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SILICON DECREASES DIMETHYLARSINIC ACID CONCENTRATION IN RICE GRAIN AND MITIGATES STRAIGHTHEAD DISORDER Matthew Alan Limmer, Patrick Wise, Gretchen E Dykes, and Angelia L. Seyfferth Environ. Sci. Technol., Just Accepted Manuscript • DOI: 10.1021/acs.est.8b00300 • Publication Date (Web): 02 Apr 2018 Downloaded from http://pubs.acs.org on April 3, 2018
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Environmental Science & Technology
SILICON DECREASES DIMETHYLARSINIC ACID CONCENTRATION IN RICE GRAIN AND
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MITIGATES STRAIGHTHEAD DISORDER
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Matthew Alan Limmer, Patrick Wise, Gretchen E. Dykes, Angelia L. Seyfferth*
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University of Delaware, Newark, DE, USA 19716
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Department of Plant & Soil Sciences
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*Correspondence: Angelia L. Seyfferth, (302) 831-4865,
[email protected] 8 9
ABSTRACT
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While root Si transporters play a role in the uptake of arsenite and organic As species dimethylarsinic
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acid (DMA) and monomethylarsonic acid (MMA) in rice (Oryza sativa L.), the impact of Si addition on the
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accumulation of DMA and MMA in reproductive tissues has not been directly evaluated, particularly in
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isolation from inorganic As species. Furthermore, DMA and MMA are suspected causal agents of
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straighthead disorder. We performed a hydroponic study to disentangle the impact of Si on
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accumulation of DMA and MMA in rice grain. At 5 µM, MMA was toxic to rice, regardless of Si addition,
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although Si significantly decreased root MMA concentrations. Plants dosed with 5 µM DMA grew well
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vegetatively but exhibited straighthead disorder at the lowest Si dose, and this DMA-induced yield loss
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reversed with increasing solution Si. Increasing Si also significantly decreased DMA concentrations in
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roots, straw, husk, and grain, particularly in mature plants. Si restricted grain DMA through competition
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for root uptake and downregulation of root Si transporters particularly at later stages of growth when Si
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uptake was greatest. Our finding that DMA causes straighthead disorder under low Si availability but not
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under high Si availability suggests Si as a straighthead management strategy.
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INTRODUCTION Arsenic in rice is a global food security concern, but different forms of As affect human toxicity
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and grain yield differently. Arsenic can be present in the porewater and plant as inorganic As (Asi) or
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organic As (Aso), with speciation ultimately affecting the overall toxicity of grain As.1 Inorganic As
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species, such as the trivalent arsenite and the pentavalent arsenate, are considered more toxic to
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humans than commonly encountered organic As species, such as pentavalent dimethylarsinic acid
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(DMA) and pentavalent monomethylarsonic acid (MMA). However, DMA and MMA are both phytotoxic
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and are thought to be causal agents of straighthead in rice,2, 3 a physiological disorder in which grains do
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not fill and the panicle remains erect, resulting in lower yields.4 Moreover, the contribution of DMA and
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MMA to plant As accumulation and effective mitigation strategies has been less well studied compared
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to inorganic As species.
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A growing body of research over the last decade has revealed that the predominant Asi species,
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arsenite, is taken up by rice roots via root silicic acid transporters Lsi1 and Lsi2,5 and one study6 has
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shown that Si transporters also may be involved in MMA and DMA root-uptake in rice. In a short-term
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study, Li et al.6 revealed that the root silicic acid transporter Lsi1, but not Lsi2, plays a role in root-uptake
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of DMA and MMA.6 In Lsi1 knock-out plants, MMA and DMA were 90% and 50% lower, respectively, in
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the xylem sap compared to the wild type after 24 hours.6 Once inside the plant, both of these organic
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arsenic species are readily translocated to grain,7, 8 despite lower root uptake efficiency than arsenite.9
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Thus, even though MMA and DMA are taken up by roots more slowly than arsenite, they may comprise
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a substantial fraction of grain As. DMA is routinely found in rice grains at concentrations of ~0.1 mg/kg
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throughout the world, with DMA becoming the predominant form of As in rice grain as grain total As
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concentration increases.10 Noteworthy, the soil conditions that correlate to higher grain DMA also
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correlate to observations of straighthead disorder. Arsenic-induced straighthead has been found
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throughout the world and most commonly occurs in fields where flooded conditions predominate, soil
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arsenic and organic matter contents are high, and fields have a history of organic arsenical herbicide
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use.4, 11-13 Rice does not have the ability to methylate As in planta, 14, 15 so DMA and MMA arise from
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microbial methylation of arsenite under the reducing conditions that develop under flooded paddy
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culture.15 While microbes can add as many as three methyl groups to Asi, DMA often dominates the Aso
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pool in porewater.13, 16 Thus in typical flooded paddy porewater, As species exist as a mixture of Asi and
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Aso, making it challenging to use soil-based experiments to disentangle the role each As species plays in
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grain accumulation and straighthead disorder, as well as the role of mitigation strategies.
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Environmental Science & Technology
The addition of Si to decrease As uptake by rice is an emerging and effective technique that
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relies on rice being an efficient Si accumulator,17 competition between Si and arsenite for uptake and/or
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downregulation of Si transporters,13, 18-21 but the role of Si addition on MMA and DMA accumulation in
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grain is unresolved. To date, only one study has directly investigated the impact of Si addition on uptake
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of MMA and DMA: after 30 minutes exposure, 0.5 mM Si did not affect rice uptake of MMA or DMA.6
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However, the impact of Si addition on uptake of DMA and MMA over longer timescales and,
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importantly, on grain accumulation has not been directly investigated in isolation from other (i.e.,
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inorganic) As species. Soil experiments have shown that Si addition can increase,19, 22-24 decrease,13 or do
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not affect19, 25 grain concentrations of DMA. These soil-based experiments differed in their
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concentrations of total soil As, extent of soil flooding, as well as sources of Si, all of which likely affected
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the extent of microbially-mediated methylation and therefore the balance of Asi and Aso species,
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thereby convoluting the impact of Si on MMA and DMA accumulation into rice.
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Here, we aimed to disentangle the impact of MMA and DMA on grain yield and the impact of Si
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addition on rice accumulation of MMA and DMA. We utilized a hydroponic approach where Si
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concentration, Aso concentration, and As speciation could be isolated and controlled, and we grew rice
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to both vegetative and mature states. Because MMA and DMA are transported into the root by Lsi1,6 we
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hypothesized that increasing Si concentrations would decrease rice uptake and accumulation of MMA
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and DMA via uptake competition and/or downregulation of Lsi1 transporters, but that the magnitude of
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this effect would change throughout the life of the plant. Our experiment allowed us to directly
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investigate the role of DMA and MMA on grain yield and plant toxicity and how these might be
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alleviated by Si addition. Thus, we measured grain yield, leaf chlorophyll, and nutrient contents for each
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treatment. Our data show that the uptake and induced toxicity of MMA and DMA in rice is alleviated by
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increasing Si due to decreased Lsi transporter expression, but the extent of toxicity and Si impacts varied
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with As species and duration of exposure to As and Si treatments.
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MATERIALS AND METHODS
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Experimental Design
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The experiment was performed using a completely randomized design with three factors. The
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first factor consisted of 3 As treatments: 5 µM DMA (as cacodylic acid, >99%, Amresco, Solon, OH, USA),
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5 µM MMA (as disodium methyl arsonate, 98.4%, Chem Service, West Chester, PA, USA) or no arsenic
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(control). As-treated plants received As as DMA or MMA throughout the entirety of the experiment after
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dosing commenced. The second factor, Si concentration, included nominal levels of 50, 250, 500, 750, 3 ACS Paragon Plus Environment
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1500 µM silicic acid (ACS grade >99.9%, Fisher Scientific). Actual As and Si concentrations were
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measured at the beginning and end of each solution change and the median Si concentration value for
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each individual was used in the statistical analyses. Arsenic concentrations in porewater remained near
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5 µM (5.22 ± 0.33 µM, n=50) for both DMA and MMA-dosed plants (Figure S1) and speciation in
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porewater and shoots was not altered throughout the entire experiment. The third factor was plant
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maturity, where plants were harvested either during the vegetative growth phase (55 days after
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germination) or grown to maturity (132 days after germination). Each treatment combination was
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performed in triplicate, giving 90 individual plants and containers.
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Experimental Conditions
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Long-grain rice (Oryza sativa L. cv. Lemont) was germinated from seeds surface-sterilized with a
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dilute bleach solution. The plants were grown in either 4-L (vegetative harvest) or 8-L (mature harvest)
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acid-washed and opaque HDPE containers. Dosing with As began 27 days after germination (10 days
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after transplanting to experimental container) and continued throughout the experiment. The plants
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were grown in hydroponic media (Table S1) which was exchanged every 4-8 days with more frequent
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changes as plants grew larger. Solution pH was measured prior to changing from representative
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containers during the experiment and remained 6.4 ± 0.2.
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The rice plants were grown in a growth chamber to resemble typical field conditions. The plants
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were grown under fluorescent light and the plant base received 100-175 µmol/m2/s of light intensity
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over a 12-hr photoperiod. Daytime and nighttime temperatures were held at 28 °C and 26 °C,
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respectively. Relative humidity was maintained at 70%. Plant location was randomized during each
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solution change.
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Chlorophyll, Yield Measurements, and Harvest
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Leaf chlorophyll was measured using a hand-held meter (CCM-200 plus, Apogee Instruments,
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Inc.) at three time-points during reproductive growth. Each chlorophyll measurement was an average of
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values obtained from three separate leaves from different tillers. During harvest of plants grown to
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maturity, several yield metrics were collected including total number of tillers, number of panicles and
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plant height. Plants were separated into roots, straw, husk and grain (unpolished). Half (longitudinally)
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of each plant’s roots and straw were flash-frozen in liquid nitrogen for transporter assays (described
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later) and the remaining half was oven-dried overnight at 70 °C to assess dry weight and for elemental
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analyses (described later). Panicles were air-dried at 20 °C for one week. Grains were removed from the
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panicles and counted using GrainScan26 and an HP Scanjet 4890 flatbed scanner operating at 300 dpi. 4 ACS Paragon Plus Environment
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The grains were described in the RGB color space and a red value exceeding 220 was used to separate
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ripe and unripe grains.
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Total Elemental Analyses
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Dried plant material was finely ground in preparation for elemental analyses following
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previously published methodology.19 Approximately 200 mg of material was transferred to a Teflon
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digestion vessel and 7 mL of trace-metal grade nitric acid was added. The mixture was digested in a
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Mars 6 microwave digester (CEM Corporation) at 200 °C for 10 minutes. Because opaline plant Si does
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not dissolve in concentrated nitric acid, the resulting solution was transferred to a conical tube and
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diluted to 35 g with deionized water and centrifuged at 500 x g for 5 minutes to separate the Si-rich
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precipitate from the acid fraction. The acid fraction was decanted into another conical tube and was
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diluted to 2 % acid for analysis via ICP-MS (As) or ICP-OES (Al, B, Ca, Cu, Fe, K, Mg, Mn, Na, P, S, Zn). The
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Si-rich precipitate remaining was washed with deionized water, centrifuged and decanted three times.
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After the final rinse and decanting, the volume was made up to 15 mL with 2 M NaOH.27 After Si
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dissolution, the solution was analyzed for Si colorimetrically using an Evolution 60S UV-visible
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spectrophotometer (Thermo Scientific) and the molybdenum blue technique, which uses the
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absorbance at 630 nm.28 Total nitrogen was measured in straw (Elementar CHNS Cube) after finely
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grinding in a ball mill (Retsch MM301). All concentrations were calculated on a dry-weight basis.
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Two standard reference materials were included in each set of digestions along with a method
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blank. NIST 1568a rice flour was used predominantly to ensure adequate recovery of arsenic (87 ± 11%,
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n=11), while WEPAL 883, carnation straw, was used to assess the recovery of other elements.
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Recoveries for both reference materials were within reported limits (Table S2).
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As Speciation Analysis
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Selected grains, husk, and straw were analyzed for arsenic speciation by HPLC-ICP-MS following
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previously published methodology.29 Briefly, 200 mg of finely ground material was extracted in 2% HNO3
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at 95°C for 10 minutes. Digestions were centrifuged, filtered and diluted 1:1 with mobile phase.
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Hydroponic solutions were diluted 1:1 with mobile phase and filtered. 20 mM NH4H2PO4 was used as the
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mobile phase with a PRP-X100 column (Phenomenex).30 An Agilent 7500cx ICP-MS was used to monitor
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m/z 75 for arsenic operating in He collision mode. The method detection limit was 0.3 µg/L for DMA,
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MMA, As(III), and As(V). Recovery of NIST 1568b was 140% for DMA, 111% for arsenite, 106% for
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arsenate, while MMA concentrations were below the detection limit.
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Transporter Expression Assays
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The relative transcript abundance of Lsi1 and Lsi2 in roots and Lsi6 in straw was measured for
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DMA and control plants. Half of each plant’s root and straw tissue were flash frozen in liquid nitrogen
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and stored at -80°C. Samples were ground to homogeneity in liquid nitrogen in autoclaved mortars and
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pestles. Total RNA was extracted using an RNeasy Mini Kit (Qiagen), and first-strand cDNA was
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generated from 1 µg RNA using random primers. PCR plate wells were loaded with 1 µL of cDNA, SYBR
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green SuperMix, and forward and reverse primers. Quantitative real-time PCR was run to quantify
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relative expression of Lsi1, Lsi2, and Lsi6 with Eukaryotic Elongation Factor 1-alpha (eEF-1α) as a
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housekeeping gene, as this has been shown to be more stably expressed in rice throughout
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development making it suitable as an internal control.31 The qPCR program parameters were: 95°C for
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30 s, 30 cycles of 95°C for 15 s, 62°C for 30 s, 68°C for 45 s, and a final incubation at 72°C for 5 min
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followed by melting curve analysis. Primers are listed in Table S3.
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Statistical Analysis
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Results were analyzed using SAS 9.4 with PROC GLM. Plant biomass normality was improved
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through a log10 transformation, while all other variables required no transformations. Post-hoc pair-wise
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least squares mean comparisons were performed using Tukey’s adjustment. ANOVA tables are available
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in Table S4. Multiple linear regression was carried out using PROC REG and stepwise selection, with
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entry α=0.5 and exit α=0.1. Residuals were examined using PROC UNIVARIATE. Outliers were excluded
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based upon heading date using the median and the median absolute deviation as a robust estimates of
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the mean and standard deviation, respectively. Plants more than 2.5 standard deviations away from the
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mean were considered outliers and were removed from the analyses.
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RESULTS
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Biomass, Yield, and Observations of Straighthead
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DMA and MMA and their interaction with Si dosing had different effects on rice biomass and
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yield. While plants dosed with 5 µM DMA were similar in appearance to control plants (Figure S2), the
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presence of DMA significantly decreased grain yield (F=7.9, p=0.011) (Figure 1). Grain development was
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most impaired for the treatment combination where plants dosed with DMA received the lowest Si
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concentration (nominally 50 µM). These plants exhibited symptoms of straighthead, where panicles
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remained erect and most grains failed to fill. Grains had a curved, ‘parrot-beak’ appearance (Figure S3),
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Increased Si dosing >50 µM reversed the DMA-induced yield impacts (Figure 1) and those plants with
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higher Si dosings did not exhibit straighthead disorder despite DMA treatment. Increasing Si dosing had 6 ACS Paragon Plus Environment
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no impact on yield in control plants (R2 = 0.0, p = 1.0), but increasing Si dosing was positively correlated
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with grain yield for DMA-treated plants (R2 = 0.47, p = 0.0072). However, overall, ANOVA revealed that
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Si concentration and the As*Si interaction did not significantly affect yield (F=1.9, p=0.180; and F=1.9,
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p=0.179, respectively). DMA did not significantly affect root biomass (F=2.5, p=0.12) or straw biomass
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(F=1.1, p=0.30), although increasing Si resulted in slight, but significant reductions in root and straw
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biomass (F=33.7, p
