Photosynthetic resPonse and tolerance of three willow sPecies to cadmium exPosure in hydroPonic culture

Three willow species (Salix alba, Salix matsudana and Salix nigra) were exposed to very high cadmium (Cd) concentrations in order to define some physiological traits related to high biomass production. Plants were grown hydroponically under semi-controlled conditions (greenhouse). It was assumed that leaves accumulate different amounts of Cd in relation to their age due to the specific occurrence of symptoms generated by Cd accumulation. The rate of photosynthesis and concentration of photosynthetic pigments in young and old leaves was correlated with biomass production in order to ascertain their significance as indicators of plant performance at sites contaminated with Cd. Changes in the photosynthetic parameters induced by treatments depended on the concentration of Cd in the nutrient solution, the species of willow and leaf age. Fv/Fm ratios were not considerably changed in treated plants, indicating preserved activity of PSII. According to results presented in this work, the Fv/Fm ratio was a less sensitive indicator of Cd stress in willows than the rate of photosynthesis and pigment concentration. High tolerance to applied treatments estimated on the basis of biomass production of S. nigra and S. matsudana irrespective of Cd concentration in the growth substrate might indicate their potential in the reclamation of Cd-contaminated sites.

introduction Cadmium (Cd) is one of the most hazardous environmental pollutants in the atmosphere, soil and water, and in excessive amounts can cause serious problems to all living organisms (Parmar et al., 2013;Tran and Popova, 2013).Although it has no known biological function (except in marine diatoms), this non-essential element is readily taken up by plants growing in Cd-enriched soils, threatening to affect metabolic processes and cause structural changes (Morel 2008;Nazar et al., 2012).Previous investigations elucidated the potential of some willow species and clones to tolerate and accumulate relatively high levels of heavy metals, and can point to good survival and biomass production (Pulford et al., 2002).In their experiment with nine clones of six willow species and one hybrid exposed to heavy metals in solution culture, Punshon and Dickinson (1997) recognized the capacity of plants to acclimate to gradually increasing concentrations of heavy metals and their potential to adapt to the toxic metals presence.Willow species exhibit different strategies to enable them to maintain optimal performance in Cd-toxic environments (Vassilev et al., 2005).
Plant productivity and plant-based extraction of heavy metals from contaminated soil and water are determined by leaf characteristics due to their importance in photosynthesis and transpiration.In the experiment with several willow and poplar clones, Zacchini et al. (2009) reported that an efficient transpiration flux necessary to drive metals from roots to shoots relies upon the efficiency of the photosynthetic apparatus.Photosynthesis is the most important process related to biomass production (Evans and von Caemmerer, 2011).Previous investigations demonstrated the harmful effect of Cd on the photosynthetic activity of many plant species exposed to either longor short-term treatment (Tran and Popova, 2013), due to structural and functional disorders.Studies have shown that excessive amounts of Cd inhibit photosynthesis both directly and indirectly through disturbance of the chloroplast structure and biosynthesis of chlorophylls and carotenoids, disruption of pigment-protein complexes, water photooxidation, CO 2 fixation, and electron transport, along with disorders in nutrient and water uptake (Seregin and Ivanov, 2001).Literature data concerning the photosynthetic activity of willow plants exposed to elevated Cd levels are inconsistent, even for the same species, probably due to differences in genotype characteristics, experimental conditions, treatment duration and Cd concentration applied.The rate of photosynthesis was not negatively affected by Cd in S. alba plants exposed to 10 µM Cd(NO 3 ) 2 added to Knop's nutrient solution (Lunáčkova et al., 2003/4).In a recent study, Pietrini et al. (2010) observed a reduction in photosynthesis of approximately 20% in the same species of plants grown in the presence of 50 µM CdSO 4 for three weeks under hydroponic conditions.A similar decline in net photosynthetic rate occurred in S. alba and S. viminalis treated with increasing Cd concentrations ranging from 0 to 15 µM due to mesophyll impairments and reduced content of chlorophyll a rather than stomatal limitation (Vassilev et al., 2005).
Chlorophyll fluorescence represents the red and far-red light emitted from de-exciting chlorophyll a molecules (Zarco-Tejada et al., 2002;Rohaček et al., 2008).This process occurs concomitantly with the other two pathways of excitation energy utilization, i.e. thermal dissipation and photochemistry (Maxwell and Johnson, 2000).The chlorophyll fluorescence parameter Fv/Fm (the ratio of variable to maximum fluorescence) is highly correlated with the quantum yield of net photosynthesis of intact leaves (Gonzalez-Mendoza et al., 2007).Under stress conditions, the decrease in Fv/Fm indicates disturbances in the photosynthetic apparatus, i.e. the decrease in the maximum quantum efficiency of PSII photochemistry (Baker and Rosenqvist, 2004).In plants exposed to heavy metal stress, a loss in PSII quantum efficiency may result from the inhibition of metabolic processes, production of chlorophyll-metal ions complexes, as well as from the slowly relaxing quenching processes and damage to the PSII reaction centers (Myśliwa-Kurdizel et al., 2002;Popovic et al., 2003).Cadmiuminduced changes in primary photochemical reactions, estimated by measuring Fv/Fm, have been observed in various plant species (Gonzalez-Mendoza et al., 2007;Parmar et al., 2013).
Leaves of different age respond differently to heavy metal stress, probably due to the specific accumulation and distribution of contaminants within and among plant organs.Research into the localization and effects of Cd in leaves of the cadmium-tolerant willow S. viminalis showed that the Cd concentration of individual leaves varied according to their insertion height on the shoot (Cosio et al., 2006).However, differences in Cd concentration in individual leaves were more evident in plants exposed to 5-50 µM Cd in relation to plants exposed to 200 µM Cd in the nutrient solution.Cosio et al. (2006) also indicated specific distribution in relation to leaf age, i.e., the heavy metal was localized mainly in the tips and edges of younger leaves, while in older leaves it was localized at the base of the leaves.Accordingly, disruption of the chloroplast structure appears differently in young and old leaves of willow plants under Cd stress, leading to a functional alteration of the photosynthetic process (Hakmaouia et al., 2007).Pietrini et al. (2010) explored the impact of Cd on photosynthesis in poplar and willow clones that had different leaf metal concentrations.Their results showed that the interaction of Cd with components of the photosynthetic process results in a specific reduction of the process in certain clones.Results from our previous work with four willow clones indicated a genotype-specific accumulation of Cd in leaves, which was positively correlated with the Cd concentration applied (Borišev et al., 2009).We assumed that dif-CADMIUM TOLERANCE AND PHOTOSyNTHETIC RESPONSE IN WILLOWS ferent concentrations of Cd in young and old leaves can affect the photosynthetic characteristics of plants and may be associated with the tolerance of species through their preserved productivity.The objectives of this study were (i) to determine the effect of Cd stress on the rate of photosynthesis (A), photosynthetic efficiency (Fv/Fm) and concentration of chlorophylls and carotenoids in young and old leaves of different species of willows, and (ii) to determine the tolerance of willow species to Cd stress.

Plant material, experimental design and growth conditions
The experiment was set up in a glasshouse of the Department of Biology and Ecology, Faculty of Sciences in Novi Sad, Serbia.Stem cuttings of four Salix clones (1 -S.alba, clone 68/53/1; 2 -S.matsudana, clone SM 4041, and 3 -S.nigra, clone 0408) were obtained from the Institute of Lowland Forestry and Environment (Novi Sad, Serbia).Plants were grown under semi-controlled conditions (natural illumination, temperature between 20 and 30 o C) using a method of water culture.The 40-l pots covered by drilled linoleum contained 18 plants, and their distribution was completely randomized.Each treatment was performed in duplicate (36 plants).Plant cuttings were rooted in deionized water.After 30 days, water was replaced by a full-strength Hoagland nutrient solution containing 0, 10 and 100 µM of Cd (supplied as CdCl 2 ).Nutrient solutions were continuously aerated and replaced every two weeks.After 70 days of exposure, photosynthetic characteristics were measured in young and old leaves.young leaves comprised the first to seventh leaf counting from the shoot top, and all other leaves were considered as old leaves.

rate of photosynthesis
The photosynthetic activity of young and old leaves was determined polarographically on leaf segments excised from the middle part of the leaves.The seg-ments were suspended in buffer pH 7.6-7.8,containing 10 mM NaHCO 3 , and the rate of photosynthetic oxygen evolution (µmol O 2 g -1 •h -1 ) was measured using the Hansatech DW1 electrode (Walker, 1987).The rate of photosynthesis of the young and old leaves of six individual plants was determined for each treatment and each clone (n=6)

In vivo chlorophyll a fluorescence
Chlorophyll a fluorescence was measured using a portable fluorometer (PSM, BioMonitor, AB, Sweeden) on the middle part of intact leaves (Öquist and Wass, 1988).Before measuring, the leaves were adapted to the dark for 30 min.The following parameters of chlorophyll a fluorescence were measured: Fo (the minimum level of fluorescence in the dark), Fm (the maximum of fluorescence in the dark), Fv (variable fluorescence, Fv= Fm-Fo), t 1/2 (half the time required to reach maximum fluorescence from Fo to Fm), Fv/ Fm (the maximum quantum efficiency of PSII photochemistry or maximum quantum yield of PSII).Chlorophyll fluorescence parameters were determined on the young and old leaves of six individual plants for each treatment and each clone (n=6).

concentration of photosynthetic pigments
The concentration of chlorophyll a (Chl a), chlorophyll b (Chl b) and total carotenoids was determined spectrophotometrically (Beckman, DU ® Series 65, Scotland) following extraction in absolute acetone in young and old leaves, and expressed as mg g -1 dry weight (Von Wettstein, 1957).The concentration of photosynthetic pigments in young and old leaves was determined in five samples for each treatment and each clone (n=5).The ratio of Chl a and Chl b concentrations, as well as of the total chlorophyll and carotenoid concentrations (chlorophyll (a+b)/carotenoids), were calculated.

tolerance index
The tolerance index (TI) was calculated using fresh weight in treated and control plants as follows: TI = fresh weight of plants treated with Cd x 100/fresh weight of control plants.This parameter is considered to be an indicator of biomass production, and it was determined for six individual plants for each treatment and each clone (n=6).

statistical analysis
Experimental data were processed with one-way analysis of variance (ANOVA) using Statistica for Windows ver.12.0.Mean values of studied parameters (rate of photosynthesis, chlorophyll fluorescence parameters Fv/Fm and t 1/2 , and concentration of photosynthetic pigments) obtained for different treatments in one plant species were compared by Duncan's multiple range test at a significance level of p<0.05.The significance of the TI changes in plants exposed to various treatments was analyzed by the t-test.Pearson's correlation coefficients were calculated between biomass production and cadmium concentrations in nutrient solution, rate of photosynthesis, photosynthetic efficiency (Fv/Fm) and total chlorophyll and carotenoid concentrations, in order to determine relationships between these parameters.

results rate of photosynthesis and chlorophyll a fluorescence
Results presented in Table 1 show a remarkable decrease in the photosynthetic rate (A) in young and old leaves of willow species exposed to different concentrations of Cd.The rate of photosynthesis was reduced more in young than in old leaves of S. alba and S. matsudana plants exposed to Cd, compared to the control.In all studied species, photosynthetic efficiency (Fv/Fm) varied between 0.70-0.76and 0.70-0.78 in young and old leaves, respectively, and changes in Fv/ Fm and t 1/2 with increasing Cd concentration were not observed (Table 1).

Concentration of photosynthetic pigments
The concentration of photosynthetic pigments varied among treatments due to leaf age in all species (Tables 2 and 3).The accumulation of Cd in leaves resulted in chlorosis of young leaves exposed to 100 µM Cd.The concentration of chlorophylls and carotenoids tended to decrease in most cases, and young leaves of S. matsudana were the least affected.However, the sensitivity of old leaves to Cd with respect to photosynthetic pigment concentration was lower than in young leaves.Chl a/b ratio changes were not uniform among species, treatments and leaves.The ratio chlorophyll (a+b)/carotenoids tended to decrease in S. alba and S. nigra (Tables 2 and 3).

tolerance of willow species to cd treatments
In order to estimate the susceptibility of willow species in relation to Cd supply, the tolerance index (TI) was calculated on the basis of plant fresh weight (Table 4).The lowest tolerance to applied Cd levels was recorded in S. alba resulting in disturbed biomass production.In S. matsudana and S. nigra, the TI was not considerably different in plants exposed to 10 and 100 µM Cd.Correlation coefficients between plant production and external Cd concentration, rate of photosynthesis (A) and concentration of chlorophylls (Chl a+b) and carotenoids (Car) in young and old leaves of the examined willow species are shown in Table 4.A significant negative correlation between biomass production and external Cd concentration was observed in S. matsudana and S. alba.Plant productivity was positively correlated with rate of photosynthesis (A) and photosynthetic pigments (Chl a+b and Car) in young and old leaves of the willow species.However, no significant correlation was found between plant productivity and rate of photosynthesis (A) in the young leaves of S. nigra.

discussion
Analysis of the in vivo photosynthetic response in treated plants is complex because of the primary and secondary effects of heavy metals and the self-regulatory system (Vassilev et al., 1995).In the studied willows, photosynthetic activity seemed to depend on the metal concentration applied, genotype (species) and leaf age.The inhibitory effect of the treatments on the rate of photosynthesis was more pronounced in young than in old leaves of S. alba and S. matsudana.In plants exposed to heavy metals, a decrease in the photosynthetic activity may be partly due to decreased chlorophyll content, as reported for other fast-growing tree species (Lunáčkováet al., 2003/4;Nikolić, 2009).In this study, the concentration of total chlorophylls (Chl a+b) and carotenoids was decreased more in young than in old leaves of S. alba, S. nigra and S. matsudana plants treated with Cd.In comparison with the other species tested in this study, S. nigra had the ability to retain a high rate of photosynthesis in young leaves (reduction by 28.6% only at 100 μM Cd treatment).Unaffected photosynthesis under stress conditions indicates a plant's ability to produce metabolites for further absorption, protection and growth (Pietrini et al., 2010).In S. alba, the rate of photosynthesis in the young leaves was decreased by 55.5 and 38.3% at 10 and 100 μM, respectively.In S. matsudana, a reduction in the rate of photosynthesis in young leaves or 55.5 and 38.3% was recorded at 10 and 100 μM, respectively.Previous studies have elucidated the importance of young leaves for determination of the growth rate of trees (Reich et al., 1990).However, experiments with Salix clones grown in the vegetation filter of a wastewater purification system confirmed the significant role of old leaves and their physiological characteristics in enabling high productivity (Aasamaa et al., 2010).
The photosynthetic efficiency (Fv/Fm ratio) is widely used as an indicator of stress in the photosynthetic apparatus.The mean value of Fv/Fm for the C3 plants is 0.832±0.004,while the optimal range of Fv/ Fm values for different plant species under optimal growth conditions varies from 0.750 to 0.850 (Bjorkman and Demmig, 1987).Since fluorescence mainly originates from PSII chlorophyll, it can indicate the status of photochemical processes in PSII (Vassilev et al., 1995).Values of Fv/Fm in this study were ether within or slightly below the optimal range for vascular plants.Figueroa et al. (1997) reported that Fv/Fm values decrease throughout the growing season to or below 0.600, so the values below 0.750 obtained in this work could be ascribed to the age of the plants.In this study, no significant changes in the chlorophyll fluorescence parameter Fv/Fm were found in treated willows, despite considerable decreases in photosynthetic rate.Stable values of the chlorophyll fluorescence parameter Fv/Fm in poplar and willow grown under polluted conditions could indicate the stability of the thylakoid structure and efficient electron flow through the photosystems (Pajević et al., 2009).These results suggest that other plant disorders, apart from those in chloroplasts, may be involved in the reduction of photosynthesis in plants exposed to heavy metals.Disturbances in enzyme activity, mineral nutrition, plant structure and water management are reported to occur in plants exposed to Cd (Seregin and Ivanov, 2001;Pietrini et al., 2010).An insignificant influence of Cd on the chlorophyll fluorescence parameters may occur along with evident disorders in thylakoid membranes and chloroplasts, implying that other metabolic units, apart from primary photochemical reactions, can also be affected (Vassilev et al., 1995).Furthermore, Fv/Fm remained high and close to that of the control leaves in Phragmites australis exposed to high Cd concentrations (0.78, 0.78 and 0.79 at 0, 50 and 100 µM Cd, respectively) (Pietrini et al., 2003).Older leaf sections, however, were most heavily affected by Cd with respect to Fv/Fm in Secale cereale seedlings (Krupa and Moniak, 1998).Generally, the values of Fv/Fm obtained in this work were similar to measurements obtained for P. australis and Secale cereale plants exposed to Cd.In Thlaspi caerulescens plants acclimated to Cd, the ratio Fv/Fm was not much affected, in contrast to a significant decline in this parameter during the initial period of stress in non-acclimated plants (Küpper et al., 2007).Since the willow plants grew in the presence of Cd for 70 days, there is a possibility of their acclimatization resulting in the maintenance of high PSII efficiency.According to the presented results, the Fv/Fm ratio was a less sensitive indicator of Cd stress in willows than photosynthesis and pigment concentration.Results obtained in this work are in agreement with the findings of Mendelssohn et al. (2001), who suggested other physiological indicators of heavy metal stress in wetland plants.
Processes that block energy transfer from the reaction centers to quinones strongly affect the chlorophyll fluorescence parameter t 1/2 (Reigosa Roger and Weiss, 2001).In this study, this parameter was not considerably changed in the willows exposed to Cd stress, although a decreasing tendency was more pronounced in young leaves.The results are in disagreement with the findings of Drążkiewicz et al. (2003), who reported a greater decrease in t 1/2 values in older leaves than in younger leaves of maize treated with 200 µmol/L of Cd.
The results show that cadmium treatments markedly changed photosynthetic pigment concentration in the willows, with young leaves being more affected than the old leaves.Leaf chlorosis and necrosis are symptoms of Cd toxicity observed in willows (Cosio et al., 2006).Chlorosis was evident in the young leaves of willow plants treated with 100 μM Cd.This concurs with previous reports on pronounced cadmium-induced chlorosis in young leaves in the hyperaccumulator Thlaspi caerulescens (Küpper et al., 2007) and poplars (Nikolić et al., 2008;Nikolić, 2009).The phenomenon could be explained by higher Cd accumulation and sensitivity of young leaves to Cd toxicity (Küpper et al., 2007).The tendency of Cd to accumulate more in young than in old leaves has been reported for S. alba and S. nigra (Borišev et al., 2009).According to Horvath et al. (1996) and Van Assche and Clijsters (1990), Cd alters chlorophyll biosynthesis by inhibiting protochlorophyllide reductase, and prevents the integration of chlorophyll molecules into pigment-protein complexes of photosystem II (PSII).Heavy metals, such as Cd 2+ , Hg 2+ , Cu 2+ , Zn 2+ and Ni 2+ are known to substitute the central Mg 2+ atom in the chlorophyll molecule, resulting in decreasing photosynthetic activity (Küpper et al., 1996).In this study, the Chl a/b ratio differed between willow species and Cd treatments indicating the differential susceptibility of Chl a and Chl b to Cd stress.The Chl a/b ratio was increased in old S. alba and S. nigra leaves at 100 μM Cd, suggesting a higher susceptibility of Chl b.Chl b content was more affected by heavy metals treatment in Zea mays (Ekmekçi et al., 2008), Brassica juncea (Ebbs and Uchil, 2008) and Sinapis alba (Fargašová, 1998).Contrary to these results, the diminished Chl a/b ratio in S. matsudana suggests that Chl a content was more affected than Chl b content in both young and old leaves.
In this study, the concentration of carotenoids in both young and old leaves was considerably lowered in treated plants with respect to the control, except in old leaves of S. matsudana treated with 10 μM Cd.The reduction in carotenoid content might be related to an intensive production of reactive oxygen species (Ghnaya et al., 2009).A slight negative effect of the treatments on chlorophyll and carotenoid concentrations was observed in S. matsudana.This implies that the influence of heavy metals stress on pigment con-tent in willows depends not only on the concentration and nature of the metal, but also on the species (genotype) used in the experiment.
Different susceptibility of leaves to cadmium toxicity was observed in the present work, probably depending on their age and the involvement of mechanisms that prevent the toxic effects of the metal on the photosynthetic apparatus.In plants exposed to heavy metals, changes in cell metabolism aimed to diminish the toxicity of metals, and the adaptation mechanisms in older plants, are not so flexible and efficient, so that the toxic effects on plant physiology and metabolism are much more pronounced (Krupa and Moniak, 1998).
Plants with high photosynthetic activity and tolerance could be efficient in the reclamation of Cdcontaminated sites.Apart from photosynthesis, other physiological characteristics, such as mineral nutrition and water regime, are important for the maintenance of biomass production in willows.For example, Merilo et al. (2006) reported larger productivity resulting from improved nutrient availability due to enhanced photosynthetic capacity.In this study, it seems that excessive Cd supply interrupted these processes in S. alba more intensively, resulting in a greater decrease in biomass production and the lowest tolerance index.Further investigations, however, are needed to confirm this hypothesis.According to our results, biomass production in S. alba, S. matsudana and S. nigra showed positive correlation with photosynthetic pigment concentration and the photosynthetic rate of both young and old leaves.
The results obtained in the present work suggest a specific response of the selected Salix species to applied Cd concentrations.According to the tolerance index scale provided by Lux et al. (2004), the studied willow species were highly tolerant to the applied Cd levels, with the exception of S. alba when exposed to 100 µM of Cd.In the willow species examined in this work, positive correlations were found between biomass production and the rate of photosynthesis and photosynthetic pigment concentration, irrespective of the different concentrations of Cd.However, biomass production was negatively affected by Cd in all species.The strongest negative effect of Cd on biomass production was found in S. alba, the species with the lowest TI.
In conclusion, the results obtained in this work suggest that the changes in photosynthetic parameters induced by Cd treatments depended on the metal concentration, plant species and leaf age.An inhibitory effect of the treatments on the rate of photosynthesis was more pronounced in young than in old leaves of S. alba and S. matsudana, while S. nigra retained a relatively high rate of photosynthesis in young leaves exposed to 10 µM Cd.The concentration of total chlorophylls (Chl a+b) and carotenoids decreased more in young than in old leaves of S. alba, S. nigra and S. matsudana treated with Cd.The chlorophyll fluorescence parameter, Fv/Fm ratio, was a less sensitive indicator of Cd stress in willows compared with the rate of photosynthesis and pigment concentration.The high tolerance to the applied treatments, judging by the biomass production, especially in S. nigra and S. matsudana, might indicate their potential in the reclamation of Cd-contaminated sites.However, further investigations are necessary to confirm these findings under field conditions.references Aasamaa, K., Heinsoo, K. and B. Holm (2010).Biomass production, water use and photosynthesis of Salix clones grown in wastewater purification system.Biomass Bioenergy.34, 897-905.

acknowledgments:
This study was conducted within the Project No. III 043007, supported by the Ministry of education, science and technological development of the Republic of Serbia.authors' contribution: All authors have been involved in designing the experiment, cultivation of plants, measurement of physiological parameters, processing and the discussion of obtained results, and writing of the manuscript.conflict of interest disclosure: The authors have no conflict of interest.

table 1 .
Effect of different concentrations of Cd on rate of photosynthesis (A, µmol O 2 g -1 h -1 ) and chlorophyll a fluorescence parameters (Fv/Fm and t 1/2 ) of young and old willow leaves.Values followed by a different letter differ significantly (p<0.05,n=6).
table 2. Concentration of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophylls (Chl a+b) and carotenoids of young willow leaves exposed to different concentrations of Cd (mg g -1 dw).Values followed by a different letter differ significantly (p<0.05,n=5).

table 3 .
Concentration of chlorophyll a (Chl a), chlorophyll b (Chl b), total chlorophylls (Chl a+b) and carotenoids of old willow leaves exposed to excessive cadmium (mg g -1 dw).Values followed by a different letter differ significantly (p<0.05,n=5).

table 4 .
Pearson's correlation coefficients between biomass production and cadmium concentrations in nutrient solution (Cd NS ), rate of photosynthesis (A), photosynthetic efficiency (Fv/Fm), total chlorophyll (Chl a+b) and carotenoid (Car) concentrations in young and old leaves, and tolerance index (TI) of willows.Marked correlations (*, **) are significant at p≤0.05 and p≤0.01, respectively, n=15.