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Applied Radiation and Isotopes 68 (2010) 444–449

Contents lists available at ScienceDirect

Applied Radiation and Isotopes

0969-80

doi:10.1

� Corr

E-m

journal homepage: www.elsevier.com/locate/apradiso

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

Priyanka Saha a, Sarmistha Sen Raychaudhuri a,�, Anindita Chakraborty b, Mathummal Sudarshan b

a Department of Biophysics, Molecular Biology and Bioinformatics, University of Calcutta, 92, APC Road, 700009 Kolkata, Indiab 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 i n f o

Article history:

Received 8 June 2009

Received in revised form

1 December 2009

Accepted 1 December 2009

Keywords:

Trace elements

Chlorophyll

Gamma irradiation

PIXE

43/$ - see front matter & 2009 Elsevier Ltd. A

016/j.apradiso.2009.12.003

esponding author. Tel.: +91 033 23508386x3

ail address: sarmistha_rc@rediffmail.com (S. S

a b s t r a c t

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.

1. Introduction

Ionizing radiation is known to induce metabolic changes invarious life forms by generating reactive oxygen species (Shirleyet al., 1992; Spitz et al., 2004; Engin et al., 2005). In itsdevelopmental and adult stages, an organism depends oninteraction of various trace elements. Earlier reports haverevealed the important role that trace elements play in themetabolism 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 theenvironment in which the plants grow or are exposed to (Bargagliet al., 1997; Pendias, 2004; Areqi et al., 2008). Changes in theenvironment can put stress on plants and, thus, affect theirconstitutional, 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 asa stress-inducing factor affecting plant growth and developmentby inducing biochemical, physiological and morphologicalchanges in plants (Salhi et al., 2004; Maity et al., 2005; Wiet al., 2007). In many cases, such response is modulated by traceelements because many of these elements play crucial roles invarious metabolic activities (Naidu et al., 1999; Yamashita et al.,2005; Zucchi et al., 2005). However, the reports describing stress-induced alteration by elemental constituents during development

ll rights reserved.

24; fax: +91 033 28661573.

en Raychaudhuri).

of plant seedlings are few. Thus, modulation of plant growth withparticipation of trace elements, which is an interesting point ofplant 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 andLian, 2003; Singh and Sujata, 2004; Toker et al., 2005). Ionizingradiation causes various phenotypic changes in plants by geneticalteration and also biochemical and physiological disorders(Roy et al., 2006; Begum et al., 2008). A number of researchersinvestigated 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 contentof manganese-stressed seedlings is also reduced drastically. Inthis perspective, the present work was designed to studyconcentrations of trace elements and chlorophyll under normaland radiation-stressed conditions during seedling stage ofPlantago ovata Forsk.

P. ovata Forsk, the common Isabgul, is an important medicinalplant. Mucilage present in the seed coat and husks of the seedsof this plant is used as laxatives. It has gained agriculturalimportance recently because of its wide use in cosmetics,pharmaceutical and food industries. P. ovata has been selectedas an in vitro test system for evaluating radiation-induced changesin trace element and chlorophyll content because of its lowgeneration time and easy germination in vitro. In this work, wehave used proton-induced X-ray emission (PIXE), which is asensitive, non-destructive analytical technique requiring smallamounts of materials. It is very effective in rapid multielementalanalyses 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; Deviet al., 2008).

2. Experimental

2.1. Germination of seedlings

Seeds of P. ovata were imbibed in 20 mL autoclaved distilledwater overnight. The next day, seeds were sterilized with 10%(v/v) liquid sodium hypochlorite (NaOCl) for about 20 min. Theexcess bleach was removed by washing the seeds 5 times withsterile 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 thestandardized method described elsewhere (Das Pal and SenRaychaudhuri, 2000). Seedlings were grown within 12–14 days.

2.2. Gamma irradiation

Individual freshly grown leaves of P. ovata were placed intoplastic bags and irradiated with 60Co gamma photons (Gammachamber 900, BRIT, Navi Mumbai, India) at the Chemical ScienceDivision of Saha Institute of Nuclear Physics (Kolkata, India). Thesamples were irradiated to 10, 20, 50 or 100 Gy at the dose rate of1 Gy/min. Five sets of the seedlings were exposed to eachradiation dose. Each of the five treatments was replicated thrice.A control set of unirradiated samples was maintained asepticallyunder laboratory conditions. All irradiated and control sampleswere grouped for carrying out PIXE and chlorophyll contentanalysis.

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 lyophi-lizer (Virtis, Gardiner, New York) at �80 1C for 48 h. After that,samples were homogenized by a brittle fracture technique underliquid nitrogen. For PIXE analysis, these were powdered by usingmortar and pestle and mixed with extra pure graphite (Merck,Mumbai, India) in the ratio of 60:40. Approximately 150 mg ofeach sample was pelletized using a Perkin Elmer press accordingto the standardized method of preparation of thick biologicalsample targets (Chakraborty et al., 2000). The resulted pelletswere 1 mm thick and 10 mm in diameter.

Blank targets, which contained standard reference materialfrom National Institute of Standards and Technology (NIST) (TraceElements in Apple Leaf Standards SRM 1515), were also preparedby the same technique.

2.4. PIXE analysis

Collimated 2.5-MeV-proton beam was delivered by a 3-MVtandem Pelletron accelerator at the Institute of Physics (IOP) inBhubaneswar (India). The diameter of the collimated proton beamwas 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 a50-mm Al filter). The characteristic X-rays emitted from thesample were extracted through a 25-mm Mylar window anddetected with a Canberra-SL 30160 Si(Li) detector. The spectrawere recorded on a PC-based MCA using WINMCA software.The generated X-ray data were stored on a disk and analyzed withGUPIX-2000 user-friendly software for PIXE analysis. The qualityof the results was checked using data obtained with the certifiedreference material.

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 seedlingswere then treated with 700mL of 80% acetone (SRL, Mumbai,India) and centrifuged at 10,000 rpm for 10 min at 4 1C. Aftercentrifugation, the supernatants were transferred into freshpolypropylene tubes, and the seedling remains were again treatedwith 80% acetone with subsequent centrifuging. This procedurewas repeated 6 or 7 times, until the supernatant became colorless.The absorbances of the extract were measured at 646 and 663 nmwith a spectrophotometer. The concentrations of chlorophyll a(Chl-a), chlorophyll b (Chl-b) and total chlorophyll (Chl a+b) inmilligram per liter were measured according to the formulaebelow and then expressed in milligram per gram fresh weight ofplant material (Ling et al., 2008):

Chlorophyll a; Chl-a¼ 2:25 ðA663Þ�2:79 ðA646Þ;

Chlorophyll b; Chl-b¼ 21:50ðA646Þ�5:10 ðA663Þ;

Total chlorophyll; Chl aþb¼ 7:15ðA663Þþ18:71 ðA646Þ:

3. Results and discussion

An analysis has shown the presence of potassium, chlorine,calcium, manganese, iron, copper, zinc, bromine and strontium inall the samples. Significant differences in the concentrations of thedetected elements were observed between unirradiated andirradiated samples (Fig. 1). The accuracy of the used analyticalmethod was confirmed by analysis of the standard referencematerial (Table 1).

3.1. Chlorine and potassium

The trends of changes in potassium and chlorine concentra-tions in the seedlings with dose were similar. The samplesirradiated to 10 Gy contained 6% more potassium and 10% morechlorine than the unirradiated ones.

3.2. Calcium and manganese

Our data show similar trends of decrease in the concentrationsof calcium (9%) and manganese (21%) in seedlings irradiated to10 Gy. Irradiation to the next dose level, 20 Gy, did not decreasethe concentrations of these two elements further. However,irradiation to 50 Gy brought down both calcium and manganeseconcentrations. 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 inunirradiated seedlings.

3.3. Iron and copper

Concentrations of iron and copper changed with the radiationdose in the opposite directions. This finding is in line with resultsof our earlier study of callus samples of P. ovata (Saha et al.,2008a, b), where we also saw an increase in iron concentrationaccompanied by a decrease in copper concentrations in irradiated42-day-old calli.

3.4. Zinc

No significant difference was observed between zinc concen-trations in unirradiated seedlings of P. ovata and seedlings

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Cl

0

2000

4000

6000

8000

CGamma dose (Gy)

Con

c (p

pm)

Ca

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c (p

pm)

Fe

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Con

c (p

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C 10 20 50 100

C 10 20 50 100

K

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Mn

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Cu

02468101214

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Zn

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Br

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Con

c (p

pm)

Sr

0

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40

60

Gamma dose (Gy)

Con

c (p

pm)

10 20 50 100 C 10 20 50 100

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).

P. Saha et al. / Applied Radiation and Isotopes 68 (2010) 444–449446

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irradiated up to 50 Gy. However, irradiation to 100 Gy resulted ina 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 allthe other trace elements in question. This is in line withobservations by Bhat et al. (2008), who found that concentrationsof copper and manganese in velvet bean seeds decreasedsignificantly after irradiation to all used doses. It has beenreported, however, that exposure of plants to UV-B radiationincreased iron concentration in maize plants (Zancan et al., 2006).This fact suggests that iron is essential for various bioprocessesneeded for growth of seedlings and is in good agreement with ourresults obtained with P. ovata. Our study also shows that calciumconcentration in unirradiated seedlings of P. ovata exceeds theconcentration of any other metal. Desai et al. (2006) have foundthat calcium accumulation at the embryo development is higher

0

0.1

0.2

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CGamma dose (Gy)

Chl

orop

hyll-

a in

mg/

gFW

∗∗∗

∗∗∗

∗∗∗

∗∗∗

10 20 50 100

0

0.1

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Gamma

Tota

l chl

orop

hyll

in m

g/g

FW ∗∗

C 10

Fig. 2. Concentrations of chlorophyll a, chlorophyll b and total chlorophyll in seedling

Table 1Comparison of determined and certified values of trace element concentrations in

Apple Leaf Standard.

Trace element Calculated value (ppm) Certified value (ppm)

Cl 618742 579723

K 15124745 160007200

Ca 15862763 150007150

Fe 9876 8375

Mn 6672 5473

Ni 371 170.1

Cu 670.5 670.2

Zn 1371 1370.3

Sr 2373 2572

than at the other developmental stages. A stimulatory effectof various levels of calcium in the medium was observed in Pinus

(Pullman et al., 2003). Calcium plays a very important role in plantgrowth and nutrition (Hepler, 2005). Also, calcium is an importantelement in cellular signaling and mediating plant response toosmotic stress (Sanders et al., 2002).

3.6. Determination of chlorophyll content

In this study, we compared chlorophyll content of seedlingsbefore and immediately after irradiation with 60Co. Figs. 2(a–c)show that all the irradiated seedlings contained less chlorophyll aand 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 totalchlorophyll, respectively. These values are significantly lowerthan chlorophyll content of unirradiated seedlings (0.39 mg/gFW). Moreover, the concentrations of chlorophyll a were higherthan the concentrations of chlorophyll b in both irradiated andunirradiated seedlings. Chlorophyll concentration varied from oneirradiated 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 belower in irradiated plants than in unirradiated ones. This is becausegamma radiation breaks chlorophyll molecules apart (Byun et al.,2002). Gamma irradiation results in various physiological andbiochemical changes in plants. It can damage or modify importantcomponents of cell walls and also disturb enzyme activity, hormonebalance and water exchange. These effects can result in changes inthe structure of plant cells and metabolism processes, such asphotosynthesis, modulation of the antioxidant system and accumu-lation of phenolic compounds. Photosynthetic pigments can bedestroyed by gamma rays with concomitant loss of photosyntheticcapacity (Strid et al., 1990). Kim et al. (2008) reported that the

0

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0.04

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0.08

0.1

0.12

Gamma dose (Gy)

Chl

orop

hyll-

b in

mg/

g FW

C 10 20 50 100

dose (Gy)

∗∗∗

∗∗∗

∗∗∗

20 50 100

s 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 withgamma rays to high doses (Wada et al., 1998).

Furthermore, as mentioned above, the chlorophyll a concentra-tions were found to be higher than the concentrations of chloro-phyll b in both irradiated and unirradiated seedlings ofP. ovata. A number of researchers (Strid et al., 1990; Ling et al.,2008) have already reported that gamma irradiation resultsin greater destruction of chlorophyll b than chlorophyll a. Thispreferential loss of chlorophyll b is due to disturbance ofits biosynthesis or degradation of its precursors (Marwoodand Greenberg, 1996). Byun et al. (2002) found that irradiation to20 kGy destroys chlorophyll b. Chlorophyll decomposition involvesrelease of chlorophyll from its protein complex with subsequentdephytolization and possibly pheophytinization (Simpson et al.,1976). However, the exact mechanism of chlorophyll breakdown isstill unknown.

As iron catalyzes biosynthesis of chlorophyll (Chereskin andCastelfranco, 1982; Spiller et al., 1982; Bollivar and Beale, 1996), itmay favor photosynthesis in leaves. Chouliaras et al. (2004) observedthat, in treatments lacking iron, net photosynthesis was reducedbecause of lower chlorophyll concentrations. Involved in chlorophyllmetabolism, 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 thechlorophyll and heme contents of leaves. There are no reports thatheme enzymes are directly involved in chlorophyll metabolism.However, Bogorad (1960) have found that the porphyrin moieties ofheme and chlorophyll are formed by the same bio-synthetic system.The point is that the chlorophyll and heme syntheses depend on anadequate iron supply. Iron also plays an important role in porphyrinbiosynthesis (Lascelles, 1955; Vogel et al., 1960). It has been found inthe earlier works that the level of activity of heme enzymes of leaftissue and its chlorophyll content are markedly influenced by ironsupply (Evans, 1959; DeKock et al., 1960). Lower activities of theheme enzymes may indirectly affect chlorophyll metabolism. Thecorrelation between chlorophyll and heme contents suggests thatiron chlorosis is an expression of a regulatory influence produced bythe supply of iron on porphyrin synthesis. Our results show thatchlorophyll concentrations in seedlings of P. ovata decreased aftergamma irradiation; however, the concentrations of iron increasedsimultaneously. Gamma radiation breaks the porphyrin ring of thechlorophyll molecule. The porphyrin ring of chlorophyll, with amagnesium atom in its center, is a part of the chlorophyll moleculethat absorbs energy of light. As the metal–porphyrin complex inseedlings of P. ovata has been decomposed by gamma rays, theincreased concentrations of iron could not be used in the synthesisof chlorophyll.

4. Conclusion

The PIXE results showed that gamma irradiation decreasesconcentrations of most of trace elements in P. ovata. The onlyexception is iron, whose concentration increased steadily withradiation dose. This increase in iron concentration did not affectthe chlorophyll metabolism of the cells because the irradiationdecreased the chlorophyll content of the plants by breaking theporphyrin rings.

Acknowledgements

Authors would like to thank Institute of Physics in Bhubanes-war, 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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