Effect of Salinity Stress on Physiological Changes in Winter and Spring Wheat

: Salinity is a leading threat to crop growth throughout the world. Salt stress induces altered physiological processes and several inhibitory effects on the growth of cereals, including wheat ( Triticum aestivum L.). In this study, we determined the effects of salinity on ﬁve spring and ﬁve winter wheat genotypes seedlings. We evaluated the salt stress on root and shoot growth attributes, i.e., root length (RL), shoot length (SL), the relative growth rate of root length (RGR-RL), and shoot length (RGR-SL). The ionic content of the leaves was also measured. Physiological traits were also assessed, including stomatal conductance ( gs) , chlorophyll content index (CCI), and light-adapted leaf chlorophyll ﬂuorescence, i.e., the quantum yield of photosystem II ( Fv (cid:48) /Fm (cid:48) ) and instantaneous chlorophyll ﬂuorescence ( Ft ). Physiological and growth performance under salt stress (0, 100, and 200 mol/L) were explored at the seedling stage. The analysis showed that spring wheat accumulated low Na+ and high K+ in leaf blades compared with winter wheat. Among the genotypes, Sakha 8, S-24, W4909, and W4910 performed better and had improved physiological attributes ( gs , Fv (cid:48) /Fm (cid:48) , and Ft ) and seedling growth traits (RL, SL, RGR-SL, and RGR-RL), which were strongly linked with proper Na + and K + discrimination in leaves and the CCI in leaves. The identiﬁed genotypes could represent valuable resources for genetic improvement programs to provide a greater understanding of plant tolerance to salt stress.


Introduction
Plants respond to environmental changes by altering metabolism, growth, and development. When climate changes are rapid, plants perceive them as stress. Among the abiotic stresses, those that most affect the productivity of agricultural crops are: extreme temperatures, osmotic stress, drought, and salinity. Many environmental conditions can lead to water stress in plants. For example, the high concentrations of salt in saline habitats germplasm in wheat. Since wheat (Triticum spp.) is a major food crop, the development and identification of salt-tolerant wheat cultivars is an important research purpose.
Fluorescence of chlorophyll (e.g., Fv /Fm , Ft) and gas exchange have been considered important physiological indicators for screening the tolerance of different cultures. There are two important exit photosystems (PSI and PSII) in the plant. PSII is found to be more prone to the hazardous effects of salinity [26]. Measuring chlorophyll fluorescence is a good indicator of salt effects in the photosynthetic apparatus [27]. Consequently, it is important to evaluate the relationship between the efficiency of PSII and CO 2 assimilation in the leaves as the measurement of fluorescence detects the differences in the response of plants to abiotic stresses by evaluating their tolerance. The use of morphological traits, along with physiological tolerance and their relationship with salinity tolerance indices, are applicable and considered sufficient to be exploited as selection criteria in the breeding of salt-tolerant germplasm [27].
Wheat is also called a salt excluder, which means it mitigates salinity stress by excluding Na + from the shoot as much as possible [28,29]. The capacity of bread wheat to exclude Na + is much stronger than that of durum wheat genotypes [10]. Moreover, salt tolerance is a polygenic trait, and its expression can be influenced by genetic, environmental, and physiological factors. In fact, in the same species it is possible to select a salt-tolerant genotype [30], suggesting that this potential may be improved through conventional breeding approaches. Furthermore, little work has been carried out to examine physiological differences in spring and winter genotypes under salt stress. In the present study, we used five different spring wheat and five durum wheat genotypes to test the different responses and adaptations to salt stress. Some of these, such as S-24, were selected for their well-known salt-tolerance; therefore, it was used as reference one [31]. The genotypes used were evaluated for the effect of salinity on some key physiological and morphological traits. The identified genotypes could represent valuable resources for genetic improvement programs to provide greater understanding of plant tolerance to salt stress, supporting agricultural production on salinized soils irrigated with brackish water.

Response of Wheat Genotypes against Different Salinity Levels
Significant differences (p ≤ 0.05) were recorded among the spring wheat genotypes (G), winter wheat genotypes, and the total wheat genotypes ( Table 1) in terms of their interactions (G * S) at different NaCl salt stress levels (S) for ionic, physiological, and seedling growth traits (Table 1). Table 1. Mean square values (p < 0.05) for root length (RL), shoot length (RL), the relative growth rate of root length (RGR-RL), the relative growth rate of shoot length (RGR-SL), leaf Na + and K + concentration (mg g −1 dry wt), K + /Na + ratio, stomatal conductance gs (mol/m 2 s), maximum quantum efficiency of PSII (Fv /Fm ), instantaneous chlorophyll fluorescence (F t ), chlorophyll content index (CCI) of spring wheat genotypes, spring wheat genotypes, and ten wheat genotypes grown under various NaCl stress levels.

Value of % Control
Significant variation (p ≤ 0.05) was observed between the spring wheat and winter wheat for ionic, seedling growth, and physiological traits (Figure 1a-l). The value of % control of ionic, physiological, and growth traits was decreased by increasing the salt stress (Figure 1a-l). Spring wheat showed a maximum value of % control for ionic (Na + , K + and K + /Na + ratio; Figure 1a-c), growth (RGR-SL), and physiological traits (gs, Ft, QY, and CCI; Figure 1i-l) compared with winter wheat.

Value of % Control
Significant variation (p ≤ 0.05) was observed between the spring wheat and winter wheat for ionic, seedling growth, and physiological traits (Figure 1a-l). The value of % control of ionic, physiological, and growth traits was decreased by increasing the salt stress (Figure 1a-l). Spring wheat showed a maximum value of % control for ionic (Na + , K + and K + /Na + ratio; Figure 1a-c), growth (RGR-SL), and physiological traits (gs, Ft, QY, and CCI; Figure 1i-l) compared with winter wheat. Figure 1. Effect of salt stress on winter and spring wheat. Diagrams are based on value of % control (salt-treated/control * 100) of Na + in leaves (a), K + in leaves (b), K + /Na + ratio (c), leaf senescence % (d), shoot length (SL) (e), root length (RL) (f), relative growth rate of root length (RGR-RL) (g), relative growth rate of shoot length (RGR-SL) (h), stomatal conductance (gs) (i), instantaneous chlorophyll fluorescence (Ft; j), quantum yield of photosystem II (Fv′/Fm′; k), and chlorophyll content index (CCI; l).

Leaf Na + and K + Contents
Significant variations in Na + and K + content were identified between the leaves of genotypes treated with salinity stress (Table 1; Figure 2a,b). Na + concentration in leaves was increased in all wheat genotypes by increasing the salinity stress (Figure 2a,b). At 100 mol/L salinity stress, the minimum Na + content in leaves was found in W4909 (8.51 mg g −1 DW; SW) followed by S-24 (8.59 mg g −1 DW; SW), W4910 (9.09 mg g −1 DW; SW), and Sakha 8 (11.03 mg g −1 DW; SW), respectively ( Figure 2a). Genotypes W4910 (11.40 mg g −1 DW; SW), W4909 (15.48 mg g −1 DW; SW), Sakha 8 (15.58 mg g −1 DW; SW), and S-24 (11.40 mg g −1 DW; SW) performed better at accumulating low leaf Na + at 200 mol/L salt stress and were considered to be salt tolerant. K + concentrations in leaves were decreased in all wheat genotypes by increasing the salinity stress ( Figure 2b). The maximum K + concentration and K + /Na + ratio were observed in Sakha 8 (23.67 mg g −1 DW; SW), followed by S-24 (21.  (Figure 2b). Furthermore, the K + /Na + ratio was also decreased by increasing the salt stress in both wheat genotypes (Figure 2c).

Shoot and Root Growth
Significant responses (p < 0.05) were observed for growth-related attributes among the genotypes for salt tolerance. Seedlings SL and RL were decreased by increasing the salt stress in all wheat genotypes (Figure 3a
The senescence of the leaves also increased in wheat genotypes by increasing the salinity stress, as shown in Figure 4c

Physiological Traits
The CCI in all wheat genotypes decreased with increasing levels of salt stress (Figure 4b Chlorophyll fluorescence, e.g., Fv′/Fm′ and Ft values, declined under salt stress. There was a significant difference (p < 0.05) in chlorophyll fluorescence concerning genotypes, salinity level, and the interaction between genotypes × salinity level (Figure 5b,c).
A significant decrease in Ft was observed in all genotypes by increasing the salt stress level (Figure 5b  Chlorophyll fluorescence, e.g., Fv /Fm and Ft values, declined under salt stress. There was a significant difference (p < 0.05) in chlorophyll fluorescence concerning genotypes, salinity level, and the interaction between genotypes × salinity level (Figure 5b,c).
A significant decrease in Ft was observed in all genotypes by increasing the salt stress level (Figure 5b

Trait Correlations
A highly significant correlation was observed among the various traits under salt stress ( Table 2). All traits were positively correlated except Na + and the senescence of leaves, which were negatively correlated with all other attributes (Table 2). Na + was positively correlated with the senescence of leaves (Table 2).

Discussion
Salt stress induces a number of negative effects including physiological and biochemical changes in plants which manifest as a reduction in plant biomass and crop yield. Different plants have different tolerance levels, as do most cereals, including wheat [17]. In the present study, five genotypes of winter wheat and five genotypes of summer wheat were considered in order to carry out a comparative study on the salt tolerance of new genotypes, with the exception of the S-24 genotype already extensively studied by Ashraf [31], that is better suited to grow on salinized soils. Overall, the results showed different responses between summer and winter genotypes in terms of physiological traits (Figure 1). Under salinity stress, all genotypes accumulated a higher Na + content in their leaves compared with non-stress conditions (Figure 2a). However, the sodium uptake in leaves was different in spring and winter wheat (Figure 1a). Winter wheat accumulated more sodium com-pared with spring wheat (Figure 1a). Most of the salt-excluder genotypes were previously recognized as salt-tolerant by many scientists [32][33][34], which is further confirmed by this study's results (Figures 1a and 2a). The salt-tolerant genotypes may possess a better ability to maintain low Na + in their leaves, as reported by Elkelish et al. [35]. Saddiq et al. [32] reported that tolerant genotypes preferred to accumulate low Na and high K in their leaves, which was also observed in this study (Figure 1a,b and Figure 2a,b). Munns and Tester [10] reported that the removal of Na + from the cytoplasm into the apoplast is due to the salt-inducible enzyme Na + /H + antiporter located at the plasma membrane. Moreover, Na + accumulation in wheat is controlled by Nax1 and Nax2 genes, located on 2A and 5A chromosomes, respectively [36,37], which are being used as molecular marker cultivars in a breeding program. TNHX1, TNHX2, and TVP1 (vascular Na + /H + antiporter) are responsible for improved seedling shoot growth by generating the pH gradient and facilitating sodium sequestration into values under salt stress [38]. Furthermore, salt-tolerant genotypes could have a sophisticated K + regulation system, such as two-pore K + channels and a shaker type, as described by Shabala and Pottosin [39], and on-selective cation channels, which aid the permeability of K + and transporters (HKT, KUP/HAK/KT, and K + /H + ). An inverse relationship exists between Na + and K + ions due to direct competition for ions in plant absorption [40].
The plants' growth performance was also decreased under salt stress conditions (Figure 1e-g, Figure 3a-c, and Figure 4a). All genotypes had lower shoot lengths and root lengths in salt-stressed conditions compared with controls (Figures 3a-c and 4a). Nevertheless, spring wheat genotypes improved their RGR-SL compared with winter wheat genotypes (Figure 1g). Janmohammadi et al. [41] reported that winter wheat had a lower root length than spring wheat under abiotic stress (e.g., cold stress), ultimately affecting the wheat's growth performance. NaCl stress induced a significant reduction in plant height, root length, and dry weight of roots and shoots in winter wheat [42]. Qiong et al. [43] reported that salinity significantly increased Na accumulation in winter wheat, which significantly reduced shoot dry weight and plant height. Na remarkably reduced the accumulation of K + , K + /Na + ratio, as well soluble proteins and proline. Brestic et al. [44] reported that chlorophyll fluorescence is a more effective method for screening PSII thermostability in winter wheat genotypes. A high concentration of salt in the soil causes water stress, which leads to a significant decrease in the yield of many crops worldwide. Zivcak et al. [45] reported that the photosynthesis efficiency of PSI of winter wheat was decreased by increasing the water stress. Damage caused by salt stress was more prominent at the donor side, rather than the acceptor side of PSII [46]. Munns and Tester [10] reported that the accumulation of Na + at toxic concentrations in the leaf negatively affects the photosynthetic mechanism, resulting in a lower intake of carbohydrates to the young leaf, reducing root and shoot growth. Thus, spring wheat was considered a tolerant crop, with a greater supply of assimilates from leaves to growing parts, e.g., root and shoot length (Figure 3a-b). This might be linked with prolonged retention of chlorophyll in the leaves of spring wheat (Figures 1l and 5b), which could stamp out Na + from leaves, and thereby prevent Na + from reaching toxic levels [41]. Poor performance in terms of growth might be linked with high cell membrane injury and senescence of leaves due to Na + toxicity in growing embryos [4,39,47,48], and this suggestion is supported by the present study (Figures 1d and 4c).
Salt stress has an adverse impact on photosynthesis by destroying chlorophyll pigments and inhibiting the PSII activity. In this study, photosynthesis efficiency declined in both winter and spring wheat genotypes under salt-stress conditions (Figure 5b,c), but winter wheat was more affected (Figure 1j,k). In fact, under saline stress, stomal closing results in a reduction in the photosynthetic rate of the plant. CO 2 assimilation in leaves, the efficiency of PSII, and their relationship allow fluorescence to be used to screen salt-tolerant germplasm against abiotic stresses [49]. Kanwal and his coworkers [26] evaluated the effects of salt stress on newly licensed wheat cultivars using gas exchange parameters and chlorophyll fluorescence. The results reported a smaller reduction in plant biomass in cultivars S-24, Saher-226, and FSD-2008.
Measuring chlorophyll fluorescence is an excellent indicator to quantify salt-induced destruction in the photosynthetic apparatus [50]. Damage to photosystem II has been studied using this technique. Reactive oxygen species (ROS) degrade various proteins (a membrane linker protein, chlorophyll protein) that are necessary for the hooking of phycobilisomes to thylakoids [35,46]. ROS burst destroys thylakoid membranes, resulting in modulations in membrane protein profiles, which leads to decreased activity of the oxygen-evolving complex (OEC) of PS II and increases the working of PS I. Salt-tolerant plants grown under a salt regime downregulate PS II in order to improve the quantum efficiency of excitation energy (Fv /Fm ) [50], as found in this study (Figure 1j,k). The maximum quantum yield of PSII, i.e., Fv /Fm , is an important parameter to discriminate wheat genotypes. Of the different physiological attributes, stomatal conductance and the chlorophyll content index have been reported to be of prime importance in screening crop plants for salt tolerance. Generally, salt stress is known to cause a marked reduction in stomatal conductance and the chlorophyll content index [35,51], as found in this study (Figure 1i,l; Figures 4b and 5a). ROS are regarded as the main source of structural damages under abiotic stresses such as drought, salinity, and heat [52]. ROS are highly cytotoxic and can seriously react with vital biomolecules such as lipids, proteins, nucleic acid, and disturb normal metabolic pathways [52,53]. It has been well documented that during salinity stress, somatically stressed plants reduced CO 2 assimilation due to the closing of stomatal pores, which generate ROS in the plant leaves [54]. In this study, spring wheat exhibited comparatively lower reductions in stomatal conductance and chlorophyll content index compared with winter wheat under salt stress (Figure 1i,l). The reduction in winter wheat might have been due to lower root water potential and the transport of plant hormone ABA from the root into different plant organs, thereby inducing stomatal closure [55]. Compared with spring wheat, winter wheat was more affected (Figure 1), which might be strongly linked with high Na levels in their leaves. The toxic concentration of Na + in leaves encourages the reduction of stomatal conductance in wheat by limiting photosynthesis efficiency [32,47]. Abiotic stress conditions caused by exposure to salinity, drought, heat, and waterlogging cause the stressed plant to produce ROS. The plant also produces antioxidants, flavonoids, and secondary metabolites that detoxify the ROS, thus protecting the plant from abnormal conditions, i.e., abiotic stress [52,53,56]. Therefore, tolerant genotypes prefer to accumulate high K + instead of Na + [57]. In this study, the influx of K + was higher in spring wheat compared with winter wheat, helping to mitigate the salinity stress. Over time, salinity causes Na + toxicity in leaves [10]. Therefore, controlling the transport of Na + in the plant through the exclusion of Na + from mesophyll cells is an important and reliable trait used to improve the salinity tolerance in many crops, i.e., durum wheat [58] and bread wheat [32,59].

Germplasm Collection
Seeds of five winter and five spring wheat genotypes (Table 3) were obtained from the USDA-ARS National Small Grains Collection, Aberdeen, ID, USA.

Hydroponic Culture
A germplasm nursery (5 spring and 5 winter wheat genotypes) was raised in November 2015 in a growth chamber by sowing 50 seeds in 8 cm × 6 cm sand-filled polythene bags at 50% relative humidity and a light intensity of 400 mol m −2 s −1 . Plants were grown with a 14-h day length and with a 20 • C/17 • C day/night temperature cycle. Fifteen plants per genotype, replicated three times, were transplanted at the two-leaf stage into hydroponic tubs filled with 50 L of aerated half-strength Hoagland solution, which was changed fortnightly [60]. Seedling root length and shoot length were also recorded before being transplanted. The experimental design was a completely randomized design (CRD) factorial with three replications. Subsequently, commercial-grade salt was added in 50 mol/L increments twice daily to create different NaCl salt stress levels (0, 100, and 200 mol/L) to avoid osmotic shock.

Determination of Leaf Na + and K + Concentrations
After applying the salt in hydroponic culture, the expanded leaves that emerged under stress conditions were collected and put into the oven for drying. Leaf dry weight was determined. Dried leaves were put into falcon tubes filled with 25 mL of 1% HNO 3 solution for digestion on a hot plate at 85 • C for 4 h. One milliliter was taken from the digested solution, and a volume of 10 mL was prepared to measure the K + and Na + concentration in the leaf samples using a flame photometer (Sherwood, U.K., Model 360) [58,61].

Morphological Traits
After 10 days in a saline environment, the performance of seedlings was assessed based on morphological traits such as seedling root length (RL), shoot length (SL), the relative growth rate of root length (RGR-RL), and the relative growth rate of shoot length (RGR-SL). The relative growth rate was calculated using the formula of Gardener et al. [62].

Chlorophyll Index and Stomatal Conductance
From the seedlings in a saline environment, the topmost fully expanded leaf was used to determine the chlorophyll index using a chlorophyll meter (Model Spad-502) [63]. Stomatal conductance was measured using a leaf photometer (Model Sc −1 ).

Chlorophyll Fluorescence
The data for the chlorophyll fluorescence were recorded based on Baker [64] and Krame et al. [65] nomenclature. Chlorophyll fluorescence parameters, i.e., instantaneous chlorophyll fluorescence (Ft) and quantum yield of photosystem II (QY), were recorded by using the portable fluorescence meter, FluorPen FP 110 (Photon systems instruments, Czech Republic). The FluorPen FP 110 was equipped with a blue LED emitter (470 nm) optically filtered and precisely focused on delivering light intensities of up to 3000 µmol m −2 s −1 to measure plant tissues. QY is a measure of the Photosystem II efficiency. QY is equivalent to Fv /Fm and F 0 is equivalent to Ft in a light-adapted leaf. Quantum yield of PSII (Fv /Fm ) was calculated as

Leaf Senescence
Three random plants in each treatment were tagged. At harvesting time, the total number of leaves and the number of green and senesced leaves were counted. A leaf was considered senesced if less than half of its area remained green.

Statistical Analysis
Quantitative observations of experiments were uploaded in SAS 9.4 (Texas A&M University, College Station, TX, USA) software to deduce the results in the form of variance analysis (ANOVA) for spring wheat genotypes, winter wheat genotypes, and all wheat genotypes. Data are presented in Table 1 with critical values to compare treatment means using the LSD test at the 5% probability level. The Statistix 8.1 package was also used to find correlations among the spring wheat genotypes, winter wheat genotypes, and all wheat genotypes for various growth, ionic, and physiological attributes ( Table 2).

Conclusions
In this study, physiological comparisons of wheat genotypes under salt regimes Sakha 8, S-24, W4909, and W4910 performed better compared with PI 94341, TX12M 4713, and TX12M 4637, depicted by improved seedling growth, CCI, which was linked with better physiology traits, i.e., Fv /Fm ; Ft and gs due to preferential K + uptake and translocation to leaves. The identified plant material can be a source for more deeper insight into determining the genes responsible for enhanced salt tolerance in wheat.