Genetics and Plant Physiology – 2018, Volume 8(1–2), pp. 68–81 ©2018 Published by the Institute of Plant Physiology and Genetics – Bulgarian Academy of Sciences Available online at http://www.ifrg-bg.com

Research Article EFFECTS OF EDTA AND CITRATE ADDITION TO THE SOIL ON C4 PHOTOSYNTHETIC ENZYMES AND BIOCHEMICAL INDICATORS FOR HEAVY METAL TOLERANCE IN TWO PAULOWNIA HYBRIDS

Miladinova-Georgieva K.1, M. Geneva1*, Y. Markovska2

1Institute of Plant Physiology and Genetics, Bulgarian Academy of Sciences, Acad. G. Bonchev str., Bl. 21,1113 Sofia, Bulgaria 2Faculty of Biology, University of Sofia, 8 Dragan Tsankov Bld., 1164 Sofia, Bulgaria

Received: 13 March 2018 Accepted: 17 May 2018

Summary: Phytoremediation is a low cost, long term, environmentally and aesthetically friendly in situ technology for toxins removal from contaminated soils (especially heavy metals and metalloids), by the roots of plants with subsequent transport to aerial plant organs. This technology is suitable for large areas, in which other approaches would be expensive and ineffective. The influence of the addition of EDTA and citrate to heavy metal contaminated soil on the activities of C4 photosynthetic enzymes, including phosphoenolpyruvate carboxylase; pyruvate, phosphate dikinase and NADP-malic enzyme in two-year-old plantlets of Paulownia tomentosa x fortunei – TF01 and Paulownia elongata x fortunei – EF02 was evaluated in a pot experiment. Treatment with 1 mM EDTA and 10 mM citrate had a protective effect against heavy metal stress through improvement of CO2 concentration mechanism and total antioxidant activity in TF01, while the application of 1 mM EDTA and 5 mM citrate enhanced antioxidant activity only in EF02. Comparative analyses of results showed that P. tomentosa x fortunei –

TF01 hybrid possessed a more effective CO2 concentration mechanism and antioxidant defence under heavy metal stress than P. elongata x fortunei – EF02 and could be successfully used for phytoremediation of polluted soils.

Keywords: C4 photosynthetic enzymes; citrate; EDTA; Paulownia; total antioxidant activity.

Abbreviations: DPPH⋅ – 2,2-diphenyl-1-picrylhydrazyl; EDTA – ethylendiaminetetraacetic acid; HEPES – 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid; MDH – malate dehydrogenase; NADH – nicotinamide adenine dinucleotide; NADP – nicotinamide adenine dinucleotide phosphate; NADP-ME – NADP-malic enzyme; PEP – phosphoenolpyruvate; PEPC – phosphoenolpyruvate carboxylase; PPDK – pyruvate phosphate dikinase.

Citation: Miladinova-Georgieva K., M. Geneva, Y. Markovska, 2018. Effects of EDTA and citrate addition to the soil on C4 photosynthetic enzymes and biochemical indicators for heavy metal tolerance in two paulownia hybrids. Genetics and Plant Physiology, 8(1–2): 68–81.

*Corresponding author: [email protected] Effect of EDTA and citrate on C4 photosynthetic enzymes 69

INTRODUCTION the metal accumulation by different plant species (Wu et al., 1999; Madrid Heavy metals can accumulate in et al., 2002). Among these chelating biological systems – humans, animals, agents, EDTA being a strong chelator microorganisms and plants, causing for metals has been suggested to be most toxicity (D`Amore et al., 2005). Metal- effective in mobility, solubility, and contaminated soils are difficult to bioavailability in the soil solution phase, remediate and the used conventional uptake by roots, and accumulation of engineering methods are expensive soil-bound metals (Evangelou et al., and non-effective (Zhou and Song, 2007; Leleyter et al., 2012). 2004). Phytoremediation (i.e. uptake The deep contamination caused by and concentration of contaminants a number of metals requires to use as an from the environment in plant biomass) alternative fast-growing woody species is proposed as an alternative low with deep root system and the ability cost technique to remediate soils to grow on nutrient-poor soil. Some contaminated with trace elements (Nouri of them (poplar, willow, black locust, et al., 2009; Kord et al., 2010). ash or alder) are successfully used for Two directions have been suggested remediation of substrates contaminated for phytoextraction of heavy metals, with inorganic and organic pollutants namely, natural phytoextraction, and (Pulford and Watson, 2003; Jensen chemically improved phytoextraction. et al., 2009; Castro-Rodríguez et al., The first one is based on utilization of the 2016). Woody species from the genus natural herbaceous hyperaccumulating Paulownia (Paulowniaceae) are native plants characterized by slow growth originating from China. Paulownia to certain areas and highest metal tomentosa has been introduced into accumulating capacity (Baker Asia, USA, Australia and Europe as a and Brooks, 1989). Low-biomass high -yielding plant species (Woods, producing herbaceous accumulators, 2008). Over the last two decades which accumulate only one specific Paulownia spp. have been extensively element and possess low depth roots studied for phytoremediation purposes are widely used for phytoremediation due to their tolerance to heavy metals of contaminated soils. The chemically in combination with outstanding growth improved phytoextraction known as rate and leaf expansion (Azzarello et induced phytoextraction is based on al., 2012; Doumett et al., 2008; 2011; high yielding plants that extract a high Stankovic et al., 2009; Wang et al., amount of metals when their mobility in 2010). the soil has been increased by chemical In Bulgaria Paulownia spp. are treatments. Several chelating agents micropropagated by the BIOTREE including organic acids (oxalic, citric, Company, according to the technology malic, succinic, tartaric, glutamic) and registered by Biotree Ltd. (Miladinova EDTA (ethylenediamine-tetraacetic et al., 2013). Two hybrid lines (P. acid), have been investigated for their tomentosa x fortunei – TF01 and P. ability to mobilize metals and increase elongata x fortunei – EF02) have been

Genetics & Plant Physiology 2018 vol. 8(1–2) 70 Miladinova-Georgieva et al. micropropagated and patented with metabolites because they the first the purpose of obtaining two types of complex organic compounds formed in plants: (i) short and branchy individuals the plant as a result of photosynthesis, and (ii) less branchy individuals for the and they also provide a major source of purpose of wood material formation. The respiratory energy. According to Anand information about heavy metal tolerance and al. (2017) the heavy metals Pb, Cd, of these hybrid lines and possibilities Hg and Zn greatly reduced the total to be used as phytoremediators of soluble sugar and protein contents of contaminated soils is still insufficient Azolla filiculoides. (Tzvetkova et al., 2013; 2015). Intensive studies have been carried

Heavy metal stress reduces CO2 out on the evolution of the physiological availability in the leaves and inhibits role of C4 metabolic enzymes in C3 carbon fixation with a consequence plants (Häusler et al., 2002). In C3 of growth reduction. Photosynthetic plants, the content and activity levels efficiency in3 C plants (grown of C4 photosynthetic enzymes are lower predominantly in temperate regions) compared with C4 plants. Different depends on the activity of the primary hypothetic schemes for the possible CO2 carboxylating enzyme ribulose-1,5- concentration mechanisms have been bisphosphate carboxylase/oxygenase considered in C3 plants under water, (Rubisco) (Lawlor and Tezara, 2009). salt, low-temperature and heavy metal

CO2 concentration mechanisms are stress conditions (Fridliand and Kaler, evolved to reduce oxygenase activity 1988). It is suggested that PEP system of Rubisco when the environmental functioning in higher plants becomes conditions are unfavourable (Sage, necessary when an organism is forced 2004). Paulownia is characterized as to reorganize its central metabolism a tree with C3 photosynthetic pathway for adaptation to changes in certain

(Ivanova et al., 2018). C4 and CAM conditions and improvement of the plants (grown predominantly in tropical metabolic conversions. Cd excess has and desert regions) are known to be a series of harmful effects, including more resistant to stress conditions than inhibition of plant growth, disorder of

C3 plants because CO2 concentration nutrient uptake, inactivation of enzymes mechanisms allow them to perform CO2 of carbon dioxide (CO2) fixation and fixation to phosphoenolpyruvate (PEP) inhibition of photosynthesis (Duo et via the secondary carboxylating enzyme al., 2016). It has also been reported phosphoenolpyruvate carboxylase that Cd generates oxidative stress in (PEPC). The metabolic cycle is connected plants through inducing the production by a pyruvate, phosphate dikinase of reactive oxygen species (ROS). To (PPDK) that catalyses the regeneration remove ROS, plants have evolved a of PEP from pyruvate (Edwards et al., series of antioxidant enzymatic and 2004; Sage, 2004). The major products non-enzymatic systems. There is a lack of carbon assimilation pathways in most of data about the relationship between of the plants are sucrose and starch. C4 photosynthetic enzyme activities and

Sugars are considered as important the antioxidant capacity of C3 Paulownia

Genetics & Plant Physiology 2018 vol. 8(1–2) Effect of EDTA and citrate on C4 photosynthetic enzymes 71 plants. to 35°C, and relative humidity varied The aim of this research was to between 40 and 65%. The plants were evaluate the influence of different harvested at the end of July 2014. concentrations of EDTA and citrate The influence of the addition of added to the soil on the activities of some complexing agents was evaluated using

C4 photosynthetic enzymes including EDTA (ethylenediaminetetraacetic di- PEPC, PPDK and NADP-malic enzyme sodium salt dihydrate, purity ≥ 97%, (NADP-ME) and reducing sugars in Fluka) and citrate (citric trisodium salt the leaves of Paulownia tomentosa x dihydrate, purity ≥ 99%, Fluka), applied fortunei (TF01) and Paulownia elongata 42 days after planting (June 2014) at 1, 5 x fortunei (EF02) hybrids in order to and 10 mM kg-1 soil dry mass, pH 8.00 (the elucidate their roles in plant adaptation natural pH of the soil). The complexing to heavy metal stress. Changes in total agents were applied to the soil in a single antioxidant capacity in the leaves of both dose via manual dispersion of 150 ml of species after addition of chelating agents their aqueous solutions at concentrations were also measured. of 0.04, 0.2 and 0.4 M. Non-treated pots were used as control. Thirty days after MATERIALS AND METHODS EDTA and citrate application plants (five replicates for each test) were harvested Sampling site, plant materials and pot for analysis. experiments

Two-year-old plantlets of P. Determination of C4 photosynthetic tomentosa x fortunei – TF01 and P. enzyme activities elongata x fortunei – EF02, derived from Extraction procedures of PEPC in vitro micropropagation of seedlings and PPDK were performed according (Miladinova et al., 2013), were cultivated to Ashton et al. (1990). About 0.2 g of in 50 plastic pots (one plants per plot) fresh material from the fourth fully filled with soil aliquots with 2.5 kg developed leaves were harvested in the dry mass, collected from the field near morning, ground into fine powder with waste depository of Kremikovtzi ferrous liquid nitrogen, then 10 ml of extraction metallurgical combine, Sofia, Bulgaria. buffer containing 50 mМ HEPES-KOH The agrochemical soil characteristics were (pH 7.5) and 10 mM dithiothreitol (DTT) described by Tzvetkova et al. (2013). The was added. For extraction of РЕРС only, soil was heavily polluted with Cd, because 5 mg ml-1 bovine serum albumin, 5 mM the content of Cd exceeded Bulgarian MgCl2 and 2 mM K3PO4 were added. The maximal permissible concentrations 1.6 mixtures were centrifuged at 10 000 x g at times. All pots were adjusted daily by 4°С for 10 min and the supernatants were weight to 60% water holding capacity applied on Sephadex G-25 (Pharmacia) with tap water to maintain vigorous plant equilibrated with HEPES-KOH buffer. growth. The experiment was conducted The activity of PEPC (EC 4.1.1.31) in a glasshouse from the beginning of was assayed at 340 nm by following the April 2014 to the end of July 2014. The reduction of oxaloacetate by nicotinamide glasshouse temperatures ranged from 15 adenine dinucleotide (NADH) in the

Genetics & Plant Physiology 2018 vol. 8(1–2) 72 Miladinova-Georgieva et al. presence of malate dehydrogenase (MDH) Reducing sugars assay using UV-VIS spectrophotometer Boeco Dry leaf material (0.50 g) was ground S-22 (Germany). Reaction mixture (2 in a mortar with 5.0 ml 80% ethanol. ml) contained 25 mM HEPES-KOH (pH The homogenous mixture obtained

8.0), 5 mM MgCl2, 2 mM DTT, 1 mM was allowed to stay for 20 min at room

NaHCO3, 5 mM glucose-6-phosphate, temperature, followed by filtration 5 mM PEP, 0.2 mM NADH, 2 U of through Whatman qualitative filter paper, MDH and 0.2 ml of the enzyme extract. Grade 4. The total content of reducing The decrease in absorbance due to the sugars was determined in the ethanol oxidation of NADH was measured for 3 extracts by using the phenol/sulphuric min (Ashton et al., 1990). acid method of Dubois et al. (1956). The activity assay of PPDK (EC An aliquot of 0.25 ml of the filtrate was 2.7.9.1) was performed at 340 nm in added and mixed with 0.75 ml distilled the forward direction by coupling the water, 0.5 ml 5% phenol and 2.5 ml 98% production of PEP to NADH via PEPC H2SO4. The resulting orange complex and MDH (Ashton et al., 1990). Reaction was then measured at 485 nm after 1 h mixture (2 ml) contained 25 mM with intermittent shaking. The content of HEPES-KOH буфер (pH 8.0), 8 mM reducing sugars was expressed as glucose -1 MgSO4, 10 mM DTT, 10 mM NaHCO3, equivalents in mg g of the sample. All

2 mM pyruvate, 5 mM (NH4)2SO4, 1 mM values were expressed as mg of glucose glucose-6-phosphate, 1 mM ATP, 2.5 mM equivalents per 1 g DW.

K3PO4, 0.2 mM NADH, 0.5 U of PEPC, 2 U of MDH and 0.2 ml enzyme extract. DPPH assay The contents were allowed to react at Free radicals scavenging activity was 30°C and the decrease in absorbance due measured from the bleaching of the purple- to the oxidation of NADH was measured coloured methanol solution of free stable for 3 min. radical (2,2-diphenyl-1-picrylhydrazyl, The activity assay of NADP- DPPH⋅) inhibition after Tepe et al. (2006). ME (EC 1.1.1.40) was performed DPPH⋅ radical is a stable radical with spectrophotometrically at 340 nm by a maximum absorption at 517 nm that following the reduction of nicotinamide can readily undergo reduction by an adenine dinucleotide phosphate (NADP) antioxidant. The inhibition of free radical in the presence of malate (Garnier– DPPH⋅ in percent (I %) was calculated in Dardart and Quieroz, 1974). The reaction the following way: mixture (2 ml) contained 50 mM HEPES- I% = (Ablank – Asampe / Ablank) × 100

KOH (pH 7.5), 3 mM MgCl2, 0.25 mM where Ablank is the absorbance of the NADP, 3 mM malate and 0.1 ml enzyme control reaction (containing all reagents extract. The increase in absorbance due except the test compound), Asample is the to the reduction of NADP was measured absorbance of the test compound, i.e. plant for 3 min. extracts. The reaction mixture consisted The determination of protein content of different concentrations from 15 to was performed by the method of Lowry 180 μg ml-1 plant methanol extract, 2.4 ml et al. (1951). methanol and 0.1 mM methanol solution

Genetics & Plant Physiology 2018 vol. 8(1–2) Effect of EDTA and citrate on C4 photosynthetic enzymes 73 of DPPH⋅. Control and tested samples plants died within a few days after EDTA were incubated in the dark for 30 min application (Fig. 1). Application of EDTA before spectrophotometric assay. at 1 and 5mM did not effect negatively plant growth. Plants treated with lower Statistical analysis EDTA concentrations and 1, 5, and 10 All data are mean values of at least mM citrate did not show any phytotoxic three to five independent experiments. The symptoms upon visual assessment, they mean values ± SD and an exact number had the same appearance as control plants. of experiments are given in the Figures. The results showed that EF02 control The significance of differences between leaves possessed 4-times higher PEPC control and treatment was analyzed activity as compared to the TF01control by Fisher`s LSD test (P≤0.05) after (Fig. 2A). A dramatic decrease in PEPC performing ANOVA multifactor analysis. activity in EF02 leaves after all treatments applied was observed, while in TF01 a RESULTS significant enhancement was established after treatment with 1 mM EDTA and 1 The 10 mM EDTA concentration mM and 10 mM citrate. All treatments is usually reported in the literature as led to a decline in PPDK activity in TF01 toxic for many herbaceous species (Wu leaves in comparison with the control as et al., 1999). Surprisingly, Doumett et the strongest impact was observed under al. (2008) did not find any phytotoxic treatments with 5 mM EDTA and 5 mM symptoms after 10 mM EDTA treatment citrate, respectively (Fig. 2B). Treatment of Paulownia tomentosa and concluded with 1 mM EDTA and 10 mM citrate that this plant possessed strong resistance caused an increase in PPDK activity in towards the presence of high chelator EF02 leaves. NADP-ME activity did not concentrations in the soil. Our results change in TF01 after treatment with 5 mM showed that application of 10 mM EDTA EDTA and 5 mM citrate, while in EF02 was harmful to both hybrid lines, in which a significant decrease was established leaves presented an extensive chlorosis after all treatments as compared to and necrotic areas, and most of the control. Lower concentrations of the

Figure 1. Paulownia tomentosa x fortunei and Paulownia elongata x fortunei grown in heavy metal polluted soil on the third day after application of 10 mM EDTA.

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Figure 2. Changes in phosphoenolpyruvate carboxylase (PEPC - A), pyruvate, phosphate dikinase (PPDK - B) and NADP-malic enzyme (NADP-ME - C) activities at the end of the experiment in mature leaves of Paulownia tomentosa x fortunei (TF01) and Paulownia elongata x fortunei (EF02) lines grown in heavy metal polluted soil supplemented with EDTA and citrate as complexing agents. Mean values ± SE (n = 3-5). Values with the same letter are not significantly different when means are separated by Fisher`s LSD test (P<0.05). chelating agents applied (1 mM) increased The lowest value was established after negligibly these values (Fig. 2C). application of the lowest concentrations Soluble sugars prevailed in EF02 of the chelating agents (1 mM EDTA) control leaves as compared to TF01 (Fig. 3). control. The highest values were measured Higher values of the total antioxidant in control plants of both hybrid lines. activity were established after all

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Figure 3. Changes in reducing sugars content at the end of the experiment in mature leaves of Paulownia tomentosa x fortunei (TF01) and Paulownia elongata x fortunei (EF02) lines grown in heavy metal polluted soil supplemented with EDTA and citrate as complexing agents. Mean values ± SE (n = 3-5). Values with the same letter are not significantly different when means are separated by Fisher`s LSD test (P<0.05). treatments in both TF01 and EF02 Cd and can be used for phytoremediation plants. No significant differences were of heavy metal contaminated soils. TF01 established for the antioxidant capacity plants accumulated higher amounts of in leaves of TF01 hybrids treated with the Pb and Zn in the aboveground parts, two complexing agents added at different while EF02 plants – only Zn. Bahri et concentrations (Fig. 4). The antioxidant al (2015) confirmed that Paulownia capacity was lower when EF02 plants tomentosa (Thunb.) Steud could be were treated with 5 mM EDTA and 1 mM used in the phytoextraction of Zn and citrate in comparison with plants treated Pb from contaminated soils, but heavy with the rest test concentrations. metals caused slight reductions in all growth parameters and deterioration of DISCUSSION photosynthetic performance. Miladinova- Georgieva et al. (2018) reported that Tzvetkova et al. (2015) have reported net-photosynthesis was enhanced in that two hybrid lines P. tomentosa x the leaves of TF01 and EF02 upon fortunei – TF01 and P. elongata x fortunei application of 1 mM EDTA, 1 mM and – EF02 are accumulators of Cu, Zn and 10 mM citrate. The obtained data showed

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Figure 4. Changes in total antioxidant activity, measured by the DPPH method at the end of the experiment in mature leaves of Paulownia tomentosa x fortunei (TF01) and Paulownia elongata x fortunei (EF02) lines grown in heavy metal polluted soil supplemented with EDTA and citrate as complexing agents. Mean values ± SE (n = 3).

that both lines responded differently in C4 plants differ from C3 plants under terms of photosynthetic enzyme activities, the CO2 concentrating mechanism, which soluble sugar levels and total antioxidant has advantages in climatic conditions activity during treatments with increasing unfavourable for growth conditions concentrations of chelating agents. TF01 such as high temperatures, low water plants reacted with increasing activities of availability, high irradiation or heavy PEPC, PPDK and total antioxidant activity metal soil contamination (Sage, 2004). at 1 mM EDTA, 1 mM and 10 mM citrate The activities of PEPC, NADP-ME and (Fig. 2A, B and Fig. 4). The highest values PPDK, which participate in the process of for PEPC, NADP-ME activities and concentrating CO2, are enhanced in plants soluble sugar content were established with the two types of photosynthesis in control EF02 plants, but these values under stress conditions (Doubnerová and declined in a different manner after the Ryšlavá, 2011). Our results showed that treatments applied (Fig. 2A, C and Fig. 3). control EF02 plants possessed higher Total antioxidant activity was enhanced PEPC, NADP-ME activities and soluble to a higher extent after treatment of EF02 sugar content as compared to TF01 control with 1 mM EDTA and 5 or 10 mM citrate plants (Fig. 2A, B and Fig. 4). PEPC and (Fig. 4). NADP-ME play a more pronounced role in

Genetics & Plant Physiology 2018 vol. 8(1–2) Effect of EDTA and citrate on C4 photosynthetic enzymes 77 the synthesis and degradation of C4 organic be helpful for TF01 to remediate metal acids and participate in the process of CO2 contaminated soils because improvement concentration, namely in EF02 control of the photosynthetic surface under stress plants than in TF01. The increase in the C4 conditions is critical to the optimum cycle enzyme activities (PEPC) caused by performance of phytoremediation. heavy metals leads to an enhancement of A reduction of reactive oxygen species the C4 acid synthesis (previously malate – after EDTA treatment in the heavy metal data not shown). CO2 involved as a result accumulating species Sedum alfredii L. of the subsequent decarboxylation of this was established (Huang et al., 2008). acid by NADP-ME is used in the Calvin EDTA reduced heavy metal impact, the cycle as a substrate for Rubisco, providing activities of POX, CAT, enzymes of the normal performance of the reactions the ascorbate-glutathione cycle, but the in the cycle. These results showed levels of ascorbate, reduced glutathione that the studied enzymes are actively and flavonoids were enhanced in the involved in the process of biochemical shoots of Tribulus terrestris (Markovska adaptation against the effects of heavy et al., 2013). Because citrate is a natural metals in EF02 control plants. PEPC and molecule, it can be metabolized by plants NADP-ME activities have undergone an more easily than EDTA. The use of induction that confirmed their key roles natural, rapidly biodegradable ligands at in the biosynthesis and degradation of much higher concentrations and with a dicarbonic acids. Because PEPC activity regular renewal of the complexing agent changed less in TF01 than in EF02, application will lead to an improvement probably non-carbohydrate pathways of plant metal accumulation without of the C-photosynthetic metabolism increasing environmental impact. The remain more stable under heavy metal activities of CAT, POX, GR and APX in stress. It is known that soluble sugar the leaves of TF01 were enhanced after production in the leaves of Paulownia rise treatment with increasing concentrations at warm conditions (Woods, 2008). Our of EDTA, while in EF02 they were results showed that EF02 control plants decreased (Miladinova - Georgieva et al., accumulated more soluble sugars than 2017). In our experiments, however, we TF01 plants (Fig. 3). The accumulation did not establish any significant changes in of sucrose could potentially play a the total antioxidant activity in the leaves direct role in osmoregulation and could of TF01 after treatment with EDTA and also provide quickly metabolizable citrate at all concentrations applied. Total carbohydrates for energy production when antioxidant activity in the leaves of EF02 carbon is diverted from growth to other plants decreased with increasing EDTA functions (Hare et al., 1998). According concentration in the soil, but it enhanced to Miladinova-Georgieva et al. (2018) 1 with increasing citrate concentration. mM EDTA and 10 mM citrate added to There is no clear correlation between the the soil enhanced leaf growth (the highest changes of antioxidant enzyme activities values for total leaf area/total dry mass and total antioxidant capacity measured ratio, total leaf area/leaf number ratio after EDTA and citrate application in both and rate of net-photosynthesis). It would plants.

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