Heavy Metal Adaptation Advanced article · 2011. 2. 25. · plants in the presence of copper and...

Heavy Metal AdaptationLuis Rafael Herrera-Estrella, Laboratorio Nacional de Genómica para la Biodiversidad,

CINVESTAV Campus Guanajuato, Irapuato, Guanajuato, Mexico

Angel Arturo Guevara-Garcı́a, Universidad Nacional Autónoma de México, Cuerna-

vaca, Morelos, Mexico

Based in part on the previous version of this Encyclopedia of Life Sciences(ELS) article, Heavy Metal Adaptation by Luis Rafael Herrera-Estrella,Angel Arturo Guevara-Garcı́a and José López-Bucio.

Fifty-three chemical elements fall into the category of heavy metal (HM) defined as the

group of elements with densities higher than 5 g cm23. In an ecological sense, any metal

or metalloid that causes environmental pollution or that cannot be biologically

degraded and is therefore bioaccumulated, should be considered as an HM. HMs are

natural components of the Earth’s crust, but as a consequence of human activities, in

many ecosystems the soil concentration of several HMs has reached toxic levels. The

adaptative responses of living organisms to HM contamined environments are efficient

processes that include genetical, physiological, anatomical and ecological traits highly

conserved in nature. These traits give certain species the ability to survive in toxic

concentrations of HM elements. Actually, using multidisciplinary strategies including

genomic, genetic, molecular, biochemical, physiological, ecological and agronomic

approaches, substantial progress is being made to elucidate the mechanism involved in

HM adaptation.

Introduction

Heavy metals have been defined as the group of elementsthat have densities higher than 5 g cm23, and 53 chemicalelements fall in this category.However, according physical,chemical or toxic properties, several definitions have beenproposed to describe a heavy metal. Interestingly, the con-cept can include elements lighter than carbon, but excludessome of the heaviest metals. In an ecological scenario, aheavy metal could be considered as any metal or metalloidelement that causes environmental pollution, that does nothave any vital function and is toxic at low concentrations(such as lead and mercury), that has a vital function but isharmful to the organisms in high concentrations (such ascopper and molybdenum) or that cannot be biologicallydegraded and tends to bioaccumulate. Wherever heavymetals are defined, living species must actively respond toprotect themselves from metal poisoning in contaminatedsites. See also: Plant Macro- and Micronutrient Minerals

Heavy metals are natural components of the Earth’scrust mainly localized as disperse components in rock for-mations. Few ecosystems present natural heavy metal tox-icity, such as aluminium, iron and manganese toxicity inacid soils. However, in a considerable number of places,heavy metal concentrations in the soil increase to toxiclevels through agriculture, manufacturing and mining orindustrial waste disposal practices. Recent findings suggestthat the adaptive response of living organisms to contam-inated environments can be a rapid and efficient process.Insights into the mechanisms involved in heavy metal ad-aptation are beginning to be obtained. See also: Adapta-tion and Natural Selection: Overview

Differences in Ecology

Soil properties and the adaptive response

The distribution of living organisms in ecosystems affectedby heavy metal toxicity depends not only on the kind,combination and concentration of metals, but also on en-vironmental factors such as soil chemistry, heterogeneityand, in particular, nutrient status (principally nitrogen,phosphorus and potassium).Gradients of soil conditions often determine areas of

increasing stress or disturbance that impose a strong se-lective pressure on the colonizing species. During evolu-tion, adaptations have developed in a very wide range oforganisms from all major taxonomic groups. This is

Advanced article

Article Contents

. Introduction

. Differences in Ecology

. Colonizers of Specific Sites

. Mechanisms of Metal Uptake, Exclusion, Transport andStorage

Online posting date: 15th March 2009

ELS subject area: Plant Science

How to cite:Herrera-Estrella, Luis Rafael; and, Guevara-Garcı́a, Angel Arturo (March

2009) Heavy Metal Adaptation. In: Encyclopedia of Life Sciences (ELS).John Wiley & Sons, Ltd: Chichester.

DOI: 10.1002/9780470015902.a0001318.pub2

ENCYCLOPEDIA OF LIFE SCIENCES # 2009, John Wiley & Sons, Ltd. www.els.net 1

reflected in the high number of species of some taxa thathave become endemic in sites polluted with heavy metals.In each case, these organismshave the ability to survive andgrow in the presence of potentially toxic concentrations ofheavy metals. See also: Natural Selection: Responses toCurrent (Anthropogenic) Environmental Changes; PlantStress Physiology

Acid soils of pH 5.5 and lower, which significantly limitcrop production worldwide, deserve special attention withrespect to the heavy metal toxicity. The acid soils problemincludes toxic levels of aluminium, iron and manganese, aswell as deficiencies of several essential mineral elements,principally phosphorous. Approximately 30% of theworld’s total soil and 50% of potentially arable land areacid soils. For this reason, revealing the biochemical mech-anisms and gene products involved in plant tolerance toacid soils, has been the objective of many laboratoriesaround the world. See also: Soils and Decomposition

Heavy metal effects on natural populations

Toxicmetals are believed to be reducing the abundance anddiversity of species, as well as selecting for proliferation ofresistant/tolerant populations. Several studies have shownthe high specificity of species distribution in metal-poisoned ecosystems. In fungi, the reduction in numberof species has been noted in soils polluted with copper,cadmium, lead, arsenic and zinc. In plants, the formationofendemisms has been documented in different ecosystemsaround theworld, for example in the tropical island ofNewCaledonia (around 16 000 km2), where serpentine soilscontaining toxic levels of nickel, chromium, cobalt andmanganese, cover about one-third of the island’s surface.These typically infertile soils contain a native flora of ap-proximately 1500 species comprising woody perennials,epiphytic orchids and Cyperaceae growing in xerophyticscrub at low altitude. One of the endemic species fromNewCaledonia is the shrub Maytenus founieri, which is one ofthe nine manganese-hyperaccumulators plant species rec-ognized worldwide (Morat et al., 1986; Fernando et al.,2008). In the copper region ofUpper Shaba in Zaire, whichcomprises about 100 copper–cobalt ore deposits totallingsome 20 km2, disseminated in a metallogenic province, anumber of distinct metalliferous habitats exist, such ascopper clearings and natural isolated copper hillocks.These areas bear specific plants, notably small annual herbsand grasses. About 220 taxa are present on these outcrops,of which 42 are endemic. Some of these are located on asingle hillock (Malaisse, 1983). In themining area of Stolb-erg (South Aachen, Germany), Betula trees grow well onhills of mining smelter ash which have a total lead contentof up to 10–20 g kg21. In each of these cases, the diversity ofplant species is severely restricted, and often edaphic eco-types, which are tolerant to the specific metal present inexcess in the soil, are selected rapidly. Endemisms are typ-ical in areas polluted for fewer than 20 years.Other habitatswhere adaptation takes place rapidly are beneath

galvanized fences and on roadsides. See also: Environ-mental Heterogeneity: Temporal and Spatial

Symbiosis events in contaminatedenvironments

Unusual ecological associations can be found amongpolluted habitats. In temperate forest ecosystems thefine roots of the majority of trees are colonized by mi-corrhyzal fungi and there are some examples that suggest acausal role of ectomycorrhizal symbiois to amelioratemetal phytotoxicity. Additionally, inoculation of coniferroots withPisolithus tinctorious improved seedling survivalon extreme acid soils with high levels of metals. In erica-ceous plants, little or no growth occurs in mycorrhiza-freeplants in the presence of copper and zinc; however, twospecies of ectomycorrhizal fungi, Amanita muscaria andPaxillus involutus, increased zinc tolerance of Betula sp.From these examples, it was suggested that the hyphalcomplexes of the mycorrhizal fungi bind metals, thus pre-venting metal translocation to the plant and the resultingtoxic symptoms (Bradley et al., 1982).See also:MutualisticSymbioses; Nutrient Acquisition, Assimilation andUtilizationArbuscular mycorrhizal fungi (AMF) are found in the

soil of both polluted and nonpolluted ecosystems. In pol-luted soils, it has been demonstrated that plant-AMF as-sociations help to alleviate plant heavy metal stress. Insome cases mycorrhizal plants show enhanced heavymetaluptake and root to shoot translocation called phytoex-traction. In other cases AMF drive soil heavy metal im-mobilization, called phytostabilization. The significanceand efficiency of each heavy metal exclusion mechanismprobably vary between different plant–fungal interactions.However, Glomus intraradices, specifically the isolate Br1,has been repeatedly reported to confer heavy metal toler-ance to several plants including important crop speciessuch asZea mayz,Medicago truncatula, Solanum licopersi-cum and Pisum sativum (Hildebrant et al., 2007). Synthesisand secretion of glomalin, an isoluble glycoprotein thatbinds heavymetals, is one of the strategies thatAMFuse toprotect themselves against heavy metal stress. See also:MycorrhizaBesides their important roles in maintenance of soil

structure and recycling plant nutrients, soil microbes alsohave been recognized as able to impact soil heavy metalbioavailability with regard to heavy metal content. Fromthe rhizosphere of Elshotia splendes, a copper-tolerantplant living on coppermining wastes, some bacteria strainswere identified as involved in metal solubility and hyper-accumulation (Chen et al., 2005). Moreover, rhizospherebacteria increased the uptake of cadmium inBrassica napusand nickel in Alyssum murale. With respect to the biore-mediation of ecosystems exposed to heavy metals, thesedata suggest that it is very important paying attention tothe dynamics of rhizosphere microorganisms (Jian et al.,2008). See also: Rhizosphere

Heavy Metal Adaptation

ENCYCLOPEDIA OF LIFE SCIENCES # 2009, John Wiley & Sons, Ltd. www.els.net2

Colonizers of Specific Sites

The ability of certain bacterial, fungal and plant species tocolonize environments polluted by heavy metals has beenwidely described. Metal-tolerant anaerobic bacteria iso-lated from contaminated habitats use toxic metals (such asselenium and chromium) as electron acceptors. The bac-terial generaThiobacillus,Streptomyces,Streptococcus andCaulobacter have been isolated from sites polluted by mer-cury ions. In these cases, the mechanism of detoxificationseems to involve the intracellular reduction of toxic forms(Hg2+) to the less toxic, relatively inert metallic form (Hg0)by the activity of specific enzymes called mercury re-ductases. The accumulation of cadmium inCitrobacter anduranium inPseudomonashas beendemonstrated. Lead andcadmium soil solubilization by Burkholderia strains hasalso been reported (Jian et al., 2008). See also: Ecophysio-logical Responses of Plants to Air Pollution

In the river Rio Tinto in Spain, which has an exception-ally low pH (around two) and high concentrations ofheavy metals including iron, arsenic, copper, zinc, lead,titanium barium, vanadium, chromium, cobalt, cadmiumand nickel, almost 1300 bacterial species have beencollected. The bacterium Thiobacillus ferrooxidans andLeptospirillum ferrooxidans are especially abundant and,surprisingly, patches of algae and masses of filamentousfungi living together with several species of yeast and pro-tist have also been found in the Rio Tinto (Ariza, 1998;Costas, et al., 2007). Interestingly, molecular studiesshowed a higher diversity of eukaryotic than prokaryoticorganisms living in this habitat. Approximately 60%of thetotal biomass is represented by eukaryotic microalgae re-lated to the neutrophilic species, such as Dyctiosphaeriumchlorelloides. Owing to the predominance of eukaryoticmicroalgae in this extreme acidic environment, it has beensuggested that, in an evolutionary timescale, the adapta-tion to this kind of conditions must be relatively fast.

In soils with highly toxic levels of copper and zinc, thefungi Geomyces and Paecilomyces have been found to bethe predominant genera, whereas Penicillium and Oidio-dendron spp. decline significantly. In samples taken fromanorganomercurial-treated golf green, the species Trichocla-dium asperum, Trichoderma hamatum, Zygorrhynchus mo-elleri and Chrysosporium pannorum are found frequently,whereas the generaChaetomium,Fusarium,Penicillium andPaecilomyces are greatly reduced. Strobilurus tenacellus,Mycena ammoniaca andArmillaria lutea are the most com-mon species of basidiomycetes present in soil contaminatedwith cadmium dust.

Although some Penicillium spp. are sensitive towardsheavy metals, one of the best examples of fungal toleranceis found in this genus, which underlines the fact that metalresponsesmay be strain-specific. For examples,Penicilliumlilacinum comprises 23% of all fungi isolated from soilpolluted by mine drainage, and Penicillium ochrochloron iscommonly present in industrial effluents. Recently, frommangroves and salterns on coastal waters of Goa, India, atleast 12 halotolerants Penicillium spp. have been isolated,

that exhibit lead, copper and cadmium tolerance(Marbaniang and Nazareth, 2007). See also: DissimilatoryMetal Transformations by Microorganisms; LichensIn the plant kingdom, it seems clear that tolerance has

arisen independently in the full spectrum of families. It iscommon to find species of Gramineae, Caryophyllaceae,Lamiaceae and Fabaceae widely distributed among heavy-metal rich ecosystems. Some plant species that have theability to accumulate 100 times higher concentrations ofmetal or metalloid elements in their shoot biomass thanother species growing in the same soil, are named hyperac-cumulators. The hyperaccumulation trait is found in 45different plant families, with the highest occurrence amongBrassicaceae, in which the genera Alyssum (50 species) andThlaspi (about 20 species) have been the most widely char-acterized (Figure 1). Interestingly, two fern species, Pterisvittata (Figure1) andSedumalfredii, have alsobeen identifiedas arsenic and zinc/cadmium hyperaccumulators, respec-tively (Wu et al., 2007). At least 418 hyperaccumulatingplant taxa have been described, of which 76% (318) accu-mulate nickel and some such as Thlaspi caerulescens andThlaspi goesingense, can hyperaccumulate multiple metals.Arabidopsis halleri (originally Cardaminopsis halleri) is azinc-hyperaccumulator with populations occurring in anumber of metal-contaminated sites across central Europe.This plant specie has been used in genetic analysis of metalhyperaccumulation and tolerance. Interestingly, Arabido-psis halleri is closely related to Arabidopsis thaliana and across-species transcriptome-global scale analysis has beensuccessfully used to identify genes involved in cellular metaluptake and detoxification (Idris et al., 2004; Ueno, et al.,2008).Native vegetation fromacid soils of the humid tropicscan grow in the presence of concentrations of aluminiumthat are toxic to species not associated with such habitats.

Figure 1 Plant heavy metal hypperaccumulators. (a) Allysum murale a nickel

hyperaccumulator. (b) Thlaspi caerulescens a cadmium hyperaccumulator. (c)

Stanleya pinnata a selenium hyperaccumulator. (d) Pteris vittata a fern arsenic

hyperaccumulator. Images taken from http://images.google.com, using

specie names as probe. Images reproduced with the permission from the

Ondrej Zicha.



Thus, organisms can evolvemechanisms to copewith excesslevels of heavy metals in their environment.

Interestingly, hyperaccumulating plants have not onlyevolved a mechanism to live in toxic concentrations ofheavy metals, but hyperaccumulation may also be func-tioning as a strategy to prevent predation. As examples, thenickel-rich leaves of Strephantus polygaloides prevent thedevelopment of the herbivorous larvae of Pieris rapae andavoid the growth of the phytopathogenic bacteriumXanthomonas campestris. A repellent effect of the plantsap from Sebertia acuminata (25% nickel dry weight) wasobserved on the fruit fly Drosophila melanogaster (Sagneret al., 1998). Stanleya pinnata (Figure 1) is a selenium hyper-accumulator which uses this trait to protect itself againstorthopterans herbivory and to increase its long-term sur-vival rate (Freeman et al., 2007).

Recently, the defensive effects of eight heavy metalsagainst the plant pest Plutella xylostella larvae have beendemonstrated. Thesemetals: copper, zinc, lead, chromium,cobalt, cadmium, manganese and nickel, are often accu-mulated by plants. Unexpectedly five of these metals (cad-mium, manganese, nickel, lead and zinc) were toxic toPlutella xylostella below accumulator levels, and three ofthem (cadmium, lead and zinc) were toxic at near normalrange concentrations. These findings suggest that metalhyperaccumulation defence mechanisms against herbi-vores may be widespread among plants (Jhee et al., 2006).

Mechanisms of Metal Uptake,Exclusion, Transport and Storage

Uptake of some metal solutes from the soil environmentmay occur through a carrier-mediated system. Alterna-tively, as in the case of cations, metal uptakemay be largelydriven by the negative potential across the plasma mem-brane, which is generated in part by proton extrusion me-diated by membrane-bound H+ ATPases (adenosinetriphosphatase). In fact, the first cell structure exposed toheavy metals is the plasma membrane, where H+ ATPasesgenerate the driving force (proton electrochemical gradi-ent) thatmediatesmetal uptake (or exclusion) from the soil.The plasma membrane H+ ATPase activity is finely reg-ulated by reversible phosphorylation in response to manyendogenous and exogenous signals, including heavy metalexposure. In plants, the mechanism of detoxification ofheavymetals implies their chelation anddeposition into thecentral vacuole. Metal transport into the vacuole is medi-ated by a tonoplast (vacuolar membrane) H+ ATPase,some times referred as ‘eco-enzyme’ due to its ability toalter its activity in response to environmental stresses.Plants use several strategies to assimilate essential metals,while at the same time, preventing metal toxicity. Plantgenomes encoded large families of metal transportersthat coordinate their activities to maintain metal home-ostasis. Transporters involved in metal efflux include theP1B-ATPase family and the cation diffusion facilitator

(CDF) family. Other transporters families involved inmetal-uptake are the natural resistance-associated ma-crophage protein (NRAMP), the ZIP (zinc–iron-regulatedtransporter) proteins and the plant-specificYellowStripe1-like (YSL) of metal uptake transporters (Kabaza et al.,2008). See also: ATPases: Ion-motive; Cell Membranes:Intracellular pH and Electrochemical PotentialBiological exudates, including microbial siderophores

and analogous compounds from plants termedphytosiderophores, are known to take part in the mobili-zation and differential uptake of certain elements. In thisway, molybdenum and copper have been shown to formstrong complexes with this class of molecules, facilitatingtheir uptake. In the majority of pathogenic and nonpath-ogenic bacteria and fungi, high-affinity iron uptake is me-diated by siderophores. Understanding the mechanisms ofsynthesis, secretion, metal scavenging and siderophore-mediatedmetal uptake, has been theobjective of the severalresearch groups. The phytosiderophores are produced byGraminaceous plants in response to iron deficiency, as partof a phytosiderophore-mediated iron uptake system. Thissystem is also effective for the transport of other metalsincluding zinc, copper, nickel and cadmium, suggesting itsparticipation in preventing heavy metal toxicity.Once in the cell, the organism must balance the intracel-

lular concentrations of potentially toxic metals. In someprokaryotes, animals and fungi, a class of small cysteine-rich metal-binding proteins named metallothioneins play arole of primary importance in metal compartmentalizationand tolerance. Animal metallothionein genes have beenused to produce heavy-metal tolerant transgenic plants. Inplants, another class ofmetal-binding ligands named phyto-chelatins has been described. These plypeptides are rich inglutamine and cysteine residues and probably act by pro-tecting sensitive enzymes by sequestering heavymetals, suchas cadmium, lead and zinc. Metallothioneins are polypep-tides encoded by small gene families. By contrast phytoche-latins are a family of peptides enzymatically synthesized.Although initially it was considered that metallothioneinswere present only in animals and fungi and phytochelatinsonly in plants, now it is clear that both types of metal-bind-ing polypeptides are expressed in some organism. Both an-imals and plants containmetallothionein and phytochelatinsynthases genes in their genomes. In any case, both cysteine-rich polypeptides have evolved as a mechanism to controlthe uptake and accumulation of heavy metals. Originally,phytochelatins were thought to only be involved in intra-cellular detoxification, transported and confining phytoche-latin-heavy metal complexes into the vacuole. Recently ithas been shown in Arabidopsis that phytochelatins are alsoimplicated inbothacropetal andbasipetal heavymetal long-distance transport (Mendoza-Cózatl et al., 2008). Heavymetal detoxification mediated by phytochelatins is an im-portant resistance mechanism in plants, yeast and protists.The principal evidence for the function of phytochelatins inheavymetal detoxification comes from the characterizationof Arabidopsis thaliana and Schizosaccharomyces pombemutants, affected in the phytochelatin synthase gene. These



mutants are highly sensitive to cadmium and arsenate con-centrations that do not affect their wild-type counterparts.The study of metallothioneins in plants has been difficult;however, when expressed in yeast and Synechococcus met-allothionein-deficient strains, the Arabidopsis thalianamet-allothioneins are able to confer copper and zinc tolerance,showing that plant metallothioneins have the biologicalfunction of confering metal tolerance (Grill et al., 1987;Cobbett and Goldsbrough, 2002). See also: Plant StressPhysiology; Transgenic Plants

In addition to the mechanisms that confer heavy metaltolerance, the majority of organisms that inhabit metallif-erous soils are known to exclude toxicmetals, preventing itsentry into the organism. The production of extracellularpolysaccharides and the excretion of chelating substances,such as organic acids by microbial and plant species, par-ticipate in the immobilization of such toxic elements. An-other mechanism exploited by bacteria is the accumulationof metals as cell-bound metal-phosphate compounds.Hyperaccumulators plants have gained considerable atten-tion owing to their potential use in biorecovery of contam-inated sites, but really little is known about the molecular,biochemical and physiological processes that result in thehyperaccumulator phenotype. However, low-molecularweight chelators, including some amino acids and organicacids (e.g. citric acid), have been shown to participate in thetransport, compartmentalization and detoxification mech-anisms. In hyperaccumulating plants only a small fractionof themetal is present as free ions; therefore, themajority ofions must be bound to low molecular mass ligands or toproteins. There are many metal-binding biomolecules thathave been reported to be involved in sequestering, trans-porting or storing heavy metals in hyperaccumulators spe-cies. Examples of plant ligands are mugineic acid,nicotinamine, citric acid and histidine. In yeast, it has beenreported that the intracellular levels of copper are control-led by chaperones that deliver the metal to specific com-partments. With respect to hyperaccumulators plants, thegenome-wide transcription analysis performed in Arabido-psis hallerideserves specialmention, such as this studymadepossible the identification of a set of novel genes involved inmetal hyperaccumulation and tolerance (Talke et al., 2006).See also: Bioremediation

In the genus Alyssum, in which the nickel concentrationcan reach 3% of leaf dry biomass, the tolerant response cor-relates with the increase in the levels of free histidine. Thesupply of this amino acid to a nonaccumulating speciesgreatly increases both nickel tolerance and transport to theshoot, confirming the role of histidine in metal tolerance(Krämer et al., 1996). Interestingly, the transcription levels ofseven of the eight genes implicated in histidine biosynthesiswere higher in the hyperaccumulatorArabidopsis lesbiacum,when compared with the nonaccumulator Arabidopsismontanum. Moreover, overexpression of the first histidinebiosynthesis enzyme (ATP-phosphoribosyltransferase) intransgenic Arabidopsis thaliana, results in nickel tolerance(Ingle et al., 2005). See also: Plant Cell: Overview

Proline has also been associated with heavy metal tol-erance. This amino acid has been extensively studied in thecontext of plant responses to salinity, water deficit, hightemperature and heavy metal stresses. Proline accumula-tion is heavy metal inducible in several plant species andthe metal-tolerant populations of three different species(Armeria maritima, Deschampsia cespitosa and Silene vul-garis) have higher proline levels, when compared with theirnontolerant relatives. Moreover, higher proline produc-tion has been demonstrated to drive metal tolerance intransgenic Chlamydomonas reinhardtii (Siripornadulsilet al., 2002). The function of proline in heavy metal tol-erance is unclear, but apparently could be related to metaltrapping or the scavenging free radicals induced by redoxmetals, such as cadmium, zinc, copper and mercury.In barley (Hordeum vulgare) a heavy metal inducible re-

ceptor-like protein kinase was recently identified. Interest-ingly, this receptor is alsoupregulatedduring leaf senescence,supporting the idea of an overlapping mechanism control-ling heavymetal homeostasis and leaf senescence. Receptor-like protein kinases perform important functions in theperception and transduction of extra- and intracellular sig-nals, but never before have any been associated with heavymetal stress response (Ouelhadj et al., 2007).The accumulation of organic acids, such as citric, iso-

citric, oxalic, tartaric, malic, malonic and aconitic, in rootsand leaves of metal-tolerant plants has been reported, im-plying that these organic acids have an important role inmetal hyperaccumulation. Formation of anionic, or un-charged, zinc–citrate complexes resulted inmore zinc pass-ing through the excised stemofPinus radiata aswith copperin Papyrus stems. In response to zinc, the roots of Arab-idopsis halleri showed an increase in the organic acids level.These complexes positively affect transport in xylem byreducing adsorption to the vessel walls and by decreasingthe rate of lateral escape. A causal relationship betweenorganic acid accumulation and metal tolerance has evenbeen proposed. This is well established for nickel-accumu-lating plants, which complex nickel with malate, malonateand citrate. In the latex of Serbetia acuminata, the mostextreme nickel-hyperaccumulating tree from New Caledo-nia,which contains approximately 25%nickel by drymass,most of the metal is bound to citrate. Apparently the or-ganic acids bind to heavy metals in the cytoplasm and thiscomplex is finally accumulated in the vacuole. However, itis likely that organic acids do not bind metal ions stronglyenough to function as long-distance transporters. See also:RhizosphereAnother way in which organic acids may confer metal

tolerance has been found in some aluminium-tolerant cult-ivars of snapbean, maize and wheat. In these plants, analuminium-resistant genotype correlates with the exuda-tion of citrate and malate. It has been shown that organicacids prevent metal toxicity by chelating the aluminiumions in the rhizosphere. The finding that the exogenousaddition of organic acids to toxic aluminium solutionsabates aluminium toxicity in the roots supports this con-clusion. According to the classical definition, aluminium



cannot be considered aheavymetal; however, as thatmetaldoes not have any physiological role, its bioaccumulationcause toxicity, which is a characteristic of heavy metals,irrespective of their atomic mass or density. Plants havetwo different classes of aluminium tolerance mechanisms;one is to exclude themetal from the root apex apoplast, andthe other to tolerate the aluminium accumulation in thecytoplasm. Interestingly, both of these mechanisms implythe participation of organic acid (malate, citrate or oxal-acetate) as chelating agents to sequester the metal. Inwheat, it has been shown that aluminium-tolerant geno-types exudate an aluminium-binding ligand (malate) thatcomplexes with the metal in the rhizosphere preventing itsentry into the root apoplast and symplast. In fact, in severalmonocot and dicot plant species, high levels of aluminium-activated organic acid exudation has been correlated withaluminium-tolerance, which strongly supports that or-ganic acid exudation is an important mechanism for heavymetal tolerance. Even more convincing is the fact that to-bacco and papaya transgenic plants expressing a bacterialcitrate synthase gene and overproducing and overexudingcitrate can grow in toxic concentration of aluminium, pre-sumably by a metal exclusion mechanism (de la Fuenteet al., 1997). In wheat, the almt1 gene that confers alumin-ium tolerance, which mediates the aluminium-activatedexcresion of malate has been recently cloned. Thealmt1 gene encodes a malate transporter, which activity isactivated by aluminium (Sasaki et al., 2004). See also:Adaptation: Genetics

Another significant metal toxicity in acid soils is man-ganese, which causes stunted growth, chlorosis and necro-tic lesions in the leaves. The physiological mechanismsconferring manganese toxicity (and tolerance) have notbeen elucidated; however, it has been suggested that man-ganese-induced oxidative stress could be the cause of itstoxicity. With respect to manganese tolerance, plant genesthat encode Mn2+ transporters have been identified,which, when overexpressed in transgenic plants, confermanganese tolerance through metal accumulation in bothwhole plant and tonoplast vesicles. Apparently this trans-porter has a broad substrate range for divalent cations andmay be implicated in tolerance to other heavy metals bysequestering them into the vacuole. See also: PlantVacuoles

The strategies for survival of heavy metal stress are di-verse. Perhaps a consequence of its sessile nature, plants inparticular have evolved several mechanisms to contendwith and to survive in heavy metal contamined habitats,which are summarized in Figure 2. Research to understandthese mechanisms and their interactions is currently beingconducted by many laboratories around the world. Thisinformation may eventually contribute to bioremediationefforts to clean-up polluted ecosystems.

Perspectives

Hyperaccumulator plants have been used as ‘indicators’ formetallic deposits for hundreds of years. Their commercial

importance formetal prospecting is indisputable.The shrubHybanthus floribundus is used as an indicator for nickel inAustralia. A wild variety of the plant Impaticus balsamina,found on lead–zinc metal dumps in India, is regarded as alocal bioindicator for these metals. In the lichen group, thespeciesCladonia convoluta, which accumulates high copperconcentrations in its tissues, has become a tool for biogeo-chemical prospecting and is used to locate mining areas(Aery and Tiagi, 1986). Erogonium inflatum (desert trum-pet), Oenothera caespitosa and some species of the genusAstragalus (Astragalus pattersoni, Astragalus presussi,Astragalus thompsonae) have been successfully used as in-dicator plants in uranium prospecting.The mechanism of metal hyperaccumulation is the ob-

ject of intensive investigations, especially with regard to theecological exploitation of such plants. Gradual depletionof ecosystems encourages researchers not only to investi-gate the natural response of organisms living on contam-inated sites, but also to suggest strategies for environmentalclean-up. The lack of approaches for heavy-metal remedi-ationhas created amajor need for the development of novelstrategies. Current research focuses on the identificationof novel players in metal tolerance, employing post-genomic approaches such as metabolic profiling, globaltranscriptome and proteomic analysis and metagenomicssequencing. Additionally, mutants need to be checkedroutinely for altered metal sensitivity. It would not be sur-prising if the list of genes and gene products involved inmetal homeostasis and heavy metal defence expand in thenear future. The applications of genetic engineering havebeen shown to be highly successful in obtaining heavy-metal resistant species. Transgenic plants tolerant tomercury, cadmium, aluminium, arsenic, lead, zinc andselenium have already been generated. With regard tomercury and arsenic tolerance, an elegant two-transgenicapproach was designed.The success of plant transgenesis to induce heavy metal

tolerance, could suggest that a single mechanism seems tobe sufficient to control heavy metal stress. However, theexperimental evidence strongly suggests that there is not asingle mechanism that can account for tolerance to a widerange of metals. Tolerance to each metal involves metal-specific mechanisms. Cotolerance is not a widespread phe-nomenon and in some cases several mechanisms operatesimultaneously to control toxicity of a particular metal(Hall, 2002). See also: Transgenic PlantsHowever, the efficiency of the transgenic strategy for en-

vironmental clean-up remains to be demonstrated on con-taminated soils. It is clear that heavy metal tolerance is amultifactor phenomenon, therefore altering the plant me-tabolism by transgenesis could result in additional benefi-cial or detrimental effects. Two reported examples help toillustrate both cases. In the first, citrate overproduction intransgenic tobacco plants resulted in aluminium tolerance,but also in a better phosphorus uptake in alkaline soil whichhas low-soluble phosphorus content, and the developmentof wild type plants was severely restricted (López-Bucio,et al., 2000). In the second example, the overexpression of



the Escherichia coli arsenate reductase (ArsC) gene intransgenic Arabidopsis thaliana plants, resulted not in ar-senic resistance, but in a hypersensitive phenotype, appar-ently due to the high affinity of arsenite for protein thiolgroups, and the consequent depletion of the cellular glutati-one pool (Dhankher et al., 2002).

Although there has been some success with heavy-metalresistant plants, the fact that we do not completely under-stand the limiting factors in increasing uptake, transloca-tion and tolerance to toxic chemical elements, makes thecontinuation of fundamental and applied research neces-sary. Substantial progress to elucidate the basis of thehomeostasis, hyperaccumulation and detoxification ofmetals in plants and microorganism has been made in re-cent years, but much work remains to be done. Improvedgenetic engineering strategies need to be devised. Powerful‘omics’ strategies have already been initiated. In particularionomics strategies deserve special mention. Ionomics in-volves the comparison of the mineral and trace elementcomposition (ionome) of an organism in response to phys-iological stimuli, developmental state or genetic modifica-tions. So, the study of the ionome in response to heavymetal stress or inmutants affected in heavymetal tolerance,will surely make important contributions to reveal genesand gene networks regulating the heavy metal responses(Salt, et al., 2008). See also: Bioremediation

In addition, hyperaccumulator species are being evalu-ated with respect to potential use as molecular geneticmodel systems. All this should eventually make it possible

to overcome, at low monetary and environmental cost, theproblem of heavy metal contamination. Finally, it is veryimportant tokeep inmind that the eventual development ofany safe environmental clean-up technology must be sup-ported by multidisciplinary, labour-intensive research fo-cused on the underlying process at the genetic, molecular,biochemical, physiological, ecological and agronomiclevels.

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HM

4

2

Glo

mal

inAr

busc

ular

myc

orrh

izal

Glomalin-HM

[HIGH HM]

[HIGH HM]

9HM

HSPsMTs

HM

HM

6

1

7

HM

Cytosol

H+

ATP

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PC-HM PC-HM-SVacuole

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heavy metal-contaminated paddy field soil and its potential in

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KabazaK, Janicka-RussakM, Burzy#skiM andKzobusG (2008)Comparison of heavy metal effect on the proton pumps of

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Penicillium species from Mangroves and Salterns and their re-

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long-distance transport of cadmiumand the effectof cadmiumon

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Sagner S, Kneer R, Wanner G et al. (1998) Hyperaccumulation,

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Sasaki T, Yamamoto Y, Katsuhara M et al. (2004) A wheat gene

enconding an aluminium-activated malate transporter. Plant

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cular mechanisms of proline-mediated tolerance to toxic heavy

metals in transgenic microalgae. Plant Cell 14: 2837–2847.

Talke IN, Hanikenne M and Krämer U (2006) Zinc-dependent

global transcriptional control, transcriptional deregulation,

and higher gene copy number for genes in metal homeostasis of

the hyperaccumulator Arabidopsis halleri. Plant Physiology

142: 148–167.

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Further Reading

Baker AJM (1987)Metal tolerance.NewPhytologist 106: 93–111.

Callahan DL, Baker AJ, Kolev SD and Wedd AG (2006) Metal

ion ligands in hyperaccumulating plants. Journal of Biological

Inorganic Chemistry 11: 2–12.

Colangelo EP andGuerinotML (2006) Put the metal to the petal:

metal uptake and transport throughout plants.Current Opinion

in Plant Biology 9: 322–330.

Cunningham SD, Berti WR and Huang JW (1995) Phytoreme-

diation of contaminated soils. Trends in Biotechnology 13:

393–397.

FordT andMitchell R (1992)Microbial transport of toxicmetals.

In: Mitchell R (ed.) Environmental Microbiology, pp. 83–101.

New York: Wiley-Liss.

Gadd GM (1993) Interactions of fungi with toxic metals. New

Phytologist 124: 25–60.

Jing YD,He ZL andYangXE (2007) Role of soil rhizobacteria in

phytoremediation of heavymetal contaminated soils. Journal of

Zhejiang University Science B 8: 192–207.

Kochian LV,HoekengaOA and PiñerosMA (2004)How do crop

plants tolerate acid soils? Mechanisms of aluminum tolerance

and phosphorous efficiency. Annual Reviews Plant Biology 55:

459–493.



KrämerU (2005) Phytoremediation: novel approaches to cleaning

up polluted soils.Current Opinon in Biotechnology 16: 133–141.

Krämer U, Talke IN and Hanikenne M (2007) Transition metal

transport. FEBS Letters 581: 2263–2272.

Mirete S, De Figueras CG and González-Pastor JE (2007) Novel

nickel resistance genes from the rhizosphere metagenome of

plants adapted to acid mine drainage. Applied Environmental

Microbiology 73: 6001–6011.

Sharma SS and Dietz KJ (2006) The significance of amino acids

and amino acid-derived molecules in plant responses and ad-

aptation to heavy metal stress. Journal of Experimental Botany

57: 711–726.

Zenk MH (1996) Heavy metal detoxification in higher plants – a

review. Gene 179: 21–30.



TRENDS in Plant Science Vol.6 No.6 June 2001

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273Review

http://plants.trends.com 1360-1385/01/$ – see front matter © 2001 Elsevier Science Ltd. All rights reserved. PII: S1360-1385(01)01961-6

21 Weichert, H. et al. (1999) Metabolic profiling ofoxylipins upon salicylate treatment in barleyleaves. FEBS Lett. 464, 133–137

22 Feussner, I. et al. (1996) Lipid-body lipoxygenaseis expressed in cotyledons during germinationprior to other lipoxygenase forms. Planta198, 288–293

23 Feussner, I. et al. (1997) Structural elucidation ofoxygenated storage lipids in cucumber cotyledons.J. Biol. Chem. 272, 21635–21641

24 Feussner, I. et al. (1995) Lipoxygenase-catalyzed oxygenation of storage lipids isimplicated in lipid mobilization duringgermination. Proc. Natl. Acad. Sci. U. S. A.92, 11849–11853

25 Höhne, M. et al. (1996) Lipid body lipoxygenasecharacterized by protein fragmentation, cDNAsequence and very early expression of the enzymeduring germination of cucumber seeds. Eur. J.Biochem. 241, 6–11

26 Hause, B. et al. (2000) Expression of cucumberlipid body lipoxygenase in transgenic tobacco.Planta 210, 708–714

27 May, C. et al. (2000) The N-terminal β-barrelstructure of lipid body lipoxygenase mediates itsbinding to liposomes and lipid bodies. Eur. J.Biochem. 267, 1100–1109

28 Hornung, E. et al. (1999) Conversion of cucumberlinoleate 13-lipoxygenase to a 9-lipoxygenating

species by site-directed mutagenesis. Proc. Natl.Acad. Sci. U. S. A. 96, 4192–4197

29 Feussner, I. et al. (1997) Do specific linoleate 13-lipoxygenases initiate β-oxidation? FEBS Lett.406, 1–5

30 Matsui, K. et al. (1999) Cucumber cotyledonlipoxygenase during postgerminative growth. Itsexpression and action on lipid bodies. PlantPhysiol. 119, 1279–1287

31 Noll, F. et al. (2000) Phospholipid monolayer of plant lipid bodies attacked by phospholipase A2 shows 80 nm holes analyzed by atomic force microscopy. Biophys. Chem. 86, 29–35

32 Balkenhohl, T. et al. (1998) A lipase specific for esterified oxygenated polyenoic fatty acids in lipid bodies of cucumber cotyledons. InAdvances in Plant Lipid Research (Sánchez, J. et al., eds), pp. 320–322, Secretariado de Publicaciones de la Universidad de Sevilla,Spain

33 Adlercreutz, P. et al. (1997) Vernonia lipase: aplant lipase with strong fatty acid selectivity.Methods Enzymol 284, 220–232

34 Huang, A.H.C. (1993) Lipases. In LipidMetabolism in Plants (Moore, J.T.S., ed.),pp. 473–503, CRC Press

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42 Millar, A.A. et al. (2000) All fatty acids are notequal: discrimination in plant membrane lipids.Trends Plant Sci. 5, 95–101

Aluminium (Al) is a light metal that makes up 7% ofthe earth’s crust and is the third most abundantelement after oxygen and silicon. Plant roots aretherefore almost always exposed to Al in some form.Fortunately, most of this Al occurs as harmless oxidesand aluminosilicates. However, when soils becomeacidic as a result of natural processes or humanactivities, Al is solubilized into the toxic trivalentcation, Al3+. Aluminium toxicity has been recognizedas a major limiting factor of plant productivity onacidic soils, which now account for ~40% of theearth’s arable land.

Micromolar concentrations of Al3+ can inhibit rootgrowth within minutes or hours in manyagriculturally important plant species1. Thesubsequent effects on nutrient and water acquisitionresult in poor growth and productivity. The molecularmechanisms underlying Al toxicity are not known,but because Al forms strong bonds with oxygen-donorcompounds2, it can interact with multiple sites in theapoplasm and symplasm of root cells. The binding ofAl with these substances is probably an importantfactor in its toxicity. Some plant species have evolvedmechanisms to tolerate Al stress, which helps them togrow on acid soils. Understanding the nature of thesetolerance mechanisms has been the focus of ongoingresearch in the area of stress physiology. Much of thecurrent evidence points to a central role for certainorganic acids that detoxify Al3+ by complexing thesecations in the cytosol or at the root–soil interface.

Organic acids detoxify Al3+ external to the rootOver a dozen Al-tolerant plant species are known tosecrete organic acids from their roots in response to Altreatment3,4. Citrate, oxalate and malate are some of

The aluminium cation Al3+ is toxic to many plants at micromolarconcentrations. A range of plant species has evolved mechanisms that enablethem to grow on acid soils where toxic concentrations of Al3+ can limit plantgrowth. Organic acids play a central role in these aluminium tolerancemechanisms. Some plants detoxify aluminium in the rhizosphere by releasingorganic acids that chelate aluminium. In at least two species, wheat and maize,the transport of organic acid anions out of the root cells is mediated byaluminium-activated anion channels in the plasma membrane. Other plants,including species that accumulate aluminium in their leaves, detoxifyaluminium internally by forming complexes with organic acids.

Aluminium tolerance in plants andthe complexing role of organic acidsJian Feng Ma, Peter R. Ryan and Emmanuel Delhaize

Jian Feng MaFaculty of Agriculture,Kagawa University,Ikenobe 2393, Miki-cho,Kita-gun, Kagawa 761-0795, Japan. e-mail: maj @ag.kagawa-u.ac.jp

Peter R. RyanEmmanuel DelhaizeCSIRO Plant Industry,GPO Box 1600, CanberraACT 2601, Australia.



274 Review

the commonly released organic acid anions that canform sufficiently strong complexes with Al3+ to protectplant roots (Fig. 1). Malate is released from the rootsof Al-tolerant cultivars of wheat (Triticum aestivum)5;citrate from Al-tolerant cultivars of snapbean(Phaseolus vulgaris)6, maize (Zea mays)7, Cassia tora8

and soybean (Glycine max)9; and oxalate frombuckwheat (Fagopyrum esculentum)10 and taro(Colocasia esculenta)11. Some plant species, such asAl-tolerant triticale (× Triticosecale Wittmack),rapeseed (Brassica napus), oats (Avena sativa), radish(Raphanus sativus) and rye (Secale cereale), releaseboth malate and citrate12–14. In some of these species,the increased secretion of organic acids by theseplants is localized to the root apex and depends uponthe presence of Al3+ in the external solution5,15. It isneither possible for all the Al3+ in soil to be detoxifiedby root exudates and nor is it necessary. The root apexis particularly sensitive to Al3+ (Ref. 16), thereforeonly the cations that immediately surround the apicalroot cells need to be detoxified. Therefore, it issensible to restrict the release of the organic acids tothis apical zone because it reduces the metabolic cost

of the Al-tolerance mechanism. Secretion needs tocontinue as the root apex moves through an acid soilto replace the organic acids that diffuse away from theroot or are broken down by microorganisms. We canimagine the organic acids forming a protective sheaththat shields the root apex from the toxic Al3+ cations.

Two patterns of organic acid secretion have beenidentified. In Pattern I, no discernible delay isobserved between the addition of Al and the onset oforganic acid release. For example, in wheat andbuckwheat, the secretion of malate or oxalate,respectively, was detectable within 15 to 30 min afterexposure to Al (Refs 5,15). In Pattern II, organic acidsecretion is delayed for several hours after exposureto Al3+. For example, in C. tora, maximal efflux ofcitrate occurs after 4-h exposure to Al (Ref. 8) and inrye, citrate and malate efflux increases steadilyduring a 10-h period14. In maize, it now appears thatAl might trigger both a rapid efflux of citrate as wellas a delayed release, which increases during a 48-hperiod (Refs 7,19). The rapidity of the Pattern Iresponse suggests that Al activates a pre-existingmechanism and that the induction of novel proteins isnot required3. In this case, Al might simply activate atransporter on the plasma membrane to initiateorganic anion efflux. By contrast, the delay observedin Pattern II-type secretion might indicate thatprotein induction is required. These induced proteinscould be involved in organic acid metabolism or in thetransport of organic acid anions (Fig. 2).

Ion channels mediate organic acid anion secretionFor organic acids to detoxify Al in the rhizosphere,they must be transported from the cytosol to theapoplasm. At the near-neutral pH of the cytoplasm,organic acids are almost entirely dissociated fromtheir protons and exist as organic acid anions. It isthese organic acid anions that are probablytransported out of the root cells. Although many typesof organic acids are found in root cells, only one or twospecific organic acids are secreted in response to Altreatment for any given species3,8 (Fig. 3). Thissuggests that a specific transport system for theorganic acid anions exist on the plasma membrane. Inwheat and maize, this transport system has beenidentified as an anion channel17–20 (Fig. 2). Anionchannels are membrane-bound transport proteinsthat allow the passive flow of anions down theirelectrochemical gradient. The large electrical-potential difference across the plasma membrane ofplant cells (inside negative) ensures that, in mostconditions, anions move from the cytosol to theapoplasm. Patch-clamp studies on protoplastsprepared from wheat roots showed that Al3+ activatesan anion channel in the plasma membrane that ispermeable to malate and chloride17,18. When thisresponse was compared in a pair of near-isogenicwheat lines that differed in Al tolerance at a singlegenetic locus, Al3+ was found to activate inwardcurrents (equivalent to anion efflux) that were both

TRENDS in Plant Science

(b)

(a)

No Al

AlCl3Al-Mal

Al-OxAl-Cit

No Al0.0

AlCl3 Al-Mal

Treatment

Al-Ox Al-Cit

0.5

1.0

1.5

2.0

Roo

t elo

ngat

ion

(cm

)

Fig. 1. Commonly secreted organic acids that detoxify aluminium (Al).Seedlings of an Al-sensitive wheat (cv. Scout 66), which secretes onlysmall amounts of malate in the presence of Al, were exposed to 0.5 mMCaCl2 solution (pH 4.5) containing either no aluminium or 10 µM of AlCl3,Al-malate (Al-Mal), Al-oxalate (Al-Ox) or Al-citrate (Al-Cit) at 1:1 ratio,respectively. (a) Root elongation during 20 h exposure. (b) Alaccumulation (pink color) stained by 0.1% solution of Eriochomecyanine R. The absence of colour indicates that the organic acid haschelated the Al and prevented its accumulation in the root apices. Thedifferent abilities of the organic acids to detoxify Al correlates with theirformation constants with Al.



275Review

larger and more frequent in protoplasts from theAl-tolerant genotype than the Al-sensitive genotype17.In a similar study on Al-tolerant maize that secretescitrate in response to Al treatment, Al was also foundto activate an anion channel on the plasmamembrane19. A second study on maize reported thatthe Al-activated anion channel is permeable to malateand citrate anions and occurs more frequently in anAl-tolerant genotype of maize than an Al-sensitivegenotype20. In addition, this study found that the Al-activated channel activity is restricted to cellslocalized in a narrow zone within the root apex.

It is not known how Al activates these anionchannels but three possibilities have been proposed21:• Al interacts directly with the channel protein to

trigger its opening.• Al interacts with a specific receptor on the

membrane surface or with the membrane itself toinitiate a secondary-messenger cascade which thenactivates the channel.

• Al enters the cytoplasm and activates the channeldirectly, or indirectly via secondary messengers(Fig. 2).

Recent evidence from maize has shown that Al is ableto activate the channel in isolated patches ofmembrane, indicating that secondary messengers areeither not required or are membrane-bound19. It isalso unclear what the differences are between theAl-tolerant and Al-sensitive genotypes that enable a large release of organic acid from the tolerantgenotype but little or none from the sensitive

genotype. There might be differences in the number ofchannel proteins in the membrane of each genotype,in their permeability to organic anions or in theiractivation by Al. Until either the channel proteins orthe genes encoding these channels have been isolated,we are reliant upon physiological approaches toanswer these questions. However, the important


OA OAOA OA

3

2

1

AnionchannelMetabolism

R

R

Pattern I Pattern IIIntermediate steps involving

soluble compounds

[Al3+:OA]

Al3+

[Al3+:OA]

Al3+

Al3+Al3+

Anionchannel

Outside pH 4.5

Cytosol pH 7

Proteinsynthesis

Geneactivation

Fig. 2. Models for the aluminium (Al)-stimulated secretion of organic acid anions (OA) from plant roots.For Pattern I-type responses, Al activates an anion channel on the plasma membrane that is permeableto organic acid anions. This stimulation could occur in one of three ways: (1) Al3+ interacts directly withthe channel protein to trigger its opening; (2) Al3+ interacts with a specific receptor (R) on the membranesurface or with the membrane itself to initiate a secondary-messenger cascade that then activates thechannel; or (3) Al3+ enters the cytoplasm and activates the channel directly, or indirectly via secondarymessengers. The Al-activated efflux from maize probably occurs by mechanism 1; the mechanismactivating malate efflux from wheat is not known. In the Pattern II response, Al interacts with the cell,perhaps via a receptor protein (R) on the plasma membrane, to activate the transcription of genes thatencode proteins involved with the metabolism of organic acids or their transport across the plasmamembrane. Organic acid anions form a stable complex with Al, thereby detoxifying Al3+ in therhizosphere. Experiments have identified some of the components shown in the model for Pattern Iwhereas the components depicted for Pattern II are entirely speculative.


0 5 10 15

Retention time (min)

Malate

Citrate

Oxalate

Abs

orba

nce

at 2

10 n

m

Root exudates (–Al)

Root exudates (+Al)

Root extracts

Fig. 3. Specific organic acids are secreted from roots in response toaluminium (Al). In this example, Cassia tora was exposed to 0.5 mMCaCl2 solution with 50 µM Al or without Al for 9 h. Organic acids in theroot tissue and exuded into the solution were determined by HPLC.Although the first peak in both HPLC profiles for the root exudates isclose to the retention time for oxalate, the peak is due to Cl– and not anorganic acid. Note that although many different organic acids arepresent in the roots, only citrate is secreted to the external medium inresponse to Al.



276 Review

conclusion from these studies is that it is the anionchannel that regulates the Al-activated secretion oforganic acids from the roots.

Changes in organic acid metabolism and secretion oforganic acids Information on how Al affects organic acidmetabolism in plants is available for only a fewspecies. Although organic acid metabolism can

change in species that exhibit the rapid Al-activatedsecretion of organic acid anions (Pattern I), thecapacity for organic acid synthesis in these plants isrelatively unaffected by Al exposure. For example, inwheat, there are no differences in the activities ofphosphoenolpyruvate carboxylase, NAD-malatedehydrogenase, citrate synthase or NADP-dependentisocitrate dehydrogenase between Al-sensitive andtolerant genotypes, with or without Al treatment,even though there is a significant loss of malate fromthe roots of the Al-tolerant plants14,22 (Fig. 4). Thesefindings suggest that in wheat, the enzymes involvedin malate biosynthesis are not rate limiting and thatthe loss of malate from the cytoplasm in the Altolerant genotype changes the activity of pre-existingenzymes to replenish internal malate pools. In someplant species that exhibit a lag between Al additionand the onset of organic acid secretion (Pattern II),changes in enzyme activities involved in organic acidbiosynthesis have been observed. For example in rye,the activity of citrate synthase in the root tipincreases 30% with Al exposure, whereas theactivities of phosphoenolpyruvate carboxylase,malate dehydrogenase and NADP-dependentisocitrate dehydrogenase are not affected14. Theincrease in citrate synthase activity occurs after 6 h ofAl exposure, just before the increase in citrate efflux.In the Al-tolerant tree species, Paraserianthesfalcataria, the activity of citrate synthase inmitochondria and the amount of citrate synthasemRNA also increases with exposure to Al (H. Osawa,pers. commun.). Although the increase in citratesynthase activity indicates an increased capacity forcitrate synthesis, to date, there is no direct evidence toshow that a change in metabolism is necessary forefflux to occur.


Oxaloacetate

Oxaloacetate

Oxaloacetate

Phosphoenolpyruvate (PEP) Cytoplasm

Acetyl CoA

Acetyl CoA

Acetyl CoA

Malate

Malate

CSCS MDH

MS

ICL

Citrate CitrateCitrateFumarate

Succinate

Succinate

Isocitrate Isocitrate Isocitrate

Glyoxyrate

Succinyl-CoA 2-Oxoglutarate

2-Oxoglutarate

SDH

SAT NAD-ICDHNADP-ICDH

AC AC

OGDH

Mitochondrion Glyoxysome

FUM

MDH Citrate

Vacuole

PEPC

Fig. 4. Key enzymes involved in citrate metabolism. Alterations in the activity of enzymes related tocitrate biosynthesis (CS, PEPC, MDH) or degradation (AC, NAD-ICDH, NADP-ICDH) might lead toaccumulation of citrate in the cytoplasm. Abbreviations: AC, aconitase; CS, citrate synthase; FUM,fumurase, ICDH, isocitrate dehydrogenase; ICL, isocitrate lyase; MDH, malate dehydrogenase; MS,malate synthase; OGDH, 2-oxoglutarate dehydrogenase; PEPC, phosphoenolpyruvate carboxylase;SAT, succinate thiokinase; SDH, succinate dehydrogenase.

High Low

Al

Fig. 5. Colours of Hydrangea sepals with different concentrations ofaluminium (Al). Hydrangea was grown in a nutrient solution with orwithout Al, or in a soil amended with or without Al. The blue colour ofHydrangea sepals is due to the formation of a complex betweendelphinidin 3-glucoside, Al and 3-caffeoylquinic acid. The Alconcentration in the sepals from pink to blue is 51, 106, 640, 804 and3959 mg Al kg–1 dry weight, respectively.



277Review

Several groups have attempted to manipulatecitrate metabolism using genetic engineering toincrease organic acid efflux. Transgenic tobacco linesexpressing the citrate synthase gene from thebacterium Pseudomonas aeruginosa reportedlyshowed increases in internal concentrations of citrate,increased citrate secretion and enhanced Altolerance23,24. However, another group that examinedthese same tobacco lines, as well as a set of additionaltransgenic lines expressing the bacterial gene at muchhigher levels, could not repeat these findings25. Thissuggests that the utility of the P. aeruginosa gene isdependent upon environmental conditions and,therefore, its use might not be a reliable strategy forincreasing the accumulation and secretion of citrate25.In a different study, overexpression of the carrotmitochondrial citrate synthase in Arabidopsisresulted in increased citrate synthase activity,increased citrate concentrations and a 60% increase in

citrate efflux. These changes were associated with asmall enhancement in Al tolerance26. The associationbetween increased citrate efflux and a greater capacityto synthesize citrate in transgenic plants suggests thatchanges in metabolism might also drive secretion.

Organic acids detoxify internal aluminiumIt is well known that some highly tolerant species canaccumulate high concentrations of Al in the above-ground herbage without showing symptoms of Altoxicity. Remarkably, in the case of Melastomamalabathricum (a tropical rainforest species thataccumulates Al), its growth is stimulated by Al(Ref. 27). Buckwheat leaves accumulate >400 mg kg–1dry weight (DW) of Al after only a short exposure (fivedays) to Al solution10 and as much as 15 000 mg kg–1

DW when grown on an acid soil. Hydrangea plants canaccumulate high concentrations of Al (>3000 mg kg–1

DW) in leaves over several months growth and thesepals of this species change from pink to blue withincreasing Al concentration28 (Fig. 5). The blue colour ofHydrangea sepals is caused by the formation of acomplex between delphinidin 3-glucoside, Al and3-caffeoylquinic acid, where Al is thought to play a rolein stabilizing an interaction between the two organiccompounds29. About 80% of the total Al in Hydrangealeaves is present in a soluble form and the Alconcentration in the cell sap can be as high as 13.7 mM(Ref. 28). The concentration of free Al3+ at the pH of thesymplasm is



278 Review

In buckwheat, changes in the chemical form of Aloccur during uptake, translocation and accumulation(Fig. 6). The form of Al taken up by the roots isprobably Al3+ because of the large inwardly directedelectrochemical gradient for this ion, but the exactmechanism for uptake is unknown31. Once taken up,Al3+ is chelated internally by oxalate to form a 1:3complex (Al-oxalate) in the root cells30. When theAl-oxalate complex is loaded to the xylem, a ligandexchange reaction occurs to form Al-citrate31. When Alis unloaded from the xylem to the leaf cells, anotherligand exchange reaction occurs to reform theAl-oxalate complex, which is then stored in thevacuole31 (R. Shen and J.F. Ma, unpublished).

ProspectsAcid soils are distributed worldwide but they areespecially prevalent in tropical regions where peoplein many developing countries rely heavily on seasonalfood production for their survival. Although it isimportant for us to understand the processes thatcause soil acidification and to minimize its advance bychanging farming practices or by applying lime toneutralize the acidity, there is also a need to providealternative strategies for managing marginal lands.One approach to maintain and even increaseproductivity on acid soils is to develop crops andpastures that are more tolerant of Al3+ stress. Thiscan be achieved by traditional breeding strategies forsome species, but for others that show little natural

variation in Al tolerance, genetic manipulation is anew approach.

Because organic acids are implicated in Al-tolerancemechanisms for a range of plant species, a logicalapproach is to manipulate organic acid biosynthesis orcatabolism. Many of the genes encoding key enzymesinvolved in organic acid metabolism have been cloned.As discussed already, mixed results have been achievedwith over-expressing citrate synthase genes in plants.However, there are other enzymes that could bemanipulated to express at a higher (e.g. PEPC, MDH)or lower (e.g. aconitase, ICDH) level to achieve thedesired result of increased organic acid accumulationand secretion (Fig. 4). Increased organic acidbiosynthesis might need to be coupled to an increasedcapacity to transport organic acid anions to theexternal solution. In some cases, expression of atransporter (for example, an anion channel) might initself be sufficient to cause increased organic acidsecretion. However, to date, a gene encoding such atransporter has not been cloned from any organismand this is a challenge for the future.

We need to understand how Al activates the anionchannel in plants showing the Pattern I response, aswell as defining the nature of the lag phase in thePattern II response. Other processes that need to beunderstood in Al-accumulating species include how Almoves across a range of membranes and the ligand-exchange reactions that occur with Al as it istransported through the plant.

References1 Kochian, L.V. (1995) Cellular mechanisms of

aluminum toxicity and resistance in plants. Annu.Rev. Plant Physiol. Plant Mol. Biol. 46, 237–260

2 Martin, R.B. (1986) The chemistry of aluminumas related to biology and medicine. Clin. Chem.32, 1797–1806

3 Ma, J.F. (2000) Role of organic acids indetoxification of aluminum in higher plants. PlantCell Physiol. 41, 383–390

4 Ryan, P.R. et al. (2001) Function and mechanism oforganic anion exudation from plant roots. Annu.Rev. Plant Physiol. Plant Mol. Biol. 52, 527–560

5 Delhaize, E. et al. (1993) Aluminum tolerance inwheat (Triticum aestivum L.) II. Aluminum-stimulated excretion of malic acid from rootapices. Plant Physiol 103, 695–702

6 Miyasaka, S.C. et al. (1991) Mechanism ofaluminum tolerance in snapbean, root exudationof citric acid. Plant Physiol. 96, 737–743

7 Pellet, D.M. et al.(1995) Organic acid exudation asan aluminum-tolerance mechanism in maize (Zeamays L.). Planta 196, 788–795

8 Ma, J.F. et al. (1997) Specific secretion of citricacid induced by Al stress in Cassia tora L. PlantCell Physiol. 38, 1019–1025

9 Yang, Z.M. et al. (2001) Aluminum tolerance isachieved by exudation of citric acid from roots ofsoybean (Glycine max). Physiol Plant. 110, 72–74

10 Ma, J.F. et al. (1997) Detoxifying aluminum withbuckwheat. Nature 390, 569–570

11 Ma, Z. and Miyasaka, S.C. (1998) Oxalate exudationby taro in response to Al. Plant Physiol. 118, 861–865

12 Ma, J.F. et al. (2000) Aluminum tolerance geneson the short arm of chromosome 3R are linked to

organic acid release in triticale. Plant Physiol.122, 687–694

13 Zheng, S.J. et al. (1998) Continuous secretion oforganic acids is related to aluminum resistanceduring relatively long-term exposure toaluminum stress. Physiol. Plant. 103, 209–214

14 Li, X.F. et al. (2000) Pattern of Al-inducedsecretion of organic acids differ between rye andwheat. Plant Physiol. 123, 1537–1543

15 Zheng, S.J. etal. (1998) High aluminum resistance inbuckwheat. I. Al-induced specific secretion of oxalicacid from root tips. Plant Physiol.117, 745–751

16 Ryan, P.R. et al. (1993) Aluminum toxicity inroots: an investigation of spatial sensitivity andthe role of the root cap. J. Exp. Bot. 44, 437–446

17 Zhang, W.H. et al. (2001) Aluminum activatesmalate-permeable channels in the apical cells ofwheat roots. Plant Physiol. 125, 1459–1472

18 Ryan, P.R. et al. (1997) Aluminum activates ananion channel in the apical cells of wheat roots.Proc. Natl. Acad. Sci. U. S. A. 94, 6547–6552

19 Pineros, M.A. and Kochian, L.V. (2001) A patchclamp study on the physiology of aluminumtoxicity and aluminum tolerance in Zea mays:identification and characterization of Al3+-inducedanion channels. Plant Physiol. 125, 292–305

20 Kollmeier, M. et al. (2001) Aluminum activates acitrate-permeable anion channel in the Al-sensitive zone of the maize root apex: acomparison between an Al-sensitive and an Al-tolerant cultivar. Plant Physiol. 126, 397–410

21 Delhaize, E. and Ryan, P.R. (1995) Aluminumtoxicity and tolerance in plants. Plant Physiol.107, 315–321

22 Ryan, P.R. et al. (1995) Characterization of

Al-stimulated efflux of malate from the apices of Al-tolerant wheat roots. Planta 196, 103–110

23 de la Fuente, J.M. et al. (1997) Aluminumtolerance in transgenic plants by alteration ofcitrate synthesis. Science 276, 1566–1568

24 Lopez-Bucio, J. et al. (2000) Organic acidmetabolism in plants: from adaptive physiology totransgenic varieties for cultivation in extremesoils. Plant Sci. 160, 1–13

25 Delhaize, E. et al. (2001) Expression of aPseudomonas aeruginosa citrate synthase gene intobacco is not associated with either enhancedcitrate accumulation or efflux. Plant Physiol.125, 2059–2067

26 Koyama, H. et al. (2000) Overexpression ofmitochondrial citrate synthase in Arabidopsisthaliana improved growth on a phosphorus-limited soil. Plant Cell Physiol. 41, 1030–1037

27 Watanabe, T. et al. (1998) Distribution andchemical speciation of aluminum in the Alaccumulator plant, Melastoma malabathricum L.Plant Soil 201, 165–173

28 Ma, J.F. et al. (1997) Internal detoxificationmechanism of Al in Hydrangea. Identification of Alform in the leaves. Plant Physiol. 113, 1033–1039

29 Takeda, K. et al. (1985) Blueing of sepal colour ofhydrangea macrophylla. Phytochemistry24, 2251–2254

30 Ma, J.F. et al. (1998) High aluminum resistance inbuckwheat. II. Oxalic acid detoxifies aluminuminternally. Plant Physiol. 117, 753–759

31 Ma, J.F. and Hiradate, S. (2000) Form ofaluminium for uptake and translocation inbuckwheat (Fagopyrum esculentum Moench).Planta 211, 355–360

AcknowledgementsWe are grateful toHirohito Ueno for help inpreparation of figures. Wealso thank Julie Hayesand Hiroyuki Koyama forcritical reading of themanuscript. Part of thiswork was supported by aGrant-in-Aid for GeneralScientific Research (grantno. 13660067 to J.F.M.)from the Ministry ofEducation, Science,Sports and Culture ofJapan), by Sunbor grants,and by the Sumitomofoundation.

27 Apr 2004 15:10 AR AR213-PP55-18.tex AR213-PP55-18.sgm LaTeX2e(2002/01/18) P1: GDL10.1146/annurev.arplant.55.031903.141655

Annu. Rev. Plant Biol. 2004. 55:459–93doi: 10.1146/annurev.arplant.55.031903.141655

Copyright c© 2004 by Annual Reviews. All rights reservedFirst published online as a Review in Advance on February 25, 2004

HOW DO CROP PLANTS TOLERATE ACID SOILS?MECHANISMS OF ALUMINUM TOLERANCE ANDPHOSPHOROUS EFFICIENCY

Leon V. Kochian, Owen A. Hoekenga,and Miguel A. PiñerosU.S. Plant, Soil, and Nutrition Laboratory, USDA-ARS, Cornell University, Ithaca,New York 14853; email: [email protected], [email protected],[email protected]

Key Words manganese tolerance, organic acid exudation, anion channel

■ Abstract Acid soils significantly limit crop production worldwide because ap-proximately 50% of the world’s potentially arable soils are acidic. Because acid soils aresuch an important constraint to agriculture, understanding the mechanisms and genesconferring tolerance to acid soil stress has been a focus of intense research interest overthe past decade. The primary limitations on acid soils are toxic levels of aluminum (Al)and manganese (Mn), as well as suboptimal levels of phosphorous (P). This reviewexamines our current understanding of the physiological, genetic, and molecular basisfor crop Al tolerance, as well as reviews the emerging area of P efficiency, whichinvolves the genetically based ability of some crop genotypes to tolerate P deficiencystress on acid soils. These are interesting times for this field because researchers are onthe verge of identifying some of the genes that confer Al tolerance in crop plants; thesediscoveries will open up new avenues of molecular/physiological inquiry that shouldgreatly advance our understanding of these tolerance mechanisms. Additionally, thesebreakthroughs will provide new molecular resources for improving crop Al tolerancevia both molecular-assisted breeding and biotechnology.

CONTENTS

INTRODUCTION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 460MECHANISMS OF ALUMINUM TOLERANCE . . . . . . . . . . . . . . . . . . . . . . . . . . . 461

Overview of Aluminum Toxicity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461Physiological Mechanisms of Al Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 461Other Potential Mechanisms of Al Exclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 470Internal Detoxification of Aluminum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 471Tolerance to Toxic Levels of Manganese . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 472Genetic Analysis of Aluminum Tolerance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473

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460 KOCHIAN ¥ HOEKENGA ¥ PIÑEROS

Tolerance Loci with Qualitative Inheritance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 473Tolerance Loci with Quantitative Inheritance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 475Genomic Analysis of Al Tolerance. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 476

PHOSPHOROUS EFFICIENCY: TOLERANCETO P DEFICIENCY STRESS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 480Root Exudates and P Mobilization from the Soil. . . . . . . . . . . . . . . . . . . . . . . . . . . 480Root Morphology and Architecture. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481Pi Homeostasis and Transport. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 481Genetic Analysis of P Efficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 482

SUMMARY . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 483

INTRODUCTION

Acid soils, which are soils with a pH of 5.5 or lower, are one of the most impor-tant limitations to agricultural production worldwide. Approximately 30% of theworld’s total land area consists of acid soils, and as much as 50% of the world’spotentially arable lands are acidic (159). The production of staple food crops, andin particular grain crops, is negatively impacted by acid soils. For example, 20%of the maize and 13% of the rice production worldwide is on acid soils (159).Furthermore, the tropics and subtropics account for 60% of the acid soils in theworld. Thus, acid soils limit crop yields in many developing countries where foodproduction is critical. In developed countries such as the United States, high-inputfarming practices such as the extensive use of ammonia fertilizers are causing fur-ther acidification of agricultural soils. Although liming of acid soils can amelioratesoil acidity, this is neither an economic option for poor farmers nor an effectivestrategy for alleviating subsoil acidity.

This paper updates our 1995 review on the same topic (67). The previousreview heavily emphasized mechanisms of aluminum (Al) toxicity, which signif-icantly impacts plant growth on acid soils. Research on the mechanisms plantsemploy to tolerate toxic levels of Al in acid soils was in its early stages in 1995.However, over the past eight years, considerable research has focused on physio-logical and biochemical mechanisms of Al tolerance, as well as the molecularbasis for tolerance. Additionally, there has been a growing awareness that thereare several factors in addition to Al toxicity that limit crop production on acidsoils. This acid soil “syndrome” includes toxic levels of Al, manganese (Mn),and iron (Fe), as well as deficiencies of several essential mineral elements, withphosphorus (P) being the major limiting nutrient on acid soils. Hence, in thisreview we highlight the key points regarding advances in our understanding ofplant responses to acid soils over the past decade. A significant portion of thisreview focuses on the physiology, biochemistry, and molecular biology of Altolerance mechanisms. We also address recent research into plant mechanismsof tolerance to phosphorus deficiency stress, a trait that is commonly termedphosphorous (P) efficiency, as well as some recent work on tolerance to Mntoxicity.

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PLANT MECHANISMS OF ACID SOIL TOLERANCE 461

MECHANISMS OF ALUMINUM TOLERANCE

Overview of Aluminum Toxicity

Due to space constraints, we focus on mechanisms of Al tolerance and not Altoxicity. The many complex mechanisms by which Al toxicity is manifested areaddressed in several reviews (7, 67, 68, 100, 102). However, to place the researchon Al tolerance mechanisms in its proper context, it is important to list some ofthe key features regarding the mechanistic basis for Al toxicity. These include:

■ When the soil pH drops below 5, Al3+ is solubilized into the soil solution andthis is the most important rhizotoxic Al species (63–65).

■ The primary symptom of Al toxicity is a rapid (beginning within minutes)inhibition of root growth, resulting in a reduced and damaged root systemand limited water and mineral nutrient uptake (see, for example, 7, 59).

■ The rapidity of this response indicates that Al first inhibits root cell expansionand elongation; however, over the longer term, cell division is also inhibited(67, 100, 102).

■ The site of Al toxicity is localized to the root apex; thus research on tolerancemechanisms also should be focused on this region of the root (138, 148, 150).

■ Because Al is so reactive, there are many potential sites including the cellwall, the plasma membrane surface, the cytoskeleton, and the nucleus thatcould be targets for injury.

■ Although most of the root-associated Al is in the apoplast, a small fractionof the Al rapidly enters the symplasm and interacts with symplastic targets(75, 146, 155).

■ Al disrupts cytoskeletal dynamics, interacts with both microtubules and actinfilaments, and could be an important component of Al-induced inhibition ofroot elongation (14, 40, 151).

■ Al interactions with signal transduction pathways, in particular Ca2+ homeo-stasis and signaling, could play a role in toxicity. Al exposure can alter cy-tosolic Ca2+ levels (58, 60, 175), and can interact with and inhibit the enzymephospholipase C of the phosphoinositide pathway associated with calcium(Ca) signaling (59). For a recent review on this topic, see Reference 129.

■ Al exposure elicits the induction of reactive oxygen species (ROS) as wellas peroxidative damage to membranes. Although lipid peroxidation is likelynot a primary mechanism of toxicity (51, 171), Al-induced ROS generationand associated mitochondrial dysfunction could be involved in Al inhibitionof root growth (170).

Physiological Mechanisms of Al Tolerance

Over the past decade, several laboratories around the world have focused theirefforts on identifying and characterizing the mechanisms employed by crop plants

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