RESEARCH ARTICLE
Effects of Exogenous Gibberellic Acid3 on Iron and Manganese Plaque Amounts and Iron and Manganese Uptake in Rice Yue Guo1, Changhua Zhu1, Lijun Gan1, Denny Ng2, Kai Xia1* 1 College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China, 2 CP Bio, Inc., 4802 Murrieta St., Chino, California, 91710, United States of America * [email protected]
Abstract
OPEN ACCESS Citation: Guo Y, Zhu C, Gan L, Ng D, Xia K (2015) Effects of Exogenous Gibberellic Acid3 on Iron and Manganese Plaque Amounts and Iron and Manganese Uptake in Rice. PLoS ONE 10(2): e0118177. doi:10.1371/journal.pone.0118177 Academic Editor: Wei Wang, Henan Agricultural Univerisity, CHINA Received: August 4, 2014 Accepted: January 5, 2015 Published: February 24, 2015 Copyright: © 2015 Xia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a grant from the National Natural Science Foundation of China (30871460). CP Bio, Inc, provided support in the form of a salary for author DN, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the Author Contributions section. Competing Interests: DN is an employee of CP Bio, Inc. There are no patents, products in development or
Gibberellins (GA) regulate various components of plant development. Iron and Mn plaque result from oxiding and hydroxiding Fe and Mn, respectively, on the roots of aquatic plant species such as rice (Oryza sativa L.). In this study, we found that exogenous gibberellic acid3 (GA3) spray decreased Fe plaque, but increased Mn plaque, with applications of Kimura B nutrient solution. Similar effects from GA3, leading to reduced Fe plaque and increased Mn plaque, were also found by scanning electron microscopy and energy dispersive X-ray spectrometric microanalysis. Reduced Fe plaque was observed after applying GA3 to the groups containing added Fe2+ (17 and 42 mg•L-1) and an increasing trend was detected in Mn plaques of the Mn2+ (34 and 84 mg•L-1) added treatments. In contrast, an inhibitor of GA3, uniconazole, reversed the effects of GA3. The uptake of Fe or Mn in rice plants was enhanced after GA3 application and Fe or Mn plaque production. Strong synergetic effects of GA3 application on Fe plaque production were detected. However, no synergetic effects on Mn plaque production were detected.
Introduction Mineral nutrients are chemical elements that plants obtain primarily from surrounding soil. They are needed for basic functions in plant metabolic, physiological, and developmental processes [1,2]. Plants depend on complex sensing and signaling mechanisms to detect external and internal concentrations of mineral nutrients [3]. Evolutionary changes in plants have included enhanced root growth [1,4,5], changes in expression and activity of ion transporters [6,7], and acidification of the surrounding soil to mobilize mineral nutrients [8]. Recent studies have identified plant hormones involved in regulation of mineral nutrient availability. Conversely, mineral nutrients influence hormone biosynthesis, suggesting a relationship between hormones and nutritional homeostasis. For instance, cytokinins and abscisic acid functioned in nitrate resupply experiments [3,9,10], ethylene acted in root hair regulation in response to low Fe supply [3], and auxin had bidirectional antagonistic effects with S deprivation signaling and upregulated K transporter accumulation [6,10–13]. GAs is essential plant hormones that
PLOS ONE | DOI:10.1371/journal.pone.0118177 February 24, 2015
1 / 18
GA3 on Iron and Manganese Plaque Changing and Fe/Mn Uptake
marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
affect nearly all aspects of higher plants growth and development [14,15]. There are also several investigations indicating that GAs is involved in the K [16, 17], P [4, 74] and Fe [1, 18, 75, 76] nutrition in plants. Fe is an essential microelement for several plant processes, particularly chlorophyll biosynthesis. Fe mainly exists as insoluble ferric ions in oxygen-rich soil and approximately neutral pH conditions and is usually in insufficient quantities for plants [18]. Thus, plants have developed two main Fe-uptake mechanisms categorized as strategy I and strategy II [19]. In most monocotyledons (strategy II), phytosiderophores (mugineic acids) are secreted into the soil to chelate with ferric ions through TOM1 [20,21]. The Fe—mugineic acid complexes are absorbed by root cells through YELLOW-STRIPE1 [22,23]. In most dicotyledons (strategy I), Fe is acquired from the soil by IRT1 (IRONREGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTION OXIDASE 2) localized in the root epidermis. Iron and Mn are oxidized and their oxide/hydroxide products, known as Fe plaque and Mn plaque, respectively, are precipitated on the root surface of aquatic plant species such as rice, Typha latifolia L., and Phragmites australis Trin. These plaque result from oxidizing Fe2+ and Mn2+ to Fe3+ and Mn3+, respectively [24,25]. In root plaque, Fe is the primary element and Mn is a secondary element. Iron and Mn usually co-exist, since the redox potentials of precipitating Fe oxides and hydroxides are lower than those of Mn oxides [26–35]. Iron and Mn plaque have diverse environmental and ecological functions in adapting to flooding and other environmental stresses. Plaque can act as a barrier to oxygen loss, which, in turn, enhances oxygen supply to root meristems [36] and affects the number of rhizosphere microorganisms [37]. Iron and Mn plaque have been shown to increase the uptake of toxic and nutrient elements [38–40]. The overall effect of Fe plaque on plant uptake of nutrients or harmful ions may depend on the amount of Fe plaque on the plant root surfaces [32,40]. The aim of the present study was to (1) investigate the effect of exogenous gibberellic acid3 (GA3) on Fe and Mn plaque, and (2) examine the response of Fe and Mn uptake to GA3 application and Fe and Mn plaque treatments.
Materials and Methods Plant materials and seedling growth The japonica rice variety Nanjing 44 was used in this study. Seeds were detoxified in 2% NaClO for 5 min and cleaned in distilled water. They were then soaked for 1 d in distilled water, followed by germination on nets. After growth at 30°C for 5 d, uniform seedlings were selected and transplanted to 300-mL pots (15 seedlings per pot) containing Kimura B nutrient solution (KB, modified from Kimura B macronutrients and Arnon micronutrients). This nutrient solution contained the macronutrients (mM): (NH4)2SO4 (0.18), MgSO47H2O (0.27), KNO3 (0.09), CaNO34H2O (0.18) and KH2PO4 (0.09), and the micronutrients (μM): Na2EDTA-Fe(II) (20), MnCl24H2O (9), H3BO3 (46), Na2MoO44H2O (9), ZnSO47H2O (0.7) and CuSO45H2O (0.3). Concentrations of Fe2+ and Mn2+ were 1.2 and 0.5 mgL-1, respectively. The pH of this solution was adjusted to 5.0 using 0.1 M HCl and 0.1 M KOH [41], and solution volume was restored daily and renewed every 3 d. The plants were cultured in a PGX-450C controlled environment growth chamber (Ningbo Sai Fu Instrument Co., Ltd., China) with a 14-h / 28°C day and 10-h / 22°C night regime, light intensity of 375 μm mol m-2s-1 and 65% relative humidity.
Application of plant hormones and their inhibitors Four-leaf seedlings were sprayed with 0.18 mM GA3, 0.12 mM uniconazole (S3307) and 100 mgL-1 sodium bisulfate (NaHSO3) aqueous solutions, respectively. Control plants were treated with distilled water. Gibberellic acid3 was purchased from Jiangxi Xinrunfeng Biochemical Co.,
PLOS ONE | DOI:10.1371/journal.pone.0118177 February 24, 2015
2 / 18
GA3 on Iron and Manganese Plaque Changing and Fe/Mn Uptake
Ltd (Ji’an, China), S3307 was purchased from Sichuan Academy of Chemical Industry Research and Design (Chengdu, China) and NaHSO3 was purchased from Sinopharm Chemical Reagent Co., Ltd. The Fe and Mn plaque dose-response design after hormone treatments. Four-leaf rice seedlings grown in KB (1.2 mgL-1 Fe2+ and 0.5 mgL-1 Mn2+, pH 5.0) were harvested at a GA3 concentration gradient (0.03, 0.06, 0.12, 0.18, and 0.24 mM) or its inhibitor, S3307 (0.04, 0.08, 0.12, 0.16, and 0.20 mM) 60 h after spraying. Treatments were compared with distilled-watertreated samples (0 mM). Fe and Mn plaque time-course design after hormone treatments. Four-leaf rice seedlings grown in KB (1.2 mgL-1 Fe2+ and 0.5 mgL-1 Mn2+, pH 5.0) were treated with 0 or 0.18 mM GA3 foliar spray for 96 h. Plants not treated with GA3 were the control.
Nutrient solution treatments Four-leaf seedlings were grown in KB solution and, prior to Fe or Mn plaque induction, all seedlings were placed in deionized water for 12 h to minimize interference from other elements. They were then transferred into 300-mL KB solution with 0, 17, and 42 mgL-1 of the Fe ion (Fe2+ as ferrous ammonium sulfate) or 0, 34, and 84 mgL-1 of the Mn ion (Mn2+ as manganese sulfate monohydrate) for 60 h after GA3 or S3307 spray. The same nutrient solutions were supplemented every day for a constant culture solution volume. Solution pH was adjusted to 5.0 with 0.1 M HCl or 0.1 M NaOH to promote root growth. Control plants were treated with distilled water and KB solution. Ferrous ammonium sulfate [(NH4)2Fe (SO4)26H2O] and manganese sulfate monohydrate (MnSO4H2O) were purchased from Shoude Apparatus Corporation (Nanjing, China).
Extraction and determination of Mn and Fe on the root surface Harvested plant material was divided into roots and shoots, and rinsed thoroughly in distilled water. Iron and Mn plaque deposited on the root surface were extracted using the dithionite-citratecarbonate (DCB) method of Taylor and Crowder [42] and McLaughlin et al. [43]. The fresh roots were incubated for 3 h at 25°C in 45 mL of a solution (pH 6.5) containing 0.27 M sodium citrate (Na3C6H5O72H2O) and 0.11 M sodium bicarbonate (NaHCO3), with the addition of 3.0-g sodium dithionite (Na2S2O4). Na2S2O4 is a strong reducer in NaHCO3 solution and can reduce Fe3+ to Fe2+, while citrate in Na3C6H5O72H2O can form a complex with Mn2+ and Fe2+. These compounds remove Fe and Mn plaque from root surfaces. After filtration, the extraction solution was analyzed for Mn and Fe content using an inductively coupled plasma optical emission spectrometer (ICP-OES) (Optima 2100, Pekin Elmer, USA) after an appropriate dilution, and roots were oven dried at 80°C for 3 d and weighed before digestion.
Scanning electron microscopy and energy dispersive X-ray spectrometric microanalysis Roots pre-treated with GA3 or distilled water (control) were harvested after 60 h and quickfrozen and freeze-dried. After applying a gold coating, root segment images and element ratio measurements were obtained using a scanning electron microscope (SEM) (model S-3000N; Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with an energydispersive X-ray detector (EDX) (Horiba Inc., Kyoto, Japan) as described by Chen et al. [41] and Xie et al. [44].
PLOS ONE | DOI:10.1371/journal.pone.0118177 February 24, 2015
3 / 18
GA3 on Iron and Manganese Plaque Changing and Fe/Mn Uptake
GA1/3 isolation and analysis by enzyme-linked immunosorbent assay (ELISA) Leaves of four-leaf rice seedlings were harvested at nine time points (0, 12, 24, 36, 48, 60, 72, and 96 h) after foliar application of exogenous GA3. The 0.5-g samples were weighed, frozen in liquid nitrogen, and maintained at—28°C before hormone extraction. GA1/3 extraction and purification prior to immunoassay were conducted according to previous reports [45–49]. The main steps were: extraction of homogenized samples at a rate of 5 ml g-1 fresh weight overnight at 4°C (with 10 mg L-1 butylated hydroxytoluene to prevent oxidation) in 80% cold (v/v) aqueous methanol. The supernatants were collected after centrifugation at 10,000 × g (4°C) for 20 min. The crude extract was passed through a C18 Sep-Pak cartridge (Waters, Milford, MA, USA), and the filtrate was collected. A 400-μl aliquot of the filtrate was removed and dried under N2. The extraction residues were dissolved in 200-μl phosphate-buffered saline (PBS) (0.01 M, pH 9.2), adjusted to pH 8.5, and separated three times with an equal volume of ethyl acetate. The aqueous phase of the remaining extract was adjusted to pH 2.5 and extracted three times with equal volumes of ethyl acetate. The ethyl acetate extracts were collected and dried under N2. Then, the residue was re-dissolved in 200-μl PBS (0.01 M, pH 7.4) to analyze GA1/3. GA1/3 levels were determined by ELISA based on a monoclonal antibody (provided by Nanjing Agricultural University, Jiangsu, Nanjing, China), as described previously [48,49].
Microscopy Whole roots of four-leaf rice seedlings were excised and photographed with a digital camera (FinePix S7000, Fujifilm, Tokyo, Japan) 60 h after a foliar application of 0.18 mM GA3.
Statistical analysis Means and standard errors were computed for three independent experiments, with at least three replicates per experiment. Treatment differences were detected using t-tests or Duncan’s multiple range tests in the SPSS 19.0 statistical software package.
Results GA3 affects Fe and Mn plaque contents in a dose/time-dependent manner We examined GA1/3 content after a GA3 foliar spray over 96 h to investigate the effects of a 0.18 mM GA3 application on endogenous biologically active GA. GA1/3 content increased significantly during the initial 48 h, followed by a sharp decline at 60 h (S1 Fig.). Then, GA1/3 content decreased slightly to its lowest level of 741.56 pmolg-1FW at 96 h. Thus, the 0.18 mM GA3 application in our experimental system strongly stimulated production of endogenous GA1/3, which corresponded to previous studies [50, 51]. Fe and Mn plaques form a reddish-brown deposition of iron oxide/hydroxide and amorphous colloidal substances on the root surfaces of aquatic plants [25]. In our experiment, rice plants were treated with exogenous GA3 foliar spray for 60 h, and the color of the rice root surface changed to light reddish-brown compared with the white roots of control plants (S1 Fig.). The root plaque image was quite consistent with previous reports [25]. However, we did not detect a difference in root length between the GA3 and control lines. Compared to the control, the application of exogenous 0.18mM GA3 resulted in a considerable decline (P
Effects of Exogenous Gibberellic Acid3 on Iron and Manganese Plaque Amounts and Iron and Manganese Uptake in Rice Yue Guo1, Changhua Zhu1, Lijun Gan1, Denny Ng2, Kai Xia1* 1 College of Life Sciences, Nanjing Agricultural University, Nanjing, 210095, China, 2 CP Bio, Inc., 4802 Murrieta St., Chino, California, 91710, United States of America * [email protected]
Abstract
OPEN ACCESS Citation: Guo Y, Zhu C, Gan L, Ng D, Xia K (2015) Effects of Exogenous Gibberellic Acid3 on Iron and Manganese Plaque Amounts and Iron and Manganese Uptake in Rice. PLoS ONE 10(2): e0118177. doi:10.1371/journal.pone.0118177 Academic Editor: Wei Wang, Henan Agricultural Univerisity, CHINA Received: August 4, 2014 Accepted: January 5, 2015 Published: February 24, 2015 Copyright: © 2015 Xia et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper and its Supporting Information files. Funding: This work was supported by a grant from the National Natural Science Foundation of China (30871460). CP Bio, Inc, provided support in the form of a salary for author DN, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of the authors are articulated in the Author Contributions section. Competing Interests: DN is an employee of CP Bio, Inc. There are no patents, products in development or
Gibberellins (GA) regulate various components of plant development. Iron and Mn plaque result from oxiding and hydroxiding Fe and Mn, respectively, on the roots of aquatic plant species such as rice (Oryza sativa L.). In this study, we found that exogenous gibberellic acid3 (GA3) spray decreased Fe plaque, but increased Mn plaque, with applications of Kimura B nutrient solution. Similar effects from GA3, leading to reduced Fe plaque and increased Mn plaque, were also found by scanning electron microscopy and energy dispersive X-ray spectrometric microanalysis. Reduced Fe plaque was observed after applying GA3 to the groups containing added Fe2+ (17 and 42 mg•L-1) and an increasing trend was detected in Mn plaques of the Mn2+ (34 and 84 mg•L-1) added treatments. In contrast, an inhibitor of GA3, uniconazole, reversed the effects of GA3. The uptake of Fe or Mn in rice plants was enhanced after GA3 application and Fe or Mn plaque production. Strong synergetic effects of GA3 application on Fe plaque production were detected. However, no synergetic effects on Mn plaque production were detected.
Introduction Mineral nutrients are chemical elements that plants obtain primarily from surrounding soil. They are needed for basic functions in plant metabolic, physiological, and developmental processes [1,2]. Plants depend on complex sensing and signaling mechanisms to detect external and internal concentrations of mineral nutrients [3]. Evolutionary changes in plants have included enhanced root growth [1,4,5], changes in expression and activity of ion transporters [6,7], and acidification of the surrounding soil to mobilize mineral nutrients [8]. Recent studies have identified plant hormones involved in regulation of mineral nutrient availability. Conversely, mineral nutrients influence hormone biosynthesis, suggesting a relationship between hormones and nutritional homeostasis. For instance, cytokinins and abscisic acid functioned in nitrate resupply experiments [3,9,10], ethylene acted in root hair regulation in response to low Fe supply [3], and auxin had bidirectional antagonistic effects with S deprivation signaling and upregulated K transporter accumulation [6,10–13]. GAs is essential plant hormones that
PLOS ONE | DOI:10.1371/journal.pone.0118177 February 24, 2015
1 / 18
GA3 on Iron and Manganese Plaque Changing and Fe/Mn Uptake
marketed products to declare. This does not alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
affect nearly all aspects of higher plants growth and development [14,15]. There are also several investigations indicating that GAs is involved in the K [16, 17], P [4, 74] and Fe [1, 18, 75, 76] nutrition in plants. Fe is an essential microelement for several plant processes, particularly chlorophyll biosynthesis. Fe mainly exists as insoluble ferric ions in oxygen-rich soil and approximately neutral pH conditions and is usually in insufficient quantities for plants [18]. Thus, plants have developed two main Fe-uptake mechanisms categorized as strategy I and strategy II [19]. In most monocotyledons (strategy II), phytosiderophores (mugineic acids) are secreted into the soil to chelate with ferric ions through TOM1 [20,21]. The Fe—mugineic acid complexes are absorbed by root cells through YELLOW-STRIPE1 [22,23]. In most dicotyledons (strategy I), Fe is acquired from the soil by IRT1 (IRONREGULATED TRANSPORTER 1) and FRO2 (FERRIC REDUCTION OXIDASE 2) localized in the root epidermis. Iron and Mn are oxidized and their oxide/hydroxide products, known as Fe plaque and Mn plaque, respectively, are precipitated on the root surface of aquatic plant species such as rice, Typha latifolia L., and Phragmites australis Trin. These plaque result from oxidizing Fe2+ and Mn2+ to Fe3+ and Mn3+, respectively [24,25]. In root plaque, Fe is the primary element and Mn is a secondary element. Iron and Mn usually co-exist, since the redox potentials of precipitating Fe oxides and hydroxides are lower than those of Mn oxides [26–35]. Iron and Mn plaque have diverse environmental and ecological functions in adapting to flooding and other environmental stresses. Plaque can act as a barrier to oxygen loss, which, in turn, enhances oxygen supply to root meristems [36] and affects the number of rhizosphere microorganisms [37]. Iron and Mn plaque have been shown to increase the uptake of toxic and nutrient elements [38–40]. The overall effect of Fe plaque on plant uptake of nutrients or harmful ions may depend on the amount of Fe plaque on the plant root surfaces [32,40]. The aim of the present study was to (1) investigate the effect of exogenous gibberellic acid3 (GA3) on Fe and Mn plaque, and (2) examine the response of Fe and Mn uptake to GA3 application and Fe and Mn plaque treatments.
Materials and Methods Plant materials and seedling growth The japonica rice variety Nanjing 44 was used in this study. Seeds were detoxified in 2% NaClO for 5 min and cleaned in distilled water. They were then soaked for 1 d in distilled water, followed by germination on nets. After growth at 30°C for 5 d, uniform seedlings were selected and transplanted to 300-mL pots (15 seedlings per pot) containing Kimura B nutrient solution (KB, modified from Kimura B macronutrients and Arnon micronutrients). This nutrient solution contained the macronutrients (mM): (NH4)2SO4 (0.18), MgSO47H2O (0.27), KNO3 (0.09), CaNO34H2O (0.18) and KH2PO4 (0.09), and the micronutrients (μM): Na2EDTA-Fe(II) (20), MnCl24H2O (9), H3BO3 (46), Na2MoO44H2O (9), ZnSO47H2O (0.7) and CuSO45H2O (0.3). Concentrations of Fe2+ and Mn2+ were 1.2 and 0.5 mgL-1, respectively. The pH of this solution was adjusted to 5.0 using 0.1 M HCl and 0.1 M KOH [41], and solution volume was restored daily and renewed every 3 d. The plants were cultured in a PGX-450C controlled environment growth chamber (Ningbo Sai Fu Instrument Co., Ltd., China) with a 14-h / 28°C day and 10-h / 22°C night regime, light intensity of 375 μm mol m-2s-1 and 65% relative humidity.
Application of plant hormones and their inhibitors Four-leaf seedlings were sprayed with 0.18 mM GA3, 0.12 mM uniconazole (S3307) and 100 mgL-1 sodium bisulfate (NaHSO3) aqueous solutions, respectively. Control plants were treated with distilled water. Gibberellic acid3 was purchased from Jiangxi Xinrunfeng Biochemical Co.,
PLOS ONE | DOI:10.1371/journal.pone.0118177 February 24, 2015
2 / 18
GA3 on Iron and Manganese Plaque Changing and Fe/Mn Uptake
Ltd (Ji’an, China), S3307 was purchased from Sichuan Academy of Chemical Industry Research and Design (Chengdu, China) and NaHSO3 was purchased from Sinopharm Chemical Reagent Co., Ltd. The Fe and Mn plaque dose-response design after hormone treatments. Four-leaf rice seedlings grown in KB (1.2 mgL-1 Fe2+ and 0.5 mgL-1 Mn2+, pH 5.0) were harvested at a GA3 concentration gradient (0.03, 0.06, 0.12, 0.18, and 0.24 mM) or its inhibitor, S3307 (0.04, 0.08, 0.12, 0.16, and 0.20 mM) 60 h after spraying. Treatments were compared with distilled-watertreated samples (0 mM). Fe and Mn plaque time-course design after hormone treatments. Four-leaf rice seedlings grown in KB (1.2 mgL-1 Fe2+ and 0.5 mgL-1 Mn2+, pH 5.0) were treated with 0 or 0.18 mM GA3 foliar spray for 96 h. Plants not treated with GA3 were the control.
Nutrient solution treatments Four-leaf seedlings were grown in KB solution and, prior to Fe or Mn plaque induction, all seedlings were placed in deionized water for 12 h to minimize interference from other elements. They were then transferred into 300-mL KB solution with 0, 17, and 42 mgL-1 of the Fe ion (Fe2+ as ferrous ammonium sulfate) or 0, 34, and 84 mgL-1 of the Mn ion (Mn2+ as manganese sulfate monohydrate) for 60 h after GA3 or S3307 spray. The same nutrient solutions were supplemented every day for a constant culture solution volume. Solution pH was adjusted to 5.0 with 0.1 M HCl or 0.1 M NaOH to promote root growth. Control plants were treated with distilled water and KB solution. Ferrous ammonium sulfate [(NH4)2Fe (SO4)26H2O] and manganese sulfate monohydrate (MnSO4H2O) were purchased from Shoude Apparatus Corporation (Nanjing, China).
Extraction and determination of Mn and Fe on the root surface Harvested plant material was divided into roots and shoots, and rinsed thoroughly in distilled water. Iron and Mn plaque deposited on the root surface were extracted using the dithionite-citratecarbonate (DCB) method of Taylor and Crowder [42] and McLaughlin et al. [43]. The fresh roots were incubated for 3 h at 25°C in 45 mL of a solution (pH 6.5) containing 0.27 M sodium citrate (Na3C6H5O72H2O) and 0.11 M sodium bicarbonate (NaHCO3), with the addition of 3.0-g sodium dithionite (Na2S2O4). Na2S2O4 is a strong reducer in NaHCO3 solution and can reduce Fe3+ to Fe2+, while citrate in Na3C6H5O72H2O can form a complex with Mn2+ and Fe2+. These compounds remove Fe and Mn plaque from root surfaces. After filtration, the extraction solution was analyzed for Mn and Fe content using an inductively coupled plasma optical emission spectrometer (ICP-OES) (Optima 2100, Pekin Elmer, USA) after an appropriate dilution, and roots were oven dried at 80°C for 3 d and weighed before digestion.
Scanning electron microscopy and energy dispersive X-ray spectrometric microanalysis Roots pre-treated with GA3 or distilled water (control) were harvested after 60 h and quickfrozen and freeze-dried. After applying a gold coating, root segment images and element ratio measurements were obtained using a scanning electron microscope (SEM) (model S-3000N; Hitachi High-Technologies Corporation, Tokyo, Japan) equipped with an energydispersive X-ray detector (EDX) (Horiba Inc., Kyoto, Japan) as described by Chen et al. [41] and Xie et al. [44].
PLOS ONE | DOI:10.1371/journal.pone.0118177 February 24, 2015
3 / 18
GA3 on Iron and Manganese Plaque Changing and Fe/Mn Uptake
GA1/3 isolation and analysis by enzyme-linked immunosorbent assay (ELISA) Leaves of four-leaf rice seedlings were harvested at nine time points (0, 12, 24, 36, 48, 60, 72, and 96 h) after foliar application of exogenous GA3. The 0.5-g samples were weighed, frozen in liquid nitrogen, and maintained at—28°C before hormone extraction. GA1/3 extraction and purification prior to immunoassay were conducted according to previous reports [45–49]. The main steps were: extraction of homogenized samples at a rate of 5 ml g-1 fresh weight overnight at 4°C (with 10 mg L-1 butylated hydroxytoluene to prevent oxidation) in 80% cold (v/v) aqueous methanol. The supernatants were collected after centrifugation at 10,000 × g (4°C) for 20 min. The crude extract was passed through a C18 Sep-Pak cartridge (Waters, Milford, MA, USA), and the filtrate was collected. A 400-μl aliquot of the filtrate was removed and dried under N2. The extraction residues were dissolved in 200-μl phosphate-buffered saline (PBS) (0.01 M, pH 9.2), adjusted to pH 8.5, and separated three times with an equal volume of ethyl acetate. The aqueous phase of the remaining extract was adjusted to pH 2.5 and extracted three times with equal volumes of ethyl acetate. The ethyl acetate extracts were collected and dried under N2. Then, the residue was re-dissolved in 200-μl PBS (0.01 M, pH 7.4) to analyze GA1/3. GA1/3 levels were determined by ELISA based on a monoclonal antibody (provided by Nanjing Agricultural University, Jiangsu, Nanjing, China), as described previously [48,49].
Microscopy Whole roots of four-leaf rice seedlings were excised and photographed with a digital camera (FinePix S7000, Fujifilm, Tokyo, Japan) 60 h after a foliar application of 0.18 mM GA3.
Statistical analysis Means and standard errors were computed for three independent experiments, with at least three replicates per experiment. Treatment differences were detected using t-tests or Duncan’s multiple range tests in the SPSS 19.0 statistical software package.
Results GA3 affects Fe and Mn plaque contents in a dose/time-dependent manner We examined GA1/3 content after a GA3 foliar spray over 96 h to investigate the effects of a 0.18 mM GA3 application on endogenous biologically active GA. GA1/3 content increased significantly during the initial 48 h, followed by a sharp decline at 60 h (S1 Fig.). Then, GA1/3 content decreased slightly to its lowest level of 741.56 pmolg-1FW at 96 h. Thus, the 0.18 mM GA3 application in our experimental system strongly stimulated production of endogenous GA1/3, which corresponded to previous studies [50, 51]. Fe and Mn plaques form a reddish-brown deposition of iron oxide/hydroxide and amorphous colloidal substances on the root surfaces of aquatic plants [25]. In our experiment, rice plants were treated with exogenous GA3 foliar spray for 60 h, and the color of the rice root surface changed to light reddish-brown compared with the white roots of control plants (S1 Fig.). The root plaque image was quite consistent with previous reports [25]. However, we did not detect a difference in root length between the GA3 and control lines. Compared to the control, the application of exogenous 0.18mM GA3 resulted in a considerable decline (P
