1 Title: Aluminum induces distinct changes in the metabolism of

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Aluminum induces distinct changes in the metabolism of reactive oxygen and nitrogen species in the roots of two wheat genotypes with different aluminum resistance Chengliang Sun, lijuan liu, Weiwei Zhou, lingli lu, chongwei jin, and Xianyong Lin J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03386 • Publication Date (Web): 10 Oct 2017 Downloaded from http://pubs.acs.org on October 11, 2017

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Journal of Agricultural and Food Chemistry

Title:

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Aluminum induces distinct changes in the metabolism of reactive oxygen and

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nitrogen species in the roots of two wheat genotypes with different aluminum

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resistance

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Running Title:

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ROS and RNS metabolism in wheat under Al stress

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Authors:

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Chengliang Sun1,2a, Lijuan Liu1a, Weiwei Zhou1, Lingli Lu1,3, Chongwei Jin1,3,

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Xianyong Lin1,3*

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Institutions:

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1

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of Natural Resource & Environmental Sciences, Zhejiang University, Hangzhou

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310058, China;

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2

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92521, USA

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3

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College of Environmental and Resource Sciences, Zhejiang University, Hangzhou

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310058, PR China.

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a

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*Corresponding author

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Prof. Dr. Xianyong Lin

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College of Natural Resource & Environmental Sciences,

MOE Key Laboratory of Environment Remediation and Ecological Health, College

Department of Environmental Science, University of California, Riverside, CA

Key Laboratory of Subtropical Soil Science and Plant Nutrition of Zhejiang Province,

Both the authors contributed equally to this work and paper.

1 ACS Paragon Plus Environment


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Zhejiang University, Hangzhou 310058, China

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Tel.: +86 571-88982476; fax: +86 571-86971395

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E-mail address: [email protected]


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TOC Graphical

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Abstract

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Aluminum (Al) toxicity in acid soils is a primary factor limiting plant growth and

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crop yield worldwide. Considerable genotypic variation in resistance to Al toxicity

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has been observed in many crop species. In wheat (Triticum aestivum L.), Al

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phytotoxicity is a complex phenomenon involving multiple physiological mechanisms

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which are yet to be fully characterized. To elucidate the physiological and molecular

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basis of Al toxicity in wheat, we performed a detailed analysis of reactive oxygen

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species (ROS) and reactive nitrogen species (RNS) under Al stress in one Al-tolerant

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(Jian-864) and one Al-sensitive (Yang-5) genotype. We found Al induced a significant

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reduction in root growth with the magnitude of reduction always being greater in

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Yang-5 than in Jian-864. These reductions were accompanied by significant

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differences in changes in antioxidant enzymes and the nitric oxide (NO) metabolism

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in these two genotypes. In the Al-sensitive genotype Yang-5, Al induced a significant

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increase in ROS, NO, peroxynitrite (ONOO-) and activities of NADPH oxidase,

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peroxidase and S-nitrosoglutathione reductase (GSNOR). A concomitant reduction in

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glutathione and increase in S-nitrosoglutathione contents was also observed in Yang-5.

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In contrast, the Al-tolerant genotype Jian-864 showed lower levels of lipid

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peroxidation, ROS and RNS accumulation, which was likely achieved through the

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adjustment of its antioxidant defense system to maintain redox state of the cell. These

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results indicate that Al stress affected redox state and NO metabolism and caused

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nitro-oxidative stress in wheat. Our findings suggest that these molecules could be

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useful parameters for evaluating physiological conditions in wheat and other crop


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species under adverse conditions.

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Key words: antioxidants, nitric oxide, nitrotyrosine, RNS, ROS, wheat.

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Introduction

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Aluminum (Al) is generally phytotoxic in acid soils (pH< 5.5) and is one of the major

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factors limiting crop growth and yield worldwide.1 It has been demonstrated that Al

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stress can have a devastating effect on plant metabolism by disrupting cellular

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homeostasis and uncoupling major physiological processes,2,3 consequently resulting

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in alterations in water and nutrient uptake and distribution. The underlying

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mechanisms of Al toxicity and tolerance in plants have been broadly focused on stress

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phytophysiology and several possible mechanisms have been proposed to explain Al

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tolerance.4,5 However, the precise mechanisms of Al toxicity still remain largely

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obscure and a topic of debate.

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A common response in plants to environmental constraints is the accumulation of

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reactive oxygen species (ROS; superoxide radicals, hydrogen peroxide, and hydroxyl

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radicals), which are can cause irreversible damage to DNA, proteins and lipids.6 It has

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been reported that NADPH oxidase plays a key role in ROS production under stress

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conditions,7 when NADPH oxidase reduces molecular oxygen by using cytosolic

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NADPH to produce O2•−, which is rapidly dismutated to H2O2 by the superoxidase

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dismutase isoenzymes.8 For example, NADPH oxidase-generated ROS seemed to be

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involved in nickel-induced oxidative stress in wheat roots.9 To avoid oxidative

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stresses, plants are well equipped with ROS scavenging such as superoxide dismutase,

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catalase, peroxidase and glutathione reductase, as well as antioxidants such as

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glutathione and ascorbate.6 In addition, maintenance of redox homeostasis appears to

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be critical to improving tolerance to Al toxicity in various plants, although the


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possible role of oxidative stress in Al toxicity is not clear.

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Recent discovery of plant cells that can generate free radical nitric oxide (NO)

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has opened new venues of research since NO and other NO-derived molecules like

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S-nitrosoglutamine (GSNO), nitrogen dioxide (·NO2) and peroxynitrite (ONOO-),

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collectively known as reactive nitrogen species (RNS), also play a role in response to

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environmental stress.10-13 Nitric oxide has been shown to be involved in a plethora of

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abiotic stresses such as low temperature,14 arsenic,15 and salinity.16 Recently, new data

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show that characteristics of NO metabolism is its interaction with O2•− to generate

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ONOO- or with protein and non-protein thiols to form nitrosothiols (SNOs), which

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can mediate post-translational modifications of different bio-molecules, mainly

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nitration and S-nitrosylation of proteins, respectively.10,11,15,16 Analysis of NO, ONOO-,

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and RSNO revealed that salinity,16 arsenic15 and water stress17 caused a general rise of

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these RNS at the cellular level, consequently inducing nitrosative stress in the plants

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under stress.

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In plant cells, metabolism of ROS and RNS must be integrated because these two

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families of molecules are characterized by rigorous metabolic interplay in higher

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plants.11,18 As a result, the antioxidant metabolism and homeostasis of ROS and RNS

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is critical for plant performance under stress conditions. Under Al stress, the concept

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of oxidative stress is commonly used to explain cellular damage caused by the

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imbalance between ROS production and scavenging antioxidant protective

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mechanisms.19-21 To our knowledge, however, little information is available on the

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endogenous metabolism of NO under Al stress conditions, although the physiological



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relevance of NO in plants under Al stress conditions has been reported.3,22 The main

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objective of our study is to elucidate the mechanisms by analyzing occurrence,

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distribution and metabolism of key components of both ROS and RNS that take place

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in roots of an Al-tolerant and an Al-sensitive genotype of wheat under Al stress.

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Materials and methods

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Plant materials and growth conditions

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Two winter wheat (Triticum aestivum L.) genotypes, Yang-5 (Al-sensitive) and

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Jian-864 (Al-resistant), were chosen for this study from a collection of 24 leading

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genotypes with good agronomic traits when grown in Zhejiang Province, China,

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where soils are strongly acidic in nature as reported in our early studies.23 Seeds were

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surface sterilized with 1% (v/v) sodium hypochlorite and placed in a glass beaker

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containing a small amount of deionized water overnight. After germination, seedlings

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were transplanted to plastic screens floating on a container filled with 2.5 l of 0.5 mM

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CaCl2 solution (pH=4.3). The solution was renewed daily. All experiments were

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conducted in a growth chamber with a 12-h/25°C day and a 12-h/22°C night regime, a

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light intensity of 300 µmol m-2 s-1, and a relative humidity of 70%. After 3 d of culture,

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uniform seedlings were transferred to 1.25 l of 0.5 mM CaCl2 (pH=4.3) containing

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AlCl3 for another 24 h.

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Evaluation of root elongation and Al accumulation

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Relative root elongation was calculated as the percentage of root elongated by various

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treatments compared to the Al-free control. Total Al content in root tips (0-1 cm) was

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analyzed by Agilent 7500A ICP-MS (Agilent, Palo Alto, CA, USA) after being 8 ACS Paragon Plus Environment

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digested with 10 ml of 2 M HCl as in Sun et al.22 Al accumulation in root apexes was

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detected by hematoxylin staining as described by Yamamoto et al.24

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H2O2 and MDA determination

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H2O2 was visually detected in root tips by using 3,3′-diaminobenzidine (DAB;

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Sigma-Aldrich) as substrate.25 Briefly, root tips were immersed in 1 mg ml-1 solution

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of DAB in 50 mM Tris-HCl (pH 3.8) for 4 h at 25°C. The roots stained with DAB

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were imaged under microscope. Membrane lipid peroxidation was estimated by

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measuring the concentration of malondialdehyde (MDA), according to the reaction

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with thiobarbituric acid, and historical staining was conducted with Schiff’s reagent

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(Sigma-Aldrich) as Yamamoto et al described.24

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Assessment of the loss of integrity of the plasma membrane

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Plasma membrane integrity was detected by staining root tips with Evans blue

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solution (0.25%, w/v) and imaged under microscope. The trapped Evans blue was

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evaluated by a spectrophotometric assay as describe in Yamamoto et al with minor

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modifications.24 After staining, the retained Evans blue was released by shaking the

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root tips in 5 ml of N, N-dimethylformamide. The absorbance was determined

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spectrophotometrically at 600 nm.

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Detection of O2•− , NO and ONOO- by epifluorescence microscope

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Superoxide (O2•−) content was detected according to Rodríguez-Serrano et al.26

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Briefly, root tips were incubated with 10 µM dihydroethidium (DHE) in 10 mM

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Tris-HCl buffer (pH 7.4) for 30 min at 37°C, and observed under a Nikon Eclipse

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E600 epifluorescence microscope equipped with a Nikon U-2A filter block (380-420



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nm excitation filter, 430 nm dichroic mirror, 450 nm barrier filter). Nitric oxide (NO)

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was visualized using the fluorescent probe diaminofluorescein-FM diacetate

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(DAF-FM DA) according to the method described by Sun et al.3 Peroxynitrite

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(ONOO-) was detected with 10 µM 3′-(p-aminophenyl) fluorescein (APF; Cayman

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Chemical, Ann Arbor, MI, USA) prepared in 10 mM Tris-HCl at pH 7.4 for 1 h as

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described by Saito et al.27

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Determination GSH, GSSG and GSNO

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The contents of reduced and oxidized glutathione were determined fluorimetrically

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according to a previous method.19 Briefly, root tips were homogenized in 200 µl of 25%

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H3PO3 and 1.5 ml of sodium phosphate-EDTA buffer (pH 8.0). For reduced

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glutathione (GSH), 100 µl supernatant was mixed with 1.8 ml of PBS-EDTA (pH 8.0)

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and 100 µl of o-phthalaldehyde (1 mg ml-1). After thorough mixing and incubation for

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15 min, the samples were measured on a fluorescence spectrophotometer (F 4600;

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Hitachi Ltd, Tokyo, Japan) at excitation 420 nm, emission 350 nm and slit width 10

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nm. For oxidized glutathione (GSSG), 0.5 ml of the supernatant was incubated with

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0.4 ml of 0.1 M NaOH and 100 µl of 0.04 M N-ethylmaleimide for 40 min before

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GSSG was measured using the procedure outlined for GSH assay.

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S-nitrosoglutathione (GSNO) content was determined by the Saville method

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according to Tanou et al.28 Briefly, root tips were frozen in liquid nitrogen and lysed

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in 50 mM Tris-HCl, pH 8.0 containing 150 mM NaCl, 1 mM phenylmethanesufonyl

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fluoride (PMSF). After centrifugation, the supernatant was incubated with 1 ml of 1%

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sulfanilamide in 3 M HCl plus 1 ml of 0.02% N-(1-naphthyl) ethylenediamine in 0.2


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M HCl with or without HgCl2 in the dark. The absorbance at 540 nm was measured

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with a spectrophotometer (Lambda 35; PerkinElmer, Waltham, MA, USA).

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Determination of NADPH oxidase activity of plasma membrane

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Plasma membrane was isolated according to Zhu et al with minor modification.29

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Briefly, root tips (about 5 g) were extracted in 10 ml of 25 mM

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Tris-2-morpholino-ethane-sulfonic acid (MES), pH 7.6, containing 0.25 M sucrose,

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10%

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ethylenediaminetetraacetic acid (EDTA), 1 mM dithiothreitol (DTT), 1 mM

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phenylmethylsulphonyl fluoride (PMSF), and 15 mM mercaptoethanol. The

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homogenate was filtered through two layers of cotton gauze and centrifuged at 13,000

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g for 20 min. Microsomal membranes were pelleted from the supernatant by

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re-centrifugation at 80,000 g for 45 min. The pellet was re-suspended in a buffer

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containing 1 mM DTT, 1 mM PMSF and 5 mM Tris-Mes (pH 6.5). The plasma

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membranes were further isolated and purified by two-phase partitioning method. The

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microsomal suspension was added to a phase system consisting of 6.1% (w:w)

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dextran T-500, 6.1% (w:w) PEG3350, 0.25 M sucrose, 3 mM KCl, 25 mM

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MES-NaOH buffer (pH 7.6). The final upper phases obtained after three successive

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rounds of partitioning were diluted with MES-NaOH buffer (pH 7.6), and centrifuged

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120,000 g for 30 min. The pellets were then resuspended in MES-NaOH buffer and

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used immediately for further analysis.

(w/v)

glycerol,

0.5%

(w/v)

polyvinylpyrrolidone,

3

mM

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The purity of the isolated plasma membrane was determined by examining the

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activity of H+-ATPase with or without inhibitors specific for different subcellular



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H+-ATPase activities.30 Compared to the H+-ATPases activity of the control treatment,

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H+-ATPases activity was mostly inhibited by Na3VO4 (inhibitor of P-type

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H+-ATPases), and seldom by NaN3 (inhibitor of F-type H+-ATPases) and NaNO3

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(inhibitors of V-type H+-ATPases) associated with plasma membrane, mitochondria

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and tonoplast, respectively (Table S1). These results were indicative of minor

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contaminations with other membrane fractions.

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Assay of enzyme activities

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For the enzyme assays, 0.15 g of root were ground with 2 ml ice-cold 50 mM

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potassium phosphate buffer (pH 7.0) containing 1 mM EDTA and 1%

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polyvinylpyrrolidone (PVP). The homogenate was centrifuged at 4°C for 20 min at

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15,000 g, and the resulting supernatant was used for the determination of enzymatic

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activity as described by Jiang & Zhang.31 An aliquot of the extract was used to

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determine the protein content by Coomassie brilliant blue G-250, using bovine serum

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albumin as a standard.

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Total superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed by

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measuring the ability to inhibit the photochemical reduction of nitro blue tetrazolium

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(NBT). Catalase (CAT; EC 1.11.1.6) activity was determined spectrophotometrically

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by measuring the rate of decomposition of H2O2 at 240 nm. Glutathione reductase

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(GR; EC 1.6.4.2) activity was determined by using oxidized glutathione, and the

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oxidation rate of NADH (ε = 6.22 mM-1 cm-1) was followed at 340 nm. Peroxidase

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(POD; EC 1.11.1.7) activity was measured by monitoring the change of absorption at

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470 nm due to guaiacol oxidation by hydrogen peroxide. The enzyme activity was


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calculated using the extinction coefficient of 26.6 mM-1 cm-1 for tetraguaiacol.

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S-nitrosoglutathione reductase (GSNOR; EC 1.2.1.1) was assayed by detecting the

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oxidation of NADH (ε = 6.22 mM-1 cm-1) at 340 nm after addition of

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S-nitrosoglutathione to the reaction mixture at a final concentration of 400 µM.15

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Total RNA isolation and semi-quantitative RT-PCR

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Total RNA was isolated from wheat root tips (0-10 mm) using 1 ml Trizol reagent

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(Life Technologies, Rockville, MD, USA) according to the manufacture’s

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recommendation. Total RNA (3 µg) was reverse transcribed using a PrimeScript™ II

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1st Strand cDNA Synthesis Kit (Takara, Dalian, Liaoning, China), following the

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manufacturer’s instructions. PCR reactions were performed using 1 µl of the cDNA,

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10 µM of each oligonucleotide primer and 1.25 U of Taq polymerase (Takara) in a 25

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µl reaction volume. Primers used were as follows: Mn-SOD (accession number

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AF092524)

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5’-GCTCCCAGACATCAATTCCCAACAAA-3’ (amplifying a 417 bp fragment);

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Cu/Zn-SOD

222

5’-CCTCTCTTCCAGGCTCCTGCC-3’

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5’-ATGAACAACAAACGCTCTCCC-3’ (amplifying a 465 bp fragment); CAT

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(accession number D86327), forward 5’-CTTCTCCTACTCCGACACGC-3’) and

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reverse 5’-TGTTGATGAATCGCTCTTGC-3’ (amplifying a 311 bp fragment); POD

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(accession number X85228) forward 5’-ACTTCCACGACTGCTTTGT-3’ and reverse

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5’-ACTGGGCCTTCCCGATG-3’ (amplifying a 643-bp fragment); for NADPH

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oxidase

forward

5’-ACCAGAAGCACCACGCCACCTAC-3’

(accession

(accession

number

U69632) and

number

AY561153),


and

reverse

forward reverse

forward


and

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5’-TAAGATGGAGGAAGAGGAGG-3’

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5’-TTGAGGCAATTTTGGCTAGG-3’ (amplifying a 404-bp fragment); for 18S rRNA

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(accession number AJ272181), forward 5’-CAAGCCATCGCTCTGGATACATT-3’

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and reverse 5’-CCTGTTATTGCCTCAAACTTCC-3’ (amplifying a 658 bp fragment).

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After 26 PCR cycles for 18S rRNA and 25-32 cycles for others, aliquots from the PCR

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reaction products were loaded on 2% agarose gels and stained with ethidium bromide.

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Statistical analysis

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Data are the means ± SD from at least three independent experiments. All data were

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statistically analyzed using the SPSS package (version 11.0; SPSS Inc., Chicago, IL,

238

USA), analysis of variance (ANOVA) was performed on the data sets and LSD (P

1 Title: Aluminum induces distinct changes in the metabolism of

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