Impact of copper toxicity on stone-head cabbage (Brassica oleracea var. capitata) in hydroponics

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1 Impct of copper toxicity on stone-hed cge (Brssic olerce vr. cpitt) in hydroponics Sjid Ali 1, Muhmmd Shhz 1,2, Ahmd Neem Shhzd 3, Hfiz Azhr Ali Khn 1, Mozzm Anees 1, Muhmmd Sleem Hider 1 nd Ammr Ftim 4 1 Institute of Agriculturl Sciences, University of the Punj, Lhore, Pkistn 2 Deprtment of Biology, Colordo Stte University, Fort Collins, Colordo, USA 3 Deprtment of Agronomy, Bhuddin Zkriy University, Multn, Pkistn 4 Deprtment of Environmentl Science, Lhore College for Women University, Lhore, Pkistn ABSTRACT Arle soils re frequently sujected to contmintion with copper s the consequence of imlnced fertiliztion with mnure nd orgnic fertilizers nd/or extensive use of copper-contining fungicides. In the present study, the exposure of stone-hed cge (Brssic olerce vr. cpitt) to elevted Cu 2+ levels resulted in lef chlorosis nd lesser iomss yield t 2 µm. Root nitrte content ws not sttisticlly ffected y Cu 2+ levels, lthough it ws sustntilly decresed t 5 µm Cu 2+ in the shoot. The decrese in nitrte contents cn e relted to lower nitrte uptke rtes ecuse of growth inhiition y Cu-toxicity. Shoot sulfte content incresed strongly t 2 µm Cu 2+ indicting n increse in demnd for sulfur under Cu stress. Furthermore, t 2 µm concentrtion, concentrtion of wter-solule non-protein thiol incresed mrkedly in the roots nd to smller level in the shoot. When exposed to elevted concentrtions of Cu 2+ the improved sulfte nd wter-solule non-protein thiols need further studies for the evlution of their direct reltion with the synthesis of metl-chelting compounds (i.e., phytocheltins). Sumitted 12 Ferury 2015 Accepted 2 July 2015 Pulished 4 August 2015 Corresponding uthor Sjid Ali, sjid.igs@pu.edu.pk Acdemic editor Xiolin Sun Additionl Informtion nd Declrtions cn e found on pge 8 DOI /peerj.1119 Copyright 2015 Ali et l. Distriuted under Cretive Commons CC-BY 4.0 OPEN ACCESS Sujects Agriculturl Science, Biochemistry, Plnt Science, Soil Science, Toxicology Keywords Lef chlorosis, Nutrient uptke, Non-protein thiol, Cu contmintion, Biomss, Sulfur, Brssic, Copper, Thiols, Hydroponics, Toxicity, Cge INTRODUCTION Trnsition metls such s copper (Cu), zinc (Zn) nd molydenum (Mo) re essentil for the growth nd development of plnts, ut they rpidly get toxic t higher levels (Kopsell & Kopsell, 2007). Cu contmintion in griculturl soils s consequence of mining metls, dispersl of sewge sludge, ritrry nd improper ppliction of grochemicls, ddition of orgnic fertilizers nd frequent use of irrigtion with low qulity wter is well-known prolem (Dch & Strmns, 2005; Yruel, 2009). Cu is redox ctive metl tht cn exist in oth Cu 2+ nd Cu + forms in living orgnisms. At the protein level, Cu serves s co-fctor for vrious enzymes such s Cu/Zn-superoxide dismutse (Cu/ZnSOD), cytochrome c oxidse, scorte oxidse, mino oxidse, lccse, plstocynin (PC), nd polyphenol How to cite this rticle Ali et l. (2015), Impct of copper toxicity on stone-hed cge (Brssic olerce vr. cpitt) in hydroponics. PeerJ 3:e1119; DOI /peerj.1119

2 oxidse (Yruel, 2005; Yruel, 2009; Pilon et l., 2006). However, redox cycling etween Cu 2+ nd Cu + could induce oxidtive stress y producing highly toxic hydroxyl rdicls (Yruel, 2005; Yruel, 2009). Plnts exposed to elevted levels of Cu show unspecific toxicity symptoms. Elevted Cu levels in soils primrily result in stunted root growth nd lef chlorosis (Kopsell & Kopsell, 2007; Shhz et l., 2010). Copper toxicity-induced reduction in chlorophyll contents hinder the development of chloroplst, thlkoid memrne nd photosystem II (PSII), which re considered s the most sensitive Cu toxicity sites (Pätsikkä, Aron & Tyystjärvi, 1998; Pätsikkä et l., 2002; Burkhed et l., 2009; Yruel, 2005; Yruel, 2009; Shhz et l., 2010). At cellulr level, toxicity my led to inding of sulfhydryl groups in proteins, insufficiency or excess of other essentil ions, oxidtive dmge nd reduced cell trnsport (De Vos et l., 1993; Yruel, 2009). Furthermore, Cu-toxicity cn chnge the minerl composition of plnts. For instnce, Fe contents my decrese in the shoot (Pätsikkä, Aron & Tyystjärvi, 1998; Pätsikkä et l., 2002; Kopsell & Kopsell, 2007; Shhz et l., 2010), C nd Mg my decrese in the root nd Zn contents my increse in oth root nd shoot upon Cu exposure t elevted levels (Shhz et l., 2010). Root growth is more severely ffected y elevted Cu thn shoot growth nd the mjor proportion of Cu uptke retins in the root. Incresed Cu contents in the plnt tissues induce the synthesis of metl-inding compounds (viz. phytocheltins), which re most likely glutthione-derived compounds. (Inouhe, 2005; Ernst et l., 2008). Inductions of phytocheltins presume tht more sulfur is needed for synthesis of these compounds, which results into higher sorption, nd incorportion of sulfte. Nonetheless, the role of phytocheltins in detoxifiction of Cu is not very cler yet (Ernst et l., 2008; Yruel, 2005; Yruel, 2009; Shhz et l., 2010). Brssic nd other vegetle crops re often grown in the surrounding res of ig cities nd industril res in developing countries like Pkistn, where they my e sujected to ir nd hevy metls pollution (Yng, Stulen & De Kok, 2006). The direct ppliction of sewge wter to vegetles is not only the source of mny nutrients, ut it is often contminted with high levels of Cu nd other hevy metls. As result of continues untreted sewge ppliction, hevy metls not only ccumulte in the soil ut lso in vegetles (Youns et l., 1998; Butt et l., 2005). High Cu content in crop plnts might not only negtively ffect plnt growth nd functioning, ut will lso enter the food chin (Brun et l., 2001). The present study used the hydroponics system which llows very close control over wter solule Cu nd other minerl concentrtions s compred to soil-grown system. Cge is very importnt vegetle in ll over the world. In Pkistn, cge is cultivted on lmost 4.9 thousnd hectres with 76.7 thousnd tonnes nnul production (FAO, 2013).The present study ws imed to investigte the response of growth, pigment contents nd sulfur metolism of stone-hed cge grown in hydroponics to copper exposure. Ali et l. (2015), PeerJ, DOI /peerj /10

3 MATERIAL AND METHODS Stone-hed cge (Brssic olerce vr. cpitt F 1 ) seeds were germinted to snd in green house. The seedlings collected t ten dys fter germintion were trnsferred on n erted 25% modified Hoglnd nutrient solution in 11 liter continer (15.8 L 10.3 W 7 H; 3 plnts set 1 nd 12 sets continer 1 ) in greenhouse for 10 dys. The nutrient solution consists of 1.25 mm C(NO 3 ) 2.4H 2 O, 1.25 mm KNO 3, 0.25 mm KH 2 PO 4, 0/0.5 mm MgSO 4.7H 2 O, 11.6 µm H 3 BO 3, 2.4 µm MnCl 2.4H 2 O, 0.24 µm ZnSO 4.7H 2 O, 0.08 µm CuSO 4.5H 2 O, 0.13 µm N 2 MoO 4.2H 2 O nd 22.5 µm Fe 3+ -EDTA with supplementl concentrtions of 0, 2, 5 nd 10 µm CuCl 2 nd ph The nutrient solution ws continuously erted with Aqu-Supreme Air Pump Model AP-4.The photoperiod ws 14 h. 30 nd 25 C (±5 C) tempertures were set for dy nd night respectively, wheres the reltive humidity ws mintined t 60 70%. Pigment contents Whole shoot ws homogenized (in 100% cetone 10 ml per g FW) followed y centrifugtion t 800 g for 20 min. Lichtenthler (1987) ws followed for the determintion of chlorophyll, nd totl crotenoid contents. Nitrte nd sulfte contents Frozen root nd shoot mteril ws homogenized in de-minerlized wter (10 ml per g fresh weight) nd one lyer of Mircloth filter ws used to filter the homogente. The superntnt ws incuted in wter th t 100 C for 10 min. The reminder ws centrifuged for 15 min (0 C) t 30,000 g. The nions were seprted y HPLC nd Ms et l. (1986) ws followed for their refrctometric determintion using Knuer differentil refrctometer (model 98.00, Bd Homurg, Germny). Wter-solule non-protein thiols Extrction medium contining 80 mm sulfoslicylic cid, 1 mm EDTA, nd 0.15% (w/v) scoric cid with n Ultr Turrx t 0 C (10 ml per g fresh weight) ws used for the homogeniztion of fresh plnt mtter. The resultnt homogenous mteril ws pssed through one lyer of Mircloth which ws then centrifuged t 30,000 g for 15 min (0 C). De Kok, Buwld & Bosm (1988) ws followed for the determintion of totl wter-solule non-protein thiol content colorimetriclly t 413 nm fter rection with 5, 5 -dithiois [2-nitroenzoic cid]. RESULTS Plnt iomss in response to Cu exposure Exposure of stone-hed cge to higher concentrtions of Cu 2+ ( 2 µm) in nutrient solution cused chlorosis of oth the shoot nd young emerging leves, tht ultimtely reduced oth root nd shoot iomss production (Fig. 1). A 10 dy exposure to incresing Cu 2+ concentrtions in nutrient medi led to significnt reduction of oth root nd shoot iomss production t 2 µm Cu 2+. Shoot to root rtio improved t 5 µm Cu 2+, demonstrting tht when exposed to copper, root growth ws more ffected thn shoot Ali et l. (2015), PeerJ, DOI /peerj /10

4 Shoot & Root Biomss (g FW) Shoot/Root Rtio Shoot Biomss Root Biomss Shoot/Root rtio c d c d Cu Concentrtion (µm) Figure 1 Impct of elevted levels of Cu 2+ on iomss production of stone-hed cge (Brssic olerce vr. cpitt). 10-dy-old seedlings of stone-hed cge (Brssic olerce vr. cpitt) were grown on 25% Hoglnd solution contining 0, 2, 5 nd 10 µm CuCl 2 in the root environment. Dt on iomss production (g FW) nd shoot/root rtio represent the men of 2 independent experiments with 9 mesurements hving 3 plnts in ech tretment (±SD). Mens with different letters differ significntly t p 0.01 (Student s t-test). growth (Fig. 1). Root dry mtter content incresed t 10 µm Cu 2+, wheres shoot dry mtter content incresed t 5 µm Cu 2+ (Fig. 2). Pigment content in response to Cu exposure The totl chlorophyll (Chl. + ) nd crotenoid contents of stone-hed cge were significntly decresed upon exposure t 2 µm Cu 2+ (Fig. 3). There were significnt decreses in chlorophyll / nd chlorophyll/crotenoid rtios when exposed to incresed Cu 2+ concentrtions (10 µm Cu 2+ ). Ten µm Cu 2+ exposure resulted in the strt of rpid development of shoot chlorosis nd significntly fster reduction in chlorophyll contents of chlorophyll nd crotenoids, ultimtely leding to significnt reduction in chlorophyll / nd chlorophyll/crotenoid rtios (Fig. 3). Sulfte nd wter-solule non-protein thiol contents in response to Cu exposure Elevted Cu 2+ levels showed significnt effect on concentrtion of the nitrte, sulfte nd wter-solule non-protein thiol in stone-hed cge. The nitrte contents of the roots showed non significnt response to the Cu exposure t different levels, however in shoots it were significntly decresed t 5 µm Cu 2+ (Fig. 4). Sulfte contents in the roots were not ffected; however, Cu 2+ tretments of 2 µm sustntilly incresed the sulfte contents of the shoot (Fig. 4). There ws slight decrese in nitrte to sulfte rtio in the root nd strong decrese in the shoot when exposed to elevted levels of Cu (Fig. 4). Further- Ali et l. (2015), PeerJ, DOI /peerj /10

5 Chlorophyll (+) nd Crotenoids (mg g -1 FW) Shoot nd Root Dry Mtter Content (%) DMC-Shoot DMC-Root Cu Concentrtion (µm) Figure 2 Impct of elevted levels of Cu 2+ on dry mtter content of stone-hed cge (Brssic olerce vr. cpitt). 10-dy-old seedlings of stone-hed cge (Brssic olerce vr. cpitt) were grown on 25% Hoglnd solution contining 0, 2, 5 nd 10 µm CuCl 2 in the root environment. Dt on dry mtter content (%) represent the men of 2 independent experiments with 9 mesurements hving 3 plnts in ech tretment (±SD). Mens with different letters differ significntly t p 0.01 (Student s t-test). Chl (+) Crotenoids c c d d Cu Concentrtion (µm) Figure 3 Impct of elevted levels of Cu 2+ on pigment content (chl. + & crotenoids) of stone-hed cge (Brssic olerce vr. cpitt). 10-dy-old seedlings of stone-hed cge (Brssic olerce vr. cpitt) were grown on 25% Hoglnd solution contining 0, 2, 5 nd 10 µm CuCl 2 in the root environment. Dt on chlorophyll content (chl. + ; mg g 1 FW) nd crotenoid content (mg g 1 FW) represent the men of 2 independent experiments with 9 mesurements hving 3 plnts in ech tretment (±SD). Mens with different letters differ significntly t p 0.01 (Student s t-test). Ali et l. (2015), PeerJ, DOI /peerj /10

6 Chl. /Chl. nd Chl./Cr. Rtio chl. /Chl. Chl./Cr c Cu Concentrtion (µm) Figure 4 Impct of elevted levels of Cu 2+ on pigment content (chl./chl. & chl./cr. rtio) of stonehed cge (Brssic olerce vr. cpitt). 10-dy-old seedlings of stone-hed cge (Brssic olerce vr. cpitt) were grown on 25% Hoglnd solution contining 0, 2, 5 nd 10 µm CuCl 2 in the root environment. Dt on chlorophyll content (chl./chl. nd chl./crotenoid rtio) represent the men of 2 independent experiments with 9 mesurements hving 3 plnts in ech tretment (±SD). Mens with different letters differ significntly t p 0.01 (Student s t-test). more, the exposure to 2 µm Cu 2+ resulted in solid rise in wter-solule non-protein thiol contents in the roots nd to smller degree in the shoots t 10 µm Cu 2+ (Fig. 5). DISCUSSION Cu exposure t elevted levels (<2 µm Cu 2+ ) to stone-hed cge significntly decresed the production of root nd shoot iomss nd rised the rtio of the shoot to the root. Copper contmintion in the root environment generlly results in retrded production of root nd shoot iomss nd reduced photosynthetic ctivity. Moreover, it cuses chlorosis, necrosis nd leching of pigments (Yruel, 2005; Yruel, 2009; Sheldon & Menzies, 2005; Shhz et l., 2010; Shhz et l., 2010). In cge, the reduced production of iomss when exposed to elevted Cu levels coincided with decresed pigment contents (chl.,, crotenoids; Fig. 3) which my hve resulted in reduced ctivity of photosynthesis nd the drk respirtion rte (Shhz et l., 2010). It hs een shown tht Cu-toxicity dmges chloroplsts either y inducing iron deficiency or y replcing Mg in the chlorophyll y Cu (Pätsikkä et l., 2002; Küpper et l., 2003). Cu exposure t elevted levels not only decresed the pigment content ut there ws lso chnge in pigment composition. Chlorophyll content decresed significntly fster thn tht of chlorophyll nd crotenoids, which resulted in decresed chlorophyll / nd chlorophyll/crotenoid rtio. Similr results were reported y Chu et l. (2006) in Trifolium repens L. It is shown tht the production of root iomss ws more influenced thn tht of the shoot iomss production. The reltively higher reduction in the root iomss upon Ali et l. (2015), PeerJ, DOI /peerj /10

7 NO 3 in Shoot & Root (µm g -1 FW) SO 4 in Root & Shoot (µm g -1 FW) Nitrte-Shoot Nitrte-Root Sulfte-Shoot Sulfte-Root c c Cu Concentrtion (µm) Figure 5 Impct of elevted levels of Cu 2+ on nitrte nd sulfte content of stone-hed cge (Brssic olerce vr. cpitt). 10-dy-old seedlings of stone-hed cge (Brssic olerce vr. cpitt) were grown on 25% Hoglnd solution contining 0, 2, 5 nd 10 µm CuCl 2 in the root environment. Dt on nitrte nd sulfte content (µmol g 1 FW) represent the men of 2 independent experiments with 9 mesurements hving 3 plnts in ech tretment (±SD). Mens with different letters differ significntly t p 0.01 (Student s t-test). exposure to metl contmintion could e due the fct tht roots come in direct contct with toxic metls (Cd, Cu). Toxic metl-induced hindered root growth lso reduces the uptke of essentil nutrients (Sheldon & Menzies, 2005). Plnts hve evolved tightly-controlled mechnism for the sorption, lloction nd ssimiltion of sulfte under norml conditions. (Hwkesford & De Kok, 2006). Enhnced exposure of cge to Cu concentrtions considerly ffects the contents nd lloction of sulfur compounds in the root nd shoot of cge (Shhz et l., 2010). The rised shoot sulfte contents might e ttriuted to Cu-toxicity induced upregultion of the sulfte suppliers in roots (Shhz et l., 2010). To mintin rpid growth rtes under stress conditions, Brssic species increse their demnd for sulfur supply (Korlewsk et l., 2008; Korlewsk et l., 2009). Exposure of stone-hed cge to elevted levels of Cu did not ffect the nitrte contents in roots, however t 5 µm Cu 2+ there ws significnt decrese in the shoot nitrte contents. The reduced production of plnt iomss t 5 µm Cu 2+ could e ttriuted to reduced supply of nitrte in the shoot. The decrese in nitrte: sulfte rtio in the shoot of stone-hed cge under elevted Cu levels my e ttriuted to enhnced sulfte contents, however the link etween uptke rtes of nitrte nd sulfte is not evident yet (Stulen & De Kok, 2012). Since oth nitrte nd sulfte re involved in mino cid nd protein synthesis, their uptke rtes re relted with growth rtes (Stulen & De Kok, 2012). Roots ccumulted slightly rised level of wter-solule non-protein thiols when compred with tht of the shoot. Only smll proportion of the rise in thiol content might e ttriuted to Cu-induced synthesis of phytocheltins in Chinese cge Ali et l. (2015), PeerJ, DOI /peerj /10

8 (Shhz et l., 2010). Previous reports suggest tht the formtion of sulfur-rich metl-chelting compounds (i.e., wter-solule non-protein thiols) when disclosed to prospective toxic metls my perhps require n enhnced demnd for sulfur, viz. the sorption rte nd incorportion of sulfte (Sirko & Gotor, 2007; Ernst et l., 2008). The possile significnce of sulfur nutrition upon elevted copper exposure needs further investigtion. CONCLUSIONS This investigtion hs shown tht the elevted copper levels in the root surroundings proved toxic for stone-hed cge. Copper exposure t 2 µm Cu 2+ negtively ffected the plnt iomss production nd pigment contents. Furthermore, elevted Cu content considerly ffected the concentrtion of sulfte nd wter-solule non-protein thiol of stone-hed cge which might e due to the induction of phytocheltins to detoxify excess copper. ADDITIONAL INFORMATION AND DECLARATIONS Funding Funding ws provided y the University of the Punj, Lhore. The funders hd no role in study design, dt collection nd nlysis, decision to pulish, or preprtion of the mnuscript. Grnt Disclosures The following grnt informtion ws disclosed y the uthors: University of the Punj, Lhore. Competing Interests The uthors declre there re no competing interests. Author Contriutions Sjid Ali conceived nd designed the experiments, wrote the pper, prepred figures nd/or tles, reviewed drfts of the pper. Muhmmd Shhz conceived nd designed the experiments, performed the experiments, wrote the pper. Ahmd Neem Shhzd nd Ammr Ftim nlyzed the dt. Hfiz Azhr Ali Khn contriuted regents/mterils/nlysis tools, wrote the pper, prepred figures nd/or tles, reviewed drfts of the pper. Mozzm Anees contriuted regents/mterils/nlysis tools. Muhmmd Sleem Hider provided the fcilities (lortory nd stff) for the reserch work. Supplementl Informtion Supplementl informtion for this rticle cn e found online t /peerj.1119#supplementl-informtion. Ali et l. (2015), PeerJ, DOI /peerj /10

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