Skip to main content
SpringerLink
Log in

Diclofenac shifts the role of root glutamine synthetase and glutamate dehydrogenase for maintaining nitrogen assimilation and proline production at the expense of shoot carbon reserves in Solanum lycopersicum L

  • Research Article
  • Published:
Environmental Science and Pollution Research Aims and scope Submit manuscript

Abstract

The continuous increase of the human population worldwide has led to an increase of pharmaceuticals’ consumption, such as diclofenac (DCF), a widely used non-steroidal anti-inflammatory drug (NSAID), that is not removed by wastewater treatment processes. Although there is some research regarding the effects of DCF on animals and aquatic invertebrates, information concerning its influence on plants’ metabolism is still scarce. Through an integrated approach, using combined biochemical and molecular biology techniques, this work aimed to evaluate the phytotoxicity of DCF in Solanum lycopersicum L., focusing on the primary plant processes: nitrogen (N) assimilation and photosynthesis. The exposure of tomato plants to increasing concentrations of DCF (0, 0.5, and 5 mg L−1) revealed that glutamine synthetase (GS) was differentially affected, in an organ-dependent manner, by this contaminant at the gene expression, protein, and activity levels, with an increased activity of 0.2-fold in shoots of plants treated with the lowest concentration of DCF although a general decrease was registered for the SlGS gene family expression, revealing that post-translational regulation was in order, since GS2 polypeptide content did not change. Glutamate dehydrogenase (GDH) activity was generally enhanced, accompanied by increases of 0.4- to 1.9-fold in proline levels, revealing GDH as an important compensatory route for both N assimilation and proline production under stressful conditions. No alterations in most photosynthetic endpoints were noticed after DCF treatments, but small decreases of 0.1- to 0.8-fold in the accumulation of RuBisCO-encoding transcripts were observed, along with a reduction in starch content. Some alterations in the soluble polypeptide profile were also detected in response to DCF, evidencing the participation of some stress-related proteins in the plant’s response to DCF.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4
Fig. 5
Fig. 6
Fig. 7
Fig. 8

Similar content being viewed by others

Abbreviations

PPCPs:

Pharmaceuticals and personal compounds

DCF:

Diclofenac

WWTPs:

Wastewater treatment plants

NSAID:

Non-steroidal anti-inflammatory drug

GS:

Glutamine synthetase

GDH:

Glutamate dehydrogenase

NR:

Nitrate reductase

RuBisCO:

Ribulose-1,5-biphosphate carboxylase oxygenase

AC:

Acetaminophen

ROS:

Reactive oxygen species

References

  • aus der Beek T, Weber FA, Bergmann A, Hickmann S, Ebert I, Hein A, Küster A (2016) Pharmaceuticals in the environment—global occurrences and perspectives. Environ Toxicol Chem 35:823–835

    CAS  Google Scholar 

  • Balakrishna K, Rath A, Praveenkumarreddy Y, Guruge KS, Subedi B (2017) A review of the occurrence of pharmaceuticals and personal care products in Indian water bodies. Ecotoxicol Environ Saf 137:113–120

    CAS  Google Scholar 

  • Bartha B, Huber C, Schröder P (2014) Uptake and metabolism of diclofenac in Typha latifolia–how plants cope with human pharmaceutical pollution. Plant Sci 227:12–20

    CAS  Google Scholar 

  • Bartrons M, Peñuelas J (2017) Pharmaceuticals and personal-care products in plants. Trends Plant Sci 22:194–203

    CAS  Google Scholar 

  • Bates L, Waldren R, Teare I (1973) Rapid determination of free proline for water-stress studies. Plant Soil 39:205–207

    CAS  Google Scholar 

  • Benotti MJ, Trenholm RA, Vanderford BJ, Holady JC, Stanford BD, Snyder SA (2008) Pharmaceuticals and endocrine disrupting compounds in US drinking water. Environ Sci Technol 43:597–603

    Google Scholar 

  • Bernard SM, Habash DZ (2009) The importance of cytosolic glutamine synthetase in nitrogen assimilation and recycling. New Phytol 182:608–620

    CAS  Google Scholar 

  • Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254

    Article  CAS  Google Scholar 

  • Brugière N, Dubois F, Limami AM, Lelandais M, Roux Y, Sangwan RS, Hirel B (1999) Glutamine synthetase in the phloem plays a major role in controlling proline production. Plant Cell 11:1995–2011

    Google Scholar 

  • Buser H-R, Poiger T, Müller MD (1998) Occurrence and fate of the pharmaceutical drug diclofenac in surface waters: rapid photodegradation in a lake. Environ Sci Technol 32:3449–3456

    CAS  Google Scholar 

  • Canton FR, Garcia-Gutierrez A, Crespillo R, Cánovas FM (1996) High‐level expression of Pinus sylvestris glutamine synthetase in Escherichia coli: Production of polyclonal antibodies against the recombinant protein and expression studies in pine seedlings. FEBS letters, 393(2-3):205–210.

  • Chen G, den Braver MW, van Gestel CA, van Straalen NM, Roelofs D (2015) Ecotoxicogenomic assessment of diclofenac toxicity in soil. Environ Pollut 199:253–260

    CAS  Google Scholar 

  • Christou A, Antoniou C, Christodoulou C, Hapeshi E, Stavrou I, Michael C, Fatta-Kassinos D, Fotopoulos V (2016) Stress-related phenomena and detoxification mechanisms induced by common pharmaceuticals in alfalfa (Medicago sativa L.) plants. Sci Total Environ 557:652–664

    Google Scholar 

  • Christou A, Agüera A, Bayona JM, Cytryn E, Fotopoulos V, Lambropoulou D, Manaia CM, Michael C, Revitt M, Schröder P (2017) The potential implications of reclaimed wastewater reuse for irrigation on the agricultural environment: the knowns and unknowns of the fate of antibiotics and antibiotic resistant bacteria and resistance genes–a review. Water Res 123:448–467

    CAS  Google Scholar 

  • Copolovici L, Timis D, Taschina M, Copolovici D, Cioca G, Bungau S (2017) Diclofenac influence on photosynthetic parameters and volatile organic compounds emission from Phaseolus vulgaris L. plants. Rev Chim 68:2076–2078

    CAS  Google Scholar 

  • Cren M, Hirel B (1999) Glutamine synthetase in higher plants regulation of gene and protein expression from the organ to the cell. Plant Cell Physiol 40:1187–1193

    CAS  Google Scholar 

  • Cullimore JV, Sims AP (1981) Glutamine synthetase of Chlamydomonas: its role in the control of nitrate assimilation. Planta 153:18–24

    CAS  Google Scholar 

  • Davies NM, Anderson KE (1997) Clinical pharmacokinetics of diclofenac. Clin Pharmacokinet 33:184–213

    CAS  Google Scholar 

  • de García SO, Pinto GP, Encina PG, Mata RI (2013) Consumption and occurrence of pharmaceutical and personal care products in the aquatic environment in Spain. Sci Total Environ 444:451–465

    Google Scholar 

  • Dubois F, Tercé-Laforgue T, Gonzalez-Moro M-B, Estavillo J-M, Sangwan R, Gallais A, Hirel B (2003) Glutamate dehydrogenase in plants: is there a new story for an old enzyme? Plant Physiol Biochem 41:565–576

    CAS  Google Scholar 

  • Fatta-Kassinos D, Meric S, Nikolaou A (2011) Pharmaceutical residues in environmental waters and wastewater: current state of knowledge and future research. Anal Bioanal Chem 399:251–275

    CAS  Google Scholar 

  • Feller U, Anders I, Demirevska K (2008) Degradation of rubisco and other chloroplast proteins under abiotic stress. Gen Appl Plant Physiol 34:5–18

    CAS  Google Scholar 

  • Food and Agriculture Organization of the United Nations, (2014) FAOSTAT Statistics Database. FAOSTAT, 2014. www.faostat.org. Accessed 5.12.19

  • Gajewska E, Skłodowska M (2009) Nickel-induced changes in nitrogen metabolism in wheat shoots. J Plant Physiol 166:1034–1044

    CAS  Google Scholar 

  • González-Naranjo V, Boltes K, de Bustamante I, Palacios-Diaz P (2015) Environmental risk of combined emerging pollutants in terrestrial environments: chlorophyll a fluorescence analysis. Environ Sci Pollut Res 22:6920–6931

    Google Scholar 

  • Gorito AM, Ribeiro AR, Almeida CMR, Silva AM (2017) A review on the application of constructed wetlands for the removal of priority substances and contaminants of emerging concern listed in recently launched EU legislation. Environ Pollut 227:428–443

    CAS  Google Scholar 

  • Gururani MA, Venkatesh J, Tran LSP (2015) Regulation of photosynthesis during abiotic stress-induced photoinhibition. Mol Plant 8:1304–1320

    CAS  Google Scholar 

  • Hájková M, Kummerová M, Zezulka Š, Babula P, Váczi P (2019) Diclofenac as an environmental threat: impact on the photosynthetic processes of Lemna minor chloroplasts. Chemosphere 224:892–899

    Google Scholar 

  • He B-s, Wang J, Liu J, Hu X-m (2017) Eco-pharmacovigilance of non-steroidal anti-inflammatory drugs: necessity and opportunities. Chemosphere 181:178–189

    CAS  Google Scholar 

  • Huber C, Bartha B, Schröder P (2012) Metabolism of diclofenac in plants–hydroxylation is followed by glucose conjugation. J Hazard Mater 243:250–256

    CAS  Google Scholar 

  • Huber C, Preis M, Harvey PJ, Grosse S, Letzel T, Schröder P (2016) Emerging pollutants and plants–metabolic activation of diclofenac by peroxidases. Chemosphere 146:435–441

    CAS  Google Scholar 

  • James D, Borphukan B, Fartyal D, Achary V, Reddy M (2018) Transgenic manipulation of glutamine synthetase: a target with untapped potential in various aspects of crop improvement, Biotechnologies of Crop Improvement, Volume 2. Springer, pp. 367-416

  • Jha AB, Dubey RS (2004) Arsenic exposure alters activity behaviour of key nitrogen assimilatory enzymes in growing rice plants. Plant Growth Regul 43:259–268

    CAS  Google Scholar 

  • K’oreje KO, Kandie FJ, Vergeynst L, Abira MA, Van Langenhove H, Okoth M, Demeestere K (2018) Occurrence, fate and removal of pharmaceuticals, personal care products and pesticides in wastewater stabilization ponds and receiving rivers in the Nzoia Basin, Kenya. Sci Total Environ 637:336–348

    Google Scholar 

  • Kalaji HM, Carpentier R, Allakhverdiev SI, Bosa K (2012) Fluorescence parameters as early indicators of light stress in barley. J Photochem Photobiol B Biol 112:1–6

    CAS  Google Scholar 

  • Kim SD, Cho J, Kim IS, Vanderford BJ, Snyder SA (2007) Occurrence and removal of pharmaceuticals and endocrine disruptors in South Korean surface, drinking, and waste waters. Water Res 41:1013–1021

    CAS  Google Scholar 

  • Krasensky J, Jonak C (2012) Drought, salt, and temperature stress-induced metabolic rearrangements and regulatory networks. J Exp Bot 63:1593–1608

    CAS  Google Scholar 

  • Kummerová M, Zezulka Š, Babula P, Tříska J (2016) Possible ecological risk of two pharmaceuticals diclofenac and paracetamol demonstrated on a model plant Lemna minor. J Hazard Mater 302:351–361

    Google Scholar 

  • Kwinta J, Cal KP (2005) Effects of salinity stress on the activity of glutamine synthetase and glutamate dehydrogenase in triticale seedlings. Pol J Environ Stud 14

  • Laemmli U (1976) SDS-PAGE to evaluate extent of hydrolysis, of proteins. Nature 227:680–685

    Google Scholar 

  • Lapworth D, Baran N, Stuart M, Ward R (2012) Emerging organic contaminants in groundwater: a review of sources, fate and occurrence. Environ Pollut 163:287–303

    CAS  Google Scholar 

  • Leclercq J, Adams-Phillips LC, Zegzouti H, Jones B, Latché A, Giovannoni JJ, Pech J-C, Bouzayen M (2002) LeCTR1, a tomato CTR1-like gene, demonstrates ethylene signaling ability in Arabidopsis and novel expression patterns in tomato. Plant Physiol 130:1132–1142

    CAS  Google Scholar 

  • Lichtenthaler HK (1987) [34] Chlorophylls and carotenoids: pigments of photosynthetic biomembranes, Methods in enzymology. Elsevier, pp. 350-382

  • Liu L, Wang J, Han Z, Sun X, Li H, Zhang J, Lu Y (2016) Molecular analyses of tomato GS, GOGAT and GDH gene families and their response to abiotic stresses. Acta Physiol Plant 38:229

    Google Scholar 

  • Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. methods 25, 402-408

  • Lonappan L, Brar SK, Das RK, Verma M, Surampalli RY (2016) Diclofenac and its transformation products: environmental occurrence and toxicity-a review. Environ Int 96:127–138

    CAS  Google Scholar 

  • Loulakakis KA, Roubelakis-Angelakis KA (1990) Plant NAD (H)-glutamate dehydrogenase consists of two subunit polypeptides and their participation in the seven isoenzymes occurs in an ordered ratio. Plant Physiol 97:104–111

  • Løvdal T, Lillo C (2009) Reference gene selection for quantitative real-time PCR normalization in tomato subjected to nitrogen, cold, and light stress. Anal Biochem 387:238–242

    Google Scholar 

  • Madikizela LM, Ncube S, Chimuka L (2018) Uptake of pharmaceuticals by plants grown under hydroponic conditions and natural occurring plant species: a review. Sci Total Environ 636:477–486

    CAS  Google Scholar 

  • Mariz-Ponte N (2017) Use of the UV-A and UV-B light supplementation in tomato producing: a perspective from plant to fruit. Dissertation, Faculty of Sciences of University of Porto. https://repositorio-aberto.up.pt/handle/10216/110653

  • Martí E, Gisbert C, Bishop GJ, Dixon MS, García-Martínez JL (2006) Genetic and physiological characterization of tomato cv. Micro-Tom. J Exp Bot 57:2037–2047

    Google Scholar 

  • Metcalfe CD, Miao XS, Koenig BG, Struger J (2003) Distribution of acidic and neutral drugs in surface waters near sewage treatment plants in the lower Great Lakes, Canada. Environ Toxicol Chem 22:2881–2889

    CAS  Google Scholar 

  • Moreira JT, Moreira TM, Cunha JB, Azenha M, Fidalgo F, Teixeira JB (2018) Differential effects of acetophenone on shoots’ and roots’ metabolism of Solanum nigrum L. plants and implications in its phytoremediation. Plant Physiol 130:391–398

    CAS  Google Scholar 

  • Nagy Z, Németh E, Guóth A, Bona L, Wodala B, Pécsváradi A (2013) Metabolic indicators of drought stress tolerance in wheat: glutamine synthetase isoenzymes and Rubisco. Plant Physiol Biochem 67:48–54

    CAS  Google Scholar 

  • Nieva FJJ, Castellanos EM, Figueroa ME, Gil F (1999) Gas exchange and chlorophyll fluorescence of C3 and C4 saltmarsh species. Photosynthetica 36:397–406

    CAS  Google Scholar 

  • Oaks JL, Gilbert M, Virani MZ, Watson RT, Meteyer CU, Rideout BA, Shivaprasad HL, Ahmed S, Iqbal Chaudhry MJ, Arshad M, Mahmood S, Ali A, Ahmed Khan A (2004) Diclofenac residues as the cause of vulture population decline in Pakistan. Nature 427:630–633

    CAS  Google Scholar 

  • Odendaal C, Seaman MT, Kemp G, Patterton HE, Patterton H-G (2015) An LC-MS/MS based survey of contaminants of emerging concern in drinking water in South Africa. S Afr J Sci 111:01–06

    Google Scholar 

  • Öllers S, Singer HP, Fässler P, Müller SR (2001) Simultaneous quantification of neutral and acidic pharmaceuticals and pesticides at the low-ng/l level in surface and waste water. J Chromatogr A 911:225–234

    Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT–PCR. Nucleic Acids Res 29:e45

    CAS  Google Scholar 

  • Pierattini EC, Francini A, Huber C, Sebastiani L, Schröder P (2018) Poplar and diclofenac pollution: a focus on physiology, oxidative stress and uptake in plant organs. Sci Total Environ 636:944–952

    CAS  Google Scholar 

  • Rabiet M, Togola A, Brissaud F, Seidel J-L, Budzinski H, Elbaz-Poulichet F (2006) Consequences of treated water recycling as regards pharmaceuticals and drugs in surface and ground waters of a medium-sized Mediterranean catchment. Environ Sci Technol 40:5282–5288

    CAS  Google Scholar 

  • Sathishkumar P, Meena RAA, Palanisami T, Ashokkumar V, Palvannan T, Gu FL (2019) Occurrence, interactive effects and ecological risk of diclofenac in environmental compartments and biota-a review. Sci Total Environ 134057

  • Schmidt W, Rainville LC, McEneff G, Sheehan D, Quinn B (2014) A proteomic evaluation of the effects of the pharmaceuticals diclofenac and gemfibrozil on marine mussels (Mytilus spp.): evidence for chronic sublethal effects on stress-response proteins. Drug Test Anal 6:210–219

    CAS  Google Scholar 

  • Sharma A, Kumar V, Shahzad B, Ramakrishnan M, Sidhu GPS, Bali AS, Handa N, Kapoor D, Yadav P, Khanna K (2019) Photosynthetic response of plants under different abiotic stresses: a review. J Plant Growth Regul:1–23

  • Soares C, Branco-Neves S, de Sousa A, Teixeira J, Pereira R, Fidalgo F (2018) Can nano-SiO2 reduce the phytotoxicity of acetaminophen?–A physiological, biochemical and molecular approach. Environ Pollut 241:900–911

    CAS  Google Scholar 

  • Soares C, Carvalho ME, Azevedo RA, Fidalgo F (2019) Plants facing oxidative challenges—a little help from the antioxidant networks. Environ Exp Bot 161:4–25

    CAS  Google Scholar 

  • Sousa B, Lopes J, Leal A, Martins M, Soares C, Valente IM, Rodrigues JA, Fidalgo F, Teixeira J (2020) Response of Solanum lycopersicum L to diclofenac–impacts on the plant’s antioxidant mechanisms. Environ Pollut 258:113762

    CAS  Google Scholar 

  • Szabados L, Savoure A (2010) Proline: a multifunctional amino acid. Trends Plant Sci 15:89–97

    CAS  Google Scholar 

  • Taiz L, Zeiger E, Møller IM, Murphy AS (2015) Plant physiology and development, Sixth ed. Sinauer Associates, Inc., Sunderland, USA

  • Teixeira J, Pereira S, Cánovas F, Salema R (2005) Glutamine synthetase of potato (Solanum tuberosum L. cv. Desiree) plants: cell-and organ-specific expression and differential developmental regulation reveal specific roles in nitrogen assimilation and mobilization. J Exp Bot 56:663–671

    CAS  Google Scholar 

  • Thalmann M, Santelia D (2017) Starch as a determinant of plant fitness under abiotic stress. New Phytol 214:943–951

    CAS  Google Scholar 

  • Thomsen HC, Eriksson D, Møller IS, Schjoerring JK (2014) Cytosolic glutamine synthetase: a target for improvement of crop nitrogen use efficiency? Trends Plant Sci 19:656–663

    CAS  Google Scholar 

  • Triebskorn R, Casper H, Heyd A, Eikemper R, Köhler H-R, Schwaiger J (2004) Toxic effects of the non-steroidal anti-inflammatory drug diclofenac: part II. Cytological effects in liver, kidney, gills and intestine of rainbow trout (Oncorhynchus mykiss). Aquat Toxicol 68:151–166

    CAS  Google Scholar 

  • Vieno N, Sillanpää M (2014) Fate of diclofenac in municipal wastewater treatment plant—a review. Environ Int 69:28–39

    CAS  Google Scholar 

  • Von Caemmerer SV, Farquhar GD (1981) Some relationships between the biochemistry of photosynthesis and the gas exchange of leaves. Planta 153:376–387

    Google Scholar 

  • Yang Y, Ok YS, Kim K-H, Kwon EE, Tsang YF (2017) Occurrences and removal of pharmaceuticals and personal care products (PPCPs) in drinking water and water/sewage treatment plants: a review. Sci Total Environ 596:303–320

    Google Scholar 

Download references

Acknowledgments

GreenUPorto is highly recognized for equipment support.

Funding

This research was supported by national funds through FCT - Foundation for Science and Technology within the scope of UIDB/05748/2020 and UIDP/05748/2020.

Author information

Authors and Affiliations

  1. GreenUPorto - Sustainable Agrifood Production Research Centre, Biology Department, Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal

    Maria Martins, Bruno Sousa, Jorge Lopes, Cristiano Soares, Fernanda Fidalgo & Jorge Teixeira

  2. Departamento de Biologia, Faculdade de Ciências, Universidade do Porto, Rua Campo Alegre s/n, 4169-007, Porto, Portugal

    Maria Martins

  3. GreenUPorto - Sustainable Agrifood Production Research Centre, Geosciences, Environment and Spatial Plannings Department, Faculty of Sciences, University of Porto, Rua do Campo Alegre s/n, 4169-007, Porto, Portugal

    Joana Machado & Susana Carvalho

Authors
  1. Maria Martins
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Bruno Sousa
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Jorge Lopes
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Cristiano Soares
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Joana Machado
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Susana Carvalho
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Fernanda Fidalgo
    View author publications

    You can also search for this author in PubMed Google Scholar

  8. Jorge Teixeira
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Maria Martins.

Additional information

Responsible editor: Gangrong Shi

Publisher’s note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Rights and permissions

Reprints and permissions

About this article

Check for updates. Verify currency and authenticity via CrossMark

Cite this article

Martins, M., Sousa, B., Lopes, J. et al. Diclofenac shifts the role of root glutamine synthetase and glutamate dehydrogenase for maintaining nitrogen assimilation and proline production at the expense of shoot carbon reserves in Solanum lycopersicum L. Environ Sci Pollut Res 27, 29130–29142 (2020). https://doi.org/10.1007/s11356-020-09136-x

Download citation

  • Received:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11356-020-09136-x

Keywords

Search

Navigation