European Journal of Experimental Biology | Archive - iMedpub

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Pelagia Research Library European Journal of Experimental Biology, 2014, 4(3):595-599

ISSN: 2248 –9215 CODEN (USA): EJEBAU

Effects of seed priming with salicylic acid and ascorbic acid on chlorophyll, carotenoids and anthocyanin content in Matricaria aurea L. under drought stress Farshad Keivan Behjou1*, Roghaieh Holghoomi2, Ehsan Zandi Esfahan3 and Mahdi Ramezani4 1

Faculty of Agricultural Technology and Natural Resources, University of Mohaghegh Ardabili, Ardabil, Iran 2 Faculty of Science, University of Mohaghegh Ardabili, Ardabil, Iran 3 Rangeland Research Division, Research Institute of Forests and Rangelands, Tehran, Iran 4 Science and Research Branch, Islamic Azad University, Tehran, Iran _____________________________________________________________________________________________ ABSTRACT In order to study the effect of salicylic acid and ascorbic acid on chlorophyll, carotenoids and anthocyanin content in Matricaria aurea L. under drought stress condition, a completely randomized experiment with four replications was conducted in plant physiology laboratory of the university of Mohaghegh-e-Ardabili. Results indicated that seed priming with optimum concentration of salicylic acid (150 ppm) and ascorbic acid (50 ppm) in three levels of drought condition (50%, 70%, 100% FC), had significant effect on chlorophyll, carotenoid and anthocyanin content in comparison to control. Key words: Ascorbic acid and Salicylic acid, Anthocyanin rate, Chlorophyll and carotenoid content, Drought stress, Seed priming. _____________________________________________________________________________________________ INTRODUCTION Drought stress is considered to be a moderate loss of water, which leads to stomata closure and limitation of gas exchange [8]. Drought stress is characterized by reduction of water content, diminished leaf water potential and turgor loss, closure of stomata and decrease in cell enlargement and growth. Severe water stress may result in the arrest of photosynthesis, disturbance of metabolism and finally the death of plant [6]. Photosynthetic pigments are important to plants mainly for harvesting light and production of reducing powers. Both the chlorophyll a and b are prone to soil drying [3]. However, carotenoids have additional roles and partially help the plants to withstand adversaries of drought. Salicylic acid (SA) is an endogenous plant growth regulator. It is involved in various physiological processes of plant growth and development [1]. Furthermore, SA significantly enhances plant tolerance to cold, heat, salt, drought and other abiotic stresses, suggesting that it has great agronomic potential in terms of improving the stress tolerance of important plants such as medicinal plants. [2];[11]. Salicylic acid and ascorbic acid and their related components have been reported to induce significant adverse effects in environment stress including drought and salinity [7];[4].

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Farshad Keivan Behjou et al Euro. J. Exp. Bio., 2014, 4(3):595-599 _____________________________________________________________________________ Priming techniques can include the effect of treatment on metabolic, biochemical and enzymatic processes in plant seeds in order to perform biological functions in the best way. Matricaria aurea’s essential oil has many therapy benefits including anti-microbial, anti-inflammatory, anti-allergic, anti-spasmodic and anti-septic properties which are used in the treatment of gastrointestinal disorders. For this reason, Matricaria aurea is one of the important medicinal plants; hence studying the ways to increase its resistance against environmental stresses such as drought stress is one of paramount importance. Pessarkli et al [9]. Stated that the continuation of photosynthesis and maintain of chlorophyll concentration under drought stress is an important physiological tolerance indicator against drought stress. According to the effect of seed priming on different physiological processes specially photosynthesis and the probability of increasing the resistance of primed seeds against drought stress after seedling establishment, the experiment of investigate the effect of seed priming with salicylic acid and ascorbic acid on chlorophylls, carotenoids and anthocyanin contents was conducted on Matricaria aurea’s leaves. MATERIALS AND METHODS Seed treatment: in this research, the seeds of Matricaria aurea, obtained from Pakanbazr Company, were used. Seeds were superficially sterilized with 5% sodium hypochlorite for about three minutes and were washed with distilled water two times. Then, seeds were dried on Whatman papers for about 24 hours and after that seeds were primed with 150ppm of SA and 50ppm of ascorbic acid and were set in room temperature for 48 hours; Afterward, seeds were planted in pots filled with a mixture of red soil, sand and the farmyard manure (FYM) at 2:2:1 ratio. Nine seeds were sown in each pot and the pots were placed in a greenhouse at the Faculty of Science, the University of Mohaghegh-e-Ardabili with a temperature ranging between about 13ºC at night and 27ºC during the day for about three months. After seedling establishment, irrigation treatments were applied: {(50% (I1), 70% (I2), and 100% FC (I0)}. Pots were weighed daily and water added to each pot according to field capacity as deficient amount of water. Experiment 1 To measuring chlorophyll and carotenoid content, 0.2 gram of fresh tissue of plant was taken in a pestle and mortar and homogenized with 15 ml of 80% Acetone. The extract was centrifuged at 4000 rpm for 10 minutes. The supernatant was used for the estimation of chlorophylls and carotenoid content was measured by Spectrophotometer (T80+ UV/VIS Spectrometer) at 470nm, 646.8nm and 663.2nm. [13]. Chlorophyll a=12.25 A663.2 – 2.798 A646.8 Chlorophyll b=21.21 A646.8 – 5.1 A663.2 Chlorophyll T= chlorophyll a + chlorophyll b Carotenoids = [1000(A470) – 1.82 (mg chlorophyll a) – 85.02(mg chl. b)]/198 Experiment 2 To measuring anthocyanin content, 0.1 gram of fresh tissue of plant was taken in a pestle and mortar and homogenized with 10 ml acidic-Methanol (pure Methanol and pure HCL in 99:1 ratio), then extraction of samples was transferred to Falcon tubes and were placed in a dark place for 24 hours in 25ºC. After that the extraction was centrifuged at 4000 rpm for 10 minutes and optical absorbance of supernatants were measured at 550nm [12]. A=εbc (b=Cuvette width, A=optical absorbance, c=samples concentration, ε=extinction coefficient) (ε=33000 1/M*cm) Data were analyzed by SPSS and mean comparisons were made by Duncan's Multiple Range Test at 5% level of probability. RESULTS AND DISCUSSION In this research, seed priming with SA and ASA cause to increasing the Chlorophylls and Carotenoids content in Matricaria aurea’s leaves.

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Farshad Keivan Behjou et al Euro. J. Exp. Bio., 2014, 4(3):595-599 _____________________________________________________________________________ According to the analysis of variance in samples and comparing their means, there was a significant difference between priming samples and control in chlorophyll and carotenoid content, so that this difference between priming samples with SA showed a significant increasing in drought stress conditions levels, indicating that increasing in photosynthetic capacity was towards the increase of plant dry matter. Also, primed samples with ASA in comparison to control had higher chlorophyll and carotenoids content. Moreover, priming with SA caused to decrease in anthocyanin content, probably referring to inhibition in ethylene synthesis [10]. There is probability that ethylene with an effect on anthocyanin and flavonoid’s biosynthetic pathway enzymes, such as phenylalanine ammonia-lyase cause to anthocyanin accumulation in plant [8]. Unlike anthocyanin decreasing, in samples with SA priming, there was an increasing rate in anthocyanin content with increasing in drought stress condition levels. This can be interpreted that anthocyanin in plants acts as free radicals acceptors and protects them against oxidative stresses. Moreover, priming with SA and ASA induces a decrease in abiotic and biotic stresses effects. This can be interpreted that increasing in anthocyanin content with increasing in stress levels, is due to priming with SA and ASA. Table.1 – ANOVA of the studied traits of M.aurea traits Chlorophyll a

Chlorophyll b

Total Chlorophyll

Carotenoid content

Anthocyanine content

Sources of variation Sum of squares df Between groups 143.505 2 Within groups 157.180 33 Total 300.684 35 Between groups 12.736 2 Within groups 66.928 33 Total 79.664 35 Between groups 226.537 2 Within groups 279.549 33 Total 506.087 35 Between groups 4.355 2 Within groups 16.368 33 Total 20.723 35 Between groups 32.060 2 Within groups 31.761 33 Total 63.821 35 * : Significant at 5% level of probability ns: not significant

Mean square 71.752 4.763 3.368 2.028 113.269 8.471 2.177 0.496 16.030 0.962 -

Sig. 0.000 * 0.056 ns 0.000 * 0.020 * 0.000* -

Fig.1 Effect of SA and ASA on Carotenoid content under different stress levels

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Farshad Keivan Behjou et al Euro. J. Exp. Bio., 2014, 4(3):595-599 _____________________________________________________________________________

Fig .2 Effect of SA and ASA on chlorophyll T under different stress levels

F ig .3 Effect of SA and ASA on Anthocyanin content under different stress levels

CONCLUSION SA and ASA have important roles in plants growth and development. According to the obtained results, it can be concluded that seed priming with SA and ASA has positive effects on chlorophylls, carotenoids and Anthocyanin content in Matricaria aurea L. under drought stress. Acknowledgements At last we acknowledge supporting provided by plant biology laboratory of University of Mohaghegh Ardabili and also thank Dr. A. Ghasemian and Dr. B. Esmaiilpour for their accompanies. REFERENCES [1] Cleland, C.F., Journal of Plant physiol., 1974, 54: 899-903. [2] Coronado, M.A.G., C. Trejo-lopez and A. Karque-Saavedra, Journal of Plant physiol Biochem., 1998, 36: 563565. [3] Farooq, M., A. Wahid, N. Kobayashi, D. Fujita and S.M.A. Basra, Plant drought stress: effects, mechanisms and management. Journal of Agronomy for Sustainable Development., 2009, 29: 185–212. [4] Farooq, M., Basra, S.M.A., Wahid, A., Cheema, Z.A., Cheema, M.A., and Khaliq., Physiological role of exogenously applied glycine betaine to improve drought tolerance in fine grain aromatic rice (Oryza sativa L.). Journal of Agronomy and Crop Science., 2008, 12: 54-59. [5] Hyodo, H., Yang, Sh., Journal of Plant physiology., 1997, 47: 765-770. [6] Jaleel, Ch., Azooz, M. M., manivannan, p., panneerselvam, R. American-eurasian journal of botany., 2008, 1: 4652.

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Farshad Keivan Behjou et al Euro. J. Exp. Bio., 2014, 4(3):595-599 _____________________________________________________________________________ [7] Khan, M.A., M.Z. Ahmed and A. Hameed, Journal of Arid Environment., 2006, 67, 535-540. [8] Kimberly, A.S., Picken, M., James, Y., Journal of HortScience., 2003, 38: 101-104. [9] Pessarkli, M., Mlicea, Dekker, 12: 45-49. 1999. [10] Qinghua, S. H., Zhujun, Z., Journal of Environment and Experimental Botany, 2008, 63, 317. [11] Rasmussen, J.B., R. Hammerschmidt and M.N. Zook., Syringae. Journal of Plant physiol, 1991, 97, 13421347. [12] Wagner, G., Content and vacuole extra vacuole distribution of neutral sugars, free amino acids, and anthocyanins in protoplast. Journal of Plant physiology, 1979, 64, 88-93. [13] .Wellburn, A.R. Journal of Plant Physiology, 1983, 144, 307.

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