Zinc tolerance and accumulation in the halophytic species Juncus acutus

1 Zinc ole ance and accumula ion in he halophy ic species Juncus acu us 1 2 En ique Ma eos-Na anjo a*, Eloy M. Cas ellanosb, Al onso Pe ez-Ma in 4 c 3 aDepa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de 5 Se illa, 1095, 41080, Se illa, Spain 6 bDepa amen o de Biología Ambien al y Salud Pública, Facul ad de Ciencias 7 Expe imen ales, Uni e sidad de Huel a, 21071, Huel a, Spain 8 c 11 Ins i u o de Recu sos Na u ales y Ag obiología de Se illa (IRNAS), CSIC, Reina 9 Me cedes 10, 41012, Se illa, Spain 10 *Co esponding au ho : 12 En ique Ma eos-Na anjo 13 Dp o. Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de Se illa, A 14 Reina Me cedes s/n, 41012 Se illa, Spain. 15 E-mail: [email protected] 16 Tel.: +34-95-4557495; ax: +34-95-4615780 17 18 19 20 21 22 23 2 ABSTRACT 24 25 The esea ch on species wi h capaci y o ole a e and accumula e zinc is o 26 pa amoun impo ance o phy o emedia ion pu poses. An expe imen was designed o 27 in es iga e he e ec o Zn om 0 o 100 mmol l-1 on he g ow h, pho osyn he ic 28 appa a us and nu ien up ake o he halophy ic species Juncus acu us. Gas exchange, 29 chlo ophyll luo escence and pho osyn he ic pigmen s concen a ion we e measu ed. 30 We also de e mined o al zinc, magnesium, po assium, phospho us and sodium 31 concen a ions, as well as C/N a io. J. acu us showed high ole ance o Zn-induced 32 s ess, since all plan s su i ed and none o hem showed any oxici y symp oms, such 33 as chlo osis, nec osis o g ow h educ ion a concen a ions up o 100 mmol l-1 Zn. The 34 in eg i y and unc ionali y o he pho osyn he ic appa a us we e una ec ed e en a zinc 35 concen a ions g ea e han 500 mg Kg-1 on ille s. Likewise, nu ien abso p ion was 36 ela i ely una ec ed. Zn ole ance was associa ed wi h he capaci y o accumula e Zn in 37 oo s (wi h alues up o 2500 mg Kg-1 41 ) and la gely a oid i s anspo o ille s. These 38 cha ac e is ics, along wi h i s abili y o es ablish in a wide a ie y o ecosys ems, ende 39 his species a use ul phy os abilize o e ege a ion o Zn-con amina ed lands. 40 42 43 Keywo ds: G ow h esponse; me al oxici y; nu ien abso p ion; pho osyn hesis; Zn-44 s ess; pho oinhibi ion. 45 46 3 1. In oduc ion 47 48 En i onmen al pollu ion by hea y me als is a se ious p oblem wo ldwide, 49 inc easing in pa allel wi h he de elopmen o human echnology. Go e nmen , he 50 indus y and he public now ecognize he po en ial dange s ha me als pose o human 51 heal h (Du uibe e al., 2007) h ough he ood chain and he heal h o e es ial and 52 aqua ic communi ies and ecosys ems (Kaba a-Pendias and Pendias, 2001). The dange 53 o oxic me als is agg a a ed by hei immu able na u e and inde ini e pe sis ence in he 54 en i onmen (Ga bisu and Alko a, 2001; Aycicek e al., 2008). Among hea y me als, 55 Zn is conside ed he main indus ial pollu an o bo h e es ial and aqua ic 56 en i onmen s (Ba ak and Helmke, 1993) and has he g ea es mobili y and 57 bioa ailabili y o all elemen s (Mo illo e al., 2004). Al hough Zn is an essen ial 58 mic oelemen wi h many oles in plan me abolism (Kaba a-Pendias and Pendias, 2001), 59 i s excess can lead o oxic e ec s in plan s (Chaney, 1993), wi h speci ic e ec s on he 60 Cal in cycle and pho osys em ac i i y (Van Assche and Clijs e s, 1986). 61 Many emedia ion s a egies ha e been conside ed o coun e he de imen al 62 e ec s o Zn excess, including physical, chemical and biological me hods ha 63 immobilize o emo e me als om he en i onmen (Ma ques e al., 2011). 64 Phy o emedia ion has ecen ly gained impo ance on accoun o i s cos -e ec i e, long-65 e m applicabili y and because i is an eco iendly, p omising clean-up solu ion o a 66 wide a ie y o con amina ed si es (Weis and Weis, 2004). This me hodology depends 67 on he use o plan s o ac upon he con aminan s, by ex ac ing, deg ading o 68 immobilizing hem (Ma ques e al., 2011). The esea ch on species which can be use ul 69 in me al phy o emedia ion has become a majo issue (Zhang e al., 2010) and hese 70 4 species should be chosen on he basis o hei capaci y o ole a e and accumula e 71 pa icula con aminan s (Ma ques e al., 2011). 72 The e exis s a wide a ia ion in sensi i i y o me al exposu e. Howe e , exis s a 73 lack o knowledge abou me al oxici y h esholds o na i e plan species (Ross and 74 Kaye, 1994) and o species used o es o e si es con amina ed by hea y me als, such as 75 sal ma shes. Species o genus Juncus ha e been employed in we land es o a ion 76 p ojec s a ound he wo ld (Spa ks e al., 2013; Ma ques e al., 2011), bu he 77 in o ma ion on he ole ance and accumula ion pa e ns o hea y me als in hese species 78 is eally sca ce. The p esen s udy is ocused on he species Juncus acu us L., a 79 halophy ic densely caespi ose plan wi h subcosmopoli an dis ibu ion ha is common 80 in Spanish coas al ma sh communi ies and can be ound g owing in sedimen s 81 con aining 100–4800 ppm Zn in se e al es ua ies o he Ibe ian Peninsula (Sáinz and 82 Ruiz, 2006). Mo eo e , his species has a wide ecological ange, ole a ing soils wi h 83 high le els o sulpha es and chlo ides (Fe nández-Ca ajal, 1982) and soils wi h a sandy 84 ex u e and hyd ic s ess du ing he d y summe season. Ou hypo hesis is ha all hese 85 ci cums ances highligh he po en ial o J. acu us o be used o me al emedia ion in 86 pollu ed a eas. Howe e , no s udies ha e analyzed i s g ow h and physiological 87 esponses o zinc excess. 88 The aim o his s udy was o e alua e he ole ance o J. acu us o ele a ed 89 concen a ion o zinc in ela ion o i s su i al, g ow h and pho osyn he ic esponse, and 90 quan i y he capaci y o his species o accumula ing his elemen . 91 92 2. Ma e ials and Me hods 93 94 5 2.1. Plan ma e ial 95 96 Seeds o J. acu us we e collec ed in Decembe 2011 om he na u al ma shes o 97 Doñana Na ional Pa k (37º 15´ N - 6º 58´W; SW Spain) and s o ed a 4ºC (in da kness) 98 o h ee mon hs. A e ha , seeds we e placed in o a ge mina o o a mon h (ASL 99 Apa a os Cien í icos M-92004, Mad id, Spain) and subjec ed o an al e na ing diu nal 100 egime o 16 h o ligh (pho on lux a e, 400-700 nm, 35 μmol m-2 s-1) a 25ºC and 8 h 101 o da kness a 12ºC. Seedlings we e hen plan ed in indi idual plas ic po s (11 cm o 102 diame e ) illed wi h pe li e and placed in a glasshouse wi h con olled empe a u e o 103 21-25ºC, 40-60% ela i e humidi y and na u al dayligh (minimum and maximum ligh 104 lux: 250 and 1000 μmol m-2 s-1 108 espec i ely). Po s we e ca e ully i iga ed wi h 20% 105 Hoagland's solu ion (Hoagland and A non, 1938) as necessa y. All he po s ecei ed he 106 same i iga ion. 107 2.2. S ess ea men s 109 110 In Oc obe 2012, a e i e mon hs o seedling cul u e, he po s (wi h be ween 5 111 and 6 ille s) we e andomly alloca ed s ill inside he glasshouse o i e Zn ea men s 112 (six po s pe ay, one ay pe Zn ea men ): 0, 10, 30, 60 and 100 mmol l-1 Zn. The 113 ea men wi h 0 mmol l-1 Zn was conside ed he con ol ea men . Zinc ea men s 114 we e es ablished by combining 20% Hoagland´s solu ion and ZnSO4·7H2O o he 115 app op ia e concen a ion. The con ol, 0 mmol l-1 Zn ea men , had exac ly 0.002 116 mmol l-1 Zn, as Hoagland´s solu ion con ains a small amoun o Zn as an essen ial ace 117 nu ien . Zn concen a ions we e chosen o co e a ia ions eco ded by Sáinz and Ruiz, 118 (2006) in he sal ma shes o he join es ua y o he Tin o and Odiel Ri e s. 119 6 A he beginning o he expe imen , 1 l o app op ia e solu ion was placed in each 120 ay (Hoagland al 20% + ZnSO4.7H2O) o a dep h o 1cm. Du ing he expe imen , he 121 le els o ays we e moni o ed and opped up o he ma ked le el wi h 20% Hoagland´s 122 solu ion (wi hou addi ional ZnSO4.7H2O) o limi he change o Zn concen a ion 123 caused by wa e e apo a ion om he nu ien solu ion. Also, he en i e solu ion 124 (including ZnSO4.7H2 126 O) was changed e e y h ee days. 125 2.3. G ow h analysis 127 128 A he beginning and he end o he expe imen (a e 50 days o ea men ) h ee 129 and i e en i e plan s, espec i ely, om each ea men we e d ied a 80ºC o 48 h and 130 weighed. Also, be o e and a e he Zn ea men , he numbe and heigh o all ully 131 de eloped ille s we e measu ed. 132 The ela i e g ow h a e (RGR) in ash- ee d y mass o whole plan s was 133 calcula ed using he o mula: 134 135 RGR = (ln B – ln Bi) · D-1 (g g-1 day-1 137 ) 136 whe e B = inal d y mass, Bi 141 = ini ial d y mass (an a e age o he h ee plan s 138 om each ea men d ied a he beginning o he expe imen ) and D = du a ion o 139 expe imen (days). 140 2.4. Gas exchange 142 143 Measu emen s we e aken on andom, ully de eloped pho osyn he ic ille s (n = 144 7 10, wo measu emen s pe plan ) using an in a ed gas analyse in an open sys em (LI-145 6400, Li-Co Inc., Lincoln, NE, USA) a e 50 days o ea men . Maximum ne 146 pho osyn he ic a e (A), in e cellula CO2 concen a ion (Ci) and s oma al conduc ance 147 o CO2 (Gs) we e de e mined a CO2 concen a ion o 400 µmol CO2 mol-1 ai , 148 empe a u e o 25-30ºC, 42.4 ± 0.4% ela i e humidi y and a pho on lux densi y o 149 1000 µmol m-2 s-1 once a s eady-s a e was eached. A, Ci and Gs we e calcula ed using 150 s anda d o mulas o Von Caemme e and Fa quha (1981). Pho osyn he ic a ea was 151 app oxima ed as he a ea o a cylinde . In insic wa e use e iciency (WUEi) was 152 calcula ed as he a io be ween A and Gs 154 . 153 2.5. Tille wa e con en 155 156 Tille wa e con en (TWC) was calcula ed a e 50 days o ea men as (n = 5, 157 one measu emen pe plan ): 158 159 TWC = (FW – DW)/FW × 100 160 161 whe e FW is he esh mass o he ille s and DW is he d y mass a e o en-162 d ying a 80ºC o 48 h. 163 164 2.6. Pho osyn he ic pigmen s 165 166 A he end o he expe imen al pe iod, pho osyn he ic pigmen s in ully 167 de eloped, pho osyn he ic ille s (n=5) om each ea men we e ex ac ed using 0.05 g 168 o esh ma e ial in 10 ml o 80% aqueous ace one. A e il e ing, 1 ml o he 169 8 suspension was dilu ed wi h a u he 2 ml o ace one and chlo ophyll a (Chl a), 170 chlo ophyll b (Chl b) and ca o enoid (Cx+c) con en s we e de e mined wi h a Hi achi 171 U-2001 spec opho ome e (Hi achi L d., Japan) using h ee wa eleng hs (663.2, 646.8 172 and 470.0 nm). Concen a ions o pigmen s (µg g w -1 175 ) we e ob ained h ough 173 calcula ion ollowing Lich en hale (1987). 174 2.7. Measu emen o chlo ophyll luo escence 176 177 Chlo ophyll luo escence was measu ed using a po able modula ed luo ime e 178 (Mini-PAM, Heinz Walz, Ge many) a e 50 days o ea men , in ille s simila o hose 179 used p e iously. Measu emen s we e made on each plan in he i e zinc ea men s (n = 180 10, wo measu emen s pe plan ). Ligh and da k-adap ed luo escence pa ame e s we e 181 measu ed a dawn (s able 75 µmol m-2 s-1 ambien ligh ) and a midday (1500 µmol m-2 182 s-1 Plan s we e da k-adap ed o 30 minu es using lea –clips designed o his 185 pu pose. The minimal luo escence le el in he da k-adap ed s a e (F ) o in es iga e whe he zinc concen a ion a ec ed he sensi i i y o plan s o 183 pho oinhibi ion (Qiu e al., 2003). 184 0) was measu ed 186 using a modula ed pulse (<0.05 µmol m-2 s-1 o 1.8 µs) oo small o induce signi ican 187 physiological changes in he plan (Sch eibe e al., 1986). The da a s o ed we e an 188 a e age aken o e a 1.6 seconds pe iod. Maximal luo escence le el in his s a e (Fm) 189 was measu ed a e applying a sa u a ing ac inic ligh pulse o 10000 µmol m-2 s-1 o 190 0.8 s (Bolhà -No denkamp and Öquis , 1993). The alue o Fm was eco ded as he 191 highes a e age o wo consecu i e poin s. Values o he a iable luo escence (F = Fm 192 - F0) and maximum quan um e iciency o PSII pho ochemis y (F /Fm) we e 193 9 calcula ed om F0 and Fm The same ille sec ion o each plan was used o measu e ligh -adap ed 196 pa ame e s. S eady s a e luo escence yield (F . This a io o a iable o maximal luo escence can be used 194 o quan i y pho oinhibi ion (Maxwell and Johnson, 2000). 195 s) was eco ded unde ambien ligh 197 condi ions. A sa u a ing ac inic ligh pulse o 10000 µmol m-2 s-1 o 0.8 s was hen used 198 o p oduce he maximum luo escence yield (Fm Using luo escence pa ame e s de e mined in bo h ligh - and da k-adap ed s a es, 201 he ollowing we e calcula ed: quan um e iciency o PSII (Φ ') by empo a ily inhibi ing PSII 199 pho ochemis y. 200 PSII = (Fm' – Fs)/ Fm') 202 (Gen y e al., 1989); pho ochemical quenching (qP = (Fm' – Fs) / (Fm' – F0'), whe e F0' 203 co esponds o open eac ion cen e aps in he ligh -acclima ed s a e), and non-204 pho ochemical quenching (NPQ = (Fm – Fm') / Fm 208 '; Sch eibe e al., 1986). 205 Pho ochemical quenching gi es an indica ion o he p opo ion o PSII eac ion cen es 206 ha a e open (Maxwell and Johnson, 2000). 207 2.8. Chemical analyses o plan issue samples 209 210 In acco dance wi h p o ocols o Ma eos-Na anjo e al. (2008), a he end o he 211 expe imen , ille and oo samples we e d ied a 80ºC o 48 h and g ound. Tille s and 212 oo s we e ca e ully washed wi h dis illed wa e be o e any u he analysis. Then, 0.5 g 213 samples om ille s and oo s ( aken om i e plan s pe ea men ) we e diges ed in 214 iplica e wi h 6 ml HNO3, 0.5 ml HF and 1 ml H2O2. Ca, Mg, K, P, Na and Zn 215 concen a ions in ille s and oo s we e measu ed by induc i ely coupled plasma (ICP) 216 spec oscopy (ARL-Fison 3410, USA). To al N and C concen a ions we e de e mined 217 o undiges ed d y samples wi h an elemen al analyze (Leco CHNS-932, Spain). 218 16 he long- e m e ec s o he highes Zn concen a ion on he g ow h a e o J. acu us 368 could be due o he di e en de elopmen o he pho osyn he ic a ea a he han o 369 a ia ions in ne pho osyn he ic a e. Hence, simila a es o CO2 assimila ion could be 370 mo e han compensa ed o by a g ea e pho osyn he ic a ea in low Zn concen a ion. 371 This esponse migh p o ide posi i e eedback, since la ge pho osyn he ic a eas would 372 induce highe g ow h a es which would in u n induce mo e pho osyn he ic a ea, 373 ampli ying he di e ence be ween plan s a di e en zinc concen a ions o e ime. 374 Thus, in ou expe imen , he la ges di e ences in RGR we e ela ed o a ia ions in he 375 numbe o ille s, which migh be ela ed o di e ences in pho osyn he ic a ea and hence 376 o educ ion in ligh in e cep ion. In line wi h ou esul s, Delpe ee and Lu s (2008) also 377 ound ha g ow h inhibi ion was no co ela ed wi h CO2 assimila ion a e o Solanum 378 lycope sicum unde cadmium s ess condi ions. This was explained by he p esence o 379 se e al mechanism o ole ance ela ed wi h oxida i e s ess con ol and he p o ec ion 380 o pho osys ems. I is possible ha J. acu us used he same p o ec ion sys em. In his 381 espec , he hype ole ance o J. acu us o Zn s ess was also e lec ed in he in eg i y 382 and unc ionali y o i s pho ochemical appa a us. Se e al s udies ha e epo ed a di ec 383 e ec o zinc on he pho osyn he ic elec on anspo chain (Vaillan e al., 2005; 384 Ma eos-Na anjo e al., 2008), which may be associa ed wi h a subs an ial s ess 385 esponse. Ou da a showed ha F /Fm alues we e always lowe a midday han a 386 dawn, a ac ha indica ed ha J. acu us expe ienced some deg ee o dynamic 387 pho oinhibi ion a he highe ligh lux. Se e al au ho s ha e de ined dynamic 388 pho oinhibi ion as a e e sible mechanism con olling he dissipa ion o excess 389 luminous ene gy by means o he mal dissipa ion (NPQ), which is in ag eemen wi h 390 he g ea e NPQ a midday han a dawn in ou da a. This was suppo ed by he lowe 391 ΦPSII a dawn han a midday, a dec ease due o he inc ease in NPQ, which indica es 392 17 ha he plan s dissipa e ligh as hea , he eby p o ec ing he lea om ligh -induced 393 damage (Maxwell and Johnson, 2000). Dawn alues o F /Fm On he o he hand, he educ ion in he abso p ion o essen ial mine al elemen s 397 has been desc ibed as one o he e ec s o hea y me als on plan s (Chaney, 1993; 398 Kaba a-Pendias and Pendias, 2001). In his ega d, ou mine al nu ien analyses 399 indica ed ha he p esence o zinc in nu ien solu ion did no gene a e la ge nu i ional 400 imbalance in J. acu us plan s, especially in ille s issues, al hough oo and ille s Ca 401 and Mg concen a ions we e lowe a he highes zinc le el. The in e ac ions Zn-Ca and 402 Zn-Mg ha e been p e iously desc ibed by se e al au ho s (Kaba a-Pendias and Pendias, 403 2001). Thus, he educ ion in Mg concen a ion in ille s could be linked wi h a 404 dec ease in chlo ophyll con en eco ded in his expe imen , since he mos amilia ole 405 o Mg in pho osyn hesis is as he cen al a om o he chlo ophyll molecule (Shaul, 406 2002). Finally, Na concen a ion o ille s and oo s inc eased wi h ex e nal Zn 407 concen a ion. Redondo-Gómez e al. (2011) de e mined ha he accumula ion o Na in 408 he issues o Spa ina densi lo a a o ed eco e y o he pho osyn he ic appa a us o 409 his species agains zinc excess. This esul is linked wi h he high in eg i y showed by 410 pho osyn he ic appa a us o J. acu us o Zn s ess. 411 we e close o op imal 394 alues o uns essed plan s (app oxima ely 0.84; Bjo kman and Demmig, 1987), his 395 ac e ealing no p esence o ch onic and i e e sible pho oinhibi ion. 396 412 5. Conclusions 413 414 J. acu us shows a high ole ance o zinc-induced s ess, as p o ed he ac ha all 415 plan s we e able o su i e and did no show any isible Zn oxici y symp oms, such as 416 chlo osis, nec osis o a s ong g ow h inhibi ion a concen a ions up o 100 mmol l-1 417 18 Zn. Likewise, una ec ed pho osyn hesis and e iciency o PSII pho ochemis y 418 appa a us migh indica e ha J. acu us is no expe iencing me al oxici y, despi e he 419 ac ha Zn concen a ions eco ded in i s ille s issues (> 500 mg Kg-1) we e g ea e 420 han oxici y h esholds eco ded o plan s. Fu he mo e, Zn excess did no a ec wa e 421 ela ions o his species and o e all abso p ion o essen ial mine al elemen s. All hese 422 esul s sugges ha J. acu us is a hype ole an species o zinc. Mo eo e , he capaci y 423 o his species o accumula e g ea amoun o Zn in i s oo s (> 2500 mg Kg-1 430 Zn) could 424 be accoun ed o by he de elopmen o such mechanisms as compa men a ion, which 425 could con ol he ion anspo in o ille s, he eby imp o ing i s ole ance o Zn. 426 Consequen ly, he hype ole ance o zinc p o ed by hese esul s, oge he wi h i s 427 abili y o es ablish in a wide a ie y o ecosys ems, e lec ha his species is sui able as 428 a phy os abilize o e ege a ion o Zn-con amina ed lands. 429 19 Acknowledgemen s 431 432 We a e g a e ul o An onio J. Ruiz Rico o e ision o he English ex o his 433 manusc ip , Ca men Ca asco o echnical assis ance and An onio Diaz-Espejo o his 434 al uis ic help in lending some o he equipmen . We also hank he Spanish Science and 435 Technology Minis y o i s suppo (p ojec CTM2008-04453) and Se ille Uni e si y 436 Glasshouse Gene al Se ices o hei collabo a ion. 437 438 439 20 Re e ences 440 441 Aycicek, M., Kaplan, O., Yaman, M., 2008. 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Bio esou ce Technol. 554 101, 2063-2066. 555 556 557 25 Figu e cap ions 558 559 Fig. 1. G ow h analysis o Juncus acu us in esponse o ea men wi h a ange o Zn 560 concen a ions o 50 d. To al d y mass (A); ela i e g ow h a e, RGR (B); numbe o 561 ille s (C) and mean heigh o ille s (D). Values ep esen mean ± SE, n = 5. Di e en 562 le e s indica e means ha a e signi ican ly di e en om each o he (Tukey es , P < 563 0.05). 564 Fig. 2. Chlo ophyll a, Chl a (A); Chlo ophyll b, Chl b (B) and ca o enoids, Cx+c (C) 565 concen a ions in andomly selec ed, ully de eloped pho osyn he ic ille o Juncus 566 acu us in esponse o ea men wi h a ange o Zn concen a ions o 50 d.Values 567 ep esen mean ± SE, n = 5. Di e en le e s indica e means ha a e signi ican ly 568 di e en om each o he (Tukey es , P < 0.05). 569 Fig. 3. Maximum quan um e iciency o PSII pho ochemis y, F /Fm (A); quan um 570 e iciency o PSII, ΦPSII Fig. 4. Concen a ion o Zn (A); calcium, Ca (B); sodium, Na (C); po assium, K (D); 576 phospho us, P (E) and magnesium, Mg (F) in ille s (○) and oo s (●) o Juncus acu us 577 in esponse o ea men wi h a ange o Zn concen a ions o 50 d. Values ep esen 578 mean, n = 5. Di e en le e s indica e means ha a e signi ican ly di e en om each 579 o he (Tukey es , P < 0.05). 580 (B) and non-pho ochemical quenching, NPQ (C) a dawn (○) 571 and a midday (●) in andomly selec ed, ully de eloped pho osyn he ic ille s o Juncus 572 acu us in esponse o ea men wi h a ange o Zn concen a ions o 50 d. Values 573 ep esen mean ± SE, n = 10. Di e en le e s indica e means ha a e signi ican ly 574 di e en om each o he (Tukey es , P < 0.05). 575 Fig. 5. C/N a io o ille s (○) and oo s (●) o Juncus acu us in esponse o ea men 581 wi h a ange o Zn concen a ions o 50 d. Values ep esen mean, n = 5. Di e en 582