Determination of some physiological parameters in spinach (Spinacia oleracea L.) cultivated in Kosovo

Researchers have given special importance to the presence of antioxidants in plants due to the increasing interest among consumers to improve the way of nutrition and prevent various chronic diseases (Moore et al., 2006). Spinach (Spinacia oleracea L.) is a leaf-producing vegetable and belongs to the Amaranthaceae family (Hassler, 2018). Spinach is consumed worldwide (Morelock and Correll, 2008). In 2016, the production of spinach in the world was 26.7 million tons (FAOSTAT, 2018), while in Kosovo, the production was 1348 tons in 2018 (KAS, 2018). Spinach is known for its low content of fats and calories and has a high content of vitamins, minerals, and fiber (Alvino and Barbieri, 2016). Spinach is also characterized by high antioxidant activity (Ismail et al., 2004; Ou et al., 2002). Various biotic and abiotic stresses inhibit plant growth and development, as well as reduce its yield and quality (Apel and Hirt, 2004). Photosynthetic pigments in plants, proline, glycine, nitrogen compounds such as proteins, sugars, phenolic compounds, flavonoids, β-carotene and lycopene, and enzymatic antioxidants such as superoxide dismutase (SOD) degrade under environmental stress conditions (Koca et al., 2007). This is due to the excessive accumulation of reactive oxygen species (ROS) containing single oxygen, superoxide (O₂⁻), hydrogen peroxide (H₂O₂), and hydroxyl radicals (Savvides et al., 2016). ROS molecules are highly reactive and cytotoxic, and they can damage DNA molecules, RNA, proteins, carbohydrates, lipids, and can result in cell death. The plant possesses enzymatic mechanisms which enable cleansing of the body from ROS. These enzymes include SOD, peroxidase (POX), catalase (CAT), glutathione reductase (GR), glutathione peroxidase (GPX), and ascorbate peroxidase (APX). SOD acts as the first line of defense against ROS, dismutating superoxide anion radical to H₂O₂. APX, GPX, POX, and CAT, subsequently decomposing H₂O₂ (Apel and Hirt, 2004). APX is a key component of the ascorbate glutathione cycle in chloroplasts. It also plays a role in ROS scavenging in cytosol, mitochondria, and peroxisomes (Miller et al., 2010). The POXs play a role in physiological processes such as auxin metabolism, lignin and suberin formation, cross-linking of cell wall components, defense against pathogens, and cell elongation (Passardi, 2005). Likewise, POXs and CATs play an important role in the fine regulation of ROS concentration in the cell through moderation of H₂O₂ deactivation. The balance of SOD, POX, and CAT activities is crucial for suppressing toxic ROS levels in the cell (Apel and Hirt, 2004). SOD also contributes to the formation of H₂O₂ and dismutation of O₂. APX and GR are involved in the ascorbate (AsA)–glutamate (Glu) cycle that occurs in chloroplasts and are responsible for converting H₂O₂ to water (Apel and Hirt, 2004). The main function of POXs and CATs in peroxisomes is to remove H₂O₂ (Miller et al., 2010). In this paper, our research had two main targets, which are to investigate (i) the influence of different locations on the content of photosynthetic pigments (Chl a, Chl b, Chl total) and (ii) the influence of different locations on the activity of enzymatic antioxidants (SOD, APX, CAT, POX, GR), total glutathione (GSH), AsA, and the level of lipid peroxidation in two different cultivars of spinach.

The results for SOD and GR activities showed that there was a statistically significant difference between the varieties at each location (Ferizaj, Prizren, and Obiliq). The highest SOD activity was found in Clipper cultivar grown in Prizren (382.60 U mg⁻¹ protein), and the highest GR (225.60 U mg⁻¹ protein) activity was found in Matador cultivar grown in the locality of Obiliq. The highest POX (3.92 U mg⁻¹ protein) activity was found in Matador and Clipper (5.75 U mg⁻¹ protein) cultivars in Obiliq, while the highest CAT activity was found in Clipper cultivar grown in Prizren locality. Results related to parameters are presented in Table 2.

The APX activities of the Matador cultivar grown in Obiliq and the Clipper cultivar grown in Prizren were significantly higher than in Ferizaj, respectively. The APX activity in Matador cultivar ranged between 1019.36 and 4673.81 U mg⁻¹ protein and in Clipper cultivar ranged from 156.43 to 3095.50 U mg⁻¹protein. The CAT activity in our research gave different results; in Matador cultivar, it ranged between 1.16 and 4.27 mg g⁻¹ protein and in Clipper, it was from 3.45 to 10.94 U mg⁻¹ protein. The differences in CAT activities between cultivars were statistically significant at a level of probability of 0.05. The SOD activity in Matador cultivar was between 61.80 and 126.67 U mg⁻¹ protein, while in Clipper, it ranged between 88.62 and 382.60 U mg⁻¹ protein. The POX activity in Matador cultivar was between 1.25 and 3.92 U mg⁻¹ protein, and in Clipper cultivar, it was from 0.58 to 5.75 U mg⁻¹ protein. The GR activity ranged between 165.67 and 225.60 U mg⁻¹ protein in Matador cultivar and from 52.25 to 135.58 U mg⁻¹ protein in Clipper. Also, the MDA activity of spinach was between 2.94 and 28.89 mol g⁻¹ FW in Matador cultivar and between 10.73 and 38.70 mol g⁻¹ FW in Clipper cultivar. The highest value of MDA, the final product of lipid peroxidation, was in Clipper variety cultivated in Obiliq. No significant difference was found in the GSH content of Clipper cultivar at different locations, but there were significant differences in Matador cultivar between Obiliq and Prizren locations. The total GSH content of spinach was between 0.44 and 1.79 μmol g⁻¹ FW in Matador cultivar and from 1.29 to 2.12 μmol g⁻¹ FW in Clipper cultivar. There was no significant effect on the total AsA content for the two spinach cultivars at different locations, but there were significant differences between the AsA contents in the two spinach cultivars grown in Obiliq. The total AsA content of spinach was between 7.47 and 9.95 μmol g⁻¹ FW in Matador cultivar and from 9.29 to 11.85 μmol g⁻¹ FW in Clipper cultivar. There was a significant difference in the total AsA content in Obiliq. We found a significant difference in the content of some pigments (total Chl a + b, Chl a, Chl b, and carotenoids) between cultivars at each location. The highest pigment content was found in Matador cultivar grown in Prizren (1.31, 0.47, 1.77, and 6.44 mg g⁻¹, respectively) and the lowest pigment content was found in Clipper cultivar grown in Obiliq (0.05, 0.04, 0.11, and 0.51 mg g⁻¹, respectively). The content of Chl a was between 0.10 and 1.31 mg g⁻¹ in Matador and between 0.05 and 0.43 mg g⁻¹ in Clipper. There was a significant difference in the pigment content of Chl a between cultivars at each location. For the cultivar Matador, the content of Chl b was between 0.12 and 0.47 mg g⁻¹, while for Clipper, it was from 0.04 to 0.19 mg g⁻¹. The total Chl a + b was found between 0.20 and 1.77 mg g⁻¹ in Matador and between 0.11 and 0.62 mg g⁻¹ in Clipper. The pattern of carotenoid content was similar to that of total Chl a + b. Results are presented in Figure 1.

Figure 1

Various biotic and abiotic stresses cause the production of ROS and lead to the appearance of oxidative stress in plants (Foyer and Noctor, 2000). Plants primarily deal with oxidative stress via an endogenous defensive mechanism consisting of different enzymatic (SOD, CAT, APX, GR) and nonenzymatic (AsA, GSH) reactions (Hasanuzzaman et al., 2020). It has been reported that the activity of enzymes that enable the purification of ROS is higher in plant genotypes that are tolerant, while in sensitive plant genotypes, lower level of enzymes is observed. Therefore, different plant genotypes react differently to biotic and abiotic stresses due to variations in the antioxidant system (Nawaz and Ashraf, 2007). Based on the results of this study on the activities of enzymes POX, APX, SOD, and CAT, as well as the content of MDA, we found that oxidative stress plays an important role as an indicator of the state of stress in the spinach plant. H₂O₂ and O₂ are highly reactive molecules and can cause serious damage to cells such as DNA molecules, membranes, and proteins. SODs are antioxidant enzymes that enable the breakdown of superoxide into H₂O₂ and molecular oxygen. McCord and Fridovich (1969) first reported this phenomenon through a cupro-zinc protein (erythrocyte) derived from bovine erythrocytes. The content of MDA, indicative of the level of lipid peroxidation, was highest in Obiliq. Due to the increase in the level of lipid peroxidation, we assume degradation in the pigment content caused by the decrease in the total AsA content and the activities of the APX and GR enzymes, which are the main enzymes of the AsA–GSH cycle in chloroplasts. These results were obtained by Fatima and Ahmad (2005) with culinary onions and Vitoria et al. (2001) with radish seedlings. Shah et al. (2001) reported similar results from a study in which they applied heavy metals to paddy plants. The soil of Obiliq region showed some differences in pH and the content of EC and OM, and may be, this was connected to physiological and biochemical parameters. Enzymatic antioxidants include SOD, APX, GPX, and CAT. These enzymes are present in almost all subcellular divisions. Usually, cellular organelles contain more than one antioxidant enzyme, which enable the clearance of ROS (Scandalios, 2005). Based on previously published results, we can say that the level of APX activity in general increases along with the activities of other enzymes, such as SOD, CAT, and GSH, at the time of environmental stress (Shigeokaet et al., 2002).

During the examination of the physiological and biochemical parameters of the Clipper cultivar of spinach plants grown in Obiliq, it was found that the content of MDA, which is an indicator of the level of lipid peroxidation, was at the highest level in this cultivar. The increase in the level of lipid peroxidation that occurred in chloroplasts was due to the increase in the level of activity of the enzymes APX and GR, which are the main enzymes of the AsA–GSH cycle. However, one reason for this result may be that Obiliq is the most polluted site in Kosovo. In general, the results of the present study clearly indicated that different levels of soil texture, that is, pH, EC, and OM, had significant effects on growth, chlorophyll, and physiological attributes of spinach plants.