Cell Mutagenic Autopolyploidy Enhances Salinity Stress Toler-Ance in Leguminous Crops

1. Introduction

Leguminous crops of the Fabaceae family such as chickpea (Cicer arietinum), cowpea (Vigna unguiculata), kidney bean (Phaseolus vulgaris), lentils (Lens culinaris), pea (Pisum sativum) and soybean (Glycine max L.) are considered the most valuable grain crops playing a key role in the diet of many people throughout the world. Grain legumes, also referred to as pulses, serve as excellent sources of essential proteins, dietary fibre, carbohydrates, mineral nutrients, and phytochemicals with very minimum-to-no fat content [1,2]. These grain crops have formed the basis of early farming culture systems, together with cereal crops centuries ago [3]. They are harvested as both dry seeds and vegetables used for human nutrition and livestock feeding. Apart from their nutritional aspect, species of the Leguminosae remain ecologically dominant across many vegetations [4], fixing atmospheric nitrogen (N) through an association with symbiotic rhizobial bacteria [5] and reducing the need for inorganic N fertilisers. Consequently, whole genome duplication events during the Cretaceous-Paleogene around 66 million years ago played a significant role in legume survival and diversification. This evolutionary process transformed legume wild taxa to forms which enabled sustainable human gains through domestication and numerous breeding programmes [3].

The domestication and production of legumes through agriculture was crucial in safeguarding food and nutritional security while enhancing environmental nitrogen cycle in terrestrial ecosystems [6,7]. Although, N-fixing microbials in association with nodulating plant species mostly grow under arid conditions as companion or dominant species, the degree in which these plants, particularly, polyploidised legumes exhibited tolerance to abiotic stress factors such as drought and salinity stress still requires further investigations. According to Rao et al. [8], the variations in gene composition and expression patterns found in homologous polyploid plants resulted in different beneficial physiological responses enhancing tolerance and optimum growth and yield under abiotic stress conditions. The study indicated that autotetraploids of Lycium ruthenicum revealed an increased abscisic acid (ABA) content of 78.4% under both normal and severe drought stress conditions compared with their diploid counterparts.

Nevertheless, molecular, and physiological variations between polyploids and diploids were mainly related to the biosynthesis and signal transduction pathways of ABA. This sesquiterpenoid hormone plays an important role in many cellular processes such as seed development, dormancy, germination, growth, and development of plants, as well as stress responses, and is subjected to complex regulations that involve the actions of a small family of 9-cisepoxycarotenoid dioxygenase (NCED) genes that indirectly produce xanthoxin, the main precursor of ABA [9]. The new challenge, however, is to understand the regulation of these biosynthetic genes and their pathways which predominantly lead to the stimulation of ABA biosynthesis and signal transduction during salinity stress.

Several studies have investigated the effects of abiotic stresses in various crop species of legumes, especially in diploid lines than polyploidised genotypes. As such, more studies must unravel how ABA induces the expression of osmotic proteins, especially in increasing stress tolerance of plants from genome to translational level as alluded by Rao et al. [8]. Under salinity stress, plants generally experience reduced growth, impaired ion or osmotic homeostasis and disruptions of metabolic processes subsequently leading to losses in crop growth and yields. This stress serves as one of the major limiting abiotic factors in agriculture, threatening food, and nutritional security. The adverse effects of this stress constraint in plant cells, organs and whole plant level take place during any stage of growth, including germination, seedling development, vegetative growth, and reproduction of plants [10]. Furthermore, salinity stress largely influences legume growth and yield by disturbing photosynthesis, hormonal regulation, nutritional imbalances, cause ion toxicity and osmotic stress effects. As reported by Farooq et al. [11], unravelling the response mechanisms and tolerance, as well as available management strategies to this abiotic stress in grain legumes may help in understanding the performance of these plants under salinity stress.

In this paper, the metabolic and genomic effects of salt stress, including tolerance mechanisms of polyploidised plants, in contrast with their diploid counterpart are discussed. This review analysed current knowledge on the application of induced autopolyploidisation to enhance plants’ adaptive traits against salinity through a multigenic response involving various compatible solutes, osmolytes, ROS, polyamines, and antioxidant activity, together with genes encoding proteins involved in the signal transduction, regulation, and response mechanisms. Altogether, the review examined whether synthetic polyploids of grain legumes exhibited enhanced response mechanisms to prevent toxic accumulation of Na⁺ and Cl^- ion levels in the cell cytosol, through a more complete genomic and physiological understanding which remains a precondition for the development of new and improved salt-tolerant leguminous varieties.

2. Role of Autopolyploidy on Legume Response to Salinity Stress

Plant ploidy level, which refers to the number of copies of the entire genome in a plant cell has implications for both physiological and molecular responses to both biotic and abiotic stresses. This process involves additional rounds of one or more genome duplications without cytokinesis to generate polyploidised mutant plants. The ploidy level can also be carried through into the germ line (gametes) to form multiple generations of polyploids [12]. Naturally, autopolyploidy or allopolyploidy can take place due to incomplete meiosis through gametogenesis and formation of unreduced chromosome number by microtubule inhibition within diploid cells [13]. Both meiotic and mitotic cell errors occur spontaneously in plants through variable frequencies depending on the species. This is a widespread phenomenon, occurring naturally and very gradually in flowering plants, but can also be rapidly synthetically induced using mutagenic chemicals such as colchicine, oryzalin, epoxomicin and nitroxide as described in Figure 1 [14,15,16].

These antimitotic agents are widely reported and comparatively evaluated in literature for their efficiency in inducing chromosome doubling through in vitro and in vivo regeneration of varying plant species [17,18,19,20,21,22,23]. As indicated on Figure 1, the influence of these chemicals on the formation of more than one haploid genomes in a single cell nucleus of plants occur as a result of the interference with kinetochore-microtubule attachments or what is widely known as microtubule depolymerisation which is extensively reported [18,19,23,24]. However, during the production of synthetic polyploids, the efficiency of genome replication depends on the concentration of the antimitotic agent and the duration of pretreatment, especially when mutagenic chemicals such as colchicine and oryzalin are applied for DNA duplication [18,19]. If successfully achieved, the marked effects of genome doubling could ameliorate unfavourable external pressures such as drought and salinity stress, including deterring pests as well as viral and bacterial induced diseases.

According to Corneillie et al. [24], morphometric analysis of polyploidised Arabidopsis plants revealed that their epidermal tissues contained increased cell sizes and reduced cell number per mesophyll compared to diploid plants. In line with these observations, the various polyploidised varieties of Arachis, Glycine wightii, Medicago sativa and Phaseolus aureus also demonstrated morphological similarities to Arabidopsis polyploids [25]. In view of the abovementioned reports, many studies presented the idea that polyploids offer adaptive advantages to environmental stress than the diploid progenitors even if the mechanism underlying stress resistance remains elusive. This somewhat holds significant truth since some of polyploids frequently showed vigorous growth, higher yield, and resistance under stressful conditions than their parent diploid species. Tetraploid plants of Medicago truncatula Gaertn and Medicago scutellate (L.) Mill. recorded up to 100% greater autumn biomass and 58% larger seed weights compared to their diploid parent lines under field conditions [26].

The M. truncatula Gaertn and M. scutellate (L.) Mill are well known to produce high protein forage content for livestock and a significant fix of atmospheric nitrogen, especially under rainfed farming systems without severe exposure to drought, heat, or salinity stress [27]. However, it is still not yet clear, especially because of complex regulatory mechanisms involved in plant development and response to environmental stresses, whether there is a common set of physiological and molecular strategies that polyploidised plants use to mitigate against abiotic stress. Some of the physiological changes that have been observed in polyploidised mutant plants compared to diploid lines as illustrated on Figure 2 include increased adaptability to environmental stress. Rao et al. [8] and Tossi et al. [28] reported that the positive impact on plant growth and net yield production of crops are due to the diversification of gene functions accompanying this evolutionary process. As expected, chromosome duplication would trigger major physiological changes that lead to notable resistance to stress, including the changing of the morphology of plant organs that confers stress tolerance.

Figure 1. Chemical structures of naturally and synthetically produced mutagenic compounds that are used to achieve the multiplication of complete sets of chromosomes and production of random mutations in the genetic materials of plant species, giving rise to the birth of new varieties. These multiple sets of chromosomes and nucleotide alterations (particularly, substitution of guanine (G)- cytosine (C) to adenine (A) alkylation [G:C to A] induced by ethyl methane sulfonate), may take place in one nucleus, and can be stably inherited to progenies [12,14,16].

Figure 1. Chemical structures of naturally and synthetically produced mutagenic compounds that are used to achieve the multiplication of complete sets of chromosomes and production of random mutations in the genetic materials of plant species, giving rise to the birth of new varieties. These multiple sets of chromosomes and nucleotide alterations (particularly, substitution of guanine (G)- cytosine (C) to adenine (A) alkylation [G:C to A] induced by ethyl methane sulfonate), may take place in one nucleus, and can be stably inherited to progenies [12,14,16].

3. Physiological and Molecular Status of Leguminous Polyploids

In legumes, polyploidy serves as a major genetic driver in regulating growth and development processes, including the legume-rhizobium symbiosis which plays an important role on global nutrient cycle and diversification of plant species as previously mentioned [29]. Thus, the metaphase inhibition illustrated in Figure 2, which may be induced through efficient, reliable, low-cost, and safe pretreatments of plant materials such as cells, organs, or seeds with the different doses of mutagenic chemicals (also demonstrated on Figure 1) for chromosome doubling could result in altered gene transcriptional activities (Figure 2a–c). Accordingly, such interactions may have further consequences that include differential expression of common and distinct gene families depending on the strength and activity of promoter sequences. Some of these expressed genes may encode proteins that are responsible for significant notable physiological and phenotypic changes that enhance the resistance of leguminous plants to abiotic stresses such as salinity, drought, and high temperature. However, at this molecular level, salinity influences the expression of genes related to the biochemical parameters associated with salt tolerance. For instance, in non-leguminous species, such as Steria rebaudiana Bertoni (an important medicinal plant native to the northeastern Paraguay and southern Brazil), the overexpression of peroxidase encoding genes (POD4, POD6 and POD9) was reported [30].

The expression of these genes virtually influenced all metabolic reactions and physiological processes, specifically, the functional pathways, nutritional imbalances and hormonal regulations eventually getting all these impeded. Although, unlike the widely reported drought triggered effects on gene regulation and expression, the effects of salinity are more physiologically pronounced, involving major biochemical metabolite synthesis, enzyme induction and membrane transport than the upregulation and downregulation of plant genes. Razzaque et al. [31] reported allelic diversity at several genetic loci that have been found in genotypes that show tolerance to salt stress, influencing growth and nutritive value of leguminous crops. However, the marked physiological effects in polyploids because of genome doubling and altered gene functions revealed increased adaptability to environmental variability. Genome duplication may lead to the improvement and/or divergence of an entire duplicated pathway among others, resulting in noticeable changes on the physiological traits related to water use efficiency and photosynthesis. In Rao et al. [8], chlorophyll and hydrogen peroxide contents, together with abscisic acid (ABA) were used as physiological indicators to judge the response of polyploidised L. ruthenicum plants against environmental stress. Under reduced soil water content, which also led to osmotic stress from dehydration, the diploids showed inhibition of chlorophyll synthesis, and excessive increase in the concentration of hydrogen peroxide (H₂O₂). As indicated by Slesak et al. [32], H₂O₂ is increased during environmental stress, interacting with thiol-containing proteins, and activating the different signalling pathways, including transcriptional factors (TFs) involved in the regulation of gene expression and cell-cycle processes in plants.

Figure 2. Summary of polyploidisation events of genome in plants and their role in abiotic stress tolerance enhanced by chromosome duplication. (a) Genome reorganisation facilitating increased allo- or auto- polyploidisation, either leading to extensive gene duplication or fractionation (loss) following polyploidy induction, (b) DNA modifications that regulate whether genes are turned on or off after polyploidisation, reprogramming gene expression networks, and (c) ploidy-related modifications on gene structure and function as a result of the depolymerisation of microtubule activities causing great alteration in the expression patterns of RNA-mediated polypeptides (d, e), which are also influenced by external stimuli such as environmental stress (d) [18,21,32].

Figure 2. Summary of polyploidisation events of genome in plants and their role in abiotic stress tolerance enhanced by chromosome duplication. (a) Genome reorganisation facilitating increased allo- or auto- polyploidisation, either leading to extensive gene duplication or fractionation (loss) following polyploidy induction, (b) DNA modifications that regulate whether genes are turned on or off after polyploidisation, reprogramming gene expression networks, and (c) ploidy-related modifications on gene structure and function as a result of the depolymerisation of microtubule activities causing great alteration in the expression patterns of RNA-mediated polypeptides (d, e), which are also influenced by external stimuli such as environmental stress (d) [18,21,32].

4. Ionic and Osmotic Stress Signalling Responses in Polyploidised Plants

To cope with salinity stress, plants have developed several adaptive strategies such as enhanced enzymatic and physiological reactions, as well as higher levels of metabolite expression such as phenols that are involved in antioxidant activities of the plant. This stress affects vital metabolic functions whereby Na⁺ and Cl^- ions interferes with several nutrients such as those highlighted in Figure 3. Salt stress affects competitive nutrient uptake, accumulation, and transport in plants [33], consequently disturbing nutritional balance, hormone interactions, causing osmotic stress effects and specific ion toxicity [34]. In soybean, the ratio of gibberellin (GA) and abscisic acid (ABA) was negatively regulated by NaCl during seed germination and post-germinative growth [35]. This report further indicated that NaCl considerably down-regulated GA₁, GA₃ and GA₄, while regulating the ABA content. As outlined in Figure 3, high soil salt content affect crops’ growth and development processes by influencing water availability in the cells, ion toxicity, oxidative stress, cell membrane organization, meiotic/ mitotic processes and causing nutritional disorders, among others.

In terms of inorganic ions, essential elements (N, P, K, S, Mg etc.) are considered intrinsic components of the structure and metabolism in plants [6,39]. The severe abnormalities that may arise due to salinity stress will result in symptoms of specific nutritional deficiencies such as chlorosis and necrosis. It remains common knowledge that salt stress influences the transfer of inorganic nutrients from the environment into a plant [40], but further insights are still required on how nonspecific osmotic stress and specific ion effects are regulated at both physiological and molecular level. Even though, the mechanisms through which legumes with altered ploidy adapted to salinity stress still need to be investigated, insights on other non-leguminous crops demonstrated novel traits and changes in existing physiological processes because of polyploidization.

As previous reported, polyploidised mutants always showed improved biosynthetic, carbolic, and signalling transduction pathways under environmental stress as indicated in Figure 2 and Figure 3. In the general scheme of signal transduction, polyploidised plants would likely show rapid environmental and developmental signal perception by specialised receptors, and effective activation of signalling cascade involving secondary messengers leading to an efficient response by the plant cells (Figure 3) [31,38]. Part of polyploids success is signalling between plants and microbes during symbiotic nitrogen fixation (SNF) which influences microbial infection of plant cell, cell division leading to nodulation, autoregulation of nodule development and intracellular accommodation of bacteria, metabolism and transport supporting SNF to prevent nodule senescence [41].

Salinity stress increases the accumulation of reactive oxygen species (ROS), however, total phenolic content, ascorbic acid content and antioxidant activity traits were enhanced in 320 tetraploids compared to 84 diploid genotypes of Solanum tuberosum L. [42]. According to the study revelations, the concentration of antioxidant compounds correlated positively with the skin tuber colour and ploidy levels. In Citrus limon (L.) Osbeck, Bhuvaneswari et al. [43] indicated that cyclic monoterpene, limonene, had significantly increased in tetraploids induced by 0.025% colchicine for 24 hours. Limonene is biosynthesised from the precussor geranyl disphosphate (GPP) by enzymatic biotransformation with d-or-l-limonene synthetase. The production of limonene monoterpenes is completed by methyl group deprotonation in the α-terpinyl cation, and this occurs in a wide variety of species including Conifers, Lamiales and Pinales, of which some are tetraploids [44,45].

Moreover, overexpression of terpenoid biosynthesis gene in soybean influenced the nodulation signalling pathways. The results indicated that the six terpenoid synthesis gene (SoTPS6, SoNEOD, SoLINS, SoSABS, SoGPS, and SoCINS) in soybean hairy roots increased nodule number, nodule root length and fresh root weight [46]. As reported in other studies, the expression of terpene in the roots was highly coordinated and cell specific. The abovementioned observations demonstrated the role of ploidy on this largest class of specialized metabolites found within different structures in plants. According to Pott et al. [47], monoterpenes like limonene plays a key role in plant’s interaction with the environment, especially in protecting plants against biotic and abiotic stresses.

Generally, polyploid plants often have improved traits than their diploid relatives and may have greater advantage in regulating both osmotic and ionic stress. The improved traits or maintained homeostasis plays an important role in many of the functions occurring in polyploidised plant cells. The accepted explanation of the influence of salinity stress is that imbalances in ion content in plant cells affect plant fitness, especially by affecting plant nutritional status, cell water potential and cause toxic effects through accumulation of ions, which disturb nutrient acquisition and result in cytotoxicity [48,49]. Under the stress, polyploidised plants show increased capacity to maintain unequal concentrations of sodium (Na⁺) and potassium (K⁺) ions both inside and outside the cells. Such improved responses occur due to tolerance genes and TFs regulating ion Na⁺/K⁺ pump and exclusion, through selective permeability of plasma membrane proteins (PMP), high sodium affinity transporter (HKT) and Na⁺/H⁺ exchangers that help to alleviate ion toxicity in the cells [50,51].

Although, information regarding polyploidised leguminous crops’ reaction to toxic ions is very scant, monocotyledous crops such as hexaploidy wheat spp. (Triticum aestivum genome BBAADD) demonstrated improved ion toxicity alleviation than both diploid counterparts and tetraploid wheat progenitors (T. turgidum) or durum wheat (T. durum). In polyploidised rice (HN2026-4x and Nipponbare-4x), Tu et al. [52] reported improved regulation of Na⁺ content and H⁺ proton content flux at root tips, with decreased Na⁺ and increased H⁺ efflux in the roots. In legume, the physiological mechanisms explaining the improvement of salt tolerance with increasing levels of ploidy remains under researched. However, genome duplication has been widely suggested to improve salinity stress resistance through enhanced proton transport pump and homeostasis in various monocot and dicot vegetable crop species [51,52,53].

5. Metabolic Changes of Legume Polyploids Exposed to Salinity Stress

Salinity stress limits crop growth and metabolism, posing major impacts on agricultural productivity. As previously mentioned, over-irrigation systems and drought stress continue to cause oversalinisation in many areas used for planting of agroeconomically important legume crops. Salinity stress also known as hyperionisation as indicated previously by Gupta and Huang [54] pose serious effects on metabolic and growth processes of plants through excessive accumulation of ions as previously indicated. Numerous reports have elaborated on NaCl effects on growth, mineral composition, proline content and antioxidant enzyme activity, which dramatically reduces the productivity of many crops. As highlighted in Figure 3, this negatively influences energy production, utilization and storage in plants generating ROS, reducing cell growths, assimilates production, and causing membrane dysfunctions [55], consequently disrupting growth, reproduction, and yield in many crop species. Hamayun et al. [56] reported significant decreases in endogenous levels of secondary metabolites, GA, jasmonic acid, salicylic acid (SA) and ABA due to salinity stress. However, abrupt changes in the osmotic and ionic state of the plant causes negative effects on crop’s metabolism and growth.

These depressive effects of NaCl were also demonstrated in a previous study conducted in our laboratory (Figure 4) where diploid plants showed inhibition to soybean, cultivar LS678 and Peking growth and yield characteristics [57]. The soybean plants were subjected to two levels of salinity stress. Similar findings were earlier reported by Amirjani [58] and Hamayun et al. [56] in soybean, cultivar Hwangkeumkong. The observations made in Figure 4a indicated that Peking and LS678 soybean plants exhibited noticeable morphological variations, particularly, in their root and stomatal density compared to the marginal but significant reductions observed in shoot length, number of branches and leaf area. The salt-stressed plants clearly indicated that yield components in both cultivars were decisively determined by the stress (Figure 5, a), especially, flowering and the number of pods produced per plant. As a consequence of the stress, lower flowering percentages and pod numbers were recorded for LS678 and Peking at 40.1%, 13.4 and 35.5%, 11.8, respectively [57]. In contrast, polyploidised plants gave improved results of 64.9% of flowering and 37.38% pods per plant using 0.8% and 0.6% EMS, respectively in mungbean (Vigna radiata L.) according to Roychowdhury et al. [59].

Additionally, the mean number of seeds per pod and 100-seed weight were increased in all the mutagenic treatments involving gamma rays and EMS in mungbean, urdbean varieties T-9 and Pant U-30 [60]. These mutation-based improvements conferred increased genetic diversity required to achieve economically important traits in both leguminous and non-leguminous crops wherein the selected mutant lines successfully contributed to diversity of the crop’s genetic base which is highly required for growth improvement against environmental stresses such as drought and salinity. Furthermore, the genetic variability induced through mutagenesis in treated populations also demonstrated some level of genotype dependency as reported by Laskar et al. [61] in lentil (Lens culinaris Medik). The genome structure in polyploids also have implications for both physiology and stress responses during vegetative and reproductive growths. As reports showed that polyploidy has occurred at least once in angiosperm lineages, the significantly altered phenotypes often influence the interactions of polyploids towards both biotic and abiotic stress constraints. According to Forrester and Ashman [29], plant-biotic interaction involving legume-rhizobia mutualism serve as an important interaction which regulates nutrient cycle, in addition to its indirect impact on vegetative (number of trifoliate leaves, leaf size, shoot height, root length, branch number etc.) and reproductive growth parameters (flower number, fruit pods, seed number and 100-seed weight) as indicated in Figure 4a,b.

Figure 4. The reproductive yield (a) and vegetative growth (V) (b) changes caused by the increase in substrate salinity (NaCl) in soybean (Glycine max L. Merr.), cultivar LS678 and Peking [57]. Soybean plants were cultivated in 25 cm plastic pots containing pre-sterilised vermiculite, and then irrigated weekly with 250 mM NaCl from V2 until V3 stage under in vivo conditions. This study revealed that soybean plants subjected to salinity stress exhibited highly reduced growth [shoot length (SL), root length (RL), number of branches (NB), leaf area (LA), stomatal density (SD) and relative water content RWC)] and yield parameters [flower number (Flw), mean pods per plant (MPP), pod length (PL), pod weight (PW), number of seeds per pod (NSP) and seed yield (SY)], in addition to biochemical features such as photosynthetic pigments, carbohydrates, phenolic content, flavonoids, and antioxidant capacity as reported in this cited report [57]. .

Figure 4. The reproductive yield (a) and vegetative growth (V) (b) changes caused by the increase in substrate salinity (NaCl) in soybean (Glycine max L. Merr.), cultivar LS678 and Peking [57]. Soybean plants were cultivated in 25 cm plastic pots containing pre-sterilised vermiculite, and then irrigated weekly with 250 mM NaCl from V2 until V3 stage under in vivo conditions. This study revealed that soybean plants subjected to salinity stress exhibited highly reduced growth [shoot length (SL), root length (RL), number of branches (NB), leaf area (LA), stomatal density (SD) and relative water content RWC)] and yield parameters [flower number (Flw), mean pods per plant (MPP), pod length (PL), pod weight (PW), number of seeds per pod (NSP) and seed yield (SY)], in addition to biochemical features such as photosynthetic pigments, carbohydrates, phenolic content, flavonoids, and antioxidant capacity as reported in this cited report [57]. .

Although, some researchers found that the notion that autopolyploids are larger than their diploid progenitors does not always hold true, such as the comparison of Arabidopsis and Musa plants, wherein obtained variations brought from haploidy to diploidy showed increases in growth parameters, but then decreased with the increase in ploidy levels in both species. Such findings were reported by Corneillie et al. [62] in A. thaliana and Brisibe and Ekanem [63] in Musa species. Quantitatively and qualitatively studying these differences in mutant plants can allow scientists to infer the functions of duplicated genes or map certain mutations on the chromosomes. Furthermore, such studies must continue selectively breeding crops to produce varieties that have higher yields, or varieties adapted to specific environmental conditions and are resistant to plant pathogens. As it is apparent from Figure 4, polyploidisation could substantially improve tolerance of legumes to salinity stress even though, this may result to losses of certain important characters. Leguminous crops such as soybean, chickpea, dry bean, cowpea, lentil, and peas are important sources of proteins and oils, so their potential improvement via ploidy duplication must rather be associated with the improvement of these useful economic traits.