Effect of thermal stress on amino acid and gene expression profiles in two local flax varieties with contrasting cold tolerance


1 Biology Department, Faculty of Sciences, Urmia University, Urmia, Iran. Institute of Biotechnology, Urmia University, Urmia, Iran.

2 Institute of Biotechnology, Urmia University, Urmia, Iran. Department of Plant Breeding and Biotechnology, Urmia University, Urmia, Iran.

3 Horticulture Crop Science Research Department, West Azarbaijan Agricultural and Natural Resources Research and Education Center, Agricultural Research, Education and Extension Organization, Urmia, Iran.


Environmental stresses such as cold and heat are adversely affecting all aspects of crop plants including yield. In this study, the contents of fourteen amino acids and expression levels of four transcription factor genes including MYB1-1, KRP2, ERF and WRKY40 were analyzed in TN-97-2 (cold sensitive) and TN-97-290 (cold tolerant) local flax varieties under cold and heat stresses conditions. Seeds of two local flax varieties were grown in growth chamber of Biotechnology Institute of Urmia University, Urmia, Iran, under control conditions for 30 days. Then, plants were subjected to three different thermal regimes including control (25± 1°C), cold (4 ± 1°C) and heat (37 ± 1 °C) for three consecutive days.  Cold stress significantly increased the Asp, His, Ala and Met amino acids contents in both varieties. TN-97-290 variety exhibited less decline in His, Val, Phe, Iso and Leu contents than TN- 97- 2 variety under heat stress. Cold stress increased expression of ERF and WRKY40 mRNAs while heat stress elevated transcript levels of KRP2 and MYB1-1 genes in TN-97-290 variety. In summary, Asp, Glu, His, Ala and Met amino acids could be exogenously applied to flax plants by either foliar spray or root supplement to tolerate cold stress conditions. In addition, application of Ser and Pro amino acids may aid conferring heat tolerance to cold sensitive flax plants. The positive response of ERF and WRKY40 genes
 (cold stress) and KRP2 and MYB1-1 (heat stress) in TN-97-290 variety suggests their over-expression assist protecting flax plants under cold and heat stresses. 


Ali, Q., Habib-ur-Rehman Athar, M. Z., Haider, S. S., Aslam, N., Shehzad, F., Naseem, J., Ashraf, R., Ali, A. and Hussain, S. M. 2019. Role of amino acids in improving abiotic stress tolerance to plants. pp. 175-203. In: M. .Hasanuzaman et al. (eds.) Plant tolerance to environmental stress: Role of Phytoprotectants. CRC Press.
Bakshi, M. and Oelmüller, R. 2014. WRKY transcription factors: Jack of many trades in plants. Plant Signal Behav. 9: e27700. DOI: 10.4161/psb.27700.
Bowne, J. B., Erwin, T. A., Juttner, J., Schnurbusch, T., Langridge, P., Bacic A. and Roessner. U. 2012. Drought responses of leaf tissues from wheat varieties of differing drought tolerance at the metabolite level. Mol Plant. 5: 418-429.
Chen, J. Q., Meng, X. P., Zhang, Y., Xia, M. and Wang, X. P. 2008. Over-expression of OsDREB genes lead to enhanced drought tolerance in rice. Biotechnol. Lett. 30: 2191-2198.
Chen, L., Song, Y., Li, S., Zhang, L., Zou, C. and Yu, D. 2012. The role of WRKY transcription factors in plant abiotic stresses. Biochim. Biophys. Acta Gene Regul. Mech. 1819: 120-128.
De Veylder, L., Beeckman, T., Beemster, G. T., Krols, L., Terras, F., Landrieu, I., Van Der Schueren, E., Maes, S., Naudts, M. and Inzé, D. 2001. Functional analysis of cyclin-dependent kinase inhibitors of Arabidopsis. Plant Cell. 13: 1653-68.
Anonymous, 1996. Growing flax: production, management and diagnostic guide, 3rd edition. Flax Council of Canada. Winnipeg, Canada. 64 pp.
Gasic, K., Hernandez, A. and Korban, S. S. 2004.  RNA extraction from different apple tissues rich in polyphenols and polysaccharides for cDNA library construction. Plant Mol. Biol. Rep. 22: 437-438.
Ghanavati, F. 2016. Domestication and introduction of flax plant, a plant with dual use of oil and fiber. Journal of Research Achievemnets for Field and Horticultural Crops 1(2): 51-62 (in Persian).
Ghoreishi, M., Rahmani, F. B., Mandoulakani, A. and Hassanzadeh Gorttapeh A. 2017. Impact of variety on resistance to cold stress at physiological levels in 'Linum usitatissimum'. Plant Omics. J. 10: 269-276.
Hildebrandt, T. M. 2018. Synthesis versus degradation: directions of amino acid metabolism during Arabidopsis abiotic stress response. Plant Mol. Biol. 98: 121-135.
Hong, S. W. and Vierling, E. 2001. Hsp101 is necessary for heat tolerance but dispensable for development and germination in the absence of stress. Plant J. 27: 25-35.
Huang, Y., Liu, Y., Zhang, M., Chai, M., He, Q., Jakada, B.H., Chen, F., Chen, H., Jin, X., Cai, H. and Qin, Y. 2020. Genome-wide identification and expression analysis of the ERF transcription factor family in pineapple (Ananas comosus (L.) Merr.). Peer J. 8: e10014. DOI:10.7717/peerj.10014.
Huis, R., Hawkins, S. and Neutelings, G. 2010. Selection of reference genes for quantitative gene expression normalization in flax (Linum usitatissimum L.). BMC Plant Biol. 10: 71. DOI:10.1186/1471-2229-10-71.
Jin, Y., Pan, W., Zheng, X., Cheng, X., Liu, M., Ma, H. and Ge, X. 2018. OsERF101, an ERF family transcription factor, regulates drought stress response in reproductive tissues. Plant Mol. Biol. 98: 51-65.
Jisha, V., Dampanaboina, L., Vadassery, J., Mithöfer, A., Kappara, S. and Ramanan, R. 2015. Overexpression of an AP2/ERF type transcription factor OsEREBP1 confers biotic and abiotic stress tolerance in rice. 
PLoS One, 10: e0127831. DOI: 10.1371/journal.pone.0127831.
Katiyar-Agarwal, S., Agarwal, M. and Grover, A. 2003. Heat-tolerant basmati rice engineered by over-expression of hsp101. Plant Mol. Biol. 51: 677-686.Katiyar, A., Smita, S., Lenka, S. K., Rajwanshi, R., Chinnusamy, V. and Bansal, K. C. 2012. Genome-wide classification and expression analysis of MYB transcription factor families in rice and Arabidopsis. BMC Genom. 13: 544. DOI:10.1186/1471-2164-13-544.
Khan, N., Bano, A., Rahman, M. A., Rathinasabapathi, B. and Babar, M. A. 2019. UPLC‐HRMS‐based untargeted metabolic profiling reveals changes in chickpea (Cicer arietinum) metabolome following long‐term drought stress. Plant Cell Environ. 42: 115-132.
Klay, I., Pirrello, J., Riahi, L., Bernadac, A., Cherif, A., Bouzayen, M. and Bouzid, S. 2014. Ethylene response factor Sl-ERF. B. 3 is responsive to abiotic stresses and mediates salt and cold stress response regulation in tomato. Sci. World J. 2014 (2). DOI:10.1155/2014/167681.
Klay, I., Gouia, S., Liu, M., Mila, I., Khoudi, H., Bernadac, A., Bouzayen, M. and Pirrello, J. 2018. Ethylene response factors (ERF) are differentially regulated by different abiotic stress types in tomato plants. Plant Sci. 274: 137-145.
Klein, D. 2002. Quantification using real-time PCR technology: applications and limitations. Trends Mol. Med. 8: 257-260.
Kovács, Z., Simon-Sarkadi, L., Vashegyi, I. and Kocsy, G. 2012. Different accumulation of free amino acids during short-and long-term osmotic stress in wheat. Sci. World J. 2012. DOI:10.1100/2012/216521.
Kurilich, A. C., Tsau, G.J., Brown, A., Howard, L., Klein, B.P., Jeffery, E.H., Kushad, M., Wallig, M.A. and Juvik, J. A. 1999. Carotene, tocopherol, and ascorbate contents in subspecies of Brassica oleracea.  J. Agric. Food Chem. 47: 1576-1581.
Lee, B. H., Henderson, D. A. and Zhu, J. K. 2005. The Arabidopsis cold-responsive transcriptome and its regulation by ICE1. Plant Cell 17: 3155-3175.
Millam, S., Obert, B. and Pret’ová, A. 2005. Plant cell and biotechnology studies in Linum usitatissimum–a review. Plant Cell Tiss. Organ. Cult. 82: 93-103.
Planchet, E., Rannou, O., Ricoult, C., Boutet-Mercey, S., Maia-Grondard, A. and Limami, A. M., 2011. Nitrogen metabolism responses to water deficit act through both abscisic acid (ABA)-dependent and independent pathways in Medicago truncatula during post-germination. J. Exp. Bot. 62 (2): 605-615.
Rehman, S. and Mahmood, T. 2015. Functional role of DREB and ERF transcription factors: regulating stress-responsive network in plants. Acta Physiol. Plant. 37 (9): 178. DOI:10.1007/s11738-015-1929-1.
Ruijter, J. M., Ramakers, C., Hoogaars, W. M., Karlen, Y., Bakker, O., Van den Hoff, M. J. and Moorman, A. F. 2009. Amplification efficiency: linking baseline and bias in the analysis of quantitative PCR data. Nucleic Acids Res. 37 (6): 45. DOI: 10.1093/nar/gkp045.
Savitch, L. V., Barker- Astrom, J., Ivanov, A. G.,  Hurry, V.,  Oquist, G.,  Huner, N. P., Gardeström, P. 2001. Cold acclimation of Arabidopsis thaliana results in incomplete recovery of photosynthetic capacity, associated with an increased reduction of the chloroplast stroma. Planta. 214 (2): 295-303.
Serra, T. S., Figueiredo, D. D., Cordeiro, A. M., Almeida, D. M., Lourenço, T., Abreu, I. A., Sebastián, A., Fernandes, L., Contreras-Moreira, B., Oliveira, M. M. and Saibo, N. J. 2013. OsRMC, a negative regulator of salt stress response in rice, is regulated by two AP2/ERF transcription factors. Plant Mol. Biol. 82: 439-455.
Sheikh, F., Raskin, A., Chu, P. H., Lange, S., Domenighetti, A. A., Zheng, M., Liang, X., Zhang, T., Yajima, T., Gu, Y. and Dalton, N. D. 2008. An FHL1-containing complex within the cardiomyocyte sarcomere mediates hypertrophic biomechanical stress responses in mice. J. Clin. Invest. 118: 3870-3880.
Shin, H., Oh, S., Arora, R., and Kim, D. 2016. Proline accumulation in response to high temperature in winter-acclimated shoots of Prunus persica: a response associated with growth resumption or heat stress? Can. J. Plant Sci. 96: 630-638.
Silvente, S., Sobolev, A. P. and Lara, M. 2012. Metabolite adjustments in drought tolerant and sensitive soybean genotypes in response to water stress. PLoS One. 7 (6): e38554. DOI:10.1371/journal.pone.0038554.
Singh, B., Chadband, W. G., Smith, C. W. and Calderwood, J. H. 1972. Pre breakdown processes in electrically stressed insulating liquids. J. Phys. D: Appl. Phys. 5 (8): 1457.
Sonju, R, and Horvath, D. P. 2005. Cloning and expression of Krp genes from adventitious buds of the perennial weed leafy spurge. pp. 18-19. In: Proceedings of 2005 Midwest American Society of Plant Biology Sectional Annual Meeting.
Taibi, K., Del Campo, A. D., Vilagrosa, A., M. Bellés, J., López-Gresa, M. P., López-Nicolás, J. M. and Mulet, J. M. 2018. Distinctive physiological and molecular responses to cold stress among cold-tolerant and cold-sensitive Pinus halepensis seed sources. BMC Plant Biol. 18: 236. DOI:10.1186/s12870-018-1464-5.
Vaisey-Genser, M., and Morris. D. H. 2003. Introduction: history of the cultivation and uses of flaxseed. CRC Press. 21 pp.
Wang, J., Yuan, B., Xu, Y. and Huang, B. 2018. Huang. Differential responses of amino acids and soluble proteins to heat stress associated with genetic variations in heat tolerance for hard fescue. J. Am. Soc. Hortic. Sci. 143: 45-55.
Wan, L., Wu, Y., Huang, J., Dai, X., Lei, Y., Yan, L., Jiang, H., Zhang, J., Varshney, R.K. and Liao, B., 2014. Identification of ERF genes in peanuts and functional analysis of AhERF008 and AhERF019 in abiotic stress response. Funct. Integr. Genomics 14: 467-477.
Yang, Y., Liu, X., Jiang, Y., Xiang, Z., Xu, Q., Zhao, N., and Shu, B. 2015. Root growth, free amino acids, and carbohydrates of tall fescue in response to soil salinity. Hort. Sci. 50: 609-614.
Yi, D., Kamei, C. L., Cools, T., Vanderauwera, S., Takahashi, N., Okushima, Y., Eekhout, T., Yoshiyama, K. O., Larkin, J., Van den Daele, H., Conklin, P., Britt, A., Umeda, M. and De Veyleder, L. 2014. The Arabidopsis SIAMESE-RELATED cyclin-dependent kinase inhibitors SMR5 and SMR7 regulate the DNA damage checkpoint in response to reactive oxygen species. The Plant Cell 26: 26-39.
You, J., Zhang, Y., Liu, A., Li, D., Wang, X., Dossa, K., Zhou, R., Yu, J., Zhang, Y., Wang, L. and Zhang, X. 2019. Transcriptomic and metabolomic profiling of drought-tolerant and susceptible sesame varieties in response to drought stress. BMC Plant Biol. 19: 267. DOI:10.1186/s12870-019-1880-1.
Zhao Z., Gitau M. M., Hu T., Xie Y., Hu L., Fu, J. 2016. Investigation of growth, free amino acids, and carbohydrate concentration in the roots of Perennial Ryegrass in response to soil salinity at subsurface soil depths. J. Am. Soc. Hortic. Sci. 141: 539-547.
Zhou, Q. Y., Tian, A. G., Zou, H. F., Xie, Z. M., Lei, G., Huang, , Wang, C. M., Wang, H. W., Zhang, J. S. and Chen, S. Y. 2008. Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants.  Plant Bioethanol. J. 6: 486-503.