Root responses to cadmium in the rhizosphere
Journal of Experimental Botany,Vol.62,No.1,pp.21–37,2011 doi:10.1093/jxb/erq281Advance Access publication20September,
2010
REVIEW PAPER
Root responses to cadmium in the rhizosphere:a review
Alexander Lux1,*,Michal Martinka1,Marek Vacul?′k1and Philip J.White2
1Department of Plant Physiology,Faculty of Natural Sciences,Comenius University in Bratislava,Mlynska′dolina B2,84215Bratislava, Slovakia
2Scottish Crop Research Institute,Invergowrie,Dundee DD25DA,UK
*To whom correspondence should be addressed:E-mail:lux@fns.uniba.sk
Received17June2010;Revised17August2010;Accepted18August2010
Abstract
This article reviews the responses of plant roots to elevated rhizosphere cadmium(Cd)concentrations.Cadmium enters plants from the soil solution.It traverses the root through symplasmic or apoplasmic pathways before entering the xylem and being translocated to the shoot.Leaf Cd concentrations in excess of5–10m g g21dry matter are toxic to most plants,and plants have evolved mechanisms to limit Cd translocation to the shoot.Cadmium movement through the root symplasm is thought to be restricted by the production of phytochelatins and the sequestration of Cd-chelates in vacuoles.Apoplasmic movement of Cd to the xylem can be restricted by the development of the exodermis,endodermis,and other extracellular barriers.Increasing rhizosphere Cd concen-trations increase Cd accumulation in the plant,especially in the root.The presence of Cd in the rhizosphere inhibits root elongation and in?uences root anatomy.Cadmium concentrations are greater in the root apoplasm than in the root symplasm,and tissue Cd concentrations decrease from peripheral to inner root tissues.This article reviews current knowledge of the proteins involved in the transport of Cd across root cell membranes and its detoxi?cation through sequestration in root vacuoles.It describes the development of apoplastic barriers to Cd movement to the xylem and highlights recent experiments indicating that their maturation is accelerated by high Cd concentrations in their immediate locality.It concludes that accelerated maturation of the endodermis in response to local Cd availability is of functional signi?cance in protecting the shoot from excessive Cd loads.
Key words:Accumulation,apoplasm,cadmium,endodermis,maize,root,suberin lamellae,symplasm,tissue asymmetry, transport.
Introduction
Cadmium(Cd)is toxic to plant cells,even at low concen-trations.Leaf concentrations greater than5–10l g Cd gà1 DM are toxic to most plants(White and Brown,2010), although some ecotypes of a few plant species have adapted to grow on soils with high Cd concentrations and can tolerate leaf concentrations in excess of100l g Cd gà1DM(Reeves and Baker,2000;Broadley et al.,2001;Verbruggen et al., 2009).These Cd-hyperaccumulator plants include ecotypes of Noccaea(formerly Thlaspi)caerulescens(J&C Presl.)FK Mey (Reeves et al.,2001),Arabidopsis(formerly Cardaminopsis) halleri(L.)O’Kane and Al-Shehbaz(Bert et al.,2002),Sedum alfredii Hance(Yang et al.,2004),Viola baoshanensis Shu, Liu et Lan.(Wei et al.,2004),Thlaspi praecox Wulf.(Vogel-Mikusˇet al.,2005),Picris divaricata Vant.(Tang et al.,2009),and Phytolacca americana L.(Liu et al.,2010b).The roots of some of these exceptional plant species proliferate in Cd-enriched patches of soil,which contrasts with the behaviour of roots of most plant species that generally avoid such patches(Whiting et al.,2000;Liu et al.,2010a).To prevent Cd accumulation in shoot tissues,plants have evolved various mechanisms to restrict the entry of Cd to the xylem.This article reviews our current knowledge of these mechanisms, which include(i)the production of Cd-chelates in the cytoplasm of root cells and the sequestration of Cd-chelates in the vacuole to restrict Cd delivery to the xylem from the symplast,and(ii)the development of physical barriers to the extracellular movement of Cd to the xylem to restrict Cd delivery to the xylem from the apoplasm.
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Cadmium in the soil
To limit Cd concentrations in edible produce,Cd concen-trations lower than3l g gà1dry soil have been recommended for agriculture and horticulture(Mengel et al.,2001). Cadmium concentrations in non-polluted soils increase with clay concentration,but are generally lower than1l g gà1dry soil(Mengel et al.,2001).Cadmium is concentrated in the topsoil,where it is associated with organic matter.Solutions extracted from non-polluted soils generally have Cd concen-trations less than40–300nM(Wagner,1993).Cadmium availability to plants is greater in acid soils(Mengel et al., 2001;Tudoreanu and Phillips,2004;Kirkham,2006),and its solubility is increased by root exudates(Zhu et al.,1999). Cadmium occurs in the soil solution predominantly as Cd2+, but also as Cd-chelates(Tudoreanu and Phillips,2004).Low Cd2+concentrations in the soil solution,in combination with low diffusion coef?cients for Cd2+in aqueous solutions, suggest that transpiration-driven mass-?ow of the soil solution will dominate the delivery of Cd2+to plant roots (Sterckeman et al.,2004).This is consistent with reports that Cd accumulation by plants grown in soil is directly related to transpiration(Ingwersen and Streck,2005).Higher soil Cd concentrations can occur either naturally or through anthro-pogenic activities(Mengel et al.,2001;He et al.,2005; Kirkby and Johnson,2008).Natural mineral outcrops can be enriched in Cd through the weathering of Cd-rich rocks. Cadmium pollution of the environment has occurred through the mining and re?ning of metal ores,and through the appli-cation of Cd-containing phosphate fertilizers,sewage sludge, and municipal composts to agricultural soils.The ability to grow on soils with high Cd concentrations is generally related to the ability of roots to exclude Cd from the plant and/or of plant tissues to chelate Cd as a non-toxic com-pound or sequester it in a non-vital cellular compartment. Plants tolerating high Cd concentrations,and especially plants accumulating this metal in their above-ground parts, have potential utility for the phytostabilization and phytor-emediation of contaminated soils(Raskin and Ensley,2000; Schwitzgue′bel et al.,2009).
Cadmium uptake by plant roots
In nature,shoot Cd concentrations vary greatly.Although much of this variation can be attributed to environmental factors,there is appreciable phylogenetic variation in shoot Cd concentrations(Broadley et al.,2001;Watanabe et al., 2007).When grown in the same environment,shoot Cd concentrations are generally highest in species from the Caryophyllales and Lamiales,and lowest in monocot species (Broadley et al.,2001).Shoot Cd concentrations are de-termined largely by Cd entry to the root,sequestration within root vacuoles,translocation in the xylem and phloem, and dilution within the shoot through growth.Cadmium concentrations are often(but not always)greater in roots than in shoots,suggesting that Cd transport to the xylem is restricted in most plants,and lowest in seeds,fruits,and tubers,suggesting that Cd is not readily translocated in the phloem(Seregin and Kozhevnikova,2008;Conn and Gilliham,2010).Grafting experiments have suggested that shoot Cd concentrations in Nicotiana(N.tabacum L.and N.rustica L.;Wagner et al.,1988),Solanum(S.integrifolium, S.melongena,and S.torvum;Arao et al.,2008),and Noccaea/ Thlaspi(Noccaeae caerulescens and Thlaspi perfoliatum; Guimara?es et al.,2009)are controlled by root properties. Shoot Cd concentration often increases to a maximum value with increasing Cd concentration in the soil(Logan et al.,1997;Zhao et al.,2003;Sterckeman et al.,2004;Vogel-Mikusˇet al.,2005;Chen et al.,2008;Peng et al.,2009;Liu et al.,2010b),bulk soil solution,or nutrient solution(Yang et al.,1995;Perriguey et al.,2008;Street et al.,2009),al-though non-saturating relationships have also been reported (Salt et al.,1995;Ingwersen and Streck,2005).The con-centration dependence of Cd uptake from hydroponic solu-tions measured over short periods into either excised roots or intact plants generally follows the sum of a single Michaelis–Menten component plus a linear component(Table1).The linear component is often attributed to tight Cd binding to cell walls,but it could also represent an apoplasmic Cd?ux to the xylem(cf.White,2001;White et al.,2002;Broadley et al.,2007).Estimates of the K m value for‘high-af?nity’Cd uptake commonly fall between20–1000nM(Table1). Cadmium uptake and accumulation by plant roots is generally inhibited by La3+,Ca2+,Cu2+,Fe2+,Zn2+or Mn2+ in the rhizosphere solution(Cataldo et al.,1983;Costa and Morel,1993,1994;Cohen et al.,1998;Lombi et al.,2001; Hart et al.,2002;Zhao et al.,2002;Berkelaar and Hale, 2003a;Han et al.,2006;Zhao et al.,2006),but Cd uptake by the Cd-hyperaccumulating Ganges ecotype of N.caerulescens was not inhibited by divalent cations nor by La3+(Lombi et al.,2001;Zhao et al.,2002).The presence of organic acid anions in hydroponic solutions increases the capacity of both the Michaelis–Menten component and the linear component of Cd uptake,but the reasons for this are unclear(Berkelaar and Hale,2003a;Han et al.,2006).
Cadmium can enter root cells as Cd2+through ZIP(Zinc-regulated transporter/Iron-regulated transporter-like Protein) transporters,such as orthologues of AtIRT1and TcZNT1/ TcZIP4,through orthologues of the wheat TaLCT1transpor-ter,or via cation channels,such as depolarization-activated calcium channels(DACC),hyperpolarization activated cal-cium channels(HACC),and voltage-insensitive cation chan-nels(VICC),all of which are relatively non-selective between cations(Fig.1;Clemens et al.,1998;Cohen et al.,1998; White and Broadley,2003;White,2005;Plaza et al.,2007; DalCorso et al.,2008;Pedas et al.,2008;Verbruggen et al., 2009).In addition,Cd might enter root cells as Cd-chelates through YSL(Yellow-Stripe1-Like)proteins(Curie et al., 2009).Cadmium can then reach the stele through a symplas-mic pathway formed by the cytoplasms of individual root cells connected by plasmodesmata.The Cd species trans-ported through the symplasm are unknown,but could include Cd2+or Cd-chelates(Verbruggen et al.,2009). Cadmium is loaded from the symplasm into the xylem by heavy metal P1B-ATPases,such as orthologues of AtHMA2and AtHMA4,and possibly also by YSL proteins
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(Mills et al.,2005;DalCorso et al.,2008;Wong and Cobbett,2008;Verbruggen et al.,2009).The Arabidopsis ABC trans-porter AtPDR8has been implicated in Cd ef?ux across the plasma membrane of root hairs and epidermal cells (Kim et al.,2007).
In many plant species,Cd tolerance is related to Cd accumulation in the vacuole (Chardonnes et al.,1998;Cosio et al.,2005;Korenkov et al.,2007;Seregin and Kozhevnikova,2008).Cadmium can be translocated across the tonoplast by H +/Cd 2+-antiporters,such as orthologues of AtCAX2and AtCAX4(Korenkov et al.,2007,2009),by heavy metal P 1B -ATPases,such as orthologues of AtHMA3(Morel et al.,2009),and as Cd-chelates by ABC transporters,such as orthologues of AtMRP3(Tommasini et al.,1998;
Table 1.Published relationships between cadmium (Cd)uptake (V )by excised roots or intact plants and Cd in the external solution ([Cd]ext ),described by Michaelis–Menten functions using the terms V max (V ,when [Cd]ext ?N )and K m ([Cd]ext ,when V ?0.5V max ),and a linear term k (V /[Cd]ext )
*Original data expressed on the basis of root surface area,V max ?400pmol m à2s à1.**Values converted from DM to FW values using the conversion factor DM/FW ?0.1.
[Cd]ext (m M)
K m (nM)V max
(nmol g à1FW h à1)K m (m M)V max
(nmol g à1FW h à1)k
(nmol g à1
FW h à1m M à1)Reference
Soybean (Glycine max (L.)Merr.)
Intact plants 0.000002.5–0.57622.9 1.2232ND Cataldo et al.,1983Maize (Zea mays L.)Intact plants 0.0063–0.16430–100*ND
ND
ND Mullins and
Sommers,1986Maize (Zea mays L.)Intact plants 0.25–5.020020.31ND ND 16Han et al.,2006
Maize (Zea mays L.)Intact plants 0.00001–100ND ND ND ND Yes Perriguey et al.,2008Maize (Zea mays L.)Excised roots 0.05–5026023.6**ND ND 3.6**Redjala et al.,2009Lupin (Lupinus albus L.)Intact plants 0.00005–5.04211.6ND
ND
ND Costa and Morel,1993Lettuce (Lactuca sativa L.)Intact plants 0.00005–5.08–3032–43**0.011–0.0542420–7740**ND Costa and Morel,1994Pea (Pisum sativum L.)[Fe-replete]Excised roots 1–10060034ND ND 5.3Cohen et al.,1998Bread wheat
(Triticum astivum L.)Intact plants 0.05–1.252026ND ND 28Hart et al.,1998Bread wheat
(Triticum astivum L.)Intact plants 0.05–1.55933ND ND Yes Hart et al.,2002Durum wheat (Triticum turgidum L.var.durum )Intact plants
0.05–1.25
40
29
ND
ND
22
Hart et al.,1998
Durum wheat (Triticum turgidum L.var.durum )Intact plants 0.05–1.56738ND ND Yes Hart et al.,2002
Durum wheat (Triticum turgidum L.var.durum )Intact plants 0.005–1.81660.87ND ND 1.69Harris and Taylor,2004
Noccaea
caerulescens (Prayon)Intact plants 0.2–5026033ND ND 6.0Lombi et al.,2001Noccaea
caerulescens (Prayon)Intact plants 0.2–5093021.8ND ND 4.2Lombi et al.,2002Noccaea
caerulescens (Ganges)Intact plants 0.2–50180160ND ND 11.2Lombi et al.,2001Noccaea
caerulescens (Ganges)Intact plants 0.2–501000187.6ND ND 3.6Lombi et al.,2002Noccaea
caerulescens (Ganges)Intact plants <5450143ND ND ND Zhao et al.,2002Noccaea
caerulescens (Nc-H)Excised roots 0.05–50390130**ND ND 1.38**Redjala et al.,2009Noccaea
caerulescens (Nc-L)Excised roots 0.05–507023.4**ND ND 2.22**Redjala et al.,2009Arabidopsis halleri Intact plants 0.2–1035039.7ND ND 15Zhao et al.,2006Rice (Oryza sativa L.)Intact plants 0.25–50254044ND ND 0.8He et al.,2007Eggplant
(Solanum melongena )Intact plants 0.04–1.2380270**ND ND ND Mori et al.,2009Solanum torvum
Intact plants
0.04–1.2
353
**
ND
ND
ND
Mori et al.,2009
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Cobbett,2000;DalCorso et al.,2008;Verbruggen et al.,2009).Within plant cells,Cd is bound to S-containing ligands,such as those present in glutathione,metallothio-neins,and phytochelatins (DalCorso et al.,2008).In many plant species,the synthesis of phytochelatins is induced by Cd exposure (see Salt et al.,1995;Schat et al.,2002;Clemens,2006;Ernst et al.,2008;Seregin and Kozhevnikova,2008;Persson et al.,2006)and Cd is often sequestered in the vacuole as Cd-phytochelatin complexes (Cobbett,2000;Clemens,2006;Ernst et al.,2008).The involvement of phytochelatins in Cd detoxi?cation is consistent with observations that mutants de?cient in phytochelatin synthesis show lower Cd tolerance than wild-type plants and mutants with enhanced phytochelatin synthesis can show higher Cd tolerance than wild-type plants (Cobbett,2000;Clemens,2006).However,differ-ences in Cd tolerance among natural ecotypes of several plant species appear to be unrelated to differences in phytochelatin synthesis,suggesting additional mechanisms of Cd-detoxi?cation (Schat et al.,2002).In some plant species,metallothioneins play an important role in Cd detoxi?cation (Ernst et al.,2008;Shim et al.,2009).The production of metallothioneins is also induced by exposure to Cd,and plants genetically engineered to produce metal-lotheioneins in greater concentrations show increased Cd tolerance (see DalCorso et al.,2008;Korenkov et al.,2009).Cadmium is thought to be exported from the vacuole by NRAMP (Natural Resistance-Associated Macrophage Pro-tein)transporters,such as orthologues of AtNRAMP3and AtNRAMP4(Thomine et al.,2003;Verbruggen et al.,2009).
It is also possible for Cd 2+and Cd-chelates to reach the xylem solely via an extracellular,apoplasmic pathway in regions of the root lacking a Casparian band (Berkelaar and Hale,2003b ;Lux et al.,2004).The uptake of cationic ele-ments reaching the xylem via an apoplasmic pathway is generally restricted to the extreme root tip and to regions in which lateral roots are being initiated (White,2001;Moore et al.,2002).Although the relative contributions of the sym-plastic and apoplasmic pathways to the delivery of Cd to the xylem are unknown,it is likely that the relative contri-bution of the apoplasmic pathway will increase as the Cd concentration in the rhizosphere solution increases,as has been proposed for both Zn and Na (White et al.,2002;Plett and M?ller,2010).
Supporting the presence of an apoplasmic pathway,are observations that the root tip is the most active region of the root for Cd 2+in?ux (Pin ?eros et al.,1998),and that Cd accumulation by wheat cultivars is positively correlated with their number of root apices (Berkelaar and Hale,2000).A linear component to the concentration dependence of Cd uptake on Cd concentration in the nutrient solution is almost universally observed in short duration studies on hydroponically-grown plants (Table 1).This phenomenon can be interpreted as the result of an apoplasmic Cd ?ux to the xylem.In challenging this view,Xing et al.(2008)showed that,among accessions of N.caerulescens with contrasting abilities to take up Cd and Zn,the amount of Cd taken up by roots over a 24h period and translocated to the shoot was inversely related to apoplasmic water ?ows assayed using a membrane-impermeable ?uorescent
dye.
Fig.1.Proteins thought to be responsible for Cd in?ux to the symplasm,sequestration in the vacuoles of root cells,and ef?ux to the rhizosphere and to the xylem (see text for details).
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However,this is inconsistent with studies of the effects of transpiration on Cd accumulation by other plant species (Ingwersen and Streck,2005).Reducing transpiration by the addition of ABA to the nutrient solution has also been shown to reduce both xylem sap Cd concentration and shoot Cd accumulation of hydroponically grown plants (Salt et al.,1995;Hsu and Kao,2003;Zhao et al.,2006;Liu et al.,2010b),but this has been interpreted as an effect of ABA on symplastic loading of Cd into the xylem since Cd uptake by roots themselves was unaffected by the presence of ABA(Salt et al.,1995;Zhao et al.,2006).More persua-sive arguments for symplasmic delivery of Cd to the xylem are(i)that there is competition between Cd2+and other cations for uptake by roots(Hart et al.,2002)and(ii)the contrasting abilities of N.caerulescens accessions to accu-mulate Cd and Zn independently in the shoot provides evidence of transport selectivity(Xing et al.,2008),both of which can be taken as evidence for protein-mediated symplasmic transport(White,2001).
Barriers to apoplasmic movement of solutes to the xylem
The isolation of the stele from the peripheral cell layers of the root is critical for the control of solute transport to the shoot.It is effected by cell wall impregnations and the most important impregnating substance in this context is suberin (Franke and Schreiber,2007).
Cadmium uptake by plant roots is generally restricted to young subapical regions of actively growing roots(Pin?eros et al.,1998).The peripheral cell layers in these regions are specialized for solute uptake.The epidermal layer,which constitutes the outermost cell layer and differs from the epidermal layer of above-ground plant parts both in lacking cutin and through the development of root hairs,is termed the rhizodermis(von Guttenberg,1968).The rest of the peripheral tissues are termed the cortex.The endodermis separates the cortex from the stele and extracellular move-ment of solutes to the xylem is restricted by suberin deposi-ted in endodermal cell walls.Suberin,together with lignins, form the impregnation material of Casparian bands de-veloped in radial and transverse endodermal cell walls (Schreiber et al.,1999;White,2001).This impregnation,the lack of intercellular spaces between endodermal cells,and a tight junction between cell walls and the plasma mem-brane form the apoplasmic barrier of the root(White, 2001).The endodermis with its Casparian bands represents a considerable,but not impenetrable,barrier to solute movement through the apoplasm(Steudle et al.,1993; White,2001;Ranathunge et al.,2005).
Casparian bands represent the?rst endodermal ontogenic stage(Stage I),which is only rarely(e.g.in some aquatic species;Seago,2002)the?nal stage.The deposition of lamellar suberin,in this case on the whole inner cell wall surface,is the commonly occurring second endodermal ontogenic stage(White,2001).Stage II endodermis presents a more complete apoplasmic barrier to the radial?ow of water and solutes to the xylem in more mature parts of the
root(Melchior and Steudle,1993;Peterson et al.,1993; Steudle and Peterson,1998;White,2001).Nevertheless,the absolute amount of suberin deposited in root cell walls affects the radial transport of water and ions,as demon-strated by the enhanced suberin1(esb1)mutant of Arabidop-
sis thaliana Heynh.(Baxter et al.,2009).This mutant has elevated amounts of suberin in the root,most likely in the endodermis,reduced water?ow to the xylem,and a decrease
in shoot Ca,Mn,and Zn accumulation(Baxter et al.,2009). However,quantitative differences in root suberin concen-trations may not be the only factor in?uencing the apo-plasmic movement of water and solutes to the xylem,and
both the chemical nature of the suberins,together with the microstructure of deposits,must also be taken into consid-eration(Schreiber et al.,2005).Differences in suberin composition along the root axis during the development of
the endodermis are indicated by an increase in fatty acid
x-hydroxylation(Thomas et al.,2007;Ho¨fer et al.,2008). However,association of these changes with functional properties of suberized barriers in the endodermis remains
to be demonstrated.
In some plant species,more distant from the root apex,in
even older parts of the root,the endodermis may pass to the
third stage(von Guttenberg,1968;White,2001).Stage III endodermis is characterized by thick cellulose secondary
walls(sometimes classi?ed as tertiary walls)deposited over
the suberin lamellae.This cell wall layer,together with the original primary wall,is often ligni?ed,and in some species impregnation of walls with silicon may occur(Sangster and Parry,1976;Lux et al.,1999).In Stage III,the function of
the endodermis is already mostly mechanical and radial transport of water and solutes is limited(Melchior and Steudle,1993;White,2001).
The gradual changes in cell wall composition of the endodermis can extend for a considerable distance along the root.The?rst stage,characterized by Casparian bands,
often starts very close to the root apex.The zone of gradual development of the second stage,the deposition of suberin lamellae,is usually very long.It may vary from several millimetres to several hundred millimetres from the root apex.Throughout this distance the number of endodermal
cells without suberin lamellae decreases,and the cells not covered by suberin lamellae are called passage cells.Passage
cells can be present even when the majority of endodermal
cells have entered the third stage of development.Indeed, passage cells can remain in the?rst state permanently, although in the majority of the plant species this does not occur.
Hypodermal layers,characterized by a gradual thickening
of cell walls,are often present in older roots,especially in monocotyledons.A suberized periderm is developed in older
roots of dicotyledons and gymnosperms.Periderm has been shown to act as a barrier preventing the movement of water
and ions(Vogt et al.,1983),gases(De Simone et al.,2003),
and pathogen incursion(Lulai and Corsini,1998).From a functional viewpoint,these older regions of the root con-tribute little to water uptake(Melchior and Steudle,1993)
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and are mostly engaged in the long-distance transport of water and solutes.This function is concentrated in inner root tissues,within the vascular cylinder,or in the secondary vascular tissues.
In the majority of angiosperms another apoplasmic barrier,the exodermis,can develop in parallel with the endodermis(Perumala et al.,1990;Peterson and Perumala, 1990;Hose et al.,2001;Ma and Peterson,2003).The exodermis develops in the same three stages as the endo-dermis.The exodermis can be uniseriate or multiseriate,in contrast to the uniseriate endodermis.The exodermis usually develops at a greater distance from the root apex than the endodermis(Ma and Peterson,2003).However, environmental conditions can modify the rate of develop-ment of the exodermis(Zimmermann and Steudle,1998) and accelerated development of the exodermis has been associated with reduced Cd uptake by roots(T Redjala and I Zelko,personal communication).In some plant species, and under speci?c environmental conditions,the exodermis may differentiate earlier than the endodermis.This has been observed in some wetland plants(Seago et al.,1999;Soukup et al.,2002),in tea(Homma et al.,2000;Tanimoto et al., 2004),and in maize(Zea mays L.)grown in soil(T Redjala and I Zelko,personal communication).The importance of the exodermis as an environmentally variable barrier to the uptake of water and ions was recognized by Peterson et al. (1993),and several subsequent studies have con?rmed this conclusion(Peterson,1997;Meyer et al.,2009).Under some conditions,such as in hydroponics,the exodermis can be absent,even in species in which it is normally present (Zimmerman and Steudle,1998).
The rhizodermis,exodermis,and endodermis have all been shown to act as barriers to the apoplasmic movement of toxic elements,including Cd(Gierth et al.,1999;White, 2001;Enstone et al.,2003;Seregin et al.,2004;Seregin and Kozhevnikova,2008).The additional peri-endodermal layer of cells with ligni?ed cell walls present in N.caerulescens may function similarly(Zelko et al.,2008).These apoplas-mic barriers develop closer to the root apex when roots are exposed to high concentrations of potentially toxic ele-ments.Accelerated development of both the endodermis and exodermis have been observed in various plant species in response to salinity(Reinhardt and Rost,1995;Karahara et al.,2004),and the multiple environmental stresses caused by cultivation in municipal solid waste slag with high salt and heavy metal content have been shown to induce extensive thickening of the inner tangential walls of maize endodermal cells(Degenhardt and Gimmler,2000).Exposure to Cd has been found to result in the formation of Casparian bands and suberin lamellae closer to the root apex in several plant species,including A.thaliana (Schreiber et al.,1999),Silene dioica(Martinka and Lux, 2004),woody shrub species such as Karwinskia humboldti-ana(Zelko and Lux,2004),and maize(Fig.2;Vacul?′k et al., 2009).Maturation of the endodermis closer to the root apex can be attributed partly to a reduction in the rate of
root
Fig.2.The development of endodermal suberin lamellae in the roots of maize plants after10d growth in Hoagland’s solution containing
no cadmium(Cd0)or5l M Cd(NO3)2.4H2O(Cd5).Three regions of the root can be distinguished:A,a region in which endodermal
suberin lamellae are fully developed(solid green lines),B,a region in which the suberin lamellae are partially developed(broken green lines),and C,a region lacking endodermal suberin lamellae.Because the length of roots grown in the absence and presence of Cd
differed,the distance from the root tip is expressed as percentage of the total root length.Endodermal suberin lamellae developed closer
to the root apex in roots exposed to Cd,when expressed in either absolute or percentage terms.White arrows indicate suberin lamellae
in the endodermis.
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extension,but it is also a consequence of accelerated development of the endodermis.Exposure of maize roots to Cd resulted in an approximately 3-fold increase of endoder-mal suberin and a 2-fold increase of endodermal lignin (Schreiber et al.,1999).It also altered the chemical com-position of endodermal suberin and lignin (Schreiber et al.,1999).All these changes can be interpreted as acclimatory responses that reduce the apoplasmic movement of Cd to the xylem and its translocation to the shoot.This hypothesis is supported by the observation that low Cd translocation to the shoot among willow clones with contrasting Cd tolerance,accumulation and translocation traits was corre-lated with the deposition of suberin lamellae closer to the root apex (Fig.3;Lux et al.,2004).
Effects of cadmium on root anatomy
In addition to the accelerated development of the endoder-mis and exodermis,many other aspects of root anatomy are altered when roots are exposed to elevated Cd concentra-tions in the rhizosphere (Seregin et al.,2004;Seregin and Kozhevnikova,2008).Elevated rhizosphere Cd concentra-tions often result in changes in the relative proportion and size of root tissues and cell types.Although many of these changes depend upon the rhizosphere Cd concentration,and appear to be species-and tissue-speci?c,they can be found both in plants permanently exposed to elevated Cd concentrations in the natural environment as well as in plants growing in arti?cial media in the laboratory.
Cadmium concentrations in the rhizosphere that inhibit root growth by about 50%have been shown to increase the production of root hairs close to the root apex in maize (Seregin and Ivanov,2001),radish (Raphanus sativus L.;
Vito
′ria et al.,2003),barley (Hordeum vulgare L.;D ˇurc ˇekova ′et al.,2007),sorghum (Sorghum bicolor L.;Kuriakose and Prasad,2008),and rhode grass (Chloris gayana Kunth.;Kopittke et al.,2010),suggesting that Cd accelerates the maturation of these cells.However,higher Cd concentra-tions in the rhizosphere can result in reduced root hair production and the disintegration of the rhizodermis and outer cortical cell layers (Seregin et al.,2004;Kuriakose and Prasad,2008;Grata ?o et al.,2009).The disintegration of cortical cells,together with a general loss of cell turgor,results in the occurrence of conspicuous intercellular air spaces and irregular-shaped epidermal and cortical cells in roots of willow (Salix alba L.),poplar (Populus 3euroamericana )and radish exposed to high rhizosphere Cd concentrations
(Luna
′c ˇkova ′et al.,2003;Vito ′ria et al.,2003).Several studies have indicated that root diameter is increased by rhizosphere Cd concentrations that do not cause signi?cant necrosis.For example,roots of willow,poplar,Miscanthus sinensis L.and maize plants grown in media containing Cd were shorter and thicker than plants grown in media lacking Cd,although their apices turned
brownish (Luna
′c ˇkova ′et al.,2003;Scebba et al.,2006;Maksimovic
′et al.,2007).Maksimovic ′et al.(2007)attrib-uted the greater diameters of maize roots exposed to Cd to an increase in the size of parenchyma cells,and proposed that the enlargement of cortical tissues had a functional role by increasing resistance to radial ?ows of water and solutes.The size of cells in the exodermis of maize roots,as well as cells in the central cylinder and vascular tissues,were
unaffected by Cd exposure (Maksimovic
′et al.,2007).By contrast,willow clones characterized by high Cd tolerance had a greater proportion of epidermal,exodermal,and endodermal tissues than Cd-sensitive clones,which had a greater proportion of mid-cortical tissues (Lux et al.,2004).Other studies have observed no effect of Cd exposure
on diameters of bean (Phaseolus vulgaris L.)roots (Va
′zquez et al.,1992),nor the diameters,lengths or speci?c surface
areas of maize roots (Florijn et al.,1993),although Va
′zquez et al.(1992)did observe an increase in the size of parenchyma cells in the cortex of bean roots exposed to Cd.Relatively little information is available about Cd-induced changes in the development of cells and tissues localized within the central part of roots.This topic requires more attention,especially considering the importance of xylem loading in regulating Cd ?uxes to the shoot (Papoyan et al.,2007;Ueno et al.,2008;Lu et al.,2009;Uraguchi
et al.,2009b ;Verbruggen et al.,2009).Vito
′ria et al.(2003)observed the proliferation of cambial cells followed by a loss of organization of the cambial region in radish roots exposed to 0.5mM Cd,suggesting that Cd accelerated root maturation,including the development of xylem elements in the central cylinder.Consistent with this interpretation,Schu ¨tzendu ¨bel et al.(2001)found that exposure to 50l M Cd caused accelerated ligni?cation of protoxylem
elements
Fig.3.Development of Casparian bands (stage 1of endodermal development;solid green lines close to the root apex)and suberin lamellae (stage 2of endodermal development;red lines)in roots of different Salix clones with various properties of Cd accumulation and Cd tolerance.Clone names refer to the property to accumu-late,translocate,and tolerate https://www.360docs.net/doc/0619309893.html,,low accumulation;Ha,high accumulation;Lt,low translocation;Ht,high translocation;
T,tolerant;S,sensitive.Note the development of suberin lamellae close to the root apex in clones with high translocation and
distant from the root apex in clones with low translocation of Cd.Modi?ed from Lux et al.(2004)with permission from Physiologia Plantarum .
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closer to the root apex in Scots pine(Pinus sylvestris L.), and Dˇurcˇekova′et al.(2007)reported premature xylogenesis in barley roots exposed to Cd.Luna′cˇkova′et al.(2003) observed that cambial activity and the formation of lateral primordia occurred closer to the root apex in plants exposed to Cd,suggesting Cd accelerated the production of lateral roots.By contrast,although Va′zquez et al.(1992) found an increase in the number of pericycle cells when roots were exposed to Cd,they observed a reduction in cell differentiation and ligni?cation in the vascular cylinder of Cd-treated bean plants.Similarly,Ederli et al.(2004) found no signi?cant changes in the structure of reed (Phragmites australis(Cav.)Trin.ex Steud.)root exposed to100l M Cd.
Root anatomy responds to local cadmium concentrations in the rhizosphere
To elucidate responses in root anatomy to local high rhizosphere Cd concentrations,as might occur in natural soils,a novel experiment was designed.The aim of this experiment was to determine whether unilateral exposure of roots to Cd could induce asymmetrical development of the endodermis or other root tissues.This would test whether individual root cells responded directly to Cd in their immediate vicinity to restrict Cd movement to the xylem through the development of appropriate apoplasmic barriers.
Maize(Zea mays L.hybrid Jose?na)seedlings with seminal roots5–6cm in length were placed between two layers of agar(Fig.4A).In the control–control treatment (Cd0–Cd0)neither agar layer contained Cd.In the control–Cd treatment(Cd0–Cd100),one agar layer contained no Cd whilst the other agar layer contained either50l mol Cd(NO3)2.4H2O or100l mol Cd(NO3)2.4H2O.Agar layers were placed in the two halves of square Petri dishes (2453245mm,Corning Incorporated,USA)?xed vertically in a growth chamber with controlled environmental con-ditions(temperature,2461°C;relative air humidity,70%; light intensity,150l mol mà2sà1).To avoid the diffusion of Cd2+from agar layers containing Cd,plastic spacers were placed between the agar layers to maintain a distance of1 mm,which corresponded to the diameter of the growing maize roots.This design ensured that only the root surface touching the agar layer containing Cd was exposed directly to Cd in the environment.Roots exposed to the Cd0–Cd0 treatment grew gravitropically and after4d they had elongated by4–5cm.By contrast,roots exposed to the Cd0–Cd100treatment exhibited limited growth and after 4d had elongated by only1.0–1.5cm.The apices of roots exposed to the Cd0–Cd100treatment bent into the agar layer containing Cd and their growth then stopped (Fig.4B).Bending was caused by the cessation of cell elongation on the side of the root exposed to the Cd-containing agar,whilst cell elongation continued on the other side of the root.
Two days after placing plants in Petri dishes,several roots were removed and transverse sections were cut at regular0.5cm distances from the root apex to the base. Transverse sections were stained with Fluorol Yellow088to identify suberin lamellae(Brundrett et al.,1991;Lux et al., 2005)and with phloroglucinol–HCl to identify
lignin
Fig.4.Effects of unilateral exposure of maize roots to100l M Cd(NO3)2.4H2O.(A)Maize seedlings with5–6cm long seminal roots
were placed between two layers of agar containing zero or100l M Cd.(B)Photograph taken4d after unilateral exposure of maize roots
to Cd showing them bending in the direction of the Cd layer.(C)Longitudinal section of the apex of a root bending toward the Cd agar
layer stained by phloroglucinol–HCl to detect lignin.A lateral root primordium has been initiated on the side opposite the Cd layer. Abbreviations:lrp,lateral root primordium;asterisk,ectopic deposition of lignin.Scale bar:500l m.
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(Figs4C,5,6).In the Cd0–Cd0treatment,endodermal cells with suberin lamellae?rst started to appear at8cm from the root tip.At9cm from the tip approximately50%of endodermal cells developed suberin lamellae and in10cm from the root tip80–100%of endodermal cells were covered by suberin lamellae(Fig.5).In the Cd0–Cd100treatment the effect of unilateral Cd exposure resulted in accelerated and irregular maturation of the endodermis.The results were similar for both Cd concentrations.Rhizodermal, cortical,and,to some extent,cells in the vascular cylinder were all affected on the side of the root directly exposed to Cd.Direct exposure to Cd resulted in the collapse of peripheral root tissues close to the apex.Endodermal cells reacted by accelerated production of suberin lamellae, which were already present at a distance of0.5cm from the root apex in tissues adjacent to agar containing Cd(Fig.5). At this distance no suberin lamellae were observed in root tissues adjacent to the Cd-free agar,which developed at a greater distance from the root apex(Fig.5).About10% of endodermal cells adjacent to Cd-free agar contained suberin lamellae at1cm from the root apex,and almost 100%of these endodermal cells had suberin lamellae at 2cm from the root apex.In the endodermal cells exposed directly to Cd,suberin lamellae were no longer detected at these distances from the root apex and ligni?cation of cells had occurred in the inner cortical tissues and in the pericycle(Fig.5).In addition,ectopic ligni?cation of protoxylem elements occurred in the half of the root directly exposed to Cd,whereas no,or only very weak, ligni?cation of cells occurred in the half of the root exposed to Cd-free agar(Figs4C,6).Primordia of lateral roots also started to develop in roots exposed to the Cd0–Cd100 treatment(Fig.4C;6A).These only appeared in the half of the root exposed to Cd-free agar.The production of lateral roots in the Cd-free agar could be a response to root bending,in a manner analogous to the initiation of lateral roots at the convex side of roots subjected to gravitropic or mechanical stimuli(Richter et al.,2009),and would constitute an adaptive avoidance response to patches of high Cd in the rhizosphere.
It is evident from the results of this study that endoder-mal development is accelerated in parts of the root exposed directly to Cd in the rhizosphere.One can hypothesize that the accelerated production of a suberized endodermis,
and
Fig.5.Schematic illustration of maize roots cultivated for2d between layers of agar containing no cadmium(Cd0–Cd0)or exposed unilaterally to100l M Cd(Cd0–Cd100;Fig.4A).(A–F)Micrographs show the gradual development of suberin lamellae in the endodermis
along the root axis visualized by Fluorol yellow088and?uorescence microscopy.In roots that were not exposed to Cd(Cd0–Cd0)all endodermal cells had developed suberin lamellae95–100mm from the root apex(A).Suberin lamellae were?rst deposited at a distance
of about80mm from the root apex,and90mm from the apex approximately50%of endodermal cells developed suberin lamellae(B).
In roots exposed unilaterally to Cd(Cd0–Cd100)the development of the endodermis was accelerated and asymmetrical(D–F).Suberin lamellae had already developed on the side of the root exposed to Cd in endodermal cells5mm from the root apex(F).At this distance
from the root apex no suberin lamellae were present on the side not exposed to Cd.At distances greater than10–15mm from the root apex,suberin was no longer detected in endodermal cells on the side exposed to Cd.However,some unidenti?ed material exhibiting red
?uorescence(excitation?lter TBP400+495+570,beamsplitter TFT410+505+585,emisssion?lter TBP460+530+610,wavelengths are
in nm)was deposited on the cell walls of endodermis,pericycle,in some xylem elements,and also in parenchyma cells of the vascular cylinder(D,E).At a distance of20mm from the root apex,suberin lamellae gradually developed on the side of the root not exposed to
Cd(E)and at a distance of25–30mm from the root apex the entire half of the root which was not exposed to Cd had deposited suberin lamellae in the endodermis(D).White arrows indicate suberin lamellae in endodermis.Scale bars:100l m.
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the ligni?cation of cell walls of inner cortical tissues and peripheral tissues of the vascular cylinder,in root tissues adjacent to a local Cd source could restrict the radial apoplasmic movement of Cd,and Cd loading to the xylem,thereby,protecting both unexposed root tissues and the shoot from Cd exposure.This phenomenon can be inter-preted,therefore,as an adaptive response to protect plants from Cd toxicity.The bending of seminal roots towards a Cd source appears to initiate the production of lateral roots on the side opposite to the Cd source,which can also be interpreted as an adaptive response of roots to avoid patches of high Cd in the rhizosphere.
It is possible that the responses to local Cd exposure are initiated through Cd-induced oxidative stress,which has been implicated in the inhibition of root initiation and elongation in various plant species (Xiong et al.,2009).This hypothesis is consistent with a large subset of signi?cant transcriptional responses to acute Cd exposure in roots of Arabidopsis thaliana being genes encoding reactive oxygen species (ROS)-scavenging enzymes and genes involved in the signal trans-duction pathway for ROS responses (Herbette et al.,2006;DalCorso et al.,2008;van de Mortel et al.,2008;Smeets et al.,2008;Zhao et al.,2009).Other genes with signi?cant transcriptional responses to acute Cd exposure in Arabidopsis roots include genes involved in sulphur assimilation/reduction and glutathione metabolism,and genes with the gene ontology (GO)classi?cations ‘response to abiotic or biotic stimulus’,
‘response to stress’,and ‘signal transduction’(Herbette et al.,2006;Weber et al.,2006;van de Mortel et al.,2008;Zhao et al.,2009).Intriguingly,the expression of genes involved in lignin biosynthesis is also up-regulated in roots of Arabidopsis thaliana and Noccaea caerulescens upon Cd exposure (Herbette et al.,2006;van de Mortel et al.,2008),suggesting candidate genes for the ectopic ligni?cation of cell walls.Recently,increased expression of genes encoding heat shock transcription factors of class A4(HsfA4a)have been associ-ated with the up-regulation of genes encoding metallothioneins and Cd tolerance in both wheat and rice (Shim et al.,2009).It has been suggested that these transcription factors co-ordinate a concerted cellular response to Cd exposure (Shim et al.,2009),but this hypothesis has not been tested.
Cadmium localization in root tissues
In most plant species,roots have higher tissue Cd concen-trations than shoots,although the opposite has been observed in many Cd-hyperaccumulating plants and in some non-hyperaccumulators,mostly from Compositae (e.g.Cichorium intybus ,Bidens frondosa ,Lactuca indica ;Abe et al.,2008).Depending on the rhizosphere Cd concentration,Cd concentrations in the root can be up to
10times higher than those in the shoot (Po 1ec
′-Pawlak et al.,2005;Wo
′jcik and Tukiendorf,2004,2005;Sol?′s-Dom?′
nquez Fig.6.Cross-sections of maize roots exposed unilaterally to Cd (Cd0–Cd100;Fig.4).Sections were cut at a distance of 20mm from the root apex and stained by phloroglucinol–HCl to detect lignin.Ligni?cation was asymmetrical,occurring primarily in the half of the root exposed to Cd (A,B).Weak ligni?cation was observed in endodermal and xylem cells in the half of the root not exposed to Cd (C).There was a gradual increase in ligni?cation from the unexposed side of the root to the side of the root directly exposed to Cd (D).Direct
exposure to Cd induced strong ligni?cation in the pericycle and inner cortical cells (D,E).Ectopic deposition of lignin was also observed in the lumen of protoxylem cells (D,E).The side of the root not exposed to Cd formed lateral root primordia (A),which were never observed on the side exposed to Cd.Abbreviations:e,endodermis;pc,pericycle;px,protoxylem;emx,early metaxylem;lmx,late metaxylem;lrp,lateral root primordium;asterisk,ectopic deposition of lignin.Scale bars:100l m (A,B);40l m (C,D,E).
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et al.,2007,Wang et al.,2009;Lukacˇova′and Lux,2010). At low rhizosphere Cd concentrations,the root retains most of the Cd entering the plant,but the fraction retained by roots decreases as rhizosphere Cd concentrations increases. The Cd concentration in the root is in?uenced by the Cd concentration in the substrate(Wo′jcik and Tukiendorf, 2004,2005;Wu et al.,2005;Van Belleghem et al.,2007),the phytoavailability of Cd(Wo′jcik and Tukiendorf,2004; Abe et al.,2008),and the duration of exposure to Cd (Va′zquez et al.,1992;Wo′jcik and Tukiendorf,2004;Isaure et al.,2006).Root Cd concentrations increase rapidly upon exposure to Cd until steady-state tissue concentrations are achieved,generally after several days exposure,and root Cd concentrations are higher when plants are grown in substrates with greater Cd concentrations or Cd phytoavailabilities.
The spatial patterns of Cd accumulation within the root have been studied in various plants,including Cd-tolerant, non-tolerant,and Cd-hyperaccumulating species(for reviews see Seregin and Ivanov,2001;Seregin and Kozhevnikova, 2008).At low rhizosphere Cd concentrations,Cd accumu-lates mostly in the apical part of the root(Arduini et al., 1996;Seregin et al.,2004)or in the proximal subapical region (e.g.3cm from the tip of maize root;Seregin et al.,2007). Cadmium penetrates into the root cap,rhizodermis,and cortex(Seregin and Ivanov,1997).In the some plant species, a well-developed root cap appears to prevent Cd incursion at the root apex(e.g.Pinus pinea and Pinus pinaster;Arduini et al.,1996).Relatively high Cd concentrations are observed at the surface of the rhizodermis(Liu et al.,2007).Tissue Cd concentrations gradually decrease from the outer cortical parenchyma to the endodermis(Va′zquez et al.,1992,2007; Ku¨pper et al.,2000).The pericycle accumulates little Cd, which may account for the continued production of lateral roots in the presence of Cd(Seregin et al.,2004).Cadmium can accumulate in the vascular cylinder,mostly in conductive elements and their adjacent parenchyma cells,presumably as a consequence of long-distance Cd transport(Seregin and Ivanov,1997;Seregin et al.,2004;Liu et al.,2007;Va′zquez et al.,2007).Signi?cantly higher Cd concentrations occur in the parenchyma cells located between the endodermis and xylem poles compared with the adjacent parenchyma cells. This patterning is probably related to the occurrence of passage cells in the endodermis,whose cell walls are likely to be more permeable to Cd than those of endodermal cells with suberin lamellae(Van Belleghem et al.,2007).High Cd concentrations occur in both the pericycle and vascular tissues in the basal part of the root or throughout the whole root when plants are exposed to high rhizosphere Cd concentrations in the medium(Wo′jcik and Tukiendorf, 2004;Isaure et al.,2006;Sol?′s-Dom?′nquez et al.,2007).This can result in a dramatic inhibition of root growth and branching(Seregin et al.,2004).
The highest Cd concentrations in root tissues are observed in the apoplasm,mainly on the outer surface of the rhizodermis and in the cell walls of the rhizodermis and cortical cells,whilst much lower Cd concentrations are found within root cells(Liu et al.,2007;Seregin et al.,2004,2007;Va′zquez et al.,2007;Wang et al.,2009).Within root cells,Cd
is mainly concentrated in vacuoles and nuclei,with lower Cd concentrations being present in the cytoplasm and plastids
(Va′zquez et al.,1992;Liu and Kottke,2004;Liu et al.,2007).
Cell fractionation studies suggest that Cd-sensitive plants
have lower Cd concentrations in cell walls,and higher vacuolar Cd concentrations,than Cd-tolerant plants (Uraguchi et al.,2009a).In barley roots,36%of the Cd is present in cell walls and51%is present in a soluble fraction
(Wu et al.,2005),whereas in metal-tolerant species,such as
S.alfredii and N.caerulescens,more Cd is present in the cell
wall/apoplasm than in the soluble fraction/vacuole(Va′zquez
et al.,1992;Ni and Wei,2003;Redjala et al.,2009).In barley roots,Cd-phytochelatin complexes account for34–50%of the soluble fraction(Persson et al.,2006).Three types of Cd-phytochelatin complexes have been reported in barley roots,
the ligands being(c Glu-Cys)n-Gly(PC n),(c Glu-Cys)n-Ser
(iso-PC n),and Cys-(c Glu-Cys)n-Gly(des-c Glu-PC n),and
a correlation between Cd tolerance and the accumulation of
Cd–PC3has been observed(Persson et al.,2006).Some Cd-tolerant plant species are able to increase the cation exchange capacity of their cell walls following exposure to Cd(Nyquist
and Greger,2009).According to transmission electron microscopy,Cd occurs in granules visualized as electron-dense aggregates.Electron-dense granules appear between the
cell wall and the plasmalemma of cells in outermost root tissues,whilst relatively few are found in the vascular cylinder
(Liu and Kottke,2004;Liu et al.,2007;Van Belleghem et al., 2007;Daud et al.,2009).These distinctive granular deposits
occur in all plant species studied,including A.thaliana(Van Belleghem et al.,2007),Iris pseudacorus(Zhou et al.,2010),
and N.caerulescens(Wo′jcik et al.,2005).In the vascular cylinder,a signi?cantly higher amount of granular precipi-
tated Cd occurs in the apoplasm surrounding the paren-chyma cells located between the endodermis and xylem poles compared with the adjacent parenchyma cells(Wo′jcik et al., 2005;Van Belleghem et al.,2007).Cadmium-containing granular deposits are also found in the middle lamellae between the endodermis and pericycle cells(Khan et al., 1984;Wo′jcik and Tukiendorf,2004).In vacuoles,electron-dense granules are aggregated and formed into larger precipitates,which increase in number and size with increasing Cd exposure(Liu and Kottke,2004;Sol?′s-
Dom?′nquez et al.,2007).Cadmium is accumulated in the vacuoles of meristematic or cortical parenchyma cells of differentiating and mature roots,but little Cd is found in the vacuoles of cells within the vascular cylinder(Liu and Kottke, 2004;Liu et al.,2007;Van Belleghem et al.,2007).In the endodermis,Cd is sequestered as very?ne and uniformly distributed granular deposits in the vacuole and as large granular deposits in the cytoplasm located near the cell wall.
In the vascular cylinder,signi?cantly more granular pre-cipitated Cd occurs in the cytoplasm of parenchyma cells located between the endodermis and xylem poles compared
with the adjacent parenchyma cells(Wo′jcik et al.,2005;Van Belleghem et al.,2007).The accumulation of Cd in the cytoplasm of the phloem and its companion cells(Khan
et al.,1984;Wo′jcik and Tukiendorf,2004)suggests the
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retranslocation of Cd from the shoot to the root in plants that restrict Cd accumulation in the shoot(Van Belleghem et al.,2007).
Conclusions and perspective
Cadmium is exceedingly toxic to plant cells.Most plants limit shoot Cd accumulation by restricting Cd movement to the xylem through both the symplasmic and the apoplasmic pathways.When plant roots are exposed to high Cd concentrations,they increase the production of phytochela-tins and sequester Cd entering root cells as Cd-chelates in the vacuole.This is likely to reduce symplasmic Cd concentra-tions and,thereby,symplasmic movement of Cd to the xylem.In tandem,they restrict apoplasmic Cd?uxes to the xylem by accelerating the maturation of the endodermis,and produce Casparian bands,suberin lamellae,and ligni?cation closer to the root apex.The maturation of the endodermis appears to respond to the local Cd concentration in the environment,and will develop asymmetrically in response to Cd gradients in the rhizosphere.We hypothesize that the accelerated maturation of the endodermis in response to local Cd availability has functional signi?cance in protecting the shoot from excessive Cd loads by reducing the entry of Cd to the xylem.The diffusion of Cd in aqueous media is slow,and endodermal suberization presents an additional barrier to Cd movement in the extracellular space. Acknowledgements
This work was supported by grants1/0472/10from the Slovak Grant Agency VEGA,COST0004-06and SK-ZA-0007-07from the Slovak Research and Development Agency APVV,and COST FA0905(AL,MM,MV),and by the Scottish Government Rural and Environment Research and Analysis Directorate(PJW).
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The way常见用法
The way 的用法 Ⅰ常见用法： 1)the way+ that 2)the way + in which（最为正式的用法） 3)the way + 省略（最为自然的用法） 举例：I like the way in which he talks. I like the way that he talks. I like the way he talks. Ⅱ习惯用法： 在当代美国英语中，the way用作为副词的对格，“the way+ 从句”实际上相当于一个状语从句来修饰整个句子。 1)The way =as I am talking to you just the way I’d talk to my own child. He did not do it the way his friends did. Most fruits are naturally sweet and we can eat them just the way they are—all we have to do is to clean and peel them. 2）The way= according to the way/ judging from the way The way you answer the question, you are an excellent student. The way most people look at you, you’d think trash man is a monster. 3）The way =how/ how much No one can imagine the way he missed her. 4）The way =because
The way的用法及其含义(二)
The way的用法及其含义（二） 二、the way在句中的语法作用 the way在句中可以作主语、宾语或表语： 1.作主语 The way you are doing it is completely crazy.你这个干法简直发疯。 The way she puts on that accent really irritates me. 她故意操那种口音的样子实在令我恼火。The way she behaved towards him was utterly ruthless. 她对待他真是无情至极。 Words are important, but the way a person stands, folds his or her arms or moves his or her hands can also give us information about his or her feelings. 言语固然重要,但人的站姿,抱臂的方式和手势也回告诉我们他(她)的情感。 2.作宾语 I hate the way she stared at me.我讨厌她盯我看的样子。 We like the way that her hair hangs down.我们喜欢她的头发笔直地垂下来。 You could tell she was foreign by the way she was dressed. 从她的穿著就可以看出她是外国人。 She could not hide her amusement at the way he was dancing. 她见他跳舞的姿势，忍俊不禁。 3.作表语 This is the way the accident happened.这就是事故如何发生的。 Believe it or not, that's the way it is. 信不信由你, 反正事情就是这样。 That's the way I look at it, too. 我也是这么想。 That was the way minority nationalities were treated in old China. 那就是少数民族在旧中
(完整版)the的用法
定冠词the的用法： 定冠词the与指示代词this ,that同源,有“那(这)个”的意思,但较弱,可以和一个名词连用,来表示某个或某些特定的人或东西. (1)特指双方都明白的人或物 Take the medicine.把药吃了. (2)上文提到过的人或事 He bought a house.他买了幢房子. I've been to the house.我去过那幢房子. (3)指世界上独一无二的事物 the sun ,the sky ,the moon, the earth (4)单数名词连用表示一类事物 the dollar 美元 the fox 狐狸 或与形容词或分词连用,表示一类人 the rich 富人 the living 生者 (5)用在序数词和形容词最高级,及形容词等前面 Where do you live?你住在哪? I live on the second floor.我住在二楼. That's the very thing I've been looking for.那正是我要找的东西. (6)与复数名词连用,指整个群体 They are the teachers of this school.(指全体教师) They are teachers of this school.(指部分教师) (7)表示所有,相当于物主代词,用在表示身体部位的名词前 She caught me by the arm.她抓住了我的手臂. (8)用在某些有普通名词构成的国家名称,机关团体,阶级等专有名词前 the People's Republic of China 中华人民共和国 the United States 美国 (9)用在表示乐器的名词前 She plays the piano.她会弹钢琴. (10)用在姓氏的复数名词之前,表示一家人 the Greens 格林一家人(或格林夫妇) (11)用在惯用语中 in the day, in the morning... the day before yesterday, the next morning... in the sky... in the dark... in the end... on the whole, by the way...
“the way+从句”结构的意义及用法
“tｈｅwａｙ+从句”结构的意义及用法 首先让我们来看下面这个句子: Ｒead the foｌloｗｉnｇpassagｅａnd talkａbout ｉt wi ｔh your classmaｔes．Try tｏｔeｌl whaｔyｏu thiｎk ｏf Tom aｎd ｏｆｔhe ｗay the chiｌｄrenｔrｅated ｈim. 在这个句子中，ｔhe waｙ是先行词,后面是省略了关系副词that或in whｉch的定语从句。 下面我们将叙述“the ｗaｙ+从句”结构的用法。 1.tｈe wａy之后，引导定语从句的关系词是tｈat而不是hoｗ,因此，<<现代英语惯用法词典＞＞中所给出的下面两个句子是错误的：Ｔhｉs is ｔｈeｗayｈowｉtｈａppened. This is the way how he ａlwａｙs treats me. 2．在正式语体中,thaｔ可被in which所代替;在非正式语体中，ｔhat则往往省略。由此我们得到theｗay后接定语从句时的三种模式：1) tｈe ｗaｙ＋ｔhａt-从句２）the way +ｉn whiｃh－从句3) the ｗay +从句 例如：Ｔｈe way(in whiｃh ，thａt) ｔhｅｓｅｃomｒade ｓloｏｋａｔｐrobｌems is wrong．这些同志看问题的方法
不对。 Ｔｈｅｗａy(that ,ｉn ｗhiｃh)yoｕ’ｒe doinｇit is comple ｔeｌy crａzy.你这么个干法,简直发疯。 Wｅａdmiｒｅd him for thｅｗay ｉｎｗhｉch he fａｃeｓdiｆficultieｓ． Waｌlａｃe ａｎd Ｄarwiｎｇreed ｏn the way ｉｎwhi ｃh ｄｉfｆｅrｅnt forms ｏf life had ｂegｕn.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 Thｉs is ｔｈe ｗaｙ（tｈａｔ) hｅdｉd it. I lｉkeｄｔhe waｙ(ｔhat) ｓｈeｏｒganized ｔhe ｍeetｉng． 3.theｗay(tｈat)有时可以与hｏw(作“如何”解）通用。例如： That’s tｈe ｗaｙ(tｈaｔ) shｅspoke. = Tｈat’s how shｅspoke.
way 用法
表示“方式”、“方法”，注意以下用法： 1.表示用某种方法或按某种方式，通常用介词in(此介词有时可省略)。如： Do it (in) your own way. 按你自己的方法做吧。 Please do not talk (in) that way. 请不要那样说。 2.表示做某事的方式或方法，其后可接不定式或of doing sth。 如： It’s the best way of studying [to study] English. 这是学习英语的最好方法。 There are different ways to do [of doing] it. 做这事有不同的办法。 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句，但是其后的从句不能由how 来引导。如： 我不喜欢他说话的态度。 正：I don’t like the way he spoke. 正：I don’t like the way that he spoke. 正：I don’t like the way in which he spoke. 误：I don’t like the way how he spoke. 4.注意以下各句the way 的用法： That’s the way (=how) he spoke. 那就是他说话的方式。 Nobody else loves you the way(=as) I do. 没有人像我这样爱你。 The way (=According as) you are studying now, you won’tmake much progress. 根据你现在学习情况来看，你不会有多大的进步。 2007年陕西省高考英语中有这样一道单项填空题： ——I think he is taking an active part insocial work. ——I agree with you_____. A、in a way B、on the way C、by the way D、in the way 此题答案选A。要想弄清为什么选A，而不选其他几项，则要弄清选项中含way的四个短语的不同意义和用法，下面我们就对此作一归纳和小结。 一、in a way的用法 表示：在一定程度上，从某方面说。如： In a way he was right.在某种程度上他是对的。注：in a way也可说成in one way。 二、on the way的用法 1、表示：即将来(去)，就要来(去)。如： Spring is on the way.春天快到了。 I'd better be on my way soon.我最好还是快点儿走。 Radio forecasts said a sixth-grade wind was on the way.无线电预报说将有六级大风。 2、表示：在路上，在行进中。如： He stopped for breakfast on the way.他中途停下吃早点。 We had some good laughs on the way.我们在路上好好笑了一阵子。 3、表示：(婴儿)尚未出生。如： She has two children with another one on the way.她有两个孩子，现在还怀着一个。 She's got five children，and another one is on the way.她已经有5个孩子了，另一个又快生了。 三、by the way的用法
The way的用法及其含义(一)
The way的用法及其含义（一） 有这样一个句子：In 1770 the room was completed the way she wanted. 1770年，这间琥珀屋按照她的要求完成了。 the way在句中的语法作用是什么？其意义如何？在阅读时，学生经常会碰到一些含有the way 的句子，如：No one knows the way he invented the machine. He did not do the experiment the way his teacher told him.等等。他们对the way 的用法和含义比较模糊。在这几个句子中，the way之后的部分都是定语从句。第一句的意思是，“没人知道他是怎样发明这台机器的。”the way的意思相当于how；第二句的意思是，“他没有按照老师说的那样做实验。”the way 的意思相当于as。在In 1770 the room was completed the way she wanted.这句话中，the way也是as的含义。随着现代英语的发展，the way的用法已越来越普遍了。下面，我们从the way的语法作用和意义等方面做一考查和分析： 一、the way作先行词，后接定语从句 以下3种表达都是正确的。例如：“我喜欢她笑的样子。” 1. the way+ in which +从句 I like the way in which she smiles. 2. the way+ that +从句 I like the way that she smiles. 3. the way + 从句(省略了in which或that) I like the way she smiles. 又如：“火灾如何发生的，有好几种说法。” 1. There were several theories about the way in which the fire started. 2. There were several theories about the way that the fire started.
way 的用法
way 的用法 【语境展示】 1. Now I’ll show you how to do the experiment in a different way. 下面我来演示如何用一种不同的方法做这个实验。 2. The teacher had a strange way to make his classes lively and interesting. 这位老师有种奇怪的办法让他的课生动有趣。 3. Can you tell me the best way of working out this problem? 你能告诉我算出这道题的最好方法吗？ 4. I don’t know the way (that / in which) he helped her out. 我不知道他用什么方法帮助她摆脱困境的。 5. The way (that / which) he talked about to solve the problem was difficult to understand. 他所谈到的解决这个问题的方法难以理解。 6. I don’t like the way that / which is being widely used for saving water. 我不喜欢这种正在被广泛使用的节水方法。 7. They did not do it the way we do now. 他们以前的做法和我们现在不一样。 【归纳总结】 ●way作“方法，方式”讲时，如表示“以……方式”，前面常加介词in。如例1； ●way作“方法，方式”讲时，其后可接不定式to do sth.，也可接of doing sth. 作定语，表示做某事的方法。如例2，例3；
the-way-的用法讲解学习
t h e-w a y-的用法
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
way的用法总结大全
way的用法总结大全 way的用法你知道多少，今天给大家带来way的用法，希望能够帮助到大家，下面就和大家分享，来欣赏一下吧。 way的用法总结大全 way的意思 n. 道路,方法,方向,某方面 adv. 远远地，大大地 way用法 way可以用作名词 way的基本意思是“路,道,街,径”,一般用来指具体的“路,道路”,也可指通向某地的“方向”“路线”或做某事所采用的手段,即“方式,方法”。way还可指“习俗,作风”“距离”“附近,周围”“某方面”等。 way作“方法,方式,手段”解时,前面常加介词in。如果way前有this, that等限定词,介词可省略,但如果放在句首,介词则不可省略。
way作“方式,方法”解时,其后可接of v -ing或to- v 作定语,也可接定语从句,引导从句的关系代词或关系副词常可省略。 way用作名词的用法例句 I am on my way to the grocery store.我正在去杂货店的路上。 We lost the way in the dark.我们在黑夜中迷路了。 He asked me the way to London.他问我去伦敦的路。 way可以用作副词 way用作副词时意思是“远远地,大大地”,通常指在程度或距离上有一定的差距。 way back表示“很久以前”。 way用作副词的用法例句 It seems like Im always way too busy with work.我工作总是太忙了。 His ideas were way ahead of his time.他的思想远远超越了他那个时代。 She finished the race way ahead of the other runners.她第一个跑到终点,远远领先于其他选手。 way用法例句
the_way的用法大全教案资料
t h e_w a y的用法大全
The way 在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或 in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的
the way 的用法
The way 的用法 "the way+从句"结构在英语教科书中出现的频率较高, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 一.在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮.
the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms. 从那天起,其他同学是夹着书本来上课,而他们却带着"失败"的思想负担来上课.
The way的用法及其含义(三)
The way的用法及其含义（三） 三、the way的语义 1. the way=as（像） Please do it the way I’ve told you.请按照我告诉你的那样做。 I'm talking to you just the way I'd talk to a boy of my own.我和你说话就像和自己孩子说话一样。 Plant need water the way they need sun light. 植物需要水就像它们需要阳光一样。 2. the way=how（怎样，多么） No one can imagine the way he misses her.没人能够想象出他是多么想念她！ I want to find out the way a volcano has formed.我想弄清楚火山是怎样形成的。 He was filled with anger at the way he had been treated.他因遭受如此待遇而怒火满腔。That’s the way she speaks.她就是那样讲话的。 3. the way=according as （根据） The way you answer the questions, you must be an excellent student.从你回答问题来看，你一定是名优秀的学生。 The way most people look at you, you'd think a trash man was a monster.从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物。 The way I look at it, it’s not what you do that matters so much.依我看，重要的并不是你做什么。 I might have been his son the way he talked.根据他说话的样子，好像我是他的儿子一样。One would think these men owned the earth the way they behave.他们这样行动，人家竟会以为他们是地球的主人。
way的用法
一．Way：“方式”、“方法” 1.表示用某种方法或按某种方式 Do it (in) your own way. Please do not talk (in) that way. 2.表示做某事的方式或方法 It’s the best way of studying [to study] English.。 There are different ways to do [of doing] it. 3.其后通常可直接跟一个定语从句(不用任何引导词)，也可跟由that 或in which 引导的定语从句 正：I don’t like the way he spoke. I don’t like the way that he spoke. I don’t like the way in which he spoke.误：I don’t like the way how he spoke. 4. the way 的从句 That’s the way (=how) he spoke. I know where you are from by the way you pronounce my name. That was the way minority nationalities were treated in old China. Nobody else loves you the way(=as) I do. He did not do it the way his friend did. 二．固定搭配 1. In a/one way：In a way he was right. 2. In the way /get in one’s way I'm afraid your car is in the way， If you are not going to help，at least don't get in the way. You'll have to move－you're in my way. 3. in no way Theory can in no way be separated from practice. 4. On the way (to……) Let’s wait a few moments. He is on the way Spring is on the way. Radio forecasts said a sixth-grade wind was on the way. She has two children with another one on the way. 5. By the way By the way，do you know where Mary lives? 6. By way of Learn English by way of watching US TV series. 8. under way 1. Elbow one’s way He elbowed his way to the front of the queue. 2. shoulder one’s way 3. feel one‘s way 摸索着向前走；We couldn’t see anything in the cave, so we had to feel our way out 4. fight/force one’s way 突破。。。而前进The surrounded soldiers fought their way out. 5.. push/thrust one‘s way（在人群中）挤出一条路He pushed his way through the crowd. 6. wind one’s way 蜿蜒前进 7. lead the way 带路，领路；示范 8. lose one‘s way 迷失方向 9. clear the way 排除障碍，开路迷路 10. make one’s way 前进，行进The team slowly made their way through the jungle.
the way的用法大全
在the way+从句中, the way 是先行词, 其后是定语从句.它有三种表达形式:1) the way+that 2)the way+ in which 3)the way + 从句(省略了that或in which),在通常情况下, 用in which 引导的定语从句最为正式,用that的次之,而省略了关系代词that 或in which 的, 反而显得更自然,最为常用.如下面三句话所示,其意义相同. I like the way in which he talks. I like the way that he talks. I like the way he talks. 如果怕弄混淆，下面的可以不看了 另外，在当代美国英语中,the way用作为副词的对格,"the way+从句"实际上相当于一个状语从句来修饰全句. the way=as 1)I'm talking to you just the way I'd talk to a boy of my own. 我和你说话就象和自己孩子说话一样. 2)He did not do it the way his friend did. 他没有象他朋友那样去做此事. 3)Most fruits are naturally sweet and we can eat them just the way they are ----all we have to do is clean or peel them . 大部分水果天然甜润,可以直接食用,我们只需要把他们清洗一下或去皮. the way=according to the way/judging from the way 4)The way you answer the qquestions, you must be an excellent student. 从你回答就知道,你是一个优秀的学生. 5)The way most people look at you, you'd think a trashman was a monster. 从大多数人看你的目光中,你就知道垃圾工在他们眼里是怪物. the way=how/how much 6)I know where you are from by the way you pronounce my name. 从你叫我名字的音调中,我知道你哪里人. 7)No one can imaine the way he misses her. 人们很想想象他是多么想念她. the way=because 8) No wonder that girls looks down upon me, the way you encourage her. 难怪那姑娘看不起我, 原来是你怂恿的 the way =while/when(表示对比) 9)From that day on, they walked into the classroom carrying defeat on their shoulders the way other students carried textbooks under their arms.
“the-way+从句”结构的意义及用法知识讲解
“the way+从句”结构的意义及用法 首先让我们来看下面这个句子： Read the following passage and talk about it with your classmates. Try to tell what you think of Tom and of the way the children treated him. 在这个句子中，the way是先行词，后面是省略了关系副词that 或in which的定语从句。 下面我们将叙述“the way+从句”结构的用法。 1．the way之后，引导定语从句的关系词是that而不是how,因此，<<现代英语惯用法词典>>中所给出的下面两个句子是错误的：This is the way how it happened. This is the way how he always treats me. 2. 在正式语体中，that可被in which所代替；在非正式语体中，that则往往省略。由此我们得到the way后接定语从句时的三种模式：1) the way +that-从句2) the way +in which-从句3) the way +从句 例如：The way(in which ,that) these comrades look at problems is wrong.这些同志看问题的方法不对。
The way(that ,in which)you’re doing it is completely crazy.你这么个干法，简直发疯。 We admired him for the way in which he faces difficulties. Wallace and Darwin greed on the way in which different forms of life had begun.华莱士和达尔文对不同类型的生物是如何起源的持相同的观点。 This is the way (that) he did it. I liked the way (that) she organized the meeting. 3.the way(that)有时可以与how(作“如何”解)通用。例如： That’s the way (that) she spoke. = That’s how she spoke. I should like to know the way/how you learned to master the fundamental technique within so short a time. 4．the way的其它用法：以上我们讲的都是用作先行词的the way，下面我们将叙述它的一些用法。
定冠词the的12种用法
定冠词the的12种用法 定冠词the 的12 种用法，全知道?快来一起学习吧。下面就和大家分享，来欣赏一下吧。 定冠词the 的12 种用法，全知道? 定冠词the用在各种名词前面，目的是对这个名词做个记号，表示它的特指属性。所以在词汇表中，定冠词the 的词义是“这个，那个，这些，那些”，可见，the 即可以放在可数名词前，也可以修饰不可数名词，the 后面的名词可以是单数，也可以是复数。 定冠词的基本用法： (1) 表示对某人、某物进行特指，所谓的特指就是“不是别的，就是那个!”如： The girl with a red cap is Susan. 戴了个红帽子的女孩是苏珊。 (2) 一旦用到the，表示谈话的俩人都知道说的谁、说的啥。如：
The dog is sick. 狗狗病了。(双方都知道是哪一只狗) (3) 前面提到过的，后文又提到。如： There is a cat in the tree.Thecat is black. 树上有一只猫，猫是黑色的。 (4) 表示世界上唯一的事物。如： The Great Wall is a wonder.万里长城是个奇迹。(5) 方位名词前。如： thenorth of the Yangtze River 长江以北地区 (6) 在序数词和形容词最高级的前面。如： Who is the first?谁第一个? Sam is the tallest.山姆最高。 但是不能认为，最高级前必须加the，如： My best friend. 我最好的朋友。 (7) 在乐器前。如： play the flute 吹笛子
Way的用法
Way用法 A:I think you should phone Jenny and say sorry to her. B:_______. It was her fault. A. No way B. Not possible C. No chance D. Not at all 说明：正确答案是A. No way，意思是“别想！没门！决不！” 我认为你应该打电话给珍妮并向她道歉。 没门！这是她的错。 再看两个关于no way的例句： （1）Give up our tea break? NO way! 让我们放弃喝茶的休息时间？没门儿！ （2）No way will I go on working for that boss. 我决不再给那个老板干了。 way一词含义丰富，由它构成的短语用法也很灵活。为了便于同学们掌握和用好它，现结合实例将其用法归纳如下： 一、way的含义 1. 路线
He asked me the way to London. 他问我去伦敦的路。 We had to pick our way along the muddy track. 我们不得不在泥泞的小道上择路而行。 2. （沿某）方向 Look this way, please. 请往这边看。 Kindly step this way, ladies and gentlemen. 女士们、先生们，请这边走。 Look both ways before crossing the road. 过马路前向两边看一看。 Make sure that the sign is right way up. 一定要把符号的上下弄对。 3. 道、路、街，常用以构成复合词 a highway（公路），a waterway（水路），a railway（铁路），wayside（路边）
way与time的特殊用法
way/time的特殊用法 1、当先行词是way意思为”方式.方法”的时候,引导定语从句的关系词有下列3种形式： Way在从句中做宾语 The way that / which he explained to us is quite simple. Way在从句中做状语 The way t hat /in which he explained the sentence to us is quite simple. 2、当先行词是time时,若time表示次数时,应用关系代词that引导定语从句,that可以省略; 若time表示”一段时间”讲时,应用关系副词when或介词at/during + which引导定语从句 1.Is this factory _______ we visited last year? 2.Is this the factory-------we visited last year? A. where B in which C the one D which 3. This is the last time _________ I shall give you a lesson. A. when B that C which D in which 4.I don’t like the way ________ you laugh at her. A . that B on which C which D as 5.He didn’t understand the wa y ________ I worked out the problem. A which B in which C where D what 6.I could hardly remember how many times----I’ve failed. A that B which C in which D when 7.This is the second time--------the president has visited the country. A which B where C that D in which 8.This was at a time------there were no televisions, no computers or radios. A what B when C which D that