Making wheat salt tolerant: What is missing?

Abstract

The growing global population presents a significant challenge to ensuring food security, further compounded by the increasing threat of salinity to agricultural productivity. Wheat, a major staple food providing 20% of the total caloric intake for humans, is susceptible to salinity stress. Developing new salttolerant wheat cultivars using wheat breeding techniques and genetic modifications is crucial to addressing this issue while ensuring the sustainability and efficiency of wheat production systems within the prevailing climate trend. This review overviews the current landscape in this field and explores key mechanisms and associated genetic traits that warrant attention within breeding programs. We contend that traditional approaches to breeding wheat for Na+ exclusion have limited applicability across varying soil salinity levels, rendering them inefficient. Moreover, we question current phenotyping approaches, advocating for a shift from whole-plant assessments to cell-based phenotyping platforms. Finally, we propose a broader use of wild wheat relatives and various breeding strategies to tap into their germplasm pool for inclusion in wheat breeding programs.

Headnote

ABSTRACT

The growing global population presents a significant challenge to ensuring food security, further compounded by the increasing threat of salinity to agricultural productivity. Wheat, a major staple food providing 20% of the total caloric intake for humans, is susceptible to salinity stress. Developing new salttolerant wheat cultivars using wheat breeding techniques and genetic modifications is crucial to addressing this issue while ensuring the sustainability and efficiency of wheat production systems within the prevailing climate trend. This review overviews the current landscape in this field and explores key mechanisms and associated genetic traits that warrant attention within breeding programs. We contend that traditional approaches to breeding wheat for Na+ exclusion have limited applicability across varying soil salinity levels, rendering them inefficient. Moreover, we question current phenotyping approaches, advocating for a shift from whole-plant assessments to cell-based phenotyping platforms. Finally, we propose a broader use of wild wheat relatives and various breeding strategies to tap into their germplasm pool for inclusion in wheat breeding programs.

Keywords:

Phenotyping

Breeding

Reactive oxygen species

Tissue tolerance

Sodium exclusion

Potassium retention

Keywords:

Phytohormones

Mitigation strategies

Microbial inoculation

Oxidative stress

(ProQuest: ... denotes formulae omitted.)

1. Introduction

Soil salinization is a significant environmental stress that adversely affects global crop production in rainfed and irrigated agricultural regions. The escalating effects of climate change and global warming are anticipated to exacerbate salinization due to higher evapotranspiration rates, especially in semi-arid and arid regions. The intrusion of saline ocean water into rivers and groundwater aquifers due to reduced river discharge and rising sea levels, necessitates the use of brackish water for irrigation [1,2]. Even irrigation water with a relatively modest electrical conductivity (0.5 dS m-1 ) can introduce sufficient salt to induce salinity stress [2]. Projections indicate that by 2050, nearly half of the total arable land will be affected by salinity, threatening global food supplies for the growing human population [3,4]. Wheat (Triticum spp.) is the predominant cereal crop worldwide, ranking first among grain-producing crops that humans consume [5]. About one-third of the global population relies on wheat as its primary nutritional source, with a significant proportion grown in saline soil [6]. In response to the escalating demands of the expanding population, wheat production must double by 2050 [7]. Despite the substantial genetic diversity for salt tolerance inherent in the wheat genome, modern elite cultivars cannot thrive in environments with high soil salinity. Most genotypes produce little grain yield when exposed to salinity levels above 150 mmol L-1 NaCl [8,9]. While durum wheat is the most salt-sensitive and bread wheat exhibits moderate tolerance [10], both are considered salt excluders. All modern cultivars have been selected for their ability to limit Na+ intake by roots. About 98% of the Na+ taken up by wheat roots is returned to the rhizosphere [11] via an active Na+ extrusion process mediated by Na+ /H+ exchangers located at the plasma membrane of epidermal root cells and encoded by the SOS1 gene [12]. While this strategy serves as a temporary defense mechanism against Na+ toxicity, it carries two significant drawbacks: (1) rapid salt buildup in the root zone (see [3] for modeling) and (2) high energy cost for osmotic adjustment at the expense of yield (discussed in subsequent sections). Hence, a salt-excluding approach may be efficient only during transient salinity events of relatively short duration and within a narrow range of salinities. Given the growing reliance on irrigation in global agriculture [3], it is unlikely that Na+ excluding cultivars will cope with impending climate scenarios. This situation necessitates a substantial shift in the fundamental principles of wheat breeding for salt tolerance, encompassing a reevaluation of the targeted genes and methodologies for plant phenotyping. These critical themes form the central focus of this review.

2. Complex physiology of salinity tolerance trait and developmental stage consideration

Salinity tolerance is a highly complex trait, both physiologically and genetically, and comprises various sub-traits. These sub-traits exhibit varying roles and contributions to overall tolerance, which varies between species and plant developmental stages [13–15]. Unfortunately, researchers and breeders often overlook this critical aspect when conducting plant screenings. One example is the misleading use of seed germination tests as a screening tool. A cursory search combining the keywords 'salinity stress', 'seed germination', and 'screening' on the Web of Science reveals 450 papers advocating this approach. However, numerous other studies have reported the absence of any meaningful correlation between seed germination under saline conditions and subsequent plant performance at later stages (e.g., [16]). These findings, though unsurprising, underscore the physiological reality that a seed's ability to germinate in the presence of salt predominantly relates to a solitary adaptive trait - osmotic stress tolerance. At this early growth stage, concerns about specific Na+ toxicity are negligible, as the few days required for wheat seed germination do not cause any major detrimental effects. Also, at the germination stage developing seedlings take most of the nutrients from the endosperm, so, the effects of Na+ on the plant's ability to uptake essential nutrients are not essential. This is not the case at the later stages of plant development when Na+ -induced depolarization of root epidermal cells has a major impact on plant's ability to acquire nutrients. As plants progress through their ontogeny, novel traits become prominent, including control of Na+ uptake and exclusion from roots, regulation of xylem Na+ loading and retrieval, its transportation, and movement to and from shoots, tissue- and organellespecific Na+ sequestration in shoots (accumulation of Na+ in vacuoles so the enzymes in cytoplasm are not inhibited), cytosolic K+ retention in mesophyll, stomata patterning and operation, and ROS signalling and detoxification [12,17,18]. Most of these traits do not exist or hold minimal importance during germination. Hence, despite the notion that the germination stage is particularly susceptible to salinity stress [19], outcomes derived from germination tests cannot be extrapolated to gauge overall plant performance through its ontogeny. While germination tests might seem appealing due to their cost and labor efficiencies, their predictive value remains limited, and thus, they should not be advocated as reliable indicators of future plant resilience and success.

3. Phenotyping dilemma: high throughput vs. predicting power

With the advent of modern genetic and molecular technologies, plant phenotyping is emerging as a significant bottleneck in wheat breeding programs. In response to salinity, wheat plants accumulate various biochemical compounds such as amino acids (AA), proteins, soluble sugars, proline, and antioxidants [20–22]. Many of these compounds have been proposed as potential biochemical markers for integration in breeding programs. However, this approach is inherently flawed as it fails to separate cause and effect. For instance, increased AA contents may occur under salt stress to increase cell osmolarity for osmotic adjustment. However, higher AA contents could also result from stress-imposed protein degradation [23], potentially negatively correlating with salinity tolerance. The same principle applies to enzymatic antioxidants (AO). The simplistic notion of 'the more AO, the better' is often misguided [24]. It has been argued that truly salt-tolerant species like halophytes do not rely on elevated AO activity as they can prevent ROS formation in the first place, sidestepping the need for detoxification [25]. Indeed, intrinsically high AO activity can interfere with ROS signaling, which is crucial for plant adaptive responses to abiotic stresses, including salinity [26].

Chlorophyll fluorescence has also been advocated as an efficient, non-destructive biomarker for assessing salt tolerance in wheat genotypes [20,21]. This method is primarily driven by its convenience for high throughput, given that each measurement takes mere seconds and is non-destructive. However, while PSII efficiency (quantified by chlorophyll fluorescence Fv/Fm ratio) does indeed respond to salinity [27], it represents only one of several components influencing CO2 assimilation and, consequently, crop biomass/yield. Moreover, using chlorophyll content as a proxy for salinity tolerance, as attempted in some studies [4,28,29], is scientifically flawed. Salinity-induced reductions in cell size may cause salt-affected leaves to appear darker due to the chlorophyll 'condensation' effect.

So, how do we navigate this conundrum? First, we must accept that relying on a single characteristic as a proxy for tolerance carries a substantial risk and is unlikely to be reliable. A multiparametric screening approach offers greater promise [30,31], though it comes at the expense of high throughput. However, a more transformative solution lies in developing and adopting cellbased (rather than whole-plant-based) phenotyping platforms. The latter can be best illustrated by using tissue Na+ content and K+ /Na+ ratio as a proxy for salt tolerance.

It is widely acknowledged that high cytosolic Na+ content is detrimental to cell metabolism [10], and it should ideally be maintained at a low level or, at the very least, complemented by high cytosolic K+ retention ability [32,33]. However, analyzing the whole shoot or leaf for Na+ content fails to account for its intracellular distribution. Some halophyte plants like Atriplex spp. can contain up to 10% Na+ within leaves (dry matter basis; reviewed by [34]) and yet function well due to the sequestration of potentially cytotoxic Na+ in mesophyll cell vacuoles or salt bladders, segregating it from active metabolic pathways. Under these circumstances, selecting wheat genotypes with lower shoot Na+ contents could inadvertently miss accessions with high tissue tolerance. Instead, cell-based screening using methods like fluorescent CoroNa Green dye [35] or high-resolution cryo-scanning electron microscopy (cryo-SEM) X-ray microanalysis [36] will differentiate between vacuolar and cytosolic/chloroplastic Na+ distribution. This methodology will establish cultivars as truly tolerant if they accumulate high amounts of leaf Na+ (measured by ICP or flame photometry) while efficiently sequestering it in vacuoles (measured by florescent dye or cryo-SEM X-ray microanalysis).

Another example demonstrating the efficiency of cell-based phenotyping is using root K+ loss as a proxy for salinity tolerance in plants. It was shown that the ability of plant roots to prevent NaCl-induced K+ loss from the root is an essential trait for salinity tolerance in many species, including wheat [37]. However, the correlation between the magnitude of salt-induced K+ loss from the root and salinity tolerance exists only in the mature root zone and is not observed in the root apex [32]. Hence, analysis of the whole-root K+ loss may compromise the sensitivity of this method while measurements of NaCl-induced K+ efflux from specific types of root cells may be used as a highly efficient screening tool (conferring over 60% of genetic variability in salinity tolerance in some species, e.g., barley; [38]).

4. Achieving salinity tolerance through breeding and genetic strategies

Breeding strategies and genetic control of salinity tolerance in wheat involve complex and polygenic approaches, where additive and dominance effects shape the inheritance of multiple traits. Significant breeding efforts and genetic modifications have contributed to identifying multiple traits and underlying genes that serve as markers for field-level salinity tolerance [39]. These avenues could expedite salinity research, improving yield quality and quantity under salt stress. Key approaches within this realm are discussed below.

Successful breeding strategies rely on identifying diverse germplasm from large donor pools, highlighting the importance of appropriate selection criteria [39]. Indirect selection based on tolerance or direct selection based on yield are commonly used breeding approaches. While indirect selection is more efficient under controlled conditions, direct selection from saltstressed fields is crucial. Indirect effects arise from multiple traits, but direct effects play a more significant role in trait selection [40]. Agronomic characteristics often take precedence as primary targets as they directly impact agricultural productivity [41]. Numerous physiological traits have been frequently highlighted as suitable indicators for wheat breeders to target salinity stress tolerance, including tissue Na+ and Cl– contents, the ability of plants to discriminate K+ over Na+ and achieve a higher tissue K+ /Na+ ratio, osmotic adjustment, oxidative tolerance, and improved transpiration efficiency [42–44]. Overall, various traits encompassing agronomic and physiological attributes contribute to salt tolerance. However, salt-tolerance mechanisms, gene environment (* E) interactions, and differing results in open field and controlled environment experiments make it difficult to establish selection criteria [44]. This complexity is not surprising, as some of these traits may be misleading, and their use as selection criteria may be counterproductive, as described in the above section. Also, depending on the severity and duration of the stress, the strategies employed by plants may be strikingly different. This can be illustrated by using stomatal operation as an example. Stomata development and behavior is a crucial component of plant adaptive mechanism to salinity [45]. Osmotic stress results in rapid closure of stomata, which reduces the influx of CO2 and ultimately biomass accumulation, and efficient stomatal operation under salinity stress involves adjustment of stomata aperture, speed of stomata response, stomata size or stomatal density [45,46]. However, there is no simple answer on what trait related to stomata behavior should be targeted in breeding programs, as the strategy employed may depend on genotype and/or duration and intensity of stress. Contrasting behavior in stomata operation in response to salt stress has been reported for wheat with salt tolerance correlating either positively [9] or negatively [47] with stomatal conductance and stomatal density. This controversy is not surprising as if the stress is mild then plants may opt to maintain stomata open to allow higher CO2 assimilation, on the provision that they can efficiently access/retain sufficient amounts of water. More severe or prolonged stresses will make this option unviable, and the better strategy would be to close the stomata and retain more water. The same applies to stomata development. Genotypes with inherently low stomata density might be efficient in reducing residual transpiration [48] but may experience yield penalties under control (non-saline) conditions.

5. Genetic approaches

5.1. Using wild relatives

Diversity for salt tolerance within wheat (including landraces) appears too narrow for salt tolerance improvements [49]. One solution is to identify new genetic sources for salt tolerance within wild relatives of wheat and subsequently introduce the relevant traits into cultivated wheat varieties. Nearly two decades ago, Colmer et al. [50] reviewed attempts to use wild relatives to improve salt tolerance in wheat, concluding that attention was focused on identifying sources with enhanced Na+ exclusion, with less attention given to other traits that contribute to salt tolerance; transfer of one trait improved salt tolerance of the progeny, but yield reduction remained a challenge. Below, we examine the progress or lack thereof in increasing salt tolerance in wheat using its wild relatives.

5.1.1. Triticum turgidum spp. dicoccoides

Triticum turgidum spp. dicoccoides (AABB; wild emmer wheat), the progenitor of tetraploid durum wheat, is distributed across the Middle East, including areas strongly affected by drought and salinity [51]. A study involving 54 wild emmer genotypes from nine geographical populations in Israel revealed substantial variation in relative shoot dry weight (37%–97%) after exposure to 300 mmol L-1 NaCl solution for ten d compared to non-saline controls. Most of these wild emmer plants had higher values than the two durum wheat (Kalka and Wollaroi) and bread wheat (Krichauff) standards (37%–43%) [52]. Interestingly, no direct link was discerned between relative dry weight (RDW) and Na+ accumulation in the third leaf, but high RDW was associated with slow growth under control conditions. Nevertheless, the salt tolerant genotype with the highest RDW had the lowest leaf sap Na+ concentration (290 mmol L-1 ). However, the leaf sap Na+ concentration surpassed that of bread wheat (180 mmol L-1 ), indicating that other traits are also needed to confer salt tolerance [50].

Feng et al. [53] evaluated 30 morphological and physiological traits in 30 wild emmer and 14 durum wheat accessions. The study revealed that significant variations in salt tolerance index in wild emmer (0.21–0.97 vs. 0.46–0.78 in durum wheat) correlated with shoot K+ /Na+ ratio and shoot Na+ concentration, but not with shoot K+ concentration, indicating that salt tolerance in wild emmer primarily stemmed from Na+ exclusion. In addition, root length, surface area, and volume were highly correlated with salt tolerance in wild emmer. Chen et al. [54] also analyzed salt tolerance mechanisms in wild emmer. The salt-tolerant wild emmer accession (18–35), selected from 400 accessions, had higher RDW, lower electrolyte leakage, and lower MDA content than cultivated wheat (Zheng 9023) when exposed to 250 mmol L1 NaCl for 4–10 d. Moreover, gene expression analysis highlighted upregulated genes in wild emmer under salt stress; however, the precise functions of these genes were not discussed [54].

5.1.2. Aegilops cylindrica

Aegilops cylindrica (CCDD; jointed goat grass) is an allotetraploid formed by hybridization between the diploid species Ae. tauschii Coss. (2n = 2x = 14; genome DD) and Ae. markgrafii (Greuter) Hammer (2n = 2x = 14; genome CC). Arabbeigi et al. [55] analyzed 44 accessions of Ae. cylindrica, concluding that Na+ exclusion is an important attribute of this species. Among the 44 accessions grown under 400 mmol L-1 NaCl for four weeks, ten exhibited less than 20% dry weight reduction (compared to the non-saline control), the lowest leaf Na+ concentration, and the highest leaf K+ /Na+ ratio [55]. Molecular marker analysis revealed that the ten most salttolerant accessions shared the same haplotype at four microsatel lite markets (Xgwm312, Xwmc170, Xgwm291 and Xgwm410) previously associated with Na+ exclusion genes (HKT1;5 and HKT1;4) [55]. The two most contrasting genotypes of Ae. cylindrica (salttolerant USL26 and salt-sensitive K44) for salinity tolerance and Na+ exclusion capacity were further used to investigate root and shoot Na+ and K+ concentrations and the expression profile of some Na+ transporter related genes (AecHKT1;5, AecSOS1, AecNHX1 and AecVP1) [56]. The salt-tolerant genotype (USL26) had significantly lower [Na+ ] but higher [K+ ] and K+ /Na+ ratios in roots and shoots than the salt-sensitive genotype (K44) when grown under 400 mmol L-1 NaCl for 7 d. Both genotypes had higher Na+ in roots than shoots. This Na+ concentration pattern correlated with gene expression profile. The salt treatment upregulated the expression of AecHKT1;5, AecSOS1, AecNHX1 and AecVP1 significantly more in USL26 than K44. At the tissue level, roots had greater AecHKT1;5 and AecSOS1 expression than shoots, but both tissues had similar AecNHX1 and AecVP1 expression. The authors suggested that the coordinated activities of AecHKT1;5 and AecSOS1 regulate shoot Na+ levels (AecHKT1;5 is involved in loading Na+ into shoot phloem for recirculation to the roots, whereas AecSOS1 involved in Na+ transport from xylem parenchyma to xylem vessels) and AecNHX1 and AecVP1 play a role in Na+ sequestration into vacuoles in roots and shoots. High homology of AecHKT1;5 to TaHKT1;5-D from wheat indicates the importance of the D genome in salt tolerance and the potential for improving salt tolerance in wheat by incorporating HKT1;5 genes from wild relatives [56].

5.1.3. Collection of Aegilops and Triticum species

Ahmadi et al. [57] screened a core collection of 179 Aegilops and Triticum accessions to identify the most tolerant wild relatives, which included six diploid species [T. boeoticum Bioss. (Ab Ab ), T. urartu Tumanian ex Gandilyan (Au Au ), Ae. speltoides Tausch. (BB), Ae. tauschii (DD), Ae. caudata L., (CC) and Ae. umbellulata Zhuk. (UU)], five tetraploid species [T. durum (AABB), Ae. neglecta L. (MMUU), Ae. cylindrica (CCDD), Ae. crassa Boiss. (DDMM), and Ae. triuncialis (CCUU)], and one hexaploid species [T. aestivum (AABBDD)]. Salinity stress (300 mmol L1 NaCl for 30 d) reduced shoot and root fresh and dry weights, shoot and root K+ /Na+ ratios and shoot [K+ ] compared to the non-saline control but increased shoot and root [Na+ ] to varying degrees, indicating a significantly high level of genetic variability. Among the diploid species, Ae. caudata (CC) had the highest leaf [K+ ] when grown under saline conditions, followed by Ae. tauschii (DD), T. boeoticum (Ab Ab ) and Ae. umbellulata (UU), whereas T. urartu (Au Au ) and Ae. speltoides (BB) had the lowest leaf [K+ ]. Ae. tauschii (DD) had the lowest leaf [Na+ ], followed by Ae. speltoides (BB), Ae. umbellulata (UU), T. boeoticum (Ab Ab ), and Ae. caudata (CC), with the highest leaf [Na+ ] in T. urartu (Au Au ). Consequently, the leaf K+ /Na+ ratios in diploid species were ranked DD > CC > UU > Ab Ab > BB > Au Au [57]. Analysis of the tetraploid and hexaploid species showed that T. aestivum (AABBDD) had the highest leaf [K+ ], followed by species containing the U (UM and CU) and AB genomes, whereas tetraploid species with D genome (DC and DM) had the lowest leaf [K+ ]. The lowest leaf [Na+ ] also occurred in hexaploid and tetraploid species with D or B genomes; T. durum (AABB) had the lowest leaf [Na+ ], followed by T. aestivum (AABBDD), Ae. crassa (DDMM), Ae. triuncialis (CU), and Ae. cylindrica (DC), whereas Ae. neglecta (UUMM) had the highest leaf [Na+ ]. The higher Na+ 'exclusion' ability in species bearing D or B genomes, combined with K+ accumulation in species harboring C or U genomes resulted in the following K+ /Na+ ratio order: ABD (T. aestivum) > AB (T. durum) > CU (Ae. triuncialis) > DC (Ae. cylindrica) > UM (Ae. neglecta) > DM (Ae. crassa). Using biplot analysis, Ahmadi et al. [57] identified Ae. triuncialis (CU), T. durum (AB), T. aestivum (ABD), and Ae. caudata (C) as the most salttolerant species, suggesting their potential for breeding salttolerant wheat.

In the same study, 12 accessions of each identified species with superior salt tolerance were further assessed for their antioxidant defense system in response to salinity [57]. Salinity stress increased antioxidant enzyme activities to varying degrees. Notably, T. aestivum (ABD), T. durum (AB), Ae. crassa (DM), and T. boeoticum (Ab ) had the highest activity of superoxide dismutase (SOD), Ae. spletoides (B) and Ae. neglecta (UM) had the highest glutathione peroxidase (GPX) activity, and T. boeoticum (Ab ) and Ae. speltoides (B) had the highest catalase (CAT) activity. However, no direct association between salinity tolerance and antioxidant activity was established. It is important to note that these findings are based on one study, and further independent validation is warranted.

5.2. Inter-specific and inter-generic hybridization

Wide hybridization, involving crossing different species or genera, has enhanced wheat salt tolerance. Introducing genes from diploid or tetraploid progenitors or wild relatives adapted to salinity stress has significantly improved wheat salt tolerance (reviewed in [50]). We identified eight papers published since 2005 (Web of Science) that used wide hybridization approaches to generate salt-tolerant wheat hybrids. Among these papers, three studies assessed salinity tolerance in wheat Hordeum marinum amphiploids [58–60]. Alamri et al. [58] used four H. marinum accessions (H21, H87, H109, H155; genome XX) to produce amphiploids (genome AABBDDXX) with Chinese spring wheat (genome AABBDD). All amphiploids maintained better relative growth rates (RGR; 78%–89% of non-saline controls) than wheat (62% of controls) when grown in a nutrient solution containing 200 mmol L-1 NaCl for several weeks. The salinity treatment increased Na+ concentrations for all genotypes, with the largest increase for wheat (2.4-fold), followed by 39%–87% for amphiploids and 15%–68% for H. marinum accessions relative to non-saline aerated controls (Table 1).

H. marinum wheat amphiploids exhibited improved Na+ and K+ regulation under saline conditions, contributing to enhanced salt tolerance [59]. Of the 15 H. marinum bread wheat (genome AABBDDXX) or H. marinum durum wheat (genome AABBXX) amphiploids, 14 had lower leaf blade [Na+ ], higher leaf blade [K+ ] and consequently higher K+ /Na+ ratios than the respective wheat parent, and several had less leaf injury when grown in the presence of high salt [59]. The authors suggested that adding the XX genome enhanced K+ /Na+ discrimination in wheat amphiploid, consistent with the notion that abiotic (specifically, salinity) stress tolerance correlates with ploidy levels [61,62]. In addition, durum wheat amphiploids had lower K+ /Na+ ratios than bread wheat amphiploids, indicating the importance of the DD genome in ion regulation under saline conditions. Similarly, transferring of salt tolerance from H. marinum to H. marinum T. aestivum amphiploids improved growth, Na+ and Cl– exclusion from young leaves, and the accumulation of compatible solutes compared to the wheat parent [60]. However, these amphiploids had lower grain production under salinity (150 mmol L-1 NaCl) than the wheat parent (95% vs. 71% for 100-grain weight and 67% vs. 54% for total grain weight per plant) due to cytoplasmic male sterility [60].

Tall wheatgrass (Lophopyrum elongatum), a diploid (genome EE) wild relative of wheat adapted to saline soils, has also been explored for enhancing wheat salt tolerance through wide hybridization. Wheat x tall wheatgrass amphiploids (genome AABBDDEE) showed greater Na+ exclusion, controlled by chromosome 3E, and have been considered a new salttolerant cereal (reviewed by [49,50]) despite their lower grain quality than the wheat parent. However, recombinant wheat lines containing small segments of tall wheatgrass chromosomes retained salt tolerance without compromising yield (reviewed by 49,50]). Mullan et al. [63] used comparative genomics to identify wheat orthologs of Arabidopsis thaliana genes regulating Na+ transport and analyze the effects of L. elongatum chromosomes on gene expression under saline conditions as the basis for using wheat x L. elongatum substitutions lines for developing wheat germplasm with reduced Na+ accumulation (Fig. 1A). The authors showed that adding the entire complement of L. elongatum to wheat decreased leaf [Na+ ] more than the substitutions lines containing chromosome 3E when grown in a nutrient solution containing 200 mmol L-1 NaCl. Another study using a set of recombinants of chromosome 3E segments from L. elongatum and homologous group 3 chromosomes in wheat identified the region controlling Na+ exclusion on the distal end of the long arm of homologs 3A and 3D replaced by tall wheatgrass chromatin and recombinant line 524–568, with the smallest portion of tall wheatgrass chromatin on the distal region of 3AL, as the most suitable for wheat germplasm development [64].

An evaluation of respiration and mitochondrial metabolism in wheat and wheat x L. elongatum amphiploids revealed that the amphiploids exhibited higher shoot biomass retention (39%) following seven weeks of 200 mmol L-1 NaCl treatment than the wheat parent (28%) and a higher abundance of mitochondrial proteins, particularly enzymes involved in detoxifying ROS, thus contributing to improved salt tolerance [65]. These authors suggested the potential for identifying and transferring genes encoding these mitochondrial proteins into salt-sensitive wheat cultivars.

Yousfi et al. [66] studied the physiological performance of four durum wheat genotypes and two related amphiploidstritordeum (durum wheat Hordeum chilense) and triticale (wheat x Secale cereale)-to salt stress during the reproductive stage. Tritordeum produced more biomass than durum wheat in all salinity treatments. For salinities up to 12 dS m-1 , tritordeum and triticale had lower Na+ accumulation and higher K+ /Na+ ratios than durum wheat, making them potential sources for introgressing salt tolerance genes.

Kiani et al. [67] used a hyper-salt-tolerant genotype of Ae. cylindrica (USL26; [48]) to produce amphiploid with Chinese Spring or Roshan wheat. The two wheat x Ae. cylindrica amphiploids had, on average, 27% and 57% of the non-saline controls' spike number and shoot dry weight when grown in the field with irrigated water containing 250 mmol L-1 NaCl (final soil EC at harvest;15 dS m-1 ), and those of wheat parents were 47% and 68% of control, respectively. The amphiploid had lower [Na+ ], higher [K+ ] and consequently higher K+ /Na+ ratios in leaves and roots than the respective wheat parents [67]. The analysis of expression profiles of some Na+ or K+ transporters genes showed higher transcript levels of HKT1;5 and SOS1 in roots, and NHX1 in shoots and roots of amphiploid than wheat parents, grown in saline conditions. In addition, the amphiploid had higher activities of ROS-scavenging enzymes such as CAT and GPX [67].

Overall, inter-specific and inter-generic hybridization have improved wheat salinity tolerance. However, plant yields were compromised under control conditions in many cases, and the range of species suitable for these purposes is somewhat limited.

5.3. Transgenic approaches

Transgenic approaches involve transferring desired genes or gene combinations from one species to another to enhance salt tolerance. We identified 124 papers published since 2005 (Web of Science) that used transgenic approaches involving wheat, although most lack quantitative comparisons of growth or yield between transformed and parental lines under saline and nonsaline conditions. Many studies were conducted under nonTable 1 Amphiploids of wheat for higher salinity tolerance. physiological conditions such as sealed Petri dishes, with limited transpiration, affecting Na+ delivery to shoots [68]). However, several studies meeting the criteria of quantitative comparisons of growth or yield between transformed and parental lines under saline and non-saline conditions showed promising results (discussed below).

As specific Na+ toxicity is the main physiological constraint affecting crop performance under long-term salinity exposure [10,69], numerous attempts have been made to prevent Na+ accumulation in plant tissues. A key target has been the HKT1 gene, which encodes for a transporter that belongs to the TrK/Ktr/HKT family. This family is divided into two sub-families based on ion selectivity [70], with the transporters in sub-family I highly selective for Na+ , while those in sub-family II can transport Na+ or K+ [71]. In durum wheat, the TmHKT1;5-A gene is located in the Nax2 QTL, which lowers leaf Na+ levels by retrieving Na+ from the xylem [72,73]. Munns and co-authors showed that a nearisogenic line of durum wheat harboring TmHKT1;5-A increased grain yield by 25% under saline conditions [74]. Introducing the Nax gene into bread wheat varieties BARI-25 and BARI-26 through conventional crossing with marker-assisted selection decreased leaf [Na+ ], with 10%–20% higher yields in the field than wild-type (WT) plants [75] (Fig. 1B). However, a closer look at this data reveals that, despite these improvements, the grain yield of transformed plants under saline conditions remained 50% of the control. The reported beneficial effects were observed only in one (or three) tested field sides (see [74]), casting doubts on the applicability of this approach for field-grown plants, which is hardly surprising. While reducing shoot Na+ load may help plants avoid Na+ toxicity, they still need to deal with the osmotic component of the stress. With less Na+ available for osmotic adjustment, plants must invest in producing compatible solutes - a strategy with an extremely high carbon cost [76], compromising plant growth and grain yield. Thus, preventing Na+ delivery to the shoots may be good for plant survival under harsh conditions but not for sustainable yield.

Another attractive target is SOS1 gene that encodes of Na+ /H+ antiporter and is considered to be a major mechanism for Na+ extrusion from roots [12]. This transporter is located in the plasma membrane of epidermal root cells. Studies have shown that endogenous SOS1 overexpression reduced Na+ accumulation and improved salinity tolerance in transgenic Arabidopsis [77], Solanum lycopersicum [synonym Lycopersicon solanum] [78], and Nicotiana tabacum [79]; however, the methods in the latter two studies do not provide sufficient confidence to support the claims of improved salt tolerance [80]. There are also reports of improved salt tolerance in transgenic Arabidopsis [81,82] and tobacco [83] transformed with either TaSOS1 from bread wheat or TdSOS1 from durum wheat. However, there are no reports on transgenic wheat overexpressing SOS1. Only one study reports correlations between the upregulation of three homologs of SOS1 (TaSOS1-A, TaSOS1-B and TaSOS1-D) and salinity tolerance in natural and synthetic hexaploid wheat accessions and their diploid parent (Ae. tauschii) treated with 200 mmol L-1 NaCl for ten d, and that TaSOS1-D playing a prominent role in salinity tolerance in transgenic Arabidopsis [82]. However, these authors indicated that the heterologous expression of TaSOS1 in Arabidopsis may not fully state the function of TaSOS1 in wheat. It should be noted that the above approach comes with a major caveat: the SOS1 exchanger is expressed in the root epidermis and the xylem–parenchyma interface [84] where it operates in xylem Na+ loading. Thus, SOS1 gene overexpression will increase the Na+ extrusion rate by roots and its delivery to shoots. The outcome of this process remains unpredictable.

Vacuolar compartmentation of ions is crucial for tissue tolerance to salinity [10,12] and is a main strategy employed by halophytes to deal with high salt load [17].

Tonoplast Na+ /H+ antiporters encoded by NHX1 mediate this process, and numerous attempts have been made to improve vacuolar Na+ sequestration by expressing NHX1 in transgenic plants [85–87]. However, only one study appears to have assessed the performance of transgenic wheat overexpressing the vacuolar Na+ /H+ antiporter [88]; the two wheat transgenic lines transformed with NHX1 from A. thaliana and exposed to 150 mmol L-1 NaCl for 30 d in a glasshouse had, on average, 75% of the shoot dry weight of non-saline controls, and the WT had 39% of its control (Fig. 1B). These transgenic wheats had reduced leaf [Na+ ] (65%) but increased leaf [K+ ] (176%) compared to WT plants. Compared to WT plants, the lower leaf [Na+ ] in transgenic plants overexpressing the NHX1 gene may be due to Na+ accumulation in root cell vacuoles and, thus, a lower rate of Na+ export from roots [88]. The yield performance of the AtNHX1 wheat lines was also assessed under field conditions where the mean ECe of soil were 1.2, 10.6, and 13.7 dS m-1 . The transgenic lines produced larger grains under saline field conditions than WT plants, with 142% and 211% higher grain weights and yields, respectively, at a mean soil salinity of 13.7 dS m-1. .

Vacuolar Na+ sequestration also requires pumping H+ to create an electro-chemical difference across the membrane, to fuel NHX1 operation [17]. This process is mediated by two H+ pumps located in the tonoplast and driven by ATP (H+ -ATPases) or pyrophosphate (PPi) (H+ -PPase) [89]. Enhanced H+ pump activity is considered to improve salinity stress tolerance. Transgenic wheat overexpressing the V-H+ -PPase gene (SeVP1 or SeVP2) from the halophyte Salicornia europea grown in a solution containing 200 mmol L-1 NaCl for two weeks had greater shoot (33%–96%) and root (2–3-fold) dry weights (DWs) and significantly higher chlorophyll content than WT plants [90] (Fig. 1B).

Osmotic adjustment by accumulating compatible solutes in the cytosol is an important component of the adaptive mechanism, especially when plants cannot use Na+ for this purpose. In addition to osmoregulation, some compatible solutes, such as glycine betaine (GB), can protect complex proteins or mitigate ROSinduced damage during salt stress. Enhancing GB synthesis in wheat, a natural accumulator of GB, might increase salinity stress tolerance. Overexpression of the betA gene encoding choline dehydrogenase from E. coli increased shoot and root DWs by about 27% in two of three transgenic wheat lines grown under saline conditions (200 mmol L-1 NaCl for 12 d) compared to WT plants [91]. These transgenic wheat lines had two-fold higher GB contents and almost double the grain yield (g per plant) in field trials compared to non-transformed plants under moderate salinity (80 mmol L-1 NaCl). Unfortunately, the performance of WT plants and transgenic lines has not been assessed under non-saline conditions, so whether the yield penalty occurs in transgenic plants is unknown.

High salt concentrations in the soil reduce plant water availability or induce water backflow from roots to soil. Regulating proteins associated with water transport, such as aquaporins, can play a role in adapting to osmotic stress [92]. Durum wheat lines overexpressing the TdPIP2;1 gene exhibited improved growth under salt stress (150 mmol L-1 NaCl) compared to WT plants, but seed yield (expressed as weight per 30 grains) significantly decreased [93]. However, recent studies indicated that some aquaporins may also operate as non-selective cation channels [94,95], permeable to Na+ . Thus, overexpressing PIP genes could increase root Na+ uptake.

Other attempts to increase salt tolerance in wheat have investigated enhancing the expression of ROS-scavenging enzymes. Transgenic wheat lines with increased antioxidant activity through the overexpression of genes encoding superoxide dismutase (TaSOD2; [96]) or peroxidase (TaPRX-2a; [97]) improved salt toler ance of transgenic lines compared to WT plants. However, the above claims were based only on differences in shoot or root lengths of young hydroponically-grown seedlings under short salinity exposures, with no biomass or yield data. Given the important role of ROS in plant signaling cascades, such constitutively high antioxidant activity may not necessarily benefit overall plant performance.

Other transgenic approaches have focused on the overexpression of stress-related proteins, ubiquitin-related proteins, or transcription factor genes. Transgenic durum wheat lines with the AISP (stress-related protein) gene from the halophyte grass Aeluropus littoralis, encoding an A20/AN1 class zinc-finger protein, exhibited improved salt tolerance through continued growth and seed set in pots under glasshouse conditions. Salt-stressed (150 mmol L-1 NaCl) plants had about 30% lower seed weights than non-saline control plants, with no grain yield in salt-stressed WT plants [98]. The improved salt tolerance of transgenic durum wheat was associated with Na+ and K+ accumulation and partitioning in young and old leaves, with young leaves accumulating higher K+ but lower Na+ concentrations than old leaves under salt stress, while non-transformed plants had similar K+ but higher Na+ in young than old leaves [98].

Improved salt tolerance due to higher selectivity of K+ over Na+ also occurred in transgenic wheat overexpressing the wheat TaPUB1 gene encoding U-box E3 ubiquitin ligase [99]. Transgenic plants grown with 200 mmol L-1 NaCl for four weeks had about 30% higher net photosynthetic rates than WT plants, although quantitative growth data was not reported [99]. In another study, transgenic wheat overexpressing the TaMYB86B gene encoding R2R3-type MYB transcription factor and grown under 150 mmol L-1 NaCl had 120% greater fresh and dry weights than WT plants [100], with this difference attributed to decreased Na+ and increased K+ accumulation. However, as the stress duration was only one week, it remains to be seen if this difference will translate into substantial yield improvements.

In conclusion, wheat transformations have included genes involved in Na+ exclusion (HKT1;5, SOS1), vacuolar sequestration (NHX1, V-H+ -PPase), osmotic adjustment, ROS scavenging stressrelated proteins, ubiquitin-related proteins, and transcription factor genes. Various studies have demonstrated higher salttolerance potential in transgenic wheat lines than WT, with three reporting improved grain yields [88,91,98], of which two were under field conditions [88,91]. Transgenic technology provides opportunities for improving salt tolerance but requires enhancing expression of multiple genes involved in salt tolerance rather than single genes.

6. Conclusions and prospects

Salinity stress is a significant constraint for wheat growth and development, reducing grain yield and quality. Addressing salinity stress in wheat requires genetic engineering and inter-specific and inter-generic hybridization techniques. Numerous attempts have been made to increase wheat growth and yield by targeting various physiological and biochemical processes, such as vacuolar Na+ sequestration, Na+ exclusion, K+ retention, osmoregulation, and enhanced photosynthetic efficiency. Despite these advances, progress in developing salt-tolerant wheat cultivars remains limited. Past strategies for breeding wheat for Na+ exclusion have limited effectiveness within a narrow range of soil salinities and may lead to yield penalties under control or moderately saline conditions. A more promising avenue seems to be targeting tissue tolerance traits that use Na+ as an economical osmotic regulator while efficiently sequestering it in vacuoles. However, this approach requires a significant shift in plant phenotyping, transitioning from whole-plant to cell-based phenotyping platforms. Furthermore, a broader utilization of wild relatives in breeding programs should be encouraged to tap into their genetic diversity and enhance salt tolerance in wheat cultivars.

CRediT authorship contribution statement

Lukasz Kotula: Conceptualization, Writing – original draft, Writing – review & editing. Noreen Zahra: Writing – original draft, Writing – review & editing. Muhammad Farooq: Writing – original draft, Writing – review & editing. Sergey Shabala: Writing – original draft, Writing – review & editing. Kadambot H.M. Siddique: Conceptualization, Writing – review & editing, Project administration.

Declaration of competing interest

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Acknowledgments

This work was supported by Australian Research Council, Australia grants to Sergey Shabala and Kadambot H.M. Siddique.

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ARTICLE INFO

Article history:

Received 18 Sptember 2023

Revised 10 October 2023

Accepted 17 January 2024

Available online 3 February 2024

Footnote

* Corresponding author.

E-mail address: [email protected] (K.H.M. Siddique).

1 These authors contributed equally to this work.

References

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