Heavy metals adversely impact not only soil and water, but also the plants existing nearby. To investigate the impact of arsenic contamination on physiological properties, antioxidant activities, and synthesis of pistachio oil, an experiment was conducted in 2021 in a CRD. Findings proved that the amounts of arsenic in different organs, bioaccumulation coefficient (BCF), and transfer factor (TF) were different in different study areas. The amount of arsenic in roots was much higher than the leaves; the highest amount of arsenic in the leaves, roots, and fruits of the studied region was 1000 to 1200 mg/kg. The highest amounts of BCF found in the leaves and fruits were 0.11 and 0.015, respectively. The TF changes were found more frequent in pistachio leaves than its fruits. An increasing trend of changes was observed in ion losses, malondialdehyde, carotenoids and flavonoids, glutathione peroxidase activity, glutathione reductase, phenolalanine amoliase, pyrroline-5-carboxylase synthase, lipoxygenase, and linoleic fatty acid which was due to an increase in total concentrations of valretic acid. Chl a and b, protein content, reduced glutathione, ascarobate peroxidase, proline dihydrogenase, oil content, oleic acid, and palmitic acid decreased linearly. Changes to malondialdehyde, Chl a, flavonoids, and pyrroline-5-carboxylase synthase showed the highest correlations with changes in arsenic level. Generally, it was found that arsenic accumulation caused damage to the structure of proteins, cell membranes, and photosynthetic pigments of the plant. These damages, in turn, led to the activation of plant's defense mechanism and had deterrent impact on the biosynthesis of the plant's oil.
Research Article
The Effects of Environmental Arsenic on the Photosynthesis, Antioxidants Systems, and Biosynthesis of Pistachio Oil
https://doi.org/10.21203/rs.3.rs-2483648/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 01 Dec, 2023
You are reading this latest preprint version
Arsenic
Protein
Pistachio oil
Plants
Pistachio (Pistacia vera L.), as an important plant of the pistachio family, is considered a unique dried fruit in Iran due to its high nutritional value, long storage life, and profitable export market (Heidari et al. 2022). One of the main environmental and health problems of modern societies is the increase in pollution caused by heavy metals due to their poisoning and accumulation in food chains (Farghali et al. 2022). In recent decades, the level of arsenic (As) in agricultural soils has significantly been increasing, especially in the eastern and central regions of Iran, which are the main locations for pistachio farming (Mitra et al. 2017). Arsenic, which is released from natural and artificial sources, is a hazardous metalloid that penetrates into environment.
One of the main effects of plant exposure to toxic concentrations of as is the production of free oxygen radicals (ROS), which damage proteins and DNA. This also reduces enzymes and lipid peroxidation, destroys cell system and diaphragm, and ultimately leads to low energy production and cell death (Gao et al. 2022).
Chloroplast is indeed one of the main sources of ROS. ROS production in mitochondria, peroxisomes, and protein oxidation are other important factors triggering stress in plants. ROS is also produced by cell wall-attached peroxidases, amine oxidases, and NADPH oxidases (Arsana et al. 2022; Doosti-Moghaddam et al. 2022; Kai et al. 2016).
Plants enhance their physiological responses to adapt to the as stress. The ROS refinement in plants occurs through enzyme and anti-enzyme systems. Anti-enzyme antioxidants play a crucial role in ROS sweeping by giving electron or hydrogen. These antioxidants include membrane and fat-soluble compounds such as alpha-tocopherols, carotenoids, and xanthophylls as well as water-soluble compounds such as GSH, ASA, and phenolic compounds. The major enzyme defense mechanisms for reducing ROS in plants are SOD, CAT, POX, ASA-GSH (Hasanuzzaman et al. 2020; Mirzaei and Shakoory-Moghadam 2022).
The main defense system against ROS in chloroplasts, cytocells, mitochondria, peroxisomes, and apoplasts Is the ASA-GSH cycle. In addition to GR, some other enzymes are involved in this cycle, i.e., APX, MDHAR, DHAR, and also GSH, ASA, and NADPH. They work together in a series of reaction cycles to reproduce ASA and GSH for detoxifying H2O2. It was related (Singh et al. 2015), the use of As in Luffa Aegyptiaca reduced the activities of some enzymes like MDHAR and DHAR. Similarly, the activities of MDHAR, DHAR, and APX decreased in a study conducted by Maresca et al. (Maresca et al. 2017) on rapeseed and cadmium. GR, as an enzyme existing in the cytoplasm and chloroplasts, converts GSSG into GSH-glutathione with the aid of NADPH. Elevation in GR activity increases the NADP+/NADPH ratio due to an increase in the amount of NADP + available for electron transfer to O2. This process, in turn, decreases the production of superoxide radicals (Couto et al. 2016).
Plants also use osmoliters such as proline, carbohydrates, and glycine betaine to counteract stress through mechanisms such as changes in osmotic pressure in the cell, involvement in the ROS detoxification, ensuring membrane stability, and regulating compositions/proteins (Ahani and Attaran 2022; Ahmadi 2022). Proline is one of these compositions whose biosynthesis is controlled by the enzyme proline-5-carboxylase synthase (P5CS). This enzyme has two activities: GK and GPR (Turchetto-Zolet et al. 2009). Prolyne hydrogenase (PRODH) is another key proline biosynthesis enzyme that oxidizes the activities of P5CS in proline biosynthesis (Meena et al. 2019).
The amounts of oil and fatty acids are also other factors affected by environmental stresses. LOXs, as one of the important enzymes affecting the oxidation of unsaturated fatty acids, has a considerable role in various developmental processes, including stress tolerance (Segura et al. 2022). Different isoforms of this enzyme play a protective role against stress through the operation of enzymes such as AOS, AOC, and HPL. This is the way these enzymes affect fatty acids and oil biosynthesis under stress conditions (Wang et al. 2022).
Considering the potential hazards and side effects of heavy metals on plants, the main objective of the present study was to investigate the accretion of As, as a heavy metal, in agricultural soils. This study also investigates the As impacts on physiological traits, enzyme/anti-enzyme systems of oxidization, and biosynthesis of pistachio oil.
Treatments and Experimental Conditions
In section one, A field experiment was conducted on the soils polluted by as in eight different districts of Kerman province (longitude; 54 ° 21 'to 59 ° 34' east, latitude; 26 ° 29 'to 31 ° 58' north), Iran, during the farming year 2021. This random experiment was replicated three times in regions with the coordinates shown in Fig. 1. To collect the pistachio samples, three farms from each region and three samples from each farm were selected. Then, each farm was divided into three sections and three locations were randomly selected from each section. At each site, 10 pistachio trees were manually picked from among the all-existing trees. Soil samples were also collected from the pistachio fields. At each stage, three soil samples (in depth of 30 cm) were collected from each field. Afterwards, the samples were dried in an oven at 105°C for 24 hours. They were then sieved with 2 mm diameter-mesh sieves to be used for measuring the as content.
Measurement of soil and plant arsenic
To prepare ash for measuring As, 2 g of the dried soil sample was placed in a kiln at 550°C for 24 hours. In the next step, acid digestion was performed by adding 5 ml of concentrated nitric acid. Then the solution was placed on a hot plate to complete the dry digestion.
After that, 10 ml of distilled water containing 1% nitric acid was added. Finally, the samples were purified by spraying the solutions with Whatman paper inside the beakers. The soil samples were dried in an oven at 105°C until uniform. At the same time, to measure the As content of the plant samples, 2 g of the samples and 12.5 mL of 4 M nitric acid were combined. The solution was then placed in an oven at 80°C for 24 hours. After acid evaporation, 10 mL of distilled water was added. Other stages were similar to the soil preparation process (Sposito 1989).
To measure As, standard solutions (Merck company) (1000 mg/L) were purchased. All samples were kept in a nitric acid solution (10%) for 24 hours and washed with distilled water. After the sample’s preparation, plasma light emission spectroscopy (ICP-OES Vista-MPX) was used to determine the as concentration in pistachios and soil and water samples.
Determination of TF and Arsenic Accumulation Coefficient (BCF)
The amounts of As transferred from soil and roots to different organs of the plant were calculated using the following relations (Grytsyuk et al. 2006):
Concentration of desired as in the growth medium / Concentration of desired as in the root or shoot = BCF
Concentration of desired as in the root / Concentration of desired As in the shoot = TF
Ion Leakage Rate
To measure the ion leakage rate, 0.3 g of the leaf sample with distilled water (25 mL) (control) and polyethylene glycol 6000 (25 mL) (PEG6000) were placed in an incubator (10°C) for 24 hours. Afterwards, the tubes were drained and the samples were washed. Then, the PEG-treated and control samples were placed in 25 mL distilled water for 24 hours. The electrical conductivity was then measured and the samples were autoclaved at 120°C and 15 atmospheres for 15 minutes. After autoclaving, the electrical conductivity was measured for the second time. The following equation was used to calculate the membrane stability:
(Eq. 1);\(CMS\left(\%\right)=\frac{\left[1-\frac{T1}{T2}\right]}{\left[1-\frac{C1}{C2}\right]}*100\)
Damage percentage = 100 - CMS (%)
where CMS, cell membrane stability and C and T represent the electrical conductivity of the polyethylene glycol treatment and control, respectively. Subscripts 1 and 2 are the initial and final electrical conductivity, respectively (Saneoka et al. 2004).
MDA
The method proposed by Heath and Packer (Heath and Packer 1968) was used in the current study to measure the concentration of MDA. According to this method, fresh leaf tissue (0.2 g) was weighed, and 5 mL of TCA was ground to 0.1%. The resultant extract was centrifuged at 10,000 rpm for 5 minutes. Then, 4 mL of a TCA (40%) solution containing TBA (0.5%) was added to 1 mL of the supernatant solution. The resultant mixture was heated to 95°C in a hot water bath for 30 minutes and then centrifuged again at 10,000 rpm for 10 minutes. The absorbability of this solution was measured with a spectrophotometer at 532 nm.
Photosynthetic pigments
In this stage, the leaf sample (0.1 g) was weighed and then thoroughly ground with acetone (5 mL of 80%) and magnesium oxide (0.1 g); then, it was centrifuged at 3000 rpm for 10 minutes. The adsorption rate at wavelengths of 646 and 645 was then read and the amount of chlorophyll type a and b was calculated using the following equations (Ashraf et al. 1994):
where V and W represent the sample size and the weight of wet sample, respectively.
Carotenoids and Flavonoids
To measure the amount of carotenoids, the absorbance of the samples was measured at the wavelength of 470 nm. The amounts of pigments in milligrams per gram were given in the following equation:
(Eq. 4);\(Carotenoids=100\left(A470\right)-3.27\left(mg chl a\right)-104 \left(mg chl b\right)\)
To measure the flavonoids, 0.5 mL of the methanolic extract prepared with methanol (1.5 ml) was first mixed with 0.1 mL of aluminium chloride (10%) in ethanol, 1M potassium acetate (0.1 ml), and distilled water (2.8 mL). The mixture was kept in a dark place for half an hour and then read at the wavelength of 415 nm. Flavonoids were measured using the Quercetin standard (Chang et al. 2002).
The amount of carbohydrates and total protein
The Nelson method (Nelson 1944) was adopted for measuring carbohydrates. For this purpose, a standard curve was established between zero and 300 µg. To measure the amount of protein, herbal sample (0.5 g ) was mixed with 3 mL of extracted buffer and then centrifuged at 2100 rpm for 21 minutes at 4°C. The upper part of the extract was centrifuged again (20,000 rpm and 20 minutes). Finally, the Bradford solution (5 mL), the extraction buffer (290 µl), and the extract (10 µl) were mixed together. The absorbance was then measured in a spectrophotometer at the wavelength of 590 nm(Bradford 1976).
Reduced glutathione
To measure the reduced glutathione, leaf tissue (0.5 g) was minced in 15% metaphosphate (4 mL) and the extract was centrifuged (30 min at 10,000 rpm and 4°C). sodium phosphate buffer (2.6 mL )(pH = 7.7) and DTNB solution (200 µl) containing 6 µM were added to 200 µl of the supernatant extracted from the centrifuging process; after 30 minutes, the samples' absorbance values were read at 412 nm (Ellman 1959).
The activities of antioxidant enzymes:
To prepare an enzymatic extract, freshly-ground leaf tissue (0.5 g), 1 mm phosphate buffer (50 mM) (acidity = 7) containing EDTA (0.5 M), and PVP (2%) were mixed. The solution was centrifuged (4°C, 20 minutes, 14,000 rpm). Then, the supernatant was used to determine the activity of the enzymes.
To evaluate APX enzyme activities, a method proposed by previous research (Aono et al. 1995) was used. 770 µl of 50 mM phosphate buffer (acidity = 7) was mixed with 100 µl of 0.1 mM EDTA, 100 µl of 5 mM ascorbic acid, and 10 µl of 0.1 mM hydrogen peroxide. Then, 20 µl of the enzyme extract was added and the curve of absorbance change at the wavelength of 290 nm was read. The activity of GPX was determined by the method of Tang and Newton (Tang and Newton 2005). The reaction mixture consisted of 2.5 mL of 50 mM phosphate buffer (pH = 7) containing 1 mL of 1% guaiacol, 1 mL of 1% hydrogen peroxide, and 0.1 mL of the extract. Afterwards, the activity of the GPX enzyme was calculated through increasing absorbance for 1 min at the wavelength of 420 nm. Saunders and McClure’s (Saunders and McClure 1974) method was adopted to measure the activity of PAL with slight modifications. The sample used for enzyme evaluation contained 250 mL enzyme extract, 250 mL 10 M sodium borate buffer (pH = 8.8), 250 mL distilled water, and 250 mL phenylalanine substrate buffer (50 mM); its absorbance was then measured at the wavelength of 290 nm. The activity of GR was evaluated based on previous research (Arora et al. 2002) method through the reduction of GSSG by GR using NADH. The reaction mixture consisted of 200 mM potassium phosphate buffer (pH = 7.5), 500 µl GSSG, 50 µl NADH, 1.5 mM MgCl2, 0.2 mM Na2EDTA, and 50 µl enzyme extract. After the addition of the enzyme to the reaction mixture, the reduction in adsorption was read at the wavelength of 340 nm for 120 seconds.
The P5CS activity was done according to the method described by previous research (Wang et al. 2007). In this method, a mixture of 50 mM Tris buffer (PH = 7.0), 20 mM MgCl2, 50 mM glutamate, 100 mM hydroxylamine HCl, and 10 mM ATP was used. The absorption rate was at the wavelength of 535 nm. The assessment of the PRODH enzyme activity was through examination of adsorption increase at the wavelength of 340 nm in the reaction mixture containing 100 mM acetate buffer (pH = 10.3), 20 mM L-proline, 10 mM NAD, and 100 µl plant extract (Rena and Splittstoesser 1974).
Measurement of lipoxygenase, the amount of oil and fatty acids
The oil of the sample was extracted through the cold extraction method and using normal hexane(99%). First, the oil was filtered using philter papers and then the solvent was removed by a rotary evaporator at 40°C and dry nitrogen gas. The LOXs enzyme activity was determined at a wavelength of 234 nm. The amounts of fatty acid in leulic acid, linoleic acid, and palmitic acid were determined by GC model 6000. Helium was used as the carrier gas. The temperature of the column was 60°C, which was raised to 198°C in 54 minutes.
Data analysis
The obtained data was analyzed using SAS 9.2 statistical software. Means were compared using the LSD test at a probability level of 5%. R was used for plotting, regression, and PCA.
Arsenic content
Findings related to the changes in the as volumes in soil and water within the studied areas showed significant differences. The As changes in irrigation water were recorded in the range of 0.62 to 483 mcg/L, while the range of these changes for the soil was between 15 to 1200 mg/kg. Based on the maximum permissible level of As in soil (30 mg/kg) and water (10 mcg/L) in Iran, it was found that the level of AS in more than 75% of the studied areas was several times higher than the permissible values. The highest amount of As in soil was observed in Shahrebabak area (1200 mg/kg), while the highest amount of Arsenic in irrigation water was in Sirjan region 483 mcg/L (Table 1).
The results obtained by analyzing the As accumulation in different organs of pistachio showed that the amounts of this metal in the leaves, roots, and fruits of this plant was significantly different (see Table 1). It was also revealed that the amount of As in the roots was much higher than the leaves, and fruits were of the least As contents among the three organs studies. The As content changes in roots were found to be ranging from 1.22 to 14.67 mg/kg, in leaves 2.17 to 8.20 mg/kg, and in fruits 0.21 to 2 mg/kg (Table 1). The highest As levels in leaves, roots, and fruits were recorded in Sirjan region (8.4, 14.6, and 2.07 mg/kg, respectively), while Shahrebabak had the highest level of environmental As (8.2, 15.4, and 2.01 mg/kg, respectively). However, in most regions, roughly similar values were recorded regarding the As content in fruits (Table 1).
The regression results of the changes in environmental as (water and soil) and tissue As presented in Fig. 2 proves a linear relationship between the environmental As and the As accumulated in the pistachio roots. The nature of the relationship between environmental As and leaf and fruit As contents was found quadratic, showing the role of As transmission system in its level of accumulation in the limbs. In pistachios, the amount of As in roots increased to 0.0082 mg/kg, but in leaves and fruits, it increased to 14 and 23 mg/kg. After reaching these values, there has been no more significant increase in the amount of this element (Fig. 2).
|
Region |
As of water (ug/L) |
As of Soil (mg/kg) |
As of pistachios (mg/kg) |
||
|---|---|---|---|---|---|
|
Root |
Leaf |
Fruit |
|||
|
Sirjan |
483.0 ± 11.9a |
1023.0 ± 42.0bc |
14.67 ± 1.09a |
8.20 ± 0.22ab |
2.07 ± 0.20a |
|
Shahr-e-Babak |
357.1 ± 20.5b |
15.42 ± 0.29a |
8.42 ± 0.23a |
2.01 ± 0.28a |
|
|
Bayaz |
177.8 ± 12.4c |
11.52 ± 0.32b |
7.92 ± 0.19bc |
1.97 ± 0.31a |
|
|
Anar |
162.4 ± 15.2c |
998.0 ± 36.1bc |
10.01 ± 0.45b |
7.62 ± 0.21cd |
1.93 ± 0.19a |
|
Kabootar-Khan |
65.7 ± 10.1d |
989.0 ± 27.0c |
8.41 ± 0.60c |
7.51 ± 0.22d |
1.82 ± 0.15ab |
|
Kazem-Abad |
28.3 ± 4.2e |
551.0 ± 27.0d |
6.12 ± 0.55d |
6.34 ± 0.22e |
1.52 ± 0.11b |
|
Zarand |
4.66 ± 0.2f |
34.0 ± 2.0e |
4.05 ± 0.54e |
4.01 ± 0.20f |
0.37 ± 0.05c |
|
Ravar |
0.62 ± 0.1f |
15.0 ± 2.0e |
1.22 ± 0.41f |
1.17 ± 0.12g |
0.21 ± 0.03c |
|
Fvalue |
** |
** |
** |
** |
** |
|
CV (%) |
4.9 |
7.2 |
11.3 |
2.0 |
3.2 |
The different letters in each column indicate a significant difference by Duncan's multiple range test at the 5% level.
BCF and TF of Arsenic
A comparison of BCF and TF of pistachios in different regions also revealed significant differences in both coefficients (Fig. 3). The plots of changes in these coefficients showed that BCF was much higher in roots and leaves than in fruits, but there was no significant difference between the two. The BCF changes in this plant was in the range of 0.09–0.12 with the highest value related to the areas with the lowest amount of environmental As (with changes ranging from 15 to 34 mg/kg). This was due to the high denominator of the BCF fraction in areas with higher as (1200 mg/kg) as compared to these areas. It was also found that BCF changes in fruits had less fluctuations in different regions; the changes ranged from 0.002 to 0.014, indicating that it was less affected by the environment (Fig. 3). The range of TF changes in pistachio leaves and fruits also showed that the rate of as transfer from root to leaf was higher than from root to fruit. Therefore, TF in this organ was 0.56–1.2, while its changes in fruit were recorded as 0.097 to 0.25 (Fig. 3).
The findings of this study showed about 75% of the studied areas had much higher As contents than the permissible limit, affecting the amount of this heavy metal in different parts of pistachio as a strategic product of the region, i.e., Kerman, Iran (Table 1). According to previous research (Gillispie et al. 2015), the existence of As in many soil samples is of tectonic origin, caused by the natural weathering of rocks and soils in contact with contaminated water sources or chemical fertilizers and pesticides. The uptake of As and other heavy metals in plant organs depends on several factors such as the type of metal, soil conditions, amount of other nutrients, plant species, and tissue type (Abbas et al. 2018). The biogeochemical properties of As in the soil-plant system (e.g., mobility, bioavailability, and toxicity) depend largely on its oxidation state. According to Joseph et al. (Joseph et al. 2015), the two main types of arsenic oxidation are arsenite (III) and arsenate As (V), which are absorbed and transferred in plant, and changes to environmental conditions (PH, soil redox, Soil microbial activity, etc.) have significant effects on the occurrence of As in different oxidation states. The form and excitability of As decreased by increasing soil pH due to the deposition of hydroxides and carbonates or the formation of insoluble organic material affecting its rate of uptake by the plant in different areas(Adra et al. 2016). BCF and TF changes also showed a decreasing trend from one region to another and from one organ to another in contrast to changes in the environmental As changes, suggesting that they are more balanced due to the influence of different environmental factors on different forms of this element. In the present study, it was observed that BCF was not significantly different in roots and leaves, while there was a big difference from fruit. However, TF, which is an indicator of the mobility of heavy metals in soil (Boularbah et al. 2006), was much more intense from root to leaf than from root to fruit (Fig. 2). In fact, the longer the transmission path of this element from different parts, the more its amount is decreased. According to the scientific reports, As mainly accumulates in the roots and its transfer to the above-ground parts is low (El-Mahrouk et al. 2019).
Membrane leakage and MDA
The leakage rate in pistachio leaves was reported to be 14%-20% (Table 2). The highest rate of ion leakage (20%) belonged to the areas with a range of 1200 mg/kg of environmental As, and 8.4 mg/kg of intra-tissue As. On the other hand, the lowest rate (14%) belonged to the area with about 15 mg/kg as (Table 2). The changes of the regression of ion leakage had a linear relationship with environmental As. The leakage rate in pistachio increased by 31% with an increase of 100 mg/kg in environmental As, indicating higher cell destruction of membrane structures due to increasing the rate of as (Fig. 4).
MDA, as a destructive compound within the tissue, was also studied in pistachio leaves, and it was found that its production was different in different regions (Table 2). The average production of this substance in pistachios was 0.65 nmol/g. The highest amount of this substance (0.86 nmol/g), similar to the ion leakage, was obtained from the regions with the As range of 1000–1200 mg/kg, while the lowest amount (0.53 nmol/g) belonged to the regions with 15 and 34 mg/kg As (Table 2). The trend of changes in the rate of MDA, similar to the ion leakage by the environmental As, was determined as a linear increase with a slope of 0.02 nmol/g per 100 mg/kg (Fig. 4).
Normally, plants can take up as in its inorganic form and with the help of transporter proteins, and the main driving force for uptake is a concentration gradient between source and reservoir. The mechanism of As uptake by plants depends on its chemical form (Abbas et al. 2018). As (V) reportedly uses Pi channels to enter the cell, and Pi transporter proteins (PHT) are key components of P channels involved in the uptake of As (V) (Abbas et al. 2018; LeBlanc et al. 2013). In contrast, plants take up as (III) via intrinsic Nodule-26 proteins (NIPs). The PHT transducers are unilateral, but NIP is bilateral, allowing as (III) to move in both directions between plant cells and the growth medium. As (III) also uses silicon transducers due to its similarity to Si (Khalid et al. 2017).
|
Region |
Membrane leakage (%) |
MDA (nM/g) |
Chl a (mg/g) |
Chl b (mg/g) |
Carotenoids (µg/g) |
Flavonoid (µg/g) |
Carbohydrates (mg/g) |
Protein (mg/g) |
|---|---|---|---|---|---|---|---|---|
|
Sirjan |
20.85 ± 0.91a |
0.86 ± 0.06a |
24.65 ± 2.20b |
6.10 ± 0.37d |
7.23 ± 0.11b |
70.00 ± 0.41b |
72.25 ± 0.48cd |
4.68 ± 0.20c |
|
Shahr-e-Babak |
19.67 ± 0.48ab |
0.76 ± 0.03ab |
24.20 ± 0.39b |
7.15 ± 0.27c |
8.78 ± 0.22a |
74.50 ± 1.32a |
76.00 ± 0 .71ab |
5.23 ± 0.28bc |
|
Bayaz |
18.82 ± 0.87abc |
0.74 ± 0.01ab |
27.43 ± 1.34a |
6.93 ± 0.08c |
8.70 ± 0.09a |
76.00 ± 0.71a |
61.00 ± 0.71e |
4.95 ± 0.74bc |
|
Anar |
17.60 ± 0.54bc |
0.69 ± 0.05bc |
29.65 ± 0.16a |
7.47 ± 0.21c |
8.43 ± 0.28a |
69.77 ± 0.63b |
74.00 ± 0.41bc |
6.03 ± 0.36ab |
|
Kabootar-Khan |
17.48 ± 0.53bc |
0.66 ± 0.06bcd |
29.15 ± 0.10a |
8.30 ± 0.19b |
6.03 ± 0.11c |
60.75 ± 0.48b |
74.00 ± 0.91bc |
5.95 ± 0.25ab |
|
Kazem-Abad |
16.52 ± 0.97cd |
0.58 ± 0.04cd |
29.58 ± 0.26a |
9.18 ± 0.15a |
6.28 ± 0.28c |
64.00 ± 0.41c |
71.25 ± 0.63d |
6.83 ± 0.38a |
|
Zarand |
16.50 ± 0.92cd |
0.56 ± 0.05cd |
30.33 ± 0.32a |
9.10 ± 0.11a |
7.33 ± 0.09b |
71.25 ± 0.48b |
77.25 ± 0.48a |
3.43 ± 0.05d |
|
Ravar |
14.90 ± 0.59d |
0.53 ± 0.03d |
29.88 ± 0.32a |
9.65 ± 0.19a |
4.25 ± 0.29d |
61.75 ± 0.48d |
60.75 ± 0.85e |
4.75 ± 0.09c |
|
Fvalue |
** |
** |
** |
** |
** |
** |
** |
** |
|
CV (%) |
17.79 |
10.5 |
6.6 |
5.3 |
5.7 |
1.97 |
13.5 |
1.8 |
The different letters in each column indicate a significant difference by Duncan's multiple range test at the 5% level.
Photosynthetic pigments
The results of the pigments (Chl a and Chl b) proved that they were affected by the region. The highest amount of Chl a (30.33 mg/kg) and Chl b (9.10 mg/kg) were observed in the regions with 15 mg/kg As, and an increase in As content in different areas decreased the rate of pigments. On the other hand, the lowest amount of pigments was observed in the regions with about 1000 to 1200 mg/kg As (Table 2). Findings of the regression models also showed that the model of changes in chlorophyll a and Chl b (as the result of As changes) was in the form of linear reduction. The slopes of these changes were 0.001 and 0.0017 mg per 1 mg/kg As, respectively (Fig. 4).
As causes oxidative stress that is destructive to plants. This stress is triggered by the overproduction of ROS, including superoxide radicals, hydroxyl radicals, and hydrogen peroxide (Rafiq et al. 2017). The ROS production causes destruction in biological membranes, chlorophyll degradation, protein denaturation, mutations in DNA molecules, and production of lipid peroxidation; it also increases membrane permeability (Banci et al., 2011). The findings of the present study (which is consistent with several other reports, e.g., it was showed (El-Mahrouk et al. 2019; Mohammadhasani et al. 2017) that the areas with the As contents ranging from 1000 to 1200 mg/kg experience more malondialdehyde and electrolyte losses. Some studies have proved that an increase in the ROS production in plants occurs through the conversion of As (V) to As (III) (Abbas et al. 2018). In addition, electron loss during the reduction of As (V) to As (III) and inhibition of key enzymes is also an important pathway for the ROS production in plants (Singh et al. 2007). Oxidative damage in plants caused by as has several other destructive consequences. Photosynthesis and photosynthetic pigments are among the other processes affected by As stress (Abbas et al. 2018). In this study, it was observed that the areas with the highest As concentration had the lowest levels of Chl a and Chl b and their changes were linear (Fig. 6). As interferes with the photosynthetic process of plants by affecting metal homeostasis due to iron deficiency, photosystem function II, chlorophyll synthesis, protein complex formation, chloroplast membrane, gas exchange, and fluorescence diffusion (Abbas et al. 2018; Basa et al. 2014). It has been also reported that different concentrations of as in corn (Emamverdian et al. 2015) and Trifolium pratense L. (Suneja et al. 2019) reduce the net photosynthesis.
Carotenoids and flavonoids
The amounts of flavonoids and carotenoids varied depending on the area studied (Table 3). The ranges of variation of these pigments were 4.25–8.78 mg/g and 60–76 µg/g for carotenoids and flavonoids, respectively. The highest contents of flavonoids were obtained in the range of 1055–1200 mg/kg As, which corresponded to about 74.5 and 76 mg/g of flavonoids. At the same time, the highest number of carotenoids was obtained in areas with a range of variation between 998 and 1200 mg/kg (Table 3). The changes of flavonoids were more affected by as compared to carotenoids. The changes in flavonoids and carotenoids were linear, and the slop of changes in carotenoids was recorded as 0.003 mg per mg/kg, while this amount was recorded as 0.0084 mg per mg/kg in flavonoids (Fig. 4).
The oxidation of proteins and their sensitivity to proteases is another as stress-induced changes in oxidation. In the current research, it was observed that in pistachio, soluble sugars showed no clear trend; however, protein levels increased due to low as concentrations, but decreased in areas with the as concentration of 1000–1200 mg/kg (Fig. 4). In a study conducted by Choudhury et al. (Choudhury et al. 2011), it was proved that protein content decreased by increasing the As concentration. The reduced protein biosynthesis under such conditions leads to changes in cell membranes, disruption of ribulose-biphosphate carbosylase reaction center, protein and sugar metabolism in plants, which reduce the plant growth (Hussain et al. 2013). The As-induced suppression of nitrate and nitrite reductase activities is also responsible for protein degradation. As (III) is also more toxic than as (V) due to its affinity for sulfhydryl groups in proteins. Normally, proteins are hydrolyzed to free amino acids and short peptides using proteases and peptidases (Abbas et al. 2018; Parkhey et al. 2014).
|
Region |
Glutathione (mg/g) |
APX (U/mg protein) |
GPX (U/mg protein) |
GR (U/mg protein) |
PAL (U/mg protein) |
P5CS (U/mg protein) |
PRODH (U/mg protein) |
LOXs (U/mg protein) |
|---|---|---|---|---|---|---|---|---|
|
Sirjan |
15.76 ± 0.26d |
0.52 ± 0.06e |
14.22 ± 0.89ab |
8.38 ± 0.84a |
34.30 ± 0.81a |
0.86 ± 0.04a |
0.11 ± 0.01e |
2.92 ± 0.51a |
|
Shahr-e-Babak |
16.42 ± 0.36cd |
0.59 ± 0.07de |
13.61 ± 0.87ab |
7.71 ± 0.56a |
33.28 ± 0.77a |
0.83 ± 0.06a |
0.16 ± 0.01ef |
2.77 ± 0.62a |
|
Bayaz |
16.94 ± 0.10cd |
0.62 ± 0.06cde |
12.97 ± 0.89ab |
6.92 ± 0.81ab |
30.51 ± 0.80bc |
0.79 ± 0.05ab |
0.19 ± 0.02def |
2.40 ± 0.25ab |
|
Anar |
17.16 ± 0.17cd |
0.69 ± 0.05bcd |
11.60 ± 0.98abc |
6.66 ± 0.92ab |
28.72 ± 0.96cd |
0.69 ± 0.05bc |
0.22 ± 0.05b − e |
2.11 ± 0.21abc |
|
Kabootar-Khan |
17.96 ± 0.34bc |
0.75 ± 0.04abc |
11.21 ± 1.20bcd |
5.52 ± 0.33bc |
27.63 ± 1.05de |
0.61 ± 0.01cd |
0.26 ± 0.04a − d |
1.91 ± 0.48abc |
|
Kazem-Abad |
18.02 ± 0.58bc |
0.82 ± 0.04ab |
9.51 ± 0.74cde |
4.80 ± 0.10bc |
26.92 ± 0.71ef |
0.53 ± 0.03d |
0.30 ± 0.04abc |
1.50 ± 0.27bc |
|
Zarand |
18.97 ± 0.54ab |
0.88 ± 0.02a |
8.71 ± 0.53de |
3.92 ± 0.57c |
25.32 ± 0.48ef |
0.49 ± 0.03de |
0.35 ± 0.07ab |
1.11 ± 0.04c |
|
Ravar |
20.25 ± 0.97a |
0.90 ± 0.03a |
7.70 ± 0.51e |
3.63 ± 0.68c |
23.44 ± 0.89f |
0.38 ± 0.04e |
0.38 ± 0.05a |
1.09 ± 0.04c |
|
Fvalue |
** |
** |
** |
** |
** |
** |
** |
* |
|
CV (%) |
4.7 |
11.5 |
13.4 |
19.1 |
4.9 |
10.8 |
18.7 |
13.7 |
The different letters in each column indicate a significant difference by Duncan's multiple range test at the 5% level.
Carbohydrates and Protein
The range of carbohydrate changes in this plant ranged from 60.7 to 77.2 mg/g, with Shahrebabak and Zarand regions (both in Kerman, Iran) having the highest rate (76-77.25 mg/kg). The fluctuation ranged from 34 to 1200 mg/kg, indicating that this trait is less affected by environmental changes in As. On the other hand, protein changes ranged from 3.4 to 6.8 mg/kg, and the highest amount (6.83 and 6.03 mg/kg) was observed in the region with a range of 555 to 998 mg/kg (Table 2). The regression results of these traits showed that the carbohydrate changes were not significantly related with the changes in environmental As. This model could predict the protein changes better, as their coefficient of determination was 0.59 that increased with a slope of 0.0023 and decreased with a slope of 0.0000728 mg per 1 mg/kg in the range of 500 mg (Fig. 4).
Plants use a complex antioxidant system with basic components of carotenoids, ascorbate, glutathione, and tocopherols to combat damage caused by oxidative stress. Antioxidant enzymes also include SOD, CAT, APX, POX, and GPX; and the enzymes involved in the ASA-GSH cycle are similar to GR (Kwon et al. 2001). In this study, it was observed that the As-induced changes of carotenoids and flavonoids in pistachios increased linearly, and the intensity of changes was greater for flavonoids than for carotenoids, indicating their effective role in pistachios (Fig. 4). Due to their ability to transfer energy to photosynthesis and the role of photo-protection of protective mechanisms, these compounds protect the plant from oxidative stresses by scavenging free radicals. Therefore, pistachio leaves produced more flavonoid in areas with higher levels of As, as confirmed by the PCA results (Jithesh et al. 2006). The activities of antioxidant enzymes also increase in the presence of plants with high as content; thus, the plant is able to reduce the ROS damage. The SOD Activity is the plant's first reaction against free radicals, converting them into H2O2 and O2. H2O2 is then neutralized by other enzymes and converted to water and O2 (Gusman et al. 2013). In the present study, the PAL, GPX, and GR enzymes played increasing roles, while APX and glutathione reduction played decreasing roles, indicating the plant reaction pathway through these enzymes and the glutathione cycle to neutralize H2O2 (Fig. 5). Begum (2016) reported an increase in the synthesis of GPX and CAT enzymes under the as stress conditions. Souri et al. (Souri et al. 2018) observed a significant increase in the activities of the GR, CAT, POX, SOD, and GPX enzymes in Isatis cappadocica Desv by increasing the As concentration. Increased activities of antioxidant enzymes in pistachios by heavy metal zinc was also reported (Mohammadhasani et al. 2017). Using SOD and ASA-GSH, It was showed (Polle 2001) that cadmium prevents the activities of enzymes such as CAT, GPX, and APX by reducing glutathione in the plants.
AsA-GSH cycle
The changes in the amounts of reduced glutathione, APX, PAL, GPX, and GR in pistachio leaves were also affected by region. The highest amounts of reduced glutathione (19.23 mg/kg) and APX activity (0.88 U/mg protein) were observed in the region with the environmental as in the range of 15–34 mg/kg, indicating a decrease in the amount of these traits by increasing the concentration of As. At the same time, the highest activities of PAL (34.3 U/mg protein), GPX (14.22 U/mg protein) and GR (8.38 U/mg protein) ranged from 1000 to 1200 mg/kg As, whereas the lowest activities were ina mount of 15 mg/kg As, indicating a different trend of changes in these enzymes in contrast to reduced glutathione and APX and increased activity due to as (Table 3). The trend of changes in regenerated glutathione and APX was linearly reduced by increasing As. With each increase of 100 mg/kg As, regenerated glutathione decreased by 0.24 mg/kg and APX decreased by 0.025 U/mg protein. The activities of PAL, GPX, and GR also increased linearly by increasing the as concentration. The slope of changes of these enzymes was recorded as 0.70, 0.46, and 0.32 per 100 mg/kg As, respectively (Fig. 5).
Proline biosynthesis enzyme
The P5CS and PRODH activities of pistachio leaves were determined in the range of 0.38–0.86 and 0.11–0.38 standard units per mg, respectively. The highest P5CS activity (0.61 standard units per mg) was corresponded to the highest environmental as (1200 mg/kg), while the highest PRODH activity (0.38 standard units per mg) was corresponded to the lowest amount of arsenic (15 Mg/kg). The trend of regression changes of these enzymes in different as concentrations also showed that the increase of as caused the growth of the P5CS activity and the decrease of PRODH activity. Changes of P5CS and PRODH activities under as conditions were found linear. The slope of P5CS changes per 100 mg/kg of as increase was 0.332 standard units per mg increase and PRODH was 0.017 standard units per mg decrease (Fig. 5).
The proline production is another mechanism of response to oxidative stress. Proline is an important amine acid that is effective in plants' growth through signaling processes. Signorli et al. (2015) reported a proline cycle that significantly helped in eliminating OH radicals. This cycle was recycled by pyrroline-5-carboxylate reductase and pyrroline-5-carboxylate with the simultaneous oxidation of NADPH to proline. The PRODH enzyme is mainly responsible for degradation and accumulation of proline in plants. The decreased activity of this enzyme was observed in areas with high as pollution (Table 3). Proline plays a key role in the osmotic potential of cells in metal-induced disorders. Proline was reported to accumulate when Triticum aestivum and Oryza sativa seedlings were treated with different levels of As (III) and As (V) (Choudhury et al. 2011; Hasanuzzaman and Fujita 2013). Proline can reduce toxicity by decreasing As uptake through altering the cell wall structure and plasma membrane protection, directly abolishing As-induced ROS production, increasing the activity of various antioxidants, and altering the expression of stress-related genes (Abbas et al. 2018).
Lipoxygenase and oil content
The LOXs enzyme and oil content were also influenced by regions. The highest percentage of oil (52.4%) belonged to fruits harvested in regions with the lowest amounts of as (between 15 and 34 mg/kg) and these fruits had the lowest amounts of LOXes (1.09 standard units per mg). The lowest amount of oil (23.1%) and the highest amount of LOXes (2.92 standard units per mg) were also observed in the areas with the lowest as contents (Tables 3 and 4). The change to the oil content trend (which occurred due to as) was linear, and as the amount of oil increased, the amount of LOXes decreased. On the other hand, as As increased, the amount of LOXes increased too, which occurred linearly in relation to As. The slope of oil changes per 1 mg/kg of as was 0.015%. The LOXs enzyme also increased by 0.008 standard units per mg per 1 mg per kilogram of as (Fig. 4 and Fig. 5).
Fatty acid
The amounts of fatty acids (oleic acid, linoleic acid, and palmitic acid) in pistachio oil varied in different regions. The ranges of changes in oleic acid and linoleic acid were 58–59% and 32.2–34%, respectively. The highest amounts of the palmitic acid and oleic acid mostly ranged from 15 to 34 mg/kg As, and the highest amount of the linoleic acid was observed in the range of 100–1200 mg/kg As, indicating a positive relationship between environmental As and linoleic acid and, on the other hand, a negative relationship between As and palmitic acid and oleic acid (Table 4). The changes occurred to the regression models of these fatty acids also revealed that the changes of the palmitic acid and oleic acid were in the form of linear reduction and the changes to the linoleic acid were in the form of linear increase. The slope of changes to the oleic acid and palmitic acid varied from 0.037 to 0.0087 per 100 mg/kg As, respectively. However, this slope was identified as 0.0055 per 100 mg/kg as (Fig. 5).
In this study, it was observed that the process of biosynthesis of pistachio fruit oil was disturbed by as; as a result, the seed oil percentage showed a linear decrease by as (Fig. 5). The reduction of oil indicates the inhibition of as biosynthesis by fatty acids. Thus, the percentages of palmitic acid and oleic acid decreased due to the increase of as (Fig. 5). The As reduction in unsaturated fatty acids may be attributed to the direct reaction of ROS with unsaturated fatty acids or the inhibition of fatty acid biosynthesis (Ahmad et al. 2015). Based on the findings of the present study and those of similar ones, heavy metals increase the activity of liposuction, which is responsible for catalyzing lipid peroxidation. In addition, such metals use the lipid components of the membrane as substrates (especially unsaturated fatty acids) and decrease biosynthesis (Youssef et al. 2005). It was reported (Sinha et al. 2010) a reduction in canola oil by three heavy metals, i.e., As, lead, and copper. Generally, the activation of plant defense responses affects the biosynthesis of many chemical compounds and processes in the plant. This increases the plant costs and negatively affects its quantitative and qualitative productivity and reduces its economic benefits.
|
Region |
Oil content (%) |
Oleic acid (%) |
Linoleic acid (%) |
Palmitic acid (%) |
|---|---|---|---|---|
|
Sirjan |
23.11 ± 1.03c |
57.99 ± 0.07c |
33.97 ± 0.03a |
8.04 ± 0.02b |
|
Shahr-e-Babak |
25.27 ± 3.60c |
57.83 ± 0.11c |
34.11 ± 0.06a |
8.06 ± 0.03b |
|
Bayaz |
25.12 ± 2.91c |
58.32 ± 0.06b |
33.53 ± 0.09b |
8.15 ± 0.03a |
|
Anar |
39.38 ± 2.04b |
58.51 ± 0.11b |
33.32 ± 0.05c |
8.18 ± 0.02a |
|
Kabootar-Khan |
33.26 ± 2.37b |
58.50 ± 0.13b |
33.31 ± 0.07c |
8.17 ± 0.02a |
|
Kazem-Abad |
33.26 ± 1.78b |
59.02 ± 0.11a |
32.87 ± 0.06e |
8.21 ± 0.03a |
|
Zarand |
39.12 ± 1.22b |
58.61 ± 0.07b |
32.20 ± 0.06d |
8.19 ± 0.02a |
|
Ravar |
52.14 ± 1.26a |
58.93 ± 0.12b |
33.16 ± 0.08c |
8.21 ± 0.02a |
|
Fvalue |
** |
** |
** |
** |
|
CV (%) |
11.0 |
2.96 |
3.42 |
5.48 |
The different letters in each column indicate a significant difference by Duncan's multiple range test at the 5% level.
PCA
The results of the PCA showed that the amounts of oil, palmitic acid, oleic acid, PRODH activity, reduced glutathione, and APX were most similar to each other and were in the same coordinate group in the zone II. It was also found that the changes of chlorophylls a and b were very similar to this group, but were located in the coordinate zone III. The activities of LOX, linoleic acid, GR, GPX, MDA, proteins, carotenoids, and flavonoids were also very similar; they were located in the coordinates of zone I. The amount of ion leakage and the activity of PAL were similar to each other and located in region IV. The results of the studied areas also showed that areas with high as such as Sirjan and Shahrebabak were in the coordinates of region IV, and areas with low as such as Ravar were in region II. Based on the level of as intensity, the coordinates of PCA diagram could be graded as IV > I > III > II, and the response of traits and regions to as could be determined based on the location in the region (Fig. 6).
The obtained results proved that pistachio was an accumulator in the fruit part of the plant; therefore, BCF was not much different in the roots and leaves of the plant, but they were much higher than the fruits, which reduced the risks of the fruits of this plant. In addition, the photosynthetic pigments and membrane stability in this plant linearly decreased due to the increase of oxidative and malondialdehyde stress in the plant. This reduced the biosynthesis of oil and some other saturated fatty acids, palmitic acid, and oleic acid in the plant. The plant activated the non-enzymatic and enzymatic reactions under these conditions, of which flavonoids were more efficient than the non-enzymatic systems, and GPX and GR were more efficient than the enzymatic systems. The biosynthesis of proline is also stimulated by increasing the concentration of As in pistachio leaves through the increase the activity of P5CS and the decrease of the PRODH activity. This can happen through the osmotic regulation of proline and plays its role in eliminating active radicals in a way to effectively improve the plant's tolerance.
AOC: Allele Cyclase
AOS: Allele Oxidase
APX: Ascorbate Peroxidase
ASA: Ascorbate
ASA-GSH: Ascorbate Cycle, and Glutathione Cycle
CAT: Catalase
Chl a: Chlorophyll a
Chl b: Chlorophyll b
CRD: Completely Randomized Design
DHAR: Dehydroascorbate Reductase
GC: Gas Chromatography
GK: Glutamate Kinase
GPR: γ-Glutamyl Phosphate Reductase
GPX: Guaiacol Peroxidases
GR: Glutathione Reductase
GSH: Glutathione
GSSG: Oxidized Glutathione
HPL: Hydroperoxidylase
LOXs: Lipoxygenase
MDA: Malondialdehyde
MDHAR: Monovalent Hydroscorbate Reductase
P5CS: Pyrroline-5-Carboxylate Synthase
PAL: Phenylalanine Ammonia Lyase
PCA: Principal Component Analysis
POX: Peroxidase
PRODH: Proline Dehydrogenase
ROS: free oxygen radicals
SOD: Superoxide Dismutase
TBA: Thiobarbituric Acid
TCA: Trichloroacetic Acid
Conflict of Interests
All authors declare no conflict of interest.
Ethics approval and consent to participate
No human or animals were used in the present research.
Consent for publications
All authors read and approved the final manuscript for publication.
Availability of data and material
All the data are embedded in the manuscript.
Authors' contributions
All authors had equal role in study design, work, statistical analysis and manuscript writing.
Informed Consent
The authors declare not used any patients in this research.
Acknowledgement
This article was achieved based on the material and equipment of Islamic Azad University, and Dr. Bahman Fazeli-Nasab (Research Institute of Zabol) that help to edit this article that the authors would like to thanks of them
Funding/Support
This study was supported by Islamic Azad University, Iran.
- Abbas G, Murtaza B, Bibi I, Shahid M, Niazi NK, Khan MI, Amjad M, Hussain M (2018) Arsenic uptake, toxicity, detoxification, and speciation in plants: physiological, biochemical, and molecular aspects. Int J Environ Res Public Health 15: 59-69. doi:https://doi.org/10.3390/ijerph15010059
- Adra A, Morin G, Ona-Nguema G, Brest J (2016) Arsenate and arsenite adsorption onto Al-containing ferrihydrites. Implications for arsenic immobilization after neutralization of acid mine drainage. Appl Geochem 64: 2-9. doi:https://doi.org/10.1016/j.apgeochem.2015.09.015
- Ahani H, Attaran S (2022) Therapeutic potential of Seabuckthorn (Hippophae rhamnoides L.) in medical sciences. Cell Mol Biomed Rep 2: 22-32. doi:10.55705/cmbr.2022.330326.1020
- Ahmad P, Sarwat M, Bhat NA, Wani MR, Kazi AG, Tran L-SP (2015) Alleviation of cadmium toxicity in Brassica juncea L.(Czern. & Coss.) by calcium application involves various physiological and biochemical strategies. PloS one 10: e0114571. doi:https://doi.org/10.1371/journal.pone.0114571
- Ahmadi S (2022) Antibacterial and antifungal activities of medicinal plant species and endophytes. Cell Mol Biomed Rep 2: 109-115. doi:https://doi.org/10.55705/cmbr.2022.340532.1042
- Aono M, Saji H, Fujiyama K, Sugita M, Kondo N, Tanaka K (1995) Decrease in activity of glutathione reductase enhances paraquat sensitivity in transgenic Nicotiana tabacum. Plant Physiol 107: 645-648. doi:https://doi.org/10.1104/pp.107.2.645
- Arora A, Sairam R, Srivastava G (2002) Oxidative stress and antioxidative system in plants. Curr Sci 82: 1227-1238
- Arsana IN, Juliasih NKA, Ayu Sauca Sunia Widyantari AA, Suriani NL, Manto A (2022) GC-MS Analysis of the Active Compound in Ethanol Extracts of White Pepper (Piper nigrum L.) and Pharmacological Effects. Cell Mol Biomed Rep 2: 151-161. doi:10.55705/cmbr.2022.351720.1051
- Ashraf M, Azmi A, Khan A, Ala S (1994) Effect of water stress on total phenols, peroxidase activity and chlorophyll content in wheat [Triticum aestivum L.]. Acta Physiol Plant 16: 185-191
- Basa B, Lattanzio G, Solti Á, Tóth B, Abadía J, Fodor F, Sárvári É (2014) Changes induced by cadmium stress and iron deficiency in the composition and organization of thylakoid complexes in sugar beet (Beta vulgaris L.). Environ Exp Bot 101: 1-11. doi:https://doi.org/10.1016/j.envexpbot.2013.12.026
- Boularbah A, Schwartz C, Bitton G, Aboudrar W, Ouhammou A, Morel JL (2006) Heavy metal contamination from mining sites in South Morocco: 2. Assessment of metal accumulation and toxicity in plants. Chemosphere 63: 811-817. doi:https://doi.org/10.1016/j.chemosphere.2005.07.076
- Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal biochem 72: 248-254
- Chang C-C, Yang M-H, Wen H-M, Chern J-C (2002) Estimation of total flavonoid content in propolis by two complementary colorimetric methods. J Food Drug Anal 10: 178-182
- Choudhury B, Chowdhury S, Biswas AK (2011) Regulation of growth and metabolism in rice (Oryza sativa L.) by arsenic and its possible reversal by phosphate. J Plant Interact 6: 15-24. doi:https://doi.org/10.1080/17429140903487552
- Couto N, Wood J, Barber J (2016) The role of glutathione reductase and related enzymes on cellular redox homoeostasis network. Free Radic Biol Med 95: 27-42. doi:https://doi.org/10.1016/j.freeradbiomed.2016.02.028
- Doosti-Moghaddam M, Miri HR, Ghahghaei A, Hajinezhad MR, Saboori H (2022) Effect of unripe fruit extract of Momordica charantia on total cholesterol, total triglyceride and blood lipoproteins in the blood of rats with hyperlipidemia. Cell Mol Biomed Rep 2: 74-86. doi:10.55705/cmbr.2022.338806.1038
- El-Mahrouk E-SM, Eisa EA-H, Hegazi MA, Abdel-Gayed ME-S, Dewir YH, El-Mahrouk ME, Naidoo Y (2019) Phytoremediation of cadmium-, copper-, and lead-contaminated soil by Salix mucronata (Synonym Salix safsaf). HortScience 54: 1249-1257. doi:https://doi.org/10.21273/HORTSCI14018-19
- Ellman GL (1959) Tissue sulfhydryl groups. Archives of biochemistry and biophysics 82: 70-77. doi:https://doi.org/10.1016/0003-9861(59)90090-6
- Emamverdian A, Ding Y, Mokhberdoran F, Xie Y (2015) Heavy metal stress and some mechanisms of plant defense response. Sci World J 2015: Article ID: 756120 doi:https://doi.org/10.1155/2015/756120
- Farghali M, Shimahata A, Mohamed IM, Iwasaki M, Lu J, Ihara I, Umetsu K (2022) Integrating anaerobic digestion with hydrothermal pretreatment for bioenergy production: Waste valorization of plastic containing food waste and rice husk. Biochem Eng J 186: 108546. doi:https://doi.org/10.1016/j.bej.2022.108546
- Gao S, Zhang A, Ma D, Zhang K, Wang J, Wang X, Chen K (2022) Enhancing pH stability of lysine decarboxylase via rational engineering and its application in cadaverine industrial production. Biochem Eng J 186: 108548. doi:https://doi.org/10.1016/j.bej.2022.108548
- Gillispie EC, Sowers TD, Duckworth OW, Polizzotto ML (2015) Soil pollution due to irrigation with arsenic-contaminated groundwater: Current state of science. Curr Pollut Rep 1: 1-12. doi:https://doi.org/10.1007/s40726-015-0001-5
- Grytsyuk N, Arapis G, Perepelyatnikova L, Ivanova T, Vynograds' Ka V (2006) Heavy metals effects on forage crops yields and estimation of elements accumulation in plants as affected by soil. Sci Total Environ 354: 224-231. doi:https://doi.org/10.1016/j.scitotenv.2005.01.007
- Gusman GS, Oliveira JA, Farnese FS, Cambraia J (2013) Mineral nutrition and enzymatic adaptation induced by arsenate and arsenite exposure in lettuce plants. Plant Physiol Biochem 71: 307-314. doi:https://doi.org/10.1016/j.plaphy.2013.08.006
- Hasanuzzaman M, Bhuyan M, Zulfiqar F, Raza A, Mohsin SM, Mahmud JA, Fujita M, Fotopoulos V (2020) Reactive oxygen species and antioxidant defense in plants under abiotic stress: Revisiting the crucial role of a universal defense regulator. Antioxidants 9: 681. doi:http://dx.doi.org/10.3390/antiox9080681
- Hasanuzzaman M, Fujita M (2013) Exogenous sodium nitroprusside alleviates arsenic-induced oxidative stress in wheat (Triticum aestivum L.) seedlings by enhancing antioxidant defense and glyoxalase system. Ecotoxicology 22: 584-596. doi:https://doi.org/10.1007/s10646-013-1050-4
- Heath RL, Packer L (1968) Photoperoxidation in isolated chloroplasts: I. Kinetics and stoichiometry of fatty acid peroxidation. Arch Biochem Biophys 125: 189-198. doi:https://doi.org/10.1016/0003-9861(68)90654-1
- Heidari F, Øverland M, Hansen JØ, Mydland LT, Urriola PE, Chen C, Shurson GC, Hu B (2022) Solid-state fermentation of Pleurotus ostreatus to improve the nutritional profile of mechanically-fractionated canola meal. Biochem Eng J 2022: 108591. doi:https://doi.org/10.1016/j.bej.2022.108591
- Hussain A, Abbas N, Arshad F, Akram M, Khan ZI, Ahmad K, Mansha M, Mirzaei F (2013) Effects of diverse doses of Lead (Pb) on different growth attributes of Zea-Mays L. Agricultural Sci 4: Article ID:32311. doi:http://www.scirp.org/journal/PaperInformation.aspx?PaperID=32311
- Jithesh M, Prashanth S, Sivaprakash K, Parida AK (2006) Antioxidative response mechanisms in halophytes: their role in stress defence. J Genet 85: 237-254. doi:https://doi.org/10.1007/BF02935340
- Joseph T, Dubey B, McBean EA (2015) Human health risk assessment from arsenic exposures in Bangladesh. Sci Total Environ 527-528: 552-560. doi:https://doi.org/10.1016/j.scitotenv.2015.05.053
- Kai K, Kasa S, Sakamoto M, Aoki N, Watabe G, Yuasa T, Iwaya-Inoue M, Ishibashi Y (2016) Role of reactive oxygen species produced by NADPH oxidase in gibberellin biosynthesis during barley seed germination. Plant Signal Behav 11: e1180492. doi:https://doi.org/10.1080/15592324.2016.1180492
- Khalid S, Shahid M, Niazi NK, Rafiq M, Bakhat HF, Imran M, Abbas T, Bibi I, Dumat C (2017) Arsenic Behaviour in Soil-Plant System: Biogeochemical Reactions and Chemical Speciation Influences. In: Anjum NA, Gill SS, Tuteja N (eds) Enhancing Cleanup of Environmental Pollutants: Volume 2: Non-Biological Approaches. Springer International Publishing, Cham, pp 97-140. doi:https://doi.org/10.1007/978-3-319-55423-5_4
- Kwon S-Y, Lee H-S, Kwak S-S (2001) Development of environmental stress-tolerant plants by gene manipulation of antioxidant enzymes. The Plant Pathol J 17: 88-93
- LeBlanc MS, McKinney EC, Meagher RB, Smith AP (2013) Hijacking membrane transporters for arsenic phytoextraction. J Biotechnol 163: 1-9. doi:https://doi.org/10.1016/j.jbiotec.2012.10.013
- Maresca V, Sorbo S, Keramat B, Basile A (2017) Effects of triacontanol on ascorbate-glutathione cycle in Brassica napus L. exposed to cadmium-induced oxidative stress. Ecotoxicol Environ Saf 144: 268-274. doi:https://doi.org/10.1016/j.ecoenv.2017.06.035
- Meena M, Divyanshu K, Kumar S, Swapnil P, Zehra A, Shukla V, Yadav M, Upadhyay R (2019) Regulation of L-proline biosynthesis, signal transduction, transport, accumulation and its vital role in plants during variable environmental conditions. Heliyon 5: e02952. doi:https://doi.org/10.1016/j.heliyon.2019.e02952
- Mirzaei AR, Shakoory-Moghadam V (2022) Bioinformatics analysis and pharmacological effect of Stevia rebaudiana in the prevention of type-2 diabetes. Cell Mol Biomed Rep: 64-73. doi:https://doi.org/10.55705/cmbr.2022.336232.1035
- Mitra A, Chatterjee S, Moogouei R, Gupta DK (2017) Arsenic accumulation in rice and probable mitigation approaches: a review. Agronomy 7: 67. doi:https://doi.org/10.3390/agronomy7040067
- Mohammadhasani F, Ahmadimoghadam A, Asrar Z, Mohammadi SZ (2017) Effect of Zn toxicity on the level of lipid peroxidation and oxidative enzymes activity in Badami cultivar of pistachio (Pistacia vera L.) colonized by ectomycorrhizal fungus. Indian J Plant Physiol 22: 206-212. doi:https://doi.org/10.1007/s40502-017-0300-5
- Nelson N (1944) A photometric adaptation of the Somogyi method for the determination of glucose. J biol Chem 153: 375-380
- Parkhey S, Naithani S, Keshavkant S (2014) Protein metabolism during natural ageing in desiccating recalcitrant seeds of Shorea robusta. Acta Physiol Plant 36: 1649-1659. doi:https://doi.org/10.1007/s11738-014-1540-x
- Polle A (2001) Dissecting the superoxide dismutase-ascorbate-glutathione-pathway in chloroplasts by metabolic modeling. Computer simulations as a step towards flux analysis. Plant Physiol 126: 445-462. doi:https://doi.org/10.1104/pp.126.1.445
- Rafiq M, Shahid M, Abbas G, Shamshad S, Khalid S, Niazi NK, Dumat C (2017) Comparative effect of calcium and EDTA on arsenic uptake and physiological attributes of Pisum sativum. Int J Phytoremediation 19: 662-669. doi:https://doi.org/10.1080/15226514.2016.1278426
- Rena AB, Splittstoesser WE (1974) Proline-Dehydrogenase from Pumpkin (Cucurbita moschata) Cotyledons. Physiol Plant 32: 177-181. doi:https://doi.org/10.1111/j.1399-3054.1974.tb03749.x
- Saneoka H, Moghaieb RE, Premachandra GS, Fujita K (2004) Nitrogen nutrition and water stress effects on cell membrane stability and leaf water relations in Agrostis palustris Huds. Environ Exp Bot 52: 131-138. doi:https://doi.org/10.1016/j.envexpbot.2004.01.011
- Saunders JA, McClure JW (1974) The suitability of a quantitative spectrophotometric assay for phenylalanine ammonia-lyase activity in barley, buckwheat, and pea seedlings. Plant Physiol 54: 412-413. doi:https://doi.org/10.1104/pp.54.3.412
- Segura PC, De Meur Q, Alloul A, Tanghe A, Onderwater R, Vlaeminck SE, Wouwer AV, Wattiez R, Dewasme L, Leroy B (2022) Preferential photoassimilation of volatile fatty acids by purple non-sulfur bacteria: Experimental kinetics and dynamic modelling. Biochem Eng J 186: 108547. doi:https://doi.org/10.1016/j.bej.2022.108547
- Singh HP, Batish DR, Kohli RK, Arora K (2007) Arsenic-induced root growth inhibition in mung bean (Phaseolus aureus Roxb.) is due to oxidative stress resulting from enhanced lipid peroxidation. Plant Growth Regul 53: 65-73. doi:https://doi.org/10.1007/s10725-007-9205-z
- Singh R, Singh S, Parihar P, Singh VP, Prasad SM (2015) Arsenic contamination, consequences and remediation techniques: a review. Ecotoxicol Environ Saf 112: 247-270. doi:https://doi.org/10.1016/j.ecoenv.2014.10.009
- Sinha S, Sinam G, Mishra RK, Mallick S (2010) Metal accumulation, growth, antioxidants and oil yield of Brassica juncea L. exposed to different metals. Ecotoxicol Environ Saf 73: 1352-1361. doi:https://doi.org/10.1016/j.ecoenv.2010.06.025
- Souri Z, Karimi N, de Oliveira LM (2018) Antioxidant enzymes responses in shoots of arsenic hyperaccumulator, Isatis cappadocica Desv., under interaction of arsenate and phosphate. Environ technol 39: 1316-1327. doi:https://doi.org/10.1080/09593330.2017.1329349
- Sposito G (1989) Soil particle surface, The Chemistry of Soils. Oxford University Press: New York, 136-141.,
- Suneja Y, Gupta AK, Bains NS (2019) Stress adaptive plasticity: Aegilops tauschii and Triticum dicoccoides as potential donors of drought associated morpho-physiological traits in wheat. Front plant sci 10: 211. doi:https://doi.org/10.3389/fpls.2019.00211
- Tang W, Newton RJ (2005) Peroxidase and catalase activities are involved in direct adventitious shoot formation induced by thidiazuron in eastern white pine (Pinus strobus L.) zygotic embryos. Plant Physiol biochem 43: 760-769. doi:https://doi.org/10.1016/j.plaphy.2005.05.008
- Turchetto-Zolet AC, Margis-Pinheiro M, Margis R (2009) The evolution of pyrroline-5-carboxylate synthase in plants: a key enzyme in proline synthesis. Mol Genet Genom 281: 87-97. doi:https://doi.org/10.1007/s00438-008-0396-4
- Wang Y, Yang H, Zhang B, Liu X, Bao J (2022) Continuous enzymatic saccharification and its rheology profiling under high solids loading of lignocellulose biomass. Biochem Eng J 186: 108543. doi:https://doi.org/10.1016/j.bej.2022.108543
- Wang Z-Q, Yuan Y-Z, Ou J-Q, Lin Q-H, Zhang C-F (2007) Glutamine synthetase and glutamate dehydrogenase contribute differentially to proline accumulation in leaves of wheat (Triticum aestivum) seedlings exposed to different salinity. Journal of plant physiology 164: 695-701. doi:https://doi.org/10.1016/j.jplph.2006.05.001
- Youssef NB, Nouairi I, Temime SB, Taamalli W, Zarrouk M, Ghorbal MH, Daoud DBM (2005) Effets du cadmium sur le métabolisme des lipides de plantules de colza (Brassica napus L.). C R Biol 328: 745-757. doi:https://doi.org/10.1016/j.crvi.2005.05.010