Study Site

The Changbai Mountain Nature Reserve (41°42’– 42°25’ N, 127°43’–128°17’ E), located in northeastern China, covers a total area of 196,465 hm², with its highest peak reaching 2744 m. [23, 24]. The annual average wind speed is 11.7 m/s. The annual precipitation in the reserve ranges from 700 to 1400 mm, 60% of which falls during the summer; winter and spring are dry, while autumn is foggy [25]. Because local average temperatures are low (as low as 2ºC), there is far less evaporation than precipitation, and the Changbai Mountain area is perennially wet, with an average annual relative humidity of 70%. The average annual temperature differential along the mountain slopes is at least 10°C [26], and the vertical landscape is typical of natural mountainous areas. Transition zones between each vegetation type are narrow, the vegetation is divided into five distinct vegetation types along the vertical gradient: broadleaved forest (<500 m), mixed coniferous and broadleaved forest (500-1100 m), conifero us forest (1100-1700 m), birch forest (1700-2100 m), and alpine tundra (>2100 m).

Sampling of Plants and Soil

Based on a preliminary investigation of L. caerulea plants growing on the northern slope of Changbai Mountain, a transect line was set along the road (Altitude gradient range of continuous distribution of L. caerulea shrubs) in July 2018 that spanned an elevation range of 800–1800 m and bisected a continuous distribution of shrubs. Along this transect, a sampling point was set up for every 200 m increase in elevation, for a total of seven sampling sites. At seven elevations (600, 800, 1000, 1200, 1400, 1600, and 1800 m), in each sampling area, 10-year-old plants (n = 30) at least 10 m apart were randomly selected among the Lonicera caerulea population (in the horizontal direction of each population per elevation level). To be included, the plant sampled had to be growing normally, with no signs of disease, insect pest infestations, or obvious structural defects. Three standard branches from each plant were randomly selected along five directions (east, south, west, north, and the top of the shrub), from which the second and third pairs of fully-grown and expanded leaves were collected from each branch tip for a total of 60 – 90 samples taken at each elevation. These leaves were removed and placed into a plastic self-sealed bag, then immediately taken to the laboratory in a portable refrigerator and stored in an ultra-low temperature refrigerator (−70ºC) for later testing. At the same time, a soil layer of 0~20 cm depth was taken from each sample plant, and the soil collected from 10 sample plants at each altitude was mixed to form a soil sample. Three soil samples were taken from each altitude, which were respectively put into cloth bags and brought back to the laboratory for analysis. Finally, at each sampling point location, plant sampling time, surface vegetation status, and meteorological data were recorded. The annual accumulated temperature of a year is calculated by adding up all the values of daily mean temperature ≥10ºC in 365 days of a year (The daily mean temperature is the average temperature for 24 hours a day. The calculation method is as follows: in meteorology, the average temperature of four times a day is usually taken as the average temperature of a day after adding together the temperature of 02, 08, 14 and 20).

Soil and Leaf Variables

The available P was extracted using sulfuric acid and hydrochloric acid and was determined using the molybdenum-antimony colorimetric method. The available K was extracted using ammonium acetate and was determined using a flame photometer, as was the extractable K. The hydrolysis N was determined using the alkali diffusion method. The total P was determined by molybdenum-antimony resistance colorimetric method. The total N was determined by automatic Kjeldahl nitrogen analyzer [26]. The pH of the soil was determined using the potential method. The potassium dichromate oxidation and reheating method was used to assess the soil organic matter. The soil samples were collected using a ring cutter with a volume of 100 cm³ to measure soil bulk density; they were then sealed in cloth bags and transported to the laboratory, where each was weighed to determine its bulk soil density.

Leaf samples (0.5 g) were homogenized in 10 ml HEPES-KOH buffer (pH 7.8) containing 0.1 mM EDTA. The homogenate was centrifuged at 15000 g for 15 min at 4ºC. In the supernatant, superoxide dismutase (SOD) activity was assayed by a photochemical method as described by Cakmak [27]. For catalase, the reaction mixture consisted of 25 mM potassium phosphate buffer (pH 6.8), 10 mM H₂O₂, and diluted enzyme extract in a total volume of 1 ml. The decomposition of H₂O₂ was followed by the decline in absorbance at 240 nm. Peroxidase activity was assayed using a reaction mixture containing 25 mM potassium phosphate buffer (pH 6.8), 10 mM H₂O₂, 0.05% guaiacol, and diluted enzyme extract. The oxidation of guaiacol was measured at 470 nm [27]. To measure ascorbate peroxidase (APX) activity, the decrease in absorbance at 290 nm due to ascorbate oxidation was measured using a reaction mixture based on the method described by Younis [28]. The reaction mixture contained 0.83 cm³ of 0.5 mM AsA in phosphate buffer (pH 7), 0.13 cm³ of 2 mM H₂O₂, and 0.04 cm³ of enzyme extract in a final volume of 1 cm³ at 25ºC [28].

Statistical Analysis

Data for the five measured leaf traits (SOD, POD, CAT, APX) were analyzed. First, one-way ANOVA was performed for each leaf trait variable. Each leaf trait variable was compared on a pairwise basis using Duncan’s multiple range test in SAS v9.1 software. To determine whether there was multicollinearity among independent variables, SPSS v19 was used, and any non-significant factors were removed. After this step (confirming no multicollinearity), the data had been rendered suitable for multiple stepwise regression analysis, and backward stepwise regression analysis was conducted.

Effects of the Altitude Gradient on Antioxidant Enzyme Activity in L. caerulea Leaves

In general, antioxidant enzyme activity in the L. caerulea leaves increased significantly with elevation up to 1600 m, followed by a significant decrease at 1800 m. Specifically, POD activity increased significantly between 800-1000 m and 1600 m, peaking at 1600 m and decreasing significantly at 1800 m (Fig. 1). Compared to POD activity at 1000 m, POD activity at 800 m, 1200 m, 1400 m, 1600 m, and 1800 m increased by 44.3%, 116.3%, 413.2%, 488.8%, and 297.5%, respectively (Fig. 1).

CAT activity increased significantly from 800 m to 1400-1600 m, followed by a significant decrease at 1800 m (Fig. 1). Compared to CAT activity at 800 m, CAT activity at 1000 m, 1200 m, 1400 m, 1600 m, and 1800 m increased by 69.8%, 144.6%, 344.3%, 363.0%, and 241.5%, respectively (Fig. 1).

APX activity levels were more variable, with significant fluctuations at 800-1400 m (Fig. 1). Nonetheless, similar to the other antioxidant enzymes tested, APX activity peaked at 1600 m, an increase of 91% compared to that at 1400 m, and declined significantly between 1600 m and 1800 m (Fig. 1). Compared to the lowest activity level (at 1000 m), activity levels at 800 m, 1200 m, 1400 m, 1600 m, and 1800 m increased by 29.5%, 48.9%, 48.7%, 184.0%, and 63.7%, respectively (Fig. 1).

Finally, SOD activity increased significantly from 800 m to 1600 m and decreased from 1600 m to 1800 m (Fig. 1), although there was no significant difference in SOD activity levels between 1400 m and 1600 m. Compared to SOD activity at 800 m, SOD activity at 1000 m, 1200 m, 1400 m, and 1600 m increased by 12.4%, 11.6%, 35.8%, and 55.2%, respectively.

Several Soil Properties Varied Along the Elevation Gradient

The properties of the soils surrounding L. caerulea plants at elevations from 800-1600 m on the northern slope of Changbai Mountain are shown in Table 1. The value of hydrolyzed N is lower at 800 m. The value of available potassium is lower at 1000~1400 m. Available phosphorus values are lower at 1600 m and 1800 m (Table 1). Lack of soil nutrients may limit plant growth.

Correlations Between Environmental Factors and Antioxidant Enzyme Activity in L. caerulea Leaves

The antioxidant enzyme activity of L. caerulea was affected by average annual temperature, annual precipitation, accumulated temperature >5ºC (The sum of the daily mean temperature over the course of one year when the daily mean temperature >5ºC), precipitation from June to September, average temperature in January, average temperature in July, frost-free period, days with snow cover, dryness index and wetness index (Table 2).

Environmental Factors Significantly Affecting Antioxidant Enzyme Activity in L. Caerulea Leaves at Different Elevations

Regression analysis showed that POD activity in L. caerulea leaves was mainly affected by precipitation from June to September (Table 3). The best-fit regression model was Y = 22.92X − 11055, where Y represented POD activity and X represented the precipitation from June to September. The correlation coefficient of this model was 0.8687.

CAT activity was mainly affected by the number of days with snow cover, soil available P, and soil total P (Table 3). Of these, the number of days with snow cover had the largest effect on CAT activity, followed by available P and total P. The best-fit regression model was Y= 5.00X₁ + 3.62X₂ + 1258.98X₃ − 742.34, where Y represented CAT activity, X₁ represented days with snow cover, X₂ represented soil effective P, and X₃ represented soil total P. The correlation coefficient of this model was 0.9999.

SOD activity was primarily affected by precipitation from June to September, followed by soil total P (Table 3). The best-fit regression model was Y = 0.09X₁ + 178.79X₂ − 23.32, where Y represented SOD activity, X₁ represented precipitation from June to September, and X₂ represented soil total P. The correlation coefficient of this model was 0.9881.

APX activity was mainly affected by soil available P (Table 3). The best-fit regression model was Y = 183.40 + 0.45X, where Y represented APX activity and X represented soil available P. The correlation coefficient of this model was 0.9073.

It is well-known that environmental conditions vary along mountain sides. CO₂ concentration, and solar ultraviolet radiation intensity. Due to these factors, plants growing at high altitudes are subjected to severe environmental stress. In particular, the low temperatures and high levels of UV-B radiation endemic to high altitudes disrupt free-radical metabolic homeostasis, leading to the production of a large number of reactive oxygen species; reactive oxygen species damage cell membranes and inhibit enzyme activity, possibly leading to cell death in extreme cases [29- 32]. Intracellular antioxidant enzymes, including POD, CAT, SOD, and APX, effectively scavenge superoxide free radicals to maintain the metabolic balance of free radicals in plants [33-34]. SOD is the first line of defense in the plant antioxidant system, scavenging residual O₂^- and other reactive oxygen free radicals in cells during photosynthesis, while POD, APX, and CAT are the main scavengers of H₂O₂ in plant chloroplasts [35-36]. Therefore, increases in the activity levels of these enzymes suggest increased scavenging activity as a result of greater production of reactive oxygen free radicals [37-38].

Fig. 1.

Enzyme activity levels in Lonicera caerulea leaves at different altitudes.

Here, POD and APX activity profiles changed similarly as elevation increased, with significantly decreased levels of activity at 1800 m as compared to 1600 m. This may be because the stronger light, increased UV-B radiation, lower temperatures, larger diurnal temperature fluctuations, and reduced partial pressures of O₂ and CO₂ found at higher altitudes have adverse effects on oxidase synthesis [39]. SOD, POD, CAT, and APX activity levels peaked at 1400-1600 m in response to changes in various environmental factors (i.e., soil and climatic characters). That is, the accumulation of reactive oxygen species in L. caerulea led to increases in enzyme activity. As POD activity levels were higher than APX or CAT or SOD activity levels, POD might better allow the plant to adapt to the adverse conditions at high altitudes [40].

In L. caerulea leaves, SOD, CAT, POD, and APX activity levels fluctuated with increasing elevation, peaked at 1600 m, and decreased significantly at 1800 m. Low temperatures and other environmental stresses are present at high altitude. These stressors promoted antioxidant enzyme activity in L. caerulea leaves and ensured that the damage associated with reactive oxygen species was minimized. In general, antioxidant enzyme activity levels were relatively high at moderate altitudes and low at lower at higher altitudes. This pattern might reflect a lack of reactive oxygen species in the L. caerulea leaves due to long-term adaptations to the environment. Environmental factors (soil properties and climate parameters) changed with altitude, leading to an excessing accumulation of reactive oxygen species in L. caerulea leaves.

This increase in reactive oxygen species promoted the synthesis of various antioxidant enzymes and increased enzyme activity [41]. Similar changes in the activity levels of antioxidant enzymes were found in the needles of Pinus massoniana growing at high altitudes in China [42]. The decrease in SOD, POD, APX and CAT activity levels at 1800 m as compared to 1600 m may indicate that the excessive accumulation of reactive oxygen species overwhelmed the protective defense capability of L. caerulea, resulting in damage to cell structure and function, leading to disruptions in the synthesis and function of various antioxidant enzymes. These results were consistent with those of a study of hawthorn plants in the Himalayas [43]. Similarly, a study of medicinal plants at different altitudes in Saudi Arabia showed that antioxidant enzyme activity steadily increased with altitude to a certain point, after which antioxidant enzyme activity decreased [44]. Although L. caerulea is found in a harsh environment, it grows and develops well, showing adaptability to adverse environmental conditions, which is inseparable from the effective operation of antioxidant systems within its body. The results of this study indicated that plant antioxidant activity levels are maximized at medium altitudes. The elevation at which L. caerulea exhibited the greatest level of antioxidant enzyme activity was identified. This result may support improvements in the growth and physiological properties of this plant.