Summer Project Report

Study of MDA (malondialdehyde) as abiotic stress marker in CSV-17 variety of Sorghum bicolor.

Submitted in partial fullfilement of the requirement for B.Tech. Biotechnology Semester VII

AMITY INSTITUTE OF BIOTECHNOLOGY AMITY UNIVERSITY RAJASTHAN JAIPUR 2011

Supervised by: Dr Ajit Kumar Sr. Research Officer S.P. Institute of Biotechnology, Jaipur

Submitted by: Ravi Pareek

DECLARATION

I hereby declare that the project report entitled “Study of MDA (malondialdehyde) as abiotic stress marker in CSV-17 variety of Sorghum bicolor” is a record of the work compiled by me under the supervision and guidance of Dr. Ajit Kumar, S.P. Institute of Biotechnology, Jaipur as a part of my 45 days summer training.

Ravi Pareek (B.TECH-BIOTECHNOLOGY) (AUR0821094)

ACKNOWLEDGEMENT

First of all with due regard to my respective god with whose kindness and blessing we could be able to accomplish the task of training. Mr. Sourabh Pareek, for his kind permission to allow me to undergo my major project at S. P. Institute of Biotechnology, Jaipur. I am overwhelmed with rejoice to take this opportunity to evince my profound sense of reverence and gratitude to my esteemed supervisor respective Dr. Ajit Kumar, for giving his regular advice and excellent suggestion which have helped us for completing the study. His regular assistance and guidance really helped me to bring formidable task in successful manner. Sincere thanks to Dr. Sonali Jana and Dr. Neha Upreti for their cooperation. I express heartiest and sincere thanks to S. P. Institute of Biotechnology, Jaipur for providing all necessary facilities without which I could not have completed my study.

Ravi Pareek (B.Tech- Biotechnology)

ABBREVATIONS:CAT – Catalase CSH - Coordinated Sorghum Hybrid CSV – Coordinated Sorghum Variety DNA - Deoxyribo Nucleic Acid GSH – Glutathione HgCl2 – Mercuric Chloride hrs. - Hours IS – Inbreed Sorghum LP – Lipid Peroxidation LTS – Low Temperature Stress MDA – Malondialdehyde ml – millilitre mM – Milimolar mol – moles NADP – Nicotinamide Adenine Dinucleotide Phosphate nm – nanometre PRX – Peroxidase PUFA‟s – Poly Unsaturated Fatty Acids SSV – Sweet Sorghum Variety TBA – Thiobarbuteric Acid

TCA – Trichloroacetic Acid TOH – Tocopherol µl – micro litre

List of Tables and Figures:Content Table 1 Table 2 Table 3 Table 4 Graph 1 Graph 2 Picture 1 Picture 2 Picture 3 Picture 4 Picture 5 Picture 6 Picture 7 Picture 8 Page No. 27 31 34 37 31 37 25 25 26 26 33 33 39 39

CONTENTS

Topic 1. Introduction 2. Aims and objectives 3. Review of Literature 4. Material and Methods 5. Results 6. Discussion 7. Conclusion 8. References

Page No. 1-7 8 9-17 18-26 27-39 40-42 43 44-46

INTRODUCTION

Sorghum (Sorghum bicolor L.) is one of the stable crops grown in arid and semi-arid countries. It is the fifth most important cereal crops grown on 44 million ha in 99 countries in Africa, Asia and the Americans. The majority of sorghum plantings are concentrated in poor countries where it constitutes a valuable source of grains for human consumption. In addition, posses high nutritional source of animal feeding.
(6)

There are many varieties available of

Sorghum Bicolor like IS 3566 , SPV 475 , CSV 13 , CSV 15 , CSV 17 , CAV 112 , IS 348 , APK 1 , TNS 340 , TNS 587 TNS 334 and many more . India is the second largest producer of this plant. And this plant ranked 5th in world in overall production (5,11,24). Sorghum exhibit excellent tolerance and yield potential to environmental stresses such as water shortage and salinity compared to millet. In such regions, salinity is impose a limiting factor for crop production, where osmotic stress, ion toxicity and mineral deficiencies are all considered as consequence of the effect of salt stress on plant growth and performance. Abiotic stresses lead to oxidative stress through increase in the production of Reactive Oxygen Species (ROS). These species are toxic and cause damage to DNA, proteins, lipids, chlorophyll and almost every other organic constituent of the living cells. (11) Abiotic and biotic stresses cause alterations in the normal physiological processes of all plant organisms, including the economically important crops. Plant damage and decrease in their productivity take place most often due to naturally occurring unfavorable factors of the environment (natural stress factors) - extreme temperatures; water deficit or abundance; increased soil salinity; high solar irradiance; early autumn or late spring ground frosts; pathogens etc. (10)

In this regard, there are many important adaptive mechanisms that plants use to cope with the adverse effects of salinity. Synthesis of compatible solutes such as: amino acid (proline), sugar alcohols (mannitol) and quaternary ammonium (glycinebetaine) that retain water within cells to combat from dehydration is one of these mechanisms. The seedlings of two forage sorghum to 0 and 100 mM of NaCl and suggested that proline accumulation is an expression of the plant reaction to the stress damage and not a salt tolerance factors. On the other hand, stated the role of glycinebetaine (GB) under a variety of unfavourable conditions. It has been shown that high concentration alleviate salt-induced destabilization of DNA helices and maintain the activity of enzymes when plants experiencing extremes of pH, high temperature and salt concentration.
(11)

Salt stress is a limiting factor of plant growth and yield, and becoming a serious problem in the world. An estimation shows that about one-thirds of irrigation sections are either saline or alkaline. Salinity is more severe in China where only the area of tidal flat is over 20,000 km 2 (China Statistical Yearbook 22). Better understanding of the mechanisms that enable plants to adapt to salt stress and maintain growth would help in the selection of stress tolerant cultivars for exploiting tidal flats. The deleterious effects of salinity on plant growth are associated with low osmotic potential of soil solution, nutritional imbalance, specific ion effect, or a combination of these factors. As a result, membrane disorganization, increase in activated oxygen species production and metabolic toxicity occur. The degree to which each of these factors affects growth depends on the plant genotype and environmental conditions. Osmotic adjustment in plants subjected to salt stress can occur by the accumulation of high concentrations of either inorganic ions or low molecular weight organic solutes. (12)

Plant species differ greatly in their ability to develop cold-tolerance through a process known as cold acclimation. Biochemical changes that have been associated with cold-acclimation include alterations in lipid composition, increased sugar and soluble protein content, expression of specific proteins, the appearance of new isozymes and so on. In this respect, enhancement of cold-tolerance of species would be of considerable interest for preventing cold damage. Therefore figuring out the mechanism of cold-acclimation of species is of a great importance even on the cultivar basis. (10)

The over production of active oxygen species such as superoxide, hydrogen peroxide result from the exposure of the plants to different environmental stimuli e.g. drought stress. Increased formation of active oxygen specie has been associated with the development of injury symptoms resulting from diverse stress conditions. (1)

Low temperature stress (LTS) is the important environmental factors that limit plant growth and productivity. Seedling establishment is a critical process to plant growth, especially under adverse environmental conditions. Seedlings adapt to stress environment by different mechanisms, including changes in morphological and developmental pattern as well as physiological and biochemical processes. Adaptation is associated with maintaining osmotic homeostasis by metabolic adjustments that lead to the accumulation of metabolically compatible compounds such as soluble sugar, malondialdehyde (MDA) and proline. It also

includes modification of related enzyme activity and cell membrane stability. Moreover, it is well known that plant structural modifications and growth pattern adjustments are useful indices of the consequences of stress environment. Therefore, plants adapt to low temperature by mediations of these substances. (5)

Both cold and drought exert common effects on plants, such as alteration of osmotic potential and accumulation of reactive oxygen species (ROS), which may induce an arsenal of analogous stress responses in plants It is well documented that plants have evolved a

multitude of physiological, biochemical, and molecular mechanisms enabling them to adapt to or tolerate harsh abiotic stresses. (15) It has been shown that under stress conditions, MDA (malondialdehyde) accumulation takes place in plants due to membrane lipid peroxidation. (5)

Lipid Peroxidation :Lipid peroxidation (LP) can be defined as the oxidative deterioration of lipids containing a number of carbon-carbon double bonds. A large number of toxic by-products are formed during LP. These have effects at a site away from area of their generation. Hence they behave as toxic „second messengers‟. Membrane lipids are particularly susceptible to LP. Since membranes form the basis of many cellular organelles like mitochondria, plasma membranes, endoplasmic reticulum, lysosomes, peroxisomes etc. The damage caused by LP is highly detrimental to the functioning of the cell and its survival. Presence of polyunsaturated fatty acids (PUFAs) in the phospholipids of the bilayer of biological membranes is the basis of their critical feature of fluidity. Since LP attacks the components that impart these properties, it affects the biophysical properties of membranes. LP decreases the membrane fluidity, changes the phase properties of the membranes and decreases electrical resistance. Also, cross-linking of membrane

components restricts mobility of membrane proteins. Peroxidative attack on PUFAs of a biological membrane will compromise one of its most important functions: its ability to act as barrier. Hence, LP causes lysosomes to have a decreased „latency‟ i.e., they become fragile or simply „leaky‟. Similarly, the leakage of cytosolic enzymes from whole cells e.g. peroxidative attack on the plasma membrane of hepatocytes causes extensive damage such that molecules as large as enzymes are able to leak out.

Peroxidation is well known to decrease activities of enzymes associated with membranes. The most extensively studied examples are enzymes of the endoplasmic reticulum, glucose-6-phosphatase and cytochrome P4501,3-5.Mitochondrion and Golgi apparatus

are also susceptible to LP. Membrane LP can result in inactivation of membrane pumps responsible for maintaining ionic homeostasis. Some enzyme activities can also be stimulated by LP e.g., increase in activity of phospholipase A2 in membranes subjected to oxidative stress, to remove and replace toxic LP products in the membrane 2,6-8. Lipid

peroxidation has been implicated in the pathogenesis of a number of diseases and clinical conditions. These include premature birth disorders, diabetes, adult respiratory distress syndrome, aspects of shock, Parkinson disease, Alzheimer disease, various chronic inflammatory conditions, ischemia- reperfusion mediated injury to organs including heart, brain, and intestine, atherosclerosis, organ injury associated with shock and inflammation, fibrosis and cancer, preeclampsia and eclampsia, inflammatory liver injury, type 1 diabetes, anthracycline-induced cardiotoxicity, silicosis and pneumoconiosis . Experimental and clinical evidence suggests that aldehyde products of LP can also act as bioactive molecules in physiological and pathological conditions. These compounds

can effect and modulate, at very low and non-toxic concentrations, several cell functions including signal transduction, gene expression, cell proliferation and more generally the response of target cells.(8)

Many of the products of LP are not overtly toxic or are

minor

products. Of major

toxicological interest are malondialdehyde (MDA), 4-hydroxynonenal (4- HNE) and various 2-alkenals. A range of alterations is known to occur upon exposure of DNA to lipid hydroperoxide (LOOH). Incubation of plasmid DNA with auto-oxidized unsaturated fatty acids (linoleic or arachidonic), results in extensive single and double strand breaks. Such strand breaks have also been detected in human lymphocytes and fibroblasts after treatment with LOOH. Exposure of calf thymus to LOOH damaged its DNA (C-8 hydroxylation of guanine residues). A spectrum of SupF mutations was produced upon error prone replication of p2189 plasmids, following treatment with autoxidized rat microsomal lipids13-15.
(8)

Products of lipid peroxidation
Oxygen- dependent deterioration of lipids, known as rancidity, has been noticed since antiquity as a major problem in the storage of oils. It was also found useful as far back as the 15th century in preparing oil paints and printing inks. The same oxidation process also occurs in the case of natural products such as fats, oils, dressings or margarines and also chemical and industrial products, such as inks, paints, resins, varnishes or lacquers. Much information concerning the mechanism of the auto-oxidation of lipid compounds has been obtained by the study of the oxidation of simple non-fatty products such as cyclohexene. Since the early 1960's, our understanding of the oxidation of

unsaturated lipids has advanced considerably as a result of the application of new analytical tools. Several research groups initiated detailed studies on the products of polyunsaturated fatty acids in the 70's to reveal more complex aspects of LP. With the help of HPLC, several hydroperoxide products could be separated after auto-oxidation of arachidonic acid.

The first demonstration of free radical oxidation of membrane phospholipids was given in 1980. LOOHs are non-radical intermediates derived from unsaturated fatty acids, phospholipids, glycolipids, esters and cholesterol. Their formation occurs in enzymatic or non-enzymatic reactions involving activated chemical species known as reactive oxygen species (ROS) that are responsible for toxic effects in the body via various types of tissue damage. The major products, LOOHs, are fairly stable molecules at physiological temperatures. Transition metal complexes catalyzed their decomposition. A reduced iron compound can react with LOOH in a way similar to H2O2 (Fenton reaction) causing fission of an O-O bond to form an alkoxyl radical. This radical also promotes the chain reaction of LP. MDA can be formed as the result of the fission of cyclic endoperoxides. With Thiobarbuteric acid (TBA), MDA readily forms an adduct. It can be measured spectrophotometrically by its characteristic pink color.

Process of lipid peroxidation
The steps involved in iron-induced reaction below. LP are described below and shown schematically in

1. 1a. 2.


LH + X L + XH LH + active-Fe L + inactive-Fe
2+ 3+

L + O2 LOO + LH LOOH+Fe


2+

LOO L + LOOH LO +Fe
3+

3. 4. 5. 6. 6a. 7.

LOOMDA and nonenal LOO + TOH LO + TOH  Inactive-Fe 
3+

LOOH + TOH LOH + TOH active-Fe
2++

+ NADPH (ascorbate)+ NADP (ascorbate-ox)

+

A peroxidative sequence is initiated by the attack of
2+

an unsaturated lipid (LH) by any

species (X or active-Fe ) that abstracts hydrogen atom from a methylene group (CH2). This leaves behind an unpaired electron on the carbon atom (reaction 1). Methylene groups adjacent to double bonds are particularly susceptible to attack, since their presence weakens the adjacent carbon-hydrogen bond. The resultant carbon radical (CH) is stabilized by molecular rearrangement to produce a conjugated diene. It can readily react with an oxygen molecule forming lipid peroxyl radical (LOO) (reaction 2). These radicals can abstract hydrogen atoms from other lipid molecules (LH) to become LOOH. When L is formed from second LH (reaction 3), LP is propagated. LOOH can further be degraded by a Fenton-type reaction in presence of Fe
2+

to another radical LO

(reaction 4). LOOis unstable and breaks down to form various products including aldehydes, such as malondialdehyde (MDA) and 4-hydroxy- nonenal (reaction 5). MDA and related aldehydes are the most commonly estimated products of lipid peroxidation.

Peroxidation can be terminated by a number of reactions. The major one involves the reaction of (reaction 6) or LO (reaction 6a) with antioxidants. Most effective of these is membrane-based  tocopherol (TOH, vitamin E) forming more stable tocopherol phenoxyl radical that can be „recycled‟ by other cellular antioxidants, such as ascorbate (vitamin C) or glutathione (GSH). Oxidation of NADPH in enzyme systems, and of

ascorbate in non-enzymic reaction regenerates the active-Fe (reaction 7). (8)

2+

Estimation of Product of Lipid Peroxidation (MDA)
All unsaturated aldehydes may undergo further changes by autoxidation producing other volatile compounds. Thus, hydroperoxy aldehydes may undergo cleavage to give

shorter chain aldehydes, sometimes with other chemical groups. Among these, MDA is of interest. Various precursors of MDA have been proposed, but the most probable and the most biochemically important seem to be the monocyclic peroxides formed from fatty acids with 3 or more double bonds. Other efficient sources have been described: hydroperoxy bis-epidioxides, hydroperoxy bis-cycloendoperoxides and dihydroperoxides. Diene fatty acids (linoleic acid) were also shown to be good precursors in some defined oxidative conditions (singlet oxygen, acid pH). As an example, 12- hydroperoxy

linolenate may undergo a cyclization, followed by an oxidation forming a 5-membered hydroperoxy epidioxide as major product. The cleavage on each side of the

endoperoxide ring was proposed as the main source of MDA. MDA may also be formed in some tissues by enzymatic processes with prostaglandin precursors as substrates. Thus, thromboxane synthetase generates MDA, with thromboxane A2, from prostaglandin endoperoxides during human platelet activation.
(8)

The accumulation of free radicals in the plants causes oxidation of poly unsaturated fatty acids in the plasma membrane , resulting in the formation of MDA an indiacation of membrane Lipid peroxidation . (7)

Aims and Objectives

1) Analysis of MDA contents of Sorghum bicolor towards salt (200mM) & Cold (4ºC). 2) Graphical representation of MDA contents of Sorghum bicolor towards salt (200mM) & Cold (4ºC).

Review of Literature

Bafeel et al., (2007) reported that chilling associated oxidative damage that enhanced the production of reactive oxygen species (ROS), slow down metabolism and modify membranes resulting in lipid peroxidation. A marked increase in the level of H2O2 was estimated in alfalfa leaves after dark chilling treatment and as a consequence, oxidation damage due to H2O2 accumulation could cause lipid peroxidation of membrane and result in a significant increase in malondialdehyde (MDA) content. After recovery period the MDA content decreased significantly due to the increase of phenolic compounds, which suppress lipid peroxidation. Also, the redox properties of α-tocopherol play an important role in adsorbing and neutralizing free radicals and provide some forms of antioxidant protection. The results indicated that cold treatment may have initially caused injury, thereafter during the recovery period leaves coordinated and enhanced the capacity of the antioxidative system, thus diminishing the potential for active oxygen species.

Gulen et al., (2008) analyzed the activity of peroxidase (PRX) isozyme, lipid peroxidation (Malondialdehyde, MDA content) and cell membrane injury were studied during low temperature treatment for different periods in strawberry (Fragaria x ananassa cv. Camarosa) leaf tissues. Seedlings were grown for six weeks (plants had 4-5 leaves) in a greenhouse then the plants were transferred to a climate chamber with constant 5 degree Celsius , 60% relative humidity, 14/10 h (light/dark) photoperiod regime and 4 LS light intensity for 1, 4, 7 or 10 days to impose a low temperature stress. In general, low temperature application during 10 days caused a linear increase in MDA content.

Li (2009) reported that the activity of anti-oxidant enzymes (Superoxide dismutase (SOD), Peroxidase (POD), Catalase (CAT) and parameters of oxidative stress malondialdehyde (MDA) of shoots were investigated in S. sieb naturally salt-resistant halophyte. The seedlings of S. sieb were treated with varying (0, 80, 160 and 240 mM) NaCl stress. The results showed that NaCl played an important role in growth of S. sieb. It made obviously promotion of certain NaCl concentration to growth of S. sieb, the seeflings of S. sieb grew best under 80 mM salt stress. MDA concentration of S. sieb obviously decreased under 80 mM salt stress then increased with salt concentration increased. The activities of SOD, POD and CAT increased with the increase of the concentration of NaCl in S. sieb. The salt tolerance of this halophyte under salt stress condition are probably due to its ability to exhibit high SOD, POD and CAT enzyme activities and Soluble Sugar (SS) concentration.

Cai et al., (2011) reports the changes in malondialdehyde (MDA) content and the activities of superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT) in endosperms and cotyledons during Jatropha curcas seed germination were investigated in the present study. MDA content in endosperms increased during 10 day of germination, while in cotyledons it increased during six day of germination and then decreased. MDA contents in endosperms increase gradually during the 10 day germination. In cotyledons, MDA content increased sharply during the first six day of germination and then decreased gradually. The peak content is about 8.5 times that of the control.

Cao et al., (2011) reported that water deficiency and low temperature are two important ecological factors which affect the distribution and cultivation of oil palm. To find out how oil palm adapts to the environmental conditions, the dynamics of a series of important physiological components derived from the leaves of potted oil palm seedlings under drought stress (DS) (water with holding) and low temperature stress (LTS) (10°C) were studied. The results showed that low temperature and water stress inhibited the growth of oil palm seedlings. The relative conductivity, injury index, malondialdehyde (MDA) and proline content in the leaves increased to different degrees with the extension of low temperature and drought stress. The variations of the earlier mentioned parameters except proline content under low temperature stress were greater than that under drought stress. Thus, oil palm possibly showed different response mechanisms under low temperature and drought stress by mediations of these substances, in order to increase plant defense capability.

Yong et al., (2008) analyzed Changes of superoxide(O2) and hydrogen peroxide (H2O2), malondialdehyde (MDA) contents and activities of enzymes involving cell defence in leaves of strawberry (Fragaria×ananassa Duch) plantlets under different time of low temperature stress were studied. With the increase of stress times, the rates of O2 generation and contents of H2O2 increased to a certain degree and then decreased. The MDA contents and the relative conductivity fluctuated were increased during the treatment. The results clearly suggested that low temperature stress triggered an increase of reactive oxygen species (ROS) and the early accumulation of ROS in plants might lead to the production of antioxidant defence system. If the stress were too strong, the defence system of plants could not remove the more production of ROS effectively and result in severely damage to plants or even death.

Ahmad et al., (2010) reported a field experiment which was conducted in Youssef ElSeddeque region, at El-Fayoum Governorate to study the influence of bioregulator arginine at the rate of 100 or 200 ppm on growth, productivity and chemical composition of two sorghum cultivars grown under limited irrigation. Spraying the plants grown under water stress with arginine at the rate of 200 ppm caused significant increase in all of the studied growth characters and yield components. It has been found that application of arginine at the two rates significantly decreased malondialdehyde (MDA) contents which in turn led to an improved cell membrane of the two sorghum cultivars grown under water shortage by reducing lipid peroxidation property of the cell membrane.

Ashraf et al., (2010) analyzed the effect of root zone salinity on two hexaploid bread wheat (Triticum aestivum L.) cultivars (S-24, salt-tolerant; MH-97, salt-sensitive) was appraised at different growth stages. Grains of the two cultivars were sown in Petri-plates at two salt levels (0 and 150 mM of NaCl). After 8 days of germination, the seedlings were transplanted into plastic tubs containing either 0 or 150 mM of NaCl in full strength Hoagland‟s nutrient solution. Changes in growth, lipid peroxidation and phenolic contents were examined in the cultivars at different growth stages (vegetative, booting and reproductive) under salt stress. Higher MDA contents were observed in cv. MH-97 as compared to that in S-24 under saline regimes at different growth stages. Salt-induced effect in terms of lipid peroxidation was more pronounced at the booting and reproductive stages as compared with that at the vegetative stage in both cultivars, however, the accumulation of leaf total phenolics was higher at the booting stage as compared with that at the other stages. A significant variability in salt response was found among different growth stages in both cultivars.

Saha et al., (2010) reported that enhancement of salt (NaCl) tolerance by pretreatment with sublethal dose (50 mM) of NaCl was investigated in V. radiata seedlings. NaCl stress caused drastic effects on roots compared to shoots. Accompanying reductions in length, number of root hairs and branches, roots became stout, brittle and brown in color. Salt stress caused gradual reduction in chlorophyll, carotenoid pigment contents and chlorophyll fluorescence intensity also. Superoxide dismutase and catechol peroxidase activities increased under stress in both roots and leaves. But catalase activity showed an increase in roots and decrease in leaves. In these seedlings, the oxidative stress has been observed under salinity stress and the level of proline, H2O2 and malondialdehyde content were increased. But pre-treatment with sub lethal dose of NaCl was able to overcome the adverse effects of stress imposed by NaCl

to variable extents by increasing growth and photosynthetic pigments of the seedlings, modifying the activities of antioxidant enzymes, reducing malondialdehyde and H2O2 content and increasing accumulation of osmolytes like proline.

Chugh et al., (2010) analyzed the seedlings of selected six genotypes of maize (Zea mays L.) differing in their drought sensitivity (LM5 and Parkash drought-tolerant and PMH2, JH3459, Paras and LM14 as drought-sensitive) were exposed to 72 h drought stress at two leaf stage. Alterations in their antioxidant pools combined with activities of enzymes involved in defense against oxidative stress were investigated in leaves. Activities of some reactive oxygen species (ROS)-scavenging enzymes, catalase (CAT) and ascorbate peroxidase (APX) were enhanced in tolerant genotypes in response to drought stress. Peroxidase (POX) activity was significantly induced in tolerant, as well as sensitive genotypes. Imposition of stress led to increase in H2O2 and malondialdehyde (MDA, a marker for lipid peroxidation) content in sensitive genotypes, while in tolerant genotypes no change was observed. Significant increase in glutathione content was observed in sensitive genotypes. Significant activation of antioxidative defence mechanisms correlated with drought-induced oxidative stress tolerance was the characteristic of the drought tolerant genotypes. These studies provide a mechanism for drought tolerance in maize seedlings.

Pandey et al., (2009) analyzed the seven species of genus Avena viz., Avena sativa, Avena strigosa, Avena brevis, Avena vaviloviana, Avena abyssinica, Avena marocana and Avena sterilis were used to study the impact of drought stress on lipid peroxidation and other antioxidant enzymes. Maximum increase in the catalase activity was recorded in A. vaviloviana (129.97%) followed by A. sativa (122.82%) and A. brevis (83.38%) at vegetative stage; however at flowering stage the maximum increase was reported in A. sativa (25.62%) followed by A. sterilis (20.46%) and A. brevis (18.53%). At vegetative stage drought, maximum increase in peroxidase activity was recorded in A. sativa (122.82%) followed by A. brevis (83.38%) and A. sterilis (49.78%). Flowering stage drought, showed maximum increase in A. sativa (27.09%) followed by A. marocana (23.50%) and A. sterilis (20.46%). A. sativa and A. sterilis showed stress tolerance at both the stages by accumulating higher percentage of peroxidase followed by A. brevis at vegetative and A. marocana at flowering stage. Level of lipid peroxidation in terms of Malondialdehyde (MDA) content was increased in the leaves when plants were subjected to moisture stress. The rate of increase in lipid peroxidation occurs irrespective of stage however; maximum increase was recorded in A.

strigosa at both the stages. Avena species which showed high level of MDA content, indicates more lipid peroxidation and more membrane permeability and are comparatively more susceptible for water stress than those which produce less Malondialdehyde (MDA) content at higher magnitude of water stress such species have better capability for moisture stress tolerance.

Khosravinejad et al., (2008) analyzed the antioxidant responses of activities of Superoxide Dismutase (SOD), Catalase (CAT), Ascorbate Peroxidase (APX) and Guaiacol Peroxidase (GPX) to saline stress in two barley varieties named Hordeum vulgare L. var. Afzal and var. EMB82-12 treated with 50, 100, 200, 300 and 400 mM NaCl for 3 days. The MDA content of Afzal plants grown under different salt regimes remained nearly constant but it largely increased in EMB82-12 plants under the same conditions. There was a linear and significant correlation in CAT, APX, SOD, GPX activities in Afzal plants in response to increased salt concentration. The strong and positive correlation between antioxidant enzymes and salt concentrations, may account for the MDA level of Afzal plants remaining constant in response to different salt regimes. In general, the activities of antioxidant enzymes were increased in the root and shoot under saline stress. But the increase was more significant and consistent in the root. Among the antioxidant enzymes, CAT activity was increased the most drastically.

Hefny et al., (2007) analyzed that the Involvement of antioxidant enzyme activities in mitigating the damage of NaCl Stress was studied in 26 genotypes of forage sorghum exhibiting different responses to salinity, including a local hybrid with unknown performance under salinity stress. The two week old sorghum seedlings were subjects to 0, 50 and 100mM NaCl for 4 weeks, which corresponds to 0.7 , 8.2 and 12.11 dsm-1 salinity levels. Salt stress resulted in significant reduction of dry weight of both tolerant and sensitive genotypes. The reduction was stronger in the later group compared with the former one at 8.2 dS m-1. In contrast, at the highest salinity level, there was severe reduction in plant dry weights; meanwhile the highest value was recorded by the local genotype. Five out of the 21 salt tolerant genotypes and the local hybrid produced the highest dry weights at 50 and 100 mM NaCl. The effect of salinity levels on antioxidant enzymes and lipid peroxidation was examined. It could be concluded that the local genotype could be considered as salinity tolerant genotype as it exhibited the same trend of tolerant genotypes.

Devasagayam et, al (2003) analyzed that among the cellular molecules, lipids that contain unsaturated fatty acids with more than one double bond are particularly susceptible to action of free radicals. The resulting reaction, known as lipid peroxidation, disrupts biological membranes and is thereby highly deleterious to their structure and function. Lipid peroxidation is being studied extensively in relation to disease, modulation by antioxidants and other contexts. A large number of by-products are formed during this process. These can be measured by different assays. The most common method used is the estimation of aldehydic products by their ability to react with thiobarbituric acid (TBA) that yield 'thiobarbituric acid reactive substances' (TBARS), which can be easily measured by spectrophotometry. Though this assay is sensitive and widely used, it is not specific and TBA reacts with a number of components present in biological samples. Zin et al., (2006) analyzed the study which was conducted in two Aloe vera cultivars (F0 and F50) to study the characters of physiology and ecology under salt stress. The results indicate decreases in tissue water, total soluble sugars and glucose, and increases in dry matter and membrane injury occurred both in F0 and F50 irrigated with 60% seawater. Less cell membrane injury were observed in F50.Moreover, total soluble sugars in F0 decreased obviously, however, no significant change in F50, while sucrose in plants had no significant change. Furthermore, F0 and F50 accumulated more inorganic cations in stems and roots. In addition, leaf K+ and Ca2+ contents were more in F50 than that in F0 to maintain normal plant growth though accumulation of Na+. F50 had a relative superiority in growth under salinity conditions due to higher K+/Na+ ratio and lower Na+/Ca2+ ratio than F0.

Gill et al., (2010) reported that various abiotic stresses lead to the overproduction of reactive oxygen species (ROS) in plants which are highly reactive and toxic and cause damage to proteins, lipids, carbohydrates and DNA which ultimately results in oxidative stress. The ROS comprises both free radical (O2.-), superoxide radicals; OH, hydroxyl radical; H2O2., perhydroxy radical and RO, alkoxy radicals) and non-radical (molecular) forms H2O2, hydrogen peroxide and (1O2, singlet oxygen). In chloroplasts, photosystem I and II (PSI and PSII) are the major sites for the production of (1O2) and (O2.-) . In mitochondria, complex I, ubiquinone and complex III of electron transport chain (ETC) are the major sites for the generation of (O2.-). ROS also influence the expression of a number of genes and therefore control the many processes like growth, cell cycle, programmed cell death (PCD), abiotic stress responses, pathogen defense, systemic signaling and development. In this review, we

describe the biochemistry of ROS and their production sites, and ROS scavenging antioxidant defense machinery.

Singh et al., (2011) analyzed different species of ferns for the modulations in the pool of nonenzymatic antioxidants in response to the maleic hydrazide treatments. Treatments at very low doses were found to trigger the accumulation of both ascorbate and proline contents. Total amount of protein and chlorophyll contents showed varying degree of sensitivity in all cultivars of ferns. Proline accumulation was found to be high in treated plants compared with control. Proline, ascorbate and flavonoid contents were found to be accumulated in all plants exposed to high doses of maleic hydrazide. All the three species showed high proneness towards the mutagen. Improved tolerance in treated plants might be explained on the basis of the elevated level of enzymatic and non-enzymatic antioxidants.

Tajdoost et al., (2007) reported that recent molecular studies show that genetic factors of salt tolerance in halophytes exist in glycophytes too, but they are not active. If these plants expose to low level salt stress these factors may become active and cause plants acclimation to higher salt stresses. So because of the importance of these findings in this research the effect of salt pre-treatment has been examined in Zea mays seedlings. To do the experiment four day old Zea mays seedlings (Var. single cross 704) pre-treated with 50 mM NaCl for the period of 20 h. Then they were transferred to 200 and 300 mM NaCl for 48 h. At the end of treatment roots and shoots of seedlings were harvested separately. The changes of K+ leakage, the amount of malondialdehyde, proline, soluble sugars and the Hill reaction rate were analyzed. The results indicated that the amount of K+ -leakage and malondialdehyde (MDA) have been increased because of salt-induced lipid peroxidation and membrane unstability.

Zou et al., (2009) analyzed the effects of different concentrations of Cr(VI) (1 μM, 10 μM, 100 μM) applied for 7, 14 or 21 days on initiation of high lipid peroxidation level (POL) and consequent changes in the enzymatic-antioxidant protective system and minimization of photosystem II (PSII) activity were studied in maize seedlings. Chromium(VI) caused an increase in the electrical conductivity of the cell membrane, and malondialdehyde (MDA) content (a peroxidation product) reflected peroxidation of membrane lipids leading to the loss of the membrane's selective permeability. It also induced distinct and significant changes in

antioxidant enzyme activity. Versus the control, superoxide dismutase (SOD, EC 1.15.1.1.), catalase (CAT, EC 1.11.1.6.) and peroxidase (POD, EC 1.11.1.11.) activity in maize seedling roots and leaves was progressively enhanced by the different Cr(VI) doses and stress periods, except for decreases in SOD and POD activity in leaves exposed to 100 μM Cr(VI) for 21 days. Terzi et al., (2006) reported that this study shows the relationship of the antioxidant enzyme system to drought stress tolerance during leaf rolling in the leaf, petiole and root of Ctenanthe setosa (Rosc.) Eichler. Chlorophyll and carotenoid content and the chlorophyll stability index decreased in the early period of drought stress but increased in later periods, approaching the control level as leaf rolling increased. Relative water content decreased, while the root:shoot ratio increased during drought stress. Lipid peroxidation also increased and then declined in the same drought period, contrary to photosynthetic pigment content. A peroxidise isoenzyme activity band present in the control leaves did not appear in leaves exposed to 32 days of drought, but in the later periods that activity increased. Tolerance of drought stress apparently is closely associated with the antioxidant enzyme system as well as leaf rolling in C. setosa.

Xin et al., (2011) analyzed the modulation of lipid per-oxidation, antioxidant enzyme activity, water status and plant growth induced by glycinebetaine (GB) applied foliarly was investigated in the plants of two maize (Zea mays L.) cultivar i.e. drought-tolerant Shaandan 9 (S9) and -sensitive Shaandan 911 (S911) under long-term mild drought stress (LMDS). Long-term mild drought stress was found to decrease dry matter (DM), grain yield (GY) and leaf relative water content (RWC), but to increase malondialdehyde (MDA) accumulation in in leaves of both cultivars. Dry matter, GR, RWC and these antioxidative enzymes activities were greater but MDA concentration was lower for S9 than those for S911 under LMDS. Additionally, exogenous GB application increased DM, GR, RWC and antioxidant enzymes activities measured, but reduced MDA accumulation in both cultivars under LMDS unlike well-watered control, which exhibited no such obvious effect with GB. The modulation induced by GB applying was more pronounced in S911 than that in S9 under LMDS. The greatest positive role of GB seemed to be found in the plants subjected to the largest MDA accumulation at mature stage. It is, therefore, concluded that GB may protect cells against oxidative damage and alleviate the negative effect of DS on water status and plant growth,

particularly in this drought sensitive cultivar and imposed to more serious damage from DS environment. D’souza et al., (2011) analyzed that drought and salinity stress is the major causes of historic and modern agricultural productivity losses throughout the world. The availability of irrigation water is a challenge for many countries that have scarce water resources, yet are highly dependent on agriculture as a means of revenue. Effect of drought on Hyacinth bean, Dolichos lablab (HA-4 cultivar) was evaluated in 10-d-old seedlings for 8 d after withholding water. The stress reduced dry and fresh weight, leaf number, surface area, root and shoot length, total chlorophyll and relative water content. Oxidative stress markers, H2O2, glutathione, malondialdehyde, proline, ascorbic acid, total phenols, and total soluble sugars were significantly elevated. Drought enhanced antioxidant enzymes, peroxidase and glutathione reductase, and reduced catalase in a time dependent manner in the leaves.. The plant showed ability to rehydrate and grow upon re-watering, and levels of antioxidant components correlated with drought tolerance of the plant.

Ormaetxe et al., (1998) reported the application of a moderate water deficit (water potential of 21.3 MPa) to pea (Pisum sativum L. cv Lincoln) leaves led to a 75% inhibition of photosynthesis and to increases in zeaxanthin, malondialdehyde, oxidized proteins, and mitochondrial, cytosolic, and chloroplastic superoxide dismutase activities. Severe water deficit (21.9 MPa) almost completely inhibited photosynthesis, decreased chlorophylls, bcarotene, neoxanthin, and lutein, and caused further conversion of violaxanthin to zeaxanthin, suggesting damage to the photosynthetic apparatus. There were consistent decreases in antioxidants and pyridine nucleotides, and accumulation of catalytic Fe, malondialdehyde, and oxidized proteins. Paraquat (PQ) treatment led to similar major decreases in photosynthesis, water content, proteins, and most antioxidants, and induced the accumulation of zeaxanthin and damaged proteins. Results also indicate that the tolerance to water deficit in terms of oxidative damage largely depends on the legume cultivar.

Material and Methods

VARIOUS REQUIREMENTS
Requirements to Grow Fresh CSV 17 Variety of Sorghum Bicolor:1. CSV 17 seeds (from IARI- DELHI) 2. Double Distilled Water 3. 0.1% HgCl2 (for surface sterilization) 4. Flask 5. Hydroponics Set up 6. Air- bubble

Requirements to give stress to plant 1) For salt Stress:- 200mM NaCl 2) For Cold Stress:- Refrigerator maintained at 4ºC

Requirements for Extraction of MDA 1) Leaf, Petiole & Root Sample – 0.25 grams. 2) 50 ml of 0.1% Trichloroacetic acid (TCA). 3) 20ml of 0.1% Thiobarbuteric acid (TBA) + 20% TCA solution. 4) 1.5ml centrifuge tube. 5) Centrifuge 6) Mortar-Pestle 7) Distilled Water 8) Tissue Paper 9) Ethanol 10) 1000 µl pipette and tips 11) Ice packs

Preparation of stock solutions:1) 0.1% HgCl2 (10 ml) For 10 ml (0.1/100) * 10

= 0.01gram of HgCl2 in 10 ml of distilled water.

2) 200mM NaCl solution (1 litre) Molecular weight of NaCl = 58.5grams 200 * 58.5 = 1000 * X X = (200 * 58.5) / 1000 X = 11.7 grams of NaCl in 1 litre of distilled water

3) Preparation of 50ml of 0.1% TCA solution In 50 ml (0.1/100) * 50

= 0.05grams of TCA in 50ml of distilled water.

4) Preparation of 20ml of 0.5% TBA + 20% TCA For 0.5% TBA In 20ml (0.5/100) * 20

= 0.1grams in 20ml For 20% TCA In 20ml (20/100) * 20

= 4 grams in 20ml

PROCEDURE
I) 1) To grow plant under hydroponics set up:Surface Sterilization of Seeds –

a. Take 90 seeds of CSV-17 in a flask.

b. Now give atleast 4 washing to these seeds; each washing is of 30 seconds with continuous shaking.

c. Now pour 0.1% HgCl2 in the flask as all 90 seeds dipped inside for 10 minutes with continuous shaking.

d. Discard the HgCl2. And again give 4 washing with double distilled water to the sterilized seed.

e. Pour double distilled water in the flask and put seeds for 24hrs soaking.

2) Hydroponics Set-up for the growth of CSV-17 variety of Sorghum bicolor

a. Take one tray, thermocol, hydroponic box (2) , distilled water etc. b. Put soaked seeds of 1st step on to the hydroponic boxes n distribute them randomly over the two boxes.

c. Place these boxes inside the tray working thermocol as median to hold on the boxes.

d. Now give oxygen supply to the Hydroponics set up via air-bubbler.

3) Check water level on daily basis inside the hydroponics set up.

4) Observe the growth daily and if any contaminated seed is there, remove that immediately to reduce effect of contamination on viable and non-contaminated seeds.

5) Growth of minimum 7 days should be there before giving any stress to the plant.

II)

To give salt tolerance to the plant

1) After 7 days of normal growth of plant, take one box of grown plant carefully in sterilized conditions.

2) Transfer this box in salinity conditions as: - Pour enough amount of NaCl in other box so that roots of plant completely dipped inside the salt solution.

3) Now observer the growth and proceed further procedure to measure MDA content on day to day basis starting with 24hrs growth in salt solution.

4) Make sure that other factors remains constant to the stress plant as compare to normal plant like light, relative humidity, oxygen supply , aeration etc.

III) Cold treatment to the plant (COLD TOLERANCE) Another type of stress given to plant is cold tolerance (4º Celsius) to measure the MDA content in stressed plant. Steps for Cold Tolerance:1) Select any one box out of two which shows they same growth duration.

2) Place the box in the fridge maintaining 4º Celsius temperature inside the fridge.

3) Observe the growth and follow the daily basis protocol.

Some important points to be considered while following procedure:  Select only complete seeds for growth.

Before the set up of hydroponics make sure that all the components are UV sterilized and contamination free because single contaminated seed affect the growth of whole seeds.



Make sure that the duration of growth of stressed plant as well as normal plant (which is working as control for the MDA content ) is exactly same i.e. if you measure stress at 24hrs , control of 24hrs is taken in its correspondence.

IV)

Procedure for MDA extraction:-

1) Cut sample from the plant with the help of sterilized forceps and weigh them to exactly 0.25 grams on weigh machine.

2) Within a minute put this samples in Mortar-pestle and add 1ml of 0.1% TCA solution as mentioned above.

3) Make the fine paste of sample and add more 0.5ml of 0.1% TCA solution to make final volume 1.5ml of sample.

4) Take this fine paste in 1.5ml centrifuged tubes and kept them on ice packs.

5) Now wipe out the Mortar-pestle first with distilled water and then with ethanol.

6) Repeat the above steps with another plant (Stressed plant).

7) Centrifuge the above paste at 15000g for 5 minutes at 4º Celsius. 8) Discard the pellet and take the supernatant in different centrifuge tube. 9) Now take 1ml of this supernatant in the test tube and add 2ml of 0.5% TBA + 20% TCA solution as prepared earlier. 10) Cover the test tube with the cotton plug. 11) Heat the above solution at 95º Celsius in water-bath for 30 minutes. 12) Divide the aliquot prepared above in two different 1.5 ml centrifuge tubes. 13) Centrifuge the above aliquot at 10000g for 10 minutes. 14) Discard the pellet and take the supernatant in centrifuge tubes.(20)

Procedure for MDA estimation :- (2, 20, 21) 1) Spectrophotometry is used to measure the MDA content present in the sample .

2) Read the absorbance of different sample including both control ant stressed sample of different duration at 532nm , 600nm & at 450nm. 3) Apply the generalized formula on the absorbance measured :-

MDA content = ∆ (A532nm – A600nm) / 1.56 * 105 nmol/ml
Here :- ∆ - mean of three triplicates of same sample. A532nm – Specific Absorbance A600nm – Non - Specific Absorbance

(2)

1.56 * 105 is the extinction coefficient. nmol/ml is the unit of MDA content present.

Picture 1:- Shows hydroponics setup of a variety in tissue culture lab.

Picture 2:- CSV-17 Seeds started germinating on a hydroponic set up.

Picture 3: Control and full germinated seeds of CSV-17 of Sorghum bicolor (7days of growth).

Picture 4: To compare with the stress (Control at 4th day after 7 days growth).

Results

Plant Growth:Plant grows properly in hydroponics setup as shown in Picture 1 under normal condition. Rooting and shooting initiated and early stage and whole plant growth is up to 7 days.

NaCl Stress:200mM NaCl stress caused a significant increase in the MDA content in shoots of plants. Because of the formation of ROS (Reactive Oxygen Species) in stressed plants it cause oxidative damage to the membrane lipids and cause LIPID PEROXIDATION (19). MDA content is measure according to the method of Rajinder et, al (1981) which is already explained in material and method section. Specific absorbance for MDA content was measured at 532 nm and non specific content was measured at 600 nm by using Spectrophotometry. MDA can be characterized by its pinkish or light yellowish colour formed during the reaction with extraction buffer (20% TCA and 0.5% TBA) (8). Table 1 shows the specific (at 532 nm) and non-specific (at 600 nm) absorbance for control and NaCl treated plant. Table 1: Absorbance of control and stressed plant at 500nm and at 600nm. Duration of plant growth (in hours) at 24 hrs at 48 hrs at 72 hrs at 96 hrs Control 0.1652 0.0699 0.1441 1.0681 Absorbance at 532nm Stress 0.1308 0.2430 0.1627 0.8502 Absorbance at 600nm Control -0.0131 -0.0062 0.0107 0.6578 Stress -0.0132 -0.0041 0.0389 0.3012

Absorbance thus obtained at 532nm shows the MDA content present, which is directly proportional to the specific absorbance. In control plant it was observed that at 24hrs, 48hrs and at 72hrs specific absorbance remains nearby constant because there is no certain change in the growth conditions and no production of LP products and H2O2 in the shoots of control plant. But after certain growth period, at 96hrs control show sudden hike in the specific absorbance which is due to higher level of LP products and MDA content, since in control

also there are some secondary stresses present. Same as in case of non-specific absorbance which is also constant up to certain period of 72hrs and increased at 96hrs. Hence, this shows that in control there is no such high production of MDA and growth will remains the same. But in case of stressed plant it was completely opposite. Chlorosis takes place in the plant. Which means wilting of leaves occur and leaf rolling observed in some parts because of the stress. Wilting is low during 24hrs of exposure and very high in prolonged exposure to stress. At 24hrs it shows high specific absorbance which was nearly doubled after prolonged exposure to same stress to 48hrs. Which proves that plant in the stress not able to grow and oxidation of membranous lipids takes place which leads to the over production of LP products hence MDA content is highly increased. At 48hrs exposure MDA content is high but when exposure leads to 72hrs specific absorbance goes on decreasing which is due to production of antioxidative enzymes which make the plant tolerant for the salt stress which cause decrease in LP process and hence its products(18). After exposure to 72hrs , when it exposed to more stress production of antioxidative enzymes decreased and inhibited and specific absorbance increased abruptly which shows high increment in the LP and MDA content in the plant and after that plant goes to death and can‟t survived much to the stress.

Estimation and Calculation of MDA content
With the obtained absorbance above MDA content can be determined using the formula

MDA content = ∆ (A532nm – A600nm) / 1.56 * 105 nmol/ml Here:∆ - mean of three triplicates of same sample. A532nm – Specific Absorbance A600nm – Non - Specific Absorbance 1.56 * 105 is the extinction coefficient. nmol/ml is the unit of MDA content present.

(2)

Calculations:1) Control at 24hrs. Put the values in the formula from the table 1 we get MDA content = (0.1652 – (-0.0131)) / 1.56 * 105 nmol/ml MDA content = 1.142 * 10-6 nmol/ml

2) Stress at 24 hrs. Put the values in the formula from the table 1 we get MDA content = (0.1308 – (-0.0132)) / 1.56 * 105 nmol/ml MDA content = 0.923 * 10-6 nmol/ml

3) Control at 48hrs. Put the values in the formula from the table 1 we get MDA content = (0.0699 – (-0.0062)) / 1.56 * 105 nmol/ml MDA content = 0.487 * 10-6 nmol/ml

4) Stress at 48hrs. Put the values in the formula from the table 1 we get MDA content = (0.2430 – (-0.0041)) / 1.56 * 105 nmol/ml MDA content = 1.583 * 10-6 nmol/ml

5) Control at 72hrs

Put the values in the formula from the table 1 we get MDA content = (0.1441– 0.0107) / 1.56 * 105 nmol/ml MDA content = 0.855 * 10-6 nmol/ml

6) Stress at 72hrs Put the values in the formula from the table 1 we get MDA content = (0.1627– 0.0389) / 1.56 * 105 nmol/ml MDA content = 0.993 * 10-6 nmol/ml

7) Control at 96hrs Put the values in the formula from the table 1 we get MDA content = (1.0681– 0.6578) / 1.56 * 105 nmol/ml MDA content = 2.63* 10-6 nmol/ml

8) Stress at 96 hrs Put the values in the formula from the table 1 we get MDA content = (0.8502– 0.3012) / 1.56 * 105 nmol/ml MDA content = 3.519 * 10-6 nmol/ml

Here, Table 2 shows MDA content (in nmol/ml) against different duration of exposure to salt stress.

TABLE 2: MDA content in CONTROL and STRESSED plant at different durations ( 10-6nmol/ml)

Duration of exposure MDA content for CONTROL (10-6 nmol/ml) MDA content for (10 nmol/ml)
-6

24hrs

48hrs

72hrs

96hrs

1.142 STRESS 0.923

0.487

0.855

2.63

1.583

0.993

3.519

Graph 1 demonstrates the same above table in graphical form for MDA content across different duration of exposure.

GRAPH 1:- Change in MDA content in Sorghum bicolor under salt stress

4 3.5

MDA content (10-6 nmol/ml)

3 2.5 2 1.5 1 0.5 0 24hrs 48hrs 72hrs 96hrs

CONTROL STRESS

DURATION IN HOURS(hrs)

The graph and table shows the MDA content which is responsible for stress in plants and product of oxidation of lipids. As it was seen in the table that in control MDA content is nearly constant for first 3 days because of no primary stress present over there. At 24hrs from the graph it was clearly seen that MDA content is more in control which means in stressed condition the plant trying to collaborate with the situation in beginning and trying to be tolerant but further exposure to salt, plant can‟t able to cope up with the stressed condition and MDA content goes on increasing very abruptly as we seen on 48hrs (where in control MDA concentration is 0.487 * 10-6 nmol/ml while in stress it touches 1.583 * 10-6 nmol/ml.) but more exposure cause further production of antioxidative enzymes in plants (for eg. Peroxidase enzyme) which reduces the effect of LP and its products (3).

Picture 5: Stressed plant showing stress at any duration.

Picture 6: Prolonged exposure to stress cause wilting and leaf rolling as shown above.

Cold Stress:4ºC stress also caused significant increase in the MDA content in the shoots of the plant as observed in the previous case and because of the same reason i.e. process called Lipid Peroxidation. Here also MDA content in nmol/ml of per gram fresh weight depicts the rate of lipid peroxidation in stressed plant. But one significant difference comes here was , in NaCl stress plant will able to survive for 4-5 days but while cold stress it will hardly survive to 3 days which shows that sold stress is more severe for CSV-17 variety of Sorghum bicolor as compare to NaCl stress. Here also for MDA content specific absorbance (at 532nm) & non-specific absorbance (at600nm) was measured by using Spectrophotometry and MDA content was calculated by according to the method of Rajinder et, al (1981) which is already explained in material and method section. Table 3 shows the specific (at 532 nm) and non-specific (at 600 nm) absorbance for control and Cold treated plant.

TABLE 3:- Absorbance of control and stressed plant at 500nm and at 600nm. Duration of plant growth (in hours) at 24 hrs at 48 hrs at 72 hrs Control 0.7488 0.7244 0.7468 Absorbance at 532nm Stress 0.848 0.6799 0.8791 Absorbance at 600nm Control 0.4717 0.4601 0.4603 Stress 0.4983 0.4925 0.4466

MDA content present is directly proportional to the specific absorbance obtained. In control it was observed that specific absorbance remains constant for all 3 days of study and growth remains as it was. At 24hrs, at 48hrs and at 72hrs in control both specific and non specific absorbance changes slightly. But in stress the results are quite same as observed in the NaCl stress. At 24hrs specific absorbance is high as compare to control but further exposure to stress for 48hrs cause the production of antioxidative enzymes which makes the plant tolerant and reduces the

membrane damage. This occurs due to formation of enzyme like peroxidase which converts H2O2 in water and oxygen which are not toxic. But due further more exposure to cold specific absorbance increased abruptly and so Lipid peroxidation and its contents (MDA). Plant unable to survive after prolonged exposure to 3 days to cold stress and leaf roiling is one of factor occurs due to cold stress in plants (10).

Estimation and Calculation of MDA content
With the obtained absorbance above MDA content can be determined using the formula

MDA content = ∆ (A532nm – A600nm) / 1.56 * 105 nmol/ml Here:∆ - Arithmetic smean of three triplicates of same sample. A532nm – Specific Absorbance A600nm – Non - Specific Absorbance 1.56 * 105 is the extinction coefficient. nmol/ml is the unit of MDA content present.

(2)

Calculations:1) Control at 24hrs Put the values in the formula from the table 3 we get MDA content = (0.7488– 0.4717) / 1.56 * 105 nmol/ml MDA content = 1.776 * 10-6 nmol/ml

2) Stress at 24hrs

Put the values in the formula from the table 3 we get MDA content = (0.848– 0.4983) / 1.56 * 105 nmol/ml MDA content = 2.241 * 10-6 nmol/ml

3) Control at 48hrs Put the values in the formula from the table 3 we get MDA content = (0.7244 – 0.4601) / 1.56 * 105 nmol/ml MDA content = 1.694 * 10-6 nmol/ml

4) Stress at 48hrs Put the values in the formula from the table 3 we get MDA content = (0.6799 – 0.4725) / 1.56 * 105 nmol/ml MDA content = 1.329 * 10-6 nmol/ml

5) Control at 72hrs Put the values in the formula from the table 3 we get MDA content = (0.7468 – 0.4603) / 1.56 * 105 nmol/ml MDA content = 1.836 * 10-6 nmol/ml

6) Stress at 72hrs Put the values in the formula from the table 3 we get MDA content = (0.8791– 0.4466) / 1.56 * 105 nmol/ml MDA content = 2.772 * 10-6 nmol/ml

Here, Table 4 shows MDA content (in nmol/ml) against different duration of exposure to cold stress. TABLE 4: MDA content in CONTROL and STRESSED plant at different durations (106

nmol/ml)

Duration of exposure MDA content for CONTROL (10-6 nmol/ml) MDA content for STRESS nmol/ml) (10-6 2.241 1.776

24hrs

48hrs

72hrs

1.694

1.836

1.329

2.772

Graph 2 demonstrates the same above table in graphical form for MDA content across different duration of exposure. GRAPH 2:- Change in MDA content in Sorghum bicolor under cold stress

3

2.5

MDA content (10-6 nmol/ml)

2

1.5

CONTROL STRESS

1

0.5

0 24hrs 48hrs 72hrs

Duration in hours(hrs)

Table 4 and Graph 2 demonstrate the same result as in case of NaCl stress. At 24hrs MDA content increased due to increase in reactive oxygen species and occurrence of process called Lipid peroxidation and hence the MDA was increased. But at 48hrs MDA content decreased to half because of the same reason we mentioned earlier i.e. formation of antioxidative enzymes which decreases the lipid peroxidation and membrane damage. But prolonged exposure to stress for 72 hrs cause abrupt increase in MDA content and hence membrane damage and cell lysis were at very high rate and cause massive injury to plant. After long exposure to 72hrs plant were not able to survive more at 4ºC.

CONTROL at 96hrs.

CONTROL at 24hrs.

Picture 7: Colored reaction for MDA content (White (Right)) shows control at 24hrs and Yellow (Left) shows control at 96hrs for NaCl /72hrs for cold).

STRESSED /Pink Color

CONTROL (at 96hrs).

Picture 8: Shows MDA reaction (STRESSED/Pink Color) in the stressed plant at 96hrs for NaCl stress. Pink color is determining color of the presence of MDA in stressed sample (with respect to control).

Discussion

Sorghum bicolor is one of the stable crops grown in the arid and semiarid countries. It is the 5th most important cereal crop grown on 44 million ha in 99 countries in Asia, Africa and Americans. Sorghum exhibit excellent tolerance and yield potential to the environmental stresses like salinity, water shortage etc. But it did not respond well in cold stress. These Abiotic stresses cause the production of Reactive oxygen species (ROS). These species are toxic in nature and cause damage to DNA, proteins, lipids, chlorophyll and almost every living constituent of the living cells. (11) The MDA content in shoots of stressed as well as controlled plant was measured as an index of rate of lipid peroxidation (19). The increased level of H2O2 is may be one of the major cause for the LP process , since H2O2 is toxic in nature and cause membranous lipids to break down under peroxidation effect and forms products of LP of which one of the major product is MDA (3). Our study documents about the MDA content present in the plant during salinity and cold stress given to plant at different duration. In salinity stress condition it show that plant‟s growth, metabolic activity affected during the stress. In salinity our plant behavior is similar to the behavior of plant as in some previous studies. For control MDA content remains constant for first three days (1.142 * 10-6 to 0.855 10-6 nmol/ml) while it increased at 4th day (2.63 * 10-6 nmol/ml) but the growth remains unaffected and reason of some hikes in MDA content is presence of some secondary stress in the environment which will overcome day to day for control. In stress from 24hrs to 48hrs MDA (0.923 * 10-6 nmol/ml to 1.583 * 10-6 nmol/ml) content increased to 150% but from 48hrs to 72hrs MDA content once again decreased (1.583 * 10-6 nmol/ml to 0.993 * 10-6 nmol/ml) which is due to tolerant capability of Sorghum bicolor as mentioned above. But due to long exposure of salinity (for 96hrs) cause abrupt increase in MDA content (3.519 * 10-6 nmol/ml) which is nearly 350% increased amount and cause excess damage to membrane, DNA etc of living cell and lipids were oxidized under process called Lipid Peroxidation which cause the cell lysis & injury to plant cells which ultimately cause the death of plant. Similar to our findings Hefny and Abdel-Kader (2007) reported the same type of tolerance and behaviour of many genotypes out of total 26 of Sorghum bicolor at Suez Canal University, Egypt
(11)

. Khosravinejad et, al (2008) reported the same pattern of increment of

MDA content in S. bicolor 4 times in response to 400mM NaCl at university of Urnia, Iran

(13)

. Similar case observed in the study of Ashraf et, al (2010) as they observed the increased

MDA content in wheat variety in leaves of plant under the salinity stress condition and concluded that MDA is responsible product of oxidation of lipids and cause the damage to cell membrane (2). Li (2009) observed that MDA content 1st increased then decreased and if it continues to more stress MDA content again increased S. sieb which is same case observed in our study and Li also proves that why MDA content goes on decreasing in between because of formation of anti-oxidative enzymes which prevents LP and hence excess amount of MDA production in plant parts (14). Saha et, al (2010) also reported same results in mungbeen seeds as MDA content increases as they exposed to more and more salinity. In their study also MDA content increased from 42% to 79% in sample part (17). The increased MDA content shows more damage in the membranes and more lysis of plant cells which cause the death of plant. For example, peroxidase enzyme converts H2O2 in H2O and oxygen which are not toxic and can‟t lead to the oxidation of lipids (Shalata et, al 2001) and membrane damage (19). Reduced contents of MDA is an important indicator of stress tolerance as shown in some earlier studies e.g., in salt tolerant cultivars of barley (Liang et al., 2003), sorghum (Brankova et al., 2005) and tobacco (Ruiz et al., 2005) (2).

During cold stress the behaviour of stressed plant is not so similar to NaCl stressed plant. Plant is not able to survive more than 3 days at 4ºC and in case it shows same tolerant effect just at the 24hrs of stress while in case of NaCl tolerancy started after exposure to 48hrs. And the same case was observed in our study. For control the MDA content is very nearly constant for all 3 days and with significant increase in growth (1.776 * 10-6 nmol/ml, 1.694 * 10-6 nmol/ml, 1.836 * 10-6 nmol/ml at 24hrs , 48hrs and 72hrs respectively), which shows that there is no such stress is present and biochemical and other activities of plant remains the same. But in case of stress at 24hrs MDA content is very high (2.241 * 10-6 nmol/ml) as compare to control which means that there is excess membrane damage and LP in plants. But plant shows tolerance against the cold just after 24hrs of stress and MDA content decreased at 48hrs (1.329 * 10-6 nmol/ml) which is even less than control at 24hrs. But similar to NaCl stress if further stress if given the MDA content increased abruptly (2.772 * 10-6 nmol/ml)

which means there is excess damage in plant living cells and Lipid peroxidation takes place at very high concentration. At cold tolerance CSV-17 variety can‟t able to survive much. Gulen et, al (2008) also reported similar results while exposed their plant to cold stress.MDA content remains same for 1st 4 days but after that it increases linearly which is result of continuous exposure. MDA content reached to its highest level at 10th day and similar to our result where MDA content is highest at 4th day before the death of plant (10). Recently Cao et, al (2011) analyzed that in oil palm seedlings MDA content increase rapidly at the last day (3.5 * 10-6 nmol/ml) which is very similar to our final day MDA content (2.772 * 10-6 nmol/ml) just prior to death of plant(5). Similarly Yong et, al also reported results in which MDA content increase with duration of time but after 48hrs of stress, their plant show tolerance and once MDA content decreased but prolonged exposure increases the MDA content in the plant. It finally increased from 51% to 141% in one of the testing specie (23). Previous studies and our study suggested that Sorghum bicolor is tolerant specie for NaCl as well as cold stress. Our study suggested that due to prolonged exposure to stress there are certain process takes place inside the cells of plant which cause injury to plant cells. And this injury produces various enzymes as well as various signals which is cause of certain tolerancy generated in the plant cells. But high concentration of salt or high salinity and remarkably continuous low temperature play a vital role in deactivation of these tolerant enzymes (for example peroxidase) and the Sorghum bicolor were not able to show the same factor for longer period. Our study deals with the MDA as abiotic stress marker which is proved b the obtained results and it greatly suggested that MDA is one of the injurious product formed suring any type of stress which cause the oxidation of membrane lipids and cause the cell lysis. To avoid these type of stress markers while growth of Sorghum bicolor, this was grown into semi-arid zones of globe as mentioned earlier. But further studies on the tolerant enzymes and their activation may lead to more growth and development of Sorghum bicolor across the whole world.

CONCLUSION

MDA (malondialdehyde) was extracted and estimated from the CSV-17 variety of Sorghum bicolor and studied as abiotic stress marker in plants. MDA is product of lipid peroxidation which formed during oxidative stress to plants (like as we given – NaCl and Cold stress) which formed by the oxidation of lipids. Its content is goes on increasing with more the plant was exposed to stress. CSV-17 show some type of tolerance to stress in between as we seen the MDA decreases after certain period of duration but prolonged exposure reduces the tolerant capacity of plant and hence MDA increases more and more and act as one of the major factor in plant death. Plant goes in abiotic stress in presence of MDA and other products of Lipid peroxidation and cause damage to cell membrane and nearly all living cells in the plant. Our plant was more tolerant to NaCl because it will able to survive for 4 days in salinity as compare to cold where it was survived only for 3 days. This proves why the agricultural practice of Sorghum bicolor limits to semi-arid zone across the globe.

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