1. Introduction
According to the FAO 2021 global map of salt-affected soils [
1], around 400 million hectares of topsoil and more than 800 million hectares of subsoil are affected by salt. Moreover, FAO reported that soil salinity per year reduces production potential by taking up to 1.5 million ha of farmland out of production and more than 30 million USD as an annual loss in agricultural productivity [
1]. Unfortunately, at such rates, 50% of total cultivated land is expected to be salinized by 2050 [
2] as a consequence of the phenomenon of “desertification,” and land degradation due to climate change and human action by poor land management [
3,
4,
5]. Projections up to 2099 assume an extreme increase in drought and the subsequent soil salt accumulation in Eurasia, Africa, and Australia [
6]. Soil salinization may occur through a natural process (primary salinization) or by human management (secondary salinization), through irrigation with salty water, excessive use of fertilizers, and intensive agriculture practices [
7,
8,
9].
Salt, mainly NaCl, in the soil slows down plant development due to two stresses. At first, the plant experiences a physiological drought (osmotic stress), which reduces leaves and root growth due to the inhibition of cell expansion, cell wall synthesis, and stomata conductance [
10,
11]. Later the plant response is related to the toxic effect of salt, where plants accumulate toxic Na
+ and Cl
− in old leaves over a long period. Due to the harmful effects of salt on germination, water uptake, transpiration, photosynthesis, enzyme activities, and metabolism of protein and lipids, a significant reduction in yield of up to 40% in agricultural lands has been reported [
12,
13]. Since the three most widely consumed kinds of cereal (maize, rice, and wheat) are susceptible to saline soil conditions [
14], it is essential to improve studies about mechanisms to induce salt stress tolerance to secure our future on food storage.
Plants could tolerate salt stress by several strategies: (i) salt exclusion, retaining the Na
+ in woody stems or roots; (ii) regulating toxic ions balance through the maintenance of a high K
+/Na
+ ratio with a mechanism of translocation of K
+ over Na
+, to reduce the Na
+ concentration in the shoot cytoplasm; and (iii) by salt compartmentalization in vacuoles, protecting the cytoplasm from the ion toxicity [
15,
16]. The root system under an excessive salt concentration in the soil due to toxic ions accumulation suffers from inhibition of nutrition supply and elements adsorption [
17]. Salinity, as drought, hurts the whole root system due to reduced cell activity in the root meristem [
18]; however, growth rate reduction was determined more in the main root than in lateral roots [
19]. As a response to stress, plants can remodel root system architecture (RSA) to adapt to adverse conditions [
20]. However, the direction of root growth as a response to external stimuli differs in salt and drought conditions. Under drought conditions, roots use positive hydrotropism, growing towards the water, but in the presence of salt, roots remodeling due to negative halotropism directs the growth away from salinity. Moreover, it was reported that the roots of Arabidopsis exhibited reduced gravitropism under salt conditions to grow against the gravity vector [
21]. In addition, the identified gene in cereal crops responsible for gravitropism versus anti-gravitropism regulatory mechanisms also controls the root angle [
22]. The narrow root angle was determined as a very important feature in selecting genotypes for drought tolerance since genotypes with a narrow root angle can grow deeper compared with varieties with a wider root angle [
23,
24,
25]. However, root system remodeling strategies to tolerate drought and salt stress differentiate in terms of tropism type, and it does not mean that a narrow root system able to adapt to drought is also capable of growing in saline soil.
In traditional breeding programs, phenotyping the above-ground plant parts was the main aspect of determining resistance to abiotic stress. However, the development of several high-throughput methods and techniques for root phenotyping in recent years has finally made it possible to determine which root systems and remodeling strategies are associated with the most successful plant development under stress conditions [
26,
27,
28,
29]. Phenotypic root traits could be used to determine genetic diversity; however, they are often influenced by environmental conditions. According to Soriano et al. [
30], the stable quantitative trait loci (QTL) and closely linked molecular markers can improve durum wheat genetically through marker-assisted selection, cloning, and pyramiding in breeding programs to create new cultivars with desired quantitative traits. Molecular markers such as simple sequence repeat (SSR) and its based association mapping analysis may allow us to determine the chromosomal regions involved in root architecture features [
30,
31,
32,
33]. Moreover, SSRs are chromosome-specific and highly effective molecular tools due to their large level of polymorphism, distributed over all of the genomes [
34,
35]. Simple sequence repeat (SSR) molecular markers closely linked with genome regions associated with root architecture features could be helpful in selecting genotypes for salt tolerance breeding programs. Moreover, the identification of closely linked candidate genes can give a better understanding of the genetic response to specific stimuli.
In this study, six durum wheat genotypes, including one high salt-tolerant J. Kethifa as check genotype, were used to evaluate differences in shoot and root traits under control and salt conditions at the seedling stage and to determine their involvement in salt tolerance. The root system was evaluated using a high-throughput phenotyping system. The genotyping was performed using 11 SSR markers closely linked with genome regions associated with root traits, and in silico, candidate gene expression analysis was performed. The study aimed to evaluate genetic diversity among genotypes based on phenotypic and genotypic variations to identify candidate parental genotypes for salt-tolerance breeding.
3. Discussion
The root system remodeling strategies under adverse conditions is a great opportunity for adaptation and optimal development of the plant, as the first organ that senses and responds to abiotic stress, and then through efficient uptake of water and nutrients. Latest studies demonstrated that root system features, especially root angle in crop plants, play a main role in adapting to adverse conditions [
20,
22,
37].
According to Pariyar et al. [
38], together with root traits, leaf-related traits should be included for more efficient selection in breeding programs for whole plant establishment. At the same time, the ideotype of the root system plays a key role in ensuring sufficient leaf area, which in turn is responsible for efficient photosynthesis, the crucial process for plant development. Genotypes that produce new leaves faster than the die of old ones and maintain a high number of leaves or shoot biomass under stress conditions could be considered tolerant. Moreover, previous studies have shown that salt-tolerant genotypes of durum wheat accumulated higher shoot biomass and maintained a bigger leaf area under salt stress compared with susceptible varieties [
39,
40,
41]. In our study, some shoot traits were associated with root angle. For example, the salt-tolerant genotype, J. Khetifa, under salt stress, maintained a narrower root angle and the highest number of leaves. Moreover, Vulci, genetically close to J. Khetifa, was characterized by a narrow root angle and a large number of leaves under salt conditions. However, in our previous drought study [
25], Pelsodur, under control and drought conditions, maintained a narrow root angle, which was associated with good shoot development and drought tolerance. While in this study, under salt stress, the Pelsodur root angle changed significantly from narrow to wide, and this caused a significant negative effect on his shoot development, especially tillers that decreased by 60%. Moreover, in genotypes with wider root angles, such as Sebatel, Cham1, and Azeghar, the number of leaves was not significantly affected by salinity. It is well known that a narrow root angle is one of the main traits for drought tolerance, but it was demonstrated that a shallower root growth angle could enhance rice yields in saline environments [
20].
Phenotypic data from our pot experiment obtained using the WinRHIZO software (version 4.0b) highlighted a few trends in plant response to salt stress at the seedling stage. Genotypes under control conditions were distinguished regarding root angle into two groups, genotypes Vulci and Pelsodur with high salt-tolerant J. Khetifa had a narrower root angle than genotypes Sebatel, Azeghar, and Cham1. It was reported that root angle depends on plant root ability to control gravitropic versus anti-gravitropic mechanisms, which is the main factor in avoiding salty soil [
20,
21,
22]. In this regard, root tips could be a good indicator for selection as they are involved in root gravity perception and regulation. In addition, the salt-tolerant genotype J. Khetifa under salt stress doubled the number of root tips and had a significantly higher number compared with the other genotypes. However, under salt conditions, the salt-tolerant J. Khetifa, with a narrow root angle, was grouped with genotypes Azeghar and Sebatel, which had a wider root angle. These results suggest that a narrow root angle is not a good indicator of salt stress tolerance.
In general, the check genotype J. Khetifa under salt stress at the seedling stage improved the number of leaves, which is a good salt tolerance indicator in terms of accumulation of toxic ions and efficient photosynthesis. Moreover, J. Khetifa improved almost all root traits under salt conditions, except the root angle, which stayed narrow, and the mean root diameter. The other two genotypes, Azeghar and Sebatel, from the same cluster of J. Khetifa under salt stress, had wide root angles, and salt did not affect shoot traits significantly. Moreover, genotype Azeghar under salt increased root volume and surface area, and the roots of Sebatel were not affected significantly. Based on present results, it can be concluded that a narrow root angle is not a good indicator of salt tolerance, moreover, exist more root system adaptation strategies that depend on the inner capacity of genotypes to maintain an efficient amount of roots.
Dendrograms based on the cluster analysis of the genetic similarity coefficients and phenotypic data under control conditions grouped genotypes mainly due to root angle. In this regard, the SSR marker wms5 can be quite informative in selecting genotypes for narrow-angle root system ideotype. It was reported that the wms5 marker was significantly associated with root angle [
42], and it was located on chromosome 3A in the durum wheat genome. In our study, genotypes grouped in the same clusters according to root angle, such as J. Khetifa, Vulci, and Pelsodur, had narrow root angles (wms5 band of 190 bp), and Sebatel and Azeghar (wms5 band of 180 bp), Cham1 (wms5 band of 185 bp) had wide root angles. Moreover, in the durum wheat genome, the location of wms5 was close to gene models representing proteins involved in plant development and response to stress. Such as thioredoxin, which is a small ubiquitous protein that reduces protein disulfides, protects cells from oxidative damage, and protects plants from the oxidative deactivation of photosynthesis-related enzymes [
43]. Moreover, homeobox leucine-zipper transcription factors are responsible for plant growth adaptation in water deficit conditions [
44,
45]. Core-2/I-branching beta-1,6-N-acetylglucosaminyltransferase family protein is involved in cell elongation during the seedling growth stage and senescence of the leaf process [
46]. Citrate-binding protein family is involved in plant development and growth, also in response to stress related to the accumulation of toxic ions in the vacuoles [
47]. DUF1677 family proteins are DUF domain-containing proteins that play a vital role in plant stress responses, including salinity [
48,
49]. Kinases superfamily proteins are well known as signal transmitters in various stresses [
2]. Members of the MADS-box gene family are involved in many processes of plant development and morphogenesis, but it was also reported to modulate root architecture [
50,
51].
Our results revealed that the root system under the salt of genotypes Azeghar and Sebatel were close to the one of salt-tolerant J. Khetifa, regardless of root angle differences, making them good candidates as parent lines in breeding programs. In this respect, some rare alleles of SSR markers could help to select salt-tolerant candidates in the segregant populations. For example, the salt-tolerant J. Khetifa and Sebatel had rare alleles near marker cfa2086, 277 bp, and 223 bp, respectively. Moreover, salt-tolerant J. Khetifa had rare alleles by SSR marker gwm234 (214 bp) and near marker gwm499 (154 bp). Moreover, genotype Sebatel had a rare allele also near marker gwm499. In the durum wheat genome, the mapped marker gwm499 was close to gene models for kinases superfamily protein, which mostly regulates transcription factors. Moreover, it was near to basic helix-loop-helix transcription factors, which are involved in a range of abiotic stress responses and adaptive responses to drought [
52]. ADP-ribosylation factor GTPase-activating protein was shown that is crucial for determining directional root hair growth in Arabidopsis [
53].
Genotypes Azeghar and Sebatel had rare alleles near marker gwm427, 201 bp, and 237 bp, respectively. The marker gwm427 position was found to overlap with previously reported durum wheat major QTL for root growth angle (qSRA-6A), where candidate gene analysis showed loci related to gravitropism, polar growth, and hormonal signaling [
54]. In the durum wheat genome (RefSeq v1.0 Chr 6A region), the mapped marker gwm427 was close to genes models for actin-related protein and plastid movement-impaired related protein. Marker gwm427’s location in the durum wheat genome was close to two related genes such as (i) the actin-related protein and (ii) the plastid movement impaired 1-related 1 G protein that regulates organelle movement [
55]. In general, the movement and positioning of the organelles play an important role in the adaptation of plants to environmental stress. Moreover, actin-related protein regulates developmental processes such as cell division and development and cell polarity [
56]. Marker gwm427 location in the durum wheat genome was close to other five gene annotations (
Table S4); (iii) cytochrome P450 from the oxidoreductases enzymes class has been reported to be involved in plant defense against various abiotic and biotic stresses [
57]. (iv) Inosine-5’-monophosphate dehydrogenase catalyzes the biosynthesis of the guanine nucleotide, which is important for DNA and RNA synthesis, signal transduction, energy transfer, glycoprotein synthesis, and cellular proliferation [
58]. (v) Proline-tRNA ligase is an enzyme involved in proline metabolism and KEGG and aminoacyl-tRNA biosynthesis pathways (BRENDA:EC 6.1.1.15). L-Proline in a stressful environment is crucial for plants due to its protective role as an osmoprotectant and stabilizes cellular structures and enzymes, and scavenging reactive oxygen species [
59]. (vi) Potassium-transporting ATPase could help plant cells to maintain a high concentration of potassium and a low concentration of sodium inside in case of high sodium concentrations in the soil. (vii) Gene containing cystathionine β-synthase (CBS) domain was found to be upregulated under high salinity, and overexpressing this gene showed higher abiotic stress tolerance in transgenic plants [
60].
Genotype Azeghar had three more rare alleles, two near marker cfa2257 (104 bp and 142 bp) and one near gwm573.2 (181 bp) (
Table S4). The marker gwm573.2 was close to gene models translated to trehalose 6-phosphate phosphatase, which regulates the activity of protein kinase, and this has a large impact on cell division, development, and function [
61]. The protein of the DNA-directed RNA polymerase belongs to complex molecular machines for the essential function of the synthesis of RNA from DNA templates, otherwise transcription [
62]. Abscisic acid is a substrate of the ABC transporter that is involved in the active transport of various molecules across biological membranes, such as heavy metals, lipids, glucosinolates, and phytohormones [
63] allowing to coordinate of physiological and developmental processes at the whole-plant level and being involved in response to several induced by biotic stresses. Other related genes (
Table S4) are (i) Phosphatidylinositol: ceramide inositol phosphotransferase, which is essential for sphingolipid biosynthesis and plays an important role in dehydration stress tolerance [
64]. (ii) Shikimate kinase catalyzes the reaction of the shikimate pathway that redirects carbon from the central metabolism pool to a wide range of secondary metabolites responsible for plant development, growth, and stress responses [
65]. (iii) G-proteins are universal signal transducers involved in many cellular responses of plants [
66]. (iv) ATP-dependent zinc metalloprotease FtsH 1 is involved in the formation of thylakoid membranes during early chloroplast development and in the proteolysis of damaged photosystem II (PSII) protein complex repair cycle, which is essential to avoid photoinhibition due to the accumulation of photodamaged PSII [
67]. (v) Aldo/keto reductase family oxidoreductase is involved in many plant metabolites reactions, such as the biosynthesis of osmolytes, reactive aldehyde detoxification, secondary metabolism, and membrane transport [
68]. (vi) Sec14p-like phosphatidylinositol transfer family protein is a plasma membrane-associated protein that has a crucial role in rooting patterning due to auxin signaling and PIN distribution in Arabidopsis [
69]. (vii) Pentatricopeptide repeat proteins regulate chloroplast gene expression and RNA metabolism in higher plants [
70]. (viii) Transcription factor bHLH involved in response and tolerance to various plant stresses, including soil salinity [
71]. (ix) The regulator of chromosome condensation (RCC1) family with FYVE zinc finger domain-containing protein is essential for regulating auxin flows toward the direction of gravity and root branch angle in Arabidopsis [
72]. (x) Transcriptomic analysis in roots revealed that the ABA-related gene encoding HVA22-like proteins was up-regulated, and considering that ABA biosynthesis is correlated with auxin transport to the root tips, their co-expression can be beneficial for the development and morphology of the roots [
73]. (xi) TCP transcription factor may activate OsNHX1 gene expression, which response to salt and PEG-induced drought stress in rice, and may be associated with abiotic stress tolerance [
74]. (xii) Photosystem (PS) II CP43 reaction center protein is the main antenna of the PS II pigment-protein complex in the thylakoid membrane and plays an essential role in the photosynthetic responses to stresses in higher plants [
75]. (xiii) BTB/POZ domain-containing proteins are associated with plant growth and development, also involved in plant responses and defense against biotic and abiotic stresses [
76]. (xiv) The enzyme Sphingoid base hydroxylase II is essential for growth and viability in Arabidopsis [
77]. The marker gwm573.2 was also close to (xv) two F-box-like protein genes models, which were described earlier as genes involved in salt tolerance, and rooting abilities.