Molecular cloning and expression under abiotic stresses and hormones of the ethylene response factor VII gene FmRAP2.12 from Fraxinus mandshurica

Abstract

RELATED TO AP2.12 (RAP2.12) is one of the Ethylene Response Factors (ERF) transcription factor and plays a key role in controlling plant root bending and responding to multiple abiotic stresses including hypoxia stress. In this study, FmRAP2.12 gene was isolated and characterized from Fraxinus mandshurica Rupr. The open reading frame (ORF) of FmRAP2.12 was 1170 bp and encoded a protein of 389 amino acids. The conserved domains, three-dimensional phylogenetic relationship of FmRAP2.12 was also investigated. Quantitative real-time (qRT-PCR) analyzed the expression of FmRAP2.12 in different tissues. The expression level of FmRAP2.12 was highest in roots followed by leaves, and lowest in male flowers. Abiotic stress and hormone signal-induced expression was established using qRT-PCR. Salt stress induced FmRAP2.12 to a double peak pattern: the first peak value was at 6 h and the second at 72 h. Drought stress also induced FmRAP2.12 to a double peak pattern: the first at 6 h and the second at 48 h. FmRAP2.12 was up-regulated after initiation of gibberellic acid (GA3) treatment, with a one peak pattern at 24 h. FmRAP2.12 may not respond to cold stress and Abscisic acid (ABA) treatment. The transient overexpression of FmRAP2.12 caused the up-expression of downstream key genes of abiotic stress response and gibberellin pathway. Our research reveals the molecular characteristic and expression patterns under abiotic stress and hormone condition of FmRAP2.12, providing support for the genetic improvement of F. mandshurica at a molecular level.

Keywords

ERF / RAP2.12 / Gene clone / Gene expression / Fraxinus mandshurica

References

[1] Achard P, Cheng H, De Grauwe L, Decat J, Schoutteten H, Moritz T, Van Der Straeten D, Peng J, Harberd NP. Integration of plant responses to environmentally activated phytohormonal signals. Science, 2006, 311: 91-94.

[2] Artlip TS, Wisniewski ME, Bassett CL, Norelli JL. CBF gene expression in peach leaf and bark tissues is gated by a circadian clock. Tree Physiol, 2013, 33: 866-877.

[3] Boutilier K, Offringa R, Sharma VK, Kieft H, Ouellet T, Zhang L, Hattori J, Liu C-M, van Lammeren AA, Miki BL. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell, 2002, 14: 1737-1749.

[4] Cao P, Azar S, SanClemente H, Mounet F, Dunand C, Marque G, Marque C, Teulières C. Genome-wide analysis of the AP2/ERF family in Eucalyptus grandis: an intriguing over-representation of stress-responsive DREB1/CBF genes. PLoS ONE, 2015, 10: e0121041.

[5] Darwish E, Testerink C, Khalil M, El-Shihy O, Munnik T. Phospholipid signaling responses in salt-stressed rice leaves. Plant Cell Physiol, 2009, 50: 986-997.

[6] Drenkhan R, Sander H, Hanso M. Introduction of Mandshurian ash (Fraxinus mandshurica Rupr.) to Estonia: is it related to the current epidemic on European ash (F. excelsior L.)?. Eur J Forest Res, 2014, 133: 769-781.

[7] Eysholdt-Derzso E, Sauter M. Root bending is antagonistically affected by hypoxia and ERF-mediated transcription via auxin signaling. Plant Physiol, 2017, 175: 00555-02017.

[8] Fukao T, Yeung E, Bailey-Serres J. The submergence tolerance regulator SUB1A mediates crosstalk between submergence and drought tolerance in rice. Plant Cell, 2011, 110: 080325.

[9] Gasteiger E, Hoogland C, Gattiker A, Duvaud S, Wilkins M, Appel R, Bairoch A, Walker J. Protein identification and analysis tools on the ExPASy server. The proteomics protocols handbook, 2005, Humana: Humana Press 571 607

[10] Gibbs DJ, Isa NM, Movahedi M, Lozano-Juste J, Mendiondo GM, Berckhan S, Marín-de la Rosa N, Conde JV, Correia CS, Pearce SP. Nitric oxide sensing in plants is mediated by proteolytic control of group VII ERF transcription factors. Mol Cell, 2014, 53: 369-379.

[11] Girardi CL, Rombaldi CV, Dal Cero J, Nobile PM, Laurens F, Bouzayen M, Quecini V. Genome-wide analysis of the AP2/ERF superfamily in apple and transcriptional evidence of ERF involvement in scab pathogenesis. Sci Hortic (Amsterdam), 2013, 151: 112-121.

[12] Giuntoli B, Shukla V, Maggiorelli F, Giorgi FM, Lombardi L, Perata P, Licausi F. Age-dependent regulation of ERF-VII transcription factor activity in Arabidopsis thaliana. Plant, Cell Environ, 2017, 40: 2333-2346.

[13] Golldack D, Li C, Mohan H, Probst N. Tolerance to drought and salt stress in plants: unraveling the signaling networks. Front Plant Sci, 2014, 5: 151.

[14] He Z, Zhan Y, Zeng F, Zhao X, Wang X. Drought physiology and gene expression characteristics of Fraxinus interspecific hybrids. Plant Growth Regul, 2016, 78: 179-193.

[15] Hinz M, Wilson IW, Yang J, Buerstenbinder K, Llewellyn D, Dennis ES, Sauter M, Dolferus R. Arabidopsis RAP2. 2: an ethylene response transcription factor that is important for hypoxia survival. Plant Physiol, 2010, 153: 757-772.

[16] Hirayama T, Shinozaki K. Research on plant abiotic stress responses in the post-genome era: past, present and future. Plant J, 2010, 61: 1041-1052.

[17] Hofmann K. TMbase-A database of membrane spanning proteins segments. Biol Chem Hoppe Seyler, 1993, 374: 166.

[18] Jung J, Won SY, Suh SC, Kim H, Wing R, Jeong Y, Hwang I, Kim M. The barley ERF-type transcription factor HvRAF confers enhanced pathogen resistance and salt tolerance in Arabidopsis. Planta, 2007, 225: 575-588.

[19] Kong DM, Preece JE, Shen HL. Somatic embryogenesis in immature cotyledons of Manchurian ash (Fraxinus mandshurica Rupr.). Plant Cell, Tissue Organ Cult, 2012, 108: 485-492.

[20] Kumar M, Choi J, An G, Kim SR. Ectopic expression of OsSta2 enhances salt stress tolerance in rice. Front Plant Sci, 2017, 8: 316.

[21] Li Y, Wang X, Wang C, Liu F, Zhang Q. Spatial variation in diurnal courses of stem temperature of Betula platyphylla and Fraxinus mandshurica and its influencing factors. Ying Yong Sheng Tai Xue Bao, 2017, 28: 3197-3207.

[22] Licausi F, Van Dongen JT, Giuntoli B, Novi G, Santaniello A, Geigenberger P, Perata P. HRE1 and HRE2, two hypoxia-inducible ethylene response factors, affect anaerobic responses in Arabidopsis thaliana. Plant J, 2010, 62: 302-315.

[23] Licausi F, Kosmacz M, Weits DA, Giuntoli B, Giorgi FM, Voesenek LA, Perata P, van Dongen JT. Oxygen sensing in plants is mediated by an N-end rule pathway for protein destabilization. Nature, 2011, 479: 419.

[24] Lim CW, Baek W, Lim S, Han SW, Lee SC. Expression and functional roles of the pepper pathogen–induced bZIP Transcription factor CabZIP2 in enhanced disease resistance to bacterial pathogen infection. Mol Plant Microbe Interact, 2015, 28: 825-833.

[25] 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.

[26] Looney CE, D’Amato AW, Palik BJ, Slesak RA. Canopy treatment influences growth of replacement tree species in Fraxinus nigra forests threatened by the emerald ash borer in Minnesota, USA. Can J For Res, 2016, 47: 183-192.

[27] Marín-de la Rosa N, Sotillo B, Miskolczi P, Gibbs DJ, Vicente J, Carbonero P, Oñate-Sánchez L, Holdsworth MJ, Bhalerao R, Alabadí D. Large-scale identification of gibberellin-related transcription factors defines Group VII ERFs as functional DELLA partners. Plant Physiol, 2014, 114: 244723.

[28] Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K. AP2/ERF family transcription factors in plant abiotic stress responses. BBA-Gene Regul Mech, 2012, 1819: 86-96.

[29] Munns R. Comparative physiology of salt and water stress. Plant, Cell Environ, 2002, 25: 239-250.

[30] Nakano T, Suzuki K, Fujimura T, Shinshi H. Genome-wide analysis of the ERF gene family in Arabidopsis and rice. Plant Physiol, 2006, 140: 411-432.

[31] Papdi C, Pérez-Salamó I, Joseph MP, Giuntoli B, Bögre L, Koncz C, Szabados L. The low oxygen, oxidative and osmotic stress responses synergistically act through the ethylene response factor VII genes RAP 2.12, RAP 2.2 and RAP 2.3. Plant J, 2015, 82: 772-784.

[32] Park HY, Seok HY, Woo DH, Lee SY, Tarte VN, Lee EH, Lee CH, Moon YH. AtERF71/HRE2 transcription factor mediates osmotic stress response as well as hypoxia response in Arabidopsis. Biochem Bioph Res Co, 2011, 414: 135-141.

[33] Patterson JH, Newbigin E, Tester M, Bacic A, Roessner U. Metabolic responses to salt stress of barley (Hordeum vulgare L.) cultivars, Sahara and Clipper, which differ in salinity tolerance. J Exp Bot, 2009, 60: 4089-4103.

[34] Rasheed S, Bashir K, Kim JM, Ando M, Tanaka M, Seki M. The modulation of acetic acid pathway genes in Arabidopsis improves survival under drought stress. Sci Rep, 2018, 8: 7831.

[35] Rehman S, Mahmood T. Functional role of DREB and ERF transcription factors: regulating stress-responsive network in plants. Acta Physiol Plant, 2015, 37: 178.

[36] Rushton DL, Tripathi P, Rabara RC, Lin J, Ringler P, Boken AK, Langum TJ, Smidt L, Boomsma DD, Emme NJ. WRKY transcription factors: key components in abscisic acid signalling. Plant Biotechnol J, 2012, 10: 2-11.

[37] Sakuma Y, Liu Q, Dubouzet JG, Abe H, Shinozaki K, Yamaguchi-Shinozaki K. DNA-binding specificity of the ERF/AP2 domain of Arabidopsis DREBs, transcription factors involved in dehydration-and cold-inducible gene expression. Biochem Biophys Res Commun, 2002, 290: 998-1009.

[38] Sen TZ, Jernigan RL, Garnier J, Kloczkowski A. GOR V server for protein secondary structure prediction. Bioinformatics, 2005, 21: 2787-2788.

[39] Shi HT, Liu W, Yao Y, Wei YX, Chan ZL. Alcohol dehydrogenase 1 (ADH1) confers both abiotic and biotic stress resistance in Arabidopsis. Plant Sci, 2017, 262: 24-31.

[40] Showalter DN, Villari C, Herms DA, Bonello P. Drought stress increased survival and development of emerald ash borer larvae on coevolved M anchurian ash and implicates phloem-based traits in resistance. Agr Forest Entomol, 2018, 20: 170-179.

[41] Thirugnanasambantham K, Durairaj S, Saravanan S, Karikalan K, Muralidaran S, Islam VIH. Role of ethylene response transcription factor (ERF) and its regulation in response to stress encountered by plants. Plant Mol Biol Rep, 2015, 33: 347-357.

[42] van Dongen JT, Licausi F. Oxygen sensing and signaling. Annu Rev Plant Biol, 2015, 66: 345-367.

[43] Villari C, Herms DA, Whitehill JG, Cipollini D, Bonello P. Progress and gaps in understanding mechanisms of ash tree resistance to emerald ash borer, a model for wood-boring insects that kill angiosperms. New Phytol, 2016, 209: 63-79.

[44] Wang H, Wang H, Shao H, Tang X. Recent advances in utilizing transcription factors to improve plant abiotic stress tolerance by transgenic technology. Front Plant Sci, 2016, 7: 67.

[45] Wang Y, Virtanen J, Xue Z, Tesmer JJ, Zhang Y. Using iterative fragment assembly and progressive sequence truncation to facilitate phasing and crystal structure determination of distantly related proteins. Acta Crystallogr D, 2016, 72: 616-628.

[46] Wang Y, Virtanen J, Xue Z, Zhang Y. I-TASSER-MR: automated molecular replacement for distant-homology proteins using iterative fragment assembly and progressive sequence truncation. Nucleic Acids Res, 2017, 45: 429-434.

[47] Wang M, Zhang WW, Li N, Liu YY, Zheng XB, Hao GY. Photosynthesis and growth responses of Fraxinus mandshurica Rupr. seedlings to a gradient of simulated nitrogen deposition. Ann Forest Sci, 2018, 75: 1123.

[48] White MD, Klecker M, Hopkinson RJ, Weits DA, Mueller C, Naumann C, O’Neill R, Wickens J, Yang J, Brooks-Bartlett JC. Plant cysteine oxidases are dioxygenases that directly enable arginyl transferase-catalysed arginylation of N-end rule targets. Nat Commun, 2017, 8: 14690.

[49] Xu D, Zhang Y. Improving the physical realism and structural accuracy of protein models by a two-step atomic-level energy minimization. Biophys J, 2011, 101: 2525-2534.

[50] Yao W, Wang S, Zhou B, Jiang T. Transgenic poplar overexpressing the endogenous transcription factor ERF76 gene improves salinity tolerance. Tree Physiol, 2016, 36: 896-908.

[51] Zeng FS, Li LL, Liang NS, Wang X, Li X, Zhan YG. Salt tolerance and alterations in cytosine methylation in the interspecific hybrids of Fraxinus velutina and Fraxinus mandshurica. Euphytica, 2015, 205: 721-737.

[52] Zhang Y, Wang Y, Wang C. Gene overexpression and gene silencing in Birch using an Agrobacterium-mediated transient expression system. Mol Biol Rep, 2012, 39: 5537-5541.

[53] Zhang P, Liu D, Shen H, Li Y, Nie Y. Proteome analysis of dormancy-released seeds of Fraxinus mandshurica Rupr. in response to re-dehydration under different conditions. Int J Mol Sci, 2015, 16: 4713-4730.

[54] Zhao T, Liang D, Wang P, Liu J, Ma F. Genome-wide analysis and expression profiling of the DREB transcription factor gene family in Malus under abiotic stress. Mol Genet Genomics, 2012, 287: 423-436.

[55] Zhou Y, He Y, Sun J, Zhang J, Zhan Y. Effects of nitrogen deficiency on physiology and growth of Fraxinus mandshurica. Pak J Bot, 2018, 50: 179-187.

[56] Zhu Z, Qi F, Yan C, Zhan Y. Sexually different morphological, physiological and molecular responses of Fraxinus mandshurica flowers to floral development and chilling stress. Plant Physiol Bioch, 2016, 99: 97-107.