Mechanisms associated with differential tolerance to Fe deficiency in okra (Abelmoschus esculentus Moench)

Accepted Manuscript Title: Mechanisms associated with differential tolerance to Fe deficiency in okra (Abelmoschus esculentus Moench) Author: Ahmad H. Kabir Mohammad M. Rahman Syed A. Haider Nishit K. Paul PII: DOI: Reference:

S0098-8472(14)00273-1 http://dx.doi.org/doi:10.1016/j.envexpbot.2014.11.011 EEB 2889

To appear in:

Environmental and Experimental Botany

Received date: Revised date: Accepted date:

20-7-2014 18-11-2014 21-11-2014

Please cite this article as: Kabir, Ahmad H., Rahman, Mohammad M., Haider, Syed A., Paul, Nishit K., Mechanisms associated with differential tolerance to Fe deficiency in okra (Abelmoschus esculentus Moench).Environmental and Experimental Botany http://dx.doi.org/10.1016/j.envexpbot.2014.11.011 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Mechanisms associated with differential tolerance to Fe deficiency in okra (Abelmoschus esculentus Moench)

Ahmad H. Kabir*[email protected], Mohammad M. Rahman, Syed A. Haider, Nishit K. Paul

Crop Physiology Laboratory, Department of Botany, University of Rajshahi, Rajshahi 6205, Bangladesh

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HIGHLIGHTS

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Phone: +880 1717134836

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 Morpho-physiological traits revealed that BARI-1 was tolerant to Fe deficiency.  Fe chelate reductase and proton extrusion in roots were greatly increased in BARI-1.

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 ZIP1 and IRT1 were greatly induced in roots under Fe deficiency in tolerant line.

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 BARI-1 was less affected by oxidative stress but showed enhanced scavenging activity.  Glutathione, cysteine, and citrate significantly increased in BARI-1 under Fe stress.

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ABSTRACT

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The study aimed at characterizing the physiological, biochemical and molecular mechanisms conferring differential tolerance to Fe deficiency in two contrasting Okra genotypes (BARI-1 and

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Orca Onamica). Fe deficiency caused greater decline in morpho-physiological traits in Orca

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Onamica compared to BARI-1, suggesting greater Fe-efficiency in BARI-1. Fe reductase, proton extrusion and phenol content in roots increased greatly in BARI-1 suggesting these responses

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contribute immensely to Fe-efficiency in this genotype. Semi-quantitative RT-PCR revealed the upregulation of ZIP1 (metal transporter) and IRT1 (iron-regulated transporter) genes in Fe-

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deficient roots of BARI-1 and Orca Onamica, albeit to a lesser extent. Also, BARI-1 was less affected by oxidative stress but showed enhanced scavenging activity that may stabilize

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antioxidant capacities and protect against oxidative damage under Fe deficiency. In addition, increased SOD and GR activities in BARI-1 suggest better capacity to detoxify ROS and to facilitate generating antioxidant metabolites, respectively. Furthermore, HPLC (Highperformance liquid chromatography) data showed increased glutathione, cysteine, citrate and ascorbic acid in roots of BARI-1 and to a lesser extent in Orca Onamica under Fe deficiency.

Elevated glutathione, ascorbic acid and cysteine suggest better protection of Okra from Fedeficiency induced oxidative stress. In addition, a significant increase in citrate and Fe-citrate was observed only in xylem sap of BARI-1 under Fe deficiency, indicating a role for citrate in long-distance transport of Fe. These findings provide first evidence on the mechanisms of differential Fe-efficiency in Okra. This will allow the improvement of Fe biofortification in Okra and other plants.

Keywords

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Okra, Fe-efficiency; Strategy I mechanisms; Fe transporters; antioxidant activity.

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Introduction

Okra (Abelmoschus esculentus L.) also known as lady’s finger, is a valuable vegetable plant.

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Okra contains proteins, carbohydrates and vitamin C (Dilruba et al., 2009), and plays a vital role in human diet (Saifullah and Rabbani, 2009). Iron (Fe)-deficiency is a common abiotic stress in

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many vegetable crops including Okra. This problem most commonly occurs in alkaline soil

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having high pH (Alcantara et al., 2002) or, less commonly, in soil with a high cation exchange

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capacity (Waters et al., 2002.). Common symptoms in Fe-stressed plants include chlorosis in

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young leaves, stunting in leaves and roots and reduced yields (Christin et al., 2009; Welch and Graham, 2004; Kabir et al., 2012). However, few plants cultivars have evolved generating

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mechanisms finely controlled to maintain Fe homeostasis and these mechanisms are different

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from species to species and genotypes to genotypes. To date, no investigation was performed on

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the differential tolerance to Fe deficiency operated in biochemical and molecular levels in Okra.

Mechanisms used by non-graminaceous monocot and dicots plants for Fe uptake are known as

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Strategy I (Curie and Briat, 2003). One of the characteristics of Strategy I plants is to reduce ferric Fe to ferrous Fe by means of a ferric chelate reductase (Walker and Connolly, 2008).

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Upregulation of the gene (FRO1) involved with FCR in a number of plants under Fe deficiency reveals that it is an obligatory step in Fe uptake as well as the primary factor in making Fe available for absorption (Waters et al. 2002; Kabir et al. 2012; (Walker and Connolly, 2008). Acidification of rhizosphere through the release of protons (H+) from an H+-ATPase pump in the root plasma membrane is believed to be another important mechanism operated in roots in

Strategy I plants (Kabir et al., 2012; Colangelo and Guerinot, 2004)). Furthermore, phenolic compounds secreted by root exudates do chelating, reducing and radical scavenging activity in response to Fe deficiency (Curie and Briat, 2003; Römheld and Maschner ,1986; Blum et al., 2000). Despite the involvement of these mechanisms with Fe-efficiency, no efforts were made to unravel these mechanisms in Okra.

Metal transporters assist in the movement of solubilised Fe through the root system. The most common metal transporters are ZIP1 (Zinc regulated transporter/iron regulated transporter) and

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IRT1 (Fe-regulated transporter) (Eide et al., 1996; Vert et al., 2001; Eckhardt et al., 2001). ZIP proteins generally contribute to metal ion homeostasis by transporting cations into the cytoplasm

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(Colangelo and Guerinot, 2006). ZIP1 is the key transporter for Zn uptake and translocation in plants, considerable progress has been achieved in cloning and characterizing its functions in

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crop plants, including soybean and rice (Xu et al., 2010; Lee et al., 2010). IRT1 is considered the main gene involved in Fe uptake and during Fe shortage (Cohen et al., 1998). Considering the

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interaction of Fe and Zn, it is therefore crucial to study the role of ZIP1 in Fe homeostasis. IRT1

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has been found to transport Fe in several plants including Arabidopsis (Eide et al., 1996; Vert et

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al., 2001), tomato (Eckhardt et al., 2001) and peas (Kabir et al., 2012). However, expression

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profile of these two transporters (ZIP1 and IRT1) is yet to be studied in Okra.

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The relationship between Fe deficiency and the possible onset of oxidative stress is becoming

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more evident, because of the dual role played by Fe in cell metabolism as either an antioxidant or a pro-oxidant factor. Fe is an integral constituent or cofactor of many antioxidant enzymes, such

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as catalase (CAT), peroxidase (POD), glutathione reductase (GR) and Fe-superoxide dismutase (Fe-SOD), but it may also act as a pro-oxidant (Minotti and Aust, 1987; Halliwell, 2006).

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Abiotic stresses induce oxidative stress in plants as a consequence of reactive oxygen species (ROS) production. Plants have developed different adaptive mechanisms to reduce oxidative

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damage resulting from altered Fe homeostasis through a cascade of antioxidative responses that stop the propagation of ROS-generating chain reactions (Jelali et al., 2013; Donnini et al., 2010). Fe deficiency has been shown to affect the expression and the activity of certain peroxidase isoenzymes and induces secondary oxidative stress in dicotyledonous species (Ranieri et al., 2001). SOD, converting O2 to H2O2, constitutes the first line of defense against ROS (Mittler

2002). However, antioxidant defense systems differ between tolerance and sensitive plant species/genotypes (Jelali et al., 2013).

Fe deficiency induces elevated amino acids (serine, glycine, citrulline, proline, lysine) and organic acids (citric acid, malic acid) in plants (Kabir et al., 2013; Rellan-Alvarez et al., 2010; Lopez-Millan et al., 2001). Another key compound shown to be part of the Fe-deficiency response in plants is glutathione (GSH). It plays multiple roles in cellular metabolism, including the scavenging of reactive oxygen species under Fe stress in plants (Zaharieva et al., 2003;

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Bashir et al. 2007, Nishizawa 2007). Plants generally promote Fe transport from roots to shoots mediated by different chelator through xylem (Kabir et al., 2013; Curie and Briat ,2003, Rellan-

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Alvarez et al., 2010). Elevated citrate and malate has been reported in the xylem under Fe deficiency in several plants (Kabir et al., 2013; Rellan-Alvarez et al., 2010; Lopez-Millan et al.,

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2000). Furthermore, increased Fe-citrate complex has been detected in tomato xylem exudates in Fe stress (Rellan- Alvarez et al., 2010). Other than Okra, plants metabolites have been shown to

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play vital role under Fe deficiency in peas (Kabir et al., 2013 Jelali et al., 2010), tomato (Lopez-

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Millan et al., 2009), soybean (Zocchi et al., 2007), etc.

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In a previous study, screening for Fe-efficiency was studied in a number of Okra genotypes based on morphological and physiological features (Rahman 2014). However, mechanisms

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conferring differential tolerance to Fe deficiency in Okra are yet to be studied. Therefore, this

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study was aimed at investigating the biochemical, molecular and metabolic analysis conferring

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Fe-deficiency tolerant in contrasting Okra genotypes (cv. BARI-1 and Orca Onamica).

2. Materials and methods

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2.1. Plant materials

Two cultivars of Abelmoschus esculentus (cv. BARI-1 and Orca Onamica), with different

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tolerance to Fe deficiency, were used in this study, the former being tolerant and latter sensitive.

2.2. Germination and growth conditions for hydroponic culture Before growing, seeds were surface sterilised in 70% ethanol and 5% sodium hypochlorite for one and 15 min, respectively. Seeds were then rinsed five times in deionised water. Seeds were

germinated on moist filter paper wetted with deionised water for three to four days in the dark at room temperature. Only healthy and uniform seedlings were transferred to solution culture. A basal nutrient solution (Hoagland and Arnon, 1950) was used with the following nutrient concentrations (µM): KNO3 (16000), Ca(NO3)2.4H2O (6000), NH4H2PO4 (4000), MgSO4.7H2O (2000), KCl (50), H3BO3 (25), Fe-EDTA (25), MnSO4. 4H2O (2), ZnSO4 (2), Na2MoO4.2H2O (0.5) and CuSO4.5H2O (0.5). Target pH value (pH 6.0) was obtained by titrating the basal solution with KOH or H2SO4. Plants were grown in 2 L of aerated solution and the environment was strictly maintained under 10 h light and 14 h dark (550–560 µmol s-1 per µA). There were 6

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plants per containers and each plant was transferred to container at the age of 4 days. Fe deficiency was induced by adding NaHCO3 (10 mM) to the treatment solutions to increase the

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pH up to 8.0 to initiate Fe deficiency as previously described (Kabir et al., 2013, Gharsalli et al.,

days. No NaHCO3 was added to the control solution.

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2.3. Preparation of glasshouse pots

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2001). However, no NaHCO3 was added to the control solution. Solution was replaced every 4

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Seeds of Okra were surface sterilized in 70% ethanol for 1min, followed by extensive washes in

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sterile distilled water. Seeds were then grown in small pots containing soil in the glasshouse. The

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soil contained all plant essential nutrients (30 mg/kg Fe and 5.8 pH). Fe deficiency was induced by mixing 1.5 g of CaCO3 with 1 kg soil followed by air drying (Kabir et al. 2013; Li-Xuan et al.

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2005). No CaCO3 was added to soil in the Fe-sufficient plants.

2.4. Measurement of morphological characters

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The number of fully expanded leaves and shoot length were counted on 2-week old plants grown on hydroponic culture. Total roots developed by each plant sample were washed in distilled

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water to remove nutrient and then quickly blotted in tissue paper before measuring the length and

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weight. There were three replicates for all these parameters measured.

2.5. Determination of chlorophyll content in leaves A chlorophyll concentration of young leaves (2-week old after treatment was imposed) was determined spectrophotometrically as

previously described

with some

modifications

(Lichtentaler and Wellburn, 1985) on hydroponically grown plants. Firstly, 0.1 g leaf was

weighted and placed in 95% acetone in a 5 mL falcon tube. The leaf sample was then grinded using mortar-pestle. The homogenate was filtered through whatman filter and was centrifuged at 2500 rpm for 10 min. The supernatant was separated and the absorbances were read at 662 nm (chlorophyll a) and 646 nm (chlorophyll b) on a spectrophotometer (UV-1650PC, Shimadzu). The amount of these pigments was calculated based on formula (Lichtentaler and Wellburn, 1985).

2.6. Determination of Fe concentration in leaves and roots

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Firstly, 1 g of leaf and root samples harvested from hydroponically grown plants (2-week old after treatment was imposed) was digested as previously described (Huang et al., 2004). Briefly,

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the dried leaf and root samples were predigested (overnight) sample and HNO3 mix is heated at 75°C for 10 min, followed by 109°C for 15 min. After cooling for 10 min, 1 mL of H2O2 was

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added to each vessel through the ventilation hole and the sample mix is heated at 109°C for a further 15 min. The samples were then analysed for Fe concentration by Flame Atomic

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Absorption Spectroscopy (AAS) outfitted with ASC-6100 auto sampler and air-acetylene

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atomization gas mixture system (Model No. AA-6800, Shimadzu). Standard solutions of Fe were

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separately prepared from their respective concentration of 1000 ppm stock solutions (Shimadzu),

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from which further serial dilutions (0.1-4 ppm) were made to cover the optimum absorbance range for the standard calibration curve. For the determination, two solutions were prepared for

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each sample. Reagent blank determinations were used to correct the instrument readings.

2.7. Fe chelate reductase assay

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Fe (III)-reductase activity was measured in excised roots using ferrozine [3-(2-pyridyl)-5, 6diphenyl-1,2, 4-triazine sulfonate] as previously described (Charalambos and Manolis, 2008) two

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weeks after treatment was imposed. Roots harvested from hydroponic culture of the two genotypes were rinsed in deionized water and root tips were then cut and placed in a beaker

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filled with ice water. About 0.1 g of root tissue was soaked in 1 mM EDTA for 5 min to eliminate apoplastic Fe and washed three times with distilled water to reduce excess EDTA. The roots were then transferred to 50 mL assay solution containing 0 .10 mM MES-NaOH (pH 5.5), 0.5 mM CaSO4, 100 mM Fe(III) EDTA, and 300 mM ferrozine. Samples and control tubes were incubated for 1 h in a shaking water bath at 14000 rpm at 23°C in the dark. After incubation, a 1

mL aliquot from each tube was transferred into a cuvette and the absorbance was read with a spectrophotometer (UV-1650PC, Shimadzu) at 562 nm wavelength. Reduced Fe [Fe(II)] was calculated with the use of an extinction coefficient of 25,200 M-1 cm-1. Control assays were also conducted to correct any unspecific chelation of ferrozine. The experiment was conducted three times to check if any wounding stress influences the results. 2.8. Determination of proton (H+) extrusion Extrusion of protons from roots was determined in plants as previously described (Kabir et al.

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2012) on 2-week old hydroponic plants. After germination, individual seedlings of the two genotypes were transplanted in small vials containing 50 mL of basal nutrient solution. NaHCO3

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(10 mM) was supplemented to the nutrient solution in order to increase the pH to 8.0, whereas no NaHCO3 was added in the control which was pH 6.0 (Kabir et al., 2012). The pH was measured

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by a digital pH meter and was maintained in subsequent days by addition of 0.1 M HCl or 0.1 M KOH. H+ efflux was estimated by measuring the titrated amount of acid or base to restore pH to

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its starting point. H+ extrusion was then calculated using the formula (Kabir et al., 2012).

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2.9. RNA isolation and semi-quantitative RT-PCR

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Expression of genes (ZIP1 and IRT1) was studied by semi-quantitative RT-PCR (reverse transcription PCR) in roots of BARI-1 and Orca Onamica harvested from 2-week old hydroponic

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plants. Around 100 mg of root tissues was ground to a fine powder in liquid nitrogen using a

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mortar and pestle. Total RNA was extracted according to the protocols given by total plant RNA isolation kit (cat. no. YTP100) manufactured by Real Biotech Corporation (RBC), Taiwan. The

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amount and purity of RNA in each sample was checked by a Spectrophotometer and RNA integrity was checked by denaturing agarose gel electrophoresis. RNA was used for first-strand

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cDNA synthesis using the FastQuant RT Ki (Cat no. KR103) supplied by Tiangen Biotech Corporation Limited, China. The primers were designed using Primer3 software and all primers

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were used to perform BLASTN searches with Arabidopsis genome database to confirm that they would specifically amplify the gene of interest. The first strand cDNA was then amplified using gene

specific

primers

(ZIP1-fw:

5’-GCATGCGGGTCATGTTCAC-3’,

ZIP1-rev:

5’-

CAACTCGGTCGAACCATGTG-3’; IRT1-fw: 5’- CGGTTGGACTTCTAAATGC-3’, IRT1rev: 5’- CGATAATCGACATTCCACCG-3’; 18S-fw: 5’-CGCTATTGGAGCTGGAATTACC-

3’, 18S-rev: 5’- AATCCCTTAACGAGGATCCATTG-3’). The PCR program used was as follows: 4 min at 95oC, 35 cycles of 30 sec at 95oC, 45 sec at 55oC, 1 min at 72oC, and 10 min at 72 oC.

2.10. Measurement of total phenol content Total phenol content in root harvested from 2-week old hydroponic plants was measured using Folin-Ciocalteu’s phenol reagent and gallic acid standard with some modifications (Kogure et al., 2004). Briefly, the calibration curve of gallic acid was prepared using different dilutions of

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stock solution, 250 μL Folin-Ciocalteu’s phenol reagents and 20% water solution of Na 2CO3 before being read the absorbance at 765 nm in spectrophotometer. The root extract (50 μL) was

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mixed with 250 μL of Folin-Ciocalteu’s phenol reagent and 20% water solution of Na2CO3. After incubation of the samples at room temperature for 30 min, their absorbance was measured

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at 765 nm. The total phenolics were measured using the gallic acid calibration curve and were

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expressed as mgL-1 Gallic acid g-1 extract (GAE).

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2.11. Enzymatic analysis

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Enzyme extraction for CAT, POD and SOD was performed in roots of hydroponic plants as

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previously described with slight modifications (Goud and Kachole, 2012) on 2-week old plants. Briefly, 0.1 g of root sample was ground in 5 mL of 100 mM phosphate buffer (KH2PO4). The

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homogenate was centrifuged for 10 min and the supernatant was used for the enzyme assay.

For CAT analysis, the reaction mixture in a total volume of 2 mL contained 100 mM potassium

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phosphate buffer (pH 7.0) mixed with 400 µL of 6% H2O2 and 100 µL root extract (Goud and Kachole, 2012). Root extract was the last component to be added and the decrease in absorbance

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was recorded at 240 nm (extinction coefficient of 0.036 mM−1 cm−1) using a UV spectrophotometer (UV-1650PC, Shimadzu) at 30s intervals up to 1 min. The specific activity of

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enzyme is expressed as µmol of H2O2 oxidized min −1 (mg protein)−1. For POD, the reaction mixture in a total volume of 2 µL contained 100 mM potassium phosphate buffer (pH 6.5) mixed with 1 µL of 0.05 M pyrogallol solution, 400 µL of 200 mM H2O2 and 100 µL root extract (Goud and Kachole, 2012). The change in absorbance was recorded at 430nm (extinction coefficient 12 mM–1cm–1) in a spectrophotometer from 30 sec up to 1.5 min. In the presence of

the hydrogen donor pyrogallol, POD converts H2O2 to H2O and O2. The specific activity of enzyme is expressed as µmol pyrogallol oxidized min−1(mg protein)−1. For SOD analysis, the assay mixture in a total volume of 1.5 mL contained 50 mM sodium carbonate/bicarbonate buffer (pH 9.8), 0.1 mM EDTA, 0.6 mM epinephrine and enzyme (Sun and Zigman, 1978). Epinephrine was the last component to be added. The adrenochrome formation in the next 4 min was recorded at 475 nm in a UV-Vis spectrophotometer. One unit of SOD activity is expressed as the amount of enzyme required to cause 50% inhibition of epinephrine oxidation under the

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experimental conditions.

The activity of GR was assayed as described by Goud and Kachole (2012) with slight

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modifications. The reaction mixture in a total volume of 2 mL contained 100 mM potassium phosphate buffer (pH 7.0), 50 µL of 0.2 mM NADPH, 100 µL of 0.5 mM oxidized glutathione,

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100 µL of 2 mM EDTA and 100 µL of root extract. Root extract was the last component to be added and the decrease in absorbance was recorded at 340 nm (extinction coefficient 6.2 mM-1

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cm-1) using a UV-Vis spectrophotometer at 10 sec intervals up to 1 min. The specific activity of

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enzyme is expressed as μmol NADPH oxidized min-1 (mg protein)-1.

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2.12. Determination of antioxidant capacity

The percentage of antioxidant activity (AA%) of each substance was assessed by DPPH (2,2-

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diphenyl-1-picrylhydrazyl) free radical assay. The measurement of the DPPH radical scavenging

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activity was performed according to a previously published method (Brand-Williams et al. 1995) in roots of hydroponic plants. The root samples were made to react with the stable DPPH radical

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in an ethanol solution. The reaction mixture consisted of adding 0.5 mL of sample, 3 mL of absolute ethanol and 0.3 mL of DPPH radical solution 0.5 mM in ethanol. When DPPH reacts

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with an antioxidant compound, which can donate hydrogen, it is reduced. The changes in color (from deep violet to light yellow) were read [Absorbance (Abs)] at 517 nm after 100 min of

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reaction using a spectrophotometer (UV-1650PC, Shimadzu). The mixture of ethanol (3.3 mL) and sample (0.5 mL) serve as blank. The control solution was prepared by mixing ethanol (3.5 mL) and DPPH radical solution (0.3 mL). The scavenging activity percentage (AA%) was then determined according to the formula previously given (Brand-Williams et al., 1995).

2.13. Analysis of plant metabolites by HPLC (High-performance liquid chromatography) Harvested roots from 2-week old hydroponic plants (1g) were grinded in mortar pestle using deionised water and were centrifuged at 12000 rpm for 10 min before storing the supernatant at 20 oC. Amino acids and organic acids in roots were then analysed by HPLC (Binary Gradient HPLC System, Waters Corporation, Milford, Massachusetts, USA) using Empower2 TM software. This comprised a Waters 515 HPLC pump and Waters In-line degasser AF. Compound separations were achieved with a C18 reverse phase-HPLC column (particle size: 5µm, pore size: 300 A, pH Range: 1.5-10, Dimension: 250 mm X 10 mm). Buffer A (water and 0.1% TFA)

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and buffer B (80% acetonitrile and 0.1% TFA) were used as mobile phase at the gradient of: 1-

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24 min 100% A, 25-34 min 100% B, 35-40 min 100% A.

Standard for each amino acid, organic acids and Fe-citrate were purchased from Sigma-

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Aldrich, Co., St. Louis and Carl Roth. All standard stock solutions (0.5 mM) were prepared in LC-MS grade water. In addition, samples were diluted 100 times in LC-MS grade water before

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injection. Both standards and samples were filtered using 0.22 µm Minisart Syringe Filters

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(Sartorius Stedim Biotech, Germany) before injection. Metabolites were detected with a Waters

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2489 dual absorbance detector (Waters Corporation, Milford, Massachusetts, USA) at 280 and

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360 nm. Peak identifications were achieved by comparing retention times and mass spectra of

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sample peaks with those of authentic standards.

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2.14. Collection of xylem sap

Xylem exudate was collected using the root pressure suction method on 2-week old soil grown

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plants (Kabir et al., 2013). Briefly, xylem exudate was obtained directly from greenhouse grown, intact Okra plants by cutting the stems 2 cm above the soil with a sharp razor blade. The exudate

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of the first 5 min was discarded to limit contamination. The surface was then washed with distilled water and blotted dry, and exudates were then collected every few minutes for 1 h using

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a micropipette. Exudate was transferred to a 1.5 mL Eppendorf tube upon collection and stored on ice until collection was finished. All exudate samples were stored at −80◦C until further analysis. Analysis of Fe-citrate and Citrate was performed by HPLC as previously described in Section 2.12.

2.15. Statistical analysis All experiments were done on complete randomized design having at least three replications for each sample. Statistical analyses and graphs were (two-tailed paired t-test) were performed using Genstat software (14th Edition) and Graphpad Prism 5 software, respectively. Statistical significance was set at P≤0.05.

3. Results 3.1. Morphological features

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Fe-deficient conditions did not significantly influence the root length and root fresh weight of BARI-1 plants. In contrast, a significant decrease was observed under Fe deficiency in Orca

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Onamica plants in the aforesaid root characteristics (Table 1). Similarly, shoot height, number of leaves and shoot fresh weight were not significantly decreased due to Fe deficiency in BARI-1;

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however, these parameters were significantly reduced in Orca Onamica under Fe deficiency

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(Table 1).

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3.2. Chlorophyll content in leaves

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Chlorophyll contents in leaves were determined 2 weeks after the imposition of Fe deficiency

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conditions. Total chlorophyll concentrations (a and b) were not significantly affected in BARI-1 under Fe deficiency compared to plants grown on Fe sufficient solution culture. In contrast, total

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chlorophyll concentrations (a and b) remarkably decreased due to Fe deficiency in Orca Onamica

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compared to controls (Fig. 1).

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3.3. Fe concentrations in leaves and roots AAS was used to determine Fe concentration in young leaves and roots of BARI-1 and Orca

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Onamica plants grown in Fe-sufficient and Fe-deficient conditions, with tissue taken 2 weeks after NaHCO3 treatment. Under Fe deficient conditions, Fe concentration in both leaves and

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roots of BARI-1 did not change significantly under Fe shortage compared to the plants grown in Fe-sufficient conditions (Fig. 2). In contrast, significant decline in Fe concentration was observed in both leaves and roots of Orca Onamica under Fe deficiency compared to controls (Fig. 2).

3.4. Ferric chelate reductase activity Ferric reductase activity was measured in roots of BARI-1 and Orca Onamica grown on Fe sufficient and Fe deficient conditions. In Fe sufficient conditions, Fe chelate reductase activity was similar in both BARI-1 and Orca Onamica. However, Fe chelate reductase activity significantly increased in BARI-1. In contrast, this activity was unchangeable in Orca Onamica under Fe deficiency compared to the controls (Fig. 3). 3.5. Proton (H+) extrusion from roots Extrusion of proton from roots was similar in both BARI-1 and Orca Onamica under Fe

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sufficient conditions. When plants were grown in Fe shortage, proton extrusion was remarkably increased in roots of BARI-1 and to a lesser extends in Orca Onamica compared to the plants

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grown on Fe sufficient conditions (Fig. 4).

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3.6. Expression of transporter genes

Semi-quantitative RT-PCR was performed in order to study the expression of ZIP1 and IRT1

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genes in roots of BARI-1and Orca Onamica grown under Fe-sufficient and Fe-deficient

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conditions. Expression of ZIP1 was highly induced by Fe shortage in roots of both BARI-1 and

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Orca Onamica. Furthermore, expression of IRT1 was also induced in roots of both genotypes due

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to Fe deficiency (Fig. 5). Importantly, expression of both genes was more pronounced in BARI-1

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than the Orca Onamica.

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3.7. Total phenolic contents in roots

Total phenolic contents were determined in roots of BARI-1 and Orca Onamica grown on both

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Fe-sufficient and Fe-deficient conditions. Phenolic contents in roots of both genotypes were significantly increased due to Fe deficiency compared to the plants grown on Fe sufficient

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hydroponic culture. However, increase of phenolic contents under Fe deficiency was greater in

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BARI-1 than that of Orca Onamica (Fig. 6).

3.8. Antioxidant enzyme activities in roots Changes in activities of antioxidant enzymes (CAT and POD) were studied in roots of BARI-1 and Orca Onamica. CAT activity was decreased both in BARI-1 (1.54-fold) and Orca Onamica (2.38-fold) due to Fe deficiency compared to Fe sufficient conditions; However, the decrease

was less in BARI-1 and not statistically significant compared to the controls. (Table 2). Similar results were also observed for POD activity where decrease of POD was observed in roots of both genotypes but not statistically significant only in BARI-1 (Table 2). SOD activity increased in roots in comparison to the control, irrespective of the cultivar though the increase was only significant in BARI-1 (1 (Table 2). Under Fe deficiency, the increase over control values was higher in the tolerant BARI-1 (2.04-fold) than in the sensitive Orca Onamica (1.11-fold). GR activity was enhanced in Fe-deficient roots of both cultivars, with a higher and statistical

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increase observed in the tolerant cultivar (1.95-fold with respect to the control).

3.9. Antioxidant capacity in roots

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DPPH free radical scavenging assay was used to evaluate antioxidant potential of BARI-1 and Orca Onamica under Fe-sufficient and Fe-deficient conditions. Under Fe-sufficient conditions,

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both BARI-1 and Orca Onamica showed similar scavenging effects in roots. However, when the plants were grown on Fe-deficient conditions, increased antioxidant activity was only observed

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in roots of BARI-1 (2-fold) whereas decreased antioxidant activity was recorded in Orca

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Onamica (Table 3).

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3.10. Comparison of metabolites between genotypes HPLC technique was employed to compare the 10 key different metabolites in roots of BARI-1

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and Orca Onamica under both Fe-sufficient and Fe-deficient conditions. Of these 10 metabolites,

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glutathione, cysteine, citrate and ascorbic acid were found to increase significantly due to Fe deficiency compared Fe sufficient controls in both genotypes; except for ascorbic acid in Orca

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Onamica. The increase of these compounds was greater in BARI-1 than Orca Onamica (Table 4). Due to Fe deficiency, oxalate was also increased in BARI-1 but decreased in Orca Onamica. No

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significant changes were observed for ascorbic acid, proline, glycine, methionine, malate and

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arginine in BARI-1 and Orca Onamica between Fe-sufficient and Fe-deficient conditions.

3.11. Analysis of xylem sap Changes of Fe-citrate and citrate in xylem exudate from BARI-1 and Orca Onamica under different Fe conditions were studied. Results showed that both BARI-1 and Orca Onamica had higher Fe-citrate and citrate due to Fe deficiency than controls though the increase was only

statistically significant in BARI-1 (Table 5). Fe-citrate and citrate increased up to 2.63-fold and 3.72-fold in BARI-1 due to Fe deficiency compared to the plants grown on Fe-sufficient conditions (Table 5).

4. Discussion Fe-efficiency is a trait of great importance in Okra given the prevalence of Fe-deficient conditions worldwide. Previous study explored the most tolerance (BARI-1) and most sensitive (Orca Onamica) genetic line to Fe deficiency in Okra (Rahman, 2014). However, mechanisms

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associated to the differential tolerance to the contrasting genotypes in Okra have not yet been studied. This study provides the novel insights of Fe-efficiency in biochemical, molecular and

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metabolic levels in Okra.

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4.1. Variations in morphology, chlorophyll and Fe concentrations in contrasting genotypes Fe deficiency caused marginal (statistically non-significant) decrease in root and shoots growth

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in BARI-1; whereas, Orca Onamica showed significant decline in root and shoot parameters

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under Fe deficiency compared to controls. These results were supported by observations of total

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chlorophyll concentrations (a and b) and Fe concentration in leaves and roots, which showed

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control plants to be healthy, but that bicarbonate treatment induced chlorosis and reduced Fe concentration in leaves and roots of Orca Onamica but not in BARI-1 (Fig. 1, Fig. 2). Fe

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deficiency-induced variations in physiological parameters have been previously reported in

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several species in several Strategy I plants (Lopez-Millan et al., 2001; Rombola et al., 2005; Kabir et al., 2012). Genotypic variations in Fe deficiency between BARI-1 and Orca Onamica

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were consistent with the previous findings (Rahman, 2014), and confirmed that an investigation of the basis of deficiency tolerance mechanisms was warranted. It further suggests that the

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genotype BARI-1 is able to contrast the negative effect of alkaline pH due to the addition of

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bicarbonate, while the Orca Onamica is not.

4.2. Biochemical mechanisms involved in Fe-efficiency To investigate the basis of differential tolerance to Fe deficiency between BARI-1 and Orca Onamica plants, we studied the Fe chelate reductase and proton extrusions in roots. Fe reductase activity through ferrozine in vitro method can be affected by wounding of root tissues. However,

we have not observed variations of results while repeated several times under same conditions. We observed that Fe chelate reductase activity occurred in both genotypes and showed similar patterns of activity under Fe sufficient conditions. However, induction of this activity was only induced in BARI-1 but not in Orca Onamica (Fig. 3) due to Fe deficiency. In case of proton extrusion, increase of this activity was more pronounced in root of BARI-1 than Orca Onamica due to Fe deficiency. Induction of Fe chelate reductase and proton extrusion activity under Fe deficiency has also been reported in several Strategy I plants (Robinson et al., 1999; Wu et al., 2011; Kabir et al., 2013; Wulandari et al., 2014). Being consistent with other Strategy I plants,

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our data suggest that the higher Fe-efficiency exhibited by BARI-1 plants could be attributable to the induction/increase of Fe chelate reductase activity and proton extrusion facilitating better Fe

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acquisition in roots.

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We also observed increase in total phenol contents in roots of both BARI-1 and Orca Onamica though the increase was more prominent in BARI-1. Phenolic compounds are the most

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frequently reported root exudates in response to Fe deficiency (Römheld and Maschner, 1986;

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Blum et al., 2000; Curie and Briat, 2003). The higher phenol contents in BARI-1 compared to

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Orca Onamica in Fe-limiting conditions suggests that BARI-1 is more efficient in utilizing

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phenolic compounds to function in Fe chelation and reduction, radical scavenging and Fe reutilization. It was also reported that Fe deficiency-induced secretion of phenolics by the roots

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of a dicot species improves plant Fe nutrition by enhancing reutilization of apoplastic Fe, thereby

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improving Fe nutrition in the shoot (Jin et al., 2007). This result gives further support to the

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notion that Fe deficiency-induced phenolics plays a key role in Fe-efficiency in Strategy I plants.

4.3. Expression of transporter genes involved with Fe-efficiency

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To investigate the molecular basis of differential Fe-efficiency between BARI-1 and Orca Onamica plants, we studied the expression of a couple transporter genes (ZIP1 and IRT1). There

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are no physiological methods available to study transporter activity, so our study of transporter activity focused at the level of gene expression. Our RT-PCR analysis showed that Okra ZIP1 gene encoding a metal transporter is upregulated in both genotypes under deficiency and has higher constitutive expression in roots of BARI-1 than Orca Onamica. To date, ZIP1 has been reported to be involved with Zn transport in several plant species (Durmaz et al., 2011; Grotz et

al., 1998). From our observation, we may conclude that the up-regulation of ZIP1 may be involved with Fe uptake into the plant. This is probably the first report on the higher expression of ZIP1 metal transporter due to Fe deficiency in plants.

Another Fe transporter, IRT1 (RIT1 homolog), had been previously shown to be upregulated in a number of Strategy 1 plants in response to Fe deficiency (Kabir et al., 2012; Henriques et al., 2002). IRT1 was expressed at higher levels in roots of BARI-1 plants than Orca Onamica plants under both Fe sufficient and Fe deficient conditions, and IRT1 expression increased in both

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genotypes in response to Fe deficiency; however, the expression was higher in BARI-1 than Orca Onamica. IRT1 is thought to function as broad specificity Fe transporter, which also

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facilitates the transport of Zn and heavy metal cations that accumulate in plants during Fe deprivation (Cohen et al., 1998). Thus, our data confirm that IRT1 has a role in Fe uptake in

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Okra, and that upregulation of IRT1 occurs in response to deficiency. In addition, our data suggest that the higher Fe-efficiency exhibited by BARI-1 plants could be attributable to higher

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expression of IRT1. Although IRT1 was originally identified as Fe transporter, it is now known

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that IRT1 is able to transport Zn along with Fe (Korshunova et al., 1999). Being consistent with

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our AAS data, it suggests that the higher Fe concentration in leaves of BARI-1 may attribute to

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the higher expression of IRT1 transporters in roots and thus pinpoint the possible correlation of

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Fe and Zn transport in Fe-efficiency in Okra plants.

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4.4. Involvement of antioxidant systems Decreased CAT activity was observed in roots of both BARI-1 and Orca Onamica due to Fe

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deficiency though the decrease was not significant in BARI-1. CAT is associated with selfprotecting many cellular components against detrimental effect of active oxygen species in

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plants. Similarly, another antioxidant enzyme, POD decreased in both genotypes due to Fe deficiency stress and decrease was less pronounced in BARI-1. These findings summarize the Fe

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deficiency causes oxidative stress in both BARI-1 and Orca Onamica; however, BARI-1 is less affect by the oxidative damage as evident by its lower decrease of CAT and POD activity under Fe deficiency. In contrast, Fe starvation may induce greater decreased capacity to CAT and POD detoxification with a consequent rise of oxidative cell status in Orca Onamica. Previously, the decrease activity of POD in maize indicated that it does not play essential roles in detoxifying

ROS (reactive oxygen species) under Fe deficiency (Sun et al., 2007). Furthermore, the decreased capacity to detoxify ROS may be the result of an unsuccessful activation and/or a reduced production of ubiquitous haem-containing POD enzyme (Ranieri et al., 2001). In parallel to these CAT and POD observations, we also studied the non-enzymatic antioxidant (DPPH radical) activity.

We also analysed the main enzyme SOD, which is involved in ROS metabolism. SOD catalyzes the conversion of the superoxide radical to molecular oxygen and H2O2, have previously been

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found to be involved in the response to Fe deficiency-induced oxidative stress (Ranieri et al. 1999; Jelali et al., 2013). In the present work, SOD activity was induced in Fe-deficient roots of

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both cultivars, but to a higher extent in tolerant BARI-1 under Fe deficiency, suggesting better efficiency in converting O2 to H2O2, thus ensuring this genotype from oxidative stress induced

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by Fe deficiency. The increment of SOD activity may account for the increased accumulation of superoxide radicals (O2-) in Fe-deficient leaves (Sun et al., 2007) and it is critical for protecting

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plants against oxidative damage (Molassiotis et al., 2006). Concerning GR activity, it increased

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particularly in BARI-1 grown under Fe deficiency. This fact suggests that the tolerant cultivar

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responds to Fe deficiency by activating not only the main antioxidant enzyme activities but also

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supported the by HPLC data.

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the ASC–GSH cycle, thus regenerating antioxidant metabolites (ASC, GSH), which are

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In this study, antioxidant activity (ROS scavenging) was only significantly enhanced in roots of Fe-efficient BARI-1 under Fe deficiency compared to controls. It suggests this genotype is

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efficient in self-protecting oxidative damage generated by Fe deficiency. Scavenging of ROS to restore redox metabolism, preservation of cellular turgor by restitution of osmotic balance, and

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associated protection and stabilization of proteins and cellular structures are among the multiple protective functions of compatible osmoprotectants during environmental stress (Mittler 2006;

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Yancey, 2005). In general, it can be inferred that in the tolerant genotype, BARI-1, Fe deficiency led to a notable increase in antioxidant activity as compared to the sensitive one, Orca Onamica. It turned out that the active involvement of ROS scavenging was related, at least in part, to the tolerance to Fe-deficiency-induced oxidative stress in Okra.

4.5. Changes of metabolite profiles due to Fe deficiency This study revealed that Fe deficiency caused major changes in some key metabolite profiles associated with differential tolerance to Fe deficiency in roots of BARI-1 and Orca Onamica. Therefore, the focus here is to explore which compounds underpin the differential Fe-deficiency tolerance in these two genotypes. Among the N-metabolites, no significant changes were observed in either genotype due to Fe deficiency. However, we observed marked increases in GSH and cysteine (S metabolites) in both genotypes due to Fe shortage. The greater increase in BARI-1 than that of Orca Onamica suggests that greater Fe-efficiency is associated with the

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elevated GSH and cysteine production in roots under Fe deficiency. One likely role for GHS is to work as an antioxidant compound to protect cells from Fe-deficiency-induced oxidative injury in

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Okra. Previous reports reveal that GHS protects plant cells from oxidative stress and confers Fedeficiency tolerance in leaves of peas (Kabir et al,. 2013), cucumber (Abadia et al., 2002) and

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beet (Zaharieva et al., 2003). Taken together, greater increase of GSH and cysteine in BARI-1 compared to Orca Onamica due to Fe deficiency suggests these compounds may possibly play a

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critical role in greater Fe-efficiency in Okra.

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Citrate, a TCA cycle metabolites had been reported to contribute higher Fe-efficiency in

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several plants (Kabir et al., 2013; Shi et al., 2002; Rellan-Alvarez et al., 2011). In our study, elevated citrate was found in roots of both Okra genotypes in Fe shortage. The increase may

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arise through anaplerotic carbon fixation in roots, prior to transport through xylem to shoots.

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This anaplerotic carbon fixation has been suggested to be crucial for short-term survival of Fedeficient plants (Lopez-Millan et al., 2000). The greater citrate level in BARI-1 than Orca

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Onamica under Fe deficiency may contribute to the enhanced Fe-efficiency of BARI-1 plants. In addition, elevated ascorbic acid in tolerant BARI-1 may confer Fe-efficiency by influencing

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ascorbate–glutathione cycle, thus regenerating antioxidant metabolites (ASC, GSH).

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In our study, changes of oxalate were quite different between BARI-1 and Orca Onamica

under Fe deficient conditions. Increase of oxalate in response to Fe deficiency has also been reported in Prunus (Jiménez et al., 2011) and Cucumber (El-Baz et al., 2004). However, the role of oxalate is still not known since its function is quite different from that of other organic acids in

plants. However, this result indicates that these genotypes have different level of oxalate that may account for their different Fe-efficiency.

4.6. Long-distance transport of Fe through xylem The finding that citrate and Fe-citrate levels increased in the xylem of BARI-1 under Fe deficiency confirms its consequence in the mechanism of Fe-deficiency tolerance. Likewise, the incapability of Orca Onamica to significantly increase these two compounds in the xylem as required by plants under Fe deficiency is likely to be a key factor leading to its low Fe-deficiency

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tolerance. Furthermore, elevated Fe-citrate in xylem in BARI-1 supports the hypothesis that long-distance transport of Fe from root to shoot is mediated by citrate in Okra under Fe

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deficiency. The role of citrate in the long-distance transport of Fe by binding to Fe in the roots and transporting it to the shoots in the xylem has been reported in a wide variety of strategy I

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plants (Ramirez-Rodriguez et al., 2001; Durrett et al., 2007; Kabir et al., 2013). Reports also revealed that citrate facilitates efficient movement of Fe from xylem into cells Green and Rogers

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(2004) and Fe–citrate enhances leaf Fe reductase prior to uptake into leaves (Bruggemann et al.,

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1993) in Arabidopsis mutants. Thus, it appears likely that citrate in Okra contributes to the

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5. Conclusion

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differential tolerance to Fe deficiency in Okra.

We investigated the contribution of a few Strategy I mechanisms, transporters, antioxidant

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capacities and metabolites leading to Fe-efficiency in two naturally occurring Okra cultivars, BARI-1 and Orca Onamica. The two cultivars exhibited marked differences in physiological

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growth and Fe content due to Fe deficiency with BARI-1 plants noticeably more tolerant to Fe deficiency than Orca Onamica. Greater Fe reductase, proton expression and phenolic secretion in BARI-1 indicated that these mechanisms are important contributors to Fe-efficiency in BARI-1 plants. Expression of two metal transporter genes (ZIP1 and IRT1) greatly induced in roots of BARI-1, suggesting the roles in Fe acquisition conferring the better Fe-efficiency in this

genotype (Fig. 7). In addition, less occurrence of oxidative stress and higher antioxidant capacities in roots of BARI-1 further implicate that this genotypes is efficient scavenging ROS generated by Fe stress. Furthermore, the results obtained on increased SOD in tolerant BARI-1 suggest better capacity to detoxify ROS. Fe shortage also resulted in stimulation of GR activities, part of the ASC-GSH pathway, which was higher in the tolerant cultivar than in the sensitive one.

HPLC results revealed that increased GSH and cysteine under Fe deficiency may function as a

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part of Fe-efficiency in BARI-1 by protecting leaves from oxidative damage. Analysis of xylem sap demonstrated the simultaneous increase of Fe-citrate and citrate only in Fe-efficiency BARI-

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1. It confirms that Fe is transported from root to shoot via xylem as a Fe-citrate complex in Okra (Fig. 7). This paper represents the first report towards understanding of Fe-efficiency in Okra.

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Findings from this study will allow more effective screens for Fe-efficiency in breeding or genetic transformation programs in Okra or other vegetables to alleviate Fe malnutrition in

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human.

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Acknowledgements

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The authors would like to thank Dr. Abu Reza of The Department of Genetic Engineering and Biotechnology, University of Rajshahi for providing assistance in HPLC analysis. We also thank

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Professor A.K.M. Rafiul Islam, Professor Monzur Hossain and Professor M. Firoz Alam for their

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support and encouragement throughout the work. We are also grateful to DNA Technology,

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Denmark, for providing primers on timely basis.

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Walker, E.L., Connolly, E.L., 2008. Time to pump iron: iron deficiency signaling mechanisms of higher plants. Curr. Opin. Plant Biol. 11, 530–535.

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Waters, B.M., Blevins, D.G., EIde, D.J., 2002. Characterization of FRO1, a pea ferric-chelate reductase involved in root iron acquisition. Plant Physiol. 129, 85–94.

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Welch, R.M., Graham, R.D., 2004. Breeding for micronutrients in staple food crops from a human nutrition perspective. J. Exp. Bot. 55, 353–364.

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Wu, T., Zhang, H.T., Wang, Y., Jia, W.S., Xu, X.F., Zhang, X.Z., Han, Z.H., 2011. Induction of root Fe(lll) reductase activity and proton extrusion by iron deficiency is mediated by auxin-based systemic signalling in Malus xiaojinensis. J. Exp Bot. 63(2), 859-70.

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Table 1 Root and shoot features in BARI-1 and Orca Onamica grown in Fe-sufficient and Fedeficient conditions. Values are the means of three independent replications with standard deviations. BARI-1

Orca Onamica

Fe (+)

Fe (-)

t-test

Fe (+)

Fe (-)

t-test

Root length (cm)

3.85±0.25

3.66±0.63

NS

4.73±1.56

2.36±0.28

*

Root fresh weight (g)

0.19±0.02

0.18±0.01

NS

0.19±0.01

0.14±0.01

*

Shoot height (cm)

8.50±1.80

8.00±1.00

NS

10.33±1.52

7.00±0.50

*

Shoot fresh weight (g)

0.41±0.01

0.36±0.02

NS

0.68±0.13

0.20±0.04

*

Number of leaves

5.00±1.00

4.33±0.57

NS

6.66±1.55

4.00±0.00

*

RI

* indicates significant (P<0.05)

PT

Parameters

SC

NS indicates non-significant (P>0.05)

Table 2 Changes in enzymatic activities in roots of BARI-1 and Orca Onamica grown in Fe-

U

sufficient and Fe-deficient hydroponic culture. The data are expressed as x-folds, where the

N

control (Fe sufficiency) is set to 1 and the treated (Fe deficiency) are x-fold (ratio) to that. Values

Genotypes

Fe (+)

CAT

D

min [(mg -1

TE

protein) ] POD

3.00±0.84 -1

EP

[(mg protein) ] 1

Fe (-)

3.05 ± 0.39 1.98 ± 0.39

-1

SOD

M

BARI-1

3.00±0.35

1.80±0.84

7.16±0.19

[U.mg- protein ]

CC

GR

[nmol.NADH.min

0.031±0.0002 0.065±0.002

-

-1

mg protein ]

A

1

A

are the means of three independent replications with standard deviations.

* indicates significant (P<0.05) NS indicates non-significant (P>0.05)

Orca Onamica

Fold (ratio) t-test 1.54

NS

Fe (+)

Fe (-)

Fold (ratio)

t-test

2.91± 0.19

1.22± 0.96

2.38

*

decrease

1.66

decrease

NS

2.40±0.69

1.03±0.84

decrease 2.04

increase

*

decrease *

5.52±0.03

6.15±0.84

increase 1.95

2.33

1.11

NS

increase *

0.051±0.000 0.056±0.0002

1.09 increase

NS

Table 3 Antioxidant activity in roots under Fe deficiency. The data are expressed as x-folds, where the control (Fe sufficiency) is set to 1 and the treated (Fe deficiency) are x-fold (ratio) to that. Values are the means of three independent replications with standard deviations. BARI-1

Orca Onamica

(x-fold±SD)

(x-fold±SD)

*

0.72±0.008

NS

PT

2.005 ±0.007

* indicates significant (P<0.05)

SC

RI

NS indicates non-significant (P>0.05)

Table 4 Changes in different metabolites in roots of BARI-1 and Orca Onamica due to Fe

U

deficiency. The data are expressed as x-folds, where the control (Fe sufficiency) is set to 1 and

N

the treated (Fe deficiency) are x-fold (ratio) to that. Values are the means of three independent

A

replications with standard deviations. (x-fold±SD)

D

Metabolites

M

BARI-1

Orca Onamica

t-test between

(x-fold±SD)

BARI-1 and Orca Onamica under Fe deficiency

25.3 ± 1.49

*

10.23 ± 1.58

*

*

Cysteine

1.63± 0.07

*

1.35± 0.89

*

*

Citrate

1.46± 0.09

*

1.35± 0.00

*

NS

Oxalate

1.76± 0.08

*

0.62± 0.14

*

*

Ascorbic acid

1.25± 0.07

*

0.88± 0.38

NS

NS

Proline

1.01± 0.43

NS

0.99± 0.56

NS

NS

Glycine

1.04± 0.16

NS

1.01± 0.32

NS

NS

Methionine

1.00± 0.20

NS

0.99±0.06

NS

NS

Malate

1.02± 0.85

NS

1.08± 0.07

NS

NS

Arginine

1.00± 0.21

NS

1.02± 0.16

NS

NS

A

CC

EP

TE

Glutathione

* indicates significant (P<0.05) NS indicates non-significant (P>0.05)

PT

Table 5 Changes in Fe-citrate and citrate in xylem sap of BARI-1 and Orca Onamica grown under Fe deficiency. The data are expressed as x-folds, where the control (Fe sufficiency) is set to 1 and the treated (Fe deficiency) are x-fold (ratio) to that. Values are the means of three independent replications with standard deviations. Orca Onamica

(x-fold±SD)

(x-fold±SD)

*

Citrate

3.72±0.50

*

A

CC

EP

TE

N

D

M

NS indicates non-significant (P>0.05)

A

* indicates significant (P<0.05)

1.08± 0.24

NS

1.16±0.14

NS

SC

2.63± 0.33

U

Fe-citrate

RI

BARI-1

Metabolites

PT

RI

Fig. 1. Chlorophyll content in young leaves of BARI-1 and Orca Onamica grown in Fe-sufficient and Fe-deficient hydroponic culture. Values are the means of three independent replications with

SC

standard deviations. Different letters indicate significant differences between means ± SD of treatments

CC

EP

TE

D

M

A

N

2 weeks after transplanting to solution culture.

U

(n = 3), comparisons were done for Fe (+) and Fe (-) conditions. Chlorophyll content was determined

Fig. 2. Fe content in young leaves and roots of BARI-1 and Orca Onamica grown in Fesufficient (Fe+) and Fe-deficient (Fe-) hydroponic culture. Values are the means of three

A

independent replications with standard deviations. Different letters indicate significant differences between means ± SD of treatments (n = 3), comparisons were done for Fe (+) and Fe (-) conditions.

Leaves were harvested 2 weeks after transplanting to solution culture.

PT

Fig. 3. Ferric chelate reductase activity in roots of BARI-1 and Orca Onamica grown in

RI

indirectly induced Fe-deficient hydroponic culture. Values are the means of three independent

SC

replicates with standard deviations. Different letters indicate significant differences between means ± SD of treatments (n = 3), comparisons were done for Fe (+) and Fe (-) conditions. Roots were harvested

CC

EP

TE

D

M

A

N

U

2 weeks after transplanting to solution culture.

Fig. 4. Proton extrusion in roots of BARI-1 and Orca Onamica grown in indirectly induced Fe

A

deficient hydroponic culture. Values are the means of three independent replicates with standard deviations. Different letters indicate significant differences between means ± SD of treatments (n = 3), comparisons were done for Fe (+) and Fe (-) conditions. Proton extrusion was estimated 2 weeks after

transplanting to solution culture.

Fig. 5. Semi-quantitative RT-PCR detection of ZIP1, IRT1 and 18S (control gene) expression in

PT

roots of BARI-1 and Orca Onamica grown in Fe-sufficient and Fe-deficient hydroponic

EP

TE

D

M

A

N

U

SC

RI

conditions. The roots were harvested from 2-week old plants.

CC

Fig. 6. Total phenolic contents in roots of BARI-1 and Orca Onamica grown in Fe-sufficient (Fe+) and Fe-deficient (Fe-) hydroponic culture. Data are means of three independent extractions

A

with standard deviations. Different letters indicate significant differences between means ± SD of treatments (n = 3), comparisons were done for Fe (+) and Fe (-) conditions.

PT RI SC U N

A

CC

EP

TE

D

M

A

Fig. 7. Mechanisms of Fe-efficiency in Okra.