions (GeneJET PlasmidMiniprep Kit, Fermentas Life Sciences, Vilnius, Lithu-ania). Plasmid pRKACC was digested with restrictionendonucleases enzymes HindIII and KpnI, and visual-ized by ethidium bromide staining following agarose gelelectrophoresis. The transconjugant S. meliloti strainwas further confirmed by amplification of the nitroge-nase (nifH) gene from its genomic DNA. The isolationof genomic DNAwas conducted using Wizard® Geno-mic DNA Purification Kit (Promega, Madison, WI,USA).

Table 1 Plasmids and bacterial strains used in this study

Plasmids and strains Relevant characteristics Reference or source

pRKACC The broad-host-range plasmid pRK415 carrying Pseudomonasputida UW4 acdS gene and its flanking regions, Tcr

Shah et al. (1998)

pRK600 pRK2013 npt::Tn9, Cmr Finan et al. (1986)

E. coli strains

DH5α SupE44ΔlacU169(φ80lacZΔM15)hsdR17 recA1 endA1gyrA96 thi-1 relA1

Green and Sambrook (2012)

MT616 Mobilizing strain, containing helper plasmid pRK600 Finan et al. (1986)

Sinorhizobium meliloti strains

CCNWSX0020 Sequenced strain with high symbiotic effectiveness andcopper resistance, Ampr

Fan et al. (2010); Li et al. (2012)

CCNWSX0020 (pRKACC) CCNWSX0020 strain containing pRKACC, Ampr, Tcr This study

Pseudomonas putida UW4 Well-studied plant growth-promoting bacterium, with theability to synthesis ACC deaminase

Glick et al. (1995)

Ampr ampicillin resistant; Cmr chloramphenicol resistant; Tcr tetracycline resistant; Gmr gentamicin resistant

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ACC deaminase activity assay

The wild-type S. meliloti CCNWSX0020 and thetransconjugant S. meliloti CCNWSX0020 (pRKACC)were tested for ACC deaminase activity as describedpreviously by Nascimento et al. (2012). S. meliloti 5356and S. meliloti 1021 were used as negative controls.R. leguminosarum bv. viciae 128C53K, Rhizobiumhedysari ATCC43676 and P. putida UW4 were usedas positive controls (Duan et al. 2009; Ma et al. 2003a).The ACC deaminase activity of P. putidaUW4was alsodetermined as described by Penrose and Glick (2003).

Plant growth conditions

M. lupulina seeds (provided by Gansu Agricultural Uni-versity, China) were surface sterilized by treatment with75 % v/v ethanol for 2 min followed by 10 min in2.5 % v/v commercial bleach. After the seeds werethoroughly rinsed with several changes of sterile dis-tilled water, they were planted in plastic pots (10 cmdiameter) filled with 200 g of sterilized Sunshine4™mix (Premier Horticulture, St. Catharines, Ontario, Can-ada). The ingredients of the plant growth medium in-clude Canadian sphagnum peat moss, coarse grade per-lite, dolomitic lime and Sun Gro’s long-lasting wettingagent. The seedling medium was supplemented withcopper in the form of CuSO4 to produce a concentrationof Cu (II) of 200 mg kg−1 (moderate Cu stress) or400 mg kg−1 (severe Cu stress). The concentration ofCu applied was based on preliminary experiments (datanot shown). After it was thoroughly mixed with the Cusolution, the soil medium was packed into the plasticpots and allowed to equilibrate for 1 week. The seed-lings were then maintained in a plant growth incubatorat 25 °C in the light at 200 μmol m−2 s−1 for 16 h, and21 °C in the dark for 8 h. Nitrogen-free mineral nutrientsolution was used to water the plants when necessary(approximately 200 ml each week). Ten seedlings wereplanted in each pot and four replicates were conductedfor each treatment. After 7 days, seedlings were inocu-lated with cell suspensions of wild-type S. melilotiCCNWSX0020 , S . me l i lo t i CCNWSX0020(pRKACC), or a co-inoculant cell suspension ofS. meliloti CCNWSX0020 with P. putida UW4, respec-tively. The bacterial cultures were standardized to anoptical density of 0.8 at 600 nm, and 1ml of the bacterialcell suspension was inoculated onto each seedling.Seedlings inoculated with the same amount of sterile

distilled water were regarded as the non-inoculatedcontrol.

Plant growth, nodulation, N content and nitrogenaseactivity

Plants were harvested at 40 days after inoculation. Thedry weight, root length, number of nodules, nodule freshweight, nitrogenase activity and N content were mea-sured and recorded. The above-ground plant tissues androots were separated, washed in distilled water and driedat 65 °C for 48 h before the dry weight was determined.Total N content in aerial plant organs and roots weremeasured by Kjeldahl method on a Kjeltec™ 8400Analyzer Unit (FOSS-Tecator AB, Hoganas, Sweden).Nitrogenase activity in nodules was measured by theacetylene reduction assay as described by Weaver andDanso (1994). Acetylene and ethylene were quantifiedthrough a HP-AL/M column (30 m, I.D. 0.53 nm,15 μm; J&W Scientific, Folsom, CA, USA) using aShimadzu GC-17A gas chromatograph (Shimadzu Cor-poration, Kyoto, Japan) and a flame ionization detector.Heliumwas used as the carrier gas with a flow rate set at6 ml/min and 36 kPa total pressure. The injector, columnand detector temperatures were 120, 100 and 150 °C,respectively. Ethylene elutes after 1.9 min, and acety-lene elutes after 3.0 min. The amount of ethylene pro-duced by each nodule sample (0.20 g) was standardizedwith a standard curve of ethylene.

Ethylene measurements

Ethylene biosynthesis rate was measured from theabove-ground plant tissues and roots of plants at 40 daysafter inoculation. Four plant samples per treatment wereharvested and the aerial parts and roots were detached,with a fresh weight of approximately 1.5–2.0 g persample (weighed after ethylenemeasurements). Accord-ing to preliminary experiments, the ethylene biosynthe-sis rate was steady for about 15 min after excision (datanot shown). Each sample was placed into a 100 ml glasscylinder for about 15 min to avoid measurement ofethylene production due to wounding, and then closedwith rubber serum stoppers and sealed with glycerin.After incubation for an additional 2 h, 5 ml of the air inthe glass cylinder was withdrawn with a syringe andinjected into a Shimadzu GC-17A gas chromatograph(Shimadzu Corporation, Kyoto, Japan) equipped with aflame ionization detector. The ethylene production by

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each sample was standardized using a 0.001–0.005 ppmstandard ethylene curve.

Cu content

The above-ground plant tissues and roots harvested at40 days after inoculation were rinsed three times withsterilized deionized distilled water (ddH2O) to removeany loosely bound Cu2+, and then dried at 65 °C for48 h. Aliquots of precisely 0.2000 g powdered planttissue samples were digested with an acid mixture (3:1HNO3: HClO4) and the Cu content was analyzed byatomic absorption spectrophotometry (Z-5000; Hitachi,Tokyo, Japan).

To evaluate the transport behavior of Cu from plantroots to above-ground plant tissues under excess Cuconditions, the translocation factor was analyzed (Singhand Agrawal 2007). The translocation factor was calcu-lated using the following formula:

Translocation factor ¼ Cuab=Cur

where Cuab and Cur are Cu content in above-groundplant tissues and roots, respectively.

Recovery of S. meliloti CCNWSX0020 (pRKACC)from nodules

The recovery of S. meliloti CCNWSX0020 (pRKACC)from nodules was conducted in order to assess thestability of plasmid pRKACC in nodules during theplant growth. Nodules were collected from plant rootsand carefully washed with running water, then surfacesterilized by immersion in 75 % ethanol for 2 min,followed by 10 min in 2.5 % v/v commercial bleach,and finally rinsed eight times in sterile distilled water.Surface-sterilized nodules were crushed on a sterileplate, and the bacteria were isolated by streaking thecrushed suspensions on yeast mannitol agar (YMA)medium (3 g yeast extract, 10 g mannitol, 0.5 gKH2PO4, 0.2 g MgSO4, 0.1 g NaCl and 18 g agar perliter; pH 7.0–7.2) plates containing Congo red, but noantibiotics. For each Cu treatment, one hundred singlecolonies were subsequently tested for the presence ofplasmid pRKACC on YMA medium with 10 μg mL−1

tetracycline. Plasmid stability was assessed by countingthe number of colonies that were resistant to tetracy-cline. For each Cu treatment, plasmid stability tests were

performed on three replicates and for two independentbiological experiments.

Quantitative real-time PCR analysis

Total RNA was separately extracted from aerial partsand roots of plants at 40 days after inoculation usingTRIzol reagent (TAKARA, Dalian, China), followingthe manufacturer’s instructions. One microgram of totalRNAwas treated with the PrimeScript™ RT reagent Kitwith gDNA Eraser (Perfect Real Time, TAKARA, Da-lian, China) in order to remove residual DNA and re-verse transcribe RNA to cDNA, according to the man-ufacturer’s instructions.

Quantitative real-time PCR was performed using theCFX96 real-time PCR system (Bio-Rad, Hercules CA,USA). SYBR Green real-time PCR assay was per-formed in a total volume of 20 μl, containing 10 μl ofFastStart Essential DNA Green Master (Roche AppliedScience, Indianapolis, IN, USA), 0.2 μM of each spe-cific primer, and 2μl of cDNA solution. For each primerset, a no-template water control was also included. ThePCR conditions consisted of denaturation at 95 °C for10 min, followed by 40 cycles of denaturation at 95 °Cfor 10 s, annealing at 60 °C for 15 s, and extension at72 °C for 20 s. A melting curve was run after each PCRreaction to confirm the uniformity of each amplifiedproduct. RT-PCR amplification for each cDNA samplewas performed in triplicate wells. The mRNA levels ofthe target genes were normalized against EF1-α(Yahyaoui et al. 2004). Specific primer pairs for thegenes cytosolic CuZnSOD, plastid CuZnSOD, plastidFeSOD, mitochondrial MnSOD, CAT, cytosolic APXand cytosolic GR were those reported by Naya et al.(2007). The specific primer pairs for acdS gene, de-signed using the Primer3 software, were as follows: 5′-GGCAAGGTCGACATCTATGC-3′ and 5′-GGCTTGCCATTCAGCTATG-3′. Quantification of gene expres-sion was performed as 2-ΔCt as previously described(Schmittgen and Livak 2008). Student’s t-test was per-formed on the qRT-PCR data to determine whether thedifference is statistically significant.

Statistical analysis

All of the statistical analyses were performed with SPSSv. 16.0 (SPSS Inc., USA) for Windows statistical soft-ware package. Physiological and biochemical measure-ments were analyzed by one-way analysis of variance

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(ANOVA) followed by Duncan test (p<0.05). All ob-tained data was analyzed using the Origin Pro v8.0(Origin Lab, Hampton, USA) to create figures.

Results

Transformation, ACC deaminase activity and recoveryof pRKACC

The wild-type S. meliloti strain was successfully trans-formed with plasmid pRKACC, carrying the ACC de-aminase gene from the plant growth-promoting bacteri-um P. putida UW4. Plasmid pRKACC was extractedfrom S. meliloti (pRKACC) cells and digested withrestriction enzymes HindIII and KpnI. After visualiza-tion with ethidium bromide following agarose gel elec-trophoresis, two fragments of approximately 3.8 and10 kb were observed as expected (Supplemental,Fig. S1a). This indicates that the plasmid pRKACCwas successfully transformed into S. meliloti cells inan intact form. The transconjugant S. meliloti strainwas further confirmed by amplification of the symbioticgene nifH, which was approximately 0.7 kb (Supple-mental, Fig. S1b).

The ACC deaminase activity was measured in bothwild-type and S. meliloti (pRKACC) strains (Fig. 1).The wild-type strain showed a moderate level of ACCdeaminase activity in comparison with the negativecontrols. On the other hand, the S. meliloti (pRKACC)

strain showed a significantly higher level of ACC de-aminase, 12.6-fold higher than the wild-type strain, butlower than the ACC deaminase activity of P. putidaUW4.

In the two experiments in which the stability ofplasmid pRKACCwas tested, 80–84% of the reisolatedbacteria under control conditions carried the plasmid,while 90–94 % and 89–93 % of the reisolated bacteriaunder 200 and 400mg kg−1 Cu2+ conditions retained theplasmid (Fig. 2). In each experiment, a significant lowerloss rate of the plasmid under Cu stress conditions thanunder control conditions was observed.

Effects of different Cu2+ concentrations on plantgrowth, nodulation and N content

Analysis of the effects of different Cu levels on plantgrowth, nodulation and N content was performed in theplants inoculated with the wild-type S. meliloti. Thebiomass production and nodulation of plants showedno significant reduction by the treatment with200 mg kg−1 Cu2+, compared to controls, while a con-centration of Cu2+ at 400 mg kg−1 induced a significantinhibition in plant growth and nodulation (Figs. 3 and5). The inhibition in plant biomass induced by400 mg kg−1 Cu2+ was more pronounced in roots thanin above-ground plant tissues (Fig. 3). There were nostatistically significant differences found in the dryweight of above-ground plant tissues in the presenceof 400 mg kg−1 Cu2+; however, the dry weight of roots

Fig. 1 ACC deaminase activitiesin various rhizobial strains andP. putida UW4. The numberslabeled indicate the mean±S.E. ofthree independent biologicalexperiments. Bars representstandard error values. WTSM:wild-type S. meliloti CCNWSX0020; RL128C53K:R. leguminosarum bv. viciae128C53K; RHATCC43676:R. hedysari ATCC43676;SM5356: S. meliloti 5356;SM1021: S. meliloti 1021; SMACC: S. meliloti CCNWSX0020(pRKACC); UW4: P. putidaUW4

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were reduced by 36.2 % compared to controls. Rootlength was significantly reduced by 24.3 and 41.4 % inthe plants treated with 200 and 400 mg kg−1 Cu2+,compared to controls (Fig. 4). Number of nodules, nod-ule fresh weight and nitrogenase activity were signifi-cantly reduced by treatment with 400 mg kg−1 Cu2+,

while in the treatment with 200 mg kg−1 Cu2+, nosignificant changes were observed in the number ofnodules or in nitrogenase activity, but a significant in-crease in nodule fresh weight were observed (Fig. 5).The N content in both aerial parts and roots of plantswas not significantly changed by treatment with 200 or400 mg kg−1 Cu2+ as compared to controls (Fig. 6).

Effect of bacterial ACC deaminase overproductionon M. lupulina plants growth and nodulation

The biomass production was significantly increased inthe plants inoculated with S. meliloti (pRKACC) or co-inoculated with wild-type S. meliloti and P. putidaUW4in comparison with the plants inoculated with wild-typeS. meliloti (Fig. 3). The dry weight of aerial parts ofS. meliloti (pRKACC)-inoculated plants were enhancedby 31.6 % in the presence of 200 mg kg−1 Cu2+, and by54.4 % in the presence of 400 mg kg−1 Cu2+, as com-pared with the plants inoculated with wild-typeS. meliloti. The dry weight of roots was also significant-ly increased by 34.6 and 39.4 % in the S. meliloti(pRKACC)-inoculated plants treated with 200 and400 mg kg−1 Cu2+. Similarly, a significant increasewas also observed in the biomass production of plantsco-inoculated with S. meliloti and P. putida UW4. Root

Fig. 2 The stability of plasmid pRKACC in bacteria recoveredfrom nodules ofMedicago lupulina plants under control, moderate(200 mg kg−1) or severe (400 mg kg−1) Cu stress conditions. Thevalues indicate the mean±S.E. of three replicates for each exper-iment. Bars carrying different letters are significantly different(Duncan test, p<0.05) from the plasmid pRKACC under controlconditions

Fig. 3 Dry weight of above-ground plant tissues (white bars) androots (grey bars) of Medicago lupulina plants under control,moderate (200 mg kg−1) or severe (400 mg kg−1) Cu stressconditions. NIC=Non-inoculated controls; WTSM=Inoculatedwith wild-type S. meliloti strain; SMACC=Inoculated withS. meliloti CCNWSX0020 (pRKACC) strain; SM+UW4=Co-

inoculated with wild-type S. meliloti and P. putida UW4. Thevalues indicate the mean±S.E. of four replicates. Bars carryingdifferent letters are significantly different (Duncan test, p<0.05)from the non-inoculated plants. *Statistically significant difference(p<0.05) for the plants inoculated with wild-type S. meliloti undercontrol and excess Cu conditions

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length of S. meliloti (pRKACC)-inoculated plants andco-inoculated plants treated with 400 mg kg−1 Cu2+ wassignificantly increased compared with those of plantsinoculated with the wild-type strain under the sameconditions (Fig. 4). On the other hand, dry weight androot length of plants inoculated with S. meliloti(pRKACC) showed no significant differences undercontrol conditions, as compared with the plants inocu-lated with the wild-type strain (Figs. 3 and 4). Inocula-tion with S. meliloti (pRKACC), as well as co-inoculation with S. meliloti and P. putida UW4, showeda remarkable increase in fresh weight of nodules in thepresence of 200 mg kg−1 Cu2+, as compared with the

plants inoculated with the wild-type strain; while asignificant increase occurred only in the co-inoculatedplants in the presence of 400 mg kg−1 Cu2+ (Fig. 5a). Nosignificant alterations were observed in the number of

Fig. 4 Root length of Medicago lupulina plants under control,moderate (200 mg kg−1) or severe (400 mg kg−1) Cu stressconditions. NIC=Non-inoculated controls; WTSM=Inoculatedwith wild-type S. meliloti strain; SMACC=Inoculated withS. meliloti CCNWSX0020 (pRKACC) strain; SM+UW4=Co-in-oculated with wild-type S. meliloti and P. putidaUW4. The valuesindicate the mean±S.E. of four replicates. Bars carrying differentletters are significantly different (Duncan test, p<0.05) from thenon-inoculated plants. *Statistically significant difference(p<0.05) for the plants inoculated with wild-type S. meliloti undercontrol and excess Cu conditions

Fig. 5 Number of nodule (a), nodule fresh weight (b) and nitro-genase activity (c) of inoculated M. lupulina plants under control,moderate (200 mg kg−1) or severe (400 mg kg−1) Cu stressconditions. NIC=Non-inoculated controls; WTSM=Inoculatedwith wild-type S. meliloti strain; SMACC=Inoculated withS. meliloti CCNWSX0020 (pRKACC) strain; SM+UW4=Co-in-oculated with wild-type S. meliloti and P. putida UW4. +=Notdetected. The values indicate the mean±S.E. of four replicates.Bars carrying different letters are significantly different (Duncantest, p<0.05) from the wild-type S. meliloti-inoculated plants.*Statistically significant difference (p<0.05) for the plants inocu-lated with wild-type S. meliloti under control and excess Cuconditions

b

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nodules or in the nitrogenase activity of plants inoculat-ed with S. meliloti (pRKACC) under Cu stress condi-tions as compared with those inoculated with the wild-type strain treated with same dose of Cu (Fig. 5b and c).Furthermore, the N content of plants inoculated withS. meliloti (pRKACC) under Cu stress conditions wassimilar to that of plants inoculated with the wild-typestrain under the same conditions (Fig. 6).

Cu content

The Cu content in both aerial parts and roots of plantsinoculated with either strain was significantly elevatedwith increased levels of Cu in the medium, an effect thatwas more pronounced in roots than in aerial plant tissues(Fig. 7a). The Cu content in above-ground plant tissuesof S. meliloti (pRKACC)-inoculated plants and co-inoculated plants were significantly reduced as com-pared with the plants inoculated with the wild-typestrain in the presence of 200 mg kg−1 Cu2+, while nosignificant changes occurred in the presence of400 mg kg−1 Cu2+. Furthermore, a significant increasewas observed when comparing the Cu content in theroots of S. meliloti (pRKACC)-inoculated plants or co-inoculated plants with that of plants inoculated with thewild-type strain in the presence of 400 mg kg−1 Cu2+.Plant biomass and Cu content were calculated to showthe total amount of Cu uptake in each plant (Fig. 7b).The results showed that the total Cu uptake in both aerialparts and roots of S. meliloti (pRKACC)-inoculatedplants and co-inoculated plants were significantly

increased as compared with the plants inoculated withthe wild-type strain in the presence of excess Cu.Concerning the transport behavior of Cu from roots toaerial parts of plants, the translocation factor of bothS. meliloti (pRKACC)-inoculated plants and co-inoculated plants increased under control conditions,compared with that of plants inoculated with the wild-type strain. On the contrary, the translocation factor ofco-inoculated plants decreased by 54.6 % when grownin the presence of 200 mg kg−1 Cu2+, in comparisonwith the plants inoculated with wild-type S. melilotistrain, although the difference was not statistically sig-nificant for the plants inoculated with S. meliloti(pRKACC) (Fig. 7c). Furthermore, a significant reduc-tion in the translocation factor of both S. meliloti(pRKACC)-inoculated plants and co-inoculated plantstreated with 400 mg kg−1 Cu2+ was observed, comparedwith that of plants inoculated with the wild-type strain.

Ethylene production

The presence of excess Cu induced a significant in-crease in ethylene production in both aerial parts androots of plants inoculated with wild-type S. meliloti(Fig. 8). On the other hand, the ethylene production inabove-ground plant tissues of plants inoculated withS. meliloti (pRKACC) or co-inoculated with wild-typeS. meliloti and P. putida UW4 showed a decrease com-pared to non-inoculated plants, under control and Custress conditions. Furthermore, the amount of ethylenebiosynthesis by the roots inoculated with S. meliloti

Fig. 6 N content of above-groundplant tissues (white bars) and roots(grey bars) of Medicago lupulinaplants under control, moderate(200 mg kg−1) or severe(400 mg kg−1) Cu stressconditions. NIC=Non-inoculatedcontrols; WTSM=Inoculated withwild-type S. meliloti strain; SMACC=Inoculated with S. melilotiCCNWSX0020 (pRKACC)strain; SM+UW4=Co-inoculatedwith wild-type S. meliloti andP. putida UW4. The valuesindicate the mean±S.E. of fourreplicates. Bars carrying differentletters are significantly different(Duncan test, p<0.05) from thenon-inoculated plants

Plant Soil (2015) 391:383–398 391

(pRKACC) and co-inoculated with wild-type S. melilotiand P. putida UW4 was significant decreased by 48.9and 37.9 % in the presence of 200 mg kg−1 Cu2+, and by22.8 and 17.1 % in the presence of 400 mg kg−1 Cu2+,compared with the plants inoculated with the wild-typestrain. No significant changes in ethylene productionwere found in the aerial parts of plants inoculated withS. meliloti (pRKACC) compared with the plants inocu-lated with wild-type S. meliloti under excess Cuconditions.

Expression analyses by quantitative real-time PCR

acdS gene: To evaluate the effect of severe Cu stress onacdS gene expression in symbiotic conditions, a quan-titative RT-PCR of the exogenous acdS gene in the

plants inoculated with S. meliloti (pRKACC) was per-formed. The mRNA level of this gene was increasedfourfold in roots in the presence of 400 mg kg−1 Cu2+

(4.02±0.27, n=3), compared with the roots under con-trol conditions, while the expression of the acdS gene inabove-ground plant tissues was not detected.

Antioxidant genes: The expression changes ofmRNAs encoding various antioxidant genes(CuZnSODc, CuZnSODp, FeSOD, MnSOD, CAT, APXand GR) involved in plant’s defense system in bothaerial parts and roots of the plants inoculated withS. meliloti (pRKACC), compared to the plants inoculat-ed with the wild-type strain, were analyzed using quan-titative real-time PCR (Fig. 9). As shown in Fig. 9a,inoculation with S. meliloti (pRKACC) induced theexpressions of CuZnSODp, FeSOD and CAT in above-

Fig. 7 Cu content (a), total Cuuptake (= dry weight×Cucontent) (b) and translocationfactor (c) of above-ground planttissues (white bars) and roots(grey bars) of Medicago lupulinaplants under control, moderate(200 mg kg−1) or severe(400 mg kg−1) Cu stressconditions. NIC=Non-inoculatedcontrols; WTSM=Inoculated withwild-type S. meliloti strain;SMACC=Inoculated withS. meliloti CCNWSX0020(pRKACC) strain;SM+UW4=Co-inoculated withwild-type S. meliloti and P. putidaUW4. The values indicate themean±S.E. of four replicates.Bars carrying different letters aresignificantly different (Duncantest, p<0.05) from the non-inoculated plants. *Statisticallysignificant difference (p<0.05)for the plants inoculated withwild-type S. meliloti under controland excess Cu conditions

392 Plant Soil (2015) 391:383–398

ground plant tissues under control conditions, but haslittle effect on expression of CuZnSODc, APX and GR.Moreover, the expressions of CuZnSODc, FeSOD, CATand APX were up-regulated in above-ground plant tis-sues in the presence of severe Cu stress, but the expres-sions of CuZnSODp, MnSOD and GR were down-reg-ulated. On the other hand, these antioxidant genesshowed different expression profiles in roots (Fig. 9b).S. meliloti (pRKACC) induced a significant increase inthe expressions of CuZnSODc, FeSOD, MnSOD, APXand GR in the absence of Cu, and of CuZnSODc,CuZnSODp, FeSOD, MnSOD, CAT, APX and GR inthe presence of Cu stress.

Discussion

The genetically engineered bacterial strain S. meliloti(pRKACC), which showed up to 12.6-fold greater ACCdeaminase activity when compared with the wild-typestrain, was tested for its plant growth-promoting char-acteristics and Cu tolerance. There were no significantalternations observed in terms of IAA production (Sup-plemental, Table S1), siderophore synthesis, or Cu tol-erance of this bacterium (Supplemental, Fig. S2, S3).According to our knowledge, rhizobia typically have

only 2–5 % of the ACC deaminase activity found infree-living plant growth-promoting bacteria. We doubtwhether the ACC deaminase geneminus (acdS-) mutantwould have an obvious effect on plant growth comparedto the wild-type strain, especially in the presence ofexcess Cu stress. Thus, it is not the presence or absenceof ACC deaminase gene that is being emphasized in thisstudy, rather the relative amounts of ACC deaminaseactivity in the wild-type S. meliloti CCNWSX0020compared to either the engineered strain S. melilotiCCNWSX0020 (pRKACC) or the co-inoculated strain.

Severe Cu stress significantly reduced the overallgrowth and nodulation of Medicago plants inoculatedwith the wild-type strain. The effect of heavy metals onthe legume-Rhizobium symbiosis has been studied ex-tensively (Balestrasse et al. 2006; Kopittke et al. 2007;Pajuelo et al. 2008; Wani et al. 2007, 2008a, b). Duringdifferent symbiotic stages, nodulation is generally moresensitive to heavy metal inhibition than the shoot or rootgrowth of legumes (Gupta et al. 2007), although thesensitivity varies among legume species, metal types,as well as experimental conditions (Pajuelo et al. 2011).A significant inhibition of nodulation was also observedin this work, however, the N content inMedicago plantsgrown under moderate or severe Cu stress showed nosignificant alternations as compared with that of plants

Fig. 8 Ethylene production of above-ground plant tissues (whitebars) and roots (grey bars) of Medicago lupulina plants undercontrol, moderate (200 mg kg−1) or severe (400 mg kg−1) Cu stressconditions. NIC=Non-inoculated controls; WTSM=Inoculatedwith wild-type S. meliloti strain; SMACC=Inoculated withS. meliloti CCNWSX0020 (pRKACC) strain; SM+UW4=Co-

inoculated with wild-type S. meliloti and P. putida UW4. Thevalues indicate the mean±S.E. of four replicates. Bars carryingdifferent letters are significantly different (Duncan test, p<0.05)from the non-inoculated plants. *Statistically significant difference(p<0.05) for the plants inoculated with wild-type S. meliloti undercontrol and excess Cu conditions

Plant Soil (2015) 391:383–398 393

grown under control conditions. This might be becausesmaller plants need less nitrogen. Moreover, since noexternal N supply was added in this work, the N contentin plants reflects the level of N fixation in root nodules.It is more likely that the wild-type Cu-resistant strainS. meliloti CCNWSX0020 is able to survive underexcess Cu, and subsequently promote a normal levelof plant nitrogen.

Plant growth under Cu stress conditions was signif-icantly enhanced when plants were inoculated with theACC deaminase-overproducing strain, or with the co-incolation of native S. meliloti and P. putida UW4,compared with plants inoculated with native

S. meliloti. The positive effects of the AcdS-overproducing strain on plant growth under Cu stressare also supported by the observation that the acdS genewas up-regulated in the roots as a result of severe Custress. However, plant growth under control conditionsshowed no significant differences when plants wereinoculated with the ACC deaminase-overproducingstrain or the wild-type strain. This may be due to thepresence of the endogenous ACC deaminase activity. Ahigher loss rate of the plasmid under control conditionsthan under Cu stress conditions could also account forthese results (Fig. 2). These results are consistent withthe previous observations that significant differences in

Fig. 9 Differentially expressedtranscripts of antioxidant genes inthe above-ground plant tissues (a)and roots (b) of plants inoculatedwith S. meliloti CCNWSX0020(pRKACC) compared to those ofplants inoculated with wild-typeS. meliloti under control andsevere Cu stress conditions. Thevalues indicate the mean±S.E. ofthree replicates. Asterisks indicatesignificant differences accordingto Student’s t-test (p<0.05)

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symbiotic performance between native and acdS-trans-formed Mesorhizobium strains were only found undersalinity (Brígido et al. 2013). These findings demon-strate that the beneficial effect of ACC deaminase-overproducing strain on plant growth is more pro-nounced when plants are grown under stress conditionsin which the ethylene production can be increased todeleterious levels. Although an increase was found innodule fresh weight under moderate Cu stress, theAcdS-overproducing strain did not promote a signifi-cant number of nodules or nitrogenase activity ofMedicago plants under Cu stress conditions. According-ly, no significant changes were found in the N content ofeither aerial parts or roots. Thus, overproduction ofACC deaminase improved plant growth under excessCu stress via mechanisms other than improved N nutri-tion, which in agreement with the findings of otherrecent studies (Brígido and Glick 2014; Reichman2007). The improvement in plant growth was morepronounced with the co-inoculation of nativeS. meliloti and P. putida UW4. The improvement ob-tained in the plant growth of the co-inoculated plants isprobably due to the higher ACC deaminase activity inP. putidaUW4 contributing to a higher alleviation of thenegative effects of ethylene. Moreover, the plantgrowth-promoting bacterium P. sp. UW4 is also Curesistant, which exhibited minimum inhibitory concen-tration (MIC) value of 7.5 mM against Cu2+ on TSBmedium (data not shown). These findings demonstratethe potential benefits of using P. putida UW4 for co-inoculation with rhizobia in legumes, which might beuseful for the remediation of heavy metal contaminatedsites.

It was previously observed that increasing or decreas-ing the amount of metal taken up by plant tissues is afunction of the bacterium, the particular host plant in-volved, the metal species, and the toxic level used(Rajkumar et al. 2009). In this study, under excess Cu,metal accumulation was considerably higher in rootsthan in above-ground plant tissues. The ability of plantsto translocate heavy metals from roots to aerial parts isevaluated by calculating translocation factor. The resultsshowed that the translocation value was considerablyless than one for plants inoculated with either strain,suggesting that Cu accumulated mainly in roots, withvery low level of Cu translocation to above-ground planttissues. This may be due to the fact that micronutrientcations, such as Cu2+, bind quite tightly to organicligand groups within the root cell walls (Kochian

1991). To obtain total Cu amount accumulated in plantorgans, dry weight of plant organs was multiplied withCu content. The resulting values showed that both theengineered strain S. meliloti CCNWSX0020(pRKACC) and the co-inoculated strain significantlyimproved total Cu accumulation per plant. The inhibi-tory or insignificant effects of S. meliloti (pRKACC) onCu content were compensated by increased biomassproduction and lead to increased total Cu accumulationper plant. As compared with the plants inoculated withwild-type S. meliloti strain, the lower translocation fac-tor for the plants inoculated with S. meliloti (pRKACC)under Cu stress conditions suggests that the ACCdeaminase-overproducing symbiotic bacteria improvethe Cu tolerance of the host plant roots with a lowerlevel of Cu transport to aerial parts. Nevertheless, furtherstudies such as metabolite and genetic analysis are re-quired to better understand the interactions betweenbacterial ACC deaminase and heavy metal translocationi n p l an t s . The s e f i nd i ng s empha s i z e t h ephytostabilization potential of the legume-Rhizobiumsymbiosis, which could avoid leaching, soil erosionand toxic metals transfer into the food chain (Ghoshand Singh 2005).

Although there are numerous literature reports thatbacterial ACC deaminase can facilitate plant growthunder a variety of environmental stresses by reducingplant ethylene levels, little is known about its otherphysiological impacts, such as the antioxidant defenseresponse in plants. In this study, the inoculation of ACCdeaminase-overproducing strain induced the expressionlevels of CuZnSODc, FeSOD, CAT and APX to a slightextent in the aerial parts of plants exposed to severe Custress. The expression levels of these antioxidant genesalso increased in roots, and was more pronounced whenplant were grown under severe Cu stress. The expres-sions of these seven antioxidant genes were all remark-ably increased by 2.8–3.6 fold in roots of plants exposedto severe Cu stress. This result was in agreement withthe decreased ethylene production observed for the rootsinoculated with S. meliloti (pRKACC) in the presence ofsevere Cu stress. Treatment of plants with the ethyleneperception inhibitor 1-methyl cyclopropene (1-MCP)has been shown to increase the activities of antioxidantenzymes in broccoli florets (Yuan et al. 2010), in mango(Wang et al. 2009) and pear (Larrigaudière et al. 2004).Application of 1-MCP has also shown to decrease eth-ylene and ROS production, enhancing antioxidant de-fense responses in soybean plants under high

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temperature stress (Djanaguiraman et al. 2011). Similar-ly, the H2O2 scavenging enzymes, i.e. CAT, POD, APXand GR in chickpea nodules were mediated by thepresence of the ethylene biosynthesis inhibitoraminoethoxyvinylglycine (Mann et al. 2002). The find-ings in the study reported here suggest that the bacteriacontaining ACC deaminase may not only reduce plantethylene levels but they may also enhance the antioxi-dant defense system directly or indirectly.

In summary, the engineered S. meliloti CCNWSX0020 (pRKACC) strain carrying an exogenous acdSgene shows a higher level of ACC deaminase activity,and improves the plant growth and Cu tolerance ofMedicago plants. The results obtained with the plantsco-inoculated with P. putida UW4 suggest that higherlevel of ACC deaminase activity may strengthen thebeneficial effects on the host plants, though it remainsto be determined whether further increase in the exoge-nous ACC deaminase gene expression (e.g. by using astrong promoter) would protect plants to a greaterextent.

Acknowledgments This work was supported by projects fromthe 863 Project of China (2012AA101402), National ScienceFoundation of China (31125007 and 31370142), the Natural Sci-ences and Engineering Research Council of Canada (NSERC),and the Fundamental Research Funds for the Central Universities(2014YQ004).

Ethical statement It is the original work of the authors. Thework described has not been submitted elsewhere for publication,in whole or in part, and all authors listed carry out the data analysisand manuscript writing. This article does not contain any studieswith human participants or animals performed by any of theauthors. Moreover, all authors have read and approved the finalmanuscript.

Conflict of interest The authors declare that they have no director indirect conflict of interest.

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