Evaluation of salt tolerance in Eruca sativa accessions based on morpho-physiological traits

Plant material, growth conditions and salt stress treatment

Twenty five E. sativa accessions were used in this study. The seeds of these accessions were kindly provided by Bio-Resources Conservation Institute, National Agricultural Research Centre, Islamabad, Pakistan. The list of accessions along with other details is given in Table S1. The seeds were surface sterilized and sown in a thick moist sheet of foam which was then placed in 72-cell seed starter trays in a growth chamber under controlled conditions of 16 h/8h day night (350 µmol/ m²/s²) at 23 °C and 60% relative humidity. After one week of growth, uniform sized seedlings were transferred to the Hoagland-type solution (Hoagland & Arnon, 1938) in 4-liter plastic containers. The set up was placed in a growth chamber under controlled conditions (as mentioned above). The solution was changed every 2–3 days. The experiment was carried out in a completely randomized design (CRD). Plants were divided into two groups; one as control (0 mM NaCl) and other as treated (150 mM NaCl). At the four-leaf stage, the salt stress was applied for 2 weeks. Before the determination of gas exchange parameters plants were kept in full sunlight for 2 h (9.00 a.m.–11.00 a.m.).

Determination of plant growth and development traits

Data from control and treated plants was collected after 14 days of growth. Morphological traits like, SL, RL, PH and leaf attributes i.e., leaf number (LN) and leaf area (LA) were recorded. The experiment was performed in six replicates for each parameter. SL, RL and PH were measured from digital images by using image analysis software Digimizer. The LA was manually measured using a grid paper. Fresh weight (FW) of control and treated plants was determined immediately after harvesting by using a digital lab scale. For determination of dry weight (DW), whole plants (root + shoot) were dried in an incubator at 75 °C for 72 h, and then weight was determined by using a digital lab scale. Plant biomass assay was carried out in three replicates.

Electrolyte leakage

Electrolyte leakage (EL) was determined following the previously described method (Yildirim, Karlidag & Turan, 2009). Briefly, ten equal size leaf discs (10 mm diameter) from fully expanded leaves of control and treated plants were prepared and washed with deionized water to remove electrolytes adhered to the surface. These were then incubated at 10 °C for 24 h in glass tubes filled with 10 ml of deionized water and the first electrical conductivity reading (EC1) was recorded. The tubes were heated at 95 °C in a water bath for 20 min to release all the electrolytes. After cooling at room temperature, final electrical conductivity reading (EC2) was recorded. The EC readings were determined by using a portable conductometer (HI-98129 Pocket EC/TDS and pH Tester). The experiment was performed in three replicates. Following equation was used for the calculation of EL: $$\mathrm{E}\mathrm{L}=\left(\frac{\mathrm{E}\mathrm{C}1}{\mathrm{E}\mathrm{C}2}\right)\times 100$$

Determination of chlorophyl content

Relative chlorophyl content was measured by using Chlorophyll Meter (CCM-200plus; Opti-Sciences, Hudson, NH, USA). Fully expanded fourth leaf from all the plants (control and treated) was selected for the reading. The data are presented in the form of chlorophyl content Index (CCI). All the measurements were recorded in six replicates.

Relative water content

Relative Water Content (RWC) was determined from the data of fresh, dry and turgid weight of control and treated plants, as described previously (Loutfy et al., 2012). The experiment was performed in three replicates. RWC was calculated by using the formula: $$\mathrm{R}\mathrm{W}\mathrm{C}\left(\mathrm{\%}\right)={\displaystyle \frac{\mathrm{F}\mathrm{W}-\mathrm{D}\mathrm{W}}{\mathrm{T}\mathrm{W}-\mathrm{D}\mathrm{W}}\times 100}$$where FW, stands for fresh weight TW for turgid weight and DW for dry weight.

Mineral ion content

For the determination of mineral ions, 100 mg of dried whole plant samples were placed in a furnace for 5 h at 520 °C for ash formation. It was then mixed with the nitric acid-perchloric acid mixture (5:1) and the final volume was raised to 15 ml with distilled water. The filtrate was used to determine the concentrations of Na⁺, K⁺, Ca²⁺ and Mg²⁺ by atomic absorption spectrometry. For standard curves, different concentrations of Na⁺, K⁺, Ca²⁺ and Mg²⁺ were prepared by diluting stock solutions of CaSO₄, KCl, NaCl and MgSO₄. The standard curve was used to determine the content of each element and the values were expressed in mg/g DW. The experiment was performed in three replicates.

Leaf gas exchange parameters

The photosynthesis rate (PR), intercellular CO₂ concentration (Ci), transpiration rate (TR), and stomatal conductance (SC) were measured with a portable gas exchange system iFL (ADC BioScientific Ltd., Hoddesdon, UK). All the measurements were conducted on a sunny day with full light intensity (11.00 a.m.–4.00 p.m.). Young fully expanded leaves (third and fourth) were used in situ for recording the above mentioned gas exchange parameters. All the measurements were recorded in four replicates. The following conditions were applied for the assay: Leaf surface diameter 6 cm, ambient atmospheric CO₂ concentration (Cref) 352 μmol mol⁻¹, PAR (Qleaf): 1,200 μmol/m²s and wide-range of chamber water vapor pressure 4.4–6.6 mbar. Three plants from each accession and treatment were analyzed for leaf gas exchange parameters.

Salt tolerance evaluation

The data of all morpho-physiological parameters (control and treatment) for each accession was converted to the salt-tolerance index (STI) which is the ratio of the value for the NaCl-treated plant/value for the control. For the categorization of E. sativa accessions according to their salt tolerance, membership function value (MFV) was applied as previously described (Chen et al., 2012). The MFV was calculated according to the following formula: $$X_p={\displaystyle \frac{X-X_{min}}{X_{max}-X_{min}}\times 100\mathrm{\%}}$$

For the traits inversely related to salt tolerance (e.g., EL), following formula for MFV calculation was used: $$X_p=1-{\displaystyle \frac{\left(X-X_{min}\right)}{\left(X_{max}-X_{min}\right)}\times 100\mathrm{\%}}$$where X_p is the MFV value of the salt stress parameter “P” in a specific accession, X is the actual value of salt tolerance parameter while X_min and X_max represent the minimum and maximum MFV values, respectively, for that parameter in all accessions. A single MFV value (X_c) was obtained for each accession by taking the mean of MFV values of all tested morpho-physiological traits. E. sativa accessions were divided into five standard groups according to the average MFV value (X_a) and S.D. The accession was considered as highly tolerant if X_c ≥ X_a + 1.64 S.D., tolerant if the X_a + 1 SD ≤ X_c < X_a + 1.64 S.D., moderately tolerant if X_a − 1 S.D. ≤ X_c < X_a + 1 S.D., sensitive if X_a − 1.64 S.D. ≤ X_c < X_a − 1 S.D., and highly sensitive if X_c < X_a − 1.64 S.D.

Statistical analysis

Differences among accessions and treatments were considered statistically significant at p < 0.05 by Duncan’s multiple-range test performed using IBM SPSS Statistics for Windows, V.20 (IBM Corporation, Armonk, NY, USA). Principal component analysis (PCA), cluster analysis, and correlation matrix analyses were performed on MFV values of studied salt tolerance traits using STATISTICA Statsoft (version 10).

Morphological traits and leaf attributes

Shoot length was significantly reduced in the salt-stressed plants compared with the control (p < 0.05). The mean SL values for the control and treatments groups were 2.30 ± 0.56 and 1.10 ± 0.48 inches, respectively (Table 1). To determine the salt stress response of accessions based on shoot growth, SL of each accession was expressed as stress index, as described in the material and methods section. Accessions showing the higher stress index were considered more salt tolerant and vise versa. SL stress index of accessions varied from 16.74 to 88.96. Es-1 and Es-15 exhibited the highest and the lowest SL stress index, respectively (Fig. 1A).

Salt stress significantly inhibited RL as compared to control (p < 0.05). Mean RL values in control and treated groups were 2.17 ± 0.42 and 0.98 ± 0.43 inches, respectively (Table 1). RL stress index ranged from 15.88 to 73.47. The highest RL stress index was exhibited by Es-12 while the lowest by Es-18 (Fig. 1B).

Plant height was also significantly reduced in salt-stressed plants compared with control (p < 0.05). Mean PH values for control and treated groups were 4.43 ± 0.77 and 2.18 ± 0.76 inches, respectively (Table 1). PH stress index varied from 19.80 to 73.98; accessions Es-1 and Es-18 showed the highest and the lowest stress index, respectively (Fig. 1C).

Leaf number and LA were also significantly reduced in salt-stressed plants compared to control (p < 0.05). Mean LN for control and treatment groups were 4.22 ± 0.78 and 2.43 ± 0.82, respectively (Table 1). LN stress index varied from 28 to 94.12; Es-19 exhibited the highest stress index while Es-17 the lowest (Fig. 1D). Mean LA was also significantly less in the treated group compared with control (p < 0.05) (Table 1). Mean LA for control and treatment groups was 0.07 ± 0.025 and 0.03 ± 0.016 inch², respectively (Table 1). LA stress index varied from 7.6 to100; Es-25 and Es-3 showed the highest and the lowest stress index, respectively (Fig. 1E). To summarize, these data show that salt stress significantly inhibited morphological traits in all the accessions. Moreover, the extent of inhibition of these traits, by salt stress, was variable in E. sativa accessions.

Plant biomass

Salt stress significantly reduced the FW and DW of E. sativa plants (p < 0.05). Mean FW values in control and treatment groups were 241 ± 164 mg and 96 ± 66 mg, respectively (Table 2). FW stress index ranged from 10.2 to 83.92 (Fig. 2A). Cultivar Es-9 showed the highest FW stress index while Es-3 the lowest (Fig. 2). Mean DW values for control and treated groups were 10.6 ± 6.9 and 6.29 ± 3.9 mg, respectively (Table 2). DW stress index varied from 24.65 to 87.42. Es-12 and Es-3 exhibited the highest and the lowest DW stress index, respectively (Fig. 2B).

RWC, EL and chlorophyl content

Mean RWC values of control and treatment groups were not significantly different, being 92.11 ± 7% and 92.83 ± 2.6%, respectively (p > 0.05) (Table 2). However, moderate but significant variations in RWC were observed between control and its treated counterpart for few accessions (p < 0.05) (Table 2). RWC, in the control group, varied from 71.6% to 98.8% while in the treated group from 88.6% to 96.3%. Among the control group, Es-2 and Es-6 showed the lowest and the highest RWC, respectively. In the treated group Es-18 showed the lowest while Es-4 showed the highest RWC (Table 2).

The EL data is presented in Fig. 3 as the ratio of treated to control for each accession. A higher ratio showed an increased EL due to salt stress. Moderate but statistically significant differences in EL were found in few accessions (p < 0.05) (Fig. 3). EL ratio of the accessions ranged from 0.949 to1.324 (mean EL ratio 1.07). The lowest ratio was recorded for accessions Es-1 while the highest for Es-5 (Fig. 3).

Compared with control, the salt-treated group had significantly less chlorophyl (p < 0.05). The mean chlorophyl content values for control and treated groups were 18.17 ± 4.79 and 14.06 ± 6.02 CCI, respectively (Table 2). Under salt stress, chlorophyl content of the accessions was highly variable. Compared to their respective control, four accessions showed a significant SPAD increase, 17 accessions showed a significant decrease while four accessions did not show any significant SPAD change under salt stress (p < 0.05) (Table 2).

Gas exchange attributes

Salt stress caused a marked and significant reduction in net PR in all accessions (p < 0.05). PR in control was 7.5 ± 4.69 µmol m⁻²s⁻¹ while in treated group it was only 1.91 ± 0.97 µmol m⁻²s⁻¹ (Table 3). A significant variation was shown by the accessions in this attribute (p < 0.05). At 0 mM NaCl, Es-3 and Es-17 had maximum and minimum PR, respectively while at 150 mM NaCl, Es-1 and Es-25 showed the maximum and minimum PR, respectively (Table 3).

Over all, no significant differences were found in mean intercellular CO₂ concentration (Ci) between control and treatment groups (p > 0.05). Mean Ci values in control and treatment groups were 281.2 ± 22.9 μmol mol⁻¹ and 284.7 ± 42.6 μmol mol⁻¹, respectively (Table 3). However, significant inter-accession variations in Ci were detected in both control and treatment groups (p < 0.05). The Ci ranged from 238 to 340 μmol mol⁻¹ in control group while from 215.5 to 361 μmol mol⁻¹ in treatment group (Table 3). Es-20 and Es-17 showed the highest and the lowest Ci respectively among control group while Es-24 and Es-15 showed the highest and the lowest Ci respectively among treatment group (Table 3).

The TR in the salt-stressed group was significantly lower compared with the control group (p < 0.05). Mean TR values for control and treated groups were 2.57 ± 1.13 mmol m⁻²s⁻¹ and 0.94 ± 0.48 mmol m⁻²s⁻¹, respectively (Table 3). In control plants TR ranged from 0.91 (Es-2) to 4.18 mmol m⁻²s⁻¹ (Es-3) while in salt-stressed plants it ranged from 0.26 (Es-15) to 1.94 mmol m⁻²s⁻¹ (Es-5) (Table 3).

The salt-stressed group exhibited significantly reduced SC compared with the control group (p < 0.05). Mean SC values for control and treated groups were 0.14 ± 0.08 mmol m⁻²s⁻¹ and 0.04 ± 0.02 mmol m⁻²s⁻¹, respectively (Table 3). Significant inter-accession variations were also observed in SC within control as well as treatment groups (p < 0.05). SC, in control group, ranged from 0.04 mmol m⁻²s⁻¹ to 0.31 mmol m⁻²s⁻¹. Es-3 and Es-2 exhibited the highest and the lowest SC, respectively (Table 3). On the other hand, SC in treatment group ranged from 0.01 mmol m⁻²s⁻¹ to 0.075 mmol m⁻²s⁻¹; Es-12 and Es-15 exhibiting the highest and the lowest SC, respectively. The accession Es-2 exhibited an interesting phenotype; SC under control and salt stress was not significantly different (Table 3). Gas exchange parameters could not be assayed in two accessions (Es-7 and Es-16) due to the unavailability of enough seeds.

Mineral ion content

Significantly higher Na⁺ accumulated in salt-stressed plants compared with control (p < 0.05). Mean Na⁺ content values for control and treated groups were 13.52 ± 5.01 and 29.55 ± 8.8 mg/g DW, respectively (Table 4). Significant inter-accession variations were found in Na⁺ content in both control and treatment groups (p < 0.05). In control group, Na⁺ content varied from 4.6 to 24.90 mg/g DW; Es-1 and Es-21 showing the lowest and the highest Na⁺ content, respectively (Table 4). Na⁺ content, in treatment group, ranged from 16.99 to 48.11 mg/g DW; accessions Es-3 and Es-20 accumulated the lowest and the highest Na⁺, respectively (Table 4).

Salt-stressed group, compared with control, exhibited a moderate but statistically significant increase in K⁺ content (p < 0.05). The mean K⁺ content values for control and treatment groups were 15.61 ± 6.08 mg/g DW and 19.44 ± 7.46 mg/g DW, respectively (Table 4). Furthermore, in both control and treated groups, accessions showed significant variability in K⁺ content. The K⁺ content in control group varied from 6.95 to 33.40 mg/g DW while in treatment group from 9.74 to 41.30 mg/g DW (Table 4). In control as well as stressed group, Es-4 and Es-9 exhibited the lowest and the highest K⁺ content, respectively (Table 4).

K⁺/Na⁺ ratio was significantly decreased in the salt-treated group as compared with control (p < 0.05). The mean K⁺/Na⁺ ratio in control and treatment groups was 1.22 ± 0.36 and 0.67 ± 0.2, respectively (Table 4). Accessions exhibited diverse trends for K⁺/Na⁺ ratio which ranged from 0.63 to 2.07 and 0.44 to 1.3 in control and treated plants, respectively (Table 4). To further distinguish the accessions in which K⁺/Na⁺ ratio was least affected by the salt stress, K⁺/Na⁺ ratio was expressed as the stress index. As shown in Fig. 4, accessions Es-23, Es-15 and Es-11 showed the highest while Es-1, Es-10 and Es-20 the lowest K⁺/Na⁺ ratio stress index (Fig. 4).

We also determined two other important cations Ca²⁺ and Mg²⁺ in E. sativa accessions. Salt-stressed group accumulated significantly more Ca²⁺ than the control group (p < 0.05) (Control mean 6.03 ± 2.63 mg/g DW vs treated mean 9.08 ± 5.34 mg/g DW) (Table 4). Accessions, growing under controlled conditions as well as under NaCl treatment, showed significant variations in Ca²⁺ content (p < 0.05) (Table 4). Ca²⁺ content, in the control group varied from 2.61 to 13.47 mg/g DW while, for the treatment group, it varied from 2.87 to 23.17 mg/g DW (Table 4).

Salt-stressed group showed significantly low Mg²⁺ content compared with control group (p < 0.05). Mean Mg²⁺ content for the control group was 5.35 ± 2.86 mg/g DW while for the salt treatment group it was 4.06 ± 1.84 mg/g DW (Table 4). Significant variations in Mg²⁺ content were observed among accessions growing under controlled conditions as well as under NaCl treatment (Table 4). Mg²⁺ content, of the control group, ranged from 2.74 to14.25 mg/g DW while in the treatment group from 2.28 to 9.95 mg/g DW (Table 4).

Principal component analysis

The principal component analysis (PCA) was performed on mean MFV values of morpho-physiological traits to understand the weightage of each trait towards observed variation. Overall, PCA1 and PCA2 represented 52.66% of the observed variation whereas individually PCA1 and PCA2 accounted for 39.96% and 12.7% of the variation, respectively (Fig. 5A). The most significant traits for PC1 were PH, SL, RL, LN, LA, FW and DW while for PC2, PR and EL were the most significant traits as these accounted for higher contributions towards PC2 (loadings ≥0.30). K⁺/Na⁺ ratio was the least significant trait for both PC1 and PC2 (Fig. 5B).

Ranking and grouping of E. sativa accessions for evaluation of salt tolerance

For determination of the extent of variation in salinity tolerance among E. sativa accessions, the data of morpho-physiological traits (for control and treatment) were standardized by calculating the STI which is the ratio of the value for the NaCl treated plant/value for the control. STI was then used for computing the MFV of E. sativa accessions. The following traits were used for MFV calculation: SL, RL, PH, LN, LA, FW, DW, SPAD, EL, K⁺/Na⁺ ratio and PR. Moreover, Pearson’s correlation analysis was done for the determination of the relationship among traits. As shown in Fig. 6A strong and highly significant correlations were found among morphological traits like RL, SL and PH (p < 0.001). Leaf attributes also showed strong correlations. LN showed significant correlation with PH, RL (p < 0.001), SL, LA, DW (p < 0.01) and FW (p < 0.05). LA exhibited a strong correlation with SL (p < 0.01) and PH (p < 0.05). FW and DW were also strongly correlated (p < 0.001). K⁺/Na⁺ ratio also exhibited significant correlations with PH, RL (p < 0.01) and SL (p < 0.05). Three of the tested traits EL, SPAD and PR did not show any significant correlation (Fig. 6).

The ranking of the degree of salt tolerance among E. sativa accessions was done based on MFV. The higher mean MFV represents higher salt tolerance and vise versa. MFV data of the accessions are presented in Table 5. Mean and standard deviation (S.D) of overall MFV data were 0.43 and 0.11, respectively. The highest MFV score was observed for Es-11 (0.62, highly tolerant) while the lowest score for Es-3 (0.18, highly sensitive). Furthermore, E. sativa accessions were categorized into five standard groups according to the previously described criteria (Chen et al., 2012). Among 25 accessions, one accession was ranked as highly tolerant (MFV ≥ 0.62), four as tolerant (0.55 ≤ MFV < 0.62), fifteen as moderately tolerant (0.33 ≤ MFV < 0.55), four as sensitive (0.26 ≤ MFV < 0.33) while one accession as highly sensitive (MFV < 0.26). Accession Es-11 was ranked as highly tolerant while accessions Es-1, Es-9, Es-12 and Es-19 as tolerant. Es-3 was ranked as highly sensitive while Es-15, Es-16, Es-17 and Es-18 as sensitive. The remaining 15 accessions were placed in moderately tolerant group (Table 5).

Furthermore, the MFV data was used to construct a similarity matrix based on Euclidean distance (Fig. 7). The accessions were separated into four distinct clusters; two smaller clusters (clusters 1 and 4) and two larger clusters (clusters 2 and 3). Clusters 1 and 4 consisted of three and five accessions, respectively, while clusters 2 and 3 consisted of eight and nine accessions, respectively. Es-9, Es-11 and Es-12 were placed in cluster 1. The members of cluster 2 included Es-5, Es-8 and Es-20 to Es-25. The largest cluster that is, cluster 3 comprised of the following members: Es-1, Es-2, Es-4, Es-6, Es-7, Es-10, Es-13, Es-14 and Es-19. Five accessions namely Es-3, Es-15, Es-16, Es-17 and Es-18 were included in cluster 4 (Fig. 7).