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Exogenous citrate and malate alleviate cadmium stress in Oryza sativa L.: Probing role of cadmium localization and iron nutrition
Ecotoxicology and Environmental Safety 166 (2018) 215–222
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Exogenous citrate and malate alleviate cadmium stress in Oryza sativa L.: Probing role of cadmium localization and iron nutrition
T
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Abin Sebastian , M.N.V. Prasad Department of Plant Sciences, University of Hyderabad, Central University P.O., Hyderabad 500046, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Cadmium Organic acids Metal chelation Iron uptake Antioxidants Photosynthesis
Organic acids play an important role in metal uptake and trafficking in plants. Therefore, the role of exogenous citrate and malate on Cd tolerance was studied in the seedlings of Oryza sativa L. cv MTU 7029. Seedlings were exposed to Cd plus organic acids in hydroponics and monitored changes in Cd accumulation, expression of metal transporters, chlorophyll fluorescence, and antioxidants. It found that organic acid supplements decrease Cd accumulation in leaf because of up-regulation of tonoplast localized heavy metal ATPase (OsHMA3) which allows vacuolar sequestration of Cd in the root. Malic acid helped Cd exclusion in the root too. A shift in Cd speciation from sulphhydryl to the carboxylic group also noticed in the roots of plants exposed to organic acids. Treatment of organic acids was effective to prevent Cd inducible Fe deficiency via up-regulation of the ironregulated transporter (OsIRT1), increase in ferric chelate reductase activity, and formation of Cd stabilized Fe3+ - organic acid complex respectively. Also, exposure to organic acids increased the accumulation of antioxidants such as anthocyanin and glutathione (GSH) under Cd stress. Above changes assisted in upholding of photosynthetic electron transport and biomass productivity during the course of Cd treatment with organic acid supplements.
1. Introduction Cadmium contamination of rice paddies is a serious health problem (Sebastian and Prasad, 2014a). Applications of phosphate fertilizers and irrigation of rice paddies with mine leachate resulted in toxic levels of Cd (0.05–7.7 mg kg−1) accumulation in rice (Chaney, 2015; Sebastian and Prasad, 2014a). Therefore, Cd minimization in rice gained attention (Sebastian and Prasad, 2014a). Metal chelators, soil dressing, and chemical reduction processes were applied to decrease plant-available Cd in paddy soils (Sebastian and Prasad, 2014a). Organic acids chelate metals, and this property utilized for the removal of toxic metals from contaminated sites (Nasciment, 2006). But organic acids confer heavy metal tolerance in plants too (Verbruggen et al., 2009). Metal-carboxylic acid complex sequester in the vacuole. This help to avoid oxidative stress come up with free metal ions in the cytosol. Secondly, organic acids secretion into rhizosphere reported as an adaptive strategy against metal toxicity and mineral nutrient deficiency (Kochian et al., 2015). Iron uptake and transport in plants take place with the help of organic acids (Kobayashi and Nishizawa, 2012). Organic acids such as muigenic acid, citrate, and malate play a critical role in Fe nutrition of plants (Kobayashi and Nishizawa, 2012). Citrate helps vacuolar ⁎
sequestration of Fe in the root as well as translocation of Fe (Kobayashi and Nishizawa, 2012). But malate acts as a carrier for the circulation of Fe in plants (Kochian et al., 2015). Malate also plays an important role in pH homeostasis, osmotic adjustment, shuttling of reducing equivalents, and aluminium tolerance in plants (Kochian et al., 2015). It is noteworthy that the synthesis of organic acids depends on photosynthesis, and the process is vulnerable to Cd stress (Sebastian and Prasad, 2014b). So Cd stress hinders the synthesis of organic acids and slows down heavy metal detoxification in plants. Transition metal transporters mediate Cd transport in plant cells (Hall and Williams, 2003). Iron-regulated transporter (IRT1) and natural resistance-associated macrophage proteins (NRAMP1) mediate Fe and Cd uptake in plants (Connolly et al., 2002; Takahashi et al., 2011). Also, cadmium tolerant 1 (OsCDT1) is a plasma membrane transporter which excludes Cd from the cell (Kuramata et al., 2012). On the other side, intracellular trafficking of Cd into plant vacuole occurs with the help of heavy metal ATPase (HMA3) (Sasaki et al., 2014). Iron deficiency responses in rice plants associated with the expression of nicotianamine synthase (OsNAS) and activity of this enzyme is critical for muigenic acids dependant Fe uptake via yellow stripe like transporter (OsYSL) (Johnson et al., 2011). So it is clear that organic acids had a critical role in Cd tolerance and Fe uptake in plants. Studies pointed out
Corresponding author. E-mail address: [email protected] (A. Sebastian).
https://doi.org/10.1016/j.ecoenv.2018.09.084 Received 8 December 2017; Received in revised form 4 September 2018; Accepted 20 September 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
Ecotoxicology and Environmental Safety 166 (2018) 215–222
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diphenyl thiocarbazone in 60.0 ml acetone, 5.0 ml glacial acetic acid, and 20.0 ml milliQ water. Staining performed on hand sections of the root tissue for 1.0 h, and after that excess stain washed away with milliQ water. The sections observed using a stereoscopic microscope, and the presence of Cd in tissues detected as dark black-brown complexes of Cd with diphenyl thiocarbazone.
that exogenous organic acids prevent Cd stress in sunflower and Changbai larch plants (Hawrylak-Nowak et al., 2015; Song et al., 2014). However, the physiological and molecular mechanisms of organic acid dependant Cd tolerance remain unexplored. More recently, studies on Brassica juncea pointed that citric acid supplements enhance Cd tolerance because of coordinated actions of metal chelation, antioxidant defense, and glyoxalase systems (Mahmud et al., 2018). Also, enhancement of Cd tolerance in Durum wheat found to associate with an increase of fumaric, malic, and succinic acids (Bolaji et al., 2010). But these studies not discussed the chance of competition in the uptake and trafficking of Fe with Cd. This situation may occur because carboxylic acids bind with a wide range of cations. Therefore, organic acids supplement dependant Cd tolerance in relation with Fe nutrition explored in the present study.
2.6. X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) analyzes the ionic state and chemical nature of metal ion in a given sample. The instrument measures the kinetic energy of electrons escape from the sample during Xray exposure (Bagus et al., 2013). XPS analysis conducted on vacuum dried root powder. Sample exposed to X-ray photoelectron spectrometer (Omicron Nano Technology, UK) using monochromatized Al Kα radiation (1486.7 eV). Measurements carried out in a constant analyzer energy mode. The intensity of the XPS peak recorded as counts per second (CPS). The chemical speciation and the chemical nature of Cd complexes determined based on the binding energy (Bagus et al., 2013). The binding energy, which indicates chemical nature of the metal ion, derived from the kinetic energy of the emitted electrons, and the photon energy used to emit these electrons.
2. Materials and methods 2.1. Hydroponics Oryza sativa cv MTU 7029 seeds were sterilized with 5.0% hydrogen peroxide for 15.0 min and washed with double distilled water. Seeds were germinated in a germination box contain wet sand under a light intensity of 50.0 µmol photons m−2 s−1. Five-day-old seedlings used for hydroponics with Hoagland media (Hoagland and Arnon, 1950). The media was diluted to half the strength using milliQ water before the experiments.
2.7. Detection of organic acids To find out the abundance of organic acids, air-dried powder of plant tissues (10.0 mg) made pellet with potassium bromide, and the pellet loaded into Fourier transform infrared (FTIR) spectrometer (Nicolet 380 FT-IR, Thermo scientific, USA) at room temperature. Spectral wave number ranges from 400 to 4000 cm−1 recorded. The peak in the Fourier transform infrared absorbance spectra at 2500–3000 cm−1 corresponds to abundance and carbonyl stretch of the carboxylic group present in the tissue (Max and Chapados, 2004).
2.2. Cadmium and organic acid treatments Cadmium treatments performed by transferring seedlings to media containing 25.0 µM CdCl2. Malate or citrate supplement carried out at a concentration of 50.0 µM in Cd containing media. A group of plants maintained without Cd or organic acid treatment as a control. Light intensity (400.0 µmol photons m−2 s−1), photoperiod (18.0 h Light / 6 h dark), temperature (25.0 ± 2.0 °C) and relative humidity (50.0 ± 10.0%) were maintained throughout the 14 days of experiment.
2.8. Exchange of metal ions bound to organic acids To study the exchange of metal ions bound with organic acids, a solution of 50.0 µM organic acid (citrate or malate), and 25.0 µM iron salt (ferrous or ferric chloride) treated with varying CdCl2, and incubated for 1.0 h at 25.0 °C. After this, the mixture treated with 10.0% hydroxylamine solution for the reduction of free Fe3+ available in the solution to Fe2+. The amount of free Fe2+ ions in the solution determined via measuring absorbance at 510 nm after reaction with OPhenanthroline (Fortune and Mellon, 1938). Changes in absorbance of the Fe-organic acid mixture against varying concentration of CdCl2 plotted.
2.3. Biomass and metal quantification Biomass quantified with the help of weighing balance, and the result expressed as mg fresh weight. For the quantitative estimation of metals, plants manually cleaned with deionized water. The material again washed with 0.5 M EDTA and dried at 80.0 °C in a hot air oven. Dried samples acid digested using HNO3-HClO4 (3:1) mixture, and the ash obtained dissolved in 0.1 M HCl for quantification of metals using standard calibrated atomic absorption spectrophotometer (GBC 932, Australia). Metal translocation factor determined as the ratio of metal in the shoot versus metal in the root (ie Metal shoot/Metal root).
2.9. Gene expression studies
2.4. Ferric-chelate reductase activity
Gene expression studies carried out using total RNA isolated from the seedlings by TRIzol reagent (Sigma), according to supplier's recommendations. The reverse-transcription reaction carried out with 100.0 ng of total RNA using reverse transcriptase (Sigma-Aldrich) in a PCR system (Eppendorf vapoprotect, Germany). Gene-specific primers designed from the 3′UTR of the rice genes. The sequences used listed in Supplementary Data. 1. The RT-PCR program was 94.0 °C denaturation for 5.0 min, then 30.0 cycles of 94.0 °C for 30.0 s, 58.0 or 60.0 °C for 30.0 s, 72.0 °C for 30.0 s, 72.0 °C extension for 5.0 min, and finally at 16.0 °C. The PCRs optimized for some cycles. This ensured product intensity within the linear phase of amplification. The products after PCR resolved via electrophoresis on a 1.0% agarose gel. This approach helps to visualize polymerase chain reaction (PCR) products unlike the real time PCR method which gives quantitative data of PCR products. The gel images digitally captured with gel documentation system (UVitec Ltd, Cambridge, UK).
Iron reductase assay carried out by submerging roots (100.0 mg) in assay solution (3.0 ml) containing 0.2 mM CaSO4, 5.0 mM 2-(N-morpholino) ethane sulfonic acid (MES) at pH 5.5, 0.1 mM Fe (III) - EDTA, and 0.2 mM 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid (BPDS) (Romera et al., 1996). The absorbance of the assay solution quantified after 30.0 min at 535 nm using a Uv–Visible spectrophotometer (GBC Cintra 5, Australia). Fe (II) - BPDS concentration determined using the extinction coefficient of 22.14 mM−1 cm−1, and the activity expressed as nmol/g/h. 2.5. Histochemical staining of Cd in the root Histochemical detection of Cd in root performed by Dithizone staining (Clabeaux et al., 2011). The stain prepared by mixing 30.0 mg 216
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acquire more Fe in roots during organic acid treatment (4.0% with citrate, 22.0% with malate) (Table 1, Fig. 1C-D). The presence of citrate and malate promoted Fe accumulation in leaves up to 11.0% and 21.0% respectively (Fig. 1D). It noticed that Cd translocation decrease (18.0–20.0%) and Fe translocation increase (15.0–20.0%) upon organic acid treatment (Table 1).
2.10. Analysis of oxidative stress markers Reduced glutathione (GSH) estimated fluorometrically using a standard graph of GSH (Hissin and Hilf, 1976). Plant material (1.0 g) grounded in 1.0 ml of 25.0% H3PO3 and 3.0 ml of 0.1 M sodium phosphate-EDTA buffer (pH 8.0). Homogenate centrifuged at 11220.0 × g for 20.0 min. The supernatant used for the estimation of GSH in a spectrofluorometer (Hitachi F-3010). Final assay mixture (2.0 ml) contained 100.0 µl of the diluted supernatant, 1.8 ml of phosphate-EDTA buffer, and 100.0 µl of O- phthalaldehyde (1.0 mg ml−1). The mixture incubated at room temperature for 15.0 min, and the fluorescence at 420.0 nm measured after excitation at 350 nm. Leaves (0.5 g) extracted with 4.0 ml of 20.0% trichloroacetic acid (TCA) containing 0.5% 2-thiobarbituric acid (Heath and Packer, 1968). The mixture heated at 95.0 °C for 30.0 min, and the homogenate centrifuged at 10000.0 rpm for 10.0 min. The absorbance of the supernatant calculated as, absorbance = 532.0–600.0 nm. Thiobarbituric acid reactive substances (TBARS) content calculated using extinction coefficient of 155.0 mM−1 cm−1.
3.2. Cadmium localization and chemical speciation Plant roots sequester heavy metals in the vacuole. Histochemical staining study pointed out more Cd accumulates in the root of citrate supplemented plants (Fig. 2A). The black-reddish precipitate of Cd with dithizone was visible inside the root cells of citrate treated plants. But the distribution of Cd-dithizone complex was in the cell wall region during malate treatment (Fig. 2A). This result points that Cd exclusion occurs in root during malate supplements. Heavy metals such as Cd bind with sulfur-containing compounds. In the present study, XPS analysis indicated that Cd exists in +2 ionic state in plants because the binding energy of Cd at 405.0 eV corresponds to Cd2+ (Fig. 2B). The increase in C1s binding energy peak at 290.0 eV during organic acid treatment indicates the chance of an increase in carboxylic Cd speciation in plant tissues. Secondly, the disappearance of S2p binding energy peak at 170.0 eV postulates vanishing of sulfur-rich compounds involved in Cd speciation during organic acids treatments. This effect was the outcome of ambient availability of organic acids for metal binding which covered up the requirement of synthesis of sulfur-rich compounds for chelation of Cd. Analysis of absorption peaks of -COOH carbon and degree of stretching of the –COOH group helps to determine the abundance of organic acids and nature of metal - carboxylic acid complexes respectively. In the present study, carboxylic group absorbance was more in the roots of citrate supplemented plants (Fig. 3A). This result indicates that more citrate entered in roots. But in the leaf, an abundance of organic acids noticed in the case of malate supplement (Fig. 3B).
2.11. Plant pigments and chlorophyll fluorescence analysis Plant pigments such as chlorophyll and carotenoids extracted from plant tissues (100.0 mg) using 5.0 ml Acetone – DMSO (1:1) extract kept in dark for 12 h. Absorbance (A) taken at 470.0, 646.0, and 663.0 nm. Amount of pigments calculated using formulae, total chlorophyll (µg/ ml) = 20.2 (A646) + 8.02 (A663), and carotenoids (µg/ml) = (1000*A470-3.27[chla]-104[chlb])/227 (Lichtenthaler and Wellburn, 1983). Anthocyanin estimated from methanol/HCl/water (90:1:1) extract (5.0 ml) of leaf powder (1.0 g). Anthocyanin content calculated using formulae, anthocyanin (µg/ml) = Absorbance 530.0 nm – Absorbance 657.0 nm, and extinction coefficient 30,000 l mol−1 cm−1 (Mancinelli, 1984), and the result expressed in per gram plant tissue. Chlorophyll fluorescence measured with a portable fluorometer (PAM2500, Heinz Walz, Germany). Fast kinetics analysis for studying polyphasic fluorescence rise carried out with Poly 300 ms. FTM mode. Fluorescence transient in the dark-adapted leaf induced by the red light of 3000.0 μmol m−2 s−1. The fluorescence data analyzed with JIP test (Stirbet and Govindjee, 2012).
3.3. Competitive binding of Cd and Fe to organic acids Organic acids are capable of binding with a wide range of metals. The results obtained after the in vitro ion exchange studies indicate that Cd tended to replace Fe2+ ions from organic acids (Fig. 3C-D). But Cd found to favor Fe3+-organic acids complex formation because there was a decrease in absorbance of the mixture (Fig. 3C-D). It is noteworthy that Fe transport to aerial part of the plants is taking place in the form of Fe3+-organic acid complex, especially as Fe3+-citrate. Thus the study on competitive nature in the binding of Fe and Cd with organic acid revealed that the presence of Cd favored aerial transport of Fe3+-organic acid complex in rice plants.
2.12. Statistical analysis Statistical analysis carried out using SPSS software. Two-way analysis of variance conducted with post hoc test namely Duncan's multiple range significance. The result expressed in the form of alphabets where a, b, c, and d that represents first, second, third, and fourth level of statistical significance. All the analysis considered significant at p < 0.05, number of samples (n) = 6.
3.4. Changes in gene expression 3. Results Semi-quantitative PCR analysis showed changes in transcript levels of Fe and Cd transporters (Fig. 4). Cadmium treatment without organic acid supplement decreased expression of Natural resistance-associated macrophage proteins (OsNramp1) and Iron-regulated transporter (OsIRT1) in both leaf and root. Plants subjected to Cd alone treatment also had a low level expression of heavy metal ATPase (OsHMA3) and nicotianamine synthase (OsNAS1) that participate in vacuolar sequestration of Cd and acquisition of Fe respectively. These changes were the outcome of Cd inducible oxidative stress which damage membranes. But organic acid treatment nullified adverse effects of Cd on the expression of above genes. This effect was the outcome of more accumulation of organic acids which chelated Cd, and thereby decreasing the toxic effect arise due to Cd2+ ions. It is noteworthy that expression of OsNAS1 was very low during malate supplement because more availability of this acid decreased requirement of nicotianamine derivatives for cation circulation. Expression of rice FRD3-like (OsFRDL1)
3.1. Metal accumulation in plants Analysis of Cd content in the present study indicates that rice seedlings tended to accumulate more Cd (6.0%) in root after treatment with citrate (Fig. 1A). But supplement of malate prevented Cd accumulation (35.0%) in the root, and the result was statistically significant (Fig. 1A). Plant root sequester metal, and hence the translocation of metal ion depend on rhizocomplexation of metal. The present study showed that Cd accumulation in the leaf decrease during exposure to citrate (24.0%) and malate (45.0%) (Fig. 1B). This effect was the outcome of Cd sequestration and exclusion in roots, respectively after citrate and malate treatments (Fig. 2A). Iron is an essential nutrient for plant growth. But Cd treatment decreased Fe accumulation in root (38.0%) and leaf (40.0%) (Fig. 1CD). It is the higher ferric chelate reductase activity which helped to 217
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Fig. 1. Total amount of Cd and Fe present in roots (A and C) and leaves (B and D). Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Alphabets over the bar correspond to results of two-way analysis of variance. Error bar represent standard error. Letters a, b, c, and d represents first, second, third, and fourth level of statistically significant difference between means during Duncan's post hoc test (p < 0.05, number of samples = 6).
3.5. Oxidative stress indicators
transporter which takes part in xylem loading of both Fe and Cd did not affect in the present study. But expression Cd excluding transporter namely cadmium tolerance 1(OsCDT1) in roots increased after malate treatment. Superoxide dismutase gene (OsSOD) which play a critical role in the detoxification of superoxide radical (O2-) produce during photosynthetic electron transport down-regulated in the presence of exogenous organic acids (Fig. 4). This effect was the result of both decrease in Cd accumulation and increase in Fe content that helped to overcome Cd inducible damage on photoelectron transport system. But the expression of catalase (OsCAT) gene requires for the conversion of hydrogen peroxide to water decreased only with citrate treatment (Fig. 4).
Thiobarbituric acid reactive substances (TBARS) are an indicator of membrane damage in plants. Cadmium stress caused accumulation of TBARS whereas the presence of organic acid decreased TBARS accumulation up to 27.0–47.0% (Table 1). Reduced glutathione (GSH) is essential for the operation of antioxidant enzymes, synthesis of phytochelatin and metal chelation in the vacuole. Plants reared under organic acid supplement had 40.0–60.0% more GSH content compare with Cd-treated plants (Table 1).
Fig. 2. Histochemical staining of cadmium in root cells (A) and X-ray photoelectron spectra (B). Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. 218
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Table 1 Effect of organic acid treatment on biomaas, Cd or Fe translocation factor (TF), ferric chelate reductase (FRO2) activity, reduced glutathione (GSH), and thiobarbituric acid reactive substances (TBARS). ID
Biomass (mg Fwt)
CdTF
Fe TF
- Cd +Cd Cd + Citrate Cd + Malate
14.4 6.7 14.8 14.7
0.00 0.35 ± 0.001a 0.29 ± 0.001b 0.28 ± 0.002b
0.20 0.20 0.24 0.23
± 0. 2a ± 0. 3b ± 0. 4a ± 0.1a
± ± ± ±
0.001b 0.001b 0.002a 0.001a
FRO2 activity
GSH (µg/g)
TBARS (mmol /g F. wt)
0.74 0.89 1.32 1.11
16.0 15.0 24.0 21.0
0.65 0.96 0.51 0.71
± ± ± ±
0.11b 0.13b 0. 12a 0.12a
± ± ± ±
0.2b 0.2b 0.1a 0.2a
± ± ± ±
0.01b 0.05a 0.02c 0.01b
Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Alphabets correspond to results of two-way analysis of variance. Sampling error is represented in the form of + or - sighn. Letters a, b, c, and d represents first, second, third, and fourth level of statistically significant difference between means during Duncan's post hoc test (p < 0.05, n (number of samples) = 6).
member MATE family responsible for Fe/Cd-citrate transport in roots, not affected in the present study (Yokosho et al., 2009). The semi quantitative RT-PCR method followed in the present study helped to visualize the PCR products. But it is noteworthy that this method had drawbacks that the proportions of dominant amplicons do not necessarily reflect the actual abundances of sequences of specific genes of interest. So the gene expression analysis conducted in the present study may be continued with real time - PCR analysis in future. Further, FTIR analysis indicated that organic acid with stretched carbonyl group increased in plant roots exposed to citrate. Stretching of the carbonyl group occur during binding of metals with –COOH group (Max and Chapados, 2004). Hence, the above results confirmed that supply of citrate enhances plant uptake of the citrate-Cd complex. Malate transporters such as cadmium tolerance 1 (OsCDT1) helps to expel Cd malate complex from roots to rhizosphere (Matsuda et al., 2009). OsCDT1 gene expressed more only after malate supplements. This result indicated that malate supplement favor exclusion of malate - Cd complex in the roots. Thus, it concluded that disparity in both chemical structure of the acids chosen for the study and transporter mediated metal uptake caused a difference in Cd accumulation in root during citrate and malate supplements. Sequestration of Cd - organic acid complex in vacuole is a major Cd detoxification mechanism operates in plants (Verbruggen et al., 2009). In the present study, heavy metal ATPase (OsHMA3) which code for a transporter involved in vacuolar sequestration of Cd in rice roots up-regulated during organic acid supply (Sasaki et al., 2014). This change increased Cd rhizocomplexation, and the result confirmed with histochemical staining of Cd as well as quantitative estimation of Cd. Also, XPS analysis revealed
3.6. Photosynthetic efficiency, plant pigments and biomass An increase in chlorophyll fluorescence indicates Cd inducible damage on photosynthetic electron transport. It found that plants reared with Cd show a rise in chlorophyll fluorescence whereas the presence of organic acids prevents it (Fig. 5A). Plant pigments considered as a biomarker of metal accumulation. Accumulation of anthocyanin decreased 54.0% during Cd treatment (Fig. 5B). But exposure to exogenous organic acids resulted in more accumulation of anthocyanin (46.0–94.0%) under Cd stress. Plants grow in the presence of organic acid also had more chlorophyll (54.0–64.0%), carotenoids (40.0–53.0%) and biomass (up to 119.0%) under Cd stress (Fig. 5C-D, Supplementary Data 2, Table 1).
4. Discussion Secretion and accumulation of organic acids imparted metal tolerance in plants (Rauser, 1999). The results of the present study showed that organic acid supplements enhance Cd tolerance in rice seedlings. Supplements of the acids also enhanced accumulation and trafficking of Fe in the plants. Metal chelators facilitate uptake of metal ions in plant roots (Rauser, 1999). These compounds showed difference in chemical structure and metal ion binding affinity. Citrate [C3H5O(COO)33−] having three carboxylic group can bind with minimum three Cd2+ ions and malate [C2H4O(COO)22-] having two carboxylic group can bind with at least two Cd2+ ions. This difference caused more plant available Cd in the presence of citrate and resulted more Cd accumulation in plants. It also noticed that expression of rice FRD3-like (OsFRDL1), a
Fig. 3. FTIR spectra of the root (A) and leaves (B) (Arrows corresponds to the absorption peak of carboxylic acid), and In vitro study on competitive binding of Cd and Fe (25.0 µM) with citrate (C) and malate (D). The increase of absorbance indicates an increase in Fe – O Phenanthroline complex due to Cd mediated displacement of Fe from carboxylic group of organic acids. Error bar represent standard error (number of samples = 6).
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Fig. 4. Gene expression analysis in roots and leaves. Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Fig. 5. Changes in chlorophyll fluorescence (A), anthocyanin (B), total chlorophyll content (C), and carotenoids (D). Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Alphabets over the bar correspond to results of two-way analysis of variance. Error bar represent standard error. Letters a, b, c, and d represents first, second, third, and fourth level of statistically significant difference between means during Duncan's post hoc test (p < 0.05, number of samples = 6).
expression of this gene during both Cd treatment and malate supply indicated that vulnerability of OsNAS to Cd stress. The result also helped to confirm that nicotinamide mediated Fe transport not participates in malate dependant Cd tolerance. But the increase of carboxylic acids observed during FTIR analysis pointed out the occurrence of carboxylic group dependant Cd tolerance in leaves during malate supplement. Cadmium accumulation induced oxidative stress in plants (Verbruggen et al., 2009). Anthocyanin and glutathione (GSH) assist both localization of Cd into vacuole and detoxification of reactive oxygen species under Cd stress (Verbruggen et al., 2009). Hence it concluded that increase in both anthocyanin and GSH also played a role in the alleviation of Cd stress during organic acid supplements. It could be channeling of carboxylic acids to secondary metabolism or amino acid synthesis that increased the content of anthocyanin and GSH during organic acid supplements (Sweetlove and Fernie, 2005). Also, Cd triggered up-regulation of chalcone synthase and glutamate–cysteine ligase account for the increase of anthocyanin and GSH respectively (Dai et al., 2012; Verbruggen et al., 2009). Low-level expression of antioxidant enzymes, especially SOD, along with less thiobarbituric acid reactive substances content in the leaves points out enhanced oxidative stress tolerance during Cd exposure with organic acid supplements. The low-level expression of SOD in these plants was the result of unaltered photosynthetic electron transport which is known to cause photo-oxidative stress during Cd stress (Sebastian and Prasad, 2014b). It was the ambient availability of Fe which helped to develop photosynthetic machinery in the course of Cd plus organic acid treatments. Polyphasic fluorescence rise indicates the efficiency of photosynthetic energy transfer in plants (Stirbet and Govindjee, 2012). In the present
enhancement in chemical speciation of Cd with carboxylic acid in the course of organic acids treatments. These results indicate that sequestering of the metal organic acid complex in the vacuole of root cells decreased Cd transport to leaf. Additionally, exclusion of Cd from root played a role in the decrease of Cd translocation during malate supplements. The extent of Cd inducible Fe deficiency lowered with exogenous organic acids in the present study. Up-regulation of the iron-regulated transporter (OsIRT1), and an increase in ferric chelate reductase activity caused more accumulation of Fe3+ in the root during organic acid treatment (Kobayashi and Nishizawa, 2012; Sebastian and Prasad, 2015). Results of the in vitro studies indicated that Cd stabilizes Fe3+ organic acid complex formation which is critical for the translocation of Fe. Ferric ion formed a tridentate complex with a carboxylic group of tricarboxylic acid, and the complex transform to bidentate complex before degradation (Francis and Dodge, 1992). But the presence of Cd favored formation of both Cd and Fe3+ containing complexes where cadmium binds to the hydroxyl group of organic acids (Francis and Dodge, 1992). This complex is thermodynamically stable, and do not undergo random structural transformations (Francis and Dodge, 1992). Thus, it confirmed that the presence of Cd favored formation of stable Fe3+ - organic acid complex, and the metal complex efficiently transported to leaves via FRD3-like transporter (OsFRDL1) resulting more Fe accumulation in leaves (Yokosho et al., 2009). Also, ambient Fe circulation in leaves assured during organic acid supplement because expression of natural resistance-associated macrophage protein 1 (OsNramp1) sustained in these group of plants. Nicotianamine synthase (OsNAS) gene expression is critical for Fe reallocation and type II Fe acquisition respectively (Kobayashi and Nishizawa, 2012). Low-level 220
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study, Cd inducible fluorescence rise decreased during organic acids supplements. The difference was prominent in the J-I and I-P phases of the polyphasic fluorescence curve. Fluorescence rise in the O-J phase also observed during Cd treatment and Cd plus malate supplement. These results indicated alteration of electron transfer at the acceptor side of PSII i.e. reoxidation of QA was not efficient in these plants. The J-I phase was markedly visible among all experimental plants except those plants rear with citrate supplements, and this confirmed blockage of electron transport due to inadequate mobile plastoquinone pool (PQ) capable of electron transport (Stirbet and Govindjee, 2012). Blockages in the photosynthetic electron transport generate reactive oxygen species (ROS) (Stirbet and Govindjee, 2012). Therefore, it predicted that citrate supplement uphold PQ content which in turn result in normal functioning of photoelectron transport chain and decrease generation of reactive oxygen species. Decreased ROS production accounts for the low-level expression of superoxide dismutase (OsSOD) and catalase (OsCAT) after citrate supplement. The more expression of antioxidant enzymes observed among –ve control plants and malate supplimented plants could be the outcome of higher light capture or Fe accumulation related Fenton reactions (Moseley et al., 2002). A prominent I-P phase fluorescence rise also observed during Cd treatment. This result indicated that Cd stress affects electron transport at the PSI end too. Photosynthetic pigments and biomass are indicators of stress tolerance (Pavlović et al., 2014). Among photosynthetic pigments, chlorophyll is essential for light harvest whereas carotenoids help to dissipate excess light energy (Pavlović et al., 2014). Cadmium detoxification via Cd chelation, Fe nutrition, and antioxidant defense accounts for unaltered pigment synthesis during Cd treatment with organic acid supplement. Secondly, ambient availability of photosynthetic pigments helped to maintain light harvest efficiency critical for photosynthesis. Thus, plants reared with organic acids supplement had a higher photosynthetic efficiency which in turn helped to produce biomass under Cd stress.
Appendix A. Supporting information Supplementary data associated with this article can be found in the online version at doi:10.1016/j.ecoenv.2018.09.084. References Bagus, P.S., Ilton, E., Nelin, C.J., 2013. The interpretation of XPS spectra: insights into materials properties. Surf. Sci. 68, 273–304. Bolaji, A.A., Mackenzie, T.B., Sheila, M.M., 2010. Production of organic acids and adsorption of Cd on roots of durum wheat (Triticum turgidum L. var. durum). Biol. Publ. 39. Chaney, R.L., 2015. How does contamination of rice soils with Cd and Zn cause high incidence of human Cd disease in subsistence rice farmers. Curr. Pollut. Rep. 1, 13–22. Clabeaux, B.L., Navarro, D.A.G., Aga, D.S., Bisson, M.A., 2011. Cd tolerance and accumulation in the aquaticmacrophyte, Charaaustralis: potential use for charophytes in phytoremediation. Environ. Sci. Technol. 45, 5332–5338. Connolly, E.L., Fett, J.P., Guerinot, M.L., 2002. Expression of the IRT1 metal transporter is controlled by metals at the levels of transcript and protein accumulation. Plant Cell. 14 (6), 1347–1357. Dai, L.P., Dong, X.J., Ma, H.H., 2012. Molecular mechanism for cadmium-induced anthocyanin accumulation in Azolla imbricata. Chemosphere 87, 319–325. Fortune, W.B., Mellon, M.G., 1938. Determination of iron with o-phenanthroline: a spectrophotometric study. Ind. Eng. Chem. Anal. Ed. 10 (2), 60–64. Francis, A.J., Dodge, C.J., 1992. Influence of complex structure on the biodegradation of iron-citrate complexes. Appl. Environ. Microbiol. 59, 109–113. Hall, J.L., Williams, L.E., 2003. Transition metal transporters in plants. J. Exp. Bot. 54 (393), 2601–2613. Hawrylak-Nowak, B., Dresler, S., Matraszek, R., 2015. Exogenous malic and acetic acids reduce cadmium phytotoxicity and enhance cadmium accumulation in roots of sunflower plants. Plant Physiol. Biochem. 94, 225–234. Heath, R.L., Packer, L., 1968. Photoperoxidation in isolated chloroplasts. 1. Kinetics and stoichiometry of fatty acid peroxidation. Arch. Biochem. Biophys. 125, 189–198. Hissin, P.J., Hilf, R., 1976. A fluorimetric method for determination of oxidized and reduced glutathione in tissues. Anal. Biochem. 74, 214–226. Hoagland, D., Arnon, D.I., 1950. The water culture method for growing plants without soils. Bull. Calif. Agric. Station. 346. Johnson, A.A., Kyriacou, B., Callahan, D.L., Carruthers, L., Stangoulis, J., Lombi, E., Tester, M., 2011. Constitutive overexpression of the OsNAS gene family reveals single-gene strategies for effective iron- and zinc-biofortification of rice endosperm. PLoS One 6 (9), e24476. Kobayashi, T., Nishizawa, N.K., 2012. Iron uptake, translocation, and regulation in higher plants. Annu. Rev. Plant. Biol. 63, 131–152. Kochian, L.V., Pineros, M.A., Liu, J., Magalhaes, J.V., 2015. Plant adaptation to acid soils: the molecular basis for crop aluminum resistance. Annu. Rev. Plant Biol. 66, 571–598. Kuramata, M., Masuya, S., Takahashi, Y., Kitagawa, E., Inoue, C., Ishikawa, S., Youssefian, S., Kusano, T., 2012. Novel cysteine-rich peptides from Digitaria ciliaris and Oryza sativa enhance tolerance to cadmium by limiting its cellular accumulation. Plant Cell Physiol. 50 (1), 106–117. Lichtenthaler, H.K., Wellburn, A.R., 1983. Determinations of total carotenoids and chlorophylls a and b of leaf extracts in different solvents. Biochem. Soc. Trans. 11, 591–592. Mancinelli, A., 1984. Photoregulation of anthocyanin synthesis.VIII.Effects of light pretreatments. Plant Physiol. 75, 447–453. Matsuda, T., Kuramata, M., Takahashi, Y., Kitagawa, E., Youssefian, S., Kusano, T., 2009. A novel plant cysteine-rich peptide family conferring cadmium tolerance to yeast and plants. Plant Signal Behav. 4 (5), 419–421. Mahmud, J.A., Hasanuzzaman, M., Nahar, K., Bhuyan, M.H.M.B., Fujita, M., 2018. Insights into citric acid-induced cadmium tolerance and phytoremediation in Brassica juncea L.: coordinated functions of metal chelation, antioxidant defense and glyoxalase systems. Ecotoxicol. Environ. Saf. 147, 990–1001. Max, J., Chapados, C., 2004. Infrared spectroscopy of aqueous carboxylic acids: comparison between different acids and their salts. J. Phys. Chem. A 108 (16), 3324–3337. Moseley, J.S., Allinger, T., Herzog, S., Hoerth, P., Wehinger, E., Merchant, S., Hippler, M., 2002. Adaptation to Fe-deficiency requires remodeling of the photosynthetic apparatus. EMBO J. 21, 6709–6720. Nasciment, C.W.A., 2006. Organic acids effects on desorption of heavy metals from a contaminated soil. Sci. Agric. 63 (3), 276–280. Pavlović, D., Nikolić, B., Đurović, S., Waisi, H., Anđelković, A., Marisavljević, D., 2014. Chlorophyll as a measure of plant health: Agroecological aspect. Pestic. Phytomed. 29 (1), 21–23. Rauser, W.E., 1999. Structure and function of metal chelators produced by plants. The case for organic acids, amino acids, phytin and metallothioneins. Cell. Biophys. 31, 19–48. Romera, F.J., Welch, R.M., Norvell, W.A., Schaefer, S.C., 1996. Iron requirement for and effects of promoters and inhibitors of ethylene action on stimulation of Fe (III)-chelate reductase in roots of strategy I species. BioMetals 9, 45–50. Sasaki, A., Yamaji, N., Ma, J.F., 2014. Over expression of OsHMA3 enhances Cd tolerance and expression of Zn transporter genes in rice. J. Exp. Bot. 65 (20), 6013–6021. Sebastian, A., Prasad, M.N.V., 2014a. Cadmium minimization in rice. A review. Agron. Sus. Dev. 34, 155–173.
5. Conclusions Supplement of organic acid enhances Cd tolerance in rice seedlings. Rhizocomplexation of Cd and lowering of Cd inducible Fe deficiency are the critical factors involved in organic acid dependant Cd tolerance. Organic acid supply enhances carboxylic speciation of Cd which in turn favors vacuolar sequestration of Cd in the root along with the overexpression of OsHMA3. Thus, rhizocomplexation of Cd decreased translocation of Cd to leaf. Also, Cd exclusion assisted with OsCDT1 expression played an important role in lowering of Cd accumulation among malate supplemented plants. The formation of a stable Fe3+organic acid complex in the presence of Cd helped the efficient transport of Fe to aerial plant parts. Also, there exists unaltered Fe circulation in the leaves of organic acid supplemented plants because of the upholding of OsNramp1 activity. It also noticed that supplement of organic acids prevent Cd related retarding of chlorophyll synthesis as well as photosystems activity with the help of anthocyanin and GSH respectively. Thus, the maintenance of ambient Fe level and antioxidant activity in the leaf helped to maintain photosynthetic efficiency and biomass productivity in the course of the organic acid supplement under Cd stress. Radioactive tracer experiments and analysis of transcription factors will provide further insights into the kinetics of Cd accumulation and molecular switching involved in organic acid dependant Cd tolerance respectively. Acknowledgment Abin Sebastian gratefully acknowledges Senior research fellowship (SRF), University Grand Commission (UGC), India for financial support, and Dr. Neha Hebalkar, International Advanced Research Centre for Powder Metallurgy and New Materials, Hyderabad, for XPS analysis. 221
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Sweetlove, L., Fernie, A.R., 2005. Regulation of metabolic networks: understanding metabolic complexity in the systems biology era. New Phytol. 168, 9–24. Takahashi, R., Ishimaru, Y., Nakanishi, H., Nishizawa, N.K., 2011. Role of the iron transporter OsNRAMP1 in cadmium uptake and accumulation in rice. Plant Signal Behav. 6 (11), 1813–1816. Verbruggen, N., Hermans, C., Schat, H., 2009. Molecular mechanisms of metal hyperaccumulation in plants. New Phytol. 181, 759–776. Yokosho, K., Yamaji, N., Ueno, D., Mitani, N., Ma, J.F., 2009. OsFRDL1 is a citrate transporter required for efficient translocation of iron in rice. Plant Physiol. 149, 297–305.
Sebastian, A., Prasad, M.N.V., 2014b. Red and blue lights induced oxidative stress tolerance promotes cadmium rhizocomplexation in Oryza sativa. J. Photoch. Photobio. B 137, 135–143. Sebastian, A., Prasad, M.N.V., 2015. Iron- and manganese-assisted cadmium tolerance in Oryza sativa L.: lowering of rhizotoxicity next to functional photosynthesis. Planta 241 (6), 1519–1528. Stirbet, A., Govindjee, 2012. Chlorophyll a fluorescence induction: a personal perspective of the thermal phase, the J–I–P rise. Photosynth. Res. 113, 15–61. Song, J., Ma, R., Huang, W., Cui, X., 2014. Exogenous organic acids protect changbai larch (Larix Olgensis) seedlings against cadmium toxicity. Fresen. Environ. Bull. 23 (12C), 3460–3468.
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Exogenous citrate and malate alleviate cadmium stress in Oryza sativa L.: Probing role of cadmium localization and iron nutrition
T
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Abin Sebastian , M.N.V. Prasad Department of Plant Sciences, University of Hyderabad, Central University P.O., Hyderabad 500046, India
A R T I C LE I N FO
A B S T R A C T
Keywords: Cadmium Organic acids Metal chelation Iron uptake Antioxidants Photosynthesis
Organic acids play an important role in metal uptake and trafficking in plants. Therefore, the role of exogenous citrate and malate on Cd tolerance was studied in the seedlings of Oryza sativa L. cv MTU 7029. Seedlings were exposed to Cd plus organic acids in hydroponics and monitored changes in Cd accumulation, expression of metal transporters, chlorophyll fluorescence, and antioxidants. It found that organic acid supplements decrease Cd accumulation in leaf because of up-regulation of tonoplast localized heavy metal ATPase (OsHMA3) which allows vacuolar sequestration of Cd in the root. Malic acid helped Cd exclusion in the root too. A shift in Cd speciation from sulphhydryl to the carboxylic group also noticed in the roots of plants exposed to organic acids. Treatment of organic acids was effective to prevent Cd inducible Fe deficiency via up-regulation of the ironregulated transporter (OsIRT1), increase in ferric chelate reductase activity, and formation of Cd stabilized Fe3+ - organic acid complex respectively. Also, exposure to organic acids increased the accumulation of antioxidants such as anthocyanin and glutathione (GSH) under Cd stress. Above changes assisted in upholding of photosynthetic electron transport and biomass productivity during the course of Cd treatment with organic acid supplements.
1. Introduction Cadmium contamination of rice paddies is a serious health problem (Sebastian and Prasad, 2014a). Applications of phosphate fertilizers and irrigation of rice paddies with mine leachate resulted in toxic levels of Cd (0.05–7.7 mg kg−1) accumulation in rice (Chaney, 2015; Sebastian and Prasad, 2014a). Therefore, Cd minimization in rice gained attention (Sebastian and Prasad, 2014a). Metal chelators, soil dressing, and chemical reduction processes were applied to decrease plant-available Cd in paddy soils (Sebastian and Prasad, 2014a). Organic acids chelate metals, and this property utilized for the removal of toxic metals from contaminated sites (Nasciment, 2006). But organic acids confer heavy metal tolerance in plants too (Verbruggen et al., 2009). Metal-carboxylic acid complex sequester in the vacuole. This help to avoid oxidative stress come up with free metal ions in the cytosol. Secondly, organic acids secretion into rhizosphere reported as an adaptive strategy against metal toxicity and mineral nutrient deficiency (Kochian et al., 2015). Iron uptake and transport in plants take place with the help of organic acids (Kobayashi and Nishizawa, 2012). Organic acids such as muigenic acid, citrate, and malate play a critical role in Fe nutrition of plants (Kobayashi and Nishizawa, 2012). Citrate helps vacuolar ⁎
sequestration of Fe in the root as well as translocation of Fe (Kobayashi and Nishizawa, 2012). But malate acts as a carrier for the circulation of Fe in plants (Kochian et al., 2015). Malate also plays an important role in pH homeostasis, osmotic adjustment, shuttling of reducing equivalents, and aluminium tolerance in plants (Kochian et al., 2015). It is noteworthy that the synthesis of organic acids depends on photosynthesis, and the process is vulnerable to Cd stress (Sebastian and Prasad, 2014b). So Cd stress hinders the synthesis of organic acids and slows down heavy metal detoxification in plants. Transition metal transporters mediate Cd transport in plant cells (Hall and Williams, 2003). Iron-regulated transporter (IRT1) and natural resistance-associated macrophage proteins (NRAMP1) mediate Fe and Cd uptake in plants (Connolly et al., 2002; Takahashi et al., 2011). Also, cadmium tolerant 1 (OsCDT1) is a plasma membrane transporter which excludes Cd from the cell (Kuramata et al., 2012). On the other side, intracellular trafficking of Cd into plant vacuole occurs with the help of heavy metal ATPase (HMA3) (Sasaki et al., 2014). Iron deficiency responses in rice plants associated with the expression of nicotianamine synthase (OsNAS) and activity of this enzyme is critical for muigenic acids dependant Fe uptake via yellow stripe like transporter (OsYSL) (Johnson et al., 2011). So it is clear that organic acids had a critical role in Cd tolerance and Fe uptake in plants. Studies pointed out
Corresponding author. E-mail address: [email protected] (A. Sebastian).
https://doi.org/10.1016/j.ecoenv.2018.09.084 Received 8 December 2017; Received in revised form 4 September 2018; Accepted 20 September 2018 0147-6513/ © 2018 Elsevier Inc. All rights reserved.
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diphenyl thiocarbazone in 60.0 ml acetone, 5.0 ml glacial acetic acid, and 20.0 ml milliQ water. Staining performed on hand sections of the root tissue for 1.0 h, and after that excess stain washed away with milliQ water. The sections observed using a stereoscopic microscope, and the presence of Cd in tissues detected as dark black-brown complexes of Cd with diphenyl thiocarbazone.
that exogenous organic acids prevent Cd stress in sunflower and Changbai larch plants (Hawrylak-Nowak et al., 2015; Song et al., 2014). However, the physiological and molecular mechanisms of organic acid dependant Cd tolerance remain unexplored. More recently, studies on Brassica juncea pointed that citric acid supplements enhance Cd tolerance because of coordinated actions of metal chelation, antioxidant defense, and glyoxalase systems (Mahmud et al., 2018). Also, enhancement of Cd tolerance in Durum wheat found to associate with an increase of fumaric, malic, and succinic acids (Bolaji et al., 2010). But these studies not discussed the chance of competition in the uptake and trafficking of Fe with Cd. This situation may occur because carboxylic acids bind with a wide range of cations. Therefore, organic acids supplement dependant Cd tolerance in relation with Fe nutrition explored in the present study.
2.6. X-ray photoelectron spectroscopy X-ray photoelectron spectroscopy (XPS) analyzes the ionic state and chemical nature of metal ion in a given sample. The instrument measures the kinetic energy of electrons escape from the sample during Xray exposure (Bagus et al., 2013). XPS analysis conducted on vacuum dried root powder. Sample exposed to X-ray photoelectron spectrometer (Omicron Nano Technology, UK) using monochromatized Al Kα radiation (1486.7 eV). Measurements carried out in a constant analyzer energy mode. The intensity of the XPS peak recorded as counts per second (CPS). The chemical speciation and the chemical nature of Cd complexes determined based on the binding energy (Bagus et al., 2013). The binding energy, which indicates chemical nature of the metal ion, derived from the kinetic energy of the emitted electrons, and the photon energy used to emit these electrons.
2. Materials and methods 2.1. Hydroponics Oryza sativa cv MTU 7029 seeds were sterilized with 5.0% hydrogen peroxide for 15.0 min and washed with double distilled water. Seeds were germinated in a germination box contain wet sand under a light intensity of 50.0 µmol photons m−2 s−1. Five-day-old seedlings used for hydroponics with Hoagland media (Hoagland and Arnon, 1950). The media was diluted to half the strength using milliQ water before the experiments.
2.7. Detection of organic acids To find out the abundance of organic acids, air-dried powder of plant tissues (10.0 mg) made pellet with potassium bromide, and the pellet loaded into Fourier transform infrared (FTIR) spectrometer (Nicolet 380 FT-IR, Thermo scientific, USA) at room temperature. Spectral wave number ranges from 400 to 4000 cm−1 recorded. The peak in the Fourier transform infrared absorbance spectra at 2500–3000 cm−1 corresponds to abundance and carbonyl stretch of the carboxylic group present in the tissue (Max and Chapados, 2004).
2.2. Cadmium and organic acid treatments Cadmium treatments performed by transferring seedlings to media containing 25.0 µM CdCl2. Malate or citrate supplement carried out at a concentration of 50.0 µM in Cd containing media. A group of plants maintained without Cd or organic acid treatment as a control. Light intensity (400.0 µmol photons m−2 s−1), photoperiod (18.0 h Light / 6 h dark), temperature (25.0 ± 2.0 °C) and relative humidity (50.0 ± 10.0%) were maintained throughout the 14 days of experiment.
2.8. Exchange of metal ions bound to organic acids To study the exchange of metal ions bound with organic acids, a solution of 50.0 µM organic acid (citrate or malate), and 25.0 µM iron salt (ferrous or ferric chloride) treated with varying CdCl2, and incubated for 1.0 h at 25.0 °C. After this, the mixture treated with 10.0% hydroxylamine solution for the reduction of free Fe3+ available in the solution to Fe2+. The amount of free Fe2+ ions in the solution determined via measuring absorbance at 510 nm after reaction with OPhenanthroline (Fortune and Mellon, 1938). Changes in absorbance of the Fe-organic acid mixture against varying concentration of CdCl2 plotted.
2.3. Biomass and metal quantification Biomass quantified with the help of weighing balance, and the result expressed as mg fresh weight. For the quantitative estimation of metals, plants manually cleaned with deionized water. The material again washed with 0.5 M EDTA and dried at 80.0 °C in a hot air oven. Dried samples acid digested using HNO3-HClO4 (3:1) mixture, and the ash obtained dissolved in 0.1 M HCl for quantification of metals using standard calibrated atomic absorption spectrophotometer (GBC 932, Australia). Metal translocation factor determined as the ratio of metal in the shoot versus metal in the root (ie Metal shoot/Metal root).
2.9. Gene expression studies
2.4. Ferric-chelate reductase activity
Gene expression studies carried out using total RNA isolated from the seedlings by TRIzol reagent (Sigma), according to supplier's recommendations. The reverse-transcription reaction carried out with 100.0 ng of total RNA using reverse transcriptase (Sigma-Aldrich) in a PCR system (Eppendorf vapoprotect, Germany). Gene-specific primers designed from the 3′UTR of the rice genes. The sequences used listed in Supplementary Data. 1. The RT-PCR program was 94.0 °C denaturation for 5.0 min, then 30.0 cycles of 94.0 °C for 30.0 s, 58.0 or 60.0 °C for 30.0 s, 72.0 °C for 30.0 s, 72.0 °C extension for 5.0 min, and finally at 16.0 °C. The PCRs optimized for some cycles. This ensured product intensity within the linear phase of amplification. The products after PCR resolved via electrophoresis on a 1.0% agarose gel. This approach helps to visualize polymerase chain reaction (PCR) products unlike the real time PCR method which gives quantitative data of PCR products. The gel images digitally captured with gel documentation system (UVitec Ltd, Cambridge, UK).
Iron reductase assay carried out by submerging roots (100.0 mg) in assay solution (3.0 ml) containing 0.2 mM CaSO4, 5.0 mM 2-(N-morpholino) ethane sulfonic acid (MES) at pH 5.5, 0.1 mM Fe (III) - EDTA, and 0.2 mM 4,7-diphenyl-1,10-phenanthrolinedisulfonic acid (BPDS) (Romera et al., 1996). The absorbance of the assay solution quantified after 30.0 min at 535 nm using a Uv–Visible spectrophotometer (GBC Cintra 5, Australia). Fe (II) - BPDS concentration determined using the extinction coefficient of 22.14 mM−1 cm−1, and the activity expressed as nmol/g/h. 2.5. Histochemical staining of Cd in the root Histochemical detection of Cd in root performed by Dithizone staining (Clabeaux et al., 2011). The stain prepared by mixing 30.0 mg 216
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acquire more Fe in roots during organic acid treatment (4.0% with citrate, 22.0% with malate) (Table 1, Fig. 1C-D). The presence of citrate and malate promoted Fe accumulation in leaves up to 11.0% and 21.0% respectively (Fig. 1D). It noticed that Cd translocation decrease (18.0–20.0%) and Fe translocation increase (15.0–20.0%) upon organic acid treatment (Table 1).
2.10. Analysis of oxidative stress markers Reduced glutathione (GSH) estimated fluorometrically using a standard graph of GSH (Hissin and Hilf, 1976). Plant material (1.0 g) grounded in 1.0 ml of 25.0% H3PO3 and 3.0 ml of 0.1 M sodium phosphate-EDTA buffer (pH 8.0). Homogenate centrifuged at 11220.0 × g for 20.0 min. The supernatant used for the estimation of GSH in a spectrofluorometer (Hitachi F-3010). Final assay mixture (2.0 ml) contained 100.0 µl of the diluted supernatant, 1.8 ml of phosphate-EDTA buffer, and 100.0 µl of O- phthalaldehyde (1.0 mg ml−1). The mixture incubated at room temperature for 15.0 min, and the fluorescence at 420.0 nm measured after excitation at 350 nm. Leaves (0.5 g) extracted with 4.0 ml of 20.0% trichloroacetic acid (TCA) containing 0.5% 2-thiobarbituric acid (Heath and Packer, 1968). The mixture heated at 95.0 °C for 30.0 min, and the homogenate centrifuged at 10000.0 rpm for 10.0 min. The absorbance of the supernatant calculated as, absorbance = 532.0–600.0 nm. Thiobarbituric acid reactive substances (TBARS) content calculated using extinction coefficient of 155.0 mM−1 cm−1.
3.2. Cadmium localization and chemical speciation Plant roots sequester heavy metals in the vacuole. Histochemical staining study pointed out more Cd accumulates in the root of citrate supplemented plants (Fig. 2A). The black-reddish precipitate of Cd with dithizone was visible inside the root cells of citrate treated plants. But the distribution of Cd-dithizone complex was in the cell wall region during malate treatment (Fig. 2A). This result points that Cd exclusion occurs in root during malate supplements. Heavy metals such as Cd bind with sulfur-containing compounds. In the present study, XPS analysis indicated that Cd exists in +2 ionic state in plants because the binding energy of Cd at 405.0 eV corresponds to Cd2+ (Fig. 2B). The increase in C1s binding energy peak at 290.0 eV during organic acid treatment indicates the chance of an increase in carboxylic Cd speciation in plant tissues. Secondly, the disappearance of S2p binding energy peak at 170.0 eV postulates vanishing of sulfur-rich compounds involved in Cd speciation during organic acids treatments. This effect was the outcome of ambient availability of organic acids for metal binding which covered up the requirement of synthesis of sulfur-rich compounds for chelation of Cd. Analysis of absorption peaks of -COOH carbon and degree of stretching of the –COOH group helps to determine the abundance of organic acids and nature of metal - carboxylic acid complexes respectively. In the present study, carboxylic group absorbance was more in the roots of citrate supplemented plants (Fig. 3A). This result indicates that more citrate entered in roots. But in the leaf, an abundance of organic acids noticed in the case of malate supplement (Fig. 3B).
2.11. Plant pigments and chlorophyll fluorescence analysis Plant pigments such as chlorophyll and carotenoids extracted from plant tissues (100.0 mg) using 5.0 ml Acetone – DMSO (1:1) extract kept in dark for 12 h. Absorbance (A) taken at 470.0, 646.0, and 663.0 nm. Amount of pigments calculated using formulae, total chlorophyll (µg/ ml) = 20.2 (A646) + 8.02 (A663), and carotenoids (µg/ml) = (1000*A470-3.27[chla]-104[chlb])/227 (Lichtenthaler and Wellburn, 1983). Anthocyanin estimated from methanol/HCl/water (90:1:1) extract (5.0 ml) of leaf powder (1.0 g). Anthocyanin content calculated using formulae, anthocyanin (µg/ml) = Absorbance 530.0 nm – Absorbance 657.0 nm, and extinction coefficient 30,000 l mol−1 cm−1 (Mancinelli, 1984), and the result expressed in per gram plant tissue. Chlorophyll fluorescence measured with a portable fluorometer (PAM2500, Heinz Walz, Germany). Fast kinetics analysis for studying polyphasic fluorescence rise carried out with Poly 300 ms. FTM mode. Fluorescence transient in the dark-adapted leaf induced by the red light of 3000.0 μmol m−2 s−1. The fluorescence data analyzed with JIP test (Stirbet and Govindjee, 2012).
3.3. Competitive binding of Cd and Fe to organic acids Organic acids are capable of binding with a wide range of metals. The results obtained after the in vitro ion exchange studies indicate that Cd tended to replace Fe2+ ions from organic acids (Fig. 3C-D). But Cd found to favor Fe3+-organic acids complex formation because there was a decrease in absorbance of the mixture (Fig. 3C-D). It is noteworthy that Fe transport to aerial part of the plants is taking place in the form of Fe3+-organic acid complex, especially as Fe3+-citrate. Thus the study on competitive nature in the binding of Fe and Cd with organic acid revealed that the presence of Cd favored aerial transport of Fe3+-organic acid complex in rice plants.
2.12. Statistical analysis Statistical analysis carried out using SPSS software. Two-way analysis of variance conducted with post hoc test namely Duncan's multiple range significance. The result expressed in the form of alphabets where a, b, c, and d that represents first, second, third, and fourth level of statistical significance. All the analysis considered significant at p < 0.05, number of samples (n) = 6.
3.4. Changes in gene expression 3. Results Semi-quantitative PCR analysis showed changes in transcript levels of Fe and Cd transporters (Fig. 4). Cadmium treatment without organic acid supplement decreased expression of Natural resistance-associated macrophage proteins (OsNramp1) and Iron-regulated transporter (OsIRT1) in both leaf and root. Plants subjected to Cd alone treatment also had a low level expression of heavy metal ATPase (OsHMA3) and nicotianamine synthase (OsNAS1) that participate in vacuolar sequestration of Cd and acquisition of Fe respectively. These changes were the outcome of Cd inducible oxidative stress which damage membranes. But organic acid treatment nullified adverse effects of Cd on the expression of above genes. This effect was the outcome of more accumulation of organic acids which chelated Cd, and thereby decreasing the toxic effect arise due to Cd2+ ions. It is noteworthy that expression of OsNAS1 was very low during malate supplement because more availability of this acid decreased requirement of nicotianamine derivatives for cation circulation. Expression of rice FRD3-like (OsFRDL1)
3.1. Metal accumulation in plants Analysis of Cd content in the present study indicates that rice seedlings tended to accumulate more Cd (6.0%) in root after treatment with citrate (Fig. 1A). But supplement of malate prevented Cd accumulation (35.0%) in the root, and the result was statistically significant (Fig. 1A). Plant root sequester metal, and hence the translocation of metal ion depend on rhizocomplexation of metal. The present study showed that Cd accumulation in the leaf decrease during exposure to citrate (24.0%) and malate (45.0%) (Fig. 1B). This effect was the outcome of Cd sequestration and exclusion in roots, respectively after citrate and malate treatments (Fig. 2A). Iron is an essential nutrient for plant growth. But Cd treatment decreased Fe accumulation in root (38.0%) and leaf (40.0%) (Fig. 1CD). It is the higher ferric chelate reductase activity which helped to 217
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Fig. 1. Total amount of Cd and Fe present in roots (A and C) and leaves (B and D). Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Alphabets over the bar correspond to results of two-way analysis of variance. Error bar represent standard error. Letters a, b, c, and d represents first, second, third, and fourth level of statistically significant difference between means during Duncan's post hoc test (p < 0.05, number of samples = 6).
3.5. Oxidative stress indicators
transporter which takes part in xylem loading of both Fe and Cd did not affect in the present study. But expression Cd excluding transporter namely cadmium tolerance 1(OsCDT1) in roots increased after malate treatment. Superoxide dismutase gene (OsSOD) which play a critical role in the detoxification of superoxide radical (O2-) produce during photosynthetic electron transport down-regulated in the presence of exogenous organic acids (Fig. 4). This effect was the result of both decrease in Cd accumulation and increase in Fe content that helped to overcome Cd inducible damage on photoelectron transport system. But the expression of catalase (OsCAT) gene requires for the conversion of hydrogen peroxide to water decreased only with citrate treatment (Fig. 4).
Thiobarbituric acid reactive substances (TBARS) are an indicator of membrane damage in plants. Cadmium stress caused accumulation of TBARS whereas the presence of organic acid decreased TBARS accumulation up to 27.0–47.0% (Table 1). Reduced glutathione (GSH) is essential for the operation of antioxidant enzymes, synthesis of phytochelatin and metal chelation in the vacuole. Plants reared under organic acid supplement had 40.0–60.0% more GSH content compare with Cd-treated plants (Table 1).
Fig. 2. Histochemical staining of cadmium in root cells (A) and X-ray photoelectron spectra (B). Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. 218
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Table 1 Effect of organic acid treatment on biomaas, Cd or Fe translocation factor (TF), ferric chelate reductase (FRO2) activity, reduced glutathione (GSH), and thiobarbituric acid reactive substances (TBARS). ID
Biomass (mg Fwt)
CdTF
Fe TF
- Cd +Cd Cd + Citrate Cd + Malate
14.4 6.7 14.8 14.7
0.00 0.35 ± 0.001a 0.29 ± 0.001b 0.28 ± 0.002b
0.20 0.20 0.24 0.23
± 0. 2a ± 0. 3b ± 0. 4a ± 0.1a
± ± ± ±
0.001b 0.001b 0.002a 0.001a
FRO2 activity
GSH (µg/g)
TBARS (mmol /g F. wt)
0.74 0.89 1.32 1.11
16.0 15.0 24.0 21.0
0.65 0.96 0.51 0.71
± ± ± ±
0.11b 0.13b 0. 12a 0.12a
± ± ± ±
0.2b 0.2b 0.1a 0.2a
± ± ± ±
0.01b 0.05a 0.02c 0.01b
Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Alphabets correspond to results of two-way analysis of variance. Sampling error is represented in the form of + or - sighn. Letters a, b, c, and d represents first, second, third, and fourth level of statistically significant difference between means during Duncan's post hoc test (p < 0.05, n (number of samples) = 6).
member MATE family responsible for Fe/Cd-citrate transport in roots, not affected in the present study (Yokosho et al., 2009). The semi quantitative RT-PCR method followed in the present study helped to visualize the PCR products. But it is noteworthy that this method had drawbacks that the proportions of dominant amplicons do not necessarily reflect the actual abundances of sequences of specific genes of interest. So the gene expression analysis conducted in the present study may be continued with real time - PCR analysis in future. Further, FTIR analysis indicated that organic acid with stretched carbonyl group increased in plant roots exposed to citrate. Stretching of the carbonyl group occur during binding of metals with –COOH group (Max and Chapados, 2004). Hence, the above results confirmed that supply of citrate enhances plant uptake of the citrate-Cd complex. Malate transporters such as cadmium tolerance 1 (OsCDT1) helps to expel Cd malate complex from roots to rhizosphere (Matsuda et al., 2009). OsCDT1 gene expressed more only after malate supplements. This result indicated that malate supplement favor exclusion of malate - Cd complex in the roots. Thus, it concluded that disparity in both chemical structure of the acids chosen for the study and transporter mediated metal uptake caused a difference in Cd accumulation in root during citrate and malate supplements. Sequestration of Cd - organic acid complex in vacuole is a major Cd detoxification mechanism operates in plants (Verbruggen et al., 2009). In the present study, heavy metal ATPase (OsHMA3) which code for a transporter involved in vacuolar sequestration of Cd in rice roots up-regulated during organic acid supply (Sasaki et al., 2014). This change increased Cd rhizocomplexation, and the result confirmed with histochemical staining of Cd as well as quantitative estimation of Cd. Also, XPS analysis revealed
3.6. Photosynthetic efficiency, plant pigments and biomass An increase in chlorophyll fluorescence indicates Cd inducible damage on photosynthetic electron transport. It found that plants reared with Cd show a rise in chlorophyll fluorescence whereas the presence of organic acids prevents it (Fig. 5A). Plant pigments considered as a biomarker of metal accumulation. Accumulation of anthocyanin decreased 54.0% during Cd treatment (Fig. 5B). But exposure to exogenous organic acids resulted in more accumulation of anthocyanin (46.0–94.0%) under Cd stress. Plants grow in the presence of organic acid also had more chlorophyll (54.0–64.0%), carotenoids (40.0–53.0%) and biomass (up to 119.0%) under Cd stress (Fig. 5C-D, Supplementary Data 2, Table 1).
4. Discussion Secretion and accumulation of organic acids imparted metal tolerance in plants (Rauser, 1999). The results of the present study showed that organic acid supplements enhance Cd tolerance in rice seedlings. Supplements of the acids also enhanced accumulation and trafficking of Fe in the plants. Metal chelators facilitate uptake of metal ions in plant roots (Rauser, 1999). These compounds showed difference in chemical structure and metal ion binding affinity. Citrate [C3H5O(COO)33−] having three carboxylic group can bind with minimum three Cd2+ ions and malate [C2H4O(COO)22-] having two carboxylic group can bind with at least two Cd2+ ions. This difference caused more plant available Cd in the presence of citrate and resulted more Cd accumulation in plants. It also noticed that expression of rice FRD3-like (OsFRDL1), a
Fig. 3. FTIR spectra of the root (A) and leaves (B) (Arrows corresponds to the absorption peak of carboxylic acid), and In vitro study on competitive binding of Cd and Fe (25.0 µM) with citrate (C) and malate (D). The increase of absorbance indicates an increase in Fe – O Phenanthroline complex due to Cd mediated displacement of Fe from carboxylic group of organic acids. Error bar represent standard error (number of samples = 6).
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Fig. 4. Gene expression analysis in roots and leaves. Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Fig. 5. Changes in chlorophyll fluorescence (A), anthocyanin (B), total chlorophyll content (C), and carotenoids (D). Abbreviations: - Cd is the negative control without Cd treatment, and + Cd is the positive control with 25.0 µM CdCl2 treatment. Additions of the name of organic acids indicate supplements of respective acids at the rate 50.0 µM along with Cd treatment. Alphabets over the bar correspond to results of two-way analysis of variance. Error bar represent standard error. Letters a, b, c, and d represents first, second, third, and fourth level of statistically significant difference between means during Duncan's post hoc test (p < 0.05, number of samples = 6).
expression of this gene during both Cd treatment and malate supply indicated that vulnerability of OsNAS to Cd stress. The result also helped to confirm that nicotinamide mediated Fe transport not participates in malate dependant Cd tolerance. But the increase of carboxylic acids observed during FTIR analysis pointed out the occurrence of carboxylic group dependant Cd tolerance in leaves during malate supplement. Cadmium accumulation induced oxidative stress in plants (Verbruggen et al., 2009). Anthocyanin and glutathione (GSH) assist both localization of Cd into vacuole and detoxification of reactive oxygen species under Cd stress (Verbruggen et al., 2009). Hence it concluded that increase in both anthocyanin and GSH also played a role in the alleviation of Cd stress during organic acid supplements. It could be channeling of carboxylic acids to secondary metabolism or amino acid synthesis that increased the content of anthocyanin and GSH during organic acid supplements (Sweetlove and Fernie, 2005). Also, Cd triggered up-regulation of chalcone synthase and glutamate–cysteine ligase account for the increase of anthocyanin and GSH respectively (Dai et al., 2012; Verbruggen et al., 2009). Low-level expression of antioxidant enzymes, especially SOD, along with less thiobarbituric acid reactive substances content in the leaves points out enhanced oxidative stress tolerance during Cd exposure with organic acid supplements. The low-level expression of SOD in these plants was the result of unaltered photosynthetic electron transport which is known to cause photo-oxidative stress during Cd stress (Sebastian and Prasad, 2014b). It was the ambient availability of Fe which helped to develop photosynthetic machinery in the course of Cd plus organic acid treatments. Polyphasic fluorescence rise indicates the efficiency of photosynthetic energy transfer in plants (Stirbet and Govindjee, 2012). In the present
enhancement in chemical speciation of Cd with carboxylic acid in the course of organic acids treatments. These results indicate that sequestering of the metal organic acid complex in the vacuole of root cells decreased Cd transport to leaf. Additionally, exclusion of Cd from root played a role in the decrease of Cd translocation during malate supplements. The extent of Cd inducible Fe deficiency lowered with exogenous organic acids in the present study. Up-regulation of the iron-regulated transporter (OsIRT1), and an increase in ferric chelate reductase activity caused more accumulation of Fe3+ in the root during organic acid treatment (Kobayashi and Nishizawa, 2012; Sebastian and Prasad, 2015). Results of the in vitro studies indicated that Cd stabilizes Fe3+ organic acid complex formation which is critical for the translocation of Fe. Ferric ion formed a tridentate complex with a carboxylic group of tricarboxylic acid, and the complex transform to bidentate complex before degradation (Francis and Dodge, 1992). But the presence of Cd favored formation of both Cd and Fe3+ containing complexes where cadmium binds to the hydroxyl group of organic acids (Francis and Dodge, 1992). This complex is thermodynamically stable, and do not undergo random structural transformations (Francis and Dodge, 1992). Thus, it confirmed that the presence of Cd favored formation of stable Fe3+ - organic acid complex, and the metal complex efficiently transported to leaves via FRD3-like transporter (OsFRDL1) resulting more Fe accumulation in leaves (Yokosho et al., 2009). Also, ambient Fe circulation in leaves assured during organic acid supplement because expression of natural resistance-associated macrophage protein 1 (OsNramp1) sustained in these group of plants. Nicotianamine synthase (OsNAS) gene expression is critical for Fe reallocation and type II Fe acquisition respectively (Kobayashi and Nishizawa, 2012). Low-level 220
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study, Cd inducible fluorescence rise decreased during organic acids supplements. The difference was prominent in the J-I and I-P phases of the polyphasic fluorescence curve. Fluorescence rise in the O-J phase also observed during Cd treatment and Cd plus malate supplement. These results indicated alteration of electron transfer at the acceptor side of PSII i.e. reoxidation of QA was not efficient in these plants. The J-I phase was markedly visible among all experimental plants except those plants rear with citrate supplements, and this confirmed blockage of electron transport due to inadequate mobile plastoquinone pool (PQ) capable of electron transport (Stirbet and Govindjee, 2012). Blockages in the photosynthetic electron transport generate reactive oxygen species (ROS) (Stirbet and Govindjee, 2012). Therefore, it predicted that citrate supplement uphold PQ content which in turn result in normal functioning of photoelectron transport chain and decrease generation of reactive oxygen species. Decreased ROS production accounts for the low-level expression of superoxide dismutase (OsSOD) and catalase (OsCAT) after citrate supplement. The more expression of antioxidant enzymes observed among –ve control plants and malate supplimented plants could be the outcome of higher light capture or Fe accumulation related Fenton reactions (Moseley et al., 2002). A prominent I-P phase fluorescence rise also observed during Cd treatment. This result indicated that Cd stress affects electron transport at the PSI end too. Photosynthetic pigments and biomass are indicators of stress tolerance (Pavlović et al., 2014). Among photosynthetic pigments, chlorophyll is essential for light harvest whereas carotenoids help to dissipate excess light energy (Pavlović et al., 2014). Cadmium detoxification via Cd chelation, Fe nutrition, and antioxidant defense accounts for unaltered pigment synthesis during Cd treatment with organic acid supplement. Secondly, ambient availability of photosynthetic pigments helped to maintain light harvest efficiency critical for photosynthesis. Thus, plants reared with organic acids supplement had a higher photosynthetic efficiency which in turn helped to produce biomass under Cd stress.
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5. Conclusions Supplement of organic acid enhances Cd tolerance in rice seedlings. Rhizocomplexation of Cd and lowering of Cd inducible Fe deficiency are the critical factors involved in organic acid dependant Cd tolerance. Organic acid supply enhances carboxylic speciation of Cd which in turn favors vacuolar sequestration of Cd in the root along with the overexpression of OsHMA3. Thus, rhizocomplexation of Cd decreased translocation of Cd to leaf. Also, Cd exclusion assisted with OsCDT1 expression played an important role in lowering of Cd accumulation among malate supplemented plants. The formation of a stable Fe3+organic acid complex in the presence of Cd helped the efficient transport of Fe to aerial plant parts. Also, there exists unaltered Fe circulation in the leaves of organic acid supplemented plants because of the upholding of OsNramp1 activity. It also noticed that supplement of organic acids prevent Cd related retarding of chlorophyll synthesis as well as photosystems activity with the help of anthocyanin and GSH respectively. Thus, the maintenance of ambient Fe level and antioxidant activity in the leaf helped to maintain photosynthetic efficiency and biomass productivity in the course of the organic acid supplement under Cd stress. Radioactive tracer experiments and analysis of transcription factors will provide further insights into the kinetics of Cd accumulation and molecular switching involved in organic acid dependant Cd tolerance respectively. Acknowledgment Abin Sebastian gratefully acknowledges Senior research fellowship (SRF), University Grand Commission (UGC), India for financial support, and Dr. Neha Hebalkar, International Advanced Research Centre for Powder Metallurgy and New Materials, Hyderabad, for XPS analysis. 221
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Sebastian, A., Prasad, M.N.V., 2014b. Red and blue lights induced oxidative stress tolerance promotes cadmium rhizocomplexation in Oryza sativa. J. Photoch. Photobio. B 137, 135–143. Sebastian, A., Prasad, M.N.V., 2015. Iron- and manganese-assisted cadmium tolerance in Oryza sativa L.: lowering of rhizotoxicity next to functional photosynthesis. Planta 241 (6), 1519–1528. Stirbet, A., Govindjee, 2012. Chlorophyll a fluorescence induction: a personal perspective of the thermal phase, the J–I–P rise. Photosynth. Res. 113, 15–61. Song, J., Ma, R., Huang, W., Cui, X., 2014. Exogenous organic acids protect changbai larch (Larix Olgensis) seedlings against cadmium toxicity. Fresen. Environ. Bull. 23 (12C), 3460–3468.
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