Biochemical and molecular tolerance of Carpobrotus acinaciformis L. halophyte plants exposed to high level of NaCl stress

Carpobrotus acinaciformis L. plant is a kind of halophyte that is able to survive in high salt conditions. It is important to determine its physiological, biochemical and molecular limit of NaCl stress if one aims to use it for phytoremediation purpose. In this study, the alkaline protocol of the modified plant comet assay were used for rapid detection of DNA damage in C. acinaciformis L. plants exposed to a series of  NaCl stress concentrations (0-, 50-, 100-, 200-, 300-, 400 and 500 mmol L-1) in hydroponic conditions for 2 weeks. DNA damage was measured as the values of percentage of DNA in tails and tail length. The halophyte C. acinaciformis L. did not show any dose response up to 400 mmol L-1 NaCl level in terms of DNA damages. DNA integrity measured via comet assay showed that DNA preserved its original shape up to 400 mmol L-1 NaCl level. However, the very high concentrations of NaCl (400 and 500 mmol L-1) caused DNA damages.  When physiological and biochemical parameters such as proline, chlorophyll a and b, peroxidase (POX), catalase (CAT), H2O2, malondialdehyde (MDA) contents were examined, oxidant molecules such as H2O2 (0.912-3.72 µmol g-1 Fwt) and MDA (7.1-34 nmol g-1 Fwt) gradually increased along with the increase of NaCl concentrations, p<0.05. On the other hand, antioxidant enzyme POX and an osmolyte molecule proline slightly increased up to 400 mmol L-1 NaCl level then slightly decreased after that. Similar issues were obtained from that of protease enzyme which indicates the power of protein hydrolysis in which a slight decrease (182-95 Unit mg-1 protein) over a dose of NaCl was evident. Chlorophyll contents and CAT activity were not affected upon increase of NaCl concentrations. This study showed that the halophyte C. acinaciformis L. can be easily used to remove salt up to 400 mmol L-1 NaCl concentrations from a saline-affected soil. Measuring DNA damage is concluded as a very useful parameter to find out what level of NaCl could be tolerated if a halophyte plant is aimed to remediate the saline soils.

NaCl stresine maruz bırakılan Carpobrotus acinaciformis L. halofit bitkisinin biyokimyasal ve moleküler tepkileri

Carpobrotus acinaciformis L. bitkisi yüksek tuz koşullarında yaşayabilen bir çeşit halofit bitkidir. Bu bitki fitoremediasyon çalışmaları için kullanılmak üzere planlandığında, bu bitkinin tuz stresine karşı fizyolojik, biyokimyasal ve moleküler sınırlarını belirlemek önem arz etmektedir. C. acinaciformis L. bitkisinde DNA hasar seviyesini belirlemek için hidroponik koşullarda 2 hafta süre ile tuz stresine (0-, 50-, 100-, 200-, 300-, 400 and 500 mmol L-1) maruz bırakılan bitkilerde modifiye edilmiş alkali bitki comet assay metodu kullanılmıştır. DNA hasarı kuyruk uzunluğu ve kuyrukta DNA yüzdesi olarak ölçülmüştür. Halofit C. acinaciformis L 400 mmol L-1 NaCl seviyesine kadar DNA hasarı ile ilgili olarak doz tepkisi göstermemiştir. Comet assay ile ölçülen yönteme göre halofit bitkilerin DNA bütünlüğünü 400 mmol L-1 NaCl  seviyesine kadar korunduğu gözlenmiştir. Fakat, çok daha yüksek NaCl konsantrasyonları (400 ve 500 mmol L-1) DNA hasarına yol açmıştır. Prolin, klorofil a ve b, peroksidaz (POX), katalaz (CAT), H2O2, malondialdehid (MDA) içerikleri gibi fizyolojik ve biyokimyasal parametreler incelendiğinde, oksidant moleküllerden H2O2 (0.912-3.72 µmol g-1 taze ağırlık)and MDA (7.1-34 nmol g-1 taze ağırlık) artan tuz konsantrasyonu ile paralel olarak sıralı artış göstermiştir, p<0.05. Diğer yandan, antioksidant enzimlerden POX ve bir osmolit olan prolin 400 mmol L-1 NaCl’ e kadar hafifçe artış göstermiş daha sonra tekrar düşmüştür. Benzer durumlar protein hidrolizini belirlemede kullanılan proteaz enzim (182-95 Unit mg-1 protein) seviyesinde de görülmüş, artan NaCl dozuna bağlı olarak enzim miktarı kademeli olarak azalmıştır. Klorofil miktarı ve CAT enzim seviyesi NaCI konsantrasyon artışına bağlı olarak değişim göstermemiştir. Bu çalışma, C. acinaciformis L. bitkisinin tuzdan etkilenmiş topraklarda 400 mmol L-1 NaCl’ e kadar olan tuz konsantrasyonunu uzaklaştırmada rahatlıkla kullanılabileceğini ortaya koymuştur. DNA hasarını ölçmek, tuzlu alanları ıslah etmede kullanılacak halofit bitkinin hangi seviyede NaCl stresine dayanabileceğini belirlemede çok kullanışlı bir parametre olarak kaydedilmiştir.

___

  • References
  • Agarwal, S., Pandey, V., 2004. Antioxidant enzyme responses to NaCl stress in Cassia angustifolia. Biologia Plantarum, 48: 555–560.
  • Anjum, N.A., Gill, S.S., Ahmad, I., Tuteja, N., Soni, P., Pareek, A., 2012. Understanding stress-responsive mechanisms in plants: an overview of transcriptomics and proteomics approaches, in Improving Crop Resistance to Abiotic Stress, Vols. 1, 2, (Eds) Tuteja, N., Gill, S. S., Tiburcio, A.F., Tuteja R., (Weinheim: Wiley-VCH Verlag GmbH and Co. KGaA; ), 337–355 pp.
  • Arnon, D.L., 1949. A copper enzyme is isolated chloroplast polyphenol oxidase in Beta vulgaris. Plant Physiol., 24: 1-15.
  • Bates, L.S., Waldren R.P., Teare, I.D., 1973. Rapid determination of free proline for water-stress studies. Plant & Soil, 39: 205-207.
  • Collins, A.R., 2004 The comet assay for DNA damage and repair. Molecular Biotechnology, 26: 249.
  • Cvikrova, M., Hrubcova, M., Vagner, M., Machackova, I., Eder, J., 1994. Phenolic acids and peroxidase activity in Alfalfa (Medicago sativa) embryogenic cultures after ethephon treatment. Plant Physiological, 91(2): 226-233.
  • Dikilitaş, M., Collins, A.R., Koçyiğit A., EL Yamani, N., Karakaş S., 2015. DNA damage in potato plants exposed tohigh level of NaCl stress. ICAW 2015 - 11th International Comet Assay Workshop. 1-4 september 2015.
  • Flowers, T.J., Colmer, T.D., 2008. Salinity tolerance in halophytes. New Phytologist, 179: 945–963.
  • Flowers, T.J., Colmer, T.D., 2015. Plant salt tolerance: adaptations in halophytes. Ann. Bot. 115(3): 327–331.
  • Gichner, T., Žnidar, I., and Száková, J. (2008). Evaluation of DNA damage and mutagenicity induced by lead in tobacco plants. Mutation Research, 652: 186-190.
  • Girard, C., Michaud, D., 2002. Direct monitoring of extracellular protease activities in microbial cultures. Analytical Biochemistry, 308: 388–391.
  • Grigore, MN, Ivanescu, L., Toma, C., 2014. Halophytes. An integrative anatomical study. Springer, Cham, Heidelberg, New York, Dordrecht, London, 1-2 pp.
  • Gupta, B., Huang, B., 2014. Mechanism of salinity tolerance in plants: physiological, biochemical, and molecular characterization. International Journal of Genomics, Article ID 701596, 18 pages.
  • He, Y., 2005. Changes in protein content, protease activity, and amino acid content associated with heath injury in creeping bentgrass. Journal of the American Society for Horticultural Science, 130(6):842-847.
  • Joshi, M., Mishra, A., Jha, B., 2011. Efficient genetic transformation of Jatropha curcas L. by microprojectile bombardment using embryo axes. Industrial Crops and Products, 33: 67-77.
  • Karakas, S., Cullu, M.A., Dikilitas, M., 2017. Comparison of two halophyte species (Salsola soda and Portulaca oleracea) for salt removal potential under different soil salinity conditions. Turkish Journal of Agriculture and Forestry, 41: 183-190.
  • Kassaye, Y.A., Salbu, B., Skipperud, L., Einset., John., 2013. High tolerance of aluminum in the grass species Cynodon aethiopicus. Acta Physiologiae Plantarum, 35: 1749-1761.
  • Lei, Y., Xu, Y., Hettenhausen C., Lu, C., Shen, G., Zhang, Cuiping., Li, J., Song, J., Lin, H., Wu, J., 2018. Comparative analysis of alfalfa (Medicago sativa L.) leaf transcriptomes reveals genotype-specific salt tolerance mechanisms. BMC Plant Biology, 18:35. Miller G, Suzuki N, Ciftci-Yilmaz S, Mittler R., Reactive oxygen species homeostasis and signalling during drought and salinity stresses. Plant,Cell and Environment, 33, 453-467.
  • Milosevic, N., Slusarenko, A.J., 1996. Active Oxygen Metabolism and Lignifications in The Hypersensitive Response in Bean. Physiological and Molecular Plant Pathology, 49: 143-158.
  • Pirasteh Anosheh, H., Ranjbar, G., Pakniyat, H., Emam, Y., 2016. Physiological mechanis of salt stress tolerance in plants; an overview. Editors: Azooz, M.M., Ahmad. P., Plant-environment interaction: responses and approaches to mitigate stress. Chichester: John Wiley & Sons; p. 141–160.
  • Pourrut B., Pinelli E., Celiz Mendiola V., Silvestre J., Douay F. (2015). Recommendations for increasing alkaline comet assay reliability in plants. Mutagenesis 30, 37–43.
  • Sairam, R.K., Sexena, D., 2000. Oxidative stress and antioxidants in wheat genotypes: possible mechanism of water stress tolerance. Journal of Agronomy and Crop Science, 184: 55-61.
  • Sairam, R.K., Srivastava, G.C., Agarwal, S., Meena, R.C. 2005. Differences in antioxidant activity in response to salinity stress in tolerant and susceptible wheat genotypes. Biol Plant, 49: 85-91.
  • Sharma, P, Jha, A.B, Dubey, R.S., Pessarakli, M., 2012. Reactive oxygen species, oxidative damage, and antioxidative defense mechanism in plants under stressful conditions. Hindawi Publishing Corporation Journal of Botany, Volume 2012, Article ID 217037.
  • Shetti, A.A., Kaliwal, B.B., 2017. Impact of imidacloprid intoxication on amylase and protease activity in soil isolate escherichia coli. Journal of Chemical and Pharmaceutical Research, 9(7):13-17.
  • Simova-Stoilova, L., Vassileva, V., Petrova, T., Tsenov, N., Demirevska, K., Feller, U., 2006. Proteolytic activity in wheat leaves during drought stress and recovery. General and Applied Plant Physioogy, Special Issue, 91-100.
  • Suo, J., Zhao, Q., David, L., Chen, S., Dai, S., 2017. Salinity Response in Chloroplasts: Insights from Gene Characterization. International Journal of Molecular Sciences, 18: 1011.
  • Tripathy, B.C., Oelmüller, R., 2012. Reactive oxygen species generation and signaling in plants. Plant Signaling Behavior, 7:12, 1621–1633. Velikova, V., Yordanov, I., Edreva, A., 2000. Oxidative Stress and Some Antioxidant Systems in Acid RainTreated Bean Plants: Protective Role of Exogenous Polyamines. Plant Science, 151, 59-66.