Abstract
Excess copper (Cu) in soils has deleterious effects on plant growth and can pose a risk to human health. In the last decade, legume-rhizobium symbioses became attractive biotechnological tools for metal phytostabilization. For this technique being useful, metal-tolerant symbionts are required, which can be generated through genetic manipulation.
In this work, a double symbiotic system was engineered for Cu phytostabilization: On the one hand, composite Medicago truncatula plants expressing the metallothionein gene mt4a from Arabidopsis thaliana in roots were obtained to improve plant Cu tolerance. On the other hand, a genetically modified Ensifer medicae strain, expressing copper resistance genes copAB from Pseudomonas fluorescens driven by a nodulation promoter, nifHp, was used for plant inoculation. Our results indicated that expression of mt4a in composite plants ameliorated plant growth and nodulation and enhanced Cu tolerance. Lower levels of ROS-scavenging enzymes and of thiobarbituric acid reactive substances (TBARS), such as malondialdehyde (a marker of lipid peroxidation), suggested reduced oxidative stress. Furthermore, inoculation with the genetically modified Ensifer further improved root Cu accumulation without altering metal loading to shoots, leading to diminished values of metal translocation from roots to shoots. The double modified partnership is proposed as a suitable tool for Cu rhizo-phytostabilization.
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References
Aebi H (1984) Catalase in vitro. Method Enzymol 105:121–126 http://www.ncbi.nlm.nih.gov/pubmed/6727660
Ahemad M, Kibret M (2014) Mechanisms and applications of plant growth promoting rhizobacteria: current perspective. J King Saud Univ Sci 26(1):1–20. doi:10.1016/j.jksus.2013.05.001
Ahmad E, Zaidi A, Khan MS, Oves M (2012) Heavy metal toxicity to symbiotic nitrogen-fixing microorganism and host legumes, In: A. Zaidi et al, (Eds), toxicity of heavy metals to legumes and bioremediation, Springer Vienna, 29–44. doi: 10.1007/978-3-7091-0730-0_2
ASTDR (Agency for Toxic Substances and Desease Register) (2004) Toxicological profile for copper http://www.atsdr.cdc.gov/PHS/PHS.asp?id=204&tid=37
Becana M, Matamoros MA, Udvardi M, Dalton DA (2010) Recent insights into antioxidant defences of legume root nodules. New Phytol 188:960–976. doi:10.1111/j.1469-8137.2010.03512.x
Beynon J, Williams MK, Cannon FC (1988) Expression and functional analysis of the Rhizohium meliloti nifA gene. EMBO J 7:7–14
Bini E (2010) Archaeal transformation of metals in the environment. FEMS Microbiol Ecol 73:1–16. doi:10.1111/j.1574-6941.2010.00876.x
Boisson-Dernier A, Chabaud M, García F, Bécard G, Rosenberg C, Barker DG (2001) Agrobacterium rhizogenes-transformed roots of Medicago truncatula for the study of nitrogen-fixing and endomycorrhizal symbiotic associations. Mol Plant-Microbe Interact 14:695–700. doi:10.1094/MPMI.2001.14.6.695
Bradford MM (1976) A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254. doi:10.1016/0003-2697(76)90527-3
Cerri MR, Frances L, Laloum T, Auriac MC, Niebel A, Oldroyd ED, Barker DG, Fournier J, Carvalho-Niebel F (2012) Medicago truncatula ERN transcription factors: regulatory interplay with NSP1/NSP2 GRAS factors and expression dynamics throughout rhizobial infection. Plant Physiol 160:2155–2172. doi:10.1104/pp.112.203190
Chen L, Jiang B, Wu C, Sun S, Hou W, Han T (2014) GmPRP2 promoter drives root-preferential expression in transgenic Arabidopsis and soybean hairy roots. BMC Plant Biol 14:245. doi:10.1186/s12870-014-0245-z
Cherian S, Oliveira MM (2005) Transgenic plants in phytoremediation: recent advances and new possibilities. Environ Sci Technol 39:9377–9390. doi:10.1021/es051134l
Cosson V, Durand P, d’Erfurth I, Kondorosi A, Ratet P (2006) Medicago truncatula transformation using leaf explants. In: Wang K. Humana Press, Agrobacterium Protocols, pp 115–128. doi:10.1385/1-59745-130-4:115
Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nature Biotechnol 20:581–586. doi:10.1038/nbt0602-581
Dary M, Chamber-Pérez MA, Palomares AJ, Pajuelo E (2010) “In situ” phytostabilisation of heavy metal polluted soils using Lupinus luteus inoculated with metal resistant plant-growth promoting rhizobacteria. J Hazard Mater 177:323–330. doi:10.1016/j.jhazmat.2009.12.035
Delgadillo J, Lafuente A, Doukkali B, Redondo-Gómez S, Mateos-Naranjo E, Caviedes MA, Pajuelo E, Rodríguez-Llorente ID (2015) Improving legume nodulation and Cu rhizostabilization using a genetically modified rhizobia. Environ Technol 36:1237–1245. doi:10.1080/09593330.2014.983990
Dimri GP, Das HK (1988) Transcriptional regulation of nitrogen fixation genes by DNA supercoiling. Mol Gen Genet 212:360–363. doi:10.1007/BF00334707
Dowling DN, Doty SL (2009) Improving phytoremediation through biotechnology. Curr Opin Biotechnol 20:204–206. doi:10.1016/j.copbio.2009.03.007
Duncan K (2009) Metallothioneins and related chelators. Metal ions in life sciences Vol. 5. Sigel A, Sigel H. Sigel RKO (Eds) Angewandte Chemie International Edition 48:7966–7967. doi:10.1002/anie.200903833
Escaray FJ, Menendez AB, Gárriz A, Pieckenstain FL, Estrella MJ, Castagno LN, Ruiz OA (2012) Ecological and agronomic importance of the plant genus Lotus. Its application in grassland sustainability and the amelioration of constrained and contaminated soils. Plant Sci 182:121–133. doi:10.1016/j.plantsci.2011.03.016
Fageria NK, Baligar VC, Clark RB (2002) Micronutrients in crop production. Adv Agron 77:185–268. doi:10.1016/S0065-2113(02)77015-6
Festa RA, Thiele DJ (2011) Copper: an essential metal in biology. Curr Biol 21:877–883. doi:10.1016/j.cub.2011.09.040
Glick BR (2010) Using soil bacteria to facilitate phytoremediation. Biotechnol Adv 28:367–374. doi:10.1016/j.biotechadv.2010.02.001
Grabbe R, Klopprogge K, Schmitz RA (2001) Fnr is required for NifL-dependent oxygen control of nif gene expression in Klebsiella pneumoniae. J Bacteriol 183:1385–1393. doi:10.1128/JB.183.4.1385-1393.2001
Guo WJ, Meetam M, Goldsbrough PB (2008) Examining the specific contributions of individual Arabidopsis metallothioneins to copper distribution and metal tolerance. Plant Physiol 146:1697–1706. doi:10.1104/pp.108.115782
Hall JL (2002) Cellular mechanisms for heavy metal detoxification and tolerance. J Exp Bot 53:1–11. doi:10.1093/jexbot/53.366.1
Hao X, Xie P, Zhu YG, Taghavi S, Wei G, Rensing C (2015) Copper tolerance mechanisms of Mesorhizobium amorphae and its role in aiding phytostabilization by Robinia pseudoacacia in copper contaminated soil. Environ Sci Technol 49:2328–2340. doi:10.1021/es504956a
Hassinen VH, Tervahauta AI, Schat H, Kärenlampi SO (2011) Plant metallothioneins—metal chelators with ROS scavenging activity? Plant Biol 13:225–232. doi:10.1111/j.1438-8677.2010.00398.x
Hattab S, Hattab S, Banni M, Boussetta H (2013) Modulation of antioxidant responses of Medicago sativa under cadmium and copper stress. Afr J Agric Res 8:2297–2306. doi:10.5897/AJAR2012.6686
Hayat R, Ali S, Amara U, Khalid R, Ahmed I (2010) Soil beneficial bacteria and their role in plant growth promotion: a review. Ann Microbiol 60:579–598. doi:10.1007/s13213-010-0117-1
Hossain MA, Piyatida P, Teixeira da Silva J, Fujita M (2012) Molecular mechanism of heavy metal toxicity and tolerance in plants: Ccentral role of glutathione in detoxification of reactive oxygen species and methylglyoxal and in heavy metal chelation J Bot 2012: Article ID 872875, 37 pages. doi: 10.1155/2012/872875
Huala E, Ausubel FM (1989) The central domain of Rhizobium meliloti NifA is sufficient to activate transcription from the R. meliloti nifH promoter. J Bacteriol 171: 3354–3365. doi: PMC210058
Huang LY , Zhang F, Qin Q, Wang WS., Zhang T, Fu BY (2015) Identification and validation of root-specific promoters in rice J Integr Agric 14: 1–10. doi: 10.1016/S2095-3119(14)60763-2
Ike A, Sriprang R, Ono H, Murooka Y, Yamashita M (2007) Bioremediation of cadmium contaminated soil using symbiosis between leguminous plant and recombinant rhizobia with the MTL4 and the PCS genes. Chemosphere 66:1670–1676. doi:10.1016/j.chemosphere.2006.07.058
Kawashima I, Kennedy TD, Chino M, Lane BG (1992) Wheat Ec metallothionein genes. Eur J Biochem 209:971–976 www.ncbi.nlm.nih.gov/pubmed/1425704
Kopittke P, Dart PJ, Menzie NW (2007) Toxic effects of low concentrations of Cu on nodulation of cowpea (Vigna unguiculata). Environ Pollut 145:309–315. doi:10.1016/j.envpol.2006.03.007
Kotrba P (2013) Transgenic approaches to enhance phytoremediation of heavy metal-polluted soils. In: Gupta DK (ed) Plant-based remediation processes, vol 35. Springer, Berlin, pp 239–271
Li Z, Ma Z, Hao X, Rensing C, Wei G (2014) Genes conferring copper resistance in Sinorhizobium meliloti CCNWSX0020 also promote the growth of Medicago lupulina in copper-contaminated soil. Appl Environ Microbiol 80:1961–1971. doi:10.1128/AEM.03381-13
Macek T, Kotrba P, Svatos A, Novakova M, Demnerova K, Mackova M (2008) Novel roles for genetically modified plants in environmental protection. Trends Biotechnol 26:146–152. doi:10.1016/j.tibtech.2007.11.009
Maiti IB, Wagner GJ, Yeargan R, Hunt AG (1989) Inheritance and expression of the mouse metallothionein gene in tobacco. Plant Physiol 91:1020–1024 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC1062111
Margui E, Queralt I, Cravalho ML, Hidalgo M (2005) Comparison of EDXRF and ICP-OES after microwave digestion for element determination in plant specimens from an abandoned mining area. Anal Chim Acta 549:197–204. doi:10.1016/j.aca.2005.06.035
Merchán F, De Lorenzo L, Rizzo SG, Niebel A, Manyani H, Frugier F, Sousa C, Crespi M (2007) Identification of regulatory pathways involved in the reacquisition of root growth after salt stress in Medicago truncatula. Plant J 51:1–17. doi:10.1111/j.1365-313X.2007.03117.x
Misra S, Gedamu L (1989) Heavy metal tolerant transgenic Brassica napus L. and Nicotiana tabacum L. plants. Theor Appl Genet 78:161–168. doi:10.1007/BF00288793
Mittler R (2002) Oxidative stress, antioxidants and stress tolerance. Trends Plant Sci 7:405–410. doi:10.1016/S1360-1385(02)02312-9
Padilla FM, Pugnaire FI (2006) The role of nurse plants in the restoration of degraded environments. Front Ecol Environ 4:196–202 http://www.jstor.org/stable/3868736
Padmavathiamma PK, Li LY (2007) Phytoremediation technology: hyper-accumulation metals in plants. Water Air Soil Pollut 184:105–126. doi:10.1007/s11270-007-9401-5
Påhlsson AM (1989) Toxicity of heavy metals (Zn, Cu, Cd, Pb) to vascular plants. Water Air Soil Poll 47:287–319. doi:10.1007/BF00279329
Pajuelo E, Rodríguez-Llorente ID, Lafuente A, Pérez-Palacios P, Doukkali B, Caviedes MA (2014) Engineering the rhizosphere for the purpose of bioremediation: an overview. CAB Reviews 9:001. doi:10.1079/PAVSNNR20149001
Pajuelo E, Rodríguez-LLorente ID, Lafuente A, M.A. Caviedes (2011) Legume–Rhizobium symbioses as a tool for bioremediation of heavy metal polluted soils In: M.S. Khan, A. Zaidi, R. Goel, J. Musarrat, (Eds.): Biomanagement of metal contaminated soils, Environmental Pollution, Vol. 20, Springer Germany, pp: 95–123.
Pajuelo E, Rodriguez-Llorente ID, Dary M, Palomares AJ (2008) Toxic effects of arsenic on Sinorhizobium-Medicago sativa symbiotic interaction. Environ Pollut 154:203–211. doi:10.1016/j.envpol.2007.10.015
Pajuelo E, Carrasco JA, Chamber MA, Romero LC, Gotor C (2007) Evaluation of the metal phytoextraction potential of crop legumes. Regulation of the expression of O-acetylserine (thiol) lyase under metal stress. Plant Biol 9:672–681. doi:10.1055/s-2007-965439
Pan A, Yang M, Tie F, Li L, Chen Z, Ru B (1994) Expression of mouse metallothionein-1-gene confers cadmium resistance in transgenic tobacco plants. Plant Mol Biol 24:341–351. doi:10.1007/BF00020172
Panda SK (2008) Impact of copper on reactive oxygen species, lipid peroxidation and antioxidants in Lemna minor. Biol Plant 52:561–564. doi:10.1007/s10535-008-0111-7
Peralta-Videa JR, De la Rosa G, Gonzalez JH, Gardea-Torresdey JL (2004) Effects of the growth stage on the heavy metal tolerance of alfalfa plants. Adv Environ Res 8:679–685. doi:10.1016/S1093-0191(03)00040-6
Posmyk MM, Kontek R, Janas KM (2009) Antioxidant enzymes activity and phenolic compounds content in red cabbage seedlings exposed to copper stress. Ecotoxicol Environ Saf 72:596–602. doi:10.1016/j.ecoenv.2008.04.024
Puckette MC, Weng H, Mahalingam R (2007) Physiological and biochemical responses to acute ozone-induced oxidative stress in Medicago truncatula. Plant Physiol Biochim 45:70–79. doi:10.1016/j.plaphy.2006.12.004
Puppo A, Groten K, Bastian F, Carzaniga R, Soussi M, Lucas MM, De Felipe MR, Harrison J, Vanacker H, Foyer CH (2005) Legume nodule senescence: roles for redox and hormone signaling in the orchestration of the natural aging process. New Phytol 165:683–701. doi:10.1111/j.1469-8137.2004.01285.x
Rajkumar M, Mab Y, Freitas H (2013) Improvement of Ni phytostabilization by inoculation of Ni resistant Bacillus megaterium SR28C. J Environ Manag 128:973–980. doi:10.1016/j.jenvman.2013.07.001
Rajkumar M, Sandhya S, Prasad MNV, Freitas H (2012) Perspectives of plant-associated microbes in heavy metal phytoremediation. Biotechnol Adv 30:1562–1574. doi:10.1016/j.biotechadv.2012.04.011
Robinson NJ, Winge DR (2010) Copper metallochaperones. Annu Rev Biochem 79:537–562. doi:10.1146/annurev-biochem-030409-143539
Rodríguez-Llorente ID, Pérez-Palacios P, Doukkali B, Caviedes MA, Pajuelo E (2010) Expression of the seed-specific metallothionein mt4a in plant vegetative tissues increases Cu and Zn tolerance. Plant Sci 178:327–332. doi:10.1016/j.plantsci.2010.01.011
Romaniuk K, Dziewit L, Decewicz P, Mielnicki S, Radlinska M, Drewniak L (2016) Molecular characterization of the pSinB plasmid of the arsenite oxidizing, metallotolerant Sinorhizobium sp. M14—insight into the heavy metal resistome of sinorhizobial extrachromosomal replicons. FEMS Microbiol Ecol 93(1):fiw215. doi:10.1093/femsec/fiw215
Rose RJ (2008) Medicago truncatula As a model for understanding plant interactions with other organisms, plant development and stress biology: past, present and future. Funct Plant Biol 35:253–264. doi:10.1071/FP07297
Sharma SS, Dietz KJ (2008) The relationship between metal toxicity and cellular redox imbalance. Trends Plant Sci 14:43–50. doi:10.1016/j.tplants.2008.10.007
Shrivastava AK (2009) A review on copper pollution and its removal from water bodies by pollution control technologies. Ind J Environ Protect 29:552–560 http://www.indiaenvironmentportal.org.in/files/A%20review%20on%20copper%20pollution.pdf
Sosa-Alderete LG, Agostini E, Medina MI (2011) Antioxidant response of tobacco (Nicotiana tabacum) hairy roots after phenol treatment. Plant Physiol Biochem 49:1020–1028. doi:10.1016/j.plaphy.2011.07.009
Sriprang R, Hayashi M, Yamashita M, Ono H, Saeki K, Murooka Y (2002) Novel bioremediation system for heavy metals using the symbiosis between leguminous plant and genetically engineered rhizobia. J Biotechnol 99:279–293. doi:10.1016/S0168-1656(02)00219-5
Srivastava S, Mishra S, Tripathi RD, Dwivedi S, Gupta DK (2006) Copper-induced oxidative stress and responses of antioxidants and phytochelatins in Hydrilla verticillata (L.f.) Royle. Aquatic Toxicol 80:405–515. doi:10.1016/j.aquatox.2006.10.006
Stern BR (2010) Essentiality and toxicity in copper health risk assessment: overview, update and regulatory considerations. J Toxicol Environ Health 73:114–127. doi:10.1080/15287390903337100
Tak HI, Ahmad F, Babalola OO (2013) Advances in the application of plant growth-promoting rhizobacteria in phytoremediation of heavy metals. In: D.M. Whitacre, Reviews of Environmental Contamination and Toxicology, Springer New York 223:33–52. doi:10.1007/978-1-4614-5577-6_2
Teng Y, Wang X, Li L, Li Z, Luo Y (2015) Rhizobia and their bio-partners as novel drivers for functional remediation in contaminated soils. Front Plant Sci 6:32. doi:10.3389/fpls.2015.00032
Tripathi BN, Mehta SK, Amar A, Gaur JP (2006) Oxidative stress in Scenedesmus sp. during short- and long-term exposure to Cu2+ and Zn2+. Chemosphere 62:538–544. doi:10.1016/j.chemosphere.2005.06.031
Trujillo ME, Willems A, Abril A, Planchuelo AM, Rivas R, Ludeña D, Mateos PF, Martínez-Molina E, Velázquez E (2005) Nodulation of Lupinus albus by strains of Ochrobactrum lupini sp. nov. Appl Environ Microbiol 71:1318–1327. doi:10.1128/AEM.71.3.1318-1327.2005
Ullah A, Heng S, Munis MFH, Fahad S, Yang X (2015) Phytoremediation of heavy metals assisted by plant growth promoting (PGP) bacteria: a review. Environ Exp Bot 117:28–40. doi:10.1016/j.envexpbot.2015.05.001
Valderrama B, Dávalos A, Girard L, Morett E, Mora J (1996) Regulatory proteins and cis-acting elements involved in the transcriptional control of Rhizobium etli reiterated nifH genes. J Bacteriol 178: 3119–3126. doi: PMC178061
Vangronsveld J, Herzig R, Weyens N, Boulet J, Adriaensen K, Ruttens A, Thewys T, Vassilev A, Meers E, Nehnevajova E, Van der Lelie D, Mench M (2009) Phytoremediation of contaminated soils and groundwater: lessons from the field. Environ Sci Pollut Res 16:765–794. doi:10.1007/s11356-009-0213-6
Wang SH, Zhang H, Zhang Q, Jin GM, Jiang SJ, Jiang D, He QY, Li ZP (2011) Copper-induced oxidative stress and responses of the antioxidant system in roots of Medicago sativa. J Agron Crop Sci 197:418–429. doi:10.1111/j.1439-037X.2011.00476.x
Wenzel WW (2009) Rhizosphere processes and management in plant-assisted bioremediation (phytoremediation) of soils. Plant Soil 321:385–408. doi:10.1007/s11104-008-9686-1
White CN, Rivin CJ (1995) Characterization and expression of cDNA encoding a seed-specific metallothionein in maize. Plant Physiol 108:831–832 http://www.ncbi.nlm.nih.gov/pmc/articles/PMC157410
Wopereis J, Pajuelo E, Dazzo FB, Jiang Q, Gresshoff PM, De Bruijn FJ, Stougaard J, Szczyglowski K (2000) Short root mutant of Lotus japonicus with a dramatically altered symbiotic phenotype. Plant J 23:97–114. doi:10.1046/j.1365-313x.2000.00799.x
Yamasaki H, Pilon M, Shikanai T (2008) How do plants respond to copper deficiency?. Plant Signal Behav 3: 231–232. doi: PMC2634185
Zurdo-Piñeiro JL, Rivas R, Trujillo ME, Vizcaíno N, Carrasco JA, Chamber M, Palomares A, Mateos PF, Martínez-Molina E, Velázquez E (2007) Ochrobactrum cytisi sp. nov., isolated from nodules of Cytisus scoparius in Spain. Int J Syst Evol Microbiol 57:784–788. doi:10.1099/ijs.0.64613-0
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This research was financed by MINECO (project BIO-2009-7766) and Junta de Andalucía (project P11-RNM-7274). P. Pérez-Palacios acknowledges a grant from the University of Sevilla.
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Pérez-Palacios, P., Romero-Aguilar, A., Delgadillo, J. et al. Double genetically modified symbiotic system for improved Cu phytostabilization in legume roots. Environ Sci Pollut Res 24, 14910–14923 (2017). https://doi.org/10.1007/s11356-017-9092-4
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DOI: https://doi.org/10.1007/s11356-017-9092-4