Sodicity stress differently influences physiological traits and anti-oxidant enzymes in pear and peach cultivars

Introduction

Presently, about one billion hectare global land area is affected by salinity and sodicity problems to varying extents (Hopmans et al., 2021). Out of this, ~76 million hectare area is impacted by the human-induced secondary salinization and sodification (Hossain, 2019). The severity of the problem is evidenced by the fact that approximately 1.5 million hectares of land become unsuitable for agricultural production every year because of salinity and related problems (Hossain, 2019). Available evidence suggests that salt sensitive horticultural crops including pear (Ahmad et al., 2022) and peach (Hernández et al., 2021) are particularly adversely affected under saline and sodic conditions. In contrast to the commonly accepted threshold of 8.5 for the pH of saturated soil paste (pH_s), pH_s >8.2 seems to be more realistic for classifying the soils as sodic under Indian conditions: soil pH_s of 8.2 is often associated with an exchangeable sodium percentage (ESP) of 15; a limit above which the soils are generally considered to be sodic (Abrol, Yadav & Massoud, 1988). Generally, soil pH_s of 8.5 roughly corresponds to ESP of ~50, high enough to suppress the crop growth (Sharma et al., 2016; Abrol, Yadav & Massoud, 1988). Although alkali salts such as sodium carbonate (Na₂CO₃) are often more detrimental to plant growth than the neutral salts such as sodium chloride (NaCl) (Yang et al., 2009), even in fairly salt tolerant crops (Abbas et al., 2021), alkali stress research continues to receive little attention. Sodic soils in the Trans-Gangetic Plains of India (study area) mostly have predominance of highly soluble Na₂CO₃ and NaHCO₃ salts, and are thus prone to the abrupt increases in the soil pH (Mandal, 2012).

In sodic soils, excessive Na⁺ causes the clay dispersion, surface crusting and deterioration in the soil physical properties (Qadir et al., 2007). Besides poor physical properties, high pH, osmotic and ionic stresses, and nutrient deficiencies are other limitations to plant growth in the sodic soils (Qadir & Schubert, 2002). Additionally, calcium carbonate (CaCO₃) concretions in the sub-soil also hamper plant establishment (Sharma et al., 2016). Considerable spatial variations in soil pH are also frequently seen in sodic soils; the more sodic parts of the field are often less congenial for crop growth (Samra, Singh & Sharma, 1988). In fruit crops, the adverse effects of sodicity stress on plant growth (Saxena & Gupta, 2006; Krishnamoorthy, 2009) are ascribed to altered plant water relations (Li et al., 2020), reduced levels of photosynthetic pigments (Krishnamoorthy, 2009; Li et al., 2020), lipid peroxidation and oxidative stress (Ahmad et al., 2014), and ionic stress (Singh et al., 2018b). Sodicity-stressed plants accumulate osmolytes such as proline for osmotic adjustment (Krishnamoorthy, 2009; Ahmad et al., 2014; Singh et al., 2018a), and also activate the antioxidant enzymes for scavenging the free radicals (Ahmad et al., 2014).

Although sodic soils display noticeable improvements in the physico-chemical properties following amendment application and salt leaching with the fresh water, such improvements are mostly transient and confined to the top soil (<15 cm) (Sharma & Singh, 2019) such that sub-soil constraints continue to persist (Kumar et al., 2019). Under such conditions, agronomic practices such as planting into amendment-treated auger-holes often give better results (Gill & Abrol, 1991; Saxena & Gupta, 2006). As a majority of fruit crops are mostly highly sensitive to salinity and sodicity stresses (Singh et al., 2018a), development of salt tolerant scion and rootstock cultivars is absolutely essential to sustain the fruit production in salt-affected soils (Mahmoud et al., 2020). Although salt tolerance is a complex polygenic trait, and is greatly influenced by the genetic and environmental cues (Flowers, 2004), there exists ample genotypic variation that needs to be explored for identifying the salt tolerant genotypes (Mahmoud et al., 2020; Singh et al., 2018a).

Although some studies have shown the adverse effects of salinity stress on tree growth and physiology of pears (Myers et al., 1995; Musacchi, Quartieri & Tagliavini, 2006; Oron et al., 2002) and peaches (Boland, Mitchell & Jerie, 1993; Karakas, Bianco & Rieger, 2000; Soliman, Abo-Ogiela & El-Saedony, 2017), their responses to sodicity stress remain elusive; excluding some preliminary observations that high soil pH and the related sub-soil constraints may suppress the plant growth (Elkins, Bell & Einhorn, 2012; Abd-Elmegeed, Nabeel & Nasr, 2013; Mestre et al., 2015; Tagliavini, Masia & Quartieri, 1995). However, the soil pH levels in these studies were rather low to draw reasonable inferences, and the plausible physiological changes accounting for the reduced plant growth were also not investigated. Plant responses to salt stress often strikingly vary with the experimental conditions; for instance, depending on experimental conditions, pears are either highly sensitive (Ebert, 1999) or moderately tolerant (Musacchi, Quartieri & Tagliavini, 2006) to the salinity stress. Interestingly, most of the aforementioned studies had used NaCl as the sole salinizing agent in relatively controlled short-term experiments; the results may be altogether different when a different salt is used to induce the salt stress (Grieve, Grattan & Maas, 2012). While an interplay among the oxidative stressors such as malondialdehyde, osmolytes, anti-oxidant enzymes and the leaf ions is known to greatly influence the plant responses to salinity stress in pears and peaches (Erturk et al., 2007; Dejampour et al., 2012; Wu & Zou, 2009; Yousefi, Naseri & Zaare-Nahandi, 2019), such responses remain uninvestigated under sodic conditions. Insights from other fruit crops (e.g., Malus halliana (Jia et al., 2019) and Morus alba (Hui-Hui et al., 2019)) also suggest that plant physiological responses to salinity and sodicity stresses remarkably vary with each other: the effects of salinity cannot be used as a reliable proxy for the effects of sodicity.

The subtropical areas in the Trans-Gangetic Plains of India (study region) usually have a frost free spring and adequate chilling availability for the cultivation of low chill cultivars of temperate fruits such as pear and peach. Pear cultivars Patharnakh (Pyrus pyrifolia (Burm F.) Nakai) and Punjab Beauty (Pyrus communis L.) (Singh et al., 2015), and peach cultivars Partap and Shan-e-Punjab (Prunus persica (L.) Batsch) (Bhatnagar & Kaul, 2002) are particularly suitable for cultivation in this area given their commercial potential, low chilling requirement and the ability to tolerate the high summer temperatures typical of the region. To our knowledge, systematic studies have not yet been carried out to evaluate the effects of sodicity stress on tree growth and physiological relations of pears and peaches. This study intended to delineate sodicity-induced changes in tree growth, leaf oxidative stress markers, proline, enzymatic anti-oxidants and leaf ions in pear and peach cultivars, and how these changes influence the overall cultivar-specific tree responses to sodicity stress.

Materials and Methods

Results

Principal component analysis

The principal component analysis (PCA) was quite efficient in reducing the dimensionality: the first two principal components (PCs) (Eigen value >1.0) alone explained 90.95% of the cumulative variance in data in pear (Table S2; Fig. 1), and 94.61% in peach (Table S2; Fig. 2). In case of pear, leaf Na⁺, K⁺ and Na⁺/K⁺ ratio alongside proline, APX and CV were the highly weighted variables on PC1; and TH, TCSA, HP, MDA and CAT on PC2 (Table S2). Likewise, for peach, TH, HP, proline and Na⁺/K⁺ ratio were the best represented variables on PC1, and TCSA, APX, POX, CAT and K⁺ on PC2 (Table S2). Interestingly, PCA achieved a clear-cut discrimination for the cultivar- and treatment-specific effects in data: while PC1 unambiguously distinguished the control and sodicity treatments, PC2 clearly separated the tested cultivars from each other (Figs. 1 and 2). The PCA also unveiled some interesting patterns in data. For pear, tight clustering of tree growth attributes, K and TC in the upper left quadrant implied a straightforward role of leaf K⁺ in osmotic adjustment in the sodicity-stressed pears (Fig. 1). Likewise, there was an apparent cultivar-specific upregulation in CAT and SOD activities in response to the oxidative (H₂O₂ and MDA) and ionic (Na⁺) stresses in Punjab Beauty trees; increased POX activity alone seemed to have alleviated salt stress in cultivar Patharnakh (Fig. 1). In the case of peach, leaf K⁺ did not have any obvious effect in alleviating the salt stress. Contrarily, osmoregulation through proline apparently increased in response to increasing HP, MDA and Na⁺ levels. Of the enzymatic anti-oxidants, APX and POX were rather specific to the cultivar Partap, and CAT and SOD to the cultivar Shan-e-Punjab (Fig. 2).

Correlation analysis

In pear, tree growth traits and total leaf chlorophyll (TC) had strong positive correlations with leaf K⁺ (r > 0.800, p = 0.000). Expectedly, leaf Na⁺/K⁺ ratio had moderate-to-strong negative correlations with TH (r = −0.623, p = 0.010), TC (r = −0.602, p = 0.014) and CV (r = −0.788 p = 0.000) (Table S3, Fig. 3). A strong positive correlation between leaf MDA and H₂O₂ (r = 0.914, p = 0.000) reflected their synergistic adverse effects on pear tree growth and physiology. Leaf proline had moderate positive correlations with MDA (r = 0.578, p = 0.019) and H₂O₂ (r = 0.519, p = 0.039). Similarly, strong positive relationships (r > 0.800, p = 0.000) were found between antioxidants (CAT and SOD) and oxidative stress indicators (MDA and H₂O₂) (Table S3, Fig. 3). In peach, leaf K⁺ had strong positive correlations with TC (r = 0.887, p = 0.000), APX (r = 0.732, p = 0.001) and POX (r = 0.791, p = 0.000), strong negative correlations with CAT (r = −0.987, p = 0.000) and SOD (r = −0.839, p = 0.000), and a moderate negative correlation with H₂O₂ (r = −0.655, p = 0.006) (Table S4, Fig. 4). In contrast to pear, elevated leaf Na⁺ had strong inhibitory effects (r > −0.880, p = 0.000) on tree growth in peach. The adverse effects of both MDA and HP on peach tree growth were also apparent (r > −0.800) (Table S4, Fig. 4).

Discussion

Sodicity stress is a serious constraint on pear and peach productivity (Elkins, Bell & Einhorn, 2012; Mestre et al., 2015). The observation that sodicity stress impairs pear and peach growth (Thind & Mahal, 2020) is anecdotal; key physiological traits underpinning sodicity tolerance remain elusive. In this backdrop, we aimed to understand the effects of sodicity stress on tree growth and physiological traits in commercially important pear and peach cultivars.

We found that peaches in general were more adversely affected than pears. Moreover, sodicity stress had a greater adverse effect on Patharnakh pear and Partap peach than other tested cultivars. The experimental soils used in this study still have pH >8.5, particularly below 30 cm depth. Additionally, a clayey sub-soil further limits the water flux and nutrient availability; hampering the plant growth (Kumar et al., 2019). This may partly explain the differential responses to sodicity stress of the crops and cultivar tested by us (Samra, Singh & Sharma, 1988). Excess salts suppress the vegetative growth and biomass production in pears (Matsumoto et al., 2006; Okubo, Furukawa & Sakuratani, 2000) and peaches (Massai, Gucci & Tattini, 1997; Massai, Remorini & Tattini, 2004); albeit in a genotype-specific manner such that some cultivars are more adversely affected than others (Krishnamoorthy, 2009). The depletion of leaf chlorophyll may be caused by the lime-induced chlorosis when pears (Ma et al., 2005) and peaches (Thomidis & Tsipouridis, 2005) are grown in the high pH soils. The sodic soils of the study area are often deficient in iron (Kaledhonkar, Meena & Sharma, 2019), a key element in chlorophyll synthesis. Furthermore, high soil pH may inhibit the uptake of Mg²⁺ and Fe²⁺ ions needed for chlorophyll synthesis (Jia et al., 2019). The presence of bicarbonate (NaHCO₃, predominant salt in the study area soils; Mandal, 2012) suppresses iron availability to the peach (Molassiotis et al., 2005) and pear (Valipour et al., 2020) plants. The increase in apoplastic pH in the presence of bicarbonates limits iron transport to the root stele and restricts its uptake (Molassiotis et al., 2005).

Sodicity stress triggered a significant increase in leaf MDA and H₂O₂ accumulation, the major indicators of oxidative injury (Sorkheh et al., 2012; Shen et al., 2021); regardless of the crop and cultivar tested. Nonetheless, the increases in leaf MDA in sodicity-stressed plants relative to controls were much higher in peach (~37.0–42.0%) than in pear (9.61–19.91%) as were the increases in leaf H₂O₂ (~22.0–27.0% and ~12.0%, respectively), suggesting that peaches in general were more adversely affected by the oxidative stress (Shahvali et al., 2020). Although comparative studies are lacking, the Prunus spp. including peach are mostly highly sensitive to the salt-induced oxidative stress and lipid peroxidation (Erturk et al., 2007; Shen et al., 2021; Toro, Pimentel & Salvatierra, 2021). Salt-induced osmotic and oxidative stresses more adversely affect osmotic-sensitive than osmotic-tolerant genotypes; the latter show relatively efficient reactive oxygen species (ROS) scavenging when exposed to these stresses (Rajabpoor et al., 2014).

Although direct evidence is lacking, insights from a related species (Pyrus betulaefolia Bunge) suggest that salt-triggered increases in leaf MDA may not be deleterious enough to cause the lipid peroxidation in pears (Wu & Zou, 2009). The overexpression of genes ‘Pp14-3-3’ (from P. pyrifolia (Burm F.) Nakai) and ‘PbrNHX2’ (from P. betulaefolia Bunge) dramatically improved the salt tolerance in transgenic tobacco and P. ussuriensis Maxim by upregulating the activity of the antioxidant enzymes (Li et al., 2014; Dong et al., 2019). Specifically, increased APX activity may account for a better redox homeostasis in pear; ‘14-3-3’ proteins interact with APX for scavenging the reactive oxygen species implicated in the oxidative damage (Li et al., 2014). Our results showed that sodicity-induced increases in APX activity were noticeably higher in pears (28.44–34.49%) than in peaches (13.49–29.41%). The cultivar differences for both MDA and H₂O₂ were highly significant (p < 0.001) in pear (Wu & Zou, 2009), but only weekly significant (MDA: p = 0.07, H₂O₂: p = 0.002) in peach (Toro, Pimentel & Salvatierra, 2021); implying quite distinct and somewhat shared responses to sodicity stress of pear and peach cultivars, respectively, in terms of lipid peroxidation. Genotypic differences for these oxidative stress markers are either pronounced or subtle in different Pyrus and Prunus species (Sorkheh et al., 2012; Rajabpoor et al., 2014; Zarafshar et al., 2014; Li et al., 2015; Toro, Pimentel & Salvatierra, 2021).

Salt-stressed plants accumulate compatible osmolytes such as proline that, in addition to osmoregulation, also reduce the cellular damage by scavenging the ROS (Dejampour et al., 2011; Rahneshan, Nasibi & Moghadam, 2018; Yousefi, Naseri & Zaare-Nahandi, 2019). The tested cultivars did not differ significantly in leaf proline under both control and sodic treatments (Wen et al., 2011). Furthermore, the increase in leaf proline in response to MDA and H₂O₂ accumulation was moderate in pear (r = 0.578 and 0.519, respectively; Fig. 3) but quite strong in peach (r = 0.870 and 0.930, respectively; Fig. 4). Leaf proline levels may not always be sufficient enough to induce the osmotic adjustment (Larher et al., 2009; Singh et al., 2022; Wen et al., 2011). Despite being a typical osmolyte, proline may not essentially lessen the osmotic potential of pear leaves (Larher et al., 2009), and other organic osmolytes such as glycine betaine may also be involved in the osmotic adjustment. The fact that MDA and H₂O₂ levels may not necessarily be toxic enough to upregulate the proline activity (Wen et al., 2011) also supports our findings as leaf MDA and H₂O₂ were two-three folds higher in peach than in pear (Table 2). Plausibly, a lower than expected increase in proline activity (Regni et al., 2019) might reflect higher sodicity tolerance in pear than in peach (Ebert, 1999; Musacchi, Quartieri & Tagliavini, 2006).

All the tested cultivars displayed increased activities of leaf antioxidant enzymes in response to sodicity stress. Enzymatic anti-oxidants efficiently protect salt-stressed plants from ROS induced oxidative stress, and are considered to be the reliable markers for discriminating the salt-tolerant and salt-sensitive genotypes (Sorkheh et al., 2012; Yousefi, Naseri & Zaare-Nahandi, 2019; Aazami, Rasouli & Panahi, 2021). The cultivar differences for anti-oxidant activities (Regni et al., 2019) can be explained by the complex nature of anti-oxidant expression in plant cells (Racchi, 2013) and the cell organelle-specific activities of anti-oxidant enzymes (Niu & Liao, 2016). The P. pashia rootstock was found to better protect the Flemish Beauty scions than clonal (Quince A and C) rootstocks against oxidative damage via enhanced CAT, POX and SOD activities (Sharma & Sharma, 2008). Similarly, peach seedling and clonal rootstocks differed considerably in leaf antioxidant levels in the presence of NaHCO₃ (Molassiotis et al., 2005). The SOD constitutes the first line of defense in alleviating the oxidative stress in plants; it dismutases the superoxide anion (O₂^–) to produce molecular oxygen (O₂) and H₂O₂. The CAT then decomposes H₂O₂ into O₂ and H₂O (Cavalcanti et al., 2004). Obviously, a balance between SOD and CAT activities and not their relative levels per se, seem crucial to maintaining the O₂^– and H₂O₂ levels in a steady-state (Azarabadi et al., 2017). The CAT and SOD activities were not only much higher (Table 3) but also had a clear synergistic effect (Figs. 1 and 2) in the sodicity-stressed Punjab Beauty pear and Shan-e-Punjab peach; enabling them to better adapt to the sodicity stress (Sorkheh et al., 2012). The decreased activity of CAT often comes at the expense of greater oxidative damage-characterized, for instance, by the increased accumulation of H₂O₂ (Molassiotis et al., 2005).

Sodicity-stressed pear and peach trees had significantly higher leaf Na⁺ and lower K⁺ than controls. In the sodic treatment, Punjab Beauty pear showed considerably lower increase in leaf Na⁺ than Patharnakh; helping it maintain a higher leaf K⁺. A more or less similar response was also seen in peach. Restricted translocation of Na⁺ to aerial plant parts (Matsumoto et al., 2006), achieved for example by Na⁺ exclusion in common pears (Musacchi, Quartieri & Tagliavini, 2006) prevents the xylem loading and translocation of Na⁺ to the leaves. The differential accumulation of leaf Na⁺ and K⁺ in response to the salt stress has also been observed in both own-rooted and grafted peaches (Massai, Gucci & Tattini, 1997; Massai, Remorini & Tattini, 2004) and interspecific Prunus hybrids (Dejampour et al., 2011), with low Na⁺ accumulators showing better salt tolerance (Massai, Gucci & Tattini, 1997). Reduced accumulation of leaf Na⁺, achieved by root exclusion (Musacchi, Quartieri & Tagliavini, 2006) or partitioning into the basal leaves (Massai, Remorini & Tattini, 2004) together with the maintenance of adequate leaf K⁺ (Massai, Remorini & Tattini, 2004) improves the salt tolerance.

In this study, the PCA was highly efficient in reducing the dimensionality, and in differentiating the cultivar- and sodicity-specific effects in data. Specifically, PCA delineated the putative traits linked to sodicity stress tolerance in the pear and peach cultivars. Previously, PCA has been used to unveiling the key responses to the salt stress in other fruit crops (Sorkheh et al., 2012; Abid et al., 2020). Multivariate techniques such as PCA are usually more suitable for detecting the key patterns in data having complex (multicollinear) variables (Julkowska et al., 2019). Additionally, graphical visualization of PCA loadings provides an easier and intuitive means to understanding the shared and contrasting physiological responses to the salt stress (Sorkheh et al., 2012; Singh et al., 2022). Based on the correlation analysis, MDA, H₂O₂ and leaf Na⁺ were found to have a greater repressive effect on tree growth in peaches than in pears. Furthermore, a strong correlation between leaf K⁺ and growth traits and leaf chlorophyll in pear, but not in peach, was indicative of leaf K⁺ mediated osmotic adjustment in pears.

Conclusions

Although sodicity stress suppressed tree growth regardless of the cultivar, strong genotypic differences were quite apparent: Punjab Beauty pear and Shan-e-Punjab peach exhibited better tolerance to sodicity stress. We found that sodicity-triggered increases in leaf malondialdehyde, hydrogen peroxide and Na⁺ had a greater repressive effect on tree growth in peaches than in pears, and induced the proline-mediated osmotic adjustment in the former. The higher activities of catalase and superoxide dismutase enzymes coupled with restricted Na⁺ uptake and the maintenance of adequate leaf K⁺ are the plausible explanations for overall better sodicity tolerance in pear. Further studies should aim to understand the effects of sodicity stress on other biochemical and transcriptional changes, and how such changes influence the fruit yield and quality in the sodicity stressed pear and peach cultivars.

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