1 Background

Plant breeders aim to increase plant production to afford food and money, this process is highly influenced by the available resources such as soil, irrigation water, and high-quality plant genotypes. The existence of different varieties coming out of one ancestor crop is evident in the efficient approaches and strategies of the crop to cope with the different environmental conditions, maintain viable functions such as photosynthesis and respiration, and develop the most tolerant varieties and lines. Selection of the plant genotypes or line (varieties); that will cope with the continuously changing environment should be supported with new research aspects. Maize (Zea mays L.) is the third strategic crop after rice and wheat. It is moderately sensitive to salinity; however, enormous genetic variation for salinity resistance exists in maize [1].

It is well known that irrigation with saline water and high evaporation increase salinity at a rate of 10% annually in agricultural areas. So, it is expected that more than 50% of arable land will turn into salty soil by 2050 [2,3,4]. The combination of climatic features and anthropogenic pressures increases salinity and cultivated soil contamination, leading to a qualitative and quantitative reduction in crop production [5]. According to Živanović [6] the flacks make up for the stomatal conductance with higher accumulation levels of free sugars and amino acids to maintain the growth rate in tomato genotypes during water shortage. In addition, the accumulation of salts in the root areas may reduce the growth of plants, and the nutrient cycle, and decline the irrigation water quality, and crop productivity [7, 8].

Salt stress also bustles the stomata closure to minimize the water loss, this stomata closure in turn increases the stress complications, raises the osmotic and oxidative pressure with excess production of reactive oxygen species (ROS) such as superoxide O2n-, hydrogen peroxide H2O2 and hydroxide radical OH., over its normal threshold [9]. Normally, most of the plants have their defense mechanisms as a natural survival instinct to equalize or neutralize the ROS burst and to reduce its harm effect on the cell survival or compartments. For example, the H2O2 molecule can cross the cellular membrane to move with water in a way distant from that of its production site [10, 11]. Many physiological and biochemical responses are included in plants under saline treatments. These changes involve a decrease in photosynthetic capacity, water status, changes in osmoregulation, and accumulation of toxic ions and ROS generation in roots and leaves. Oxidative damage was much higher in salt-sensitive rice cultivars than in salt-tolerant cultivars which have a greater antioxidant capacity [12]. High salinity levels impede the plant water uptake and the ionic (Na + and Cl −) accumulation in the cytosol leads to ion toxicity. Because of the reduction in photosynthesis excessive ROS will accumulate [13,14,15]. As a biodefense mechanism to the ionic stresses, plants close their stomata to minimize water loss and reduce all the physiological processes and growth as well their growth rates [16, 17]. The ROS over-exceed production and send signals to all cell compartments; the strong signal may devastate the molecular and cellular components due to the bimolecular oxidation and may lead to plant death. Plant immunity tends to equalize and neutralize the damages, via the production of enzymatic and non-enzymatic antioxidants which quench the excess ROS, thereby providing a shield against oxidative stress [18]. High accumulation of ROS products works in two different ways; either by the intercellular and/or intracellular signaling (cross talks) to fortify the plant fitness or by oxidative burst leads to apoptosis or complete plant death. ROS directly demonstrates that treatment with compounds inducing oxidative stress resulted in increased autophagy gene expression and autophagosome formation [19].

The enzymatic antioxidant system includes enzymes such as Superoxide dismutase (SOD) exist in all oxygen-metabolizing cells and subcellular compartments like mitochondria, chloroplasts, nuclei, cytoplasm, peroxisomes, apoplasts, etc. [20]. Since that helps the plants to get rid of and sweep the ROS that arose during stress conditions successfully [21]. Another enzyme in the plant defense system is ascorbate peroxidase (APX) enzymes in many isoforms play a catalyzing role in the transforming of H2O2. Different APX isoforms are also present in distinct cytoplasmic organelles, such as chloroplasts, mitochondria, peroxisome, and cytosol [22]. Additionally, Phenylalanine ammonia-lyase (PAL) has a very vital role in plant defense against pathogens and abiotic stress. PAL accumulation triggers cinnamic acid production which enhances the plant's performance under such stresses [23]. Another way to scavenge the excess of ROS is the secondary metabolites which are produced through plant growth to serve some of the cellular functions related to physiological processes against stress and defense response signaling. The species and genotype of plants affect the type and concentration of secondary metabolites [24]. Focusing on the balance between ROS species and the antioxidant enzymes as a biomarker can explain the plant's performance and fitness under stress; this can help in the selection of the most tolerant plant. Transmission electron microscopy (TEM) is a very useful tool for imaging cellular structures with nanometer resolution and it can be used to figure out the cellular organelle's alterations if exist to character the plant response to stress and to localize the point of damage of organelles.

Therefore, the objectives of the present study were to evaluate the effects of salinity stress on different white maize inbred lines differing in their sensitivity by measuring the level of biochemical mechanisms and their correlation with changes in antioxidant enzyme activities. In addition, the effect of salinity on some subcellular compartments in meristematic root cells to understand the mechanism of salt tolerance in these cultivars.

2 Materials and methods

This designed experiment was conducted during the year 2021–2022. Pure grains of white maize from five inbred line (P4, P8, P12, P15, and P17) released seven years ago; were kindly provided by the head of research team of corn breeders- corn breeding program- in the Genetics and Cytology Department. NRC, Giza, Egypt. These lines showed respectable tolerance against drought; they were studied for their responses to salt stress. The study went on two parallel steps as follows:

1- A field experiment was conducted in the greenhouse of the National Research Centre (NRC) to take the morphological and biochemical data and all of the chemical analyses were performed at the biochemistry lab Faculty of Agriculture, Cairo University.

2- The lab experiment was conducted in the cytology and cytogenetic lab of “National Research Centre, Egypt (NRC)” and processed for transmission electronic microscope investigation and image capture in TEM lab CURP (Cairo University Research Park—Faculty of Agriculture), to study the ultrastructural components of salt treated root meristems after direct treatment for three hours with the transmission electronic microscope.

3 Experimental design

The experiment was designed in a randomized complete design with three replicates. The seeds of the same size for maize inbred lines (P4, P8, P12, P15, and P17) were cultivated with three seeds in each pot. Pots were filled with normal soil (loamy sand) pH of 7.6. During cultivation, the control plants were irrigated with tap water as usual to maintain the optimum moisture level. Four levels of salinity (1000, 2000, 3000, and 10000 mg/L) were used, which were prepared using NaCl and distilled water [25]. Two weeks after seedling emergence, plants subjected to saline stress were irrigated with a saline solution three times per week to maintain the desired level of salinity. Three plants of uniform size were maintained in each pot in the greenhouse with 70% relative humidity, natural temperature, and day 16 h photoperiod throughout the experimental period, after two weeks of irrigation with saline water, data for the shoot lengths were recorded in percentage.

3.1 Biochemical analysis

  • Total Chlorophyll content

Total Chlorophyll was extracted by 80% (v/v) acetone from leaves of three biological replicates of the above-inbred lines and determined spectrophotometrically at wavelengths 645 nm and 663 nm, and the amount of chlorophyll a, chlorophyll b, and total chlorophyll were calculated [26].

  • Total free amino acids and Total soluble sugars

Total free amino acids were extracted from leaf tissues (100 mg) in three biological replicates of inbred lines and determined [27] using ninhydrin reagent. The purplish-blue color was read at 570 nm and the quantity of amino acids was calculated from a reference curve prepared using glycine. The total soluble sugars were quantified from leaf tissues (100 mg) of inbred lines using the phenol–sulphuric acid method [28] and calculated using a glucose standard curve.

  • Measurement of Antioxidant enzyme activity and Hydrogen peroxide content

All estimated enzymes were extracted from fresh leaf tissues of the above-inbred lines (0.5 g) by 4 ml phosphate buffer (100 mM, pH = 7.8), 0.5% Triton-100, and 0.1 g PVPP for grinding. The temperature was maintained at 4 °C overnight. The extract was centrifuged for 15 min at 10,000 rpm at 4 °C [29].

Superoxide dismutase (SOD) activity was estimated using UV–a visible spectrophotometer at pH 8.8, 37 ℃, in 50 mM air-saturated 2-amino-2-methyl-1,3- propanediol buffer containing 3 mM boric acid and 0.1 mM diethylenetriaminepentaacetic acid. The kinetic measurement at 560 nm for each sample extract and expressed as U.g−1FW.min−1 [30].

Ascorbate peroxidase (APX) activity was determined by measuring a decrease in optical density at the wavelength of 290 nm as ascorbate was oxidized (U.g−1Fw.min−1) [31].

Hydrogen peroxide (H2O2) was extracted from plant leaf tissues and estimated [32], 0.5 g fresh leaves were ground together with 5 ml of 5% TCA and 0.15 activated charcoal. The mixture was centrifuged at 10,000 g at 4 °C for 20 min. Supernatant was taken and adjusted to pH 8.4 with (17 M) ammonia solution and filter. 1 ml of filtrate was added 8 µg of catalase then kept for 10 min at room temperatures. Prepare the blank without catalase, to both aliquots, 1 ml of colorimetric reagent was added. The reaction solution was incubated at 30 °C for 10 min. The absorbance of the solution was calculated at 505 nm and used to determine the H2O2 concentration.

  • Phenylalanine ammonia lyase (PAL) activity and total phenolic content

PAL enzyme extraction was achieved according to the method developed by [33] with some modifications. PAL was estimated in 2 ml of reaction mixture [0.4 ml of enzyme extract made up to 1.5 ml by the addition of 0.1 mol L−1 sodium borate buffer (pH 8.8) and 0.5 ml of 12 mol L− phenylalanine]. After incubation for an hour at 25 ℃, the reaction was stopped by incubation at 47 °C for 10 min. Conversion of L-phenylalanine to trans-cinnamic acid was measured at 290 nm [34].

The total phenolic content was determined spectrophotometrically at 760 nm, using the Folin–Ciocalteu reagent [35].

3.2 The cytological experiment

Grains of nearly equal weight and size were grown for five days on filter paper rolls in beakers filled with running water till have 1.5–2 cm root length, seedlings in three replicates for each inbred line were then directly treated with 0, 2000, and 3000 mg/L of NaCl solutions for period 3 h (the average of the clay soil–water holding capacity), then immediately fixed in 5.0% glutaraldehyde in 0.1 M sodium cacodylate buffer, 7.3 pH; post-fixed in 1% osmium tetroxide in 0.1 ml sodium cacodylate buffer; immersed in a mixture of resin and propylene oxide; stained in toluidine blue for light microscope examination before final trimming; stained for ultra-thin sections: uranyl acetate 30 min followed by lead citrate for 15 min, the sections of tissue were stained with uranyl acetate and lead citrate [36]. The ultra-thin sections were then examined by transmission electron microscope JEOL (JEM-1400 TEM) at the candidate magnification. Images were captured by CCD camera model AMT, an optronics camera with 1632 × 1632 pixels format as a side mount configuration.

4 Statistical evaluation

Analysis of variance (ANOVA) one-way, followed by SMD (standard mean deviation) was used. The parameters were significantly different found at P ≤ 0.001, and the significance of the main results obtained from treatments was compared to the control [37].

5 Results

5.1 Effect of irrigation with saline water in different concentrations on maize plants’ shoot length and total chlorophyll contents

The obtained data showed that, the salinity causes several physiological deteriorations in plants and yields losses in major grain crops like maize. The adverse effects of salinity on shoot lengths of maize plants are illustrated in Table 1; the means of shoot lengths reflect the plant response to saline on the morphological level by a concentration-dependent decrease in shoot length relative to control. In normal watering conditions (control), line P8 scored the highest shoot length (23.5 cm), followed by line P17 scored 21.76 cm. Raising the salt concentration induces a decrease in shoot length to reach 7.1 cm after treating line P4 with 10000 mg/L.

Table 1 Effect of irrigation with saline water in different concentrations on the shoot length scored in different white maize inbred lines
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The total chlorophyll content of fresh leaves is shown in Table 2. The maximum scored amount reached 1.71 mg/g in P8 after irrigation with normal tap water as a control for the experiment, the amount of total chlorophyll was reduced as a response to the salinity stress in all genotypes to reach the lowest 0.61 mg/g in P17 after treatment with 10000 mg/L of NaCl. The reported data in Tables 1 and 2 clearly showed a decrease in shoot length and total chlorophyll contents in the salinity-treated plants in all maize cultivars, whereas the mean value of P8 reported the highest significant shoot length and total chlorophyll compared with all other genotypes followed by P17, on contrary, the treatment P4 reported a significant decrease in the shoot length and the total chlorophyll amount comparing with all treatments but, the inbred lines P12 and P15 reported the moderated levels.

Table 2 Effect of irrigation with saline water in different concentrations on the Total chlorophyll contents (mg/g FW) scored in different white maize inbred lines
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5.2 Estimation of total soluble sugars and total free amino acids

Tables 3 and 4 showed that total soluble sugars and total free amino acids scored the highest accumulation values for genotype 8 to reach double scored in control (37.12 mg/g FW for soluble sugar) and four times scored in control (14.08 mg/g FW for free amino acid) all after the highest salinity treatment with 10000mg/L. P17 came in the second place to score (27.98 and11.46mg/g) for soluble sugar and free amino acid respectively. Among the tested lines; the P4 scored the less accumulation of total soluble sugars (20.23 mg/g FW) and total free amino acids levels (9.10 mg/g FW. The white maize inbred lines P12 and P15 reported moderated levels.

Table 3 The mean effect of irrigation with saline water in different concentrations on Total soluble sugars (mg/g FW) scored in different white maize inbred lines
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Table 4 The mean effect of irrigation with saline water in different concentrations on total free amino acids (mg/g FW) scored in different white maize inbred lines
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5.3 Effect of irrigation with saline water in different concentrations on the white maize inbred lines on biomarkers SOD, APX and H2O2 levels

The accumulation of ions resulting in salt stress enhances metabolic impairment and oxidative stress through ROS generation. Tables 5, 6, and 7 showed that the mean values of Superoxide dismutase (SOD), Ascorbate peroxidase (APX), and H2O2 in the salinity-treated plantsthe maximum SOD 351.33 U.g−1FW.min−1 was scored in P17, the maximum APX 60.31 U.g−1FW.min−1 was scored in P8 and the maximum H2O2 501.33 μ mol.g−1 FW scored in P8., on contrary, the treatment P4 reported the least significant increase in the production levels after treatment with highest NaCl (311.33, 28.20 and 276.47) for SOD,APX and H2O2 respectively, comparing with all treatments but the P12 and P15 reported the moderate levels.

Table 5 Effect of irrigation with saline water in different concentrations on the Superoxide dismutase activity (U.g−1Fw.min−1) scored in different white maize inbred lines
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Table 6 Effect of irrigation with saline water in different concentrations on the ascorbate peroxidase activity (U.g−1FW.min−1) scored in different white maize inbred lines
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Table 7 The mean effect of irrigation with saline water in different concentrations on the H2O2 accumulation (μ mol.g−1 FW) scored in different white maize inbred lines
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6 Effect of irrigation with saline water in different concentrations on the white maize inbred lines on phenyl alanine ammonia lyase activity and total phenolic content

Table 8 revealed the rising in the PAL activity as response to salinity stress to reach its maximum value 87.37 µg min−1 g −1 FW in P8 nearly about three times what scored in control 27.64 µg min−1 g −1 FW after the highest salinity stress. On the other hand, the P4 scored the least rising in PAL activity 29.58 µg min−1 g −1 FW among the five lines.

Table 8 Effect of irrigation with saline water in different concentrations on PAL activity in different white maize inbred lines
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Table 9 revealed the rising in the phenolic compounds to reach it maximum value 17.33 mg/g DW in P8 after the highest NaCl concentration. For more time, the P4 scored the least rising in the phenolic compounds among the five tested lines to reach 7.93 mg/g DW.

Table 9 Effect of irrigation with saline water in different concentrations on total phenolic content (mg/g DW) in different white maize inbred lines
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On contrary, the treatment P4 reported the lesssignificant PAL activity and total phenols contents compared with all treatments. The inbred lines P12 and P15 reported the moderately significant PAL activity comparing with other treatments. This is evidence for the relationship between high PAL and total phenolic content levels and the occurrences of high salt concentration that represent the significant activation of PAL inducing high accumulation of phenolic content supporting the defense against salinity in tolerant P8 and P17.

6.1 The balance between the ROS accumulation and antioxidant productions to refer the plant performance under stress

The balance between the ROS production and the antioxidant enzymes activity points to the plant fitness under abiotic stresses was represented in Fig. 1, and the ROS dual role within the cell compartments as signals from and to antioxidant enzymes were represented in Fig. 2. Also, PAL and its phenolic compounds' productivity represented the highest accumulation as non-enzymatic antioxidant compounds that support the rebalance of the oxidative /reductive ratio that enhances the acclimatization of tolerant P8. On the contrary, the most sensitive P4 reported the lowest PAL enzyme activity and lowest phenolic accumulation that imbalance the high accumulation of ROS. The obtained biochemical results showed that the inbred line P8 was the most responded to the salt stress at 2000 and 3000 mg/L by increasing the SOD and APX antioxidant enzymes activity and announced this genotype fitness under salt stress over the other genotypes. P4 is the most sensitive among the other genotypes for its least SOD and APX activities which reflect he failure of antioxidants production in adequate quantities to neutralize the ROS compounds reflected the plants’ sensitivity.

Fig. 1
figure 1

The equilibrium balance between ROS and the detoxification system refers to the plant’s defense system response

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Fig. 2
figure 2

The ROS dual role within the cell compartments. (Red arrows represent effect of ROS on the organelles’ structure. Blue arrows represent the signals send to the antioxidant enzymes)

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Here comes the cytological studies to follow up the consequences of the ROS on the cytoplasmic organelles’ morphology and function.

7 Cytological evaluation

The transmission electronic microscope investigations focused on the descriptive study of the salinity stress at 2000 and 3000 mg/L on the ultrastructural cytoplasmic organelles in the five investigated maize cultivars, root tip meristems, these two concentrations represent moderate salinity stresses (Figs. 3, 4, 5, 6 and 7) all relative to the untreated sample as control for zero salinity (Fig. 8).

Fig. 3
figure 3

TEM micrographs show the effects of: 2000 (a) and 3000 mg/L (b, c and d) of NaCl on cytoplasmic organelles of white maize inbred line P4 in the root meristem cells (N nucleus, M mitochondria, dM degraded mitochondria, D dictyosome, rM ring mitochondria, L lysosome, Pl plastid, P peroxisome, ER endoplasmic reticulum, CW cell wall, ob swollen vascular body with oil, V vacuole, Av autophagic vacuole, G gap)

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Fig. 4
figure 4

TEM micrographs of the effects of 2000 mg/L (a, c and d) and 3000 mg/L(b and d) of NaCl on cytoplasmic organelles of white maize inbred line P8 in the root meristem cells (N nucleus, M mitochondria, dM degraded mitochondria, D dictyosome, rM ring mitochondria, L lysosome, Pl plastid, P peroxisome, ER endoplasmic reticulum, CW cell wall, ob swollen vascular body with oil, V vacuole, Av autophagic vacuole, G gap, vb vascular bodies)

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Fig. 5
figure 5

TEM micrographs of the effects of 2000 mg/L (a and c) and 3000 mg/L (b and d) of NaCl on cytoplasmic organelles of white maize inbred line P12 in root meristem cells (N nucleus, M mitochondria, dM degraded mitochondria, rM ring mitochondria, eM elongated mitochondria, fM fused mitochondria, arrow mitochondria with protuberances of outer membrane, D dictyosome, L lysosome, Pl plastid, P peroxisome, ER endoplasmic reticulum, CW cell wall, ob swollen vascular body with oil, V vacuole, Av autophagic vacuole, G gap, vb vascular bodies)

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Fig. 6
figure 6

TEM micrographs of the effects of 2000 mg/L (a, e and h)) and 3000 mg/L (b, c, d, f and g)) of NaCl on cytoplasmic organelles of white maize inbred lines P15 in root meristem cells (N nucleus, M mitochondria, dM degraded mitochondria, rM ring mitochondria, eM elongated mitochondria, arrow mitochondria with protuberances of outer membrane, D dictyosome, Llysosome, Pl plastid, P peroxisome, ER endoplasmic reticulum, CW cell wall, ob swollen vascular body with oil, V vacuole, Av autophagic vacuole, G gap, vb vascular bodies, T tight junction between cells)

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Fig. 7
figure 7

TEM micrographs of the effects of 2000 mg/L (a, c, and e) & 3000 mg/L (b, d and f) of NaCl on cytoplasmic organelles of white maize inbred lines P17 in root meristem cells (N nucleus, M mitochondria, dM degraded mitochondria, rM ring mitochondria, eM elongated mitochondria, arrow mitochondria with protuberances of outer membrane, D dictyosome, L lysosome, Pl plastid, P peroxisome, ER endoplasmic reticulum, CW cell wall, ob swollen vascular body with oil, V vacuole, Av autophagic vacuole, G gap, vb vascular bodies, T tight junction between cells)

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Fig. 8
figure 8

TEM micrograph shows the cytoplasmic organelles of non-treated white maize root meristem cells of inbred lines P17 as control

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The most tolerant white maize inbred lines P8 responded to salt stress on the level of the ultrastructural investigation of the cytoplasmic organelles. Mitochondria were found normally or with very slight degradation, endoplasmic membranes were found normally folded, vacuoles were found in different sizes but mostly small in size, peroxisomes were found normally and cell walls were found slightly deformed with a gap junction between cells as a reflection on the osmotic alteration. These observations have been approved by the biochemical evaluation of total soluble sugars and total soluble amino acids that, reported the highest accumulation in tolerant P8 increases the cytoplasmic osmotic pressure and reflects a very slight alteration in the tolerant P8. The most sensitive P4 was found to be affected by salt stress. The ring-shaped mitochondria with the new autophagic function showed up, the endoplasmic reticulum showed normally folded and also found to be ruptured, many starch droplets showed up to reflect the plant cells struggling under the salt stress, and peroxisomes were also found.

8 Results can be summarized as the following

Partially degraded mitochondria were detected mostly after all the salt stress in concentrations on white maize inbred lines under investigation. The new ring-shaped mitochondria with new engulphed function were observed and imaged in P4 after treatment with 2000 mg/L (Fig. 3a) and after 3000 mg/L (Fig. 3b, c and d). Elongated mitochondria were observed in most of the studied genotypes elongated mitochondria were observed in P12 (Fig. 5d), elongated mitochondria with protuberances of the outer membrane were found and imaged after the P15 (Fig. 6g). Fused mitochondria (interconnect mitochondria to network) were detected in P12 under the salinity stress 2000 mg/L (Fig. 5a and c). Plastids were found to rupture after the highest salt concentration of 3000 mg/L in P12 (Fig. 3) and found partially degraded plastids with engulphed undefined bodies were imaged after P15 (Fig. 6f). The endoplasmic reticulum (ER) is a net that extends from the nuclear envelope to the Golgi bodies.

It was found folded in a bundle around the cytoplasmic organelles to do its function normally after genotype 8 which showed high tolerance to the salinity stress. Normal folded, gathered in bundles and ruptured reticulum were also detected after 2000 and 3000 mg/L stress on P4 (Fig. 3a and c), P12 (Fig. 5a) and P17 (Fig. 7a, b, c and f). The cell wall was observed to be irregular after treatment with NaCl (2000 mg/L) in P12 and P15 which are moderate in their tolerance toward salinity. A very tight junction and thin cell wall were observed after treatment with 3000 mg/L in P12 (Fig. 5b). Peroxisome organelles cross-communicate with other intercellular organelles (nucleus, mitochondria, endoplasmic reticulum (ER), and lysosomes), and this cross-talk communication reflects the cell response to the affecting stress to proliferate the cell performance. Peroxisomes were detected after all genotypes, with variations in numbers. Line P17 (Fig. 7c and d) showed a notable large number of peroxisomes to reach 25 in one cell after treatment with 3000 mg/L of salt concentration. P12 also showed a high number of peroxisomes arranged near the cytoplasmic membrane after treatment with 2000 mg/L (Fig. 5e and d). Figures 3, 4, 5, 6 and 7 recorded the peroxisomes are frequently seen wrapped around the ER membrane, to emphasize the cross-connection between the two organelles (peroxisome—ER); Dictyosomes were detected with swelling at the edge of cisterna in P17 after treatment with 3000 mg/L (Fig. 7b) and P15 after treatment with 2000 and 3000 mg/L (Fig. 6a and  h) (starch granules They were recorded in P4 after treatment with 3000 mg/L (Fig. 3c). Vacuoles were detected in different sizes and numbers, large vacuoles were observed in P17 after both used concentrations 2000 and 3000 mg/L (Fig. 7a, d and e).

9 Discussion

Plants normally have a strong tolerance mechanism related to several; morphological, biochemical, molecular, and physiological processes work together synergistically to equalize the external stresses and cope/adapt with the environment [38]. The reduction scored in the shoot length and chlorophyll content as a consequence of salinity came in agree with [39,40,41,42]. This reduction was justified by the osmotic effect, ion toxicity, the nutritional imbalance leading to a reduction in photosynthetic efficiency, less stomatal conductivity, limited C-fixation capacity and destroy photosynthetic pigments due to suppression of the specific enzymes responsible for the synthesis of green pigments. Salinity stress is considered as complexed stress resembles the heat and drought stresses as they all affect the water content, retention and uptake. Generally, the trend and magnitude of adverse changes varied in species, varieties, and genotypes according to the level of stress and the plants’ adaptation mechanism, based on that fact, the different white maize inbred lines responses to salinity stress can be counted to configure the plant sensitivity or tolerance.

Our data also, revealed that the tolerant lines P8 and P17 which reported to have the least rate of shoot shortening as response to salinity stress; recorded significantly higher antioxidant defense enzymes’ production (SOD, APX) levels which in turn neutralize and decrease the elevated ROS contents and directly decrease the destructed chlorophyll at moderate and high salinities and increase the nutrient uptake and plant energy level and in consequence the plant shoot length in a tolerant plant. The decrease in the shoot length and chlorophyll content were recorded earlier by Ali [43] when study the salinity stress on rice plant and referred these results to decrease in the plant ability to water-uptake and rises the toxicity of Na+ and Cl− ions in the plant cells, and photosynthesis reduction. While the Reduction in chlorophyll content may be due to the suppressing effect of the accumulated ions on the biosynthesis of the different chlorophyll fractions [44, 45].

The scored increase in the amino acid content came in agree with [46] who approved that, the unexpected accumulation of free amino acids content under low saline conditions in the tested Chickpea Cultivars may be explained by the salinity stress mediated various signaling mediators inducing various cellular biochemical responses that significantly affect the plant anabolism and catabolism for maintain the cell alive under different stresses. Stress affected the carbohydrates metabolism by activating the hydrolysis into low molecular weight glucose units that rebalance the osmotic pressure to maintain the simple diffusion for stuff to pass through the plasma membrane. Also, increase the accumulation of cytoplasmic free amino acids as osmolytes protecting the antioxidant defense enzymes.

In accordance, Sofo et al. [47] the salinity stress significantly induced oxidative burst by accumulation of H2O2 over a threshold and caused cell death in tested plants through induction of severe damage to biomolecules such as lipids and proteins. Plants under stress evolves regulatory mechanisms to adapt, increasing the H2O2 metabolizing enzymes such as catalases, APX, glutathione/thioredoxin peroxidases, and glutathione sulfotransferase is one of the defense mechanisms. In particular, APX has a higher reduction in chloroplasts, cytosol, mitochondria, and peroxisomes, as well as in the apoplastic space, utilizing ascorbate as a specific electron donor. In addition, the salt stress concentration is accompanied by accumulated hydrogen peroxide which works as a signaling molecule starts the hydrolyzing the storage starch and proteins to soluble sugars and free amino acids that, responsible for re-balancing the cellular osmotic pressure against salinity. ROS, in particular, hydrogen peroxide is a key component of the signaling pathways’ network, and acts as the main regulator of cellular responses and cell physiology of plants to environmental factors [48, 49]. So, the H2O2 reported as a biomarker of the extent of oxidative stress that happened during stress may cause severe damage to biomolecules and direct the cell to death [50]. From the results, the reported tolerant inbred lines P8 and P17 represented a highly significant bio-marked antioxidant defense enzymes activity APX and SOD that could be activated by highly accumulated H2O2 for re-balancing the oxidative /reductive homeostasis for cell survival. The balance of enzymatic mechanisms represents the main scavenging mechanism in plants, which is vital for the suppression of toxic ROS levels in the cell [51]. On the same track; Monsur et al. [12] recommended studying the importance of antioxidants in different cellular parts (chloroplast, mitochondria, peroxisomes) in scavenging ROS, and what type of cellular redox signaling occurs to modulate other biochemical processes to enhance salt tolerance in rice plants.

In accordance, [52] the salt stress stimulates PAL expression (12–18 times) and subsequently increases PAL activity (42–45%) and total phenolic accumulation (35%-43%). Also, Petridis et al. [53] reported that salinity stimulated the biosynthesis of phenols and oleuropein in leaves, whereas the hydroxy-tyrosol concentration level was either negatively or not affected by the salt stress. Also, several studies reported that one of the major consequences of salinity stress is the oxidative burst mediated by the accumulation of ROS which tend to interact with many cellular constituents, causing significant damage [54]. Plants normally produce many antioxidants aimed to detoxify the ROS [55, 56]. So, the high accumulation of phenolic compounds in tolerant cultivars P8 and P17 represents the biomarker indication of elevated levels of phenolic content.

Mitochondria represent the cell’s lung and reflect the energy level of the cell; it mainly converts the ADP to ATP through the proton-pum** electron transport chain (ETC)which participates in the generation of O2•– which on dismutation by SOD produces H2O2 and to produce the energy required for the vital processes. Mitochondria also have a role in the maintenance of the ionic balance, and ROS and cytochrome c (cyt c) production. Different deformations in the mitochondria (degraded, ring-shaped, elongated) were observed in maize lines under salinity stress. The mitochondrial observation supported and explained by the biochemical analysis, as the free amino acids as the effective nonenzymatic antioxidant molecules supporting the dismutation of accumulated ROS, the Glutathione (GSH) contents in the most sensitive maize line P4 was much lower than its amount accumulated in the much tolerant line P8, thus, in turn, reduce the cellular defense against oxidative stress under salinity conditions and in sequence quenching all deformation in the mitochondria (degraded, ring shaped, elongated) that have been observed with sensitive P4.

Mitochondria degradation is known as the “Autophagy defense mechanism” functioning in the degradation of proteins, protein aggregates, and whole organelles’ constituents to recycle damaged or unwanted proteins and organelles and reuse the building units to renew themselves [57]. The more sensitive genotypes of maize showed the most alteration in the mitochondria structure which may shed its effect on the mitochondrial suspected function as a consequence of the ROS production and effect on the lipids that constitute membrane and matrix. The same was observed by [58] when testing the salinity stress on the rice leaves and suggested that highly generated H2O2 is mainly responsible for the degradation of mitochondrial cristae. The ROS production in the mitochondria over the normal threshold leads to lipid peroxidation and programmed cell death (PCD). This results in deformation in the membrane integrity leading to the release of cyt c from the inter-membrane space to the cytosol [84].

The cytological investigation on vital organelles with TEM, show that our results and findings came in great match and agreement with Rai et al. [85], who stated that; the inter-cellular crosstalk among the plant phytohormones and the tripeptide thiol molecule (glutathione GSH) which are synthesized in cytosols, mitochondria, peroxisome, and chloroplasts; could lower H2O2 and ROS production during oxidative stress under rapidly changing climatic conditions as an immunity and self-defense in crop plant.

10 Conclusion

This study will enable the outcome of crop plants more able to tolerate future climatic requirements and support production possibilities. White maize inbred line P8 followed by P17 was the highest cope with the irrigation of saline water in different ranges of concentrations (0–10000 mg/L) of NaCl. The biomarker analysis supports the root ultrastructure observation and vice versa in inter and intra-cellular signaling reflecting the tolerance detoxification mechanism and pushing the equilibrium balance between the ROS and the detoxification systems. The biochemical analysis for the detoxification system including the enzymatic and the non-enzymatic systems, in addition to the accumulation of sugar, free amino acids, and the physical alteration in the cytoplasmic organelles reflects the consequence response to the stress. Based on the discussed experiment, among the five maize inbred lines, the line P8 followed by the P17 line are highly recommended to be crossed and planted in the newly reclaimed lands irrigated with salty water. The tolerant plant lines intense the signal to enhance performance under stress treatment. It is recommended that future studies consider the effect of phenolic compounds on plant resistance to salinity stress.