Physiological adaptations to osmotic stress and characterization of a polyethylene glycol-responsive gene in Braya humilis

Wang Lirong, Zhao Pengshan, Zhao Xin, Wang Xiaopeng, Ma Xiaofei, Li Yi


Braya humilis (Brassicaceae) is a widely distributed plant in arid and semi-arid regions of northern Asia. This plant is well adapted to extremely arid conditions and is a promising candidate species to discover novel drought tolerance strategies. However, not much information about the mechanism(s) mediating drought resistance in this species is currently available. Therefore, the present study aimed to characterize the physiological traits and expression patterns of a polyethylene glycol (PEG)-responsive gene in B. humilis responding to different levels of osmotic stress induced by PEG-6000. Several important physiological parameters were examined, including the levels of relative water content, soluble protein, malondialdehyde, and antioxidant enzyme activity. A tolerance threshold between 20 and 30% PEG-6000 was identified for B. humilis. The water status and oxidative damage below this threshold were maintained at a relatively constant level during the 12 h of treatment. However, once the threshold was exceeded, the water status and oxidative damage were obviously affected after treatment for 4 h. The soluble protein results suggest that B. humilis maintains a vigorous resistance to osmotic stress and that it may play a greater role in osmotic regulation at late stages of stress. Moreover, superoxide dismutase and catalase may be important at preventing oxidative damage in plants at early stages of stress, while peroxidase may be more involved in some biological processes that resist osmotic stress at the late stage, especially in severely damaged plants. Furthermore, a PEG-responsive gene, BhCIPK12, was identified by differential display reverse transcription-polymerase chain reaction (PCR), cloned, and characterized by quantitative real-time PCR. We hypothesized that this gene may play an important role in mediating osmotic stress or drought resistance in plants. Altogether, these results provide valuable insights into the mechanism(s) mediating drought tolerance in B. humilis.


Braya humilis; drought; polyethylene glycol; physiological response; BhCIPK12; expression pattern

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Morte A, Lovisolo C, Schubert A. Effect of drought stress on growth and water relations of the mycorrhizal association Helianthemum almeriense–Terfezia claveryi. Mycorrhiza. 2000;10:115–119.

Maarouf HE, Zuil-Fodil Y, Gareil M, d’Arcy-Lameta A, Pham-Thi AT. Enzymatic activity and gene expression under water stress of phospholipase D in two cultivars of Vigna unguiculata L. Walp. differing in drought tolerance. Plant Mol Biol. 1999;39(6):1257–1265.

Shinozaki K, Yamaguchi-Shinozaki K, Seki M. Regulatory network of gene expression in the drought and cold stress responses. Curr Opin Plant Biol. 2003;6:410–417.

Hasanuzzaman M, Nahar K, Gill SS, Fujita M. Drought stress responses in plants, oxidative stress, and antioxidant defense. In: Tuteja N, Gill SS, editors. Climate change and plant abiotic stress tolerance. Hoboken: Wiley Online Library Press; 2014. p. 209–250.

Hameed A, Goher M, Iqbal N. Drought induced programmed cell death and associated changes in antioxidants, proteases, and lipid peroxidation in wheat leaves. Biol Plant. 2013;57:370–374.

Haugen R, Steffes L, Wolf J, Brown P, Matzner SD, Siemens DH. Evolution of drought tolerance and defense: dependence of tradeoffs on mechanism, environment and defense switching. Oikos. 2008;117(2):231–244.

Bai LP, Sui FG, Ti-Da G, Sun ZH, Lu YY, Zhou GS. Effect of soil drought stress on leaf water status, membrane permeability and enzymatic antioxidant system of maize. Pedosphere. 2006;16:326–332.

Gajewska E, Skłodowska M, Słaba M, Mazur J. Effect of nickel on antioxidative enzyme activities, proline and chlorophyll contents in wheat shoots. Biol Plant. 2006;50:653–659.

Simova-Stoilova L, Demirevska K, Petrova T, Tsenov N, Feller U. Antioxidative protection and proteolytic activity in tolerant and sensitive wheat (Triticum aestivum L.) varieties subjected to long-term field drought. Plant Growth Regul. 2009;58(58):107–117.

Zhang W, Fei W, Pan XJ, Tian, ZG, Zhao XM. Antioxidant enzymes and photosynthetic responses to drought stress of three Canna edulis Cultivars. Korean Journal of Horticultural Science and Technology. 2013;31(6):677–686.

Bai J, Gong CM, Chen K, Kang HM, Wang G. Examination of antioxidative system’s responses in the different phases of drought stress and during recovery in desert plant Reaumuria soongorica (Pall.) Maxim. J Plant Biol. 2009;52:417–425.

Lepeduš H, Gaća V, Viljevac M, Kovač S, Fulgosi H, Šimić D, et al. Changes in photosynthetic performance and antioxidative strategies during maturation of Norway maple (Acer platanoides L.) leaves. Plant Physiol Bioch. 2011;49:368–376.

Song Y, Ci D, Tian M, Zhang D. Comparison of the physiological effects and transcriptome responses of Populus simonii under different abiotic stresses. Plant Mol Biol. 2014;86:139–156.

Zhou J, Wang X, Jiao Y, Qin Y, Liu X, He K, et al. Global genome expression analysis of rice in response to drought and high-salinity stresses in shoot, flag leaf, and panicle. Plant Mol Biol. 2007;63:591–608.

Aprile A, Mastrangelo AM, DeLeonardis AMD, Galiba G, Roncaglia E, Ferrari F, et al. Transcriptional profiling in response to terminal drought stress reveals differential responses along the wheat genome. BMC Genomics. 2009;10:279.

Cohen D, Bogeat-Triboulot MB, Tisserant E, Balzergue S, Martin-Magniette ML, Lelandais G, et al. Comparative transcriptomics of drought responses in Populus: a meta-analysis of genome-wide expression profiling in mature leaves and root apices across two genotypes. BMC Genomics. 2010;11:630.

Deeba F, Pandey AK, Ranjan S, Mishra A, Singh R, Sharma YK, et al. Physiological and proteomic responses of cotton (Gossypium herbaceum L.) to drought stress. Plant Physiol Bioch. 2012;53:6–18.

Torres GAM, Stephanie P, Fabienne CM, Christelle M, Caroline H, Christine LB. Identification of novel drought-related mRNAs in common bean roots by differential display RT-PCR. Plant Sci. 2006;171:300–307.

Kavar TMM, Kidri M, Ultar-Vozli J, Meglic V. Identification of genes involved in the response of leaves of Phaseolus vulgaris to drought stress. Molecular Breeding. 2008;21:159–172.

Padmalatha KV, Dhandapani G, Kanakachari M, Kumar S, Dass A, Patil DP, et al. Genome-wide transcriptomic analysis of cotton under drought stress reveal significant down-regulation of genes and pathways involved in fibre elongation and up-regulation of defense responsive genes. Plant Mol Biol. 2012;78:223–246.

Kolukisaoglu Ü, Weinl S, Blazevic D, Batistic O, Kudla J. Calcium sensors and their interacting protein kinases: genomics of the Arabidopsis and rice CBL–CIPK signaling networks. Plant Physiol. 2004;134:43–58.

Yong X, Yuemin H, Lizhong X. Characterization of stress-responsive CIPK genes in rice for stress tolerance improvement. Plant Physiol. 2007;144:1416–1428.

Girdhar KP, Poonam K, Amita P. Distribution and expression in plants. In: Girdhar KP, Poonam K, Amita P, editors. Global comparative analysis of CBL–CIPK gene families in plants. London: Springer Briefs in Plant Science; 2014. p. 19–23.

Zhou GY, Chen GC, Chen ZG, Si Zhen MA, Han YJ. Response of the characteristics of Alpine meadow plant community to disturbance gradient of human along Qinghai–Tibet railway: a case study in the Alpine meadow in Fenghuoshan area. J Glaciol Geocryol. 2006;28:240–248.

Liu B, Wenjin LI. Influence of nurse effect on the diversity of desert Reaumuria soongorica community under arid environment in the Loess Plateau. Journal of Arid Land Resources and Environment. 2012;26(10):117–120.

Warwick SI, Al-Shehbaz IA, Sauder C, Harris JM. Phylogeny of Braya and Neotorularia (Brassicaceae) based on nuclear ribosomal internal transcribed spacer and chloroplast trnL intron sequences. Can J Bot. 2004;82:376–392.

German DA, Friesen N, Neuffer B, Al-Shehbaz IA, Hurka H. Contribution to ITS phylogeny of the Brassicaceae, with special reference to some Asian taxa. Plant Syst Evol. 2009;283:33–56.

Zhou TY, editor. Brassicaceae. Beijing: Chinese Academy of Sciences; 1987. (Flora of China; vol 33).

Jin X, Yin H, Wang W, Qin M, Liao X, Xia L. Identification of Cd-responsive genes of Solanum nigrum seedlings through differential display. Plant Mol Biol Rep. 2009;27:563–569.

Yildiztugay E, Ozfidan-Konakci C, Kucukoduk M. Exogenous nitric oxide (as sodium nitroprusside) ameliorates polyethylene glycol-induced osmotic stress in hydroponically grown maize roots. J Plant Growth Regul. 2014;33:683–696.

Marok MA, Tarrago L, Ksas B, Henri P, Abrous-Belbachir O, Havaux M, et al. A drought-sensitive barley variety displays oxidative stress and strongly increased contents in low-molecular weight antioxidant compounds during water deficit compared to a tolerant variety. J Plant Physiol. 2013;170:633–645.

Jung S. Variation in antioxidant metabolism of young and mature leaves of Arabidopsis thaliana subjected to drought. Plant Sci. 2004;166(2):459–466.

Bradford MM. A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem. 1976;72:248–254.

H Mete T, Turgay S, Mine I, Fahrettin A, Kubilay U, Ergul S. Effect of quercetine and glutathione on the level of superoxide dismutase, catalase, malonyldialdehyde, blood pressure and neonatal outcome in a rat model of pre-eclampsia induced by NG-nitro-L-arginine-methyl ester. Eur J Obstet Gynecol. 2005;118(2):190–195.

García A. Hypoxia, reoxygenation and cytosolic manganese superoxide dismutase (cMnSOD) silencing in Litopenaeus vannamei: effects on cMnSOD transcripts, superoxide dismutase activity and superoxide anion production capacity. Dev Comp Immunol. 2010;34(11):1230–1235.

Wei X, Li D, Liu G. Anti-oxidative responses of Elodea nuttallii (Planch.) H. St. John to short-term iron exposure. Plant Physiol Bioch. 2010;48(10–11):873–878.

Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible WR. Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol. 2005;139(1):5–17.

Nopo-Olazabal C, Condori J, Nopo-Olazabal L, Medina-Bolivar F. Differential induction of antioxidant stilbenoids in hairy roots of Vitis rotundifolia treated with methyl jasmonate and hydrogen peroxide. Plant Physiol Bioch. 2014;74:50–69.

Hong SY, Seo PJ, Yang MS, Xiang F, Park CM. Exploring valid reference genes for gene expression studies in Brachypodium distachyon by real-time PCR. BMC Plant Biol. 2008;8(1):112.

Expósito-Rodríguez M, Borges AA, Borges-Pérez A, Pérez JA. Selection of internal control genes for quantitative real-time RT-PCR studies during tomato development process. BMC Plant Biol. 2008;8(6):443–447.

Nygard AB, Jørgensen CB, Cirera S, Fredholm M. Selection of reference genes for gene expression studies in pig tissues using SYBR green qPCR. BMC Mol Biol. 2007;8(16):67.

Livak KJ, Schmittgen TD. Analysis of relative gene expression data using real-time quantitative PCR and the 2−ΔΔCT method. Methods. 2001;25:402–408.

Zhang JY, Cruz de Carvalho MHC, Torres-Jerez I, Kang Y, Allen SN, Huhman DV, et al. Global reprogramming of transcription and metabolism in Medicago truncatula during progressive drought and after re-watering. Plant Cell Environ. 2014;37(11):2553–2576.

Dhindsa RS. Drought stress, enzymes of glutathione metabolism, oxidation injury, and protein synthesis in Tortula ruralis. Plant Physiol. 1991;95:648–651.

Rahman MA, Ren L, Wu W, Yan Y. Proteomic analysis of PEG-induced drought stress responsive protein in TERF1 overexpressed sugarcane (Saccharum officinarum) leaves. Plant Mol Biol Rep. 2014;33(3)716–730.

Skutnik M, Rychter AM. Differential response of antioxidant systems in leaves and roots of barley subjected to anoxia and post-anoxia. J Plant Physiol. 2009;166:926–937.

Hare PD, Cress WA, Van Staden J. Proline synthesis and degradation: a model system for elucidating stress-related signal transduction. J Exp Bot. 1999;50:413–434.

Mostajeran A, Rahimi-Eichi V. Effects of drought stress on growth and yield of rice (Oryza sativa L.) cultivars and accumulation of proline and soluble sugars in sheath and blades of their different ages leaves. Am Eurasian J Agric Environ Sci. 2009;5(2):264–272.

Sairam RK, Srivastava GC. Water stress tolerance of wheat (Triticum aestivum L.): variations in hydrogen peroxide accumulation and antioxidant activity in tolerant and susceptible genotypes. Journal of Agronomy and Crop Science. 2001;186:63–70.

Selote DS, Khanna-Chopra R. Drought acclimation confers oxidative stress tolerance by inducing co-ordinated antioxidant defense at cellular and subcellular level in leaves of wheat seedlings. Physiol Plant. 2006;127:494–506.

Mittler R, Vanderauwera S, Gollery M, van Breusegem F. Reactive oxygen gene network of plants. Trends Plant Sci. 2004;9(10):490–498.

Gill SS, Tuteja N. Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Bioch. 2010;48(12):909–930.

Tattini M, Loreto F, Fini A, Guidi L, Brunetti C, Velikova V, et al. Isoprenoids and phenylpropanoids are part of the antioxidant defense orchestrated daily by drought-stressed Platanus acerifolia plants during Mediterranean summers. New Phytol. 2015;207(3).

Rossi CF, Abreu OJT, Simone MA, Almeida VR. Superoxide dismutase, catalase and peroxidase activities do not confer protection against oxidative damage in salt-stressed cowpea leaves. New Phytol. 2004;163(3):563–571.

Chen XF, Gu ZM, Liu F, Ma BJ, Zhang HS. Molecular analysis of rice CIPKs involved in biotic and abiotic stress responses. Rice Science. 2010;6:3.

Hu W, Xia ZQ, Yan Y, Ding ZH, Tie WW, Wang LZ, et al. Genome-wide gene phylogeny of CIPK family in cassava and expression analysis of partial drought-induced genes. Front Plant Sci.