Trees (2009) 23:277285 DOI 10.

1007/s00468-008-0275-y

ORIGINAL PAPER

Photosynthetic changes and oxidative stress caused by iron ore dust deposition in the tropical CAM tree Clusia hilariana
Eduardo Gusmao Pereira Marco Antonio Oliva Kacilda Naomi Kuki Jose Cambraia

Received: 5 February 2008 / Revised: 11 August 2008 / Accepted: 8 September 2008 / Published online: 2 October 2008 Springer-Verlag 2008

Abstract The effect of iron solid particulate matter (SPMFe) deposited onto soil and leaves on photosynthesis and oxidative stress was evaluated in Clusia hilariana, a CAM tropical tree of high occurrence in Brazilian restingas. Signicant increases in iron content were found in plants exposed to SPMFe applied onto leaf and soil surfaces. However, only the application of SPMFe on leaves of C. hilariana caused signicant reductions in some evaluated characteristics such as photosynthetic rate, stomatal conductance, transpiration, organic acid accumulation, potential quantum yield of PSII, and changes in daily CAM photosynthesis pattern. Increase in relative membrane permeability and reduction in catalase and superoxide dismutase activities in the leaves of plants exposed to SPMFe also were observed; however, lipid peroxidation did not change. These responses seem to be due to the combination of physical effects such as increase of leaf temperature, reduction in light absorption, obstruction of stomatal pores, and biochemical effects triggered by oxidative stress. Keywords Antioxidant enzymes Crassulacean acid metabolism (CAM) Particulate matter Photosynthesis Quantum yield

Introduction Clusia hilariana Schltdl. is a CAM plant (Franco et al. 1999) of high occurrence in Brazilian restingas, an ecosystem which may be exposed to various environmental stresses. C. hilariana is a key species to the dynamics of restinga plant communities characterized by high solar irradiance, high air and soil temperatures, low availability of nutrients and a low water-holding capacity of the sandy soils (Liebig et al. 2001). It is found as a central tree of the open Clusia scrub formation, islands of vegetation fragment surrounded by sand strips. As a pioneer and nurse plant, a large diversity of plant species grow around and under its canopy due to the accumulation of litter, humus and nutrients, increased water-holding capacity and protection from high soil temperatures, and high light intensity. Clusia is the only genus of species performing CAM with secondary growth characteristics of dicotyle donous trees characterized so far (Luttge 2006). The open Clusia scrub formation is one of the few vegetational systems that have a CAM plant as central species (Dias et al. 2006) and contributes to quality and quantity of the restinga oristic biodiversity (Liebig et al. 2001). The Brazilian coastline hosts an increasing number of iron ore processing and beneciation factories. These industrial activities near the restinga emit iron solid particulate matter (SPMFe), causing changes in the structure, dynamics, and diversity of the exposed vegetation, affecting the growth and development of sensitive species, reducing their occurrence in the vegetation, or even leading to their disappearance (Grantz et al. 2003; Kuki et al. 2008). SPMFe emissions can affect several organization levels of a vegetational system. At individual level, these emissions can affect processes such as photosynthesis, mineral nutrition, biomass accumulation and reproduction,

Communicated by T. Grams. E. G. Pereira (&) M. A. Oliva K. N. Kuki Departamento de Biologia Vegetal, Universidade Federal de Vicosa (UFV), Vicosa, Minas Gerais 36570-000, Brazil e-mail: egpereira@gmail.com J. Cambraia Departamento de Biologia Geral, UFV, Vicosa, Minas Gerais 36570-000, Brazil

123

278

Trees (2009) 23:277285

either by physical effects such as light blocking, abrading plant surface, leaf warming, obstruction of stomatal pores, or by chemical effects as oxidative stress, changes in pH, nutritional status, and nutrient leaching from leaves. In a more severe degree of ecological risk, and at population level, particulate matter can cause changes in nutrient cycling or energy ow (Grantz et al. 2003). Iron is an essential micronutrient to higher plants. Nevertheless, an excessive absorption and accumulation of this metal by plants can enhance the formation of reactive oxygen intermediates (ROIs; Becana et al. 1998). These intermediates are common by-products of aerobic metabolism participating in cellular signaling. However, as result of several stresses, such as iron in excess, the ROIs may lead to the unspecic oxidation of proteins and membrane lipids or may cause DNA injury (Schutzendubel and Polle 2002). Plants, however, are able to eliminate most of these ROIs by enzymatic and non-enzymatic mechanisms. Nonenzymatic antioxidants include ascorbate and glutathione (GSH), as well as tocopherol, avonoids, alkaloids, and carotenoids. The enzymatic mechanisms include enzymes like superoxide dismutase (SOD), ascorbate peroxidase (APX), glutathione peroxidase (GPX), and catalase (CAT) capable to eliminate most of the ROIs produced under oxidative stress (Apel and Hirt 2004). Clusia hilariana, as a CAM plant, is capable of enduring varied environmental stresses, probably by having a better control of carbon and water balances than C3 plants (Black and Osmond 2003). However, CAM plants are dependent on the limited supply of malate that is accumulated overnight in the vacuoles as a source of CO2 during the daylight deacidication period (Franco et al. 1999). The amplitude of daily balance of organic acid contents and the amounts of day versus night CO2 xation are very sensitive to environmental changes such as variation in light intensity, temperature, photoperiod, O2, and CO2 levels, plant development, salinity, or water availability (Black and Osmond 2003). We believe that high plant iron content due to SPMFe deposition from polluting sources close to restinga fragments possibly affect CAM photosynthesis and oxidative stress in C. hilariana. Injuries to this species can lead to changes in the plant communities in which it is inserted. Due to its signicant ecological role, the purpose of this study was to evaluate the effect of SPMFe applied onto the soil and leaves on biochemical and physical factors that may affect photosynthesis and oxidative metabolism in C. hilariana.

Materials and methods Plants of C. hilariana Schlechtendal were obtained by cuttings from individuals provided from the nursery of

Paulo Cesar Vinha State Park, Guarapari, Espirito Santo state, Brazil. After adequate root development in Hoaglands nutrient solution, plants were transplanted to 5-L pots containing 2:2:1 soil:sand:organic matter as substrate, and acclimatized for 3 months at these conditions, up to the beginning of the experiments. The experiments were performed under greenhouse conditions (annual average temperature of 27.6C, and average air relative humidity of 72%) at the Universidade Federal de Vicosa, Brazil (20450 S, 42520 W). Before application of SPMFe treatment, plants were selected for uniformity in size, height, leaf number, and phytosanitary status. In a rst experiment 0, 17.5, and 52.5 g of SPMFe per plant, were manually applied, in a uniform way, weekly onto soil surface for 3 months, avoiding contact of SPMFe to the plant. The 17.5 g dose corresponds approximately to the amount deposited on a polluted area, 6 mg cm-2 day-1 SPMFe, according to Lopes et al. (2000). The soil was acidied twice a week by adding 200 mL of H2SO4 solution, pH 3. In a second experiment 2.4 mg cm-2 day-1 SPMFe was applied onto leaf surface during 45 days, either at the beginning (8:00 h) or at the end of the day (17:00 h) by a chamber built according to Hirano et al. (1995) with modications. The chamber consisted of a wire dome covered with transparent polypropylene, connected to a dust generator and delivery system. The SPMFe was applied to the plants under natural light conditions, and the amount of SPMFe was enough to create a lm of dust on the leaves. Before each SPMFe application, the soil surface in the pots was completely covered with plastic lm. The amount of SPMFe deposited onto the leaves and the light transmittance through this deposited layer of SPMFe was estimated by gravimetry or by a photo-radiometer, respectively, using Petri dishes of known area. Gas exchanges were measured using a Portable Photosynthesis System LI-6400 (LI-COR Incorporation, Lincoln, NE, USA), at 29, 44, 75, 93, and 110th day and 3, 8, 28, 39, and 45th day after the beginning of SPMFe application to the soil or to the leaf surface, respectively. The measurements were made in a pair of opposite leaves of the third node. In the rst leaf, the SPMFe was removed from its surface 6 h before each measurement, avoiding gradual SPMFe accumulation. In the corresponding opposite leaf, the SPMFe was not removed, allowing its accumulation over the entire experimental period, i.e., up to the 45th day. At the end of the experiments, and after SPMFe removal, the net photosynthesis (A), stomatal conductance (gs), transpiration (E), and intercellular to atmospheric CO2 concentration ratio (Ci/Ca) were simultaneously measured in both leaves. Measurements were always done between 0:00 and 2:00 am. Daily courses of A and gs were performed at the end of the experiments. Leaf temperature was

123

Trees (2009) 23:277285

279

measured at 31, 38, and 45 days after the beginning of SPMFe application on the leaves, always at midday, using an infra-red thermometer (Model AG42, Telatemp). At the end of the experiment (45th day), instant lightresponse curves of uorescence were measured in the early morning (CAM II phase), using the light curve program of the Mini-PAM uorometer (Heinz Walz, Effeltrich, GmbH). The leaves were dark adapted for 30 min, and then the actinic light was applied in an increasing intensity varying from 0 to 3,500 lmol m-2 s-1 in nine steps of 30 s. At each light intensity the effective quantum yield of PSII (DF/Fm0 ), apparent electron transport rates through PSII (ETR) and non-photochemical quenching (NPQ) were measured. The potential quantum yield of PSII (Fv/Fm) was evaluated in the dark adapted state. All measurements were taken in fully expanded leaves on which the SPMFe was not periodically removed, in areas without apparent damages. The cleaning of those leaves was performed only before the beginning of the measurements. Iron content in leaves was determined by atomic absorption spectrophotometry (Kampfenkel et al. 1995). Chlorophyll a, b, and carotenoid determination followed the protocol established by Wellburn (1994), using dimethylsulphoxide (DMSO) as extractant. In these same leaves, titratable acidity was determined at the beginning and the end of the day (Eastmond and Ross 1997) and membrane permeability by electrolyte leakage (Tarhanen et al. 1999). The antioxidant enzyme activities were determined in plant tissue taken from the same leaves used in uorescence measurements and pigment determination, collected at noon, homogenized in an extraction medium constituted of 0.3 M phosphate buffer, pH 6.8, 0.1 mM ethylenediaminetetraacetic acid (EDTA), 1 mM phenylmethylsulfonyl uoride (PMSF), 1 mM dithiothreitol (DTT), and insoluble polyvinylpolypyrrolidone (1% of the sample fresh weight). The

homogenate was centrifuged at 15,0009g for 15 min and the supernatant used for enzyme activity determination: superoxide dismutase (SOD; EC 1.15.1.1; Del Longo et al. 1993), catalase (CAT; EC 1.11.1.6; Havir and McHale 1987), total peroxidase (POX; EC 1.11.1.7; Shevyakova et al. 2002) and ascorbate peroxidase (APX; EC 1.11.1.11; Nakano and Asada 1981). Lipid peroxidation was evaluated by malonaldehyde production according to method of Heath and Packer (1968). The experiments followed a randomized block design, with ve replicates. The data were subjected to an analysis of variance (ANOVA) and the means compared by Tukeys test at 5% of probability using the software SAEG 9.0, UFV. Regression analysis was carried out when required.

Results Iron contents in leaves of C. hilariana were about 2.6 and 4.9 times higher in plants in which SPMFe was applied onto the soil and onto the leaves, respectively (Table 1). Application of 6 mg cm-2 day-1 of SPMFe onto the soil did not change leaf iron content signicantly. Titratable acidity was reduced only when SPMFe was applied onto leaf surfaces (Table 1). Reductions of 46 and 35% in titratable acidity were recorded at the beginning and at the end of the light period, respectively. These plants also showed signicant reductions in chlorophyll a and b, while carotenoid contents remained constant in both SPMFe treatments. No signicant differences were found in leaf gas exchanges when the SPMFe was applied onto the soil (data not shown), whereas there were decreases of about 37, 40, and 45% in photosynthesis, stomatal conductance and transpiration, respectively, as early as 3 days after SPMFe application onto leaf surfaces (Fig. 1).

Table 1 Iron content, titratable acidity determined at the beginning (initial) and the end (nal) of the day and photosynthetic pigments (chlorophyll aChl a, chlorophyll bChl b and carotenoidCar) in SPMFe (mg cm-2 day-1) Soil 0 6 18 Leaf 0 2.14 0.09 B 0.44 A 480.9 Aa 259.4 Ba 0.11 B 0.14 B 0.29 A 444.7 Aa 452.3 Aa 465.0 Aa Leaf iron content (mg g-1 DW)

leaves of Clusia hilariana after application of different doses of SPMFe onto the soil and leaf surface Photosynthetic pigments (lg cm-2) Chl a Chl b Car

Titratable acidity (lmol g-1 FW) Initial Final

150.7 Ab 144.8 Ab 111.2 Ab 253.1 Ab 165.7 Bb

51.87 A 42.47 A 45.25 A 48.57 A 34.29 B

29.00 A 24.84 A 23.55 A 34.12 A 27.30 B

14.95 A 16.18 A 13.75 A 21.32 A 19.12 A

Means followed by the same capital letters in the columns and small letters in the rows are not signicantly different by Tukeys test at 5% of probability

123

280

Trees (2009) 23:277285

Measurements done at the 45th day in different leaves exposed to SPMFe showed reductions in photosynthesis (A), stomatal conductance (gs), and transpiration (E) in the following descending order: control [ leaves in which SPMFe was periodically removed [ leaves in which SPMFe was accumulated up to the end of the experiment. In the last case, these parameters were reduced by approximately 100, 85, and 83%, respectively, while Ci/Ca ratio increased 112% relative to control plants (Fig. 1). Application of SPMFe onto the soil, did not affect photosynthesis rate over the daily course, while the deposition of SPMFe onto leaf surfaces of C. hilariana strongly reduced it. The sudden increase in stomatal conductance at the beginning of the day was much lower in plants treated with SPMFe. There was an increase in A with the increase in gs over the night (Fig. 2). Leaf temperature increased in both treatments during the experiment in response to the semi-controlled conditions, and was higher in plants exposed to SPMFe than in the control plants during all the evaluation period, showing differences up to 3C between treatments (Fig. 3). Leaf daily deposition was about 2.14 mg cm-2 of SPMFe, which leads to approximately 55% blocking of incident light per day according to an experimental procedure using Petri dishes (Fig. 4).

The effective quantum yield of PSII (DF/Fm0 ) was reduced by SPMFe application onto leaf surfaces in all photosynthetic photon ux densities (PPFD). Strong photoinhibition was observed by the reduction of the Fv/Fm values in the dark adapted state from 0.8 in control plants to 0.3 in treated plants (Fig. 5). Even under low irradiance (250 lmol m-2 s-1 imposed by the modulated uorometer) there was reduction in the apparent electron transport rates through PSII (ETR) in leaves exposed to the SPMFe. This reduction was more severe with the increase of the light intensity, lowering the maximal electron transport rate in 56% at 2,000 lmol m-2 s-1. The light-response curves of the non-photochemical quenching (NPQ) and the DF/ Fm0 were altered by the SPMFe deposition onto leaf surface (Fig. 5). None of these uorescence parameters were affected by application of SPMFe onto the soil. Peroxidase (POX) and ascorbate peroxidase (APX) activities were not changed by either SPMFe applications (Table 2). When SPMFe was applied onto soil surface there were increases of about 73 and 28% in catalase (CAT) and superoxide dismutase (SOD) activities, respectively. Application of SPMFe onto the leaves, however, lead to reductions of about 66 and 42% in CAT and SOD activities, respectively. The relative membrane permeability of

Fig. 1 Changes in photosynthetic rate (A), stomatal conductance (gs), transpiration (E) and intercellular to atmospheric CO2 concentration ratio (Ci/Ca) in C. hilariana as a function of time of application of SPMFe onto leaf surface. Open circle control, lled circle ?SPMFe. Gray bars represent data evaluated after 45 days of continued SPMFe application. Error bars represent standard deviation

a
6

b
0.05

A (mol m-2 s-1)

4 0.03

0.02

0.01 0 0.00

c
0.5

d
1.0 0.8 0.6 0.4 0.2 0.0

E (mmol m-2 s-1)

0.4 0.3 0.2 0.1

0.0 0 10 20 30 40 0 10 20 30 40

Time (Days)

Time (Days)

123

Ci/Ca

gs (mol m-2 s-1)

0.04

Trees (2009) 23:277285 Fig. 2 Time course of photosynthesis (A) and stomatal conductance (gs) in C. hilariana: after SPMFe application onto leaf surface (a, c), and onto the soil (b, d). Open circle control, lled circle SPMFe applied on the leaf surface, inverted triangle 6 and lled square 18 mg cm-2 day-1. The black bars, at the bottom of the graphs, indicate the night period

281

5 4

A (mol m-2 s-1)

3 2 1 0 -1 -2

c
0.08

gs (mol m-2 s-1)

0.06

0.04

0.02

0.00 22 3 8 13 18 22 3 8 13 18

Hour of the day

Hour of the day

40

100
30

Light radiation blocked (%)

Leaf temperature (C)

80

y = 25.02Ln(x) + 36.414
R2 = 0.92**

20

60

40

10

20
0 0 30 35 40 45

Time (Days)
Fig. 3 Leaf temperature in C. hilariana subjected to the application of SPMFe onto leaf surface as a function of time of treatment. Open circle control, lled circle SPMFe. Error bars represent standard deviation

0 0 5 10 15
-2

20

SPMFe (mg cm )
Fig. 4 Light radiation blocked as a function of the amount of SPMFe deposited onto Petri dishes (** P \ 0.01)

123

282 Fig. 5 Effective quantum yield of PSII (DF/Fm0 ), apparent electron transport rates (ETR) and non-photochemical quenching (NPQ) under different photosynthetic photon ux densities (PPFD) in C. hilariana after SPMFe application onto leaf surface (a, c, e) and onto the soil (b, d, f). Open circle control plants, lled circle SPMFe applied onto leaf surface, inverted triangle 6 mg cm-2 day-1 and square 18 mg cm-2 day-1 of SPMFe applied onto soil. Data were subjected to regression analysis and grouped within the treatments with iron applied to the soil according to the analysis of variance. Quadratic model for ETR, NPQ and DF/Fm0 in both the treatments, except for the DF/Fm0 curve in plants with application of SPMFe on the leaves, which showed squaredroot model. (*** P \ 0.001)

Trees (2009) 23:277285

0.8

R : 0.99***
0.6

a
2

R : 0.94***

F/Fm'

0.4

R : 0.99***
0.2

0.0

c
250 200 150 100 50 0
2

R : 0.99***

R : 0.99***

ETR

R : 0.99***

3.0 2.5 2.0

R : 0.99***

R2: 0.98***

NPQ

1.5 1.0 0.5 0.0 0 1000 2000 3000 0 1000 2000 3000

R : 0.99***

PPFD

PPFD

C. hilariana increased only when SPMFe was applied onto the leaves, though no changes in membrane lipid peroxidation were observed.

Discussion Iron accumulation in leaves of plants exposed to SPMFe was considerably higher than in control plants, exceeding

the usual nutrient requirements and reaching values close to 0.5 mg g-1 DW, which is considered to be phytotoxic for most plants (Marschner 1995). This iron accumulation brought changes to physiological evaluated characteristics when SPMFe was applied onto leaf surface but not when it was applied onto soil. The main effect of SPMFe on C. hilariana leaves seems to be a combination of factors: obstruction of stomatal pores (Figs. 1, 2), increase in leaf temperature (Fig. 3),

123

Trees (2009) 23:277285

283

Table 2 Catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX) and superoxide dismutase (SOD) activities, relative membrane permeability and malondialdehyde (MDA) concentration in Clusia hilariana after application of different doses of SPMFe onto the soil or leaf surface SPMFe (mg cm-2 day-1) Soil 0 18 Leaf 0 2.14 51.71 A 17.63 B 1.78 A 1.80 A 0.38 A 0.54 A 54.07 A 31.08 B 26.83 B 31.79 A 131.82 A 131.20 A 18.43 B 31.95 A 0.58 A 0.62 A 0.35 A 0.25 A 38.93 B 49.69 A 26.82 A 23.83 A CAT (lmol min-1 g-1 FW) POX (lmol min-1 g-1 FW) APX (lmol min-1 g-1 FW) SOD (lmol min-1 g-1 FW) Relative permeability (%) MDA (nmol g-1 FW)

Means followed by the same capital letters in the columns are not signicantly different by Tukeys test at 5% of probability

blockage of light radiation (Fig. 4) and oxidative stress (Table 2). The limited light radiation that effectively reaches the leaf (Shari et al. 1997) and the oxidative stress generation can lead to reduction in pigment contents as observed here. The increase in temperature, due the dark color SPMFe heat absorption, and transference to leaf, in combination with reduction in stomatal conductance could be an explanation for the observed changes in gas exchange parameters. Moreover, the enzyme antioxidant system was not capable to alleviate the effects of the application of SPMFe onto leaf surface. The observed activity loss on SOD and CAT defense enzymes is suggestive of a strong oxidative stress (Schutzendubel and Polle 2002). Nevertheless, when SPMFe was applied onto soil the enzyme apparatus seemed to be capable to scavenge the oxygen reactive species produced (Table 2). Increases in SOD activity after exposure to iron has also been found by other authors (Sinha et al. 1997) and indicates that Clusia has some tolerance to intermediate levels of iron. This ability of CAM plants to tolerate oxidative stress increasing the expression of the antioxidative response system has been recognized by Luttge (2002). The direction of the response in the antioxidant system is dependent on the plant species, the metal and the intensity of the stress (Schutzendubel and Polle 2002). ROIs at low concentrations are key signaling molecules that act at the interface between abiotic and biotic stresses and function as central coordinators of cell biology and responses to numerous developmental and environmental stimuli (Apel and Hirt 2004). However, under high levels of iron, near or in the toxicity zone, the amount of ROIs produced lead to enzyme inhibition (Schutzendubel and Polle 2002), as was observed in this experiment when SPMFe was applied onto leaf surface. SPMFe application onto leaves, mainly in those leaves from which SPMFe was not periodically removed, drastically blocked sunlight (Fig. 4). The light blockage and the oxidative stress caused by SPMFe, besides reducing

chlorophyll contents, contributed to a lower DF/Fm0 and CO2 uptake. At high irradiances, both the processes of photochemical and non-photochemical quenching, mainly by thermal dissipation, were impaired (Fig. 5). SPMFe leaf surface deposition reduced ETR drastically. Fv/Fm and ETR depletion caused by particulate deposition was also reported by Naidoo and Chirkoot (2004) in Avicennia marina, a typical mangrove plant. Impairment of light absorption and energy conversion in C. hilariana caused by the SPMFe can limit the establishment of this species in areas of restinga exposed to SPMFe deposition. The titratable acidity reduction observed at the beginning of the day is related to a lower photosynthetic rate in leaves of plants exposed to SPMFe deposition. Even the deposition of inert materials affects gas exchanges when they are accumulated on leaves (Hirano et al. 1995). Despite the blockage of stomata, the Ci/Ca ratio tend to increase, reaching at 45th day of continuous SPMFe application onto the leaves a value two times higher than in control plants, probably due an reduction on carboxylation capacity, indicated by A rate near zero (Fig. 1). Some reports show that an additional absorption of the incident radiation by the layer of deposited particulate matter can result in an increase in the leaf temperature (Borka 1984; Hirano et al. 1995). This increase in temperature could be the result of reduced transpiration levels, probably due to pore obstruction by iron ore dust. Plants of Hellianthus annus and Triticum aestivum also showed reduction in transpiration rates with increase in leaf temperature due to cement material deposition, or even iron ore on leaf surfaces (Borka 1980, 1984). The photochemical mechanism may have been impaired by damages to cell membranes, as indicated by an increase in electrolyte leakage. This cell damage, however, did not reect an increase in membrane lipid peroxidation (Table 2). Nevertheless, lipid peroxidation has been reported for several plant species exposed to iron in excess (Fang et al. 2001; Souza-Santos et al. 2001). In spite of

123

284

Trees (2009) 23:277285 CO2-exchange and organic acid decarboxylation in the tropical CAM tree Clusia hilariana. Tree Physiol 19:635644 Grantz DA, Garnerb JHB, Johnson DW (2003) Ecological effects of particulate matter. Environ Int 29:213239. doi:10.1016/S01604120(02)00181-2 Havir EA, McHale NA (1987) Biochemical and developmental characterization of multiple forms of catalase in tobacco leaves. Plant Physiol 84:450455 Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplast. I. kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125:189198. doi:10.1016/0003-9861(68) 90654-1 Hirano T, Kiyota M, Aiga I (1995) Physical effects of dust on leaf physiology of cucumber and kidney bean plants. Environ Pollut 89:255261. doi:10.1016/0269-7491(94)00075-O Kampfenkel K, Montagu MV, Inze D (1995) Effects of iron excess on Nicotiana plumbagnifolia plants: implications to oxidative stress. Plant Physiol 107:725735 Kuki KN, Oliva MA, Pereira EG (2008) Iron ore industry emissions as a potential ecological risk factor for tropical coastal vegetation. Environ Manage 42:111121. doi:10.1007/s00267-0089093-7 Liebig M, Scarano FR, Mattos de EA, Zaluar HLT, Luttge U (2001) Ecophysiological and oristic implications of sex expression in the dioecious neotropical CAM tree Clusia hilariana Schltdl. Trees (Berl) 15:278288. doi:10.1007/s004680100096 Lopes SA, Oliva MA, Martinez CA (2000) Impacto das imissoes de dioxido de enxofre e deposicao de material particulado de ferro em especies vegetais de restinga: avaliacao ecosiologica. In: Espndola E, Paschoal C, Rocha O, Bohrer M, Oliveira Neto A (eds) Ecotoxicologia. RiMa Artes e Textos, Sao Carlos, pp 5371 Luttge U (2002) CO2-concentrating: consequences in crassulacean acid metabolism. J Exp Bot 53:21312142. doi:10.1093/jxb/ erf081 Luttge U (2006) Photosynthetic exibility and ecophysiological plasticity: questions and lessons from Clusia, the only CAM tree, in the neotropics. New Phytol 171:725. doi:10.1111/j.14698137.2006.01755.x Marschner H (1995) Mineral nutrition of higher plants, Second edn. Academic Press, London, p 889 Naidoo G, Chirkoot D (2004) The effects of coal dust on photosynthetic performance of the mangrove, Avicennia marina in Richards Bay, South Africa. Environ Pollut 127:359366. doi: 10.1016/j.envpol.2003.08.018 Nakano Y, Asada K (1981) Hydrogen peroxide is scavenged by ascorbate specic peroxidase in spinach chloroplasts. Plant Cell Physiol 22:867880 Schutzendubel A, Polle A (2002) Plant responses to abiotic stresses: heavy metal-induced oxidative stress and protection by micorrhization. J Exp Bot 53:13511365. doi:10.1093/jexbot/53. 372.1351 Shari MR, Gibson AC, Rundel PW (1997) Surface dust impacts on gas exchange in Mojave Desert shrubs. J Appl Ecol 34:837846. doi:10.2307/2405275 Shevyakova NI, Stetsenko LA, Meshcheryakov AB, Kuznetsov VV (2002) The activity of the peroxidase system in the course of stress-induced CAM development. Russ J Plant Physiol 49:598 604. doi:10.1023/A:1020224531599 Sinha S, Gupta M, Chandra P (1997) Oxidative stress induced by iron in Hydrilla verticillata (l.f.) Royle: response of antioxidants. Ecotoxicol Environ Saf 38:286291. doi:10.1006/eesa.1997. 1598 Souza-Santos P, Ramos RS, Ferreira ST, Carvalho-Alves PC (2001) Iron-induced oxidative damage of corn root plasma membrane H?-ATPase. Biochim Biophys Acta 1512:357366. doi: 10.1016/S0005-2736(01)00341-8

that, the changes in SOD and CAT activities observed here indicate the occurrence of oxidative stress, contributing to the photosynthetic impairment. Clusia hilariana showed high sensitivity to SPMFe, especially when it was applied onto the leaf surface. Since leaf, the organ directly exposed to the metal, is also responsible for uptake and transforming of light into energy in the plant, it is probable that even in plants with CAM metabolism, which show great resistance to a variety of environmental stresses, all factors affecting directly or indirectly the process of photosynthesis would be potentially harmful. However, to reinforce the results of the semi-controlled experiments, tests under eld conditions are being considered in ongoing research. The SPMFe emissions can limit the establishment of C. hilariana in areas of restingas exposed to SPMFe deposition. So, special care should be taken with plant communities like those in Brazilian restingas subjected to SPMFe emissions by iron processing industries nearby.

References
Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373399. doi:10.1146/annurev.arplant.55.031903.141701 Becana M, Moran JF, Iturbe-Ormaetxe I (1998) Iron-dependent oxygen free radical generation in plants subjected to environmental stress: toxicity and antioxidant protection. Plant Soil 201:137147. doi:10.1023/A:1004375732137 Black CC, Osmond CB (2003) Crassulacean acid metabolism photosynthesis: working the night shift. Photosynth Res 76:329341. doi:10.1023/A:1024978220193 Borka G (1980) The effect of cement dust pollution on growth and metabolism of Helianthus annuus. Environ Pollut 22:7579. doi: 10.1016/0143-1471(80)90084-7 series A Borka G (1984) Effect of metalliferous dusts from dressing works on the growth, development, main metabolic processes and yields of winter wheat in situ under controlled conditions. Environ Pollut 35:6773. doi:10.1016/0143-1471(84)90131-4 series A Del Longo OT, Gonzales CA, Pastori GM, Trippi VS (1993) Antioxidant defenses under hypergenic and hyper osmotic conditions in leaves of two lines of maize with differential sensitivity to drought. Plant Cell Physiol 34:10231028 Dias ATC, Mattos EA, Vieira SA, Azeredo JV, Scarano FR (2006) Above ground biomass stock of native woodland on a Brazilian sandy coastal plain: estimates based on the dominant tree species. For Ecol Manage 226:364367. doi:10.1016/j.foreco. 2006.01.020 Eastmond PJ, Ross JD (1997) Evidence that the induction of Crassulacean acid metabolism by water stress in Mesembryanthemum crystallinum (L.) involves root signalling. Plant Cell Environ 20:15591565. doi:10.1046/j.1365-3040.1997.d01-50.x Fang WC, Wang JW, Lin CC, Kao CH (2001) Iron induction of lipid peroxidation and effects on antioxidative enzyme activities in rice leaves. Plant Growth Regul 35:7580. doi:10.1023/ A:1013879019368 Franco AC, Herzog B, Hubner C, Mattos de EA, Scarano FR, Ball E, Luttge U (1999) Diurnal changes in chlorophyll a uorescence,

123

Trees (2009) 23:277285 Tarhanen S, Metsarinne S, Holopainen T, Oksanen J (1999) Membrane permeability response of lichen Bryoria fuscescens to wet deposited heavy metals and acid rain. Environ Pollut 104:121129. doi:10.1016/S0269-7491(98)00157-2

285 Wellburn AR (1994) The spectral determination of chlorophylls a and b, as well as total carotenoids, using various solvents with spectrometers of different resolution. J Plant Physiol 144:307 313

123