Changes in the MDA content
MDA is one of the main products of membrane peroxidation and can strongly react with various components in the cell, causing damage to enzymes and plasma membranes, leading to membrane structure damage and impaired physiological function . Fig. 1 shows that during the freezing period, the MDA content in the F group and the combined stress group increased and peaked at -3°C. The MDA content of the A+F group, the D+F group and the A+D+F groups was higher than that of the blank control group, i.e., by 8.03~61.40%, 45.55~107.25% and 61.01~138.07%, respectively. This indicates that combined stresses cause more intense stress conditions, which resulted in more MDA in the alfalfa plants.
During the freeze-thaw period, the MDA content measured in the seedlings of the combined stress and freeze-thaw groups showed a downward trend. When the temperature was increased to 10°C, the content of MDA in the D+F, A+F and A+D+F groups decreased by 31.87%, 57.77% and 69.73%, respectively, compared to that measured 142 at -3°C. It also can be observed from Fig. 1 that the combined stresses resulted in a more significant effect than the individual low-temperature stress treatment, which is consistent with published experimental results on the effects of low-temperature stress on plant physiological indexes [6, 7].
Changes in the protein content
According to Fig. 2, the protein content in the combined stress group and the freeze-thaw group was significantly lower than that in the blank control group throughout the whole freeze-thaw cycle. This may be due to the adverse conditions that reduced the activity of protein synthetase or increased the activity of the enzyme in the plant, resulting in a decrease in the protein content. This result is similar to that presented by Gilmour et al. . During the freezing period, the protein content of the combined stress group and the freeze-thaw group reached a peak at 0°C, and that of each test group showed a downward trend from 0 to -3°C, indicating that low temperature decreased the plant protein content. This is consistent with the existing research results .
During the freeze-thaw period, the protein content of the A+D+F group decreased initially and then slowly increased, reaching the lowest value at 0°C. The reason may be that plants start to grow slowly and consume protein when warming up. At a temperature higher than 0°C, the protein content of each group showed a steady or slightly decreasing trend, which is similar to the results of Fleck et al., who investigated protein accumulation in winter wheat leaves and their freeze resistance . During this period, higher protein content was measured in the freeze-thaw group than in the combined stress group, indicating that the combined stress caused more damage to the plants, resulting in slower recovery when the temperature increased.
Changes in the soluble sugar content
It can be observed from Fig. 3 that the soluble sugar content of each test group was significantly higher than that of the blank control group throughout the freeze-thaw cycle. This may have occurred because plants under stress conditions protect themselves by accumulating soluble sugars, similar to the findings of Kaufmann and Blanke . During the freeze-thaw period, the soluble sugar content of each test group increased, reaching a peak at -3°C. The results show that in the low-temperature environment, the soluble sugar content in the plants increased significantly, and the plants protected themselves by accumulating a large amount of soluble sugar. The highest soluble sugar content was measured in plants subjected to the freeze-thaw+ deicing salt + acid rain combined stress condition.
During the freeze-thaw period, the soluble sugar content of each test group showed a downward trend. Specifically, during the temperature change from -3°C to 0°C, the soluble sugar contents of the F group and the A+D+F group differed significantly (by 26.68 ~ 30.12%), but the soluble sugar content of the two other groups did not differ significantly from that of the freeze-thaw group. The findings indicated that the conditions resulting from the combination of the three stress factors caused the most damage to the plants.
Changes in the proline content
As shown in Fig. 4, the proline content in the test group was higher than that in the control group throughout the whole freeze-thaw period. This is consistent with the research results of Hare et al., who analyzed the cumulative effect of osmotic pressure under stress conditions . The resistance of alfalfa seedlings to stress caused by acid rain and deicing salt resulted in an increase in the proline content in vivo. During the freeze-thaw period, the proline content in the freeze-thaw group and in the combined stress group increased and peaked at -3°C; the following values were measured: F group: 64.1 µg/g; A+F group: 70.4 µg/g; D+F group: 69.2 µg/g; A+D+F group:76.4 µg/g, and compared with 10°C, the proline content these respective values were 91.34%, 86.24%, 96.59% and 96.40% higher. This is consistent with the research results of Xin and Browse, that is, plants resist low-temperature damage by regulating the proline content .
During the freeze-thaw period, the proline content decreased with increasing temperature. Specifically, the proline content was19.97%, 18.46%, 19.80% and 8.38% lower in the respective test groups (according to the above-mentioned order) at -3°C compared with that measured at 0°C. During the freeze-thaw period, the proline content was 16.10~40.38% higher in the A+D+F group than that in the group subjected to only freeze-thaw. This indicates that compared with freeze-thaw stress, the combined stresses resulted in more severe conditions, to which the plants responded by producing more proline to protect themselves.
Changes in the chlorophyll content
As shown in Fig.5, during the freeze-thaw period, the chlorophyll content of each experimental group exhibited an initial decrease followed by an increase. The chlorophyll content of the alfalfa decreased under low-temperature stress. During the freeze-thaw period, the chlorophyll content in all groups exhibited a downward trend and reached the minimum value at-3°C, i.e., in the F group, the A+F group, the D+F group, and the A+D+F group, the values were 22.38%, 12.76%, 11.08% and 17.79% lower, respectively, than those measured at 10°C.
During the freeze-thaw period, the chlorophyll content in the control group and the experimental groups showed an upward trend. Compared to the values measured at -3°C, significantly higher values, i.e., increases of 41.64%, 25.59%, 25.76%, and 20.64%, respectively, were measured at 10°C. However, there was no significant difference in the chlorophyll content among the experimental groups throughout the whole freeze-thaw period. Low temperature is the main factor that affects alfalfa photosynthesis. This is consistent with the published results of the effect of low-temperature stress on the chlorophyll content of herbage [14-16].
Correlation analysis between indexes
Table 1 shows that under the freeze-thaw condition, MDA and proline were significantly positively correlated. MDA was positively correlated with soluble sugar, proline was positively correlated with soluble sugar. Chlorophyll was negatively correlated with all three indexes. There was no significant correlation between protein and the other indicators. This indicates that both proline and the soluble sugar content increased with the accumulation of MDA in plants under external stress, while the chlorophyll content decreased.
The correlations between the indexes of the freeze-thaw + acid rain + deicing salt group were similar to those of the freeze-thaw group, but all correlations were highly significant in the former group. The results showed that the combined stresses of freeze-thaw, acid rain and deicing salt resulted in more obvious changes in the plant physiological indexes, and the correlations between indexes were highly significant.