Changes in Chlorophyll and carotenoids content
The content of Chlorophyll (Chl) a showed a strong increase at 48 h during the salt stress treatment and then significantly decrease in the end of the salt stress treatments (Fig. 1a). During the recovery condition the Chl a abundance tends to go back to control levels, without reaching this prior value. It appears that the Chl a content is stable during the recovery phase. In contrast to this a significantly increase in Chl b content (Fig.1b) and subsequent total Chl/Cars ratio (Fig 1c) were observed at S3 and R2 time-points (p<0.01). In the Chl a/Chl b ratio, the significant difference was observed among R1 time-point and others.
The detectable amount of |Cars showed a strong dynamic in response to the change of condition. After an increase at the time-point S2 during stress a strong decrease at the later stress time-pint was detectable. During the regeneration phase the opposite pattern was detectable. A reduction at R2 was shown, which returned back to the level similar to control condition.
The amount of total Chl content was increasing over the entire stress treatment compared to control (Fig. 1F). This increase is based on the early accumulation of Chl a (S1 and S2) and late accumulation of Chl b (S3). During the regeneration phase R1 and R3 showed a decrease in their contents and in the middle of the regeneration (R2) a strong peak of total Chl based on Chl b abundance was detected.
ICP–OES Analysis
ICP-OES technique was done to determine the composition of elements in treated samples using plasma absorption emission spectroscopy technology. Sodium ion (Na+) in salt stressed plant significantly increased in S2 and S3 time-points, and were decreased in R2 and R3 condition as expected (Fig. 2a). The most variation in Na+ concentration were observed in root tissue by highest and lowest level of Na+ in salt stress and recovery condition, respectively. No significant differences were observed in Na+ concentrations between leaf and stem in recovery condition. In contrast, the accumulation of potassium ion (K+) in leaf and stem were decreased in salt stress, and were increased in recovery condition (Fig. 2b). K+ accumulations in root significantly increased after 48 h treatment with 600 mM NaCl in compare to control, and then back to control level after one week. By transferring the plant to control solution, the K+ concentration was declined significantly compare to control. Increasing in ratio of Na+/K+ were observed in S2 and S3 in while the decreasing trend were seen in recovery conditions (Fig. 2e). Despite the decreasing trend which was observed, their ratio was still higher than to control.
The calcium ion (Ca2+) content significantly declined in leaf, root and stem during salt stress compare to control, whereas it increased significantly in leaf and stem during the recovery condition (Fig. 2c). It should be noted that, the no significant difference was observed in root Ca2+ content in salt stress compared to recovery condition. The trend of Na+/ Ca2+ ratio was similar to Na+/K+ ratio with minor difference (Fig. 3f).
The magnesium (Mg2+) concentration in the leaf gradually declined in salt stress, and after transferring to recovery condition it was gradually increasing (Fig. 2d). In the analyzed stem tissue, a sharp decrease was observed during salt stress (with no significant difference among stress time-points), and then the gradually increased were observed during recovery condition. Although the accumulation of Mg2+ in root were significant among control and other samples, but its fluctuation was not high except the R1 that the maximum level of Mg2+ accumulation has been observed in this time-point.
Proline content, Total protein and ROS enzyme activities
Proline as a stress‐responsive amino acid plays a crucial role in plants, by protecting the plants from various kinds of abiotic stresses. The proline content was significantly lower in leaf than root during salt stress and recovery condition (Fig. 3a). The proline content in salt stressed plant significantly increased rather than control, whereas a reduction was observed in recovery condition in both leaves and roots. In leaf tissue, the proline content increased at S1 time-point, then it decreased gradually in S2 and S3 stressed time-points and recovery condition. In roots, a significant enhancement was observed at S1 time-point, then decreased suddenly at S2 time-point (Fig. 3a).
Total protein in salt stressed plant and recovery condition significantly decreased rather than control in both leaves and roots (Fig. 3b). An increase was observed in leaf tissue at R1 condition in comparison to the stressed time-points, while the lowest amount of protein was obtained at R2 condition. As the results showed in Fig. 3b, the protein content in leaves was higher than roots. The same pattern was observed in roots in stressed plants, however the difference was in R1 and R2 condition.
In the recovery condition, the CAT activity was higher than control and stressed condition in leaf (Fig. 3c). The highest and the lowest CAT activity occurred at R1 and S2 condition, respectively. In roots, the CAT activity increased at S1, S2 and R1, R2 condition, but suddenly decreased significantly in S3 and R3 time-points. The SOD activity increased in both leaf and root compare to the control (Fig. 3d). Stressed condition caused a profound increase in SOD activity to maximum level at S3 time-point, however it reduced suddenly in recovery condition in leaf. In recovery condition, there was no significant difference from R1 to R3 for SOD activity in root, but an increase was observed in stressed plant.
The increasing changes of APX activity in leaf during stressed condition showed that the highest APX activity occurred at S3 time-point (Fig. 2e). APX activities decreased significantly in recovery condition. In root tissues, the highest APX activity was observed at S2 time-point, while the lowest was at R1 condition. In stressed plant, the POD activity significantly enhanced up to the highest level at S3 time-point, while it decreased in recovery condition in both leaf and root (Fig. 2f). In recovery condition, the POD activity gradually decreased in leaf from R1 to R3, but in roots it did not follow the specific pattern.
RT-qPCR analysis of Antioxidant related genes
The levels of mRNA encoded by genes with a related function to antioxidant activity were tested by real-time qPCR. The four genes CAT, SOD, cAPX and pAPX were tested for their relative mRNA abundance under salt and recovery conditions. As in our experimental system the salt is taken up via the root system and extruded via the leaves, the analysis was performed in these two tissues. A strong decrease in CAT mRNA level was detectable in leaf tissue at time-point S2 (Fig. 4 a). Even during stress conditions the CAT mRNA level returned to control niveau at S3 and stayed manily unchanged during the recovering phase. In roots an early increase of CAT mRNA is detectable in line with the early root response and uptake of salt via the root system. The mRNA level remained on a higher niveau, compared to control, but does not show further modulations.
According to the results of transcript levels in leaf, the SOD transcript level in R3 condition was the lowest and the highest SOD transcript level presented in S2 time-point (Fig. 4b). In roots, the SOD transcript level were up-regulated significantly compare to the control, but in recovery condition it gradualy decreased rather than stressed one. The pattern of cAPX transcript level was highly similar to SOD mRNA: both in leaf and roots. The cAPX transcript levels of leaf tended to decrease in duriation of recovery condition rather than stressed plant (Fig. 4c). The pattern of pAPX transcript level in roots and leaves indicated an increase during salt application and a strong reduction during the recoveryphase (Fig. 4d)