Effects of exogenous zinc on the physiological characteristics and enzyme activities of Passiflora edulis Sims f. edulis seedlings

Experimental design

A 2-month pot experiment was carried out at the experimental site of Guizhou Minzu University (University City Campus) in Huaxi District, Guiyang City, Guizhou Province, China (106°37′25.08″ E, 26°22′32.43″ N). The experimental area has a typical highland monsoon humid climate, with sufficient annual rainfall, an annual average temperature of 15.7 °C, an extreme maximum temperature of 34 °C, and an extreme minimum temperature of −3.1 °C. P. edulis Sims f. edulis seedlings were used as experimental materials. Soil from which stones and grass roots were removed was added to a plastic basin with a height of 31.5 cm and inner diameter of 48 cm, and the soil was evenly turned (tested soil: pH 6.90, organic matter 190.57 g kg⁻¹, cation exchange capacity 161 mg kg⁻¹). Well-growing and uniform passionflower plants were transplanted into a large pot and adapted to culture for 7 days. Exogenous Zn was uniformly added to the soil in the form of a ZnSO₄ 7H₂O aqueous solution. Studies have shown that the effectiveness of ZnSO₄ 7H₂O in soil can persist for 200 d (Zhao et al., 2011), so ZnSO₄ fertilizer was effective in this experimental cycle. According to the soil Zn background value of 99.5 mg kg⁻¹ in Guizhou Province (Ma et al., 2019), the Zn concentration concentrations were set to 200, 400 and 800 mg kg⁻¹ (calculated by soil dry weight). Each treatment was repeated three times, there was one plant per pot, and a blank control (CK) was set. During the experiment, water was added to keep the soil moisture consistent, and 1‰ urea was applied every 7 days to maintain plant growth. A transparent plastic cover was used to seal the top, and holes allowed for the plants to pass through while reducing the leaching of rain water. There was a diversion tube and a kettle at the bottom of the basin, which was checked regularly, and water was poured back into the pot to reduce the loss of Zn.

Index determination

The fifth leaf from the top of the passionflower plants with good growth was removed, and the leaf structure index was measured immediately by a Yaxin-1241 leaf area meter (Beijing Yaxin Liyi Technology Co., Ltd., China). The leaf area (LA), leaf perimeter (LP), leaf length (LL) and leaf width (LW) were recorded, and then the photosynthetic pigment content was determined. A total of 0.2 g of chopped fresh leaves was weighed, added to a small amount of quartz sand and an appropriate amount of 95% ethanol, ground into a homogenate, filtered into a 25 mL brown volumetric bottle and fixed in 95% ethanol. The chloroplast pigment extract with a constant volume was transferred to a quartz colorimetric plate, and 95% ethanol was used as a blank. The absorbances at wavelengths of 665 nm, 649 nm and 470 nm were measured, and the chlorophyll a (Chl a), chlorophyll b (Chl b), chlorophyll a+b (Chl a+b), carotenoid (Car) contents and chlorophyll a/chlorophyll b (Chl a/b) values were calculated. The following equations were calculated (Cai, 2013): (1)${\displaystyle C_{\mathrm{a}}\left(\mathrm{mg}{L}^{-1}\right)=\left(13.95A_{665}-6.88A_{649}\right)}$ (2)${\displaystyle C_{\mathrm{b}}\left(\mathrm{mg}{L}^{-1}\right)=\left(24.96A_{649}-7.32A_{665}\right)}$ (3)${\displaystyle C_{\mathrm{x}}\left(\mathrm{mg}{L}^{-1}\right)=\left(1000A_{470}-2.05C_{\mathrm{a}}-114.8C_{\mathrm{b}}\right)/245}$ (4)${\displaystyle \text{Photosynthetic pigment content}\left(\mathrm{mg}{\mathrm{g}}^{-1}\right)=\left(C\times V\times N\right)/\left(W\times 1000\right).}$

In the above equations, C_a, C_b and C_x denote the concentrations of Chl a, Chl b and Car, respectively, in the extracts, C is the corresponding pigment concentration (mg L⁻¹), V is the volume of the extract (ml), N is the dilution multiple, and W is the fresh mass of the sample (g).

The stretched leaves of passionflower plants with the same tip and good growth in different treatments were used to determine the biochemical indexes immediately after extraction. The contents of malondialdehyde (MDA), proline (Pro), soluble sugars (SS) (Li, 2000), superoxide dismutase (SOD), catalase (CAT) and peroxidase (POD) were determined by the thiobarbituric acid method, ninhydrin colorimetric method, anthrone sulfuric acid method and enzyme labelling method. The kits were purchased from Suzhou Mengxi Biomedical Science and Technology Co., Ltd. The SOD kit refers to the method of Ukeda et al. (1999), the CAT kit refers to the method of Peng et al. (2009), and the POD kit refers to the method of Wang et al. (2014). The steps for CAT, POD and SOD activity determination were the same: 0.1 g fresh sample was weighed in a mortar, one mL extract was added followed by grinding into a homogenate in an ice bath, centrifugation (12000 r min⁻¹ at 4 °C for 10 min) was conducted, the supernatant was placed on ice to be tested, the reagents were added according to the method in the kit and then placed in a 96-well plate, the absorbance was determined by a Thermo Scientific Multiskan FC enzyme labelling instrument, and the enzyme activity was calculated.

Statistical analysis

Excel 2016 was used to collate the data, and SPSS 26.0 was used for one-way ANOVA and Pearson’s correlation. The significance of multiple comparative differences was analysed by the Duncan method. The data that did not satisfy the normality and homogeneity of variance were tested by the Mann–Whitney method. OriginPro 2021 was used to produce the graphics. The data in the charts are the mean ± standard error.

Plant growth

Leaves are the main organs of plant photosynthesis and can also reflect the growth status of plants. As shown in Fig. 1, the leaf growth of passionflower was inhibited with increasing Zn concentration. Except for leaf width, the addition of 200 mg kg⁻¹ Zn had no significant effect on the structural indexes of the P. edulis Sims f. edulis seedling leaves (P > 0.05). The addition of 400 mg kg⁻¹ and 800 mg kg⁻¹ Zn significantly inhibited the growth of the P. edulis Sims f. edulis seedling leaves; the LA decreased by 19.75% and 23.36%, the LP decreased by 9.26% and 8.32%, and the LW decreased by 11.97% and 11.18% (P > 0.05), respectively.

Chl and Car are important photosynthetic pigments in plant leaves and indispensable substances for plant photosynthesis (Tie et al., 2020). As shown in Table 1, exogenous Zn effectively promoted the synthesis of Chl and Car in P. edulis Sims f. edulis seedlings, which is beneficial to photosynthesis. Compared with CK, the addition of 200 mg kg⁻¹ Zn significantly increased the contents of Chl a (37.65%), Chl b (41.22%), Chl a+b (38.59%) and Car (29.74%) (P < 0.05), and the value of Chl a/b decreased significantly by 2.46% (P < 0.05). The contents of Chl and Car increased with the addition of 400 mg kg⁻¹ and 800 mg kg⁻¹ Zn (P > 0.05).

SS, Pro and MDA contents

SS and Pro are important osmoregulatory substances in plants, and stress leads to changes in the contents of SS and Pro (Zhang et al., 2018). As shown in Fig. 2, the SS content of P. edulis Sims f. edulis seedlings decreased with increasing Zn concentration. The 200 mg kg⁻¹ and 400 mg kg⁻¹ Zn treatments had no significant effect on the SS content (P > 0.05), while the 800 mg kg⁻¹ Zn treatment significantly reduced the SS content of P. edulis Sims f. edulis seedlings (36.67%; P < 0.05). The addition of different concentrations of Zn significantly increased the Pro content of P. edulis Sims f. edulis seedlings by 332.14%, 116.84%, and 202.56%, respectively (P < 0.05).

Under stress, unsaturated fatty acids in the protoplasmic membrane of plant cells will be oxidized and produce MDA, which reflects the degree of membrane lipid peroxidation (Zhang et al., 2018). As shown in Fig. 2, the addition of 200 mg kg⁻¹ Zn had no significant effect on the MDA content of P. edulis Sims f. edulis seedlings (P > 0.05). The addition of 400 mg kg⁻¹ and 800 mg kg⁻¹ Zn significantly increased the MDA content of P. edulis Sims f. edulis seedlings (42.69%, 26.7%; P < 0.05).

Antioxidant enzyme activity

CAT, POD and SOD are important antioxidant enzymes in plants that can reduce the damage caused by excess free radicals to the cell membranes (Chowdhury & Choudhuri, 1985). As shown in Fig. 3, the CAT activity of P. edulis Sims f. edulis seedlings increased with the addition of exogenous Zn, which increased the POD activity of P. edulis Sims f. edulis seedlings. With increasing Zn concentration, the POD activity increased gradually, and the POD activity of P. edulis Sims f. edulis seedlings treated with 800 mg kg⁻¹ Zn significantly increased by 67.00% (P < 0.05). Compared with CK, the SOD activity of P. edulis Sims f. edulis seedlings treated with different concentrations of Zn decreased significantly by 30.03%, 47.20% and 32.86% (P < 0.05).

Correlation and principal component analyses

The correlation analysis of the physiological indicators of P. edulis Sims f. edulis seedlings is shown in Fig. 4A. LA was significantly positively correlated with LP, LL, LW, and SOD. There was a significant negative correlation between LA and MDA, a significant positive correlation between LP and LW and SOD, an extremely significant positive correlation between Chl a, Chl b, Chl a+b, Car and Pro, a significant negative correlation between POD and SS, and a significant negative correlation between SOD and MDA.

Principal component analysis is shown in Fig. 4B. PC1 and PC2 explained 39.60% and 28.80%, respectively, of the variation and accounted for 68.40% of the variation, indicating that the first two principal components reflected the degree of variation in each physiological index. Chl a, Chl b, Chl a+b, Car and Pro together had a large positive load on PC1, indicating that these indexes can be used as important indicators to reflect the growth status of P. edulis Sims f. edulis seedlings. SS, SOD, LA, LP, LL and LW had positive loads on PC2, while MDA, CAT and POD had negative loads on PC2. In the principal component analysis, different physiological indexes aggregated to different treatment points, indicating that different concentrations of Zn had different physiological effects on P. edulis Sims f. edulis seedlings.