where does all the carbon go? the missing sink

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Commentary

Pisolithus

– death of the pan-global super fungus

1977 saw this author in his first undergraduate year – it wasfairly uneventful, notable mainly as the year that ElvisPresley consumed his ultimate deep fried peanut butter andbanana sandwich, and Kilmarnock Football Club wasrelegated from the Scottish Premier League. Those interestedin the biology of

Pisolithus

may recall it also as the year thatD. H. Marx’s oft-cited review, ‘Tree host range and worlddistribution of the ectomycorrhizal fungus

Pisolithus tinctorius

’,was published. Based on literature citations, herbarium recordsand personal communications, Marx (1977) concludedthat

P. tinctorius

(Pers.) Coker and Couch occurred in 33countries on six continents and formed mycorrhizas withsome 51 tree species.

P. tinctorius

, it seemed, was somethingof a pan-global super fungus that might enhance forestproduction world-wide. Reasonable conclusions from theavailable information but, as recent work has revealed, it isnot quite that straightforward.

P. tinctorius sensu lato

represents a complex of species, and

P. tinctorius sensu stricto

is far more limited in both its host range and geographicaldistribution than was envisaged by Marx.

‘The ease with which DNA sequence data can be

compared between studies has facilitated rapid progress’

I encountered

Pisolithus

for the first time 10 years afterpublication of that review, and found myself an unwittingcontributor to the growing pan-global

P. tinctorius

myth. Asmany like me had done previously (and have done since), Iunquestioningly accepted that the

Pisolithus

isolate I hadreceived was

P. tinctorius

as described by Coker and Couch(1928). The paper published from this work indicates that

P. tinctorius

readily forms ECM with

Eucalyptus pilularis

andthat it obtains a considerable amount of recently fixed carbonfrom this host (Cairney

et al.

, 1989). To read between the linesof this paper is to believe that

P. tinctorius

occurs in Australiaand forms functionally compatible ECM with a

Eucalyptus

host.Not so, it turns out. The paper by Martin

et al

. (pp. 345–357in this issue) confirms that a number of

Pisolithus

species existworldwide, that

P. tinctorius

does not occur in Australia andthat

Eucalyptus

spp. are not natural hosts of this taxon.

Growing evidence for multiple

Pisolithus

species

To be fair to those who have variously contributed in thearea, the writing has been on the wall for some time. Severalauthors described heterogeneity in basidiospore ornamenta-tion within

Pisolithus

collections (Grand, 1976; Kope &Fortin, 1990). Isolates from certain hosts were found to bepoorly compatible with other host taxa, notably

Eucalyptus

and

Pinus

(Burgess

et al.

, 1994), and some inter-isolate matingincompatibilities were identified (Kope & Fortin, 1990).Burgess

et al

. (1995) also separated a range of

Pisolithus

isolateson the basis of expressed polypeptide patterns and basidiosporeornamentation, emphasising the existence of considerablevariation. Together, these observations pointed to the like-lihood that not all assumed

P. tinctorius

were conspecific.However, the relatively limited scope of individual studies,along with the global distribution of the

Pisolithus

complex,served to reduce the impact of these observations.

DNA-based analyses provided further convincing evidencefor multiple

Pisolithus

species, and the ease with which DNAsequence data can be compared between studies has facilitatedrapid progress. Anderson

et al

. (1998, 2002) separated threeputative species from Australian native sclerophyll forests byITS sequence comparison, two of which appeared to fit therecent descriptions of

P. albus

(M. C. Cooke & G. E. Massee)M. J. Priest

nom. prov.

and

P. marmoratus

(M. J. Berkeley)M. J. Priest

nom. prov.

(Bougher & Syme, 1998). Martin

et al

. (1998) proposed three phylogenetic

Pisolithus

speciesbased on combined ITS and IGS1 sequence comparisons ofbasidiome collections from Kenya. Several other groups haveused combined ITS-RFLP and isozyme (Sims

et al.

, 1999)or ITS sequence analyses (Gomes

et al.

, 2000; Díez

et al.

,2001) to separate other isolates from Asia, North America,Europe and Australia into several phylogenetic groups.Furthermore, these studies alluded to possible host and/orecological specificity for certain of the putative

Pisolithus

species, and inferred that Australian and northern hemisphere

Pisolithus

have been disseminated globally via exotic eucalyptand pine plantations, respectively.

How many species?

The paper by Martin

et al

. confirms each of these observations.It further provides a singularly broad view of phylogeneticrelationships among

Pisolithus

collections from a range of foresttypes worldwide. ITS sequence data generated by several ofthe above investigations have been combined with animpressive array of 102 new ITS sequences to leave no doubt

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that


comprises several species. Thequestion now is, how many? Martin

et al

. suggest 10 (11 if

P. aurantioscabrosus

is included). Delimiting species boundariesvia phylogenetic analysis is, of course, a subjective process,especially where based on data from only a single locus.According to Taylor

et al

. (2000), subjectivity can be reducedconsiderably by comparative analysis of data from two ormore loci. This, however, remains to be done for

Pisolithus

.For this reason, I believe that the species boundariesdescribed by Martin

et al

., particularly in lineages AII andBI wherein isolates were from pine/oak and eucalypt/acaciahosts, respectively, must at present be regarded with a degreeof caution. Moreover, as Bruns (2001) has recently highlighted,even a lack of ITS sequence variation cannot be taken asunequivocal evidence of conspecificity. Inclusion of comparativedata from other loci, along with data from further

Pisolithus

populations, may thus alter these putative species boundariessignificantly.

Using neighbour-joining analysis of ITS sequence datafor a limited number of

Pisolithus

isolates from central andeastern Australia, Anderson

et al

. (2002) observed two ter-minal groups that were equivalent to the species 7 (putative

P. albus

) and 9 (putative

P. microcarpus

) proposed by Martin

et al

. Anderson

et al

. (2002), however, considered that allprobably represented a single species (putative

P. albus

),based largely on the absence of any clear difference in basid-iospore ornamantation/size between the two groups. Prelim-inary data from my lab (C. J. Hitchcock

et al.

, unpublished)indicate that isolates from these two groups can be separatedby polymorphisms in two short simple sequence repeat-richregions. It is not clear if these represent alleles that havebecome fixed within two species or simply reflect polymorph-isms within the population of a single species. Screening ofmore isolates and comparison of polymorphisms at furtheralleles will help to clarify this point. Detailed morphologicalcomparisons between the various isolates should also aid inthis respect. Indeed, mating incompatibility tests may ulti-mately be required to clarify boundaries between putativephylogenetic/morphological

Pisolithus

spp. With all of theabove comments in mind, there may be merit at this stage inadopting a more circumspect approach to species delimita-tion and assignment of putative names to isolates within themajor lineages than taken by either Anderson

et al

. (2002)or Martin

et al

. Perhaps simple reference to

Pisolithus

isolates according to their placement in the lineages (e.g.AI, BII) identified by Martin

et al

. may prevent unnecessaryconfusion and back-tracking at a later date.

Final comments

Regardless of the number of species that arise from furthermolecular and morphological analyses of

Pisolithus

collections,the data presented by Martin

et al

. represent an excellentresource that should be used by all concerned with the

biology of

Pisolithus

. For the cost of obtaining an ITSsequence (these days not that much), we can place our isolateswithin the proposed lineages of the

Pisolithus

species complex.Not only will this allow better informed physiological andfunctional comparisons between isolates (and studies) but,by adding further ITS sequences to the databases, will increasethe available data. Placement of the sequence alignment on awebsite to allow direct downloading would be a good develop-ment – and regularly updating the alignment to accommodatenew ITS sequences submitted to the databases would furtherenhance its value. With such a resource at hand, it should bepossible to start making sense of the many reports in theliterature of considerable physiological variation betweenisolates of


(Chambers & Cairney, 1999).Martin

et al

. have clearly demonstrated that host preferenceof isolates is correlated with placement within the major

Pisolithus

lineages. The exciting process of investigatingecological adaptation and functional variation within andbetween the various

Pisolithus

lineages can now begin.

John W. G. Cairney

Mycorrhiza Research Group, Centre for Horticulture &Plant Sciences, Parramatta Campus, University of Western

Sydney, Locked Bag 1797, PENRITHSOUTH DC NSW 1797, Australia

(tel +61 29685 9903; fax +61 29685 9915;email [email protected])

References

Anderson IC, Chambers SM, Cairney JWG. 1998.

Molecular determination of genetic variation in

Pisolithus

isolates from a defined region in New South Wales, Australia.

New Phytologist

138

: 151–162.

Anderson IC, Chambers SM, Cairney JWG. 2002.

ITS-RFLP and ITS sequence diversity in

Pisolithus

from central and eastern Australia.

Mycological Research

(In press.)

Bougher NL, Syme K. 1998.

Fungi of Southeastern Australia

. Perth, Australia: University of Western Australia Press.

Bruns TD. 2001.

ITS reality.

Inoculum

52

: 2–3.

Burgess T, Dell B, Malajczuk N. 1994.

Variation in mycorrhizal development and growth stimulation by 20 Pisolithus isolates inoculated onto

Eucalyptus grandis

W.Hill ex Maiden.

New Phytologist

127

: 731–739.

Burgess T, Malajczuk N, Dell B. 1995.

Variation in

Pisolithus

based on basidiome and basidiospore morphology, culture characteristics and analysis of polypeptides using 1D SDS-PAGE.


99

: 1–13.

Cairney JWG, Ashford AE, Allaway WG. 1989.

Distribution of photosynthetically fixed carbon within root systems of

Eucalyptus pilularis

plants ectomycorrhizal with


.

New Phytologist

112

: 495–500.

Chambers SM, Cairney JWG. 1999.

Pisolithus

. In: Cairney JWG, Chambers SM, eds.

Ectomycorrhizal fungi: key genera in profile

. Berlin, Germany: Springer-Verlag, 1–31.

Coker WC, Couch JN. 1928.

The Gasteromycetes of Eastern United

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States and Canada

. Chapel Hill, NC, USA: University of North Carolina Press.

Díez J, Anta B, Manjón JL, Honrubia M. 2001.

Genetic variation of

Pisolithus

isolates associated with native hosts and exotic eucalyptus in the western Mediterranean region.

New Phytologist

149

: 577–587.

Gomes EA, de Arbeu LM, Borges AC, Araújo EF. 2000.

ITS sequence and mitochondrial DNA polymorphism in

Pisolithus

isolates.


104

: 911–918.

Grand LF. 1976.

Distribution, plant associates and variation in the basidiocarps of


in the United States.

Mycologia

68

: 672–678.

Kope HH, Fortin JA. 1990.

Germination and comparative morphology of basidiospores of

Pisolithus arhizus

.

Mycologia

82

: 350–357.

Martin F, Delaruelle C, Ivory M. 1998.

Genetic variability in intergenic spacers of ribosomal DNA in

Pisolithus

isolates associated with pine, eucalyptus and

Afzelia

in lowland Kenyan forests.

New Phytologist

139

: 341–352.

Martin F, Díez J, Dell B, Delaruelle C. 2002.

Phylogeography of the ectomycorrhizal

Pisolithus

species as inferred from nuclear ribosomal DNA ITS sequences.

New Phytologist

153

: 345–357.

Marx DH. 1977.

Tree host range and world distribution of the ectomycorrhizal fungus


.

Canadian Journal of Microbiology

23

: 217–223.Sims KP, Sen R, Watling R, Jeffries P. 1999. Species and population

structures of Pisolithus and Scleroderma identified by combined phenotypic and genomic marker analysis. Mycological Research 103: 449–458.

Taylor JW, Jacobson DJ, Kroken S, Kasuga T, Geiser DM, Hibbett DS, Fisher MC. 2000. Phylogenetic species recognition and species concepts in fungi. Fungal Genetics and Biology 31: 21–32.

Key words: Pisolithus, Pisolithus tinctorius, ectomycorrhizas, forest production, phylogeography, nuclear ribosomal DNA ITS sequences.

Letters

LettersLetters

Does zinc move apoplastically to the xylem in roots of Thlaspi caerulescens?Thlaspi caerulescens can accumulate very large amounts of zincin its tissues, which is of significant interest in commercialstrategies for obtaining plants able to remove Zn from con-taminated land – phytoremediation (Chaney, 1993; Salt et al.,1998; Schat et al., 2000). An understanding of the mechanismscontrolling Zn entry into the root and the accumulation ofZn within the shoot of such hyperaccumulators can informboth genetic modification and conventional breeding strategiesto obtain plants with improved phytoremediation potential(Lasat & Kochian, 2000). However, at present these mechan-isms are not clear. Zinc may reach the xylem (and hence theshoot) either through the symplast or apoplast. Currenthypotheses suggest that all Zn reaches the xylem through asymplastic pathway and that hyperaccumulation in T. caerulescensis the result of enhanced unidirectional influx of Zn2+ into rootcells, coupled to a greater Zn efflux to the xylem. Here, twoarguments are presented that suggest that Zn movement tothe xylem cannot be solely symplastic when roots are exposedto high Zn concentrations in the rhizosphere ([Zn]ext). First, therate of delivery of Zn to the xylem may exceed Zn influx toroot cells. Second, Zn influx to root cells cannot supply sufficientZn for hyperaccumulation with the combination of relativegrowth rates and shoot : root ratios observed in T. caerulescens.

Zinc hyperaccumulation

A Zn hyperaccumulator is defined as a plant with a shootZn content (tissue concentration) in excess of 10 mg Zn g−1

d. wt when growing in its natural habitat (Baker & Brooks,1989). They appear to have unusually active mechanismsfor Zn uptake and translocation to the shoot, as well as theability to detoxify excessive Zn2+ within the shoot. ElevenZn hyperaccumulators have been recorded, of which Thlaspicaerulescens is the most studied (Baker et al., 1994; Brooks,1998; Reeves & Baker, 2000; Broadley et al., 2001). This short-lived perennial occurs mainly on calamine soils (enriched withZn, Pb and often Cd), but also occurs on serpentine (enrichedin Co, Cr, Fe, Mg, Ni) and nonmineralised soils. It isextremely tolerant of Zn and, when grown hydroponically,can accumulate up to 25–30 mg Zn g−1 shoot d. wt withoutshowing toxicity symptoms (Baker et al., 1994; Brown et al.,1995; Pollard & Baker, 1996; Shen et al., 1997; Zhao et al.,1998). It is thought that T. caerulescens detoxifies Zn2+ in theshoot by decreasing its physiological availability within thecytoplasm, either by chelation or by sequestration withinthe vacuole (Shen et al., 1997). Studies using energy-dispersiveX-ray microanalysis to determine the spatial distribution ofZn within leaves of T. caerulescens concur with these conclu-sions (Frey et al., 2000). The characteristics of Zn toleranceand Zn hyperaccumulation in T. caerulescens are geneticallydetermined and genetically independent (Baker et al., 1994;Pollard & Baker, 1996; Meerts & Van Isacker, 1997; Schatet al., 2000). Thus, the selection and breeding for optimalcombinations of these traits could lead to improved varietiesfor phytoremediation.

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Delivery of zinc to the xylem

Zinc may traverse the root to the xylem either through thecytoplasmic continuum of root cells linked by plasmodesmata(symplast) or through the extracellular spaces between cells(apoplast). The symplastic pathway selects between cations,since it involves specific transporters in the plasma membraneof root cells. The apoplastic cation flux is largely determinedby the cation exchange properties of the cell wall and bywater flows (Sattelmacher, 2001). Despite decades of research,remarkably little is known about the relative contributionof the symplastic and apoplastic pathways to the delivery ofparticular cations to the xylem (White, 2001).

Recently, Lasat & Kochian (2000) presented a schematicmodel of Zn fluxes across root cell membranes to accountfor the phenomenon of hyperaccumulation in T. caerulescens(Box 1). This model contrasts the magnitude of the Zn fluxesacross the cell membranes of T. caerulescens with those acrossthe cell membranes of the related nonhyperaccumulatorspecies T. arvense. It is postulated that the entry point for Znaccumulation in the plant is across the plasma membraneof root cells and that all Zn reaches the xylem through asymplastic pathway in both species (Lasat et al., 1996; Lasat& Kochian, 2000). It is proposed that hyperaccumulationcan be explained by a fourfold greater unidirectional influx(φoc) of Zn2+ into root cells, coupled to a greater Zn effluxto the xylem (φcx), in T. caerulescens than in T. arvense. Inthe xylem sap of T. caerulescens much Zn is complexed toorganic acids (Lasat et al., 1998; Salt et al., 1999) or histidine(Schat et al., 2000); Schat et al. (2000) speculate that inter-specific variation in the xylem concentrations of these solutesmay also contribute to the trait of Zn hyperacumulation. Inaddition, it is proposed that T. arvense sequesters more Znin the vacuoles of root cells, having a greater unidirectionalinflux (φcv) and reduced unidirectional efflux (φvc) from thevacuole than T. caerulescens. The unidirectional efflux (φco)of Zn from the root cytoplasm to the solution was found tobe similar in the two species.

The model presented by Lasat & Kochian (2000) con-siders only the symplastic movement of Zn across the root.However, Zn may also reach the xylem via an apoplasticpathway. It is noteworthy that, when supplied with Zn, theZn content of shoots of T. caerulescens reaches that of majornutrient elements, such as Ca. Furthermore, like Ca2+, a lowcytoplasmic Zn2+ concentration in root cells is maintained(Brune et al., 1994; Reid et al., 1996). The total cytoplasmicZn concentration in plant cells appears to be homeostatic-ally maintained at approximately 55 µM (Brune et al., 1994;Reid et al., 1996). To deliver Zn to the xylem stream, whichmay contain in excess of 500 µM Zn2+ (Lasat et al., 1998),requires active transport across the plasma membrane of cellswithin the stele. This may be effected by a CPx-ATPase orby a H+/Zn2+ antiport (Williams et al., 2000; Clemens, 2001).It has been argued that such mechanisms are kinetically

(a) Nomenclature

(b) Thlaspi caerulescens

(c) Thlaspi arvense

Øoc Øcv

Øco Øvc

Joc JcvVacuole

Symplast

Øcx

Xylem

Box 1 Nomenclature and schematic representation of Zn fluxes across the root of Thlaspi caerulescens and Thlaspi arvense according to the model of Lasat & Kochian (2000). (a) The symplastic pathway across the root can be represented by unidirectional (φ) and net (J) fluxes across the plasma membrane between the cytoplasm (c) and the external solution (o) or xylem (x) and across the tonoplast between the cytoplasm (c) and the vacuole (v) of root cells. The apoplast of cortex and stele are hydraulically separated by the Casparian band. (b,c) The trait of hyperaccumulation is thought to result from greater Zn influx across the plasma membrane (φoc), reduced Zn influx to the vacuole (φcv) and greater Zn efflux to the xylem (φcx) in roots of T. caerulescens than in roots of T. arvense.


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challenged for Ca2+ delivery to the xylem (White, 1998,2001), and this is likely also to be true for Zn2+. Thus, byanalogy with Ca2+, it is likely that an apoplastic pathwaycontributes to the delivery of Zn2+ to the xylem. If this is thecase, then differences in the apoplastic movement of Zn acrossthe root may impact on the trait of hyperaccumulation.

Here, a simple test of the hypothesis that all Zn is deliveredto the xylem by a symplastic pathway is presented: if thesymplastic pathway operates exclusively, it is impossible forthe Zn flux to the xylem (φcx) to exceed the unidirectionalZn influx to root cells (φoc). This test employs published data.In addition, the physiological characteristics (relative growthrate, RGR, and shoot : root ratio) that would be required ifall the Zn accumulated by the shoot of T. caerulescens weredelivered to the xylem by a symplastic pathway across the roothave been determined. These are compared with publishedRGR and shoot : root ratios for T. caerulescens.

Not all zinc is delivered symplastically to the xylem

The rate of delivery of zinc to the xylem may exceed zinc influx to root cells

Unidirectional influx (φoc) has been estimated by allowingintact plants to accumulate Zn from solutions containingthe radioisotope 65Zn for a period of 20 min and thenwashing briefly in solutions containing nonradioactive Zn(Lasat et al., 1996; Pence et al., 2000; Lombi et al., 2001).Following this procedure, the dependence of Zn accumula-tion by T. caerulescens (A) on the solution Zn2+ concentration([Zn]ext) can be fitted to the sum of a saturatable (Michaelis–Menten) and a linear component:

A = ( (Vmax[Zn]ext)/(Km + [Zn]ext) ) + k[Zn]ext

(Km, the Michaelis constant; Vmax, the maximal rate of Zninflux through the Michaelian component; and k, theproportionality constant of the linear component.) It is arguedthat the linear component reflects 65Zn bound to the cellwall rather than influx to the root symplast (Lasat et al.,1996; Lombi et al., 2001). Thus, only the Michaeliancomponent reflects Zn influx (φoc) to the root symplast. TheMichaelian component saturates at low micromolar [Zn]ext(Table 1). This is a significant observation when one considersthat Zn translocation to the shoot does not saturate even atmillimolar [Zn]ext when plants are grown hydroponically(Brown et al., 1995; Tolrà et al., 1996, 2001; Shen et al.,1997; Zhao et al., 1998; Schat et al., 2000) or with 5 mg g−1

extractable Zn in the soil (Robinson et al., 1998; Küpperet al., 1999). (Since experiments suggest a maximum valueof about 1–3% for shoot Zn accumulation in T. caerulescens,it should be noted that the [Zn]ext at which the shootapproaches this maximal content will be determined bygrowth characteristics, such as RGR and shoot : root ratio,in addition to the kinetics of Zn translocation from the root(Fig. 2): this may explain the observations of Knight et al.(1997), who reported saturation of Zn translocation to theshoot at lower [Zn]ext.) Since the Vmax for Zn influx (φoc) toroots decreases in plants grown at higher [Zn]ext (Table 1), theapparent Km is even lower under steady state conditions (Fig. 1).

In addition to studying Zn influx to roots, Lasat et al.(1996) obtained data (i) on the accumulation of Zn in theshoot of 35 day-old T. caerulescens seedlings transferred toa solution containing 10 µM [Zn]ext over a 96-h period(14.7 nmol h−1 g−1 f. wt shoot), which allows an estimate ofthe rate of Zn translocation to the shoot at this [Zn]ext, and(ii) on the Zn content of the shoot of 32-d-old T. caerulescensseedlings that had been transferred to solutions containingvarious [Zn]ext for a 10-d period. The latter data allow thecalculation of the rate of Zn translocation to the shoot at

Table 1 Dependence of zinc accumulation (A) by intact Thlaspi species grown at different zinc concentrations on the zinc concentration in the assay solution ([Zn]ext)

Thlaspi speciesEcotype/Growth [Zn]ext

Vmax (nmol g−1 f. wt h−1)

Km (µM)

k (nmol g−1 f. wt h−1 µM−1) Reference

T. caerulescens Ganges/5 µM 176 0.30 8.6 Lombi et al. (2001)T. caerulescens Prayon/5 µM 239 0.92 10.0 Lombi et al. (2001)T. caerulescens Prayon/1 µM 270 8 3.15 Lasat et al. (1996)T. arvense Wisconsin/1 µM 60 6 3.57 Lasat et al. (1996)T. caerulescens Prayon/0 µM 244 4 nd Pence et al. (2000)T. caerulescens Prayon/1 µM 271 6 nd Pence et al. (2000)T. caerulescens Prayon/50 µM 76 5 nd Pence et al. (2000)T. arvense Wisconsin/0 µM 80 2 nd Pence et al. (2000)T. arvense Wisconsin/1 µM 43 2 nd Pence et al. (2000)T. arvense Wisconsin/10 µM 43 2 nd Pence et al. (2000)

Original data obtained at [Zn]ext ≤ 100 mM were fitted to the the sum of a saturatable (Michaelis–Menten) and a linear component: A = ((Vmax [Zn]ext)/(Km + [Zn]ext) ) + k[Zn]ext, where Km is the Michaelis constant, Vmax is the maximal rate of Zn influx through the Michaelian component and k is the proportionality constant of the linear component.


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other [Zn]ext provided that the shoot mass, shoot f. wt : rootf. wt ratio and tissue f. wt : d. wt ratios did not change with[Zn]ext, which seems reasonable under the conditions of theseexperiments (Brown et al., 1995; Shen et al., 1997; Zhao et al.,1998). Assuming a shoot f. wt : root f. wt ratio of 3.1 for T.caerulescens growing hydroponically (S. N. Whiting, unpub-lished), the rate of Zn translocation to the shoot at 10 µM[Zn]ext approximates 45.6 nmol h–1 g–1 FW root (Fig. 1),and increases substantially at higher [Zn]ext. This is consistentwith direct experimental observations (Brown et al., 1995; Tolràet al., 1996, 2001; Shen et al., 1997; Zhao et al., 1998; Schatet al., 2000). At [Zn]ext greater than about 27 µM the predictedrate of Zn translocation to the shoot exceeded that of Zninflux to the roots (Fig. 1). Thus, at [Zn]ext > 27 µM anapoplastic pathway must contribute to the delivery of Zn tothe xylem. For comparison, soils from seven European sitescontaminated by industrial activity or the disposal of sewagesludge had up to 20 µM Zn in solution when determined at80% field water holding capacity (Knight et al., 1997).

If additional, low-affinity Zn transporters contributed toZn influx across the plasma membrane of root cells, then thesecalculations would underestimate the potential symplasticZn flux to the xylem, especially at higher [Zn]ext. It is possiblethat part of the (substantial) linear component to thedependence of Zn accumulation by roots of intact plants and[Zn]ext (Lasat et al., 1996; Lombi et al., 2001) is due to low-affinity Zn influx into root cells. However, this linear com-ponent principally suggests the presence of a large apoplasticreservoir for Zn, which does not differ between T. caerulescensand T. arvense (Lasat et al., 1996). It would seem worthwhiletherefore to probe the relationship between this componentand any apoplastic pathway for Zn movement to the xylem.

Since the symplastic pathway selects between divalentcations, but the apoplastic pathway does not, an estimate ofthe relative importance of symplastic and apoplastic pathwayscan be obtained by comparing the accumulation factors (shootcation content/solution cation concentration) for differentdivalent cations. At low [Zn]ext (< 20 µM) the accumulationfactor for Zn was far greater than for Ca (Knight et al.,1997). However, the Zn concentration factor decreased as[Zn]ext increased (Knight et al., 1997), and the accumula-tion ratio for Zn approached the accumulation ratio for Cain plants grown hydroponically at [Zn]ext between 100 and500 µM (Tolrà et al., 1996). These data suggest an increas-ing contribution of the apoplastic pathway to the delivery ofZn to the xylem as [Zn]ext increases.

Relative growth rates and shoot : root ratios are incompatible with a symplastic pathway

An assessment of the contribution of the apoplastic pathwayto the delivery of Zn to the xylem in roots of T. caerulescenscan also be approached from a separate perspective. Thephysiological characteristics of T. caerulescens that would be

0

50

100

150

200

250

0 20 40 60 100[Zn]ext (µM)

Zn

influ

x (n

mol

h– 1

g–1

FW

)

80

Fig. 1 The relationship between unidirectional Zn influx (φoc) to roots, or Zn translocation to the shoot (φcx), in intact Thlaspi caerulescens growing hydroponically and the Zn concentration of the solution ([Zn]ext). Values for Zn influx (o) were calculated from the data presented in Table 1 from studies by Lasat et al. (1996) and Pence et al. (2000). The curve for Zn influx is interpolated to 1 µM [Zn]ext based on a Km of 7 µM and a Vmax of 270 nmol g–1 f. wt h–1 and extrapolated from 50 µM [Zn]ext based on a Km of 5 µM and a Vmax of 76 nmol g–1 f. wt h–1. Values for Zn translocation to the shoot (closed circles) were calculated from the data supplied by Lasat et al. (1996) as described in the text.

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ol g

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(a)

(b)

Fig. 2 The relationships between the maximum shoot Zn content ([Zn]shoot) of plants with relative growth rates of 0.03, 0.05 and 0.15 d−1 and (a) shoot f. wt : root f. wt ratio, assuming Zn influx to roots (φoc) of 80 nmol h−1 g−1 f. wt root, or (b) Zn influx to roots (φoc), assuming a shoot f. wt : root f. wt ratio of 3. The minimal Zn content of 32 µmol g−1 f. wt shoot for a Zn hyperaccumulating Thlaspi caerulescens is indicated by the horizontal line.


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required for a given shoot Zn content ([Zn]shoot) if all the Znwere delivered to the xylem by a symplastic pathway can bedetermined using simple allometric relationships.

If it is assumed that the root Zn content of T. caerulescensgrowing exponentially at a constant [Zn]ext does not change,and that all the Zn destined for the xylem traverses the rootthrough the symplastic pathway, then the maximal rate atwhich Zn could be translocated to the shoot (φcx), in theabsence of the recirculation of Zn within the plant, equalsthe rate of Zn influx (φoc) to the root symplast (Box 1). Bothφoc and φcx are conventionally expressed on the basis of rootFW (R). At a given [Zn]ext, the rate at which Zn is trans-located to the shoot (T) at time t is given by the equation:

Tt = φcxRt = φocRt

If it is also assumed that φcx is constant during exponentialgrowth, then the accumulation of Zn in the shoot followsthe exponential equation:

Znt = Zn0ebt

(Zn0, initial amount of Zn in the shoot; Znt, amount ofZn in the shoot after an interval t; and b, the relative accu-mulation rate of Zn in the shoot.) The rate at which Zn istranslocated to the shoot in the absence of Zn recirculationwithin the plant equals the rate at which Zn is accumulatedin the shoot and is given by:

Tt = bZnt = bZn0ebt

If it is assumed that both root and shoot have identicalrelative growth rates (RGR) and that these are identical tothe relative accumulation rate for Zn in the shoot (i.e. thatthe root f. wt : shoot f. wt ratio, R/S, and the shoot Zn con-tent are constant) then:

Tt = φcxRt = φcxR0ebt = φcxR/SS0e

bt

Finally, assuming that the shoot Zn content remains con-stant ([Zn]shoot = Zn0/S0), then:

[Zn]shoot = (R/S)(φoc/b)

Using this equation, the effect of shoot f. wt : root f. wtratio, RGR and φoc on the maximal [Zn]shoot supported byexclusively symplastic Zn transport across the root can beassessed in T. caerulescens.

Under most soil conditions it is unlikely that φoc exceeds80 nmol g−1 f. wt h−1 (Fig. 1; Pence et al., 2000) and thisvalue has been used for the calculations shown in Fig. 2(a).The shoot FW : root FW ratio of T. caerulescens growinghydroponically at low [Zn]ext approximates 3.1 (S. N.Whiting, unpublished). This value is consistent with the

shoot d. wt : root d. wt ratios observed in T. caerulescensgrowing hydroponically at [Zn]ext ≤ 500 µM (2.7–4.8;Shen et al., 1997; Zhao et al., 1998, 2001), although theshoot d. wt : root d. wt ratio increases markedly at higher[Zn]ext and the shoot d. wt : root d. wt ratio of plantsgrowing in contaminated soil may be more than triplethis value (McGrath et al., 1997; Whiting et al., 2000). (Thef. wt : d. wt ratio of shoots approximates 4.8 and the f.wt : d. wt ratio of roots approximates 5.7 in T. caerulescensgrowing hydroponically at low [Zn]ext (S. N. Whiting,unpublished)). The RGR of T. caerulescens growing hydro-ponically can be calculated from the data of Shen et al.(1997) as lying between 0.049 and 0.055 d−1, which isconsistent with the RGR of slow-growing, stress-tolerantplant species (Grime & Hunt, 1975). The RGR of plantsgrowing under suboptimal field conditions may, of course,be lower. Based on the definition that a Zn hyperaccumulatorhas a shoot content in excess of 10 mg Zn g−1 d. wt(Baker & Brooks, 1989) and assuming a f. wt : d. wt ratioof 4.8 for shoots of T. caerulescens grown hydroponically,a possible shoot Zn content of 32 µmol g−1 FW for T. caeru-lescens growing in Zn rich soils can be calculated. This shootZn content can be obtained only in plants combining alow shoot f. wt : root f. wt ratio with a low RGR (Fig. 2a),which may be the reason why metal hyperaccumulatorsgenerally grow slowly. However, published data suggest thatT. caerulescens does not possess an appropriate combinationof shoot f. wt : root f. wt ratio and RGR to allow exclusivelysymplastic delivery of Zn to the xylem at high [Zn]ext.Indeed, a maximal φoc of 80 nmol g−1 FW h−1 (Fig. 1;Pence et al., 2000) could not sustain the symplastic Znflux required for Zn hyperaccumulation in plants with ashoot FW : root FW ratio of 3.0 even at RGR as low as0.03 d–1 (Fig. 2b). An RGR of < 0.02 d−1 would be requiredif plants with a shoot f. wt : root f. wt ratio of 3.0 and aφoc of 80 nmol g−1 f. wt h−1 were to hyperaccumulateZn through a symplastic pathway. Thus, the perfectlycontrolled and selective hyperaccumulation of Zn in theshoot of T. caerulescens seems improbable. Some apoplastictransport of Zn to the xylem is likely to occur at high[Zn]ext and future studies should attempt to ascertain therelative contributions of the symplastic and apoplasticpathways to the delivery of Zn to the xylem in roots ofT. caerulescens.

Implications for phytoremediation

Previously, it was assumed that all Zn entering the xylem hadtraversed the root through a symplastic pathway. The argu-ments presented here challenge this assumption and suggestthat some Zn may reach the xylem through an apoplasticpathway, especially at higher [Zn]ext. This has importantimplications for identifying the targets for improving phyto-remediation potential: targets of the symplastic pathway are


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the Zn transporters in cell membranes, but targets of theapoplastic pathway may be more diverse.

Molecular targets for the manipulation of symplasticZn fluxes across the root to the xylem are beginning tobe identified. Genes for several Zn transporters likely tomediate Zn influx to root cells of T. caerulescens have beencloned. These include the high-affinity Zn transportersZNT1 and ZNT2 (Pence et al., 2000; Assunção et al.,2001). The expression of both ZNT1 and ZNT2 is up-regulated by Zn deficiency, which is consistent with anincrease in the Vmax for Zn influx to roots of plants with areduced Zn status (Table 1), and far greater in T. caerulescensthan in T. arvense, which is consistent with the greaterZn influx capacity of T. caerulescens (Table 1). The efflux ofZn to the xylem across the plasma membrane of cellswithin the stele of the root is likely to be mediated by aCPx-ATPase or by a H+/Zn2+ antiport (Williams et al., 2000;Clemens, 2001), but little is known about these transportmechanisms.

Molecular and physiological targets for the manipulationof apoplastic Zn fluxes to the xylem have yet to be identi-fied. However, experimental strategies for the identificationof such targets could be based on the apparent lack of cationselectivity and competition between cations following a pre-dominantly apoplastic route to the xylem (White, 2001).The trait of cation discrimination could be correlated withappropriate anatomical traits, such as the development of theendodermis, within a phylogenetic framework (cf. Broadleyet al., 2001). Alternatively, quantitative trait loci (QTL) impact-ing on Zn hyperaccumulation could be identified in mappingpopulations developed for this purpose between ecotypesdiffering in Zn hyperaccumulation (Pollard & Baker, 1996;Meerts & Van Isacker, 1997). These approaches would pro-vide independent verification of the cations sharing anapoplastic pathway, insight into the physiology of apoplasticcation fluxes and candidate genes impacting on the trait ofZn hyperaccumulation.

Acknowledgements

This work was supported by the Biotechnology and Bio-logical Sciences Research Council (UK). We thank Ian Burns,Abraham Escobar-Gutiérrez, Ken Manning and Brian Thomas(HRI) for comments.

Philip J. White1,3, Steven N. Whiting2, Alan J. M.Baker2 and Martin R. Broadley1

1Horticulture Research International, Wellesbourne,Warwick CV35 9EF, UK; 2School of Botany,

University of Melbourne, Victoria 3010, Australia3(Author for correspondence:

tel +44 (0) 1789 470382; fax +44 (0) 1789 470552;email [email protected])

ReferencesAssunção AGL, Da Costa Martins P, De Follter S, Vooijs R,

Schat H, Aarts MGM. 2001. Elevated expression of metal transporter genes in three accessions of the metal hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 24: 217–226.

Baker AJM, Brooks RR. 1989. Terrestrial higher plants which hyperaccumulate metallic elements – A review of their distribution, ecology and phytochemistry. Biorecovery 1: 81–126.

Baker AJM, Reeves RD, Hajar ASM. 1994. Heavy metal accumulation and tolerance in British populations of the metallophyte Thlaspi caerulescens J. & C. Presl (Brassicaceae). New Phytologist 127: 61–68.

Broadley MR, Willey NJ, Wilkins J, Baker AJM, Mead A, White PJ. 2001. Phylogenetic variation in heavy metal accumulation in angiosperms. New Phytologist 152: 9–27.

Brooks RR. 1998. Geobotany and hyperaccumulators. In: Brooks RR, ed. Plants that hyperaccumulate heavy metals. Wallingford, UK: CAB International, 55–94.

Brown SL, Chaney RL, Angle JS, Baker AJM. 1995. Zinc and cadmium uptake by hyperaccumulator Thlaspi caerulescens grown in nutrient solutions. Soil Science Society of America Journal 59: 125–133.

Brune A, Urbach W, Dietz K-J. 1994. Compartmentation and transport of zinc in barley primary leaves as basic mechanisms involved in zinc tolerance. Plant, Cell & Environment 17: 153–162.

Chaney RL. 1993. Zinc phytotoxicity. In: Robson AD, ed. Zinc in soil and plants. Dordrecht, The Netherlands: Kluwer Academic Publishers, 135–150.

Clemens S. 2001. Molecular mechanisms of plant metal tolerance and homeostasis. Planta 212: 475–486.

Frey B, Keller C, Zierold K, Schulin R. 2000. Distribution of Zn in functionally different leaf epidermal cells of the hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 23: 675–687.

Grime JP, Hunt R. 1975. Relative growth-rate: Its range and adaptive significance in a local flora. Journal of Ecology 63: 393–422.

Knight B, Zhao FJ, McGrath SP, Shen ZG. 1997. Zinc and cadmium uptake by the hyperaccumulator Thlaspi caerulescens in contaminated soils and its effects on the concentration and chemical speciation of metals in soil solution. Plant and Soil 197: 71–78.

Küpper H, Zhao FJ, McGrath SP. 1999. Cellular compartmentation of zinc in leaves of the hyperaccumulator Thlaspi caerulescens. Plant Physiology 119: 305–311.

Lasat MM, Baker AJM, Kochian LV. 1996. Physiological characterization of root Zn2+ absorption and translocation to shoots in Zn hyperaccumulator and nonaccumulator species of Thlaspi. Plant Physiology 112: 1715–1722.

Lasat MM, Baker AJM, Kochian LV. 1998. Altered Zn compartmentation in the root symplasm and stimulated Zn absorption into the leaf as mechanisms involved in hyperaccumulation in Thlaspi caerulescens. Plant Physiology 118: 875–883.

Lasat MM, Kochian LV. 2000. Physiology of Zn hyperaccumulation in Thlaspi caerulescens. In: Terry N, Banuelos G, eds. Phytoremediation of contaminated soil and water. Boca Raton, FL, USA: CRC Press LLC, 159–169.

Lombi E, Zhao FJ, McGrath SP, Young SD, Sacchi GA. 2001. Physiological evidence for a high-affinity cadmium transporter highly expressed in a Thlaspi caerulescens ecotype. New Phytologist 149: 53–60.


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McGrath SP, Shen ZG, Zhao FJ. 1997. Heavy metal uptake and chemical changes in the rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils. Plant and Soil 188: 153–159.

Meerts P, Van Isacker N. 1997. Heavy metal tolerance and accumulation in metallicolous and non-metallicolous populations of Thlaspi caerulescens from continental Europe. Plant Ecology 133: 221–231.

Pence NS, Larsen PB, Ebbs SD, Letham DLD, Lasat MM, Garvin DF, Eide D, Kochian LV. 2000. The molecular physiology of heavy metal transport in the Zn/Cd hyperaccumulator Thlaspi caerulescens. Proceedings of the National Academy of Sciences, USA 97: 4956–4960.

Pollard AJ, Baker AJM. 1996. Quantitative genetics of zinc hyperaccumulation in Thlaspi caerulescens. New Phytologist 132: 113–118.

Reeves RD, Baker AJM. 2000. Metal-accumulating plants. In: Raskin I Ensley BD, eds. Phytoremediation of toxic metals: using plants to clean up the environment. New York, USA: John Wiley & Sons, 193–229.

Reid RJ, Brookes JD, Tester MA, Smith FA. 1996. The mechanism of zinc uptake in plants. Characterisation of the low-affinity system. Planta 198: 39–45.

Robinson BH, Leblanc M, Petit D, Brooks RR, Kirkman JH, Gregg PEH. 1998. The potential of Thlaspi caerulescens for phytoremediation of contaminated soils. Plant and Soil 203: 47–56.

Salt DE, Prince RC, Baker AJM, Raskin I, Pickering IJ. 1999. Zinc ligands in the metal hyperaccumulator Thlaspi caerulescens as determined using X-ray absorption spectroscopy. Environmental Science and Technology 33: 712–717.

Salt DE, Smith RD, Raskin I. 1998. Phytoremediation. Annual Review of Plant Physiology and Plant Molecular Biology 49: 643–668.

Sattelmacher B. 2001. The apoplast and its significance for plant mineral nutrition. New Phytologist 149: 167–192.

Schat H, Llugany M, Bernhard R. 2000. Metal-specific patterns of

tolerance, uptake, and transport of heavy metals in hyperaccumulating and nonhyperaccumulating metallophytes. In: Terry N, Banuelos G, eds. Phytoremediation of contaminated soils and water. Boca Raton, Florida, USA: CRC Press LLC, 171–188.

Shen ZG, Zhao FJ, McGrath SP. 1997. Uptake and transport of zinc in the hyperaccumulator Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum. Plant, Cell & Environment 20: 898–906.

Tolrà RP, Poschenrieder C, Alonso R, Barceló D, Barceló J. 2001. Influence of zinc hyperaccumulation on glucosinolates in Thlaspi caerulescens. New Phytologist 151: 621–626.

Tolrà RP, Poschenrieder C, Barceló J. 1996. Zinc hyperaccumulation in Thlaspi caerulescens. I. Influence on growth and mineral nutrition. Journal of Plant Nutrition 19: 1531–1540.

White PJ. 1998. Calcium channels in the plasma membrane of root cells. Annals of Botany 81: 173–183.

White PJ. 2001. The pathways of calcium movement to the xylem. Journal of Experimental Botany 52: 891–899.

Whiting SN, Leake JR, McGrath SP, Baker AJM. 2000. Positive responses to Zn and Cd by roots of the Zn and Cd hyperaccumulator Thlaspi caerulescens. New Phytologist 145: 199–210.

Williams LE, Pittman JK, Hall JL. 2000. Emerging mechanisms for heavy metal transport in plants. Biochimica et Biophysica Acta 1465: 104–126.

Zhao FJ, Hamon RE, McLaughlin MJ. 2001. Root exudates of the hyperaccumulator Thlaspi caerulescens do not enhance metal mobilization. New Phytologist 151: 613–620.

Zhao FJ, Shen ZG, McGrath SP. 1998. Solubility of zinc and interactions between zinc and phosphorus in the hyperaccumulator Thlaspi caerulescens. Plant, Cell & Environment 21: 108–114.

Key words: apoplast, hyperaccumulation, phytoremediation, symplast, Thlaspi caerulescens, zinc.

Meetings

Where does all the carbon go? The missing sink

Tracing carbon in elevated CO2 experiments: a workshop on isotopic analyses of where the carbon is going, Durham, NC, USA, October 2001

As 164 nations (with the notable exception of the USA) signthe Kyoto Protocol to limit CO2 emissions to the atmo-sphere, fundamental questions about the carbon cycle remainunresolved. Elevated CO2 experiments can address such ques-tions, and following the early experiments demonstratingincreased growth – with ‘greening planet earth’ headlines –

these are now focussing on growth limitations. This shiftof focus has stimulated new generations of technologiesand research approaches to identify the ‘missing’ sink(s) inecosystems.

‘Stable isotope techniques are emerging as a critical

component in elevated CO2 experiments, without

which changes in soil carbon cycling would often be

impossible to detect’


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Growth stimulation and limitation

Experiments simulating elevated atmospheric CO2 concen-trations in terrestrial ecosystems may help track the fate of‘missing’ C that is emitted by anthropogenic activities, butwhose flux into known or unknown reservoirs is unquantified.These experiments were initially driven by plant scientistsfocused on phyotosynthetic and growth responses, and gar-nered national attention with reports of greatly enhancedcrop yields. Over the past 10–15 yr, as experimental approacheshave grown out of constant environment chambers to open-top chambers (OTCs) to Free-Air CO2 Enrichment (FACE)technology (Norby et al., 2001), the emphasis has shiftedto ecosystem level responses, and to interactions and feed-backs that may limit enhanced plant growth (DeLucia et al.,1999).

Recent literature reviews suggest that plant growth is almostuniversally stimulated (in C3 plants) at higher CO2 levels, aslong as other resources are not limiting. For example, wheatcrop yields, averaged over 156 experiments at doubled-ambientCO2 with adequate water and nitrogen, increased by c. 30%(Amthor, 2001). Tree seedlings and saplings exposed to doubledCO2 also average c. 30% more biomass, but these growthenhancements can be negated if other resources are limiting(Curtis & Wang, 1998), and may not be applicable tomature trees. Large uncertainties still exist, particularly withrespect to forest ecosystem responses and feedbacks to elevatedCO2. Although interactions with other factors (e.g. nutri-ents and water) are likely to prevent maximal C assimilationby plants, the potential for any enhanced assimilation andconsequent sequestration in plants and soils has stimulatednew approaches.

Where is the carbon going?

A recent symposium in Durham, NC, USA addressed thetopic of tracing carbon in elevated CO2 experiments, focussingon isotopic analyses. Stable isotopes (and 14C) are emergingas valuable tools to help quantify pools and fluxes of recentlyassimilated C in elevated CO2 experiments (Leavitt et al.,1994, 2001). Measurements of above-ground and below-ground net primary productivity (NPP), and net ecosystemCO2 exchange (NEE, e.g. with open-top chambers that serveas cuvettes) have been unable to resolve the fate of the ‘extra’C taken up (Drake et al., 1996; Cheng et al., 2000; Niklauset al., 2000). Although some techniques used to estimateNEE may consistently overestimate uptake (Niklaus et al.,2000), it is inherently difficult to detect ‘locally missing’ C.At roughly three times the size of the above-ground biomass,the soil C pool is an enormous reservoir, and incrementalchanges in absolute amounts resulting from elevated CO2are difficult to detect at high confidence levels with standardmeasurements of soil organic C content (Hungate et al., 1996).For instance, despite a 25% increase in loblolly pine NPP

during 3 yr of FACE, C content in bulk mineral soil did notincrease when variability in bulk density was accounted for(C concentration did increase significantly; Schlesinger &Lichter, 2001).

Stable C isotopes offer the potential to increase the resolu-tion in quantifying input of new C into soil reservoirs. TheCO2 used for fumigation in many elevated CO2 experimentsis derived from fossil fuels, and is strongly depleted in 13Crelative to background air. Therefore, plants in elevated CO2plots are incorporating a 13C tracer that can be tracked intoecosystem pools and fluxes. If the C isotope ratio (δ13C) ofnew soil organic matter (SOM) inputs is several permilledifferent from bulk SOM, a small change in the inventory ofthe pool may be detectable (Leavitt et al., 2001). However,a 13C tracer has not often been used for ambient treat-ments. δ13C values of SOM in the loblolly pine experimentwere significantly different between treatments, but no directcomparison in C input rate could be made owing to the lackof change in ambient δ13C inputs (Schlesinger & Lichter,2001). Nonetheless, during the workshop, the isotope tracerwas shown to be invaluable for shedding light on below-ground C cycling processes. Chris Van Kessel (UC-Davis,CA, USA) highlighted the use of δ13C to quantify new SOMinputs at the Swiss FACE experiment on managed pasture-land. The rate of change of new C in the soil was fitted to anexponential function to calculate turnover rates, input ratesand pool sizes under elevated CO2 at contrasting N levels andfor two different pasture species. Input rates did not varywith N treatment, but turnover rates were faster in the Loliumperenne than the Trifolium repens sward (Van Kessel et al.,2000b). Miquel Gonzales-Meler (U. of Illinois-Chicago,IL, USA) and Roser Matamala (Argonne National Laborat-ory, IL, USA) used this approach to demonstrate thatroot turnover rates of loblolly pine in the Duke ForestFACE experiment were slower than previously estimated byother techniques.

Experiments in controlled environment chambers or mes-ocosms are well suited for tracing C in both ambient andelevated treatments because a soil that contrasts isotopicallywith the plants of interest can be utilized (Cheng et al.,2000; Cardon et al., 2001). The situation is obviously moredifficult in large-scale FACE or OTC experiments. Leavittet al. (2001) demonstrate two new approaches to overcomethis problem: using subplots of soil with δ13C values thatcontrasted with the wheat crop; and pulse-labeling subplotsof the crop with 13CO2 in canopy gas exchange enclosuresat intervals throughout the growing season (labeling ofambient plots at a large scale or continuously would be pro-hibitively expensive). While the ‘transplanted soil’ producedambiguous results, pulse-labeling resulted in significant changesin δ13C of ambient SOM, and therefore detection of 4–6%new C input, comparable to 6% new C for FACE SOM. Atransplanted C4 soil did successfully allow new C inputs tobe calculated at elevated and ambient CO2 after 4 yr on the


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Swiss FACE (Van Kessel et al., 2000a). At the workshop,Leavitt presented results from the FACE sorghum experiment,in which ‘new’ C produced by the C4 plants had δ13C valuesthat contrasted with SOM values, and enabled quantificationof changes in below-ground inputs on all treatments with ahigh degree of certainty.

Field-scale elevated CO2 experiments in forests and otherC3 ecosystems cannot readily take advantage of contrastingC3–C4 isotope signals or pulse-labeling to trace new C inputson ambient treatments. An alternative approach to quantify-ing SOM dynamics in these cases involves the use of bombspike and natural abundance 14C signals. Jim Ehleringer(University of Utah, UT, USA) presented preliminary dataof Margaret Torn (Lawrence Berkeley Laboratory, CA, USA)demonstrating the utility of measuring both 13C and 14Con density separations of SOM, for greater resolution ofthe effects of elevated CO2 on turnover rates of active andslow C pools. Rolf Siegwolf (Paul Scherrer Institute, Villigen,Switzerland) was able to utilize the ‘dead’ CO2 (no 14C inthe geologically derived CO2) emitted from a natural springin Italy to estimate the CO2 enrichment in nearby vegeta-tion. These results were combined with 13C data to calculatethe relationship between photosynthetic discrimination of13C and CO2 enrichment (M. Saurer et al., unpublished).All of these approaches are still undergoing development,but will certainly lead to better quantification of changes insoil C sequestration under elevated CO2.

Partitioning soil respiration into decomposition and rhizosphere respiration

Quantifying the individual components of the soil respirationflux (i.e. rhizosphere respiration (including root respirationand microbial turnover of recent rhizodeposits) and organicmatter decomposition) is critical for predicting soil C sequestra-tion potential (Edwards & Norby, 1999). These two com-ponents will probably respond to climatic changes and feedback on atmospheric CO2 in contrasting ways. Increasedrates of rhizosphere respiration under elevated CO2 owingto increased root growth and turnover would have no neteffect on C storage in soil (Edwards & Norby, 1999). However,stimulation of SOM decomposition in response to a sub-strate induced priming effect (e.g. by enhanced availabilityof labile substrates) may cause net losses of soil C if inputsdo not keep pace (Zak et al., 1993). Stable C isotopes allownondestructive partitioning of respiration components if thereis a large isotopic difference between SOM and growing plants.Similar limitations apply to respiration flux partitioningand estimating new C inputs into SOM, in that ambienttreatments require a tracer (Hungate et al., 1997; Pendallet al., 2001).

A fortuitous disequilibrium between currently growing plantsand SOM at the Shortgrass Steppe OTC experiment hasallowed partitioning of soil respiration on all three treatments,

elevated and ambient chambers, as well as non-chamberedcontrol plots. Recent changes in grazing regime favored C3plants at this OTC site, where SOM δ13C reflects the 80%C4 component of nearby grasslands. Elevated CO2 in thisexperiment has resulted in a doubling of decompositionrates, but no change in rhizosphere respiration, relative toambient conditions (E. Pendall et al. unpublished). Elise Pendall(University of Colorado, CO, USA) presented preliminaryresults suggesting that despite enhanced decomposition ofslow pool C, new inputs are increasing at a faster rate, result-ing in net C accumulation under elevated CO2.

Partitioning the soil respiration flux allows estimation ofturnover of ‘old’ or slow pool C, but changes in turnover ratesof ‘new’ or labile pool C are difficult to quantify because theisotopic composition of rhizodeposits is indistinguishablefrom that of root respiration. Conversely, using 13C isotopesto estimate inputs of ‘new’ C into SOM pools neglects changesin turnover of ‘old’ C. Evaluation of the dynamics of boththe pools and fluxes of soil C is required to predict currentand future C sequestration in soils under elevated CO2.

Isotopic indicators of interactions

Examining interactions between elevated CO2 and otherfactors is increasingly important for understanding feedbacksthat affect ecosystem processes. At the workshop, the roleof N, water, O3, temperature, and multiple interactingfactors including food web and ecosystem structural changeswas discussed. Of particular interest was the possibility ofusing 15N as an indicator of feedbacks on C and N cyclingmediated by elevated CO2. For example, elevated CO2 resultedin soil water conservation in a growth chamber experimentwith Panicum coloratum; large changes in δ15N of soil nitratewere attributed to enhanced denitrification at the high CO2treatment, although no N2O flux measurements were reported(Robinson & Conroy, 1999). Changes in plant δ15N valuesare somewhat more complex to interpret, but Dave Evanspresented results from Sharon Billings (both at Universityof Arkansas, AR, USA) showing that the desert ecosystem Ncycle in the Nevada FACE experiment is being perturbed,with a possible increase in microbial activity.

Summary

Stable isotope techniques are emerging as a critical com-ponent in elevated CO2 experiments, without which changesin soil C cycling would often be impossible to detect. Newapproaches, many being developed by workshop participants,are overcoming limitations of the technique, such as the lackof a tracer in ambient CO2 treatments. Isotope studies ofcomplex interactions and feedbacks, such as perturbations ofsoil food webs, the N cycle and the soil water balance, willadd insights not otherwise apparent with more conventionalapproaches. Collecting appropriate samples and archiving them


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properly is essential for ensuring the possibility of addressingfuture research questions and conducting cross-site compar-isons. David Ellsworth (University of Michigan) and workshopparticipants have outlined sampling and archiving protocolsto be followed across the Elevated CO2 Network, and thesewill soon be publicly available (see http://www.gcte.org).

Acknowledgements

The workshop was sponsored by the Global Change in Ter-restrial Ecosystems (GCTE) core project of the InternationalGeosphere-Biosphere Programme (IGBP). The workshopparticipants are grateful to organizers David Ellsworth andDiane Pataki, and for funding from the Department of Energy’sOffice of Biological and Environmental Research and theFinnegan Corporation.

Elise Pendall

Institute for Arctic and Alpine Research, 450UCB,University of Colorado, Boulder, CO 80309, USA

(email [email protected])

References

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Leavitt SW, Pendall E, Paul EA, Brooks T, Kimball BA, Pinter PJ Jr, Johnson HB, Matthius A, Wall GW, LaMorte RL. 2001. Stable-carbon isotopes and soil organic carbon in wheat under CO2 enrichment. New Phytologist 150: 305–314.

Niklaus PA, Stocker R, Korner C, Leadley PW. 2000. CO2 flux estimates tend to overestimate ecosystem C sequestration at elevated CO2. Functional Ecology 14: 546–599.

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Pendall E, Leavitt SW, Brooks T, Kimball BA, Pinter PJ Jr, Wall GW, LaMorte R, Wechsung G, Wechsung F, Adamsen F, Matthias AD, Thompson TL. 2001. Elevated CO2 stimulates soil respiration in a FACE wheat field. Basic and Applied Ecology 2: 193–201.

Robinson D, Conroy JP. 1999. A possible plant-mediated feedback between elevated CO2, denitrification and the enhanced greenhouse effect. Soil Biology and Biochemistry 31: 43–53.

Schlesinger WH, Lichter J. 2001. Limited carbon storage in soil and litter of experimental forest plots under increased atmospheric CO2. Nature 411: 466–469.

Van Kessel C, Horwath WR, Hartwig U, Harris D, Luscher A. 2000a. Net soil carbon input under ambient and elevated CO2 concentrations: isotopic evidence after 4 years. Global Change Biology 6: 435–444.

Van Kessel C, Nitschelm J, Horwath WR, Harris D, Walley F, Luscher A, Hartwig U. 2000b. Carbon-13 input and turn-over in a pasture soil exposed to long-term elevated atmospheric CO2. Global Change Biology 6: 123–135.

Zak DR, Pregitzer KS, Curtis PS, Teeri JA, Fogel R, Randlett DL. 1993. Elevated atmospheric CO2 and feedback between carbon and nitrogen cycles in forested ecosystems. Plant and Soil 151: 105–117.

Key words: carbon, tracing carbon, elevated CO2, isotopic analyses, Kyoto Protocol.


Books


Forum 211

2002153(no issue no.)000000

Books

A holistic view of rhizosphere ecology

The rhizosphere: biochemistry and organic substances at the soil–plant interface

Ed. by Pinton R, Varanini Z & Nannipieri P. 1st edn. 424 pages. New York, USA: Marcel Dekker, Inc, 2001. $175.00 h/b. ISBN 08247 0427 4

This is the third book that aimsto present a holistic view ofrhizosphere ecology. The twoprevious texts (Curl & Trueglove,1986; Lynch, 1990) were good, sothe question arises as to whetherthis book can provide a worth-while addition to the library.The answer is a wholehearted yes.Unlike the previous volumes, thisbook attempts to assign a pivotalrole to root exudates driving therhizosphere. For this reason, most

of the book is dedicated to understanding the ecologicalsignificance of root exudates from various perspectives.The book is an edited collection of chapters, each of whichis written by an expert in its respective area. In most casesthe selection of authors appears well justified and this is borneout by the quality of texts that have emerged. However,through the multiauthored nature of the book it lacks a cer-tain degree of the coherence that is found in single-authoredtexts such as ‘Mineral nutrition of higher plants ’ (Marschner,1986). For example, the basic constituents of root exudates,and some of their interactions in soil, are repeated in manyof the chapters. Despite this, however, most of the chaptersare very well written, can be read in isolation and containup-to-date literature. In its entirety, the book brings togethermore than 1200 references on rhizosphere biology and so

provides a good synthesis of published material. Typically,each page contains 5–6 citations providing ample room forthe authors’ views to be expressed. The literature is criticallyassessed, in contrast to many books where the tendency issimply to bring information together without synthesizingit. The ‘future prospects ’ at the end of each chapter are also anindication of level of thought given by authors to this exercise.The book contains sufficient illustrations to highlight thekey points from each chapter.

With respect to readership, the book contains manychapters that will be beyond the capability of most under-graduate students as the book, quite rightly in my view,spends little time covering introductory material dealingwith soil science, plant physiology, microbiology, etc. Themain market for this book therefore will be researchers forwhom it provides a fundamental rhizosphere text. The bookis somewhat expensive, which will prevent adoption as ageneral undergraduate text and will also discourage purchaseby most individuals. This is a particular shame, as the truevalue of the book will probably be lost by its absence frommost researchers’ bookshelves.

In conclusion, I can thoroughly recommend this book asa worthwhile purchase for any science library and forresearchers with money left in the grant at the end of thefinancial year.

Davey Jones

School of Agricultural & Forest Sciences,University of Wales, Bangor, Deiniol Road, Bangor

Gwynedd Ll57 2UW, UK(tel +44 1248382579; fax +44 1248354997;

email [email protected])

References

Curl EA, Trueglove B. 1986. The rhizosphere. Berlin, Germany: Springer–Verlag.

Lynch J. 1990. The rhizosphere. London, UK: Wiley Interscience.Marschner H. 1986. Mineral nutrition of higher plants. London, UK:

Academic Press.



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where does all the carbon go? the missing sink

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