Semina: Ciências Agrárias ISSN: 1676-546X [email protected] Universidade Estadual de Londrina Brasil
Brito de Abreu, Claudia; Lima do Sacramento, Bárbara; Teixeira Alves, Andréia; Cardim Moura, Silvany; Santos Pinelli, Milena; Dias de Azevedo Neto, André Nutritional and biochemical changes induced by lead in sunflower (Helianthus annuus L.) Semina: Ciências Agrárias, vol. 37, núm. 3, mayo-junio, 2016, pp. 1229-1242 Universidade Estadual de Londrina Londrina, Brasil
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How to cite Complete issue Scientific Information System More information about this article Network of Scientific Journals from Latin America, the Caribbean, Spain and Portugal Journal's homepage in redalyc.org Non-profit academic project, developed under the open access initiative DOI: 10.5433/1679-0359.2016v37n3p1229 Nutritional and biochemical changes induced by lead in sunflower (Helianthus annuus L.)
Distúrbios nutricionais e bioquímicos induzidos pelo chumbo em girassol (Helianthus annuus L.)
Claudia Brito de Abreu1*; Bárbara Lima do Sacramento2; Andréia Teixeira Alves3; Silvany Cardim Moura2; Milena Santos Pinelli4; André Dias de Azevedo Neto5
Abstract
The aim of this study was to evaluate the effects of increasing concentrations of lead (Pb) in the nutrient solution on growth and macronutrient and organic solute contents of Helianthus annuus plants. The experimental design was completely randomized with four replications. The dry mass yield was not affected by the treatments up to a Pb concentration of 0.6 mM, indicating that H. annuus was tolerant to this Pb concentration in the growth medium. The reductions in leaf, stem and root dry masses at a Pb concentration of 0.8 mM were 74, 84 and 85%, respectively. Lead stress did not reduce the levels of nitrogen (N), phosphorus (P) and potassium (K), indicating that the growth reduction observed at 0.8 mM Pb could not be attributed to deficiencies of these nutrients. On the other hand, Pb significantly reduced calcium (Ca) and magnesium (Mg) contents in leaves, stems and roots, which might, at least in part, explain the Pb-induced growth reduction in the H. annuus plants. Pb increased soluble carbohydrate, free amino acid and proline contents in leaves, and soluble protein and proline contents in roots, showing stress-induced changes in cell metabolism. The data also suggest that Ca and Mg concentrations may be used as nutritional indicators and the proline content may be used as a biochemical indicator of Pb toxicity in H. annuus. Key words: Lead. Helianthus annuus. Mineral nutrition. Heavy metal. Organic solutes.
Resumo
Objetivou-se com esse trabalho, avaliar os efeitos de doses crescentes de chumbo (Pb) na solução nutritiva sobre o crescimento e teores de macronutrientes e solutos orgânicos em plantas de Helianthus annuus, em casa de vegetação. O delineamento experimental foi o inteiramente casualizado, com quatro repetições. A produção de massa seca não foi afetada até o tratamento de 0,6 mM indicando que o H. annuus é tolerante até esta concentração de Pb no meio de cultivo. As reduções nas massas secas das folhas, caule e raízes foram de 74, 84 e 85%, respectivamente. O estresse por Pb não reduziu os teores de nitrogênio (N), fósforo (P) e potássio (K), indicando que a redução do crescimento induzida pelo Pb não pode ser atribuída às deficiências destes nutrientes. Por outro lado, o Pb reduziu significativamente os teores de cálcio (Ca) e magnésio (Mg) nas folhas, caule e raízes, o que pode, ao menos em parte,
1 M.e em Solos e Qualidade de Ecossistemas, Universidade Federal do Recôncavo da Bahia, UFRB, Cruz das Almas, BA, Brasil. E-mail: [email protected] 2 Discentes do Curso de Mestrado em Solos e Qualidade de Ecossistemas, UFRB, Cruz das Almas, BA, Brasil. E-mail: [email protected]; [email protected] 3 Bióloga, UFRB, Cruz das Almas, BA, Brasil. E-mail: [email protected] 4 Discente do Curso de Doutorado em Química, Universidade Federal da Bahia, UFBA, Salvador, BA, Brasil. E-mail: milanelly@ hotmail.com 5 Prof. Associado, UFRB, Cruz das Almas, BA, Brasil. E-mail: [email protected] * Author for correspondence Recebido para publicação 12/05/15 Aprovado em 09/10/15 1229 Semina: Ciências Agrárias, Londrina, v. 37, n. 3, p. 1229-1242, maio/jun. 2016 Abreu, C. B. et al.
explicar a redução do crescimento induzida pelo Pb nas plantas de H. annuus. O Pb aumentou os teores de carboidratos solúveis, aminoácidos livres e prolina nas folhas e os de proteínas solúveis e de prolina nas raízes, evidenciando as alterações no metabolismo celular decorrentes do estresse. Os dados também sugerem que os teores de Ca e Mg podem ser utilizados como indicadores nutricionais e os de prolina como indicador bioquímico da toxidez por Pb em H. annuus. Palavras-chave: Chumbo. Helianthus annuus. Nutrição mineral. Metal pesado. Solutos orgânicos.
Introduction physiological and biochemical changes, such as impairments of the membrane function, enzyme Heavy metal pollution has become one of the activity, hormonal balance, mineral nutrition, major environmental problems worldwide (MALAR et al., 2014). It is particularly difficult to remediate photosynthesis, translocation and water relations, soil, water and air of metal pollutants because, which lead to metal-induced growth reduction unlike organic pollutants, which may be degraded (POSCHENRIEDER; BARCELÓ, 2006). into harmless small molecules, toxic elements, such Thus, several authors have reported changes in as lead (Pb), mercury (Hg), cadmium (Cd), copper macronutrient contents in plants grown under Pb (Cu) and zinc (Zn), are immutable by biochemical stress (AKINCI et al., 2010; AUGUSTO et al., 2014; reactions (MALAR et al., 2014). The heavy metals BERTOLI et al., 2011; HUANG; CUNNINGHAM, are defined as metals with a density higher than 5 g 1996; KIBRIA et al., 2009; LAMHAMDI et al., 2013; -3 cm (GASIC; KORBAN, 2006). PAIVA et al., 2002, 2003) and in organic solutes in Excess lead in plants can cause various plants grown in the presence of Pb (BHARDWAJ et symptoms of toxicity, such as reduced growth, al., 2009; LAMHAMDI et al., 2013). However, little chlorosis, browning of the root system, inhibition research has been conducted on the damage caused of photosynthesis and changes in mineral nutrient by Pb to physiological and biochemical processes in concentrations (SHARMA; DUBEY, 2005). As sunflower (Helianthus annuus L.). a result of toxicity caused by the pollutant action, H. annuus is a dicot of the family Compositae, inhibition of cell activity or damage to the cell originally from North America, which possesses structure may occur due to heavy metal interference important agronomic traits, such as tolerance with some essential nutrients (COUTINHO; to high and low temperatures and adaptation to BARBOSA, 2007). Responses to stress range different soil and climate conditions (CASTRO widely, depending on the plant species, the toxic et al., 1997). The global production of H. annuus element, and the environmental conditions (SOUZA grain has grown in recent years, reaching in 2013- et al., 2011). 2014 42.9 million tons. This is due to sunflower At high soil concentrations, Pb replaces seed and oil characteristics, which have a number elements such as potassium (K) and calcium (Ca) of applications in the food industry. In 2013-2014, in aluminosilicates, particularly feldspar, metals the cultivated area for sunflower was 145,700 ha, and sulfides (BOSSO; ENZWEILER, 2008). When and the production reached 232,700 tons in Brazil soil contains large amounts of metals, their uptake (CONAB, 2014). The entire plant can be used for by plants is mainly affected by the bioavailability forage, silage, and green manure (EVANGELISTA; fraction (VAMERALI et al., 2010). In the case of LIMA, 2001). lead, the uptake occurs either in an ionic form (Pb2+) Determination of heavy metals in soil, plants, or via a passive mechanism (KABATA-PENDIAS; PENDIAS, 2011). and human beings, and knowledge of their toxicity are of great importance for the utilization of food Heavy metal ions are toxic to plants at the and economic crops (MACÊDO; MORRIL, 2008). micromolar level and can induce a variety of 1230 Semina: Ciências Agrárias, Londrina, v. 37, n. 3, p. 1229-1242, maio/jun. 2016 Nutritional and biochemical changes induced by lead in sunflower (Helianthus annuus L.)
Thus, this study aimed to evaluate the effect of analytical balance. The dried plant material was increasing concentrations of lead on growth and ground to be analyzed for nitrogen (N), phosphorus contents of macronutrients and organic solutes in (P), potassium (K), calcium (Ca), magnesium (Mg), H. annuus plants to gain a better understanding of and lead (Pb). the nutritional and biochemical effects of Pb on this crop. Extract preparation and nutrient and biochemical analyses Materials and Methods For analysis of nutrient concentrations, extracts Growth and treatment conditions were prepared by wet acid digestion in a mixture of concentrated sulfuric acid (H SO ) and 30% The experiment was carried out in a greenhouse 2 4 hydrogen peroxide (H O ), as described by Jones of the Universidade Federal do Recôncavo da Bahia 2 2 (2001). The digested samples were adjusted to a (UFRB), Cruz das Almas, Brazil, from April to May volume of 100 mL with deionized water to carry out 2013. the analysis of N, P, K, Ca, Mg, and Pb. Seeds from H. annuus genotype Olisun-05 were Determination of N and P was performed sown in 200-mL plastic cups containing washed sand spectrophotometrically by the phenol-hypochlorite and irrigated daily with distilled water. Seedlings (WEATHERBURN, 1967) and molybdo-vanadate (12-day-old) were transferred for acclimatization (FAITHFULL, 2002) methods, respectively. to trays containing 12 L of aerated Hoagland and Concentrations of Pb, K, Ca, and Mg were Arnon (1950) nutrient solution. Treatments were determined simultaneously by Inductively Coupled applied five days later and consisted of a no-lead Plasma Optical Emission Spectrometry (ICP-OES, control and four different lead concentrations in the Varian, VISTA-PRO). nutrient solution (0.2, 0.4, 0.6 or 0.8 mM), referred to thereafter as Pb0.0 through Pb0.8. Lead was applied For biochemical analysis, extracts were prepared as Pb(NO3)2, and each treatment contained four by homogenization of 1.0 g of lyophilized leaf and replicates, resulting in a total of 20 experimental root powders with a mortar and pestle in 5 mL of plants. The experiment was performed in duplicate 100 mM potassium phosphate buffer, pH 7.0, 0.1 in order to obtain samples for biochemical and mM EDTA. The homogenate was filtered through growth analyses, respectively. Lead added to the muslin cloth and then centrifuged at 12,000 ×g for nutrient solution was chelated with EDTA to avoid 15 min. The supernatant fraction was kept in an precipitation with sulfate and phosphate ions. The ultra freezer (-80 °C) and used for the determination volume of the nutrient solution was made up daily of organic solutes. with distilled water, and the pH was maintained at Soluble carbohydrates, free proline, free amino 5.5 ± 0.5 by adding NaOH or HCl. acids, and soluble proteins were determined After 16 days of Pb stress, leaf and root samples according to the methods described by Dubois et of four plants from each group were cut, frozen al. (1956), Bates et al. (1973), Yemm and Cocking in liquid nitrogen, and lyophilized for further (1955), and Bradford (1976), respectively. biochemical analyses. The remaining four plants were harvested, separated into leaves, stems and roots, put in paper bags and dried in an oven at 65 °C Experimental design and statistical analysis for 72 h for the determination of leaf (LDM), stem The experimental design was completely (SDM) and root (RDM) dry masses using a semi- randomized, with five treatments of four replicates
1231 Semina: Ciências Agrárias, Londrina, v. 37, n. 3, p. 1229-1242, maio/jun. 2016 Abreu, C. B. et al.
each. The treatment effects were evaluated by concentration of 0.6 mM. However, it abruptly analysis of variance (p < 0.05) and, in the case of decreased in the treatment with 0.8 mM Pb, and the significance, a regression study was performed reductions in the LDM, SDM, and RDM were 74, using the statistical program SISVAR (FERREIRA, 84, and 85%, respectively (Table 1). Compared with 2003). the other plant organs, the roots showed the highest reduction in the dry mass as a result of Pb stress, probably because they are the only part directly Results and Discussion exposed to the toxic metal. The reduction in root Growth growth can affect the growth of the whole plant due to limited water and nutrient absorption (PEREIRA The dry mass production in all parts of the H. et al., 2013). annuus plants remained unchanged up to a Pb
Table 1. Leaf (LDM), stem (SDM) and root (RDM) dry mass yields of H. annuus plants after 16 days of growth in
a greenhouse with different concentrations of Pb(NO3)2 in the nutrient solution. The values indicate the mean of four replicates ± SD. The values in parentheses represent the percentage of reduction relative to the control.
Levels of Pb (mM) LDM SDM RDM 0.0 3.995 ± 0.811 2.832 ± 0.160 2.443 ± 0.463 0.2 3.649 ± 0.283 (9) 2.599 ± 0.150 (8) 1.827 ± 0.248 (25) 0.4 3.458 ± 0.494 (13) 2.383 ± 0.432 (16) 1.766 ± 0.482 (28) 0.6 3.647 ± 0.221 (9) 2.605 ± 0.253 (8) 1.965 ± 0.116 (20) 0.8 1.054 ± 0.225 (74) 0.455 ± 0.098 (84) 0.372 ± 0.069 (85)
Lead concentrations the roots were about three times as high as those in the stems and leaves. The leaf and root Pb concentrations increased linearly, while in the stems they showed a quadratic Greater accumulation of Pb in roots than in relationship with the Pb levels in the nutrient other parts of the plant suggests that roots play an solution (Figure 1). The Pb concentrations in the important role in the lead storage (AZAD et al.,
H. annuus leaves ranged from 0.067 (Pb0.0) to 2011). It has been shown that lead can be retained in -1 5.22 mmol g of dry weight (DW, Pb0.8). The stem the root cell wall, particularly in the pyrophosphate
concentrations varied from 0.51 (Pb0.0) to 5.55 mmol form (MARSCHNER, 2012). Lead accumulation in -1 g DW (Pb0.66), and the root concentrations ranged roots is considered to be a factor that increases plant -1 from 1.74 (Pb0.0) to 20.18 mmol g DW (Pb0.8). tolerance to Pb toxicity, because it prevents the Thus, the Pb concentrations in the leaves and roots metal translocation to leaves (AZAD et al., 2011). In exposed to 0.8 mM Pb were, respectively, 76 and 11 this study, it could also be observed that the stem Pb times higher than those found in the control. In the concentrations showed a stabilization trend, starting stems, the highest Pb concentration was detected from the treatment with 0.2 mM Pb, indicating a at a Pb concentration of 0.66 mM in the solution, saturation of the Pb retention mechanism in the stem corresponding to a 9.78-fold increase relative to the and, consequently, a finite capacity of this organ to control. Comparison of the Pb contents in different act as a metal reservoir, preventing Pb transport to parts of the plant showed that the concentrations in leaves.
1232 Semina: Ciências Agrárias, Londrina, v. 37, n. 3, p. 1229-1242, maio/jun. 2016 Figure 1. LeadNutritional (Pb) content and biochemical in leaves, changes stems, induced and roots by lead of in H. sunflower annuus (Helianthus plants after annuus 16 L.) days of growth in a greenhouse with different concentrations of Pb(NO3)2 in the nutrient solution. *Significant (p ≤ 0.05); Figure**significant 1. Lead (Pb)(p ≤ content0.01). in leaves, stems, and roots of H. annuus plants after 16 days of growth in a greenhouse with different concentrations of Pb(NO3)2 in the nutrient solution. *Significant p( ≤ 0.05); **significant p( ≤ 0.01).
Greater GPbreater accumulation accumulation in rootsof Pb compared in roots thanto in othera cationic parts form, of the it plantis likely suggests that thethat absence roots play of an shootsimportant was rolealso in reported the lead by storage Romeiro (AZAD et al. et (2007) al., 2011) changes. It has been in N shownconcentrations that lead observedcan be retained in this instudy the root in Canavalia ensiformis and by Andrade et al. resulted from the lack of competition between this cell wall, particularly in the pyrophosphate form (MARSCHNER, 2012). Lead accumulation in roots is (2009) in Paspalum notatum. In general, apparent nutrient and the toxic metal. This may explain concentrationsconsidered to beof aPb factor in abovegroundthat increases tissuesplant tolerancewhy little to Pbinformation toxicity, isbecause available it inprevents the literature the metal decreasetranslocation as the to transportleaves (AZAD distance et fromal., 2011) the root. In thisregarding study, itchanges could alsoin Nbe concentrations observed that inthe plants stem Pb increases.concentration Thiss isshowed due to a higherstabilization concentration trend, starting of grown from in the the treatmentpresence ofwith Pb. 0.2 mM Pb, indicating a Pb in the root cell walls than in other parts of plants, saturation of the Pb retention mechanism in the stem andThe, consequently leaf and stem, a finite P concentrations capacity of this increased organ to act showing that the transport is restricted by the root quadratically with the Pb concentration increase in endodermisas a metal reservoir(SHARMA;, preventing DUBEY, Pb 2005). transport to leaves. the culture medium (Fig. 2). Thus, the estimated P Greater Pb accumulation in roots compared to shoots was also reported by Romeiro et al. (2007) in concentrations in the leaves and stems of the plants Canavalia ensiformis and by Andrade et al. (2009) in exposedPaspalum to notatum0.8 mM. InPb general, were, respectively, apparent concentrati 87 and ons Macronutrient (N, P, K, Ca, and Mg) concentrations of Pb in aboveground tissues decrease as the transport67% distance higher than from those the rootin the increases control.. ThisThis increase is due to a Fig. 2 shows the macronutrient (N, P and K) in the P concentration in shoot tissues, in conjunction higher concentration of Pb in the root cell walls than in other parts of plants, showing that the transport is levels in the leaves, stems, and roots of the H. with the growth reduction, indicates a concentration annuusrestricted plants by theat differentroot endodermis Pb concentrations (SHARMA; in DUBEY, the effect 2005). (IMO, 2012). In contrast, the mathematical nutrient solution. simulation indicated that the P content in the MacronutrientIt was found, (N, usingP, K, Ca the, and regression Mg) concentrations equation, plant roots subjected to higher Pb concentration that Pb did not induce substantial changes in the N decreased by only 8% (Fig. 2), suggesting that Pb content in differentFig. 2 shows plant the parts macronutrient (Figure 2). Nitrogen(N, P and K)stress levels did in notthe leaves,affect the stems, P translocation and roots of from the H. roots annuus isplants a constituent at different of variousPb concentrations components in ofthe the nutrient plant solution.to shoots in the H. annuus plants. Contrasting results were reported by (BERTOLI et al., 2011; cell, includingIt was amino found, acids,using thenucleic regression acids, equation,and that Pb did not induce substantial changes in the N chlorophylls (TAIZ; ZEIGER, 2013). It is absorbed HUANG; CUNNINGHAM, 1996; PAIVA et al., content in different plant parts (Figure 2). Nitrogen is a constituent of various components of the plant cell, - 2003), respectively, for Lycopersicon esculentum, by roots in the form of nitrate (NO3 ) and ammonium including+ amino acids, nucleic acids, and chlorophyllsAmbrosia (TAIZ; ZEIartemisiifoliaGER, 2013)., and It isseedlings absorbed of by Tabebuia roots in the (NH4 ), with preferential uptake in the anionic form - - + + impetiginosa. These authors reported that Pb (nitrate).form of Consideringnitrate (NO 3 that) and the ammonium NO3 /NH4 ratio(NH4 in), with preferential uptake in the anionic form (nitrate). Hoagland solution is 14:1 - and that+ Pb is absorbed applications increased P concentrations in roots. Considering that the NO3 /NH4 ratio in Hoagland solution is 14:1 and that Pb is absorbed in a cationic form, 1233 it is likely that theSemina: absence Ciências of changes Agrárias, in Londrina, N concentrations v. 37, n. 3, p. observed 1229-1242, in maio/jun.this study 2016 resulted from the lack of competition between this nutrient and the toxic metal. This may explain why little information is available in Abreu, C. B. et al. the literature regarding changes in N concentrations in plants grown in the presence of Pb. FigureFigure 2. Nitrogen 2. Nitrogen (N), (N),phosphorus phosphorus (P), and (P), potassium and potassium (K) contents (K) contents in leaves, in stems, leaves, and stems, roots ofand H. rootsannuus of plants H. annuus after 16 days of growth in a greenhouse with different concentrations of Pb(NO ) in the nutrient solution. *Significant plants after 16 days of growth in a greenhouse with different concentrations3 2 of Pb(NO3)2 in the nutrient (p solution.≤ 0.05); **significant *Significant p( (≤p 0.01).≤ 0.05); **significant (p ≤ 0.01).
The leaf and stem P concentrations increased quadratically- with the Pb concentration increase in It has been suggested that increased root P and the H2PO4 anions, the predominant form of P concentrationsthe culture medium might be(Fig. due 2). to Thus,the formation the estimated of Puptake concentrations (KABATA-PENDIAS; in the leaves PENDIAS, and stems 2001). of the In plants a exposedPb-P (lead-phosphate) to 0.8 mM Pb were complex,, respectively, an insoluble 87 and 67%this higher study, than there those was in no the increase control. in This the increaseP levels in the P form of Pb unavailable for translocation from the roots, which might be due to the application of concentration in shoot tissues, in conjunction with the growth reduction, indicates a concentration effect roots to shoots (HUANG; CUNNINGHAM, 1996). Pb chelated with EDTA to avoid metal precipitation Formation(IMO, 2012). of this In complex contrast, is theexpected mathematical due to the simulation with P inind theicated form that of thethe Pb-P P content complex. in the plant roots tendencysubjected of tothe higher reaction Pb concentrationbetween the Pb decreased2+ cations by only 8% (Fig. 2), suggesting that Pb stress did not affect the P translocation from roots to shoots in the H. annuus plants. Contrasting results were reported by 1234 (BERTOLI et al.Semina:, 2011; CiênciasHUANG; Agrárias, CUNNINGHAM, Londrina, v. 37, 1996 n. 3,; PAIVAp. 1229-1242, et al., maio/jun.2003), respectively 2016 , for Lycopersicon esculentum, Ambrosia artemisiifolia, and seedlings of Tabebuia impetiginosa. These authors reported that Pb Nutritional and biochemical changes induced by lead in sunflower (Helianthus annuus L.)
The stress caused by 0.8 mM Pb significantly levels of this nutrient in roots and stems of Tabebuia increased the K content in all parts of the H. annuus impetiginosa seedlings (PAIVA et al., 2003). Azad et plants; however, this effect was more pronounced in al. (2011) observed that Pb stress decreased the Ca the roots. In the leaves, the increase was linear, while content in leaves of H. annuus seedlings. Augusto quadratic patterns were observed in both stems and et al. (2014) reported that Ca contents in roots roots (Figure 2). Thus, the equations indicated that in and leaves of Brassica juncea were significantly the treatment with 0.8 mM Pb the K content increased affected at lower Pb levels. Małkowski et al. (2005) by 31, 17 and 65% in the leaves, stems and roots, found that a higher absorption of Ca occurred with respectively, compared to the control. The data from increasing Pb accumulation in root tips of Zea mays. this study indicated that the increases in the potassium According to Marschner (2012), divalent cations, concentration were the result of a concentration effect such as Pb, can compete with other cations, such as (IMO, 2012), when taking into account the significant Ca. The Pb inhibition of the Ca channel can be the growth reduction in this treatment. Increases in the K result of either the Pb-blockage of the channel or content in two Amaranthus genotypes grown in the competitive transport of these cations through the presence of high Pb concentrations were also reported channel (MARSCHNER, 2012; SHARMA; DUBEY, by Kibria et al. (2009). 2005). Regardless of the cause, the data from this study Considering that Pb stress did not affect the uptake indicated that in H. annuus the deleterious effect of and translocation of N, P, and K, the data from this Pb on Ca nutrition only occurred at Pb concentrations study indicated that the Pb-induced growth reduction equal to or greater than 0.6 mM. in the 0.8 mM Pb treatment could not be due to Calcium is a key element for maintaining cell the changes in concentrations of these elements in integrity, because as a divalent ion Ca can form different plants parts. intramolecular complexes and bind molecules In Figure 3, it can be observed that the Ca to form intermolecular complexes (PILBEAM; concentrations in different plant parts did not change MORLEY, 2007). Pb-induced Ca deficiency may up to a Pb concentration of 0.6 mM. However, the Ca cause disturbances in cell division and elongation concentrations were reduced by 21 and 28% in the processes (SHARMA; DUBEY, 2005). Calcium leaves and stems, respectively, of the plants treated is required for the formation of the mitotic spindle with 0.8 mM Pb, showing a negative quadratic effect. during cell division and for synthesis of the new cell wall in newly divided cells. It is also necessary for the In Zea mays, Huang and Cunningham (1996) physical integrity and normal function of membranes observed that 20 µM Pb reduced Ca concentrations and, more recently, has been considered as a second by 12% in roots and more than 40% in shoots. messenger for various plant responses to hormonal However, no changes were observed in the Ca and environmental signals (HOPKINS; HÜNER, content of Ambrosia . artemisiifolia (HUANG; 2009; TAIZ; ZEIGER, 2013). The data from this CUNNINGHAM, 1996). The application of 288 µM study suggested that reduced calcium levels could, at Pb decreased by 35.6 and 18.2%, respectively, Ca least partly, explain the Pb-induced growth reduction concentrations in roots and leaves of Cedrela fissilis in the H. annuus plants. seedlings (PAIVA et al., 2002), but did not affect
1235 Semina: Ciências Agrárias, Londrina, v. 37, n. 3, p. 1229-1242, maio/jun. 2016 Abreu, C. B. et al.
Figure 3. Calcium (Ca) and magnesium (Mg) contents in leaves, stems, and roots of H. annuus plants after 16 days
of growth in a greenhouse with different concentrations of Pb(NO3)2 in the nutrient solution. *Significant p( ≤ 0.05); **significant p( ≤ 0.01).
A linear negative effect of increasing Pb in shoots of Z. mays and A. artemisiifolia plants. concentrations was also observed on the Mg contents Lamhamdi et al. (2013) reported that Pb concentrations in the leaves and stems; thereby, the levels of Mg higher than 1.5 mM significantly decreased Mg were, respectively, 31 and 46% lower at 0.8 mM Pb contents in shoots and roots of Triticum aestivum and than those in the control (Fig. 3). In the roots, the Mg Spinacia oleracea seedlings. contents showed a negative quadratic behavior and In plant cells, Mg ions have a specific role decreased dramatically (by 58%) only in the 0.8 mM of activating a number of enzymes involved in Pb treatment. This reduction in the Mg levels could be respiration, photosynthesis and DNA and RNA related to a reduction in the levels of chlorophyll a and synthesis (TAIZ; ZEIGER, 2013). In addition, they b, observed in several species grown in the presence of have a structural role in the chlorophyll molecule, Pb (GOMES et al., 2014). stabilization of proteins, ribosomes, nucleic acids Similar to this study, treatment with Pb and membranes and are critical in the reactions concentrations below 0.3 mM did not affect the Mg involving ATP (HOPKINS; HÜNER, 2009). content in the roots of T. impetiginosa seedlings Since Mg is an integral component of chlorophyll (PAIVA et al., 2003). Huang and Cunningham and enzyme processes of energy metabolism, its (1996) observed that Pb reduced Mg concentrations deficiency directly affects the carbon assimilation 1236 Semina: Ciências Agrárias, Londrina, v. 37, n. 3, p. 1229-1242, maio/jun. 2016 Nutritional and biochemical changes induced by lead in sunflower (Helianthus annuus L.) and energy transformations (MERHAUT, 2007). accumulation of soluble carbohydrates (CHAI et al., Considering that both growth and Mg contents 2012; ELLOUMI et al., 2014) and free amino acids were affected by Pb present in the culture medium, (BHARDWAJ et al., 2009). In this work, the stress- the reduction in the Mg content in all plant parts induced increases in leaf soluble carbohydrate, indicated an antagonistic effect between Mg and free amino acid and proline contents suggested an Pb at the absorption sites (IMO, 2012). Thus, it is osmotic adjustment resulting from a decrease in likely that the reduction in the levels of this nutrient water availability induced by Pb stress. In contrast, is an important factor affecting the metabolism of the observation that the levels of these solutes in the H. annuus plants and, consequently, reducing their roots did not increase may indicate limited water growth. absorption by the roots and its flux to the shoots, affecting the water balance in the Pb-stressed plants. The increase in the leaf amino acid content also Organic solutes suggested a detoxification mechanism through Pb Changes in the soluble carbohydrate, free amino chelation by these compounds. Chelating agents acid, soluble protein and free proline levels in the contribute to detoxification and increase plant leaves and roots are shown in Figure 4. tolerance to toxic levels of metal ions (GASIC; In the leaves, the soluble carbohydrate levels KORBAN, 2006), and it is already well established showed a positive quadratic response and increase that amino acids can be mobilized in response to by 42% at the 0.8 mM Pb level. In the roots, no Pb toxicity to form complexes with this metal effect of Pb was observed on the levels of these (MAESTRI et al., 2010; MANARA, 2012; compounds. Free amino acids increased linearly POURRUT et al., 2011). in the leaves and decreased linearly in the roots. The observation that the increase in leaf amino Therefore, at 0.8 mM Pb the equations indicated acids occurred simultaneously with the decrease an increase (by 76%) of amino acids in the leaves in leaf soluble protein suggested the occurrence and a reduction (by 28%) of amino acids in the of Pb-induced proteolysis. Bhardwaj et al. (2009) roots. The soluble protein levels showed a quadratic also reported that increases of Pb and Cd decreased behavior in the leaves and decreased by 42% at soluble protein concentrations in leaves of 0.8 mM Pb, in contrast with the roots where Pb Phaseolus vulgaris. On the other hand, the increase stress induced a linear, about 10-fold, increase in root soluble protein occurred in parallel with the compared to the control. The free proline contents decrease in root amino acids, suggesting that protein quadratically increased in the leaves and roots. synthesis was increased in the roots. Considering Thus, the proline concentrations in the leaves and that roots are directly exposed to the contaminant, it roots were, respectively, 80 and 1.5 times higher is likely that this increase is the result of the induction at a Pb concentration of 0.8 mM than those in the of stress proteins (LAMHAMDI et al., 2010). In control. this context, metallothioneins and phytochelatins Several studies have reported reduction of water represent the main classes of heavy metal-chelating content in plants grown under lead stress (PATRA peptides in plants (MANARA, 2012; POURRUT et et al., 2004; SHARMA; DUBEY, 2005). In this al., 2011). Pb stress has also been shown to increase scenario, an exposure to potentially toxic metal the protein content, including phytochelatins, in ions induces an osmotic adjustment, mainly due to Ceratophyllum demersum L. (MISHRA et al., 2006).
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Abreu, C. B. et al. Figure 4. Soluble carbohydrate, free amino acid, soluble protein, and free proline contents in leaves, stems, Figureand 4.roots Soluble of H.carbohydrate, annuus plants free amino after acid,16 days soluble of protein,growth andin afree greenhouse proline contents with differentin leaves, concentrationsstems, and roots of
of H.Pb(NO annuus3)2 plantsin the afternutrient 16 days solution. of growth *Significant in a greenhouse (p ≤ 0.05); with **significantdifferent concentrations (p ≤ 0.01). of Pb(NO3)2 in the nutrient solution. *Significant p( ≤ 0.05); **significant p( ≤ 0.01).
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Among the organic solutes studied, the proline mM in the culture medium and may be a viable levels were increased most with increasing Pb alternative for phytoremediation programs where concentrations in the nutrient solution, indicating soil contamination with Pb does not exceed that proline accumulation can be a good biochemical this limit. The data also showed that Pb toxicity indicator of Pb stress in H. annuus. Increased changed the nutritional balance of Ca and Mg and proline biosynthesis has been considered to play the concentrations of major groups of cell organic an important role in plant tolerance to Pb stress solutes, indicating that the plant growth reduction (POURRUT et al., 2011; QURESHI et al., 2007; was the result of Pb-induced metabolic changes. SHARMA; DUBEY, 2005). Lamhamdi et al. (2013) Based on the results, Ca and Mg may be used as found that proline contents increased in leaves of nutritional markers and proline may be used as a T. aestivum and S. oleracea when the plants were biochemical marker of lead toxicity in H. annuus. exposed to increasing Pb concentrations. According to Verbruggen and Hermans (2008), proline contents may vary among species, and their values are up to Acknowledgements 100 times higher in stressed plants than those in The authors thank to Coordenacão de control ones. Proline accumulation is also related Aperfeiçoamento de Pessoal de Nível Superior to stress tolerance, and concentrations of this amino (CAPES) and Universidade Federal do Recôncavo acid are usually higher in tolerant than in sensitive da Bahia (UFRB) for the financial support over the plants (ASHRAF; FOOLAD, 2007). years. There are different opinions regarding the potential mechanisms by which proline reduces References the toxic effects of metals, including detoxification of excess ammonia (KAVI KISHOR et al., 2005), AKINCI, I. E.; AKINCI, S.; YILMAZ, K. Response of tomato (Solanum lycopersicum L.) to lead toxicity: osmoprotection and stabilization of proteins Growth, element uptake, chlorophyll and water content. (SHARMA; DUBEY, 2005), metal chelation African Journal of Agricultural Research, Lagos, v. 5, n. (MANARA, 2012), inhibition of lipid peroxidation 6, p. 416-423, 2010. and removal of free radicals (WHITE, 2012). ANDRADE, M. G.; MELO, V. F.; GABARDO, J.; Proline can also contribute as an available source of SOUZA, L. C. P.; REISSMANN, C. B. Metais pesados carbon and nitrogen. em solos de área de mineração e metalurgia de chumbo. I – fitoextração. Revista Brasileira de Ciência do Solo, Our data showed that the presence of Pb in Viçosa, MG, v. 33, n. 6, p. 1879-1888, 2009. the nutrient solution significantly changed the ASHRAF, M.; FOOLAD, M. R. Roles of glycine betaine concentrations of the major groups of cell organic and proline in improving plant abiotic stress resistance. solutes, indicating significant metabolic changes Environmental and Experimental Botany, Elmsford, v. 59, n. 2, p. 206-216, 2007. due to the presence of this toxic metal in the cellular environment. These changes in the metabolism AUGUSTO, A. S.; BERTOLI, A. C.; CANNATA, M. G.; CARVALHO, R.; BASTOS, A. R. Bioacumulação may, at least in part, explain the Pb-induced growth de metais pesados em Brassica juncea: Relação de reduction. toxicidade com elementos essenciais. Revista Virtual de Química, Niterói, v. 6, n. 5, p. 1221-1236, 2014. AZAD, H. N.; SHIVA, A. H.; MALEKPOUR, R. Conclusions Toxic effects of lead on growth and some biochemical and ionic parameters of sunflower (Helianthus annuus The results of this study showed that H. L.) seedlings. Current Research Journal of Biological annuus tolerates Pb up to a concentration of 0.6 Sciences, Taiwan, v. 3, n. 4, p. 398-403, 2011.
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