Plant and Soil215: 115–122, 1999.© 1999Kluwer Academic Publishers. Printed in the Netherlands.

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The significance of ectomycorrhizal fungi for sulfur nutrition of trees

Heinz RennenbergInstitut für Forstbotanik und Baumphysiologie, Professur für Baumphysiologie, Albert-Ludwigs-UniversitätFreiburg, Am Flughafen 17, D-79085 Freiburg i.Br., Germany∗

Received 17 April 1998. Accepted in revised form 14 December 1998

Key words:allocation, beech, cysteine,Fagus sylvatica, glutathione,Laccaria laccata, methionine, mycorrhiza-tion, oak, phloem,Quercus robur, sulfate, sulfur, uptake, xylem, xylem loading

Abstract

Sulfur nutrition of plants is largely determined by sulfate uptake of the roots, the allocation of sulfate to the sitesof sulfate reduction and assimilation, the reduction of sulfate to sulfide and its assimilation into reduced sulfur-containing amino acids and peptides, and the allocation of reduced sulfur to growing tissues that are unable tofulfill their own demand for reduced sulfur in growth and development. Association of the roots of pedunculateoak (Quercus roburL.) and beech (Fagus sylvaticaL.) trees with ectomycorrhizal fungi seems to interact withthese processes of sulfur nutrition in different ways, but the result of these interactions is dependent on both theplant and the fungal partners. Mycorrhizal colonisation of the roots can alter the response of sulfate uptake tosulfate availability in the soil and enhances xylem loading and, hence, xylem transport of sulfate to the leaves. Asa consequence, sulfate reduction in the leaves may increase. Simultaneously, sulfate reduction in the roots seemsto be stimulated by ectomycorrhizal association. Increased sulfate reduction in the leaves of mycorrhizal treescan result in enhanced phloem transport of reduced sulfur from the leaves to the roots. Different from herbaceousplants, enhanced phloem allocation of reduced sulfur does not negatively affect sulfate uptake by the roots of trees.These interactions between mycorrhizal association and the processes involved in sulfur nutrition are required toprovide sufficient amounts of reduced sulfur for increased protein synthesis that is used for the enhanced growthof trees frequently observed in response to ectomycorrhizal association.

Abbreviations:fw – fresh weight; mbbr – monobromobimane

Introduction

Association of roots with ectomycorrhizal fungi hasfrequently been observed to enhance the growth oftrees (e.g. Frank, 1884; 1885; Le Tacon and Bouchard,1986; Chalot et al., 1989; Marschner and Dell, 1994;Seegmüller and Rennenberg, 1994). This enhance-ment has mainly been attributed to a protection ofthe trees against soilborn root pathogens, an improveduptake of water, and an extended acquisition andassimilation of growth-limiting nutrients such as phos-phorus and nitrogen (Harley and Smith 1983; Bücking

∗ FAX No: +49 761 203 8302.E-mail: here@ruf.uni-freiburg.de

and Heyser, 1997; Martin and Lorillou, 1997). En-hanced growth and biomass accumulation is largelydependent on an increased rate of protein synthesis.This is of particular significance in annual plants andyoung trees where additional biomass is mainly in-vested into living, protein-containing tissue. But alsoin mature trees that preferentially accumulate wood,enhanced protein synthesis is required to adapt thecellular metabolism to an increased need for precurs-ors of structural components of the cell wall such ascellulose and lignin. Since reduced sulfur in the sulfur-containing amino acids cyst(e)ine and methionine isan essential constituent of protein, enhanced proteinsynthesis not only relies on an improved availabilityof organic nitrogen, but also of sulfur in the oxidation

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state -II. As a consequence, enhanced acquisition andassimilation of nitrogen in ectomycorrhizal symbiosisalone is not sufficient to improve growth. Even if sul-fur is present in the soil in excess of the tree’s demandrather than in growth-limiting amounts, sulfur uptakeand assimilation has to be adapted to an enhanced ac-quisition of nitrogen. Thus, interaction of mycorrhizalsymbiosis not only with nitrogen acquisition and as-similation, but also with sulfur nutrition of trees is tobe expected.

The present understanding of sulfur nutrition atthe whole plant level is mainly derived from invest-igations with herbaceous species (Rennenberg, 1995;Rennenberg and Herschbach, 1996; Herschbach andRennenberg, 1997). From these studies it appearsthat sulfate taken up by the roots is loaded into thexylem, transported to the leaves with the transpira-tion stream, unloaded from the xylem in the leavesand transported into the chloroplasts of the mesophyllcells. In this organelle, sulfate is reduced and incor-porated into cysteine. This sulfur containing aminoacid serves as central metabolic precursor of othercompounds which contain sulfur in the oxidation state-II, such as methionine and the tripeptide glutathione(γ -glutamylcysteinylglycine). Especially glutathioneis distributed within the plant via long-distance trans-port in phloem and xylem, in order to meet the wholeplant’s need for reduced sulfur in growth and devel-opment. In this way reduced sulfur is supplied totissues unable to reduce and assimilate sulfate in suf-ficient amounts. Glutathione allocated from the leavesto the roots by phloem transport is considered to ful-fill an additional function. If allocation of glutathioneto the roots exceeds the roots’ demand for reducedsulfur, glutathione may accumulate in the roots andmay inhibit further uptake and/or xylem loading ofsulfate. As a result the flux of sulfate to the shootcan be adapted to the requirement of the whole plantfor reduced sulfur (Rennenberg, 1995; Rennenbergand Herschbach, 1996; Herschbach and Rennenberg,1997). Thus, the glutathione content of the phloemis thought to be indicative of the sulfur nutritionalstatus of herbaceous plants (Lappartient and Touraine,1996).

In trees, sulfur nutrition and its regulation may bemuch more complex, because (a) storage and mobil-ization processes may determine sulfur nutrition, (b)shoot-to-root signaling by phloem-allocated reducedsulfur may not be efficient as a consequence of thelong distances to cover, and (c) sulfate reduction maybe distributed between the leaves and the roots (Her-

Table 1. Effect of the association of oak (Quercus roburL) rootswith the ectomycorrhizal fungusLaccaria laccataon sulfate trans-port processes. Oak seedlings were cultivated in autoclaved soil andin soil inoculated withLaccaria laccataunder controlled environ-mental conditions as previously described (Seegmüller et al., 1996).Sulfate transport processes were determined with excised mycor-rhizal and non-mycorrhizal roots (Seegmüller et al., 1996). The datashown are means of 72 independent experiments with one tree each.Standard error is given in parentheses. Different indices indicate sig-nificant differences between mycorrhizal and non-mycorrhizal rootsat p≤ 0.05

Mycorrhizal Sulfate uptake Xylem loading Relative xylem

colonisation (nmol SO2−4 (nmol SO2−4 loading

g−1 fw h−1) g−1 fw h−1) (% of uptake)

– 34.0a (38) 1.2a (39) 3.7a (25)

+ 38.4a (34) 2.2b (47) 5.6b (32)

schbach and Rennenberg, 1997). Recent field studieswith spruce trees growing at a nitrogen-limited sitederived at similar conclusions for nitrogen nutrition.Initially ammonium fertilization of the trees resultedin a strongly enhanced uptake of ammonium and ni-trate that was down-regulated only more than one yearafter fertilization (Rennenberg, unpublished results).During the period of enhanced nitrogen uptake, ni-trogen storage pools of the root and the shoot wereexpanded as a consequence of long-distance trans-port of amino-compounds; but when down regulationof nitrogen uptake was observed, soluble nitrogencompounds had accumulated specifically in the roots(Weber et al., 1998). Thus, plant internal nitrogen cyc-ling mediated both, storage of nitrogen and regulationof nitrogen uptake. Similar studies with trees growingunder sulfur-limitation are lacking.

In addition to tree internal processes, associationof the roots of trees with ectomycorrhizal fungi mayinfluence sulfur nutrition of trees at the level of (a)sulfate uptake and xylem loading and the regulation ofthese processes, (b) sulfate reduction and assimilation,and (c) allocation of reduced sulfur. The signific-ance of ectomycorrhizal fungi for sulfur nutrition oftrees has been addressed in recent studies with beech(Fagus sylvaticaL.) and oak (Quercus roburL.) as-sociated withLaccaria laccataand other ectomycor-rhizal fungi. In the present report, the results achievedin these studies are summarized and compared withpublished literature.

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Table 2. Kinetic parameters of sulfate uptake by excised roots of herbaceous and woodyplants. HAUS, high affinity uptake system; LAUS, low affinity uptake system. Data from(1) Kreuzwieser et al. (1996, 1998); (2) Seegmüller (unpublished results); (3) Herschbachand Rennenberg (1991); (4) Clakrson et al. (1983)

Species/mycorrhizal colonisation KM (µmol l−1) Vmax (nmol g−1 fw h−1)

Fagus sylvatica/ – 1 15 31

Fagus sylvatica/Laccaria laccata1

HAUS 15 19

LAUS 160 68

Fagus sylvatica/Paxillus involutus1

HAUS 13 25

LAUS 150 82

Quercus robur/Laccaria laccata2

HAUS 1.6 16

LAUS 3180 132

Nicotiana tabacum3 12 657

Macroptilium atropurpureum4 5 337

Sulfate uptake

Since plant-available sulfate constitutes only a minorfraction of sulfate in the soil (Mengel, 1993), act-ive sulfate transport is required at the soil–root in-terface for an appropriate sulfate supply of plants(Rennenberg, 1984; Cram, 1990; Clarkson et al.,1993). Active sulfate uptake by the roots has not onlybeen demonstrated for herbaceous plants but also fortrees (Herschbach and Rennenberg, 1997). At a givensulfate concentration mycorrhization did not influencethe rates of sulfate uptake by the roots of conifers,beech and oak trees (Morrison, 1962, 1963; Seeg-müller et al., 1996; Kreuzwieser et al., 1997; Table1). Depending on the fungal partner significant differ-ences in the kinetics of sulfate uptake were observedbetween mycorrhizal and non-mycorrhizal beech roots(Kreuzwieser, 1997; Table 2). Irrespective of my-corrhization, sulfate uptake showed bi-phasic kineticswith similar KM and Vmax for non-mycorrhizal rootsand roots inoculated withPaxillus involutus, but muchlower KM and Vmax for roots inoculated withLac-caria laccata (Kreuzwieser et al., 1998; Table 2).Apparently, the kinetics of sulfate uptake of mycor-rhizal beech roots largely depend on the fungal partner.At the relatively low sulfate concentrations frequently

found in the aqueous phase of forest soils (Geßler etal., 1998), sulfate uptake by the roots of trees mayalmost exclusively be mediated by high affinity up-take systems (HAUS, Table 2). Apparent Km valuesof HAUS for sulfate of trees were found to be sim-ilar to (beech), or lower than (oak) those found forthe roots of herbaceous plants (Table 2). By contrast,apparent Vmax values of HAUS for sulfate of treeswere generally lower (Table 2). This finding is con-sistent with low rates of sulfate uptake by the rootsof trees (Kreuzwieser and Rennenberg, 1998). It mayreflect the relatively slow growth of trees as comparedto annual plant species.

Recently, HAUS and LAUS have also been foundfor the thiol-containing tripeptide glutathione in my-corrhizal oak roots (Seegmüller, unpublished results).These uptake systems for organic sulfur operate at Km

and Vmax values similar to those for sulfate (Table 2).The physiological significance of these uptake systemsis obscure, since information on the concentration oforganic sulfur compounds in the aqueous phase of thesoil is lacking.

Regulation of sulfate uptake was found to dependon sulfur and nitrogen nutrition in non-mycorrhizalbeech, but to a lesser extend in mycorrhizal plants(Kreuzwieser, 1997). Nitrogen depletion diminished

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the rates of sulfate uptake by both mycorrhizal andnon-mycorrhizal beech roots. As previously foundin experiments with tobacco roots (Herschbach andRennenberg, 1991), sulfate uptake of non-mycorrhizalbeech roots was enhanced in response to sulfur de-ficiency and reduced when sulfur was supplied inexcess. This effect was not observed in short-termexperiments with mycorrhizal roots associated withPaxillus involutusor Laccaria laccata. Apparently, thefungal partner in mycorrhizal symbiosis contributessignificantly to the regulation of sulfate uptake. Thisview was not supported, however, by experiments inwhich mycorrhizal and non-mycorrhizal beech rootswere externally supplied with compounds that are con-sidered to regulate sulfate uptake (Kreuzwieser, 1997).Irrespective of mycorrhization, glutathione which in-hibits sulfate uptake by the roots of herbaceous plants(Herschbach and Rennenberg, 1991; Lappartient andTouraine, 1996), did not affect sulfate uptake by beechroots. L-Cysteine inhibited, whereas its metabolic pre-cursor O-acetylserine stimulated, sulfate uptake bymycorrhizal and non-mycorrhizal beech roots. Theseresults clearly demonstrate that observations on theregulation of sulfate uptake cannot readily be trans-ferred from herbaceous species to trees.

Xylem loading of sulfate

Sulfate taken up by the roots has to be loaded into thexylem in order to become available to xylem transportto, and sulfate reduction in the leaves. The relat-ive amount of the sulfate taken up that was loadedinto the xylem (relative xylem loading) was low inexcised mycorrhizal and non-mycorrhizal beech (10–15%; Kreuzwieser et al., 1996; Kreuzwieser, 1997)and oak roots (2.5–7%; Seegmüller et al., 1996; Table1) as compared to tobacco roots (25–50%; Herschbachand Rennenberg, 1991, 1994). In beech, absolute andrelative xylem loading were not affected by mycor-rhizal association withPaxillus involutusor Laccarialaccata (Kreuzwieser, 1997), whereas in oak bothparameters were enhanced when roots were inoculatedwith Laccaria laccata(Seegmüller et al., 1996; Table1). It may therefore be concluded that an interaction ofboth partners in ectomycorrhizal symbiosis determinesulfate nutrition at the level of xylem loading. Still thepositive effect of mycorrhizal association withLac-caria laccataon xylem loading of sulfate in oak rootsis surprising, since the hyphae of ectomycorrhizalfungi generally do not extend past the endodermis and,

Table 3. Sulfur compounds in the xylem sap of shootsand roots of young pedunculate oak trees (Quercus roburL) as affected by inoculation with the ectomycorrhizalfungus Laccaria laccata (Seegmüller, unpublished res-ults). Oak seedlings were cultivated in autoclaved soil andin soil inoculated withLaccaria laccataunder controlledenvironmental conditions as previously described (Seeg-müller et al., 1996). Xylem sap was collected as reportedby Rennenberg et al. (1996). Sulfate was determined byion chromatography (Kreuzwieser, 1997), reduced sulfurcompounds were quantified as mbbr-derivatives after sep-aration by reversed-phase HPLC (Schupp and Rennenberg,1988). The data shown are means of 24 independent ex-periments with one tree each. Standard error is given inparenthesis. Different indices indicate significant differ-ences between mycorrhizal and non-mycorrhizal roots atp≤ 0.05. n.d., not detected

Compound Mycorrhizal colonisation

(nmol ml−1) − +

Shoot

Sulfate 564a (48) 4697b (54)

Cysteine 2.1a (25) 0.4b (27)

γ -Glutamylcysteine 2.6 (87) n.d.

Glutathione 9.0a (50) 31.2b (150)

Root

Sulfate 2163a (45) 12378b (59)

Cysteine 7.2a (65) 10.4b (89)

γ -Glutamylcysteine 0.1 (27) n.d.

Glutathione 35.6a (79) 328.6b (79)

therefore, cannot interact directly with xylem loading.An explanation for the positive effect of mycorrhiza-tion on xylem loading may be a rapid reduction andassimilation of sulfate in the fungal compartment. Re-duced sulfur derived from sulfate reduction in thefungal compartment that is loaded into the xylem willthen simulate enhanced xylem loading of sulfate inlabeling experiments with35S-sulfate supplied to my-corrhizal roots. The finding of enhanced amounts ofreduced sulfur in the xylem sap of mycorrhizal as com-pared to non-mycorrhizal oak trees (Table 2) supportsthis assumption. Still it cannot be excluded that a reg-ulatory compound, so far unknown, is produced in thefungal compartment of mycorrhizal roots and trans-ported to the plant compartment in order to regulatexylem loading. In this case, ectomycorrhizal associ-ation should result in enhanced sulfate contents in thexylem sap.

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Table 4. Sulfur compounds in phloem exudates ofthe shoot and the root of young pedunculate oaktrees (Quercus roburL) as affected by inoculationwith the ectomycorrhizal fungusLaccaria laccata(Seegmüller, unpublished results). Oak seedlingswere cultivated in autoclaved soil and in soil inocu-lated with Laccaria laccataunder controlled envir-onmental conditions as previously described (Seeg-müller et al., 1996). Phloem exudates were collectedas reported by Herschbach et al. (1998), reduced sul-fur compounds were quantified as mbbr-derivativesafter separation by reverse-phase HPLC (Schupp andRennenberg, 1988). The data shown are means of 12independent experiments with one tree each. Standarderror is given in parenthesis. Different indices indic-ate significant differences between mycorrhizal andnon-mycorrhizal roots atp≤ 0.05

Compound Mycorrhizal colonisation

(nmol ml−1) − +

Shoot, acropetal

Cysteine 1.3a (86) 1.0a (119)

γ -Glutamylcysteine 3.6a (184) 1.9a (191)

Glutathione 8.5a (51) 13.5b (81)

Shoot, basal

Cysteine 1.1a (64) 0.9a (93)

γ -Glutamylcysteine 2.1a (175) 3.7a (181)

Glutathione 6.1a (74) 9.9b (77)

Root

Cysteine 1.2a (67) 1.3a (109)

γ -Glutamylcysteine 2.1a (192) 1.4a (212)

Glutathione 7.2a (81) 25.6b (144)

Sulfur allocation from the root to the shoot

Xylem sap analysis of young pedunculate oak treesshowed a 5- to 6-fold increased sulfate content in rootxylem sap of plants inoculated withLaccaria laccataas compared to xylem sap of non-mycorrhizal roots(Table 3). During xylem transport to and within theshoot, xylem sap sulfate content declined by morethan 50%. From studies with other species it maybe assumed that this decline is due to xylem-to-phloem exchange (Biddulph, 1956) and/or unloadingof sulfate for storage in the wood (Rennenberg etal., 1994; Herschbach and Rennenberg, 1996). Asin other tree species (Herschbach and Rennenberg,1997), considerable amounts of reduced sulfur werepresent in the xylem sap of pedunculate oak trees(Table 3). Different from beech, but similar to spruceand poplar, glutathione was the dominant reduced

sulfur compound in the xylem sap of pedunculateoak (Table 3). Cysteine andγ -glutamylcysteine werealso detected, but in much smaller amounts. Mycor-rhization withLaccaria laccataenhanced glutathionecontents in root xylem sap by almost a factor of10, whereas cysteine andγ -glutamylcysteine contentswere not significantly affected (Table 3). Like sulfate,glutathione was removed from the xylem sap duringlong-distance transport from the roots to the shoot.In mycorrhizal trees this reduction amounted to ca.90% (Table 3). Therefore, it appears unlikely thatglutathione in the xylem sap of pedunculate oak treesoriginates from glutathione produced in the leaves andsubjected to phloem-to-xylem exchange during basi-petal transport. This view is supported by the findingthat glutathione contents of phloem exudates showedonly minor differences between the root and the shoot(Table 4). Apparently, glutathione unloaded from thexylem during long-distance transport does not undergoa xylem-to-phloem exchange that previously has beenobserved in spruce (Schneider et al., 1994; Blaschke etal., 1996); rather it is used either for growth of, or stor-age in axial tissue. Glutathione in the xylem may eitheroriginate from sulfate reduced in the leaves, transpor-ted to the roots, unloaded in the root from the phloem,and re-loaded into the root xylem. Alternatively, gluta-thione loaded into the root xylem may originate fromsulfate reduction in the root. In this case sulfate reduc-tion and assimilation in the roots of pedunculate oakmust be strongly enhanced by mycorrhization withLaccaria laccata.

Distribution of sulfur reduction and assimilationbetween the roots and the shoot

Direct information on the rates of sulfate reductionand assimilation in the leaves and/or the roots of treespecies has not been published. The distribution ofindividual sulfur compounds (sulfate, reduced sulfur)and the enzymes of sulfate reduction and assimila-tion between the fungal and the plant compartmentof mycorrhizal roots is unknown. Information on thesubcellular distribution of these compounds and en-zymes within fungal and plant tissues of mycorrhizalroots is lacking. Still several pieces of evidence sug-gest that - probably different from annual plant species- a significant portion of the sulfate reduction in treestakes place in mycorrhizal and non-mycorrhizal roots.In spruce, the activities of enzymes involved in sulfatereduction and assimilation were found in mycorrhizal

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and non-mycorrhizal roots and were stimulated whenthe roots were exposed to cadmium (Galli et al., 1993).Stimulation of sulfate reduction may be required underthese conditions for enhanced production of gluta-thione, the metabolic precursor of cadmium chelatingphytochelatins (Rauser, 1990). Transport of reducedsulfur from the shoot to the roots has not been ob-served in spruce (Schupp et al., 1992; Schneider etal., 1994; Blaschke et al., 1996), but high amounts ofreduced sulfur are found in the xylem sap of spruceroots (Köstner et al., 1998). During most of the year,cysteine in the xylem sap of beech trees does not ori-ginate from protein breakdown in storage tissue orfrom phloem-to-xylem exchange. It, therefore, hasbeen assumed that sulfate reduced and assimilated inthe roots may meet the root’s and the trunk’s demandfor reduced sulfur in growth and development of beechtrees (Herschbach and Rennenberg, 1997). The con-tribution of ectomycorrhizal fungi to sulfate reductionand assimilation is still obscure, but the present find-ing of almost 10-fold enhanced glutathione contents inroot xylem sap of pedunculate oak as a consequence ofinoculation withLaccaria laccatastrongly suggest avital role of ectomycorrhizal association in sulfate re-duction and assimilation. As to whether reduced sulfuris exchanged between the fungal and the plant part-ner of mycorrhizal roots or whether sulfate reductionand assimilation of the plant partner of mycorrhizalroots is stimulated by the mycorrhizal fungus, remainsto be elucidated. The observation that inoculation ofpedunculate oak with the ectomycorrhizal fungusLac-caria laccataenhanced allocation of sulfate from theroots to the shoot (Table 3) suggests that mycorrhizalassociation may also improve sulfate reduction in theleaves.

Allocation of reduced sulfur from the shoot to theroot

Enhanced sulfate reduction in the leaves in responseto ectomycorrhizal association may either increasethe reduced sulfur content in the leaves and/or maystimulate allocation of reduced sulfur from the leavesto the growing tissues of the shoot and roots to fa-cilitate improved growth. Phloem exudate analysisof young pedunculate oak trees supports the latterpossibility (Table 4). Thiol contents of phloem ex-udate were somewhat higher in the upper shoot ascompared to the lower shoot and the root, indic-ating thiol loading in the upper stem sections and

partial unloading during long distance transport inthe phloem. Cysteine,γ -glutamylcysteine and gluta-thione contributed to phloem exudate thiol content,with glutathione dominating all along the transportpath. Mycorrhization withLaccaria laccataenhancedphloem exudate glutathione contents, particularly inthe roots, but did not affect the contents of cysteineand γ -glutamylcysteine significantly (Table 4). Ap-parently, phloem loading of glutathione in the upperpart of the stem and its allocation to the roots ex-ceeded phloem unloading in the roots. Since sulfateuptake of the roots was not affected (Table 1), phloemallocation of glutathione cannot be considered as asignal in demand-driven control of sulfate uptake ofpedunculate oak as suggested for herbaceous plants(Rennenberg, 1995; Lappartient and Touraine, 1996).Whether phloem allocated glutathione enters a pool ofsulfur that has been reduced in the roots remains to bedetermined.

Labeling experiments with35S-glutathione fed tothe leaves of oak trees showed that mycorrhizationwith Laccaria laccata does not affect glutathioneexport from the leaves; but the distribution of ra-diolabeled glutathione within the plant was altered inpreference of the stem (Schulte et al., 1998). Elevatedlevels of glutathione in phloem exudates in response tomycorrhization (Table 4) may therefore be consideredthe consequence of a reduced demand for organic sul-fur compounds in mycorrhizal roots. Enhanced sulfatereduction and assimilation in mycorrhizal roots maybe responsible for such a reduced organic sulfur de-mand as compared to non-mycorrhizal roots. Appar-ently, the stem is able to gain organic sulfur underthese conditions (Schulte et al., 1998).

Conclusion and future research needs

In contrast to previous assumptions (Morrison, 1962,1963), association with ectomycorrhizal fungi can sig-nificantly affect sulfur nutrition of trees, at the levelof xylem loading and its regulation, sulfate and re-duced sulfur allocation in xylem and phloem, as wellas sulfate reduction and assimilation. The mechanismsinvolved in these interactions are largely unknownand, therefore, should be analyzed in future studieson the significance of plant–fungal – interactions inectomycorrhizal symbiosis. In addition, the followingquestions have to be addressed to improve the presentunderstanding of sulfur nutrition of ectomycorrhizaltrees and its significance for the improvement of tree

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growth in response to ectomycorrhizal association: (1)Does the enhanced reduced sulfur content in the xy-lem of ectomycorrhizal trees originate (a) from sulfatereduction and assimilation in the fungal part of my-corrhizal roots, (b) from sulfate reduction and assimil-ation of the plant part of mycorrhizal roots, or (c) fromre-translocation of sulfate assimilated in the leaves andallocated to the roots by phloem transport? (2) Doesthe enhanced reduced sulfur content in the phloem ofectomycorrhizal trees (a) contribute to the growth ofthe fungal part of mycorrhizal roots, (b) contribute tothe growth of the plant part of the mycorrhizal roots,or (c) reflects an improved sulfur nutritional status as aconsequence of fungal sulfate reduction and assimila-tion? There is also an urgent need (a) to further explorethe significance of differences between ectomycor-rhizal fungi in sulfur nutrition, (b) to comprehensivelyanalyze the significance of sulfur metabolism, esp. ofglutathione biosynthesis, during the infection process,and (c) to further link studies of nitrogen and sulfurnutrition in mycorrhizal trees.

Studies with ectomycorrhizal systems in whichsulfate uptake, reduction and/or assimilation can bemodified in both the plant and the fungal partners bythe application of plant molecular biology techniquesmay be required to address most of these questions.At present tree–fungus combinations that allow suchan approach are not available. Recently, transforma-tion of poplar byAgrobacterium tumefacienshas beenachieved by routine techniques of plant molecular bio-logy and transformed trees were successfully used tostudy regulatory processes of sulfur metabolism (Noc-tor et al., 1998). It may therefore be useful to identifymycorrhizal fungi that are suitable for symbiosis withpoplar roots and can be transformed in the activityof sulfate uptake systems and regulatory enzymes ofsulfate reduction and assimilation.

Acknowledgements

Financial support of studies performed by the authorby grants of the Deutsche Forschungsgemeinschaft(Re 515/4) and the Commission of the EuropeanCommunities (EC EV5V-CT92-0093) is gratefully ac-knowledged. The author thanks S Seegmüller forproviding data so far unpublished (Tabs. 2 – 4).

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