Frontiers in molecular mycorrhizal research – genes, loci, dots and spins

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Frontiers in molecular mycorrhizal research – genes, loci, dots and spins

Functional genomics is revolutionizing our understandingof biological mechanisms, nowhere more than in complexsymbiotic systems. Functional genomics entails the analysis ofall of the genetic material (the genome) of an organism, andthen relating it to the form and function of that organism.Through the application of the cutting-edge tools of genomeanalysis at several levels – those of the genome, transcriptome,proteome and metabolome – remarkable progress hasbeen made in understanding the mechanisms that controlthe development and functioning of nitrogen-fixing sym-bioses (Harrison, 1999; Downie & Bonfante, 2000; Györgyey

et al.

, 2000). A surge of investigations based on these novelapproaches (large scale gene sequencing, cDNA array analysisof gene expression, proteomics) have recently allowed anassessment of the development and functioning of arbuscularendomycorrhizal (AM) and ectomycorrhizal (ECM) sym-bioses (Box 1) on a larger scale (Harrison, 1999; Lapopin

et al.

, 1999; Bago

et al.

, 2001; Voiblet

et al.

, 2001). Similarly,PCR-based detection of genome polymorphisms have revolu-tionized our perception of the ecology of mycorrhizal fungi(Peter

et al

., 2001). However, the limitations and ultimateutility of genomics to determine the ecological role ofthe mycorrhizal symbiosis in the real world remain to bedetermined. The reviews featured in this issue of

NewPhytologist

have been selected with all these issues in mind.In addition to providing the ‘state-of-the-art’ of currentknowledge of molecular mechanisms driving the develop-ment, physiology and ecology of the symbiosis, the reviewsalso provide a glimpse of things to come.

A new conceptual framework

Understanding the complexity of the interactions betweenmycorrhizal symbionts and how these mutualistic associ-ations adapt and respond to changes in the biological,chemical and physical properties of the rhizosphere remainsa significant challenge for plant and microbial biologists.Identification of the primary genetic determinants control-ling the development of the symbiosis and its metabolicactivity (e.g. P and N scavenging) will open the door tounderstanding the ecological fitness of the mycorrhizalsymbiosis. The symbiosis provides an additional layer of

complexity to the challenge of designing experimentalsystems and framing questions, and thus it has been relat-ively difficult to study these primary genetic determinants.For many years, most physiological analyses were focused onlimited sections of the complex metabolic networks (e.g.N and P acquisition and assimilation) taking place in sym-bionts (Hampp

et al.

, 1995; Harrison, 1999), which limitedthe degree to which symbiosis behaviour could be under-stood. Molecular details of mycorrhiza development andfunctioning are now becoming experimentally tractableowing to simultaneous advances in various fields. Themolecular identity of recently discovered symbiosis-regulated(SR) genes, the analysis of mycorrhiza mutants, new experi-mental approaches, such as cDNA arrays, and innovativeapplications of older technologies, such as NMR, are togethergenerating a conceptual framework for understanding theformation and physiology of mycorrhizas in which modelpredictions can now be tested at the molecular level. Whilefamiliar with the practicalities of making a PCR or abiochemical analysis, many scientists lack a ‘nuts-and-bolts’appreciation of the pros and cons of functional genomics.Detailed and thorough understanding of the options avail-able improves the odds that one’s choice will be vindicated;an appreciation of the biological and ecological contexts ofgenetic (and genomic) questions can also be critical to thesuccess of genome-based analysis.

Dissection of the steps leading to mycorrhizas

At present, we do not know either the molecular mechanismsunderlying overall symbiotic tissue patterning nor thespecific signals ensuring fungus–root-coordinated develop-ment and metabolic continuity between symbiotic partnersand between the different hyphal networks (i.e. Hartignet and extraradical hyphal web). Marsh & Schultze (seepp. 525–532, in this issue) emphasize that distinct, butpartially overlapping, sets of fungal and plant factors arerequired and produced at each stage of AM development.Similar developmental stages can be identified during theformation of ECM (Martin & Tagu, 1999). These symbioticstages are probably controlled by specific signalling pathwaysand regulatory genes (loci that control the expression of othergenes). These genes probably encode sequence-specific DNA-binding transcriptional activators and components of thecell–cell signalling networks. Identification of these symbiosisregulatory factors is a daunting task. It involves the typicalgene-to-phenotype approach (i.e. identification of traitsthrough characterization of gene expression and, subsequently,gene inactivation). Although genetic transformation of ECM

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and AM mycorrhizal fungi has been reported (Bills

et al.

,1995; Forbes

et al.

, 1998), these species are not yet amenableto gene inactivation. Therefore, a mutant-based geneticapproach would allow an unbiased dissection of the stepsleading to the symbiosis formation in both AM and ECM.Direct inspection for mycorrhizal mutants in fungi andplants is labour intensive. Genetic analysis is thus scarce andmost mutants currently available have been identified inleguminous plants, owing to the fact that the AM symbiosisshares common steps with the plant–

Rhizobium

symbiosis.Duc

et al

. (1989) were the first to report that, in pea, somemutants defective in nodulation also did not form the AMsymbiosis. Several authors have expanded on that observation(e.g. Albrecht

et al.

, 1998; Resendes

et al.

, see pp. 563–572)and, in the present issue, Marsch & Schultze summarizethe current state of knowledge concerning the existingcollection of Myc

–

mutants and discuss the future prospectsfor isolating mycorrhizal specific mutants, as well as thetechniques that may be used to generate and identify them.The mutant lines reviewed by these authors representnearly 40 mutations in at least 7–10 separate loci. Geneticanalysis is, however, far from saturation point. Consequently,there is no reliable estimate of the number of genes involvedin mycorrhiza development. However, given that mycorrhizalsymbioses are widespread, a significant number of mycorrhiza-specific genes must exist. Several of the Nod

–

Myc

–

mutantsappear to be involved in cell–cell signalling. In view of theobservation that some Nod

–

Myc

–

mutants induce calciumspiking in root hairs, one of the earliest responses torhizobial nodulation factors, but others do not, Walker

et al

.(2000) have speculated that calcium spiking may becommon to both mycorrhizal and nodulation signalling andthat the pathways could diverge after calcium spiking.Catoira

et al

. (2000) and Marsch & Schultze proposed aworking model with parallel pathways that control differentprocesses required during the development of the complex

symbiotic association. Future work on AM leguminous sym-bioses should concentrate on identification of the componentsof this signalling network. Direct screens for mycorrhizalmutants in nonleguminous plants are not biased byassociated nodule or Nod factor perception defects andmight thus identify further signalling mechanisms. The onlymutant available so far in a nonleguminous plant wasdetected in tomato (Barker

et al.

, 1998a). Large collections oftransposon insertion lines in

Populus

spp. (Mathias Faldung,pers. comm.) and UV-generated

Hebeloma cylindrosporum

mutants (Gilles Gay, pers. comm.) may give a reasonablechance of finding plant and fungal knock-out mutants forECM development.

Arraying the symbionts to sketch the complexity

For optimal development of the symbiosis, partners needto coordinate complex developmental processes (e.g. theformation of intraradicular symbiotic structures) and, at thesame time, sense and respond to novel physiological factorsand environmental cues (Barker

et al.

, 1998b; Martin &Tagu, 1999, Nehls

et al.

, 2001a, see pp. 533–542 in this issue).In both AM and ECM symbioses, development involves atleast five main events: survival of hyphae in the rhizosphere;primary attachment of hyphal tips to host roots; invasion ofroot tissues; coordinated construction of symbiotic struc-tures; and bilateral transfer/acquisition of assimilates. All ofthese steps are dependent of the generation and transductionof rhizospheric and intracellular signals. An understandingof the mechanisms that underlie the temporal and spatialcontrol of genes involved in symbiosis development is nowwithin reach, as more sophisticated techniques of functionalgenomics are applied to mycorrhizal interactions. In addi-tion to genetic screens for mycorrhiza mutants, Franken &Requena (see pp. 517–523 in this issue) describe how thedevelopment of the technology to sequence expressed genes on

Box 1 The mycorrhizal symbiosis

The rhizosphere hosts a large and diverse community of microorganisms that compete and interact with each other and with plantroots. Within this community, mycorrhizal fungi are almost ubiquitous. Their vegetative mycelium and root tips form a mutualisticsymbiosis. The novel composite organ is the site of nutrient and carbon transfer between the symbionts. The various mycorrhizalassociations allow terrestrial plants to colonize and grow efficiently in suboptimal environments. Among the various types of mycorrhizalsymbioses, arbuscular endomycorrhiza (AM), ectomycorrhiza (ECM) or ericoid associations are found on most annual and perennialplants (probably > 90%). About two-thirds of these plants are symbiotic with AM glomalean fungi. Ericoid mycorrhizas are ecologicallyimportant, but mainly restricted to heathlands. While a relatively small number of plants develop ECM, they dominate forest ecosystemsin boreal, temperate and mediterranean regions. In the different mycorrhizal associations, extramatrical and intraradicular hyphalnetworks are active metabolic entities that provide essential nutrient resources (e.g. phosphate and amino acids) to the host plant.These nutrient contributions are reciprocated by the provision of a stable carbohydrate-rich niche in the roots for the fungal partner,making the relationship a mutualistic symbiosis. The ecological performance of mycorrhizal fungi is a complex phenotype affected bymany different genetic traits and by biotic and abiotic environmental factors. Without doubt, anatomical features (e.g. extensionof the extramatrical hyphae) resulting from the development of the symbiosis are of paramount importance to the metabolic (andecophysiological) fitness of the mature mycorrhiza.


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a large scale and to analyse this DNA through bioinformaticsand mRNA profiling will have enormous impacts on theway we think about the biology of mycorrhizal associations.PCR-suppression subtractive hybridizations, transcriptomeprofilings and proteomics have been used to identifypatterns of gene expression underlying the development andphysiology of AM and ECM symbioses. Mycorrhiza develop-ment affects not only genes involved in cell differentiationand organ development, but also genes controlling nutrientscavenging and assimilation and plant defence reactions.

Until recent times, it has been impossible to study morethan one, two, or perhaps a handful of genes and how theyinteract in complex biological systems, such as symbioses.Franken & Requena, and our own investigations on ECM(Voiblet

et al.

, 2001), show that instead of studying oneparameter and one gene at a time, we can use cDNA arrays(macro- and microarrays) to study, within a single experi-ment (Fig. 1), hundreds to thousands of the host genes andthose of the fungal partner whose expression is modifiedduring the host–symbiont interaction: the global picture ofthe dialogue between the mycobiont and the host in oneexperiment. Transcriptome analyses, based on cDNA arrays,promise great strides in our understanding of gene expres-sion and interactions, and how overlapping signalling net-works simultaneously regulate developmental processes insymbiotic partners. Combined studies applying detailedanalysis of key genes, such as phosphate and hexose trans-porters (Harrison, 1999; Nehls

et al.

, 2001b, see pp. 583–589)and comprehensive transcript profilings by cDNA arrays willallow the prospect, in the future, of linking the regulation ofa particular metabolic step to the overall physiological com-plexity of the symbiosis. Applied to expression analysis, thisapproach facilitates the measurement of RNA levels forthe complete set of transcripts of an organism. Arrays con-taining 1000–10 000 genes are already in common use for

Arabidopsis

and yeast, and current protocols allow reliabledetection of messages present at several copies per cell.Genome-wide expression in

Arabidopsis

is currently beingreleased at a fast pace and has dealt with circadian rhythms(Schaffer

et al.

, 2001), impact of cold- and drought-stresses(Seki

et al.

, 2001), and both pathogenesis and wounding(Maleck

et al.

, 2000). By differential screening of arrayscarrying about 500 ectomycorrhizal cDNAs, we found 65genes differentially expressed during the formation of theectomycorrhizal mantle in the

Eucalyptus globulus–Pisolithu

ssp. association (Voiblet

et al.

, 2001). The number of SRgenes displaying similarity to genes involved in cell walland membrane synthesis, stress/defence responses, proteindegradation (in plant cells) and protein synthesis (in hyphae)suggested a highly dynamic cellular environment in whichboth partners are sending and receiving a varied set of cues,including high levels of stress conditions. Hierarchical clusteranalysis defines groups of genes having both similar regula-tion patterns and expression amplitudes (Eisen

et al.

, 1998)

(Fig. 1). For example, several genes that clustered with thesymbiosis-regulated hydrophobin genes were known cell wallproteins, such as 32 kDa-symbiosis-regulated acidic poly-peptides (SRAP32), but also newly identified structural genes,such as proline-rich proteins and proteophosphoglycans(Duplessis & Martin, unpublished). Many sequences in thiscluster of coordinately expressed genes (regulon) showed nosignificant similarity to known genes and are candidates forfurther biochemical analyses.

The challenge is no longer in the use of expression arraysthemselves, but in developing data mining tools to exploitthe full power of a global perspective (e.g. identifying thecoregulation of N, P and C metabolic pathways). Some ofthe most important biological applications involve studyingvery small target tissues – for example, the endomycorrhizalarbuscules or different regions of ectomycorrhizal tissues; ora particular class of cells in the root cortex. It is hoped thatstudies based on functional genomics (cDNA arrays andproteomics) will be carried out in the near future on a widerrange of mycorrhizal symbioses. In an optimistic scenario,identification of several alternative functional symbioticpatterns generated in a given genetic background (i.e. samehost interacting with different AM and ECM fungi, or samemycobiont colonizing different hosts) could soon provide amolecular understanding of both the evolution and develop-ment of mycorrhizal symbioses.

A maze of metabolic pathways

The nutritional benefits that mycorrhizal symbioses conferon each partner hinge on the structural and physiologicalintricacy between the symbionts, and therefore detailedinformation on metabolism, transport (Nehls

et al.

, 2001a)and functional anatomy of intact systems is required. Inaddition, elucidation of the function of the hundreds ofknown and unknown genes analysed by transcript profil-ings and proteomics may be nicely supplemented by thesimultaneous analysis of the metabolome, the latter changingaccording to the physiological and developmental state of acell, tissue, organ, or organism. Although metabolic profil-ing using gas chromatography-mass spectrometry (GC-MS)technologies (Roessner

et al.

, 2001) can give valuable informa-tion and is often more sensitive than NMR, it yields muchless quantitative information on the position of isotopicenrichments within each molecule. In their review, Pfeffer

et al

. (see pp. 543–553 in this issue) demonstrate that theNMR approach has been used to trace, in detail, the bio-chemical pathways by which a given molecule was made inAM and ECM symbioses. NMR spectroscopy yields label-ling information directly and quantitatively and this is asignificant advantage for metabolic studies. The potential todifferentiate, spectroscopically, host from fungal metabolites

in vivo

or in crude extracts without the need for separationor chemical derivatization is another useful aspect of NMR


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measurements in studying the biochemistry of mycorrhizas.NMR has therefore contributed significantly to such questionsas the biochemistry of polyphosphates and the pathways andregulation of C and N metabolism in mycorrhizas, as well asthe identification of secondary metabolites made in responseto the synthesis of mycorrhizas and/or to xenobiotics (Bago

et al.

, 2001; Pfeffer

et al.

, 2001). High throughput techniques,such as high-resolution NMR and GC-MS, are already usedfor comprehensive phenotyping of model systems in con-junction with transcriptome analysis (Roessner

et

al

., 2001;Raamsdonk

et al.

, 2001). They may be equally useful inanalysing known AM and ECM mutants and future genetic-ally modified plant and fungal systems.

Bridging genomics and molecular ecology

During the past decade, many PCR-based molecularmethods have been developed to identify mycorrhizal fungi,and their potential for mycorrhizal ecology has been proven.Dahlberg (see pp. 555–562 in this issue) emphasizes the factthat the application of these molecular methods has pro-vided detailed insights into the complexity of ECM fungalcommunities and offers exciting prospects to elucidateprocesses that structure ECM fungal communities. Theywill improve our understanding of plant ecology, such asplant interactions and ecosystem processes. About 50 suchECM community studies have been published over the pastfive years. Molecular studies have shown the following:• Any single mycorrhiza can potentially be identified to spe-cies either by PCR-RFLP of the nuclear ribosomal DNAinternal transcribed spacers or by DNA sequencing.• Sporocarp production is unlikely to reflect below-groundsymbiont communities. Not all ectomycorrhizal fungi produceconspicuous epigeous sporocarps and of those fungi that doproduce conspicuous sporocarps, a species’ sporocarp produc-tion does not necessarily reflect its below-ground abundance.• A few fungal ECM taxa account for most of the mycor-rhizal abundance and are widely spread, whereas the majorityof species are only rarely encountered.• The spatial variation of ECM fungi is very high andmost species show aggregated distributions. As stressed byDahlberg, little is known about the relative importance ofvegetative spread and longevity of genotypes vs novel coloniza-tion from meiospores for any ECM fungal species: this deserves

attention if we want to understand the dynamics and struc-ture of ECM communities/populations. Similar advancesare being made in our understanding of AM communities.

Despite the fact that mycorrhizal fungi play an importantrole in N, P and C cycling in ecosystems in decomposingorganic materials, the detailed function of fungi in nutri-ent dynamics is still unknown. Mycorrhizal fungi differ intheir functional abilities and the different mycorrhizas theyestablish thus offer distinct benefits to the host plant. Somefungi may be particularly effective in scavenging organic N,and may associate with plants for which acquisition of N iscrucial (Peter

et al.

, 2001); others may be more effective at Puptake and transport. An important goal is therefore todevelop approaches by which the functional abilities of thesymbiotic guilds are assessed in the field. In any case, it isnecessary to look below ground to see what is really happen-ing (Lilleskov & Bruns, 2001). Analyses of

13

C and

15

Nisotopic signatures have a significant potential to provideinformation in this area (Hobbie

et al.

, see pp. 601–610),although further investigation is required to understand theisotopic enrichment phenomenon (Henn & Chapela, 2000).Combined community/population structure and functionstudies applying genomics may, in the future, significantlypromote our understanding of the interactions betweenmycorrhizal fungal species with their hosts and with theirbiotic and abiotic environment. A first step toward thisenvironmental genomics is to explore fungal communityfunctioning under simulated forest conditions using micro-cosm systems (Timonen

et al.

, 1997). In these systems, intactmycorrhizal root systems comprising individual species(e.g. abundant taxa likely to be functionally important) ornatural communities can be manipulated and analysed todetermine, for example, C and N relations and host root–fungus metabolic activities that contribute to plant growthor plant community productivity. As a consequence, withinthe next decade, determining large sets of gene sequences ofsymbionts will become a prerequisite for truly detailedanalyses of gene expression and its regulation in response toenvironmental changes.

What can we expect to dig up next?

The genomics era for mycorrhizas is not yet in full swing,but it is clear from recent studies, highlighted in the reviews

Fig. 1

A typical cDNA array experiment to study mycorrhizal development. (a) RNA is prepared from symbionts or free-living partners and converted into radioactive or fluorescent cDNA probes by reverse transcription. (b) Arrays are usually robotically printed by using each of the mycorrhizal genes of known or unknown sequences, which have been previously amplified by PCR. (c) The labelled cDNA probes are used to hybridize cDNA macro- or microarrays containing (mycorrhizal) genes. (d) After hybridization, laser scanning, image detection, and analysis of each array provides a precise indication of the relative expression of the genes present on the arrays. (e) The final results of the comparative analysis are the expression ratio, where a red dot indicates gene activation upon mycorrhiza development and a green dot means downregulation of the gene, whereas a black dot indicates no change in gene expression. Hierachical clustering (e) allows the identification of coordinately expressed genes or regulons (Eisein

et al.

, 1998). In (e), transcripts of

Pisolithus

sp. coding for hydrophobins, symbiosis-regulated acidic polypeptides (SRAPs), proline-rich proteins, proteophosphoglycans, and membrane proteins are clustered according to their temporal expression patterns (S. Duplessis and F. Martin, unpublished).

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in this issue, that functional genomics and molecularecology have already made significant contributions to ourunderstanding of developmental and metabolic mechanismsleading to the formation and functioning of mycorrhizas.What will the future bring? We are likely to see imminentadvances in understanding of the molecular and cellularmechanisms of the coordinated regulation of developmentaland metabolic gene expression in symbiotic partners,although the equally important mechanisms that modulatecell growth rates and shape during mycorrhiza developmenthave not generated a similar intensity of interest. Thecurrent compendium of mycorrhiza-regulated genes willprovide the basis for a more precise molecular dissection ofthe complex genetic networks that control symbiosisdevelopment and function. The symbiosis regulated-genesidentified might be especially interesting targets for futuregene disruption technology. Further studies are now neededto delineate the functions of both the known and novelgenes that are differentially expressed during mycorrhizadevelopment. Understanding how mycorrhiza regulated-genes exert their developmental effects at the cellular andsupracellular levels will be a difficult challenge. In thisregard, one anticipates that increased attention will be givento the role of N, P and C transporters (Harrison, 1999;Nehls

et al.

, 2001a) in mycorrhiza function. There willcertainly be automation of hybridization assays based onmicroarray/DNA chip technology.

It will be far harder to define genes that influencessymbiont fitness. Any gene that provides the mycobiont witha growth advantage could easily influence the benefits of aparticular strain within a given host. Therefore, dissectingthe molecular mechanisms of symbiosis requires both identi-fication of the functions of individual genes as well asknowledge of how genes interact to form complex traitssuch as those expressed in a mutualistic symbiosis. Thereare more than 5000 different ECM associations and 150species described in the Glomales, and each and everymycorrhizal type may express a specific set of genes. Despitethis, a variety of morphological and molecular parameterscan be used to classify mycorrhizal symbioses into discretemolecular classes for further investigation. Symbioticmolecular phenotypes may in the future be correlated toecological phenotypes. It will be rewarding to compareplant and fungal gene expression profiles among differentAM and ECM associations, mycorrhizal symbioses andother plant–fungus interactions. This will certainly identifyoverlaps in the genetic make up of plant–fungus interactions.

Environmental samples will soon be probed withhundreds to thousands of different DNA probes to tackleecological questions. Single nucleotide polymorphisms(SNPs) and high-density DNA arrays usher in the pos-sibility of determining allelic imbalance at hundreds ofthousands of loci from hundreds of DNA samples, allowing

the contemplation of whole genome association studies todetermine the genetic contribution to complex polygenicprocesses (Rothberg

et al.

, 2000). Applied to screening ofgenetic variation (allelic heterogeneity), the use of micro-arrays is likely to bring typing of individuals, or even entirepopulations, into the realm of practical reality (Mei

et al.

,2000). A combination of the analysis of community/population composition of mycorrhizal fungi by DNA prob-ing (e.g. Bruns & Gardes, 1993) with

in situ

assessment offunction by transcript profiling (environmental genomics)will have a substantial effect on the kinds of questions thatcan be addressed, particularly for model symbionts wheregenome analyses have already been initiated (e.g.

Pisolithus

,

Hebeloma

,

Glomus

,

Medicago

and

Populus

).

Acknowledgements

I would like to thank Denis Tagu and Sébastien Duplessis(INRA-Nancy) for valuable discussions on functional genomicsof ectomycorrhiza. Genomics studies carried out in mylaboratory were supported by grants from the INRA (Pro-grammes ‘Functional Genomics of Poplar’ and ‘LIGNOME’).

Francis

Martin

UMR INRA–UHP Interactions Arbres/Micro-Organismes, Centre INRA de Nancy, 54280

Champenoux, France(tel +33 383 39 40 80; fax +33 383 39 40 69;

email [email protected])

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Key words: genomics, transcript profiling, plant–microbe interactions, functional ecology, cDNA arrays, symbiosis-regulated genes, fungal populations, community structure.150no issue no.2001145CommentaryCommentaryCommentary000000Graphicraft Limited, Hong Kong


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Forum506

Variability of inorganic nutrient concentrations in leaves

Nutrient supply and acquisition are two of the most import-ant factors that control plant productivity and diversity, asgrowth is generally limited by the availability of inorganicnutrients in the soil. From a biochemical point of view, allplant species should need the same quantity of nutrients toconstruct a given amount of tissue. However, differences doexist in tissue concentrations because of heterogeneous dis-tribution of nutrients in the soil and varying uptake effici-ency. In addition, plants respond differently to environmentalvariability, including nutrient shortage, and have differentphysiological needs that cause differences in metabolism andconstitution (Chapin, 1980). Adaptation to variation ofnutrient availability in soils leads to nutrient concentrationsthat differ widely between organs, species and ecosystems.Alonso & Herrera (see pp. 629–640 in this issue) looked atnutrients in a novel way. They considered concentrations ofleaf nutrients as traits that respond plastically to environ-mental variability and tested whether a consistent pattern ofnutrient covariation among populations could be expectedas a result of the plant’s acclimation to a given environment.

The role of nutrients

Traditionally, agronomists looked for an equilibrium amongnutrients to optimize yield and thereby establish basic valuesfor optimal plant growth. Critical nutrient ratios (e.g. N : P,

N : K, P : K) were soon established that could increase theproductivity of crops – nonspecific interactions among mineralnutrients can cause a deficient concentration of one nutrientto become sufficient as the concentration of other nutrientschange. There are synergies and antagonisms among nutrientsthat could cause unexpected responses (Marschner, 1986).

Mineral nutrients have different functions within theplant, and their concentration in the leaves may differ byorders of magnitude – from nitrogen, which is the base ofstructural and soluble proteins (such as Rubisco) and isfound in the range of 0.5–10 g g−1 of leaf dry mass, to traceelements like molybdenum, which acts as an enzyme cofactorand is needed in minute amounts in the order of a few mg kg−1

of leaf (Table 1). In addition to their different functions,nutrients also differ in their concentration with time.Mineral nutrient concentrations show seasonal changes –those with an active metabolic function (N, P and K) increasewhen the leaf is developing and decline afterwards, partlybecause of the increasing proportion of cell wall structures andpartly because there is translocation of nutrients out of leavesbefore senescence (Chapin & Kedrowsky, 1983; Pugnaire& Chapin, 1993). By contrast, nutrients such as Ca andMg are less mobile and monotonically increase with age.

Plants in natural systems show high inter- and intra-specific variability in nutrient concentrations and criticalratios have been used to analyse growth and predict whichelements are limiting at the community level (Verhoeven et al.,1996), although to a lesser extent than in agronomy. In spiteof the natural variability of nutrient concentrations in thesoil, plants from adverse environments (e.g. arid or coldregions) show more consistent tissue concentrations ofnutrients and a conservative strategy in the use of resources(Valladares et al., 2000).

Concentration

Element Symbolµmol g−1 dry mass

mg kg−1 dry mass

% mass mass−1

Relative number of atoms

Molybdenum Mo 0.001 0.1 – 1Copper Cu 0.10 6 – 100Zinc Zn 0.30 20 – 300Manganese Mn 1.0 50 – 1 000Iron Fe 2.0 100 – 2 000Boron B 2.0 20 – 2 000Chlorine Cl 3.0 100 – 3 000Sulphur S 30 – 0.1 30 000Phosphorus P 60 – 0.2 160 000Magnesium Mg 80 – 0.2 80 000Calcium Ca 125 – 0.5 125 000Potassium K 250 – 1.0 250 000Nitrogen N 1000 – 1.5 1 000 000

1Reproduced courtesy of E. Epstein, in Marschner (1986).

Table 1 Average concentrations of mineral nutrients in plant shoot dry matter that are sufficient for adequate growth1


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Forum 507

Nutrient variability

Originally interested in the potential consequences of vari-ability of tissue nutrient concentration to habitat selectionby herbivores, Alonso & Herrera (this issue) focused on theintraspecific patterns of nutrient covariation at the individualand population levels in Prunus mahaleb. They found no con-sistent pattern of relatedness among concentrations of differ-ent nutrients at the individual level; individual P. mahalebshrubs living in the same site differed vastly in their tissueconcentrations of nutrients with no apparent patterns ofcovariation. They did find, however, consistent pairwisecorrelations across all sites for Ca and Mg, but that otherrelationships, such as B–Ca or N–P, were significantlycorrelated in only one or a few populations. The latter resultis striking because many reports over the years have con-sistently found positive correlations of N and P concentra-tions. In addition, correlations between nutrients did not holdamong populations. Covariation of macronutrients (N, P, K,Ca and Mg) show that they were closely related, and respon-sible for the main gradients of variation observed across trees.The results show that covariance among nutrients was mutu-ally independent and had no significant spatial trends.Differences between shrubs in some elements (N, K, Cuand Fe) were responsible for most of the intraspecific variation,while concentrations of other elements (P and Ca) caused thelarge variation among populations. Variability in nutrient con-centration was generally greater among individual trees livingin the same site than among populations from different sites.

Variations within a population could be attributable tomicrosite characteristics as much as genotype differences.The important high levels of variation across populations didnot mirror within-population variation, and probably reflecteddifferent local soil characteristics (e.g. different proportionsof dolomite and calcite under each population). Altogether,the data show that phenotypic integration of nutrient con-centrations in P. mahaleb is weak, and blurred by plasticityin nutrient uptake and transport (Sultan & Bazzaz, 1993).

There seems to be no ecological reasons for the high intra-specific variation in the concentration of some elements,which often equals or exceeds interspecific variation (Ohlson,1988). Nutrients frequently covary nonrandomly acrossspecies because they share functional similarities and/orchemical behaviour, though the ultimate reason for randomcovariance is not really known. Statistically significant patternsof nutrient covariation may also be strongly affected by thenature of the sampling units and by patterns of allocation sothat environmental or sampling effects may conceal inherentphysiological or chemical associations between elements.

Nutrient concentration has a strong effect on herbivorybecause feeding is affected by leaf quality (McNaughton,1988). A nutrient imbalance may affect resistance or toleranceto herbivores (Hartley & Jones, 1997). Nonetheless, thepaper by Alonso & Herrera shows that the predictability of

nutritive quality is low and, as the authors conclude, diffusepatterns of nutrient covariation show a lack of commonground for selective processes exerted by herbivores on plants,whereas local, specific factors provide ways of adaptive adjust-ment of herbivores to host plants.

Summary

Alonso & Herrera clearly show that the highly variable nutrientenvironment experienced by plants favours plasticity rather thangenetic specialization, and that the ability to grow at both lowand high nutrient supply may be an important aspect of adjust-ment of individual plants to the environment. Other factors suchas irradiance, soil moisture, or herbivory similarly influencephenotypic responses in plants (Sultan & Bazzaz, 1993).

Francisco I. Pugnaire

Estación Experimental de Zonas Áridas, CSIC, GeneralSegura 1, 04001 Almería, Spain

(tel +34 950 281 045; fax +34 950 277 100;email [email protected])

ReferencesAlonso C, Herrera CM. 2001. Patterns made of patterns: variation

and covariation of leaf nutrient concentrations within and between populations of Prunus mahaleb. New Phytologist 150: 629–640.

Chapin III FS. 1980. The mineral nutrition of wild plants. Annual Review of Ecology and Systematics 11: 233–260.

Chapin FS III, Kedrowsky RA. 1983. Seasonal changes in nitrogen and phosphorus fractions and autumnal retranslocation in evergreen and deciduous taiga trees. Ecology 64: 376–391.

Hartley SE, Jones CG. 1997. Plant chemistry and herbivory, or why the world is green. In: Crawley M, ed. Plant ecology. Oxford, UK: Blackwell Scientific, 284–324.

Marschner H. 1986. Mineral nutrition of higher plants. London, UK: Academic Press.

McNaughton SJ. 1988. Mineral nutrition and spatial distribution of African ungulates. Nature 334: 343–345.

Ohlson M. 1988. Variation in tissue element concentration in mire plants over a range of sites. Holarctic Ecology 11: 267–279.

Pugnaire FI, Chapin III FS. 1993. Controls over nutrient resorption from leaves of evergreen mediterranean species. Ecology 74: 124–129.

Sultan SE, Bazzaz FA. 1993. Phenotypic plasticity in polygonum persicaria. III. The evolution of ecological breath for nutrient environment. Evolution 47: 1050–1071.

Valladares F, Martínez-Ferri E, Balaguer L, Pérez-Corona E, Manrique E. 2000. Low leaf-level response to light and nutrients in Mediterranean evergreen oaks: a conservative resource-use strategy? New Phytologist 148: 79–91.

Verhoeven JTA, Koerselman W, Meuleman AFM. 1996. Nitrogen- or phosphorus-limited growth in herbaceous, wet vegetation: relations with atmospheric inputs and management regimes. Trends in Ecology and Evolution 11: 494–497.

Key words: nutrient variability, nutrient dynamics, nutrient supply, phenotypic plasticity.


Frontiers in molecular mycorrhizal research – genes, loci, dots and spins

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