Soil moisture content
The soil moisture content of the four oak control groups ranged from 28.0–31.9%. With continuous drought stress, the soil moisture content of the treatment group decreased gradually, and the range of the decrease was similar. The soil moisture content reached the lowest point of 3.1% ~ 3.5% at day 31 of drought stress and returned to the control level after rewatering (Fig. 1).
Water Potential And Rwc
The RWC and water potential of the leaves of all four oak species tended to decrease under continuous drought stress compared to those of the control group. The values of the treated groups were not significantly different from those of the control group on the first day, but all decreased and reached the lowest values, which were significantly lower than those of the control group, on day 31 (P < 0.05). After rewatering, the leaf water potential increased rapidly, and the recovery rate of the leaf water potential was faster than that of RWC on day 36. The RWC and water potential of Q. fabri leaves decreased from 81.13% and -1.52 MPa (day 1) to 41.10% and -2.84 MPa (day 31), with decrease rates of 49.47% and 87.25%, respectively. After rewatering, the RWC and water potential recovered to 73.02% and -1.67 MPa (day 36), respectively, but did not recover to the normal level. The RWC and water potential of Q. serrata leaves decreased from 81.67% and -1.48 MPa (day 1) to 48.67% and -2.45 MPa (day 31), with decrease rates of 40.41% and 66.14%, respectively. After rewatering, the RWC and water potential recovered to 76.02% and -1.52 MPa (day 36), respectively. The RWC and water potential of Q. acutissima leaves decreased from 81.72% and -1.43 MPa (day 1) to 43.23% and -2.46 MPa (day 31), with decrease rates of 47.10% and 72.03%, respectively. After rewatering, the RWC and water potential recovered to 72.72% and -1.54 MPa (day 36), respectively. The RWC and water potential of Q. variabilis leaves decreased from 81.02% and -1.52 MPa (day 1) to 38.07% and -2.48 MPa (day 31), with a decrease rate of 53.10% and 63.16%, respectively. After rewatering, the RWC and water potential recovered to 73.67% and -1.57 MPa (day 36), respectively. The leaf water potential of Q. serrata, Q. acutissima and Q. variabilis returned to normal levels, but the RWC remained lower than the normal level (Fig. 2).
Osmotic Regulating Substances
Under continuous drought stress, the levels of soluble protein, SS and Pro in the four oak species first increased, then decreased, and then increased after rehydration. However, GB level increased continuously and decreased after rehydration. The SS levels of Q. fabri, Q. acutissima and Q. variabilis (Day 6) peaked earlier than that of Q. serrata (day 11). Compared with the control group, the SS level of the four oak species increased by 1.50, 1.28, 1.15 and 1.11 times, respectively. Then, the SS levels of the four oak species were the lowest on day 31 and were significantly lower (P < 0.05) than that of the control group, decreasing by 43.74%, 48.75%, 42.52% and 51.60%, respectively. After rewatering, only Q. fabri returned to a normal level, while the levels in the other three oak species were significantly lower than normal (P < 0.05) (Fig. 3A). There were no significant differences in the SS levels when comparing between the four oak species and the control group at the early stage (day 1 and day 6) (P > 0.05). On day 26, the SS level in each of the four oak species reached a peak, which was 1.46, 1.48, 1.35 and 1.37 times higher than that of the control group, respectively. Then, the levels decreased and were the lowest on day 31, decreasing by 15.44%, 12.79%, 10.62% and 14.22%, respectively. After rewatering, the SS level of Q. serrata was significantly lower than the normal level (P < 0.05), but the other three oak species returned to normal levels (Fig. 3B). The Pro level in the four oak species reached a peak on day 26 and was significantly higher than that in the control group (P < 0.05), increasing by 1.89, 1.66, 1.98 and 1.87 times, respectively. Then, the Pro level decreased but was still significantly increased compared with the level in the control group (day 31) (P < 0.05) and returned to the normal level after rehydration (Fig. 3C). The GB level in the four oak species showed a continuous increasing trend, which was significantly higher than that in the control group (P < 0.05), increasing by 1.56, 2.05, 1.96 and 2.29 times, respectively. After rehydration, the GB level decreased sharply but was still significantly higher than that in the control group (P < 0.05), increasing by 1.28, 1.50, 1.65 and 1.98 times, respectively (Fig. 3D).
Antioxidant Enzyme Activities And Mda
Under continuous drought stress, the activities of POD, SOD and CAT of the four oak species first increased, then decreased, and then increased after rehydration. The MDA level increased continuously and decreased after rehydration. The POD activity of Q. fabri, Q. acutissima and Q. variabilis (day 16) reached a peak later than that of Q. serrata (day 11), which was 1.36, 1.11, 1.34 and 1.18 times higher than that of the control group, respectively. The POD activity levels of the four oak species were the lowest on day 31 and were significantly lower than that of the control group (P < 0.05). The POD activity levels were decreased by 18.34%, 32.21%, 34.74% and 36.65%, respectively, and did not return to normal levels after rehydration (Fig. 4A). The SOD activity level of the four oak species was significantly different from that of the control group at day 5 (P < 0.05) and peaked at day 21. The SOD activity levels were 2.14, 2.33, 1.93 and 2.28 times higher than that of the control group, respectively. Then, the SOD activity levels were the lowest at day 31 and were significantly lower than that in the control group (P < 0.05). The SOD activity levels were reduced by 32.55%, 29.26%, 37.10% and 20.73%, respectively, and did not recover to normal levels after rehydration (Fig. 4B). The CAT activity levels of Q. fabri, Q. acutissima and Q. variabilis (day 21) reached a peak later than that of Q. serrata (day 16). The CAT activity levels were 1.54, 1.48, 1.52 and 1.39 times higher than that of the control group, respectively. The CAT activity levels of the four oak species were the lowest on day 31 and were significantly lower than that of the control group (P < 0.05). The CAT activity levels decreased by 25.13%, 20.61%, 8.12% and 11.43%, respectively. After rehydration, only Q. fabri returned to the normal CAT activity level, but the levels of the other three oaks were significantly lower than normal (P < 0.05) (Fig. 4C). The MDA levels of the four oak species showed a continuous growth trend, and there were no significant differences in the levels at the early stage (day 1 and day 6) when comparing between the four oak species and the control group (P > 0.05). The MDA levels peaked on day 31 and were significantly higher than that in the control group (P < 0.05), increasing by 2.71, 2.15, 2.23 and 2.06 times, respectively. The MDA levels decreased sharply after rehydration but were still significantly higher than that in the control group (P < 0.05), increasing by 1.38, 1.41, 1.36 and 1.45 times, respectively (Fig. 4D).
Photosynthetic Parameters (Dup: 5 ?)
Compared with the control group, the Pn, Gs and Tr values for the four oak species showed a decreasing trend under continuous drought stress. The values were the lowest on day 31 and rose rapidly after rehydration. As drought stress was prolonged, the Pn values of the four oak species decreased continuously. The Pn of Q. serrata at day 6 was significantly different from that of the control group (P < 0.05). The Pn values of the four oak species decreased to the lowest level on day 31 and were significantly lower than that of the control group (P < 0.05). The Pn values decreased by 72.10%, 63.29%, 63.67% and 60.16%, respectively. After rehydrating, the Pn of Q. serrata was significantly lower than normal (P < 0.05), but the Pn values of the other three oak species returned to normal (Fig. 5A). There were no significant differences in the Gs values when comparing between the four oak species and the control group at the early stage (day 1 and day 6) (P > 0.05). The Gs values of the four oak species were the lowest on day 31 and were significantly lower than that of the control group (P < 0.05). The Gs values decreased by 71.14%, 57.45%, 50.03% and 45.24%, respectively, but returned to normal after rehydration (Fig. 5B). There were significant differences in the Tr values when comparing between the four oak species and the control group at day 5 (P < 0.05). The Tr values were the lowest on day 31 and were significantly lower than that of the control group (P < 0.05). The Tr values decreased by 62.37%, 60.08%, 61.28% and 60.72%, respectively, and did not return to normal after rehydration (Fig. 5C).
Multivariate Statistical Analysis
The eigenvalues and contribution rates of principal components were the basis for selecting principal components. The 13 physiological and biochemical indexes of the leaves from the 4 oak species were analysed by PCA. Two principal components with eigenvalues greater than 1 were obtained, and their contribution rates were 64.24% and 19.70%, respectively. The cumulative contribution rate was 83.94%, and most of the information on the original characteristics was retained (Table 1). Therefore, the first two principal components could be selected as the important principal components of the drought resistance of the four oak species. The factors with higher loading capacity in the first principal component were Ψw, RWC, Pro, SP, GB, MDA, Pn, GS and Tr, which were mainly related to leaf water status, osmoregulatory substances and photosynthesis. The second principal component was mainly related to antioxidant enzymes. The treatments of the 4 oaks under drought stress and during rehydration were completely separated. In addition, Q. acutissima, Q. serrata and Q. variabilis highly overlapped under drought stress and during rehydration. In contrast, Q. fabri varied greatly with the other three species. To further understand the relationship between leaf water status, osmotic regulatory substances, photosynthesis and antioxidant enzymes, Pearson correlation analysis was used to analyse the data. The results showed that Pn, GS and Tr positively correlated with each other, negatively correlated with MDA, GB and Pro levels and positively correlated with SP, Ψw and RWC. Ψw and RWC levels were significantly negatively correlated with Pro, GB and MDA levels. MDA levels were significantly positively correlated with GB and Pro levels and negatively correlated with SP levels and POD activities. Pro levels were significantly negatively correlated with SP levels and positively correlated with SS and GB levels. In addition, SOD, POD and CAT activities were significantly positively correlated (Fig. 6). To comprehensively evaluate the drought resistance of the four oak species, 13 physiological and biochemical indexes of the four oak species under continuous drought stress and rehydration conditions were analysed by membership functions (Table 2). The membership function value of each index was calculated according to a formula. The degree of correlation between different indexes and drought resistance was different. The arithmetic mean of the membership value of each index did not fully reflect the level of drought resistance in the four oak species. Therefore, the proportion of eigenvalues corresponding to each principal component to the sum of the total eigenvalues of the extracted principal components was taken as the weight. The comprehensive evaluation value of the four oak species was calculated. Higher comprehensive evaluation values were positively correlated with stronger drought-resistance ability. The results showed that the order of drought resistance of the four oak species were as follows: Q. serrata > Q. fabri > Q. variabilis > Q. acutissima.
Table 1
Eigenvalue and cumulative contribution rate of each index of four oak species
Measured index | Principal component |
PC1 | PC2 |
Relative water content | 0.335 | -0.014 |
Water potential | 0.326 | 0.018 |
Soluble sugar | -0.174 | 0.365 |
Soluble protein | 0.276 | 0.084 |
Glycine betaine | -0.309 | -0.134 |
Pro | -0.322 | 0.099 |
SOD | -0.083 | 0.551 |
POD | 0.150 | 0.447 |
CAT | -0.142 | 0.540 |
MDA | -0.311 | -0.176 |
Pn | 0.331 | 0.041 |
Gs | 0.334 | 0.045 |
Tr | 0.328 | -0.024 |
Eigenvalue | 8.352 | 2.562 |
Cumulative contribution rate (%) | 64.24 | 83.94 |
Table 2 Membership function values and evaluation index of the drought resistance of four oak
species
Item | Q. fabri | Q.serrata | Q.acutissima | Q.variabilis |
Relative water content | 0.3340 | 0.2399 | 0.2314 | 0.2325 |
Water potential | 0.4529 | 0.5773 | 0.4193 | 0.3608 |
Soluble sugar | 0.3002 | 0.2288 | 0.3272 | 0.2844 |
Soluble protein | 0.0900 | 0.0883 | 0.0863 | 0.0823 |
Glycine betaine | 0.3613 | 0.3867 | 0.1349 | 0.1464 |
Pro | 0.1980 | 0.2321 | 0.3348 | 0.3919 |
SOD | 0.0777 | 0.1053 | 0.0780 | 0.1094 |
POD | 0.1384 | 0.0909 | 0.1013 | 0.0920 |
CAT | 0.1125 | 0.1110 | 0.1236 | 0.1305 |
MDA | 0.4770 | 0.5235 | 0.4702 | 0.5055 |
Pn | 0.4322 | 0.4225 | 0.4932 | 0.5193 |
Gs | 0.4354 | 0.4725 | 0.5329 | 0.4984 |
Tr | 0.3608 | 0.3990 | 0.3285 | 0.3118 |
Comprehensive evaluation | 0.2900 | 0.2983 | 0.2817 | 0.2819 |
Sequencing | 2 | 1 | 4 | 3 |