Functional traits of okra cultivars (Chinese green and Chinese red) under salt stress

This experiment was conducted in a greenhouse at Jiangsu University, Zhenjiang, Jiangsu, China (32.20ºN, 119.45ºE) from April to June 2017. Okra (Abelmoschus esculentus L.) was studied in a greenhouse maintained at a temperature of 28/22 ± 5ºC day/night, 60% relative humidity and 16 h photoperiod throughout the experimental period. Seeds of two okra cultivars, namely Chinese green (var. Blondy) and red (var. Red Velvet) were bought from the open market and were germinated in seedling trays containing a vermiculite, perlite and peat moss mixture with the ratio 1:1:1 (v/v/v). Germination trays were placed in an incubator with the controlled temperature of 25 ± 5ºC during day and night. Relative humidity was 60%, and artificial light was installed on the roof of the incubator, operating 12 h over the day time. During the germination stage, the seeds were watered daily with full strength of Hoagland solution (Hoagland and Arnon, 1950). After 2 weeks, when the seedlings had grown enough, we transferred them into plastic pots having four drain holes at the bottom with 9 cm inner diameter and 12 cm height containing vermiculite as a growing medium. These plants in pots were placed under a small tub, where they received 1 L of nutrient solution of every treatment. On every alternating day, we drained all the remaining nutrient treatment solution in the tub to avoid salt accumulation and added new solution of every treatment. Four treatments of saline irrigation were made, adding 1:1 ratio of NaCl and CaCl₂ into full-strength Hoagland solution to get the following treatments: T1 (20.8 mM), T2 (103.3 mM), T3 (180.0 mM) and T4 (257.0 mM) (Table 1).

The experiment was arranged in a complete randomised block design with four replicates of each treatment for every week, with 384 pots (4 × 2 × 4 × 12). One week after transplanting, every plant was treated with the corresponding saline water treatment; plants were exposed to saline irrigation for 12 weeks. After the end of every week, the plants in each treatment were randomly selected to measure the gas exchange and growth parameters with four replicates. After measuring these parameters, the plants were harvested by removing all vermiculate particles very carefully with the help of water to avoid root and shoot damage. Subsequently, the root length was measured with a measuring scale; then, the fresh weight of the plant was measured using a weighing balance. These plants were oven-dried at ≤80°C for 48 h (Azeem et al., 2020) to measure the dry weight.

The statistical analysis of the data collected for the growth and gas exchange parameters was performed in different steps. Assumptions of parametric statistics were tested to verify normality and homogeneity of variance using the Shapiro–Wilk normality test and the Levene test before further analysis. Two-way analysis of variance (ANOVA) was performed between salinity levels and salt duration, and the means of the results were compared by using the Tukey test at 95% probability levels. Then, three-way ANOVA was used to compute the differences in the measured response variables among cultivars, salinity levels, salt durations (weeks) and their interaction effects. Partial correlation was used to determine the relationship among salinity, salt duration and physiological traits. All statistical analyses were performed using SPSS 22, and graphs were drawn on OriginPro 9.0.

All physiological parameters of both cultivars (Chinese green and Chinese red) were significantly different among cultivars, salinity levels, salt durations and their interactions (Table 2). We found that P_N of both Chinese green and Chinese red decreased significantly in every salinity level; the reduction was increasingly evident in every level of salinity with increase in the duration of salinity (Figure 1). At T2, the P_N of both cultivars was non-significant at Weeks 1–4 than in Weeks 5–8. With the T3 treatment, both cultivars showed different results according to their tolerance ability; Chinese green maintained non-significant P_N from Week 1 to Week 5 than in Weeks 6–8, and the remaining 4 weeks also yielded non-significant results. The same trend was shown by Chinese red for the first 5 weeks, but at Weeks 6–12, the P_N was maintained, showing more tolerance than Chinese green (Figure 1). Both cultivars tolerated T4 salinity till the third week; after that, the plants were under severe stress and could not maintain reasonable P_N value. The interaction effect of salt durations and cultivars was non-significant because the plants’ maintenance of P_N continued for 3–4 weeks (Table 2 and Figure 1).

Figure 1

Stomatal conductance (Gs) of both cultivars was significantly different among salinity levels, cultivars, salt durations and their interactions (Table 2). The trend of Gs for both cultivars was the same as for P_N. The Gs was reduced more with increase of salinity level and salt duration (Figure 1). Chinese green, at T2, maintained the Gs from Week 1 to Week 6, which then decreased from Week 7 to Week 12. At T3, the Gs level was maintained between Week 1 and Week 3, then decreasing at the fourth week compared with the first week. At T4, after Week 2, the Gs level decreased greatly, showing that the plants were under severe salt stress and found it difficult to survive. For this variable, the Chinese red cultivar showed a different response from that of Chinese green due to its tolerance ability. The Gs was reduced at T2 salinity, but differences were non-significant throughout the whole salt stress period under T2. A non-significant reduction in Gs was observed at T3 salinity, during the period between Week 1 and Week 8, but after this period, a significant reduction was detected. After Week 3, the Gs under T4 decreased drastically, showing a severe salt stress effect on the plants (Figure 1).

Significant differences in the transpiration (E) of both cultivars were detected among salinity treatments, salt durations, cultivars and their interactions (Table 2). The value of E of both cultivars under different salinity levels decreased with longer salt stress durations (Figure 1). These decreasing E values indicated that at the beginning of salt stress, the plants tried to withstand that stress and maintained a constant rate, but when the salt stress duration became longer, the plant's tolerance ability was affected and the E value increased (Figure 1). Chinese green showed increasing E after Weeks 8, 5 and 4 at salinity levels T2, T3 and T4, respectively. Chinese red reflected increases in E after Weeks 10, 6 and 3 at salinity levels T2, T3 and T4, respectively. At the same stage, the value of E increased in both cultivars after Week 8 in every salinity level, suggesting that both cultivars were under high salt stress, losing their ability to use the water for their development, with consequent water losses due to continuous transpiration.

Significant differences in water use efficiency (WUE) were detected between cultivars, salt durations, salinity treatments and their interactions (Table 2). Nevertheless, salinity treatments and cultivar interactions were non-significant. The WUE was calculated with the help of P_N and E; so, according to these parameter results, the WUE was also affected with respect to salt durations and salinity levels. At T2, T3 and T4, Chinese green WUE increased at Weeks 7, 5 and 4; subsequently, the WUE was decreasing because at that stage, the value of E was increasing. The WUE of Chinese red also decreased with longer duration of salt stress, but Chinese red showed more tolerance compared with Chinese green. At T4, T3 and T2 salinity levels, the WUE of Chinese red decreased at Weeks 9, 5 and 4. When the WUE started to decrease, the results were similar and non-significant for both cultivars; such condition reflected the clear tendency of being under a severe stress (Figure 2).

Figure 2

The water potential (Wp) was significantly different between cultivars, salinity treatments, salt durations and their interactions (Table 2). The Wp of both cultivars between salt stress durations and salinity levels was significantly decreased (Figure 2). At T2, T3 and T4, the Wp values were non-significantly increased in Weeks 8, 6 and 2, respectively. After this period, the results were also non-significant in the remaining weeks, indicating that at the first stage, the plants bear these stresses and maintain good water status in the plant leaf, which helps the plant to develop in growth (Figure 2). Thereafter, the plants lose their withstanding ability and continue to lose water in the form of E, and only salts are present in the plant leaf tissue, which shows decreasing water potential. Similar results were shown by Chinese red and Chinese green under these salinity levels, but Chinese red tried to bear these salinity treatments 1 week longer in every treatment level compared with Chinese green.

The main effects of cultivars, salinity treatments, salt durations and their interactions were significant in terms of most of the growth parameters (Table 3). Plant height (PH) increased smoothly under the control condition for both cultivars (Tables 5 and 6). Salinity levels and salt durations affected growth differently in both cultivars. At T2, the PH of Chinese green increased significantly from Week 1 to Week 8, and after 8 weeks, the PH was non-significant. Chinese green showed the same trend: at T3 and T4, the PH increased significantly from Week 1 to Weeks 5 and 3, respectively, and, thereafter, stayed non-significant for the remaining duration of salt stress. At T2, the PH increased significantly every week, indicating that Chinese red can bear the salt stress level even with significant reduction in growth, as compared to the control (T1). At T3 and T4, the PH was increased significantly from Week 1 to Weeks 5 and 4, respectively. Subsequently, there was a non-significant increase in the PH for the remainder of the salt stress duration (Table 6).

The stem diameters (SD) of both cultivars were significantly affected by cultivars, salinity treatments, salt durations and their interactions (Table 3). The SD values of both cultivars were decreased significantly as compared to the control (T1). The cultivars’ effect on SD was found to be non-significant, indicating that both cultivars behave the same under these salinity treatments. At T2 and T3 treatments, both cultivars showed significant increment in the SD from Week 1 to Weeks 8 and 5, respectively; thereafter, there was an increment but this was non-significant, indicating greater demonstration in plant growth. The SD for Chinese green significantly increased from Week 1 to Week 4 (Table 5) and, for Chinese red, from Week 1 to Week 3 (Table 6); subsequently, both cultivars showed non-significant increment.

The root is the main part for plant growth because it gets connected first with the salt water; it then transfers water to the stem of the plant. The root lengths (RLs) of both cultivars were affected significantly by salinity treatments, salt durations, cultivars and their interactions (Table 3). The RL significantly decreased in all salinity treatments of both cultivars (Tables 5 and 6). The RLs of Chinese green at T2, T3 and T4 increased significantly from Week 1 to Weeks 8, 6 and 3, respectively. The RL of Chinese red, at T2, T3 and T4, increased significantly from Week 1 to Weeks 8, 5 and 3, respectively.

The fresh weight (FW) traits differed significantly among salinity treatments, salt durations, cultivars and their interactions (Table 3). The FWs of both cultivars were decreasing every week with respect to the control (Figure 3). In the seventh week, >50% reduction was found for Chinese green with treatment levels T2 and T3 compared with the control. For individual levels, the T2 FW was increasing slowly throughout the whole salt duration period. At treatment levels T2 and T3, the FW of Chinese green increased significantly from Week 1 to Weeks 7 and 5, respectively. After that, the FW under these treatments decreased significantly until the harvest (Figure 3). Chinese red also showed reduction in FW in every treatment when plotted against salt duration as compared to the control treatment.

Figure 3

Chinese red showed a similar trend as Chinese green with the salinity level T2. At T3 salinity level, the Chinese red FW increased significantly between the first and the fourth weeks. Thereafter, during Weeks 4–8, the increment was non-significant; in the remaining 4 weeks, constant FW was found, showing the tolerance ability of Chinese red. At T4, the FW increased tremendously from Week 1 to Week 4; then, the FW decreased throughout the remaining salt duration period, which implies that the plant was under severe salt stress, losing its original weight, with eventual lethality.

The dry weight (DW) of both cultivars was significantly changed with respect to individual factors, but their interactions were non-significant (Table 3). Both cultivars showed reduced DW at every salinity level, as compared to the control (T1). At T2, both cultivars’ DW increased significantly between these levels throughout the salt duration period. At T4 level, both cultivars’ DW decreased after 4 weeks of treatment and continued to decrease until the harvest. At T3, Chinese green DW increased from Week 1 to Week 7, and Chinese red DW increased from Week 1 to Week 11 significantly, which also indicated that Chinese green is sensitive in nature and Chinese red has the ability to bear higher stresses for long durations.

The partial correlation coefficients for the relationship between different parameters of both okra cultivars are shown in Table 4. In both cultivars, a significant positive correlation between gas exchange parameters and growth parameters on the one hand and a negative correlation with the salinity treatment levels on the other hand were observed. The duration of the salt stress period reflected a positive significant correlation with the growth parameters in both cultivars, but it was negatively correlated with Wp and WUE. The negative correlation between water status parameters and salt duration explains that continually receiving higher salts in the nutrient solution over a long duration causes osmotic pressure, which creates a negative impact on plant water status.

Screening of crops against salt stress treatments is considered of high potential value under the water-saving irrigation perspective (Munns et al., 2012). The studied cultivars reflected a high variation in salt tolerance, according to their physiological and growth-related responses to salt stress and duration of the salt stresses.

Photosynthesis, known as one of the most complex and fundamental physiological processes, could be severely affected by salt stress in several crops (Qiu et al., 2017; Allel et al., 2018). A large variation in P_N was observed in our study with respect to the salinity levels, salt durations and their interactions (Table 2). It seems that the photosynthetic apparatus of both okra cultivars could tolerate all salt stress treatments for different salt durations (Figure 1). Both cultivars tolerated salt stress at T4 for only the first 3 weeks; afterwards, a continued decrease of gas exchange activities was observed but it was also tolerated. At T2 and T3, the Chinese red cultivar tolerated salt stress from Week 1 to Week 8 or 9, respectively, and Chinese green, from Week 1 to Week 5 or 6, respectively, maintaining adequate gas exchange parameters (Figure 1). Saline irrigation may limit the P_N activity by a reduction of CO₂ supply, arising from a partial closure of stomata or by altering the biochemical CO₂ fixation mechanism or by both procedures at once (Senguttuvel et al., 2014). The reduction in CO₂ availability due to the limitation of stomatal diffusion is considered as the starting effect of salt stress on P_N, which is caused either by a decline of guard cell turgidity or by abscisic acid accumulation, mediating stomatal closure (Chaves et al., 2009). We found high variability in salt tolerance of okra cultivars in terms of stomatal conductance depending on salt stress levels and their durations. Accordingly, we conclude that the change in P_N appeared to be associated with stomatal conductance. The state of the stomatal aperture is a compromise between water loss and assimilation of atmospheric CO₂. In this study, stomatal closure was a response to reduce water loss through transpiration, trying to maintain an adequate water status within plant cells and maintaining reasonable WUE. At T2 and T3 levels, Chinese green and Chinese red maintained WUE from Week 1 to Week 9 or 8, respectively, and Week 4 or 5, respectively. Afterwards, the value of E increased and P_N decreased, which affect the WUE because WUE has a strong relationship with them (Table 4). Increase in E indicated that the plant was under severe salt stress and was not able to maintain its growth due to continuous water loss (Allel et al., 2018).

At the beginning of the salt stress period, both cultivars significantly reduced their growth rate with respect to the control (T1), but afterwards, they sustained and developed a significant growth rate (Tables 5 and 6). This showed that both cultivars have tolerance ability to bear these salt stresses for some duration (Adem et al., 2014). Chinese red was the most successful cultivar, giving significant production under T2 for 1–9 weeks and, at T3, for 1–5 weeks; after that, the increment in the growth was non-significant with respect to the previous week. On the other hand, Chinese green showed significant changes in plant growth at T2 from Week 1 to Week 8, and at T3, from Week 1 to Week 4; thereafter, the growth parameters did not show sufficient increase, and at this stage, the plants were under their threshold limit of bearing salt stresses (Javed et al., 2017). The presentation of plant growth is linked with plant water status because continuous use of saline irrigation increases the salt concentrations in the root zone, thus decreasing soil water potential (Javed et al., 2019a). Under these conditions, dehydration occurs at the cellular level, which ultimately causes osmotic stresses (Javed et al., 2019b). These osmotic stresses produce ionic imbalance by increasing the amounts of Na⁺ and Cl⁻ and decreasing the amounts of K⁺, Ca²⁺ and Mn²⁺ (Chen et al., 2016). When these toxic salts reach the leaf, turgor loss and the death of leaf cells and tissues happen (Azeem et al., 2017a).

A strong relationship was found between the gas exchange parameters and plant growth under salt stress conditions. The interaction effect of salinity levels and their durations significant affected P_N, Gs and E in every salinity treatment, due to which plant growth was reduced, as shown in Tables 2 and 3. The reduction in gas exchange parameters was due to the increase in salt concentration in the nutrient solution, creating osmotic potential within the soil and the plant, which causes loss in the ability of the plant to get water as it normally does from a non-saline solution (Shahid et al., 2011). The increasing concentration of salt in the nutrient solution creates a negative effect on plant water status by reducing WUE and Wp (Xing et al., 2018), which causes higher solute concentration in the leaf cytosol (Zhang et al., 2015). Under these conditions, plants try to save water by means of stomatal closure. Then, E decreases due to stomatal closure, forcing the plant to maintain a reasonable P_N only for a short time, because CO₂ assimilation is disturbed because of stomatal closure (Javed et al., 2017; Qiu et al., 2017). This response was detected in both Chinese red and Chinese green at T2, T3 and T4, which maintained the gas exchange and growth parameters under these different salinity treatments from Week 1 to Weeks 9, 5 and 3. After these periods, both cultivars reflected non-significant changes in behaviour in terms of physiological and growth variables, due to which the fresh weight of both cultivars clearly decreased (Figure 3). This suggested that plants cannot withstand these stresses for longer duration because their cells are lethally damaged (Brodribb et al., 2005). It may suggest that Weeks 3 and 9 are the tolerance threshold limits for both cultivars to sustain their gas exchange activities and growth under T4 and T2 salinity treatments, respectively. Similarly, at T3, the threshold limit for bearing this salinity treatment in the case of Chinese red was Week 8 and, Chinese green, Week 5. At these threshold durations, both cultivars needed to reduce salt concentrations for their survival (Javed et al., 2018). In all previous studies, the researchers overcame the salt stress effect with different techniques and methods, such as using halophyte crops (Belkheiri and Mulas, 2013) and by removing salt in the root zone by scraping, flushing and leaching (Jouyban, 2012). Some of them gave fresh water while re-watering (Javed et al., 2018), and many used biochar in the soil to overcome salinity effects (She et al., 2018; Huang et al., 2019). According to the response of both cultivars, we suggest using dilute water for re-watering, i.e. T4 salinity treatment would receive water of T3 on the suggested threshold week; similarly, for T3 and T2 treatments.