Insights into acetate-mediated copper homeostasis and antioxidant defense in lentil under excessive copper stress

Journal Pre-proof Insights into acetate-mediated copper homeostasis and antioxidant defense in lentil under excessive copper stress Md. Shahadat Hossain, Mostafa Abdelrahman, Cuong Duy Tran, Kien Huu Nguyen, Ha Duc Chu, Yasuko Watanabe, Mirza Hasanuzzaman, Sayed Mohammad Mohsin, Masayuki Fujita, Lam-Son Phan Tran PII:

S0269-7491(19)33910-7

DOI:

https://doi.org/10.1016/j.envpol.2019.113544

Reference:

ENPO 113544

To appear in:

Environmental Pollution

Received Date: 23 July 2019 Revised Date:

29 September 2019

Accepted Date: 29 October 2019

Please cite this article as: Hossain, M.S., Abdelrahman, M., Tran, C.D., Nguyen, K.H., Chu, H.D., Watanabe, Y., Hasanuzzaman, M., Mohsin, S.M., Fujita, M., Tran, L.-S.P., Insights into acetatemediated copper homeostasis and antioxidant defense in lentil under excessive copper stress, Environmental Pollution (2019), doi: https://doi.org/10.1016/j.envpol.2019.113544. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. 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. © 2019 Published by Elsevier Ltd.

Cu stress

Cu accumulations in roots and shoots

Antioxidant defense

ROS production Photosynthetic damage

Osmoprotection Growth

Acetate + Cu stress

Cu accumulations in roots and shoots

Antioxidant defense

ROS production Photosynthetic damage

Osmoprotection Growth

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Insights into acetate-mediated copper homeostasis and antioxidant defense in lentil under

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excessive copper stress

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Md. Shahadat Hossain1, Mostafa Abdelrahman2,3, Cuong Duy Tran4,5, Kien Huu Nguyen6, Ha

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Duc Chu5, Yasuko Watanabe4, Mirza Hasanuzzaman7, Sayed Mohammad Mohsin1, Masayuki

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Fujita1 and Lam-Son Phan Tran4,8*

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1

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2393, Miki–cho, Kita gun, Kagawa, 761-0795, Japan

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2

Arid Land Research Center, Tottori University, Tottori 680-0001, Japan

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3

Botany Department, Faculty of Science, Aswan University, Aswan 81528, Egypt

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4

Stress Adaptation Research Unit, RIKEN Center for Sustainable Resource Science, 1-7-22,

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Suehiro-cho, Tsurumi, Yokohama 230-0045, Japan.

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5

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Agricultural Science, Pham Van Dong str., Hanoi, 100000, Vietnam

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6

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Academy of Agricultural Sciences, Pham Van Dong Str., Hanoi, 100000, Vietnam

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Dhaka 1207, Bangladesh

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8

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Vietnam

Laboratory of Plant Stress Responses, Faculty of Agriculture, Kagawa University, Ikenobe

Department of Genetic Engineering, Agricultural Genetics Institute, Vietnamese Academy of

National Key Laboratory for Plant Cell Technology, Agricultural Genetics Institute, Vietnam

Department of Agronomy, Faculty of Agriculture, Sher-e-Bangla Agricultural University,

Institute of Research and Development, Duy Tan University, 03 Quang Trung, Da Nang,

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Running title: Acetate-induced copper toxicity tolerance in lentil

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*Corresponding authors:

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Lam-Son Phan Tran

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E-mail: [email protected] (L.-S.P. Tran)

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Abstract

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Gradual contamination of agricultural land with copper (Cu), due to the indiscriminate uses of

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fungicides and pesticides, and the discharge of industrial waste to the environment, poses a high

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threat for soil degradation and consequently food crop production. In this study, we combined

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morphological, physiological and biochemical assays to investigate the mechanisms underlying

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acetate-mediated Cu toxicity tolerance in lentil. Results demonstrated that high dose of Cu (3.0

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mM CuSO4. 5H2O) reduced seedling growth and chlorophyll content, while augmenting Cu

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contents in both roots and shoots, and increasing oxidative damage in lentil plants through

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disruption of the antioxidant defense. Principle component analysis clearly indicated that Cu

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accumulation and increased oxidative damage were the key factors for Cu toxicity in lentil

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seedlings. However, acetate pretreatment reduced Cu accumulation in roots and shoots, increased

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proline content and improved the responses of antioxidant defense (e.g. increased catalase and

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glutathione-S-transferase activities, and improved action of glutathione-ascorbate metabolic

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pathway). As a result, excess Cu-induced oxidative damage was reduced, and seedling growth

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was improved under Cu stress conditions, indicating the role of acetate in alleviating Cu toxicity

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in lentil seedlings. Taken together, exogenous acetate application reduced Cu accumulation in

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lentil roots and shoots and mitigated oxidative damage by activating the antioxidant defense,

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which were the major determinants for alleviating Cu toxicity in lentil seedlings. Our findings

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provide mechanistic insights into the protective roles of acetate in mitigating Cu toxicity in lentil,

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and suggest that application of acetate could be a novel and economical strategy for the

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management of heavy metal toxicity and accumulation in crops.

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Keywords: Acetate; antioxidant defense; copper homeostasis; heavy metal toxicity; oxidative

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stress

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Capsule: This study showed that acetate application can protect lentil plants from the toxicity of

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excess Cu by enhancing photosynthetic capacity, activating antioxidant defense, maintaining

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osmotic adjustment and reducing Cu accumulation in both shoots and roots. The acetate

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treatment could be recommended to the farmer as low-cost and effective method for the

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management of heavy metal toxicity in lentils and other crop plants.

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1. Introduction

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Copper (Cu) is one of an essential micronutrients required for normal growth and development

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of plants; however, it can be toxic for plants at higher concentrations (Wuana and Okieimen

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2011). Moreover, Cu in excess is more toxic for plants than other heavy metals, such as

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cadmium (Cd), nickel (Ni), manganese (Mn) and zinc (Zn) (Gajewska and SkŁodowska 2010;

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Adrees et al. 2015a). Unfortunately, the abundance of Cu has been increasing in the soils due to

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the excessive use of Cu-containing fungicides or pesticides, and due to the release of industrial

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waste into the environment (Adrees et al. 2015a), posing a potential threat to crop production. Cu

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is naturally present in the soil, ranging from 2.0 to 100 mg kg−1 (Adriano, 2001). Furthermore,

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Cu concentrations in long-contaminated soils can be varied, ranging from 500 to 3000 mg kg-1,

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depending on the severity of the polluted areas (Brun et al. 1998; McBride and Martínez 2000;

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Yurela, 2009; Adrees et al. 2015b).

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Like other heavy metals, Cu in excess disturbs seed germination, photosynthesis and

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nutrient uptake, causing reduced plant growth, biomass accumulation and yield (Yruela 2009;

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Adrees et al. 2015a). Physiological studies have revealed that excess Cu induces severe oxidative

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damage (Adrees et al. 2015a), which occurs in plants when a high amount of reactive oxygen

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species (ROS) are produced in the cells due to extreme adverse conditions (Hasanuzzaman et al.

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2017a). ROS include singlet oxygen (1O2), superoxide radical (O2•–), hydroxyl radical (•OH) and

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hydrogen peroxide (H2O2) (Demidchik 2015; Abdelrahman et al. 2016). At higher

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concentrations, ROS are toxic for plants, and cause oxidative damage by oxidizing vital

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constituents of the cells, such as DNA, lipids and proteins (Demidchik 2015). Production of a

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basal level of ROS also occurs under optimal growing conditions, and these ROS can act as

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signaling molecules for proper plant growth and development (Hasanuzzaman et al. 2017a;

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Waszczak et al. 2018). Thus, plants possess antioxidant defense system to restrict the levels of

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ROS, particularly under stress conditions. Antioxidant defense system includes enzymatic (e.g.,

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catalase, CAT; ascorbate peroxidase, APX; monodehydroascorbate reductase, MDHAR;

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dehydroascorbate reductase, DHAR; glutathione reductase, GR; and glutathione-S-transferase,

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GST) and non-enzymatic (e.g., ascorbate, AsA; and reduced glutathione, GSH) components

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(You and Chan, 2015; Choudhury et al. 2017; Nguyen et al. 2019). These components act in

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sequence to detoxify excessive ROS, thereby enhancing plant tolerance to various abiotic

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stresses, including Cu toxicity (Choudhary et al. 2012; Choudhury et al. 2017)

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Numerous studies on understanding of Cu stress tolerance mechanisms in plants have

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suggested that reduction in Cu uptake, chelation of Cu with phytochelatin and subsequent storage

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in vacuoles, and induction of antioxidant defense are the major ways to alleviate Cu toxicity in

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plant cells (Choudhary et al. 2012; Adrees et al. 2015a). The use of chemicals for management of

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abiotic stresses, including Cu stress, in different crops, such as radish (Raphanus sativus), rice

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(Oryza sativa), maize (Zea mays), wheat (Triticum aestivum) and soybean (Glycine max), has

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been shown as a common and effective approach (Choudhary et al. 2012; Savvides et al. 2016;

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Nguyen et al. 2018). For example, exogenous applications of salicylic acid (Moravcová et al.

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2018), indole acetic acid (Massoud et al. 2018), gibberellic acid (Massoud et al. 2018),

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castasterone (Yadav et al. 2018), proline (Noreen et al. 2018) enhanced Cu stress tolerance in

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plants like wheat, maize and mustard (Brassica juncea). Recently, acetate, a low-cost chemical,

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was reported to enhance drought tolerance in Arabidopsis (Arabidopsis thaliana), wheat,

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rapeseed (B. napus) (Kim et al. 2017) and cassava (Manihot esculenta) (Utsumi et al. 2019), as

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well as salinity tolerance in lentil (Lens culinaris) plants (Hossain et al. 2018), suggesting the

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potential applications of this chemical in the management of a wide range of environmental

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stresses in various plant species.

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However, acetate-mediated mitigation of Cu stress has not been investigated yet,

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particularly in the important legume crop lentil. This pulse crop is very popular in many

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developing countries like Bangladesh, and serves as an inexpensive source of proteins for the

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local people. In addition, lentil improves soil health by adding atmospheric nitrogen to the soil

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through its nitrogen fixation ability (Hossain et al. 2017, Abdelrahman et al. 2018). Being

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sensitive to Cu stress, lentils grown in Cu-contaminated areas suffer severe losses in biomass and

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yield due to oxidative burst resulted from excess Cu accumulation in the cells (Islam et al. 2016).

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Therefore, in the present study, we examined the potential of acetate in alleviating Cu toxicity in

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lentil with the aim to propose an effective and economical approach for the management of

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excess Cu. According to our knowledge, this is the first report showing the positive role of

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acetate in mitigating Cu-induced damage in lentil seedlings.

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2. Materials and methods

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2.1. Plant research materials and treatments

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Lentil (Lens culinaris Medik cv. BARI Lentil-7) seeds were sterilized with 70% ethanol for 5

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min and soaked for 1 day in distilled water, before they were placed on six-layered water-

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moistened paper towels preset in Petri plates to allow germination. After incubating for 3 days

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under dark conditions, 40 germinated seedlings were kept in each Petri plate, and the plates were

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transferred to the growth chamber (350 µmol m–2 s–1 photon flux density, 25 ± 1°C, continuous

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illumination). Subsequently, each Petri plate was flushed with 30 mL of 5000-time-diluted

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Hyponex nutrient solution (Tokyo, Japan). The pH of the nutrient solutions with or without

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acetate was adjusted to 6.5. At 5th day after soaking (DAS), two sets of seedlings were grown in

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nutrient solution supplemented with 10 mM Na-acetate (CH3COONa) for 2 days, whereas three

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sets of seedlings were grown in Na-acetate-free nutrient solution. Acetate dose was selected

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based on a previous report (Hossain et al. 2018), where 10 mM Na-acetate was shown to be

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effective in enhancing salt stress tolerance of lentil. Subsequently, 6-day-old seedlings (two Na-

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acetate-treated sets and two Na-acetate-non-treated sets) were exposed to 0.3 or 3.0 mM copper

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sulfate (CuSO4. 5H2O) added to nutrient solution, except a control group which was treated with

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nutrient solution only. Treatment solutions were changed every alternate day. After 4 days of

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stress treatment, roots and shoots of 10-day-old lentil seedlings were separately taken for further

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analyses. Each treatment had three biological replications grown under the same experimental

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conditions, which were used for physiological and biochemical analyses. For selecting Cu doses,

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preliminary trial with a series of Cu doses (0.1, 0.3, 1.0, 1.5, 2.0, 2.5 and 3.0 mM CuSO4. 5H2O)

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with or without 10 mM Na-acetate was performed. Clearer phenotypic differences between

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acetate-treated and untreated plants were observed at higher Cu concentrations, particularly at

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3.0 mM Cu. The 3.0 mM Cu (190.65 mg L-1) mimicked a slightly contaminated soil condition,

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since natural soils contain 2.0 to 100 mg Cu kg−1 soil (Adriano, 2001), while long-contaminated

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soils may contain 500 to 3000 mg Cu kg-1 soil (Brun et al. 1998; McBride and Martínez 2000;

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Yurela, 2009; Adrees et al. 2015b). Thus, based on our optimized conditions and published

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literature, 0.3 and 3.0 mM Cu concentrations (19.65 and 190.65 mg L-1, respectively) were

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selected as representative concentrations in this study for low and high Cu doses, respectively.

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2.2. Measurement of root dry weight (RDW), shoot dry weight (SDW) and relative water

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fraction (RWF)

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To measure RDW and SDW, roots and shoots were separated and kept in paper towel to remove

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surface water after washing with distilled water. Roots and shoots were subsequently dried for 48

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h at 80°C until their weight became constant. Finally, DW of each sample was measured using

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an electric balance in three replications, with each replication was calculated as average of 10

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roots (RDW) or shoots (SDW) from 10 seedlings (Mettler AE 240, USA). RWF was determined

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according to Negrão et al. (2017).

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2.3. Measurement of chlorophyll (Chl) and carotenoid (Car) contents

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To measure Chl a and b contents, 0.1 g of each leaf sample was taken in a tube containing 10 mL

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dimethyl sulfoxide (DMSO) solution. The tubes were then heated for 1 h at 65°C to extract Chls.

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After the solution was cooled to room temperature, absorbances of the samples were taken at 645

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nm and 663 nm. Chl a and b contents were determined according to the methods of Hiscox and

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Israelstam (1979), and Chl (a+b) content was expressed as mg g–1 FW. Car content was

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determined according to Brito et al. (2011) and expressed as mg g–1 FW.

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2.4. Determination of proline (Pro) content and electrolyte leakage (EL)

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Pro contents were determined in shoots according to Bates et al. (1973), and were expressed as

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µmol g–1 FW. EL of shoot samples was assessed according to the previous method (Dionisio-

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Sese and Tobita 1998).

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2.5. Determination of the contents of malondialdehyde (MDA), other aldehydes, H2O2, AsA,

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and GSH and oxidized GSH (GSSG) contents

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Shoot samples (0.5 g/each) were homogenized in 3 mL of 5% (w/v) trichloroacetic acid (TCA).

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After centrifugation, the supernatant was used to determine MDA and other aldehyde contents

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following the method of Heath and Packer (1968) and Keramat et al. (2010), respectively. H2O2

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content was determined following the method of Yang et al. (2007); while AsA, and total GSH

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and GSSG contents were measured as described by Noctor et al. (2016). GSH content was

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calculated using the formula: GSH = (Total glutathione – GSSG).

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2.6. Determination of total soluble protein content and enzyme activity assays

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Total soluble proteins were extracted from 0.5 g of lentil shoots according to Nahar et al. (2016).

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Total soluble protein content was measured and calculated using bovine serum albumin as

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standard (Bradford 1976). CAT, DHAR, APX, GR and MDHAR activities were determined and

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calculated following the methods described in Noctor et al. (2016). GST activity was determined

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according to Nahar et al. (2016).

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2.7. Determination of Cu content in plants

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Dry shoot and root (0.1 g) samples were independently digested using an acid mixture [nitric

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acid and perchloric acid (5:1)] at 80°C for 48 h (Rahman et al. 2016). Subsequently, Cu contents

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were measured in root and shoot samples using an inductively coupled plasma optical emission

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spectrometry (Agilent 5110 ICP-OES VDV, USA).

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2.8. Gene expression analysis

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Leaf samples (top 2.0 cm) from each treatment were collected for extraction of total RNA using

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RNeasy Plant Mini Kit (Qiagen, Hildren, Germany). Expression analysis of GR and APX genes

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using real-time quantitative polymerase chain reaction (RT-qPCR) was performed according to

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Le et al. (2011), using their specific primers (Supplementary Table 1). Relative expression values

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were calculated as described in Le et al. (2011), with tubulin gene being used as the reference

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gene (Sinha et al. 2019).

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2.9. Statistical analysis

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The analysis of variance (ANOVA) and the differences of means from 3 biological replications

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(n = 3) were evaluated by Tukey’s honest significant difference (HSD) test using the XLSTAT

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v.2019 software. Using the same software, student’s t-test were also performed between ‘Cu

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(0.3)’ and ‘Ac + Cu (0.3)’ or ‘Cu (3.0)’ and ‘Ac + Cu (3.0)’, when necessary. Mean differences

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at P ≤ 0.05 were denoted significant. Principal component analysis (PCA) was performed using

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factoextra package in R software (v3.5.1) (https://www.r-project.org/). For PCA analysis, all the

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variable trait data derived from three biological replicates were normalized using the Z-score

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method, and the obtained data matrix was further used for generating the PCA plots. Heatmap

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clustering was produced by ‘gplots’, ‘RColorBrewer’ and ‘gdata’ packages in the R v.3.5.1 using

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the generated Z-score data matrix.

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3. Results

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3.1. Acetate improves growth parameters of lentil seedlings under Cu stress

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To investigate the role of acetate in mitigating the negative effect of Cu stress on the growth of

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lentil, we first examined whether acetate pretreatment could improve the biomass of lentil plants

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grown under 0.3 and 3.0 mM Cu stress. Excess Cu reduced the DWs of both shoots and roots of

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lentil plants in a dose-dependent manner. SDW declined by 41.76% at Cu (3.0 mM) stress, and

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RDW reduced by 41.07 and 68.75% under Cu (0.3 mM) and Cu (3.0 mM) stress levels,

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respectively, compared with that of non-stressed control (Table 1). However, acetate-pretreated

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seedlings showed slight improvement, albeit non-significant, in SDW and RDW as compared

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with that of Cu-stressed seedlings (Table 1).

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3.2. Effects of acetate pretreatment on water relation, Pro accumulation and contents of

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photosynthetic pigments in lentil seedlings exposed to Cu stress

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Like other heavy metals, excess amount of Cu in the growth medium perturbs the water relation

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in plants by lowering the water uptake and transport through roots (Rucińska-Sobkowiak 2016).

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Thus, we evaluated the role of acetate in restoring water balance in lentil seedlings under Cu

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stress conditions. In comparison with non-stressed control, the RWF in lentil plants reduced in a

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Cu dose-dependent manner. The two levels of Cu stresses, 0.3 and 3.0 mM, declined the RWF in

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lentil seedlings by 41.10 and 79.19%, respectively, relative to non-stressed control (Table 1). On

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the other hand, acetate-pretreated seedlings grown under 3.0 mM Cu stress exhibited 125.91%

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increase in RWF, when compared with that of acetate-non-pretreated plants grown under the

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same Cu stress level (Table 1).

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Plants synthesize Pro under stress conditions to adjust osmotic balance at cellular level

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(Hayat et al. 2012). A sharp increase in Pro content was observed, displaying 270.52% rise of

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Pro level in the shoots of lentil seedlings exposed to medium containing 3.0 mM Cu over that of

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control (Table 1). Interestingly, Pro content was augmented by 71.82% at 3.0 mM Cu in acetate-

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pretreated seedlings in comparison with acetate-non-pretreated corresponding Cu-stressed

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seedlings (Table 1). We then measured the contents of key photosynthetic pigments in lentil

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leaves to understand the mitigating effect of acetate on the excess Cu-induced damage on the

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photosynthetic performance of lentil seedlings. Chl (a + b) and Car contents were diminished in

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lentil leaves by 12.13 and 23.82% with Cu (3.0 mM) treatments relative to that of control

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seedlings (Table 1). However, pretreatment of the seedlings with acetate improved the Car (by

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16.34%) and Chl (a + b) contents (by 29.96%) in leaves of the lentil seedlings exposed to 0.3 and

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3.0 mM Cu, respectively, as compared with the corresponding value of acetate-non-pretreated

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plants grown under the respective Cu stress level (Table 1).

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3.3. Acetate pretreatment improves phenotypic appearance, and reduces the oxidative

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stress-induced damage and Cu contents in lentil seedlings grown under excess Cu

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At 3.0 mM Cu stress, severe chlorosis occurred on the lentil seedlings, with yellow-greyish spots

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being observed on the lentil leaflets (Fig. 1A). Gladly, acetate pretreatment reduced the

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development of characteristic yellow-greyish spots on the leaflets (Fig.1A), indicating the role of

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acetate in attenuating Cu toxicity in lentil seedlings. Like other heavy metals, excess Cu induces

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oxidative stress in plants (Choudhary et al. 2012; Mostofa et al. 2015). To understand the level of

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Cu stress-induced ROS accumulation and oxidative damage, and to evaluate whether acetate

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could alleviate this adverse effect imposed on plants, we measured MDA content and other

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aldehydes content (indicator of oxidative stress-induced lipid peroxidation), EL (an indicator of

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oxidative stress-induced membrane damage) and H2O2 content in lentil plants grown under Cu

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stress conditions with and without acetate pretreatment. A significant surge in MDA content (by

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229.63%), other aldehyde content (by 293.37%), EL (by 197.26%) and H2O2 content (by

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509.79%) was observed in Cu stressed seedlings (3.0 mM) in comparison with non-stressed

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control (Fig. 1B-E). We also observed a significant increase in MDA content (by 63.89, 124.52

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and 161.96%) in lentil seedlings exposed to 1.5, 2.0 and 2.5 mM Cu concentrations, respectively

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(Supplementary Fig. 1A). In contrast, application of acetate to the lentil seedlings prior to

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treatment of plants with excess Cu reduced their other aldehyde content (by 34.60) at 0.3 mM

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Cu, and MDA content (by 30.34%), EL (by 26.63%) and H2O2 content (by 70.40%) at 3.0 mM

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Cu concentration, relative to that of the corresponding Cu-stressed alone seedlings (Fig. 1B-E).

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To examine whether the oxidative stress and subsequent membrane damage of lentil

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seedlings were associated with Cu accumulation, the Cu contents were measured in Cu-stressed

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seedlings. Cu uptake increased in both lentil roots and shoots dependently on the dose applied

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(Fig. 1F, G; Supplementary Fig. 1E, F). Specifically, in comparison with non-stressed control a

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significant rise in root-Cu content by 1874.53 and 16761% was noted at 0.3 mM and 3.0 mM Cu,

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respectively, while a significant increase in shoot-Cu content was observed at 3.0 mM Cu (Fig.

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1F, G). On the other hand, acetate pretreatment reduced the Cu accumulation in roots by 28.62 at

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0.3 mM Cu concentration, whereas it decreased Cu accumulation in shoots by 66.50% at 3.0 mM

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Cu stress level, in comparison with the respective Cu-stressed alone seedlings (Fig. 1F, G).

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3.4. Enhancement of antioxidant defense by exogenous acetate in lentil seedlings under Cu

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stress conditions

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To gain an insight into the mechanisms of acetate-mediated attenuation of excess Cu-induced

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oxidative stress and membrane damage in lentil seedlings, we explored the responses of several

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key components of the antioxidant defense system in lentil plants to different levels of Cu with

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or without acetate pretreatment. As shown in Fig. 2A-C, CAT activity decreased (by 70.69%),

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APX activity increased (by 180.8%) and MDHAR decreased (no activity) in seedlings at 3.0 mM

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Cu stress levels as compared with that of non-stressed control. Interestingly, CAT activity was

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enhanced by 58.74, 61.21 and 40.64% in lentil seedlings grown in 1.5, 2.0 and 2.5 mM Cu-

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containing solution (Supplementary Fig. 1D). Furthermore, acetate-pretreated seedlings showed

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higher CAT activity, by 317.30% at 3.0 mM Cu concentration than Cu-stressed alone seedlings

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(Fig. 2A). The MDHAR activity gained a remarkable increase from undetected level under 3.0

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mM Cu stress in the seedlings pretreated with acetate when compared with that in seedlings

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without acetate pretreatment (Fig. 2C). There was a decrease of 47.02% in DHAR activity in

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seedlings exposed to 0.3 mM Cu stress relative to that of non-stressed control (Fig. 2D). In

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comparison with acetate-non-pretreated Cu-stressed seedlings, acetate pretreatment reduced

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DHAR activity in the seedlings by 37.96% at 3.0 mM Cu stress (Fig. 2D). GR activity in lentil

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seedlings augmented by 315.40 % at 3.0 mM Cu stress as compared with that of non-stressed

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seedlings, whereas acetate pretreatment did not increase the GR activity in comparison with

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acetate-non-treated lentil plants exposed to the same Cu stress level (Fig. 2E). In comparison

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with Cu-stressed seedlings, acetate-pretreated seedlings exhibited 105.30% upregulation in GST

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activity at 3.0 mM Cu stress (Fig. 2F).

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AsA content dropped in lentil seedlings exposed to high Cu concentrations. Specifically,

299

it decreased by 62.76, 97.44, 99.68% and 99.84% at 1.5, 2.0, 2.5 and 3.0 mM Cu stress levels

300

versus non-stressed control (Fig. 2G; Supplementary Fig. 1C). However, an increasing tendency

301

in GSH content (by 905.75%), GSSG content (by 799.92%) and GSH/GSSG ratio (by 27.20%)

302

was observed in lentil seedlings at 3.0 mM Cu stress level, in relation to non-stressed control

303

(Fig. 2H-J). Acetate pretreatment showed increased levels in all these examined non-enzymatic

304

antioxidants in lentil seedlings grown under excess Cu conditions. More specifically, significant

305

elevations in AsA (by 392.59%), GSH (by 66.45 %) and GSSG (by 34%) contents were recorded

306

in acetate-pretreated seedlings grown under 3.0 mM Cu stress, as compared with that of the Cu-

307

stressed alone seedlings (Fig. 2G-I). However, there was no significant difference in GSH/GSSG

308

ratio between acetate-treated and acetate-non-treated Cu-stressed plants (Fig. 2J).

309

Since the AsA-GSH cycle forms an important part of the antioxidant defense (Wang et

310

al., 2018), in which we had a particular interest, we were then curious whether there was a

311

correlation between enzyme and gene expression levels under our experimental conditions. Thus,

312

we used RT-qPCR to determine the transcript levels of GR and APX genes encoding GR and

313

APX enzymes, respectively, in the leaves of lentil seedlings subjected to all kinds of treatments.

314

Results shown in Fig. 2K-L indeed revealed a positive correlation between the expression levels

315

of the GR and APX genes and their corresponding enzyme activities.

316 317

3.5. Insights into treatment-variable interactions through hierarchical clustering and PCA

318

analysis

319

The heatmap hierarchical clustering of the growth, physiological and biochemical traits in 10-

320

day-old lentil seedling pretreated with or without acetate under different Cu-stress levels showed

321

two major clusters (Supplementary Fig. 2A). Specifically, the growth-related parameters (RDW,

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SDW and RWF), photosynthetic pigments [Chl (a + b) and Car contents], and enzymatic and

323

non-enzymatic antioxidants (GST, CAT, MDHAR and DHAR activities, and AsA content) were

324

grouped in the cluster I (Supplementary Fig. 2A). On the other hand, oxidative stress markers

325

(H2O2, MDA and EL), Cu contents in roots and shoots, APX activity, and Pro, GSH and GSSG

326

contents were grouped in cluster II (Supplementary Fig. 2A). All growth, physiological and

327

biochemical traits in cluster I exhibited a decreasing trend in 10-day-old lentil seedling grown

328

under high Cu stress [Cu (3.0)], in comparison with non-stressed (C) and acetate-pretreated and

329

moderately Cu-stressed [Ac + Cu (0.3)] plants (Supplementary Fig. 2A). In contrast, all of the

330

examined oxidative stress markers (H2O2, MDA and EL) and shoot-Cu content grouped in

331

cluster II exhibited an increasing tendency in 10-day-old lentil seedlings grown under ‘Cu (3.0)’

332

in relation to ‘C’ plants (Supplementary Fig. 2A). However, 10-day-old ‘Ac + Cu (3.0)’ lentil

333

seedlings exhibited reductions in these oxidative stress markers and shoot-Cu content, but

334

increases in Pro, GSH, GSSG, GST and CAT levels when compared with that in ‘Cu (3.0)’

335

seedlings (Supplementary Fig. 2A).

336

In order to get further insights into the interrelated effects of Cu stress on the growth,

337

physiological and biochemical variable traits in the 10-day-old lentil seedlings pretreated with or

338

without acetate, we performed a PCA analysis (Supplementary Fig. 2B-D). All the variable traits

339

and treatment conditions were loaded into the two major PC1 and PC2 components, explaining

340

85.70% of the total variance (Supplementary Fig. 2B-D). The majority of the examined variable

341

traits were differentiated by PC1, and thus indicated by the larger proportion (69.70%) of

342

variance, while the lower proportion of variance (16.0%) was indicated by PC2 (Supplementary

343

Fig. 2B). The loading plot discriminated the variable traits based on their contribution to the PC1

344

and PC2 axes. For instance, the traits with green color and located within the circle displayed

345

high positive loading into PC1 (Supplementary Fig. 2B), whereas growth, photosynthetic

346

pigment, AsA, MDHAR and CAT variables were negatively loaded into PC1 (Supplementary

347

Fig. 2B). Similarly, the PCA score plot showed clear distinct separation of lentil seedlings on the

348

basis of their growth conditions. Lentil seedlings grown under ‘Cu (3.0)’ and ‘Ac + Cu (3.0)’

349

conditions were positively loaded into PC1, whereas lentil seedlings grown under ‘C’, ‘Cu (0.3)’

350

and ‘Ac + Cu (0.3)’ conditions were negatively loaded into PC1 (Supplementary Fig. 2C). In

351

addition, the PCA biplot clearly indicated the interrelationship between variable traits and

352

treatment conditions (Supplementary Fig. 2D). For instance, lentil seedlings grown under ‘Cu

353

(3.0)’ were closely connected with oxidative stress markers H2O2, MDA and EL, as well as

354

shoot-Cu content (Supplementary Fig. 2D). The ‘Ac + Cu (3.0)’ lentil seedlings were linked with

355

antioxidant and osmoprotectant variables, such as GST, GSSG, GSH and Pro (Supplementary

356

Fig. 2D), whereas the ‘C’, ‘Cu (0.3)’ and ‘Ac + Cu (0.3)’ lentil seedlings were strongly linked

357

with growth, photosynthetic pigment, AsA and MDHAR variables (Supplementary Fig. 2D).

358 359

4. Discussion

360

Several approaches have been used to increase the productivity of crops grown under different

361

abiotic stress conditions, among which the exogenous applications of chemical substances, such

362

as nitric oxide, silicon, selenium and melatonin, have been widely reported to be effective

363

(Manivannan et al. 2016; Yu et al. 2018; Zahedi et al. 2019). Exogenous substances could be a

364

promising tool for crop stress management, because they can enhance plant tolerance to multiple

365

stresses without genetic modifications (Savvides et al. 2016; Nguyen et al. 2018). Therefore,

366

searching for novel chemical agents that can more effectively and economically confer stress

367

tolerance to crops, in comparison with the existing chemicals, is the main focus of many plant

368

researchers (Savvides et al. 2016; Nguyen et al. 2018). In this study, we investigated the

369

potentiality of acetate in alleviating Cu toxicity in lentil seedlings. Our results demonstrated that

370

acetate pretreatment enhanced lentil tolerance against Cu stress by lowering the Cu accumulation

371

in the plant shoots and roots, maintaining the levels of photosynthetic pigments, improving

372

osmoprotection and inducing antioxidant defense.

373

We first investigated whether acetate could alleviate Cu-induced damage in lentil

374

seedlings based on their growth parameters and phenotypic appearance (Table 1 and Fig. 1A).

375

Excess Cu caused a severe reduction in the levels of RDW, SDW and photosynthetic pigments in

376

‘Cu (0.3)’ and ‘Cu (3.0)’ plants relative to non-stressed ‘C’ plants (Table 1). Our results are in

377

line with the Cu-induced growth reduction previously reported in maize, rice and lentil (Mostofa

378

et al. 2015; Islam et al. 2016; Moravcová et al 2018). Growth reduction is a common response of

379

plants under Cu stress conditions, which might be associated with Cu-induced perturbation in

380

Chl synthesis, oxidative stress and disruption in Cu homeostasis in plants (Adrees et al. 2015a).

381

Under 0.3 and 3.0 mM Cu stress levels, acetate pretreatment improved the contents of

382

photosynthetic pigments Cars and Chl (a+b), respectively, in acetate-pretreated Cu-stressed lentil

383

seedlings, in comparison with acetate-non-pretreated Cu-stressed seedlings (Table 1), indicating

384

a positive role of acetate in protecting photosynthetic pigments and subsequently giving better

385

growth performance (Table 1). PCA results also indicated a positive relationship of the

386

photosynthetic pigment contents and growth-related parameters with the ‘Ac + Cu (0.3)’

387

treatment, providing further evidence for the protective role of acetate against Cu stress in lentil

388

plants (Supplementary Fig. 2D).

389

Heavy metals, including Cu, negatively affect the water balance in different plant species

390

(Barceló and Poschenrieder 1990; Kholodva et al. 2011; Rucińska-Sobkowiak 2016). Our

391

findings also showed disturbance in the water relation in lentil plants exposed to excess Cu

392

(Table 1). Acetate-pretreatment improved RWF in lentil seedlings grown under Cu stress relative

393

to acetate-non-pretreated Cu-stressed plants (Table 1), indicating a positive role of acetate in

394

maintaining the water content in the Cu-stressed plants. In support of our results, previous

395

reports demonstrated that acetate was able to maintain water content in various plant species

396

under drought (e.g. in Arabidopsis and cassava) (Kim et al. 2017; Utsumi et al. 2019) and salt

397

stress (e.g. in lentil) (Hossain et al. 2018). We hypothesized that the role of acetate in reducing

398

cellular dehydration under Cu stress might be associated with its ability to increase the levels of

399

osmoprotection-related compounds like Pro. Pro content increased in lentil seedlings grown

400

under ‘Cu (3.0)’ stress (Table 1) relative to ‘C’ plants, which was in agreement with the result

401

previously reported in rice plants exposed to Cu stress (Mostofa et al. 2015). More importantly,

402

acetate-pretreated lentil plants exhibited much higher level (71.82%) of Pro than ‘Cu (3.0)’

403

plants (Table 1 and Supplementary Fig. 2D). This result indicated an acetate-induced increase in

404

Pro content, explaining the role of acetate in osmoprotection of lentil plants under excess Cu. Pro

405

is a well-known osmoprotectant and antioxidant agent, and Pro homeostasis is essential for

406

generating energy by transporting Pro from aerial part (source) to the roots (sink) to improve root

407

growth, subsequently better water uptake (Kishor and Sreenivasulu 2014). However, Pro

408

synthesis is also a costly process, and may affect plant growth as a trade-off survival (Munns and

409

Tester 2008).

410

Upon exposure to the ‘Cu (3.0)’ treatment, lentil seedlings showed damaging symptoms

411

(yellow-greyish spot) on leaves (Fig. 1A), which might be associated with a high level of

412

oxidative stress triggered by the increased accumulation of Cu in the shoots (Fig. 1B-G). Indeed,

413

the examined oxidative stress markers, including H2O2, MDA and other aldehyde contents, and

414

EL, were strongly augmented in the ‘C (3.0)’ relative to that of ‘C’ plants (Fig. 1B-E). In

415

agreement with this finding, PCA analysis demonstrated a positive relationship of ‘Cu (3.0)’

416

with shoot-Cu content and oxidative stress-related markers (Supplementary Fig. 2D). These

417

results indicated a severe oxidative damage occurred in lentil plants due to excess Cu, which was

418

also reported in different crops like radish, rice and mustard (Choudhary et al. 2012; Mostofa et

419

al. 2014; Yadav et al. 2018). However, lower MDA and H2O2 contents, and EL in ‘Ac + C (3.0)’

420

plants resulted in slightly or no yellowish damage symptoms on the leaves under Cu stress,

421

indicating an ameliorative role of acetate against the excess Cu-induced oxidative stress (Fig.

422

1A). We assumed that the healthy phenotype observed in acetate-pretreated lentil plants under

423

excess Cu might be attributed to the reduced accumulation of Cu in shoots, and better ROS-

424

scavenging capacity to reduce oxidative damage. Our results indeed indicated that ‘Ac + Cu

425

(3.0)’ lentil plants exhibited lower Cu content in the shoots compared with that of ‘Cu (3.0)’

426

plants (Fig. 1G). The acetate-induced reduction in shoot-Cu content might enable the aerial part

427

to maintain proper photosynthetic and osmoprotection functions as evidenced by the increased

428

Chl, Car and Pro contents (Table 1), and might also reduce the accumulation of excess Cu in

429

grains or edible parts of plants, which is an important factor for food safety (Clemens and Ma

430

2016). Thus, our findings indicated that acetate pretreatment is an effective approach for the

431

maintenance of Cu homeostasis in crop plants, particularly in aboveground part, enabling plant

432

survival under excess Cu conditions.

433

In plants, antioxidant defense system controls the ROS homeostasis under both favorable

434

and extreme conditions (Gill and Tuteja, 2010; You and Chan 2015). We observed severe

435

reductions in CAT and MDHAR activities, and in the AsA content in the ‘Cu (3.0)’ versus the

436

non-stressed ‘C’ seedlings, indicating an impairment of the antioxidant defense associated with

437

these parameters under such severe Cu stress conditions (Fig. 2A, C and G). Our findings are in

438

line with that of Islam et al. (2016), who reported a decrease in CAT activity in lentil plants

439

under Cu stress. In ROS-scavenging process, CAT degrades H2O2 into H2O and O2, while

440

MDHAR participates in the AsA-GSH cycle and regenerates AsA from monodehydroascorbate (

441

Foyer and Noctor 2011; Ighodaro and Akinloye 2018). Due to their relatively high cellular

442

concentrations, AsA and GSH can scavenge ROS, by donating electron to free radical molecules,

443

acting as scavengers or sacrificial nucleophiles, which consequently leads to interruption of the

444

radical chain reaction in various biological membranes (Foyer and Noctor 2011). In addition,

445

AsA is an essential cofactor in several biosynthetic pathways, while GSH acts as a sulfur source

446

necessary for biosynthesis, transport and detoxification process (Foyer and Noctor 2011). In the

447

present study, ‘Ac + Cu (3.0)’ plants exhibited an increase in CAT and MDHAR activities, while

448

showing a decrease in H2O2 content in comparison with the ‘Cu (3.0)’ plants (Figs. 1E, 2A and

449

2C). Furthermore, AsA contents were also increased by acetate treatment in Cu-stressed

450

seedlings (Fig. 2G). Consistently, the PCA analysis demonstrated a negative relationship

451

between the components of antioxidant defense (CAT, MDHAR and AsA) and oxidative stress-

452

related markers (Supplementary Fig. 2D). These results collectively indicated that CAT and AsA

453

were vital components for enhancement of Cu-induced oxidative stress tolerance in lentil plants

454

by acetate treatment.

455

Under heavy metal stress, GSH and GST play important roles not only in ROS

456

detoxification but also in metal chelation (Hasanuzzaman et al. 2017b; Nianiou-Obeidat et al.

457

2017). For instance, GST forms complex with GSH to detoxify xenobiotics and noxious

458

compounds (Hossain et al. 2012; Kumar and Trivedi et al. 2018). In the present study, ‘Ac + Cu

459

(3.0)’ lentil plants exhibited an increase in GST activity and GSH content in comparison with

460

‘Cu (3.0)’ plants (Fig. 2F and H), as also evidenced by the positive relationship between ‘Ac +

461

Cu (3.0)’ and GST and GSH detected by PCA (Supplementary Fig 2D). Therefore, the increased

462

GST activity and GSH content in ‘Ac + Cu (3.0)’ seedlings might contribute to (i) enhanced

463

antioxidant scavenging efficiency to reduce the oxidative damage and (ii) increased Cu chelation

464

to maintain the Cu homeostasis. Our finding was supported by the observations of similar

465

protective roles of GST and GSH in trehalose-induced Cu stress tolerance in rice (Mostofa et al.

466

2015) and castasterone-induced Cu stress tolerance in mustard (Yadav et al. 2018). The response

467

of antioxidant defense to a given stress is greatly dynamic, depending on dose, duration and type

468

of stress, as well as age and plant species (Adrees et al. 2015a; Mostofa et al. 2015; Abdelrahman

469

et al. 2019a; Abdelrahman et al. 2019b). In addition, the upregulation of one or two components

470

of the antioxidant defense system in plants could also be sufficient to improve their stress

471

tolerance (Abogadallagh 2010; Chiang et al. 2014; Nguyen et al. 2019). Therefore, we proposed

472

that exogenous acetate treatment modulated the activities of CAT, MDHAR and GST, as well as

473

the contents of AsA and GSH to enhance lentil tolerance to excessive Cu stress.

474

In conclusion, Cu stress caused severe reduction in growth parameters and photosynthetic

475

pigment contents, increased oxidative stress damage, disruption in antioxidant defense and

476

increased Cu uptake in roots and shoots of lentil plants. The application of acetate increased the

477

content of the osmoprotectant Pro, reduced root- and shoot-Cu accumulations, and enhanced the

478

antioxidant defense, which in turn reduced Cu-induced cellular oxidative damage, and

479

subsequently improved Cu stress tolerance in lentil seedlings (Fig. 3). Based on these results, we

480

suggest that utilization of acetate could be an economical (compared with hormones),

481

environmentally friendly (as acetate is rapidly degraded by microorganisms in soil), easy and

482

effective approach for the farmers to mitigate heavy metal stress in plants. To unravel the

483

acetate-mediated Cu stress tolerance mechanisms in detail, we need to explore how acetate

484

regulates the Cu uptake and transport at molecular level in a future study.

485

486

Conflicts of interest

487

The authors declare no conflicts of interest.

488 489

Acknowledgements

490

This research was funded by the Ministry of Education, Culture, Sports, Science and Technology

491

(MEXT), Japan. We thank Yoji Makita and Akinari Sonoda, National Institute of Advanced

492

Industrial Science and Technology, Kagawa, Japan for measuring the Cu contents in plants.

493 494

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Table 1. Effects of Cu stress on the biomass, physiological and biochemical traits in 10-day-old lentil seedlings pretreated with or Treatment C Cu (0.3) Ac + Cu (0.3) Cu (3.0) Ac + Cu (3.0) Treatment C Cu (0.3) Ac + Cu (0.3) Cu (3.0) Ac + Cu (3.0)

RDW (mg seedling–1)

SDW (mg seedling–1)

a

a

without Na-acetate.

RWF (%) a

3.73 ± 0.24 2.20 ± 0.10bc ns 2.67 ± 0.30b 1.16 ± 0.07d ns

10.07 ± 0.58 8.06 ± 0.54ab ns 8.46 ± 0.95ab 5.87 ± 0.22b

1.53 ± 0.18cd

6.83 ± 0.32b

Pro (µmol g–1 FW)

Chl (a + b) (mg g–1 FW)

Car (mg g–1 FW)

6.54 ± 0.24c 13.62 ± 0.99bc

4.65 ± 0.06a 4.39 ± 0.06ab

2.07 ± 0.02ab

bc

12.04 ± 0.62

b

24.24 ± 2.49 41.65 ± 5.66a

ns

4.72 ± 0.13

a

c

3.11 ± 0.02 4.04 ± 0.23b

ns

1.00 ± 0.00 0.59 ± 0.06b

ns

0.66 ± 0.14ab 0.21 ± 0.07c * 0.47 ± 0.05bc

bc

ns

1.82 ± 0.06

2.12 ± 0.07a 1.58 ± 0.04c 1.69 ± 0.09c

ns

Values represent the means ± standard errors calculated from three biological replicates (n = 3) for each treatment. For RDW and SDW, each replication was calculated as average of 10 roots (RDW) or shoots (SDW) from 10 seedlings. Different letters within the same column show significant differences at P ≤ 0.05 among treatments following a Tukey’s honest significant difference (HSD) test. “*” indicates significant difference, whereas “ns” indicates non-significant difference between ‘Cu (0.3)’ and ‘Ac + Cu (0.3)’ or ‘Cu (3.0)’ and ‘Ac + Cu (3.0)’ following Student’s t test. Root dry weight (RDW), shoot dry weight (SDW), relative water fraction (RWF), shoot proline (Pro) content, and leaf chlorophyll (a + b) [Chl (a + b)] and carotenoid (Car) contents. Control, C; 0.3 mM CuSO4, Cu (0.3); 10 mM Na-acetate + 0.3 mM CuSO4, Ac + Cu (0.3); 3.0 mM CuSO4, Cu (3.0); 10 mM Na-acetate + 3.0 mM CuSO4, Ac + Cu (3.0).

Fig. 1. Effects of Cu stress on the morphological appearance, oxidative stress markers and Cu uptake in 10-day-old lentil seedlings pretreated with or without Na-acetate. (A) Phenotypic appearance. (B-E) Malondialdehyde (MDA) content (B), other aldehyde content (C), electrolyte leakage (D), and hydrogen peroxide (H2O2) content (E) in shoots. (F-G) Root-Cu (F) and shootCu (G) contents. Bar charts show the means ± standard errors calculated from three biological replicates (n = 3) for each treatment. Different letters within the same graph show significant differences at P ≤ 0.05 among treatments following a Tukey’s honest significant difference (HSD) test. “*” indicates significant difference, whereas “ns” indicates non-significant difference between ‘Cu (0.3)’ and ‘Ac + Cu (0.3)’ or ‘Cu (3.0)’ and ‘Ac + Cu (3.0)’ following Student’s t test. Control, C; 0.3 mM CuSO4, Cu (0.3); 10 mM Na-acetate + 0.3 mM CuSO4, Ac + Cu (0.3); 3.0 mM CuSO4, Cu (3.0); 10 mM Na-acetate + 3.0 mM CuSO4, Ac + Cu (3.0). 25

Fig. 2. Effects of Cu stress on the responses of antioxidant defense systems of 10-day-old lentil seedlings pretreated with or without Na-acetate. (A) Catalase (CAT) activity, (B) ascorbate peroxidase (APX) activity, (C) monodehydroascorbate reductase (MDHAR) activity, (D) dehydroascorbate reductase (DHAR) activity, (E) glutathione reductase (GR) activity, (F) glutathione-S-transferase (GST) activity, (G) ascorbate (AsA) content, (H) reduced glutathione (GSH) content, (I) oxidized glutathione (GSSG) content, and (J) GSH/GSSG ratio in the shoots. 26

(K-L) Expression levels of GR (K) and APX (L) genes in leaves. Bar charts show the means ± standard errors calculated from three biological replicates (n = 3) for each treatment. Different letters within the same graph show significant differences at P ≤ 0.05 among treatments following a Tukey’s honest significant difference (HSD) test. “*” indicates significant difference, whereas “ns” indicates non-significant difference between ‘Cu (0.3)’ and ‘Ac + Cu (0.3)’ or ‘Cu (3.0)’ and ‘Ac + Cu (3.0)’ following Student’s t test. Control, C; 0.3 mM CuSO4, Cu (0.3); 10 mM Na-acetate + 0.3 mM CuSO4, Ac + Cu (0.3); 3.0 mM CuSO4, Cu (3.0); 10 mM Na-acetate + 3.0 mM CuSO4, Ac + Cu (3.0); nd, not detected.

27

Fig. 3. A probable model of acetate-induced mechanisms underlying Cu stress tolerance in lentil seedlings. High dose of Cu increased Cu accumulation in both roots and shoots, induced reactive oxygen species (ROS) production in shoots, decreased photosynthetic pigments, disturbed water balance and disrupted antioxidant defense by decreasing ascorbate (AsA) content. As a result, severe chlorosis, oxidative damage [e.g. increased malondialdehyde (MDA) content] and membrane damage [e.g. increased electrolyte leakage (EL)] were observed. On the other hand, acetate pretreatment reduced Cu accumulations in roots and shoots, maintained photosynthetic pigments and controlled ROS generation by improving AsA and glutathione (GSH) contents in the antioxidant defense system. As a result, acetate-pretreated lentil seedlings experienced lower oxidative stress and less chlorosis at excess Cu level, showing mitigation of Cu toxicity. Moreover, acetate application increased water balance by maintaining osmotic adjustment through proline (Pro) accumulation. The colored scheme for each trait was developed based on fold changes obtained from ‘acetate-treated Cu-stressed plants/control plants’ and ‘acetate-nontreated Cu-stressed plants/control plants’ comparisons. Catalase, CAT; glutathione disulfide, GSSG.

28

Highlights
Effects of acetate on improvement of lentil performance under Cu stress were studied.
Exogenous acetate improved photosynthetic pigments and osmoprotection in Cu-stressed plants.
Acetate alleviated Cu-induced oxidative damage by modulating antioxidant system of lentil.
Cu accumulations in both roots and shoots of lentil were decreased by acetate treatment.
Acetate application is a low cost solution for farmers to manage Cu toxicity in plants.

Conflicts of interest The authors declare that there are no conflicts of interest.