Similar and divergent responses to salinity stress of jamun (Syzygium cumini L. Skeels) genotypes

Introduction

Salinity stress adversely affects most fruit crops (Denaxa et al., 2022; Niu, Davis & Masabni, 2019) which abruptly begin to decline due to cumulative impacts of osmotic and ion-specific stresses (Meir et al., 2014). While some fruit crops thrive under saline conditions, prolonged exposure to salt stress eventually suppresses their growth and yield (Rohit Katuri, Trifonov & Arye, 2019). Salt-induced lowering of soil water potential initially impairs root’s ability for water uptake, which then decreases the turgor pressure and hinders shoot and root growth (Zhao et al., 2020). Salt-stressed plants accumulate organic and inorganic solutes for osmotic adjustment (Ahmed et al., 2012). Stomatal closure brought on by osmotic stress diminishes the plant’s capacity to assimilate CO₂ (Chartzoulakis, 2011). Ionic imbalance, caused by the build-up of Na⁺ and Cl⁻, disrupts the metabolic processes by interfering with the activities of essential ions such as NO₃⁻ and K⁺ (Zhao et al., 2020). This eventually leads to the destruction of cell membranes, reduced activity of enzymes, decreased levels of chlorophyll pigments, and suppression of photosynthesis (Singh & Sharma, 2018). Reactive oxygen species (ROS) such superoxides and singlet oxygen build-up abnormally under saline conditions, leading to oxidative stress and lipid peroxidation (Singh et al., 2023). Plants activate their antioxidant defense to offset the negative effects of ROS (Moradbeygi et al., 2020).

Although intra-specific variation has greatly aided to the selection of salt-tolerant cultivars, the genetic diversity for salt tolerance within many species still remains underutilized (Ismail & Horie, 2017). Fruit crops exhibit distinct intra-specific variations when exposed to salt (Denaxa et al., 2022; Etehadpour et al., 2020). Identifying such genetic differences may lead to the use of salt-tolerant genotypes as rootstock for salt-affected soils. Comprehending the morpho-physiological mechanisms underlying salinity tolerance is also crucial for the development of appropriate breeding techniques aimed at enhancing salt tolerance (Ismail & Horie, 2017). Salt-induced changes in biomass allocation and ion uptake differ greatly among genotypes, and serve as valuable indicators for selecting the salt-tolerant cultivars (Kchaou et al., 2010). Genetic differences are known for salt-induced declines in biomass (Pandey et al., 2014; Kchaou et al., 2010). While salt stress typically restricts the availability of essential nutrients (e.g., K⁺ and Ca²⁺), their levels are little affected in certain genotypes (Mahouachi, 2018; Papadakis et al., 2018), probably because of preferential uptake (Liu et al., 2020).

Jamun (Syzygium cumini Skeels) is widely distributed in many Asian and African countries (Singh et al., 2019; Tewari, Singh & Nainwal, 2017). Various parts of the jamun tree are used as constituents in traditional medicine. Jamun fruit pulp and seed contain a variety of bioactive substances with strong anti-glycaemic, anti-oxidant and chemoprotective properties (Kapoor, Ranote & Sharma, 2015; Shrikanta, Kumar & Govindaswamy, 2015). Genetic variations for salt tolerance in Jamun remain elusive, notwithstanding some anecdotal evidence suggesting that this species flourishes under saline conditions (Sarvade et al., 2016; Tewari, Singh & Nainwal, 2017). This is because the prior salinity experiments in Jamun did not even examine the salt-induced changes in biomass allocation and ion partitioning (Chhabra & Kumar, 2008; Tomar et al., 2003). Jamun exhibits polyembryony, characterized by the emergence of more than one seedling from a single seed (Sivasubramaniam & Selvarani, 2012). Despite the fact that polyembryonic saplings may perform better under salt stress (Nimbolkar et al., 2019), the comparative reactions to salt stress of mono- and poly-embryonic seedlings of jamun are not known.

Considering the previously highlighted gaps in research, this experiment was conducted to assess the effects of incremental rise in salinity on biomass allocation and ion partitioning in 20 monoembryonic and 28 polyembryonic genotypes of jamun with the aim of identifying major traits that underpin salt tolerance. The polyembryonic seedlings were distinguished from the monoembryonic types on the basis growth habit i.e., the presence of multiple seedlings per seed. The shared and contrasting responses to salt of mono- and poly-embryonic seedlings were also examined. Six randomly chosen genotypes were additionally assessed to elucidate salt-induced changes in gas exchange attributes and antioxidant enzymes.

Materials and Methods

Results

Comparative responses under control and saline conditions

The polyembryonic types showed significantly higher LDM (t = 5.77, p = < 0.001) and PDM (t = 3.30, p = 0.001) than monoembryonic types under control treatment. Similarly, they also exhibited significantly higher (p < 0.001) LDM, RDM and PDM (16.53, 9.41 and 31.26 g/plant, respectively) than monoembryonic types (14.35, 7.97 and 27.31 g/plant, respectively) when treated with salt. Shoot: root ratio was significantly higher (p = 0.009) in monoembryonic types under salt treatment (Table S4; Fig. 1). Of the leaf ions, the two groups differed in Na⁺ only in control and in Cl only in salt treatment. Comparably, K⁺ and Ca²⁺ were different under both fresh and salt treatments. Leaf Mg⁺ did not differ significantly under both the conditions (Fig. 2A). Of the stem ions, differences between mono- and poly-embryonic types for Na⁺ were significant only in control and for K⁺ and Ca²⁺ in salt treatment (Fig. 2B). In roots, only Na⁺ (10.57%) and Ca²⁺ (32.50%) were significantly higher in polyembryonic types under salt treatment; the differences were non-significant for other ions under both control and salt treatments (Table S4; Fig. 2C).

Linear discriminant analysis

Table S6 and Fig. 3 display the results of LDA. The first two discriminant functions alone explained approximately 98.0% of the cumulative variance in the data, indicating that LDA efficiently reduced the dimensionality. We found that while LD-1 was mainly a construct of stem Cl⁻ and root Cl⁻, LD-2 had root Na⁺, leaf K⁺ and leaf Cl⁻ as the highly weighted variables. The differences between mono- and poly-embryonic types for stem and root Cl⁻ contents were non-significant under both control and salinity treatments. Conversely, the differences between two groups were significant for root Na⁺, leaf K⁺, and leaf Cl⁻ under salt treatment (Table S6). Therefore, we infer that while stem and root Cl^- contents accounted for shared responses, root Na⁺, leaf K⁺ and leaf Cl⁻ explained the divergent responses to salinity stress. A perusal of the LDA biplot showed that while LD1 effectively discriminated the control and salinity treatments, LD2 could fairly reasonably distinguish monoembryonic and polyembryonic types from each other (Fig. 3). The confusion matrix estimates for predicted group membership from LDA are presented in Table S7. The overall classification accuracy (jacknifed) of LDA was 80.21%. In the case of monoembryonic types, 25.0% and 18.75% of the instances were mislabelled as polyembryonic in the control and salt treatments, respectively. Similarly, 19.64% and 13.39% of the polyembryonic instances were incorrectly classified as monoembryonic under control and salinity treatments, respectively. Interestingly, a small proportion of the polyembryonic instances (3.57%) were also incorrectly labelled as monoembryonic in the salt treatment.

Discussion

Little is known about traits underlying salinity tolerance, as well as genetic variations for salt tolerance, in Jamun. In this backdrop, our study aimed to elucidate the effects of salinity stress on biomass allocation and ion uptake in 48 diverse genotypes of jamun. Similar and contrasting responses to salt stress of mono- and poly-embryonic seedlings were also analyzed to identify the mechanisms underlying salinity tolerance. In order to avoid salt shock, irrigation water salinity was increased gradually (Singh et al., 2024). Since ‘osmotic’ rather than ‘salinity’ stress usually triggers early reactions under saline conditions, long-term studies are probably more dependable for analyzing salt tolerance (Zhu et al., 2016). By using this method, we were also able in better mimicking the field conditions where salinity typically peaks in the summer (Sadder et al., 2021).The genetic variability for salt tolerance remains elusive in Jamun because prior studies had examined the effects of salt stress only on one genotype (Chhabra & Kumar, 2008; Patil & Patil, 1983; Tomar et al., 2003). Crop genotypes differ markedly in salt tolerance (Liu et al., 2020; Mousavi et al., 2019), suggesting that a sizeable number of genotypes must be evaluated to reasonably assess the variability for salt tolerance. We noticed substantial genotypic differences (p < 0.001) for salt-induced declines in the dry mass of different plant parts. In monoembryonic types, while CSJ-28 was most adversely affected, genotypes CSJ-5, CSJ-18 and CSJ-38 exhibited the lowest drops in leaf, stem and root biomass. Contrasting genetic variation was also observed within polyembryonic types for the reductions caused by salt in LDM, SDM and RDM. Salt stress suppresses the leaf area, damages the cell membranes, impairs the water relations, causes oxidative stress and hampers the photosynthetic assimilation, which then adversely impact plant growth (Gholami et al., 2023; Moula et al., 2020). The repressive effects of salinity on shoot and root growth vary with genotype in a particular fruit crop (Liu et al., 2020; Moula et al., 2020). We found that while several monoembryonic genotypes (e.g., CSJ-2, CSJ-18 and CSJ-28) showed comparable reductions, a few (e.g., CSJ-5, CSJ-12 and CSJ-45) showed higher decreases in root mass, and the remaining showed greater declines in shoot mass. Likewise, salt-induced reductions in root mass were either lower or substantially lower than decreases in shoot mass in most polyembryonic genotypes. Preferential allocation of biomass to shoots or roots may imply genotype-specific adaptations to overcome excessive salt accumulations. Maintenance of root biomass may also improve water and nutrient uptake in saline soils (Perica, Goreta & Selak, 2008; Rasheed, Bakhsh & Qadir, 2020).

Salinity (10.0–12.0 dS/m) had little effects on Na⁺, K⁺, Ca²⁺ and Mg²⁺ contents in jamun plants (Patil & Patil, 1983; Yousaf et al., 2020). In our study, leaf, stem and root Na⁺ increased to varying degrees, irrespective of the seedling type, following salt treatment. Salt-induced upticks in leaf Na⁺ (<100.0%) suggested comparatively efficient Na⁺ exclusion in some genotypes (mono: CSJ-2, CSJ-5, CSJ-29, CSJ-42, and CSJ-43; poly: CSJ-3, CSJ-6, CSJ-10, CSJ-13 and CSJ-26) (Brumos et al., 2010; Etehadpour et al., 2020; Mahouachi, 2018). Both mono- (20.55 and 27.37%, respectively) and poly-embryonic (22.19 and 19.36%, respectively) types showed relatively minor decreases in leaf, stem and root K⁺ under salt stress. Leaf K⁺ was hardly different between control and salinity treatments in some genotypes (mono: CSJ-4, CSJ-5, CSJ-28 and CSJ-32, poly: CSJ-13, CSJ-14, CSJ-22 and CSJ-23). Some mono- (CSJ-16, CSJ-28, CSJ-31, CSJ-35, CSJ-38 and CSJ-45), and poly-embryonic (CSJ-6, CSJ-11, CSJ-14, CSJ-25, CSJ-27 and CSJ-40) genotypes also exhibited strong capacities for Cl^- exclusion from leaves. Such variations in Na⁺, K⁺ and Cl⁻ levels in various plant parts were likely because of distinct ion uptake and partitioning mechanisms (Gengmao et al., 2015; Lovelli et al., 2012), and implied genotypic differences for ion retention in roots (Zarei et al., 2016). Despite significant increases in Na⁺ and Cl⁻, lack of salt-specific injuries in most genotypes suggested tissue tolerance to the excess Na⁺ and Cl⁻ (Munns et al., 2016). Salinity up to 12.0 dS/m did not cause leaf scorch and stem dieback in jamun plants (Patil & Patil, 1983). Pomegranate (Sun et al., 2018) and sapota (Kumar et al., 2023) plants did not exhibit leaf tip burn and marginal scorch even after extended exposure to salinity.

Ion translocation from roots to shoots may also account for genetic differences in salt tolerance (Khoshbakht, Ramin & Baninasab, 2015; Calzone et al., 2020). Under salinity stress, leaf Na⁺ was lower than root Na⁺ in certain genotypes (mono: CSJ-16, CSJ-21, CSJ-42 and CSJ-43; poly: CSJ-3, CSJ-8, CSJ-13, CSJ-14, CSJ-39 and CSJ-40). Similarly, leaf and root Na⁺ contents were fairly similar in some genotypes (CSJ-1, CSJ-11, CSJ-21, CSJ-22, CSJ-30 and CSJ-39) with relatively higher Na⁺ (>3.0 mg/g DW) in roots. Leaf Cl⁻ was also lower than root Cl⁻ in many genotypes (mono: CSJ-21, CSJ-28, CSJ-32 and CSJ-42; poly: CSJ-22, CSJ-23, CSJ-24, CSJ-25, CSJ-27, CSJ-39 and CSJ-40). Such genotypes seemed to limit Na⁺ and Cl⁻ translocation from roots to shoots (Musacchi, Quartieri & Tagliavini, 2006). Interestingly, leaf, stem and root Cl⁻ levels, regardless of the seedling type, were consistently lower than those of Na⁺ under salt treatment, implying a better efficiency for Cl⁻ exclusion (Mehdi-Tounsi et al., 2017). This may also enhance tolerance to salt since excessive Cl⁻ commonly causes leaf chlorosis and plant mortality, even when Na⁺ levels are low (Liu et al., 2018). Only a few genotypes displayed noticeable declines in leaf (mono: CSJ-2, CSJ-21, CSJ-29, CSJ-38; poly: CSJ-34, CSJ-41, CSJ-44) and stem (mono: CSJ-42, CSJ-43; poly: CSJ-10, CSJ-11, CSJ-17, CSJ-36, CSJ-37) K⁺ when treated with salt. Leaf K⁺ levels under the salt treatment were particularly little affected in genotypes with very high leaf Na⁺ (i.e., > 4.0 mg/g DW). Sufficient K⁺ levels could have enabled these genotypes in sequestering excess Na⁺ in vacuoles (Zarei et al., 2016). It also implied a strong selectivity for K⁺ uptake over Na⁺ (Musacchi, Quartieri & Tagliavini, 2006; Teixeira & Carvalho, 2009). Salt stress did not significantly alter leaf, stem and root Ca²⁺ in the monoembryonic types. Salt-triggered decreases in leaf Ca²⁺ were modest (~15.0%) in most genotypes. Similarly, while leaf Mg²⁺ slightly increased, the differences in stem and root Mg²⁺ were hardly discernible between salt-free and salt-stressed plants. In polyembryonic types, salt stress caused significant upticks only in stem and root Ca²⁺. Numerous crops preferentially accumulate Ca²⁺ and Mg²⁺, or at the very least, maintain their levels when exposed to salt (Liu et al., 2020; Mahouachi, 2018; Teixeira & Carvalho, 2009). Sufficient Ca²⁺ and Mg²⁺ levels improve the osmotic adjustment (Mahouachi, 2018). Adequate Ca²⁺ probably also enhances selective uptake of K⁺ over Na⁺ (Gengmao et al., 2015), and improves cell membrane integrity (Cimato et al., 2010). Interestingly, leaf, stem and root Ca²⁺ and Mg²⁺ were substantially greater than Na⁺ and Cl⁻ contents, irrespective of treatment and genotype, indicating that jamun plants can overcome nutritional constraints imposed by salt (Cuiyu et al., 2020; Liu et al., 2020).

When treated with salt, the polyembryonic types had significantly higher LDM, RDM and PDM than monoembryonic types. While the differences in leaf and stem Na⁺ were not statistically significant, polyembryonic types retained significantly more Na⁺ in their roots under salinity stress. Comparably, leaf Cl⁻ was noticeably lower in salt-stressed monoembryonic types. This suggested comparatively greater capacity for Cl⁻ exclusion in monoembryonic seedlings, and for Na⁺ exclusion in polyembryonic types (Dayal et al., 2014; Hussain et al., 2012). This is probably because different mechanisms regulate the absorption and partitioning of Na⁺ and Cl⁻ in salt-stressed plants (Saleh et al., 2008). Importantly, polyembryonic types maintained significantly higher levels of leaf K⁺, leaf Ca²⁺, stem K⁺, stem Ca²⁺, and root Ca²⁺ when exposed to salt. In addition to boosting osmotic adjustment (Mahouachi, 2018) and improving cell membrane stability (Cimato et al., 2010), this may have also restricted Na⁺ uptake (Gengmao et al., 2015), leading to higher leaf and root biomass in the polyembryonic types. Our results broadly concur with earlier findings in mango (Pandey et al., 2014) and citrus (Hussain et al., 2012), which suggest that polyembryonic genotypes may perform better under salt stress.

Correlation analysis indicated that Na⁺ probably had a stronger restrictive effect on leaf, stem and root biomass than Cl⁻, regardless of the seedling type. The greater inhibitory effects of Na⁺ on plant growth are known in citrus (Balal et al., 2012) and olive (Perica, Goreta & Selak, 2008). In our study, salt stress caused increased build-up of Na⁺ in different plant parts. Na⁺ transport in plants is primarily unidirectional with little recirculation from shoots to roots, which causes Na⁺ to gradually build-up in shoots. The higher Na⁺ levels then cause metabolic toxicity by competing with K⁺ in cellular functions (Tester & Davenport, 2003). Contrarily, phloem recirculation seems to limit Cl⁻ accumulation in aerial plant parts (Godfrey et al., 2019). Thus, we suppose that phloem recirculation might shield jamun plants against Cl⁻ toxicity (Brumos et al., 2010). Significant positive correlations between biomass attributes and leaf K⁺ implied that enhanced accumulation of K⁺ may contribute to osmotic adjustment (Pérez-Pérez et al., 2009) and facilitate sequestration of excess Na⁺ into vacuoles (Zarei et al., 2016). Approximately 98.0% of the cumulative variance in the data was described by the first two linear discriminant functions alone, demonstrating the robustness of LDA in reducing the dimensionality (Ye & Ji, 2010). Because the variables (stem and root Cl^-) loaded heavily on first linear discriminant function (LD-1) were not significantly different between both the groups under control and saline conditions, we assume that this discriminant function represented the shared responses to salinity stress. Similarly, we assume that LD-2 represented the divergent responses to salinity stress, since the major variables on LD-2 (root Na⁺, leaf K⁺ and leaf Cl⁻) significantly differed between the two groups under salt treatment. Earlier, the most significant features driving the responses of grape (Bari et al., 2021) and olive (Boshkovski et al., 2022) genotypes to salt stress were reliably delineated by discriminant analysis. In our study, the overall classification accuracy of LDA was 80.21%, quite similar to the values reported in Boshkovski et al. (2022).

Salinity-triggered declines in P_n varied between 20.47% (CSJ-18) and 83.37% (CSJ-1). Interestingly, the genotypes showing the largest salt-induced decreases in P_n (CSJ-1 and CSJ-13) also had the highest P_n rates in the absence of salt (Mousavi et al., 2019). The decreases in P_n also seemed to be largely independent of leaf Na⁺ and Cl⁻ contents. For instance, despite quite similar increases in leaf Na⁺ (109.16 and 97.89%, respectively), genotypes CSJ-18 and CSJ-42 showed remarkably different reductions in P_n (i.e., 20.46 and 58.37%, respectively). Similarly, genotypes CSJ-1 and CSJ-18 showed 83.38 and 20.46% decreases in P_n, respectively, despite salt-induced increases in leaf Cl⁻ being 19.23 and 80.77%, respectively (Alipour, 2018; Hussain et al., 2012). The tested genotypes showed varying degrees of reductions in transpiration rate (E) and internal CO₂ (C_i). Despite being crucial for regulating ion absorption, reduced transpiration can impede plant growth by lowering the photosynthesis (Negrão, Schmöckel & Tester, 2017). The activity of antioxidant enzymes also showed significant genotypic differences. For instance, while APX activity increased markedly (>70.0%) in response to salt in CSJ-18 and CSJ-19, it increased by only ~28.0% in CSJ-1. CSJ-1 also displayed the lowest (~8.0%) increase in CAT activity while it was the highest (63.48%) in CSJ-18. When exposed to salt, genotypes CSJ-18 and CSJ-19 also showed significantly higher levels of POX and SOD than other genotypes. This implies that jamun genotypes react differently in terms of antioxidant enzyme activity to oxidative stress brought on by salt (Ayaz et al., 2021; Singh et al., 2023). The antioxidant enzymes shield the salt-stressed plants from oxidative damage by detoxifying the ROS and regulating ROS formation (Moradbeygi et al., 2020), and are believed to be potential markers for identifying the salt-tolerant genotypes (Sorkheh et al., 2012). We noticed that genotypes CSJ-18 and CSJ-19 were particularly efficient in upregulating anti-oxidant enzymes. Certain genotypes are frequently better at fending-off oxidative damage caused by salt because they have stronger antioxidant defenses (Abid et al., 2020; Ayaz et al., 2021; Etehadpour et al., 2020). Genotypic variations in antioxidant activities can be attributed to their intricate expression (Racchi, 2013) and organelle-specific activities (Niu & Liao, 2016) within plant cells.

Conclusions

Our results demonstrated distinct genotypic responses to salt within both mono- and poly-embryonic types. The decreases brought on by salt in leaf, stem, root and whole plant biomass were relatively greater in monoembryonic than in polyembryonic types. Furthermore, most polyembryonic genotypes exhibited lower or much lower reductions in root than in shoot mass when treated with salt, suggesting that they might be more adept at absorbing water and nutrients in saline soils. Despite significant increases in Na⁺ and Cl^- in different plant parts, leaf tip burn and marginal scorch were not seen in the majority of the genotypes. While this raised the possibility of tissue tolerance, which assists in storing excess Na⁺ and Cl⁻ in vacuoles, we also assume that preferential accumulation of K⁺, Ca²⁺ and Mg²⁺ may have played a role in osmotic adjustment and decreased Na⁺ uptake. Discriminant analysis suggested that while stem and root Cl^- were likely responsible for the common reactions, root Na⁺, leaf K⁺ and leaf Cl⁻ accounted for the divergent responses to salt of the mono- and poly-embryonic types. Some polyembryonic genotypes (CSJ-7, CSJ-8, CSJ-14 and CSJ-19), found to be least affected by salt treatment, could be used as salt-tolerant rootstocks. All the genotypes evaluated by us, including the promising polyembryonic types which propagate true-to-type from seeds, are being maintained in a salt-affected field for further investigation and use. Future studies should aim at delineating the plausible factors accounting for tissue tolerance to excess Na⁺ and Cl⁻, and preferential uptake of K⁺, Ca²⁺ and Mg²⁺.

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