The responses of growth, photochemical and antioxidant defence of safflower species (Carthamus oxyacantha M. Bieb. and Carthamus tinctorius L. exposed to nickel (Ni) toxicity were investigated in the study. Fourteen-day-old seedlings were treated with excessive Ni levels [control, 0.50, 0.75 and 1.00 mM] for 7 days. The results of chlorophyll a fluorescence indicated that toxic nickel exposure led to changes in specific, phenomenological energy fluxes and quantum yields in thylakoid membranes, and activities of donor and acceptor sides of photosystems. These changes resulted in a significant decrease in the photosynthetic performance of the species, but these negative effects of Ni were not in a level to destroy the functionality of the photosystems. At the same time, toxic Ni affected membrane integrity and the amount of photosynthetic pigments in the antenna and active reaction centers. Additionally, the accumulation of Ni was higher in roots than in stem and leaves for both species. Depending on Ni accumulation, a significant reduction in dry biomass of root and shoot was observed in both species. Two species could probably withstand deleterious Ni toxicity with better upregulating own protective defence systems such as antioxidant enzymes. Among of them, SOD and POD activities were increased with increasing Ni concentrations. The POD activities of both species were most prominent and consistently increased in toxic Ni levels and may be protected them from damaging effect of H2O2. When all results are evaluated as a whole, Carthamus species produced similar responses to toxicity and also both species have Bioconcentration (BCF) and Bioaccumulation factors (BF) > 1 and Translocation factor (TF) < 1 under Ni toxicity may be regarded a good indication of Ni tolerance. Consequently, it is possible to use the Carthamus species in the remediation (phytostabilization) of soils contaminated with nickel, because of their roots accumulating more nickel.
Research Article
Physiological, Photochemical and Antioxidant Responses of Wild and Cultivated Carthamus Species Exposed to Nickel Toxicity and Evaluation of Their Usage Potential in Phytoremediation
https://doi.org/10.21203/rs.3.rs-475470/v1
This work is licensed under a CC BY 4.0 License
Journal Publication
published 18 Aug, 2021
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Carthamus species
safflower
nickel
chlorophyll fluoresence
antioxidant enzymes
accumulation
Heavy metal pollution is one of the most worldwide threats to the environment organisms. Nickel is emitted in the environment from a variety of natural (such as serpentine soils) and anthropogenic sources including chemical, food, metallurgical industries (Chen et al., 2009; Sachan and Lal, 2017). Ni is an essential micronutrient for growth and development and some cellular processes of higher plants. Although the concentration of nickel that is required for plant growth is very low, higher Ni concentrations cause toxicity and could lead to several deleterios alterations in plants (Shahzad et al., 2018).
The main way to uptake Ni from the soil is absorption by the plant roots. Some metals that accumulate in the soil are attached to their organic components and become inaccessible for plants, whereas metal ions can enter the roots easily. Ni+ 2 uptake and translocation by the plant roots, same as of other metals, take place both passive diffusion and active transport (Seregin and Kozhevnikova, 2006). Nickel uptake and ratio of transport form (active/passive) may vary depending on the species of the plant, oxidation state, pH, concentrations of other metal ions and availability of nickel in the growth medium (Sachan and Lal, 2017; Salinitro et al., 2020). Moreover, Ni, a divalent cation, may compete with other cations with a similar charge/size ratio such as Fe, Cu, Zn, and Mn, therefore Ni toxicity causes the deficiency of these elements (Shahzad et al., 2018; Hassan et al., 2019).
Most of the deleterious effects of nickel toxicity are associated with the photosynthetic process of the plant via direct and/or indirect ways. Toxic level of Ni indirectly affects stomatal opening though alteration in ion fluxes (K+) across membranes (Ahmad and Ashraf, 2011). Stomatal limitations allow plants to limit transpiration, but it also limit CO2 absorption, which leads to reduced photosynthetic activity. In addition to that, limitations to CO2 absorption may provoke an imbalance between photochemical activity of photosystems and the electron requirement of the Calvin reactions, leading to an excess of absorbed excitation energy and subsequent photoinhibition (Baker and Rosenqvist, 2004). The action of excess Ni in photosynthesis may primarily target the reaction center of photosystem II (PSII). The inhibition of electron transport of Ni is mainly on the donor side of PSII and the binding site for QB, the secondary quinone acceptor of PSII (Mohanty et al., 1989; Bhalerao et al., 2015; Khaliq et al., 2016). Thus, electron transport is restricted in PSII and this restriction leads to nutrient deficiencies in plant as a result of negative affected of assimilation (Hassan et al., 2019). Many photochemical parameters could be calculated from obtained data and provide valuable information about the PSII and PSI functionality. Photosynthetic activity measurements (especially Chl a fluorescence parameters) have been widely used to screen and reveal the effects of metal stress on photosynthetic behaviour of plants (Öz et al., 2014; Sitko et al., 2017; Ekmekçi et al., 2020).
Excessive Ni causes reduction of chlorophyll and carotenoid contents, generation of free radicals, lipid peroxidation, disruption of cell structure, reduction in physiological functions, alteration of many enzymatic activities, destruction of photosynthetic protein complexes and chloroplast structure, dehydration (wilting), and consequently lower biomass production and yield (Sachan and Lal, 2017; Batool, 2018; Amjad et al., 2020). Generation of reactive oxygen species (ROS) enchanced by nickel at elevated levels and this over-production and accumulation of ROS that could not be scavenged may cause damage to several critical bio-molecules like lipids, proteins and nucleic acids in plant tissues (Gajewska and Skłodowska, 2007). Members of defense mechanisms such as enzymatic (SOD, POD, GR, APX, etc.) and non-enzymatic (glutathion, ascorbate, anthocyanin, flavonoid, etc.) antioxidants play an important role in the reducing oxidative damage in plants. Changes in the activity of antioxidant enzymes may vary depending on the plant species as well as nickel concentration (Gajewska and Skłodowska, 2007; Zaid et al., 2019; Amjad et al., 2020).
Carthamus oxyacantha (wild safflower) and Carthamus tinctorius (cultivated safflower) species are an oilseed bioenergy crops which are a member of the family Asteraceae. Safflower is a prominent oilseed crop with its tolerance capacity against such as drought, salt, metal stress etc. (Al Chami et al., 2015; Pourghasemian et al., 2019; Çulha-Erdal et al., 2021). It has also been stated that C. tinctorius can be grown on metal-contaminated soils (Al Chami et al., 2015). Production of safflower is not only for food, cosmetic, pharmaceutical and dye industries but also safflower is a substrate for multi-biofuel (ethanol, biogas, and biodiesel) production in a biorefinery approach (Pourghasemian et al., 2019; Hashemi et al., 2020). Moreover, it has been reported that safflower also may be used in phytoremediation (Al Chami et al., 2015; Pourghasemian et al., 2019), but it is poorly known the physiological responses of C. oxyacantha and C. tinctorius to heavy metal stress, especially nickel. To our knowledge, the effects of nickel stress on photosynthetic efficiency by using chlorophyll a fluorescence technique and antioxidant activity of safflower were studied for the first time with present research.
Finally, this study was designed to determine physiological differences, responses and tolerance capacity between two safflower species exposed to increased Ni concentrations using the following approaches: 1) to explore underlying physiological and metabolic mechanisms responsible for Ni toxicity 2) to evaluate the effect of Ni toxicity on photochemical activity by using polyphasic chlorophyll a fluorescence kinetics, 3) to elucidate possible protective role of antioxidant enzyme activity against the Ni induced oxidative stress 4) to assess whether or not the potential utilize of Carthamus species as a suitable phytoremediator for Ni-contaminated areas.
2.1. Plant materials, growth and treatment conditions
Wild (Carthamus oxyacantha M.Bieb.) and cultivated (Carthamus tinctorius L.) species of safflower used in this study and seeds were provided from Trakya Agricultural Research Institute.
The seeds of safflower were sterilized with 5% sodium hypochlorite for 5 min. After sterilization, seeds were germinated under dark conditions at 23±2 °C for 4 days. Then, seedlings were transferred to hydroponic culture containing Hoagland’s nutrient solution and were grown for 10 days in control condition. At the 10th day of the growth, nickel stress was treated by applying Hoagland's nutrient solution containing 0.50, 0.75 and 1.00 mM NiCl2.6H2O for next 7 days. The concentration and duration of toxicity groups were decided by preliminary tests on the 1st, 3rd, 5th, 7th, 9th days from the beginning of nickel stress by using polyphasic chlorophyll a fluorescence measurements.
The experiments were carried out in a controlled growth chamber at 25°C, 16-h light and 8-h dark photoperiod, 180–200 µmol.m− 1s− 1 light intensity and 40–50% humidity. On the 21st day, plants were harvested and 2nd and 3rd leaves of plants were used for analysis. The dry biomass of roots and shoots of treatment groups were determined as g. plant− 1 DW.
2.2. Ni Accumulation
The plant parts (leaves, stems, roots) were properly rinsed with distilled water and then tissues were oven-dried at 80°C for 48 h and ground powder. The powder of tissues (0.2g) was ashed in a muffle furnace at 550°C for 5 hours. The residue was brought to a standard volume with 1 M HNO3 and then filtered trough whatmann paper. The Ni concentration of the extract was determined by atomic absorption spectrometer (Varian AA2420FS). The Ni concentration [Ni] of tissues [root, shoot (stem + leaf)] was calculated in terms of mg.kg− 1 DW. Translocation (TF), Bioconcentration (BCF) and Bioaccumulation Factors (BF) were calculated according to Yoon et al. (2006) and Roccotiello et al. (2010):
2.3. Water status and membran integrity
The water status of leaves was evaluated by calculating the relative water content (RWC) (Farrant, 2000). Electrolyte leakage was measured according to Redmann et al. (1986) with minor modifications.
2.4. Polyphasic chlorophyll a fluorescence kinetics measurement
Chlorophyll a polyphasic fluorescence (OJIP) measurements were performed using the HandyPEA (Hansatech Instruments Ltd., Norfolk, UK) fluorimeter on selected fully expanded 2nd and 3rd leaves. Following a 30 min dark adaptation, samples were illuminated with continuous light [650 nm; 3.000 µmol (photon) m− 2 s− 1 for 1 s] provided by three LEDs. The fluorescence intensity at 20 µs (F0), 100 µs, 300 µs (FK), 2 ms (FJ), 30 ms (FI), and maximum fluorescence (FM) were recorded. The recorded data were analyzed with BiolyzerHP3 to prove the physiological state of the safflower plants. Additionally, the relative variable fluorescence [Vt: between the steps O and K, VOK = (FK – F0)/(FM – F0); O and J, VOJ = (FJ – F0)/(FM – F0); and I and P, VIP = (FM – FI)/(FM – F0)] (Strasser et al., 2004; Oukarroum et al., 2007) were calculated to elucidated the differences between safflower species in response to nickel toxicity. VK/VJ and ΔVIP, gives information about the PSII donor side and acceptor side of PSI respectively, were also calculated (Ceppi et al., 2012; Ripoll et al., 2016). Some selected JIP test parameters were used in this study: Maximum quantum yield of primary PSII photochemistry (FV/FM), specific energy fluxes [the absorption/antenna size (ABS/RC), maximum trapping (TRo/RC), electrons transferred (ETo/RC) and dissipation (DIo/RC) per active reactive center (RC)], phenomenological energy fluxes [the absorption (ABS/CSo), maximum trapping (TRo/CSo), electron fluxes transferred (ETo/CSo), dissipation (DIo/CSo) and amount of active PSII RCs (RC/CSo) per leaf cross-section (CS)], performance indexes (PIABS and PITOTAL) and structure-function index [SFI(ABS)] (Strasser et al., 2004, 2010; Goltsev et al., 2016; Stirbet et al., 2018). The equations for calculation of JIP-test some parameters are also given in Goltsev et al. (2016).
2.5. Pigment analysis
The amount of chlorophyll (a + b) and carotenoid (xanthophyll + β-carotene) (mg. ml− 1 cm− 2) pigments in leaf tissues of species were determined using method of Lichtenthaler (1987). The anthocyanin content was calculated as described by Mancinelli et al. (1975) and was expressed as mg g FW− 1 and flavonoid content was measured according to method of Mirecki and Teramura (1984).
2.6. Hydrogen peroxide (H2O2) content
The content of H2O2 of leaves was determined as µmol g FW− 1 according to Esterbauer and Cheeseman (1990).
2.7. Protein determination and antioxidant enzyme activities
Fresh leaf samples (0.5 g) were ground with liquid nitrogen and soluble protein was extracted by homogenizing in related buffer. The protein concentrations of extracts were determined according to Bradford (1976).
Superoxide dismutase (SOD; EC 1.15.1.1) activity was assayed as described by Beyer and Fridovich (1987). One unit of SOD activity was defined as the amount of enzyme required to cause 50% inhibition of NBT photoreduction. The activity was expressed in units mg protein− 1. Ascorbate peroxidase ( APX; EC 1.11.1.11) activity was assayed according to the method of Wang et al. (1991). The enzyme activity was calculated from the initial rate of the reaction using the extinction coefficient of ascorbate (ɛ = 2.8 mM cm− 1) at 290 nm. Glutathione reductase (GR; EC 1.6.4.2) activities were determined according to the method of Rao et al. (1995). The enzyme activity was calculated from the initial rate of the reaction after subtracting the nonenzymatic initial oxidation rate using the extinction coefficient of NADPH (ɛ = 6.2 mM cm− 1) at 340 nm. Guaiacol peroxidase (POD; EC 1.11.1.7) activity was based on the determination of guaiacol oxidation (ɛ = 26.6 mM cm− 1) at 470 nm by H2O2 (Bergmeyer, 1974). A unit of peroxidase activity was defined as nmol H2O2 decomposed per minute per milligram of protein.
2.8. Statistical analysis
The experiments were performed in a completely randomized design with 3 replicates. Analysis of variance (ANOVA) of the data was performed using SPSS 20.0 software (IBM SPSS Statistics) and the significance of differences between treatments and spesies was compared using LSD test at p < 0.05 level.
3.1. Response of plant growth to excessive Ni toxicity
Nickel has an adverse effect on plant growth and development at toxic levels. Increasing nickel accumulation of plants leads to alterations in physiological, biochemical and metabolic processes that causes a reduction in growth and biomass production. While low concentrations of Ni play important role in plant growth and development (Chen et al., 2009), high concentrations of Ni reduce shoot and root lenght and dry biomass production in crops (Seregin et al., 2003; Schaaf et al., 2006; Ahmad and Asraf, 2012). A primary phenotypic effect of Ni toxicity induced generally growth inhibition in both safflower species (Fig. 1A-D). The reduction in the root and shoot dry biomass of both species was determined in all Ni treatments compared to the their controls (Fig. 1A and B). It was noticed that the most common response of plants to nickel toxicity was growth inhibition depending on reduction dry matter yield (Khaliq et al., 2016; Batool, 2018). Also, the reduction in the shoot dry biomass of C. oxyacantha was more drastic than that of C. tinctorius. In addition to that, the roots of both species have exhibited blackening and stunted growth behaviour, in concordance with the results of study on C. tinctorius of Al Chami et al. (2015). The stunted growth symptom of Ni toxicity inhibits root elongation and growth to a greater extent than shoot growth (Seregin and Kozhevnikova, 2006). Furthermore, it was observed that a lateral root development was reduced depending on increased nickel concentration for both species. It has been stated that the inhibition of lateral root formation is a characteristic of Ni toxicity compared to other heavy metals and this response has been explained by the accumulation of nickel in the pericyclic cells (Seregin et al., 2003). Previous studies have showed an excess Ni concentrations causes metabolic toxicity that alters the function of root system, thereby inhibiting the uptake of essensial nutrients and mitotic activity of root meristem (Seregin and Kozhevnikova, 2006; Bhalerao et al., 2015; Rizwan et al., 2017). Schaaf et al. (2006) have observed that toxic Ni levels did not affect mitotic activity in root meristem, conversely limits longitudinal root cell expansion and also promotes radial cell expansion. Lescova et al. (2020) and Merlot (2020) have suggested that increase in ROS in the root cells exposed the highly toxic Ni regulates the activities of PIN2 transporters, so affecting the auxin homeostasis and subsequent root growth. However, the mechanism of adverse effect of Ni on plant growth has not been adequately clarified.
Ammen et al. (2019) stated that one of the possible reasons of the reduction in dry biomass is the loss of water content under toxic Ni levels. Heavy metals like Ni considerably reduce the movement of water from roots to upper parts of plant, thereby leading to shoot dehydration (Chen et al., 2004). Excessive Ni levels induced wilting due to gradually reduced RWC of leaves in safflower species (Fig. 1E). Many researchers reported that high amount of Ni accumulation in plants caused a reduction in water content and transpiraton rate of the leaves (Pandey and Sharma, 2002; Seregin and Koznevnikova, 2006; Gajewska et al., 2006; Chen et al., 2009; Hassan et al., 2019). At the highly toxic Ni concentration (1.00 mM), the RWC for C. oxyacantha was decreased by 56.3%, while for C. tinctorius 43.0% compared to controls. The reason of decrease in leaf RWC of both species grown under excessive Ni levels could be disruption of water uptake from roots due to the higher negative osmotic potential of hydroponic medium. Severe dehydration in safflower leaves could be due to an increase in electrolyte leakage in the leaves exposed to Ni toxicty (Fig. 1F). The membrane is the first part of the cell that comes in contact with toxic metals that destroy membrane integrity and fluidity (Hassan et al., 2019). RLR of C. oxyacantha leaves was increased with increasing Ni concentrations by 2.40, 2.67 and 4.67 folds, while for C. tinctorius 2.07, 2.99 and 5.27 folds, respectively. Based on the electrolyte leakage results, a greater extent of membrane damage was observed at the highly toxic Ni level in both species.
3.2. Nickel accumulation and tolerance to Ni toxicity
Nickel accumulation in roots, stems and leaves are shown in Table 1. Carthamus species accumulated Ni in descending order of root > stem > leaf. Under all toxic treatments, Ni contents in root and shoot tissues of both species progressively increased in a concentration-dependent manner. These results demonstrated that large amounts of nickel were accumulated primarly in the roots than the other part of plant, while a small amount of nickel gradually translocated to the above-ground organs for safflower species. Many studies on the other plant species have also reported that greater amount of nickel accumulates in the roots (Al Chami et al., 2015; Gonzales et al., 2015; Syam et al., 2016; Rizwan et al., 2017; Parlak, 2020). Differences between nickel accumulation in the root and stem of both genotypes were determined significant in toxic Ni concentrations, except 0.50 mM, whereas it is not significant in leaves. The highest Ni accumulation was determined at the 1.00 mM Ni treatment in the roots of C. oxyacantha and C. tinctorius by 4662 and 3385 mg kg− 1 DW respectively. However, C. oxyacantha transferred generally less amounts of nickel to stem and leaves compared to C. tinctorius. BCF, BF and TF factors for species under toxic Ni conditions are calculated and given in Table 1. TF values of both species were determined below one (TF < 1). The low TF (less than 1) for Ni might indicate that both species stored nickel in the roots, so roots were the primary regulation parts and then were translocated less amount of Ni to shoot. The translocation of the essensial metals as Ni from roots to leaves was lower compared to non essensial metals (Takarina and Pin, 2020). The BCF is a important value to reflect the capacity of plant to accumulate heavy metals from the soil (Wang et al., 2018). BCF and BF values (> 1) represents the ability of plants to accumulation of Ni in the roots and shoots, respectively (Netty et al., 2013). In present study, BCF values of both species were higher than 1. Yoon et al. (2006) and Roccotiello et al. (2010) have stated that plants can be defined phytostabilizers if they possess BCF and BF > 1 and TF < 1 values. These results have shown that safflower species are Ni tolerant plant and they can be used for the phytostabilisation of Ni contaminated soils.
3.3. Photochemical response to Ni toxicity
Many abiotic stress such as metal toxicity directly or indirectly affect the photosynthetic activity of plants and as a consequence alter the chlophyll a fluorescence kinetics (Sitko et al., 2017). Nickel toxicity also inhibits photosynthesis (Velikova et al., 2011), but its effects on photochemical activity are still not fully understand. The polyphasic chlorophyll a fluoresence transients (OJIP), provides detailed information about photosynthetic fluxes analyses using JIP test, can be used as a tool to answer the question about stress induced alterations in plant (Oukarroum et al., 2007; Kalaji et al., 2016). Typical shape of OJIP transient curves has three main phases including O-J, J-I and I-P represents the changes in the redox state of photosystems and very sensitive to environmental stresses (Strasser et al., 2004; Umar et al., 2009). Both safflower species exposed to toxic nickel treatments differed obviously regarding the shape of the OJIP fluoresence transient curves and changes of amplitude from those of their controls (Fig. 2A and B). Highly toxic Ni level induced marked increase in O-J fluoresence rise of C. tinctorious at compared to other treatments. O to J rise which is a photochemical phase, represents QA reduction to QA- in PSII and also gives information about an antenna size (Kalaji et al., 2016; Küpper et al., 2019). Toxic Ni concentration caused largest increases in J step in C. tinctorius. Increase in J step and also I step reflects the start of QA re-oxidation by QB and PQ (Strasser et al., 1995), so an increase in J step would suggest a problem in QA re-oxidation and build up reduced QA. Meanwhile, the increased value of VJ and VI of species at nickel toxicity confirmed the accumulation of reduced QA and PQ which was blocked further electron transport. The J-I and I-P rise represent non-photochemical phases (Strasser et al., 1995). Most pronounced decrease in I step was determined in C.oxyacantha at highly toxic Ni level. The decline in the I-P amplitute of polyphasic rise in both species was found to be significant in toxic Ni treatments compared to their controls but this effect was most remarkable C. oxyacantha especially 1.00 mM Ni concentration. The decline in the I-P phase reflects the reduction of the acceptor side of PSI (Kalaji et al., 2016). The amplitute of the relative variable fluoresence of the I to P rise (ΔVIP) decreased in both species under toxic Ni conditions (bottom insert in Fig. 2A and B). ΔVIP is a an important parameter to measure the ratio of PSII/PSI and can be used as an indicator for changes in PSI content (Ceppi et al., 2012; Umar et al., 2019). These results showed that Ni toxicity decreased PSI content of safflower species, the lower values of ΔVIP expressed a smaller pool of electron acceptor in PSI. Meanwhile, increased in VK/VJ indicated that Ni toxicity significantly affected donor side of PSII in species (upper insert in Fig. 2A and B), which may imply to possible damage and inactivation of the oxygen evolving complex (OEC). This response were supported by the changes in VOK and VOJ, which also indicate either limitation of the OEC or/and an increase of PSII antenne size of both species.
The JIP test is based on the theory of energy fluxes across thylakoid membranes (Strasser et al., 2000). JIP parameters representing the energy fluxes are divided into specific and phenomenological. Specific energy fluxes parameters (ABS/RC, TR0/RC, ET0/RC, RE0/RC) calculated per reaction center (RC) of both species were affected from toxic Ni treatments (Fig. 3A and B). The flux of absorption and trapping per reaction center of PSII, defined as ABS/RC and TR0/RC respectively, were significantly increased in both species at only highly toxic (1.00 mM) Ni treatment. Increase in ABS/RC value means that, either fraction of reaction centers is inactivated or the apparent antenna size increased (Ceurters et al., 2019). The increase of ABS/RC was accompanied by increase of trapping per reaction centers (TR0/RC) in both species at 1.00 mM Ni treatment. It has been suggested that the accumulation of inactive reaction centers is associated with the increased efficiency of dissipation of absorbed light as heat. The higher values of dissipation energy flux per reaction center (DI0/RC) of both species indicated that the energy dissipation (φD0 and DI0/RC) is enhanced in order to protect plants exposed to stress from over-excitation and photo-oxidative damage, so excess absorbed light energy was converted to heat dissipation. The value of φD0 (the quantum yield at t = 0; F0/FM) of both species was increased significantly with increasing Ni concentrations (0.75 and 1.00 mM). Also the reduction in the TR0/RC at Ni treatments (except 1.00 mM) resulted in reduced electron transport per reaction center (ET0/RC) as well as RE0/RC due to lower amount of trapped energy. The electron transport from QA- to PSI electron aceptor (RE0/RC) in both species decreased in all treatments compared to their controls, but it increased in C. tinctorius in only higly toxic Ni level compared to other treatments. At the 1.00 mM Ni induced a significant decrease in the maximum PSII quantum yield (FV/FM) of both species compared with their controls. The decline in the FV/FM value was connected with reduced effectiveness of ET0/RC, increased of dissipated energy and disruption of antenna complex of PSII (Kalaji et al., 2012; Umar et al., 2019). SFI(ABS), a structure and functioning indicator of PSII, was significantly decreased depending on increasing in Ni levels (especially more toxic than 0.50 mM) (Fig. 3A and B). This pointed out that Ni caused damage in photosynthetic process. Stirbet et al. (2018) have suggested that decrease in SFI(ABS) and increase in energy dissipation might be due to the increase in inactive reaction centers and decrease in quantum yield of photochemistry. Ni toxicity adversely affected the photosynthetic performance indexes (PIABS and PITOTAL) and their companents. Decrease in PI’s in stress treatments suggested decrease in overall photosynthetic performance associated with generally decrease of electron transport capacity (Oukarroum et al., 2007).
The phenomenological parameters are calculated per sample cross section (CS) and can be plotted as energy pipeline leaf model (Kalaji et al., 2016). The model demostrated the proportion of active and inactive PSII reaction centers per cross-section (Fig. 4). Related parameters such as efficiency of light absorption (ABS/CS0), trapping (TR0/CS0) and electron transport (ET0/CS0) decreased at toxic nickel levels while dissipation per cross section of PSII (DI0/CS0) increased in response to nickel toxicity. Nickel toxicity caused increasing in inactive reaction centers and producing dissipative sinks for excitation energy, and resulting in decrease in ET0/CS0. These changes were somewhat more in C.oxyacantha than in C. tinctorius.
JIP test analyses showed that, the decrease of photochemical efficiency related to increase in dissipation and reduced of active reaction centers were found in both species affected highly toxic Ni treatments. It was determined that the Ni toxicity were affected more acceptor side of PSI and its components than donor side of PSII. But these adversely effects of Ni on photosystems were not in a level to cessation the functionality of them. This was consistent with the electrolyte leakage results (Fig. 1F).
3.4. Effect of Ni toxicity on pigment contents
Nickel toxicity leads to leaf chlorosis and necrosis (Singh and Pandey, 2010; Hassan et al., 2019). The decrease in total chlorophyll and carotenoid content of both species leaves at toxic Ni levels resulted in an inhibition on photochemical activity (Table 2 and Fig. 3). The leaves of both safflower were significantly affected by Ni treatments, which resulted in the devastation of chlorophyll including chlorosis and necrosis. The chlorophyll pigments of both genotypes leaves remarkably declined (about 40–60%) with increasing Ni concentrations compared with controls. Consequently, these changes were reflected to chlorophyll a/b ratio and the ratio was decreased with increasing toxicity (Table 2). Çiçek and Çakırlar (2008) have suggested that this ratio indicates the antenna size of photosystems. The core antenna contain only chl-a, whereas the outher antenna contains both chlorophlls. So, a smaller chl a/b ratio points out larger antenna size, higher ratio smaller antenna size. This finding is consistent with ABS/RC results (Fig. 3). In addition to that, decrease in the chlorophyll pigment under excessive Ni conditions are generally proposed to be increased oxidative stress and competition with essential microelements, including iron (Seregin and Kozhevnikova, 2006; Leskova et al., 2017). It could be suggested that the high concentration of Ni competed with iron in root cell then it prevent uptake of iron and induced the appearance of chlorosis symptoms that resembling iron deficiency. Chlorophyll pigments perform most of the light harvesting operations, while carotenoids provide protection against excessive light energy as well as light harvesting (Yao and Liu, 2007; Ekmekci et al., 2008). It was showed that carotenoid content was significantly declined in Ni treatments for both species.
Anthocyanin belong to the flavonoid group of polyphenolic compounds, which are water-soluble non-photosynthetic pigments in plants. Anthocyanin and flavonoid act as antioxidants and protect the photosynthetic apparatus from damage due to high energy fluxes as an optical filter (Baskar et al., 2018; Stetsenko et al., 2020). Anthocyanin content of the safflower species increased under Ni stress. The anthocyanin content of C. oxyacantha at 1.00 mM Ni level was approx. fifteen folds higher than that of the control. In contrast to wild species, anthocyanin content of C. tinctorius was around six folds higher than that of the control (Fig. 5A). Our results are coherence with the results of previously carried out studies (Singh et al., 2011; Rizwan et al., 2017). Flavonoids can also enhance plant defence against heavy metal stress by chelating transition metal ions which trigger the ROS production (Khalid et al., 2019). Flavonoid content of both species increased with increasing Ni levels (Fig. 5B). This increase was nine times higher at highly toxic level in C. oxyacantha, while the four times higher in C. tinctorius compared with their controls. However, it was clear that flavonoid content of wild type in all Ni levels was higher than that of cultivated type. Kováĉik et al. (2009) also reported that the increased concentration of flavonoids was found in the leaves under high Ni2+. Increased of anthocyanins and flavonoids could be correlated with Ni induced ROS generation which evidence through enhanced membrane leakage and H2O2 generation (Fig. 1F and Fig. 5C).
3.5. Response of antioxidant enzyme activities to Ni toxicity
Ni is a redox inactive metal which could indirectly initiate oxidative stress by disrupting the balance of formation and destruction of ROS associated with normal cellular metabolism (Chen et al., 2009). The accumulation of ROS in plants damages cell membrane, lipids, pigments, enzymes, chloroplasts, and nucleic acids etc. (Hassan et al., 2019). The antioxidant defence system plays an important role in scavenging harmful ROS in order to protect plant cell from oxidative damage (Ameen et al., 2019). Carthamus species were exposed to toxic Ni levels which could potentially increase the probability of generating ROS due to excess excitation, resulting in possible damage and/or deterioration of membrane integrity (Fig. 1F and Fig. 2). To cope with ROS plant cells possess an antioxidative system consisting of both enzymatic and non-enzymatic antioxidants. In this study, some antioxidant enzymes exhibited different responses to excess Ni (Fig. 6).
SOD is considered to be the first step of defense against ROS (Gajewska and Skłodowska, 2007). The activity of SOD increased with elevated Ni levels in leaves and roots of both species (Fig. 6A and B). These increases were by 10.2 and 8 folds in roots of C. tinctorius and C. oxyacantha at a highly toxic Ni levels compared to their controls, respectively. In contrast, the activity of SOD increased by 5.7 and 11.2 folds in leaves of C. tinctorius and C. oxyacantha at 1.00 mM Ni levels, respectively. Many studies have confirmed that nickel toxicity causes increase in SOD activity of various plants (Ali et al., 2003; Gajewska and Sklodowska, 2006; 2007). As a result of the reaction catalyzed by the SOD, hydrogen peroxide (H2O2) is produced. The amounts of H2O2 were significantly increased in all nickel concentrations in leaves of C. tinctorius, while changes of the H2O2 amounts in those of C. oxyacantha were significant only at 0.50 Ni treatment compared to controls (Fig. 5C). The H2O2 are scavenged by APX catalyzes a reduction of H2O2 using ascorbate as an electron donor. It was found that there were no significant changes in the activities of APX in the root of both species, but the activity in the leaves increased for both species in all Ni levels when compared to their controls (Fig. 6C and D). Previous studies have shown that APX enzyme activity increases (Kumar and Sharma, 2012) or does not change (Gajewska and Sklodowska, 2007) under nickel stress. There were also no significant changes in the GR activity of roots in both species; however, C. oxyacantha showed significantly higher GR activity in leaves, while an insignificant change in GR activity was observed in C. tinctorius compared to their controls (Fig. 6E and F). It has been reported in other studies that GR activities in both organs were increased under Ni toxicity (Kumar et al., 2012; Abdelgawad et al., 2020). The activities of GR and APX, enzymes taking part in the Asada-Halliwell cycle, were determined remarkably little responsible for H2O2 detoxcification in both species exposed to Ni treatments (Fig. 6C-F). On the other hand, POD is also capable of scavenging H2O2. These enzymes constitute second step of defense against the ROS production (Ahmad and Ashraf, 2012). Excessive Ni levels were increased POD activities in the roots for C. oxyacantha and C. tinctorius (Fig. 6G and H). The highest increase in POD activity was determined at higly toxic Ni concentration (1.00 mM) in the roots of both species. These increases were by 1.9 and 2.2 folds in the roots of C. tinctorius and C. oxyacantha at a highly toxic Ni levels compared to their controls, respectively. Our results showed that Ni-induced POD activity increased in root and leaf tissues of both species, and this increase was much more in the their roots. The removal of harmfull concentration of H2O2 was probably achieved by POD enzymes. The POD activity of both species was most prominent and consistently increased in excessive Ni levels and may be protected from oxidative damage. Also many researchers have stated that, POD, which are stress enzymes in plants, were used a potential biomarker for metal toxicity in plants (Radotic el al., 2000; Ekmekçi et al., 2008; Wang et al., 2010; Nadgorska-Socha et al., 2013).
The results of study demonstrated that large amounts of Ni were accumulated in the roots more than upper parts of safflower species when exposed to Ni toxicity. The excessive Ni levels caused the limitation of growth and water uptake, loss of chlorophyll, accompany by reduce in photochemical activity. The decline in photochemical performance of species under elevated Ni concentrations might correlated with increasing in inactive reaction centers and decreasing in electron transport capacity. Down-regulation of photosynthetic efficiency together with a significant increase in energy dissipation could play a vital role in protecting both species suffered from Ni toxicity from pressure of over-excitation energy and photoinhibition. Additionally, Carthamus species may have cope with Ni toxicity by upregulating antioxidant defence capacity as well as increasing anthocyanin and flavonoid contents and especially POD activities may protect species from oxidative damage. It was not possible to distinguish two species from each other in terms of their similar responses to toxicity. Concequently, Carthamus species that exhibited better tolerance response to Ni toxicity can be considered as potential Ni-phytostabilizator contaminated areas.
Acknowledgements
Authors are thankful to Prof. Dr. Nuran Çiçek, University of Hacettepe for scientific redaction and suggestion of the manuscript. This study was submitted to the Graduate School of Science and Engineering at the Hacettepe University as a MSc thesis of Uğurcan Baran (2018YL59102).
Author’s contributions
Uğurcan Baran performed all the experiments and drafted the manuscript. Yasemin Ekmekçi supervised the overall work and provided critical comments, and reviewed and edited the manuscript.
Ethical Approval and Consent for Participate Not applicable
Consent for Publish Not applicable
Availability of data and materials All data generated or analysed in the study are included in this manuscript.
Competing Interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
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Due to technical limitations, table 1,2 is only available as a download in the Supplemental Files section.
