Changes in MDA content
Fig. 1 shows that the MDA content in seedlings of groups under combined stresses (A-F, D-F and A-D-F) was higher than that of the group under single freeze-thaw stress (F) by 7.87~62.60%, 63.40~120.96% and 69.48~136.40% [see Additional file 1], respectively. This indicates that combined stresses cause more intense stress conditions, resulting in an accumulation of MDA in the alfalfa plants. During the thawing period (8h~14h), the MDA content measured in seedlings of groups under either combined stresses or single freeze-thaw stress decreased. When the temperature rose to 10°C (14h), the content of MDA in seedlings of groups A-F, D-F and A-D-F decreased by 57.58%, 42.10% and 40.20% [see Additional file 1], respectively. It also can be observed from Fig. 1 that under freeze-thaw stress, the MDA content in seedlings of group A-D-F was significantly higher than that of group A-F (P < 0.05), while showed no significant difference compared with that of group D-F (P > 0.05). The above results indicated that the combined stresses had a more significant effect on the MDA content in seedlings than single freeze-thaw stress, and the deicing salt stress had a greater impact on MDA content than acid precipitation and freeze-thaw stress.
Changes in soluble protein content
According to Fig. 2, the soluble protein content in seedlings of the combined stresses groups showed a trend of increasing initially and then decreasing throughout the whole freeze-thaw cycle, while that of the single freeze-thaw stress group showed a fluctuant decreasing. When the temperature dropped to 0°C (6h), the soluble protein content in seedlings of groups under combined stresses reached its peak. At thawing stage (8h~14h), the soluble protein content in seedlings of groups under freeze-thaw stress was significantly lower than that of group CK (P < 0.05) [see Additional file 2], which may attribute to the adding of freeze-thaw stress. However, the content of soluble protein in seedlings of groups under compound stresses showed no significant difference compared with that of group under single freeze-thaw stress, indicating either acid precipitation stress or deicing salt stress had less impact on soluble protein content. Moreover, during this period, higher soluble protein content was measured in the freeze-thaw group than in the combined stresses groups, indicating that the combined stresses caused more damage to the plants.
Changes in 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 group CK during the freeze-thaw cycle. When the temperature decreased, the soluble sugar content in seedlings of all groups except CK increased and reached its peak at -5°C (8h). The results demonstrated 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 combined stresses of freeze-thaw, deicing salt and acid precipitation. During the thawing period (8h~14h), the soluble sugar content in seedlings of all groups except CK showed a downward trend with the temperature increased. Notably, during the temperature rose from -5°C (8h) to 0°C (10h), the soluble sugar contents in seedlings of group F was significantly lower than that of group A-D-F by 17.13% (8h) and 14.79% (10h) [see Additional file 3] (P < 0.05), but the soluble sugar content in seedlings of groups A-F and D-F did not differ significantly from that of group F (P > 0.05). The findings indicated that the conditions resulting from the combination of the three stress factors caused the maximum accumulation of soluble sugar in plants.
Changes in proline content
As shown in Fig. 4, the proline content in seedlings of the test groups was higher than that of group CK throughout the whole freeze-thaw period, indicating that stresses resulted from acid rain and deicing salt caused an increase in proline content in plants. During the freezing period, the proline content in seedlings of groups F, A-F, D-F and A-D-F increased and peaked at -5°C (8h) with respectively 91.34%, 86.24%, 96.59% and 96.40% higher than those measured at 10°C (2h) [see Additional file 4]. During the thawing period (8h~14h), the proline content of groups F, A-F, D-F and A-D-F at -5°C (8h) decreased by 19.97%, 18.46%, 19.80% and 8.38% respectively, compared with those measured at 0°C (10h) [see Additional file 4]. Fig. 4 also showed that the proline content was significantly higher in group A-D-F than that in the group subjected to only freeze-thaw (P < 0.05). Besides, except CK, the proline content in seedlings of groups under acid rain stress was significantly higher than that of groups under non-acid-rain stress (P < 0.05), which indicated that freeze-thaw stress accompanied by acid rain stress resulted in more proline produced in plants to protect themselves.
Changes in chlorophyll content
During the freeze-thaw period, the chlorophyll content in seedlings of each experimental group exhibited an initial decrease followed by an increase (Fig. 5). At the freezing stage, the chlorophyll content in groups F, A-F, D-F and A-D-F showed a downward trend and reached the minimum value at -5°C (8h) that were 22.38%, 12.73%, 11.11% and 17.79% lower than those measured at 10°C (2h) [see Additional file 5], respectively. During the thawing period (8h~14h), compared with the chlorophyll content measured at -5°C (8h), the contents measured at 10°C (14h) in seedlings of groups F, A-F, D-F and A-D-F were significantly increased by 42.32%, 25.60%, 25.77% and 20.65% (P < 0.05) [see Additional file 5]. However, there was no observed significant difference in the chlorophyll content among the experimental groups throughout the whole freeze-thaw period (P > 0.05).
Correlation analysis between indexes
Table 1 shows that under the freeze-thaw condition, MDA and proline were significantly positively correlated (P < 0.01), both of which was positively correlated with soluble sugar (P < 0.05). Chlorophyll was negatively correlated with MDA, proline and soluble sugar. However, there was no significant correlation between protein and the other indicators. 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 (P < 0.01). These findings indicated that both proline and the soluble sugar content increased with the accumulation of MDA in plants under external stress, while the chlorophyll content decreased.