Physiological and biochemical responses of two precious Carpinus species to high-concentration NO2 stress and their natural recovery

Morphological changes of the leaves

The influence of NO₂ stress on the plants was first reflected by the morphological changes of the leaves (Fig. 1)^20,22. Slight NO₂ injury was manifested by slight green deficiency and light color. Moderate NO₂ injury was manifested by irregular watery spots between leaf veins, which gradually developed into yellow necrotic spots followed by lesions at the leaf stalk and margins. When the exposure time extended to 72 h, the leaves turned yellow, and irreversible injury occurred, which led to leaf death. The damaged areas of the leaves of the two species at different time points of NO₂ exposure are summarized in Table 1.

Changes in MDA content

The changes in the MDA content of C. betulus and C. putoensis at different time points of NO₂ stress are shown in Fig. 2. With the prolongation of NO₂ stress, the MDA content of C. betulus showed an increasing tendency with the variation range from 0.016 to 0.029 µmol g⁻¹ fw. However, no significant differences were observed at different time points of NO₂ exposure.

As NO₂ fumigation time extended, the MDA content of C. putoensis also showed an increasing tendency, with the variation range from 0.015 to 0.034 µmol g⁻¹ fw. Compared with the control group, a significant difference was observed in the MDA content from 24 h, which peaked at 72 h. In C. putoensis, although the recover group and the control group did not show a significant difference, the MDA content of the former lay between 0.015 (6 h) and 0.019 µmol g⁻¹ fw (12 h), which suggests that the plant did not recovered completely from the stress damage.

Compared with C. putoensis, C. betulus exhibited a smaller variation amplitude in the MDA content under NO₂ stress. The MDA content of C. betulus did not show noticeable changes at 1, 6, and 12 h, and it was till 24 h when a rapid increase occurred. These findings indicate a delayed injury response of C. betulus. In contrast, with the prolongation of NO₂ stress, the MDA content of C. putoensis noticeably increased, which indicates an increase in the membrane lipid peroxidation activity of C. putoensis under NO₂ stress.

Changes in POD activity

The changes in POD activity of C. betulus and C. putoensis at different time points of NO₂ stress are shown in Fig. 3. With the prolongation of NO₂ stress, the POD activity of C. betulus showed an increasing tendency, with a variation range from 323 to 663 U (g * min)⁻¹ fw. After 30-d self recovery, the POD activity returned to 409 U (g * min)⁻¹ fw, which was comparable to that of the control.

As NO₂ fumigation time extended, the POD value of C. putoensis also showed an increasing tendency, with a variation range from 385 to 596 U (g * min)⁻¹ fw. The recovery group did not show a significant difference compared with the control group.

In C. betulus, the POD activity value rapidly increased at 72 h of NO₂ stress, which showed a significant difference compared with any other group (adjusted p < 0.05). In C. putoensis, however, a significantly increased POD value appeared from 24 h. These findings indicate that C. putoensis presented with injury response earlier than C. betulus.

Changes in soluble protein content

The changes in the soluble protein content of C. betulus and C. putoensis under NO₂ stress at different time points are shown in Fig. 4. Despite that the soluble protein content of C. betulus slightly decreased at 1 h and 6 h compared with the control (0 h), no significant differences were observed among them. As the fumigation time extended, the soluble protein content showed an increasing trend, with the variations ranging from 2.32 to 4.65 mg g⁻¹ fw. The soluble protein contents did not show a significant difference between the recovery group and the control group (adjusted p > 0.05).

In C. putoensis, the soluble protein content also showed an increasing trend as the fumigation time prolonged. The variations ranged from 2.61 to 3.27 mg g⁻¹ fw. Compared with the control group, the recovery group exhibited a lower soluble protein content, although no significant difference was observed between them.

As shown in Fig. 4, the maximum difference in the soluble protein content of C. betulus was 2.33 mg g⁻¹ fw, which was greatly larger than that of C. putoensis (0.66 mg g⁻¹ fw). Particularly, C. betulus exhibited a rapid increase in the soluble protein content from 12 h of fumigation, which indicates that C. betulus increased protein synthesis when encountered with NO₂ stress, whereas C. putoensis showed only weak resistance against the stress.

Changes in NR

At 0 h of NO₂ treatment, the NR activity of C. betulus was 1.43 ± 0.14 µmol NO₂⁻·g⁻¹fw·h⁻¹. With the prolongation of NO₂ exposure, the NR activity of C. betulus exhibited a gradual increase followed by a gradual decrease, and a significant difference (adjusted p < 0.05) was observed from 24 h. After 30-d recovery, the NR activity returned to a normal level (adjusted p > 0.05). In C. putoensis, the NR activity of the control group was 0.58 ± 0.06 µmol NO₂⁻·g⁻¹fw·h⁻¹. As the NO₂ exposure time prolonged, NR activity exhibited a rapid increase (adjusted p < 0.05) followed by a fast decrease. After 30-d recovery, the index returned to a normal level (adjusted p > 0.05). The results were shown in Fig. 5.

Changes in NO₃
⁻N

As the NO₂ treatment time extended, the NO₃⁻N content of C. betulus exhibited an increase followed by a gradual decrease, and a significant difference (adjusted p < 0.05) was observed from 24 h. After 30-d recovery, the gradual returned to a normal level (adjusted p > 0.05). In C. putoensis, the NO₃⁻N content also exhibited an increase followed by a decrease after NO₂ exposure. However, a significant difference was observed from 12 h. After 30-d recovery, the index returned to a normal level (adjusted p > 0.05). The results were shown in Fig. 6.

Changes in mineral elements

The changes in the mineral elements of C. betulus and C. putoensi under NO₂ stress and after self recovery are summarized in Table 2.

Macroelements

(1) N. At 1 h of NO₂ stress, the total nitrogen content of C. betulus increased slightly to 1.68 ± 0.17 g/kg; this value was higher than that of control (1.4 ± 0.13 g/kg), but no significant difference was observed (adjusted p > 0.05). With the prolongation of the stress, the content decreased, with the variations ranging from 0.84 to 1.68 g/kg and the maximum difference of 0.84 g/kg. The recovery group did not show a significant difference compared with the control group (1.53 ± 0.15 vs. 1.4 ± 0.13; adjusted p = 1.00).

Overall, the changes in the total nitrogen content of C. putoensis showed a similar trend with those of C. betulus. At 1 h of NO₂ stress, the total nitrogen content of C. putoensis significantly increased compared with that of the control (1.68 ± 0.15 g/kg vs. 1.12 ± 0.11 g/kg; adjusted p < 0.001). With the prolongation of NO₂ fumigation, the content gradually decreased, with the variations ranging from 0.46 to 1.68 g/kg and the maximum difference of 1.22 g/kg. No significant difference was observed between the recovery group and the control group (adjusted p = 1.00). Although both species showed noticeable changes in the total nitrogen content compared with their corresponding control, the variation amplitude of C. putoensis was much greater than that of C. betulus (1.22 g/kg vs. 0.84 g/kg).

(2) P. As the NO₂ stress prolonged, the P content of C. betulus increased, showing significant differences compared with the control. The variations ranged from 0.72 to 4.69 ppm dw, with the maximum difference of 3.97 ppm dw. No significant difference was observed between the recovery group and the control group (0.72 ± 0.06 vs. 0.93 ± 0.08; adjusted p = 1.00). Compared with the control, the P content of C. putoensis undergoing NO₂ stress showed an increase followed by a decrease. The variations ranged from 1.85 to 4.78 ppm dw with the maximum difference of 2.93 ppm dw.

(3) K. With the prolongation of NO₂ exposure, the K content of C. betulus gradually decreased, and a significant difference was observed from 12 h. The variations in the K content ranged from 11.4 to 21.6 µg L⁻¹ dw, with maximum difference of 10.2 µg L⁻¹ dw. The K content of C. putoensis showed a similar trend to that of C. betulus. The variations in the K content ranged from 9.8 to 30.2 µg L⁻¹ dw. Compared with C. betulus, C. putoensis exhibited a relatively greater amplitude of the variations in the K content (10.2 µg L⁻¹ vs. 20.4 µg L⁻¹ dw). In both species, the recovery groups did not show a significant difference compared with the control (adjusted p > 0.05).

(4) Ca. With the prolongation of NO₂ exposure, the Ca content of C. betulus exhibited an increase followed by a gradual decrease, and the variations ranged from 84 to 243 µg L⁻¹ dw. A significant difference was observed at 72 h of NO₂ exposure. In C. putoensis, significant differences in the Ca content were observed during NO₂ exposure, except at 12 h. In both species, the Ca content of the recovery group did not show a significant difference compared with the control group. The variation amplitude of the Ca content of C. betulus (159 µg L⁻¹ dw) was noticeably greater than that of C. putoensis (68 µg L⁻¹ dw).

(5) Mg. As the NO₂ stress prolonged, the Mg content of C. betulus did not show a significant drop, except at 48 h. The variations ranged from 21.4 to 31.3 µg L⁻¹ dw. In C. putoensis, the variations ranged from 12.2 to 32.2 µg L⁻¹ dw. In both species, the Ca content of the recovery group did not show a significant difference compared with the control group. The variation amplitude of the Ca content of C. betulus (9.9 µg L⁻¹ dw) was remarkably smaller than that of C. putoensis (20 µg L⁻¹ dw).

Microelements

(1) Zn. With the prolongation of NO₂ exposure, the Zn content of C. betulus exhibited an increase followed by a gradual decrease. Compared with the control, significant differences were observed at 1, 6, and 12 h. The variations anged from 7.1 to 10.6 µg L⁻¹ dw. In C. putoensis, significant differences in the Zn content were observed at 6 h and 48 h, and the variations ranged from 5.7 to 11.2 µg L⁻¹ dw. The variation amplitude of the Zn content of C. betulus (3.5 µg L⁻¹ dw) was smaller than that of C. putoensis (5.5 µg L⁻¹ dw). In each species, the Zn content of the recovery group showed a significant difference compared with the control group.

(2) Mn. At 1 h of NO₂ fumigation, a sharp drop was observed, compared with the control. Afterwards, the Mn content of C. betulus exhibited a general increase followed by a gradual decrease. However, at any time point during NO₂ exposure, a significant lower Mn content was observed when compared to the control. The variations of the Mn content ranged from 11.2 to 78.1 µg L⁻¹ dw. In C. putoensis, the Mn content during NO₂ exposure significantly decreased compared with control, and the variations ranged from 9.4 to 85.5 µg L⁻¹ dw. The variation amplitude of the Mn content of C. betulus (66.9 µg L⁻¹ dw) was slightly smaller than that of C. putoensis (76.1 µg L⁻¹ dw). In each species, the Mn content of the recovery group did not show a significant difference compared with the control group.

Correlation analysis

The correlations between the investigated indices and NO₂ exposure time were analyzed using the Pearson’s method (Table 3). POD and soluble protein had a strong positive correlation with NO₂ exposure time (correlation coefficient: 0.891 and 0.799, respectively), and NR, NO₃⁻N, N, K, and Ca had a strong negative correlation with NO₂ exposure time (correlation coefficient: -0.691, -0.805, -0.744, -0.606 and -0.696, respectively). MDA and the Zn content were not correlated with the exposure time.

Source: Ecology - nature.com