1. Introduction
Rice
(Oryza sativa L.) is the primary source of caloric intake for more than half of the world population [
1,
2]. Drought, salinity, heavy metals, cold, and high temperatures are many forms of abiotic stress that rice experiences during the growth stage [
3]. Drought has mainly reduced rice yields by 25.4% over the previous two decades [
4], making it one of the most severe stress factors that prevent rice grain yields to reach optimal production. Rice is a crop that is very sensitive to drought stress and notoriously consumes a large amount of irrigation water [
5]. Rice’s vulnerability to drought stress has hindered efforts to minimize water use related to this semi-aquatic crop [
6]. Thus, the greatest method to boost rice output is to create new drought-resistant rice genotypes with high yield potential [
7].
According to the USDA’s Foreign Agricultural Services (FAS) in Cairo, Egypt’s rice output is expected to exceed 4.0 million metric tons (MMT) in 2020/2021, with cultivated land covering 451,164 hectares [
8]. The amount of irrigation water accessible from the Nile River is not only limited; it is also annually declining. In addition, a shortage of water reduced the yields of 15.2% of the rice fields [
9]. As a result, breeding for drought-tolerant rice lines is critical for Egypt’s rice cultivation.
Drought resistance is a multifaceted feature that is regulated by four main physiological mechanisms: escape, avoidance, tolerance, and recovery [
10]. The characteristics of drought tolerance might be morphological, physiological, or yield-related. Drought-tolerant genotypes may be easily identified by testing for many drought-tolerance-related traits simultaneously [
11]. One of the main goals of drought tolerance is to breed a new genotype with the best yield potential [
12,
13].
Rice has a relatively efficient antioxidant defense system to counter ROS-induced oxidative damage [
14]. There is a greater increase in antioxidant enzyme and metabolite concentrations in tolerant cultivars than in susceptible cultivars [
15]. Abscisic acid (ABA), which is transported from roots to the canopy, causes water shortages in the leaf organs [
16]. Rice cultivars with varying levels of drought tolerance should possess better antioxidant defense systems and proline contents.
Water deficiency can alter anatomical traits at the level of damage and trigger a successful adaptive response [
17]. Drought-exposed leaf, stem, and root tissues exhibit variations in their responses [
18]. The likely contributions of leaf, stem, and root structures to rice genotypes’ drought resistance may be addressed succinctly by analyzing variations in tissue structures from distinct genotypes under normal and drought circumstances [
19].
Molecular methods indirectly aid in determining complicated features without the requirement for extensive and labor-intensive phenotypic assessments by facilitating the identification of genomic regions connected to traits of interest [
20]. ISSR markers are PCR-based markers that are useful in various phases of the breeding process and are very informative and cost-effective in finding genetic linkages between different rice genotypes [
21,
22]. ISSR markers have been employed to investigate cereal genetic diversity and evolutionary relationships [
23] as well as gene tagging in molecular assisted selection [
24] and DNA fingerprinting [
25].
Given the above, the biochemical and anatomical reactions in the new promising rice crosses were used to investigate the degree of tolerance under drought stress. Furthermore, ISSR markers and gene expression were recruited to evaluate genetic diversity and identify new promising crosses with distinct responses to drought.
4. Discussion
Rice, which requires a lot of water for growth and development [
41], has a significant challenge during a water shortage. As a result, one of the most significant environmental variables restricting plant growth and development is drought [
42]. The lack of knowledge of the genetics and inheritance of drought tolerance characteristics, alongside a full misunderstanding of physiological drought tolerance features and drought processes, is a huge setback in drought tolerance breeding. Selecting secondary features that contribute to drought tolerance in breeding programs may also boost the production and roots in water-limited conditions. To find the true potential of drought-resistant genotypes, we combined the findings of phenotypic, physiological, and grain yield research with genomic data and genetic diversity studies.
When plants are subjected to drought, their generation of reactive oxygen species (ROS) rises, resulting in lipid peroxidation, protein denaturation, DNA mutation, and cellular oxidative damage [
43]. Induced antioxidant enzyme activities in plants are a natural way for plants to combat oxidative damage in a hostile environment [
44]. Herein, we measured antioxidant enzyme activities. In a drought setting, APX, SOD, and CAT exhibited a greater degree of induction. The level of TSP was similarly increased in the promising crosses. Antioxidant enzymes and phenols are critical for scavenging H
2O
2 toxicity. The joint action of CAT and SOD turns the lethal superoxide radical (O
2) and hydrogen H
2O
2 into the water and molecular oxygen under unfavorable conditions such as drought stress (O
2) [
45,
46].
Additionally, the drought stress dramatically increased the content of proline in rice crosses’ leaves. These findings indicated that plants’ production of these osmotic modifications is a widespread response to drought. Osmotic adjustment via buildup of a cellular solute, such as proline, has been proposed as one of the probable ways to overcome osmotic stress produced by a decrease in cellular water [
47]. Proline is a non-protein amino acid that develops in most tissues when they are exposed to water stress and is quickly reduced when a drought is terminated [
48]. Proline is important for maintaining cell membrane integrity, the stability of enzymes, and proteins [
49].
The observed changes in Chl agreed well with a previous study [
50], which revealed that the synthesis of photosynthetic pigments being lost or reduced under drought stress is a common avenue and is directly associated with plant biomass and yield reduction. The two crosses Sakha 107 × Sakha super 300 and Sakha 107 × M206 had the greatest Chl among all genotypes when drought stress occurred. According to these results, drought stress reduced the chlorophyll variability in rice leaves in possibly tolerant genotypes [
51].
Anatomical root, stem, and leaf alterations are required to explain the differences between parents and crosses under water stress, and therefore, to comprehend the processes utilized by promising crosses to cope with drought. The obtained results indicated that the root and xylem vessel diameters significantly increased in promising crosses. These findings fit well with those of Al-Khalifah et al. (2006) [
52], who illustrated a link between the xylem channel diameter and water conductivity maintenance. Drought-tolerant genotypes may grow bigger xylem vessels and roots with greater diameters only on rare occasions to enhance water intake when it becomes available. The thickness of the epidermis (μ), cortex (μ), and vascular cylinder (μ) were all enhanced compared with their parents. Recently, the root cortex thickness has been shown to be decreased in sensitive rice cultivars exposed to water deprivation [
53]. In the promising crosses subjected to water shortage, the cross-sectional stem area, pith, and xylem vessel diameter all increased. The epidermis, cortex thickness, and number of vascular bundles were also significantly increased in the crosses compared to the parents. Most leaf anatomical structures are strongly impacted by water deprivation in plants [
54]. Since leaves are the principal organs of internal water removal, drought-tolerant rice genotypes perform leaf anatomical modifications to conserve water. Similarly, it was discovered that the mid-vein thickness of leaves, mesophyll, and bundle sheaths for the promising crosses significantly increased compared with the parents’ values. Overall, from the anatomical point of view, stems were more resistant to water shortage than roots and leaves. However, the rice cultivars with greater stem areas and higher stem xylem diameters maintained high leaf water potential under water restriction [
55]. There was a positive link between the total stem and leaf xylem areas of drought-stressed rice [
56]. All these qualities gave the morphological foundation for drought tolerance in promising rice genotypes via the capacity to modify their root, stem, and leaf structures. Consequently, they may live and develop in water-stressed circumstances.
For drought improvement, several studies have relied on phenotypic selection. As a result, developments in DNA molecular markers and their capacity to identify genomic areas linked with significant features would be more beneficial (e.g., drought tolerance). ISSR markers are useful because they offer a rapid, accurate, and comprehensive method that may also be utilized to develop genetic and genomic fingerprinting [
57]. ISSR markers were employed herein to measure the genetic diversity and to identify the prospective crosses by comparing these parents at the molecular level.
The obtained results indicated that molecular polymorphisms were detected among the selected genotypes using 10 ISSR markers. Six of the ten tested primers showed polymorphisms for genotypes. A total of 44 amplified polymorphic fragments were detected, and the highest numbers of alleles were detected with the primers ISSR 6, 10, 9, 18, 3, and 5, with 12, 10, 9, 7, 3, and 3 alleles, respectively. These polymorphic bands were used to determine the drought tolerance in rice genotypes. The clustering pattern and similarity index indicated that there was a close relationship between Sakha 107 × Sakha super 300 and Sakha 107 × M206, mostly because they were developed from the tolerant parent (Sakha 107). On this premise, the crosses Sakha 107 × Sakha super 300 and Sakha 107 × M206 are proposed as the most acceptable crosses for drought tolerance, which have the greatest similarity value and clustered in a unique cluster.
These findings also suggest that ISSR markers may be a superior tool for studying drought tolerance. A relationship between GA repeats and rice variety abiotic stress resistance (salinity, drought, and flood) was previously claimed [
58]. The results indicate that ISSR markers linked to GA 8 YG may be used to identify genes/new alleles associated with the three abiotic stresses in rice germplasm and can be used to differentiate three groups of stress-tolerant genotypes. ISSR markers based on AG and GA repeats were also employed to differentiate geographically different
Oryza nivara accessions [
59]. As previously reported for rice and tomato [
58,
59], the primer (GATA) might assist in recognizing all kinds and is acceptable for fingerprinting.
Drought tolerance is a multiplex plant trait with broad integrated molecular responses, which mainly comprise two main steps: (i) stress sensing/signaling and (ii) promoting molecular, physiological, and phonological adaptive changes [
60]. The fine-tuning of hormone homeostasis is crucial for creating an adaptive and efficient link among different molecular responses to drought stress in a spatio-temporal manner [
61]. Ethylene (C
2H
2) is a well-acknowledged gaseous plant stress hormone that contributes to plant development and the response to drought, flooding, low temperature, and salinity stress [
62]. Under drought stress mimicked by osmotic stress, the expression of OsACS2 (encodes 1-aminocyclopropane-1-carboxylic acid synthase, a key ethylene biosynthesis enzyme) was significantly elevated in the two most drought-tolerant rice genotypes (Sakha 107 × M206 and Sakha 107 × Sakha super 300). Ethylene was reported to be associated with enhancing plant antioxidative machinery under stress. The two drought-tolerant rice genotypes obviously accumulated higher amounts of proline [
63]. Furthermore, the ethylene-responsive factor OsWR1 was induced in transgenic rice lines and enhanced drought tolerance by increasing wax production in rice [
64].
In this study, the contribution of calcium signaling in rice under drought stress was noted. Like
OsACS2, the most drought-sensitive genotypes showed the highest level of
OsCML31 gene expression.
OsCML31 was reported to be significantly responsive under drought, salt, and alkalinity stress in rice [
36]. Calcium ions are crucial second messengers in plants and are essential for efficient stress sensing and signal transduction, the process that eventually could trigger an adaptive response to drought stress in terms of early stomatal closure and H
2O
2 production [
65,
66]. This assumption could be supported by the significant up-regulation of
OsSRO1c (related to stomatal closure) in the two drought-tolerant crosses Sakha 107 × M206 and Sakha 107 × Sakha super 300 in response to osmotic stress compared to other rice genotypes.
OsROC1c is a rice homologue of SRO (similar to RCD one), which was identified as a direct target gene of SNAC1 (stress-responsive NAC 1), which is involved in the regulation of the stomatal aperture and the oxidative response [
67,
68].
Finally, the catabolic turnover of jasmonic acid was found to be associated with rice’s tolerance to abiotic stress [
37].
OsCYP94C2a, which encodes the jasmonic-acid-degrading enzyme Cytochrome P450 of the subfamily CYP94 subclade C member 2a, was highly expressed in rice under osmotic and salt stress in the Egyptian rice genotype Sakha 101 [
38]. In this study,
OsCYP94C2a was strongly expressed in the most drought-sensitive rice genotypes. Jasmonic acid (JA) is a growth-inhibiting stress hormone associated with retarded root and shoot growth [
68]. It is believed that the regulation of JA biosynthesis/turnover is essential in setting up the balance between growth and adaptation in plants under abiotic stress. Yang et al. (2012) [
69] reported that jasmonic acid could interfere with the gibberellic acid signaling cascade and inhibit growth in favor of defense by re-allocating carbon energy towards adaptation or resistance mechanisms. It could be assumed that drought-tolerant genotypes could efficiently degrade jasmonic acid using the enzyme
CYP94C2A more than other rice genotypes under drought stress [
70].
It could be concluded that crossing genotypes with varying degrees of drought tolerance, as shown in this study, may have resulted in valuable transgressive segregates with improved drought resistance. The results have significant implications for rice breeding, particularly in terms of choosing drought-tolerant genotypes at the molecular level in the lab and facilitating drought breeding programs. Drought stress has become a severe threat to food security in the developing world as well as in Egypt. Although water is required during all growth periods of the rice plant, there are some critical growth stages when drought stress has a serious impact and creates massive reductions in the quantities of yields.
Rice’s responses to drought stress and its tolerance level can be measured by monitoring different anatomical, biochemical, and molecular changes during a drought period. The responses of plants to drought are complex, and different morphological and biochemical mechanisms are involved within plants during drought.
These mechanisms can occur via different avenues. One avenue was the genetic background of the parental lines that were used in the crosses to develop new combinations that were more tolerant to abiotic stress, especially water stress. According to the yield trails, the parents Sakha 108 and M206 registered higher numbers for panicles/plant, 1000-grain weight, and grain yield/plant under normal irrigation, while under drought stress the parents Sakha 107 and M206 recorded the highest values for all yield attributes. The cross of Sakha 108 × M206 occupied the first position for all studied yield trails under normal conditions. Similarly, under stress, the cross combinations Sakha 107 × M206 and Sakha 107 × Sakha super 300 ranked first and second for all yield attributes, respectively. With regards to the annual average temperature fluctuation during the life span of rice plants, it increased by 2 0, as the temperature was higher in 2022 than in the previous season, which resulted in earlier flowering and led to a greater yield. Secondly, the selected superior crosses showed remarkable evidence for their anatomical, biochemical, and molecular advanced differentiation and tended to be more tolerant to drought stress. Thirdly, drought is escaped by rapid development, which allows plants to finish their life span before severe water stress. Fourthly, drought is avoided by increasing water uptake and reducing the transpiration rate, stomatal conductance, and leaf area. Fifthly, drought tolerance is increased by maintaining tissue turgor during water stress via osmotic adjustment, allowing plants to maintain growth under water stress. The biosynthesis of the most famous amino acid (proline, which is related to the enzyme pyrroline-5-carboxylate synthetase) as an osmo-protectant indicates water stress.