Effect of plant growth
The morphological characteristics of the oilseed rape plants exposed to tetracycline were assessed. Tetracycline inhibited oilseed rape plant growth in a dose-dependent manner.In this study, the fresh weights of plants exposed to tetracycline at concentrations of 0.25, 0.50, 1, 2, 4, and 8 mg/L were 117.71%, 94.53%, 87.82%, 58.97%, 41.85%, and 29.40%, respectively, of the fresh weights of the control group plants(Fig. 1). Tetracycline at a concentration of 0.25 mg/L clearly promoted growth.These results show that tetracycline can promote growth at low concentrations. But as the concentration increases, it would inhibit the growth of oilseed rape.
The chlorophyll contents of plants are often measured to assess the effects of environmental stress(Ibrahim et al. 2017).The effects of tetracycline on the chlorophyll a, chlorophyll b, and total chlorophyll (chlorophyll a + chlorophyll b) contents and the chlorophyll a/chlorophyll b ratios for oilseed rape plants exposed to tetracycline for 14 d are shown in Table 1. The total chlorophyll contents of the plants exposed to tetracycline were 33–96% lower than the total chlorophyll contents of the control plants. The chlorophyll a/chlorophyll b ratio was also lower for plants exposed to tetracycline than for the control group plants. This agrees with an analogous reduction in chlorophyll contents in ryegrass plants treated with tetracycline(Han et al. 2019).
Table 1. The effect of tetracycline on Chl a, Chl b, Chl a+b, and Chl a/b of oilseed rape
Treatment concentration(mg/L)
|
|
Chlorophyll a (mg/g)
|
Chlorophyll b (mg/g)
|
Chlorophyll a+b (mg/g)
|
Chlorophyll a/b
|
0
|
1.139±0.009a
|
0.262±0.009a
|
1.401±0.017a
|
4.351±0.113a
|
0.25
|
0.753±0.012b
|
0.185±0.014ab
|
0.938±0.024b
|
4.074±0.250a
|
0.5
|
0.589±0.040c
|
0.156±0.027ab
|
0.712±0.015c
|
3.816±0.368a
|
1
|
0.456±0.081d
|
0.156±0.059ab
|
0.646±0.023d
|
3.162±1.014ab
|
2
|
0.409±0.075d
|
0.134±0.064b
|
0.542±0.015e
|
3.317±0.815ab
|
4
|
0.453±0.041d
|
0.145±0.057ab
|
0.460±0.026f
|
3.331±0.840ab
|
8
|
0.253±0.084e
|
0.143±0.120ab
|
0.276±0.021g
|
2.258±0.897b
|
Data are the means of three replicates (±SE). Different letters indicate remarkable differences at P < 0.05.
Changes in secondary metabolite contents
Secondary metabolism in plants is key to mediating responses to environmental stress. Phenolic acidandflavonoids are very important specialized metabolites that are involved in plant signaling and defense(Jiang et al. 2016), therefore, we first tested the effect of tetracycline on thetotal phenolic acid flavonoid content in plants.The total phenolic acid and flavonoid contents gradually increased as the tetracycline concentration increased(Fig. 2A,B). For example, the phenol and flavonoid contents were 118.46% and 99.67% higher, respectively, in the plants exposed to tetracycline at a concentration of 8 mg/L than in the control group plants.Phenolic acids act as antioxidative compounds by acting as reducing agents, hydrogen donors, singlet oxygen quenchers, and superoxide radical scavengers(Heleno et al. 2015, Riaz et al. 2017).Flavonoids can decrease oxidative damage caused by ROS(Dong &Lin 2021) and therefore protect a plant from oxidative stress.
Besides, sinapine is an important secondary metabolite in cruciferous plants(Fang et al. 2012).Sinapine provides sinapic acid and choline. Coincidentally, sinapic acid is the precursor for the biosynthesis of phenolic compounds such as flavonoids(Milkowski &Strack 2010).As shown in Fig. 2C, all of the treatments stimulated sinapine production. The sinapine contents of the plants exposed to tetracycline at concentrations of 0.25, 0.5, 1, 2, 4, and 8 mg/L were 5.12%, 12.82%, 19.23%, 26.92%, 36.54%, and 93.07% higher, respectively, than the sinapine contents of the control group plants. The phenolic group in sinapine has marked antioxidant properties and is effective at scavenging hydroxyl radicals(Thiyam et al. 2006).
The photosynthesis pigment content is a recommended indicator of environmental contamination(Rydzynski et al. 2017).Carotenoids are important lipid-soluble antioxidants. Carotenoids are also integral parts of pigment-binding complexes and are involved in light harvesting and quenching excess energy(Opris et al. 2013).In addition, carotenoids can buffer the number of ROS in plants(Rogers &Munne-Bosch 2016).Exposure to tetracycline for 14 d significantly decreased the carotenoid content (Fig. 2D). The carotenoid contents were markedly higher in the control group plants than in the plants exposed to tetracycline. The plants treated with tetracycline at concentrations of 0.25, 0.5, 1, 2, 4, and 8 mg/L had carotenoid contents 33.01%, 46.99%, 58.99%, 64.01%, 73.00%, and 76.47% lower, respectively, than the carotenoid contents of the control group plants. This was consistent with the results of a study performed by Han et al(Han et al. 2019).The responses of carotenoids to tetracycline in this paper suggested that they are involved in oxidativestress.
In addition, we quantified the PAL activity in the oilseed rape plants to improve our understanding of how tetracycline increases the secondary metabolite contents. We found that tetracycline significantly induced PAL activity(Fig. 3). Maximum PAL activity was found at a tetracycline concentration of 8 mg/L.PAL is generally recognized as a marker of environmental stress(He et al. 2020).In addition, the PAL activity followed a similar trend to the flavonoid and total phenolic acid contents. These results were consistent with the results of a study performed by Jahan(Jahan et al. 2020). The results indicated that tetracycline can increase the phenolic acid and flavonoid contents of oilseed rape plants by inducing PAL enzyme activity. This suggests that PAL is a key enzyme involved in the production of flavonoids and phenols.
Changes in nutritional value
The effects of tetracycline on the dietary fiber, soluble sugar, and trace element contents are shown in Figs. 4 and 5.Soluble fiber decreases cholesterol concentrations in the blood through several mechanisms. Insoluble fiber causes rapid gastric emptying, so may decrease the intestine transit time and promote digestive regularity(Soliman 2019). The soluble dietaryfiber (SDF) and Insoluble dietaryfiber (IDF)contents both decreasedas the tetracycline concentrationincreased (Fig. 4). The SDF and IDF contents were 15.14% and 9.22% lower, respectively, in plants exposed to tetracycline at a concentration of 8mg/L than in the control group plants.Dietary fiber can influence the bioavailabilities of secondary metabolites by interfering with lipids(Palafox-Carlos et al. 2011).The decrease in DF contents in this experiment mayreflect the decrease in the transport of fat-soluble secondary metabolites, such as carotenoids, as a response to the stress caused bytetracycline.
Soluble sugars can help protect plants before or during oxidative stress(Xiang et al. 2011)because they can directly or indirectly trigger the production of ROS scavengers and/or repair enzymes (Van den Ende &Valluru 2009).In addition, sugar acts as a respiratory substrate to provide energy for a variety of physiological activities in plants(Wang et al. 2019).The soluble sugar contents of leaves were higher in plants exposed to tetracycline at low concentrations than in the control group plants(Fig. 5A).However, the soluble sugar contents of leaves decreased as the tetracycline concentration increased.Both decreases and increases in sugar contents can disturb respiratory metabolism and increase ROS production(Keunen et al. 2013). Zhou et al. (Zhou et al. 2021)found that the sugar content of pakchoidecreased as the Cd and sulfamethazine concentrationsincreased. The changes in soluble sugar contentswere primarily caused by tetracycline affectingthe activitiesof enzymes involved in sugar synthesis, conversion, and metabolism(Sil et al. 2019). Moreover, we hypothesized that the decrease of soluble sugars could provide the substance orenergy for phenolic accumulation, which attributed to the phenylpropanoidmetabolism pathway and played a critical role in antioxidant capacity.
The Fe, Mn, and Zn contents were significantly lower in the plants exposed to tetracycline than in the control group plants (Fig. 5B, C, D). For example, the Zn contents of the plants exposed to tetracycline at concentrations of 0.25, 0.5, 1, 2, 4, and 8 mg/L were 11.98%, 19.28%, 32.93%, 47.31%, 53.77%, and 61.8% lower, respectively, than the Zn contents of the control group plants.Fe deficiency can impair electron transportand inducethe production of ROS, leading tooxidative stress(Graziano &Lamattina 2005). Moreover, the leaves of plants turn yellow which is also a typical sign of iron deficiency in plants(Ahammed et al. 2020).This was consistent with our results that plants continue to turn yellow as their iron content decreases. In a previous study, the herbicide imazethapyr decreased trace elements of Arabidopsis thaliana and negatively affected the distributions of various nutrients in the plants (Chen et al. 2015). The concentration of nutrient elements may provide valuable information regarding the health and disease status.The decrease of trace elements indicates that oilseed rape is under stress under the interference of tetracycline. These results suggested that tetracycline reduced the nutritional value of oilseed rape.
Changes in oxidative stress and antioxidant responses
Total phenols and flavonoids act as non-enzyme participants in ROS-scavenging mechanisms and share the roles of antioxidant enzymes(Ma et al. 2019).The phenolic group in sinapine has marked antioxidant properties and effectively scavenges hydroxyl radicals(Thiyam et al. 2006).The ROS contents of leaves can be buffered by carotenoids(Rogers &Munne-Bosch 2016). Arab also found that secondary metabolites can contribute to ROS scavenging in plants exposed to O3(Arab et al. 2022).Therefore, the effects of tetracycline on the ROS level, MDA content, and antioxidant enzyme activities are investigated.
As shown in Fig. 6A, the ROS level increased as the antibiotic concentration increased. The MDA content washigher in plants exposed to tetracycline at concentrationsin the range 0.25–4mg/L than in the control group plants. The MDA content was 49.96% higher in plants exposed to tetracycline at a concentration of 4 mg/L than in the control group plants. The MDA content was somewhat lower in plants exposed to tetracycline at a concentration of 8 mg/L (the maximum concentration) than in plants exposed to tetracycline at a concentration of 4 mg/Lbut was still significantly higher than the MDA content of the control group plants (Fig. 6B). The remarkable changes in the MDA content of the plants exposed to tetracycline were indicative of extensive oxidative damage. If the amount of ROS produced exceeds the scavenging capacity of the antioxidant system, ROS will accumulate in the plant cells and MDA production will be induced.This can damage the cell membranes and organelles and even cause apoptosis(Amjad et al. 2015).
The CAT activity increased significantly as the tetracycline concentration increased. The CAT activity was 2.51 times higher for the plants exposed to tetracycline at a concentration of 8 mg/L than for the control group plants (Fig. 7A). The POD activity followed a similar pattern (Fig. 7B).Similar results were found in a previous study(Han et al. 2021). The increases in the POD and CAT activitiesindicated that both enzymes effectively scavenged H2O2. POD and CAT can directly catalyze the conversion of H2O2 to H2O and O2(Gill &Tuteja 2010, Mittler 2002).The SOD activity decreased as the tetracycline concentration increased and reached a minimum at a tetracycline concentration of 8 mg/L (Fig. 7C). The SOD activity may have decreased because of increased consumption or inactivation of the detoxifying enzymes(Mates 2001). SOD is a special enzyme and the main scavenger of O2−(Holley et al. 2014, Peng et al. 2018).The synergistic actions of SOD, POD and CAT can maintain relatively low free radical concentrations in a plant and therefore decrease the damage caused by ROS to plant cell membranes. With the increase of treatment dose, changes in antioxidant enzymes showed that oilseed rape underwent oxidant stress.