1. Introduction
The shortage of fresh water for irrigation, combined with the ever-increasing population, is a major hindrance to fulfilling world food demand. It is expected that this situation will become worse for researchers in the near future due to the changing environment, as well as patterns of rainfall that have further increased the aridity problem in many areas of world [
1]. Agriculturalists are facing the problem of, by the middle of 21st century, meeting the requirements for the food demands of nine billion people [
2]. The production of more food with less availability of water, especially in semi-arid and arid zones, is also an emerging challenge for agriculturalists nowadays [
3]. Different agricultural areas of the world are facing the problem of land degradation due to the shortage and scarcity of water [
4,
5]. A total of 80% of the world’s agriculture depends on rain-fed land, which produces 80% of the food globally [
6,
7]. Approximately 95% of land in North Africa and West Asia is rain-fed, and 40% of the land in Uzbekistan faces water shortages, causing the problem of despoiled fields [
8,
9]. In Pakistan, approximately 12.53 million hectares are irrigated with underground water, 6.35 million hectares of agriculture land are cultivated with canal water, and no water is available for the remaining 3.59 million hectares, from a total of 22.45 million hectares [
10,
11].
Under such challenging conditions, to fulfill the demand for food at a global level, it is either necessary to increase the cultivated land or to produce high-yielding stress-tolerant crop varieties. The former option seems unfeasible due to limitations of fresh water for irrigation. In crop plants, the stress tolerance is the function of alteration in different physiological and biochemical mechanisms, which leads to clear morphological adaptation [
12]. These mechanisms are better water content, the increased accumulation of photosynthetic pigments leading to a better photosynthetic activity, and efficient antioxidative defense mechanisms (enzymatic and non-enzymatic antioxidants). Different physiological and biochemical changes are studied in agronomic plants including the bread wheat and found genotype-specific in their responses to drought stress [
13,
14,
15,
16]. The plant species with better performances in all (or a few) of the above-mentioned mechanism are categorized as drought-sensitive or tolerant species. The better performance in the yield of crop plants, especially under adverse environmental conditions, is the function of physio-biochemical mechanisms.
The physio-biochemical traits of plants are considered an important selection tool under stressful environmental conditions, especially water deficit stress, because they have a direct relationship with the plant adaptations to stressful conditions [
17]. Plant physio-biochemical responses under water deficit stress include a decreased photosynthetic activity due to membrane disruptions by oxidative stress, the accumulation of toxic metabolites, and perturbations in cellular water relations, leading to a loss of turgidity, and reduction in growth rates and productivity. It is reported that, in crop plants, different physiological responses are linked with their resistance functions under drought, such as a high amount of relative water content, water potential and integrity of membranes [
18,
19,
20]. Perturbations in biomass production are considered as one such important process which is sensitive to drought and linked with the disturbances in cellular water relations [
21].
The performance of crop plants regarding physio-biochemical mechanisms is responsible for survival under drought; that are found plant species and/or cultivar-specific [
12]. Furthermore, the mechanism for plant survival also changes under different environmental conditions that clearly adhere to the genetic potential of plants [
22]. In this regard, the selection of high-yielding crop varieties with rapidly changing conditions of environment is considered a complex mechanism and plant-species-specific [
18]. In the case of cereals, regarding to that of wheat that is hexaploid in nature, the stress tolerance mechanism likely becomes more complex in exploring and understanding. Therefore, to address the present challenging environmental conditions, the selection from available germplasms, or the development of new cultivars through breeding or genetic engineering for stress tolerance, trait selection is indeed of prime importance to fulfill future world food demand [
12].
Mostly in crop plants, their selection for better production under drought is mainly based on the different agronomic traits, including biomass and yield traits, and very few cases are reported, considering the different physiological and biochemical attributes in parallel with agronomic traits, which are the main enigma behind the stress tolerance of crop plants [
12]. Though the agronomic traits are considered as the most important traits in field crops for their selection for better production under drought [
12], it is necessary to select the cultivars with a better yield under stressful environments by considering the physiological and biochemical traits, along with agronomic traits. Work is already in progress regarding the aforementioned aspects, but the mechanism is still unclear. The world’s most major food dependence is on cereals, especially wheat, including in Pakistan where wheat has a complex mechanism due to its hexaploidy nature. Pakistan is an agricultural country and 70% of its population is dependent on agricultural crops for food. Being a staple food, wheat is most consumed crop among the important cereal crops and helps to solve food security problems. Pakistan is in sixth position worldwide for global wheat production and in eighth position for cultivation, producing approximately 3.5% wheat annually [
23,
24].
World demands more food due to a fast increase in human population and is expected that it will reach to 40% by 2030. Therefore, crop production must be increased to meet increased food demands and attain the goals of sustainable food security [
12,
25]. The global population is estimated to reach 9.8 billion people by 2050 [
26]. Therefore, there is a dire need to increase crop productivity by 70% to fulfill the projected demand by the middle of the 21st century [
27]. In this regard, the major focus of the breeders is to increase the productivity of wheat at a global level as it faces the serious threat of ongoing and increasing adverse environmental conditions. Due the various abiotic stress constraints, including drought, wheat production was compromised [
19,
28]. Therefore, the primary goal of wheat breeders is developing drought-tolerant wheat cultivars.
Wheat is one of the most important founder crops in agricultural, started about 10,000 years ago in Fertile Crescent. It is developed through long evolutionary process, involving the different ancestors [
26,
27,
29]. The genetics of hexaploidy wheat is very complicated due of its polyploidy. It results into genes duplication and triplication that leads to complex epistatic effects and segregational patterns that are difficult to analyse Sentenseand to resolve the effects of component genes. Therefore, the genes description and location in wheat lags is similar to diploid crop barley [
30]. Therefore, the objectives of the study were the selection of drought-tolerant wheat genotypes from a set of experimental lines developed from a cross of high-yielding, stress-tolerant wheat varieties based on the agronomic traits, such as plant biomass production and yield attributes, in relation to biochemical and physiological traits, including leaf photosynthetic pigments, water relations of plant, lipid peroxidation and antioxidative defense mechanisms. Moreover, the studies of stress tolerance indices were also under consideration.
4. Discussion
Breeding to enhance the yield of crops with abruptly changing environmental conditions is the prime focus for breeders/researchers to meet the increasing demand for food caused by a rapidly increasing world population. The behavior of plants under such changing environmental conditions is a challenge to the production of consistent, high-yielding crop varieties, especially regarding cereals, which are considered to be the most important crops needed to fulfill the world food demand. These crops demonstrate the abrupt and unexpected changing behavior of the stressful environment [
12,
26]. In crop plants, the selection of high-yielding crop varieties by agronomists for growth and better production under such abruptly changing environmental conditions is mainly based on agronomic traits [
56]. However, very little importance was given to physiological and biochemical mechanisms; their activity appears in phenotypic characteristics, including agronomic traits, due to their firm genetic attachment, especially under changing environmental conditions [
19,
57,
58]. The morphological adaptations under water deficit stress include denser, deeper, and extended roots; reduced shoot growth to maintain a high root/shoot ratio; and delayed senescence, while the high content of chlorophyll, lower osmotic potential, and waxy leaf coverings indicate biochemical adjustments. Different physiological adaptations include the balanced photosynthetic activity and production of fatal metabolites [
59]. Aside from these, other changes that occur under water deficit stress include damages to photosynthetic apparatus and components, disruptive effects on the enzymes of the Calvin cycle, leading to crop yield reductions due to oxidative stress, the latter of which is also considered to be a key factor responsible for reduced photosynthesis, leading to yield losses [
60].
In crop plants, the effects of adverse environmental conditions, such as those of water stress, are mainly measured by focusing on reductions in growth and yield that are strongly and primarily linked with plant–water relations and should be considered of prime importance in selection breeding. Other physiological mechanisms, such as plant photosynthetic efficiency, membrane integrity, antioxidative defense mechanisms, and nutrient acquisition are also strongly dependent on the adjustment of plant–water relations. However, in the development of stress-tolerant genotypes through breeding techniques, there is very little focus on the interactive roles to such mechanisms, along with other physiological mechanisms [
57]. The crop genotypes that show tolerance against the adverse environmental conditions have a better capacity of cellular osmotic adjustment for maintaining plant–water relations, as well as reduced lipid peroxidation by maintaining the antioxidative defense mechanism. Therefore, the tolerance in crop plants against stressful environments is the combined effect of genotypic expression and physiological alterations that lead to phenotypic modulations with better agronomic performance [
19,
61].
Plant biomass production under water deficit conditions is linked with the better adjustment of water relations to maintain physiological and biochemical processes [
19,
28,
62]. It is known that, to survive under water deficit stress, wheat experiences phenotypic changes and altered dry matter partitioning, e.g., a reduced biomass production and leaf area, a smaller decrease in root growth and reduced injury [
63] that shows the adoptability for balancing the water status of plant tissues [
64,
65]. Moreover, the leaf longevity, shrunken leaf size and decreased number of leaves are also the adaptabilities for survival and better production under water deficit stress [
66], which are genetically controlled and genotype-specific [
67,
68,
69]. In the present findings, the significant negative impacts of limited water supply were found for different morphological traits, biomass production and yield-related attributes of all the studied wheat genotypes. However, the impacts were genotype-specific, and the extent of decrease shows the relative tolerance of wheat genotypes to water deficit stress. Genotypes MP1, MP8, MP5, and MP10 performed better in relation to the studied growth attributes under stressful environmental conditions. Significant reductions were recorded in FLA, PLA, SFW, SDW, RFW, RDW and PL in all 12 of the studied genotypes, but a significantly lower reduction was recorded in genotypes MP1, MP8, MP5 and MP10, as compared with other genotypes that showed their better ability to tolerate water deficit stress. Moreover, wheat genotypes MP1, MP8, MP5, MP10, followed by MP3, also performed better regarding the different yield attributes, such as the number of Spkt/Spk, NOG/Spk, GW/Spk, 100 GW and GY/plant, and showed a lower decrease in these attributes as compared with other genotypes. It is well known that genotypes which perform better in maintaining the biomass under water stress show better results in producing better seed yield [
70,
71]. Moreover, these wheat genotypes were also found better when studied for different STIs in relation with yield attributes, as has been presented in
Table 4. However, very little is reported regarding the use of STI for the selection of drought-tolerant wheat genotypes in combination with the physio-biochemical mechanisms. It was reported that genotypes with higher values of STI are considered to be stress-tolerant genotypes [
72]. In view of the reports made by Nouraein et al. [
73], the STI were found to be better for the selection of stress-tolerant crop genotypes for growth under water deficit stress. In the present study, genotypes MP1, MP3, MP5, MP8 and MP10 were also found to be categorized as stress-tolerant genotypes based on STI in combination with the physio-biochemical mechanisms.
The maintenance of better growth and yield in genotypes MP1, MP5, MP10 and MP8 under water deficit stress is also well-correlated with the maintenance of better leaf photosynthetic pigments. An increase was recorded in Chl.
a for genotypes MP5 and MP8, in Chl.
b for genotypes MP1 and MP8, but a lower decrease was recorded in Chl.
a for genotypes MP1 and MP10 and Chl.
b for genotype MP5 and MP10. Similarly, regarding T. Chl., an increase was found in genotype MP8, a lower decrease in MP5 and MP3, and no effect was recorded in genotype MP1. Therefore, the genotypes, MP1, MP5, MP10 and MP8, tend to maintain the leaf photosynthetic pigments under water stress, which shows their relative stress tolerance ability. Leaf chlorophyll is also considered as one of the most commonly used metrics for assessing the severity of drought stress [
74,
75]. It is stated that, in crop plants, the green leaf area corresponds well with their photosynthetic capacity and better productivity, as well as phenological and physiological status, especially under water deficit stress [
76,
77,
78] and the leaf photosynthetic efficiency is the function of light capturing ability and leaf stomatal regulation. The first case is dependent on the better photosynthetic pigments and the latter case is dependent on plant water status. Studies revealed that leaf chlorophyll contents correlated well with yield-related attributes and can detect the health status of the plant under water deficit stress [
79,
80,
81]. Drought-tolerant varieties maintain better chlorophyll contents and vice versa [
82]. The maintenance of proper chlorophyll contents under water deficit stress is a purely under genetic control in crop plants [
83]. It was reported that the content of leaf chlorophyll also affects yield traits, such as grain yield and number of grains [
84,
85], which signify the importance of leaf chlorophyll contents, especially under water deficit stress [
86,
87,
88]. In the present findings the better performance of genotypes MP1, MP5, MP10, and MP8, in relation with the growth and yield attributes, is correlated well with their better maintenance of photosynthetic pigments. This means that there is a greater light-capturing ability with the maintenance of better photosynthetic pigments [
89,
90,
91]. The reduction in photosynthetic pigments in other genotypes can be correlated with the increased oxidative stress and lipid peroxidation [
78,
92], which are typical symptoms of chlorophyll reduction under water deficit stress. It was found that changes in photosynthetic pigments and components, along with the damages to photosynthetic apparatus, resulted in crop yield losses [
60] and the similar was found in the present findings.
The primary impact of limited water supply is on the water relations of plants that determine the ability of a plant to tolerate water-deficit conditions regarding growth, yield and photosynthetic efficiency. In screening, the drought-tolerant genotypes LRWC are considered the most important parameter. This indicates the extent of membrane stability and balance between evapotranspiration and water supply. Drought-tolerant genotypes retain more cellular water content when compared with drought-sensitive ones [
19]. The most effective genotypic variation, under water deficit conditions and regarding the better LRWC, is having the potential to uptake more water from the soil or the plant’s ability to maintain a lower osmotic potential for the maintenance of the tissue turgor, leading to better physiological activities for a better seed yield [
19,
93]. The genotypes MP1, MP5, MP8 and MP10 maintained their better LRWC in the present study, showing their better ability to tolerate water deficit conditions with better growth, photosynthetic pigments and yield, as compared with other genotypes.
It is known that tolerant genotypes acquire their better LRWC by solute accumulation and metabolites, known as cellular osmoregulation [
94,
95]. The maintenance of better LRWC under water stress is dependent on better cellular osmotic adjustment due to the accumulation of inorganic nutrients, metabolites, amino acids, and less lipid per oxidation due to reduced leaky membranes [
94,
95,
96,
97]. The genotypes MP8, MP1, and MP5 accumulated more FAA; genotypes MP8, MP5, and MP10 accumulated more RS; genotypes MP8, MP10 and MP5, including the parental genotypes, accumulated more NRS and TSS; and genotypes MP8, MP5, and MP3 accumulated more TSP. This better osmotic adjustment in genotypes MP8, MP10, and MP3 is positively correlated with their better LRWC, which shows their greater ability to tolerate water deficit conditions by maintaining better water contents.
In view of the cellular osmotic adjustment under water deficit stress the accumulation of sugars is also considered as an organic osmolyte and has been reported in many plant species [
98,
99,
100]. While working on wheat, it was found by Chen et al. [
101] that sugar accumulation is considered to be a physiological marker for comparing different wheat genotypes because a high sugar accumulation maintains the protein structure, enzyme activities, osmotic adjustments, signal transduction, cell structure stability functions and cellular redox balance [
102]. Cellular sugar accumulation also plays a crucial role in coping with moisture deficiency by maintaining LRWC in a genotypic association manner [
84], as was also found in the present study. Moreover, in the present findings, the maintenance of better chlorophyll pigments can be associated with the higher accumulation of sugars, which also act as osmoprotectant and maintain better cellular activities. This helps to tolerate drought stress with a better cell membrane in tolerant wheat genotypes when grown with a limited water supply [
101]. In the present study, LRMP was also lower in genotypes, MP10, MP5 and MP1, as compared with other genotypes that maintained a better cell turgidity, necessary for better growth. In these genotypes, it was also linked with better biomass production, photosynthetic pigments and seed-yield-related attributes. Similar trends were also recorded in previous studies, where the maintenance of better water relations in wheat genotypes was associated with the better accumulation of free amino acids, proline, and total soluble protein, along with less membrane damages, leading to better biomass production and seed yield [
95].
The better performance of the plants under water deficit stress is also found to be associated with a better performance of the antioxidative defense system, maintaining the integrity of membranes through a better scavenging mechanism for overly produced ROS [
21,
103]. This means that the cultivars with better antioxidative defense mechanisms not only show reduced damages to chloroplastic membranes in maintaining electron transport, but also less leaky cellular membranes that maintain a better cell turgidity, essential for better growth and water relations [
104]. Damages to cellular membranes due to overly produced ROS are studied in terms of MDA accumulation, and the extent of MDA production represents the severity of oxidative stress along with the potential of antioxidative defense [
105,
106]. The oxidative stress results in reductions in growth and yield through several disturbances in normal metabolic activities. Plants evolved a well-developed antioxidative defense system to counteract the damaging impacts of oxidative stress. However, the antioxidative defense system is plant-species- and even cultivar-specific [
107,
108]. It includes the enzymatic activities of POD, SOD, APX and CAT, and the accumulation of non-enzymatic antioxidants such as AsA, phenolics, carotenoids, flavonoids, and anthocyanin. Both of these types of antioxidantive mechanisms work together in a supportive way [
19,
106,
109,
110,
111]. However, this antioxidative mechanism is complicated and genetically controlled [
111,
112,
113]. In wheat, this antioxidative mechanism is more complicated under adverse environmental conditions due to its complex genome, where most of its traits are multigenic [
17,
114,
115].
In present study, genotypes MP1, MP5, MP8 and MP10, including parental genotypes performed better regarding the activities of antioxidant enzymes such as CAT, POD, SOD and APX, as well as the accumulation of non-enzymatic antioxidants, including AsA, phenolics, flavonoids, and anthocyanin. The results of the present study can be correlated with previous studies, where the activities of different antioxidants were increased significantly in different wheat genotypes under stressful environmental conditions but the increase was genotype-specific [
107,
108,
116,
117]. Upadhyay et al. [
118] reported that improved activities of different enzymatic antioxidants, in combination with non-enzymatic antioxidants, helped to alleviate the damages of ROS to the cellular membranes in four wheat genotypes. Similar results to those found in the present study were reported by Chakraborty and Pradhan [
116] in different wheat cultivars, where increased activities of different antioxidative enzymes and the content of non-enzymatic antioxidants in response to oxidative stress resulted in less leaky membranes due to overly produced ROS; the efficiency of the oxidative stress tolerance mechanism is measured based on MDA accumulation and the levels of ROS such as H
2O
2. The genotypes with better antioxidative defense mechanisms in terms of decreased MDA accumulation were considered tolerant. Such genotypes have better enzymatic and non-enzymatic antioxidative defense mechanisms [
106,
110]. The overly produced ROS damages the cellular membranes, which become leaky and result in a decrease in cell turgor potential, leading to disturbed cellular water relations. Damages to thyllakoid membranes because of overly produced ROS under limited water supply is also well known, causing a reduction in leaf photosynthetic pigments and its components. These damages to the photosynthetic membranes lead to their reduced functioning [
60].
In the present study, low levels of H
2O
2 were found in genotypes MP10, MP8, MP5 MP1 and MP3 with a reduced lipid peroxidation that is linked with their better antioxidative defenses regarding the increased activities of antioxidant enzymes (CAT, POD, SOD and APX) and a higher accumulation of non-enzymatic antioxidants (AsA, anthocyanin, carotenoids, phenolics and flavonoids). It can also be correlated well with better photosynthetic pigments in these genotypes due to less degradation of lipids per oxidation of chloroplastic membranes, resulting in the maintaining of a better chlorophyll content. Moreover, the H
2O
2 contents in these genotypes were lower, confirming their better antioxidation capacity to capture the overly produced ROS that led to a reduction in lipids per oxidation and resulting in better plant growth and yield. Moreover, the reduced LRMP in parallel with better LRWC, further conferred their excellent antioxidative defense mechanisms. It was found that less H
2O
2 content and MDA accumulation in different wheat varieties was dependent on a better antioxidative defense potential. This might be due to the maintenance of better cellular water content that resulted in better growth [
119].