REGULAR ARTICLE

Molybdenum improves antioxidant and osmotic-adjustmentability against salt stress in Chinese cabbage (Brassicacampestris L. ssp. Pekinensis)

Mu Zhang & Chengxiao Hu & Xiaohu Zhao &

Qiling Tan & Xuecheng Sun & Anyong Cao &

Min Cui & Ying Zhang

Received: 5 December 2011 /Accepted: 14 December 2011 /Published online: 5 January 2012# Springer Science+Business Media B.V. 2011

AbstractAims A pot experiment was conducted to determinethe effects of molybdenum on antioxidative defenseand osmotic-adjustment systems of Chinese cabbageunder salt stress.Methods Molybdenum fertilizer was applied at threelevels (0, 0.15, 0.3 mg kg−1). Ten days after sowing,500 ml 136.8 mM of NaCl solution was added to halfof the plants for each treatment every 10th day forthree consecutive times.Results The results revealed that with the applica-tion of molybdenum in Chinese cabbage under saltstress the fresh weight significantly increased; ac-tivities of antioxidant enzymes such as superoxidedismutase (SOD), peroxidase (POD) and catalase(CAT) were dramatically improved; the contents ofnon-enzymatic antioxidants such as glutathione(GSH), carotenoid (CAR) and ascorbic acid(ASA) were significantly increased. There was alsoan significant increase in low molecular osmotic-adjustment products such as soluble sugar, solubleprotein and proline. Moreover, molybdenum signif-icantly increased potassium ion (K+) content and

reduced sodium ion (Na+) contents, which eventu-ally improved the K+/Na+ ratios.Conclusions The present study suggests that the ap-plication of molybdenum enhances the salt stress tol-erance in Chinese cabbage by increasing the capacityto eliminate active oxygen and the ability of osmotic-adjustment.

Keywords Molybdenum . Antioxidant . Osmotic-adjustment . Salt stress . Chinese cabbage (Brassicacampestris L. ssp. Pekinensis)

AbbreviationsMo MolybdenumSOD Superoxide dismutasePOD PeroxidaseCAT CatalaseASA AscorbateGSH Reduced glutathioneCAR Carotene

Introduction

Molybdenum (Mo) is a component of nitrate reductaseand nitrogenase which are involved in nitrogen me-tabolism of plants (Mulder 1948), and Mo is alsoinvolved in phosphorus and sulphur metabolism(Mendel and Hansch 2002; Liu et al. 2010). In addi-tion, Mo also plays an important role in resisting

Plant Soil (2012) 355:375–383DOI 10.1007/s11104-011-1109-z

Responsible Editor: Timothy J. Flowers.

M. Zhang : C. Hu (*) :X. Zhao :Q. Tan :X. Sun :A. Cao :M. Cui :Y. ZhangMicro-element Research Center,Huazhong Agricultural University,Wuhan 430070, Chinae-mail: hucx@mail.hzau.edu.cn

various environmental stresses. Mo was reported tohave a positive impacts on photosynthetic rates andphotosynthetic products in winter wheat under coldstress (Yaneva et al. 1996) and drought stress(Zakhurul et al. 2000). Mo also enhanced the chillingresistance of turf grass by escalating activities of anti-oxidative enzymes (Yu et al. 2005). Xiao et al. (2009)discovered that Mo decreased cadmium concentrationunder cadmium stress. Furthermore, it was reportedthat the biomass, seed yield and product quality wereall declined due to Mo deficiency or excess (Nautiyaland Chatterjee 2004). But there were no reports abouteffects of applying Mo fertilizer on plants particularlyin Chinese cabbage resistance to salt stress. However,it is important to clarify that due to more and morevegetable field are becoming salty caused by exces-sive fertilization, poor quality water for irrigation andpoor drainage.

Previous studies showed that salt stress couldinduce oxidative stress with excess formation ofactive oxygen such as superoxides, hydroxy andperoxy radicals (Xue and Liu 2008; Liang 1999).Plants scavenge these oxyradicals by increasingactivities of antioxidative enzymes such as super-oxide dismutase (SOD), peroxidase (POD) andcatalase (CAT), which crucially abolish active ox-ygen. In addition, non-enzymatic systems are alsoinvolved in protecting plants from oxyradical tox-icity such as ascorbic acid (ASA), glutathione(GSH) and carotenoid (CAR) (Lu 1994). More-over, plants enhanced their osmotic-adjustmentability to resist salt stress. Meanwhile, osmotic-adjustment would account for the accumulationof organic solutes. Among the organic osmotic-adjustment-accumulating products, soluble sugar,soluble protein and proline are single-minded forresisting osmotic stress (Empadinhas and da Costa2008). Additionally, the abilities of plant to main-tain high cytosolic potassium and low sodiumconcentrations are both critical factors associatedwith the ability of plants to withstand salt stress(Blumwald 2000).

Chinese cabbage, an important cruciferous vegeta-ble, has been widely planted in China, and it is sensi-tive to Mo deficiency. Mo deficiency often results inlow production and poor quality of Chinese cabbage.The main objective of this experiment was to investi-gate the effects of Mo application on biomass, antiox-idant enzymes activities, non-enzymatic antioxidants

contents and osmotic-adjustment-accumulating prod-ucts in Chinese cabbage under salt stress.

Materials and methods

Materials and experimental treatments

A pot experiment was conducted in the greenhouse ofthe Micro-element Research Center in Huazhong Ag-ricultural University in Wuhan, China(30°28′26″N,114°20′15″E), from 10 September to 15 October2010. The meteorological conditions of the green-house were: 30/20°C day/night. Chemical propertiesof the soil were as follows: pH 4.60 (soil water ratio of1:5), organic matter 20.6 g kg−1, alkaline hydrolysis N72.37 mg kg−1, Olsen-P 31.38 mg kg−1, available K48.86 mg kg−1 and Tamm reagent extractable Mo0.09 mg kg−1. Chinese cabbage seeds (shanghaiqing)were obtained from the Jiangxi Hangcheng Seed Com-pany. The seedings were thined to eight seedings perpot 5 days later of planting.

Three Mo treatments (Mo0, Mo0.15 and Mo0.3) withdifferent Mo concentrations (0, 0.15 and 0.3 mg kg−1)in this experiment (0.15 mg kg−1 is the threshold ofsoil Mo deficiency). (NH4)2 MoO4·4H2O of analyticalgrade was used as Mo fertilizer. Each pot was filledwith 6 kg of soil and was fertilized with the followingmacroelements (g kg−1 soil): N 0.2, P2O5 0.15 andK2O 0.2 supplied in the form of CO(NH2)2,NH4H2PO4 and KCl, respectively. The microelementssupplied were 0.025 mg Fe-EDTA, 1.81 mgMnCl2·4H2O, 0.08 mg CuSO4·5H2O, 0.22 mgZnSO4·7H2O and 2.86 mg H3BO3 per kg soil. Tendays after sowing, 500 ml 136.8 mM of NaCl solutionwas added to half of the plants for each treatmentevery 10th day for three consecutive times, to createa soil salt-stressed environment. Each treatment wasreplicated eight times, and the expertment design wasshown in Table 1.

Table 1 Experiment design

Conditions −NaCl +NaCl

Mo levels (mg kg−1) 0 0.15 0.3 0 0.15 0.3

−NaCl and +NaCl represent normal and salt stress conditionsrespectively

376 Plant Soil (2012) 355:375–383

Thirty-five days after sowing, plant leaves from dif-ferent treatments were collected, immersed in N2 liquidimmediately, and then stored at −80°C for further anal-ysis. The other four replicates were oven-dried at 65°Cafter determining fresh weight. Oven-dried samples ofplants were sieved through a 1 mm nylon sieve for theanalysis of Mo, K+ and Na+ contents.

Determination of Mo

Concentrations of Mo in Chinese cabbage were deter-mined by polarographic catalytic wave analysis meth-od with a Jp-4000 oscilloscope polarograph (Wan etal. 1988).

Analysis of antioxidant enzyme activities

SOD activity was determined according to the methodof Tandy et al. (1989). In brief, fresh 0.5 g leaf sampleswere ground in 5 ml extraction buffer containing50 mM potassium phosphate (pH7.8), and the homog-enate was centrifuged at 10,000×g for 15 min at 4°C.A 3 mL reaction mixture contained 13 mM methio-nine, 75 μM NBT, 2 μM riboflavin, 0.1 mM EDTAand 100μL enzyme extract. The reaction mixture wasilluminated for 15 min and the sample absorbance wasdetermined at 560 nm. One unit of SOD activity wasdefined as the amount of enzyme corresponding to50% inhibition of the NBT reduction.

POD activity was measured based on the method ofPinhero et al. (1997). In brief, fresh 0.5 g of cabbageleaves was extracted by 5 mL 50 mM potassiumphosphate buffer (pH 5.5). The extracts were thencentrifuged at 10,000×g for 15 min at 4°C. The reac-tion mixture contained 1 mL extraction buffer, 5 μL30% H2O2, 5 μL guaiacol and 15 μL supernatant. Themolar extinction coefficient of 26.6 mM−1 cm−1 wasused for the enzyme activity calculation.

CAT activity was tested by monitoring the decreaseof absorbance at 240 nm for 3 min (Zhang and Kirkham1994). In brief, approximately 0.5 g of leaves wasground in 5 mL 50 mM potassium phosphate buffer(pH7.0). The homogenate was centrifuged at 10,000×gfor 15 min at 4°C. The assay mixture contained 50 mMpotassium phosphate buffer, 10 mM H2O2 and 200 μLenzyme extract. The extinction coefficient of39.4 mM−1 cm−1 was used for the calculation of CATactivity. One CAT unit was defined as the amountdecomposing 1 μmol of H2O2 in 1 min.

Analysis of non-enzymatic antioxidants

ASA was extracted from 1 g cabbage leaves with10 g L−1 oxalic acid. ASA was determined spectropho-tometrically following the 2, 4-dinitrophenylhydrazinecolorimetry method described by Bao (2002). Samplesof 1 g was homogenized with 3% metaphosphoric acid,and the homogenate was centrifuged for 10 min at10,000×g. The supernatant was used for GSH assays(Song and Ge 2001). CAR assay followed the methodof Lichtenthaler (1987). Approximately 0.2 g of cabbageleaves was extracted by 80% acetone, and the absorptionwas determined by spectrophotometry.

Analysis of osmotic-adjustment-accumulatingproducts

Proline was assayed by the method of Li (2000). Inbrief, approximately 0.5 g leaves was homegenizedin 5 mL of 3% aqueous sulphosalicylic acid andfiltered. The reaction mixture contained 2 mL ofthe filtrate, 2 mL of glacial acetic acid and 2 mLof acid ninhydrin. The reaction mixture was heatedin a boiling water-bath for 30 min and cooled toroom temperature. The absorption was determinedby spectrophotometry at 520 nm. The standardcurve was used to calculate the concentration ofProline.

Soluble sugar and Soluble protein concentrationswere determined by anthrone colorimetry and coomas-sile brilliant blue methods, separately (Gao 2005).Total K+ and Na+ contents in the samples were deter-mined using a Flame Photometer (Sherwood 410,UK).

Statistical analysis

The data was statistically analyzed by SPSS 12.0software and the mean values of each treatment un-derwent multiple comparisons using the LSD-test atthe p<0.05 level.

Results

Fresh weight and Mo contents of Chinese cabbage

As shown in Fig. 1, NaCl dramatically decreased thefresh weight of Chinese cabbage, but the application

Plant Soil (2012) 355:375–383 377

of Mo caused a significant increase in the fresh weightin both normal and salt treatments. Compared with nosalt application treatments, the contents of Mo in saltapplication treatments were significantly (p<0.05) de-creased, while the content of Mo in the Mo-fertilizedChinese cabbage were higher than those in Mo defi-ciency Chinese cabbage whether under normal or saltstress condition.

Antioxidant enzyme activities of Chinese cabbage

The effects of Mo on the three antioxidative enzymessuch as SOD, POD and CAT were represented inFig. 2. The application of Mo significantly increasedthe activities of SOD, POD and CAT, with the highestvalues all in Mo0.3 treatment under salt stress. How-ever, there had no dramatic effects on the activities ofthese enzymes in normal condition.

Non-enzymatic antioxidants contents of Chinesecabbage

The ASA contents in Chinese cabbage were dramati-cally increased by application of Mo and the highestcontent of ASA occurred in Mo0.15 treatment, in-creased by 16.7% compared with those of Mo0 treat-ment under salt stress (Fig. 3), while there was nosignificant difference in ASA content under normalcondition. Similarly, with the increase of Mo concen-tration, ASA contents declined both in normal and salt

stress conditions. Mo application led to a sharp in-crease in GSH contents both in Mo0.15 and Mo0.3treatments under salt stress, with 52.0% and 64.0%increased respectively, compared with the control.However, no greater effects were observed under nor-mal condition. The same trend of CAR content alsooccurred in Chinese cabbage, CAR content in Mo0.3treatment was significantly greater than those in Mo0treatment under salt stress, and there was no obviousdifference in normal condition.

Osmotic-adjustment-accumulating products contentsof Chinese cabbage

Application of Mo significantly affected solublesugar contents of Chinese cabbage both in normaland salt stress conditions, and the highest yields ofsoluble sugar were both observed in Mo0.3 treat-ment (Fig. 4). Mo application had an increasingtrend on soluble protein content in Mo0.15 treatmentunder salt stress, but there was a significant in-crease in the Mo0.15 treatment under normal condi-tion. With the rates of Mo application furtherincreased, soluble protein content had a decreasingtrend whether under salt stress or normal condition.Compared with normal condition, addition of NaClsignificantly increased proline content. Besides,proline contents were notably increased by applica-tion of Mo and were improved by 41.7% and37.5%, respectively, under salt stress. However,

Fre

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Fig. 1 Effects of Mo on Fresh weight and Mo content of Chinese cabbage. Bars indicate standard error of the mean. Mean value foreach treatment with different lowercase letters indicate significant differences by the LSD-test (p<0.05 n04)

378 Plant Soil (2012) 355:375–383

ASA

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Fig. 3 Effects of Mo on AsA, GSH and CAR contents ofChinese cabbage. Bars indicate standard error of the mean.Mean value for each treatment with different lowercase lettersindicate significant differences by the LSD-test (p<0.05 n04)

SOD

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bc bc

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b

ab

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Fig. 2 Effects of Mo on SOD, POD and CAT activities ofChinese cabbage. Bars indicate standard error of the mean.Mean value for each treatment with different lowercase lettersindicate significant differences by the LSD-test (p<0.05 n04)

Plant Soil (2012) 355:375–383 379

Mo application had no significant effect on prolinecontent under normal condition.

K+ , Na+ contents and K+/ Na+ ratios of Chinesecabbage

Mo application affected K+ and Na+ contents of Chi-nese cabbage under salt stress (Fig. 5). K+ contents ofChinese cabbage in the Mo treatments were signifi-cantly greater than those in Mo0 treatment under saltstress, and which were positively correlated (r00.768,P<0.01) with the rates of Mo application. Conversely,Na+ contents were decreased by application of Mo andwere negatively correlated (r0−0.866, P<0.01) with

Solu

ble

suga

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% )

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cc c

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Fig. 4 Effects of Mo on Soluble sugar, Soluble protein andProline contents of Chinese cabbage. Bars indicate standarderror of the mean. Mean value for each treatment with differentlowercase letters indicate significant differences by the LSD-test(p<0.05 n04)

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-1 )

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-1 )

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cb

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Fig. 5 Effects of Mo on K+ and Na+ contents of Chinesecabbage. Bars indicate standard error of the mean. Mean valuefor each treatment with different lowercase letters indicate sig-nificant differences by the LSD-test (p<0.05 n04)

380 Plant Soil (2012) 355:375–383

the rates of Mo application under salt stress. The K+/Na+ ratios were significantly increased by applicationof Mo under salt stress, and the highest value occurredin Mo0.3 treatment (Fig. 6). However, Mo applicationhad no significant effects on both K+ and Na+ contentsunder normal condition.

Discussion

Mo application increases fresh weight and Mocontents of Chinese cabbage under salt stress

High levels of salt in the soil can severely limit plantgrowth and crop production, and an increase in freshweight is widely used as an indicator for salt stresstolerance in all plants (Munna 2005). In our study, theapplication of Mo has dramatically improved the freshweight by 12.4% under salt stress. In addition, ourstudy shown that, salt caused a significant decreasein Mo contents, which indicated that salt may lead toMo deficiency but application of Mo can improveplant Mo nutrition. It has been reported that enhancingsalinity resistance in plants will directly improve theirfresh weight (Cheong and Yun 2007). Hence, it couldbe concluded that the salinity tolerance in Chinesecabbage was enhanced by Mo application under saltstress.

Mo application enhances antioxidant enzymeactivities and non-enzymatic antioxidants contentsof Chinese cabbage under salt stress

Salt stress results in oxidative stress because of theoverproduction of active oxygen such as superoxides,hydroxy and peroxy radicals. The active oxygen couldcause membrane dysfunction and cell death. SOD,POD and CAT, the antioxidative enzymes, are crucialfor alleviate the oxidative stress, and enhance salinitytolerance in many plants (Bohnert and Jensen 1996).Here, SOD, POD and CAT activities were enhancedwith the increase of the concentration of molybdenumused under salt stress. However, Mo application hadno obvious effect on the activities of those enzymesunder normal condition, which indicated that applica-tion of Mo could protect Chinese cabbage from lipidperoxidation under salt stress. These results corre-spond with the previous study, e.g. Liu and Yang(2000) found that molybdenum application can in-crease activities of SOD, POD and CAT in soybeanleaves.

In addition, non-enzymatic antioxidants such asASA, GSH and CAR also play the key role in oxida-tive stress defense. ASA is one of the important indi-ces of quality for vegetables, though it also play animportant part in the protection of plants against mem-brane lipid peroxidation. In our experiment, appropri-ate application of Mo increased ASA contents undersalt stress, whereas excessive Mo might not be condu-cive to ASA increase. The similar effect of applicationof Mo on Chinese cabbage ASA content was alsoreported by Nie et al. (2007). Mo application enhancedASA content due to the regeneration of ascorbateoxidase and ascorbate peroxidase activities (Nie et al.2008). GSH is also a major antioxidant, serving twofunctions as an H2O2 eliminator and in the repair ofcellular injury. Our study has shown that Mo applica-tion significantly enhanced GSH contents under saltstress and were positively correlated(r00.821, P<0.01)with the levels of Mo application. CAR as a pivotalaccessory pigment can enhance the ability of plants tocapture light energy. In association with other compo-nents of the non-enzymatic antioxidant system, CARalso protects plants against oxidative stress. It has beenreported that application of molybdenum couldincrease CAR contents in winter wheat under normalor low temperatures (Sun et al. 2006; Yu et al. 2006).Similar results were also obtained in our experiment.

K+ / N

a+

0.3

0.4

0.5

0.6

0.7

0.8

0.9

1.0

0 0.15 0.3

+NaCl

Applied Mo (mg kg-1)

c

b

a

Fig. 6 Effects of Mo on K+/Na+ rations of Chinese cabbageunder salt stress. Bars indicate standard error of the mean. Meanvalue for each treatment with different lowercase letters indicatesignificant differences by the LSD-test (p<0.05 n04)

Plant Soil (2012) 355:375–383 381

For instance, the increase of CAR was associated withthe increase in the rates of Mo application under saltstress. The increased antioxidant enzymes activities andnon-enzymatic antioxidants contents in Mo treatmentsmay protect Chinese cabbage against oxidative damageunder salt stress.

Mo application enhances osmotic-adjustment abilityof Chinese cabbage under salt stress

The plants’ ability to resist salt is determined by mul-tiple biochemical functions and facilitating acquisitionof water is named as one of the most crucial features(Flexas et al. 2006). It has been reported that there wasa close relationship between water deficits and osmot-ic stress, for example, high concentrations of salt insoil, plant roots struggle to extract water. To accom-modate osmotic potential in the vacuoles, plants accu-mulate low molecular mass compounds, which notonly enhance osmotic potential but also replace waterin biochemical reaction, such as soluble sugar, solubleprotein, proline et al. (Parida and Das 2005). In ourexperiment, with the application of Mo, soluble sugarand soluble protein were all significantly increased un-der salt stress. It is worth noting that proline markedlyaccumulated in Chinese cabbage exposed to oxidativestress caused by salt stress, while application of Mosignificantly increased proline contents under salt stresscondition. Proline have diverse roles under osmoticstress, such as stabilization of proteins, subcellular andmembranes structures (Vanrensburg et al. 1993).

Salinity tolerance also depends on limiting Na+

accumulations and maintaining K+ concentrations inthe cytosol and high K+/Na+ ratio is a key determinantof salt tolerance (Munns and Tester 2008; Shabala andCuin 2007). The Na+ contents rely on Na+/ H+ anti-portes, which are the products of the GhNHX1 geneexpression. However, the gene is strongly induced byABA (Wu et al. 2004; Fukuda et al. 2004). Applica-tion of Mo has a positive effects on the biosynthesis ofABA under stress conditions (Sun et al. 2009; Xionget al. 2001). Hence, we speculated that Mo affected theNa+ contents may through ABA regulate the expres-sion levels of GhNHX1 gene, and then lead to Na+

being expelled from plant. Moreover, the phytohor-mone ABA plays a significant role in plant growth andnot only reduces the Na+ contents but also increasesplant roots ability to extract K+ (Zhang et al. 2008).Our study revealed that K+ contents were kept high

level with the molybdenum application in condition ofNa+ stress. Accordingly, the K+/Na+ ratios were in-creased with the rates of Mo application under saltstress. Associated with the results ahead, we can getthe deduction that Chinese cabbage had a superiorcapacity of osmotic-adjustment by application of Mounder salt stress.

Conclusions

In our experiment, application of Mo increased freshweight of Chinese cabbage significantly under salt stress.The activities of antioxidant enzyme and contents of non-enzymatic antioxidants were all remarkably improved byMo application under salt stress. Osmotic-adjustment-accumulating products were also dramatically increasedby Mo application under salt stress. Furthermore, the K+/Na+ ratios were dramatically increased and were posi-tively correlated with the rates of Mo application. There-fore, we can speculated that the application of Moenhanced salinity tolerance by increasing the ability ofeliminating active oxygen and the tolerance of osmoticstress.

Acknowledgements The authors acknowledge Dr Di (Soil,Plant and Ecological Sciences Division, Lincoln University,Canterbury, New Zealand) for critical reviewing and revisionof the manuscript. Financial support by the Fundamental Re-search Funds for the Central Universi t ies(ProgramNo.2010PY025, 2010PY150)and Innovation Funds of Huaz-hong Agricultural University (52902-0900206044) are greatlyacknowledged.

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