PIXE analysis of trace elements in relation to chlorophyll concentration in Plantago ovata Forsk

ARTICLE IN PRESS Applied Radiation and Isotopes 68 (2010) 444–449

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PIXE analysis of trace elements in relation to chlorophyll concentration in Plantago ovata Forsk Priyanka Saha a, Sarmistha Sen Raychaudhuri a,, Anindita Chakraborty b, Mathummal Sudarshan b a b

Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, 92, APC Road, 700009 Kolkata, India UGC-DAE consortium for Scientific Research, Kolkata Centre, Radiation Biology Division, 3/LB Bidhannagar, Salt Lake, 700098 Kolkata, India

a r t i c l e in f o

a b s t r a c t

Article history: Received 8 June 2009 Received in revised form 1 December 2009 Accepted 1 December 2009

Plantago ovata Forsk – an economically important medicinal plant – was analyzed for trace elements and chlorophyll in a study of the effects of gamma radiation on physiological responses of the seedlings. Proton-induced X-ray emission (PIXE) technique was used to quantify trace elements in unirradiated and gamma-irradiated plants at the seedling stage. The experiments revealed radiation-induced changes in the trace element and chlorophyll concentrations. & 2009 Elsevier Ltd. All rights reserved.

Keywords: Trace elements Chlorophyll Gamma irradiation PIXE

1. Introduction Ionizing radiation is known to induce metabolic changes in various life forms by generating reactive oxygen species (Shirley et al., 1992; Spitz et al., 2004; Engin et al., 2005). In its developmental and adult stages, an organism depends on interaction of various trace elements. Earlier reports have revealed the important role that trace elements play in the metabolism of plants (Obiajunwa et al., 2002; Queralt et al., 2004; Devi et al., 2008; Lamari et al., 2008; Saha et al., 2008a, b). Concentrations of elements in plants vary depending upon the environment in which the plants grow or are exposed to (Bargagli et al., 1997; Pendias, 2004; Areqi et al., 2008). Changes in the environment can put stress on plants and, thus, affect their constitutional, structural, and, ultimately, functional processes (Braam et al., 1997; Mora et al., 1999; Stenstrom et al., 2002; Gostin and Ivanescu, 2007). Radiation has been widely studied as a stress-inducing factor affecting plant growth and development by inducing biochemical, physiological and morphological changes in plants (Salhi et al., 2004; Maity et al., 2005; Wi et al., 2007). In many cases, such response is modulated by trace elements because many of these elements play crucial roles in various metabolic activities (Naidu et al., 1999; Yamashita et al., 2005; Zucchi et al., 2005). However, the reports describing stressinduced alteration by elemental constituents during development

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E-mail address: [email protected] (S. Sen Raychaudhuri). 0969-8043/$ - see front matter & 2009 Elsevier Ltd. All rights reserved. doi:10.1016/j.apradiso.2009.12.003

of plant seedlings are few. Thus, modulation of plant growth with participation of trace elements, which is an interesting point of plant science research, has barely been studied. However, radiation is used to stimulate seedling growth in certain plants (Charbaji and Nabulsi, 1999; Vilela and Ravetta, 2000; Hua and Lian, 2003; Singh and Sujata, 2004; Toker et al., 2005). Ionizing radiation causes various phenotypic changes in plants by genetic alteration and also biochemical and physiological disorders (Roy et al., 2006; Begum et al., 2008). A number of researchers investigated the effect of gamma radiation on chlorophyll content (Dale et al., 1997; Byun et al., 2002; Kiong et al., 2008; Ling et al., 2008). Moroni et al. (1991) have reported that chlorophyll content of manganese-stressed seedlings is also reduced drastically. In this perspective, the present work was designed to study concentrations of trace elements and chlorophyll under normal and radiation-stressed conditions during seedling stage of Plantago ovata Forsk. P. ovata Forsk, the common Isabgul, is an important medicinal plant. Mucilage present in the seed coat and husks of the seeds of this plant is used as laxatives. It has gained agricultural importance recently because of its wide use in cosmetics, pharmaceutical and food industries. P. ovata has been selected as an in vitro test system for evaluating radiation-induced changes in trace element and chlorophyll content because of its low generation time and easy germination in vitro. In this work, we have used proton-induced X-ray emission (PIXE), which is a sensitive, non-destructive analytical technique requiring small amounts of materials. It is very effective in rapid multielemental analyses of a wide range of biological and environmental samples

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(Owicz et al., 2004; Naga Raju et al., 2006; Tylko et al., 2007; Devi et al., 2008).

2. Experimental 2.1. Germination of seedlings Seeds of P. ovata were imbibed in 20 mL autoclaved distilled water overnight. The next day, seeds were sterilized with 10% (v/v) liquid sodium hypochlorite (NaOCl) for about 20 min. The excess bleach was removed by washing the seeds 5 times with sterile distilled water. Seeds were aseptically inoculated in agar– sucrose medium [3% sucrose (SRL, Mumbai, India) and 0.9% agar– agar (SRL, Mumbai, India)] for germination according to the standardized method described elsewhere (Das Pal and Sen Raychaudhuri, 2000). Seedlings were grown within 12–14 days.

2.5. Determination of chlorophyll content Each of the irradiated and unirradiated seedling samples (200 mg each) was placed into a pre-chilled mortar. The seedlings were then treated with 700 mL of 80% acetone (SRL, Mumbai, India) and centrifuged at 10,000 rpm for 10 min at 4 1C. After centrifugation, the supernatants were transferred into fresh polypropylene tubes, and the seedling remains were again treated with 80% acetone with subsequent centrifuging. This procedure was repeated 6 or 7 times, until the supernatant became colorless. The absorbances of the extract were measured at 646 and 663 nm with a spectrophotometer. The concentrations of chlorophyll a (Chl-a), chlorophyll b (Chl-b) and total chlorophyll (Chl a+b) in milligram per liter were measured according to the formulae below and then expressed in milligram per gram fresh weight of plant material (Ling et al., 2008): Chlorophyll a; Chl-a ¼ 2:25 ðA663 Þ2:79 ðA646 Þ;

2.2. Gamma irradiation

Chlorophyll b; Chl-b ¼ 21:50ðA646 Þ5:10 ðA663 Þ;

Individual freshly grown leaves of P. ovata were placed into plastic bags and irradiated with 60Co gamma photons (Gamma chamber 900, BRIT, Navi Mumbai, India) at the Chemical Science Division of Saha Institute of Nuclear Physics (Kolkata, India). The samples were irradiated to 10, 20, 50 or 100 Gy at the dose rate of 1 Gy/min. Five sets of the seedlings were exposed to each radiation dose. Each of the five treatments was replicated thrice. A control set of unirradiated samples was maintained aseptically under laboratory conditions. All irradiated and control samples were grouped for carrying out PIXE and chlorophyll content analysis.

Total chlorophyll; Chl a þ b ¼ 7:15ðA663 Þ þ 18:71 ðA646 Þ:

2.3. Sample preparation for PIXE analysis All irradiated and unirradiated samples of the seedlings, approximately 1 g each, were freeze-dried with a vertex lyophilizer (Virtis, Gardiner, New York) at 80 1C for 48 h. After that, samples were homogenized by a brittle fracture technique under liquid nitrogen. For PIXE analysis, these were powdered by using mortar and pestle and mixed with extra pure graphite (Merck, Mumbai, India) in the ratio of 60:40. Approximately 150 mg of each sample was pelletized using a Perkin Elmer press according to the standardized method of preparation of thick biological sample targets (Chakraborty et al., 2000). The resulted pellets were 1 mm thick and 10 mm in diameter. Blank targets, which contained standard reference material from National Institute of Standards and Technology (NIST) (Trace Elements in Apple Leaf Standards SRM 1515), were also prepared by the same technique. 2.4. PIXE analysis Collimated 2.5-MeV-proton beam was delivered by a 3-MV tandem Pelletron accelerator at the Institute of Physics (IOP) in Bhubaneswar (India). The diameter of the collimated proton beam was 2 mm. The beam current varied from 2 to 5 nA for low-Z elements and from 20 to 40 nA for higher-Z elements (with a 50-mm Al filter). The characteristic X-rays emitted from the sample were extracted through a 25-mm Mylar window and detected with a Canberra-SL 30160 Si(Li) detector. The spectra were recorded on a PC-based MCA using WINMCA software. The generated X-ray data were stored on a disk and analyzed with GUPIX-2000 user-friendly software for PIXE analysis. The quality of the results was checked using data obtained with the certified reference material.

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3. Results and discussion An analysis has shown the presence of potassium, chlorine, calcium, manganese, iron, copper, zinc, bromine and strontium in all the samples. Significant differences in the concentrations of the detected elements were observed between unirradiated and irradiated samples (Fig. 1). The accuracy of the used analytical method was confirmed by analysis of the standard reference material (Table 1). 3.1. Chlorine and potassium The trends of changes in potassium and chlorine concentrations in the seedlings with dose were similar. The samples irradiated to 10 Gy contained 6% more potassium and 10% more chlorine than the unirradiated ones. 3.2. Calcium and manganese Our data show similar trends of decrease in the concentrations of calcium (9%) and manganese (21%) in seedlings irradiated to 10 Gy. Irradiation to the next dose level, 20 Gy, did not decrease the concentrations of these two elements further. However, irradiation to 50 Gy brought down both calcium and manganese concentrations. An irradiation to an even higher dose, 100 Gy, lowered concentrations of both the elements almost equally (by 24% for Ca and 20% for Mn) as compared with their levels in unirradiated seedlings. 3.3. Iron and copper Concentrations of iron and copper changed with the radiation dose in the opposite directions. This finding is in line with results of our earlier study of callus samples of P. ovata (Saha et al., 2008a, b), where we also saw an increase in iron concentration accompanied by a decrease in copper concentrations in irradiated 42-day-old calli. 3.4. Zinc No significant difference was observed between zinc concentrations in unirradiated seedlings of P. ovata and seedlings

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Cl

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Fig. 1. PIXE-determined concentrations of trace elements in seedlings of P. ovata under normal and gamma-stressed conditions. C stands for control (zero dose).

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irradiated up to 50 Gy. However, irradiation to 100 Gy resulted in a 30% increase in zinc concentration.

3.5. Bromine and strontium Concentration of Br and Sr decreased with dose. Our results show that only the concentration of iron grows with the radiation dose; radiation decreases concentrations of all the other trace elements in question. This is in line with observations by Bhat et al. (2008), who found that concentrations of copper and manganese in velvet bean seeds decreased significantly after irradiation to all used doses. It has been reported, however, that exposure of plants to UV-B radiation increased iron concentration in maize plants (Zancan et al., 2006). This fact suggests that iron is essential for various bioprocesses needed for growth of seedlings and is in good agreement with our results obtained with P. ovata. Our study also shows that calcium concentration in unirradiated seedlings of P. ovata exceeds the concentration of any other metal. Desai et al. (2006) have found that calcium accumulation at the embryo development is higher Table 1 Comparison of determined and certified values of trace element concentrations in Apple Leaf Standard. Trace element

Calculated value (ppm)

Certified value (ppm)

Cl K Ca Fe Mn Ni Cu Zn Sr

618 742 15124 745 15862 763 98 76 66 72 3 71 6 70.5 13 71 23 73

5797 23 160007 200 150007 150 837 5 547 3 17 0.1 67 0.2 137 0.3 257 2

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3.6. Determination of chlorophyll content In this study, we compared chlorophyll content of seedlings before and immediately after irradiation with 60Co. Figs. 2(a–c) show that all the irradiated seedlings contained less chlorophyll a and b than the unirradiated plants. Seedlings irradiated to 10, 20, 50 and 100 Gy had 0.36, 0.28, 0.34, and 0.30 mg/g FW of total chlorophyll, respectively. These values are significantly lower than chlorophyll content of unirradiated seedlings (0.39 mg/g FW). Moreover, the concentrations of chlorophyll a were higher than the concentrations of chlorophyll b in both irradiated and unirradiated seedlings. Chlorophyll concentration varied from one irradiated sample to another. Our data are in agreement with the results of earlier works (Schwimmer and Weston, 1958; Dale et al., 1997; Kim et al., 2008; Ling et al., 2008), where chlorophyll concentrations were found to be lower in irradiated plants than in unirradiated ones. This is because gamma radiation breaks chlorophyll molecules apart (Byun et al., 2002). Gamma irradiation results in various physiological and biochemical changes in plants. It can damage or modify important components of cell walls and also disturb enzyme activity, hormone balance and water exchange. These effects can result in changes in the structure of plant cells and metabolism processes, such as photosynthesis, modulation of the antioxidant system and accumulation of phenolic compounds. Photosynthetic pigments can be destroyed by gamma rays with concomitant loss of photosynthetic capacity (Strid et al., 1990). Kim et al. (2008) reported that the

0.12

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than at the other developmental stages. A stimulatory effect of various levels of calcium in the medium was observed in Pinus (Pullman et al., 2003). Calcium plays a very important role in plant growth and nutrition (Hepler, 2005). Also, calcium is an important element in cellular signaling and mediating plant response to osmotic stress (Sanders et al., 2002).

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Gamma dose (Gy) Fig. 2. Concentrations of chlorophyll a, chlorophyll b and total chlorophyll in seedlings of P. ovata before and immediately after gamma exposure. C stands for control.

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chlorophyll content of plants decreased gradually after irradiation. Chlorophyll degradation was also observed in plants irradiated with gamma rays to high doses (Wada et al., 1998). Furthermore, as mentioned above, the chlorophyll a concentrations were found to be higher than the concentrations of chlorophyll b in both irradiated and unirradiated seedlings of P. ovata. A number of researchers (Strid et al., 1990; Ling et al., 2008) have already reported that gamma irradiation results in greater destruction of chlorophyll b than chlorophyll a. This preferential loss of chlorophyll b is due to disturbance of its biosynthesis or degradation of its precursors (Marwood and Greenberg, 1996). Byun et al. (2002) found that irradiation to 20 kGy destroys chlorophyll b. Chlorophyll decomposition involves release of chlorophyll from its protein complex with subsequent dephytolization and possibly pheophytinization (Simpson et al., 1976). However, the exact mechanism of chlorophyll breakdown is still unknown. As iron catalyzes biosynthesis of chlorophyll (Chereskin and Castelfranco, 1982; Spiller et al., 1982; Bollivar and Beale, 1996), it may favor photosynthesis in leaves. Chouliaras et al. (2004) observed that, in treatments lacking iron, net photosynthesis was reduced because of lower chlorophyll concentrations. Involved in chlorophyll metabolism, iron is essential for growth of higher plants. Marsh et al. (1963) tried to investigate the role of iron in chlorophyll metabolism, but without much success. Evans (1959) and DeKock et al. (1960) found a correlation between the iron content of the medium and the chlorophyll and heme contents of leaves. There are no reports that heme enzymes are directly involved in chlorophyll metabolism. However, Bogorad (1960) have found that the porphyrin moieties of heme and chlorophyll are formed by the same bio-synthetic system. The point is that the chlorophyll and heme syntheses depend on an adequate iron supply. Iron also plays an important role in porphyrin biosynthesis (Lascelles, 1955; Vogel et al., 1960). It has been found in the earlier works that the level of activity of heme enzymes of leaf tissue and its chlorophyll content are markedly influenced by iron supply (Evans, 1959; DeKock et al., 1960). Lower activities of the heme enzymes may indirectly affect chlorophyll metabolism. The correlation between chlorophyll and heme contents suggests that iron chlorosis is an expression of a regulatory influence produced by the supply of iron on porphyrin synthesis. Our results show that chlorophyll concentrations in seedlings of P. ovata decreased after gamma irradiation; however, the concentrations of iron increased simultaneously. Gamma radiation breaks the porphyrin ring of the chlorophyll molecule. The porphyrin ring of chlorophyll, with a magnesium atom in its center, is a part of the chlorophyll molecule that absorbs energy of light. As the metal–porphyrin complex in seedlings of P. ovata has been decomposed by gamma rays, the increased concentrations of iron could not be used in the synthesis of chlorophyll.

4. Conclusion The PIXE results showed that gamma irradiation decreases concentrations of most of trace elements in P. ovata. The only exception is iron, whose concentration increased steadily with radiation dose. This increase in iron concentration did not affect the chlorophyll metabolism of the cells because the irradiation decreased the chlorophyll content of the plants by breaking the porphyrin rings.

Acknowledgements Authors would like to thank Institute of Physics in Bhubaneswar, India, for the beam time provided to perform the PIXE

experiments and Saha Institute of Nuclear Physics in Kolkata, India, for providing the 60Co source.

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