11/29/12 11th Meeting of the Inter-Regional Cooperative Research Network on Cotton for the Mediterranean and Middle East Regions Antalya, Turkey, November 05-07, 2012 The antioxidant potential: factor of abiotic stress tolerance in cotton 1
11/29/12 A. Gurel, A. Edreva Ege University, Engineering Faculty, V. Velikova Bioengineering Department, Izmir, Turkey T. Tsonev E. Gesheva L. Yildiz-Aktas Institute of Plant Physiology and Genetics, Ege University, Science Faculty, Bulgarian Academy of Sciences, Sofia, Bulgaria Biology Department, Izmir, Turkey S. Dagnon B. Yagmur P.Hilendarski Plovdiv University, Plovdiv, Bulgaria H. Hakerlerler Ege University, Agriculture Faculty, D. Stoyanova-Koleva Soil and Plant Nutrition Department, Izmir, Turkey St. Kl.Ohridsky Sofia University, Sofia, Bulgaria B. Izci Çanakkale Onsekiz Mart University, Agriculture Faculty, Field Crops Department, Çanakkale, Turkey Y. Oren Tari ş Figs, Raisins, Cotton and Oil Seeds Agricultural Sales Cooperatives Unions Izmir, Turkey H. Akdemir Ege University, Ödemi ş Vocational Training School, Ödemi ş -Izmir, Turkey It is commonly accepted that the primary event induced by various stress factors in plants is the burst of reactive oxygen species ROS i.e. a state of oxidative stress that can have deleterious effect on cell function and structure. Plants elaborated a diversified network of antioxidants (ROS scavengers) to regulate the oxidative stress. The antioxidant potential is deployed as a response to stress inflict. 2
11/29/12 Stress factors ROS ROS Temperature extremes Antioxidants Pathogens Nutrient imbalance Metals Water deficit High light UV Enzymes Non-enzymatic compounds Carotenoids Peroxidase Catalase Tocopherols Superoxidedismutase Polyphenols Proline Glutathione reductase Polyamnines Etc. Etc. The antioxidant defense of plants involves compounds of diverse chemical types 3
11/29/12 In our long-term research on cotton stress physiology we examined two cases of cotton- stress factor interactions: � Nutrient (K/Na) imbalance (leaf reddening) � Water deficit In both cases abiotic constraints induce a state of oxidative stress Cotton reddening 4
11/29/12 Red cotton leaves • Three locations in Aegean region: Söke, Menemen, Bergama • Cotton plants: Nazilli 84 cv. ♦ Leaves of green plants (controls) Leaves with symptoms of reddening : ♦ Light symptoms ♦ Severe symptoms • Soils: ♦ Under green plants ♦ Under reddening plants 5
11/29/12 We have established that reddening of cotton leaves is provoked by K deficiency in the soil and K/Na imbalance leading to overaccumulation of Na in the leaves 2000 Soils under: Na green plants reddening plants 1500 ppm 1000 K 500 0 K and Na content (ppm) of soils on which green and reddening plants are grown 6
11/29/12 2.0 green plants reddening plants K 1.5 1.0 % Na 0.5 0.0 K and Na content (%) in leaves of green and reddening plants Plants make better use of Na in case of K deficiency Excess of Na induces over-accumulation of toxic OH • free radicals in plants, i.e. a state of oxidative stress (Alia et al. 1993). 7
11/29/12 Biochemical changes related to cotton reddening Green Light red Strong red 300 450 400 250 350 % of controls % of controls 200 300 250 150 200 100 150 100 50 50 0 0 Peroxidase Proline Increase of non-enzymatic (proline) and enzymatic (peroxidase) antioxidants in reddening leaves 8
11/29/12 Green Light red Strong red 700 500 450 600 400 500 350 % of controls % of controls 300 400 250 300 200 200 150 100 100 50 0 0 Anthocyanins Total phenols Dramatic increase of anthocyanins and total phenols in the reddening leaves Reddening leaves Green leaves Retention time (min) HPLC of anthocyanins (C6-C3-C6+) in green and reddening cotton leaves Peaks 4 and 5 are identified as cyanidin glycosides 9
11/29/12 Green leaves Reddening leaves OCH 3 OH OH OH + + B B O O HO HO OCH 3 H A A OH OH OH OH Cyanidin Malvidin • High antioxidant activity • Low antioxidant/antiradical activity (due to the o-OH grouping in the B-ring) • Predominant aglycone in green leaves • Predominant aglycone in reddening leaves � The shift from malvidin to cyanidin in reddening leaves determines a stronger protective potential against oxidative stress. The efficacy of the antioxidant defense in reddening leaves is evidenced by the low damage of membrane integrity as shown by the � Malonyl dialdehyde (MDA) test � Transmission electron microscopy 10
11/29/12 120 100 80 % of controls 60 40 20 0 Malonyl dialdehyde MDA MDA test Low membrane damage in reddening leaves Green leaves Reddening leaves Transmission electron microscopy Preserved membrane integrity in reddening leaves 11
11/29/12 ROS Green leaves Red leaves Shortage of Accumulation Accumulation Increase of Accumulation Shift of Burst of ROS K of Na of proline peroxidase of aglycons activity anthocyanins Scavenging of ROS Antioxidant protection in reddening cotton leaves: general proposed scheme Drought tolerance 12
11/29/12 Experimental design Ş ahin 2000 Nazilli 84-S Drought tolerant (T) Drought sensitive (S) Irrigation regimes ♦ Field capacity (normal water supply) ♦ 1/3 field capacity (drought stress) Locality ♦ Söke, Aegean region of Turkey 13
11/29/12 Biochemical • Non-enzymatic antioxidants Parameters - Polyphenols - Proline - Carotenoids • Markers of membrane damage - Malonyldialdehyde (MDA) Physiological • Photosynthesis • Water use efficiency (WUE) • Max photochemical activity of PSII • Relative water content (RWC) Non-enzymatic antioxidants � Polyphenols � Proline � Carotenoids 14
11/29/12 HPLC pattern of polyphenols in the leaves of cotton genotype Nazilli 84-S. 1, 2, 3 – isomers of chlorogenic acid: 1 – 5-O-caffeoyl quinic acid 2 – 3-O-caffeoyl quinic acid 3 – 4-O-caffeoyl quinic acid 4 – 10 – flavonoids: 4 – isoquercitrin glycoside 6 – rutin 8 – quercitrin 9 – kaempferol-3-rutinoside 10 – quercetin Main polyphenols in cotton leaves COOH OH H 2 H 2 OH O CH CH CO H H OH H HO Chlorogenic acid OH OH OH Cinnamic acid B O derivatives Flavonoids: A quercetine OH derivatives O Quercetin Both types have high antioxidant activity due to the presence of o-dihydroxy grouping 15
11/29/12 Quercetin derivatives (rutin, isoquercitrin) are the major flavonoids in cotton leaves Nazilli 84 (S) Nazilli 84 (S) Nazilli 84 (S) Sahin 2000 (T) Sahin 2000 (T) Sahin 2000 (T) Rutin Isoquercitrin Kaempferol -3-rutinoside 3 2 0,5 2,5 0,4 1,5 2 mg g -1 FM mg g -1 FM mg g -1 FM 0,3 1,5 1 0,2 1 0,5 0,1 0,5 0 0 0 Normal water supply Drought stress Normal water supply Drought stress Normal water supply Drought stress Nazilli 84 (S) Sahin 2000 (T) Total flavonoids 5 4,5 Higher content of flavonoids in the drought-tolerant ( ) 4 than in the sensitive ( ) genotype: 3,5 -at normal water supply mg g -1 FM 3 -at drought 2,5 2 1,5 1 0,5 0 Normal water supply Drough tstress 16
11/29/12 Nazilli 84 (S) Nazilli 84 (S) Nazilli 84 (S) Sahin 2000 (T) Sahin 2000 (T) Sahin 2000 (T) 4-0-caffeoyl quinic acid 5-0-caffeoyl quinic acid 3-0-caffeoyl quinic acid 1,5 0,5 0,2 0,4 0,15 1 mg g -1 FM mg g -1 FM 0,3 mg g -1 FM 0,1 0,2 0,5 0,05 0,1 0 0 0 Normal water supply Drought stress Normal water sullpy Drought stress Normal water supply Drought stress Nazilli 84 (S) Sahin 2000 (T) Total chlorogenic acid 2 Higher content of chlorogenic acid isomers in the drought-tolerant ( ) than in the sensitive ( ) 1,5 genotype: mg g -1 FM -at normal water supply 1 -at drought 0,5 0 Normal water supply Drought stress Non-enzymatic antioxidants H 2 C CH 2 Proline Carotenoids H 2 C CH COOH N H Proline β -carotene 17
11/29/12 Nazilli 84 (S) Nazilli 84 (S) Sahin 2000 (T) Sahin 2000 (T) Proline Carotenoids 1,6 5 1,2 4 μ M g -1 FM -1 FM 3 0,8 mg g 2 0,4 1 0 0 Normal water content Drought stress Normal water supply Drought stress Higher content of proline and carotenoids in the drought-tolerant ( ) than in the sensitive ( ) genotype: -at normal water supply -at drought The efficacy of the antioxidant defense in the drought-tolerant genotype is evidenced by: � lower membrane damage � better physiological performance as compared to the sensitive genotype 18
11/29/12 Nazilli 84 (S) Sahin 2000 (T) MDA 0,06 0,05 0,04 mol g-1 FM 0,03 0,02 0,01 0 Normal water supply Drought stress Lower membrane damage (malonyl dialdehyde content, MDA) in the drought-tolerant ( ) than in the sensitive ( ) genotype at: - normal water supply - drought 100 Nazilli 84 (S) Sahin 2000 (T) 80 60 % 40 20 0 Photosynthesis Max PS2 activity WUE Photosynthetic parameters in drought-subjected plants as % of the plants grown at normal water supply Better photosynthetic performance in the drought-tolerant ( ) than in the sensitive ( ) genotype 19
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