Understanding the development of roots exposed to contaminants and the potential of plant-associated bacteria for optimization of growth

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1 Annls of Botny 110: , 2012 doi: /o/mcs105, ville online t RESEARCH IN CONTEXT: PART OF A SPECIAL ISSUE ON ROOT BIOLOGY Understnding the development of roots exposed to contminnts nd the potentil of plnt-ssocited cteri for optimiztion of growth Tony Remns*, Sofie Thijs, Ssch Truyens, Nele Weyens, Kerim Schellingen, Els Keunen, Heidi Gielen, Ann Cuypers nd Jco Vngronsveld Environmentl Biology, Hsselt University, Agorln Building D, 3590 Diepeneek, Belgium * For correspondence. E-mil tony.remns@uhsselt.e Received: 30 Novemer 2011 Returned for revision: 28 Ferury 2012 Accepted: 28 Mrch 2012 Pulished electroniclly: 25 My 2012 Bckground nd Scope Plnt responses to the toxic effects of soil contminnts, such s excess metls or orgnic sustnces, hve een studied minly t physiologicl, iochemicl nd moleculr levels, ut the influence on root system rchitecture hs received little ttention. Nevertheless, the precise position, morphology nd extent of roots cn influence contminnt uptke. Here, dt re discussed tht im to increse the moleculr nd ecologicl understnding of the influence of contminnts on root system rchitecture. Furthermore, the potentil of plnt-ssocited cteri to influence root growth y their growth-promoting nd stress-relieving cpcities is explored. Methods Root growth prmeters of Aridopsis thlin seedlings grown in verticl gr pltes re quntified. Mutnts re used in reverse genetics pproch to identify moleculr components underlying quntittive chnges in root rchitecture fter exposure to excess cdmium, copper or zinc. Plnt-ssocited cteri re isolted from contminted environments, genotypiclly nd phenotypiclly chrcterized, nd used to test plnt root growth improvement in the presence of contminnts. Key Results The moleculr determinnts of primry root growth inhiition nd effects on lterl root density y cdmium were identified. A verticl split-root system reveled locl effects of cdmium nd copper on root development. However, systemic effects of zinc exposure on root growth reduced oth the voidnce of contminted res nd coloniztion of non-contminted res. The potentil for growth promotion nd contminnt degrdtion of plnt-ssocited cteri ws demonstrted y improved root growth of inoculted plnts exposed to 2,4-di-nitro-toluene (DNT) or cdmium. Conclusions Knowledge concerning the specific influence of different contminnts on root system rchitecture nd the moleculr mechnisms y which this is chieved cn e comined with the exploittion of plnt-ssocited cteri to influence root development nd increse plnt stress tolernce, which should led to more optiml root systems for ppliction in phytoremedition or sfer iomss production. Key words: Aiotic stress, metls, orgnic contminnt, plnt-ssocited cteri, cdmium, copper, zinc, 2,4-DNT, root morphology, endophyte, green revolution, systemic response. INTRODUCTION Optimiztion of plnt growth is required to ensure food nd feed supply, nd to respond to the need for iomss for renewle energy production nd industril feedstock pplictions (Weyens et l., 2009, ; Vngronsveld et l., 2009). However, plnt productivity is gretly ffected y environmentl stresses such s drought nd nutrient-deficiency (de Dorlodot et l., 2007; Den Herder et l., 2010). Plnt roots hve recently gined ttention in strtegies for improving plnt growth nd yield. In the so-clled second green revolution, roots re considered s meditors of incresed nutrient uptke efficiency nd drought tolernce, which is essentil s wter nd nutrients will ecome incresingly limiting in the future (Lynch, 2007; Den Herder et l., 2010; Ghnem et l., 2011; Hinsinger et l., 2011). Another prolem is tht food supply is endngered due to the use of lrge res of griculturl lnd for the production of energy iomss (Weyens et l., 2009; Den Herder et l., 2010). Indeed, it is estimted tht griculturl lnd in the Europen Union devoted to iomss production for iofuel/energy use will increse to km 2 y 2030 (Europen Biofuels Technology Pltform 2010, Innovtion driving sustinle iofuel industry, With incresed nutrient uptke efficiency nd drought or slt tolernce, mrginl lnds [ndoned frmlnd, degrded (sline, rid, etc.) nd (diffusely) contminted lnd, excluding conservtion res] my e tken into use to counter this prolem (Weyens et l., 2009, ). Contminted lnds my e unsuitle for food production due to possile ccumultion of toxic sustnces in the food chin, ut tking modertely contminted lnd into use for non-food iomss production my further relieve the prolem, lthough oviously plnt growth would e ffected due to toxicity of the contminnts. There re up to 3 million potentilly contminted sites in the EU-15 countries nd t lest sites require urgent remedil ction (Brdos et l., 2008). Moreover, diffuse contmintion ffects lrge res cross the world, minly s result of mining ctivity, fllout from industril processes such s smelting, griculture, trffic, res elevted with contminted # The Author Pulished y Oxford University Press on ehlf of the Annls of Botny Compny. All rights reserved. For Permissions, plese emil: journls.permissions@oup.com Downloded from

2 240 Remns et l. Development of roots exposed to contminnts dredged sediments, former lndfill sites nd ndoned industril sites. Re-using such contminted lnd for non-food crops during nd fter soil ( phyto)remedition could ring them ck into eneficil nd sustinle use nd reduce detrimentl environmentl, socil nd economic impcts on ffected communities (Mench et l., 2009; Vngronsveld et l., 2009). Reserch towrds etter understnding nd improving plnt growth in the presence of contminnts could contriute to future use of these contminted res for sfe nd efficient iomss production. Plnt growth nd stress tolernce need to e improved to enhnce iomss production. Uptke, trnsloction, sequestrtion nd possile degrdtion of the contminnts need to e controlled ccording to the ppliction of the iomss produced. For sfe iomss production on modertely contminted lnds, minimum uptke nd influence on iomss qulity prmeters is desirle (Shute nd Mcfie, 2006). For phytoremedition purposes, mximl uptke, trnsloction nd, if possile, rekdown of the products is desired, while mintining miniml iomss qulity for downstrem vloriztion (Vngronsveld et l., 2009). Root system rchitecture is mjor fctor tht cn e influenced to increse tolernce to iotic stresses such s nutrientdeficiency, drought nd slinity (de Dorlodot et l., 2007; Den Herder et l., 2010; Glvn-Ampudi nd Testerink, 2011; Ghnem et l., 2011; Hinsinger et l., 2011). Indeed, s roots re the uptke system for wter nd nutrients, sensing of the locl environment nd root growth in the vicinity of essentil resources re essentil. An exmple of the intimte link etween trnsport systems nd root development is functioning of the AtNRT1.1 trnsporter in oth nitrte uptke nd in nitrte-sensing mechnism tht directs root growth towrds locl nitrte enrichments (Remns et l., 2006; Krouk et l., 2010). Root growth in response to slt stress hs een well studied (Glvn-Ampudi nd Testerink, 2011), ut the role of root system rchitecture to iotic stresses relted to excess metls or orgnic sustnces hs not een investigted. Yet, root trnsporter proteins unvoidly tke up excess essentil elements, s well s toxic non-essentil elements vi the trnsport systems for essentil elements (Plmer nd Guerinot, 2009; Verruggen et l., 2009). A stress-induced morphogenic response, consisting of reduced primry root elongtion nd incresed lterl root density, hs een oserved in plnts exposed to numer of iotic stress conditions, for exmple excess metls, nd it ws postulted tht this response redirects plnt growth to diminish stress exposure (Potters et l., 2007). Therefore, these kinds of morphogenic responses should e considered s determining fctor in the voidnce of contminted soil ptches. However, eyond the voidnce mechnism, the ility to colonize noncontminted res when root systems re prtly exposed to contminnts needs to e evluted. It cn e ssumed tht the comintion of voidnce nd coloniztion would enle the plnt to grow nd produce sfe iomss. On the other hnd, for phytoremedition purposes, positioning of roots in contminted zones is desirle. Much reserch hs een dedicted to identify moleculr nd physiologicl trits of metl uptke nd sequestrtion in hyperccumultors, with the im to trnsfer these trits to high-iomss plnts in phytoremedition strtegies (Hssn nd Arts, 2011). Some hyperccumultors re lso le to direct root growth towrds metls in the soil (Whiting et l., 2000; Alford et l., 2010; Liu et l., 2010). However, with regrd to non-ccumultors, only limited numer of studies hve delt with the influence of metls on root system rchitecture, nd the underlying mechnisms remin lrgely unexplored (Lequeux et l., 2010; Petó et l., 2011; Zho et l., 2011). Symiosis etween mycorrhizl fungi nd plnts hs een shown to llevite the dverse effects on plnt growth y stress fctors in the soil, such s excess metls (reviewed y Mirnsri, 2010). Here, we focus on non-mycorrhizl plnts, with Aridopsis thlin s model, for understnding root development in the presence of contminnts. Root system rchitecture hs een intensively studied in A. thlin. Its rther smll root system is esy to quntify, nd the reltively erly vilility of genome informtion nd nnottion, nd development of moleculr tools hs yielded fundmentl insight into root development in this species (Mlmy, 2005; Péret et l., 2009; Blušk et l., 2010; Den Herder et l., 2010). Becuse of the vst knowledge on intrinsic root developmentl progrmmes, A. thlin lso remins n excellent model to study environmentl influences on the sic root developmentl pthwys (Remns et l., 2006; Pérez-Torres et l., 2008; Niu et l., 2008; Monshusen nd Gilroy, 2009; Den Herder et l., 2010; Krouk et l., 2010). Although it cnnot e considered generl rule tht selected A. thlin genes hve similr effect in other species, exmples exist of genes selected for positive yield effects in A. thlin (e.g. one promoting root growth, Prk et l., 2005) tht hve similr effect in other species (Gonzlez et l., 2009). Beneficil plnt-ssocited cteri cn ply key role in supporting nd/or enhncing plnt helth nd growth (Zhung et l., 2007; Tghvi et l., 2009; vn der Lelie et l., 2009; Weyens et l., 2009, ; Yng et l., 2009; Frncis et l., 2010). The presence nd ctivity of microorgnisms in the rhizosphere cn hve mjor effect on the nutrient uptke efficiency nd stress tolernce of the plnt (Weyens et l., 2009, ; Ghnem et l., 2011). Plnt growthpromoting (PGP) ctivity cn e cused y direct or indirect mechnisms. Direct PGP mechnisms my involve nitrogen fixtion y dizotrophs (oth rhizospheric nd endophytic), incresed vilility of highly unville nutrients such s phosphorus, iron nd other minerl nutrients, production of plnt growth regultors such s uxins, cytokinins nd gierelines, nd suppression of ethylene production y 1-minocyclopropne-1-croxylic cid (ACC) deminse ctivity (Bloemerg nd Lugtenerg, 2001; Cocking, 2003; Ryu et l., 2005; Mntelin et l., 2006; Contesto et l., 2008; Onofre-Lemus et l., 2009; Weyens et l., 2009; Rshid et l., 2012; Gmlero nd Glick, 2012). Plnt-ssocited cteri cn indirectly enefit the growth of their host plnt y preventing the growth or ctivity of plnt pthogens through competition for spce nd nutrients, ntiiosis, production of hydrolytic enzymes, inhiition of pthogen-produced enzymes or toxins, nd induction of plnt defence mechnisms (Selosse et l., 2004; Rijmkers et l., 2009). Plnt-ssocited cteri cn thus e very importnt in enling plnts to estlish or to grow etter on mrginl nd contminted lnd, nd could contriute to more economic nd environmentlly friendly production of iomss (Mstrett Downloded from

3 Remns et l. Development of roots exposed to contminnts 241 et l., 2006; Sleem et l., 2007; Zhung et l., 2007; Weyens et l., 2009). Here, experiments imed to increse our moleculr nd ecologicl understnding of chnges in root system rchitecture in the presence of excess metls re discussed. Exmples demonstrte the potentil of plnt-ssocited cteri to positively influence root growth nd to llevite the negtive effects of excess metls or orgnic contminnts. A lrge mount of moleculr dt on root development under norml conditions nd stress conditions exists for A. thlin. A fundmentl prt of our reserch is imed t identifying interference of contminnts with these known moleculr prmeters of root development. In this regrd, mutnts for genes potentilly involved cn e selected from the lrge collection of ville mutnts of A. thlin. This re of reserch will yield fundmentl knowledge concerning the environmentl impcts on root development, nd cn e trnsferred to strtegies for optimiztion of plnt growth in the presence of contminnts using economiclly interesting plnts. UNDERSTANDING ROOT DEVELOPMENT IN THE PRESENCE OF CONTAMINANTS It ws postulted tht different iotic stresses trigger similr morphologicl outcomes, nd tht t lest some of the underlying moleculr determinnts re interchngele etween stress conditions (Potters et l., 2009). The underlying determinnts of the stress-induced morphogenic responses (SIMRs) would e relted to ltered orgnisml grdients of plnt hormones (e.g. uxin nd ethylene), rective oxygen species (ROS) nd ntioxidnts, which t the cellulr level result in ltered cell division, elongtion nd/or differentition (Potters et l., 2009; Ghnem et l., 2011). The impliction of the ove postultion is tht, if ny stress-specific perception nd/ or signlling exists, this would t some point e integrted into the generl pthwy tht triggers the SIMRs cusing reduced primry root growth nd incresed density of lterl roots. Description of the similrities etween SIMRs cused y different stress conditions is useful for determining downstrem events leding to similr chnges in morphogenesis. These events re often linked to the influence on intrinsic root developmentl progrmmes. For exmple, similr chnges in uxin grdients hve een reveled for numer of stress conditions tht re linked to the sme morphogenic response (Potters et l., 2007). Auxin is key hormone tht is prt of intrinsic root developmentl pthwys (Niu et l., 2008; Fukki nd Tsk, 2009; Hodge, 2009) nd it is therefore not surprising tht chnges in root rchitecture re likely to e connected to chnges in uxin grdients nd sensitivity. However, much remins to e lerned out more upstrem pthwys of perception nd signlling of the stress fctor nd the ctul interference mechnisms with the generl intrinsic root developmentl pthwys. Furthermore, the description of similrities etween SIMRs hs often een limited to primry root growth inhiition nd increses in lterl root densities in juvenile plnts (Potters et l., 2007), ut lterl root outgrowth hs received little ttention, lthough it is mjor component defining root system rchitecture. Here, we investigte the following questions relting to root development in the presence of contminnts: (1) Wht re the upstrem components tht led to chnges in intrinsic root development progrmmes? (2) Wht is the mechnism of interference with intrinsic root developmentl pthwys? (3) To wht extent re these components stress-specific nd do they led to stress-specific chnges in root system rchitecture? We elorte on possile experimentl procedures tht cn e used to solve these questions, in prticulr using A. thlin in verticl gr plte system. A. thlin hs reltively simple root system for which root growth prmeters cn e esily quntified, nd numer of progrmmes for semi-utomtic nlysis of root systems hve een developed (Armengud et l., 2009; Loet et l., 2011). Furthermore, lrge mount of knowledge on intrinsic root development is ville, which is essentil to our im of connecting environmentl triggers to the intrinsic pthwys. As iotic stress fctors, we pply toxic mounts of cdmium (Cd), copper (Cu) nd zinc (Zn) ions (s sulphte slts) to the roots nd mesure morphologicl responses of wildtype plnts to descrie the stress-specificity of the responses, nd of selected mutnts to discover underlying mechnisms in reverse genetics pproch. These three metls hve different properties in plnts: Cd is non-essentil nd non-redox ctive, Zn is essentil ut not redox-ctive, nd Cu is essentil nd redoxctive. Nevertheless, the three metls ll led to oxidtive stress, ut the underlying mechnisms of oxidtive stress genertion re different, with Cu eing le to trigger direct production of ROS vi Fenton-rections, nd Cd nd Zn indirectly vi inhiition of nti-oxidtive defence, electron trnsport chins, or stimultion of pro-oxidtive ctivities such s NADPH oxidse or lipoxygense (Drzkiewicz et l., 2004; Brodley et l., 2007; Burkhed et l., 2009; Cuypers et l., 2009, 2011; Smeets et l., 2009; Remns et l., 2010). By compring the effect of metls with different properties we imed to increse the likelihood of encountering stress-specific effects. As such, we oserved tht exposure of A. thlin roots to excess Cd, Cu nd Zn in verticl gr plte system led to inhiition of primry root growth, nd to metl-specific chnges in lterl root development (see Fig. 2A; T. Remns, unpul. res.). Cd nd Cu cused n incresed lterl root density (similr to descried SIMRs) ut the lterl root elongtion ws much less inhiited y Cu thn y Cd t concentrtions tht cused similr inhiition of primry root growth. When Zn ws pplied t the sme effect concentrtion, oth lterl root density nd lterl root elongtion were negtively ffected. Thus, these three metls cused three different morphologicl outcomes, which contrdicts the postultion y Potters et l. (2009) tht different stresses cuse similr morphologicl outcomes. Experiments with these three metls re used here to unrvel the moleculr mechnisms ehind the oserved responses nd to gin more ecologiclly relevnt insight into the responses. This is chieved y using A. thlin mutnts in reverse genetics pproch, nd exmples re given of the involvement of lipoxygense gene nd gene involved in ethylene signlling in primry root growth inhiition nd in the regultion of lterl root density, respectively, when plnts re exposed to Cd. Discovering metl-specific moleculr determinnts of primry root growth inhiition A first question is whether the primry root growth inhiition is determined y the sme mechnisms, or whether Downloded from

4 242 Remns et l. Development of roots exposed to contminnts Reltive primry root length A * Wild-type lox1-1 mutnt * B CdS0 4 (µm) CuS0 4 (µm) F IG. 1. Primry root growth of A. thlin wild-type nd lox1-1 mutnt seedlings (s indicted) during 7 d exposure on verticl gr pltes to CdSO 4 (A) or CuSO 4 (B), expressed reltive to the control (0 mm) for ech genotype. Significnt genotype effects within tretments re indicted (*P, 0.05, t-test; n ¼ 20). metl-specific determinnts contriute to the sme outcome. Becuse exposure to excess Cd nd Cu cuses oxidtive stress, we tested numer of mutnts of genes involved in oxidtive stress genertion nd oxidtive signlling. These included NADPH oxidse mutnts rohc (Foremn et l., 2003), rohd nd rohf (Torres et l., 2002), nd lipoxygense mutnts lox1-1, lox5-2 (Vellosillo et l., 2007) nd lox3d (Cldelri et l., 2011). Seedlings were germinted on control pltes in 50 diluted Gmorg s B5 nutrient ckground for 7 d; susequently, homogeneous set of plnts ws trnsferred to tretment pltes covering concentrtion rnge of Cd nd Cu. Detiled methods for ll originl dt re given in the Appendix. NADPH oxidse mutnts rohc, rohd nd rohf did not show significntly ltered sensitivity of primry root growth to Cd or Cu, nd neither did lox3d or lox5-2 (dt not shown). However, primry root growth of the lox1-1 mutnt ws less inhiited y moderte concentrtions of Cd thn wild-type plnts, wheres no significnt difference ws oserved for Cu (Fig. 1). This shows tht the sme morphologicl effect ( primry root growth inhiition) cn involve different stress-specific moleculr determinnts, which ws shown here for the involvement of LIPOXYGENASE1 in Cd-induced primry root growth inhiition, ut not in Cu-induced primry root growth inhiition. Two hypotheses cn e posted concerning the possile metlspecificity of LOX1: (1) oxidtive stress induces primry root growth inhiition (Potters et l., 2009) nd this oxidtive stress cn e generted directly y Cu, ut indirect oxidtive stress genertion y Cd needs LOX1; (2) lterntively, Cd-specific LOX1-derived oxylipin signlling my contriute to the onset of primry root growth inhiition. The fct tht Cd nd Cu oth strongly induce LOX1 gene expression in roots (Remns et l., 2010; Cuypers et l., 2011) my fvour the first hypothesis. Moleculr determinnts of lterl root growth effects Under iotic stress conditions, lterl root development is lso modified. Plnts exposed to incresing CdSO 4 concentrtions showed n incresed lterl root density tht returned ck to control levels t higher concentrtions (Fig. 2B). In our reverse genetics pproch, t higher Cd exposure concentrtions, the incresed lterl root density is mintined in the ein3-1 mutnt (Solno et l., 1998), wheres it returns to norml levels in control plnts (Fig. 2B). This suggests tht ethylene signlling is involved in the decrese of lterl root outgrowth under high Cd exposure. Incresing the lterl root density would llow the plnt to explore the soil more thoroughly for non-contminted zones, ut t high Cd concentrtions the response would e lost gin due to high toxicity levels or generl growth inhiition, y which investment of the plnt in lterl roots in these zones would e voided. The upstrem regultion of the EIN3 trnscription fctor nd its downstrem trgets tht regulte lterl root development when plnts re exposed to Cd remin to e reveled to confirm the involvement of ethylene in this response. Further studies using reverse genetics pproch could revel more fctors involved in lterl root responses, nd evlution of selected mutnts under multiple stress conditions could revel stress-specific determinnts. Focus should e given to oth lterl root density (numer of lterl roots) nd lterl root outgrowth (elongtion), s oth fctors determine root rchitecture nd whether plnt will e le to colonize non-contminted zones. Indeed, s illustrted in Fig. 2A, lterl root outgrowth my e crucil to rech non-contminted zones. In this regrd, the lterl root growth response oserved for Cu my e more eneficil thn the response to Cd or Zn. Clerly, determintion of the moleculr prmeters underlying oserved morphologicl responses is importnt to link environmentl fctors to intrinsic root developmentl pthwys. However, understnding root developmentl responses is nother spect tht deserves ttention. The next section descries our experimentl set-up imed t deciphering responses in more ecologicl context. Wht more cn the gr plte system revel? Mimicking heterogeneous conditions to study voidnce nd coloniztion responses Identifying the moleculr determinnts of morphologicl responses is worthwhile for gining fundmentl knowledge on how environmentl triggers interct with intrinsic root development pthwys. However, focusing only on root growth effects in homogeneous conditions my not e sufficient to Downloded from

5 Remns et l. Development of roots exposed to contminnts 243 A control Cd Cu Zn 1cm B Lterl root density (cm 1 ) Wild-type ein3-1 mutnt CdS0 4 (µm) * * F IG. 2. (A) Seven-dy-old A. thlin plnts, with primry root length equl to the white mrk, were exposed for nother 7 d to 5 mm CdSO 4,10mM CuSO 4 or 75 mm ZnSO 4. At similr inhiition of primry root growth, lterl root density ws incresed y Cd nd Cu, ut decresed y Zn exposure. Lterl root elongtion ws less ffected y Cu thn y Cd. The effect on lterl root density nd elongtion my ffect the ility of the lterl root tips to rech non-contminted zones, for which the response to Cu my e more optiml. (Note: imge contrst ws enhnced to visulize roots, nd shoot colour my look rtificil). (B) Lterl root density (se to pex) of A. thlin wild-type nd ein3-1 mutnt seedlings (s indicted) fter 7 d exposure on verticl gr pltes to CdSO 4. Significnt genotype effects within tretments re indicted (*P, 0.05, non-prmetric Kruskl Wllis test, n ¼ 6). understnd plnt responses in more ecologicl context. Root forging responses to nutrients hve een discovered in conditions of heterogeneous nutrient distriution (reviewed y Hodge, 2009) nd hs led to the identifiction of the moleculr prmeters involved (Niu et l., 2008). By nlogy, we pplied heterogeneous growth conditions to study voidnce/ coloniztion responses, i.e. the ility of the roots to void contminted zones nd t the sme time to colonize noncontminted zones. Stress voidnce only without efficient coloniztion could e insufficient for survivl of the plnt. We use verticl split-root system to mimic heterogeneous growth conditions, y positioning 8-d-old seedlings such tht the growing root tip experiences the opposite condition (contminted or non-contminted) thn the rest of the plnt (Fig. 3A). This llows us to interpret the responses in context tht is more ecologiclly relevnt s it permits (1) distinguishing locl nd systemic effects, nd (2) descriing voidnce nd coloniztion responses. The effect on primry root growth rte of heterogeneous ppliction of Cd, Cu nd Zn to different prts of the root system ws studied (Fig. 3B). Homogeneous conditions where the full root system ws exposed or not served s controls. The concentrtions of the metls were chosen such tht the homogeneous exposure hd similr inhiiting effect on primry root growth (Figs 3B nd 4A). In heterogeneous conditions, exposure of the primry root tips only to 5 mm CdSO 4 or 10 mm CuSO 4 ws sufficient to induce n voidnce response consisting of complete inhiition of primry root growth tht occurred etween dys 1 nd 4 fter trnsfer, depending on the metl nd the exposure condition of the top zone (Fig. 3B). However, primry root tips exposed to 75 mm Zn did not stop growing when the rest of the plnt experienced control conditions, ut continued t reduced ut stedy growth rte (Fig. 3B; Zn 0 75). This suggests tht the voidnce mechnism induced y Zn is not s strong s tht induced y Cd or Cu, something which ws not found in homogeneous conditions s similr growth inhiition is oserved for plnts exposed to homogenous medium contining 5 mm Cd or 75 mm Zn (Fig. 3B; Cd 5-5 nd Zn 75-75). In the inverse exposure of only the top prt of the root system to Cd or Cu, primry root growth in the non-contminted zone ws unffected. However, the primry root of Zn-exposed plnts grew more slowly (Fig. 3B, Zn 75-0), resulting 7 d fter trnsfer in smll ut sttisticlly significnt inhiitory effect (Fig. 4A; Zn 75-0). In summry, Cd nd Cu exert locl effect on primry root growth, leding to mximl voidnce nd coloniztion responses, wheres exposure of plnts to Zn reveled systemic effects tht led to decresed voidnce nd coloniztion responses. As mentioned ove, nother importnt prmeter determining the root rchitecturl responses re lterl roots. For those plnts whose primry roots encounter contminted res, will the upper lterl roots e le to continue their coloniztion of the uncontminted zone (Fig. 3A)? Totl lterl root length in the uncontminted upper zone when primry root tips encounter metl-contminted res seems unffected y Cu, slightly ffected y Cd nd strongly ffected y Zn (Fig. 3A). Indeed, quntittive dt show tht totl lterl root length in the upper control zone ws unffected when primry root growth ws inhiited y 10 mm Cu (Fig. 4B; Cu 0-10). For plnts whose primry root tips were exposed to 5 mm Cd smll ut sttisticlly insignificnt reduction ws oserved (Fig. 4B; Cd 0-5). However, for plnts exposed to Zn, totl lterl root length in the upper control zone ws significntly reduced, to one-third of the control level (Fig. 4B; Zn 0-75). In fct, the totl lterl root length in the upper zone ws the sme regrdless of which prt of the root system ws exposed to Zn, suggesting tht Zn exerts complete systemic effect on totl lterl root length (cf. Zn 0-75, 75-0 nd 75-75; Fig. 4B). This systemic effect reduces the coloniztion response in the non-contminted zone, something which ws not oserved for Cu nd Cd. Totl lterl root length cn e dissected into lterl root numer nd men lterl root length to revel which of these prmeters is systemiclly ffected y excess Zn. The design of these experiments does not llow us to study ny inhiitory Downloded from

6 244 Remns et l. Development of roots exposed to contminnts A 8d 15d control Cd Cu Zn B Primry root growth rte (cm d 1 ) 0 9 Upper zone Lower zone (µm) Cu 10 0 Cd 5 0 Zn Zn 0 75 Cd 0 5 Cd 5 5 Zn Cu 0 10 Cu Dy fter trnsfer FIG. 3. (A) In the verticl split root-system, 8-d-old seedlings grown under control conditions were positioned such tht the primry root tip ws exposed to different tretment from the rest of the root system (control conditions hve the sme tretment in the upper nd lower zone). Visulized here re plnts exposed t the growing root tip only to 5 mm CdSO 4,10mM CuSO 4 or 75 mm ZnSO 4. Lterl root growth in the upper non-contminted zone is visully compromised y Zn exposure of the primry root tip, which is not the cse for plnts exposed to Cd or Cu t the primry root tip. (B) Primry root growth rtes of A. thlin wildtype seedlings 7 d fter trnsfer of the seedlings to verticl split-root pltes contining heterogeneous conditions of CdSO 4, CuSO 4 or ZnSO 4. Indicted in the legend is the metl concentrtion (mm) in the top zone nd the concentrtion in the ottom zone (n ¼ 6 14). effects of the metls on lterl root initition, s the lterl root primordi hd lredy een initited during pre-culture. The numer of visile lterl roots would therefore originte from the stimulted or inhiited outgrowth of pre-initited lterl root primordi. The numer of emerged lterl roots in the upper control zone ws not significntly ffected when primry root tips were exposed to 75 mm Zn (Fig. 4C; Zn 0-75), ut the men length of these lterl roots ws strongly reduced (Fig. 4D; Zn 0-75), suggesting tht the systemic effect of Zn cts on lterl growth rte rther thn on lterl root outgrowth. Also for Cd lower ut sttisticlly significnt systemic effect on lterl root elongtion ws oserved (Fig. 4D; Cd 0-5). Compring the effects of the tretments on lterl root numer (Fig. 4C) nd lterl root length (Fig. 4D) shows tht lterl root lengths re more significntly ffected, gin emphsizing the importnce of quntifying lterl root lengths longside lterl root density in the description of morphologicl responses to iotic stress. It is lso remrkle tht homogeneous exposure to Cu triggers morphologicl response similr to tht of phosphorus (P)-deprivtion, yet the P-deprivtion response cn e systemiclly triggered y exposure of the primry root tip only to low P(Svistoonoff et l., 2007), wheres the effect of Cu is entirely locl s contct of the primry root tip with excess Cu did not chnge lterl root numer or lterl root length in the upper control zone (Fig. 4C, D). Screening for the moleculr determinnts of root rchitecturl chnges relted to efficiency of voidnce nd coloniztion We hve previously mde the ssumption tht efficient voidnce mechnisms of contminted zones, comined with n efficient coloniztion mechnism of non-contminted zones, would enle the plnt to produce sufficient nd sfe Downloded from

7 Remns et l. Development of roots exposed to contminnts 245 Primry root length (cm) Bottom zone Top zone Totl LR length (cm) Bottom Top zone LR numer Men LR length (cm) Top zone Bottom Top zone Bottom A B C D c,c,d c,e c, c,e Cd Cu Zn µm Top Bottom FIG. 4. Primry root length (A), totl lterl root (LR) length (B), numer of lterls (C) nd men lterl root length (D) 7 d fter trnsfer of 8-d-old A. thlin seedlings to verticl split-root pltes, in which the primry root tip ws positioned in the ottom zone nd the rest of the root system in the top zone. The x-xis vlues indicte the exposure concentrtions (mm) s top zone ottom zone. Plnts were exposed to CdSO 4, CuSO 4 or ZnSO 4, colour-coded on the figure; lck indictes zones with zero exposure. Different letters indicte sttisticlly significnt differences (P, 0.05) fter one-wy ANOVA nd Tukey correction (n ¼ 6 14) (ng, no growth detected). iomss. A systemic inhiitory response to contminnts would decrese this efficiency, nd Zn exposure is n exmple of this. Future reserch to revel the moleculr ng,,c c,d ng e c c,e c,e c c,e prmeters involved in voidnce/coloniztion responses should e conducted in split-root systems, using forwrd or reverse genetics, lthough the ppliction of forwrd genetics will depend on utomted root growth nlysis (de Dorlodot et l., 2007; Iyer-Pscuzzi et l., 2010) nd high-throughput system. Reverse genetics my t first instnce e more fesile nd cn e conducted with selection of mutnts used to test hypotheses. For exmple, the involvement of signl trnsduction pthwys cn e investigted to revel the underlying mechnism of the systemic Zn effect. As such, growth of A. thlin roots in two dimensions in the verticl gr plte system, with incresed sophistiction of heterogeneous zones, nd in comintion with the lrge collection of mutnts, cn e used to unrvel growth-inhiiting nd growth-ctivting mechnisms in voidnce nd coloniztion responses of the root system. The responses will e evluted in reltion to plnt fitness nd provide understnding towrds more sfe nd more efficient iomss production or phytoremedition pplictions. IMPROVING THE DEVELOPMENT OF ROOTS EXPOSED TO CONTAMINANTS On contminted soils, nturl popultions of plnt species or specific ecotypes cn exist tht re tolernt to the contmintion. However, these tolernt plnts re often low in iomss (Vngronsveld et l., 2009). Nevertheless, the tolernce mechnisms in these plnts re n interesting suject of study, nd genetic determinnts of tolernce hve een trnsferred to higher iomss species using trnsgenic technology (Kärenlmpi et l., 2000; Pilon-Smits, 2005; Seth et l., 2012). Although this my e worthwhile in some instnces, the technology hs numer of disdvntges. Overexpression of trnsgene my increse tolernce to the stress condition, ut my hve metolic impct leding to decresed growth (Wojs et l., 2010). Also, the trnsgene my not e le to overcome multiple stress conditions, in which cse introduction of multiple genes would e necessry. Economic vloriztion of contminted lnd my demnd specific species nd not ll of these my e esily menle to genetic modifiction. Another wy of ssisting plnts to overcome the contminnt-ssocited stress is y exploiting the properties of nturlly occurring plnt-ssocited cteri. These properties cn improve the resistnce of the plnt to the contminnt, ut lso to other stress fctors such s drought nd nutrient deficiency. In the following, two exmples re presented of the ppliction of plnt-ssocited cteri to improve growth under stress conditions perceived y the roots. The use of plnt-ssocited cteri to improve root growth of A. thlin exposed to Cd Plnt endophytic cteri cn e trnsferred to susequent genertions vi the seeds (Mstrett et l., 2009), through direct vsculr connections from the mternl prent (Block et l., 1998), y coloniztion of the meristems (Pirttilä et l., 2000) or trnsferred through gmetes directly (Mdmony et l., 2005). It cn e expected tht those endophytes tht contriute to the incresed ility of the plnt to tolerte certin Downloded from

8 246 Remns et l. Development of roots exposed to contminnts Primry root length (cm) 8 A µm Cd Root growth (cm) C 2 µm Cd Control Cd Inoculted ** ** ** ** E 2 5 B Primry root length (cm) µm Cd Root growth (cm) 0 5 D µm Cd * * * ** 0 0 Control Cdmium Inoculted Time (d) c FIG. 5. Inocultion with seed endophytes cn enhnce root growth under Cd stress. Aridopsis thlin control seeds (from plnts tht were never exposed to Cd) s well s Cd seeds (from plnts tht were exposed to 2 mm Cd for severl genertions) were grown on verticl gr pltes contining 0, 2 or 10 mm CdSO 4 during the entire experiment. Hlf of the 7-d-old control plnts were inoculted y trnsferring them to pltes on which cteril suspension ws streked out (108 c.f.u. ml 21 ) representing the endophyte popultion isolted from Cd seeds (see text for species composition). (A, B) Primry root length of control plnts, Cd plnts nd inoculted control plnts fter 21 d exposure to (A) 2 mm Cd or (B) 10 mm Cd (P, 0.001, one-wy ANOVA with Tukey correction, n ¼ 20). (C, D) Primry root growth rte for control plnts, Cd plnts nd inoculted plnts upon exposure to (C) 2 or (D) 10 mm Cd. Dy 1 on the grphs corresponds to the first dy fter inocultion (*P, 0.05, **P, 0.01, P, within-dy one-wy ANOVA with Tukey correction, n ¼ 20). (E) Verticl gr plte with control plnt (), Cd plnt () nd inoculted plnt (c). stress would e trnsferred preferentilly, proly s result of the inility of the non-resistnt endophytes to survive the stress. Therefore, seed endophytes cn e considered s source for isoltion of plnt-ssocited cteri to improve plnt growth under unfvourle conditions. It ws oserved tht the cultivle species composition nd phenotypic chrcteristics of these species ws different fter cultivting A. thlin for severl genertions on 2 mm CdSO 4. The microil composition in these seeds differed from the A. thlin line grown in prllel for the sme numer of genertions on control soil (dt not shown). Some of the cteril strins isolted from the Cd seeds were used to inoculte A. thlin nd test their ility to improve growth under Cd stress. For this, A. thlin control seeds (originting from plnts tht were never exposed to Cd) s well s Cd seeds (originting from plnts tht were exposed to 2 mm Cd for severl genertions) were sown on verticl gr pltes. After 1 week, hlf of the control plnts were inoculted with cteril suspension (10 8 c.f.u. ml 21 ) representtive for the endophytic popultion isolted from Cd seeds. It consisted of four non Cd-resistnt seed endophytes (4.28 % Bcillus sp., 13.7% Bcillus rsenicus, 1.59 % Bcillus nicini, % Bcillus circulns) nd one Cd-resistnt seed endophyte (0.53 % Bcillus rsenicus). The non-cd-resistnt endophytes my possess stress-relieving properties improving plnt growth under Cd stress. Inocultion ws crried out over 1 week. The plnts were exposed to 0, 2 or 10 mm Cd during the entire experiment nd primry root lengths were mesured. In the sence of Cd, totl root length nd growth rte of the root were the sme for control plnts, Cd plnts nd inoculted control plnts. Upon exposure to 2 or 10 mm Cd, totl root length (Fig. 5A, B, E) s well s growth rte (Fig. 5C, D) were significntly higher for inoculted control plnts thn for non-inoculted control plnts or Cd plnts. Apprently, the plnts only enefited from the inocultion in the cse of Cd stress. Under optiml conditions, when plnts were not exposed to Cd stress, there ws no mesurle PGP effect, which my e due to the cteri possessing chrcteristics tht re stress-relieving nd thus only promote growth under stress conditions. The lck of growth promotion in the Cd plnts, even though contining the sme cteri s the inoculted control plnts, cn e explined y the fct tht the growth-promoting effect only ecomes pprent when the seed endophytes re present in high numers, s is the cse in the inoculted control plnts (10 8 c.f.u. ml 21 ). This study provides evidence tht inocultion of A. thlin with seed endophytes cn led to the presence of cteri in the plnt tht hve positive effect on root growth upon exposure to Cd. Downloded from

9 Remns et l. Development of roots exposed to contminnts 247 B 120 A Root growth (mm) ,4-DNT concentrtion (mg L 1 ) Root growth (mm) 120 Non-inoculted control C Geocillus sp. Rlstoni sp. 100 Bcillus sp. Sphingomons sp. 80 Burkholderi sp ** D c ,4-DNT concentrtion (mg L 1 ) d e f FIG. 6. Bcteri isolted from n explosives-polluted soil re cple of enhncing root growth in presence of 2,4-DNT contmintion. (A) 2,4-DNT inhiits primry root growth when 7-d- old A. thlin seedlings re grown over 9 d on pltes mended with different 2,4-DNT concentrtions (0 10 mg L 21,dtre men + s.e.; P, 0.001, non-prmetric Kruskl Wllis test; n ¼ 12). (B) Aridopsis thlin growth on 0 mg L 21 () nd 1 mg L 21 () 2,4-DNT. (C) Primry root length 9 d fter trnsfer of 7-d-old seedlings in response to 2,4-DNT (0 nd 1 mg L 21 ) nd in the presence or sence of cteri tested for 2,4-DNT degrdtion nd plnt-growth promoting chrcteristics (IAA production nd ACC-deminse ctivity, see text) c.f.u. ws spred on plte to inoculte the trnsferred seedlings (dt re men + s.e.; P, 0.001, **P, 0.01, non-prmetric Kruskl Wllis test; n ¼ 12). (D) Visuliztion of root hir formtion y stining with Crystl Violet in 15-d-old A. thlin seedlings exposed to 1 mg L 21 DNT nd in the presence or sence of cteri. () Non-inoculted control, () Burkholderi, (c) Sphingomons sp., (d) Bcillus sp., (e) Rlstoni sp., (f) Geocillus sp. Growth promotion nd degrdtion of orgnic contminnts 2,4-Di-nitro-toluene (2,4-DNT) is phytotoxic to A. thlin seedlings, cusing severe reduction in primry root length t concentrtions s low s 0.5mgL 21. An 80 % reduction in root length ws oserved t 2,4-DNT concentrtion of 1mgL 21. Seedlings on the pltes with 5 mg L 21 or higher 2,4-DNT concentrtions did not grow t ll (Fig. 6A). Primry root growth ws inhiited in the presence of the nitroromtic nd highly curled growth of the min root ws lso oserved. Lterl root density ws visully incresed in the presence of 2,4-DNT (Fig. 6B) ut the numer of lterl roots nd lterl root length could not e mesured due to the dense growth. Numerous studies hve investigted the toxicity of 2,4,6- trinitrotoluene (TNT) to plnts using soil-sed pproches, lthough little is known out specific root responses of plnts towrds romtics (Gong et l., 1999; Trvis et l., 2008). Active uptke of TNT in the roots hs een oserved nd little is trnsported to the eril prts of the plnts (Sens et l., 1999; Yoon et l., 2006). Becuse of its resemlnce to TNT, similr trnsformtion process for 2,4-DNT in plnts is suggested. Bcteri re descried to minerlize DNT (Spnggord et l., 1991; Snellinx et l., 2003) nd RDX (hexhydro-1,3,5-trinitro-1,3,5-trizine; Seth-Smith et l., 2002) ut not TNT, nd plnts should e le to Downloded from

10 248 Remns et l. Development of roots exposed to contminnts minerlize RDX, ut not TNT or DNT (Vn Aken et l., 2004; Rylott nd Bruce, et l., 2009; Rylott et l., 2011). TNT nd DNT re nitroromtics while RDX is nitrmine. Detoxifiction of nitroromtics y plnts involves conjugtion with glutthione or trnsformtions ctlysed y cytochrome P450 ccording to the green liver model (Burken, 2003; Gndi-Herrero et l., 2008). The glutthione conjugtes re then trnsported to the vcuole or cell wll, protecting the plnt from the toxic chemicl. Metolites of 2,4-DNT trnsformtion hve een reported s minonitrotoluenes nd unknown ound metolites, suggesting its incorportion in cellulose nd lignin (Yoon et l., 2006). A collection of soil cteri isolted from n explosivescontminted site ws tested for 2,4-DNT trnsformtion ilities. Four cteri showed high 2,4-DNT trnsformtion cpcity, with % reduction in 2 weeks (Tle 1). These cteri were tested for their indole-3-cetic cid (IAA) production nd ACC-deminse ctivity. Four of five were positive for IAA production However, it is importnt to mention tht Slkowski s regent detects lmost ll indole ring compounds, including IAA nd other IAA-like molecules. All of the cteri displyed high levels of ACC-deminse ctivity, rnging from 32 to 648 mm -ketoutyrte (KB) (mg protein) 21 h 21 (Tle 1). When the 2,4-DNT-trnsforming strins were inoculted on plnt growth medium, min root length of the plnts ws significntly reduced in comprison with the non-inoculted control, except for Burkholderi sp. (Fig. 6C). Despite the high level of ACC-deminse ctivity, these strins could not promote root growth etter thn the non-inoculted control under unpolluted conditions. However, when plnts were exposed to 1 mg L 21 2,4-DNT, ll strins except Geocillus promoted primry root growth (Fig. 6C). The est root growth promotion ws oserved for Burkholderi sp., with 150 % increse in min root length in comprison with the non-inoculted exposed control. This strin ws most efficient in trnsforming 2,4-DNT in the flsk cultures nd displyed high level of ACC-deminse production [398 mm KB (mg protein) 21 h 21 ]. A generl trend ws oserved for IAA production, ACC-deminse ctivity, 2,4-DNT-trnsforming ilities nd the stimultory effect on min root growth under DNT-polluting conditions. Only Geocillus did not enhnce root growth in control nd exposed conditions despite its moderte level of ACC-deminse ctivity. However, this strin did not produce IAA in vitro. At the end of the growth study, roots were stined to visulize root hir morphology. Most of the PGP cteri incresed root hir density nd root length nd the root hir formtion strted closer to the root tip (Fig. 6D). This effect is generlly oserved for PGP cteri (Doelere et l., 1999; Contesto et l., 2008). As mentioned erlier, PGP rhizocteri colonize the rhizosphere of mny plnts nd stimulte plnt growth either y fixing tmospheric nitrogen, mking nutrients more ville to plnts or relesing phytohormones (Weyens et l., 2009, ; Bloemerg nd Lugtenerg, 2001). In ddition, PGP rhizocteri cn lso ffect hormone levels in the plnt. For exmple, ACC-deminse ctivity of cteri lowers ethylene levels in the host plnt, relesing the growth-inhiiting effect exerted y this molecule (Glick et l., 1998; Glick, 2005). Much of the plnt ACC produced in the roots is exuded in TABLE 1. In vitro 2,4-DNT trnsformtion rte nd production rte of IAA nd ACC-deminse ctivity of the cteril strins Txon 2,4-DNT trnsformtion (% reduction fter 2 weeks) IAA [mm IAA (mg protein) 21 h 21 ] ACC deminse [mm KB (mg protein) 21 h 21 ] Geocillus Rlstoni 21.3, Bcillus 10.3,10 37 Sphingomons Burkholderi the rhizosphere where it is susequently tken up y the cteri nd hydrolysed y ACC-deminse. This genertes strong sink, therey lowering internl ACC levels in the root. It hs een demonstrted tht trnsgenic ACC-deminse plnts show higher tolernce to flooding nd metl stress (Grichko nd Glick, 2001; Sterns et l., 2005; Onofre-Lemus et l., 2009). In this study, we show tht cteri with ACC-deminse ctivity nd 2,4-DNT-degrding ilities promote root length of 2,4-DNT-stressed plnts, proly due to lowering of ethylene levels nd trnsformtion of 2,4-DNT to less toxic compounds. However, pleiotrophic effects, including simultneously signlling pthwys elicited y the microe, ply role in root elongtion (Tnimoto et l., 1995; Pitts et l., 1998; Co et l., 1999). Recently, two mechnisms were descried to explin the effect of PGP rhizocteri on root hir elongtion, one ethylene-dependent signlling mechnism involving cteril ACC-deminse ctivity nd one ethylene-independent mechnism explining proly the lrgest prt of the effect (Contesto et l., 2008; Desrosses et l., 2009). Hormone-mutnt studies cn provide further insight into the regultion mechnisms of root growth under PGP nd pollution conditions. CONCLUSIONS Understnding the moleculr sis of root development cn led to the development of strtegies tht interfere with developmentl nd stress-relted pthwys with the im to optimize root development. However, this my not e esily chieved s root growth seems to e determined y smll-effect loci environment interctions (Ghnem et l., 2011), nd therefore plnts selected with the est ility to dpt root development to one environmentl condition my not e optiml for nother condition (Den Herder et l., 2010). We hve discussed here the potentil of plnt-ssocited cteri to improve root growth. Is it still worthwhile studying the underlying moleculr mechnisms of root developmentl stress responses? Unrvelling the moleculr ckground of root development under stress conditions is worthwhile, oth from fundmentl iologicl point of view nd lso from n pplied point of view. The potentil of plnt-ssocited cteri to improve root development is first estimted y screening phenotypic chrcteristics of production of PGP sustnces (e.g. IAA), stress-relieving properties (e.g. ACC-deminse), incresed nutrient uptke (orgnic cids, siderophores, nitrogen fixtion, etc.) nd sequestrtion or rekdown of the contminnt(s). Regrding the influence of the ssocited cteri on plnt Downloded from

11 Remns et l. Development of roots exposed to contminnts 249 growth, knowledge of the mechnisms y which plnts lter root rchitecture my enhnce the likelihood of finding the est plnt cterium interction y designing pproprite phenotypic screens. Fundmentl knowledge gined in in vitro systems will hve to e vlidted in growth systems nd species tht re more relevnt to the eventul ppliction in the field. Nevertheless, etter understnding of (1) the fundmentl moleculr sis nd mechnisms of root development, (2) plnt cteri interctions, (3) the interctions etween different PGP cteri (quorum sensing) nd (4) n integrtion of environmentl effects on ll prtners nd their interctions is needed to optimize the pproch nd exploit the right plnt cteri interction. ACKNOWLEDGEMENTS Funding ws provided y Hsselt University Methuslem project 08M03VGRJ, nd from the Reserch Foundtion Flnders (FWO) doctorl (E.K., S.Tr., S.Th.) nd post-doctorl (T.R. nd N.W.) fellowships, nd n FWO Individul Reserch Grnt ( ) to T.R., nd y Hsselt University BOF funding (Bijzonder Onderzoeksfonds). LITERATURE CITED Alford E, Pilon-Smits E, Pschke M Metllophytes view from the rhizosphere. Plnt nd Soil 337: Armengud P, Zmux K, Hills A, et l EZ-Rhizo: integrted softwre for the fst nd ccurte mesurement of root system rchitecture. Plnt Journl 57: Brdos P, Chpmn T, Andersson-Sköld Y, et l Biomss production on mrginl lnd. Biocycle 49: Blušk F, Mncuso S, Volkmnn D, Brlow P Root pex trnsition zone: signlling-response nexus in the root. Trends in Plnt Science 15: Belimov AA, Hontzes N, Sfronov VI, et l Cdmium-tolernt plnt growth-promoting cteri ssocited with the roots of Indin mustrd (Brssic junce L. Czern.). Soil Biology & Biochemistry 37: Block CC, Hill JH, McGee DC Seed trnsmission of Pntoe stewrtii in field nd sweet corn. Plnt Disese 82: Bloemerg GV, Lugtenerg BJJ Moleculr sis of plnt growth promotion nd iocontrol y rhizocteri. Current Opinion in Plnt Biology 4: Brodley M, White P, Hmmond J, Zelko I, Lux A Zinc in plnts. New Phytologist 173: Burken JG Uptke nd metolism of orgnic compounds:green liver concept. In: McCutcheon SC, Schnoor JL. eds. Phytoremedition: trnsformtion nd control of contminnts. New Jersey, John Wiley & Sons, Burkhed J, Reynolds K, Adel-Ghny S, Cohu C, Pilon M Copper homeostsis. New Phytologist 182: Cldelri D, Wng G, Frmer E, Dong X A. lox3 lox4 doule mutnts re mle sterile nd defective in glol prolifertive rrest. Plnt Moleculr Biology 75: Co XF, Linsted P, Berger F, Kieer J, Doln L Differentil ethylene sensitivity of epiderml cells is involved in the estlishment of cell pttern in the Aridopsis root. Physiologi Plntrum 106: Cocking EC Endophytic coloniztion of plnt roots y nitrogen-fixing cteri. Plnt nd Soil 252: Contesto C, Desrosses G, Lefoulon C, et l Effects of rhizocteril ACC deminse ctivity on A. indicte tht ethylene medites locl root responses to plnt growth-promoting rhizocteri. Plnt Science 175: Cuypers A, Smeeets K, Vngronsveld J Hevy metl stress in plnts, Weinheim, Germny: Wiley-VCH Verlg GmH & Co. KGA. Cuypers A, Smeets K, Ruytinx J, et l The cellulr redox stte s modultor in cdmium nd copper responses in A. thlin seedlings. Journl of Plnt Physiology 168: de Dorlodot S, Forster B, Pges L, Price A, Tueros R, Drye X Root system rchitecture: opportunities nd constrints for genetic improvement of crops. Trends in Plnt Science 12: Den Herder G, Vn Isterdel G, Beeckmn T, De Smet I The roots of new green revolution. Trends in Plnt Science 15: Desrosses G, Contesto C, Vroquux F, Gllnd M, Tourine B PGPR A. interctions is useful system to study signling pthwys involved in plnt developmentl control. Plnt Signling & Behvior 4: Doelere S, Croonenorghs A, Thys A, Vnde Broek A, Vnderleyden J Phytostimultory effect of Azospirillum rsilense wild type nd mutnt strins ltered in IAA production on whet. Plnt nd Soil 212: Drzkiewicz M, Skorzynsk-Polit E, Krup Z Copper-induced oxidtive stress nd ntioxidnt defence in A. thlin. Biometls 17: Foremn J, Demidchik V, Bothwell J, et l Rective oxygen species produced y NADPH oxidse regulte plnt cell growth. Nture 422: Frncis I, Holsters M, Vereecke D The Grm-positive side of plnt microe interctions. Environmentl Microiology 12: Fukki H, Tsk M Hormone interctions during lterl root formtion. Plnt Moleculr Biology 69: Glvn-Ampudi C, Testerink C Slt stress signls shpe the plnt root. Current Opinion in Plnt Biology 14: Gmlero E, Glick BR Ethylene nd iotic stress tolernce in plnts. In: Ahmd P, Prsd MNV. eds. Environmentl dpttions nd stress tolernce of plnts in the er of climte chnge. New York: Springer, Gndi-Herrero F, Lorenz A, Lrson T, et l Detoxifiction of the explosive 2,4,6-trinitrotoluene in A.: discovery of ifunctionl O- nd C-glucosyltrnsferses. Plnt Journl 56: Ghnem M, Hichri I, Smigocki A, et l Root-trgeted iotechnology to medite hormonl signlling nd improve crop stress tolernce. Plnt Cell Reports 30: Glick BR Modultion of plnt ethylene levels y the cteril enzyme ACC deminse. FEMS Microiology Letters 251: 1 7. Glick BR, Penrose DM, Li J A model for the lowering of plnt ethylene concentrtions y plnt growth-promoting cteri. Journl of Theoreticl Biology 190: Gong P, Wilke BM, Fleischmnn S Soil-sed phytotoxicity of 2,4,6-trinitrotoluene (TNT) to terrestril higher plnts. Archives of Environmentl Contmintion nd Toxicology 36: Gonzlez N, Beemster G, Inze D Dvid nd Golith: wht cn the tiny weed A. tech us to improve iomss production in crops? Current Opinion in Plnt Biology 12: Grichko VP, Glick BR Ameliortion of flooding stress y ACC deminse-contining plnt growth-promoting cteri. Plnt Physiology nd Biochemistry 39: Hssn Z, Arts M Opportunities nd fesiilities for iotechnologicl improvement of Zn, Cd or Ni tolernce nd ccumultion in plnts. Environmentl nd Experimentl Botny 72: Hinsinger P, Brumn A, Devu N, et l Acquisition of phosphorus nd other poorly moile nutrients y roots. Where do plnt nutrition models fil? Plnt nd Soil 348: Hodge A Root decisions. Plnt Cell nd Environment 32: Hodge A, Bert G, Doussn C, Merchn F, Crespi M Plnt root growth, rchitecture nd function. Plnt nd Soil 321: Iyer-Pscuzzi A, Symonov O, Mileyko Y, et l Imging nd nlysis pltform for utomtic phenotyping nd trit rnking of plnt root systems. Plnt Physiology 152: Kärenlmpi S, Scht H, Vngronsveld J, et l Genetic engineering in the improvement of plnts for phytoremedition of metl polluted soils. Environmentl Pollution 107: Krouk G, Lcome B, Bielch A, et l Nitrte-regulted uxin trnsport y NRT1.1 defines mechnism for nutrient sensing in plnts. Developmentl Cell 18: Lequeux H, Hermns C, Lutts S, Verruggen N Response to copper excess in A. thlin: impct on the root system rchitecture, hormone distriution, lignin ccumultion nd minerl profile. Plnt Physiology nd Biochemistry 48: Downloded from