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