Effects of aminolevulinic acid on growth, antioxidant activity and anthocyanin accumulation of apple explants under salinity stress in in vitro culture conditions

Document Type : Full Paper


1 Former Ph.D. Student, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

2 Associate Professor, Faculty of Agriculture, University of Tabriz, Tabriz, Iran

3 Assistant Professor, Faculty of Agriculture, University of Tabriz, Tabriz, Iran


The present study was carried out to evaluate the effect of 5-aminolevulinic acid (ALA) on physiological and biochemical changes of in vitro-cultured Budaguvsky 9 (Bud 9) apple cultivar under NaCl induced salinity stress. Virus-free shoots and callus  of Bud 9 apple  were cultured on MS medium containing different levels of NaCl (0, 30, 60 and 90 mM) and ALA (0, 2.5, 5, 10 and 20 µM). Four weeks later phytochemical variations of regenerated plantlets with or without NaCl and/or ALA treatments were recorded.  Shoot explants and callus treated with 5-aminolevolinic acid at 2.5 to 20 µM concentrations demonstrated reduction in catalase and peroxidase and enhancement in superoxide dismutase and ascorbate peroxidase enzyme activity. The highest chlorophyll a and b values were observed at shoot explants treated with concentration of 10 μm of aminolevolinic acid. Also, salinity stress was performed an effective role on increasing the amount of anthocyanins of explants and the highest amount was obtained in 90 mM salinity stress. Increasing the intensity of salinity stress and amino acid concentration (2.5-10 μm) in culture media had synergic effect on the antioxidant activity, total phenol as well as flavonoids content of studied explants.  


Main Subjects

  1. Akram, N. A., Ashraf, M. & Al-Qurainy, F. (2012). Aminolevulinic acid-induced changes in some key physiological attributes and activities of antioxidant enzymes in sunflower (Helianthus annuus L.) plants under saline regimes. Scientia Horticulturae, 142, 143-148.
  2. Akram, N. A. & Ashraf, M. (2013). Regulation in plant stress tolerance by a potential plant growth regulator, 5-aminolevulinic acid. Journal of Plant Growth Regulation, 32(3), 663-679. DOI 10.1007/s00344-013-9325-9.
  3. Alizadeh, A., Xlilova, X. & Eivazi, A. (2011). Biochemical response of apple dwarf rootstock to salinity stress.   Technical journal of Engineering and applied sciences, 1, 118-124.
  4. Al-Khateeb, A. A, Al-Khateeb, S. A. Okawara, R. & Al-Abdoulhady, I. A. (2006).  Promotive effects of 5-aminolevulinic acid on fruit yield and quality of date palm cv Khs. Biological Science, 6, 1118-1121.
  5. Balestrasse, K. B., Tomaro, M. L., Batlle, A. & Noriega, G. O. (2010). The role of 5-aminolevulinic acid in the response to cold stress in soybean plants. Phytochemistry, 71, 2038-2045.
  6. Beaudion-Eagan, L.D. & Thorpe, T.A. (1989). Tyrosine and phenylnine ammonialyase activities during shoot initiation in tobacco callus cultures. Plant Physiology, 78, 438-441.
  7. Beyer, W. F. & Fridovich, I. (1987). Assaying for superoxide dismutase activity: some large consequences of minor changes in conditions. Analytical Biochemistry,161, 559-566.
  8. Bradford, M. M. (1976). A rapid and sensitive method for the quantification of microgram quantities of protein utilizing the principle of protein-dye binding. Analytical Chemistry, 72, 248-254.
  9. Brand-Williams, W., Cuvelier, M. E. & Berset, C. (1995). Use of a free radical method to evaluate antioxidant activity. Lebenson Wiss Technologie, 28, 25-30.
  10. Chance B. & Maely, A. C. (1955). Assay of catalase and peroxidases. Methods in Enzymology. 2, 764 -775
  11. Chang, C., Yang, M., Wen, H. & Chern, J. (2002). Estimation of total flavonoid content in propolis by two complementary colorimetric methods. Food and Drug Analysis, 10, 178-182.
  12. Feierabend, J. & Dehne, S. (1996). Fate of the porphyrin cofactors during the light dependent turnover of catalase and of the photosysem II reaction center protein DI in mature rye leaves. Planta, 198(3), 413-422.
  13. Flurky, W. H. (1986). Polyphenol oxidase in higher plants. Plant physiology, 81, 614-618.
  14. Folin, O. & Ciocalteu, V. (1927). On tyrosine and tryptophan determination in proteins. Journal of Biology and Chemistry, 27, 627-650.
  15. Gibon, Y., Sulpice, R. & Larher, F. (2000). Proline accumulation in canola leaf discs subjected to osmotic stress is related to the loss of chlorophylls and to the decrease of mitochondrial activity. Physiologia Plantarum, 4, 469-476.
  16. Guisti, M. M. & Wrolstad, R. E. (2001). Characterization and measurement of anthocyanins by UV-Visible spectroscopy. Current Protocols in Food Analytical Chemistry, (F1.21-F1.2.13). New York: John Wiley & Sons.
  17. Lichtenthaler,H. K. (1987). Chlorophylls and carotenoids, the pigments of photosynthetic biomembranes. In: Douce R, Packer L (eds) Methods Enzymol 148. Academic Press Inc. New York: pp. 350-382
  18. Hashemi, S., Asrar, Z. & Pourseyedi, S. (2010). Effects of seed pretreatment by salicylic acid on growth and some physiological and biochemical parameters in Lepidium sativum. Iranian Journal of Plant Biology, 2(2), 1-10.
  19. Hichema, H., Mounir, D. & Naceurc, E. A. (2009). Differential responses of two maize (Zea mays L.) varieties to salt stress: Changes on polyphenols composition of foliage and oxidative damages. Industrial Crops and Products, 30, 144-151.
  20. Jakopic, J., Veberic, R. & Stampar, F. (2007). The effect of reflective foil and hail nets on the lighting, color and anthocyanins of ‘Fuji’. Scientia Horticulturae, 115, 40-46.
  21. Jamil, M., Rehman, S. H. & Rha, E. S. (2007). Salinity effect on plant growth, psii photochemistry and chlorophyll content in sugar beet (Beta vulgaris L.) and cabbage (Brassica Oleracea Capitata L.). Pakistan Journal of Botany, 39, 753-760
  22. Kanto, U., Jutamanee, K., Osotsapar, Y., Chai-arree, W. & Jattupornpong, S. (2015). Promotive effect of priming with 5- aminolevulinic acid on seed germination capacity, seedling growth and antioxidant Enzyme activity in rice subjected to accelerated ageing treatment. Plant Production Science, 18, 443-454.
  23. Ksouri, R., Megdiche, W., Debez, A., Falleh, M., Grignon, C. & Abdelly, C. (2007). Salinity effect on polyphenol content and antioxidant activities in leaves of the halophyte Cakile maritime. Journal of Plant Physiology and Biochemistry, 45, 244-248.
  24. Liu, D. M., Zhang, J., Sun, W. J., Li, Q., Dai, A. H. & Bai, J. G. (2011). 5-Aminolevulinic acid pretreatment mitigates drought stress of cucumber leaves through altering antioxidant enzyme activity. Scientia Horticulturae, 130, 820-828.
  25. Lim, J. H., Park, K. J., Jeong, J. W. & Kin, K. J. (2012). Effect of salinity stress on phenolic compounds and carotenoids in buckwheat (Fagopyrum esculentum M.) sprout. Food chemistry, 135, 1065-1070.
  26. Lobon, N. C. Gallego, J. C. A., Diaz, T. S. & Garcia, J. C. E. (2002). Allepopathyic potential of Cistrus landanifer chemicals in response to variation of light and temperature. Chemoecology, 12, 139-143.
  27. Meratan, A., Ghaffari, S. M. & Niknam, V. (2008). Effects of salinity on growth, proteins and antioxidant enzymes in three Acanthophyllum species of different ploidy levels. Journal of Undergraduate Science & Technology, 33, 1-8.
  28. Murashige, T. & Skoog, F. (1962). A revised medium for rapid growth and bioassays with tobacco tissue cultures. Plant Physiology, 15, 473-497.
  29. Nahar, S. J. & Shimazaki, K. (2014). Application of 5-aminolevulinic Acid for the in vitro Micropropagation of Cymbidium as a potential novel plant regulator. Environmental Control in Biology, 52, 117-121
  30. Nakano, Y. & Asada, K. (1981). Hydrogen peroxide is scavenged by ascorbate specific peroxidase in spinach chloroplasts. Plant and Cell Physiology, 22, 867-880.
  31. Navarro, J. M., Flores, P., Garrido, C. & Martinez, V. (2006). Changes in the contents of antioxidant compounds in pepper fruits at different ripening stages, as affected by salinity. Food Chemistry, 96(1), 66-73.
  32. Nikolskii-Gavrilov, I., Landeros-Sanchez, C., Pcios-Velez, O. L. & Henandez-Perez, J. M. (2015). Impact of climate change on salinity and drainage of irrigated lands in Mexico. Agriucultural Science, 7, 197-204.
  33. Noreen, Z. & Ashraf, M. (2009). Assessment of variation in antioxidative defense system in salt treated pea (Pisum sativum L.) cultivars and its putative use as salinity tolerance markers. Plant Physiology, 166, 1764-1774.
  34. Pattanayak, G. K. & Tripathy, B. C. (2011). Overexpression of protochlorophyllide oxidoreductase C regulates oxidative stress in Arabidopsis. PLoS ONE, 6: e26532.
  35. Putter, J. (1974). “Peroxidase” Methods of enzymatic analysis, Academic Press New York, pp. 685-690.
  36. Rai, M. K., Kalia, R. K., Singh, R., Gangola, M. P. & Dhawan, A. K. (2011). Developing stress tolerant plants through in vitro selection- An overview of the recent progress. Environmental and Experimental Botany,71, 89-98.
  37. Rosso, V. V. & Mercadante, A. Z. (2007). Evaluation of colour and stability of anthocyanins from tropical fruits in an isotonic soft drink system. Innovative Food Science and Emerging Technologies, 8, 347-352.
  38. Samadi, S., Ghasemnezhad, A. & Alizadeh, M. (2014). Investigation on phenylalanine ammonia-lyase activity of artichoke (Cynara scolymus L.) affected by methyl jasmonate and salicylic acid in in-vitro conditions. Journal of Plant Production Research, 21 (4), 135-148.
  39. Shiyab, M. S., Shibli, R. A. & Mohammad, M. M. (2003). Influence of sodium chloride salt stress on growth and nutrient acquisition of sour orange in vitro. Plant Nutrition, 26, 985-996.
  40. Singh, R., Rastogi, S. & Dwivedi, U. N. (2010). Phenylpropanoid metabolism in ripening fruits. Comprehensive Reviews in Food Science and Food Safety, 9, 398-416.
  41. Sun, Y. P., Zhang, Z. P. & Wang, L. J. (2009). Promotion of 5- aminolevulinic acid treatment on leaf photosynthesis is related with increase of antioxidant enzyme activity in watermelon seedlings grown under shade condition. Photosynthetica, 47, 347-354.
  42. Tanaka, Y., Tanaka, A. & Tsuji, H. (1992). Stabilization of apoproteins of light-harvesting chlorophyll-a/b protein complex by feeding 5-aminolevulinic acid under intermittent illumination. Plant Physiology and Biochemistry, 30, 365-370
  43. Van Nocker, S., Berry, G., Najdowski, J., Michelutti, R., Luffman, M., Forsline, P., Alsmairat, N., Beaudry, R., Nair, M. G. & Ordidge, M. (2012). Genetic diversity of red-fleshed apples (Malus). Euphytica, 185(2), 281-293.
  44. Wang, J. J., Jiang, W. B. & Huang, B. J. (2004). Promotion of 5-aminolevulinic acid on photosynthesis of melon (Cucumis melo) seedlings under low light and chilling stress conditions. Plant Physiology, 121, 258-264.
  45. Wang, J. W., Zheng, L. P., Wu, J. Y. & Tan, R. X. (2008). Involvement of nitric oxide in oxidative burst phenylnine ammonia-lyase activation and taxol production induced by low-energy ultrasound in Taxus yunnanensis cell suspension cultures. Nitric Oxide Biology and Chemistry, 15, 351-358.
  46. Xie, L., Wang, Z. H., Cheng, X. H., Gao, J. J., Zhang, Z. P. & Wang, L. J. (2013). 5-Aminolevulinic acid promotes anthocyanin accumulation in Fuji apples. Plant Growth Regulation, 69, 295-303.
  47. Xu, F., Chang, J., Cheng, S.Y., Zhu, J., Li, L. L., Wang, Y. & Cheng, H. (2013). Promotive effect of 5-aminolevulinic acid on the antioxidant system in Ginkgo biloba leaves. African Journal of Biotechnology, 8, 3769-3776.
  48. Yang, Z., Chang, Z., Sun, L., Yu, J. & Huang, B. (2014). Physiological and metabolic effects of 5- aminolevulinic acid for mitigating salinity stress in creeping bentgrass. PLoS ONE, 9(12), e116283.
  49. Yoshida, R., Fukuta, Y., Watanabe, S. & Tanaka, T. (2003). Physiological function of 5-ALA in leaf vegetable is related to AsA-GSH pathway. Japan Society of Coordination Chemistry, 38, 70-77.
  50. Youssef, T. & Awad, M. A. (2008). Mechanisms of enhancing photosynthetic gas exchange in date palm seedlings (Phoenix dactylifera L.) under salinity stress by a 5-aminolevulinic acid-based fertilizer. Plant Growth Regulation, 27, 1-9.
  51. Zhen, A., Bie, Z. L., Huang, Y., Liu, Z. X. & Fan, M. L. (2012). Effects of 5 aminolevulinic acid on the H2O2-content and antioxidative enzyme gene expression in NaCl-treated cucumber seedlings. Biologia Plantarum, 56(3), 566-570.
  52. Zhao, G. Q., Ma, B. L. & Ren, C. Z. (2007). Growth, gas exchange, chlorophyll fluorescence, and ion content of naked oat in response to salinity. Crop Science, 47, 123-131.