tissue fractions of cadmium in two hyperaccumulating …ir.yic.ac.cn/bitstream/133337/8581/1/tissue...

Research ArticleTissue Fractions of Cadmium in Two HyperaccumulatingJerusalem Artichoke Genotypes

Xiaohua Long1 Ni Ni12 Zhaopu Liu1 Zed Rengel3 Xin Jiang2 and Hongbo Shao14

1 Jiangsu Provincial Key Laboratory of Marine Biology College of Resources and Environmental SciencesNanjing Agricultural University Nanjing 210095 China

2 State Key Laboratory of Soil and Sustainable Agriculture Institute of Soil Science Chinese Academy of Sciences71 East Beijing Road Nanjing 210008 China

3 School of Earth and Environment The University of Western Australia 35 Stirling Highway Crawley WA 6009 Australia4Key Laboratory of Coastal Biology amp Bio-Resources Utilization Yantai Institute of Coastal Zone Research (YIC)Chinese Academy of Sciences (CAS) Yantai 264003 China

Correspondence should be addressed to Zhaopu Liu zhaopuliu163com and Hongbo Shao shaohongbochu126com

Received 15 March 2014 Accepted 26 March 2014 Published 14 April 2014

Academic Editor Xu Gang

Copyright copy 2014 Xiaohua Long et al This is an open access article distributed under the Creative Commons Attribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

In order to investigate the mechanisms in two Jerusalem artichoke (Helianthus tuberosus L) genotypes that hyperaccumulate Cda sand-culture experiment was carried out to characterize fractionation of Cd in tissue of Cd-hyperaccumulating genotypes NY

2

and NY5 The sequential extractants were 80 vv ethanol (FE) deionized water (FW) 1M NaCl (FNaCl) 2 vv acetic acid (FAcet)

and 06M HCl (FHCl) After 20 days of treatments NY5had greater plant biomass and greater Cd accumulation in tissues than

NY2 In both genotypes the FNaCl fraction was the highest in roots and stems whereas the FAcet and FHCl fractions were the highest

in leaves With an increase in Cd concentration in the culture solution the content of every Cd fraction also increased The FWand FNaCl ratios in roots were lower in NY

5than in NY

2 while the amount of other Cd forms was higher It implied that in high

accumulator namely NY5 the complex of insoluble phosphate tends to be shaped more easily which was much better for Cd

accumulation Besides translocation from plasma to vacuole after combination with protein may be one of the main mechanismsin Cd-accumulator Jerusalem artichoke genotypes

1 Introduction

Cd is one of biotoxic metal elements which has strongchemical activity and long-term toxicity and is relativelymobile in plants [1 2] It is also one of the major envi-ronmental pollutants Moderate Cd contamination of arablesoils can result in considerable Cd accumulation in edibleparts of crops [3ndash5] Cd can be present in plant tissues inconcentrations that are nontoxic to crops but can contributeto substantial Cd dietary intake by humans [6]

Existing methods of cleaning up Cd-contaminated soilsare expensive such as mechanical removal and chemicalengineering [7] Comparatively phytoextraction has a greatpotential in ameliorating Cd-contaminated soils because it isa cost-effective environmentally friendly approach applica-ble to large areas [8 9]

Plant resistance tometal toxicity stress includes avoidanceand tolerance [10] Avoidance frequently results in exclusionwhereas accumulation in plant tissues must be linked withinternal tolerance mechanisms These tolerance mechanismsmight rely on metal being retained mainly in roots withtransport to photosynthetically active above-ground tissuesimpeded In addition tolerance may be underpinned bymetals existing in nonactive (nontoxic) forms in plant tissuesSuch nontoxic forms may for example include binding ofmetals in the cell wall or complexation with organic acid andproteins mostly in the vacuole [11]

Wehave previously reported that two Jerusalem artichokegenotypes NY

2and NY

5 when grown in Cd contami-

nated soils did not suffer from Cd toxicity even thoughCd concentration not only in roots but also in leaves andstems exceeded 100mg kgminus1 dry weight [12] which is the

Hindawi Publishing Corporatione Scientific World JournalVolume 2014 Article ID 421249 6 pageshttpdxdoiorg1011552014421249

2 The Scientific World Journal

Table 1 Extractants and relevant extracted chemical forms of Cd

Extract ion reagent Code Predominant forms of extracted Cd80 vv ethanol FE Cd-nitrate Cd-chloride Cd-amino acid complexesDeionized water FW Soluble Cd-organic acid complexes Cd(H2PO4)21M NaCl FNaCl Cd-pectates Cd-polypeptide or Cd-protein complexes2 vv acetic acid FAcet Sparingly soluble CdHPO4 Cd3(PO)2 andor other Cd-phosphate complexes0 6M HCl FHCl Cd-oxalate

main feature of hyperaccumulators Hence NY2and NY

5

genotypes showed a potential to be used in phytoremediationof Cd-contaminated soils via phytoextraction However theresearch so far has mainly concentrated on Cd accumula-tion and plant physiological properties rather than on Cdchemical forms in Jerusalem artichoke genotypes The Cdchemical forms in plant tissues are expected to be linkedto Cd tolerance via detoxication mechanisms An under-standing of Cd-tolerancemechanisms in Jerusalem artichokegenotypes is a prerequisite for quick screening ofgermplasmfor improved Cd accumulation and toleranceThis study wasaimed at characterizing the distribution of Cd chemical formsin Jerusalem artichoke genotypes that hyperaccumulate Cd

2 Materials and Methods

21 Plants Two Jerusalem artichoke (Helianthus tubero-sus L) genotypes NY

2and NY

5 were selected from the

Nanjing Agricultural University Experimental Station (ldquo863Programrdquo) at Laizhou County in Shandong Province ChinaPrevious work [12] indicated that these two genotypes havethe capacity to hyperaccumulate Cd

22 Experimental Setup Thetestswere carried out in a green-house at Nanjing Agricultural University (N32∘ 21015840 62510158401015840E18∘ 501015840 234710158401015840) Nanjing China The average temperaturethroughout the test period was between 266 plusmn 44∘C (day-time) and 220 plusmn 24∘C (night) and the relative humiditywas 615 plusmn 13 (daytime) and 680 plusmn 19 (night) Tuberslices with buds were germinated on sand moistened with12 Hoagland nutrient solution in an incubator The nutrientsolutionwas replaced every seconddayAt trefoil stage youngplants were transplanted into porcelain pots About one weeklater Cd treatments were imposed (0 25 50 or 10mg Lminus1 asCdCl2sdot25H

2O) Each Cd treatment was replicated in three

pots and two uniform plants were allowed to grow in eachpot at a uniform spacing Sampling was carried out after 3-week treatment duration

23 Plant Sampling and Analysis Roots were washed indeionized water and then shoots and roots were separatedweighed and used for sequential extraction to determinechemical forms of Cd [13 14] Briefly 1 gram fresh leaf stemor root material was cut into pieces of 1-2mm2 transferredinto a beaker with 10mL of extractant (Table 1) and kept at25∘C overnight (20ndash24 h) on a shaker [15] The following daythe solutes were saved and the residues were extracted again

overnight with the next extractant In total there were fivesequential extractions

The extracts were digested with a concentrated acidmixture of HNO

3ndashHClO

4(3 1 vv) and heated at 160∘C for

5 h After cooling the extracts were diluted filtered andmade up to 25mL with 5 vv HNO

3 The Cd concentration

in the extract was determined by inductively coupled plasmaatomic emission spectroscopy (ICP-AES IRIS Intrepid IIXSP Thermo Electron Company USA) The analyses werecarried out in triplicate

24 SymbolMeanings FE FW FNaCl FAcet and FHCl show theamounts of the Cd-containing fractions extracted by ethanolwater NaCl acetic acid and HCl respectively

25 Statistical Analysis All statistical tests were performedusing SPSS 130 Two-way ANOVA was used to determinethe significance of genotype and Cd treatment effects on CdformsMean treatment differences were separated by the leastsignificant difference (LSD

005) test if119865-tests were significant

(119875 le 005 Fisherrsquos protected test)

3 Results

31 Effects of Cd Treatments on the Biomass and Its Compo-nents of Two H tuberosus Genotypes Even though the twoJerusalem artichoke genotypes showed good tolerance to Cdtoxicity NY

2showed somewilting in the high-Cd treatments

Compared to control the Cd treatment (25 and 10mg kgminus1)decreased leaf stem root and total biomass for both NY

2

and NY5(Table 2) In contrast the 5mg kgminus1 Cd treatment

increased the biomass and its components for NY2 In every

Cd treatment the biomass and its components of NY5were

lower than in the 0mg kgminus1 Cd supply but there was nosignificant difference

32 Chemical Forms of Cd in Plants

321 Effects of Cd Treatments on Cd Chemical Forms ofRoots in Two Jerusalem Artichoke Genotypes As can be seenfrom Table 3 the distribution ratios raised with increased Cdsupply in both NY

2and NY

5 In control group the difference

between the five forms was not remarkable In treatmentgroup the Fw ratio increased and was higher than the FRratio in NY

2andNY

5 FNACl occupied themost proportion in

both two Jerusalem artichoke genotypes secondly FAcet andFE were the least

The Scientific World Journal 3

Table 2 Effects of different cadmium treatments on the fresh biomass of two H tuberosus genotypes

Genotype Cd supply (mg Lminus1) Leaves (g plantminus1) Stem (g plantminus1) Root (g plantminus1) Whole plant (g)

NY2

0 243a 107ab 233a 582a

25 246a 111ab 211a 568a

5 285a 130a 284a 699a

10 154b 83b 111b 349b

NY5

0 298a 130a 258a 685a

25 291a 128a 171b 589a

5 301a 139a 209ab 649a

10 275a 127a 169b 571a

Different letters within a column indicate the significant differences among the treatments (119875 le 005 119899 = 3)

Table 3 Fractionation of Cd in roots of two Jerusalem artichoke genotypes

Genotype Cd supply (mg Lminus1) FE (120583g gminus1) FW (120583g gminus1) FNaCl (120583g g

minus1) FAcet (120583g gminus1) FHCl (120583g g

minus1)

NY2

0 11a 84a 11a 12a 83a

25 41c 48c 223a 127b 35c

5 38b 103b 655a 127b 54b

10 62b 150b 1675a 329b 91b

NY5

0 37c 49bc 13a 79b 78b

25 51c 36c 304a 113b 33c

5 55b 83b 469a 259ab 63b

10 80b 117b 1280a 387b 245b

Different letters within a row indicate significant differences among the fractions (119875 le 005 119899 = 3)

Table 4 Cd chemical forms of stems in two Jerusalem artichoke genotypes

Genotype Cd supply (mg Lminus1) FR (120583g gminus1) FW (120583g gminus1) FNaCl (120583g gminus1) FAcet (120583g g

minus1) FHCl (120583g gminus1)

NY2

0 70a 58a 66a 87a 64a

25 17c 14c 126a 66b 35b

5 18b 28b 278a 48b 34b

10 22b 43b 315a 91b 57b

NY5

0 29c 53b 74a 65ab 64ab

25 13c 13c 99a 54b 31bc

5 16a 17a 195a 92a 47a

10 32c 49bc 393a 135b 131b

Different letters within a row indicate the significant differences among the forms (119875 le 005 119899 = 3)

There were some differences of the five main chemicalforms in roots between NY

2and NY

5 The Fw ratio was

higher in NY2than that in NY

5 Water extracts the soluble

Cd organic acid complex Cd (H2PO4)2 which is poisonous

and tends to cause harm to plants The FAcet ratio of highaccumulator was higher than low accumulator while the Fwratio was lower Ethylic acid extracts unsoluble CdHPO

4

Cd3(PO)2 or Cd-phosphate complexes which may be better

for Cd accumulation in high accumulator

322 Effects of Cd Treatments on Cd Chemical Forms ofStems in Two Jerusalem Artichoke Genotypes We can seefrom Table 4 that the distribution ratios were raised withincreased Cd supply in both NY

2and NY

5 Compared

to the roots every proportion of the five chemical formsin stems decreased greatly Similarly FNaCl occupied the

most proportion in both two Jerusalem artichoke genotypessecondly FAcet and FR were the least The Fw ratio was higherin NY

2than that in NY

5 The FAcet ratio of high accumulator

was higher than low accumulator while the Fw ratio waslower

323 Effects of Cd Treatments on Cd Chemical Forms ofLeaves in Two Jerusalem Artichoke Genotypes The distribu-tion ratios were raised with increased Cd supply in both NY

2

and NY5(Table 5) Compared to stems the five forms were

further reduced in different degrees especially FNaCl FAcetcovered the most part 38 and 41 respectively in NY

2and

NY5The FW ratio was the lowest in leaf instead of the FR ratio

in NY2 Ethanol extracts Cd-nitrate Cd-chloride and Cd-

amino acid The lowest FNaCl ratio might be beneficial for


Table 5 Cd chemical forms of leaves in two Jerusalem artichoke genotypes



NY2

0 87a 52a 59a 94a 89a

25 13c 69c 20c 70a 47b

5 83b 12b 17b 44a 42a

10 15bc 9c 20b 56a 45a

NY5

0 14c 53b 73a 65ab 75a

25 74b 66b 14b 45a 47a

5 10b 89b 14b 61a 78a

10 15b 15b 41b 129a 112a


protecting leaves because NaCl extracts mainly Cd-pectatesCd-polypeptide or Cd-protein

4 Discussion

41 Cd Distribution in Cells of Roots Cd concentrationshowed the same order (root gt stem gt leaf gt tuber) andCd accumulation in plant components showed the order ofstem gt leaves gt roots for both NY

2and NY

5in previous

work [12] Cd chemical forms in plants were linked withCd transporting activity among which FR and FW werethe strongest secondly FNaCl FAcet and FHCl were theweakest [16] Most of Cd enriched in roots after absorbingby plants and the amount transported up to shoots wasusually a little [17] Cd concentrated in roots which mightbe related to Cd that formed stable large molecule complexwith protein polysaccharide ribose and nuclein in rootsdeposited [18] and then lightened poisoning to organs inshoots Cd accumulation in roots is usually accredited to cellwall [19] Cell wall is the first protective screen protecting cellprotoplast from being harming by heavy metals Cellulosehemicelluloses xylogen and pectic substance which consistof cell wall have abundant active perssads such as carboxyloxhydryl and aldehyde group A part of external Cd beingpassed through will combine with these perssads It preventslarge amount of Cd from going into plasma and reducestoxicity [16] Especially in the condition of short time andlow concentration this tolerance system of the combinationof Cd and cell wall is most important [20] However somescholars consider oppositely that the amount of Cd combinedwith root cell wall is much less than that in the cell [19 2122] Vacuole is a Cd accumulating place in higher plantsbut not the main point only in the condition of high Cdconcentration [19 23] As can be seen from Table 3 theabsolute advantage laid with the FNaCl ratio showing thatCd mainly adheres to protein This is because that Cd hasvery strong affinity with protein or hydrosulphonyil in otherorganic compounds [24] On the one hand the combinationof Cd and protein in plants can decrease the amount offree Cd reducing its availability and mobility and avoidingharm to plants Cd also may be combined with enzymesand functional proteins disturbing their regular function anddisordering physiological and biochemical metabolism [25]Because of the higher Fw ratio and the lower FAcet ratio the

amount of Cd is more poisonous and is transported morequickly in NY

2than that in NY

5 with NY

2showing fewer

biomass of roots in some degree and NY5showing normal

for the growth of 20 days

42 Cd Transporting from Roots to Shoots The transportingofCd from roots to shoots and accumulated in shoots is a verycomplicated process There are many studies on it [26] It isusually considered that Cd absorbed by roots is transportedto other components in plants through the xylem Rootmetalions go into root vascular bundle through endoderm andthe inner casparian strip Passing through casparian stripis difficult so this translocation is mainly carried out inyoung roots in which casparian strip has not been formedcompletely [27] Then mental ions may be transported upto shoots by transpiration There are many reports on long-distance translocation and the system of Cd long-distancetranslocation in plants is controversial The Fw and FNaClratios were reduced substantially in both NY

2and NY

5 so

compared to control stems did not suffer toxicity in NY5

while except when at the Cd concentration of 10mg Lminus1 andthe biomass of stem in NY

2decreased by small degrees

43 Cd Accumulation and Distribution in Leaves Cd in leafcells mainly comes from the water translocation from vas-cular bundle to leaf tissue which indicates that transpirationplays an important role in heavy mental accumulation [19]Similar to root cells the combination of leaf cell wall andCd decreases Cd concentration in cell sap lightening toxicityto leaf cells However the interception of cell wall plays asecondary role and the main detoxication mechanism is inthe vacuole [19] There are rich small molecule substancessuch as GSH oxalic acid histidine citrate and phosphoricacid in vacuole Cd avoids contacting with organelle torealize Cd detoxication through chelation or laydown withthose small molecule substances [28 29] Compared to otherchemical forms the FAcet ratios have absolute advantagein both two Jerusalem artichoke genotypes showing that aconsiderable part of Cd tends to form insoluble phosphate inleaves accordingly the amount of free Cdwhich is poisonousbecomes low (Table 5)Therefore from the appearance thereis almost no remarkable difference between the biomass oftreatment group and that of control group in NY

2and NY

5

leaves Previous studies have documented that Cd exists and


transports in ion form in some certain plants [30 31] Butin some Cd-accumulators Cd existence is mostly in organiccombination [31]

5 Conclusions

In summary Cd toxicity and tolerance mechanism are mostcomplex Different plants even different strains of the sameplant or different ecological types may show diverse Cdtolerance ability and mechanism According to the previousstudy compared with NY

2 genotype NY

5may be a better

candidate for phytoremediation of and biofuel productionon Cd-contaminated soils The present study implied thatin high accumulator namely NY

5 the complex of insoluble

phosphate tends to be shaped more easily which is muchbetter for Cd accumulation Besides translocation fromplasma to vacuole after combination with protein may beone of the main mechanisms in Cd-accumulator Jerusalemartichoke genotypes

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Xiaohua Long and Ni Ni contributed to the paper equally

Acknowledgments

The authors are grateful for the financial support of NationalNatural Science Foundation of China (no 31201692) theNational Key Projects of Scientific and Technical SupportPrograms funded by the Ministry of Science and Technologyof China (no 2011BAD13B09) the Project of a Special Fundfor PublicWelfare Industrial (Agriculture) Research of China(no 200903001-5) the Ministry of Science and Technol-ogy of Jiangsu Province (no BE2011368) and FundamentalResearch Funds for Central Universities (no Y0201100249)

References

[1] M Halim P Conte and A Piccolo ldquoPotential availabilityof heavy metals to phytoextraction from contaminated soilsinduced by exogenous humic substancesrdquoChemosphere vol 52no 1 pp 265ndash275 2003

[2] M B Kirkham ldquoCadmium in plants on polluted soils effects ofsoil factors hyperaccumulation and amendmentsrdquo Geodermavol 137 no 1-2 pp 19ndash32 2006

[3] T Arao and N Ae ldquoGenotypic variations in cadmium levels ofrice grainrdquo Soil Science and Plant Nutrition vol 49 no 4 pp473ndash479 2003

[4] G Ondrasek D Romic Z Rengel M Romic and M ZovkoldquoCadmium accumulation by muskmelon under salt stress incontaminated organic soilrdquo Science of the Total Environmentvol 407 no 7 pp 2175ndash2182 2009

[5] G Ondrasek Z Rengel D Romic and R Savic ldquoSalinitydecreases dissolved organic carbon in the rhizosphere and

increases trace element phyto-accumulationrdquo European Journalof Soil Science vol 63 pp 685ndash693 2012

[6] G J Wagner ldquoAccumulation of cadmium in crop plants and itsconsequences to human healthrdquo Advances in Agronomy vol 51pp 173ndash212 1993

[7] I D Pulford and C Watson ldquoPhytoremediation of heavymetal-contaminated land by treesmdasha reviewrdquo EnvironmentInternational vol 29 no 4 pp 529ndash540 2003

[8] N T H Ha M Sakakibara S Sano R S Hori and K SeraldquoThe potential of eleocharis acicularis for phytoremediationcase study at an abandoned mine siterdquo CleanmdashSoil Air Watervol 37 no 3 pp 203ndash208 2009

[9] D Lin Y Ouyang C H Huang and D Y Huang ldquoCharacteri-zation of heavy metals from banana farming soilsrdquo CleanmdashSoilAir Water vol 38 no 5-6 pp 430ndash436 2010

[10] L Chen X Long Z Zhang X Zheng Z Rengel and Z LiuldquoCadmium accumulation and translocation in two Jerusalemartichoke (Helianthus tuberosus L) cultivarsrdquo Pedosphere vol21 no 5 pp 573ndash580 2011

[11] T Ni and Y Wei ldquoSubcellular distribution of cadmium inmining ecotype Sedum alfrediirdquo Acta Botanica Sinica vol 45no 8 pp 925ndash928 2003

[12] X H Long N Ni L Wang et al ldquoPhytoremediationof cadmium-contaminated soil by two Jerusalem artichoke(Helianthus tuberosusL) genotypesrdquoCleanmdashSoil AirWater vol41 no 2 pp 202ndash209 2013

[13] J L Xu Z P Bao J R Yang H Lu and W C Song ldquoChemicalforms of Pb Cd and Cu in cropsrdquo Chinese Journal of AppliedEcology vol 2 no 3 pp 244ndash248 1991

[14] F Wu J Dong Q Q Qiong and G Zhang ldquoSubcellulardistribution and chemical form of Cd and Cd-Zn interactionin different barley genotypesrdquo Chemosphere vol 60 no 10 pp1437ndash1446 2005

[15] Z M Yang S J Zheng and A T Hu ldquoAccumulaion chemicalforms and physiological characteristic of cadmium in plantsaffected by phosphorusrdquo Chinese Journal of Applied and Envi-ronmental Biology vol 6 no 2 pp 121ndash126 2000

[16] Y Sun Q Zhou W Liu J An Z Xu and L Wang ldquoJointeffects of arsenic and cadmium on plant growth and metalbioaccumulation a potential Cd-hyperaccumulator and As-excluder Bidens pilosa Lrdquo Journal of Hazardous Materials vol165 no 1ndash3 pp 1023ndash1028 2009

[17] J M Kelly G R Parker and W W McFee ldquoHeavy metalaccumulation and growth of seedlings of five forest species asinfluenced by soil cadmium levelrdquo Journal of EnvironmentalQuality vol 8 no 3 pp 361ndash364 1979

[18] YUysal and F Taner ldquoBioremoval of cadmiumby Lemnaminorin different aquatic conditionsrdquo CleanmdashSoil Air Water vol 38no 4 pp 370ndash377 2010

[19] M Wojcik J Vangronsveld J DrsquoHaen and A TukiendorfldquoCadmium tolerance inThlaspi caerulescens II Localization ofcadmium in Thlaspi caerulescensrdquo Environmental and Experi-mental Botany vol 53 no 2 pp 163ndash171 2005

[20] G Wu H Kang X Zhang H Shao L Chu and C Ruan ldquoAcritical review on the bio-removal of hazardous heavy metalsfrom contaminated soils issues progress eco-environmentalconcerns and opportunitiesrdquo Journal of Hazardous Materialsvol 174 no 1ndash3 pp 1ndash8 2010

[21] W E Rauser and C A Ackerley ldquoLocalization of cadmium ingranules within differentiating andmature root cellsrdquoCanadianJournal of Botany pp 643ndash646 1987


[22] M D Vzquez C Poschenrieder and J Barce ldquoUltrastructuraleffects and localization of low cadmium concentrations in beanrootsrdquo New Phytologist vol 120 no 2 pp 215ndash226 1992

[23] V S Bezel T V Zhuikova and V N Pozolotina ldquoThe structureof dandelion cenopopulations and specific features of heavymetal accumulationrdquo Russian Journal of Ecology vol 29 no 5pp 331ndash337 1998

[24] J R Yang J Q He and G X Zhang ldquoTolerance mechanism ofcrops to Cd pollutionrdquo Journal of Applied Ecology vol 6 no 1pp 87ndash91 1995

[25] G Q Chen and X J Zhang ldquoCd accumulation chemical formandphysiology characteristic in pepper byZincrdquoEnvironmentalScience vol 31 no 7 pp 1657ndash1662 2010

[26] U Kramer ldquoCadmium for all mealsmdashplants with an unusualappetiterdquo New Phytologist vol 145 no 1 pp 1ndash5 2000

[27] R T Hardiman and B Jacoby ldquoAbsorption and translocation ofCd in bush beans (Phaseolus vulgaris)rdquo Physiologia Plantarumvol 61 no 4 pp 670ndash674 1984

[28] H Nishizono H Ichikawa S Suziki and F Ishii ldquoThe roleof the root cell wall in the heavy metal tolerance of Athyriumyokoscenserdquo Plant and Soil vol 101 no 1 pp 15ndash20 1987

[29] H J Weigel and H J Jager ldquoSubcellular distribution andchemical form of cadmium in bean plantsrdquo Plant Physiologyvol 65 no 3 pp 480ndash482 1980

[30] C B Jagna L Isabelle L Stanley and D Joanna ldquoShortterm signaling responses in roots of young soybean seedlingsexposed to cadmium stressrdquo Journal of Plant Physiology vol 170pp 1585ndash1594 2013

[31] A P G C Marques H Moreira A R Franco A O S SRangel and P M L Castro ldquoInoculating Helianthus annuus(sunflower) grown in zinc and cadmium contaminated soilswith plant growth promoting bacteriamdasheffects on phytoreme-diation strategiesrdquo Chemosphere vol 92 pp 74ndash83 2013

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Table 1 Extractants and relevant extracted chemical forms of Cd

Extract ion reagent Code Predominant forms of extracted Cd80 vv ethanol FE Cd-nitrate Cd-chloride Cd-amino acid complexesDeionized water FW Soluble Cd-organic acid complexes Cd(H2PO4)21M NaCl FNaCl Cd-pectates Cd-polypeptide or Cd-protein complexes2 vv acetic acid FAcet Sparingly soluble CdHPO4 Cd3(PO)2 andor other Cd-phosphate complexes0 6M HCl FHCl Cd-oxalate

main feature of hyperaccumulators Hence NY2and NY

5

genotypes showed a potential to be used in phytoremediationof Cd-contaminated soils via phytoextraction However theresearch so far has mainly concentrated on Cd accumula-tion and plant physiological properties rather than on Cdchemical forms in Jerusalem artichoke genotypes The Cdchemical forms in plant tissues are expected to be linkedto Cd tolerance via detoxication mechanisms An under-standing of Cd-tolerancemechanisms in Jerusalem artichokegenotypes is a prerequisite for quick screening ofgermplasmfor improved Cd accumulation and toleranceThis study wasaimed at characterizing the distribution of Cd chemical formsin Jerusalem artichoke genotypes that hyperaccumulate Cd

2 Materials and Methods

21 Plants Two Jerusalem artichoke (Helianthus tubero-sus L) genotypes NY

2and NY

5 were selected from the

Nanjing Agricultural University Experimental Station (ldquo863Programrdquo) at Laizhou County in Shandong Province ChinaPrevious work [12] indicated that these two genotypes havethe capacity to hyperaccumulate Cd

22 Experimental Setup Thetestswere carried out in a green-house at Nanjing Agricultural University (N32∘ 21015840 62510158401015840E18∘ 501015840 234710158401015840) Nanjing China The average temperaturethroughout the test period was between 266 plusmn 44∘C (day-time) and 220 plusmn 24∘C (night) and the relative humiditywas 615 plusmn 13 (daytime) and 680 plusmn 19 (night) Tuberslices with buds were germinated on sand moistened with12 Hoagland nutrient solution in an incubator The nutrientsolutionwas replaced every seconddayAt trefoil stage youngplants were transplanted into porcelain pots About one weeklater Cd treatments were imposed (0 25 50 or 10mg Lminus1 asCdCl2sdot25H

2O) Each Cd treatment was replicated in three

pots and two uniform plants were allowed to grow in eachpot at a uniform spacing Sampling was carried out after 3-week treatment duration

23 Plant Sampling and Analysis Roots were washed indeionized water and then shoots and roots were separatedweighed and used for sequential extraction to determinechemical forms of Cd [13 14] Briefly 1 gram fresh leaf stemor root material was cut into pieces of 1-2mm2 transferredinto a beaker with 10mL of extractant (Table 1) and kept at25∘C overnight (20ndash24 h) on a shaker [15] The following daythe solutes were saved and the residues were extracted again

overnight with the next extractant In total there were fivesequential extractions

The extracts were digested with a concentrated acidmixture of HNO

3ndashHClO

4(3 1 vv) and heated at 160∘C for

5 h After cooling the extracts were diluted filtered andmade up to 25mL with 5 vv HNO

3 The Cd concentration

in the extract was determined by inductively coupled plasmaatomic emission spectroscopy (ICP-AES IRIS Intrepid IIXSP Thermo Electron Company USA) The analyses werecarried out in triplicate

24 SymbolMeanings FE FW FNaCl FAcet and FHCl show theamounts of the Cd-containing fractions extracted by ethanolwater NaCl acetic acid and HCl respectively

25 Statistical Analysis All statistical tests were performedusing SPSS 130 Two-way ANOVA was used to determinethe significance of genotype and Cd treatment effects on CdformsMean treatment differences were separated by the leastsignificant difference (LSD

005) test if119865-tests were significant

(119875 le 005 Fisherrsquos protected test)

3 Results

31 Effects of Cd Treatments on the Biomass and Its Compo-nents of Two H tuberosus Genotypes Even though the twoJerusalem artichoke genotypes showed good tolerance to Cdtoxicity NY

2showed somewilting in the high-Cd treatments

Compared to control the Cd treatment (25 and 10mg kgminus1)decreased leaf stem root and total biomass for both NY

2

and NY5(Table 2) In contrast the 5mg kgminus1 Cd treatment

increased the biomass and its components for NY2 In every

Cd treatment the biomass and its components of NY5were

lower than in the 0mg kgminus1 Cd supply but there was nosignificant difference

32 Chemical Forms of Cd in Plants

321 Effects of Cd Treatments on Cd Chemical Forms ofRoots in Two Jerusalem Artichoke Genotypes As can be seenfrom Table 3 the distribution ratios raised with increased Cdsupply in both NY

2and NY

5 In control group the difference

between the five forms was not remarkable In treatmentgroup the Fw ratio increased and was higher than the FRratio in NY

2andNY

5 FNACl occupied themost proportion in

both two Jerusalem artichoke genotypes secondly FAcet andFE were the least




NY2

0 243a 107ab 233a 582a

25 246a 111ab 211a 568a

5 285a 130a 284a 699a

10 154b 83b 111b 349b

NY5

0 298a 130a 258a 685a

25 291a 128a 171b 589a

5 301a 139a 209ab 649a

10 275a 127a 169b 571a





minus1)

NY2

0 11a 84a 11a 12a 83a

25 41c 48c 223a 127b 35c

5 38b 103b 655a 127b 54b

10 62b 150b 1675a 329b 91b

NY5

0 37c 49bc 13a 79b 78b

25 51c 36c 304a 113b 33c

5 55b 83b 469a 259ab 63b

10 80b 117b 1280a 387b 245b





NY2

0 70a 58a 66a 87a 64a

25 17c 14c 126a 66b 35b

5 18b 28b 278a 48b 34b

10 22b 43b 315a 91b 57b

NY5

0 29c 53b 74a 65ab 64ab

25 13c 13c 99a 54b 31bc

5 16a 17a 195a 92a 47a

10 32c 49bc 393a 135b 131b



2and NY

5 The Fw ratio was





4




2and NY

5 Compared



2than that in NY




2



2and








NY2

0 87a 52a 59a 94a 89a

25 13c 69c 20c 70a 47b

5 83b 12b 17b 44a 42a

10 15bc 9c 20b 56a 45a

NY5

0 14c 53b 73a 65ab 75a

25 74b 66b 14b 45a 47a

5 10b 89b 14b 61a 78a

10 15b 15b 41b 129a 112a



4 Discussion


2and NY

5in previous



2than that in NY

5 with NY

2showing fewer




2and NY

5 so





2and NY

5




5 Conclusions


2 genotype NY

5may be a better








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology




NY2

0 243a 107ab 233a 582a

25 246a 111ab 211a 568a

5 285a 130a 284a 699a

10 154b 83b 111b 349b

NY5

0 298a 130a 258a 685a

25 291a 128a 171b 589a

5 301a 139a 209ab 649a

10 275a 127a 169b 571a





minus1)

NY2

0 11a 84a 11a 12a 83a

25 41c 48c 223a 127b 35c

5 38b 103b 655a 127b 54b

10 62b 150b 1675a 329b 91b

NY5

0 37c 49bc 13a 79b 78b

25 51c 36c 304a 113b 33c

5 55b 83b 469a 259ab 63b

10 80b 117b 1280a 387b 245b





NY2

0 70a 58a 66a 87a 64a

25 17c 14c 126a 66b 35b

5 18b 28b 278a 48b 34b

10 22b 43b 315a 91b 57b

NY5

0 29c 53b 74a 65ab 64ab

25 13c 13c 99a 54b 31bc

5 16a 17a 195a 92a 47a

10 32c 49bc 393a 135b 131b



2and NY

5 The Fw ratio was





4




2and NY

5 Compared



2than that in NY




2



2and








NY2

0 87a 52a 59a 94a 89a

25 13c 69c 20c 70a 47b

5 83b 12b 17b 44a 42a

10 15bc 9c 20b 56a 45a

NY5

0 14c 53b 73a 65ab 75a

25 74b 66b 14b 45a 47a

5 10b 89b 14b 61a 78a

10 15b 15b 41b 129a 112a



4 Discussion


2and NY

5in previous



2than that in NY

5 with NY

2showing fewer




2and NY

5 so





2and NY

5




5 Conclusions


2 genotype NY

5may be a better








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology





NY2

0 87a 52a 59a 94a 89a

25 13c 69c 20c 70a 47b

5 83b 12b 17b 44a 42a

10 15bc 9c 20b 56a 45a

NY5

0 14c 53b 73a 65ab 75a

25 74b 66b 14b 45a 47a

5 10b 89b 14b 61a 78a

10 15b 15b 41b 129a 112a



4 Discussion


2and NY

5in previous



2than that in NY

5 with NY

2showing fewer




2and NY

5 so





2and NY

5




5 Conclusions


2 genotype NY

5may be a better








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



5 Conclusions


2 genotype NY

5may be a better








Acknowledgments


References









































Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology



















Volume 2014

Zoology






















Advances in

Virolog y



Volume 2014




Enzyme Research



Microbiology

tissue fractions of cadmium in two hyperaccumulating …ir.yic.ac.cn/bitstream/133337/8581/1/tissue...

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