Introduction

Salinity is one of the major abiotic stressors limiting agricultural production and plant distribution with NaCl as the predominant salt (Jin et al. 2009). Many crop species are sensitive to the presence of NaCl (Szalai and Janda 2009). The responses of plants to excess NaCl are very complex and involve changes in morphology and metabolism (Kováčik et al. 2009; Yousfi et al. 2010). Also, elevated NaCl evokes the formation of reactive oxygen species (ROS) depending not only on the developmental stage and plant species/organ (Jamal et al. 2011; Radl et al. 2013; Jabeen et al. 2015) but also on the mode of NaCl application (Spanò and Bottega 2016).

Barley (H. vulgare L.) is widely cultured in arid and semiarid regions. It is considered a moderately tolerant forage crop and a highly tolerant grain crop, but a great variability exists among cultivars in response to salinity (Jin et al. 2009; Kamboj et al. 2015).

Soil quality may be influenced by organic materials, which improves its chemical, physiological, and biological properties (Khaled and Fawy 2011). Humin, humic, and fulvic acids, commonly known as humic substances, are the main organic soil compounds.

Despite extensive research, the metabolic response of plants to either increased salinity or humic substances have only rarely been reported. The aim of our work was to study the impact of humic acid (HA) on the metabolic response of barley exposed to salinity stress. To achieve this, selected metabolites were quantified by combined liquid chromatography/mass spectrometry, and fluorescence microscopy was used aimed at visualizing changes of ROS and nitric oxide in root tips. Foliar and hydroponic applications of HA were also compared.

Materials and methods

Plant culture and experimental design

Barley (Hordeum vulgare L., cv. Radegast) grains were surface-sterilized in 70 % ethanol, rinsed in deionised water, and germinated in Petri dishes on wet filter paper for 72 h in a growth chamber. Thereafter, uniform seedlings were placed in 1/10-strength Hoagland solution in dark plastic boxes (Kováčik et al. 2014a) and 7 days-old ones were exposed to either 100-mM NaCl, 6.0-mg HA/L (technical humic acid, Sigma-Aldrich, Cat. No. 53680) or a combination (NaCl + HA). Also, foliar application of HA (6.0 mg/L) to plants cultured with 100-mM NaCl was tested by applying 5 mL of test solution per shoot daily (Triton X-100, 0.1 mL/L, was added to the stock solution to increase solubility, and the hydroponics was protected from contamination by droplets). The dose of HA was selected based on the preliminary experiments (impact of HA on Na accumulation, data not shown). Control plants were cultured in Hoagland solution with no additions. Unless otherwise specified, plant material was harvested 7 days after treatment, but time dynamics of some antioxidants was monitored (1, 3 and 7 days of exposure), and selected metabolites were measured in the shoots only. All experiments were performed in a growth chamber under conditions as reported earlier (Kováčik et al. 2014a). Spectrophotometry was carried out with Agilent/HP DAD UV/Vis 8453 spectrophotometer. Metabolites were quantified by liquid chromatography with mass spectrometry detection (LC–MS/MS) using device Agilent 1200 Series Rapid Resolution LC system coupled on-line to Agilent 6460 Triple quadrupole detector with Agilent Jet Stream Technologies. Fluorescence microscopy was performed by Axioscop 40 (Carl Zeiss, Germany) equipped with appropriate set of excitation/emission filters (Kováčik et al. 2014a).

Measurement of growth, tissue water content, soluble proteins, and free amino acids

After 7 days of exposure to treatments, plants were harvested, separated into roots and shoots, and dried. Their length, fresh, and dry mass were also recorded. For parameters measured in fresh samples, whole shoots or roots were powdered using liquid N2 and assayed as described below. Plant water content [100 − (dry mass × 100/fresh mass)] was determined to recalculate parameters measured in fresh samples. Dry samples (dried at 75 °C to constant weight) were ground to a fine powder and analyzed for mineral nutrients, free amino acids, and phenolic acids. Soluble proteins were quantified using Bradford method and bovine serum albumin as standard (in extracts prepared using 50-mM potassium phosphate buffer) and free amino acids by HPLC (Hewlett Packard, Waldbronn, Germany) with fluorometric detector FLD HP 1100 and precolumn derivatization as reported earlier (Kováčik et al. 2014a).

Assay of antioxidants and antioxidative enzymes

Extraction of antioxidants (ascorbic acid—AsA, reduced glutathione—GSH, phytochelatin 2—PC2) was done in 0.1 M HCl (fresh biomass) followed by centrifugation, and the supernatant from these crude extracts was analyzed by LC–MS/MS system at specific m/z values (Kováčik et al. 2014b). Activities of antioxidative enzymes were determined in potassium phosphate buffer extracts mentioned above. Ascorbate peroxidase (APX), glutathione reductase (GR), catalase (CAT), and superoxide dismutase (SOD) activities were evaluated spectrophotometrically (Kováčik et al. 2014a, b, 2015).

Determination of phenolic and organic acids

Selected cinnamic and benzoic acid derivatives from dried shoots only were extracted in 80 % methanol, while aliphatic organic acids were quantified in the 0.1-M HCl extracts from fresh samples mentioned above; identification and quantification was performed by LC–MS/MS system using specific m/z values and available standards (Kováčik et al. 2014a, 2015).

Quantification of mineral nutrients

Mineral nutrients (Na, K, Ca, Mg, Fe and Zn) were determined by atomic absorption spectrometer AA30 (Varian Ltd., Mulgrave, Australia) and the air-acetylene flame. Samples were prepared by digestion of dried samples in the mixture of concentrated ultrapure HNO3 and water using microwave decomposition (Ethos Sel Microwave Extraction Labstation, Milestone Inc.) as noted earlier (Kováčik et al. 2014b, 2015).

Fluorescence microscopy

Only root tips were observed owing to their direct contact with the culture medium. They were excised from similar positions and immediately stained. ROS were visualized using CellROX® Deep Red Reagent (644ex/665em) and nitric oxide (NO) using 2,3-diaminonaphthalene (365ex/415em). Stock solution of CellROX® Deep Red Reagent in DMSO was diluted by phosphate buffered saline (PBS) buffer (0.05 M, pH 6.8) to final concentration of 5 μM, while stock solution of 2,3-diaminonaphthalene in 0.62 M HCl was diluted by PBS buffer to the final concentration of 250 μM. Root tips were stained for 60 min at 37 °C in the dark, washed three times with PBS buffer, and observed (Kováčik et al. 2014a, b).

Statistical analyses

Homogeneity of variance was verified by Levene’s test, and data were evaluated using ANOVA followed by Tukey’s test (MINITAB Release 11, Minitab Inc.; State College, PA, USA) at P < 0.05. Number of replications (n) in tables/figures denotes individual plants measured for each parameter. At least three boxes were cultured for each treatment.

Results and discussion

Effect of HA and NaCl on physiology and mineral content of barley plants

In accordance with previous findings in Triticum aestivum grown for 55 days with 80- or 120-mM NaCl (Jamal et al. 2011), we found that 100-mM NaCl decreased shoot and root length compared with control plants (Table 1). Protective effect of humic acid has been demonstrated in many cereals, e.g., in maize and wheat under salt stress (Khaled and Fawy 2011; Aydin et al. 2012). Our results showed that HA was able to relieve the growth inhibition induced by NaCl in shoots but not in roots (Table 1). Only shoot water content was affected by salinity (Table 1) as previously reported in barley (Yildiz and Terzi 2013). HA altered the water content neither alone nor in combination with NaCl (Table 1), which could be related to a lower HA dose used in this experiment in comparison with other works (Khaled and Fawy 2011). Soluble protein content was unaffected by NaCl or HA, and only foliar HA was able to compensate for its decrease induced by NaCl in roots (Table 1). In comparison, more extensive depletion of soluble proteins was observed in salt-tolerant T. aestivum roots exposed to 80-mM NaCl over 16 days (Radl et al. 2013).

Table 1 Shoot and root length, water, soluble protein, and mineral nutrients content in barley (H. vulgare, cv. Radegast) plants after 7 days of treatments, as mentioned in Fig. 1
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As might be expected, Na strongly accumulated in both shoots and roots of barley after the addition of NaCl (ca. 100- and 7-times more when compared with control; Table 1), which is in accordance with data from various barley cultivars exposed to 150-mM NaCl (Kamboj et al. 2015). It was an interesting finding that HA reduced Na accumulation in both above- and below-ground tissues (Table 1). This is in contrast with data from corn plants where the level of Na was elevated after exposure to 60-mM NaCl with foliar application of 0.1 % HA (Khaled and Fawy 2011).

The decrease in K+ content under salinity detected in barley tissues (Table 1) may be related to the apparent restriction in elongation growth owing to K+ importance for many biochemical processes. Besides, NaCl also negatively affected the accumulation of Ca, Fe, and Zn but decreased Mg content in barley roots only. This negative effect on mineral nutrient uptake was observed, e.g., in cultivars of Triticum durum exposed to 60-mM NaCl for 35 days (Aşik et al. 2009). Compared with accumulation of Na, the interaction between NaCl and HA did not affect majority of macro- and micronutrients in the present study (Table 1).

Effect of HA on metabolic responses in NaCl-exposed barley

Amount of free amino acids in barley plants exposed to NaCl revealed that NaCl significantly decreased the content of Asp, Gly, Thr, Arg, and Ala (Table 2). Consequently, NaCl depleted the total amino acid amount except for histidine and proline: strong decrease in total shoot amino acid content was also observed in barley plants at 100-mM NaCl (Yousfi et al. 2010). In the present study, HA has an elevating effect on the majority of monitored free amino acids except for His, Tyr, Val, Leu, and Ile (Table 2). Interaction between HA and NaCl elevated the accumulation of Asp, Glu, Gly, and Ala. Moreover, foliar unlike to hydroponic application enhanced the content of phenylalanine, which is a crucial precursor of the phenylpropanoid pathway. Proline is known to be involved in osmoregulation, acts as a free radical scavenger (Yildiz and Terzi 2013) and is typically elevated by NaCl in various species (Haddadi et al. 2016). In both combined treatments, amount of proline was higher compared with HA alone but lower in comparison with NaCl. These results indicate that proline appears to be related to salt tolerance as its content changed similarly to shoot Na amount (Table 1).

Table 2 Accumulation of free amino acids (µmol g−1 DW) in barley (H. vulgare, cv. Radegast) shoots after 7 days of treatments, as mentioned in Fig. 1
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Among 11 monitored phenolic derivatives, NaCl depleted the content of p-hydroxybenzaldehyde, p-coumaric, p-hydroxybenzoic, protocatechuic, salicylic, sinapic, and vanilic acids in the shoot (Table 3). These data are in contrast with the results found in other plants, such as chamomile (Kováčik et al. 2009), in which phenolic acids rather increased. These differences might be related to given species and experimental conditions. HA coapplied with NaCl had a significant ameliorating effect on some acids and their sum when compared with NaCl-treated plants (Table 3): this could play a role in diminishing the oxidative stress. Other phenols may also be involved in the protection against salinity, for example, the expression of some flavonoid genes along with the accumulation of metabolites was detected in Solanum nigrum exposed up to 150-mM NaCl over 3 weeks (Ben Abdallah et al. 2016).

Table 3 Accumulation of phenolic acids (µg g−1 DW) and organic acids (mg g−1 DW) in barley (H. vulgare, cv. Radegast) shoots after 7 days of treatments, as mentioned in Fig. 1
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Salt treatment induced a decrease in the majority of organic acids, except for succinic acid, in shoot tissue (Table 3). Slight accumulation of succinic acid was also observed in NaCl-treated Arabidopsis thaliana: γ-aminobutyric acid metabolism seems to be a source of succinate mobilized under stress conditions to help in buffering the tricarboxylic acid cycle (Renault et al. 2013). Depletion of the majority of monitored organic acids, such as citric, fumaric, and malic acids, indicates a deleterious impact on respiration. It may lead to the inhibition of cellular respiration and, consequently, to disruption of shoot development. Addition of HA to the culture medium evoked a decrease in citric, malic, and pyruvic acids but enhanced lactic acid accumulation which is evidence of induction of fermentative processes. However, HA coapplied with NaCl stimulated mainly citric acid accumulation (compared with NaCl alone), which is further evidence supporting a positive effect of HA against salinity.

HA modulates NaCl-induced oxidative stress and antioxidants

The most intensive increase in ROS was observed under the NaCl treatment (Fig. 1). Application of HA alone had a negligible impact on ROS generation, while HA in combination with NaCl attenuated ROS formation in particular in the treatment with foliar HA application (Fig. 1). NO formation was also visibly affected by HA coapplication mainly after 7 days of exposure and in the apical part of root tips—ROS were suppressed, but NO enhanced (Fig. 1). Changes of NO evoked by HA may, therefore, be involved in the protective effect of HA observed in our study, and further experiments with the manipulation of NO level are needed.

Fig. 1
figure 1

Fluorescence microscopy of reactive oxygen species (CellROX® Deep Red Reagent, red signal) and nitric oxide (2,3-diaminonaphthalene, blue signal) in H. vulgare root tips cv. Radegast exposed to NaCl (100 mM), humic acid (HA, 6.0 mg/L), NaCl + humic acid applied hydroponically (NaCl + HA), or NaCl + foliar application of humic acid (NaCl + HA/foliar) over 1 or 7 days. C control, bar indicates 100 µm. HA technical humic acid (Sigma-Aldrich, Cat. No. 53680)

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Salinity induced a time-dependent decrease in the GSH and AsA content in barley shoots (Fig. 2): this contrasts with report that NaCl-induced an increase in AsA content in barley leaves cultured under 160-mM NaCl (Pérez-López et al. 2010). In the roots, NaCl-induced depletion of AsA or GSH (if any) was less visible. HA alone had either no effect or stimulated an increase in AsA or GSH amount time-dependently. In the combined treatments, foliar and hydroponic HA coapplication (with NaCl) showed a typically similar impact in the shoots (Fig. 2a) and rather stimulated AsA or GSH accumulation (in comparison with NaCl alone) in the roots (Fig. 2b). A more pronounced impact of HA coapplication in the roots could be related to the above-mentioned attenuation of the ROS formation (see Fig. 1). Phytochelatin (PCs) accumulation in humate-treated plants has only rarely been reported (Figueroa et al. 2007). In the present study, NaCl caused a time-dependent decrease in PC2 content in barley shoots and roots (Fig. 2). This contradicts the data from Cajanus cajan roots, where the level of PC2 was not affected after 60 days with 60-mM NaCl (Garg and Chandel 2012). Surprisingly, HA applied alone to the culture medium induced the biosynthesis of PC2 in roots (Fig. 2), which could be related to the ability of HA to stimulate mineral nutrients uptake through eventual formation of PC2-metal complexes (Garg and Chandel 2012).

Fig. 2
figure 2

Time course of quantitative changes of non-enzymatic antioxidants (AsA ascorbic acid, GSH reduced glutathione, PC2 phytochelatin 2) in barley (H. vulgare, cv. Radegast) shoots (a) and roots (b) cultured in treatments as mentioned in Fig. 1. C control. Data are means ± SDs (n = 6). Values for each harvest day, followed by the same letter(s), are not significantly different according to Tukey’s test (P < 0.05)

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The activities of APX, GR, CAT, and SOD were significantly higher in barley roots after 7 days of exposure to NaCl (Fig. 3), which corresponds to the increase in ROS formation (Fig. 1). In accordance, the activities of APX, GR, CAT, and SOD increased very rapidly (within 1 day) under salt conditions (200-mM NaCl) in barley, and then variously elevated levels were maintained (Kim et al. 2005). Increase in SOD activity observed here was also detected in barley shoots exposed to 100-mM NaCl (Jin et al. 2009). NaCl depleted the activity of GR in the shoots which may be related to the observed decline of GSH content. Changes in the activity of antioxidative enzymes under the influence of HA have only rarely been reported (García et al. 2012). HA alone elevated the activities of GR and CAT in shoots (Fig. 3). This is in accordance with data where HA (20–80 mg L−1) increased CAT activity in rice (García et al. 2012). We also found that applied HA decreased the activities of APX, CAT, and SOD in the roots (in comparison with NaCl-treated roots), and this may be related to depleted signal of ROS (Fig. 1). It is concluded that HA reduces the oxidative damage in NaCl-treated roots by modulating oxidative balance; however, these effects are certainly dose-dependent (if compared present data with other works discussed above).

Fig. 3
figure 3

Changes in the activity of antioxidative enzymes (APX ascorbate peroxidase, GR glutathione reductase, CAT catalase, SOD superoxide dismutase) in barley (H. vulgare, cv. Radegast) plants after 7 days of treatments as mentioned in Fig. 1. Data are means ± SDs (n = 6). Values for leaves (shoots) or roots, followed by the same letter(s), are not significantly different according to Tukey’s test (P < 0.05)

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Conclusions

Metabolic analyses of hydroponically cultured barley confirmed that low dose of humic acid (6.0 mg/L) may positively affect shoot growth and accumulation of selected metabolites including antioxidants (ascorbate, thiols), while the protective impact of HA in combination with NaCl was rather visible at the level of Na uptake and oxidative stress appearance. Foliar and hydroponic HA application revealed roughly similar efficiency. Our data suggest the potential usefulness of HA to protect the sensitive, but valuable barley cultivar Radegast against NaCl excess and urge for further field research.

Author contribution statement

MJ and JK designed the experiments, performed enzymatic measurements, data analyses, and wrote the manuscript. BK, PB and JH performed HPLC, fluorescence microscopy and mineral nutrients assay, respectively.