Melatonin alleviates iron stress by improving iron homeostasis, antioxidant defense and secondary metabolism in cucumber

Scientia Horticulturae 265 (2020) 109205

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Melatonin alleviates iron stress by improving iron homeostasis, antioxidant defense and secondary metabolism in cucumber

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Golam Jalal Ahammed, Meijuan Wu, Yaqi Wang, Yaru Yan, Qi Mao, Jingjing Ren, Ronghui Ma, Airong Liu, Shuangchen Chen* College of Forestry, Henan University of Science and Technology, Luoyang, 471023, PR China

ARTICLE INFO

ABSTRACT

Keywords: Melatonin Iron deficiency Iron toxicity Antioxidant enzyme Oxidative stress Secondary metabolism

Iron is an essential element for plants and animals; however, deficit or excess iron can result in stress on plants. In this study, we unveiled the mechanisms of shoot-based tolerance to iron toxicity by melatonin (N-acetyl-5methoxytryptamine) in cucumber plants. Both low and high iron (Fe) supply in hydroponics decreased growth and biomass accumulation, induced chlorosis and oxidative stress, and reduced chlorophyll content, photosynthesis rate and transpiration rate in cucumber leaves. Notably, the negative effect of low-Fe was more profound than that of high-Fe treatment. However, exogenous melatonin application alleviated those inhibitions in growth, biochemical and physiological parameters, which entailed an elevation of endogenous melatonin content and a reduction of electrolyte leakage, reactive oxygen species accumulation and lipid peroxidation by improving the activity and transcripts of antioxidant enzymes and secondary metabolism-related enzymes, and concentrations of phenols and flavonoids under low and high iron conditions after melatonin treatment. Analysis of iron content in leaves and roots revealed that melatonin significantly increased the iron content under low-Fe conditions, but it decreased the same under high-Fe conditions. Moreover, melatonin increased the transcript levels of FRO2 and IRT1 under low-Fe, but it decreased those transcripts under high-Fe, suggesting that melatonin plays a dual role in iron uptake under low and high iron conditions. This study expands the stress ameliorative role of melatonin in plants and may have potential implications in agronomic management of crops in low and high iron prone soils.

1. Introduction Iron is an essential micronutrient, playing a vital role in a number of plant physiologic and metabolic processes (Mahender et al., 2019; Zhu et al., 2019). It is involved in photosynthesis, respiration (mitochondrial), nucleic acid synthesis, protein functions and chlorophyll structure (Zhou et al., 2016; Wu et al., 2017; Li et al., 2019; Mahender et al., 2019;). However, iron deficit or excess, either is harmful to plants for normal growth and development, and causes a significant yield penalty in terms of quantity as well as quality (Mahender et al., 2019). Improper iron homeostasis in plants eventually affects human health through dietary pathway (He et al., 2013a, b). Therefore, understanding the mechanisms of iron homeostasis and developing strategies to overcome iron stress have been a key challenge to plant scientists (Li et al., 2019). Nonetheless, the complexity of iron stress tolerance and inadequate knowledge of underlying gene networks restrict target breeding approaches for iron acquisition and tolerance (Mahender et al., 2019). Thus, development of efficient agronomic management ⁎

strategies coupled with exogenous application of plant growth regulators for mitigating iron stress has emerged as a promising research avenue (Pavlovic et al., 2013; Kong et al., 2014; Lei et al., 2014; Zhou et al., 2016; Chaiwong et al., 2018; Dos Santos et al., 2019; Kaya et al., 2019). Despite being the 4th most abundant element in the earth’s crust, iron deficiency has been as a major problem in many soils, particularly in soils with high pH (alkaline soils or calcareous soils) (Wang et al., 2012). In general, iron deficiency in plants is not due to the absence of iron in soils, rather because of the low Fe solubility and unavailability of ferrus (Fe2+) iron at high soil pH (Wu et al., 2017; Li et al., 2019). Plants use multiple strategies to adapt to the Fe-deficient conditions, which are broadly classified into two categories, strategy I (in dicots and non-graminaceous monocots) and strategy II (graminacesous monocots) (Mahender et al., 2019). The strategy I includes both morphological (such as abundant root hairs in sub-apical root zones, transfer cells) and physiological (rhizosphere acidification, exudation of phenolic compounds) adaptations that enhance solubility as well as

Corresponding author. E-mail address: [email protected] (S. Chen).

https://doi.org/10.1016/j.scienta.2020.109205 Received 1 December 2019; Received in revised form 30 December 2019; Accepted 17 January 2020 0304-4238/ © 2020 Elsevier B.V. All rights reserved.

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availability of Fe (ferrous) in the rhizosphere (Wu et al., 2017). Then ferrus (Fe2+) is transported via the plasma membrane by the action of specific metal transport mechanism involving the membrane-bound FERRIC REDUCTASE-OXIDASE2 (FRO2) and divalent metal ion transporter IRON-REGULATED TRANSPORTER1 (IRT1). Interestingly, iron deficiency induces the expression of FRO2 and IRT1 in strategy I plants including Arabidopsis thaliana and Cucumis sativus (Wang et al., 2012; Pavlovic et al., 2013; Zhou et al., 2016). Moreover, iron starvation causes chlorosis, particularly leaf interveinal chlorosis, which is a typical sign of iron deficiency in plants (Zhou et al., 2016; Mahender et al., 2019). Iron deficiency-induced chorosis is attributed to a significant reduction in leaf chlorophyll content, which also affects photosynthetic capacity of leaves (Pavlovic et al., 2013; Zhou et al., 2016; Kaya et al., 2019). Moreover, iron deficiency impairs ion homeostasis and induces production of reactive oxygen species (ROS), leading to oxidative stress (Kong et al., 2014). ROS overload can damage lipids, proteins and nucleic acids (Ahammed et al., 2019). As a response, plants elevate the activity of antioxidant enzymes to remove toxic-levels of ROS. Therefore, enhancing plant antioxidant potential is considered as one of the useful strategies to mitigate abiotic stress caused by deficiency or excess of metal ions (Hasanuzzaman et al., 2019). Iron toxicity is caused by excess iron uptake, a major problem in Asia and West Africa, mainly in the flooded soils (Wu et al., 2017; Li et al., 2019). Under iron excess conditions, catalytic Fe2+ gives rise to highly toxic ROS, such as hydroxyl radical (•OH) from hydrogen peroxide (H2O2) through Fenton reaction (Mahender et al., 2019; SiqueiraSilva et al., 2019). ROS accumulation due to excess iron induces oxidative stress, leading to distinct symptoms, such as leaf bronzing (especially in rice), chlorosis and growth reduction in plants (Quinet et al., 2012; Chalmardi et al., 2013; Wu et al., 2017). Iron excess also interrupts other nutrient acquisition and causes nutritional disorder (Zhao et al., 2018; Bresolin et al., 2019). Moreover, iron toxicity results in water stress as a secondary stress (Majerus et al., 2007). Plants use different mechanisms to enhance tolerance to Fe toxicity (Wu et al., 2017; Siqueira-Silva et al., 2019). These include root-based mechanisms, such as inhibiting excessive Fe absorption by forming a physical barrier namely iron plaque on roots and retaining Fe in specific root compartments as metabolically inactive forms (Mahender et al., 2019). On the other hand, the shoot-based tolerance mechanism entails the strengthening of antioxidant system for efficient ROS scavenging by antioxidants, such as glutathione, ascorbate, phenolics as well as antioxidative enzymes, such as, superoxide dismutase and ascorbate peroxidase etc. (Majerus et al., 2007; Wu et al., 2017). Melatonin (N-acetyl-5-methoxytryptamine) is a ubiquitous regulatory molecule with multiple functions in animals and plants (Arnao and Hernandez-Ruiz, 2018; Hasan et al., 2019; Kanwar et al., 2018; Ahammed et al., 2019). It is involved in specific plant physiological and developmental processes, such as root formation (mainly lateral root), flowering, ripening of fruits and senescence of leaves (Kanwar et al., 2018). In addition to its vital role in plant growth and development, melatonin can enhance plant adaptation to biotic and abiotic stresses, such as extreme temperatures, drought, salinity, UV radiation and heavy metals (Arnao and Hernandez-Ruiz, 2018; Hasan et al., 2018; Ahammed et al., 2019; Zhan et al., 2019). Melatonin acts as a strong antioxidant in plants. Moreover, melatonin-induced enhancement in antioxidant enzyme activity confers tolerance to oxidative stress in plants under abiotic stresses, such as high temperature, nutrient deficiency and cadmium stress (Ahammed et al., 2019; Hasan et al., 2018, 2019). Under heavy metal stress, melatonin inhibits metal uptake and translocation to aerial plant parts in tomato (Hasan et al., 2019). However, under Fe-deficit conditions, melatonin improves iron acquisition in A. thaliana (Zhou et al., 2016) and Capsicum annuum (Kaya et al., 2019), suggesting the complexity of melatonin actions under different levels of metal ions. Nonetheless, the role of melatonin in iron excess conditions remains unclear in plants. Cucumber is one of the widely cultivated vegetable crops, highly

popular throughout the world. Previous studies have revealed the sensitivity of cucumber plants to iron deficiency (Wang et al., 2012; Pavlovic et al., 2013); however, its response to excess iron remains elusive. Considering the multifarious stress protective roles of melatonin, we administered exogenous melatonin in cucumber seedlings to unveil its role in iron deficiency and iron toxicity by growing the plants under low iron and high iron conditions, respectively. Our results revealed a positive role of melatonin in mitigating iron stress (both deficiency and toxicity)-induced chlorosis, growth inhibition and oxidative stress by modulating the activity of antioxidant enzymes and secondary metabolism. Moreover, the stimulatory and inhibitory effects of melatonin on iron uptake under low-Fe and high-Fe conditions, respectively, indicate far more complexity in melatonin action under different levels of an essential element (i.e. Fe). The study unraveled a stress ameliorative role of melatonin in iron stress, which could be useful for agronomic management of crops. 2. Materials and methods 2.1. Plant materials and treatments Cucumber seeds (Cucumis sativus L. cv. Jinyou 28) were germinated in vermiculite and allowed to grow until the cotyledons were fully expanded. Then each batch of 6 seedlings were transferred to a plastic tank to hydroponically grow them in half-strength Hoagland’s nutrient solution. Seedlings were grown in growth chambers maintaining the temperature at 25/18 °C (day/night), a 14 h light period (600 μmol m−2 s-1 PPFD) and a 10 h dark period. After five days, the seedlings (foliage) were sprayed with either 100 μM melatonin or distilled water, and 24 h after the melatonin treatment, the seedlings were divided into 6 groups and treated with 3 levels of iron (normal Fe, low Fe and high Fe) in Hoagland’ nutrient solution, which resulted in 6 treatments in total, such as (1) control (normal Fe supply and sprayed with distilled water), (2) melatonin (MT; normal Fe supply and sprayed with 100 μM melatonin), (3) low-Fe, (4) low-Fe + MT, (5) high-Fe, and (6) high-Fe + MT. There were 24 cucumber plants per treatment. The concentration of low-Fe was 1/10th of normal i.e. Fe-EDTA 3 mg/L, and that of high-Fe was 3-times of normal Fe supply i.e. Fe-EDTA 90 mg/L. The modified Hoagland’s nutrient solution included: Ca(NO3)2∙4H2O, 1200 mg/L; KNO3, 799 mg/L; KH2PO4, 207 mg/L; MgSO4∙7H2O, 366 mg/L; NaFeIIIEDTA, 30 mg/L; MnSO4∙4 H2O, 5 mg/L; CuSO4∙5 H2O, 0.9 mg/L; ZnSO4∙7 H2O, 1.1 mg/L; (NH4)6Mo7O24∙4H2O, 0.9 mg/L; H2BO3, 4.1 mg/L. The pH of the nutrient solution was adjusted daily at 6 ± 0.2 and nutrient solutions with different levels of Fe were replaced with freshly prepared nutrient solutions at 5-day intervals. Melatonin concentration was chosen based on previous studies (Hasan et al., 2019; Sun et al., 2019), and our preliminary experiment (data not shown) and the treatment was repeated at 5-day intervals. After 3 weeks, samples were harvested for different analyses. 2.2. Determination of biomass, iron content, chlorophyll content and leaf gas exchange Three weeks after the imposition of iron stress, leaf gas exchange and the lengths and fresh weights of shoots and roots were measured. After measuring the fresh weights of shoots and roots of 12 cucumber plants from each treatment, plant samples (from six plants) were kept in an oven at 75 °C for 72 h, and then dry weights were measured with an electrical balance. Dried plant samples (approximately 100 mg per sample unit) were digested with HNO3:HClO4 (4:1, v/v) and the total Fe content was analyzed with an inductively coupled plasma mass spectrometer (ICP-MS, Agilent 7500a, Agilent Technologies Inc., Santa Clara, CA, USA) and the content was expressed on a dry-weight (DW) basis (Zhang et al., 2019). Chlorophyll (Chl) such as Chl a and Chl b contents were measured in the fully expanded leaves. About 0.1 g leaf tissue was placed in a tube containing 96 % ethanol. The tubes were 2

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kept in the dark for 24 h. The absorbance of the pigment extract was measured at 470 nm, 649 nm, and 665 nm wave length with a spectrophotometer (UV-2450, Shimadzu, Japan) and chlorophyll contents were calculated as previously described (Lichtenthaler and Wellburn, 1983). Gas exchange parameters, such as CO2 assimilation rate, stomatal conductance, intercellular CO2 concentration, and transpiration rate were measured in the second fully expanded cucumber leaves with a LI-6400 Portable Photosynthesis System (LI-6400; LICOR, Lincoln, NE, United States) following the conditions described previously (Ahammed et al., 2018).

homogenates were centrifuged at 15,000 g for 15 min, the supernatants were collected. PAL activity was determined by measuring the formation of cinnamic acid at 290 nm as described previously (Li et al., 2018). The PPO activity was determined by recording the reduction in absorbance of 2-nitro-5-thiobenzoic acid at 412 nm following reaction with quinones produced from enzymatic oxidation of 3,4-dihydroxyphenylalanine (Ahammed et al., 2017).

2.3. Root activity, electrolyte leakage, ROS, and membrane lipid peroxidation assay

Total RNA was extracted from ∼100 mg plant tissues using total RNA extraction kit (Tiangen, Shanghai, China). A 1 μg aliquot of total RNA was reverse-transcribed to generate cDNA using a ReverTra Ace qPCR RT Kit (Toyobo, Japan). The SYBR Green PCR Master Mix (Takara, Tokyo, Japan) was used for qRT-PCR assay and the analysis was performed with LightCycler@ 480 II Real-Time PCR Detection System (Roche, Basel, Swiss) under the default thermal cycling conditions with an added melting curve. Gene-specific primers for qRT-PCR were designed for the genes Fe-SOD, POD, CAT, PAL, FRO2 and IRT1; and the marker gene actin was used as internal control (Supplementary Table S1). The PCR was run at 95 °C for 3 min, followed by 40 cycles at 95 °C for 30 s, then 30 s at 58 °C, and 30 s at 72 °C. Relative transcript levels were calculated as previously described (Livak and Schmittgen, 2001)

2.6. Extraction of total RNA and quantitative real-time PCR (qRT-PCR) assay

The triphenyl tetrazolium chloride (TTC) method was used to determine root activity as previously described (Chen et al., 2017). Malondialdehyde (MDA) content was measured as an index of lipid peroxidation in leaves according to the thiobarbituric acid reaction method as described previously (Ahammed et al., 2019). Leaf H2O2 content was analyzed spectrophotometrically by a peroxidase assay according to Willekens et al. (1997) as described in detail previously (Ahammed et al., 2019). Superoxide (O2−%) accumulation in leaves was visualized by nitroblue tetrazolium chloride (NBT) staining as previously described (Zhang et al., 2019). O2−% production rate in leaves was assayed according to Elstner and Heupel, 1976) using the sulfanilamide method by measuring the reaction at 530 nm. O2−% production rate was calculated from a standard curve of NaNO2 reagent. Electrolyte leakage (%) was measured recording the electrical conductivity (EC) of leaf suspension before (EC1) and after autoclaving (EC2) as described previously (Ahammed et al., 2019). The percentage electrolyte leakage was calculated as follows: Electrolyte leakage (%) = 100×EC1/EC2.

2.7. Statistical analysis All data were subjected to the analysis of variance and analyzed with SPSS 20.0 statistical software package. To determine the treatment effect, we used Tukey’s least significant difference test. The difference was considered significant at P < 0.05 and indicated by different letters.

2.4. Melatonin content and secondary metabolite assay Endogenous melatonin content in cucumber leaves was quantified by HPLC analysis as previously described (Ahammed et al., 2019). Total phenol content was analyzed using a method based on Folin-Ciocalteu’s reagent reduction. Total flavonoid concentration was assayed spectrophotometrically (Li et al., 2018). Briefly, flavonoids were extracted by homogenizing leaf samples in 70 % ethanol (v/v) at 100 °C, and the absorbance of aqueous extract was measured at 510 nm for total flavonoid concentration by using AlCl3 method and rutin was used as the standard to calculate total flavonoid concentrations. For the determination of free radical scavenging activity in leaves, the 2,2-diphenyl-1picrylhydrazyl (DPPH) was used as previously described (Tadolini et al., 2000).

3. Results 3.1. Melatonin improves plant growth and biomass accumulation Both iron deficit and excess conditions resulted in significant growth inhibitions (Fig. 1). Low Fe supply decreased shoot length and root length by 30.7 % and 42.6 %, whereas high Fe supply decreased those parameters by 27.9 % and 39.25 %, respectively compared with the control (Fig. 1A). However, exogenous melatonin application on stressed plants significantly increased the lengths of shoots (+35.7 %) and roots (+55.15 %) under low-Fe supply. Likewise, melatonin also increased the lengths of shoots (+29.5 %) and roots (+62.5 %) under high-Fe supply compared with the respective iron treatments without melatonin. Similarly, low-Fe supply decreased the fresh weights of shoots and roots by 58.8 % and 65.7 %, whereas high-Fe supply resulted in 30.2 % and 32.1 % decreases in fresh weights of shoots and roots, respectively compared with the control (Fig. 1B). Consistent with the fresh biomass, shoot dry weight and root dry weight decreased by 63.4 % and 49.2 % in low-Fe, and 33.8 % and 29.3 % in high-Fe supply, respectively compared with the control (Fig. 1C). However, exogenous melatonin significantly increased biomass accumulation under low-Fe and high-Fe conditions. More precisely, melatonin increased shoot and root dry weight by 81.8 % and 37.7 % under low-Fe supply, and 27.8 % and 23.4 % under high-Fe supply, respectively compared with the only iron stress without melatonin application. All these results suggest that melatonin could improve growth and biomass accumulation in cucumber seedlings under both iron deficit and iron excess conditions.

2.5. Determination of activity of enzymes For the extraction of antioxidant enzymes, 0.3 g leaf tissues were homogenized with 3 ml ice-cold 25 mM HEPES buffer (pH 7.8) containing 0.2 mM EDTA, and 2% (w/v) insoluble polyvinylpyrrolidone (PVP). The homogenates were centrifuged at 12,000 g at 4 °C for 20 min. The supernatants obtained from the homogenates were used for the assay of antioxidant enzyme activity. Superoxide dismutase (SOD) activity was assayed by measuring its ability to inhibit the photoreduction of nitro blue tetrazolium (Giannopolitis and Ries, 1977). Peroxidase (POD) as well as catalase (CAT) activity was measured according to the method described previously (Cakmak and Marschner, 1992). The activity of enzymes were expressed on the basis of protein content which was estimated using bovine serum albumin (BSA) as described elsewhere (Bradford, 1976). To extract phenylalanine ammonia-lyase (PAL) and polyphenol oxidase (PPO) from leaves, leaf samples (0.3 g) were homogenized in 50 mM sodium phosphate buffer (pH 8.8) containing 2 % (w/v) PVP, 2 mM EDTA, 18 mM g-mercaptoetanol and 0.1 % v/v Triton X-100. The

3.2. Melatonin enhances photosynthetic pigment content, endogenous melatonin content and photosynthesis rate Both iron deficiency and iron toxicity caused leaf chlorosis; 3

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Fig. 1. Effect of low- and high- iron (Fe) supply on growth and biomass accumulation as influenced by melatonin (MT) in cucumber seedlings. (A) Shoot length, (B) Root length, (C) Shoot fresh weight (FW), (D) Root FW, (E) Shoot dry weight (DW), and (F) Root DW. Cucumber seedlings were treated with 3 mg L−1 Fe-EDTA (lowFe) and 90 mg L−1 Fe-EDTA (high-Fe) in hydroponics for twenty one days and leaves were sprayed with 100 μM melatonin at 5-day intervals. Control plants were supplied with Hoagland’s nutrient solutions containing 30 mg L−1 Fe-EDTA. Data are presented as the mean and the error bars indicate standard deviation (n = 12 for length and fresh weight analysis, and n = 6 for dry weight analysis). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test. Fig. 2. Effect of different levels of iron (Fe) and melatonin (MT) on leaf phenotype, photosynthetic pigment content and endogenous melatonin content in cucumber seedlings. (A) Leaf phenotype, (B) Chlorophyll a (Chla), (C) Chlorophyll b (Chl b) and (D) melatonin content in leaves. Twenty one days after the exposure of roots to different levels of Fe, leaf samples for Chlorophyll assay were collected and photographs were taken. Scale bars =1 cm. Data are presented as the mean of 3 replicates (± standard deviation). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test.

however, the symptom was more profound under low-Fe supply than that in high-Fe supply. Interestingly, exogenous melatonin application remarkably alleviated chlorosis under iron stress. Consistent with the leaf phenotypes, low-Fe supply decreased the chlorophyll a (chl a) and chl b content by 51.0 % and 54.3 %, respectively (Fig. 2B-C). High-Fe supply also decreased the chl a and chl b content by 20.9 % and 29.3 %, respectively compared with the control. Interestingly, exogenous melatonin application significantly increased the chl a and chl b content by 34.6 and 47.9 % in low-Fe supply, and 18.3 % and 35.9 % in high-Fe supply, respectively compared with the respective only iron treatments. Analysis of melatonin content revealed that both iron deficit and excess

conditions induced endogenous melatonin levels in cucumber leaves; however, the content of melatonin in low-Fe conditions was significantly higher than that in high-Fe conditions (Fig. 2C). More precisely, the low- and high-Fe conditions resulted in a 2.4- and 1.8-fold increase in leaf melatonin content, respectively compared with the control. Interestingly, exogenous melatonin application caused a further increase in endogenous melatonin levels under both low-and highFe conditions, suggesting that exogenous melatonin application stimulated endogenous melatonin content under iron stress. Analysis of gas exchange parameters revealed that low-Fe and highFe conditions significantly decreased CO2 assimilation rate (Pn) by 62.8 4

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Fig. 3. Leaf gas exchange as influenced by melatonin (MT) and different levels of iron (Fe) treatments. (A) CO2 assimilation rate (Pn), (B) Stomatal conductance (Gs), (C) Intercellular CO2 concentration (Ci), and (D) Transpiration rate (Tr) in cucumber leaves. Twenty one days after the exposure of roots to different levels of Fe, the gas exchange parameters were measured in the second fully expanded cucumber leaves (n = 6). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test.

% and 40.5 %, stomatal conductance (Gs) by 48.3 % and 27.8 %, and transpiration rate (Tr) by 42.0 % and 24.6 %, respectively compared with the control (Fig. 3). Notably, the reduction in Gs and associated changes in the Pn and Tr were more profound under low-Fe conditions than that in high-Fe conditions. However, exogenous application of melatonin significantly improved the Pn, Gs and Tr under both low- and high-Fe conditions compared with the sole treatment of low-Fe and high-Fe in cucumber plants. Notably, intercellular CO2 concentrations were not significantly altered by the low- and high-Fe stress, suggesting that non-stomatal factors were mainly involved in iron stress-induced reduction in photosynthesis (Fig. 3C)

3.4. Melatonin activates antioxidant enzymes As shown in Fig. 5A, the activity of antioxidant enzymes, such as SOD, POD and CAT, all were inhibited by either low-Fe or high-Fe stress. For instance, SOD activity was decreased by low-Fe and high-Fe stress by 48.5 % and 51.1 %, respectively (Fig. 5A). The activity of POD was also reduced by those iron treatments by 40.5 and 59.6 %, respectively and the POD activity in high-Fe was significantly lower than that in low-Fe. The CAT activity was dramatically suppressed by low-Fe by 61.3 %. Although high-Fe supply also caused a significant decrease in CAT activity, it was higher than that in low-Fe. Interestingly, exogenous melatonin application significantly elevated the activity of these enzymes under both deficit and excess iron conditions (Fig. 5A). Meanwhile, the transcript levels of Fe-SOD, POD and CAT were significantly suppressed by both low- and high-Fe conditions (Fig. 5B). However, POD expression was not significantly reduced by low-Fe compared with the control, but the transcript levels of Fe-SOD and CAT in low-Fe conditions were significantly lower than that of high-Fe treatment. Notably, exogenous melatonin application on low- and highFe-stressed plants increased the transcript levels of Fe-SOD by 3.2- and 3.3- fold, POD by 2.4- and 2.9-fold, and CAT by 4.8 and 1.9-fold, respectively compared with the respective only low-Fe and high-Fetreated cucumber plants. Like antioxidant enzyme activity, DPPH free radical scavenging capacity was significantly inhibited by low-Fe followed by high-Fe supply (Supplementary Figure S2). However, exogenous melatonin application significantly increased the DPPH by 131.3 % and 55.6 % in low-Fe and high-Fe conditions, respectively.

3.3. Melatonin alleviates iron stress-induced oxidative stress Iron stress triggers ROS production, which can cause damage to biological membranes. NBT staining showed that O2•− accumulation remarkably increased under low- and high-Fe conditions in leaves (Fig. 4A). In addition, low- and high-Fe supply significantly increased H2O2 content by 177.8 % and 153.6 %, respectively (Fig. 4B). Consistent with NBT staining results, O2•− production rate increased by 103.5 % and 112.6 % under low- and high-Fe conditions, respectively (Fig. 4C). However, exogenous melatonin application significantly decreased the iron stress-induced ROS accumulation in cucumber leaves. To assess the injury to roots due to impaired or excess iron supply, we analyzed the root activity. The results showed that the root activity was significantly decreased by 36.7 % and 32.0 % under low-Fe and high-Fe supply, respectively (Supplementary figure S1). Although exogenous melatonin had no effect on root activity under control conditions, melatonin significantly improved the root activity in Fe-depleted and Fe excess conditions. On the other hand, electrolyte leakage (%) significantly increased by 74.8 % and 80.1 % under low-Fe and high-Fe conditions, respectively (Fig. 4D). However, exogenous melatonin application significantly decreased the electrolyte leakage (%), more profoundly in high-Fe stressed plants. Consistent with the ROS accumulation, the MDA content was also elevated by low-Fe and high-Fe by 172.1 % and 138.2 %, respectively compared with the control, suggesting that iron deficiency and iron toxicity both induced oxidative stress in cucumber (Fig. 4E). However, exogenous melatonin application significantly decreased the accumulation of ROS and MDA under low-Fe and high-Fe conditions. These results suggest that melatonin could alleviate iron stress-induced oxidative stress in cucumber.

3.5. Melatonin promotes secondary metabolism The PAL activity was significantly reduced by both low-Fe and highFe supply compared with the control. Exogenous melatonin not only increased PAL activity under control condition, but also improved PAL activity under low-Fe and high-Fe stress (Fig. 6A). Consistently, the transcript levels of PAL were suppressed by low-Fe, but not by high-Fe; however, exogenous melatonin application significantly increased the transcript levels of PAL by 2.9 and 2.2-fold under low- and high-Fe conditions, respectively (Supplementary Figure S3). Meanwhile, low-Fe condition significantly decreased PPO activity by 52.2 % compared with the control (Fig. 6B). Although PPO activity was slightly higher in high-Fe conditions, it was also significantly lower than the control. 5

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Fig. 4. Effect of low- and high- iron (Fe) supply on membrane stability and reactive oxygen species accumulation as influenced by melatonin (MT) in cucumber seedlings. (A) In situ O2•− accumulation, (B) H2O2 content, (C) O2•− content, (D) electrolyte leakage (%), and (E) malondialdehyde (MDA) content in leaves. Twenty one days after the exposure of roots to different levels of Fe, leaf samples were harvested for the assays. Foliage was sprayed with melatonin before administration of iron stress, and melatonin was reapplied at 5-day intervals. Data are presented as the mean of 3 replicates (± standard deviation). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test.

in leaves and roots, respectively. qRT-PCR assay of FRO2 and IRT1 expression revealed that the transcript levels of FRO2 and IRT1 were significantly increased by both low-and high-Fe supply (Fig. 7C,D). Iron deficiency caused a 4.3- and 10.2-fold increase in FRO2 and IRT1 expression, while the iron toxicity increased those transcripts by 12.6- and 28.4-fold, respectively compared with the control. Interestingly, exogenous melatonin application significantly increased the transcript levels of FRO2 and IRT1 under low-Fe stress; however, a reverse effect of melatonin on the transcript levels of FRO2 and IRT1 was observed under high-Fe conditions. These results suggest that melatonin might play a dual role in iron homeostasis under iron deficiency and iron toxicity in cucumber seedlings.

However, exogenous melatonin application significantly increased the PPO activity in both low-Fe and high-Fe conditions. Consistent with the enzyme activity, total phenol and flavonoid concentrations were significantly decreased by low-Fe and high-Fe treatments (Fig. 6C, D). High-Fe supply caused the maximum decrease (165.1 %) in flavonoid concentrations (Fig. 6D). However, melatonin application significantly increased the phenols and flavonoid concentration in low and high iron stress in cucumber leaves. Briefly, exogenous melatonin increased the concentration of flavonoids by 57.2 % and 165.1 % in low-Fe and highFe conditions, respectively compared with the respective only iron treatments. 3.6. Melatonin alters Fe acquisition

4. Discussion

While iron concentrations in both leaves and roots were decreased by low-Fe supply, the iron concentrations in those tissues were significantly increased by high-Fe supply. Leaf Fe content decreased by 52.6 % in low-Fe and increased by 113.2 % in high-Fe compared with the control (Fig. 7A). Likewise, root Fe content decreased by 58.6 % in low-Fe supply and increased by 96.5 % in high-Fe supply, respectively (Fig. 7B). Although exogenous melatonin application did not alter the Fe content in leaves and roots under the control conditions, the melatonin administration significantly increased the iron content under lowFe supply and decreased the iron content under high-Fe supply. More precisely, melatonin increased leaf and root iron content by 61.1 and 59.2 %, respectively under low-Fe supply, whereas under high-Fe conditions, melatonin decreased the iron content by 26.1 % and 23.8 %

Iron is an essential element for all organisms including plants, but Fe deficit and Fe excess, both result in physiological and developmental disorders in plants (Li et al., 2019; Mahender et al., 2019). Despite enormous research efforts towards development of iron toxicity tolerant crop cultivars, the achievements remains far away from the goals due to largely unknown gene networks responsible for plant tolerance to iron stress (Li et al., 2019). Apart from breeding, different growth regulators and signaling molecules, such as silicon, salicylic acid, nitric oxide, polyamines and abscisic acid have been shown to improve plant tolerance to nutrient deficiency, including low iron stress, indicating their potential uses in agronomic management of crops (Pavlovic et al., 2013; Kong et al., 2014; Lei et al., 2014; Zhou et al., 2016; Kaya et al., 2019; 6

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Fig. 5. Activity and gene expression of antioxidant enzymes in cucumber leaves as influenced by melatonin (MT) and different levels of iron (Fe) treatments. (A) Superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) activity, and (B) relative transcript levels of Fe-SOD, POD and CAT. Twenty one days after the exposure of roots to different levels of Fe, leaf samples were harvested for the assays. Foliage was sprayed with melatonin before administration of iron stress, and melatonin was reapplied at 5-day intervals. Data are presented as the mean of 3 replicates (± standard deviation). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test.

Zhang et al., 2019). Here we showed that melatonin, a multifunctional molecule in plants, can confer tolerance to low-Fe and high-Fe supplyinduced stress in cucumber plants. Melatonin-induced enhanced tolerance to iron stress was attributed to increased accumulation of photosynthetic pigments, enhanced CO2 assimilation rate, reduced lipid peroxidation and ROS accumulation, enhanced antioxidant enzyme activity and secondary metabolism, and modulation of iron homeostasis in leaves and roots of cucumber plants (Fig. 1–7).

4.1. Melatonin-induced photosynthesis contributes to biomass production Growth retardation is the ultimate result of any abiotic stress, mostly because of the stress-induced physiological, metabolic and nutritional disorders. Previous studies have revealed that both iron deficit or excess could result in growth inhibition, leading to significant crop yield losses (Pavlovic et al., 2013; Zhou et al., 2016; Wu et al., 2017; Kaya et al., 2019; Li et al., 2019). Consistent with this, we found that both low-Fe and high-Fe conditions drastically reduced cucumber growth and biomass accumulation (Fig. 1). However, the comparison of the fresh and dry weights of shoots and roots suggests that low-Fe Fig. 6. Activity of secondary metabolism-related enzymes and content of secondary metabolites in cucumber leaves as influenced by melatonin (MT) and different levels of iron (Fe) treatments. (A) Phenyl ammonia-lyaze (PAL) activity, (B) Polyphenol oxidase (PPO) activity, (C) Total phenols, and (D) Total flavonoids content in cucumber leaves. Twenty one days after the exposure of roots to different levels of Fe, leaf samples were harvested for the assays. Foliage was sprayed with melatonin before administration of iron stress, and melatonin was reapplied at 5-day intervals. Data are presented as the mean of 3 replicates (± standard deviation). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test.

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Fig. 7. Iron accumulation and expression of iron acquisition-related genes in cucumber plants as influenced by melatonin (MT) and different levels of iron (Fe) treatments. (A) Fe content in leaves, (B) Fe content in roots, (C) Relative expression of FERRIC REDUCTASE-OXIDASE2 (FRO2) and (D) IRON-REGULATED TRANSPORTER1 (IRT1) in roots. Twenty one days after the exposure of roots to different levels of Fe, plant samples were harvested for the assays. Foliage was sprayed with melatonin before administration of iron stress, and melatonin was reapplied at 5-day intervals. Data are presented as the mean of 3 replicates (± standard deviation). Means denoted by the same letters do not significantly differ at P < 0.05 according to a Tukey's test.

treatment was more deleterious than high-Fe in terms of biomass accumulation. This could be possibly due to disturbance in key physiological and metabolic processes, as Fe acts as a co-factor of numerous enzymes and is involved in the maintenance of photosynthesis and respiration (Pavlovic et al., 2013; Mahender et al., 2019). Consistent with this, the CO2 assimilation rates were significantly decreased by low- and high-Fe stress, which were possibly attributed to non-stomatal limitations (Fig. 3; Zhang et al., 2020). Notably, chloroplasts, the key organelle of photosynthesis, are the store-house of cellular iron, which localize about 80 % of total iron in plants. Thus iron deficiency or toxicity interrupts chloroplast functions, by increasing the vulnerability of the photosystem II to photoinhibtion, leading to a reduction in the photosynthetic rate (Quinet et al., 2012). Here, we found a significant reduction in photosynthetic pigment content under low-Fe or high-Fe stress; nonetheless, leaf chlorosis was more obvious in low-Fe than that in high-Fe conditions (Fig. 2). Chlorophylls are the vital molecule in photosynthetic light reaction because of their role in light harvesting. Decreased chlorophyll accumulation is expected to reduce overall photosynthetic activity of cucumber plants, which would also affect biomass accumulation as a result.

antioxidant enzymes. Melatonin is well-recognized as an important antioxidant and it also stimulates plant antioxidant systems under stressful conditions (Ahammed et al., 2019). Previous studies showed that exogenous melatonin application drastically reduced ROS and MDA accumulation under high temperature, cold, drought, salinity, nutrient deficiency and heavy metal stress in a range of plant species (Arnao and Hernandez-Ruiz, 2018; Kanwar et al., 2018; Ahammed et al., 2019; Hasan et al., 2019; Kaya et al., 2019). Consistent with this, we found that exogenous melatonin application significantly increased endogenous melatonin content but decreased the H2O2 and O2•− accumulation, electrolyte leakage and MDA content in cucumber leaves in the current study (Fig. 2D and 4). Moreover, melatonin application increased activity of antioxidant enzymes, such as SOD, POD and CAT and the transcript levels of their biosynthetic genes, such as Fe-SOD, POD and CAT under low- and high-Fe conditions, suggesting that melatonin-induced enhanced antioxidant enzyme activity potentially functioned in scavenging ROS and minimizing oxidative stress in cucumber plants. These results are in good agreement with the role of melatonin in alleviating oxidative stress by improving the antioxidant enzyme activity in many previous studies (Arnao and Hernandez-Ruiz, 2018; Kanwar et al., 2018).

4.2. Melatonin alleviates oxidative stress by improving antioxidant potential

4.3. Melatonin stimulates secondary metabolism to alleviate iron stress

Reactive oxygen species (ROS) are the byproduct of aerobic metabolism in cells and their production is triggered in response to abiotic stress (Arnao and Hernandez-Ruiz, 2018; Ahammed et al., 2019). Despite a well-established role of ROS in stress signaling, excessive ROS accumulation could induce oxidative stress (Wu et al., 2017). We found that H2O2 and O2•− accumulation significantly increased under lowand high-Fe conditions, which was well corroborated with the corresponding increases in relative electrolyte leakage (%) and MDA content (Fig. 4). MDA is an indicator of membrane lipid peroxidation and its higher concentrations under low- and high-Fe conditions indicate the induction of oxidative stress (Ahammed et al., 2019, 2020). Our results are consistent with a recent report that showed that iron deficiency could induce ROS accumulation and oxidative stress in pepper plants (Kaya et al., 2019). Notably, excess Fe conditions also enhance ROS accumulation due to the catalytic effect of Fe2+ through Fenton reaction (Wu et al., 2017). Moreover, the activity of antioxidant enzymes was attenuated by low- and high-Fe treatment, suggesting that ROS overload might either suppress their biosynthesis or inactivate those

In plants, secondary metabolism provides a large number of compounds that basically function to enhance plant resistance or tolerance to biotic and abiotic stressors (Li et al., 2016, 2017). Phenyl propanoid pathway is the key biosynthetic route for the production of numerous secondary metabolites, in which PAL acts as the first line rate-limiting enzyme. In the current study, low- and high-Fe conditions suppressed the activity of PAL, which was consistent with the reduced accumulation of phenol and flavonoids in leaves under iron stress (Fig. 6). Similarly, it was found that expression of genes related to biosynthesis of flavonoids and phenolics were significantly affected by iron toxicity in rice roots (Wu et al., 2017). However, iron deficiency in cucumber plants resulted in differential responses depending on the duration of stress as the catechin (a kind of flavonoids) concentrations decreased at 3 and 5 days after Fe depletion, but it was significantly increased after 7 days of Fe deficiency treatment (Pavlovic et al., 2013). Meanwhile, melatonin application significantly increased the activity of PAL and PPO alongside increased concentrations of phenols and flavonoids 8

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antioxidant enzyme activity and secondary metabolism, and impaired iron homeostasis in cucumber plants. On the other hand, exogenous melatonin application alleviated oxidative stress induced by low- and high-Fe conditions, which was attributed to melatonin-induced elevation in endogenous melatonin content, photosynthetic pigment content, photosynthesis rate, activity and transcripts of antioxidant- and secondary metabolism-related enzymes and accumulation of phenols and flavonoids in cucumber plants. In addition, melatonin altered the expression of Fe acquisition related genes, FRO2 and IRT1, leading to increased and decreased iron uptake under low- and high-Fe conditions, respectively. The study explored a novel role of melatonin in mitigating iron toxicity and this knowledge might be useful to developing efficient agronomic management practices in low- and high- iron prone soils for sustainable crop production. CRediT authorship contribution statement

Fig. 8. A working model showing the mechanism of melatonin-mediated alleviation of iron stress as revealed in the present study. Briefly, exposure of cucumber plants to low- or high-Fe conditions induced oxidative stress through excessive accumulation of reactive oxygen species (ROS). ROS overload caused chlorosis and suppressed antioxidant capacity, photosynthesis and secondary metabolism, leading to growth inhibitions. However, exogenous melatonin application alleviated the impaired Fe homeostasis-induced oxidative stress by enhancing antioxidant potential and secondary metabolism. Melatonin also inhibited Fe acquisition under high-Fe conditions and improved tissue Fe content under low-Fe conditions, resulting in an enhanced tolerance to iron stress in cucumber plants.

Golam Jalal Ahammed: Conceptualization, Methodology, Formal analysis, Investigation, Writing - original draft, Writing - review & editing, Supervision, Funding acquisition. Meijuan Wu: Formal analysis, Investigation. Yaqi Wang: Investigation. Yaru Yan: Investigation. Qi Mao: Investigation. Jingjing Ren: Investigation. Ronghui Ma: Formal analysis, Investigation. Airong Liu: Supervision, Resources, Funding acquisition. Shuangchen Chen: Writing - review & editing, Supervision, Resources, Funding acquisition, Project administration.

(Fig. 6), suggesting that melatonin-induced enhanced activity of PAL and PPO stimulated biosynthesis of secondary metabolites like flavonoids. Flavonoids possess strong antioxidative potential and might function in scavenging ROS produced by low- and high-Fe supply in cucumber plants (Ahammed et al., 2017). Moreover, it is believed that excretion of phenol and flavonoids from plant roots is an important adaptive strategy of plants to deal with iron toxicity (Wu et al., 2017). Thus melatonin-induced stimulation of plant secondary metabolism might contribute to alleviate oxidative stress caused by low- and highFe treatments in cucumber.

Declaration of Competing Interest The authors declare no conflict of interest. Acknowledgements This work was funded by the National Natural Science Foundation of China (31950410555), the National Key Research and Development Program of China (2018YFD1000800), the National Natural Science Foundation of China(31872092, 31872157), Program for Science and Technology Innovation Talents in Universities of Henan Province (19HASTIT009), Henan Natural Science Foundation (182300410046, 182300410090), Henan University of Science and Technology Research Start-up Fund for New Faculty (13480058), the Key Laboratory of Horticultural Crop Growth and Quality Control in Protected Environment of Luoyang City.

4.4. Melatonin improves iron homeostasis Iron stress at seedling and early vegetative stages causes severe growth inhibition and can result in a total crop failure (Quinet et al., 2012). However, plant tolerance to iron deficit and excess conditions largely depends on the ability to increase and decrease iron acquisition under low- and high-Fe conditions, respectively (Mahender et al., 2019). Previously, we showed that exogenous melatonin could enhance sulfur uptake and metabolism under deficit sulfur conditions in tomato (Hasan et al., 2018). Similarly, melatonin could increase iron uptake in Arabidopsis by enhancing the expression of some genes involved in Fe acquisition, such as FRO2, FIT1 and IRT1 (Zhou et al., 2016). On the other hand, our previous study also revealed that melatonin could also restrict cadmium translocation to shoot by enhancing its sequestration in roots (Hasan et al., 2019). Consistent with this assumption, here we found that exogenous melatonin application significantly decreased the iron content in leaves and roots under high-Fe conditions, which was associated with a significant downregulation in the transcript level of FRO2 and IRT1, suggesting that melatonin could reduce iron acquisition as well as inhibit iron translocation to aerial parts, possibly by modulating the expression of genes involved in iron acquisition and transport.

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5. Conclusions In conclusions, we found that iron deficit or excess conditions drastically inhibited plant growth and biomass accumulation by inducing chlorosis and oxidative stress (Fig. 8). Iron stress also suppressed 9

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