Environ Sci Pollut Res (2015) 22:574–585 DOI 10.1007/s11356-014-3375-9
RESEARCH ARTICLE
Photosynthesis light-independent reactions are sensitive biomarkers to monitor lead phytotoxicity in a Pb-tolerant Pisum sativum cultivar Eleazar Rodriguez & Maria da Conceição Santos & Raquel Azevedo & Carlos Correia & José Moutinho-Pereira & José Miguel Pimenta Ferreira de Oliveira & Maria Celeste Dias
Received: 5 February 2014 / Accepted: 23 July 2014 / Published online: 6 August 2014 # Springer-Verlag Berlin Heidelberg 2014
Abstract Lead (Pb) environmental contamination remains prevalent. Pisum sativum L. plants have been used in ecotoxicological studies, but some cultivars showed to tolerate and accumulate some levels of Pb, opening new perspectives to their use in phytoremediation approaches. However, the putative use of pea plants in phytoremediation requires reliable toxicity endpoints. Here, we evaluated the sensitivity of a large number of photosynthesis-related biomarkers in Pbexposed pea plants. Plants (cv. “Corne de Bélier”) were exposed to Pb concentrations up to 1,000 mg kg−1 soil during 28 days. The photosynthetic potential biomarkers that were analyzed included pigments, chlorophyll (Chl) a fluorescence, gas exchange, ribulose-1,5-bisphosphate carboxylase/ oxygenase (RuBisCO) activity, and carbohydrates. Flow cytometry (FCM) was also used to assess the morpho-functional status of chloroplasts. Finally, Pb-induced nutrient disorders were also evaluated. Net CO2 assimilation rate (A) and RuBisCO activity decreased strongly in Pb-exposed plants. Plant dry mass (DM) accumulation, however, was only reduced in the higher Pb concentrations tested (500 and 1,000 mg kg−1 soil). Pigment contents increased solely in
plants exposed to the largest Pb concentration, and in addition, the parameters related to the light-dependent reactions of photosynthesis, Fv/Fm and ΦPSII, were not affected by Pb exposure. In contrast to this, carbohydrates showed an overall tendency to increase in Pb-exposed plants. The morphological status of chloroplasts was affected by Pb exposure, with a general trend of volume decrease and granularity increase. These results point the endpoints related to the lightindependent reactions of photosynthesis as more sensitive predictors of Pb-toxicity than the light-dependent reactions ones. Among the endpoints related to the light-independent photosynthesis reactions, RuBisCO activity and A were found to be the most sensitive. We discuss here the advantages of using these parameters as biomarkers for Pb toxicity in plants. Finally, we report that, despite showing physiological disorders, these cultivar plants survived and accumulated high doses of Pb, and their use in environmental/decontamination studies is open to debate. Keywords Biomarkers . Lead toxicity . Pisum sativum . Photosynthesis . RuBisCO . Net CO2 assimilation
Responsible editor: Elena Maestri E. Rodriguez : R. Azevedo : J. M. P. Ferreira de Oliveira : M. C. Dias Department of Biology, Laboratory of Biotechnology and Cytometry, Campus Universitário de Santiago, University of Aveiro, CESAM, 3810 Aveiro, Portugal M. da Conceição Santos (*) Laboratory of Biotechnology and Cytometry, University Aveiro & CESAM, Campus Universitário de Santiago, 3810 Aveiro, Portugal e-mail:
[email protected] C. Correia : J. Moutinho-Pereira Department of Biology and Environment, University of Trás-os-Montes e Alto Douro, CITAB, Apartado 1013, 5001-801 Vila Real, Portugal
Introduction The Environmental Protection Agency (EPA) highlights that lead (Pb) is the most common “heavy metal” contaminant in the environment, and the negative impacts on environment, agriculture, and humans have been widely studied. In particular, plumbemia episodes and Pb intoxication, due to soil/water contamination by Pb, food chain transfer, or other types of contamination, still persist worldwide, as highlighted by the World Health Organization (e.g., Prüss-Üstün et al. 2003; WHO 2010). Several affected soils contain Pb in the range of 400–500 mg kg−1 soil (Devi et al. 2013), while contaminations
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above 1,000 mg kg−1 (e.g., Hardison et al. 2004; Berti and Cunningham 1997; Tawinteung et al. 2005; Gupta et al. 2013) have currently been reported for heavily contaminated soils in industrial areas. Several sites near lead/zinc mine tailings reached levels of Pb contamination around 100,000 mg kg−1 soil (Li et al. 2005; for review, see Kumar et al. 2013). The potential mobilization of Pb in soil depends primarily on the metal content, its solubility in water, soil pH, redox potential, and other soil characteristics (e.g., Bertrand and Poirier 2005). Once in contact with plants, Pb is transported by CPx-type ATPases, a subgroup of P-type ATPases that pump essential and non-essential metals such as Cu2+, Zn2+, Cd2+, and Pb2+ across the plasma membrane (Kučera et al. 2008), affecting the nutritional status of the plant (e.g., Monteiro et al. 2009; Dias et al. 2013). Pb phytotoxicity involves a decrease of seed germination and root and shoot growth, impairment of cell division and DNA synthesis, and oxidative stress (e.g., Capelo et al. 2012). We also have recently demonstrated that high levels of Pb may induce genetic instability in pea plants, in a region that may be linked to amino acid metabolism and detoxification processes (Rodriguez et al. 2013a). Pb has also been reported to have deleterious effects on photosynthesis-related parameters (e.g., Prasad 2002; Malecka et al. 2012; Romanowska et al. 2012), but the comparative sensitivities of both light-dependent vs. light-independent photosynthetic reactions to this metal have never been addressed. Using maize thylakoids from mesophyll and bundle sheath chloroplasts, Romanoswska and collaborators (2012) reported that Pb increased phosphorylation of the photosystem II (PSII) core proteins. John et al. (2009) reported for Brassica juncea that Pb exposure impaired growth and decreased Chl content; Kosobrukhov et al. (2004) working with Platago major showed that Pb affected stomatal conductance (gs) and pigment contents; Bibi and Hussain (2005) demonstrated that A, transpiration rate (E), and gs of Vigna mungo plants were significantly affected when exposed to Pb. Using both pea detached leaves and protoplasts, Parys et al. (1998) reported that Pb(NO3)2 negatively affected the gas exchange and transpiration intensity after 24 h of exposure, but these in vitro studies lack the functional information at the individual level, necessary in ecotoxicological tests. Data on ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) expression and activity upon Pb exposure is less abundant and may depend on species and exposure conditions. In Thypha latifolia (commonly used in phytoremediation approaches), Pb induced the expression for RuBisCO large subunit and RuBisCO activase (Bah et al. 2010). However, other studies highlight that Pb decreases the activity of several enzymes, including RuBisCO (reviewed by Pourrut et al. 2011), as occurred in pea (Rodriguez et al. 2012) and lettuce (Dias et al. 2013). Plant tolerance to metals is the “ability to survive in a soil that is toxic to other plants and is manifested by an interaction
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between a genotype and its environment” (Hall 2002). Recommended quantitative markers/endpoints to assess toxicity and tolerance remain very general, including survival, plant/organ growth, biomass production, or qualitative endpoints (e.g., ISO 1993; ISO 1995; OECD 2006; for details, see Oehha 2009). However, for example, seed germination was found to be less sensitive or even insensitive to metal toxicity (e.g., Cd), and its use as an endpoint to assess metal toxicity in soils is discouraged by several authors (e.g., An 2004). Therefore, there is an increasing interest in finding sensitive physiological and biochemical parameters as alternative biomarkers, e.g., direct photosynthetic parameters (gas-exchange parameters and Chl a fluorescence), pigment and sugar contents, and enzymatic activities (Monteiro et al. 2007; 2009; 2012), to accurately assess and predict the phytotoxicity of metals. Moreover, photosynthesis is strictly linked with plant productivity and primary production of a population. Therefore, the recommendation of adequate photosynthetic biomarkers is indispensable in ecotoxicological studies. Nevertheless, photosynthesis is one of the most complex physiological processes in plants, involving biochemical and biophysical processes, multiple cell compartments, and interdependence on several other physiological events as transpiration, nutrition, and respiration. The photosynthetic output also depends on the experimental conditions. Therefore, the selection of the most representative biomarker for photosynthesis in ecotoxicological studies remains a question of debate, as it must be carefully analyzed within a large battery of available parameters, and the results should always be discussed within a comprehensive physiological framework. Despite the strong need to assess Pb effects on plant metabolism, including photosynthetic reactions, no study to date has evaluated photosynthetic parameters as suitable biomarkers for Pb toxicity. Pisum sativum is an important crop and has several characteristics typical of an ideal model test species, e.g., it typically exhibits fast growth rates, is amenable to genetic manipulation, and can be used in reproduction studies. This species has been selected by many ecotoxicological laboratories to assess metal toxicity (e.g., Päivöke and Simola 2001; Rodriguez et al. 2013b). However, pea susceptibility to metals depends on the cultivar, and “Kwestor,” “Little Marvel,” “Perfection,” and “Alderman” have even been pointed as interesting models in soil decontamination programs (Piechalak et al. 2003; Alzandi 2012). Recently, we used OECD endpoints and reported that “Corne de Bélier” plantlets were tolerant to Pb (and other metals), showing only occasional genetic instability (Rodriguez et al. 2011; 2012; 2013b). Moreover, these plants maintained a relatively high biomass production compared to the biomass of some tolerant species. Due to this fact, the usefulness of tolerant pea cultivars in phytoremediation programs deserves therefore further attention and to be compared with those hyperaccumulator species
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with much low biomass. In this study, we use P. sativum as model species for evaluating the sensitivity of a toolbox of Pb effects in photosynthesis. To our best knowledge, this is the most comprehensive study that provides information on the impact of Pb on specific targets of both photosynthesis phases and compares a wide range of candidate photosynthesisrelated biomarkers for Pb toxicity in plants. For this, P. sativum plants were exposed to Pb solutions ranging from 10 to 1,000 mg kg−1 soil. After 28 days, plant growth (dry mass (DM) and fresh mass (FM)), carbohydrates, pigments content, mineral content, photosynthetic performance, and RuBisCO activity were analyzed. Moreover, and for the first time, flow cytometry (FCM) was used to evaluate the effect of metals on the morphology and fluorescence emission of chloroplasts extracted from plants exposed to Pb. With the data presented by this investigation, we give a better insight into the phytotoxicity of Pb, select the most sensitive biomarkers for Pb-induced toxicity (including the suitability of FCM as a powerful in this type of studies), and discuss the use of pea plants in decontamination strategies.
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a CaSO4 solution to remove Pb2+ adsorbed to the root surface; then, tissues were dried to constant weight at 60 °C and treated for later analysis, as described (Silva et al. 2010). Pigment quantification Leaf disks (0.5 cm2) were ground in cold acetone/50 mM Tris, pH 7.8 (80:20, v/v) and centrifuged at 2,800×g during 5 min. The absorbance at 470, 537, 647, and 663 nm was determined with a Genesys 10 spectrophotometer (Thermo Fisher Scientific Inc., Waltham, USA). The contents of chlorophyll (Chl) a, Chl b, and carotenoids were calculated using the formulae of Sims and Gamon (2002). According to Lichtenhaler (1987), the molecular weights used to convert gram units to mole units were as follows: Chl a = 893.5 g mol−1, Chl b=907.5 g mol−1, and carotenoids= 550.0 g mol−1. Chl fluorescence and gas exchange measurements
Pea seeds (P. sativum L., cv. Corne de Bélier, IPSO BP 301, 26401 Crest, France) were hydrated for 48 h and then sown in pots containing around 200 mg of 4:1 of peat/perlite mixture (commonly used in commercial crop production in greenhouses). Pb was added as PbCl2 (Sigma-Aldrich, St. Louis, MO, USA) dissolved in 1:10 Hoagland’s solution (SigmaAldrich, St. Louis, MO, USA), in the concentrations of 0; 20; 200; 1,000; and 2,000 mg L−1. These concentrations corresponded to, respectively, 19.76; 201.92; 998.73; and 1,988.36 mg L−1 as determined by inductively coupled plasma atomic emission spectroscopy (ICP-AES) (Jobin Yvon, JY70 Plus, Longjumeau Cedex, France). Pots were irrigated twice a week with 100 mL of the above-mentioned contaminated solutions, resulting in calculated Pb soil doses of 10; 100; 500; and 1,000 mg kg−1 soil, respectively. Plants were grown in a climate chamber during 28 days at 24±1 °C, under light intensity of 250 μmol m−2 s−1 and a photoperiod of 16/ 8 h (light/dark). Afterward, the plants were collected, rinsed thoroughly to remove substrate adhered to roots, and tissue was sampled for analysis.
Chl a fluorescence parameters were measured in situ in the adaxial side of the leaf with a pulse-amplitude-modulated FMS 2 fluorimeter (Hansatech Instruments, Norfolk, England). Maximum quantum efficiency of photosystem II (PSII) was calculated as Fv/Fm = (Fm −F0) /Fm by measuring the fluorescence signal from a dark-adapted leaf when all reaction centers are open using a low-intensity pulsed measuring light source (F0) and during a 0.7-s saturating light pulse when all reactions centers are closed (Fm). Leaves were dark-adapted for 30 min using dark-adapting leaf clips for these measurements. Following Fv/Fm estimation, after a 20-s exposure to actinic light (500 μmol m−2 s−1), light-adapted steady-state fluorescence yield (Fs) was averaged over 2.5 s, followed by exposure to saturating light for 0.7 s to establish F′m. From these measurements, quantum effective efficiency of PSII was calculated as ΦPSII = ((F′ m − Fs) /F′ m). In situ leaf gas exchange measurements A, E, and the ratio of intercellular to atmospheric CO2 concentration (Ci/Ca) were performed using a portable infrared gas analyzer (LCpro+, ADC, Hoddesdon, UK), operating in open mode under growth chamber conditions. The stomatal conductance (g s ) was automatically calculated according to von Caemmerer and Farquhar (1981). Measurements were always performed in the middle of the photoperiod at growth temperature and atmospheric CO2 concentration in the youngest fully developed leaf.
Pb and nutrient accumulation
RuBisCO activity
Accumulation of Pb and of some photosynthetic important nutrients (K, Mg, Mn, P, Zn, and Fe) in aerial parts and roots was verified by ICP-AES (Jobin Yvon, JY70Plus, Longjumeau Cedex, France) (Loureiro et al. 2006). Roots were washed with
Leaf disks of 0.5-cm2 size were homogenized with 1 mL extraction buffer according to Dias and Brüggemann (2010). The extraction buffer consisted of 50 mM Tris/HCl, Ph 7.9, 8 mM MgCl2, 5 mM Na-pyruvate, 1 mM EDTA, 2 mM
Material and methods Plant growth conditions and exposure to Pb
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K2HPO4, 20 mM dithiothreitol, and 0.3 % (m/v) bovine serum albumin. The homogenate was centrifuged at 9,000 × g. Immediately after extraction, total RuBisCO (EC 4.1.1.39) activity was assayed as described by Lilley and Walker (1974). Briefly, after incubation of the homogenate in 20 mM MgCl2 and 10 mM NaHCO3 for 20 min, NADPH oxidation was measured spectrophotometrically at 340 nm.
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SigmaStat program, and correlations were considered significant for p
