Estimation of Mda, Total Chlorophyll And

International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in ESTIMATION OF MDA, TOTAL CHLOROPHYLL AND METHIONINE CONTENT IN RELATION TO MALFORMATION DISORDER OF MANGO (Mangifera indica L.) UNDER LOW TEMPERATURE AND HIGH RELATIVE HUMIDITY CONDITION PREVAILING IN DIFFERENT STATES OF NORTH INDIA

1V. RANI, 2G.BAINS

Department of Plant Physiology, College of Basic science and Humanities, G.B Pant University of Agriculture & Technology, Pantnagar-263145, Uttarakhand. E-mail: [email protected]

Abstract - Malformation disorder of mango occurs as a result of surge of ethylene synthesis in plants following biotic and abiotic stresses like pathogen attack, chilling, high relative humidity etc. The increased level of ethylene brings about physio- biochemical alteration in plants. In malformed tissue samples of five commercial mango varieties collected from different states of north India recorded an increase in MDA (malondialdehyde) and methionine while a reduced content of chlorophyll.

Keywords - Mango malformation, MDA, methionine, total chlorophyll, stress ethylene, low temperature, high R.H

I. INTRODUCTION climatic statistics pertaining to maximal and minimal temperature, RH and Wind velocity, have been Mango (Mangifera indica L.) is an important fruit gathered for a year i.e. 2016, for the months of crop of tropical and subtropical areas of the world. It February and March (during flower initiation to is extensively cultivated in many mango growing flowering period). Five varieties of mango namely provinces, but due to a deadly disorder of mango Amrapali, Dasheri, Langra, Chausa and Bombay known as malformation, its productivity has dropped Green were selected. The experimental material used to a meager value, causing heavy economic losses on was healthy and malformed leaf tissues and was global basis (NHB, Indian Horticulture Database, collected from Mango orchards of Pantnagar, 2011). Uttarakhand; Ranchi, Jharkhand; New Delhi, The malady appears in two forms viz, vegetative and Allahabad, Uttar Pradesh and Patna, Bihar. floral. In vegetative malformation there is multi branching of shoot apex with scaly leaves leading to Malondialdehyde (MDA) Content Bunchy Top or Witch’s broom (Bhatnagar and Procedure Beniwal, 1977). Floral malformation shares the same The amount of MDA produced by thiobarbituric acid symptom of multi branching of rachis and bears reaction was used to determine lipid peroxidation, by mostly male flowers and rarely bisexual. In method given by Heath and packer (1968), 0.2 gm of malformed panicles flowers are sterile and thus bear fresh sample was homogenized in 3mL 0.1% TCA. no fruits, leading to yield loss (Schlosser, 1971). The centrifugation of homogenized mixture was done With a lot of controversies over its etiology such as at 10,000 rpm for 10 min. 1.2mL of (0.5% TBA in fungi (Crespo and Cazorla, 2012), virus (Satter, twenty percent TCA) was added to 0.3mL 1946), mites (Sternlicht and Goldenberg, 1976) and supernatant and it was then incubated in water bath physiological factors (Ansari et al., 2013), most of for 30 min at 950C. Ice was used to terminate the the scientific communities have expressed their reaction. Absorbance was read at 532 and 600 nm. reliance on the fact that surge of ethylene produced in The concentration of MDA was estimated using the the plant elicited due to several biotic and abiotic extinction coefficient of 155mM-1cm-1, after stresses is the main causal agent of malformation. As subtracting the non specific absorbance at 600 nm a result of stress condition , several biochemical compounds accumulate in plants. The present study Chlorophyll Content was undertaken to estimate the amount of MDA, total Procedure chlorophyll and methionine in abiotic stressed plants Chlorophyll content was determined in leaves by a in five states of northern India. method described by Hiscox et al. (1979). For this 50 mg of finely chopped leaf tissues were taken in test II. MATERIAL AND METHODS tubes. 10 mL of dimethyl sulfoxide was added to each test tubes and it was incubated at 650C for three hours The study of weather parameters was conducted in in an oven. Incubation of three hours was then five states of India namely Bihar, Jharkhand, Uttar followed by determining the absorbance of DMSO Pradesh, Uttarakhand and Delhi. For these location

Estimation of MDA, Total Chlorophyll and Methionine Content in Relation to Malformation Disorder of Mango (Mangifera indica L.) under Low Temperature and High Relative Humidity Condition Prevailing in Different States of North India

75 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in containing chlorophyll at 663 nm and 645 nm using a fresh weight of leaf tissues in Bihar, Jharkhand, spectrophotometer. Pure DMSO was used as blank. Uttar Pradesh, Uttarakhand and Delhi, respectively.

Methionine Content Similarly in the healthy vegetative tissue of cultivar, Procedure Dasheri, it was found to be 62.23, 61.24 , 55.75 , Methionine content was determined by method 53.25 and 55.25 mg phenol/ 100 g of leaf tissues in prescribed by Horn et al. (1946), with some Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and modifications. 0.5 gms deffated samples were Delhi respectively and that in malformed vegetative weighed in fifty millilitre conical flasks. Six milli tissue was 82.25 , 75.25 , 78.15, 78.25 and 66.28 litre of 2N hydrogen chloride was added and was mgphenol/ 100 g of leaf tissues in Bihar, Jharkhand, autoclaved at 15lb pressure for 1 hr and small amount Uttar Pradesh, Uttarakhand and Delhi , respectively. of activated charcoal was mixed and the hydrosylate In the healthy vegetative tissue of Langra it was 53.25 was boiled and washed with warm water. The filtrate , 70.16 , 56.93 , 60.75 and 56.75 mg phenol/ 100 g was neutralized with 10N sodium hydroxide to pH of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, 6.5. The volume was made to 50 ml with water after Uttarakhand and Delhi, respectively, and that in lowering to air temperature and 25 ml of it was malformed vegetative tissue was 67.60 , 69.10 , 95.28 transferred to 100ml conical flask. 3ml of ten percent , 94.68 and 92.47 mg phenol/ 100 g of leaf tissues in sodium hydroxide was added to it followed by 0.15ml Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and sodium nitroprusside (10%). After 10 min 1ml of Delhi, respectively. glycine (3%) solution was added. After another 10 min 2ml of orthophosphoric acid was added and Similarly, in the healthy vegetative tissue of cultivar, shaken vigorously. Intensity of red color after 10 min Chausa it was found to be 71.80 , 52.25, 61.72 , was read at 520 nm against a blank prepared in the 61.82 and 58.73 mg phenol/ 100 g of leaf tissues in same way without nitroprusside. Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, respectively, and that in malformed Statistical analysis vegetative tissue was 82.25 , 75.25 , 78.15, 78.25 and 66.28 mg phenol/ 100 g of leaf tissues in Bihar, The data were analyzed statistically by two factor Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, Randomized Block Design (RBD). respectively. In the healthy vegetative tissue of Bombay Green it was 57.00 , 58.60 , 70.96 , 72.96 III. RESULTS and 61.85mg phenol/ 100 g of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, The study of weather parameters in five states of respectively, and that in malformed vegetative northern India revealed that in the entire region tissue was 76.00 , 78.00 , 94.10 , 69.45 and 76.02 during flower initiation to flowering period mg phenol/ 100 g of leaf tissues in Bihar, Jharkhand, (February- March) the weather variables were highly Uttar Pradesh, Uttarakhand and Delhi, favorable for malformation disorder of mango. Low temperature (maximum and minimum), high relative Total chlorophyll content (mg g-1 fw) in humidity and feeble wind favored the occurrence of malformed and healthy leaf tissue samples malformation (Table 10). The total chlorophyll content in healthy and malformed tissues was estimated over two months Malondialdehyde content ( mol g-1 fresh weight) (February-March) during flower initiation to in malformed and healthy leaf tissue samples flowering period in Amrapali, Dasheri, Langra, Malondialdehyde content in healthy and malformed Chausa and Bombay Green cultivars of mango in tissues was estimated over two months (February- different states (Table 2 and Fig 2). March) during flower initiation to flowering period in Amrapali, Dasheri, Langra, Chausa and Bombay As may be observed, the Total chlorophyll content in Green cultivars of mango in different states (Table 1 malformed tissue was lower as compared to healthy and Fig 1). tissue in all the cultivars in different states. Total chlorophyll content in healthy vegetative tissue of As may be observed, the Malondialdehyde content in Amrapali was 3.15, 3.15, 3.12, 3.12 and 3.12 mg g-1 malformed tissue was higher as compared to healthy fw of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, tissue in all the cultivars in different states. Uttarakhand and Delhi, respectively and that in Malondialdehyde content in healthy vegetative tissue malformed vegetative tissue was 2.34, 2.35, 2.51, of Amrapali was 0.114 , 0.116 , 0.115 , 0.121 and 2.48 and 2.35 mg g-1 fw of leaf tissues in Bihar, 0.117  mol g-1 fresh weight of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, respectively. respectively and that in malformed vegetative tissue was 0.436 , 0.475 , 0.425 , 0.417 , 0.435  mol g-1 Similarly in the healthy vegetative tissue of cultivar, Dasheri, it was found to be 3.17, 3.32, 3.24, 3.15 and

76 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in 3.15 mg g-1 fw of leaf tissues in Bihar, Jharkhand, In the healthy vegetative tissue of Langra it was Uttar Pradesh, Uttarakhand and Delhi respectively 64.72, 64.35, 66.34, 63.00 and 62.15 g g-1 fw of and that in malformed vegetative tissue was 2.38, leaf tissues in Bihar, Jharkhand, Uttar Pradesh, 2.74, 2.63, 2.51 and 2.45 mg g-1 fw of leaf tissues in Uttarakhand and Delhi, respectively, and that in Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and malformed vegetative tissue was 100.72, 99.95, Delhi respectively. In the healthy vegetative tissue of 100.75, 96.00 and 100.02 g g-1 fw of leaf tissues in Langra it was 3.20, 3.11, 3.11, 3.18 and 3.17 mg g-1 Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and fw of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, Delhi, respectively Uttarakhand and Delhi, respectively, and that in In the healthy vegetative tissue of Chausa it was malformed vegetative tissue was 2.97, 2.15, 2.72, 63.89, 63.61, 65.42, 65.22 and 63.15 g g-1 fw of -1 2.75 and 2.57 mg g fw of leaf tissues in Bihar, leaf tissues in Bihar, Jharkhand, Uttar Pradesh, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, Uttarakhand and Delhi, respectively, and that in respectively . malformed vegetative tissue was 98.72, 100.21, 99.45, 100.25 and 99.65 g g-1 fw of leaf tissues in Similarly, in the healthy vegetative tissue of cultivar, Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Chausa it was found to be 3.30, 3.35, 3.443.15 and -1 Delhi, respectively 3.13 mg g fw of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, respectively, In the healthy vegetative tissue of Bombay green it and that in malformed vegetative tissue was 2.48, -1 -1 was 62.76, 62.74, 62.15, 63.10 and 66.70 g g fw 2.60, 2.80, 2.45 and 2.40 mg g fw of leaf tissues in of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Uttarakhand and Delhi, respectively, and that in Delhi respectively. In the healthy vegetative tissue of malformed vegetative tissue was 95.34, 96.35, 95.26, Bombay Green it was 3.18, 3.11, 3.12, 3.17 and 3.16 -1 -1 99.72 and 99.50 g g fw of leaf tissues in Bihar, mg g fw of leaf tissues in Bihar, Jharkhand, Uttar Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, Pradesh, Uttarakhand and Delhi respectively, and respectively. that in malformed vegetative tissue was 2.53, 2.25, -1 2.56, 2.78 and 2.45 mg g fw of leaf tissues in Bihar, IV. DISCUSSION Jharkhand, Uttar Pradesh, Uttarakhand and Delhi, respectively In an another finding, higher content of

-1 malondialdehyde was recorded in maize genotypes Methionine content (g g fresh weight) in under water stress condition as compared to control malformed and healthy leaf tissue samples ones (Helal Ragab Moussa and Samir Moustafa The Methionine content in healthy and malformed abdel-Aziz, 2008). Increase in MDA content under tissues was estimated over two months (February- stress condition advocates that stress causes harm to March) during flower initiation to flowering period in membrane, due to lipid peroxidation by means of Amrapali, Dasheri, Langra, Chausa and Bombay ROS (Sairam et al., 2000). Drastic increase in free Green cultivars of mango in different states (Table 3 radicals leads to leaky membrane which causes lipid and Fig 3). peroxidation (Smirnoff, 1993). Result of a finding suggested that salinity stress affects lipid peroxidation As may be observed, the Methionine content in in leaves and roots of groundnut (Jain et al., 2001) malformed tissue was higher as compared to healthy tissue in all the cultivars in different states. Stability of chlorophyll depends on the stability of Methionine content in healthy vegetative tissue of membrane. Being a membrane bound pigment, Amrapali was 62.65, 62.11, 63.14, 62.15 and 62.15 chlorophyll is lost due to breakdown of membrane -1 g g fw of leaf tissues in Bihar, Jharkhand, Uttar under stress condition. Under salinity stress Pradesh, Uttarakhand and Delhi, respectively and that condition, reduction in chlorophyll content was in malformed vegetative tissue was 94.35, 95.75, observed (Molazem et al., 2010). In an another -1 96.00, 95.00 and 95.62 g g fw of leaf tissues in finding it was reported that there is decrease in Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and chlorophyll content under stress condition (Ommen et Delhi, respectively. al., 1999). In an another study, large decline in the content of chlorophyll a, chlorophyll b and total Similarly in the healthy vegetative tissue of cultivar, chlorophyll was reported under water stress condition Dasheri, it was found to be 65.35, 63.72, 62.81, 62.82 in Sunflower (Manivannan et al., 2007). Similar and 60.70 g g-1 fw of leaf tissues in Bihar, result was observed in genotypes of bread wheat Jharkhand, Uttar Pradesh, Uttarakhand and Delhi under lead stress condition as compared to normal respectively and that in malformed vegetative tissue ones (Awaad et al., 2010). -1 was 2.38, 2.74, 2.63, 2.51 and 2.45 mg g fw of leaf tissues in Bihar, Jharkhand, Uttar Pradesh, As methionine is precursor of ethylene, increase in its Uttarakhand and Delhi respectively content is an indication of stress in plant tissues

77 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in (Yang and Adams, 1980). Leaves and fruits under [7] Heath, R.L.; and Packer, L. (1968). Photo peroxidation in stress condition liberates ethylene (Yang and isolated chloroplasts. I. kinetics and stoichiometry of fatty acid peroxidation. Archives in biochemistry and biophysics. Hoffman, 1984). In a similar study high level of 125:189-198. ethylene was reported in malformed tissues of mango [8] Hiscox, J.D; and Israelstam, G.F.(1979). A method for the , as compared to healthy ones (Singh and Dhillon, extraction of chlorophyll from leaf tissue without maceration. 1990). Similarly (Bains et al., 2003) reported high Canadian Journal of Botany. 57:1332-1334. [9] Horn, M.J.;Jones B.; and Blum, A.E (1946). Calorimetric ethylene level in malformed panicles and shoots determination of methionine in protein and food. Journal of bearing them at all developmental stages than Biological chemistry. 166:313-320. healthier ones. [10] Jain, M.; Mathur, G.; Koul, S.; and Sarin, N. (2001). CONCLUSION Ameliorative effects of proline on salt stress-induced lipid peroxidation in cell lines of groundnut (Arachis hypogaea L.). Plant Cell Reports. 20:463-468. The data recorded in present study shows that low [11] Manivannan, P.; Abdul C.; Sankar, B.; Koshorekumar, A.; temperature and high R.H prevails in northern belt of Somasundaram, R.; Lakshmanan, G.M.A.; and India in the months of February and March during Panneerselvam, R. (2007). Growth, biochemical modifications and proline metabolism in Helianthus annuus flower initiation and flowering stages in mango. L. as induced by drought stress. Colloids and Surfaces B: These weather parameters are known to cause Biointerfaces, 59: 141-149. malformation disorder in the crop. The analysis of [12] Molazem, D.; Qurbanov, E.M.; and Dunyamaliyev, S.A. malformed vegetative tissues collected from different (2010). Role of Proline, Na and Chlorophyll Content in Salt Tolerance of Corn (Zea mays L.). American Eurasian J. northern states revealed rise in the content of MDA Agric. & Environ. Sci., 9 (3): 319-324. and methionine This is an indicator of stress in plants [13] Moussa, H.R.; and Abdel-Aziz, S.M. (2008). Comparative as MDA is produced due to peroxidation of lipids and response of drought tolerant and drought sensitive maize methionine being precursor of ethylene leads to its genotypes to water stress. Australian Journal of crop science. 1(1):31-36. synthesis which is an indicator of stress in plants. [14] NHB (2011).Indian horticulture database, 2011. National Besides this, reduced content of total chlorophyll Board of Horticulture,India. recorded, indicates the breakdown of membranes due (http://assamagribusiness.nic.in/database 2011.pdf). to stressed condition. [15] Ommen, O.E.; Donnelly, A.; Vanhoutvin, S.; Van Oijen, M.; and Manderscheid, R. (1999). Chlorophyll content of spring

wheat flag leaves grown under elevated Co2 concentrations REFERENCES and other environmental stresses within the ESPACE-wheat project. Eur. J. Agron., 10: 197-203. [1] Adams, D.O.; and Yang, S.F. (1981). Ethylene the gaseous [16] Sairam, R.K.; Srivastava, G.C.; and Saxena, D.C. (2000). plant hormone: mechanism and regulation of biosynthesis. Increased antioxidant activity under elevated temperature: a Trends in Biochemical Sciences.6:161-164. mechanism of heat stress tolerance in wheat genotypes. Biol. [2] Ansari, M.W.; Bains, G.; Shukla, A.; Pant, R.C.; and Tuteja, Plant. 43: 245-251. N. (2013). Low temperature stress ethylene and not [17] Sattar, A. (1946). Diseases of mango in Punjab. Punjab Fruit Fusarium, might be responsible for mango malformation. J. 10:56-58. Plant physiol. and Biochem.1-5. [18] Schlosser, S.E. (1971). Mango malformation: Symptoms, [3] Awaad, H.A.; Youssef, M.A.H.; and Moustafa, E.S.A. occurrence and varietal susceptibility. FAO. Plant Prolect. (2010). Identification of genetics variation among Bread Bull.19:12-14. Wheat Genotypes for Lead Tolerance Using Morpho- [19] Singh, Z.; and Dhillon, B.S. (1990). In vivo role of Indole-3- physiological and Molecular Markers. Journal of American acetic acid, gibberellic acid, zeatin, abscisic acid and ethylene Science 6: 10. in floral malformation of mangifera indica L. J. Phytopathol. [4] Bains, G.; and Pant, R.C. (2003). Mango malformation: 128:235-245. etiology and preventive measures. Physiol. Mol. Biol. Plants. [20] Smirnoff, N. (1993). The role of active oxygen in the 9:41-61. response of plants to water deficit and dessication. New [5] Bhatnagar, S.S.; and Beniwal, S.S. (1977).Involvement of phytologist. 125:27-58. Fusarium oxysporum in causation of mango malformation. [21] Sternlicht, M.; and Goldenberg, S. (1976). Mango eriophyid Plant Dis Rep.,61:894-898. mites in relation to inflorescence. Phytoparasitica. 4:45-50. [6] Crespo, M.; And Cazorla, F.M. (2012). First Report of [22] Yang, S.F.; and Hoffman, N.E. (1984). Ethylene biosynthesis Mango Malformation Disease caused by Fusarium and its regulation in higher plants. Annual review of plant mangiferae in Spain. Plant Dis.96:286. physiology, 35(1):155-189.

Table 10: Meterological data of five states (Bihar, Jharkhand, Uttar Pradesh, Uttarakhand and Delhi) of northern India.

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Table 1. Malondialdehyde (MDA) content ( mol g-1 fw) in malformed and healthy leaf tissues of different mango varieties from different states

Fig 1: Malondialdehyde content ( mol g-1 fw) in malformed and healthy leaves of different mango varieties from different states

79 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in

Table 2: Total chlorophyll content (mg g-1 fw) in malformed and healthy leaf tissues of different mango varieties from different states

Fig 2: Total chlorophyll content (mg g-1 fw) in malformed and healthy leaves of different mango varieties from different states

80 International Journal of Advances in Science Engineering and Technology, ISSN: 2321-9009 Volume-5, Issue-2, Aprl.-2017 http://iraj.in

Table 3: Methionine content (g g-1 fw) in malformed and healthy leaf tissues of different mango varieties from different states

Fig 3: Methionine content (g g-1 fw) in malformed and healthy leaves of different mango varieties from different states

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