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. 2022 Jul 27;28(7):1467–1476. doi: 10.1007/s12298-022-01214-3

Proteomic and physiological analyses to elucidate nitric oxide-mediated adaptive responses of barley under cadmium stress

Kübra Alp 1, Hakan Terzi 1,✉, Mustafa Yildiz 1
PMCID: PMC9424405  PMID: 36051236

Abstract

Nitric oxide (NO) is known to induce plant resistance for several environmental stresses. The protective roles of NO in cadmium (Cd) toxicity have been well documented for various plant species; nevertheless, little information is available about its molecular regulation in improving Cd tolerance of barley plants. Therefore, we combined a comparative proteomics with physiological analyses to evaluate the potential roles of NO in alleviating Cd stress (50 μM) in barley (Hordeum vulgare L.) seedlings. Exogenous application of NO donor sodium nitroprusside (SNP, 100 μM) decreased the Cd-mediated seedling growth inhibition. This observation was supported by the reduction of lipid peroxidation as well as the improvement of chlorophyll content and inhibition of hydrogen peroxide accumulation. Activities of the superoxide dismutase and guaiacol peroxidase were reduced following the application of SNP, while ascorbate peroxidase activity was enhanced. In this study, a total of 34 proteins were significantly regulated by NO in the leaves under Cd stress using a gel-based proteomic approach. The proteomic analysis showed that several pathways were noticeably influenced by NO including photosynthesis and carbohydrate metabolism, protein metabolism, energy metabolism, stress defense, and signal transduction. These results provide new evidence that NO induce photosynthesis and energy metabolism which may enhance Cd tolerance in barley seedlings.

Supplementary Information

The online version contains supplementary material available at 10.1007/s12298-022-01214-3.

Keywords: Barley, Cadmium stress, Nitric oxide, Proteomics

Introduction

Agricultural lands have become polluted by expanding industrial sector releasing high concentration of heavy metals. Heavy metal contamination of ecosystems is known to induce highly deleterious effects on the biological activity of soil, vital metabolic pathways in plants, and the human health (Hussain et al. 2021). Among metals, Cd easily accumulates in edible parts of plants due to its high bioavailability and mobility (Choppala et al. 2014). Even at low concentrations, Cd leads to chlorosis and stunted growth, inhibition of the carbon fixation and photosynthetic activity, disruption of water balance and uptake of minerals, and over-production of reactive oxygen species (ROS) in plants (Haider et al. 2021). A number of studies have reported that Cd-induced oxidative stress can easily be detected by an elevated accumulation of ROS and lipid peroxidation (Zhang et al. 2019; Qin et al. 2020; Hussain et al. 2021). Plants have evolved many complex defense mechanisms to withstand the oxidative stress (Tian et al. 2016). Moreover, plants also produce signaling molecules including nitric oxide (NO) which is involved in responses to various environmental stresses (Wani et al. 2021).

Nitric oxide acts as a multi-functional signaling molecule influencing a range of metabolic pathways such as growth regulation, flowering, senescence, and cell death (Domingos et al. 2015). NO is becoming increasingly significant as a regulator of various physiological processes in plants grown under Cd toxicity. The protective roles of NO against Cd toxicity have been reported in various crops including tomato (Ahmad et al. 2018), pepper (Kaya et al. 2019), maize (Liu et al. 2020a), and mustard (Khator et al. 2021). In wheat, exogenously applied NO can alleviate Cd toxicity by reducing the accumulation of Cd and ROS, and enhancing antioxidative defense system (Kaya et al. 2020). Moreover, NO has been shown to induce the activities of antioxidative enzymes, and production of non-enzyme antioxidants to reduce the ROS accumulation and membrane injury (Romeropuertas and Sandalio 2016). Additionally, the participation of NO in mitigating Cd toxicity has been documented in certain aspects including amplification of hormone signaling (Xu et al. 2010; Wang et al. 2013), mobilization of secondary messengers (Zhang et al. 2012), and modification and modulation of proteins (Gill et al. 2012; Wang et al. 2015). Although numerous studies have been performed to reveal the roles of NO in coping with Cd toxicity, molecular studies such as proteomics are required to unravel the NO-mediated Cd tolerance in plants.

Recently, the omics approaches have provided high-throughput analyses of biomolecules to address the precise molecular mechanisms related to stress tolerance in plants. Proteomics has a great potential to identify the main regulators of stress responses (He et al. 2018). Liu et al. (2020b) identified numerous differentially regulated proteins in rice seedling and suggested that proteins involved in redox reactions and chlorophyll metabolism facilitate the adaptation to Cd toxicity. Gong et al. (2017) suggested that NO treatment helped the cucumber seedlings to combat Cd stress by modulating photosynthesis, Cd transport and localization, glutathione-mediated Cd detoxification, redox homeostasis, and Ca2+ signaling. Yang et al. (2016) demonstrated that NO regulated the expression of plasma membrane proteins which were ATPases, transporters, metabolic enzymes, kinases, phospholipases, and phosphatases in rice leaves exposed to Cd. However, only a limited number of researches have studied the roles of NO at proteome level in plants under Cd toxicity.

In the present study, we aimed to derive new insight into the molecular mechanism of NO induced Cd tolerance response in the leaves of barley seedlings. Therefore, a gel-based proteomic analysis was used to identify protein functioning in the plant’s response to NO and Cd stress. Moreover, we also analyzed the seedling growth, chlorophyll content, ROS accumulation, lipid peroxidation as well as antioxidant enzyme activities in NO-treated barley seedlings under Cd stress conditions.

Materials and methods

Plant materials and growth conditions

Barley seeds (Hordeum vulgare L. cv. Akar) were surface-sterilized using 1% NaClO for 15 min and rinsed with sterile distilled water. Barley seeds were placed in petri dishes containing filter paper wetted with dH2O at 25 °C for 48 h in the dark. The seedlings with uniform size were transferred into hydroponic pots containing 1 L of modified Hoagland solution (pH 6.0). The seedlings were grown under controlled conditions. The light intensity was 250 μmol photons m−2 s−1 with 14/10 h photoperiod, and the temperature was 23 °C with 60% relative humidity. After 3 d of acclimation period, sodium nitroprusside (100 μM, NO donor) and Cd (50 μM, as CdCl2) treatments were given to the five-day-old seedlings. Thus, the following treatments were made: (1) control; 0 µM SNP and 0 µM Cd, (2) SNP; 100 µM SNP and 0 µM Cd, (3) Cd; 0 µM SNP and 50 µM Cd, and (4) SNP + Cd; 100 µM SNP and 50 µM Cd. The SNP and Cd applications were made for 10 days. The nutrient solution was replaced after every two days. After this, seedlings were harvested and leaf tissues were used for physiological, biochemical and proteomic analyses.

Measurement of growth attributes

Approximately 90 seedlings from three independent biological replicates for each treatment sample were used for the determination of fresh and dry weights. Fresh biomass of shoot and root tissues of treated seedlings were weighed after separation of seedlings into root and shoot. For the determination of dry weights, shoot and root tissues were oven-dried for 48 h at 80 °C.

Determination of chlorophyll and malondialdehyde (MDA) contents

The total chlorophyll content was determined following the method of Wellburn (1994). About 100 mg of tissues were collected from the second leaves and pigments were extracted with absolute methanol. The absorbance of supernatant was measured at 666 and 653 nm.

The degree of lipid peroxidation was determined by measuring the MDA content. MDA concentration was determined according to the protocol of Heath and Packer (1968). The extinction coefficient (155 mM−1 cm−1) was used to estimate the MDA content.

Histochemical analysis

Using 3′3′-diaminobenzidine (DAB) assay, hydrogen peroxide (H2O2) accumulation was visualized in the leaves (Christensen et al. 1997). For histochemical detection of lipid peroxidation, the leaf tissues were stained in Schiff’s reagent for 60 min (Pompella et al. 1987). Leaves were then decolorized by immersing in boiling 0.15% TCA in ethanol:chloroform (4:1). A digital camera was used to record the images of leaves.

Determination of antioxidant enzyme activities

The homogenates of leaf tissues (0.5 g) were prepared in 50 mM phosphate buffer (pH 7.0) containing 1% (w/v) polyvinylpyrrolidone (PVP) and 1 mM EDTA, and supernatants were used for enzyme assays after centrifugation at 14,000 rpm for 20 min at 4 °C. Ascorbate (5 mM) was added to extraction buffer for ascorbate peroxidase (APX) assay. Then, the homogenates were centrifuged at 14,000 rpm and + 4 °C for 20 min. The amount of protein in the supernatants was determined according to Bradford (1976). The superoxide dismutase (SOD) activity was estimated following the method of Beauchamp and Fridovich (1971). The estimation of total catalase (CAT) activity was done according to the method of Aebi (1984). The guaiacol peroxidase (POD) activity was determined according to Mika and Lüthje (2003). The method of Nakano and Asada (1981) was used for the determination of the ascorbate peroxidase (APX) activity.

Protein extraction and 2-D electrophoresis

Fine powdered leaf samples (1 g) were homogenized in 10% trichloroacetic acid and 0.07% β-mercaptoethanol (β-ME) in acetone. The homogenate was kept at -20 °C for 1 h and the precipitate was collected by centrifugation at 13,000 rpm for 15 min. The pellet was washed twice with cold acetone containing 0.07% β-ME. After centrifugation, the washed pellets were vacuum dried. Protein concentration in pellets solubilized in a buffer containing 2 M thiourea, 7 M urea, 4% CHAPS (3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate), 0.2% biolyte pH 3–10, and 40 mM dithiothreitol (DDT) was quantified according to Bradford (1976).

Prior to isoelectric focusing (IEF), approximately 80 µg protein sample was loaded to a 17 cm immobilized gradient IPG strips (pH 4–7). IEF was performed in Protean i12™ IEF System. After IEF, second dimension electrophoresis was performed on a 12.5% gel using a Protean II XL Cell System. Proteins in gels used for mass spectrometry were visualized according to Candiano et al. (2004). The analytical gels were stained with silver to visualize protein spots (Sinha et al. 2001).

Image analysis and protein identification

Briefly, a ChemiDoc™ MP System was used for imaging of the protein spots on the analytical gels, and images were evaluated with PDQuest 8.01. The volume of each protein spot was normalized against total spot volume of gels. Proteins exhibiting significant and reproducible abundance changes (at least 2.0-folds) were subjected to one-way analysis of variance (ANOVA). Only the spots showing significant (P < 0.05) alterations were designated to mass spectrometry.

The target protein spots cut manually from the preparative gels were subjected to in-gel digestion. A commercial kit was used for trypsin digestion (ThermoFisher Scientific). Resulting peptides were desalted and concentrated in ZipTipC18 pipette tips (Millipore). Purified peptides were mixed with α-cyano-4-hydroxycinnamic and eluted onto a MALDI plate. Tryptic-digested peptide masses were acquired using an AB Sciex MALDI-TOF/TOF 5800 plus MS. The MS/MS data were used to protein identification using the MASCOT search engine. The following parameters were used: enzyme trypsin, green plants taxonomic group, methionine as oxidized, carbamidomethylation of cysteine, mass tolerance of 50 ppm, ± 0.4 Da of peptide MS tolerance, and allowance one miscleavage.

A spot was regarded as a credibly identified protein when the protein score confidence interval (CI) was > 95%. The online analysis tool STRING was used to explore the protein–protein interactions (Szklarczyk et al. 2011). Molecular functions and biological processes were estimated by the BiNGO 3.0 plug-in of the Cytoscape (Maere et al. 2005).

Statistical analysis

The experiment was performed using completely randomized design with four treatments and three replicates per treatment. The Duncan’s multiple range test (DMRT) was performed to prove the significance at the 95% confidence level using SPSS software (version 22). The data are given as the mean ± standard deviation.

Results

SNP exerts positive effect on seedling growth

The effects of exogenous 100 μM SNP and 50 μM Cd stress on shoot and root growth parameters of barley seedlings are given in Table 1. Cd stress decreased shoot and root fresh weights by 38.1 and 53.4%, respectively, and dry weights by 29.5 and 41.9%, respectively (P < 0.05). SNP + Cd application caused a significant increase in seedling growth compared to Cd alone. This increase was 23.8% in root dry weight and 27.6% in shoot dry weight.

Table 1.

Effect of exogenous SNP (100 µM) on fresh weights (FW, mg.plant−1) and dry weights (DW, mg.plant−1) of barley seedlings exposed to Cd stress (50 µM)

Parameters Treatments
Control SNP Cd SNP + Cd
Shoot FW 437.2 ± 9.5a* 456.2 ± 16.0a 270.5 ± 12.1c 342.8 ± 11.2b
Shoot DW 40.0 ± 1.1a 42.0 ± 1.2a 28.2 ± 1.3c 36.0 ± 1.2b
Root FW 91.0 ± 3.0a 93.6 ± 2.9a 42.4 ± 3.4b 48.6 ± 2.4b
Root DW 8.55 ± 0.25b 9.32 ± 0.14a 4.97 ± 0.26d 6.15 ± 0.30c
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*Different letters indicate a significant difference according to DMRT

SNP protect chlorophyll content

The effect of exogenous 100 μM SNP and 50 μM Cd stress on leaf chlorophyll content is given in Table 2. Cd stress caused significant decrease (51.9%) in chlorophyll content (P < 0.05). SNP + Cd application increased the chlorophyll content by 58.7% compared to when Cd was applied alone. Additionally, SNP supplementation caused a significant increase in chlorophyll content compared to the control.

Table 2.

Effect of exogenous SNP (100 µM) on chlorophyll content, lipid peroxidation, and activities of antioxidant enzymes in the leaves of barley seedlings subjected to 50 µM Cd stress

Parameters Treatments
Control SNP Cd SNP + Cd

Chlorophyll content

mg.g−1 FW

 1.58 ± 0.06b* 1.73 ± 0.07a 0.76 ± 0.05d 1.13 ± 0.09c

MDA content

nmol g−1 FW

 15.3 ± 1.5a 14.1 ± 1.9a 23.7 ± 1.8c 18.1 ± 1.8b

SOD activity

Unit mg−1 protein

 8.32 ± 1.2a 12.6 ± 1.2b 16.9 ± 1.7c 11.3 ± 1.3ab

POD activity

μmol min−1 mg−1 protein

 5.54 ± 1.7a 7.94 ± 1.6a 75.6 ± 5.1c 28.5 ± 3.1b

APX activity

μmol min−1 mg−1 protein

 1.44 ± 0.18a 2.04 ± 0.14b 2.62 ± 0.19c 3.52 ± 0.23d

CAT activity

μmol min−1 mg−1 protein

 1.62 ± 0.07a 1.63 ± 0.06a 1.56 ± 0.05a 1.63 ± 0.09 a
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*Different letters indicate a significant difference according to DMRT

SNP mitigated Cd-mediated oxidative stress

The effect of 100 μM SNP and 50 μM Cd stress on leaf malondialdehyde (MDA) content is given in Table 2. When compared to the control, Cd stress caused a 1.5-fold increase in MDA content (P < 0.05). SNP + Cd application decreased the MDA content by 23.6% compared to Cd alone. The effect of 100 μM SNP and 50 μM Cd stress on hydrogen peroxide accumulation and lipid peroxidation is depicted in Fig. 1. Cd stress significantly increased hydrogen peroxide accumulation and lipid peroxidation. However, SNP + Cd application resulted in a significant decrease in these parameters compared to the Cd treatment.

Fig. 1.

Fig. 1

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In vivo detection of hydrogen peroxide accumulation and lipid peroxidation in the leaves of barley seedlings subjected to 100 µM SNP and/or 50 µM Cd stress

SNP regulated the activities of antioxidant enzymes under Cd stress

The effect of exogenous 100 μM SNP and 50 μM Cd stress on the activities of superoxide dismutase (SOD), guaiacol peroxidase (POD), ascorbate peroxidase (APX), and catalase (CAT) are given in Table 1. While Cd stress resulted in a significant increase in SOD and POD activities compared to control, SNP + Cd application reduced this increase. A gradual increase in APX activity was detected in SNP, Cd and SNP + Cd treatments compared to control which was 1.4, 1.8, and 2.4-fold, respectively (P < 0.05). CAT activity did not exhibit any significant alteration in Cd and/or SNP treatments.

SNP modulated proteome alterations under Cd stress

Alterations in leaf protein patterns were analyzed using two-dimensional (2-D) gel electrophoresis (IEF/SDS-PAGE) (Fig. 2). Approximately 500 protein spots were reproducibly detected on the gels. Thirty-nine protein spots with a 2.0-fold or greater increase in abundance were identified by image analysis. Thirty-four spots were successfully identified by spectrometric analysis (Supplementary Table S1).

Fig. 2.

Fig. 2

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The 2-DE pattern of leaf proteins from barley seedlings under control and Cd stress with and without exogenous SNP. The identified protein spots (1–34) are marked by the arrows (Supplementary Table S1)

Identified proteins which responded to SNP and/or Cd were categorized into different groups according to their biological roles (Supplementary Table S1). The categories with a high level of expression variations were those involved in photosynthesis and carbohydrate biosynthesis (47.1%), protein metabolism (23.5%), primer metabolism (11.8%), energy metabolism (11.8%), stress defense (5.9%), and signal transduction (2.9%). Totally, 30 proteins were significantly regulated by Cd compared to control. Of those, 24 proteins were up-regulated, and 6 proteins were down-regulated in abundance. Exogenous SNP application mitigated the Cd-mediated reduction in abundance of these proteins. On the other hand, 14 protein spots were significantly up-regulated by SNP under control or Cd stress conditions. Additionally, SNP reduced the Cd-mediated increase in the level of 15 proteins.

In order to explore protein–protein interactions, STRING clustering was performed (Fig. 3). It has been determined that photosynthesis and energy metabolism-related proteins such as glyceraldehyde-3-phosphate dehydrogenase (GAPB), transketolase (AT3G60750), ATP synthase proteins (ATP1, ATPB, ATPE) and cytosolic enolase 1 (ENOC) are important interaction points. Additionally, proteins such as heat shock 70 kDa proteins (HSC70 and HSP70), ATP-dependent Clp protease ATP-binding subunit clpA homolog (CLPC1), protein disulfide-isomerase (PDI), and 1-Cys peroxiredoxin PER1 (PER1) formed another interaction group. Proteins identified in this study were analyzed using the web-based BiNGO to determine the biological pathways and molecular functions associated with exogenous SNP and Cd applications (Fig. 4). Cadmium stress and SNP treatments intensely induced stress response related proteins and less intensely to photosynthesis related proteins.

Fig. 3.

Fig. 3

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Protein interaction map acquired by STRING tool indicates the complex interaction network

Fig. 4.

Fig. 4

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Gene ontology enrichment for differentially accumulated proteins in the leaves of barley seedlings

Discussion

It has been established that nitric oxide (NO) can regulate various physiological and biochemical responses of plants exposed to environmental stresses (Domingos et al. 2015; Prakash et al. 2019). In our study, Cd toxicity significantly decreased the seedling growth of barley plants and the SNP application mitigated the Cd-mediated reduction in seedling growth. This SNP-induced amelioration of growth may be related to the improvement in chlorophyll content as demonstrated in this study. NO-induced improvement in chlorophyll content may result in increased photosynthesis and plant growth which is important for plant Cd tolerance. Singh et al. (2020) suggested that NO-induced Cd tolerance is associated with the modulation of the antioxidative defense system to minimize the formation of oxidative damage in macromolecules, the organization of the cell wall to reduce metal accumulation, and up-regulation of the stress responsive genes. It has been shown that NO can significantly increase the Cd tolerance by reducing Cd uptake and ROS accumulation as well as increasing the antioxidant defense system and nutrient assimilation in wheat (Kaya et al. 2020). Additionally, it has been noted that NO interacts with molecules such as hydrogen peroxide and salicylic acid to activate important defense mechanisms and thus provide the elimination of oxidative damage (Mostofa et al. 2015). Our findings showed that SNP application significantly reduced Cd-mediated H2O2 production and thus causing a reduction in lipid peroxidation and MDA content. Similarly, previous studies showed that NO prevents ROS production and lipid peroxidation under Cd stress conditions (Ahmad et al. 2018; Kaya et al. 2020; Khator et al. 2021). These findings suggest that NO plays a role in reducing oxidative damage caused by Cd toxicity. The decrease in lipid peroxidation and H2O2 level in NO-treated barley seedlings may have resulted from the differential modulation of the antioxidative defense system.

To minimize oxidative damage caused by cadmium stress, plant cells generally have the ability to induce antioxidative defense mechanisms (Nabi et al. 2019). Re-establishment of cellular redox status by antioxidant machinery is one of the resistance strategies of plants to minimize ROS-induced toxicity under Cd stress (Kaya et al. 2020). In our study, SOD, POD and APX activities enhanced in leaf tissues of barley seedlings under Cd stress, while CAT activity remained unchanged. However, exogenous SNP significantly reduced the activities of SOD and POD enzymes compared to Cd alone while it increased APX activity. It has been showed that the reduction in the activities of antioxidant enzymes with SNP application is due to the fact that NO plays an important role in countering heavy metal-mediated oxidative stress (Kaur et al. 2015). A decrease in the antioxidant enzyme activities may be related to a putative role of NO in alleviating Cd-mediated oxidative stress. Additionally, the increase in APX activity indicates that NO has an important role in prevention of over-production of H2O2 and maintaining photosynthetic electron transport by providing NADH concentration.

Impairment in the plant growth and deterioration of the photosynthetic apparatus under Cd stress directly affected the level of proteins related to photosynthesis. Cadmium seriously affects photosynthetic machinery by diminishing the chlorophyll content, slowing down the photosynthetic rate, and directly damaging photosynthetic enzymes. Therefore, it is not surprising that Cd causes alterations in the abundance of proteins predominantly involved in photosynthesis. In the present study, the proteomic analyses revealed that the expression levels of RuBisCO small chain, ATP synthase epsilon chain and ferredoxin-NADP reductase (FNR) proteins decreased under Cd stress. SNP application resulted in higher levels of these proteins under Cd stress. Although the small subunit is not catalytic, it is required for maximum activity. In particular, it has been reported that the RuBisCO small subunit is required for the catalytic efficiency of carboxylation and the specificity of CO2/O2 (Krech et al. 2012). ATP synthase protein was differentially regulated in plants under Cd stress and may adjust ATP synthesis according to the plant's need for survival under Cd toxicity. These results suggest that exogenous SNP induces the abundance of photosynthesis-related proteins to improve resistance to Cd stress.

Increased expression levels of protein functioning in photosystems and the Calvin cycle may be a regulated feature to overcome Cd damage. The proteomic analyses showed that SNP application reduced Cd-induced increase in expression levels of plastid-lipid-associated protein 2 (fibrillin), RuBisCO activase A, RuBisCO activase B, glyceraldehyde-3-phosphate dehydrogenase B, ATP synthase beta subunit, transketolase, and glucose-1-phosphate adenylyltransferase which function in different stages of photosynthesis. Fibrillins are a large family of chloroplastic structural proteins related to stress resistance (Singh et al. 2010). RuBisCO activase is a key regulatory enzyme that catalyzes the activation of RuBisCO (Ashraf and Harris 2013), and previous studies have reported that Cd stress increases its abundance (Rao et al. 2017; Bagheri et al. 2015). Regulation of the Calvin cycle enzymes such as GAPDH and transketolase is required not only to enhance the production of ATP, NADH and NADPH depending on the energy demand of cells, but also to provide production of carbon skeletons for the synthesis of chelators involved in Cd detoxification (Sarry et al. 2006). Differential modulation of these proteins depending on the SNP application indicates the regulatory role of NO on the photosynthetic mechanism.

Eight proteins were found to be associated with protein metabolism and divided into three functional groups. The first group includes the 50S ribosomal protein L12, which play a role in the initiation of polypeptide chain synthesis in chloroplasts. Cadmium stress significantly reduces the expression level of 50S ribosomal protein L12 indicating that protein biosynthesis in chloroplasts is inhibited under Cd stress. However, exogenous SNP application increased the expression level of this protein under Cd stress. In the second group, protein disulfide isomerase, heat shock cognate 70 kDa protein 1 and heat shock 70 kDa protein are involved in protein folding and assembly. The proteomic analyses revealed that the expression levels of these proteins increased under Cd stress. SNP + Cd application reduced the abundance of these proteins compared to Cd application. It has been noted that HSPs play vital roles in restoring the natural structures of damaged proteins by maintenance of normal protein folding (Finka et al. 2016). These results may suggest that Cd stress negatively affects protein metabolism and that NO protects proteins against Cd-induced damage, possibly by different mechanisms.

In the third group related to protein metabolism, there are three proteins associated with protein degradation. It is known that Cd disrupts photosynthesis and causes over-production of ROS (Schützendübel and Polle 2002). It has been shown that ROS can oxidize proteins and increase protease activity (Sandalio et al. 2001). In our study, Cd stress up-regulated the chloroplastic cell division protease ftsH homolog 1 and ATP-dependent Clp protease ATP-binding subunit clpA homolog proteins. ATP-dependent Clp protease has been reported to help remove abnormal or damaged proteins (Lionaki and Tavernarakis 2013). These proteases may have a crucial function in protein conversion to prevent the accumulation of dysfunctional proteins, thereby increasing the available amino acid pool necessary for the synthesis of defense-related proteins.

Carbohydrate and energy metabolisms are very important biological processes in terms of meeting the increasing energy needs of plants under stress. Adenylate kinases stabilize adenylate levels by reversibly forming ADP through the transfer of a phosphate group from ATP to AMP (Dzeja and Terzic 2009). In the current study, the NO-promoting increased level of mitochondrial adenylate kinase may provide more adenylate substrate for ATP synthesis in mitochondria under Cd stress. Expression levels of mitochondrial ATP synthase alpha subunit and enolase 1 proteins were upregulated in SNP + Cd application. NO-induced up-regulation of these enzymes may be necessary to meet the increased energy demand under stress conditions.

An up-regulation of oxidative stress related proteins is one of the important responses of plants under heavy metals. In this study, while Cd stress decreased the expression level of 1-Cys peroxiredoxin PER1 protein, exogenous SNP application recovered this effect. Besides being a thiol specific peroxidase that detoxifies peroxides, peroxiredoxins (Prx) have important roles in redox sensing and enzyme activation (Medina et al. 2009). Gupta et al. (2018) proposed that the role of Prx in Cd detoxification is based on being part of the peroxiredoxin/thioredoxin (Prx/Trx) pathway that detoxifies hydrogen peroxide. This pathway includes reduction of hydrogen peroxide with Prx and recovery of catalytic activity of Prx using Trx (Zhong et al. 2017).

Plants must be able to sense Cd stress in order to generate cellular responses to tolerate Cd. Sensing cadmium stress and generating tolerance responses requires the coordinated functioning of intertwined biological processes such as changes in gene expression and protein modification (Urano et al. 2010). The 14–3-3 protein group contains multifunctional regulators related to various processes in the cell. 14–3-3 proteins interact with several signaling molecules such as mitogen-activated protein kinase and calcium-dependent protein kinase (Ormancey et al. 2017). It is known that 14–3-3 proteins play a role in the formation of cellular responses to various stresses including Cd stress (Du et al. 2015). In our study, SNP + Cd application increased the expression level of 14–3-3 protein. An increase in relative abundance of 14–3-3 protein in barley leaves suggested that it may be involved in the activation of defense against Cd stress.

Conclusions

In this study, Cd stress negatively affected seedling growth by causing a significant reduction in chlorophyll content, and an increase in lipid peroxidation and H2O2 content. However, SNP application significantly alleviated the Cd-mediated reductions in chlorophyll content and helped to maintain the growth of barley seedlings under Cd stress by regulating the activity of antioxidant enzymes and causing a decrease in lipid peroxidation. Increased expression levels of crucial proteins related to photosynthesis, carbohydrate metabolism and glycolysis provided a main energy source for growth and metabolism of barley seedlings under Cd stress. However, it can be argued that exogenous SNP application contributes to the development of Cd tolerance by regulating photosynthesis, energy, and protein metabolism. These findings will provide a better understanding of the molecular mechanisms associated with NO-induced Cd tolerance in plants and will form the basis for further functional studies.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file1 (DOCX 162 kb) (162.6KB, docx)

Acknowledgements

The work was funded by the Scientific Research Projects Coordination Unit (Project No: 19.FEN.BİL.43) of Afyon Kocatepe University. The authors gratefully acknowledge the Medicinal Genetics Laboratory of Afyonkarahisar Health Sciences University and the DEKART Proteomics Laboratory of Kocaeli University for their technical help. The authors are grateful to Afyon Kocatepe University’s Foreign Language Support Unit for language editing.

Author contributions

All authors have contributed equally to this work.

Funding

This study has been funded by Afyon Kocatepe University Scientific Research Projects Coordination Unit (Project No: 19.FEN.BİL.43).

Data availability

The submitted work is original and has not been published elsewhere in any form or language.

Code availability

Not applicable.

Declarations

Conflict of interest

The authors have declared that they have no conflict of interest.

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