Chlorophyll Fluorescence Spectra as an Indicator of X-Ray + EMS-Induced Phytotoxicity in Safflower

The present investigation deals with the study of in vivo laser-induced chlorophyll florescence spectra (LICF) of safflower leaves (Carthamus tinctorius L.) for X-rays + EMS-treated plants. Seeds were treated with different doses of Xray + EMS (5, 8, 12, 25, and 30 Kr + 0.5% EMS) and were grown in the green house. The effects of the concerned treatment on chlorophyll (Chl) contents and Chl fluorescence were investigated after 7 days of germination. Results obtained revealed that the values of Chl contents, intensity of Chl fluorescence spectra, and fluorescence intensity ratio (FIR) F685/F730 are directly correlated with the treatment doses monitored. The treatment sets of 8, 12, and 25 Kr + 0.5% EMS doses showed an increase in FIR and thereby a decrease in the Chl contents. However, the lowest treatment dose of 5 Kr + 0.5% showed a decrease in FIR and thereby an increase in chlorophyll contents. Safflower seeds treated with 30 Kr + 0.5% EMS were proved to be lethal as they showed no germination. Thus, our study demonstrates early detection of chlorophyll damage caused by various physical and chemical mutagens through the application of LICF spectra.


Introduction
Laser-induced fluorescence (LIF) is a powerful tool for plant investigation, and it can illustrate a lot of information about plant health and identity of plants.Leaf pigments emit fluorescence after irradiation with laser light [1].The in vivo chlorophyll fluorescence spectra of plant leaves shows two fluorescence maxima, one in the spectral region near 685 nm and other in the region near 730 nm [2].The shape of the fluorescence spectra and the value of the fluorescence intensity ratio (FIR) up to a great extent depend upon the Chl contents and absorbance of the leaves [3].Fluorescence intensity ratios show a good correlation with pigment contents and pigment ratios [4].The intensity of the red and far-red chlorophyll fluorescence is inversely related to the photosynthetic activity.With the decrease in the photosynthesis owing to various stress conditions, the FIR increases.The increase in chlorophyll content in plants results in a decrease in the value of the FIR.
Safflower is one of the world's oldest oilseed crops that have been grown commercially for edible oil and natural dye sources around the world [5].Safflower petals besides being a source of dye are medicinally important in curing several chronic diseases like hypertension, coronary heart ailments, rheumatism, and male-and female-fertility-related problems [6,7].It is an important alternative plant that can be used to increase edible oil sources.It is a highly tolerant crop that can be safely grown under arid and saline sodic conditions [8,9].
X-rays are nonparticulate electromagnetic radiations with a wavelength of 0.001-10 Å.These are high-energy radiations and consist of photons, that is, small packets of energy.X-rays are produced when very fast moving electrons strike a high-melting-point element like Tungsten in X-ray tubes.X-rays are often referred to as hard (0.001-0.1 Å) or soft (1-10 Å) depending upon their wavelength.X-rays are highly penetrating and sparsely ionizing.Ionizing radiations produce a wide range of effects on DNA through either free radical effects or direct action on DNA.It causes breaks in sugar phosphate backbone of one or both strands of DNA, consequently, leading to the rearrangements through tautomerization, deletions, chromosome loss, and so forth.Mutations are also caused by damage or loss of bases.Sometimes the effect may result in cross-linking of DNA to itself or proteins, breaking of H-bonds of bases, blockage of cell division, organelle failure, or cell death [10][11][12].
EMS is a widely used chemical mutagen that is a nonfunctional agent with one reactive group.It causes ethylation of bases in DNA.EMS is a monofunctional alkylating agent that reacts with DNA at the 7-N and 6-0 positions.Alkylation of ring N causes depurination, which leads to backbone breaks.When 7-ethylguanine is produced, it pairs with thymine to cause G:C → A:T transitions.The lethality of EMS is due to alkylation of proteins.Alkylating agents interact with DNA causing changes in its structure.This may result in the loss, addition, or replacement of bases, thus, altering their sequence in the DNA and affecting the fidelity of the genetic message.Relative frequencies of mutation depend on the reactivity of the agents involved.Deletion and insertion leading to producing frameshift mutations.The inactivating alterations include removal of bases, dimmer formation, cross-linking of the two DNA strands, and single or double strand breaks [13,14].
X-ray and EMS both are highly toxic for plant and animal health.In order to evaluate the mutagenic efficiency of X-rays and EMS on seeds, the present study has been conducted on safflower.Here, we investigate the combined effect of highly toxic chemical mutagen EMS and mutagenic ioning radiation X-ray treatment of seeds on pigment contents and chlorophyll fluorescence response of safflower leaf and to suggest the most appropriate dose for further mutation breeding programs.

Procurement of Seeds and Chemical
Seeds of safflower (Carthamus tinctorius L. var.A1) was obtained from NBPGR, New Delhi, India, and EMS was obtained from Merck, India.

Plant Growth and X-Ray + EMS Treatment
The dry seeds of safflower were exposed to five different doses of X-ray irradiation, that is, 5, 8, 12, 25, and 30 Kr, respectively.X-ray irradiation was delivered at 230 kV for 84 rad/min at room temperature.X-ray-irradiated seeds were presoaked in 0.5% solution of ethylmethane sulphonate (EMS) for 5 hours.Then, the X-ray + EMS-treated seeds of safflower after washing well in running water were sown in 3 replicates with 10 seeds in each pot.Seeds treated with distilled water were kept as control and were also sown in their respective pot simultaneously in greenhouse conditions to a raise the M 1 generation.All the treated sets except for 30 Kr + 0.5% EMS showed germination, which depicts that this dose is lethal for safflower.

Determination of Pigment
Plant leaves (20 mg) from control and X-ray + EMS-treated safflower plants were extracted in 3 mL 80% acetone (v/v, in double distilled water), and the extract was used for the measurement of pigment contents.The pigment contents were determined from the transparent, centrifuged acetone extract solution by measuring the absorbance in the region 380-700 nm using the UV/VIS spectrometer (Perkin Elmer lambda 35).The pigment contents were determined according to the method of Lichtenthaler and Wellburn [15].

Laser-Induced Chlorophyll Fluorescence Spectra
LICF spectra were recorded using computer control Acton 0.5 M triple grating monochromator, Hamamatsu R928 PMT, as a detector, excited with 405 nm violet diode laser (Oxxus CE, made in France, Modal PS-001) light.The beam expander was aligned to obtain 4.0 cm 2 expanded laser light on leaves.The fluorescence radiation was collected on the entrance slit of monochromator.
LICF spectra were recorded in the region of 650-780 nm with 1800 grooves/mm grating blazed at 500 nm wavelength using survey mode of spectra sense software.These spectra were analyzed using GRAMS 32 software with Curve-Fit Array Basic program.Spectral correction was made from the response curve of PMT and grating of monochromator.

Curve Fitting
Interactive nonlinear curve fitting was made using the Levenberg-Marquardt algorithm method.After choosing the Gaussian spectral function, the individual component peaks were selected.Peak widths were adjusted so as to obtain approximately the line shape of the spectrum.It provides a reasonable matching fit of the spectral data with good F-statistics, standard error for peak amplitude, peak center and bandwidth (full width at half-intensity maximum).

Photosynthetic Pigments
Treated the Plants showed better growth than the control plants for 5 Kr + 0.5% EMS treatment as the photosynthetic pigments, that is, Chl a, Chl b, and carotenoid contents were increased by 7.39, 11.36, and 1.30%, respectively, over the control plants (Table 1).Except the dose of 5 Kr + 0.5% EMS, with increasing dose of X-ray + EMS treatment, the leaf Chl contents decreased continuously and that decrease was recorded up to 22.22% for 25 Kr + 0.5% EMS as compared to the control plants (Table 1), whereas carotenoid contents increased continuously for all used treatment doses and this increase was up to 49.35% for 25 Kr + 0.5% EMS.The decrease in the Chl b content was higher in comparison to the Chl a for the doses 8, 12, and 25 Kr + 0.5% EMS; thus the ratio of Chl a/b increased for these doses and it increased maximally up to 27.51% for 25 Kr + 0.5% EMS.The Chl a/b ratio decreased for 5 Kr + 0.5% EMS as the increase in the Chl b was higher at this dose.As the carotenoids contents increased for all used doses, the Chl/Car ratio decreased for all used doses except the 5 Kr + 0.5% EMS because at this dose the increase in the carotenoid contents was lower than the increase in the Chl contents.The decrease in the pigment contents for 8, 12,and 25 Kr + 0.5% EMS doses clearly reflects the effect of mutagenic treatment on safflower plants and obviously the maximum inhibition observed at the maximum dose of treatment with minimum Chl contents (or maximum decrease in the Chl contents).The inhibition response decreases with the decrease in the intensity of treatment doses, and it shows a positive response for 5 Kr + 0.5% EMS dose.Lower value of the Chl a/b indicates the presence of more light-harvesting Chl complexes of LHC2 [16,17], thus we can assume that a lower number of light harvesting Chl complexes in the case of 8, 12, and 25 Kr + 0.5% EMS-treated plants and it decreases with the increase in the dose of treatment, whereas light-harvesting Chl complexes may be increasing for treatment of 5 Kr + 0.5% EMS dose.Carotenoids are essential constituents of Chl-binding proteins in all higher plants and they have two key roles in plants and algae: firstly they absorb light energy for use in photosynthesis, and secondly they protect chlorophyll from photodamage [18].Increase in the carotenoid contents may be due to X-ray + EMS-induced damage in photosystem of plant, which has

Conclusion
The effect of treatment with X-ray + EMS can be distinctly observed by in vivo Chl fluorescence spectroscopy.The intensities of the Chl fluorescence at short-wavelength red fluorescence (near 685 nm) and long-wavelength far-red fluorescence (near 730 nm) and the ratio of fluorescence intensities at 685 and 730 nm (FIR) depend upon the dose of X-ray + EMS treatment.The FIR is the lowest (1.66) in the case of 5 Kr + 0.5% EMS-treated set, which depicts that this dose has biostimulatory effect on plant and thus could be safely employed for breeding purposes as compared to control plants where it was recorded to be 1.79.The applied FIR method has several advantages.It is a nondestructive/in vivo and noncontact/remote sensing technique, and the plant leaves remain intact during the measurement because cutting induces additional stress.Additional measurements can be performed with the same plant at any time.It can be used with chlorophyll-fluorescence LIDAR techniques for remote monitoring of vegetation and damage assessment.

Figure 1 :
Figure 1: Gaussian curve-fitted laser-induced chlorophyll fluorescence spectra of control as well as X-ray + EMS treated safflower plant leaves excited with 405 nm violet diode laser.

Table 1 :
Photosynthetic pigment contents and pigment ratios of control and X-ray + EMS-treated safflower plants.
± Values indicate standard deviation (mean n = 3).The values in parenthesis show percent decrease/increase over control plant.

Table 3 :
Fluorescence intensity ratio (F 685 /F 730 ) of control and X-ray + EMS-treated safflower plants excited by 405 nm violet diode laser.