Introduction

Rice is an important cereal for human consumption, supplying most of the calories to a large portion of the world’s population. Various environmental factors can directly interfere with the productivity of this staple food, including the availability of nutrients. Both a deficiency and an excess of nutrients may equally result in drastic reductions in grain production.

Iron is insoluble and unavailable to most plants because it is mostly present in the soils as trivalent iron oxides (Guerinot and Yi 1994). Rice plants have developed strategies that allow absorption of iron in both divalent (Fe+2) and trivalent (Fe+3) forms (Bughio et al. 2002; Ishimaru et al. 2006). Fe+2 is soluble and readily available to plants, and it is absorbed by specific transporters (Eide et al. 1996; Yang et al. 2009; Ishimaru et al. 2010). However, Fe3+ ions must be reduced by Fe+3-chelate reductase (Robinson et al. 1999) or complexed to phytosiderophores released by the roots (Higuchi et al. 2001; Bashir et al. 2006; Inoue et al. 2009) to be absorbed. This dual character of rice in terms of iron uptake allows the cultivation of this crop in two different systems: under constant flooding or partial periods of water retention in the soil (lowland rice) or in the absence of flooding (upland rice) (Azambuja et al. 2004). In flooded conditions, owing to an anaerobic environment and low pH, iron, initially Fe3+, can be reduced to Fe2+, which, under excess conditions, can reach toxic levels (Bode et al. 1995a; Sahrawat 2005).

Iron toxicity is a complex disorder that can affect different physiological aspects of the plant. Iron excess inside the plant can lead to the enhancement of oxidative stress, as it increases the production of reactive oxygen species (Fang et al. 2001; Majerus et al. 2009), unleashing havoc on most metabolic processes, including photochemical or biochemical obstructions of photosynthesis, with a consequent reduction in the rate of carbon assimilation (Suh et al. 2002; Nenova 2009; Pereira et al. 2013). These biochemical and physiological changes, coupled with sequential nutritional disorders (Olaleye et al. 2001; Onaga et al. 2013) may lead to a loss of productivity (Audebert and Fofana 2009; Chérif et al. 2009; Sahrawat 2010). Also, anatomical alterations, such as the disruption of roots cells and cellular components, are described as results of iron toxicity and undoubtedly affect plant performance (Zhang et al. 2011).

Different mechanisms may confer to rice plants the ability to overcome stress due to iron excess. The formation of an iron plaque, as an exclusion mechanism in the roots of rice plants, can limit the absorption of minerals, such as iron itself (Snowden and Wheeler 1995; Deng et al. 2009). Protection strategies against iron toxicity within the plant may involve enzymatic mechanisms that include the actions of antioxidant enzymes, such as superoxide dismutase, catalase and ascorbate peroxidase (Majerus et al. 2007b; Saikia and Baruah 2012). Non-enzymatic mechanisms can neutralize oxygen reactive species through organic compounds, such as reduced glutathione, α-tocopherol, ascorbic acid and carotenoids (Smirnoff 2005). Besides these factors, the compartmentalization of organelles and/or specific proteins is an important step in the metabolism of minerals. Iron storage may occur in the apoplast, vacuoles and plastids, and within the central cavity of a specialized protein known as ferritin (Silveira et al. 2009; Stein et al. 2009a, b; Briat et al. 2010). This macromolecule acts in favor of iron homeostasis in the cells, as observed in rice plants exposed to excess amounts of this metal (Majerus et al. 2009).

In this work the differential responses and sensitivities to iron excess from three iron sources were studied in two rice cultivars. We assessed the effects of iron excess on tissue anatomy, photochemical reactions, absorption and accumulation of nutrients. Further, we investigated the expression of genes involved in iron absorption, transport and homeostasis.

Material and methods

Plant material, growth conditions and treatments

Rice (Oryza sativa L.) lowland cultivar BR-IRGA 409 (Cordeiro and Medeiros 2008), previously characterized as sensitive to iron excess (SOSBAI 2007), and Canastra (Soares et al. 1997), an upland cultivar which can be cultivated on lowland system, were used in this study. Seeds were sterilized with commercial sodium hypochlorite (10 %) for 10 min and germinated in rolled paper towel substrate wetted with distilled water in a growth chamber (27 ± 2 °C) for 14 days. For the first 7 days the seeds were in the dark, followed by 7 days of a 12-h photoperiod with a light intensity of 200–250 μmol m−2 s−1. Seedlings were then transferred to Hoagland nutrient solution (pH 4.0, full strength), in pots with 3.8 L, without intermittent aeration, under greenhouse conditions. Twenty seedlings per pot were transferred and thinning was performed after 7 days of acclimation leaving only eight seedlings per pot. The Hoagland’s nutrient solution used to acclimate the plants and for the control treatments was composed by 1 mM NH4H2PO4, 6 mM Ca(NO3)2•4H2O, 4 mM KNO3, 2 mM MgSO4•7H2O, 46 μM H3BO3, 9 μM MnCl2•4H2O, 0.78 μM ZnSO4•7H2O, 0.32 μM CuSO4•5H2O, 0.14 μM MoO3 and 18 μM FeSO4-EDTA. After approximately 30 day, plants at the V5 developmental stage (Counce et al. 2000) were exposed to excess amounts of ferrous, Fe+2, or ferric iron, Fe+3, with chelating agents, in a non-aerated hydroponic system. Iron treatments consisted of 7 mM iron sulfate, which was conjugated with ethylenediamine tetraacetic acid (EDTA) (w/w), 7 mM iron chloride and 7 mM iron citrate. These concentrations had been previously determined to be toxic to these cultivars (Pereira et al. 2013). A control treatment consisting of a nutritive solution with chelated iron sulfate at physiological concentrations (0.018 mM FeSO4-EDTA, 5 ppm) was conducted in parallel. Each treatment consisted of four replicates, with one pot per replicate. The pH (4.0) was adjusted every 2 days with NaOH or HCl, and the complete solution was renewed weekly. Measurements were performed after 7 days of exposure to excess iron, when the temperature was between 24 and 28 °C and the relative humidity between 53 and 66 % inside the greenhouse.

The iron concentration and speciation in the nutrient solution was calculated using the Visual Minteq software version 3.1 (Gustafsson 2014). The concentration of components of the modified Hoagland solution occupied for each treatment was used as input for the Visual Minteq. The Visual Minteq speciation results showed that the major iron species in each solution corresponded to what was expected for each treatment (Table 1). For the iron sulfate 88.9 % was in Fe2+ complexed to EDTA, for the iron chloride treatment more than 90 % was in the form of Fe3+ complexed to EDTA and for the iron citrate treatment 98 % was in the form of Fe3+ complexed to citrate.

Table 1 Iron chemical species in solutions with different sources of iron as predicted by the MinteqA2 software
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Variables analyzed

Mineral composition

The concentrations of iron and other nutrients were determined in shoots and roots, after 7 days of exposure to treatments. Prior to the quantification of the nutrient contents of the roots, samples of fresh materials were washed for 70 min in a dithionite-citrate-bicarbonate solution containing sodium dithionite (3 %, w/v), sodium citrate (0.3 M) and sodium bicarbonate (1.0 M) as described by Liu et al. (2008), to extract possible iron plaque deposits on the root’s surface, allowing for the analysis of merely symplastic iron. Then, roots and leaves were dried, ground, digested in concentrated nitric acid (118 °C for 4 h), diluted in water and analyzed by an inductively coupled plasma mass spectrometry (ICP-MS) according to Lahner et al. (2003). The mineral concentration (mg kg−1) was determined for each of the following elements: iron (Fe), magnesium (Mg), phosphorus (P), sulfur (S), potassium (K), calcium (Ca), boron (B), cobalt (Co), manganese (Mn), nickel (Ni), copper (Cu) and zinc (Zn). The allocation of iron assimilated in the different organs was evaluated according to Bao et al. (2009), as being bioconcentration factor or accumulation in the roots (BCF) and translocation factor (TF):

$$ \begin{array}{l}\mathrm{B}\mathrm{C}\mathrm{F}\left(\%\right)=\left[{\mathrm{Fe}}_{\mathrm{root}}/\left({\mathrm{Fe}}_{\mathrm{shoot}}+{\mathrm{Fe}}_{\mathrm{root}}\right)\right]*100\kern0.5em \mathrm{and}\hfill \\ {}\mathrm{T}\mathrm{F}\left(\%\right)=\left[{\mathrm{Fe}}_{\mathrm{shoot}}/\left({\mathrm{Fe}}_{\mathrm{shoot}}+{\mathrm{Fe}}_{\mathrm{root}}\right)\right]*100,\hfill \end{array} $$

where Feshoot and Feroot is the iron concentration (mg Kg−1) in shoots and roots, respectively.

Leaf anatomy characterization

The structural characterization of samples from the leaf blade subapical region (third expanded leaf) was carried out using optical microscopy. Leaf samples were fixed in Karnovsky’s solution (2.5 % glutaraldehyde and 4.0 % paraformaldehyde in sodium cacodylate buffer, pH 7.2, plus 5 mM CaCl2; Karnovsky 1965) for 48 h and stored in ethanol (70 %). Subsequently, they were dehydrated in an ethylic series and embedded in hydroxyethyl methacrylate resin (Leica Historesin, Nussloch/Heidelberg, Germany). Transverse sections (5 μM thick) were made using an automatic rotary microtome (model RM2155, Leica Microsystems Inc. Deerfield, IL, USA) stained with toluidine blue, pH 4.0 (O’Brien et al. 1964) and mounted in Permount. Observations and photographic documentation were performed using a photomicroscope (model AX70 TRF, Olympus Optical, Tokyo, Japan) equipped with photo-U system, coupled to a camera (model SPOT Insight color 3.2.0, Diagnostic Instruments Inc., Sterling Heights, MI, USA).

Malondialdehyde (MDA) measurement

The level of lipid peroxidation was assessed by measuring the MDA concentration (Hodges et al. 1999). MDA was extracted from approximately 160 mg of fresh leaves with 2.0 mL of trichloroacetic acid (TCA; 0.1 %). After centrifugation (10 000×g for 15 min at 4 °C), 1.5 mL of a mixture of thiobarbituric acid (TBA; 0.5 %) and TCA (20 %) solutions was added to 500 μL of the extract’s supernatant. A control for each sample was obtained without the addition of TBA (-TBA). The samples were incubated in a water bath (90 °C) for 20 min, and the reactions were stopped on ice. Absorbance values were read at 440, 532 and 600 nm. The MDA concentration was calculated from the differences in absorbance at 440, 532 and 600 nm using the extinction coefficient 157 mM−1 cm−1, as shown in the following equations:

$$ \begin{array}{l}\mathrm{A}=\left({\mathrm{Abs}}_{532+\mathrm{T}\mathrm{B}\mathrm{A}}-{\mathrm{Abs}}_{600+\mathrm{T}\mathrm{B}\mathrm{A}}\right)-\left({\mathrm{Abs}}_{532-\mathrm{T}\mathrm{B}\mathrm{A}}-{\mathrm{Abs}}_{600-\mathrm{T}\mathrm{B}\mathrm{A}}\right),\hfill \\ {}\mathrm{B}=\left({\mathrm{Abs}}_{400+\mathrm{T}\mathrm{B}\mathrm{A}}-{\mathrm{Abs}}_{600+\mathrm{T}\mathrm{B}\mathrm{A}}\right)\times 0.0571\kern0.5em \mathrm{and}\hfill \\ {}\mathrm{M}\mathrm{D}\mathrm{A}\left(\mathrm{nmol}\kern0.5em {\mathrm{mL}}^{-1}\right)=\left(\mathrm{A}-\mathrm{B}/157000\right)\times 106,\hfill \end{array} $$

where MDA values were expressed as relative to fresh matter.

Photochemical reactions

Images of chlorophyll fluorescence were obtained using a Modulated Imaging-PAM fluorometer (Heinz Walz, Effeltrich, Germany). The pixel value images of the fluorescence variables were displayed with the help of a color code ranging from black (0.000) to pink (1.000). For the measurements, leaves were initially dark-acclimated so that the reaction centers were fully opened to obtain the minimal (F 0) and maximal chlorophyll fluorescence (F m) values; from these, the maximal photosystem II (PSII) quantum yield was calculated (F v/F m = (F m−F 0)/F m) (Kitajima and Butler 1975). After sample illumination, saturation pulses were applied to determine the light-acclimated maximal fluorescence (F m’) and the steady-state fluorescence yield (F s). From these parameters, it was possible to calculate the quantum yield of photochemical energy conversion in PSII (YII = (F m’−F s)/F m’) according to Genty et al. (1989).

Gene expression

Total RNA was extracted from fresh leaf materials using homemade TRIzol reagent [38 % phenolic acid (pH 4.5), 0.8 M guanidine thiocyanate, 0.4 M ammonium thiocyanate, 0.1 M sodium acetate (pH 5.0), 5.0 % glycerol and diethylpyrocarbonate-treated water] (modified from Chomczynski and Sacchi 1987). After extraction, sodium chloride solution (150 μL, 5 N) and chloroform (450 μL) were added, and the mixture was centrifuged (16 000×g for 10 min at 4 °C). RNA was precipitated from the aqueous phase using an equal volume of isopropanol and washed in ethanol (75 %). Total RNA was quantified in a nanocell coupled to a spectrophotometer and analyzed by agarose gel electrophoresis (0.8 %, w/v) stained with GelRed (Biotium™). cDNA synthesis was performed with 2 μg of total RNA, pre-treated with DNAse (1 μL, 50 U μL−1, Amplification GradeDNase I, Invitrogen™) and incubated at 37 °C for 15 min. The first-strand cDNA was synthesized using the kit SuperScript™ First-Strand Synthesis System for RT-PCR (Invitrogen™), in a thermomixer, and stored at −20 °C.

Four biological replicates with three technical replicates were performed for the quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) experiment. Gene expression was analyzed using a StepOnePlus™ Real-Time PCR System (Applied Biosystems®, CA, USA), with a fluorescence detection system for SYBR Green I Mix (Applied Biosystems). The reaction was performed with first-strand cDNA solution (0.5 μL), primers (forward and reverse, 10 μM each), dNTPs (5 mM), 10X PCR buffer (Invitrogen), MgCl2 (50 mM), SYBR Green I (1:10000; Invitrogen), Platinum Taq DNA polymerase (5 U μL−1), ROX (Invitrogen) and sterile water. The amplification conditions were as follows: hot start at 95 °C, 40 cycles of 95 °C for denaturation, annealing temperature between 58 and 60 °C, depending on the primer Tm, and an extension temperature of 72 °C. Ct values were calculated from the values obtained from the fluorescence reactions using Real-time PCR Miner v 2.2 software (Zhao and Fernald 2005; http://www.miner.ewindup.info). The data were normalized to the following housekeeping genes selected from the rice genome using geNorm v. 3.5 software: actin (OsACT), β-1,3-glucanase (OsGLU), tubulin (OsTUB4) and ubiquitin_5 (OsUBQ5). The comparative algorithm proposed by Zhao and Fernald (2005) was used to compare RNA transcript abundance. The forward and reverse primer sequences are provided in Table 2.

Table 2 Oryza sativa genes analyzed and corresponding primers
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Statistical analysis

The experimental design consisted of randomized blocks with four replicates. Differences in MDA concentrations, gas exchange rates, chlorophyll fluorescence levels, mineral concentrations and gene expression were analyzed by ANOVA and Duncan’s test, with a 5 % level of significance, using SAS v 9.0 software.

Results

Differences in planta iron concentrations and translocation rates in response to divalent and trivalent iron sources

A higher iron concentration in the hydroponic solution resulted in higher iron concentrations in rice plants. However, different iron sources had different effects. Use of chelated iron sulfate, resulted in higher root iron concentrations in both cultivars, but higher values were found in the lowland rice cultivar (BR-IRGA 409). No differences in roots’ iron concentrations were observed between cultivars when iron chloride and iron citrate were supplied (Fig. 1a). A higher leaf iron concentration in the upland cultivar (Canastra) was observed when iron citrate was used (Fig. 1b and c).

Fig. 1
figure 1

Iron concentrations in roots (a) and in leaves (b) and iron uptake by roots (c, ) and iron translocation factor to shoots (c, ) of the Oryza sativa L. upland cultivar (Canastra) and lowland cultivar (BR-IRGA 409), after 7 days exposure to different sources of iron (7 mM) or to 0.018 mM iron sulfate (FeSO4-EDTA, control) in a hydroponic system. Data are presented as the means ± SE. Means followed by the same letter do not differ significantly from each other, as determined by Duncan’s test (p ≥ 0.05)

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The iron accumulation by the roots, and its translocation to the shoots, represents the allocation of iron assimilated by the different organs (Fig. 1a). Iron translocation was the lowest for both cultivars (25 % for upland and 17 % for lowland) when iron sulfate was applied. The highest iron translocation rate (62 %) was observed in the upland cultivar when the rice plants were cultivated in a solution containing iron citrate.

Toxic levels of iron result in morpho-anatomical leaf symptoms and oxidative damage

Visual symptoms of iron toxicity were observed after 3 days of exposure to iron sulfate or iron chloride in both cultivars. The most marked symptom was the bronzing, which initiated at the leaf apex and progressively expanded to the leaf blade mid-region (Fig. 2). After 7 days, both cultivars showed necrosis without visual symptoms of iron toxicity in treatment controls and iron citrate (Fig. 2). By light microscopy, necrotic lesions were more pronounced in leaf blade subapical regions, mainly on the leaf edges of plants receiving the iron sulfate treatment (Fig. 3b and d) for both cultivars. Iron toxicity was linked to the collapse of epidermal and mesophyll cells, resulting in a reduced leaf blade thickness (Fig. 3b and d). The leaves of control plants (both cultivars) presented normal tissues (Fig. 3a and c).

Fig. 2
figure 2

Oryza sativa L. leaves from the upland cultivar (Canastra) and lowland cultivar (BR-IRGA 409), after 7 days exposure to different sources of iron (7 mM) or to 0.018 mM iron sulfate (FeSO4-EDTA, control) in a hydroponic system

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Fig. 3
figure 3

Leaf blade of Oryza sativa L. upland cultivar, Canastra (a and b) and lowland cultivar, BR-IRGA 409 (c and d), in transverse sections (light microscopy). Control treatment (a and c). Treatment with iron excess (b and d), after culturing 7 days in a nutrient solution containing FeSO4-EDTA (7 mM). a and c Whole leaf border. b and d Necrotic leaf border. Legend: AdE adaxial leaf surface, AbE abaxial leaf surface, VB vascular bundle, BC bulliform cell, Me mesophyll, VE vessel element, and N necrotic tissue. Bars: 100 μm (a–d)

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Lipid oxidation in leaves was evaluated by quantifying MDA. The treatments with iron sulfate and iron chloride led to an average increase of 134 % (38.1 nmol mg−1 gFW; Fig. 4b) in the MDA concentrations of leaves from both upland and lowland cultivars compared with control plants. In roots, the highest MDA concentration was achieved in both cultivars when plants were treated with iron citrate (9.9 nmol mg−1 gFW; Fig. 4a).

Fig. 4
figure 4

Malondialdehyde (MDA) levels in roots (a) and leaves (b) of the Oryza sativa L. upland cultivar (Canastra) and lowland cultivar (BR-IRGA 409), after 7 days exposure to different sources of iron (7 mM) or to 0.018 mM iron sulfate (FeSO4-EDTA, control) in a hydroponic system. Data are presented as the means ± SE. Means followed by the same letter do not differ significantly from each other, as determined by Duncan’s test (p ≥ 0.05)

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High levels of iron affect photochemical reactions

Both rice cultivars showed a reduction in the YII in PSII under high iron levels. The reductions were more pronounced (up to 57 %) in the upland cultivar exposed to a high concentration of iron sulfate (Fig. 5c). Reductions were observed in the lowland cultivar only when excess iron sulfate (31 %) or iron chloride (41 %) was supplied (Fig. 5c). No changes were observed in F0 or Fv/Fm of either cultivar (Fig. 5a and b).

Fig. 5
figure 5

Minimal fluorescence (F0; a), maximal PSII quantum yield (Fv/Fm; b) and quantum yield of photochemical energy conversion in PSII (YII; c) of the Oryza sativa L. upland cultivar (Canastra) and lowland cultivar (BR-IRGA 409), after 7 days exposure to different sources of iron (7 mM) or to 0.018 mM iron sulfate (FeSO4-EDTA, control) in a hydroponic system. Data are presented as the means ± SE. Means followed by the same letter do not differ significantly from each other, as determined by Duncan’s test (p ≥ 0.05). The false color code depicted at the bottom of images ranges from 0.0 (black) to 1.0 (pink)

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Iron source impacts nutrient concentrations of rice plants although subordinate to the cultivar

Iron excess in hydroponic solution affected only leaf Ca and Mg concentrations and not their levels in roots (Fig. 6a and c). Ca concentrations in leaves were reduced, under iron sulfate and iron citrate treatments, by approximately 27 % in both cultivars (Fig. 6a). Leaf Mg levels were reduced by 26 % in both cultivars when iron sulfate was applied (Fig. 6c). The root concentration of K decreased at a high iron dosage in both cultivars; however, the decrease was more pronounced when iron sulfate was applied (Fig. 6b). No effects on leaf K concentrations were observed in any treatment (Fig. 6b). P concentrations were reduced (25 %) in roots of the lowland cultivar under iron chloride supply (Fig. 6d). However, in leaves, lower P concentrations (40 and 50 %) and Mn (43 and 54 %) were observed in the upland and lowland cultivars, respectively (Fig. 6d and g), with all iron sources. An incremental increase in Co concentrations was observed in both cultivars, reaching approximately 200 % in roots of plants exposed to ferric iron forms (iron chloride and iron citrate; Fig. 6e). In leaves, Co concentrations increased in both cultivars (Fig. 6e) in response to iron chloride (38 % in Canastra and 64 % in BR-IRGA 409) and iron citrate (around 125 % for both cultivars). Cu concentrations in roots increased 535 and 342 % (in plants from the upland and lowland cultivars, respectively, under iron chloride excess (Fig. 6f). In leaves, an increase of 67 %, on average, in the Cu concentration was observed in both cultivars exposed to iron chloride (Fig. 6f). The Zn concentration was marginally increased in roots (20 %), but only in the lowland cultivar grown under iron sulfate excess (Fig. 6h). However, iron citrate led to a decrease (18 %) in the Zn concentration in the roots of the same cultivar. In leaves, the Zn concentration increased (37 %) only in the upland cultivar when iron sulfate was supplied (Fig. 6h).

Fig. 6
figure 6

Mineral nutrient concentration (mg kg−1 DW) of roots and shoots of the Oryza sativa L. upland cultivar (Canastra) and lowland cultivar (BR-IRGA 409), after 7 days exposure to different sources of iron (7 mM) or to 0.018 mM iron sulfate (FeSO4-EDTA, control) in a hydroponic system. Data are presented as the means ± SE. Means followed by the same letter do not differ significantly from each other, as determined by Duncan’s test (p ≥ 0.05)

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Iron excess affects the relative expression of different genes

The relative expression of the genes that encodes ferritin (OsFER1 and OsFER2), protein responsible for the storage of iron, increased in the lowland cultivar in all treatments (up to 70-fold, Table 3). The primers used in this work matched for the two ferritin genes present in rice genome (Stein et al. 2009b). Expression of these same genes in the upland cultivar increased by 46-fold when plants were grown in iron sulfate, 30-fold when grown in the presence of iron chloride and 18-fold in the presence of iron citrate, compared with the respective control. Expression of OsPsbA, which encodes protein D1 in PSII, increased up to 2.6-fold in both cultivars when the plants were treated with iron sulfate and iron citrate (Table 3). Expression of the gene that encodes glutamate synthase (OsGS1), an enzyme related to photorespiration in plants, increased in the upland cultivar (3.8-fold) and lowland cultivar (2.6-fold) when treated with a high level of iron sulfate. Expression of the same gene increased by 5.5-fold in plants from the lowland cultivar exposed to excess iron chloride. The gene expression of transporters was differentially affected in rice plants grown in iron excess. The expression of nicotianamine synthetase, OsNAS2, necessary for the biosynthesis of phytosiderophores (PS), was higher in the control treatment than under the different iron excess treatments in the upland rice cultivar (Table 3). Expression of this gene was increased in the lowland cultivar treated with iron sulfate (2.6-fold), iron chloride (2.9-fold) and iron citrate (1.8-fold), compared with the control. The gene expression of Yellow Stripe-Like 1, OsYSL1, a transporter of Fe-PS, increased 3.4- and 2.1-fold in the upland cultivar grown in iron sulfate and iron citrate, respectively. The lowland cultivar had a 2.2-fold increase in OsYSL1 expression when the plants were treated with iron sulfate or iron chloride. The relative gene expression of the Phosphate-limitation I nducible Gene 1 (OsPI1), a phosphorus transporter, was increased 2.1- and 1.8-fold under the iron sulfate treatment in the lowland and upland cultivars, respectively (Table 2). There were changes in expression of the phosphate 1 limitation transcription factor OsPTF1, which is involved in tolerance to inorganic phosphate (Pi) starvation, only in the upland rice cultivar grown under iron sulfate excess.

Table 3 qRT-PCR analysis on mRNA relative level (fold) of the genes: ferritin (OsFER), protein chloroplastic Q-B (OsPsbA), cytosolic glutamine synthetase (OsGS1), nicotianamine synthetase (OsNAS2), Yellow Stripe Like 1 (OsYSL1), tonoplast monosaccharide transporter inducible by phosphate 1 limitation (OsPI1), inducible bHLH by phosphate 1 limitation transcription factor (OsPTF1), in leaves of a rice upland cultivar (Canastra) and a lowland cultivar (BR-IRGA 409), after 7 days of exposure to different sources of iron (7 mM) or to 0.019 mM (FeSO4-EDTA, control) in nutrient solution
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Discussion

Iron excess caused morphological, anatomical and physiological changes in plants of the upland and lowland rice cultivars. The effects were visually observed as the bronzing of leaves from the third day of treatment (Fig. 2). The bronzing is caused by an increase in polyphenol content, which is normally oxidized in leaves with high levels of iron above 300 mg kg−1 DW (Tanaka et al. 1966; Mehraban et al. 2008; Saikia and Baruah 2012), as observed in this study in all treatments (Fig. 1). Plants treated with iron-citrate did not show visual symptoms at the end of the treatment indicating that this complex can be better metabolized.

The high iron accumulation in roots of both rice cultivars grown with excess of iron sulfate (Fig. 1a) may be due to the maintenance of solubility and availability of iron in its divalent form (Fang et al. 2001; Sahrawat 2005). The addition of EDTA may have also favored the bioavailability by minimizing re-oxidation of iron and iron plaque formation (Taylor et al. 1984; Snowden and Wheeler 1995). Indeed, iron plaque formation was induced in Brazilian upland and lowland cultivars when iron sulfate was provided without the use of the chelating agent (Pereira et al. 2014). Unfortunately in this work we did not measured iron plaque formation.

Rice plants cultivated with iron chloride excess had lower levels of iron accumulation in roots than plants from the iron sulfate treatment (Fig. 1a). This may be because Fe+3 requires the reduction or complexation of the metal ions into phytosiderophores, as mugenic acid, to enable iron uptake (Bashir et al. 2006; Ishimaru et al. 2006). Conversely, Co and Cu showed an increase in rice plants exposed to trivalent iron form sources (Figs. 6e and 6f). The Fe+3-mugenic acid complex is structurally analogous to the Co+3 complex (Mino et al. 1983). A possible increase in the synthesis of mugineic acid and other phytosiderophores due to Fe+3 excess treatment could favor Co+3 uptake. In fact, an upregulation of OsNAS2 gene expression that codifies for an enzyme of phytosiderophore biosynthetic pathway was observed in the lowland rice cultivar treated with excess of iron chloride and iron citrate (Table 3). Previous studies had shown that the expression of genes related to phytosiderophore biosynthetic pathway remains constant or decreases in response to iron excess, in contrast to genes related to the transport of metal-phytosiderophore complexes that are upregulated which could be the case for the upland cultivar (Quinet et al. 2012). On the other hand, the copper (Cu) and Fe share the same plasma-membrane reductase system (Mukherjee et al. 2006), which can generate synergy in the absorption of Fe and Cu by plants grown with excess Fe+3. Regarding the sources of trivalent iron, however, the application of ferric chloride promoted a sharp increase in the copper content of the roots, which can be explained by ferric chloride solubilizes copper (Khandpur 2005), promoting its absorption.

The highest level of iron translocation to shoots of rice plants was observed when iron was provided in the iron-citrate complex (Fig. 1c). This iron form is immediately available because rice roots possess citrate transporters necessary for the translocation of iron-citrate directly to the shoots (Cataldo et al. 1988; Yokosho et al. 2009). However, it is interesting to note that despite the increased translocation of iron, this condition was less toxic than the others. The high iron accumulation associated with a high biomass production indicates a tissue tolerance mechanism (Elec et al. 2013). In other experiments using iron sulfate in similar concentrations but in the absence of chelators it was observed much higher shoot iron concentrations than in our conditions (Pereira et al. 2014; Wu et al. 2014). In our experiments EDTA was used as an iron chelator preventing its precipitation in the nutrient solution. Due to the differences in iron shoot concentrations observed, probably EDTA maintain iron in a more stable form in solution being more difficult for rice roots to mobilize and absorb the mineral. The lowest MDA concentrations and highest YII in PSII were observed under this condition (Figs. 4b and 5c). Some rice genotypes are able to accumulate high iron levels in leaves without showing leaf toxicity symptoms (Asch et al. 2005; Stein et al. 2009a; Engel et al. 2012). This may occur because the plants can oxidize the large amounts of iron translocated to shoots. This oxidation occurs after iron is released from chelator molecules in the apoplast, preventing its entry into the symplast, as a possible exclusion mechanism (Majerus et al. 2007a; Engel 2009). Alternatively, intracellular iron may be incorporated into organic compounds, such as the protein ferritin (Silveira et al. 2009). The increase in the expression level of OsFER was observed in both cultivars tested in this work, indicating that this biochemical mechanism for counteracting iron toxicity was not cultivar specific (Table 3). Although the primers used in this work matched for the two ferritin genes present in rice genome, the transcript increase observed is probably from OsFER2 that was previously reported to be responsive to iron excess (Stein et al. 2009b). The scavenging action of ferritin also plays an important role in plant defenses against oxidative stress induced by iron, preventing the metal’s reaction with oxygen (Majerus et al. 2009; Briat et al. 2010).

The accumulation of various nutrients in leaves was affected by excess iron in the solution. This was previously observed in other studies and the degree of mineral nutrient variation may depend on iron dosage, time of exposure, developmental stage and genotype (Dorlodot et al. 2005; Pereira et al. 2014; Silveira et al. 2007). However this is the first time that different types of iron sources are evaluated. We observed that accumulation of P and Mn was significantly reduced in rice plants, regardless of the sources of iron or the cultivar evaluated (Fig. 6d and g). The decrease in Mn levels in leaves when under iron excess may be linked to the antagonistic effects between these elements (Tanaka and Navasero 1966). Since Mn is required in PSII (Brudvig 1987), this nutrient deficiency can affect photosynthetic processes in rice plants. Consequently, the symptoms of Mn deficiency correspond to the symptoms of iron toxicity (Somers and Shive 1942). Decreases in P concentrations in leaves of rice grown under iron excess have also been reported, suggesting that there is a limitation in the uptake of P from the root apoplast to the symplast under this condition, which results in limited translocation to shoots (Silveira et al. 2007). In fact, large amounts of iron oxides that accumulate in the roots, as part of the iron root plaque, strongly interact with P, and the iron plaque on the rice roots can be considered a P reservoir (Zhang et al. 1999). The increase in OsPI1 and OsPTF1 expression, which are, respectively a P transporter and a transcription factor related to P starvation (Table 3), also indicates that excess iron is affecting P nutrition.

Iron excess in leaf tissues caused prominent morpho-anatomical changes in the rice leaves. The leaf bronzing observed in both cultivars studied is a classic symptom of iron toxicity (Engel et al. 2012; Santana et al. 2014). Cellular collapse due to injuries of the epidermal and mesophyll cells, and the reduction in the thickness of leaf blade (Fig. 3), were followed by foliar necrosis (Fig. 2). These symptoms, which are indicative of oxidative stress in leaves (Peng and Yamauchi 1993; Bode et al. 1995b; Engel et al. 2012), occurred markedly in plants treated with iron sulfate or iron chloride. The severe stress can cause a cellular imbalance in favor of pro-oxidants to the detriment of antioxidants, triggering oxidative stress, as confirmed by the high concentration of MDA in leaves of rice grown under an excess of iron sulfate or iron chloride (Fig. 4b). Thus, intrinsic antioxidant defense systems can also be important factors in the resistance to toxicity (Suh et al. 2002). The iron-citrate complex, supplied in one of the treatments, may have contributed to a lower availability of free iron to react with reactive oxygen species and to promote oxidative stress in both cultivars (Fig. 4b). Plants from the upland cultivar, when cultivated under flooded conditions, did not exhibit a potential exclusion mechanism for iron-citrate and allowed for greater iron accumulation in leaves (Fig. 1b). Despite the high iron concentration in leaves, MDA levels were low in these plants. These results suggest that mechanisms to prevent cellular damage as iron cellular compartmentalization were at play.

The YII in PSII in rice plants was also affected by iron excess in the nutrient solutions. YII is of crucial importance in plants and is highly sensitive to stresses. The upland cultivar showed a more drastic decrease in YII than the lowland cultivar, when exposed to iron sulfate (Fig. 5c). The reduction in YII may have been due to the oxidation of constituents of the photosynthetic complexes, such as psbA protein (D1) present in PSII. The greater increase in expression of the OsPsbA gene in the lowland cultivar (Table 3) suggests a possible protective mechanism of regeneration and repair, since the psbA protein is a critical component for the functionality of PSII (Bhagwat and Bhattacharjee 2005).

In addition to photochemical electron dissipation, other cellular processes have alternative actions in the dissipation of excess energy under stress conditions, including the water-water cycle (Asada 2000; Weng et al. 2007) or cyclic electron flow (Makino et al. 2002), the maintenance of electron transport in alternative ways and photorespiration (Wingler et al. 2000), which avoids the formation of reactive oxygen species once reducing power is consumed. The increase in expression of the OsGS1 gene has a key role in photorespiration (Oliveira et al. 2012) and may indicate a protective mechanism in both cultivars under iron stress. Gene expression of the cytosolic glutamine synthetase increased in both cultivars; however, it was more pronounced in the lowland cultivar (Table 3), which may indicate an attempt to avoid and/or minimize the more severe damage observed by the higher level of lipid peroxidation (Fig. 4) and YII reduction (Fig. 5c). Increased expression of OsGSl, which was less pronounced in the upland cultivar, may have been due to another mechanism for efficient energy dissipation in these plants. YII is also competitive with other processes that can act in non-photochemical dissipation and may occur to minimize the damage caused by iron, such as thermal energy dissipation by the xanthophyll cycle (Wilson et al. 2007; Pereira et al. 2013). Similar findings were also revealed by the imaging of the fluorescence parameters, indicating that the YII in PSII was the most susceptible parameter, especially under the iron sulfate treatment.

We showed in this work that the divalent iron source was more toxic to the two rice cultivars even though the iron source was translocated to a lesser degree in the plant. When the plants were treated with this iron source, the photosynthetic parameters decreased the most. Despite the toxicity to both cultivars, the levels of accumulation in roots and the translocation of iron were different among the sources of iron evaluated. Under cultivation with ferrous sulfate, the symplastic iron was mainly accumulated in the roots of rice plants. This can be considered an exclusion mechanism. The iron citrate however, was highly translocated to the shoots in the upland cultivar but still showed a lower toxicity compared with ferrous sulfate, which indicates an internal tolerance mechanism to iron excess in the shoots. The upland cultivar was more sensitive to excess iron under anaerobic conditions than the lowland cultivar. This study shows that even though ferrous sulfate showed a greater toxicity in both cultivars in relation to sources of trivalent iron, different cultivars may also present different mechanisms to deal with excess iron. This differential response of Brazilian lowland and upland rice cultivars to iron excess is currently being assessed in our laboratory.