Effect of various abiotic stressors on some biochemical indices of Lepidium sativum plants

In this study, the regulation of ascorbate peroxidase (APX) specific activity, anthocyanin, carotenoid, hydrogen peroxide, lipid peroxidation, and protein levels in cress leaves in response to different abiotic stresses were investigated. The total APX specific activity was significantly elevated after 9 days of drought treatment, short-term (2 h) exposure to 10, 100 and 370 µE of light, long-term exposure (at least 6 days) to 100 mM NaCl versus the specific APX activity in the controls. Furthermore, a significant change in total APX activity was detected in response to treatment with different temperatures; this change was an early response to 4 °C and 30 °C for a maximum of 4 h, while short-term exposure to 35 °C did not change total APX activity. The results of the present study revealed that plants have a wide range of mechanisms to cope with different stresses that possibly involve morphological changes. The results indicated that Lepidium sativum plants launch common protective pathways only under drought, salinity and high light stresses, while other protective mechanisms/strategies could be responsible for increasing the plants tolerance towards temperature and low light. Future studies will investigate changes in the photosynthetic quantum yield and specific target metabolites, proteins, and nonenzymatic antioxidants.

www.nature.com/scientificreports/ are a group of antioxidant enzymes in plants. All the forms of APX are thought to function as scavengers of H 2 O 2 , which is generated continuously in cells 13 . Superoxide anion (O 2 − ) is formed in the chloroplasts of photosynthetic organisms and in mitochondria by reactions of the electron transport chain. In both cases, superoxide dismutase converts O 2 − to H 2 O 2 , which can then be removed by APX or catalase 6 . Various environmental stimuli, such as drought, salt stress, high light levels, high and low temperatures, H 2 O 2 and abscisic acid, modulate the expression of APX-encoding genes 14 .
This study aimed to identify changes in certain biochemical parameters such as APXs (APX; EC 1.11.1.7), which play a protective role, as well as the levels of nonenzymatic antioxidants, including anthocyanins and carotenoids, in L. sativum leaves under different abiotic stress conditions. Moreover, this study intended to investigate the effects of various abiotic stresses on the levels of hydrogen peroxide, proteins and lipid peroxidation in L. sativum leaves.

Materials and methods
Plant materials and growth conditions. L. sativum seeds (Vilmorin, France) were obtained from local distributer and germinated in Molecular Biology Research Laboratory/ Department of Biological Sciences/ Mutah University, Jordan. The germinated seeds grown under controlled conditions (14 h under ~ 54 µE light at 21 °C/10 h in dark at 20 °C; 55-60% relative humidity) then transplanted to 2/1/1 (vol/vol/vol) mixture of peat moss, perlite and vermiculite. After 6 weeks of growth, plants were either subjected to one of the abiotic stress treatments for different time periods or kept in the plant growth chamber under the previously specified controlled conditions as control experiments.

Abiotic stress treatments.
To induce abiotic stress, 6-week-old growing seedlings were used in abiotic stress treatments as designed previously 7 . For drought treatment, plants were introduced to a water deficit by withholding water for 3, 6 or 9 days. Control samples were irrigated continually three times a week for the same time periods (3, 6 or 9 days). For salinity treatment, 6-week-old plants were irrigated with a 100 mM NaCl solution three times per week for up to 14 days. Interleaves samples were collected at 2, 6, 10 and 14 days of treatment. Control samples were irrigated with tap water three times a week for an additional 2, 6, 10 and 14 days under controlled growth conditions. Heat shock was imposed by transferring the 6-weeks plants to a growth chambers adjusted at 4, 25, 30 or 35 °C. Samples were collected from interleaves after 2, 4 and 6 h of incubation at the studied temperatures. In order to evaluate the impact of light quantity, 6-week-old plants were grown under light at ≈54 μmol photon m 2 s −1 (control); then, plants subjected to light quantity treatment under 10 μmol photon m −2 s −1 , 100 μmol photon m −2 s −1 , or 370 μmol photon m −2 s −1 of light. Samples were collected from interleaves after 2, 6, 12 and 24 h of growth under the studied light quantity. At the end of each specific abiotic treatment, leaves collected from the treated plants were directly frozen in liquid nitrogen and stored at − 80 °C until further analysis. Untreated control plants for all four-stress conditions were grown in parallel with the treated plants.
Quantification of anthocyanin and carotenoid contents. Twenty mg of the frozen leaves, treatment and control experiments, were grinded into fine powder in liquid nitrogen. Anthocyanin and carotenoid pigments were extracted from the grinded tissue using 1 mL of cold buffer containing methanol/HCl/water (90/1/1, vol/vol/vol). After homogenization, the suspension was incubated for 1 h in the dark. The samples were centrifuged for 15 min at 16,240 × g. The methods of Sims and Gamon 15 were used to spectrophotometrically quantify the pigments. H 2 O 2 assay. A total of 100 mg of frozen leaf material was lysed with 0.1% (wt/vol) trichloroacetic acid, followed by centrifugation at 15,000 × g for 15 min at 4 °C. Then, 0.5 mL supernatant was added to 1.5 mL of assay solution composed of 0.5 mL of 10 mM potassium phosphate buffer (pH 7.0) and 1 mL of 1 M KI, mixed gently and the absorbance of the assay mixture was read at 390 nm 16 . Lipid peroxidation assay. Aerial plant tissue (50 mg) was lysed in 1 mL 80% (vol/vol) ethanol on ice. The homogenate was centrifuged at 16,000 × g for 20 min at 4 °C. The supernatant (0.5 mL) was mixed with 0.5 mL 20% (w/v) trichloroacetic acid (TCA) containing 0.65% (wt/vol) thiobarbituric acid (TBA). After incubation at 95 °C for 30 min, the reaction was immediately cooled in an ice bath. Following centrifugation at 10,000 × g for 10 min, the absorbance of the supernatant at 532 and 600 nm was measured. The absorbance value at 600 nm, indicating nonspecific absorption, was subtracted from the absorbance of the supernatant at 532 nm to determine the level of malondialdehyde (MDA) as final product of lipid peroxidation 17 . The MDA concentration was calculated with an extinction coefficient of 155 mM −1 cm −1 .
Proteins quantification. Protein was extracted from aerial tissue (50 mg) using 1 mL 100 mM HEPES buffer (pH 7.6) on ice and centrifuged at 16,240 × g for 10 min at 4 °C. The protein content of 0.2 mL supernatant was determined following Bradford method 18 . Bradford reagent (1 ml) was added to the supernatant, and the absorbance of the mixture at 595 nm was measured.
APX specific activity. Aerial plant tissue (50 mg) was extracted in 125 µL 100 mM HEPES-NaOH (pH 7.6). The homogenate was centrifuged at 16,240 × g and 4 °C for 5 min. Crude extract (50 µL) was added to 1 mL of the assay solution composed of 50 mM HEPES-NaOH buffer (pH 7.6), 50 µL of 5 mM ascorbate and 100 µl of 3 mM H 2 O 2 to initiate the reaction. The absorbance of the reaction mixture at 290 nm was measured spectrophotometrically. The APX activity was standardized according to the protein content 9 .
Scientific Reports | (2020) 10:21131 | https://doi.org/10.1038/s41598-020-78330-1 www.nature.com/scientificreports/ Statistical analysis. Samples were analyzed in triplicate in all experiments, and all the assays were carried out in triplicate. The results expressed as the mean ± SD. The results of each analysis were compared using analysis of variance (ANOVA) with Tukey's HSD, differences between exposure treatments and the corresponding controls were considered statistically significant if P < 0.05.

Results
Anthocyanin and carotenoid contents. In this study, the anthocyanin content changed immediately after exposure to different abiotic factors. There were significant increases in the anthocyanin content following drought and light quantity treatments. Six and nine days of drought treatment induced a 2.2-and 2.6-fold increase in anthocyanin content, respectively. Exposure to 10 µE light for 12 h caused a 2.7-fold increase in anthocyanin content, and exposure to 370 µE light for 2, 6, 12 and 24 h increased the anthocyanin content 2.7-, 3.2-, 4.6-and 2.8-fold, respectively, compared to the anthocyanin content of the controls (Fig. 1). In parallel to the increased anthocyanin content, the carotenoid content was significantly higher in response to the same abiotic factors versus the carotenoid content of the controls (Fig. 2). Figure 3 shows the effects of different abiotic factors on H 2 O 2 content in the examined cress leaves. H 2 O 2 levels in the leaves increased depending on the type and duration of abiotic stress treatment. The H 2 O 2 levels were significantly higher following drought, light quantity and salinity treatment. The H 2 O 2 levels in drought-treated plants were 1.4-, 1.7-and 2.6-fold higher than those in wellwatered plants after 3, 6 and 9 days of treatment, respectively. However, significant changes in H 2 O 2 production after 24 h exposure to all light quantities were observed. After 24 h of exposure to 10 µE, 100 µE and 370 µE light, the H 2 O 2 level peaked and was 1.6-fold higher than the H 2 O 2 level in control plants. The H 2 O 2 levels increased in salinity-treated plants by 1.5-fold and 1.2-fold compared to the H 2 O 2 levels in control plants after 6 and 10 days of treatment, respectively.

Hydrogen peroxide accumulation.
Effects on lipid peroxidation level. Given that drought, light and salinity stresses induced H 2 O 2 production, experiments were carried out to examine whether this increase in H 2 O 2 was related to oxidative damage in the leaves. For this purpose, the MDA level was analyzed (Fig. 4). MDA levels were significantly increased by 2.3-, 2.5-and 3.8-fold after 3, 6 and 9 days of drought treatment, respectively. However, treatment with 10 µE, 100 µE and 370 µE light for 24 h significantly increased the levels of MDA, which peaked and was 2.2-, 4.4-and  Effects on the total protein content. The protein content was significantly increased by 1.7-, 1.8-and 1.9-fold after 3, 6 and 9 days of drought treatment, respectively, compared to the protein content in the controls. The protein content in salinity-treated plants was significantly increased by 1.3-, 1.4-, 1.6-and twofold after 2, 6, 10 and 14 days of treatment, respectively, compared to the protein content in the controls, as shown in Fig. 5.
Effects on total APX specific activity. A significant increase in APX specific activity was observed in drought-, light-, salinity-and temperature-stressed plants compared to the APX specific activity in control plants (Fig. 6). Drought treatment resulted in a 3.7-, 4-and 4.6-fold increase in total APX specific activity after 3, 6 and 9 days of treatment, respectively. Exposure to 10 µE light increased the total APX specific activity by 1.

Discussion
The currently reported results of Lepidium sativum (cress) plant in response to most of the applied stressors (drought, salinity, low light quantity and temperature) indicated common biochemical reactions observed as increment in all the studied stress indicators. However, the exposure of the plant to low light quantity and high temperature exhibited some uncommon reactions. Under low light, anthocyanin, carotenoids and H 2 O 2 contents increased. On the contrary, MDA, protein and APX decreased. The most divergent responses were observed in the case of low and high-temperature. Under the latter stressor, anthocyanin and carotenoids contents, MDA and protein content decreased, while under low temperature anthocyanin and carotenoids contents increased and the other indices were decreased. www.nature.com/scientificreports/ Similar to most of our results, it was reported that the levels of endogenous anthocyanins in foliage are stimulated by different abiotic factors 10,19 . However, contrast results of elevated anthocyanin content in response to long-term exposure (6 weeks) to temperature 20 or to the synergetic effect of high temperature and duration of cultivation 21 was reported. Chalker-Scott 19 also reported different finding from the present study where low temperatures in the absence of either visible or ultraviolet B (UVB) light prevent anthocyanin biosynthesis. Anthocyanin accumulation has been proposed to have a role in protection against photoinhibition, in which anthocyanins serve as osmotically active solutes and antioxidants for ROS in addition to their function as a UV screen [22][23][24][25] . In addition, accumulated anthocyanins might act as signaling markers of plant stress induced by salt 26 enhancing peroxidase activity and anthocyanin content 26,27 . Similarly, elevated carotenoids levels allow plants to cope with oxidative stress 28,29 by scavenge ROS to prevent the oxidation of membrane lipids, and ultimately mitigate oxidative stress. Thus, plants can tolerate stressful conditions by maintaining a higher or invariable level of total carotenoids. The results of the present study regarding carotenoids accumulation are in agreement with those of previous studies 30-32 on plant acclimations to stress.
Increased production of ROS is one of the earliest responses of plant cells to abiotic stress 33 . Among ROS, H 2 O 2 has been found to trigger a variety of plant responses 33,34 . Drought and light stress found to be linearly induces H 2 O 2 accumulation [35][36][37] . H 2 O 2 is versatile and plays a series of roles that range from orchestrating physiological processes to the stress response 37,38 . In the present study, our results on the effects of salinity stress on the H 2 O 2 content in leaves are in agreement with those from previous reports 39 . H 2 O 2 can induce antioxidant enzymatic defenses to reduce the deleterious effects of salinity since H 2 O 2 is a signaling molecule that mediates crosstalk between signaling pathways and contributes to protection against other sources of stresses 40 .
Plants tolerance to temperature stress depending upon the plant type, duration and intensity of the stress have been reviewed by Awasthi et al. 41 . Furthermore, the variations in the levels of MDA and H 2 O 2 levels might be temperature-specific 42 , which might explain the reduction of the levels of these molecules during low and high temperature stress. Lipid peroxidation induced by a variety of stresses is involved in diverse signaling processes to protect plants from oxidative stress [43][44][45][46] . The overproduction of ROS increases the content of MDA, which is an indicator of oxidative damage 47 . Our results show a correlation between H 2 O 2 accumulation and the increase in MDA level in response to drought, salinity and light stress. Salt stress results in extensive lipid peroxidation, which is often used as a marker of stress-induced cellular damage. Therefore, plants that tolerate salt stress are better protected from leaf oxidative damage 48,49 . The significant change in protein content in response to the severity of abiotic stress might be a mechanism to enhance plant stress tolerance through the increased abundance of proteins involved in energy production, amino acid synthesis, protein synthesis and the antioxidant www.nature.com/scientificreports/ defense system [50][51][52][53] . Our results showing changes in protein content in response to salt stress are consistent with those previously reported findings, in which an increased leaf protein concentration was directly associated with stress tolerance 54,55 . Gülen and Eris 56 reported similar results showing that the total protein content is decreased by heat stress. This reduction in total protein content might be due to the occasional production of specific proteins to reduce stress severity, such as peroxidases and ROS scavenger enzymes, as an acclimation response 57 . APX is ROS-scavenging enzyme 58 , that minimize the effects of oxidative stress by cooperating with other proteins to maintain the integrity of photosynthetic membranes under oxidative stress through direct scavenges ROS or producing a nonenzymatic antioxidant 58 . Because of this, APX plays a key role in the acclimation of plants to stress 59 via the metabolism of H 2 O 2 which leads to increasing plant cellular tolerance to oxidative stress 38,60 . Regulation of the activities and levels of APX isoenzymes and other antioxidant enzymes offers additional stress defense capabilities 11,61 . In agreement with our results, previous studies on the influence of salt stress, high and low temperatures reported increment in total specific APX activity, which might help in the reduction of stress severity as an acclimation response 56,57,62 . In particular, at 30 °C our findings are in agreement with the observations of Kumar et al. 42 for the changes in total specific APX activity to different temperatures. According to Pandey et al. 63 , H 2 O 2 at low levels acts as a secondary messenger in initiating scavenging system including increasing APX activity. The considerable increase in APX activity could not stop the deleterious effects of heat stress but reduced stress severity as an acclimation response.
The current finding suggests that L. sativum launch common protective pathways under drought, salinity, and high light stresses, while another protective mechanism could be responsible for increasing the plants tolerance towards high temperature and low light. Further investigation is required to clarify the cascade of biochemical changes starting from the molecular level. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creat iveco mmons .org/licen ses/by/4.0/.