Antioxidant properties of two alfalfa (Medicago sativa L.) ecotypes in response to sodium chloride salinity stress

Document Type : Research Paper


1 Department of Biology, College of Sciences, Yadegar-e- Imam Khomeini (RAH) Shahre Rey Branch, Islamic Azad University, Tehran, Iran.

2 Department of Biology, Tonekabon Branch, Islamic Azad University, Tonekabon, Iran.

3 3Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran, Iran.


Biochemical and physiological responses of alfalfa under salinity stress were comparatively studied in a factorial experiment based on randomized complete block design by using Yazdi as the tolerant and Diabolourde as the sensitive ecotypes. Salt levels of 100, 150 and 200 mM were prepared by adding sodium chloride to the Hoagland half-strength culture medium. Total phenolics content, polyphenol oxidase (PPO), β-glucosidase and antiradical activities of leave′s extract, stomata properties and chlorophyll fluorescence parameters of the leaves including Fv/Fm and Fv/Fo were measured in response to salinity stress. Stomata characters were reduced in both ecotypes but chlorophyll fluorescence parameters only declined in Diabolourde but not in the Yazdi ecotype.  The PPO and β-glucosidase activities increased in both ecotypes. The phenolics content and antiradical activities increased in the Yazdi ecotype at all salt levels but those of Diabolurde increased only at the higher salinity levels. Our observations indicated that the Yazdi ecotype manipulated biochemical and physiological responses more efficiently to alleviate the reduction of growth parameters under salinity.  


Article Title [Persian]

ویژگی های آنتی اکسیدانی دو اکوتیپ یونجه (Medicago sativa L.) در پاسخ به تنش شوری کلرید سدیم

Authors [Persian]

  • سید افشین حسینی بلداجی 1
  • بابک باباخانی 2
  • رضا حسن-ساجدی 3
  • مهدیه هوشنی 2
1 گروه زیست شناسی، دانشکده علوم پایه، واحد یادگار امام خمینی (ره) شهر ری، دانشگاه آزاد اسلامی، شهر ری
2 گروه زیست شناسی، دانشکده علوم زیستی، واحد تنکابن دانشگاه آزاد اسلامی، تنکابن.
3 گروه بیوشیمی، دانشکده علوم زیستی، دانشگاه تربیت مدرس، تهران.
Abstract [Persian]

پاسخ ­های بیوشیمیایی و فیزیولوژبک یونجه در یک آزمایش فاکتوریل بر پایه بلوک ­های کامل تصادفی با استفاده از اکوتیپ­ های یزدی و دیابلورده به ترتیب به عنوان اکوتیپ­ های مقاوم و حساس بررسی شد. سطوح شوری 100، 150 و 200 میلی مولار با اضافه کردن نمک کلرید سدیم به محیط کشت نیم-قدرت هوگلند تهیه شد. اثر تنش شوری بر ویژگی­ های بیوشیمایی و فیزیولوژیک شامل محتوی فنلی کل، فعالیت آنزیم­ های پلی فنل اکسیداز و بتاگلوکوزیداز در عصاره برگی، رفتار روزنه­ ای و پارامترهای فلورسانس کلروفیل (Fv/Fm وFv/F0 ) تعیین شد. نتایج نشان داد که ویژگی­ های روزنه ­ای در هر دو اکوتیپ کاهش یافت ولی فلورسانس کلروفیل فقط در دیابلورده کاهش نشان داد و در اکوتیپ یزدی تغییری مشاهده نشد. میزان کاهش این پارامترها بین دو اکوتیپ متفاوت بود. فعالیت آنزیم­ های پلی فنل اکسیداز و بتاگلوکوزیداز در هر دو اکوتیپ افزایش یافت. محتوی فنلی کل و فعالیت آنتی اکیسیدانی در تمامی تیمارها در اکوتیپ یزدی افزایش نشان داد در حالی که در اکوتیپ دیابلورده تنها در تیمارهای بالای شوری افزایش یافت. میزان افزایش این پارامترها در دو اکوتیپ با یکدیگر متفاوت بود. این مشاهدات نشان می­ دهد که اکوتیپ یزدی از پاسخ­ های بیوشیمیایی و فیزیولوژیک با کارایی بالاتر برخوردار بود تا اثرات کاهشی شوری بر پارامترهای رشد را کم کند.  

Keywords [Persian]

  • بتاگلوکوزیداز
  • پلی فنل اکسیداز
  • محتوی فنل تام
  • ویژگی های روزنه ها
  • یونجه

Abbruzzese G, Beritognolo I, Muleo R, Piazzai M, Sabatti M,  Mugnozza GS and Kuzminsky E, 2009. Leaf morphological plasticity and stomatal conductance in three Populus alba L. genotypes subjected to salt stress. Environmental and Experimental Botany 66(3): 381-388.

Acosta-Motos JR, Ortuno MF, Bernal-Vicente A, Diaz-Vivancos P, Sanchez-Blanco MJ and Hernandez JA, 2017. Plant responses to salt stress: adaptive mechanisms. Agronomy 7(1): 1-38.

Agastian P, Kingsley SJ and Vivekanandan M, 2000. Effect of salinity on photosynthesis and biochemical characteristics in mulberry genotypes. Photosynthetica 38: 287-290.
Ahmed IM, Nadira UA, Bibi N, Cao F, He X, Zhang G and Wu F, 2015. Secondary metabolism and antioxidants are involved in the tolerance to drought and salinity, separately and combined, in Tibetan wild barley. Environmental and Experimental Botany 111: 1-12.
Ayaz FA, Kadioglu A and Turgut A, 2000. Water stress effects on the content of low molecular weight carbohydrates and phenolic acids in Ctenanthe setosa (Rosc.) Eichler, Canadian Journal of Plant Science 80: 373-378.
Babakhani B, Hosseini-Boldaji SA and Hassan-Sajrdi R, 2017. Biochemical and physiological responses of alfalfa (Medicago sativa L.) cultivars to osmotic stress. Journal of Plant Physiology and Breeding, 7(1): 87-97.
Babakhani B, Khavari-Nejad RA, Hassan-sajedi R, Fahimi H and Saadatmand S, 2001. Biochemical responses of Alfalfa (Medicago sativa L.) ecotypes subjected to NaCl salinity stress. African Journal of Biotechnology 10(55): 11433-11441.
Baker NR and Rosenqvist E, 2004. Applications of chlorophyll fluorescence can improve crop production strategies: an examination of future possibilities. Journal of Experimental Botany 55(403): 1607-1621.
Bartels D and Sunkar R, 2005. Drought and salt tolerance in plants. Critical Reviews in Plant Sciences 24: 23-58.
Bettaieb T, Mhamdi M, Ruiz-de GJI and Du JP, 2007. Relation between the low temperature stress and catalase activity in gladiolus somaclones (Gladiolus grandiflorus Hort.). Scientia Horticulturae 113: 49-51.
Ceulemans R and Mousseau M, 1994. Tansley review no 71. Effects of elevated atmospheric CO2 on woody plants. New Phytologist 127: 425-446.

Chunlong C,  Song L,  Rongsu L,  Fengping W and Junqing L, 2008. Concentration of phenolic compounds of Populus euphratica and soil water contents in Ejina oasis, Inner Mongolia, China. Acta Ecologica Sinica, 28(1): 69-75.

Curtis PS and Lauchli A, 1987. The effect of moderate salt stress on leaf anatomy in Hibiscus cannabinus (kenaf) and its relation to leaf area. American Journal of Botany 74: 538-542.
Dai J and Mumper RJ, 2010. Plant phenolics: extraction, analysis and their antioxidant and anticancer properties. Molecules 15: 7313-7352. 
De-Beer D, Joubert E, Gelderblom WCA and Manley M, 2003. Antioxidant activity of South African red and white ecotype wines: free radical scavenging. Journal of Agricultural and Food Chemistry 51: 902-909.

Delfanian M, Esmaeilzadeh Kenari R and Sahari MA, 2015. Antioxidant activity of loquat (Eriobotrya japonica Lindl.) fruit peel and pulp extracts in stabilization of soybean oil during storage conditions. International Journal of Food Properties, 18(2): 2813-2824.

Dietz KJ, Sauter A, Wichert K, Messdaghi D and Hartung W, 2000.  Extracellular β-glucosidase activity in barley involved in the hydrolysis of the ABA glucose conjugate in leaves. Journal of Experimental Botany 51(346): 937-944. 
Dziedzic, SZ and Hudson BJF, 1983. Polyhydroxy chalcones and flavanones as antioxidants for edible oils. Food Chemistry 12: 205-212.
Falleh H, Oueslati S, Guyot S, Ben-Dali A, Magne C, Abdelly C and Ksouri R, 2011. LC/ESI-MS/MS characterization of procyanidins and propelargonidins responsible for the strong antioxidant activity of the edible halophyte Mesembryanthemum edule L. Food Chemistry 127: 1732-1738.
Foyer CH, Descourvieres P and Kunert KJ, 2006. Photoxidative stress in plants. Physiologia Plantarum 92(4): 696-717.
Foyer CH and Noctor G, 2005. Oxidant and antioxidant signalling in plants: a re-evaluation of the concept of oxidative stress in a physiological context. Plant, Cell & Environment 28: 1056-1071.
Francisco RB, 2011. Biochemistry of grape berries: post genomic approaches to uncover the effects of water deficit on ripening. Ph.D. thesis in Plant Physiology, Universidade NOVA de Lisboa, Portugal.
Fricke W and Peters WS, 2002. The biophysics of leaf growth in salt-stressed barley: a study at the cell level. Plant Physiology 129: 374-388.
Garcia C, Hernandez T, Costa F, Ceccanti B and Gianni A, 1993. Hydrolases in the organic matter fractions of sewage sludge: changes with composting. Bioresource Technology 45: 47-52.
Ghassemi-Golezani K, Taifeh-Noori M, Oustan Sh, Moghaddam M and Seyyed-Rahmani S, 2011. Physiological performance of soybean cultivars under salinity stress. Journal of Plant Physiology and Breeding 1(1): 1-8.
Han S, Tang N, Jiang H, Yang LT, Li Y and Chen LS, 2009. CO2 assimilation, photosystem II photochemistry, carbohydrate metabolism and antioxidant system of Citrus leaves in response to boron stress. Plant Science 176: 143-153.
Hanato T, Kagawa H, Yasuhara T and Okuda T, 1988. Two new flavonoids and other constituents in licorice root: their relative astringency and radical scavenging effects. Chemical and Pharmaceutical Bulletin 36: 2090-2097.
Hosseini-Boldaji SA, Khavari-Nejad RA, Hassan-Sajedi R, Fahimi H and Saadatmand S, 2012. Water availability effects on antioxidant enzyme activities, lipid peroxidation, and reducing sugar contents of alfalfa (Medicago sativa L.). Acta Physiologiae Plantarum 34: 1177-1186.
Hura T, Hura K and Grzesiak M, 2007. Effect of long-term drought stress on leaf gas exchange and fluorescence parameters in C3 and C4 plants. Acta Physiologiae Plantarum 29: 103-113.
Jaleel CA, Manivannan P, Lakshmanan GMA, Gomathinayagam M and Panneerselvam R, 2008. Alterations in morphological parameters and photosynthetic pigment responses of Catharanthus roseus under soil water deficits. Colloids and Surfaces. B: Biointerface 61: 298-303.
Jamil A, Riaz S, Ashraf M. and Foolad MR, 2011. Gene expression profiling of plants under salt stress. Critical Reviews in Plant Sciences 30: 435-458.
Johnson DW, Smith SE and Dobrenz AK, 1992. Genetic and phenotypic relationships in response to NaCl at different development stages in alfalfa. Theoretical and Applied Genetics 83: 833-838.
Kato-Noguchi H and Tanaka Y, 2008. Effect of ABA-β-D-glucopyranosyl ester and activity of ABA-β-D-glucosidase in Arabidopsis thaliana. Journal of Plant Physiology 165: 788-790.
Keutgen AJ and Pawelzik E, 2008. Quality and nutritional value of strawberry fruit under long term salt stress. Food Chemistry 107: 1413-1420.
Khan AM, Ahmed MZ and Hameed A, 2006. Effect of sea salt and L-ascorbic acid on the seed germination of halophytes. Journal of Arid Environments 67: 535-540.
Kumar KB and Khan PA, 1982. Peroxidase and polyphenol oxidase in excised ragi (Eleusine coracana cv. PR 202) leaves during senescence. Indian Journal of Experimental Botany 20: 412-416.

Kwang HL, Hai LP, Ho-Youn K, Sang MC, Fan J, Wolfram H, Ildoo H, June MK, In-Jung L and Inhwan H, 2006. Activation of glucosidase via stress-induced polymerization rapidly increases active pools of Abscisic acid. Cell 126:1109-1120.

Li H, Lin F, Wang G, Jing R, Zheng Q, Li B and Li Z, 2012. Quantitative trait loci mapping of dark-induced senescence in winter wheat (Triticum aestivum). Journal of Integrative Plant Biology 54(1): 33-44.

Michałowicz J, Posmyk M and Duda W, 2009. Chlorophenols induce lipid peroxidation and change antioxidant parameters in the leaves of wheat (Triticum aestivum L.). Journal of Plant Physiology 166(6): 559-568.

Msilini N, Oueslati S, Amdouni T, Chebbi M, Ksouri R and Lachaal M, 2012. Variability of phenolic content and antioxidant activity of two lettuce varieties under Fe deficiency. Journal of the Science and Food and Agriculture 93(8): 2016-2021.

Muthukumarasamy M, Gupta SD and Pannerselvam R, 2000. Enhancement of peroxidase, polyphenol oxidase and superoxide dismutase activities by tridimefon in NaCl stressed Raphanus sativus L. Biologia Plantarum 43: 317-320.
Namiki M, 1990. Antioxidants/antimutagens in food. CRC Critical Reviews in Food Science and Nutrition 29: 273-300.
Navarro JM, Flores P, Garrido C and Martinez V, 2006. Changes in the contents of antioxidant compounds in pepper fruits at ripening stages, as affected by salinity. Food Chemistry 96: 66-73.
Niknam V, Razavi N, Ebrahimzadeh H and Sharifizadeh B, 2006. Effect of NaCl on biomass, protein and proline contents, and antioxidant enzymes in seedlings and calli of two Trigonella species. Biologia Plantarum 50: 591-596.
Popovic BM, Stajner D, Zdero-Pavlovic R, Tumbas-Saponjac V, Canadanovic-Brunet J and Orlovic S, 2016. Water stress induces changes in polyphenol profile and antioxidant capacity in poplar plants (Populus spp.). Plant Physiology and Biochemistry 105: 242-250.
Proteggente AR, Pannala AS, Paganga G, Van-Buren L, Wagner E, Wiseman S, De-Put F, Dacombe C and Rice-Evans C, 2002. The antioxidant activity of regularly consumed fruit and vegetable reflects their phenolic and vitamin C composition. Free Radical Research 36: 217-233.
Rengasamy P and Olsson KA, 1993. Irrigation and sodicity. Australian Journal of Soil Research 31(6): 821-837.
Robinson MF, Very AA, Sanders D and Mansfield TA, 1997. How can stomata contribute to salt tolerance? Annals of Botany 80: 387-393.
Singh RP, Murthy KNC and Jayaprakasha GK, 2002. Studies on the antioxidant activity of pomegranate (Punica granatum) peel and seed extracts using in vitro models. Journal of Agricultural and Food Chemistry 50: 81-86.
Song LJ, Di Y and Shi B, 2000. The significance and development trend in research of plant polyphenols. Progress in Chemistry 12(2): 161-170.
Sreenivasulu N, Grimm B, Wobus U and Weschke W, 2000. Differential response of antioxidant compounds to salinity stress in salt-tolerant and salt-sensitive seedlings of foxtail millet (Setaria Italica). Physiologia Plantarum 109: 435-442.
Stepien P and Johnson GN, 2009. Contrasting responses of photosynthesis to salt stress in the glycophyte Arabidopsis and the halophyte Thellungiella: role of the plastid terminal Oxidase as an alternative electron sink. Plant Physiology 149: 1154-1165.

Valifard M, Mohsenzadeh S, Kholdebarin B and Rowshan V, 2014. Effects of salt stress on volatile compounds, total phenolic content and antioxidant activities of Salvia mirzayanii. South African Journal of Botany 93: 92-97.

Vysotskaya L, Hedley PE, Sharipova G, Veselov D, Kudoyarova G, Morris J and Jones HG, 2010. Effect of salinity on water relations of wild barley plants differing in salt tolerance. AoB Plants, doi: 10.1093/aobpla/plq006.