*2.2. E*ff*ect of Temperature Decrease on ROS-Scavenging Enzyme Activities in P. tobira Cultivars*

The natural occurrence of leaf variegation in plants suggests that the trait might have adaptive functions [32]. In line with this, we investigated the enzymatic changes with respect to cold stress response in leaves of "Variegatum" and "Green Pittosporum" cultivars over a period of three months (from August to November) when the ambient temperature decreases from optimal condition (20 ◦C) to cold condition (10 ◦C). The results showed that the two cultivars respond similarly to the temperature decrease (Figure 4). The activities of all the three ROS-scavenging enzymes including peroxidase (POD), catalase (CAT), superoxide dismutase (SOD), were increased over the assayed period. Notably, CAT and POD displayed a sharp increase in response to the temperature decrease. At the lowest temperature (10 ◦C, November 15th), POD and CAT activities were significantly higher (*P* < 0.01) in "Variegatum" as compared to "Green Pittosporum" (Figure 4A–C), denoting a stronger response to cold in "Variegatum". We extended the investigation on the MDA contents in both cultivars in order to record the stress levels induced by the temperature decrease. As expected, MDA levels also increased in both cultivars with the temperature decrease, but "Variegatum" seems to suffer less from cold stress. This is evidenced by the significantly higher (*p* < 0.01) MDA in "Green Pittosporum" when the temperature reached 10 ◦C (Figure 4D).

Overall, our results indicated that "Variegatum" is endowed with an efficient ROS-scavenging enzymatic system, which is mainly triggered under low temperature. Hence, leaf variegation trait plays a low temperature protective function in *P. tobira*.

**Figure 4.** Antioxidant enzymatic activities during temperature decrease. (**A**) Peroxidase activity (POD), (**B**) Superoxide dismutase activity (SOD), (**C**) Catalase activity (CAT), (**D**) malonaldehyde content (MDA). \*\* above the lines represents significant difference between the two cultivars at *p* < 0.001, using Tukey HSD test.
