Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effects of...

Plant, Cell and Environment

(2002)

25,

75–84

©

2002 Blackwell Science Ltd

75

Blackwell Science, LtdOxford, UK

PCEPlant, Cell and Environment0016-8025Blackwell Science Ltd 2002251January 2002810Local ion fluxes along ectomycorrhizal roots of maritime pineC. Plassard et al.10.1046/j.0016-8025.2001.00810.xOriginal ArticleBEES SGML

Correspondence: C. Plassard. Fax: +33

467545

708; E-mail: [email protected]

Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effects of ectomycorrhizal symbiosis

C. PLASSARD,

1

A. GUÉRIN-LAGUETTE,

1

* A.-A. VÉRY,

2

V. CASARIN

1

† & J.-B. THIBAUD

2

1

Sol et Environnement, UMR 388, AgroM-INRA, and

2

Biochimie et Physiologie Moléculaire des Plantes, UMR 5004, AgroM/CNRS/INRA/UM2, Place Viala, 34060 Montpellier cedex 1, France

ABSTRACT

In order to assess the actual role of ectomycorrhizae in ionuptake by the ectomycorrhizal root system, we used amicroelectrode ion flux estimation methodology that pro-vided access to local values of net fluxes. This made it pos-sible to investigate the heterogeneity of ion fluxes along thedifferent types of roots of

Pinus pinaster

associated or notwith ectomycorrhizal species. We compared two fungi ableto grow with nitrate in pure culture,

Rhizopogon roseolus

and

Hebeloma cylindrosporum

, the former having a posi-tive effect on host tree shoot growth (

c.

+30%) and the lat-ter a negative effect (

c.

−−−−

30%). In non-mycorrhizal plants(control), NO

3–

was taken up at higher rates by the shortroots than by the long ones, whereas K

+

uptake occurredmainly in growing apices of long roots. In mycorrhizalplants,

H. cylindrosporum

did not modify K

+

uptake andeven decreased NO

3–

uptake at the level of ectomycorrhizalshort roots, whereas

R. roseolus

strongly increased K

+

andNO

3–

fluxes at the level of ectomycorrhizal short roots with-out any modification of the fluxes measured along the fun-gus-free long roots. The measurement of ion influxes at thesurface of the ectomycorrhizal roots can provide a wayto reveal actual effects of mycorrhizal association on iontransport in relation to mycorrhizal efficiency in naturalconditions.

Key-words

: Ectomycorrhizae;


;ion-selective microelectrodes; nitrate flux;

Pinus pinaster

;potassium flux;

Rhizopogon roseolus

; root structure.

INTRODUCTION

In natural conditions, the association of conifer species withectomycorrhizal fungi is the rule. This association takesplace only at the level of short roots through morphologicaland anatomical modifications resulting in the symbiotic

organs – the ectomycorrhizae – where complex exchangesbetween the fungus and the host-plant occur (Smith &Read 1997). It is generally assumed that this associationimproves the growth and the mineral nutrition of the host-plant (Smith & Read 1997). However, some studiesreported negative effects of mycorrhizal symbiosis on thegrowth of the host-plant (Colpaert, Van Assche & Luitjens1992; Conjeaud, Scheromm & Mousain 1996; Plassard,Bonafos & Touraine 2000). Similarly, reports dealing withmycorrhizal influence on the uptake of major ions such asNO

3–

or K

+

are controversial. In mycorrhizal plants, NO

3–

uptake was found to be either decreased (Sarjala 1991;Vézina

et al

. 1989; Kreuzwieser

et al

. 2000), increased (Box-man & Roelofs 1988; Rygiewicz, Bledsoe & Zasoski 1984;Plassard

et al

. 1994; Eltrop & Marschner 1996) or not mod-ified (Ingestad, Arveby & Kähr 1986; Scheromm, Plassard& Salsac 1990; Rygiewicz

et al

. 1984). K

+

uptake was lessstudied than NO

3–

but the effect of mycorrhizal symbiosiswas found to be either nil (Boxman & Roelofs 1988) or pos-itive (Rygiewicz & Bledsoe 1984). When performed inintact plants, most of these studies carried out for measur-ing uptake rates of nitrate or potassium relied upon varia-tion of ion concentration in the bulk solution bathing wholeroot systems, or upon root accumulation of

86

Rb as a tracerfor K

+

(Rygiewicz & Bledsoe 1984). Therefore, the uptakerates calculated from these measurements corresponded toaverage values for the whole root system (Robinson 1986).The root system generally consisted of various proportionsof ectomycorrhizae and roots free of fungus, whose func-tioning may be modified by the symbiosis, and even extra-radical hyphae. Thus, apart from the functional differencesthat may exist between fungal species leading to a positiveor a negative effect on the growth, the great heterogeneityof the mycorrhizal root system probably contributes to thevariability of reported data.

Local measurements of NO

3–

and K

+

fluxes along rootsare therefore necessary to improve our understanding ofmineral acquisition by woody plants and the actual role ofectomycorrhizal association in this acquisition. Such localmeasurements have been obtained in annual plants usingion-selective microelectrodes for NO

3–

(Henriksen, Bloom& Spanswick 1990; McClure

et al

. 1990; Henriksen

et al

.1992; Bloom 1997; Colmer & Bloom 1998; Taylor & Bloom1998) and for K

+

(Newman

et al

. 1987; Kochian, Schaff &Lucas 1989; Kochian

et al

. 1992). They made it possible todetermine ion fluxes at any place, on a micrometric scale,

Present addresses: *Laboratory of Forest Botany, Graduate Schoolof Agricultural and Life Sciences, The University of Tokyo, Yayoi1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan and †Soil ScienceDepartment, São Paulo University, ESALQ, C. P. 9, 13418–900,Piracicaba (SP), Brazil.

76

C. Plassard

et al.

© 2002 Blackwell Science Ltd,

Plant, Cell and Environment

,

25

, 75–84

along the root system under study (Henriksen

et al

. 1992;Newman 2001). So far, this methodology has never beenused in woody plants.

In this study, we aimed to assess the actual role of ecto-mycorrhizal symbiosis in NO

3–

and K

+

fluxes occurring inany part of intact, complex, ectomycorrhizal root systems,using a methodology providing access to local fluxes. Thiswas carried out on

Pinus pinaster

plants, whether associatedor not with two ectomycorrhizal species,

Rhizopogonroseolus

and

Hebeloma cylindrosporum.

These fungal spe-cies were chosen because they lead to opposite effects onplant growth.

MATERIALS AND METHODS

Plant and fungal culture conditions

Seedlings of

Pinus pinaster

Soland

in

Ait. were obtainedfrom germination of seeds carried out in sterile conditions(Plassard

et al

. 1994). In some experiments, we used 3-week-old non-mycorrhizal plants grown in sterile condi-tions in vertical tubes filled with vermiculite moistenedwith distilled water. We also used 2-month-old plants,whether non-mycorrhizal or associated with two ectomyc-orrhizal Basidiomycete species,

Rhizopogon roseolus

(Corda) Th. Fr. (strain 2) and


Romagn. (strain 9). Mycorrhizal synthesis were carried outas previously described (Plassard

et al

. 1994). Fungal spe-cies were also grown in pure culture in liquid medium(Plassard

et al

. 1994). All media contained NO

3–

as the solesource of N.

Plants were pre-treated before measurement of ionfluxes in order to obtain

P. pinaster

seedlings fully inducedfor NO

3–

uptake and maximal K

+

uptake. Each intact plantwas incubated in the growth chamber successively in100 mL of an aerated solution containing 0·2 mol m

−

3

CaSO

4

, 0·1 mol m

−

3

KNO

3

and 0·05 mol m

−

3

KH

2

PO

4

, pH5·7 for 3 d and then in 0·2 mol m

−

3

CaSO

4

, pH 5·7 for 16 h.In the case of ion flux measurement with ion-selectivemicroelectrodes, the plant was tied with a sewing threadonto a Perspex holder then placed in the experimentaldevice, as described in Plassard

et al

. (1999). The myceliagrown

in vitro

were pre-treated using the same solutions asfor plants, except that they contained 110 mol m

−

3

glucose.Pre-treatment of fungi was carried out in the dark.

In order to study the impact of ectomycorrhization onplant growth, mycorrhizal and control

Pinus pinaster

plantsgrown in tubes for 2 months were transferred into contain-ers containing natural soil (Cazevieille, near Montpellier,France). Analysis of this soil indicated that it contained, perkg of dried soil: 3 mg N-NH4

+

, 6 mg soluble organic-N, 2·5 gtotal organic-N, 33·5 g N-NO

3–

and 45 mol K

+

. Before use,the soil was previously enriched with insoluble mineralphosphorus (350 mg of P kg

−

1

soil), homogenized and ster-ilized by autoclaving. The plants were supplied with a nutri-tive solution (1·0 mol m

−

3

KNO

3

and microelements as inPlassard

et al

. 1994). The solution (50 mL per plant) wasadded each week and the plants were allowed to grow for

three more months in a growth chamber, as previouslydescribed (Plassard

et al

. 1994).

Root structure assessment

Main roots and secondary long roots were cut into samplesapproximately 2 cm long and embedded in 5% agar inorder to make longitudinal- and cross-sections using ahand-held razor blade. After discarding the agar, the sec-tions were bleached for 10 min with commercial sodiumhypochlorite 12% and rinsed with deionized water. Sec-tions were then incubated for 5 min with CH

3

COOH 1%,rinsed with deionized water and stained for 10 min with car-mine-green aqueous solution, producing a pink colour withcellulose and a green colour with suberin and lignin(Langeron 1949). This solution was made of 15 kg m

−

3

drycarmine red 40 for histology (Code International (C.I)75470) and 30 kg m

−

3

aluminium-potassium sulfate(AlKO

8

S

2

, 12 H

2

O). These first two compounds were dis-solved in deionized water (half of the final volume) andgently heated before adding the remaining deionized waterand 1 kg m

−

3

iodo-green (C.I. 42556). The solution wasboiled and, after cooling, it was filtered before use. The sec-tions were rinsed before observation. The variation oftransmembrane potential of cortical cells as a function ofthe root zone was determined by impaling the root cortexwith microelectrodes filled with 2 kmol m

−

3

KCl. The intactplant was placed in the experimental device (Plassard

et al

.1999) and bathed by flowing solution (usually 3 mL min

−

1

flow rate) containing 0·2 mol m

−

3

CaSO

4

and 20 mmol m

−

3

KNO

3

, pH 5·7. The microelectrode was impaled into theroot by 30 10

−

6

m steps between the surface and a quarter ofthe root diameter.

Ion-selective microelectrodes

K

+

and NO

3–

microelectrodes were made as previouslydescribed in Bertrand

et al

. (2000) using glass capillariescontaining a filling fibre (Clark Electromedical, GC150F).After pulling and silanization, electrodes were back-filledwith 0·4

µ

L of the appropriate ion-selective sensor. K

+

sen-sor was purchased ready to use (Fluka, ref. 60398), whereasNO

3–

sensor was adapted from Zhen, Smith & Miller (1992)and contained 0·5% methyltridodecylammonium nitrate(MTDDA NO

3–

), 0·084% methyltriphenylphosphoniumbromide (MTPPB) and 99·4% n-phenyloctyl ether(NPOE). This modified NO

3–

sensor enabled us to increasethe linearity range to 10 mmol m

−

3

NO

3–

. The pipettes werethen backfilled with 2·0 kmol m

−

3

KCl (K

+

microelectrodes)or 0·1 kmol m

−

3

KCl + 0·1 kmol m

−

3

KNO

3

(NO

3–

micro-electrodes). Tips were broken to a diameter of 5–10

µ

m toreduce microelectrode resistance to 1–3 G

Ω

in solutionscontaining 20 mmol m

−

3

K

+

or NO

3–

. Both microelectrodeswere used in the same experimental device as described inPlassard

et al

. (1999) and calibrated in KNO

3

solutions con-taining 0·2 mol m

−

3

CaSO

4

. They gave linear responsesdown to 10

−

5

kmol m

−

3

with slopes of linear regressions

© 2002 Blackwell Science Ltd, Plant, Cell and Environment, 25, 75–84

Local ion fluxes along ectomycorrhizal roots of maritime pine 77

varying between 56 and 59 mV per logarithmic unit of con-centration.

Determination of net ion fluxes with ion-selective microelectrodes

Ion fluxes were determined using the same experimentaldevice as described in Plassard et al. (1999). The room tem-perature was maintained at 20 °C. The plant was bathed byflowing experimental solutions (usually 3 mL min−1 flowrate) containing 0·2 mol m−3 CaSO4 and 20 or 65 mmolm−3 KNO3, pH 5·7.

Net ion fluxes were estimated from the measurements ofradial gradients of ion activity in the unstirred layer nearthe root surface as described in herbaceous plants (New-man et al. 1987; McCLure et al. 1990; Henriksen et al. 1992;Plassard et al. 1999). Net fluxes (J) were calculated accord-ing to the equation:

where (J) is the net ion flux (in µmol h−1 g−1 FW), D is theself-diffusion coefficient for the ion concerned (1·69 ×10−5 cm2 s−1 for K+, Newman et al. 1987, and 1·92 × 10−5 cm2

s−1 for NO3–, McClure et al. 1990), C1 and C2 are the activ-

ities of the ion concerned (mmol m−3) at radial distances r1

and r2 (cm) from the root axis, A is the cross-section area ofthe root (cm2), d is the density of root tissue (g cm−3) and kis a conversion factor for units (Newman et al. 1987). C1 wasmeasured at 10 µm from the root surface and C2 at 400 µmfrom the root surface. These radial distances were locatedin the depleted unstirred layer extending 900 µm from theroot surface for both ions in all types of roots (long or shortroots) (for an example, see Fig. 2).

Determination of net uptake by ion depletion from the medium

NO3– or K+ net uptake rates were estimated by measuring

ion depletion from the medium following the incubation ofintact 3-week-old non-mycorrhizal P. pinaster seedlings ormycelia grown in vitro. Plants were incubated in thegrowth chamber for 1 h in an aerated solution containing0·2 mol m−3 CaSO4 and 20 or 65 mmol m−3 KNO3, pH 5·7.Mycelia were incubated for 45 min in non-shaken condi-tions (Plassard et al. 1994) in pH 5·7 solution containing110 mol m−3 glucose, 0·2 mol m−3 CaSO4 and 20 or 65 mmolm−3 KNO3. Uptake rates were calculated from the differ-ence in ion concentration measured after 10 and 60 minincubation of the root systems, or after 15 and 45 min incu-bation of the mycelia. Concentrations of NO3

– and K+

were assayed using colorimetry (Plassard et al. 1994) andemission flame spectrometry (Scheromm & Plassard1988), respectively.

Analysis of the data

Means were submitted to multiple comparisons (one-wayANOVA, Fisher PLSD test, P < 0·05) using Statview SE +

J D C C Ad r r( ) = -( ) ( )[ ]2 2 1 2 1p ln

graphics® (Abacus Concepts, Inc., Berkeley, CA, USA) forMacintosh.

RESULTS

Root morphology of Pinus pinaster

Pinus pinaster forms complex and heterorhizic root sys-tems, comprising long roots with undefined growth thatbear short roots with defined growth. Individual long rootschange morphologically along their length as they age.However, from their colour, three root zones always can bedistinguished in long roots (main or lateral). As shownschematically in Fig. 1 for the main root of 3-week-old seed-lings, the root appeared first white in its apical zone (zone 1,0–5 mm), then pink (zone 2, up to 60 mm) and finally brown(zone 3). According to Triboulot, Pritchard & Tomos(1995), zone 1 consisted of the root cap, the meristem andthe beginning of the cell expansion region ending at c. 7 mmfrom the root tip, in zone 2. Therefore, most of zone 2 cor-responded to the mature region of the root. The rootbrowning was not linked to root suberization, as root-crosssections stained with carmine-green showed the presence ofcellulose and the absence of suberin in walls of epidermaland cortical cells of root zones 2 and 3 from long roots,whether main or lateral (data not shown). Measurements ofmembrane potential of the cortical cells in the differentroot zones showed that the highest values of potential weremeasured 50 mm from the apex and that the values mea-

Figure 1. Variation of root zones (a) and of membrane potential values of cortical cells (b) along the main root of non-mycorrhizal Pinus pinaster. Three-week-old seedlings were incubated in a solution containing 0·2 mol m−3 CaSO4 and 20 mmol m−3 KNO3 (pH 5·7). From their external colour, three main root zones were distinguished: white (1), pink (2) and brown (3). Membrane potentials were recorded after impaling a microelectrode by steps of 30 10−6 m between the surface and a quarter of the root diameter. Each point represents the average of at least three independent determinations (± SE).

78 C. Plassard et al.


sured in the white root tip were equal to those in the brownroot zone (Fig. 1).

Effect of inoculation on plant growth

Pinus pinaster plants, whether associated or not with Rhizo-pogon roseolus or Hebeloma cylindrosporum, were grownin pots containing natural soil as solid substrate and weresupplied with a nutrient solution containing NO3

– as thesole N source. After 3 months, root and shoot dry weights ofplants (Table 1) indicated that root growth was not modi-fied by the mycorrhizal association. On the contrary, shootgrowth was increased by 30% in R. roseolus mycorrhizalplants compared to the control, whereas it was decreasedby 30% in H. cylindrosporum mycorrhizal plants. Becauseof their opposite effects on plant growth, these two fungalspecies appeared as suitable experimental models forstudying the effects of mycorrhizal association on rootuptake of NO3

– and K+, two major nutrients supplied to theplant growing in the soil.

Nitrate uptake by the fungal species grown in pure culture

We measured the ion uptake capacities of both fungal spe-cies grown in pure culture. We focused on NO3

– uptakerates because, in our culture conditions, it may be suggestedthat uptake and assimilation of NO3

– is the main limitingfactor of the fungal growth. The uptake rates were esti-mated by measuring ion depletion from the medium con-taining initial KNO3 concentrations of 20 or 65 mol m−3. Asshown in Table 2, the two fungal species were able to takeup NO3

– from the medium. However, H. cylindrosporumincubated in KNO3 20 mol m−3 took up NO3

– at a lower ratethan R. roseolus.

NO3– and K+ net fluxes along main roots of Pinus

pinaster with ion-selective microelectrodes

Given the macroscopic complexity of P. pinaster root sys-tem, ion fluxes should vary as a function of root morphol-ogy and this aspect was investigated using ion-selective

microelectrodes. We used this methodology first in non-mycorrhizal Pinus pinaster with a very simple root systemconsisting of a single main root. Three-week-old seedlingswere incubated in a solution containing KNO3 20 mmolm−3. Concentrations of NO3

– and K+ were measured in theradial direction from the root surface to the bulk solution.The data demonstrated the occurrence of a large depletedunstirred layer (Fig. 2a & b), enabling us to estimate localion fluxes (Henriksen et al. 1992; Newman 2001). Figure 2(c& d) shows that net nitrate and potassium fluxes varied withrespect to the distance from the root tip. For NO3

–, netinfluxes occurred all along the root, whereas for K+, a netefflux was observed in the apical zone, between 0 and10 mm from the root tip, corresponding to zone 1 and thebeginning of zone 2 (see Fig. 1). For both ions, the highestuptake rates occurred between 20 and 50 mm from the roottip, corresponding to zone 2. The net fluxes were lower inthe brown zone starting 60 mm from the root tip.

Integrating the values of local fluxes determined withion-selective microelectrodes along the single root of theseyoung plants made it possible to estimate the rates of NO3

–

and K+ uptake per plant. The integrated values were com-pared with NO3

– and K+ uptake rates estimated by measur-ing ion depletion from liquid media. Both types ofmeasurements carried out on roots of the same length gavesimilar values of NO3

– and K+ uptake rates (Fig. 3a & b).This suggested that, in our experimental conditions, thedata obtained with ion-selective microelectrodes could pro-vide accurate estimation of net ion fluxes. This methodol-ogy can therefore be used to investigate the effect of themycorrhizal symbiosis on ion fluxes occurring either alongthe lateral long roots or at the surface of ectomycorrhizalshort roots.

NO3– and K+ net fluxes in fungus-free lateral

long roots

Subsequent experiments were carried out on 2-month-oldplants, whether mycorrhizal or not, with R. roseolus or H.cylindrosporum. All the plants exhibited a root systemcomposed of a main root with secondary lateral long roots.In the laterals, zone 2 ended at c. 45 mm from the apex andtertiary lateral short roots, whether mycorrhizal or not,were apparent 50–60 mm from the root tip, in zone 3.

Table 1. Biomass production of Pinus pinaster plants, whether non-mycorrhizal (control) or associated with R. roseolus or H. cylindrosporum. At time of harvest, plants were 5 months old and were previously grown in natural soil amended in P (350 mg P kg−1 soil) for 3 months. Plants were fertilized weekly with 1 mmol m−3 NO3

–. The values are the means ± SE (n ≥ 4). In each column, asterisks indicate data that are significantly different from the reference value (control) at P <0·05

Root biomass(g FW plant−1)

Shoot biomass(g FW plant−1)

Control 1·00 ± 0·07 0·69 ± 0·06R. roseolus 0·98 ± 0·17 0·90 ± 0·12*H. cylindrosporum 0·98 ± 0·18 0·47 ± 0·08*

Table 2. Net uptake of NO3– in mycelia of R. roseolus and H.

cylindrosporum grown in pure culture. Intact mycelia were incubated in solutions containing 0·2 mol m−3 CaSO4, 110 mol m−3 glucose, 20 or 65 mmol m−3 KNO3 (pH 5·7). Uptake rates of NO3

– were estimated from ion depletion from the medium. Values are means ± SE (n = 6). For each line, asterisk indicate significant difference between species at P ≤ 0·05

[KNO3] (mmol m-3)

NO3– uptake rate (nmol h−1 g−1 FW)

R. roseolus H. cylindrosporum

20 391 ± 62* 165 ± 1365 870 ± 80 600 ± 102



Observation of roots showed that in mycorrhizal plants, theapical root zone extending before appearance of short rootswas free of fungus. We first checked whether the mycor-rhizal association could affect ion fluxes in this fungus-freepart of the root system. NO3

– and K+ fluxes occurred mainlyat the level of zone 2 in all tested conditions, as found in themain long root of 3-week-old seedlings (see Fig. 2). In zone3, NO3

– influxes were 5-fold lower than the maximal influxin zone 2, whereas K+ fluxes were either nil or negative (notshown). In all the conditions tested, the longitudinal varia-tions of net NO3

– and K+ fluxes measured in control plantsdid not differ significantly from those measured in R. roseo-lus and H. cylindrosporum (not shown). Integration of thelocal flux values determined with ion-selective microelec-trodes all along the active 40 mm apical region led to similarvalues in control and mycorrhizal systems (Table 3). Thus,R roseolus and H. cylindrosporum scarcely affected theNO3

– and K+ exchanges in the fungus-free part of the rootsystem.

NO3– and K+ net fluxes in short roots from

ectomycorrhizal or control plants

In non-mycorrhizal Pinus pinaster plants, the lateral shortroots had an apex without any root cap. In mycorrhizalplants, the formation of ectomycorrhizae occurred exclu-

sively at the level of the lateral short roots that becameentirely covered by a continuous layer of fungal hyphaeconstituting the fungal sheath. Rhizopogon roseolusformed a thicker fungal sheath around the root than H.cylindrosporum, leading to values of root diameter greaterthan those of H. cylindrosporum and control ones (Table4). Approximating short roots to cylinders, it is possible tocalculate the amount of fresh matter per length unit of alltypes of root. For the same root length, the differencebetween the biomass of control and mycorrhizal rootsshould be due to fungal hyphae. Based on this hypothesis,R. roseolus and H. cylindrosporum represented, respec-tively, 62 and 23% of the total biomass of ectomycorrhizalroots (Table 4).

In our conditions, all short roots, whether mycorrhizal ornot, were 1·0–1·5 mm long. Ion fluxes were measured at thesurface of the ectomycorrhizal short roots in mycorrhizalplants and at the surface of non-mycorrhizal short roots incontrol seedlings at c. 0·7 mm from the root tip, in a regioncorresponding to non-elongating root cells (Harley &Smith 1983). Whatever the mycorrhizal status of the shortroots, radial measurements of concentrations of NO3

– andK+ from the root surface to the bulk solution showed alsothe occurrence of a large depleted unstirred layer (as in Fig.2, data not shown), enabling us to estimate local ion fluxes.

As shown in Fig. 4(a), NO3– influxes were measurable at

Figure 2. Estimation of NO3– and K+ net fluxes in roots of non-mycorrhizal maritime pines using ion-selective microelectrodes. Three-

week-old seedlings were incubated in a solution containing 0·2 mol m−3 CaSO4 and 0·02 mol m−3 KNO3 (pH 5·7). (a), (b) Concentration gradients of NO3

– (a) and K+ (b) near the root surface: the ion activity is plotted against ln(r) where r is the radial distance of measurement from the root axis (r in cm). The plain line shows a linear regression drawn using local concentrations probed between the root surface and 700 µm from the root surface. As described by Henriksen et al. (1992), the point of intersection of this line with that of 99% bulk ion concentration (horizontal dotted line) can be used to estimate the width of the depleted unstirred layer (vertical dotted line). In this example, the root radius is 1050 µm and the width of the unstirred layer is approximately 900 µm from the root surface for both ions. (c), (d) Longitudinal variation of net NO3

– (c) and K+ (d) fluxes estimated from ion concentrations measured at 10 µm from the root surface and 400 µm from the root surface, corresponding, respectively, to ln (r) values of −2·25 and −1·93 in (a) and (b). Each point represents the average of three independent determinations ± SE.



the surface of all types of short roots, whether mycorrhizalor not. Flux data were obtained in external solutions con-taining 20 or 65 mmol m−3 KNO3. The effect of mycorrhizalassociation depended largely on the fungal species. NO3

–

fluxes in R. roseolus ectomycorrhizae were always higherthan those obtained in control plants at both studied exter-nal KNO3 concentrations, whereas fluxes in H. cylin-drosporum ectomycorrhizae were lower than in the control.

In contrast to NO3– influxes, low and variable K+ influxes

were measured at the surface of non-mycorrhizal shortroots (Fig. 4b). In H. cylindrosporum ectomycorrhizae, datawere not significantly different from the control. By con-trast, a significant K+ influx was observed in short rootsassociated with R. roseolus in both solutions containing 20or 65 mmol m−3 KNO3.

DISCUSSION

Our aim was to determine the actual contribution of ecto-mycorrhizal association to ion acquisition by woody plantsby evaluating real values of NO3

– and K+ fluxes in maritimepine roots using ion-selective microelectrodes. In fast-grow-ing annual plants, this methodology made it possible thedetermination of ion fluxes on a micrometric scale (Hen-riksen et al. 1992; Newman 2001). However, Pinus rootsshowed a slow growth rate compared to young roots ofannual seedlings (e.g. maize primary roots) and, accord-ingly, low-rate ion exchanges with the surrounding solution.Despite this, in our experimental conditions, Pinus rootswere able to deplete the unstirred layer around themenough to allow detection of concentration gradients byion-selective microelectrodes (Fig. 2a & b). It should benoticed that a slowly flowing solution did not hamper theoccurrence of a large unstirred layer, thereby making it pos-sible to estimate local ion fluxes in these conditions. In addi-tion, as a strictly controlled rate of flowing solution wasmaintained during the experiments, all data were obtainedin a quasi steady state. This resulted in good reproducibilityof flux determination with essentially time-independentflux. Therefore, in our experimental conditions and withinthe time of our experiments, the fluxes that were obtaineddepended essentially on the location along the root system,thereby making it possible to investigate the contributionby the different parts of the root system to mineral acqui-sition. Moreover, the comparison of uptake rates estimatedusing either conventional methods (bulk medium deple-

Figure 3. Net uptake rates of NO3– (a) and K+ (b) determined in

whole roots of young non-mycorrhizal P. pinaster. Three-week-old plants presenting only one root, whose length varied from c. 50 to c. 160 mm, were incubated in a solution containing 0·2 mol m−3 CaSO4 and 20 mmol m−3 KNO3 (pH 5·7). Uptake rates were measured either from ion depletion from the medium (open symbols) or using ion-selective microelectrodes (closed symbols). In the latter case, local flux data obtained along the root were integrated over the whole root length to obtain net uptake rates per plant.

IonConcentration(mmol m−3)

Ion uptake rate (µmol h−1 g−1 FW) in:

Control R. roseolus H. cylindrosporum

NO3– 20 0·085 ± 0·007 0·068 ± 0·016 0·075 ± 0·008

65 0·200 ± 0·005 0·186 ± 0·009 0·180 ± 0·011K+ 20 0·089 ± 0·021 0·092 ± 0·011 0·097 ± 0·015

65 0·196 ± 0·038 0·191 ± 0·008 0·194 ± 0·011

Table 3. NO3– and K+ fluxes in fungus-free

lateral long roots of Pinus pinaster plants, whether non-mycorrhizal (control) or associated with R. roseolus or H. cylindrosporum. Local fluxes of NO3

– and K+ were first determined along the root region extending 40 mm from the apex before integrating them to get net uptake rates per unit of root fresh weight. The mean value of root fresh weight corresponding to this region was 85 ± 10 × 10−3 g. The values are the means of four measurements ± SE. In each line, no significant differences were found between the values of control and mycorhizal roots at P <0·05



tion) or NO3–- or K+-selective microelectrodes (Fig. 3)

assessed the relevance of the latter method.In Pinus pinaster seedlings, the long roots, main or lat-

eral, always displayed three different coloured zones. Asobserved in other Pine species (McKenzie & Peterson1995; Peterson et al. 1999), we found that root browningwas not linked to deposit of suberin in cortical cell walls.According to McKenzie & Peterson (1995), root browningin Pinus pinaster roots was therefore probably due to tan-nin deposit in the walls of the cortical cells. These authorsalso studied the vitality and the wall permeability of thecortical cells in roots of Pinus banksania by following cyto-plasm or nucleus staining with fluorescein, and the accumu-lation of berberine thiocyanate crystals, respectively. Incortical cells from the tannin zone, no cytoplasmic stainingwith fluorescein was apparent, whereas accumulation ofberberin thiocyanate crystals was observed in the cell wallsand at the inner face of the cortical cells (McKenzie &Peterson 1995). From these observations, the authors con-cluded that the cortical cells from the tannin zone were notalive but that the tannin-impregnated walls were perme-able to water and ions. In Pinus pinaster, however, wefound that cells from the tannin zone were able to maintainthe same values of membrane potential as those found inthe root tip, suggesting that these cells might be able toparticipate in nutrient uptake. Our results showed thatlocal fluxes varied greatly in relation to the root zone. NO3

–

uptake occurred mainly in the subapical zone 2 of the longroots, main or lateral. NO3

– influxes decreased strongly butwere, however, still measurable in zone 3, when the rootwas brown. Our data could therefore explain the results ofKronzucker, Siddiqi & Glass (1995), who showed that thespecific 13NO3

– uptake rates by old roots were typically 20–30% of those by young roots of non-mycorrhizal Piceaglauca. Contrary to NO3

– uptake, K+ uptake could bedetected only in zone 2, corresponding to the matureregion of P. pinaster root. Essentially no K+ uptake, andeven K+ release, was measured at the surface of zone 3,corresponding to completely brown roots. This differentialpattern with root differentiation could explain why the val-ues of N to K accumulation increased with time and rootdevelopment of non-mycorrhizal Pinus pinaster plants

Root type

Diameter(d)(10−6 m)

Estimatedroot biomassa

(g F wt m−1)

Estimatedfungalbiomassb

(g F wt m−1)

Ratiofungal/totalbiomass (%)

Control 506 ± 25 0·200 – –R. roseolus *826 ± 16 0·536 0·336 62H. cylindrosporum *575 ± 24 0·260 0·060 23

aRoot biomass was estimated from the root diameter approximating short roots to cylinders.bFungal biomass was estimated from the difference between mycorrhizal and control root biomass.

Table 4. Diameter, estimated total and fungal biomass of short roots of Pinus pinaster plants, whether non-mycorrhizal (control) or associated with R. roesolus or H. cylindrosporum. Diameter data were determined before NO3

– and K+ fluxes measurements carried out using ion-selective microelectrodes. The values are the means ± SE (n = 5). Asterisks mark diameter significantly different from the reference value (control) at P < 0·05

Figure 4. Net local fluxes of NO3– (a) and K+ (b) in short roots of

Pinus pinaster plants measured with ion-selective electrodes. Two-month-old intact plants, whether non-mycorrhizal (open bars) or associated with R. roseolus (closed bars) or H. cylindrosporum (grey bars) were incubated in a solution containing 0·2 mol m−3 CaSO4 and 20 or 65 mmol m−3 KNO3 (pH 5·7). The bars correspond to means of five measurements (± SE). Asterisks mark data significantly different from the reference value (non-mycorrhizal root) at P < 0·05. Total length of these short roots varied from 1·0 to 1·5 mm and ion concentrations were probed c. 0·7 mm from the root tip.



grown with NO3– as the sole N source (Scheromm & Plas-

sard 1988). We observed that a large although localized K+

efflux occurred 0–5 mm from the apex of long roots, corre-sponding to the division zone (Fig. 2c). This agrees with aprevious report of lower K+ uptake rates measured in the0–15 mm region when compared with older parts includingthe elongation zone of Picea abies roots (Häussling et al.1988). The mechanism underlying this K+ efflux, as well asits physiological function, remains to be determined. Inter-estingly, a similar pattern of net K+ flux was observed in theapical region of maize roots (Véry & Thibaud, unpub-lished), and is currently under detailed investigation on thisannual model. It should be emphasized that mycorrhizalassociation did not modify the uptake rates measured inthe apical part of the long root that was free of myceliumalthough bearing ectomycorrhizae in its basal part (Table2). These results demonstrated that the symbiosis did nothave any remote effect on the activity of the fungus-freepart of the root system.

In addition to long roots, P. pinaster plants have shortroots with a defined growth, which can be associated withectomycorrhizal fungi through the formation of ectomyc-orrhizae. In non-mycorrhizal plants, our data demonstratedthat these short roots are able to take up NO3

– even fasterthan the active part of a long root. By contrast, theyscarcely took up K+, compared to the active zone 2 of longroots, suggesting that the main sites of K+ uptake were theapices of long roots. In ectomycorrhizal plants, our resultsshowed that the mycorrhizal association dramaticallychanged ion fluxes measured at the surface of these shortroots (Fig. 4). The association with R. roseolus resulted inion fluxes significantly higher than those determined at thesurface of non-mycorrhizal roots. From the data given inTables 2 and 4 and Fig. 4, it is possible to calculate a theo-retical contribution of each partner from their amounts offresh matter and their individual NO3

– uptake rates. Theratios between the actual value of NO3

– fluxes measured inmycorrhizal roots and the sum of potential plant and fun-gal contributions were 2·0 and 1·8 in 20 and 65 mol m−3

KNO3, respectively, suggesting that a synergistic effect dueto the mycorrhizal association should occur. In contrast, H.cylindrosporum decreased the net NO3

– fluxes and did notmodify K+ fluxes measured at the surface of ectomycor-rhizae. Based on the same calculations as above, the ratiosbetween the actual value of NO3

– fluxes measured in H.cylindrosporum mycorrhizal roots and the sum of potentialplant and fungal contributions were 0·5 in both KNO3 solu-tions. These calculations suggest that a negative effect dueto the mycorrhizal association could occur when H. cylin-drosporum is associated with Pinus pinaster. As a result ofthese possible positive or negative synergistic effects, therewould be no clear relationship between the uptake capaci-ties measured in the hyphae grown in pure culture andthose measured in the hyphae associated with the roots inectomycorrhizae. This would agree with recent datareported by Voiblet et al. (2001) showing that the expres-sion level of several genes originating from the fungus or

the plant was altered in ectomycorrhizae compared to thelevel found in the symbionts in pure culture.

By contrast, it was shown that NO3– uptake rates of P.

pinaster plants associated with the same isolate of H. cylin-drosporum were always higher than those of control plants(Plassard et al. 1994). However in this latter study, NO3

–

uptake rates were measured by following NO3– depletion

from the medium after incubation of the whole root system.Therefore, it was not possible to distinguish between NO3

–

uptake of extra-radical hyphae (representing up to 30% ofthe total root dry weight) from that of ectomycorrhizalroots or roots free of mycelium. These uncertainties areeliminated using ion-selective microelectrodes and, takentogether, it may be suggested that the increased NO3

–

depletion previously observed in H. cylindrosporum-P. pin-aster plants (Plassard et al. 1994) was probably due to NO3

–

uptake in hyphae developing outside of the root.Taken as a whole, our data indicate that despite its com-

plexity, the root system of Pinus pinaster could be consid-ered as a sum of repeated root units consisting of a longroot mainly active in the pink zone (zone 2) and bearingshort roots, whether mycorrhizal or not. Our data made itpossible to calculate the contributions of the apical zone ofa long root and that of short roots to total root NO3

– uptake.In this study, 2-month-old plants presented n lateral longroots, each bearing an average of 10 short roots (10−3 mlong). We estimated the mean value of root fresh weightcorresponding to the active region of these laterals to be 85± 10 × 10−3 g. Using estimations of short root biomass (Table4), it is possible to calculate the ratio of biomass containedin 10 short roots to that contained in the active region of alateral root. These ratios were 0·06, 0·03 and 0·02 in R.roseolus, H. cylindrosporum and control plants, respec-tively. Using the data given in Table 2 and Fig. 4, it is alsopossible to calculate NO3

– uptake in short roots and activeroot zone. The sum of these latter values represents thetotal uptake of the long root, whether mycorrhizal or not.The ratios between the total uptake of a mycorrhizal longroot and a control root in 20 and 65 mmol m−3 KNO3 were,respectively, 1·31 and 1·28 for R. roseolus and 0·88 and 0·78for H. cylindrosporum. These calculations indicate that,despite their low proportion of biomass, mycorrhizal shortroots could quantitatively modify the uptake rates occur-ring in a whole lateral root. Interestingly, it should benoticed that the results of our calculations agree with theeffects of mycorrhizal association on plant growth, espe-cially for R. roseolus (Table 1). Although the effect of myc-orrhizal symbiosis on plant growth depends on manyfactors, such as the ability of the fungus to take up, to immo-bilize and/or to transfer mineral nutrients toward the hostplant, we suggest that the differential effect of ectomycor-rhizal symbiosis on plant growth could be related to differ-ences in ion uptake capacities occurring in ectomycorrhizalshort roots. Consequently, the measurement of ion influxesat the surface of the ectomycorrhizal roots with ion-selec-tive microelectrodes could be a good indicator of the actualmycorrhizal efficiency.



ACKNOWLEDGMENTS

This work was supported by the MYCOMED AIR2-CT94-1149 European Contract (EC DG XII). The authors thankDr H. Sentenac (INRA, Montpellier) for helpful discussionand constructive comments on the manuscript.

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Received 30 May 2001; received in revised form 21 September2001; accepted for publication 21 September 2001

Local measurements of nitrate and potassium fluxes along roots of maritime pine. Effects of...

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