The activities of superoxide dismutase (SOD), peroxidase (POD), catalase (CAT), and ascorbate peroxidase (APX), along with reduced glutathione (GSH), hydrogen peroxide (H₂O₂) and malondialdehyde (MDA) contents were determined in the leaves to study the effects of Mn²⁺ on the antioxidant system of leaves of “Hong yang” tissue culture seedlings. POD activity was determined by the guaiacol oxidation method [17]. CAT activity was determined using visible light spectrophotometry [18]. The SOD activity was determined by the nitrogen blue tetrazole (NBT) photoreduction method [19]. The APX activity was determined by determining the oxidation rate of ascorbic acid [20]. The GSH content was determined using 2-nitrobenzoic acid (DTNB) [21]. The MDA content was determined by the thiobarbituric acid (TBA) method [22]. The H₂O₂ content was determined by the titanium salt-based spectrophotometry method [23].

The relative expression of the genes was determined using real-time quantitative PCR (qRT-PCR). The reverse transcription product cDNA synthesized from the total RNA of the “Hong yang” seedling leaves was used as the template for qRT-PCR. According to the conserved region sequences of POD27 and POD64 genes of the Mn-SOD and POD gene family members in the kiwifruit genome database, combined with the transcriptome sequencing results of “Hong yang” Kiwifruit, gene-specific PCR primers were designed accordingly (see Table 1), which were then synthesized by Shanghai Weiqing Biotechnology Co., Ltd. (China). Using cDNA as a template and polyubiquitin (UBQ) as the internal reference gene, qRT-PCR was performed.

SOD is a widely existing metalloenzyme in organisms. It can catalyze the dismutation of superoxide anions and is vital in the biological antioxidant system. As shown in Table 2, as compared with the control treatment (water spraying), spraying MnCl₂^.4H₂O solution on the leaf can improve the SOD activity of “Hong yang” seedlings. Under the same measurement time, with the increase in the MnCl₂^.4H₂O concentration, the SOD activity of leaves of “Hong yang” seedlings first increased and then decreased. Under 50 μM MnCl₂^.4H₂O, the SOD activity was significantly higher than that of other concentrations, especially, 12 h post treatment, the SOD activity was the highest, with the difference being the most significant. Furthermore, at the same treatment concentration and different determination times, the SOD activity also showed a trend of first increasing and then decreasing, with the SOD activity at 12 h being the highest. In conclusion, the SOD activity of “Hong yang” leaves can be improved by using 50 μM MnCl₂^.4H₂O, with it being the most significant at 12 h post treatment.

POD catalyzes diverse oxidative reactions involving H₂O₂, and is closely related to various physiological and biochemical processes, including respiration, photosynthesis, and auxin oxidation. It is vital in the cellular redox metabolism. As shown in Table 3, as compared with the control treatment, foliar spraying with MnCl₂^.4H₂O improved the POD activity of “Hong yang” seedlings. POD activity at 50 μM MnCl₂^.4H₂O was significantly higher than those of the control group and 5 μM treatment groups, with the most significant difference observed at 12 h. However, when MnCl₂^.4H₂O was (≤50 μM), the POD activity in the leaves first increased, and then decreased with the increasing determination time, but it only decreased at 100 μM. With the increasing concentration, the POD activity of the leaves of “Hong yang” showed a trend of first increasing and then decreasing during the same determination time, with its peak being observed at 50 μM at 12 h post treatment. Therefore, MnCl₂^.4H₂O at 50 μM was the most beneficial for improving the POD activity of the “Hong yang” leaves.

CAT assists SOD in removing superoxide free radicals. In plants, the vital coordination between SOD, CAT, and POD maintains the cellular redox homeostasis. Table 4 shows that the CAT activity in the “Hong yang” leaves first increased and then decreased with the increasing MnCl₂^.4H₂O treatment concentration at the same determination time. At 50 μM, the CAT activity of leaves was the highest among all concentrations (p < 0.05). Under different determination times, the CAT activity showed an “N” type trend under control treatment, with the maximum being 4.391 U·g⁻¹ at 24 h post treatment. At 5 μM MnCl₂^.4H₂O, the CAT activity first decreased and then increased, with the minimum value of 3.121 U·g⁻¹ being reached at 12 h post treatment. Although the opposite trend was seen at 10 or 50 μM, the maximum CAT activities were 7.158 and 11.213 U·g⁻¹, respectively, at 12 h post spraying. However, for 100 μM, the CAT activity decreased with the increasing time. Thus, the CAT activity of leaves was significantly affected by the MnCl₂^.4H₂O treatment. Therefore, spraying MnCl₂^.4H₂O at 50 μM can promote and improve the CAT activity, with the effect being best at 12 h after spraying.

APX is a specific peroxidase with ascorbic acid as the electron donor, which directly affects the ascorbic acid content in plants. As one of the most important antioxidant enzymes involved in the metabolism of reactive oxygen species (ROS) in plants, it can remove the damaging H₂O₂ that accumulated and protect chloroplasts and other cellular components. As can be seen from Table 5, at the same measurement time, with the increasing MnCl₂^.4H₂O concentration, the APX activity in the “Hong yang” leaves first increased and then decreased. The same trend was also seen for the same concentration and different determination times. At 50 μM, the APX activity showed the maximum value, and was significantly higher than the control group (p < 0.05). The APX activity in the leaves also increased first and then decreased when the same concentration of MnCl₂^.4H₂O was sprayed under different. When the treatment concentration was ≤10 μM, the APX activity was the maximum at 24 h post treatment, which when compared with other treatment times, the increase was not significant (p < 0.05). Although when the treatment concentration was (>10 μmol·L⁻¹), the APX activity peaked at 12 h post treatment, which was not significant as compared with other treatment times. Therefore, the APX activity in leaves was significantly affected by MnCl₂^.4H₂O treatment, which significantly differed from the control group. Therefore, spraying 50 μM MnCl₂^.4H₂O can improve the APX activity, with the effect being the best at 12 h post spraying.

GSH is an important antioxidant participating in the ASA-GSH cycle that is vital to abiotic stress tolerance. It helps maintain the plant immune system, with its antioxidative and integrated detoxification effects. Table 6 shows that, at the same determination time, with the increasing MnCl₂^.4H₂O concentration, the GSH content in “Hong yang” first increased and then decreased. At 50 μM, the GSH content was the highest, which significantly differed from the control group. However, under the same concentration at different determination times, although it showed a trend of first increasing and then decreasing, it tended to stabilize and was not significantly different. Therefore, the GSH content of leaves was significantly affected by the MnCl₂^.4H₂O, which was improved by spraying at a concentration of 50 μM, and reached the highest (62.22 µg·g⁻¹) at 12 h after spraying.

MDA is the end product of membrane lipid peroxidation, which inhibits the cell protective enzymes. Since the MDA content can reflect the stress-induced cellular stability, increased plant MDA content indicates increased plant cell membrane damage. As can be seen from Table 7, within the same determination time, the MDA content in “Hong yang” showed a trend of first decreasing and then increasing with the increasing MnCl₂^.4H₂O treatment concentration, with the minimum observed at 50 μM. Meanwhile, as compared with the control group, the MDA content in the “Hong yang” leaves had significantly decreased by 16.4%, 13.3%, 19.2%, and 24.89%. Under the same treatment concentration of MnCl₂^.4H₂O and different determination times, the MDA content in leaves also showed a trend of first decreasing and then increasing. It can be seen that the MDA content in the leaves is affected by the concentration of MnCl₂^.4H₂O. Foliar spraying of 50 μM MnCl₂^.4H₂O can significantly reduce the MDA content, with the best effect (5.312 nmol^.g⁻¹) seen at 12 h post treatment.

H₂O₂ is the most common reactive oxygen molecule in living organisms and is the hub of mutual transformation of reactive oxygen species. It is mainly generated as a byproduct by SOD and xanthine oxidase (XOD), which is then degraded by CAT and POD. It can not only directly or indirectly oxidize nucleic acid, protein, and other biological macromolecules, but also damage the cell membrane, thereby accelerating cellular aging and disintegration. As can be seen from Table 8, H₂O₂ content in the leaves of the “Hong yang” seedlings first decreased and then increased with increasing treatment concentration during the same determination time. At 50 μM MnCl₂^.4H₂O, the H₂O₂ content in leaves was 98.02%, 96.63%, 97.47% and 97.54% of that in control group, and significantly decreased (p < 0.05). Under the same treatment concentration and different determination times, the H₂O₂ content of leaves also showed a trend of first decreasing and then increasing. When the treatment concentration was ≤10 μM, the minimum H₂O₂ content was at 24 h post treatment, with the difference not being significant as compared to other determination times. However, when the concentration was >10 μM, the H₂O₂ content was the minimum at 12 h post treatment, which did not significantly differ from other determination times. Therefore, at the same treatment concentration and the increasing determination times, the H₂O₂ content was unaffected, as its enzymatic decomposition remained similar irrespective of the increasing determination time post MnCl₂^.4H₂O treatment. Therefore, when the spraying time of MnCl₂^.4H₂O is 12 h, the minimum H₂O₂ content was 72.167 μmol^.g⁻¹ when the spraying concentration was 50 μM.

Mn-SOD mainly exists in prokaryotic cells and a few plant cells, where most are located in their mitochondria. Besides chloroplasts, plant mitochondria are the second site producing ROS, with Mn-SOD located in them being vital in clearing ROS. As can be seen from Fig. 1, with the increase in the Mn²⁺ treatment concentration, the Mn-SOD expression level first increased and then decreased under different determination times. However, when the Mn²⁺ was >50 μM), the Mn-SOD expression showed a decreasing trend under different determination times. Therefore, with the increasing Mn²⁺ concentration, the Mn-SOD expression was significantly up-regulated under the same determination time, as compared to the control group. However, when the Mn²⁺ concentration was too high, it had greater toxicity to the leaves of “Hong yang” seedlings.

At the same concentration of Mn²⁺, the Mn-SOD expression in the leaves also showed a trend of first increasing and then decreasing with the increasing determination time, while peaking at 12 h post treatment, which was higher than other treatments. Therefore, at 50 μM MnCl₂^.4H₂O and 12 h post treatment, the Mn-SOD expression was the highest. But when the determination time was >12 h, the promoting effect of Mn²⁺ on the antioxidant system of “Hong yang” seedling leaves would decline, and the effect would be gradually weakened with the extension of time.

POD27 and POD64 are mainly involved in hydrogen peroxide decomposition, auxin metabolism, lignin synthesis, and stress response in intercellular spaces and vacuoles. Fig. 2 shows that with the increasing Mn²⁺ concentration, the POD27 expression in leaves first increased and then decreased when the determination time was ≤24 h. However, when Mn²⁺ concentration ≥50 μM, the expression level of the POD27 expression in leaves showed a decreasing trend at all determination times. Moreover, when the determination time was (>24 h), it showed a downward trend even with the increasing Mn²⁺ concentration. Therefore, the POD27 expression was the highest when the determination treatment time was 12 h and the Mn²⁺ concentration was 50 μM.

With the increasing Mn²⁺ concentration, the POD64 expression showed an “M” shaped trend. At the same Mn²⁺ concentration, it first increased and then decreased with the increasing determination time. However, at 12 h and 50 μM, the POD64 expression level was the highest (see Fig. 3).