Changes of biomass, REC and lipid peroxidation in wheat seedlings under different treatments
As indicated in Fig. 1a, the shoots and roots of wheat seedlings were significantly shorter when wheat seedlings were subjected to 150 mM NaCl stress. In comparison with the control, 25 mg L− 1 TP treatment resulted in the notable reduction of shoot length and the insignificant change of root length. Shoot and root lengths in 100 mg L− 1 TP-treated seedlings increased to 111% and 129% of the control, respectively. When 25 or 100 mg L− 1 TP was added, the length of shoots and roots in salt-treated seedlings increased significantly compared with those of the seedlings treated with salt stress alone.
Compared with the control, the Fw of leaves and roots in response to salt treatment decreased by 61% and 47%, and the Dw decreased by 50% and 30%, respectively (Fig. 1b and 1c). 25 mg L− 1 TP treatment alone did not affect the Fw of leaves and roots, but increased the Dw. By contrast, these parameters increased to 1.18, 1.13, 1.14 and 1.16 times of the control, respectively, under 100 mg L− 1 TP treatment alone. Compared with salt treatment alone, the Fw of leaves and roots as well as the Dw of leaves increased significantly in salt stressed seedlings with the addition of TP at different concentrations. However, TP application had no obvious effect on the Dw of roots under NaCl treatment (Fig. 1b and 1c).
A significant elevation of REC due to 150 mM NaCl stress was observed, whereas different TP concentrations resulted in the notable decrease, with about 37% and 27% reduction due to 25 mg L− 1 or 100 mg L− 1 TP treatment, respectively, in wheat leaves in comparison with the control. The addition of 25 mg L− 1 or 100 mg L− 1 TP effectively weakened the salinity-induced enhancement of REC, by about 34% and 19% decrease compared with salt stress alone, respectively (Fig. 2a).
As shown in Fig. 2b, MDA content increased by about 78% in the leaves of salt-stress seedlings, in comparison with untreated seedlings, and this parameter was not affected by different TP concentrations (25 and 100 mg L− 1). Leaf MDA content due to 25 mg L− 1 TP + 150 mM NaCl and 100 mg L− 1 TP + 150 mM NaCl treatments decreased by about 17% and 20% in comparison with salt stress alone.
Changes of Na + , K+ and Ca2+ content in wheat leaves under different treatments
Compared with the control, leaf Na+ content increased to 2.74 times in wheat seedlings treated with 150 mM NaCl. Additionally, low TP concentration did not affect leaf Na content, whereas a large amount of Na+ was found due to high TP concentration (Fig. 3a). Compared with salt stress alone, the addition of 25 and 100 mg L− 1 TP effectively alleviated the salt-induced increase in leaf Na+ level, and the reduction was about 31% and 24%, respectively.
150 mM NaCl or 25 mg L− 1 TP treatment alone decreased K+ content in wheat leaves by 71% and 66% of the control, respectively. Nevertheless, 100 mg L− 1 TP treatment alone did not affect K+ content. Compared with salinity-alone-treated seedlings, leaf K+ content in NaCl-stressed ones enhanced significantly by about 38% and 36% in the presence of 25 or 100 mg L− 1 TP, respectively (Fig. 3b).
In comparison with the untreated seedlings, leaf Ca2+ content decreased by 35% under 150 mM NaCl stress. Differently, 25 and 100 mg L− 1 TP treatment resulted in the significant elevation of Ca2+ content by 13% and 38%, respectively. In addition, leaf Ca2+ level in response to 25 mg L− 1 TP + 150 mM NaCl and 100 mg L− 1 TP + 150 mM NaCl treatments notably increased by about 45% and 51% compared with that under salt stress alone (Fig. 3c).
Changes of antioxidant enzyme activities in wheat leaves under different treatments
As shown in Fig. 4a, leaf SOD activity increased initially, followed by decrease with the extension of growth time of wheat seedlings. Compared with the control, the activity of SOD was not affected by salt stress alone on the 2 days, and obviously increased to 123% and 113% of the control group on the 4 and 6 days. This enzyme activity in response to 100 mg L− 1 TP treatment alone significantly decreased in comparison with the control, but did not alter under 25 mg L− 1 TP treatment on the 2 and 4 days, whereas notably reduced for 6 days. Compared with salt treatment alone, the presence of two TP concentrations had no significant effect on the change of leaf SOD activity induced by salt stress.
Compared with the control, leaf CAT activity decreased by about 30% and 14%, respectively, when the seedlings were stressed with 150 mM NaCl on the 2 and 4 days, but there was no evident change on the 6 days (Fig. 4b). In addition, leaf CAT activity under 25 mg L− 1 TP treatment alone for 2 days significantly decreased, but notably increased by about 28% and 23% under the treatment with 100 mg L− 1 TP for 4 and 6 days. In comparison with salt stress alone, the addition of 25 mg L− 1 TP did not affect salt-induced change of CAT activity, while the further decrease in this parameter was found in wheat leaves treated by salinity together with 100 mg L− 1 TP, which was reduced by about 10%, 19% and 38% on the 2, 4 and 6 days, respectively (Fig. 4b).
In comparison with the control, the activity of POD in wheat leaves reduced with the extension of salt treatment time, and decreased by about 31%, 40% and 11% on the 2, 4 and 6 days, respectively (Fig. 4c). Moreover, the changes of leaf POD activity under TP treatment were similar to those under salt stress. For example, this enzyme activity under 25 mg L− 1 TP treatment on the 2, 4 and 6 days decreased by about 31%, 53% and 32%, respectively, compared with the control. The presence of two TP concentrations did not affect salinity-induced change of POD activity on the 2 days, but this enzyme activity significantly increased by about 45% and 40% at 4 days as well as 27% and 28% on the 6 days, respectively, due to NaCl + 25 mg L− 1 TP and NaCl + 100 mg L− 1 TP treatment in comparison with salt stress alone.
Changes of antioxidant enzyme gene expression in wheat leaves under different treatments
As shown in Fig. 5a, the expression level of TaCu/Zn-SOD significantly increased when the seedlings exposed to NaCl stress alone on the 2 and 4 days, which were 1.68 and 1.34 times higher than those of the control, whereas its expression was significantly lower than that of the control on the 6 days. Compared with the control of the same period, the expression of TaCu/Zn-SOD did not change significantly in response to 100 mg L− 1 TP treatment. Compared with salt stress alone in the same period, this parameter expression increased by about 19% on the 2 days after NaCl + TP treatment, and the change of the gene expression on the 4 days was not significant, but it was significantly lower at the 6 days of treatment.
It can be seen from the Fig. 5b that the expression of TaMn-SOD under salt treatment alone increased significantly to 149%, 238% and 156% of the control on the 2, 4 and 6 days, respectively. This gene expression increased to 1.08 times as much as that of the control in wheat leaves when treated with TP on the 2 days, whereas the effect of TP treatment of this gene significantly reduced on the 4 and 6 days. NaCl + TP treatment had no effect on the expression of TaMn-SOD on the 2 days, while it was reduced by about 53% and 67% of the salt treatment alone on the 4 and 6 days.
Compared with the control, the expression of TaCAT in wheat leaves reduced by about 12%, 22% and 53% on the 2, 4 and 6 days of salt treatment alone, respectively (Fig. 5c). This parameter expression tended to decrease with the increase of TP treatment time, but it was remarkably higher than control 1.98 and 1.39 times on the 2 and 4 days, respectively, whereas reduced about 41% on the 6 days. In comparison with the control, the presence of TP did not affect the expression of TaCAT on the 2 days, but resulted in notable reduction of this gene expression on the 4 and 6 days by about 23% and 39%.
As shown in Fig. 5d, exposure to salt for 2 days did not affect TaPOD expression, but remarkably inhibited on the 4 and 6 days, with reduction by about 51% and 77% compared with the control, respectively. The TaPOD expression in wheat leaves evidently decreased due to 100 mg L− 1 TP treatment, which was 0.71, 0.72 and 0.34 times of that of the control on the 2, 4 and 6 days. Compared with salt treatment alone, the application of 100 mg L− 1 TP significantly increased this gene expression on the 2, 4 and 6 days by about 41%, 86% and 143%, respectively.
Changes of ROS content in wheat leaves under different treatments
As can be seen from the Table 2, with the extension of growth time, leaf O2·− production in unstressed seedlings decreased gradually. The amount of O2·− in wheat leaves increased with salt stress alone, which was 1.58, 1.16 and 1.53 times of the control values on the 2, 4 and 6 days, respectively. Compared with the control, leaf O2·− content increased by about 11% when the seedlings were exposed to 25 mg L− 1 TP on the 2 days, while did not significantly change on the 4 and 6 days. Differently, this parameter in response to 100 mg L− 1 TP treatment significantly rose, with about 56%, 39% and 15% enhancement on the 2, 4 and 6 days, respectively. In addition, 25 mg L− 1 TP did not affect NaCl-induced changes of O2·− production in wheat leaves, whereas the addition of 100 mg L− 1 TP remarkably reduced O2·− accumulation on the 4 and 6 days of salt treatment.
Table 2
Changes of reactive oxygen species (ROS) content in wheat leaves under different treatments
|
Treatments
|
Two days
|
Four days
|
Six days
|
O2·− content
(µmol g− 1 Fw)
|
CK
|
2.05 ± 0.15a
|
1.60 ± 0.10a
|
1.43 ± 0.01a
|
150 mM NaCl
|
3.23 ± 0.09c
|
1.86 ± 0.13b
|
2.19 ± 0.01c
|
25 mg L− 1 TP
|
2.28 ± 0.12b
|
1.59 ± 0.15a
|
1.51 ± 0.02ab
|
100 mg L− 1 TP
|
3.20 ± 0.05c
|
2.22 ± 0.15c
|
1.65 ± 0.03b
|
150 mM NaCl + 25 mg L− 1 TP
|
3.02 ± 0.17c
|
1.83 ± 0.17b
|
2.25 ± 0.03c
|
150 mM NaCl + 100 mg L− 1 TP
|
3.06 ± 0.25c
|
1.63 ± 0.12a
|
1.43 ± 0.01a
|
H2O2 content
(ng g− 1 Fw)
|
CK
|
347.05 ± 1.96b
|
453.36 ± 1.11a
|
536.17 ± 2.05a
|
150 mM NaCl
|
601.23 ± 1.36d
|
872.62 ± 1.25e
|
819.34 ± 5.80d
|
25 mg L− 1 TP
|
310.38 ± 1.82a
|
779.36 ± 1.67d
|
613.21 ± 3.29b
|
100 mg L− 1 TP
|
466.05 ± 1.19c
|
681.48 ± 1.26c
|
587.30 ± 2.24ab
|
150 mM NaCl + 25 mg L− 1 TP
|
345.66 ± 1.57b
|
753.67 ± 2.09d
|
708.12 ± 6.59c
|
150 mM NaCl + 100 mg L− 1 TP
|
498.44 ± 1.04cd
|
552.14 ± 2.57b
|
614.60 ± 5.60b
|
·OH content
(nmol g− 1 Fw)
|
CK
|
27.94 ± 1.39a
|
53.31 ± 1.79a
|
72.23 ± 2.36b
|
150 mM NaCl
|
34.23 ± 1.62b
|
69.12 ± 1.85bc
|
93.91 ± 3.72c
|
25 mg L− 1 TP
|
31.20 ± 1.06b
|
65.04 ± 1.72b
|
54.68 ± 2.40a
|
100 mg L− 1 TP
|
28.62 ± 1.09ab
|
75.98 ± 1.95c
|
52.32 ± 2.92a
|
150 mM NaCl + 25 mg L− 1 TP
|
30.80 ± 1.64ab
|
67.82 ± 1.56bc
|
87.06 ± 2.05c
|
150 mM NaCl + 100 mg L− 1 TP
|
28.28 ± 1.10ab
|
72.28 ± 2.21c
|
55.26 ± 2.96a
|
Values represent mean ± SE of at least three experiments with replicated measurements; Different small letters mean significant difference at 0.05 levels. TP: Tea polyphenols |
Leaf H2O2 level due to salt or TP treatment alone showed a trend of increased initially, followed by decrease with the extension of seedlings growth time, and reached the peak on the 4 days of growth (Table 2). In comparison with the control, 150 mM NaCl stress induced a notable elevation in H2O2 content, which was 173%, 192% and 153% of the control on the 2, 4 and 6 days, respectively. After the seedlings were exposed to 25 mg L− 1 TP alone on 2 days, the amount of H2O2 showed no obvious change, while on the 4 and 6 days, this parameter enhanced to 1.72 and 1.14 times of the control. Differently, leaf H2O2 content due to 100 mg L− 1 TP treatment on the 2 and 4 days increased to 1.34 and 1.50 times, whereas 100 mg L− 1 TP was treated on the 6 days, there was no significant change in this parameter, in comparison with that to the control. Additionally, when 25 mg L− 1 TP was added on the 2, 4 and 6 days, leaf H2O2 content in salt-stressed seedlings decreased by about 43%, 14% and 14%, respectively, in comparison with salinity-alone stressed ones. Similar, the application of 100 mg L− 1 TP on the 2, 4 and 6 days, also decreased this parameter in salinity-stressed seedlings.
Leaf ·OH production in untreated seedlings enhanced with the extension of plant growth time. This parameter due to 150 mM NaCl treatment enhanced by about 23%, 30% and 30% on the 2, 4 and 6 days, respectively, in comparison with the control. Similarly, leaf ·OH content elevated by about 12% and 22% due to 25 mg L− 1 TP treatment on the 2 and 4 days, respectively, as well as increased by about 43% to 100 mg L− 1 TP on the 4 days. Wheat seedlings treated by both TP concentrations on the 6 days, exhibited in the reduction of leaf ·OH content. In addition, the application of TP on the 2 and 4 days did not affect leaf ·OH production in salt-treated seedlings, but salt-induced ·OH generation significantly reduced at 6 days of 100 mg L− 1 TP treatment, compared with salt treatment alone (Table 2).
Changes of DAO and PAO activities in wheat leaves under different treatments
As shown in Table 3, DAO activity in response to 150 mM NaCl stress alone notably increased on the 4 and 6 days, but no obviously changed on the 2 days, in comparison with the control. In the early and middle stage of 25 mg L− 1 TP treatment (2 and 4 days), leaf DAO activity decreased initially, followed by an increase. Differently, in the middle and late stage of 100 mg L− 1 TP treatment (4 and 6 days), this parameter showed the changes of increased initially, followed by decrease, in comparison with the control. Compared with the salt treatment alone, leaf DAO activity in salt-treated seedlings decreased by 51%, 33% and 35% in the presence of 25 mg L− 1 TP on the 2, 4 and 6 days, respectively. After the seedlings were treated with NaCl + 100 mg L− 1 TP on the 2 days, the activity of DAO reduced by about 32%, but did not significantly alter on the 4 and 6 days, compared to salt treatment alone.
Table 3
Changes of diamine oxidase (DAO) and polyamine oxidase (PAO) activities in wheat leaves under different treatments
|
Treatments
|
Two days
|
Four days
|
Six days
|
DAO activity
(U mg− 1 protein)
|
CK
|
0.84 ± 0.07c
|
1.07 ± 0.02a
|
2.97 ± 0.09c
|
150 mM NaCl
|
0.91 ± 0.01c
|
1.79 ± 0.01c
|
3.39 ± 0.01d
|
25 mg L− 1 TP
|
0.67 ± 0.02b
|
1.71 ± 0.09c
|
2.95 ± 0.04c
|
100 mg L− 1 TP
|
0.83 ± 0.04c
|
1.92 ± 0.06c
|
1.95 ± 0.02a
|
150 mM NaCl + 25 mg L− 1 TP
|
0.45 ± 0.02a
|
1.20 ± 0.01b
|
2.27 ± 0.03b
|
150 mM NaCl + 100 mg L− 1 TP
|
0.62 ± 0.04b
|
1.85 ± 0.01c
|
3.38 ± 0.03d
|
PAO activity
(U mg− 1 protein)
|
CK
|
5.79 ± 0.44e
|
3.73 ± 0.30c
|
1.76 ± 0.02bc
|
150 mM NaCl
|
5.65 ± 0.27de
|
4.62 ± 0.29cd
|
2.31 ± 0.01d
|
25 mg L− 1 TP
|
4.63 ± 0.17c
|
2.70 ± 0.24a
|
2.05 ± 0.04cd
|
100 mg L− 1 TP
|
5.16 ± 0.19d
|
3.15 ± 0.33b
|
1.28 ± 0.03a
|
150 mM NaCl + 25 mg L− 1 TP
|
3.04 ± 0.25a
|
4.01 ± 0.33d
|
1.60 ± 0.07b
|
150 mM NaCl + 100 mg L− 1 TP
|
4.09 ± 0.10b
|
3.85 ± 0.12c
|
2.08 ± 0.03cd
|
Values represent mean ± SE of at least three experiments with replicated measurements; Different small letters mean significant difference at 0.05 levels. TP: Tea polyphenols |
Leaf PAO activity of 150 mM NaCl stress on the 2 days showed no significant change, but significantly increased by about 24% and 31% on the 4 and 6 days, compared with that of the control. Treatment with 25 mg L− 1 TP alone on the 2 and 4 days resulted in significant inhibition of leaf PAO activity, while this parameter decreased to 89%, 84% and 73% of the control on the 2, 4 and 6 days of 100 mg L− 1 TP treatment, respectively. The application of TP effectively alleviated salinity-induced effect on the activity of PAO. For example, when 25 mg L− 1 TP was added on the 2, 4 and 6 days, leaf PAO activity in salt-treated seedlings decreased by about 46%, 13% and 31%, respectively, in comparison with salt-alone-stressed ones (Table 3).