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Zinc ole ance and accumula ion in he sal -ma sh sh ub
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Halimione po ulacoides
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J. Camb ollé*, J.M. Mancilla-Ley ón, S. Muñoz-Vallés, T. Luque & M.E. Figue oa
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Depa amen o de Biología Vege al y Ecología, Facul ad de Biología, Uni e sidad de
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Se illa, Apa ado 1095, 41080 - Se illa, España
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*Co esponding au ho .
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Pos al add ess: Jesús Camb ollé Sil a, Dp o. Biología Vege al y Ecología, Facul ad de
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Biología, Uni e sidad de Se illa, A . Reina Me cedes 6, 41012 Se ille, Spain.
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Tel.: +34-95-4557165; ax: +34-95-4615780. E-mail add ess: [email p o ec ed]
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Abb e ia ions: A, ne pho osyn he ic a e; Chl a, chlo ophyll a; Chl b, chlo ophyll b;
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Ci, in e cellula CO2 concen a ion; Cx+c, ca o enoids; F0, minimal luo escence le el
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in he da k-adap ed s a e; Fm, maximal luo escence le el in he da k-adap ed s a e; Fs,
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s eady s a e luo escence yield; F , a iable luo escence le el in he da k-adap ed s a e;
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F /Fm, maximum quan um e iciency o PSII pho ochemis y; ΦPSII, quan um
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e iciency o PSII; Gs, s oma al conduc ance; NPQ, non-pho ochemical quenching;
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RGR, ela i e g ow h a e; SLA, speci ic lea a ea.
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Abs ac :
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The halophy ic sh ub Halimione po ulacoides is known o be capable o g ow h
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in soils con aining ex emely high concen a ions o Zn. This s udy e alua ed in de ail
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he ole ance and accumula ion po en ial o H. po ulacoides unde mode a e and high
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ex e nal Zn le els. A g eenhouse expe imen was conduc ed in o de o in es iga e he
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e ec s o a ange o Zn concen a ions (0 o 130 mmol l-1) on g ow h and pho osyn he ic
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pe o mance by measu ing ela i e g ow h a e, o al lea a ea, speci ic lea a ea, gas
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exchange, chlo ophyll luo escence pa ame e s and pho osyn he ic pigmen
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concen a ions. We also de e mined he o al zinc, ni ogen, phospho us, calcium,
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magnesium, sodium, po assium, i on and coppe concen a ions in he plan issues.
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H. po ulacoides demons a ed hype ole ance o Zn s ess, since i su i ed wi h lea
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concen a ions o up o 2300 mg Zn kg-1 d y mass, when ea ed wi h 130 mmol Zn l-1.
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Zinc concen a ions g ea e han 70 mmol l-1 in he nu ien solu ion nega i ely a ec ed
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plan g ow h, in all p obabili y due o he eco ded decline in ne pho osyn hesis a e.
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Ou esul s indica e ha he Zn-induced decline in he pho osyn he ic unc ion o H.
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po ulacoides may be a ibu ed o he ad e se e ec o he high concen a ion o he
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me al on pho osyn he ic elec on anspo . G ow h pa ame e s we e i ually una ec ed
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by lea issue concen a ions as high as 1500 mg Zn kg-1 d y mass, demons a ing he
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s ong capabili y o H. po ulacoides o p o ec i sel agains oxic Zn concen a ions.
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The esul s o ou s udy indica e ha his sal -ma sh sh ub may ep esen a aluable ool
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in he es o a ion o Zn-pollu ed a eas.
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Keywo ds: Halimione po ulacoides; pho osyn hesis; phy o emedia ion; Zinc.
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1. In oduc ion
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Due o hei oxici y and capaci y o bioaccumula ion, pollu ion by hea y
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me als is a se ious p oblem. Zinc is a me al cha ac e ized by i s high mobili y and
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bioa ailabili y (Mo illo e al., 2004) and is conside ed o be a majo indus ial pollu an
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o he e es ial and aqua ic en i onmen (Ba ak and Helmke, 1993). Al hough Zn is an
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essen ial mic oelemen in ol ed in nume ous physiological p ocesses (Rengel, 1999),
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ele a ed concen a ions o his me al in soils can lead o oxici y symp oms in mos
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plan s: A high concen a ions, Zn causes lea chlo osis, nu ien imbalances and
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inhibi ion o pho osyn hesis a a ious s ages, esul ing in impai men o plan g ow h
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(Chaney, 1993; Kaba a-Pendias and Pendias, 2001; Van Assche and Clijs e s, 1986).
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Despi e his, ce ain plan species ha e e ol ed hea y me al ole ance and can g ow in
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Zn-con amina ed soils, emaining una ec ed by he ele a ed Zn le els (E ns e al.,
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2000). Fo example, i is well known ha sal -ma sh plan s can ole a e and accumula e
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high con en s o hea y me als (e.g. Ma eos-Na anjo e al., 2008a; Ma heus e al., 2005).
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Halimione po ulacoides (L.) Aellen is a halophy ic sh ub equen ly ound on
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sandy and muddy sea-sho es and sal ma shes a ound he coas s o Eu ope, No h A ica
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and Sou h-Wes Asia. The species is equen ly he physiognomic dominan on well-
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d ained and uppe ma shes, o en inging channels and pools ha a e looded a high
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ide (Chapman, 1950). In se e al es ua ies o he Ibe ian Peninsula, H. po ulacoides
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g ows in sedimen s ea u ing ex emely high concen a ions o me als. In he join
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es ua y o he Tin o and Odiel i e s (SW Spain), one o he wo ld´s mos pollu ed a eas
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in e ms o hea y me als, H. po ulacoides can be ound g owing in sedimen s ha
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con ain 100-4800 ppm Zn (Camb ollé e al., 2008; Nelson and Lamo he, 1993; Sáinz
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and Ruiz, 2006). Mo eo e , in he Tagus es ua y sal ma shes (Lisbon, Po ugal), his
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species has demons a ed i s abili y o ole a e and seques a e high le els o Zn
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(Caçado e al., 2000). In iew o his abili y, se e al ecen s udies ha e explo ed he
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phy o emedia ion po en ial o H. po ulacoides (Almeida e al., 2009; Dua e e al.,
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2007; Sousa e al., 2008). To da e, he physiological impac o ele a ed concen a ions
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o me als on his species emains unknown; howe e , his knowledge is necessa y in
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o de o unde s and i s limi s o phy o oxici y and, ul ima ely, i s po en ial o use in he
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phy o emedia ion o a eas con amina ed by me als.
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The p esen s udy was unde aken o e alua e he ole ance and accumula ion
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po en ial o H. po ulacoides unde exposu e o mode a e and high Zn le els. The
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speci ic objec i es we e: (1) o de e mine he Zn phy o oxici y h esholds o he s udy
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species by analyzing he g ow h o plan s in a ange o ex e nal Zn concen a ions, om
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0 o 130 mmol l-1 Zn; (2) o asce ain he ex en o which Zn de e mines plan
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pe o mance, in e ms o in luence on he pho osyn he ic appa a us (PSII chemis y),
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gas exchange cha ac e is ics and pho osyn he ic pigmen s; and (3) o examine he
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possible ela ionship be ween he e ec s o Zn on g ow h and he N, P, Ca, Mg, Na, K,
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Fe and Cu concen a ions in plan issues.
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2. Ma e ials and Me hods
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2.1. Plan ma e ial and s ess ea men s
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Seeds o Halimione po ulacoides we e collec ed in he sal ma shes o “La
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Ma a-To e ieja” (Alican e, SE Spain). The collec ed seeds we e subsequen ly
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ge mina ed in pe li e mois ened wi h dis illed wa e , and main ained a 25 ºC o 30
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days. The esul ing seedlings we e sown in indi idual plas ic po s (diame e 11 cm)
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illed wi h pe li e, and placed in a glasshouse wi h minimum-maximum empe a u es o
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21-25ºC, 40-60% ela i e humidi y and na u al dayligh (minimum and maximum ligh
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lux: 200 and 1000 μmol m-2 s-1, espec i ely). Po s we e ca e ully i iga ed wi h 20%
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Hoagland's solu ion (Hoagland and A non, 1938) as equi ed.
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When seedlings we e be ween 20 and 25 cm in heigh (a e 4 mon hs o
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g ow h), he po s we e alloca ed o i e di e en Zn concen a ion ea men s: 0, 10, 30,
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70 and 130 mmol l-1 Zn, applied in shallow ays wi hin he same glasshouse ( en po s
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pe ay, one ay pe Zn ea men ). Zn ea men s we e p epa ed by mixing 20%
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Hoagland's solu ion wi h ZnSO4·7H2O o he app op ia e concen a ion. The con ol
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ea men , 0 mmol l-1 Zn, in ac con ained 0.002 mmol l-1 o Zn, since Hoagland’s
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solu ion con ains a small amoun o Zn as an essen ial ace nu ien . These Zn
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concen a ions we e chosen in o de o e lec he ange o le els ound by se e al
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au ho s in s udies o he sal -ma shes o di e en me al pollu ed es ua ies o he
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sou he n Ibe ian Peninsula, a ound 100-5000 mg Zn kg-1 pe o al soil DW (Camb ollé
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e al., 2008; Nelson and Lamo he, 1993; Rebo eda and Caçado , 2007b; Sáinz and Ruiz,
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2006), as well as in p e ious expe imen s o de e mine he phy o oxici y h esholds o
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H. po ulacoides.
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A he beginning o he expe imen , a 3 L olume o he app op ia e solu ion was
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placed in each o he ays o a ma ked dep h o 1 cm. Th oughou he expe imen ,
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solu ion le els in he ays we e moni o ed and opped up o he ma ked le el wi h 20%
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Hoagland's solu ion, (wi h no addi ional ZnSO4·7H2O) in o de o limi he change in
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Zn concen a ion due o e apo a ion o he wa e in he nu ien solu ion. In addi ion,
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he en i e solu ion (including ZnSO4·7H2O) was changed on a weekly basis.
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2.2. G ow h
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F om each ea men , ou comple e plan s ( oo s and shoo s) we e ha es ed a
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he beginning, and he emaining six a he end o he expe imen (i.e. ollowing 75 days
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o ea men ). These plan s we e d ied a 80ºC o 48 h and hen weighed.
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The ela i e g ow h a e (RGR) o whole plan s was calcula ed using he o mula:
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RGR = (ln B – ln Bi) · D-1 (g g-1day-1)
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whe e B = inal d y mass, Bi = ini ial d y mass (a e age o he ou plan s om each
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ea men d ied a he beginning o he expe imen ) and D = du a ion o expe imen
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(days).
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Lea a ea was de e mined om he p ojec ed a ea by scanning and digi alising
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he lea es (Epson V30, Seiko Epson Co p., Nagano, Japan), and using app op ia e
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so wa e (MideBMP . 4.2.; O diales-Plaza, 2000) o p ocessing and analysis. Speci ic
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lea a ea (SLA) was calcula ed as he a io o lea a ea o lea d y mass.
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2.3. Chemical analysis o plan samples
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A he end o he expe imen al pe iod, lea and oo samples we e d ied a 80ºC
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o 48 h and g ound, in acco dance wi h he p o ocols o Redondo-Gómez e al. (2007).
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Lea es and oo s we e ca e ully washed wi h dis illed wa e p io o u he analysis.
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Then, 0.5 g samples we e diges ed wi h 6 ml HNO3, 0.5 ml HF and 1 ml H2O2.
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Measu emen s o Ca, Cu, Fe, K, Mg, Na, P and Zn we e conduc ed by induc i ely
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coupled plasma (ICP) spec oscopy (ARL-Fison 3410, USA). To al N concen a ion
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was de e mined om undiges ed d y samples using an elemen al analyze (Leco CHNS-
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932, Spain).
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2.4. Gas exchange
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Gas exchange measu emen s we e aken om andomly selec ed, ully
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expanded lea es (n = 10, one measu emen pe plan plus ou ex a measu emen s
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aken andomly), ollowing 75 days o ea men , using an in a ed gas analyze in an
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open sys em (LI-6400, LI-COR Inc., Neb., USA). Ne pho osyn he ic a e (A),
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in e cellula CO2 concen a ion (Ci) and s oma al conduc ance o CO2 (Gs) we e
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de e mined a an ambien CO2 concen a ion o 360 µmol mol-1, empe a u e o
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20/25ºC, 50 ± 5% ela i e humidi y and a pho on lux densi y o 1000 µmol m-2 s-1.
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Values o he pa ame e s A, Ci and Gs we e calcula ed using he s anda d o mulae o
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Von Caemme e and Fa quha (1981).
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(2000 and 2006) ound EC50-plan alues o be ween 1.2 and 3.4 mmol l-1 Zn o i e
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eclama ion g ass species and six es o a ion o bs.
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Sal ma sh plan s gene ally accumula e di e en pe cen ages o me als wi hin
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hei below- and abo e-g ound biomass, wi h a highe pe cen age o me als ound in he
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oo s a he han in he shoo s (Fi zge ald e al., 2003; Ma hews e al., 2004). In ou
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g eenhouse expe imen , H. po ulacoides issue concen a ions o Zn we e highe in he
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below-g ound biomass han in he lea es a ex e nal Zn concen a ions o 10 and 30
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mmol l-1; he ans e ac o (TF), which is de ined as he a io o he me al
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concen a ion in shoo s o ha in he oo s (Sun e al., 2009), was he e o e below 1.0 in
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hese Zn ea men s. P e ious s udies showed ha he oo s o his sal -ma sh sh ub
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accumula e mo e me als han he abo e-g ound biomass (Caçado e al., 2000;
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Rebo eda and Caçado , 2007a; Rebo edo, 1991). Sousa e al. (2008) ound ha his
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species is able o e ain a high quan i y o me als in he oo cell wall, and s a ed ha
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compa men a ion and de oxi ica ion mechanisms a e c ucial o allow H. po ulacoides
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o ole a e high le els o hea y me als. Ou s udy shows ha
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H. po ulacoides could limi Zn anspo in o he shoo s up o an ex e nal concen a ion
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o 30 mmol l-1, since lea me al le els became highe han hose in oo s when he
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ex e nal Zn exceeded his concen a ion; TF alues we e he e o e highe han 1.0 a 70
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and 130 mmol l-1 ex e nal Zn. Kaba a-Pendias and Pendias (2001) indica ed ha ,
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al hough oo s o en con ain much mo e Zn han shoo s, a ex emely high le els o Zn
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in soils his me al can be ansloca ed om he oo s and accumula ed wi hin he shoo s.
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Zinc is ela i ely ac i e in biochemical p ocesses and is known o be in ol ed in
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se e al biological and chemical in e ac ions wi h se e al o he elemen s. In addi ion, he
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supply o Zn is expec ed o dec ease he up ake o mos nu ien s (Chaney, 1993;
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Fage ia, 2001). In ou expe imen , Zn-Cu, Zn-Fe and Zn-Mg in e ac ions we e no
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ound, despi e he ac ha such in e ac ions ha e been widely obse ed and epo ed
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(Kaba a-Pendias and Pendias, 2001). Howe e , he p esence o Zn did al e he
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concen a ions o he mac oelemen s, N, P, Ca, Na and K. The endency o N
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concen a ion in H. po ulacoides oo s o inc ease sligh ly wi h inc easing ex e nal Zn
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could be due o a highe binding o Zn by p o eins and amino acids in oo issues
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(Olsen, 1972). Sousa e al. (2008), in a ield s udy wi h H. po ulacoides de e mining
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me al concen a ions in se e al ac ions o plan ma e ial, ound ha a high pe cen age
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o oo Zn was being s o ed in he p o eic ac ion.
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A zinc-phospho us in e ac ion has been widely obse ed and epo ed in many
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c op plan s (Fage ia, 2001) al hough he speci ic mechanism o his in e ac ion is no
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ye known. In ou g eenhouse expe imen , he obse ed phospho us dis ibu ion
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be ween oo s and lea es, wi h espec o inc easing ex e nal Zn, sugges ed ha he
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upwa d ansloca ion o P could be being a ec ed by he excess o he me al. Howe e ,
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despi e se e al s udies epo ing he an agonis ic e ec o hese elemen s (Kaba a-
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Pendias and Pendias, 2001), he P concen a ions eco ded in issues o H. po ulacoides
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in his s udy did no su e a ma ked dec ease wi h inc easing ex e nal Zn. Päi öke
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(2003) also ound a educ ion o oo P con en wi h inc easing Zn supply in seedling
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and adul plan s o Pisum sa i um, while shoo concen a ions emained unal e ed.
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Mo eo e , me als can eplace Ca ions a essen ial si es on he cell memb anes, and
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me al-induced changes in memb ane p ope ies a ec K+ and H+ ex usion, as well as
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he unc ion o memb ane ca ie s and ion channels (B eckle and Kahle, 1991; El-Enany
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e al., 2000; Janicka-Russak e al., 2008). All hese e ec s, leading o an inc ease in
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non-speci ic memb ane pe meabili y, could be esponsible o he epo ed imbalances
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in Ca, Na and K concen a ions in issues o H. po ulacoides. Se e al ield and
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g eenhouse s udies ha e ound simila ly ele a ed Na concen a ions in plan s g owing
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in high me al condi ions (e.g. Päi öke, 2002; S einnes e al., 2000).
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Zinc oxici y inhibi s pho osyn hesis a a ious s ages and h ough di e en
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mechanisms (Chaney, 1993; Van Assche and Clijs e s, 1986). The ne pho osyn hesis
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a e (A) in H. po ulacoides was unal e ed when exposed o Zn le els o up o 70 mmol
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l-1, a alue ha is conside ably highe han hose eco ded by se e al au ho s. Fo
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example, Vaillan e al. (2005) epo ed ha he pho osyn hesis o ou Da u a species
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dec eased a 2.5 mmol l-1 Zn in he nu ien solu ion, and Ma eos-Na anjo e al. (2008b)
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ound ha ex e nal Zn concen a ions om 10 mmol l-1 caused ma ked educ ions in A
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in he sal -ma sh co dg ass Spa ina densi lo a. The e we e no clea e ec s o Zn on
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s oma al conduc ance in ou expe imen , hus indica ing ha he decline in A may be
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a ibu ed o non-s oma al limi a ions. Zinc may dec ease he ac i i y o enzymes
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in ol ed in C ixa ion (Mysliwa-Ku dziel e al., 2004), and hus he inc ease o
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in e cellula CO2 concen a ion (Ci) ound in plan s exposed o he highes ex e nal
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concen a ions o Zn in his s udy may be explained by modi ica ions in RuBisCO
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ac i i ies. Inhibi ion o enzyme ac i i y in he p esence o hea y me als could be due o
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subs i u ion o Mg2+ in he ac i e si e o RuBisCO subuni s by me als ions (Siedlecka
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and K upa, 2004). In his s udy, we eco ded a modi ica ion in he Zn/Mg concen a ion
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a io, caused by an inc ease in Zn concen a ion while Mg emained cons an , which
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could be ela ed o a dec ease in RuBP ca boxylase.
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Se e al s udies ha e epo ed a di ec e ec o zinc on he pho osyn he ic
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elec on anspo chain (e.g. Di Baccio e al., 2009; Vaillan e al., 2005), which may be
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associa ed wi h a subs an ial s ess esponse. In ou expe imen , maximum quan um
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e iciency o PSII (F /Fm) did show a signi ican educ ion a midday compa ed o he
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alues eco ded a dawn. A midday, he measu ed educ ion in F /Fm alues indica ed
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ha H. po ulacoides had expe ienced pho oinhibi ion a he highe ligh lux. The
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quan um e iciency o PSII (ΦPSII) also showed his di e ence be ween sampling
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imes. ΦPSII dec eased as a consequence o he inc ease in NPQ, indica ing ha he
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plan s dissipa ed ligh as hea , hus p o ec ing he lea om ligh -induced damage
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(Maxwell and Johnson, 2000). F /Fm and ΦPSII we e signi ican ly a ec ed by Zn
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concen a ions abo e 70 mmol l-1, sugges ing ha an excess o zinc enhances he
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pho oinhibi ion induced by ligh s ess.
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Pho oinhibi ion is caused by damage o pho osyn he ic componen s; he e ec
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can be sho - e m and e e sible (dynamic pho oinhibi ion), o long- e m and
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i e e sible (ch onic pho oinhibi ion; We ne e al., 2002). Values o dawn F /Fm a he
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highes Zn ex e nal concen a ion emained lowe han con ol pa ame e s o uns essed
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plan s (Bjö kman and Demmig, 1987), e ealing ch onic pho oinhibi ion o
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pho odamage. This decline in F /Fm was caused by lowe alues o Fm, which indica ed
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a dec ease in he p opo ion o open eac ion cen es (Maxwell and Johnson, 2000),
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possibly due o he eco ded dec ease in he concen a ion o chlo ophyll. The
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in ol emen o ace me als in hylakoyd eac ions and Chl syn hesis has been
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documen ed by se e al au ho s (Küppe e al., 1998, 2002; on We s ein e al., 1995).
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Se e al s udies ha e simila ly demons a ed educ ions in pho osyn he ic pigmen
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con en caused by ele a ed me al concen a ions (e.g. Camb ollé e al., 2011b;
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Redondo-Gómez e al., 2011). Ou esul s sugges ha he Zn-induced decline in he
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pho osyn he ic unc ion o H. po ulacoides may be a ibu ed o he ad e se e ec o
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he excess o his me al on pho osyn he ic elec on anspo , as a consequence o ei he
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he dec ease in chlo ophyll syn hesis o he inc ease in i s deg ada ion.
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5. Conclusions
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The pho osyn he ic unc ion o H. po ulacoides was nega i ely a ec ed by Zn
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concen a ions abo e 70 mmol l-1 in he nu ien solu ion. In his manne , and despi e he
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ac ha Zn concen a ions o be ween 100 and 500 mg Zn kg-1 DW in ma u e lea
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issue a e gene ally conside ed excessi e o oxic (Kaba a-Pendias and Pendias, 2001),
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g ow h pa ame e s we e p ac ically una ec ed by lea issue concen a ions as high as
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1500 mg Zn kg-1 d y mass. Mo eo e , his species su i ed wi h lea concen a ions o
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up o 2300 mg Zn kg-1 d y mass, when ea ed wi h 9000 mg Zn l-1 (130 mmol l-1). This
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s udy o he physiological esponses o H. po ulacoides o expe imen al Zn ea men s
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anging om 0 o 130 mmol l-1 (0-9000 ppm) has p o ided new insigh s in o he hea y
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me al ole ance o his sal -ma sh sh ub. Ou esul s indica e ha his species could be
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use ul in he eco e y o soils in Zn-con amina ed a eas, since he plan can ole a e
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high issue concen a ions o Zn wi hou su e ing ad e se physiological e ec s, and
486
can p oduce a signi ican amoun o biomass while seques a ing high concen a ions o
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