Melatonin-related mitochondrial respiration responses are associated with growth promotion and cold tolerance in plants

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Melatonin-related mitochondrial respiration responses are associated with growth promotion and cold tolerance in plants Hulya Turk a, *, Mucip Genisel b a b

East Anatolian High Technology Application and Research Center, Ataturk University, Erzurum, Turkey Department of Pharmaceutical Botany, Faculty of Pharmacy, Agri Ibrahim Cecen University, Agri, Turkey

A R T I C L E I N F O

A B S T R A C T

Keywords: Melatonin Cold stress Mitochondrial respiration Plant growth parameters Oxidative stress

Melatonin has the ability to improve plant growth and strengthened plant tolerance to environmental stresses; however, the effects of melatonin on mitochondrial respiration in plants and the underlying biochemical and molecular mechanisms are still unclear. The objective of the study is to determine possible effects of melatonin on mitochondrial respiration and energy efficiency in maize leaves grown under optimum temperature and cold stress and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance. Melatonin and cold stress, alone and in combination, caused significant increases in activities and gene expressions of pyruvate dehydrogenase, citrate synthase, and malate dehydrogenase, indicating an acceleration in the rate of tricarboxylic acid cycle. Total mitochondrial respiration rate, cytochrome pathway rate, and alternative respiration rate were increased by the application of melatonin and/or cold stress. Similarly, gene expression and protein levels of cytochrome oxidase and alternative oxidase were also enhanced by melatonin and/or cold stress. The highest values for all these parameters were obtained from the seedlings treated with the combined application of melatonin and cold stress. The activity and gene expression of ATP synthase and ATP concentration were augmented by melatonin under control and cold stress. On the other hand, cold stress reduced markedly plant growth parameters, including root length, plant height, leaf surface area, and chlorophyll content and increased the content of reactive oxygen species (ROS), including superoxide anion and hydrogen peroxide and oxidative damage, including malondialdehyde content and electrolyte leakage level; however, melatonin significantly promoted the plant growth parameters and reduced ROS content and oxidative damage under control and cold stress. These data revealed that melatonin-induced growth promotion and cold tolerance in maize is associated with its modulating effect on mitochondrial respiration.

1. Introduction Melatonin (N-acetyl-5-methoxy-tryptamine), a versatile effective molecule with receptor-mediated and receptor-independent multifar­ ious effects, is present throughout the plant and animal kingdoms [4]. Its impacts on physiological indices and metabolic processes have been largely elucidated in animals since it was first identified in 1958 [47,52]. Subsequently, the identification of melatonin in plants in 1995 pio­ neered to begin intense investigations to reveal its primary functions on plant metabolism [4]. In those studies, melatonin has been demon­ strated to be a plant growth regulator with modulating effects on numerous physio-biochemical and metabolic processes including carbon metabolism, photosynthesis, nitrogen assimilation, fruit formation and maturation, senescence, circadian rhythms, and etc. Moreover, as in

mammals, there are many reports showing its direct role as antioxidant and free radical scavenger and indirect resistance-improving impact in plants exposed to different biotic and abiotic stress factors, such as extreme temperatures, osmotic stress, UV radiation, salinity, heavy metals, nutrient deficiencies, and pathogen infections [4,5,57,58,60]. Among abiotic stresses, cold stress is a major environmental factor that limits agricultural production by inducing physiological and metabolic disproportions causing to hormonal imbalance, nutritional disorders, reduced photosynthetic ability, membrane dysfunctioning, reactive oxygen species (ROS) accumulation, and etc. [9,19,21,38,57]. Also, cold stress causes an energy shortage in plants due to reduction in ATP production resulted from mitochondrial damage, inhibition of en­ zymes involved in mitochondrial respiration, activation of alternative respiration pathway, and substrate shortage for respiration as a result of

* Corresponding author., E-mail addresses: [email protected], [email protected] (H. Turk). https://doi.org/10.1016/j.cryobiol.2019.11.006 Received 3 September 2019; Received in revised form 10 November 2019; Accepted 18 November 2019 Available online 20 November 2019 0011-2240/© 2019 Elsevier Inc. All rights reserved.

Please cite this article as: Hulya Turk, Mucip Genisel, Cryobiology, https://doi.org/10.1016/j.cryobiol.2019.11.006

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reduced photosynthesis efficiency [6,7]. Excessive expenditure of ATP for the synthesis of defensive substances to cope with the stress in­ tensifies more this energy shortage. Mitochondrial respiration, where metabolic intermediates involved in various metabolic pathways occur and energy production is completed, is crucial for maintaining cellular processes. Adjustment of mitochondrial respiration rate has a critical importance in the control of plant growth and adaptation to environ­ mental stresses [38]. In mammals, the relationship between melatonin and mitochondria has been widely investigated in recent years. Increasing evidence shows the ability of melatonin to influence mitochondrial respiration both in vivo and in vitro [1,10,51]. Melatonin was found to enhance activity of complexes I and IV of mitochondrial electron transport chain (ETC) in brain and liver tissues of rats in vivo in a time-dependent manner. In the same study, melatonin counteracted the inhibitory effect of ruthenium red on ETC and ATP synthesis [51]. Similarly, in vitro experiments showed that melatonin increased ATP synthesis by increasing activity of complexes I and IV in a dose-dependent manner and counteracted cyanide-induced inhibition of complex IV. These effects of melatonin on ETC and ATP synthesis was attributed to both its ability to donating and accepting electrons through an interaction with ETC complexes and its limiting impact on electron leakage and ROS generation because of its antioxidant role [1]. Today one of the major questions that remains unclear is whether melatonin has any direct or indirect effect on mitochondrial respiration, one of the most important crossroads of metabolism, in plants grown under control and stressed-conditions. Covey-Crump et al. reported that while mitochondrial respiration rate is mainly delimited by capacities of enzymes involved in this pathway under cold conditions, substrate supply and adenylate restriction have a major role under moderate temperature conditions [14]. Similarly, it was suggested that respiratory rate is modulated by enzyme capacity instead of extra-mitochondrial reactions in cold conditions [6]. Pyruvate dehydrogenase (PDH), cit­ rate synthase (CS), malate dehydrogenase (MDH), cytochrome oxidase (COX), alternative oxidase (AOX), and ATP synthase are among main enzymes involving in stages of mitochondrial respiration in plants [30]. In this context, determining possible effects of melatonin on activities and gene expressions of respiration-related these enzymes will contribute to be elucidating its mode of action on this pathway under optimum temperature and cold stress. To the best of our knowledge, no study has been carried out to evaluate the effect of melatonin on mitochondrial respiration in plants grown under stress-free and stressed conditions. Therefore, the objective of this study was to investigate the effect of melatonin on mitochondrial respiration in maize leaves and to reveal the relationship between melatonin-induced possible alterations in mitochondrial respiration and cold tolerance.

resulted the greatest positive effect on the growth and cold tolerance of maize seedlings (Data not shown). While the control and cold group were sprayed with deionized water, melatonin and melatonin plus cold group were sprayed with 100 μmol L 1 melatonin. Tween-20 (0.01%) was used as a surfactant. All treatment groups were held in dark con­ ditions for about 3 h to provide complete absorption of the solutions without deteriorating from light and then transferred in different climate cabinets. Control and melatonin groups were transferred a climate chamber set to 25–27/20-22 � C (day/night) until plants were harvested. Cold and melatonin plus cold groups were moved to a climate cabinet set to 10/7 � C (day/night) for 2 days without changing photo­ period, light intensity, and humidity. The cold temperature range � et al. applied was chosen based on the previous reports [19,25,56]. Hola [32] reported that temperatures below 12–15 � C induces chilling stress in maize. Stone et al. [53] informed that leaf growth is controlled by soil temperatures rather than air temperature. Nguyen et al. [45] showed that 6 � C temperature application for 2 days induced expression of a lot of genes in maize. Thus, in the present study, cold stress was induced in maize by exposure to 10/7 � C (day/night) for 2 days from the beginning of the photoperiod. 2.2. Mitochondria isolation Mitochondria of maize leaves was isolated using differential centri­ fugation and Percoll gradient [29]. Briefly, the maize leaves were extracted in grinding buffer (50 mM MOPS (pH 7.2), 4 mM L-cysteine, 2 mM EGTA, 0.4 M mannitol, 0.5%, (w/v) BSA, 0.6% (w/v) PVP and 20 mM β-mercaptoethanol) according to Chien et al. [13]. The homoge­ nates were centrifuged using differential centrifugation steps. Then the obtained raw mitochondrial pellet was homogenized in rinse medium (20 mM MOPS (pH 7.2), 1 mM EGTA 0.3 M mannitol, and 0.1% (w/v) BSA), and once again the samples were centrifuged at different centri­ fugation steps. Finally, the crude mitochondrial pellet on the bottom of the tube was washed with rinse buffer and Percoll gradient was used for purified mitochondria. All processes were performed at 4 � C. 2.3. Pyruvate dehydrogenase (PDH) activity The isolated mitochondria samples were incubated in the medium of 50 mM Tris–HCl buffer (pH 7.5), 20 mM MgCl2, and Triton X-100 (0.1%) at 4 � C for 30 min. The samples were centrifuged at 12.000�g for 10 min. The 30 μg of proteins was mixed with 900 μL of the reaction me­ dium (70 mM TES–KOH (pH 7.5), 0.2 mM TPP, 2 mM MgCl2, 0.12 mM CoA, 2 mM NADþ, and 20 mM Cys). To determine the enzyme activity, 100 μL of pyruvate (1 mM) in Triton X-100 (0.05% (w/v)) was added and incubated at 25 � C for 5 min. The absorbance changes were deter­ mined as the production of NADH at 340 nm [69].

2. Materials and methods

2.4. Citrate synthase (CS) activity

2.1. Plant material, growth conditions and applications

CS activity was measured using the Citrate Synthase Assay Kit (Catalog Number CS0720 Sigma-Aldrich) according to the producer’s instruction and the activity was calculated according to the coefficient of Ɛ412 ¼ 13.6 cm 1 mM 1 [37].

The seeds of maize (Zea mays L.) were surface-sterilized with ethanol (96%) and NaClO (5%), respectively, and then they were rinsed with running tap water. The seeds were cultured on moistened filter papers for 2 days. The germinated uniform seedlings were selected and sown in flowerpots containing a concoction of air-dried soil and sand in the ratio of 1:1 (v/v). The seedlings were grown in a climate chamber set to 25–27/20-22 � C (day/night) temperature, 14 h photoperiod at 450 μmol quanta m 2 s 1 and 60–70% relative humidity. In three-leaf stage, twelve-day-old seedlings were separated four different groups as a control, cold, melatonin plus cold, and melatonin for making treatment. Initially, different concentrations of melatonin solution were prepared (10 μmol L 1, 50 μmol L 1, 100 μmol L 1, 250 μmol L 1, 500 μmol L 1, and 1000 μmol L 1) based on prior investigation and the optimum concentration was determined to be 100 μmol L 1, in which melatonin

2.5. Malate dehydrogenase (MDH) activity The malate dehydrogenase activity was determined based on the reduction in absorbance of NADH at 340 nm. Mitochondrial MDH ac­ tivity was measured the reduction of oxaloacetate at 25 � C using the method defined by Lü et al. [41]. The activity assay was conducted in 50 mM potassium phosphate buffer (pH 7.5), 0.15 mM NADH, 1 mM oxaloacetate, and an appropriate amount of the mitochondrial piece. The reaction was initiated by the supplementation of oxaloacetate for determining the specific NADH oxidation [71]. 2

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2.6. Mitochondrial respiration measurements

μg isolated mitochondria. To initiate the reaction, 1 mM phosphoenol pyruvate was added to the medium. The medium did not contain cyto­ chrome c because its addition inhibit the coupled enzymatic reaction.

Total respiration rate (Vt), cytochrome pathway capacity (Vcyt), and alternative respiration pathway (Valt) were defined using an oxygen electrode (Clark-type, Hansatech, UK) in isolated mitochondria samples [8]. The samples were placed in oxygen electrode cuvettes and were incubated in a standard reaction buffer at the room temperature. After adding 200 μM ATP into reaction buffer, 500 μM ADP and 10 mM suc­ cinate were added as substrate. Vcyt was defined using potassium cya­ nide (KCN, 1 mM) as inhibitor, and Valt was defined using salicylhydroxamic acid (SHAM, 2 mM) as inhibitor. KCN and SHAM were added to the buffer for 2 min intervals, respectively. Vt was determined as O2 consumption rate before KCN addition to the buffer, and Valt was defined as O2 consumption rate before SHAM addition to the buffer. Vcyt was determined as the difference between Vt and Valt. Residual respiration was defined as O2 consumption rate in the presence of both KCN and SHAM [56].

2.10. ATP concentration ATP concentration was assayed based on fluorescence intensity, which was measured with a Fluorescence Spectrophotometer (Agilent Technologies) and calculated based on an ATP standard curve using the ATP Assay Kit (Catalog Number MAK190 Sigma-Aldrich) accordingly the producer’s instructions. 2.11. Plant growth parameters The root length and plant height of 14-day-old maize seedlings were measured by using a tap-measure. The results were given as the mean of the values obtained. Total chlorophyll content was determined based on the method of Crocker [15]. Leaf surface area was measured, using automatic leaf area meter [35].

2.7. RNA isolation and gene expression analyses The gene expression analyses were determined using SYBR green quantitative Real-Time PCR technique. The RNeasy plant mini kit provided by Qiagen Company (Hilden, Germany) was used for total RNA isolation. The content of purity of RNA was measured using the Qiaexpert apparatus. According to manufacturer’s directive, cDNA synthesis was carried out from 2 ng total RNA using Nanoscript 2 RT kit (Primer Design) [23]. β-actin was considered as housekeeping gene for cytochrome oxidase (COX-19-afterwords named as COX), alter­ native oxidase (AOX) citrate synthase (CS), pyruvate dehydrogenase (pdh1), malate dehydrogenase (MDH) and ATP synthase (ATP6). Gene expression analyses were made using Qiagen Data Analysis Center. Gene-specific primers for AOX; 50 - AGGTGCTTTTCTGGCGTTT-30 and 50 - CGATATTAGCGAGCCCAATTC-30 , for COX-19; 50 - CATGAG­ TGCGACTTGGAGAA-30 and 50 - TCAGGAGATGTACCCGCTTC-30 ; for CS; 50 - TGCTCACAGTGGAGTTTTGC-30 and 50 -AACACTCTTC­ GGCCTCTCAA-30 ; for MDH: 50 -CCTGCCATTCTTCGCATCAA-30 and 50 -TCAAGTGCCTTGGCCTCATA-30 ; for Pdh1; 50 - CTCAACATTTCGGC­ CCTCTG-30 and 50 -CATAGTCGCCACGCTTGTAG-30 ; for ATP6; 50 -CACTTAACGAGCACCACCAG-30 and 50 -GGATCCTGCAGACTCT­ CTCC-30 ; and for β-actin; 50 -AAAGTGGCGGAATGCTCTC-30 and 50 -TCTACGGATTTCACCCCTACA-30 , were designed, respectively.

2.12. Reactive oxygen species (ROS) and oxidative damage parameters The content of superoxide anion was assessed according to Elstner and Heupel [17] and the results were calculated by using a standard calibration curve used sodium nitrite as a standard. The content of hydrogen peroxide was determined according to the method of Velikova et al. [61] and the results were calculated by using a standard calibration curve. The malondialdehyde (MDA) content was measured according to the method of Velikova et al. [61] and the results were calculated by using the molar extinction coefficient 155 (mmol cm 1). Electrolyte leakage (EL) was assessed according to the methods of Lutts et al. [42]. The leaf samples were placed in a closed vial containing ddH2O and incubated on a shaker for 1 d. Then, the firt electirical conductivity of the solution (EC1) was measured. After autoclaved at 120 C for 20 min, the solution was cooled at room temperature and the second electirical conductivity of the solution (EC1) was measured. The results were calculated using the following formula: EL (%) ¼ (EC1/EC2) x 100. 2.13. Statistical analysis All experiments were set up in a factorial arrangement using completely randomized design with six independent experiments. The data was analysed by ANOVA with Duncan’s multiple range test with SPSS 20.0. The significant differences were appreciated at p < 0.05. The data were also assessed by principal component analysis (PCA) to comprehensively assessed the impact of melatonin treatment on the cold-induced changes in mitochondrial respiration using XLSAT 2019 software.

2.8. Immuno-blot analysis The crude mitochondrial proteins were separated using SDSpolyacrylamide gel (10%). Thereafter, the gel transferred to PDVF western blotting membrane (0.45-mm pore size) at 2.5A in semi-dry Western blotting for 7 min using Bio-Rad Trans-Blot® Turbo™ Trans­ fer System. While the antibody obtained from Sauromatum guttatum [18] was preferred as the monoclonal antibodies (1:5000 dilution), the anti-mouse secondary antibody (1:20,000 dilution) bonded to horse­ radish peroxidase was preferred as a secondary antibody. COX protein bands were determined by using the method described by Rurek et al. [48]. The all process was performed using the same procedure like AOX protein. Unlike the AOX protein, to determine the COX protein bands was used the polyclonal antibody in the rate of 1:1000. The relative density of protein bands was measured using Vilber Lourmat’s Chem­ iluminescence (Fusion FX) software.

3. Results 3.1. Effect of melatonin on the activities of matrix-related enzymes in maize leaves under cold stress The activities of PDH, CS, and MDH were presented in Table 1. Cold stress led to significant increases in the activities of all of the enzymes studied in comparison to their controls. The increases in the activities of PDH, CS, and MDH were 22.2, 73.5, and 18.6%, respectively. Similarly, melatonin application alone caused marked increments in the activities of these enzymes. The increments in the activities of PDH, CS, and MDH were 40.7, 68, and 33.1%, respectively, as compared with their controls. The highest activities of these enzymes were determined in cold stress plus melatonin-treated seedlings. The increase rates were 74.1, 116, and 50.4%, respectively, in comparison to their controls.

2.9. ATP synthase activity ATP synthase activity was determined based on coupling the pro­ duction of ADP to the oxidation of NADH via the pyruvate kinase-lactate dehydrogenase assay [11]. The reaction medium of 1 ml (pH 7.2) 4 units of pyruvate kinase, 50 μM nPG, 2 units of lactate dehydrogenase, 0.2 mM NADH, 1 mM KCN, 1.5 mM ATP, 5 μM FCCP) was mixed with 100 3

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Table 1 Effect of melatonin on the activities of pyruvate dehydrogenase (PDH), citrate synthase (CS), and malate dehydrogenase (MDH) in the leaves of maize seed­ lings grown under cold stress.Different letters in the same column indicate sta­ tistically significant differences (P � 0.05).

Control Melatonin Cold Cold þ Melatonin

PDH activity (μmol min 1 mg 1 protein FW)

CS activity (μmol ml 1min 1 FW)

MDH activity (μmol min 1 mg 1 protein FW)

0.027 � 0.038 � 0.033 � 0.047 �

20.44 34.33 35.46 44.22

23.49 � 31.27 � 27.86 � 35.32 �

0.0008d 0.0014b 0.0009c 0.0013a

� 0.44c � 0.67b � 0.78b � 0.81a

0.49d 1.11b 0.75c 1.56a

3.2. Effect of melatonin on gene expression levels of mitochondrial respiration-related enzymes in maize leaves under cold stress Fig. 2. Effect of melatonin on total mitochondrial respiration (Vt), cytochtome pathway capacity (Vcty), and alternative respiration capacity (Valt) in the levaes of maize seedlings grown under control and cold conditions. Twelve-dayold maize seedlings were sprayed with distilled water or 100 μM melatonin. The treated seedlings were grown under control or cold conditions for two more days. The experimental materials were obtained from six growth media, and leaves were taken from each medium for the measurement of mitochondrial respiration parameters. The data was analysed by ANOVA using Duncan’s multiple range test (SPSS 20.0) with a significance level of p < 0.05. Each value was represented as means � SE. The result indicated in figures in such a way that alphabets a, b, c, and d represent the first, second, third, and fourth levels of statistical significance, respectively.

PDH, CS, MDH, COX, AOX, and ATP synthase are among the main enzymes catalysing the matrix and inner membrane reactions of mito­ chondrial respiration. Determining gene expression and protein levels of these enzymes will contribute to elucidating at molecular level the mode of action melatonin on mitochondrial respiration. The separate appli­ cations of melatonin and cold stress significantly upregulated gene ex­ pressions of all of enzymes studied in comparison to their controls (Fig. 1). The expression levels of PDH, CS, MDH, COX, AOX, and ATP synthase were 2.7-fold, 2.9-fold, 2.3-fold, 2.5-fold, 1.8-fold, and 2.4fold, respectively, in melatonin-treated seedlings and 2.4-fold, 2.7fold, 1.7-fold, 1.9-fold,2.9-fold, and 1.6-fold, respectively, in coldstressed seedlings relative to their controls. The maximum level of gene expressions of PDH, CS, MDH, COX, AOX, and ATP synthase were recorded as 3.6-fold, 4.3-fold, 2.9-fold, 3.2-fold, 3.9-fold, and 3.1-fold in melatonin plus cold stress-treated seedlings.

Vcyt was significantly increased by melatonin and cold stress ap­ plications. Vcyt in treatment with melatonin was higher than that with cold stress. The increase ratio of Vcyt was 34% for melatonin-treated seedlings and 14% for cold-stressed seedlings. Melatonin plus cold stress-treated group did not differ from melatonin-treated group alone with regard to Vcyt. Melatonin plus cold stress application resulted in similar an increase of 38% in comparison to the control. Compared to control, cold stress led to an increase of 3.27-fold in Valt in maize leaves. Melatonin-induced increase of 2.19-fold in Valt in comparison to the control was lower than that of cold stress. The com­ bined application of melatonin and cold stress gave the maximum in­ crease of 4.82-fold of Vt, compared with the control.

3.3. Effect of melatonin on mitochondrial respiration rate in maize leaves under cold stress The cold stress and/or melatonin-induced alterations in Vt, Cyt pathway capacity, and Alt pathway capacity were presented in Fig. 2. Vt was measured as 97 nmol O2 min 1 mg 1 protein in the leaves of control seedlings. The separate applications of melatonin and cold stress significantly increased Vt by 35 and 42%, respectively, in comparison to the control. The maximum value of Vt was recorded as 167 nmol O2 min 1 mg 1 protein at melatonin plus cold stress-treated seedlings.

Fig. 1. Melatonin and cold stress, alone or in combination, upregulate the expressions of mRNAs of pyruvate dehydrogenase (PDH), citrate synthase (CS), malate dehydrogenase (MDH), cytochrome oxidase (COX), alternative oxidase (AOX), and ATP synthase. Quantitative RT-PCR (qPCR) analysis of the expression levels of the indicated genes in 14-day-old maize leaves after pretreatment with 100 μM melatonin under normal and cold conditions. 4

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3.4. Effect of melatonin on plant growth parameters under cold stress

respectively, compared with their controls. The highest level of COX and AOX proteins were determined in the combined application of mela­ tonin and cold stress as 96 and 240%, respectively, compared with their controls. These results were agreement with increases in the activities of these enzymes studied and suggested that melatonin had a stimulating effect at biochemical and molecular level on mitochondrial respirationrelated enzymes in the leaves of maize seedlings grown under control and cold stress.

Growth parameters, including root length, plant height, leaf surface area, and chlorophyll content were measured to determine the reducing effect of cold stress on plant growth and the alleviating effect of mela­ tonin on cold-stressed maize seedlings (Table 2). Cold stress significantly reduced root length, plant height, and leaf surface area by 17.3, 15.3, and 17%, respectively, compared with the control. Foliar spray of melatonin alleviated the inhibition of cold stress on root length, plant height, and leaf surface area by 8, 6.1, and 9%, respectively. On the other hand, melatonin application alone gave rise to a significant in­ crease in these parameters by 12.6, 9.6, and 13%, respectively, compared with the control. Similar results were observed on chlorophyll content. While chlo­ rophyll content of cold-stressed seedlings diminished by 24.8%, compared with control, melatonin pre-treatment caused a significant increase of 17.7% in leaf chlorophyll content, comparatively to cold stress alone. Under control conditions, melatonin significantly enhanced chlorophyll content by 14%, compared with the control.

3.7. Effect of melatonin on ATP synthase activity and ATP concentration in maize leaves under cold stress As shown in Fig. 5, ATP synthase activity significantly decreased in leaves of maize seedlings under cold stress. The reduction in ATP syn­ thase activity was about 40%. On the contrary, melatonin application alone significantly elevated by 11% ATP synthase activity compared with the control. When combined with cold stress, melatonin applica­ tion significantly decreased by 23.1% cold-induced reduction in ATP synthase activity, compared to cold stress alone. As seen in Fig. 5, as compared to the control, ATP concentration was slightly decreased by 5.5% under the cold stress; however, this decrease was not statistically significant. On the other hand, melatonin applica­ tion led to marked elevation in ATP concentration by up to 27.6 and 14.45% under stress-free and cold stress, respectively.

3.5. Effect of melatonin on ROS content and oxidative damage parameters in maize leaves under cold stress ROS content of maize leaves was profoundly affected by cold stress (Table 3). Cold stress led to increment of 22.8% and 30.9% in superoxide anion and hydrogen peroxide contents in maize leaves, respectively, when compared with the control. Melatonin pre-treatment significantly reduced the cold-induced enhanced content of superoxide anion and hydrogen peroxide by 10.5 and 12.6%, respectively. In comparison to the control, melatonin pre-treatment alone remarkably reduced the content of superoxide anion and hydrogen peroxide by 10 and 14%, respectively. Cold application dramatically increased oxidative damage in­ dicators, MDA content and EL in maize leaves, compared with the control (Table 3). These increases reached to 54.2 and 20.3%, respec­ tively. These harmful effects were significantly mitigated by melatonin pre-treatment. Melatonin-induced reductions in these parameters reached to 27.9 and 12.1%, respectively. On the other hand, melatonin application significantly reduced the MDA content and EL by 17.4 and 4.3, respectively, in comparison to the control.

3.8. Hierarchical clustering with heat map As shown in Fig. 6, non-supervised hierarchical clustering with heat map visualization demonstrated co-regulation of genes involved in mitochondrial respiration and revealed that melatonin and cold stress, alone and in combination, significantly upregulated the expressions of all of the genes studied. There was a strong positive correlation among the gene expressions of PDH, CS, and MDH, matrix-related enzymes of mitochondrial respiration. Similarly, it was found that a strong positive correlation among the gene expressions of AOX, COX, and ATP synthase, ETS-related enzymes of mitochondrial respiration. 3.9. Biplot 2-D graphical analysis

3.6. Effect of melatonin on protein levels of COX and AOX in maize leaves under cold stress

The data were also analysed with PCA to exhibit the correlation of plant response to melatonin and cold stress (Fig. 7). Biplot analysis was fulfilled with the help of two main principle factors (F1 and F2) for each application group. The treatments and variables were merged in a single Biplot graph to further facilitate the visualization. The PCA accounts for 98.68% variation under cold stress with melatonin treatment (Fig. 6). All treatments were displayed a significant separation with control (p < 0.05). Biplot analysis clearly shows that root length, plant height, leaf surface area, chlorophyll content, and ATP synthase activity exhibited significant positive correlations among each other and these parameters were positively correlated with melatonin application; however, su­ peroxide anion, hydrogen peroxide, MDA, and EL displayed consider­ able negative correlations with these parameters in varying degrees and these parameters were positively correlated with cold stress. Similarly, there was a positive correlation among Vcyt, Vt, COX protein level, ATP synthase expression, MDH activity and expression, CS activity and expression, PDH activity and expression, and COX expression and these parameters were positively correlated with cold stress plus melatonin application. In conclusion, PCA of growth parameters, ROS content, oxidative damage indicators, and mitochondrial respiration-related re­ actions in maize seedlings displayed a clear discrimination between application groups and control group, thus, indicating that melatonininduced growth promotion and cold tolerance of maize seedlings is associated with stimulation of mitochondrial respiration.

On the other hand, Fig. 3 and Fig. 4 demonstrates that melatonin significantly enhanced the protein level of COX and AOX by 83 and 80%, respectively, in comparison to their controls. Similarly, cold stress alone caused marked elevation in level of these proteins by 39 and 160%,

Table 2 Effect of melatonin on the root length, plant height, leaf surface area, and chlorophyll content of maize seedlings grown under cold stress. Leaf surface area was considered as 100% in control plants.Different letters in the same column indicate statistically significant differences (P � 0.05).

Control Melatonin Cold stress Cold þ Melatonin

Root length (cm)

Plant height (cm)

Leaf surface area (%)

Chlorophyll content (mg g 1 FW)

20.33 � 0.51b 22.89 � 0.59a 16.81 � 0.39d 18.44 � 0.47c

31.64 � 0.72b 34.68 � 0.80a 26.80 � 0.59d 28.72 � 0.66c

100 � 2.4b

2.78 � 0.11b

113 � 3.1a

3.17 � 0.19a

83 � 3.2d

2.09 � 0.14d

92 � 2.6c

2.46 � 0.13c

5

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Table 3 Effect of melatonin on the contents of superoxide anion and hydrogen peroxide, and malondialdehyde and electrolyte leakage in the leaves of maize seedlings grown under cold stress.Different letters in the same column indicate statistically significant differences (P � 0.05). Superoxide anion content (μg.g Control Melatonin Cold Cold þ Melatonin

13.72 12.36 16.86 15.42

� 0.39c � 0.43d � 0.58a � 0.46b

1

FW)

Hydrogen peroxide content (μg.g 20.31 � 17.46 � 26.59 � 24.03 �

0.53c 0.44d 0.77a 0.63b

1

FW)

Malondialdehyde content (ng.g 8.36 � 0.34c 6.91 � 0.38d 12.89 � 0.61a 10.56 � 0.43b

1

FW)

Electrolyte leakage (%) 14.31 � 0.056 9.02 � 0.04 34.63 � 0.11 22.56 � 0.08

Fig. 4. Effect of melatonin on alternative oxidase (AOX) protein level and band intensity in the leaves of maize seedlings grown under control and cold con­ ditions. AOX protein levels were monitored by immunoblot analysis, and band intensities were calculated as percent compared to control using Vilber soft­ ware. The protein level was considered as 100% in control plants. Experimental data are presented as means � SE for six independent experiments. Significant differences were determined by one-way ANOVA with Duncan’s multiple range test at p < 0.05 and marked with different small letters.

Fig. 3. Effect of melatonin on cytochrome oxidase (COX) protein level and band intensity in the leaves of maize seedlings grown under control and cold conditions. COX protein levels were monitored by immunoblot analysis, and band intensities were calculated as percent compared to control using Vilber software. The protein level was considered as 100% in control plants. Experi­ mental data are presented as means � SE for six independent experiments. Significant differences were determined by one-way ANOVA with Duncan’s multiple range test at p < 0.05 and marked with different small letters.

4. Discussion Mitochondrial respiration providing the driving force for maintain­ ing many cellular events, such as active transport and various anabolic and catabolic reactions, is one of the first metabolic pathways affected by cold stress [31]. Cold-induced alterations on mitochondrial respira­ tion in plants have been widely investigated by many researchers [26, 31,36,58]; however, to our best knowledge, there is no detailed report exhibiting effect of melatonin on mitochondrial respiration in plants under stress-free and stressed conditions. The effect of cold stress on mitochondrial respiration are dependent on a number of factors, including the severity and duration of the stress as well as the cold sensitivity of the plant species [28,59]. Research findings on this subject, therefore, show differences from each other. Some studies demonstrated that cold stress causes a decline in mitochondrial respiration rate because of multiple dysfunctions and activity and capacity limitations of the related components of mitochondrial ETS and TCA cycle [39,44,46]. The others indicated that respiration rate elevates upon exposure to cold stress in order to develop adaptation mechanisms through multiple structural and biochemical alterations in the cell [21,27,31,52,68]. In the present study, effect of melatonin alone and in combination with cold stress on mitochondrial respiration was investigated step by step by being determined the alterations in activities and/or gene expressions of PDH, TCA cycle-related enzymes, ETC-related enzymes, and ATP synthase.

Fig. 5. Effect of melatonin on ATP synthase activity and ATP concentration in the leaves of maize seedlings grown under control and cold conditions. The data was analysed by ANOVA using Duncan’s multiple range test (SPSS 20.0) with a significance level of p < 0.05. Each value was represented as means � SE. The values were expressed as nmol ATP min 1 mg 1 protein for ATP synthase ac­ tivity and nmol g 1 FW for ATP concentration.

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Fig. 6. Unsupervised hierarchical clustering was performed on expression profiles of genes of mitochondrial respiration-related six enzymes of maize leaves grown under control and cold stress. The heat map of differentially expressed genes based on clustering is shown in the figure. Each col­ umn represents an independent experiment and each row represents a gene. For each gene, “red” indicates high relative expres­ sion, and “green” indicates low relative expression. (For interpretation of the refer­ ences to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 7. Combined comparative biplot-based principal component analysis (PCA) with first two principal components for different parameters related with growth parameters, oxidative stress indicators, and mitochondrial respiration in maize seedlings treated with melatonin under control and cold conditions. (PH: Plant height; RL: Root length; Chl: Chlorophyll content; LSA; Leaf surface area; ATP synthase-GE: ATP synthase gene expression level; MDH: Malata dehy­ drogenase: MDH-GE: Malate dehydrogenase gene expression level; CS: Citrate synthase; CS-GE: Citrate synthase gene expression level; PDH: Pyruvate dehydroge­ nase: PDH-GE: Pyruvate dehydrogenase gene expression level; AOX: Alternative oxidase; AOX-GE: Alternative oxidase gene expression level; EL: Elecktrolyte leakage; MDA: Malandialdehyde content; O2 : Superoxide anion content; H2O2: Hydrogen peroxide content).

PDC catalyses the conversion of pyruvate into acetyl-CoA, thus linking glycolysis with the tricarboxylic acid (TCA) cycle. Activity of mitochondrial PDC is modulated by reversible phosphorylation cata­ lysed by intrinsic kinase and phosphatase components in addition to subcellular compartmentation, control of gene expression, product in­ hibition, and metabolic effectors. PDH is the first component enzyme of pyruvate dehydrogenase multienzyme complex (PDC) [34,55]. Alma­ danim et al. [2] reported that cold stress increased phosphorylation of PDH E1 component subunit alpha-1 in wild-type rice. Jan et al. [34]

informed that activity and gene expression of mitochondrial PDH was regulated by gibberellin and this regulation had an important role on rice growth and development. In concert with this report, we found that cold stress significantly upregulated activity and expression of PDH (Table 1 and Fig. 1). Similarly, melatonin application, alone and in combination with cold stress, markedly increased activity and gene expression of PDH in comparison to their controls. These data meant that under control and cold stress, melatonin stimulated PDH activity at the biochemical and molecular level, thus contributing to acceleration of 7

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TCA cycle by increasing formation of acetyl-CoA from pyruvate. How­ ever, because acetyl-CoA is also involved in the synthesis of some plant growth regulators, fatty acids, and secondary metabolites [40], it is needed to evaluate together the alterations in TCA cycle and ETS to confirm that increased PDH activity is related with mitochondrial respiration pathway. TCA cycle is highly flexible and is firmly modulated in changing mitochondrial actions to counteract and adapt to environmental stresses. TCA-related enzymes are differentially affected by cold stress [16]. CS, a thermally unstable, is responsible enzyme for catalysing the first reaction of TCA cycle, in which it is rate-limiting factor [64]. CS has been detemined to be effective in regulating carbon-nitrogen interaction and increasing tolerance to environmental stresseses in broccoli, Ara­ bidopsis, beans, rice, maize, and tobacco [20,44,50,58,66]. Sanchez-Bel et al. [49] reported that CS expression increased at 10 � C in bell pepper. The other TCA-related enzyme, MDH, catalyses reversible oxidation of malic acid to oxaloacetate (OAA) coupled to the reduction of the NADþ pool, after which OAA re-enters the TCA cycle along with another acetyl-CoA in order to form citric acid [3,37,63,66]. Vitamvas et al. [63] reported that cold stress increased accumulation of MDH in cold-tolerant wheat genotypes. Similarly, it was determined an increase in CS and MDH protein levels in cold-sensitive strawberry cultivars but not in cold-tolerant ones [50,63]. Consistently with previous studies, we found that cold stress signifantly increased the activity and gene expression of CS and MDH (Table 1 and Fig. 1). Melatonin application also resulted in similar increases in the activity and gene expressions of these enzymes. These data conformed with melatonin-induced enhanced activity of PDH and suggested that melatonin and cold stress, alone and in combination, accelerated the TCA cycle, thus leading to the production of metabolic intermediates and high-energy electrons in a higher levels. The high-energy electrons in the form of NADH and FADH2 derived from glycolysis, PDC complex, and TCA cycle is converted to potential energy stored in a proton gradient across the mitochondrial membranes, which is then used to drive ATP generation through ATP synthase [31, 38]. In the present study, melatonin and cold stress, alone and in com­ bination, significantly increased Vt compared with the control (Fig. 2). These data were consistent with increased-activities of PDH, CS, and MDH. In plants, ETC consists of the phosphorylating pathway (named as Cyt pathway) and non-phosphorylating pathway (named as Alt pathway). COX and AOX are the terminal enzymes Cyt pathway and Alt pathway, respectively [21,22]. Mitochondrial ETS is modulated by adjustment of partitioning between Cyt pathway and Alt pathway and alterations in the activities and relative abundance of individual enzymes-related with these pathways [31]. In the present study, when compared to controls, Vcyt was significantly increased by cold stress. This data was accordance with Erdal and Genisel [21] who reported that cold stress led to an increase in Cyt pathway activity in maize seedlings. Similarly, melatonin application, alone and in combination with cold stress, significantly enhanced Vcyt in comparison to their controls. In addition to Vcyt, melatonin and cold stress applications had also a marked effect on gene expression of COX (Fig. 1). These genes that encode mitochondrial and nuclear-encoded subunits of Complex IV are highly responsive to cold conditions. Previous research has shown that COXs respond to cold stress in plants [31,72]. Cold-acclimated and non-acclimated Arabidopsis plants exhibited a remarkable increase in COX6 transcript abundance [65,70]. Similarly, protein abundances of COX6 isoforms and the other COX genes in different plants exposed to cold stress were higher than in those of control [43]. As parallel to previous studies, we found that cold stress resulted in a significant upregulation in the gene expression and protein level of COX (Figs. 2 and 3). Similarly, melatonin application also resulted in a similar rise in the expression level of COX compared with control under control and cold stress. These data put forward that while melatonin stimulated Cyt pathway to supply high level of ATP needed for melatonin-induced enhanced plant growth in stress-free conditions, when applied in

combination with cold stress, it activated Cyt pathway more to supply generation of ATP that is needed at a higher rate for alleviation of cold-induced growth reduction and for improvement of defence mech­ anisms to counteract the stress. The findings obtained from growth pa­ rameters clearly confirmed this assumption. We found that while melatonin application significantly promoted plant growth parameters, including root length, plant height, dry weight, and chlorophyll content in comparison to control, it significantly mitigated cold-induced re­ ductions observed in these parameters (Table 2). Alt pathway plays an important role in the adaptation of plants to abiotic stresses. AOX is more tolerant to short-term temperature changes than COX. When environmental stresses limit Cyt pathway, AOX can prevent the blockade of electron flow by receiving electrons directly from ubiquinone, and thus delimits excessive production of ROS [13,22, 56,62]. In the present study, melatonin and cold applications resulted in a significant increase in Valt and gene expression and protein level of AOX (Figs. 1, 2 and 4). In accordance with the current findings, many studies report that cold stress contributes to adaptation by stimulating Alt pathway in plants (such as, cucumber, maize, chickpea, and etc.) [21,22,29,33]. These data can be interpreted that melatonin upregu­ lated Alt pathway to prevent cold-induced blockade of electron flow in ETS and excessive production of ROS. Melatonin-induced reductions determined in superoxide anion, hydrogen peroxide, and hydroxyl radical content under control and cold stress strongly supported this assumption (Table 3). To date, many researchers have attributed the decrease in ROS content to melatonin-induced increased antioxidant activity under stress-free and stressed conditions [24,54,57,60]. This is the first study showing that the reducing effect of melatonin on ROS content is also associated with its stimulating effect on Alt pathway. ATP synthase, the central bioenergetics engine of all organisms, directly generates ATP, main energy molecule used in cells, during the process of cellular respiration [38,48]. To date, increasing reports have proven that ATP synthase play an important role in stress response of plants [31]. Prior studies demonstrated that cold stress affected activity, gene expression level, and protein abundance of ATP synthase depending on duration and severity of stress and cold sensitivity of plant species [38]. Zhang et al. [72] reported that overexpression of ATP synthase small subunit confer tolerance to salt, drought, and low tem­ perature stresses in Arabidopsis. Besides, many researchers informed that cold conditions increased abundance of ATP synthase proteins [12, 67]. In the present study, although expression level of ATP synthase was upregulated by cold stress, its activity decreased significantly (Fig. 1). This data suggested that plants tried to counteract cold-induced reduc­ tion in ATP synthase activity by increasing its protein level. Moreover, in spite of high activity and expression of COX under cold stress, deter­ mining lower activity of ATP synthase data put forward that activity of ATP synthase is more sensitive than Cyt pathway to cold stress. On the other hand, despite of significant decline in ATP synthase activity, ATP concentration did not change statistically in cold-stressed seedlings compared to control (Fig. 5). This was probably due to cold-induced activation of Cyt pathway and high expression of ATP synthase. Even though the activity value for per mg of protein was reduced, the total ATP level was not significantly affected due to the high expression level of ATP synthase. Different from cold stress alone, melatonin application significantly augmented activity of ATP synthase under both control and cold stress in comparison to their controls. Consistently with the results obtained from Cyt pathway and ATP synthase activity, melatonin resulted also in similar increases in ATP concentration. This meant that melatonin coordinated mitochondrial respiration to supply higher level of ATP needed for maintaining plant growth and improving the stress tolerance. The hierarchical cluster with heap map clearly demonstrated the co-regulating effect of melatonin on the gene expressions of mito­ chondrial respiration-related enzymes (Fig. 6). Moreover, biplot analysis put also forward that melatonin resulted in a co-regulation among plant growth, ROS content, oxidative damage, and mitochondrial respiration under control and cold stress (Fig. 7). 8

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In conclusion, the present study showed that melatonin application has a stimulating effect on mitochondrial respiration of maize leaves grown under optimum temperature and cold stress. Melatonin carries out its stimulating effect by activating both matrix and ETC-related re­ actions in a balanced manner. In addition, the effect of melatonin is at the level of not only enzyme activity biochemically but also regulating expression of the corresponding genes.

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