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Genetics and Plant Physiology – 2018, Volume 8(1–2), Pp 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 in 3C 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
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