Biomass and accumulation of proline
After 21 days of Ce or Sm exposure, the growth parameters of the wheat plants showed significant alternations when treated with various concentrations of REE (Table 1,2). Plant FW increased significantly by 56.43% in Arta and 25.4% in Baharan at 2500 µM Ce treatment, respectively, as compared to control. Other levels of Ce treatment declined plant FW in two cultivars and the rate of reduction was the highest levels in 15000 µM Ce-treated plants. Ce treatment at 2500 µM enhanced (68.42%) plant DW in Arta cultivar, but 15000 µM Ce decreased (47.37%) this parameter as compared to control. Plant DW enhanced with 2500 µM Ce by 32.14% in Baharan cultivar, but plant DW of Baharan cultivar reduced by 7.14, 7.14, 39.28 and 67.85% with the application of 5000, 7500, 1000 and 15000 µM Ce, respectively, in comparison with the control. However, only with 15000 µM was there significant difference with respect to the control. In Arta cultivar, the length of the plants treated with 2500 to 10000 µM Ce were not significantly affected. However, the length plant was significantly reduced in Arta cultivar for applied 15000 µM Ce concentration when compared to the control. Plant length declined in Baharan cultivar with increasing Ce concentration. Root length diminished in Arta cultivar at 7500, 10000 and 15000 µM Ce, but different concentrations of Ce did not induce any significant changes in root length. The root length in Baharan cultivar decreased significantly with the application of all levels of Ce, in comparison with the control. Contrary to the effect on root length, Ce treatment had no influence on shoot length in two cultivars (Table 1).
Table 1
Impact of Ce on growth parameters in wheat plants.
Ce (µM)
|
Cultivar
|
Plant FW
(g)
|
Plant DW
(g)
|
Plant length
(cm)
|
Root length
(cm)
|
Shoot length (cm)
|
0
|
Arta
|
2.02 ± 0.460 bc
|
0.196 ± 0.040 c-f
|
48 ± 9.165 b-d
|
19.6 ± 6.70 bc
|
28.4 ± 2.468 b-d
|
2500
|
|
3.16 ± 0.238 a
|
0.326 ± 0.039 ab
|
50.5 ± 5.392 bc
|
22 ± 2.516 b
|
28.5 ± 3.5 b-d
|
5000
|
|
1.43 ± 0.194 c-e
|
0.206 ± 0.013 c-e
|
44.5 ± 2.753 b-e
|
12.5 ± 1.755 b-d
|
32 ± 1.040 a-c
|
7500
|
|
1.25 ± 0.151 c-f
|
0.210 ± 0.208 cd
|
41.833 ± 1.691 b-e
|
9.433 ± 0.788 d
|
32.4 ± 1.248 a-c
|
10000
|
|
1.09 ± 0.226 d-f
|
0.213 ± 0.033 cd
|
35.666 ± 2.682 de
|
8.266 ± 1.134 d
|
27.4 ± 1.646 c-e
|
15000
|
|
0.55 ± 0.066 f
|
0.106 ± 0.017 ef
|
32.266 ± 3.266 e
|
9.033 ± 1.583 d
|
23.23 ± 2.411 de
|
0
|
Baharan
|
2.56 ± 0.185 ab
|
0.286 ± 0.043 a-c
|
74 ± 6a
|
41.9 ± 5.901 a
|
32.1 ± 0.152 a-c
|
2500
|
|
3.21 ± 0.375 a
|
0.370 ± 0.058 a
|
56 ± 2.309 b
|
19.766 ± 0.788 bc
|
36.233 ± 1.920 a
|
5000
|
|
1.73 ± 0.321 cd
|
0.266 ± 0.029 b-d
|
47.166 ± 3.678 b-d
|
14 ± 2.516 b-d
|
33.166 ± 1.301 a-c
|
7500
|
|
1.34 ± 0.148 c-e
|
0.266 ± 0.020 b-d
|
44.733 ± 2.395 b-e
|
10.5 ± 0.763 cd
|
34.233 ± 1.794 ab
|
10000
|
|
0.856 ± 0.103 ef
|
0.176 ± 0.017 d-f
|
41.666 ± 0.666 c-e
|
10.5 ± 0.763 cd
|
31.166 ± 0.833 a-c
|
15000
|
|
0.496 ± 0.029 f
|
0.096 ± 0.008 f
|
30.666 ± 4.226 e
|
8.833 ± 1.922 d
|
21.833 ± 2.315 e
|
Values are means ± SE of three replicates. Different letters indicated significant (p<0.05) differences
Table 2
Impact of Sm on growth parameters in wheat plants.
Sm (µM)
|
Cultivar
|
Plant FW
(g)
|
Plant DW
(g)
|
Plant length
(cm)
|
Root length
(cm)
|
Shoot length (cm)
|
0
|
Arta
|
2.506 ± 0.199 ab
|
0.303 ± 0.032 cd
|
53.8 ± 0.435 a-c
|
22.333 ± 1.013 bc
|
31.466 ± 1.278 a-c
|
2500
|
|
3.266 ± 0.135 a
|
0.360 ± 0.01 bc
|
57.6 ± 3.780 ab
|
26.666 ± 3.527 ab
|
30.933 ± 1.484 a-c
|
5000
|
|
2.666 ± 0.377 ab
|
0.333 ± 0.029 b-d
|
53.566 ± 4.330 a-c
|
19.9 ± 3.044 b-d
|
33.666 ± 1.847 ab
|
7500
|
|
2.18 ± 0.740 a-c
|
0.29 ± 0.011 c-e
|
54.6 ± 1.908 a
|
18.766 ± 1.398 c-e
|
35.833 ± 0.935 a
|
10000
|
|
0.83 ± 0.061 d
|
0.166 ± 0.024 fg
|
38.566 ± 1.433 de
|
11.4 ± 1.242 ef
|
27.166 ± 0.666 c
|
15000
|
|
0.723 ± 0.127 d
|
0.126 ± 0.0371 g
|
35.4 ± 1.594 e
|
9.233 ± 0.338 f
|
26.166 ± 1.540 c
|
0
|
Baharan
|
2.856 ± 0.112 ab
|
0.33 ± 0.01 b-d
|
56.5 ± 1.322 a-c
|
21.933 ± 1.212 bc
|
34.566 ± 1.026 ab
|
2500
|
|
3.396 ± 0.362 a
|
0.42 ± 0.02 ab
|
64.166 ± 5.783 a
|
29.833 ± 4.693 a
|
34.333 ± 1.092 ab
|
5000
|
|
3.47 ± 0.625 a
|
0.47 ± 0.052 a
|
55.5 ± 2.886 a-c
|
18.666 ± 1.369 c-e
|
36.833 ± 1.540 a
|
7500
|
|
1.913 ± 0.624 b-d
|
0.256 ± 0.039 c-f
|
48.166 ± 5.333 b-d
|
17.333 ± 1.589 c-e
|
30.833 ± 3.982 a-c
|
10000
|
|
1.156 ± 0.386 cd
|
0.236 ± 0.063 d-f
|
45.666 ± 4.977 c-e
|
16.333 ± 2.587 c-f
|
29.333 ± 2.803 bc
|
15000
|
|
0.856 ± 0.076 d
|
0.183 ± 0.028 e-g
|
42.5 ± 1.755 de
|
13.666 ± 1.424 d-f
|
28.833 ± 1.641 bc
|
Values are means ± SE of three replicates. Different letters indicated significant (p<0.05) differences
In Arta cultivar, plant FW and plant DW were augmented by 17.8 and 20% for 2500 µM Sm, respectively. The reduction in Arta cultivar treated with 7500, 10000 and 15000 µm Sm was 12.8, 66.8 and 71.2% in plant FW and 3.3, 46.6 and 60% in plant DW compared to untreated plants, respectively. Plant FW in Baharan cultivar enhanced by 28 and 21.4% with application of 2500 and 5000 µM Sm. Other concentrations of Sm (7500, 10000 and 15000 µm Sm) caused 32.14, 58.93 and 69.64% decreases in plant FW of Baharan cultivar in compared with control groups, respectively. In Baharan cultivar, there was an increment of 27.3 and 42.42% in the plant DW with the addition of 2500 and 5000 µM Sm. Plant DW in Baharan cultivar declined in plants treated with high levels of Sm, in comparison with the control. These reductions were 24.24% for 7500 µM, 30.30% for 10000 µM and 45.45% for 15000 µM. In Arta cultivar, the plant length and root length were not significantly affected by the application of 2500–7500 µM Sm, compared with the control. On the other hand, the application of 10000 and 15000 µM Sm reduced the plant length and root length in Arta cultivar in comparison with the control Conversely, the shoot length in Arta cultivar showed a significant trend with the application of Sm. The plant length and root length in Baharan cultivar did not change significantly at Sm concentrations up to 10000 µM, while these parameters dropped with highest level of Sm (15000 µM). Also, no significant difference was found in the shoot length between the Sm-treated and control plants in Baharan cultivar (Table 2).
No significant impacts on proline content were detected in all treatment of Ce in Arta cultivar, except for plants treated with 15000 µM Ce. The 15000 µM Ce treatment enhanced proline accumulation by 844 fold in Arta cultivar. In Baharan cultivar, low and moderate concentrations of Ce (2500–7500 µM) had no significant impact on proline content as compared to that of control, whereas high Ce concentrations (10000 and 15000 µM) caused a significant rise by 54.2 and 101.2 fold in proline content, respectively (Table 3). Proline content was not significantly affected by 2500–7500 µM Sm treatment in Arta cultivar relative to that of the control treatment. The exposure of 10000 and 15000 µM Sm caused significant elevation by 16 and 33.12 fold of proline content in Arta cultivar, displaying an increasing trend with exposure dose. Treatments with 7500 µM Sm in Baharan cultivar induced a significant enhancement in proline content, whereas other treatments did not cause any significant impact on proline content (Table 4).
Table 3
Impact of Ce on proline content, H2O2 content and MDA content in wheat plants.
Ce (µM)
|
Cultivar
|
Proline content
(µg g− 1FW)
|
H2O2 content
( µg g− 1FW )
|
MDA content
(nmol g− 1FW)
|
0
|
Arta
|
0.817 ± 0 d
|
8.552 ± 0.034 e
|
0.0077 ± 0 c
|
2500
|
|
1.295 ± 0.670 d
|
9.043 ± 0.045 d
|
0.0092 ± 0 c
|
5000
|
|
0.817 ± 0 d
|
10.208 ± 0.055 a
|
0.0151 ± 0.0001 b
|
7500
|
|
32.915 ± 3.064 d
|
7.408 ± 0.019 f
|
0.0256 ± 0.0008 a
|
10000
|
|
4.061 ± 1.810 d
|
7.457 ± 0.034 f
|
0.0251 ± 0.0003 a
|
15000
|
|
689.530 ± 0 a
|
5.911 ± 0.047 h
|
0.0275 ± 0.0002 a
|
0
|
Baharan
|
3.036 ± 0.171 d
|
9.946 ± 0.077 b
|
0.0085 ± 0 c
|
2500
|
|
5.097 ± 1.911 d
|
8.400 ± 0.032 e
|
0.0072 ± 0.0004 c
|
5000
|
|
3.469 ± 2.031 d
|
9.513 ± 0.036 c
|
0.0149 ± 0.0002 b
|
7500
|
|
2.883 ± 0.305 d
|
5.304 ± 0.072 i
|
0.0189 ± 0.0004 b
|
10000
|
|
163.823 ± 2.801 c
|
5.204 ± 0.142 i
|
0.0249 ± 0.0046 a
|
15000
|
|
308.327 ± 131.283 b
|
6.794 ± 0.072 g
|
0.0240 ± 0.0002 a
|
Values are means ± SE of three replicates. Different letters indicated significant (p<0.05) differences
Table 4
Impact of Sm on proline content, H2O2 content and MDA content in wheat plants.
Ce (µM)
|
Cultivar
|
Proline content
(µg g− 1FW)
|
H2O2 content
( µg g− 1FW )
|
MDA content
(nmol g− 1FW)
|
0
|
Arta
|
12.643 ± 1.030 d
|
11.454 ± 0.051 c
|
0.0397 ± 0.0006 g
|
2500
|
|
20.258 ± 0.381 d
|
10.504 ± 0.046 d
|
0.0412 ± 0.0007 g
|
5000
|
|
11.960 ± 0.591 d
|
10.095 ± 0.035 e
|
0.0536 ± 0.0009 f
|
7500
|
|
18.596 ± 2.037 d
|
8.820 ± 0.031 f
|
0.0880 ± 0.0027 d
|
10000
|
|
202.243 ± 1.431 bc
|
7.882 ± 0.073 i
|
0.1286 ± 0.0011 b
|
15000
|
|
418.696 ± 93.619 a
|
6.496 ± 0.110 k
|
0.1443 ± 0.0005 a
|
0
|
Baharan
|
122.006 ± 3.527 c
|
13.540 ± 0.035 a
|
0.0445 ± 0.002 g
|
2500
|
|
32.505 ± 0.870 d
|
12.137 ± 0.054 b
|
0.0428 ± 0.0026 g
|
5000
|
|
21.373 ± 0.290 d
|
8.182 ± 0.023 h
|
0.0778 ± 0.0022 e
|
7500
|
|
223.926 ± 6.280 b
|
8.442 ± 0.015 g
|
0.0847 ± 0.0011 d
|
10000
|
|
23.696 ± 1.453 d
|
8.005 ± 0.0788 i
|
0.0999 ± 0.0045 c
|
15000
|
|
162.813 ± 2.578 bc
|
6.845 ± 0.077 j
|
0.1266 ± 0.0010 b
|
Values are means ± SE of three replicates. Different letters indicated significant (p < 0.05) differences |
H2O2 and MDA content
In Arta cultivar, H2O2 content augmented at 2500 and 5000 µM Ce and then decreased to values lower than that of control at other doses of Ce treatment. H2O2 content in Baharan cultivar reduced significantly following treatment with Ce at all concentrations as compared to control (Table 3). Application of varying Sm doses to Arta and Baharan cultivar significantly declined H2O2 content and maximum decrease showed at 15000 µM (Table 4).
The 2500 µM Ce treatment did not cause any significant impact on MDA content in both cultivars. The MDA content at 5000, 7500, 10000 and 15000 µM Ce treatment was 114.28, 265.71, 258.57 and 285.71% in Arta cultivar and 75, 125, 211.25, 200% in Baharan cultivar larger than the control (Table 3). Treatment with 2500 µM Sm had no significant effect on the MDA content in both cultivars. The MDA content showed a significant rise by 35.89, 125.64, 228.20 and 269.23% in Arta cultivar and 75, 90.90, 125, 186.36% in Baharan cultivar at 5000, 7500, 10000 and 15000 µM Sm treatment as compared to control (Table 4).
Enzymatic antioxidant system
Ce treatment boosted protein content in both cultivars (Fig. 1a). Sm treatment also caused an increase in the protein content in Arta cultivar, whereas this was not caused significant change trend in Baharan cultivar (Fig. 1b).
The activities of the ROS scavenging enzymes, the antioxidant enzymes SOD, POX and PPO are shown in Fig. 1,2. Ce treatment considerably declined the SOD activity in both cultivars as compared to that of control and no significant difference was found in the SOD activity between various levels of Ce. The reduction in SOD activity of Baharan Cultivar was more than Arta cultivar (Fig. 1c). No significant difference was found between the POX activity in Ce-treated and control plants in Arta cultivar. A decrease was seen in POX activity of Baharan cultivar under Ce treatment as compared to control (Fig. 2a). PPO activity was significantly reduced in the two cultivars at all Ce doses (Fig. 2c).
All of Sm treatments dropped SOD activity in both cultivars, but this reduction was higher in the Arta cultivar when compared to the Baharan (Fig. 1d). A decrement in POX activity occurred in all levels of Sm treatment in two cultivars and no significant changes in activity of POX were observed between different concentrations (Fig. 2b). PPO activity enhanced in Arta cultivar by 2500 µM Sm and then decreased at higher concentrations. Treatments with all tested Sm concentrations induced no significant variations in PPO activity of Baharan cultivar (Fig. 2d).
Non-Enzymatic antioxidant system
The effect of Ce and Sm on the non-enzymatic antioxidants of two wheat cultivars is displayed in Fig. 3,4. Total phenol content was heightened under Ce treatment up to 2500 µM in both cultivars; the effect of Ce on total phenol content was more pronounced in Baharan cultivar (Fig. 3a). Flavonoid content in both cultivars boosted in plants treated with 2500 µM Ce, but in the plants treated with other doses of Ce a significant decline was observed (Fig. 3b). Anthocyanin content only enhanced by 2500 µM Ce whereas it reduced in both cultivars at 5000–15000 µM Ce as compared to non-treated plants (Fig. 4a).
The total phenol content in Arta cultivar was significantly reduced and lower than that of the control under the Sm treatments. Total phenol content was higher in Baharan cultiva exposed to 2500 and 5000 µM Sm comparison with the control, whereas higher doses caused significant decrease in this content (Fig. 3b). A significant decrease in flavonoid content in Arta cultivar was detected under Sm treatments. Treatment with 7500–15000 µM Sm declined flavonoid content in Baharan cultivar, while no significant differences were found at other Sm treatments, compared with the control (Fig. 3d). Similar results were observed for anthocyanin content in both cultivars under Sm treatment. In both cultivars, anthocyanin content increased at 2500 and 5000 µM Sm, then decreased up to 15000 µM Sm to values higher than that of control (Fig. 4b).
Investigation of correlation between various Ce and Sm levels and studied parameters by PCA analyze
The potential correlations among the studied variables under the different treatments of Ce and Sm were analyzed based on Pearson's correlation coefficient. According to this analyze, DW or growth in wheat plants treated with Ce and Sm had more positive correlation with non- enzymatic antioxidant system than that enzymatic antioxidant system. Thus, these findings confirmed that that non-enzymatic antioxidant mechanism is more important for cope on destructive effects of Ce and Sm in wheat plants. In both treatments, growth negatively correlated with MDA level. The control treatment adjudged as the best value giving treatments. Its impact was followed by that of 2500 µM of Ce and Sm. This displayed the stimulatory effect of 2500 µM. The other doses caused growth limitations in wheat plants as these treatments were clustered on the other side of loading plot (Fig. 5a,b).