Radiation modiﬁes the effect of water availability on the ... · SW, 36–58%). Soil proﬁles are characterized as terra fusca– rendzina derived from limestone (Weißjura beta

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Research

Blackwell Science Ltd

Radiation modifies the effect of water availability on the

carbon isotope composition of beech (

Fagus sylvatica

)

Arthur Geßler, Stefan Schrempp, Andreas Matzarakis, Helmut Mayer, Heinz Rennenberg and Mark A. Adams

Institute of Forest Botany and Tree Physiology, University of Freiburg, Am Flughafen 17, 79110 Freiburg, Germany

Summary

• Water availability and flux and carbon isotope and other chemical compositionsof wood, foliage and phloem sap were quantified in a replicated field test examiningthe effects of exposure (north-east, south-west) and stem density on beechecophysiology (

Fagus sylvatica

).• Standard techniques for quantifying water potential, water flux in stems andcarbon isotope composition of wood and foliage were complemented with chemicalanalysis of phloem sap collected using the ‘phloem bleeding’ technique.• Phloem sap was similar in composition to other hardwoods. The

δ

13

C signatures ofsap reflected short-term fluctuations in water availability and intercepted radiation,whereas foliar

δ

13

C was a poor reflection of current environmental conditions.•

δ

13

C of tree rings suggested that the south-west-facing site experienced a greatershortage of water during summer than the paired north-east-facing site. However,phloem

δ

13

C analysis showed that interpreting the effects of water availability using

δ

13

C might be confounded by radiation. Better integration of hydrological modelswith reliable indices of water stress in beech will be required for reliable predictionsof growth, especially if (when) climates change.

Key words:

Fagus sylvatica

,

carbon isotope discrimination

,

wood

,

foliage

,

phloem

,

water availability

,

light

.

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Author for correspondence:

Mark A. Adams Tel: +61 89380 3445 Fax: +61 89380 1001 Email: [email protected]

Received:

21 August 2000

Accepted:

10 January 2001

Introduction

Owing to their effects on internal concentrations of carbondioxide (CO

2

) (

C

i

), intercepted radiation and atmosphericand soil water deficits modify the ratio of

13

C :

12

C in plantcarbon (

δ

13

C, e.g. Leavitt & Long, 1986; Livingston &Spittlehouse, 1996; Korol

et al.

, 1999). For example, rates ofphotosynthesis are generally greater in sunlit foliage than inshaded foliage, leading to a greater reduction in

C

i

and agreater depletion in

13

C (Leavitt & Long, 1986). Even morewell described is the reduction in

C

i

caused by stomatalclosure in response to water deficits (e.g. Guehl

et al.

, 1995;Stewart

et al.

, 1995; Lauteri

et al.

, 1997; Panek & Waring,1997).

The relatively large body of literature dealing with

δ

13

C inconifers shows that a number of other factors (e.g. altitude,nutrition, hydraulic architecture) also play roles in determin-ing

δ

13

C (e.g. Walcroft

et al.

, 1996; Korol

et al.

, 1999; Hultine& Marshall, 2000; Warren & Adams, 2000) in wood andfoliage. For example,

δ

13

C varied with shoot water potential

at a rate of –0.18‰ MPa

–1

in

Quercus

spp. (Damesin

et al.

,1998) and with altitude at a rate of approx. 2‰ km

−

1

innumerous species and studies (e.g. Körner

et al.

, 1991). Mostof the

δ

13

C studies have been ‘observational’ and there are fewfield-based, controlled tests of the effects of environmentalconditions (e.g. availability of radiation, water, nutrients) on

δ

13

C in plant tissues (e.g. Morecroft & Woodward, 1990).From one controlled field experiment, Högberg

et al

. (1993)provided evidence for the effects of nitrogen fertilization on

δ

13

C signatures in foliage of

Picea abies

and

Pinus

sylvestris

.As for conifers, both simple assessments of water avail-

ability (e.g. Macfarlane & Adams, 1998) and modelled waterbalances (Dupouey

et al.

, 1993) show moderately strongrelationships with

δ

13

C signatures of the wood in recent(e.g. < 10 yr) annual growth rings of hardwoods. Over thelonger term, increases in atmospheric concentrations of CO

2

,and variations in the leaf-to-air humidity gradient and in leaftemperature will all influence

δ

13

C (Comstock & Ehleringer,1993). For both conifers and hardwoods, modelled waterbalances for the growing-season, and sometimes for periods as

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short as 1 month, provide the strongest relationships (up to93% of variance in

δ

13

C explained) between

δ

13

C of woodand indices of environmental conditions (Dupouey

et al.

,1993; Livingston & Spittlehouse, 1996). Like most, thesemodels were developed using data from more or less ‘ideal’sites with homogeneous vegetation and soil.

Recently, Yoneyama

et al

. (1997) provided a first report ofthe isotope ratios in phloem sap for wheat that was followedby Pate & Arthur (1998) and Pate

et al

. (1998) for

Eucalyptusglobulus

. Part of the attention on phloem sap originates froma desire to obtain a better estimate of the

δ

13

C signatures ofleaf sugars – some of the first products of carbon fixation –and thus an estimate of the effects on

δ

13

C of current environ-mental conditions. Hence the determination of

δ

13

C in phloemsap, containing carbon assimilated in a period of hours todays before sampling, is useful to assess short-term variationsin

C

i

/

C

a

(Brugnoli

et al.

, 1998; Pate & Arthur, 1998). Suchdata are required to help explain why the relationshipbetween foliar

δ

13

C signatures and current environmentalconditions varies so greatly among species, genera and climates.In the case of beech, new season’s foliage, like that of manyother species, is formed largely from stored carbon andnutrients (e.g. Millard & Proe, 1993; Kozlowski & Pallardy,1997) and thus more likely to carry a

δ

13

C signature thatreflects the environmental conditions for carbon fixation inprevious seasons than the current one. Clearly, the determina-tion of

δ

13

C in phloem sap provides a strong guide to

C

i

/

C

a

during the present growing-season. Yoneyama

et al

. (1997)used aphids to sample phloem sap, whereas Pate

et al

. (1998)relied on phloem sap that bled from shallow incisions inthe bark. Irrespective of the method, the ability to obtainphloem sap, will ‘enable more rigorous tests of prediction andreality for

δ

13

C and

δ

15

N, and determination of the uses ofnatural abundance measurements in improving our under-standing of C and N metabolism and transport’ (Yoneyama

et al.

, 1997).Water availability is a limiting factor for growth of beech

(

Fagus sylvativa

) in central Europe (Ellenberg, 1995). Climatemodels for central Europe predict an increase in temperatureand longer periods of drought during the growing-season(Enquete Kommission, 1994). Reductions in soil water avail-ability will, arguably, strongly influence patterns of com-petition between drought-sensitive beech and less sensitivespecies. Both mature and regenerating stands of beech growon the shallow, rendzina soils derived from limestone that arewidespread in southern Germany (e.g. Schwäbische Alb).These soils often have poor water-holding capacity. Equallyimportantly, there is strong political and social support for thetransformation of spruce monocultures into site-adapteddeciduous forests with beech as the dominant species. At thepresent time, we lack the necessary physiological basis for reli-able prediction of either the likely success of re-establishmentof beech on shallow, rendzina soils or effective managementstrategies, especially the management of stand density.

Advantage was taken of the opportunity presented by areplicated field trial in southern Germany to characterize theinteracting effects of radiation and water availability on

δ

13

Csignatures in phloem sap, foliage and wood, in order to obtaininformation about temporal variation in

C

i

. By combiningthese data with the determination of water potential of twigsand soil, tree water use and stand transpiration, it was hopedto validate

δ

13

C signatures of different tissues as indices ofwater availability and incident radiation in beech forests thatwere sensitive to typical management treatments. The trialincluded paired sites that differed mainly in aspect on eitherside of a narrow valley and, within each site, replicated plotsof differing stand density. The broad hypotheses, based onpast records of growth and vegetation analysis, were that themore south-facing site would have less available water andgreater radiation than the more north-facing site, and thatthinning would increase the availability of water. Additionally,an aim was to characterize ‘phloem bleeding’ as a means ofsampling phloem sap from beech. This sampling technique isa prerequisite to characterizing

δ

13

C signatures in the phloemsap of adult beech trees, since other techniques are difficult touse in the field (aphid technique) or introduce C compoundsinto the sampling solution (Schneider

et al.

, 1996), therebyconfounding interpretation of

δ

13

C signatures.

Materials and Methods

Study site, climate and experimental design

The experimental sites are located in the south of Germany,about 100 km south-south-west from Stuttgart (longitude:8

°

40

′

E; latitude: 48

°

00

′

N) in a low mountain range(Schwäbische Alb, 740–760 m above sea level). The meanannual air temperature at the site is about 6.6

°

C, and themean temperature during the growing-season (April toOctober) is about 11.5

°

C. The average annual precipitation is810 mm, with monthly maxima in June and July. In 1999both mean air temperature and total precipitation weregreater than long-time averages (Table 1). In July and August,potential evaporation (

Vp

) exceeded precipitation.The experimental sites are located on the two opposing

sides (not more than 500 m apart) of a single, narrow valley.On one site, a set of experimental plots face the north-east(NE), while on the other site the plots face the south-west(SW). Rainfall does not vary significantly across the valley.The slope of both sites is moderately steep (NE, 58–100%;SW, 36–58%). Soil profiles are characterized as terra fusca–rendzina derived from limestone (Weißjura beta and gammaseries) and on both sites (SW and NE) are shallow, averagingless than 20-cm depth of topsoil before becoming dominatedby parent rock interspersed with pockets of organic matterand mineral soil. The soil profile at the SW site is especiallyrocky, containing more than 40% (volumetric basis) rocksand stones (> 63-mm diameter) in the top 20 cm of the soil,


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rising to 80% below 0.5 m. The soil of the NE site contains15% rocks and stones in the uppermost 20 cm of the soil andapprox. 30% below 0.5 m. The soil pH (water) is 5.7 in thesurface organic layer and 7.5 at 0.6-m depth. On both sitesbeech (

Fagus sylvatica

L.) is the dominant species, makingup > 90% of the total basal area of trees.

In 1999, trees on both sites had reached an average age of70–80 yr. Also in 1999, and before experimental treatments(see below) were applied, the trees on the NE site had a meanheight of 27.3 m. The mean diameter at breast height was23.9 cm and the stocking amounted to approx. 530 stems ha

−

1

.The trees on the SW site had a mean height of 25.4 m, a meandiameter at breast height of 21.5 cm and a stocking of approx.530 stems ha

−

1

. The understorey vegetation of the two sitesdiffers according to the radiation budget (see below) and leadsto a classification of the stand on the NE site as

Hordelymo-Fagetum

, with that on the (putatively drier) SW site classifiedas

Carici-Fagetum

(Oberdorfer, 1992).On each site, two silvicultural (thinning) treatments plus

controls (unthinned) were established in 1999. The experi-mental design consisted of two blocks, each containing asingle plot (each approx. 0.53 ha in size) of the two silviculturaltreatments plus a control plot. The total basal area (BA) oftrees on the untreated control plots varied between sites – onthe NE site the mean BA in control plots was close to27 m

2

ha

−

1

, while on the SW site the control plot BA wasabout 20 m

2

ha

−

1

. The two thinning treatments on bothsites were BA = 15 and 10 m

2

ha

−

1

. Thinning decreasedthe leaf area index (LAI) from 5.16 (control) to 3.15 (BA =15 m

2

ha

−

1

) and 1.68 (BA = 10 m

2

ha

−

1

) on the NE site, andfrom 5.12 to 3.24 and 2.12 on the SW site.

The difference in aspect (NE or SW) produces a cleardifference in radiation interception. Calculation of globalradiation according to Keding (1984) and Fritsch (1998)shows that the maximum daily radiation for the NE siteamounts to 79% of radiation available on the SW site in Julyand only 47% of that available in October (Fig. 1). This dif-ference between sites is reflected in rates of evaporation andhence available soil water. Fig. 2 illustrates the variation in soilwater potential for a 10-d period (20 September to 1 October1999). At the beginning of the period, the lack of recent rain-fall greatly reduced water potential in soils on both sites. Thereduction was more severe for the SW site, and here soil waterpotential reached minimum values of –0.08 MPa. After rainsstarting in the middle of September, soil water potentialincreased at both sites, and more quickly at the SW site. Shootpressure potential (

ψ

, MPa) and water use (ml cm

−

2

s

−

1

) bytrees were strongly influenced by water availability. Forexample, in 1999 shoot pressure potentials (measured usingstandard techniques with a Scholander-type pressure bomb(Rennenberg

et al.

, 1996) ) were similar at both sites in

Table 1 Precipitation (P), potential evapotranspiration (Vp) and mean air temperature (T ) for the experimental sites in 1999. Precipitation and mean air temperature were recorded at a climate station of the Deutscher Wetterdienst in Tuttlingen, approx. 5-km distance from the experimental plots. Vp was modelled according to Matzarakis et al. (1999)

Month P (mm) Vp (mm) T (°C)

January 55.2 19.2 0.1February 97.5 16.7 –2.2March 48.0 32.4 4.0April 77.4 44.5 7.2May 130.2 72.7 13.0June 110.7 76.1 13.7July 87.9 94.8 17.1August 71.4 81.3 15.8September 77.4 63.7 14.4October 50.6 36.3 7.3November 42.9 20.2 0.6December 134.1 18.5 0.1

Sum/average* 983.3 576.3 7.6

*Sums for P and Vp; average for T.

Fig. 1 Diurnal course of global radiation on a horizontal area (G, thick solid line) and on the north-east-facing (GNE, dashed line) and the south-west-facing (GSW, thin solid line) experimental sites near Tuttlingen, southern Germany, for cloudless days in July (top) and October (bottom). GSW > G since the surface of the south-west slope is irradiated by the incident parallel sunlight with a less acute angle than the horizontal area.


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mid-July (when there had been good recent rains). However,shoot pressure potentials declined, and more strongly at theSW site (Table 2), during August when almost no rain fell inthe week preceding the sampling (Table 2) and soil waterpotentials were strongly negative (Fig. 2). These patterns werereflected in those of tree water use.

The prevailing conditions of water availability and climatewere established by measuring xylem (water) flow densitiesusing Granier-style probes. Briefly, flow densities in thewater-conducting sap wood of beech were determined usingthe constant-heating method according to Granier (1985)and Köstner et al. (1996). Flux densities (ml cm−2 sapwoodarea s−1) were determined every 5 min in seven to nine beechtrees per plot and calculated as means of every 30 min.Thus in July 1999 xylem flow densities in trees on the SWsite (control and 10 m2 ha−1 treatments) were greater thanthose in trees on the NE site (Fig. 3a). However, 1 monthlater when the soil water potential became more negative(Fig. 2), this pattern was reversed and trees on the NE siteshowed higher xylem flow densities than those on the SW site(Fig. 3b).

Flux densities can be converted to estimates of water useusing the stand sapwood area according to the followingequation:

ST = SA × FD Eqn 1

(ST, stand transpiration (mm); SA, sapwood area (cm2 m−2);FD, mean flow density in the xylem (ml cm−2 sapwood areas−1) ). Half-hourly rates of stand transpiration were summedfor 24 h. Sapwood area was determined according to thetechnique of Glavac et al. (1989). A solution of berberinechloride was used to displace the xylem sap from sections(cut from the height at which the sensors had been installed)of representative beech trees. The infiltration of berberine

chloride also stained the sapwood area and allowed sub-sequent estimation of the depth of xylem tissue that wasactive in water transport. About 80% of the total cross-sectional area of the trunks of adult trees was active in watertransport.

In July 1999 the mean transpiration of the stand on theNE control site (within the period shown in Fig. 3a)amounted to 1.3 mm day−1 and was comparable to the trans-piration of the SW control site (1.4 mm day−1). Thinning (to10 m2 ha−1: Fig. 3a) decreased stand transpiration to about60% of the control on the SW site and to about 45% on theNE site. At the NE site, mean daily transpiration in August/

Fig. 2 Water potential of soils in control plots of the south-west- and north-east-facing sites in the autumn of 1999, and daily sums of precipitation. Soil water potential was measured using tensiometers placed at depths of 40 cm (dashed line, SW; dotted line, NE) and 60 cm (thin solid line, SW; bold solid line, NE).

Table 2 Xylem water potential in beech for consecutive months in summer 1999. Rainfall in the month (week in parentheses) preceding the July sampling was 108 (19.3) mm, and rainfall in the month preceding the August sampling was 71 (0.5) mm. Similarly, there were approx. 58 h of full sunlight in the week preceding the July sampling, and approx. 69 h preceding the August sampling. The modelled, plant-available soil water content (water balance model WBS3: Schmidt, 1990; Fritsch, 1998) was some 55% of field capacity at the north-east site in July and 45% in August

AspectTreatment (basal area, m2 ha–1) Month

ψ (Mpa) SE

North Control (approx. 25) July –1.24 –0.1415 –1.23 –0.1210 –1.38 –0.16

South Control (approx. 25) July –1.34 –0.4115 –1.18 –0.4110 –1.20 –0.11

North Control (approx. 25) August –0.54 –0.2215 –0.89 –0.3210 –0.71 –0.22

South Control (approx. 25) August –2.45 –0.1115 –2.35 –0.1710 –2.40 –0.12


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Research 657

September (during the period shown in Fig. 3b) was com-parable to that in July for both control (1.4 mm day−1) andthinned (10 m2 ha−1, 0.6 mm day−1) stands. However, duringthis period both control and thinned stands on the SW sitestranspired between 55% and 65% of the water transpired inJuly, and significantly less water than transpired by the standson the NE site. Hereafter, the data are reported as xylem fluxdensities rather than rates of transpiration, since relative ratesof water use rather than absolute rates are of more concern.

Sampling and analysis of wood, foliage and phloem

The δ13C of wood was characterized from both the NE andSW sites, as described in previous studies of F. sylvatica(Dupouey et al., 1993) or of other hardwoods such asEucalyptus (Macfarlane & Adams, 1998) or softwoods (Saureret al., 1995; Panek & Waring, 1997). Trees for these analyseswere felled in March 1999, before the growing-season hadstarted. In brief, wood samples were collected from discs cut

Fig. 3 Effects of site (south-west aspect, SW; north-east aspect, NE) and treatment (control and 10 m2 ha–1) on sap flow densities of beech growing at the experimental sites near Tuttlingen, southern Germany, in July (a) and August/September (b). Sap flow densities were determined in the sapwood of seven to nine beech trees per site and per treatment, and displayed in the figure as mean (solid line) ± SD (dotted line). The trees chosen were representative for the forest stand and covered the range of diameters (at breast height, over bark) at the experimental sites.



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at breast height from felled trees (four co-dominant treesper site). For determination of mean δ13C in growth rings,a radial–tangential section was taken from each disc and asample of wood from each whole growth ring was oven-driedat 65°C and then ground into a fine, homogeneous powderof which 2.0–2.5 mg per sample were analysed. As notedby Dupouey et al. (1993), earlywood–latewood transitions‘cannot be unambiguously located’ in beech and, moreover,are ‘unlikely to represent the same date for each year’. Wholerings were thus analysed, excluding material obviouslystrongly contaminated with lignin or resin, such as that inresin ‘pockets’ or at tree ring boundaries. Furthermore, holo-cellulose was not separated from the whole wood samples.This introduces a slight error but one that is generally stronglysystematic (e.g. Livingston & Spittlehouse, 1996; Macfarlaneet al., 1999; Warren & Adams, 2000) over the measurementrange – the measured δ13C for wood in tree rings is generallyless than 1‰ depleted in 13C vis-à-vis holocellulose or cellulose.

Phloem sap was collected, as described by Pate et al. (1998)for E. globulus, at approximately monthly intervals from atleast three trees in each plot on each site. The bark of eachsample tree was cut at heights between 1.3 and 1.8 m to thedepth of the wood, at about 15° to the horizontal, usinga single-sided razor blade or scalpel. The ‘bleeding’ phloem sapwas always exuded either immediately after the incision wasmade or not at all. Trees bled poorly or not at all in latespring/early summer, and increasingly readily through thesummer into autumn (Table 3). Different trees were used ateach sampling date in order to avoid cumulative effects ofbark damage.

On selected sampling dates, four trees were climbed in eachplot and foliage was sampled from one to two branchesexcised from the sunlit part of the tree crowns. Different treeswere used for phloem sap and foliage sample in order to avoidartefacts in the composition of phloem sap as a consequenceof the harvest of twigs for foliage samples. However, materialfrom neighbouring trees of the same social class (co-dominantor dominant) was collected.

Finely ground samples of foliage and wood were com-busted to CO2 under a surplus of oxygen (O2) using an

elemental analyser (NC 2500, Carlo Erba Instruments, Milan,Italy) before passing via a Conflo II interface (ThemoQuestFinnigan Instruments, Bremen, Germany) into an isotoperatio mass spectrometer (Deltaplus ThermoQuest, FinniganInstruments, Bremen, Germany). An aliquot of undilutedphloem sap (10 µl) was pipetted into tin capsules and oven-dried for 15 min at 70°C, and then analysed for 13C : 12C.δ13C (in ‰ units) was calculated with respect to the Pee DeeBelemnite (PDB) standard, [(13C : 12C)sample/(

13C : 12C)standard– 1] × 1000.

Phloem sap was further analysed for sugar content asfollows. Diluted (1 : 250) aliquots of 100 µl of sap were injectedinto a HPLC system (Dionex DX 500: Dionex, Idstein,Germany). Separation was achieved on a CarboPac 1 separa-tion column (250 × 4.1 mm: Dionex, Idstein, Germany) with36-mM sodium hydroxide as an eluent at a flow rate of1 ml min−1. Carbohydrates were measured by means of apulsed amperometric detector equipped with a gold workingelectrode (Dionex DX 500: Dionex). Individual carbohy-drates were eluted 8–16 min after injection and were identi-fied and quantified by internal and external standards.

Statistical analysis

All data were analysed using the routines contained withinthe commercial packages StatView, SuperAnova (AbacusConcepts, Berkeley, CA, USA) and SAS (SAS Institute Inc.,Cary, NC, USA). The effect of site (NE, SW) and thinningtreatment on δ13C signature and sugar content of phloem sapand on δ13C in foliage was assessed using a two-way ANOVAprocedure. The same test was applied to differentiate theeffects of site and sampling location (top or bottom oftreatment plots) on δ13C signature of the phloem sap.

Results

Retrospective analysis of climate and δ13C in wood

Patterns of rainfall and radiation during the growing-seasonover the 20-yr period 1978–98 were more or less in keepingwith longer-term trends. Rainfall in the growing-seasonaveraged more than 600 mm or 75% of annual rainfall andthere were typically more than 1100 h of full sunlight forthe same period (Fig. 4a). Growing-season temperaturesaveraged 10.8°C (the long-term mean being 11.5°C). Theyears 1985, 1989 and especially 1991 were relatively dry andrainfall was less than 500 mm in the growing-season; only425 mm of rain fell in this period in 1991.

Wood from the SW site was significantly less depleted in13C (δ13C was less negative) than the wood from the NE site(Fig. 4b) from 1979 to 1991. None the less, and after anabrupt change in δ13C at the SW site in 1991–92, this trendwas reversed for the years 1992–95, probably associated withboth the below-average rainfall in 1991, and a minor thinning

Table 3 Qualitative analysis of the rate of phloem exudation from incisions (approx.10-cm length) in bark at a height of 1.3 m above ground level

Month Rate of phloem bleeding*

May NoneJune SlightJuly ModerateAugust HeavySeptember Heavy

*Slight, < 5 µl per bleeding incision; moderate, 5–10 µl; heavy, > 20 µl.



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at the SW site in the subsequent year. Rates of radial growthof trees at the two sites were generally closely matched over the20-yr period, despite generally greater year-to-year variationthan exhibited by δ13C (Fig. 4b,c). As for δ13C, there was amarked change in radial growth at the SW site in the latterpart of the study period, albeit beginning 2 yr later (1993–94)than the change in δ13C. Hence the decrease in δ13C in thewood of the trees from the SW site in 1991–92 was followedby an increase in radial growth in 1993–94. None of thedirectly measured climatic variables, nor any of a number ofsimple indices of water balance (e.g. ratio of precipitationto evapotranspiration), was strongly related to growth or toδ13C (results not shown). On the other hand, radiation (hoursof full sunlight during the growing-season) was significantlyrelated (P < 0.05, r = 0.62) to δ13C, but only at the SW site.

Phloem and foliage analysis

Phloem sap collected via the ‘bleeding’ technique was domin-ated by sucrose (generally > 80% of total sugar concentration)with smaller concentrations of glucose and fructose (Fig. 5).Several other sugars were present at minor concentrations butthese were not identified or quantified. Raffinose is oftenfound in phloem sap and during the analytical techniqueemployed (see Materials and Methods) may have co-elutedwith fructose and been quantified thereas.

Owing to the relatively large size of each sample plot andthe steep slope, an objective was to characterize the effect ofsampling location (top or bottom of plot) on the δ13C signa-ture in the phloem sap. This comparison showed that, acrossall thinning treatments, there was a strongly significant effect

Fig. 4 Historical data for rainfall and sunshine, δ13C and yearly radial growth of beech. (a) Sum of rainfall (solid diamonds) and sunshine hours (shaded bars) during the growing-season measured at the permanent weather stations of the Deutscher Wetterdienst in Tuttlingen and Möhringen between 1982 and 1998. (b) Weighted mean δ13C of wood in tree rings of beech growing on north-east-facing (NE, open circles) and south-west-facing (SW, filled circles) sites near Tuttlingen, southern Germany, for the period from 1979 to 1998. (c) Yearly radial growth of beech from the north-east-facing (NE, open triangles) and south-west-facing (SW, filled triangles) experimental sites. δ13C and the radial growth data are means ± SE (for δ13C) from measurements of four and 16 trees per aspect, respectively.



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of site (NE or SW) on δ13C, whereas sampling location (topor bottom of plot) had no effect. The mean values of δ13C forphloem sap collected at the bottom and the top of the NEplots amounted to –26.43 ± 0.46‰ and –26.95 ± 0.43‰,respectively. For the SW plots the same quantities were –20.61 ± 0.2‰ (top) and –20.80 ± 0.31‰ (bottom). Con-sequently, on all sampling dates, samples were collected fromtrees randomly selected within, alternately, either the top orbottom third of each plot. Sampling was alternated so as toavoid repetitive sampling and possible injury (and thusartefacts generated by wound responses) of individual trees.

Over the 3-month period in the peak of the growing-season( July–September), the δ13C of phloem sap varied by about13‰ (Fig. 6) across all sampling dates, sites and treatments;a much greater variation than seen between years in the δ13Cof wood. In July, there was no effect of thinning treatment,but phloem sap from the SW site was consistently and signi-ficantly (P < 0.05) more depleted in 13C than sap from theNW site. In August, the driest month of the 1999 growing-season, there were both highly significant (P < 0.001) siteeffects and treatment (P < 0.01) effects. In contrast to July,phloem sap from the NW site was now always more depleted

in 13C than sap from the SE site, by approx. 7‰ in controlplots, 6.3‰ in plots with a BA of 15 m2 ha−1, and 5.4‰ inplots with a BA of 10 m2 ha−1. By the end of September afterintensive rainfall, phloem sap from both sites and all treat-ments had a δ13C signature < −32‰ but only the effect of sitewas significant (P < 0.05), the SW site again producing sapthat was more depleted in 13C than sap from the NW site.

Foliage of beech carried a δ13C signature that varied littleover the growing-season (Fig. 6) and was not significantlydifferent either between sites or among treatments. The meanδ13C signature of foliage from both sites was −28.1 ± 0.1‰,whereas that of wood of the most recent (1996–98) threeannual rings was −25.7‰ for the NE site and −25.9‰ forthe SW site.

The sugar content of phloem sap and its δ13C signature arecompared in Fig. 7. Only August and September sampleswere analysed for both sugar and carbon isotope composition,owing to the small samples of sap that could be collected inJuly. For both sites in September and for the SW site inAugust, sugar concentrations were unrelated to δ13C. For theNE site in August, sugar concentrations were weakly andinversely related to δ13C (P < 0.01, n = 35, r2 = 0.29). Shootpressure potential (SWP, see Materials and Methods) was alsoclosely related to phloem δ13C signatures: δ13C(‰) = −3.93(‰ MPa−1) × SWP( Mpa) − 30.65(‰) (r2 = 0.83, P < 0.01),although the relationship was not robust since data werelacking for the intermediate range.

Discussion

The first hypothesis (i.e. that aspect would determine wateravailability) was broadly supported by the data. On the basisof previous studies of beech (Saurer & Siegenthaler, 1989;Schleser, 1990, 1992; Dupouey et al., 1993), the 20-yr recordin tree rings shows that water availability was less at the SWsite since wood from this site was significantly less depletedin 13C than wood from the NE site. Equally, August wasthe driest month during the 1999 growing-season, and in thismonth phloem sap from the SW site was also much lessdepleted in 13C than sap from the NE site. The relationbetween soil water availability and δ13C signature is illustratedin Fig. 2, and soil water potential was distinctly more negativeat the SW site than at the NE site at the time of the Augustsampling. Only slight differences in δ13C signatures of thephloem were observed between sites (SW and NE) in Sep-tember when soil water potential had increased because ofheavy rainfall. Pate & Arthur (1998) and Pate et al. (1998)showed recently that the δ13C signature of phloem sap fromE. globulus was an excellent predictor of current wateravailability. Here, phloem δ13C was closely (albeit notrobustly) related to water potential, although the slope of theregression line (approx. −3.9‰ MPa−1) was much greaterthan that recorded for Quercus spp. (−0.18‰ MPa−1,Damesin et al., 1998) and still greater than that recorded for

Fig. 5 Effects of site (north-east, NE; south-west, SW) and treatment (control, 15 m2 ha–1 and 10 m2 ha–1) on sugar composition of phloem sap collected from beech growing near Tuttlingen, southern Germany, in summer/autumn 1999. The significance of the main effects from analysis of variance is shown (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001), as are standard errors for each treatment. In the histograms, solid black denotes glucose, no shading denotes fructose, and stippled shading denotes sucrose. See the text for details of experimental design and replication.



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Pinus spp. in much more seasonally arid environments inAustralia (Warren et al., 2001). The analysis of the relation-ship between δ13C and water potential remains speculative,and further work is needed to confirm the linear range forbeech.

As with previous studies where growth (measured aseither BA increment or radial growth) has been comparedwith δ13C, there was reasonable agreement between the twoparameters (see Fig. 4) in the present study. However, both

growth and δ13C exhibited a variable ‘time-lag’ with environ-mental conditions (rainfall, radiation) that precluded a signi-ficant correlation. For growth, the lag effect has been at leastpartly explained by Sass & Eckstein (1995). The mean area ofthe first xylem vessels formed in each growth ring of beech isstrongly influenced by water availability in the previous sum-mer. Sass & Eckstein (1995) concluded that vessel formationat the annual beginning of cambial activity was mainly con-trolled by ‘internal factors’ with little influence of rainfall,

Fig. 6 Effects of site (north-east, NE; south-west, SW) and treatment (control, 15 m2 ha–1) on carbon isotope composition δ(13C) of foliage and phloem sap. The significance of the main effects from analysis of variance is shown (n.s., not significant; *, P < 0.05; **, P < 0.01; ***, P < 0.001), as are standard errors for each treatment. In the histograms, no shading denotes foliage, and solid black denotes phloem sap.

Fig. 7 The relationship between carbon isotope signatures and sugar concentrations of beech phloem sap sampled in consecutive months in 1999. Filled circles and filled upward-pointing triangles, control; open circles and open squares, 15 m2 ha–1 treatment; filled downward-pointing triangles and open upward-pointing triangles, 10 m2 ha–1 treatment; open downward-pointing triangles, all treatments.



Research662

while towards the end of cambial activity vessel formation wasstrongly influenced by recent rainfall.

Based on Pate & Arthur (1998), it could be proposed thatwater availability would be the major influence (or even thesole determinant) of the δ13C signature of carbon in beechtissues and phloem sap contents. If this were the case thenthinning, which almost universally increases the amount ofwater available to remaining trees (e.g. Breda et al., 1995),should produce tissues that are more depleted in 13C thancontrols (the second hypothesis). However, in August whenwater supply on both sites was strongly limited by climaticconditions as indicated by the increase in soil water potential(Fig. 2), plots with the least tree density and BA (10 m2 ha−1)produced phloem that was the least depleted in 13C. Analternative explanation is that thinning – which caused adrastic decrease in LAI – has substantially increased theradiation intercepted by remaining trees, thereby increasingrates of photosynthesis and reducing C i (e.g. Ehleringer et al.,1986; Leavitt & Long, 1986; Pearcy & Pfitsch, 1994; Israeliet al., 1996; Berry et al., 1997). A significant though weakrelationship was also observed between hours of sun-light (a measure for the plant-available radiation) in thegrowing-season and δ13C in wood for the SW site. In part, theexpression of an irradiance effect in δ13C only when waterwas in limited supply may stem from the rather low lightcompensation point of beech (approx. 350 µmol m−2 s−1,Kreuzwieser et al., 1997). Only when Ci is already constrainedby partly closed stomata will the response to light (reductionowing to increased rates of carbon fixation) be realized.

Apart from direct tests of the hypotheses, the data allowuseful comment on several other aspects of the interactions ofclimate and soils/landforms. It can be argued that, for anygiven climatic conditions, soil/landform characteristics holdsoil water more ‘tightly’ at the NE site, against both evapo-transpiration and evaporation, than at the SW site. This ismostly due to the poor water storage capacity (see soil descrip-tions and soil water potential, Fig. 2) of the soil at the SW siterelative to that on the NE site. When water was in plentifulsupply (e.g. in July), trees on the SW site transpired freely(Fig. 3a), at higher rates than trees on the NE site, and phloemcarbon was significantly less depleted in 13C at the SW site(Fig. 6). A similar pattern of δ13C was seen in September.According to the literature, the higher rate of depletion ofavailable soil water at the SW site (Fig. 2) should cause at leastpartial closure of stomata and a lower δ13C. Indeed, this wasconfirmed in August when water flux densities in the xylemof trees at the SW site fell by more than half. In addition SWPdoubled and phloem δ13C was increased by at least 5‰. Thispattern was partially reversed for the NE site – water fluxes inthe xylem of trees at the NE site were greater than at the SWsite during the dry month of August (Fig. 3b), which is sup-posed to be a consequence of the open stomata. In addition,SWP actually increased between July and August (Table 2),and δ13C signatures of phloem sap were little changed from

those in July (Fig. 6). These results are strongly suggestive thatduring the dry month of August, the supply of available waterat the NE site had been moderately well maintained, whereasit had been substantially depleted at the SW site (see Fig. 2).

In particularly dry years and the year immediately there-after, there were few differences in δ13C of wood between theSW and NE sites. For example, the mean δ13C of wood fromthe years 1985, 1986 and 1989–92 was −25.5‰ at the NEsite and −25.3‰ at the SW site. It is concluded that the soil/landform characteristics set ‘lower limits’ to water availabilitythat are not particularly different between the two sites butthat, in any given period, availability is largely determined byexposure to radiation and thence by vapour pressure deficit.The results are consistent with the findings of Walcroft et al.(1997) that the observed close relationships between soilwater availability (e.g. Stewart et al., 1995) or cumulativetranspiration (e.g. Livingston & Spittlehouse, 1996) andδ13C owe much to the integration of a number of environ-mental variables (e.g. precipitation, irradiance, temperatureand air saturation deficit) by Ci, and to the direct relationshipof transpiration and δ13C through stomatal conductance.

It was not intended to model δ13C and water availability atthe available sites and, in any event, the required long-termclimatic data (radiation and evaporation) were lacking. More-over, these sites pose considerable difficulties for water balancemodelling (in contrast to most sites for which models havebeen generated) owing to their shallow surface soils, rockysubsurface soils that quickly grade to ‘blocky’ parent material,and steep slopes. It must also be noted that water balance–δ13C relationships have seldom been tested beyond sites atwhich they were generated. It remains to be seen if on-goingresearch can develop a successful water availability model forthese sites and, then, if it has any predictive capacity for δ13C.Given the observed strong influence of radiation on phloemδ13C (and on wood δ13C), inclusion of radiation in the latterwill probably improve sensitivity (Walcroft et al., 1997).

The sugar and carbon isotope analysis of the phloem sap ofbeech proved instructive, first for the range of δ13C valuesobserved, and secondly for the relative concentrations ofcarbon isotopes and sugars.

Over the growing-season in 1999, the mean δ13C ofsap from the 12 experimental plots (six per site) varied bymore than 13‰: from approx. –20‰, values close to thoseobserved for foliage from desert plants (e.g. Ehleringer &Cooper, 1986), to approx. –33.5‰, values typical of plants inhigh-rainfall, tropical forests (e.g. Jackson et al., 1993). Thisrange is perhaps best interpreted in light of the soil profile atthe SW site which, when coupled with the steep slope, clearlyholds little water for plants for any length of time. Growth ofbeech on this site is probably a fine balance between evapo-transpiration and rainfall during the growing-season, andis not buffered by a huge soil water storage pool. Changes tothis balance via global warming may influence growth andvitality of beech, and may cause changes in the vegetation



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composition owing to a shift in competition patterns. On theother hand, the extreme values of δ13C for phloem sap areintegrated within the tree to produce wood and foliage withδ13C signatures close to those found in other studies of beech(e.g. Schleser, 1990, 1992; Dupouey et al., 1993). The abilityto sample phloem sap from beech, as demonstrated here, shouldenable future studies to elucidate relationships among carbontransport and metabolism and account for the differences inδ13C signatures among recently fixed carbon, transportedsugars, and developing wood and foliage.

Phloem sap from beech has a sugar ‘profile’ comparableto that of other hardwoods for which there are data (e.g.Zimmerman & Ziegler, 1975; Pate & Arthur, 1998; Pateet al., 1998). While the sap was notably rich in sucrose(> 200 mM), it also contained glucose and fructose at concen-trations in the range 0–10 mM. Sugar concentrations variedgreatly between trees within treatments and sites, and neveras predictably with δ13C as shown by Pate et al. (1998) forE. globulus in late summer. There, sugar concentrations inphloem sap increased as water availability (as indicated byδ13C) decreased. While there is some suggestion (Fig. 7) thatsugar concentrations in beech phloem sap may be relatedto δ13C, the relationship is tenuous and largely obscured bytree-to-tree variation. Even when only those data from treesunder the greatest water stress are considered (SW site inAugust), sugar concentrations were independent of δ13C. Pate& Arthur (1998) attributed their result to ‘independentresponses of the sugar-loading systems of the mesophyll :minor vein system to current water status of the leaf, xylemand parent plant’. The differences between the two studiescan be attributed to plot-level climate or (given the hightree-to-tree variability) even individual tree microclimate.Severe shortages of water for beech (as indicated by δ13Cof −20‰) are restricted to days, or at most weeks, whereasin western Australia the ‘summer drought’ may last for3 months or more.

This study has shed some light on past accounts of theabundance of carbon isotopes in beech, and should stimulatefurther attempts to model the relationships among environ-mental variables and δ13C. δ13C signatures in the phloem ofbeech are sensitive to seasonal changes in water availability,whereas δ13C signatures in the foliage are far less sensitive. Inaddition, the data suggest an interactive effect of low wateravailability and increased radiation on δ13C in the phloemsap. The demonstrated ability to sample phloem sap ofmature beech in the field, both directly and easily, opens thedoor to many future field studies – most obviously of carbonand nutrient transport but also of hormone transport betweenshoots and roots.

Acknowledgements

The authors thank the DFG (SFB 433) for financial support.MAA acknowledges the support of an Alexander von

Humboldt Fellowship. The authors thank S. Hauser,H. Spiecker, S. Augustin and E. Hildebrand for providingdata on radial growth, LAI and soil water potential.

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Radiation modiﬁes the effect of water availability on the ... · SW, 36–58%). Soil proﬁles are characterized as terra fusca– rendzina derived from limestone (Weißjura beta