Dynamics of bioavailable rhizosphere soil phenolics and photosynthesis of Arum maculatum L. in a lime-beech forest

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Flora 203 (2008) 590–601 www.elsevier.de/flora

Dynamics of bioavailable rhizosphere soil phenolics and photosynthesis of Arum maculatum L. in a lime-beech forest Lola Djurdjevic´, Zorica Popovic´, Miroslava Mitrovic´, Pavle Pavlovic´, Snezˇana Jaric´, Ljiljana Oberan, Gordana Gajic´ Department of Ecology, Institute for Biological Research ‘‘Sinisˇa Stankovic´’’, University of Belgrade, Bulevar Despota Stefana 142, 11060 Belgrade, Serbia Received 21 May 2007; accepted 31 August 2007

Abstract In this article, the dynamics of phenolics in the soil originated from dominant trees and the photosynthetic performance and growth of the forest floor species Arum maculatum is firstly reported. Assimilation of CO2, chlorophyll fluorescence and chlorophyll concentration in the leaf tissue of A. maculatum as well as natural concentrations of total bioavailable phenolics and phenolic acids in the rhizosphere of this herb were estimated simultaneously during the growing season. Additionally, growth dynamics of A. maculatum were assessed by determination of instantaneous growth rate and leaf area index. The dominant species Fagus moesiaca and Tilia tomentosa were the main sources of the total phenolics and phenolic acids in plant litter and soil. The amounts of bioavailable phenolics and phenolic acids in rhizosphere soil were several times lower than in the litter or in freshly fallen leaves of lime and beech. In the rhizosphere soil of A. maculatum, the amount of total phenolics decreased rapidly from March to May. All of five phenolic acids present in leaves of dominant trees were identified in the A. maculatum rhizosphere soil, with characteristic turnover dynamics shown by ferulic and vanillic acid. Dynamics of the photosynthetic performance of A. maculatum was assessed as net photosynthetic rates and chlorophyll fluorescence, which had opposite courses. PN decreased continuously during the growing season (from 9.9171.41 mmol m2 s1 at the beginning of March to 4.3670.86 mmol m2 s1 at the end of May). Photosynthetic rate, growth rate and chlorophyll a:b ratio were positively correlated with total soil phenolics, and also with the available derivatives of cinnamic and benzoic acids (po0.05). Photosynthetic efficiency, total chlorophyll content and leaf area index were negatively correlated with total soil phenolics and derivatives of benzoic acid, and positively correlated with the derivatives of cinammic acid (po0.05). These results indicate that there was a high correlation between total bioavailable rhizosphere soil phenolics and phenolic acids originated from dominant trees on one side, and the photosynthetic performance and growth parameters of A. maculatum on the other side. r 2008 Elsevier GmbH. All rights reserved. Keywords: Allelopathy; Arum maculatum; Disturbed ecosystems; Fv/Fm; Photosynthesis; Soil phenolics

Abbreviations: PN, net photosynthesis; Fv/Fm, photosynthetic efficiency; R, instentenous growth rate; LAI, leaf area index; chl a:b, chlorophyll a and chlorophyll b ratio; PS II, photosystem II. Corresponding author. Tel.: +381 11 2078 359; fax: +381 11 2761 433. E-mail address: [email protected] (L. Djurdjevic´). 0367-2530/$ - see front matter r 2008 Elsevier GmbH. All rights reserved. doi:10.1016/j.flora.2007.08.006

Introduction Regarding their abundance and primary productivity, dominant trees in forests can have a great allelopathic

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significance in forest communities (Al Naib and Rice, 1971; Gonza´les et al., 1995; Hane et al., 2003; Lodhi, 1975; Lodhi and Rice, 1971; Souto et al., 1994, 1995). Allelopathic effects of trees on understorey plants are caused by phenolic phytotoxins that are present in all plant parts, but the greatest content of these compounds is accumulated in leaves (Djurdjevic´ et al., 1998b, 1999; Lodhi, 1975, 1978; Lodhi and Killingbeck, 1980; Loponen et al., 2001; Ossipov et al., 1995a; Rice and Pancholy, 1973). In an effort to investigate the interrelationships among different plants in their natural growing sites, special attention has been paid to the presence of phenolics in dominant plants, plant litter and in the soil. Accumulation of phenolic toxins in soil leads to the inhibition of seed germination as well as it impedes growth of roots and seedlings, photosynthesis, water and ion uptake of target plants, etc. (Aliotta et al., 1993; Barkosky and Einhellig, 1993; Barkosky et al., 2000; Li et al., 1992; Lodhi, 1975; Lodhi and Rice, 1971; Lyu and Blum, 1990; Muller, 1966; Rice, 1974, 1979; Souto et al., 1994). As consequences, either a reduction of number of plant individuals can be found or complete elimination of some herbaceous plants and tree seedlings may occur. Due to their allelopathic activity, phenols therefore can strongly affect the structure and floristic composition of plant communities (Chou and Muller, 1972; Djurdjevic´ et al., 2004; Lodhi, 1978; Wardle et al., 1998). The photosynthetic apparatus, particularly photosystem II, is rather sensitive to allelochemicals, as it was documented from laboratory experiments (Rice, 1974, 1979). However, there is no evidence about the relationships between photosynthetic performance, and consequently biomass production of plants, with changes in the phenolics content and composition of the soil under field conditions. The need for more data obtained from natural ecosystem conditions was emphasized, e.g., by Inderjit and Weston (2000) and Inderjit and Callaway (2003). In a complex ecosystem such as a deciduous forest, plants have to cope with multiple abiotic factors in surplus or deficiency (e.g., nutrient availability, light levels, soil moisture), also being exposed to biotic interactions among co-existing species, competitive pressures within and between their populations, and allelopathic effects of dominant trees. Vernal forest ephemeroids complete their aboveground vegetation in the short spring period, which is characterized by the dynamic environmental changes during canopy closure and by mass production of herbaceous layer. Photosynthetic intensity and efficiency of forest spring ephemeroids and their growth dynamics respond to these changing conditions, being most strongly affected by changes in light and water availability (Jovanovic´, 2002; Jovanovic´ et al., 1998; Masarovicˇova and Eliasˇ , 1986; Popovic´ et al., 2005, 2006; Rothstein and Zak, 2001). In addition, the influence of allelochemicals of

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donor plants to the ecophysiology of target plants can modify these essential processes which determine whole plant production (Reigosa et al., 1999). This may become obvious from the performance of herb layer species under influence of seasonal changes of phenolics and phenolic acids with a proved allelopathic potential. The present study was conducted in order to identify and quantify the bioavailable phenolic compounds in the soil of a lime-beech forest and to clarify the correlation of these with the photosynthetic performance and growth of the understorey ephemeroid Arum maculatum. For achieving this aim: (1) total phenolics and phenolic acids in fallen leaves and litter of dominant trees were measured, and also bioavailable phenolics and phenolic acids in the herb rhizosphere soil; and (2) net photosynthesis, photosynthetic efficiency, chlorophyll content, instantaneous growth rate and leaf area index of the target herb A. maculatum were determined.

Materials and methods Study site The investigation was performed on Avala Mountain (511 m a.s.l.), 18 km south of Belgrade, Serbia. This mountain is characterized by a great richness of flora and vegetation, with many different forest communities coexisting in small distance from each other. The research area was situated on the mountains’ southern side, at 300–440 m a.s.l. It included several forest types, from a mesophilous forest with lime and beech (TilioFagetum submontanum Jank and Misˇ , 1960; Misˇ ic´, 1972) to the most thermophilous forest with virgilius oak and manna ash (Orno-Quercetum virgilanae Gajic´, 1952) (Borisavljevic´ et al., 1955).

Description of forest community The hilly lime-beech forest is situated at 440 m a.s.l. on the northern slope of a local depression with a slope of 15–201 and covers an area of 0.18 ha. The forest is 50–60 years old, degraded and periodically cut, so it is considered as disturbed ecosystem. In the highest canopy floor of trees (18–22 m height, stem diameter 20–60 cm), the following species are present: Fagus moesiaca, Tilia tomentosa, Carpinus betulus, and Quercus petraea. In the shrub layer are present: T. tomentosa, Sambucus nigra, Acer campestre and C. betulus. The herbaceous layer is rich (covering index 80%) and composed of the following species: A. campestre juv., Galium aparine, Rubus hirtus, A. maculatum, Euphorbia amygdaloides, Circaea lutetiana, Hedera helix, Corydalis cava, Corydalis solida and others.

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Meteorological data from March to May 2004, when this research was conducted, were obtained at the ‘Avala’ meteorological observation station, which is about 1.5 km distant to the study site (Republic Hydrometeorogical Service of Serbia, 2004), whereas the actual light conditions were evaluated periodically, always at the same five sites within the forest stand, 0.5 m above the ground, using a PPFD sensing selenium cell which is a component of LI-6200 equipment. Average monthly meteorological data were as followed: March (temperature 6.4 1C, precipitation 53.3 l m2, relative humidity 89%, light intensity 12817120 mmol m2 s1); April (temperature 10.7 1C, precipitation 60.8 l m2, relative humidity 77.4%, light intensity 908.4726.6 mmol m2 s1); May (temperature 16.8 1C, precipitation 77.6 l m2, relative humidity 56.5%, light intensity 482.99765.3 mmol m2 s1). The soil is brown and acidic (Dystric cambisol, F.A.O.; Typic dystrochrept, USA) (Duchaufour 1976). Chemical properties of the soil at the stand, regarding to humusaccumulative layer, are as following: pH (H2O) 6.7, humus 9.28%, N 0.48%, P2O5 15 mg/100 g of soil, K2O 425 mg/100 g of soil, C 5.38%, C/N 11.21. Total weight of litter was 5380 kg ha1 yr1.

Sampling of plant material, litter and soil The experimental area was divided into three transects: along the upper edge of forest slope, at the middle part and at the lower edge of the forest slope. Recently fallen leaves of dominant trees and litter (mostly resulted from partially decomposed leaves of the same species) were uniformly collected along the transects during the October 2003. Along each transect, three samples of fallen leaves of beech and lime were taken (9  0.5 kg; n ¼ 9 for both species). Litter was sampled in the same way (9  0.5 kg; n ¼ 9). The collected plant material and litter were air dried at room temperature, milled and sieved through a sieve with 0.5 mm diameter holes. The rhizosphere soil was taken under the same individuals of A. maculatum which were subjected to the ecophysiological survey. Rhizosphere soil of A. maculatum was sampled simultaneously with the measurement of photosynthesis and growth parameters, i.e. during March, April and May 2004, in 10 days intervals. Taking the soil samples in short intervals gives a better insight into the real concentrations of rhizosphere soil phenolics. After removal of partially decomposed litter, soil samples were uniformly collected from the surface topsoil (up to 20 cm depth) including the ‘‘fermentation’’ (Of) and the organo-mineral ‘‘humic’’ (Ah) horizons. In each transect, seven samples of rhizosphere soil of A. maculatum were collected (each sample had an equal weight of 500 g), and a mixed sample was prepared so that each transect was

represented with one mixed sample. During the experiment, total 27 mixed samples were taken (nine 10-days intervals  three mixed samples from each transect; n ¼ 9). After removal of visible plant remains, the soil was air dried at room temperature, milled and sifted through a sieve with 0.5 mm diameter holes.

Extraction of plant and litter phenolics Both phenolic acids and total phenolics were extracted from dry fallen leaves of both lime and beech, and litter (9  2 g for each) with 30 ml of distilled water by shaking 24 h at room temperature. Water extracts were adjusted to pH 2.0 with 2N HCl and phenolics were transferred to ethylacetate (3  50 ml). The mixture was evaporated to dryness and the residue dissolved in 4 ml of 80% (v/v) HPLC grade methanol (water soluble-free phenolics) and used for HPLC analysis or stored at 20 1C until use.

Extraction of soil phenolics Bioavailable soil phenolics were extracted from 9  30 g per sample of dried soil in distilled water (100 ml) by shaking during a 24 h period. Water extracts were evaporated to 30 ml, adjusting pH to 2.0 with 2 N HCl and phenolics were transferred to ethylacetate (3  50 ml). The mixture was evaporated to dryness and the residue dissolved in 4 ml of 80% (v/v) HPLC grade methanol. Samples were used for immediate HPLC analysis or stored at 20 1C until use (Hennequin and Juste, 1967; Katase, 1981a, b).

Determination of total phenolics Total phenolics were measured spectrophotometrically (Shimadzu UV 160 spectrophotometer) using the Folin–Ciocalteu reagent (Feldman and Hanks, 1968). A standard curve was constructed with different concentrations of ferulic acid. Units of total phenolics were expressed in mg of ferulic acid equivalent per gram of plant material resp. soil dry weight.

Determination of phenolic acids by HPLC Phenolic acids were detected between 210 and 360 nm using a Hewlett Packard diode array detector (HP 1100 HPLC system). The separation was achieved with a Nucleosil 100–5 C18 column; 5 mm; 4.0  250 mm (Agilent Technologies, USA). A step-gradient of acetonitrile in water was used: 15% acetonitrile (5 min, gradient), 30% acetonitrile (20 min, gradient), 40% acetonitrile (25 min, gradient), 60% acetonitrile (30 min, gradient), 60% acetonitrile (35 min, gradient) and 100% acetonitrile (45 min, isocratic) at a flow rate of 1.0 ml min1, the injection volume being 5.0 ml. In

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order to avoid tailing of the phenolic acids, 0.05% o-phosphoric acid was added to the solvents. Individual phenolic acids were identified by comparing the retention times and absorption maxima with those of p-hydroxybenzoic and syringic acid (Acros Organics, USA), ferulic, vanillic and p-coumaric acid (Serva, Germany), serving as phenolic standards. The content of phenolic acids was expressed in mg g1 of plant material resp. soil dry weight.

Steady state fluorescence was determined with a Plant Stress Meter (BioMonitor S.C.I. AB, Sweden) by the method of induced fluorometry (Powels, 1984; O¨quist and Wass, 1988). The photosynthetic function was assessed by the rate of basic fluorescence, i.e. the ratio of variable to maximal fluorescence (Fv/Fm ¼ (FmFo)/ Fm, where Fo and Fm are initial and maximal fluorescence of dark-adapted leaves). Each leaf was illuminated with saturating low light (100 mmol m2 s1) for 2 s, after having been in darkness for at least 20 min (n ¼ 10).

Photosynthetic measurements

Chlorophyll determination

The object of our study was A. maculatum L. (Araceae), the cuckoopint, a spring-flowering rhizomatous geophyte inhabiting temperate broadleaved deciduous forests. It optimally grows on a moist, loose soil, well supplied with humus. Its leaves sprout from the last-year’s leaf buds on a stout stem tuber in mid-March. Flowering period is typically from 15 April to 1 May, and the flowers disappear until the mid-May, while leaves stay fresh until end of May. Seeds, new shoots and new tubers are set every year (Sowter, 1949). Maximum of leaf expansion and biomass accumulation occurs after forest canopy closure (Popovic´ et al., 2005). All photosynthetic measurements were carried out under the field conditions using a LI-6200 closed photosynthesis system (Li-Cor Inc. Lincoln, NE, USA). Microclimate was measured and recorded for each sample using the sensors housed in the leaf chamber. After routine calibration of the instrument at the start of each sampling date, all measurements were conducted with a leaf chamber CO2 concentration of 350 ml l1, at a chamber temperature of 20 1C and a relative humidity of 55%. Photosynthetic photon flux density (PPFD) was measured with a selenium cell mounted on the leaf chamber. Measurements were performed from the beginning of March to the end of May, in 10 days intervals (15 measurements three times monthly, n ¼ 45). As the data from three monthly measurements were not significantly different, these were summarized as mean monthly photosynthesis. Sampling was usually conducted between 9:00 and 12:00 h, and only the data on CO2 uptake above the light saturation level were considered (PN at PPFD 4550 mmol m2 s1). Shaded leaves and leaves under direct sunlight were measured separately at each sampling location and in their natural orientation to minimize disturbance and reduce variation due to light and temperature acclimation responses. The averaged value from at least five measurements made evenly over a 5 min timespan was taken as the final value for CO2 exchange of each leaf and expressed on a leaf area basis (leaf area was determined by scanning harvested leaves and using the ‘‘Areameter’’ software, Karadzˇic´ et al., 1999).

Chlorophyll amount in the leaf tissue was spectrophotometrically determined, based on light absorption of a solution obtained after extraction with dimethyl sulfoxide (DMSO, Hiscox and Israelstam, 1979). One disc (1 cm diameter) per leaf was harvested from 10 leaves and used to extract chlorophyll with 5 ml of DMSO (n ¼ 10). After incubation at 65 1C until fully extraction of chlorophyll was reached, the absorbance of each sample in 1.00 cm cuvettes was measured at 647.0 and 664.5 nm using a Shimadzu UV 160 spectrophotometer. Chlorophyll amount was recalculated related to dry leaf mass according to formulas given by Arnon (1949).

Biometric measurements The plant material of 25 plants from five randomly selected plots (each 0.25 m  0.25 m) (n ¼ 75) was sampled simultaneously with measurements on photosynthesis. Each plant was collected using a shovel, keeping the most of its root system intact. Plants were overwashed and oven-dried at 75 1C to a constant weight. For each harvested plant total plant dry mass (W) was determined. The growth rate for the growing season was calculated using the formulas given by South (1995). R ¼ (1/W)  (dW/dt) (where dW is the increase in plant biomass per unit of plant biomass, i.e. dW ¼ W2W1, where W2 and W1 are the dry weights at the end and at the beginning of each sampling period, respectively; per unit of time dt). To estimate total leaf area index (LAI), six plots (each 0.25 m  0.25 m) were randomly selected, and all leaves from these plots were scanned in order to determine total leaf area (m2 of leaf per m2 of ground area) (Karadzˇic´ et al., 1999).

Statistical analyses The statistical evaluation of differences in the total content of phenolics and the composition of phenolic acids in plant material and soil samples was performed with two-sided ANOVA tests. Differences among average monthly values of PN (CO2 assimilation above the light

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Results Free phenolics and phenolic acids in fallen leaves and litter of dominant trees Total contents of phenolics and phenolic acids found in fallen tree leaves and litter are given in Table 1. Higher contents of total phenolics were detected in freshly fallen beech leaves than in such leaves of lime whereas the lowest content was detected in litter. Phenolic acids were not uniformly distributed among samples: both lime and beech leaves contained lower amounts of p-coumaric and p-hydroxibenzoic acid than litter; beech leaves contained higher amounts of vanillic and syringic acids (not detected in lime leaves) compared to both lime leaves and litter (po0.001); lime leaves contained the highest amount of ferulic acid (po0.01). When the percentages of individual phenolic acids were calculated in relation to the content of total phenolics (content of total phenolics ¼ 100%), the share of each of phenolic acids examined in total phenolic content were clearly distinct in comparison to the amounts of individual phenolic acids only. Regarding to their participation in total phenolics, free phenolic acids were present with 1.22% in fallen lime leaves and with 3.10% in fallen beech leaves, whereas their share in litter was 10.82%. In general, litter contained higher percentages of derivatives of cinnamic acid (p-coumaric and ferulic) and derivatives of benzoic acid (p-hydroxybenzoic, vanillic and syringic) in comparison with fallen leaves of lime and beech.

Dynamics of total soil phenolics and phenolic acids The amount of total phenolics in the rhizosphere soil of A. maculatum decreased from March to May. All five phenolic acids present in leaves of dominant trees were identified in A. maculatum rhizosphere soil, with characteristic dynamics shown by ferulic and vanillic acids (Fig. 1). During the growing season derivatives of cinnamic acid made up 5.13–8.89%, while derivatives of benzoic acid made up 7.59–9.37% of total soil phenolics.

Dynamics of PN, Fv/Fm, Chl, R, LAI; relations between the photosynthetic and growth parameters with phenolic content Dynamics of photosynthetic performance of A. maculatum was assessed through net photosynthetic rate and chlorophyll fluorescence, which had opposite courses. Based on previously established photosynthetic light–response curves of A. maculatum (light saturation range reached at approx. 400 mmol m2 s1, Popovic´ et al., 2006), all PN values reached at light level above 100

p-coumaric (R) ferulic (R) p-hydroxybenzoic (R) vanillic (R) syringic (R) free soil phenolic (L)

90 80

12 10 8

70 6 60 4 50

2

Concentration of phenolic acids (µg g-1)

compensation point), Fv/Fm, total Chl, Chl a:b, R and LAI were taken as significant if po0.05, tested by one-way break down ANOVA test. Data on PN, Fv/Fm, chlorophyll content, chlorophyll a:b ratio, R and LAI were correlated with total soil phenolics. All correlation coefficients (r) are Pearson coefficients. Relationships were considered as significant at po0.05. Data were processed using the statistical package Statistica 6.0 for Windows.

Concentration of total soil phenolics (µg g-1 )

594

40 March

April

May

Fig. 1. Dynamics of total phenolics and phenolic acids in rhizosphere soil of A. maculatum in lime-beech forest; mean7S.D., R is for right Y and L is for left Y axis, respectively.

Table 1. The content of free phenolics and phenolic acids in fallen leaves of dominant trees and litter (mg g1)

Total phenolics p-Coumaric acid Ferulic acid p-Hydroxybenzoic acid Vanillic acid Syringic acid

Tilia argentea

Fagus moesiaca

Litter

7825.337346.00 b*** 24.8674.44 ans 45.5374.72 a,b** 11.8372.34 ans 13.1471.51 –

15009.77592.81 a,c*** 19.0471.40 21.9973.01 12.3472.41 189.46710.58 a,c*** 222.13717.80 a,c***

2065.607205.56 47.8776.45 b**c* 23.9775.30 cns 19.9473.30 b,c* 47.6974.98 b*** 83.9779.11 b***

Compared are: (a) Tilia argentea/Fagus moesiaca; (b) Tilia argentea/litter; (c) Fagus moesiaca/litter (ANOVA; ns, not significant; *po0.05; **po0.01; *** po0.001; n ¼ 9). Mean7S.D.

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14

0.80

(a)

12 12

0.76

0.72

6

b

B

Fv/Fm

a

A 8

0.68 C

4

PN (µmol m-2 s-1 )

c

10

10 8 6

0.64

2

r=0.769

4

0

0.60 April

2 3.4

May

Fig. 2. Light saturated photosynthesis (filled squares, filled line) and chlorophyll fluorescence (empty circles, dashed line) of A. maculatum during the growing season. Different letters show significant differences tested by ANOVA (po0.05) in PN (upper case) and Fv/Fm (lower case), respectively.

550 mmol m2 s1 were considered as light-saturated photosynthesis. PN decreased continuously through the growing season (from 9.9171.41 mmol m2 s1 at the beginning of March to 4.3670.86 mmol m2 s1 at the end of May; Fig. 2), being positively correlated with bioavailable soil phenolics (r ¼ 0.769, po0.05; Fig. 3a). An increase in photosynthetic efficiency occurred from March to May (Fv/Fm 0.7370.02 and 0.7670.01, respectively; po0.05; Fig. 2), with a minimal value noted in April. A negative correlation was found between this parameter and total soil phenolics (r ¼ 0.605, po0.05; Fig. 4a). Changes of total chlorophyll content, chlorophyll a:b, R and LAI are given in Table 2. Total chlorophyll amount in leaves and leaf area index of A. maculatum were negatively correlated with total soil phenolics (r ¼ 0.780 and 0.898, respectively; po0.05; Fig. 4b and c). Opposite relations were established between total soil phenolics and the relative growth rate and chlorophyll a:b (r ¼ 0.707 and 0.794, respectively; po0.05; Fig. 3b and c).

Correlations between photosynthetic and biometric parameters and phenolic acids Photosynthetic rate, chl a:b ratio and relative growth rate were significantly positively correlated with the derivatives of cinnamic and benzoic acids (po0.05). Photosynthetic efficiency, total chlorophyll content and leaf area index were significantly correlated in a positive relation with the derivatives of cinnamic acid, and negatively with the derivatives of benzoic acid (po0.05, Table 3).

(b)

3.2 3.0 Chl a / Chl b

March

2.8 2.6 2.4 2.2

r=0.707

2.0 100

(c)

80 R (mg g-1 d-1)

PN (µmol m-2 s-1)

595

60 40 20

r=0.794

0 40

50

60

70

80

90

100

Total soil phenolics (µg g-1)

Fig. 3. Correlations between total soil phenolics and net photosynthesis (a), chlorophyll a/b ratio (b) and growth rate (c) of A. maculatum, respectively, with correlation coefficients.

Discussion Free phenolics and phenolic acids in fallen leaves and litter of dominant trees Phenolics may be released into the litter and soil from the donor plant by leaf falling, foliar and stem leaching, through glandular trichomes and root exudation. The phenolics are accumulated in soil also by microbial decomposition of plant remains, mostly due

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596

0.78

(a)

Table 2. Average monthly values for chlorophyll content and growth parameters of Arum maculatum

0.76

March

0.74 Fv/Fm

April

May

1

21.6172.05 a 25.2072.60 b 30.7672.28 c Chl (mg g ) Chl a:b 3.0170.14 a 2.6670.28 b 2.4970.20 b R (mg g1 day1) 79.1275.22 a 43.1672.98 b 27.1472.55 c LAI (m2 m2) 0.0670.01 a 0.2170.02 b 0.8770.04 c

0.72 0.70

Chl, chlorophyll content per leaf dry mass (n ¼ 10); Chl a:b, chlorophyll a:b ratio (n ¼ 10); R, instantenous growth rate (n ¼ 75); LAI, leaf area index (n4100). Mean7S.D.; different letters mark significant differences at po0.05.

0.68 r=-0.605 0.66 36

(b)

Total chlorphyll (mg g-1)

32

28

24

20 r=-0.780 16 1.0

(c)

LAI (m2 m-2)

0.8 0.6 0.4 r=-0.898 0.2 0.0 40

50

60

70

80

90

100

Total soil phenolics (µg g-1)

Fig. 4. Correlations between total soil phenolics and chlorophyll fluorescence (a), total chlorophyll content in leaves (b) and leaf area index (c) of A. maculatum, respectively, with correlation coefficients.

to degradation of lignin (Alexander, 1977; Carballeira and Reigosa, 1999; Hyder et al., 2002; Ko¨gel-Knabner, 2002; Reigosa et al., 1999; Sterling et al., 1987; Tang and Young, 1982; Whitehead et al., 1983). Our analysis showed that fallen leaves of dominant lime and beech as starting material contained 7.82 and 15.01 mg g1 of total free phenolics, respectively. This is in accordance with previous reports regarding different tree species where content of phenolics varied from 2.94 to 77.00 mg g1 (Djurdjevic´ et al., 1998b; Feeny and

Bostock, 1968; Ishikura, 1976; Ossipov et al., 1995a, b; Rice and Pancholy, 1973; Soumela et al., 1995). Litter contained several times lower concentrations of total phenolics in comparison to the fallen leaves of lime and beech, which can be caused by the leaching of watersoluble phenolics during the decomposition process. Similar results were obtained in litter of Pinus contorta, Acacia longifolia, Alnus glutinosa and fallen willow leaves, where the insoluble and less degradable compounds (such as lignin, cellulose and hemicellulose) prevailed during the later stages of decomposition, as the consequence of the free phenols leaching (Pereira et al., 1998; Schofield et al., 1998; Yavitt and Fahey, 1986). Five phenolic acids made 3% of total phenolics in fallen leaves of dominant trees, which is in accordance to our previously reported data regarding different tree species (Djurdjevic´ et al., 1998b, 1999, 2005). The data showed that litter contained a higher percentage of derivatives of cinnamic and benzoic acids than fallen leaves of lime and beech. An increase of amount of phenolic acids could be explained by the microbial decomposition of plant remains, especially by degradation of lignin. Microorganisms could contribute to overall amount of phenolics by synthesizing some of these compounds as well (Alexander, 1977; Crawford and Crawford, 1980; Ko¨gel, 1986; Lewis and Yamamoto, 1990; Martin and Haider, 1969).

Dynamics of total soil phenolics and phenolic acids The share of total free phenolics in the soil was negligible (0.39% of total amount) in comparison to their amounts in fallen leaves of dominant trees, which caused the decrease of bioavailable phenolics in the rhizosphere soil of A. maculatum during the growing season. Phenolics originated from the dominant trees became bound to soil particles or incorporated into polymerized compounds which are resistent to decomposition. It was previously reported that total bound phenolics were the main part of total soil phenolics in different forest ecosystems (up to 96.7%), whereas only

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Table 3.

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Correlations of soil phenolic acids with photosynthetic and growth parameters of Arum maculatum

p-Coumaric Ferulic p-Hydroxybenzoic Vanillic Syringic Cinnamic Benzoic Cinnamic+benzoic

PN (mmol m2 s1)

Fv/Fm

Chl (mg g1)

Chl a:b

R (mg g1 day1)

LAI (m2 m2)

0.42 +0.66 0.08 +0.67 +0.18 +0.17 +0.64 +0.67

+0.60 +0.18 +0.23 0.73 0.73 +0.45 0.76 0.58

+0.38 0.55 +0.07 0.80 0.22 +0.14 0.76 0.78

0.23 +0.64 +0.12 +0.58 +0.33 +0.29 +0.60 +0.68

0.38 +0.75 0.05 +0.70 +0.05 +0.26 +0.65 +0.71

+0.67 0.43 +0.22 0.96 0.38 +0.13 0.92 0.84

 Marked correlations are significant at po0.05.

the smallest part was attributed to the free phenolics (Djurdjevic´ et al., 1998a, b, 2000). Additionally, phenolics are leached from the soil solution into deeper layers of soil, and removed by plant uptake and by degradation by soil microorganisms (An et al., 2001; Gallet and Pellissier, 1997; Inderjit and Bhowmik, 2004; Katase, 1981b; Souto et al., 2000). In the rhizosphere soil of A. maculatum the amount of total bioavailable phenolics decreased from 89.22 at the beginning to 54.11 mg g1 at the end of growing season, whereas the amounts of individual phenolic acids increased from 1.16 to 4.18 mg g1. Kuiters and Denneman (1987) detected slightly lower amounts of water soluble ferulic, p-coumaric, p-hydroxybenzoic and syringic acids (from 0.56 to 2 mg g1) in a humic layer of the soil under Fagus sylvatica, with the same seasonal trend. Also Gallet and Pellissier (1997) found a decrease of total soil phenolics and phenolic acids content during the vegetation period in a coniferous forest and similar contents of these compounds as detected in rhizosphere soil of A. maculatum. The target plant A. maculatum was exposed to the different concentrations of bioavailable phenolics in the rhizosphere soil during the short growing period. The root system of target plants is the primary way for receiving the phenolic acids. So the mobility and persistence of these chemicals are the key processes for allelopathic effects (Cecchi et al., 2004). Reigosa et al. (1999) underlined that more information about seasonal changes in soil concentration of allelochemicals are essential, especially when the rapid changes in shorter periods occur. Moreover, Weidenhamer (2005) emphasized that the demonstration that fluxes of allelochemicals measured in the rhizosphere must not prove that allelopathic interactions are occurring. Namely, allelopathic interactions without data on allelochemical dynamics in soil will remain problematic. In order to gain reliable information, the phenology of vernal ephemeroids, such as A. maculatum, requires frequent observation and short sampling intervals for the bioavailabile rhizosphere soil phenolics.

Dynamics of PN, Fv/Fm, Chl, R, LAI; relations between the photosynthetic and growth parameters with phenolic content Photosynthetic CO2 assimilation is the measure of plant primary production sensu lato, whereas Fv/Fm ratio indicates the intrinsic efficiency of photosystem II photochemistry in the absence of light. PN of A. maculatum decreased during the growing period, as expected for semi-sciophilous forest herbs with a short life-span of leaves, which mostly are photosynthetically acclimated to shade conditions (Popovic´ et al., 2006; Rothstein and Zak, 2001). However, photosynthetic efficiency of PSII had the opposite tendency, being the highest in the period when the photosynthetic assimilation was the lowest. The high values of Fv/Fm ratio demonstrate that leaves of cuckoopint possess a high efficiency of photon utilization in PSII, despite the low light conditions in the forest. PN and Fv/Fm were also oppositely correlated both with soil phenolics and phenolic acids. Sa´nchez-Moreiras and Reigosa (2005) reported that photosynthesis and Fv/Fm were oppositely affected by allelochemicals in laboratory conditions. In an environment where a continuous decrease of light occurs during the vegetation period due to leaf development of the overstorey, this fact could indicate that carbon reactions and dark metabolism of A. maculatum are more sensitive to identified allelochemicals than the light phase of photosynthesis. Moreover, clearly distinct relations between the various parameters and soil phenolics were found. Namely, photosynthetic rate and growth rate were positively correlated with bioavailable phenolics. At the whole plant level, photosynthetic capacity determines the growth rate, and it is expected that factors which depress photosynthesis have the same effect to plant growth. On the other hand, both physiology and morphology of plant assimilative organs are affected by environmental changes (Poorter, 1989), contributing to the effectiveness of plant adaptive responses in the natural stand. Fv/Fm presents the efficiency of photosynthetic apparatus, which can be achieved by a favorable chlorophyll a:b ratio, even if

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a decrease of total chlorophyll content occurs. Leaf maturation and aging are particularly important processes in whole plant physiology, contributing to a significant degree to decreasing of photosynthesis, respiration and nitrogen content (Bond, 2000, and references therein). However, some authors suggested that seasonal decline in photosynthesis and nitrogen content might be an adaptation to changing light levels rather than just and inevitable consequences of aging, especially in forest communities (Field, 1981; Field and Mooney, 1983). It is also important to consider that the rate of photosynthesis is related with developmental stages in species with storage organs (Popovic´ et al., 2005; Usuda and Shimogawara, 1998). Therefore, the effects of phenolics on photosynthesis of A. maculatum should be considered as one among several photosynthesis-depressing impacts, such as age-associated disorders, light availability and sink demands. In previous reports, it has been already indicated that the influence of phenolic compounds on the photosynthetic reactions is rather indirect (concerning the metabolism of chlorophyll, see Gniazdowska and Bogatek, 2005; Inderjit and Duke, 2003; Yang et al., 2004). In laboratory conditions, phenolics showed negative effects on the porphyrins and chlorophyll content, net photosynthesis, and ion uptake; they caused stomatal closure, and finely retarded the growth of seedlings. Phenolics concentrations in above mentioned experiments were up to 100 mg g1 or 5  104 to 103 M (Alsaadawi et al., 1986; Einhellig and Kuan, 1971; Einhellig et al., 1970; Yang et al., 2002, 2004). In natural conditions, we found that concentrations of analytically detected phenolic compounds were rather low. During the growing season of A. maculatum the amount of total rhizosphere soil phenolics was up to 89.22 mg g1, whereas soil phenolic acids amount was up to 4.18 or 2.49  105 M. It is very well known that phenolic compounds may exhibit synergistic effects (Einhellig et al., 1970), which could be an explanation for inhibition of certain ecophysiological processes of A. maculatum. Gradually decreasing of leaf chlorophyll during the vegetation season is mostly affected by disintegration of chloroplasts and changes in photosynthetic pigments due to cumulative oxidative stress (Munne-Bosch and Alegre, 2002). Changes in chlorophyll a:b ratio, which is the main biochemical adaptation to light levels, suggests that chlorophyll a and chlorophyll b containing complexes were not degraded at the same time. Our findings also suggest that phenolics are related with chlorophyll biosynthesis in general, and with chlorophyll a:b ratio. Total chlorophyll content decreased, and chlorophyll a:b ratio increased, which could indicate that the biosynthesis pathways of chlorophyll b were particularly affected by the increased amount of phenolics. Hejl et al. (1993) also reported the lowered total chlorophyll content in duckweed grown in the presence of juglone,

but no alternations in chlorophyll a:b ratio. Finally, LAI is a measure of partitioning of photosynthates into leaves with a resulting positive feedback to total biomass production (Hirose et al., 1997), and for that reason a good predictor of morphological adaptations of plants to maintain biomass production even if the photosynthetic carbon gain per leaf area is reduced. Further laboratory investigations with dose-dependent experiments would allow an objective comparison of field patterns with bioassay results and may characterize more precisely the participation of phenolic compounds in the ecophysiological processes occuring in the forest herbs. From the present field study we conclude that dominant trees as the main source of phenolic compounds can influence photosynthesis and related metabolic processes of the vernal ephemeroid A. maculatum via the bioavailable rhizosphere soil phenolics.

Acknowledgments This work was supported by the Ministry of Science of Serbia no.143025. We thank the referees for their valuable comments for the improvement of this paper.

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