Abstract
Previous microarray analyses of Arabidopsis roots identified two closely related WRKY transcription factors (WRKY25 and WRKY33) among the transcripts that increased in abundance following treatment with NaCl. Here, we report further characterization of these genes, which we found to be inducible by a variety of abiotic stresses in an SOS-pathway independent manner, although WRKY33 induction was dependent on ABA signaling. Transcripts of both genes were detected in roots and leaves, while specific patterns of enrichment were observed in stems and floral buds for WRKY25 and WRKY33, respectively. We also identified upstream intergenic regions from each gene that were sufficient to confer stress-inducible expression on a reporter gene. However, the stress sensitivity of wrky25 null mutants did not differ from wild-type under any assay condition, while wrky33 null mutants and wrky25wrky33 double mutants showed only a moderate increase in NaCl-sensitivity, suggesting functional redundancy with other transcription factors. Nevertheless, overexpression of WRKY25 or WRKY33 was sufficient to increase Arabidopsis NaCl tolerance, while increasing sensitivity to ABA. Through microarray analyses of relevant genotypes, we identified 31 and 208 potential downstream targets of WRKY25 and WRKY33, respectively, most of which contained a W-box in their upstream regions.
Similar content being viewed by others
References
Alonso JM, Stepanova AN, Leisse TJ, Kim CJ, Chen H, Shinn P, Stevenson DK et al (2003) Genome-wide insertional mutagenesis of Arabidopsis thaliana. Science 301:653–657. doi:10.1126/science.1086391
Andreasson E, Jenkins T, Brodersen P, Thorgrimsen S, Petersen NH, Zhu S, Qiu JL, Micheelsen P, Rocher A, Petersen M, Newman MA, Bjorn Nielsen H, Hirt H, Somssich I, Mattsson O, Mundy J (2005) The MAP kinase substrate MKS1 is a regulator of plant defense responses. EMBO J 24:2579–2589. doi:10.1038/sj.emboj.7600737
Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399. doi:10.1146/annurev.arplant.55.031903.141701
Apse MP, Blumwald E (2002) Engineering salt tolerance in plants. Curr Opin Plant Biotechnol 13:146–150
Aukerman MJ, Sakai H (2003) Regulation of flowering time and floral organ identity by a microRNA and its APETALA2-like target genes. Plant Cell 15:2730–2741. doi:10.1105/tpc.016238
Baima S, Possenti M, Matteucci A, Wisman E, Altamura MM, Ruberti I, Morelli G (2001) The Arabidopsis ATHB-8 HD-zip protein acts as a differentiation-promoting transcription factor of the vascular meristems. Plant Physiol 126:643–655. doi:10.1104/pp.126.2.643
Blein JP, Coutos-Thévenot P, Marion D, Ponchet M (2002) From elicitins to lipid transfer proteins: a new insight in cell signaling involved in plant defence mechanism. Trends Plant Sci 7:293–296. doi:10.1016/S1360-1385(02)02284-7
Cavalier DM, Keegstra K (2006) Two xyloglucan xylosyltransferases catalyze the addition of multiple xylosyl residues to cellohexose. J Biol Chem 281:34197–34207. doi:10.1074/jbc.M606379200
Chinnusamy V, Schumaker K, Zhu J-K (2004) Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants. J Exp Bot 55:225–236. doi:10.1093/jxb/erh005
Chinnusamy V, Jagendorf A, Zhu J-K (2005) Understanding and improving salt tolerance in plants. Crop Sci 45:437–448
Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743. doi:10.1046/j.1365-313x.1998.00343.x
Czechowski T, Stitt M, Altmann T, Udvardi MK, Scheible W (2005) Genome-wide identification and testing of superior reference genes for transcript normalization in Arabidopsis. Plant Physiol 139:5–17. doi:10.1104/pp.105.063743
Eulgem T, Somssich IE (2007) Networks of WRKY transcription factors in defense signaling. Curr Opin Plant Biol 10:366–371. doi:10.1016/j.pbi.2007.04.020
Eulgem T, Rushton PJ, Schmelzer E, Hahlbrock K, Somssich IE (1999) Early nuclear events in plant defence signalling: rapid activation by WRKY transcription factors. EMBO J 18:4689–4699. doi:10.1093/emboj/18.17.4689
Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206. doi:10.1016/S1360-1385(00)01600-9
Finkelstein RR, Gampala SS, Rock CD (2002) Abscisic acid signaling in seeds and seedlings. Plant Cell 14:S15–S45
Fukuda Y (1997) Interaction of tobacco nuclear proteins with an elicitor responsive element in the promoter of a basic class I chitinase gene. Plant Mol Biol 34:81–87. doi:10.1023/A:1005737128339
Gadjev I, Vanderauwera S, Gechev TS, Laloi C, Minkov IN, Shulaev V, Apel K, Inze D, Mittler R, Van Breusegem F (2006) Transcriptomic footprints disclose specificity of reactive oxygen species signaling in Arabidopsis. Plant Physiol 141:436–445. doi:10.1104/pp.106.078717
Gilmour SJ, Zarka DG, Stockinger EJ, Salazar MP, Houghton JM, Thomashow MF (1998) Low temperature regulation of the Arabidopsis CBF family of AP2 transcriptional activators as an early step in cold-induced COR gene expression. Plant J 16:433–442. doi:10.1046/j.1365-313x.1998.00310.x
Hiraga S, Sasaki K, Ito H, Ohashi Y, Matsui H (2001) A large family of class III plant peroxidases. Plant Cell Physiol 42:462–468. doi:10.1093/pcp/pce061
Horton P, Park K-J, Obayashi T, Nakai K (2006) Protein subcellular localization prediction with WoLF PSORT. Proceedings of the 4th annual Asia Pacific bioinformatics conference APBC06, Taipei, Taiwan, pp 39–48
Ichimura K, Mizoguchi T, Yoshida R, Yuasa T, Shinozaki K (2000) Various abiotic stresses rapidly activate Arabidopsis MAP kinases ATMPK4 and ATMPK6. Plant J 24:655–665. doi:10.1046/j.1365-313x.2000.00913.x
Jiang Y-Q, Deyholos MK (2006) Comprehensive transcriptional profiling of NaCl-stressed Arabidopsis roots reveals novel classes of responsive genes. BMC Plant Biol 6:25. doi:10.1186/1471-2229-6-25
Jiang Y-Q, Yang B, Harris NS, Deyholos MK (2007) Comparative proteomic analysis of NaCl stress-responsive proteins in Arabidopsis roots. J Exp Bot 58:3591–3607. doi:10.1093/jxb/erm207
Johnson CS, Kolevski B, Smyth DR (2002) TRANSPARENT TESTA GLABRA2, a trichome and seed coat development gene of Arabidopsis, encodes a WRKY transcription factor. Plant Cell 14:1359–1375. doi:10.1105/tpc.001404
Kilili KG, Atanassova N, Vardanyan A, Clatot N, Al-Sabarna K, Kanellopoulos PN, Makris AM, Kampranis SC (2004) Differential roles of Tau class glutathione S-transferases in oxidative stress. J Biol Chem 279:24540–24551. doi:10.1074/jbc.M309882200
Koornneef M, Léon-Kloosterziel KM, Schwartz SH, Zeevaart JA (1998) The genetic and molecular dissection of abscisic acid biosynthesis, signal transduction in Arabidopsis. Plant Physiol Biochem 36:83–89. doi:10.1016/S0981-9428(98)80093-4
Lagace M, Matton DP (2004) Characterization of a WRKY transcription factor expressed in late torpedo-stage embryos of Solanum chacoense. Planta 219:185–189. doi:10.1007/s00425-004-1253-2
Ledger S, Strayer C, Ashton F, Kay SA, Putterill J (2001) Analysis of the function of two circadian-regulated CONSTANS-LIKE genes. Plant J 26:15–22. doi:10.1046/j.1365-313x.2001.01003.x
Li J, Brader G, Palva ET (2004) The WRKY70 transcription factor: a node of convergence for jasmonate-mediated and salicylate-mediated signals in plant defense. Plant Cell 16:319–331. doi:10.1105/tpc.016980
Lippok B, Birkenbihl RP, Rivory G, Brümmer J, Schmelzer E, Logemann E, Somssich IE (2007) Expression of AtWRKY33 encoding a pathogen- or PAMP-responsive WRKY transcription factor is regulated by a composite DNA motif containing W box elements. Mol Plant Microbe Interact 20:420–429. doi:10.1094/MPMI-20-4-0420
Liscum E, Reed JW (2002) Genetics of Aux/IAA and ARF action in plant growth and development. Plant Mol Biol 49:387–400. doi:10.1023/A:1015255030047
Ma SS, Gong QQ, Bohnert HJ (2006) Dissecting salt stress pathways. J Exp Bot 57:1097–1107. doi:10.1093/jxb/erj098
Magome H, Yamaguchi S, Hanada A, Kamiya Y, Oda K (2004) Dwarf and delayed-flowering 1, a novel Arabidopsis mutant deficient in gibberellin biosynthesis because of overexpression of a putative AP2 transcription factor. Plant J 37:720–729. doi:10.1111/j.1365-313X.2003.01998.x
Maleck K, Levine A, Eulgem T, Morgen A, Schmid J, Lawton K, Dangl JL, Dietrich RA (2000) The transcriptome of Arabidopsis thaliana during systemic acquired resistance. Nat Genet 26:403–410. doi:10.1038/82521
Mare C, Mazzucotelli E, Crosatti C, Francia E, Stanca AM, Cattivelli L (2004) Hv-WRKY38: a new transcription factor involved in cold- and dehydration-response in barley. Plant Mol Biol 55:399–416. doi:10.1007/s11103-004-0906-7
Medina J, Bargues M, Terol J, Pérez-Alonso M, Salinas J (1999) The Arabidopsis CBF gene family is composed of three genes encoding AP2 domain-containing proteins whose expression is regulated by low temperature but not by abscisic acid or dehydration. Plant Physiol 119:463–470. doi:10.1104/pp.119.2.463
Miao Y, Laun T, Zimmermann P, Zentgraf U (2004) Targets of the WRKY53 transcription factor and its role during leaf senescence in Arabidopsis. Plant Mol Biol 55:853–867
Miller G, Shulaev V, Mittler R (2008) Reactive oxygen signaling and abiotic stress. Physiol Plant. doi:101111/j1399-3054200801090x
Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45. doi:10.1093/nar/29.9.e45
Pnueli L, Hallak-Herr E, Rozenberg M, Cohen M, Goloubinoff P, Kaplan A, Mittler R (2002) Molecular and biochemical mechanisms associated with dormancy and drought tolerance in the desert legume Retama raetam. Plant J 31:319–330
Rizhsky L, Davletova S, Liang H, Mittler R (2004) The zinc finger protein Zat12 is required for cytosolic ascorbate peroxidase 1 expression during oxidative stress in Arabidopsis. J Biol Chem 279:11736–11743. doi:10.1074/jbc.M313350200
Robatzek S, Somssich IE (2001) A new member of the Arabidopsis WRKY transcription factor family, AtWRKY6, is associated with both senescence- and defence-related processes. Plant J 28:123–133. doi:10.1046/j.1365-313X.2001.01131.x
Rushton PJ, Torres JT, Parniske M, Wernert P, Hahlbrock K, Somssich IE (1996) Interaction of elicitor-induced DNA binding proteins with elicitor response elements in the promoters of parsley PR1 genes. EMBO J 15:5690–5700
Schwacke R, Fischer K, Ketelsen B, Krupinska K, Krause K (2007) Comparative survey of plastid and mitochondrial targeting properties of transcription factors in Arabidopsis and rice. Mol Genet Genomics 277:631–646. doi:10.1007/s00438-007-0214-4
Seki M, Kamei A, Yamaguchi-Shinozaki K, Shinozaki K (2003) Molecular responses to dehydration, salinity and frost: common and different paths for plant protection. Curr Opin Biotechnol 14:194–199. doi:10.1016/S0958-1669(03)00030-2
Sessions A, Burke E, Presting G, Aux G, McElver J, Patton D, Dietrich B, Ho P, Bacwaden J, Ko C, Clarke JD, Cotton D, Bullis D, Snell J, Miguel T, Hutchison D, Kimmerly B, Mitzel T, Katagiri F, Glazebrook J, Law M, Goff SA (2002) A high-throughput Arabidopsis reverse genetics system. Plant Cell 14:2985–2994. doi:10.1105/tpc.004630
Skriver K, Olsen FL, Rogers JC, Mundy J (1991) cis-acting DNA elements responsive to gibberellin and its antagonist abscisic acid. Proc Natl Acad Sci USA 88:7266–7270. doi:10.1073/pnas.88.16.7266
Stockinger EJ, Gilmour SJ, Thomashow MF (1997) Arabidopsis thaliana CBF1 encodes an AP2 domain-containing transcriptional activator that binds to the C-repeat/DRE, a cis-acting DNA regulatory element that stimulates transcription in response to low temperature and water deficit. Proc Natl Acad Sci USA 94:1035–1040. doi:10.1073/pnas.94.3.1035
Sun C, Palmqvist S, Olsson H, Boren M, Ahlandsberg S, Jansson C (2003) A novel WRKY transcription factor, SUSIBA2, participates in sugar signaling in barley by binding to the sugar-responsive elements of the iso1 promoter. Plant Cell 15:2076–2092. doi:10.1105/tpc.014597
Suzuki Y, Kawazu T, Koyama H (2004) RNA isolation from siliques, dry seeds, and other tissues of Arabidopsis thaliana. Biotechniques 37:542–544
Taylor CB (1997) Promoter fusion analysis: an insufficient measure of gene expression. Plant Cell 9:273–275
Tian Q, Reed JW (1999) Control of auxin-regulated root development by the Arabidopsis thaliana SHY2/IAA3 gene. Development 126:711–721
Ülker B, Somssich IE (2004) WRKY transcription factors: from DNA binding towards biological function. Curr Opin Plant Biol 7:491–498. doi:10.1016/j.pbi.2004.07.012
van der Graaff E, Hooykaas PJJ, Keller B (2002) Activation tagging of the two closely linked genes LEP and VAS independently affects vascular cell number. Plant J 32:819–830. doi:10.1046/j.1365-313X.2002.01470.x
Verslues PE, Agarwal M, Katiyar-Agarwal S, Zhu J, Zhu J-K (2006) Methods and concepts in quantifying resistance to dehydration, salt and freezing, abiotic stresses that affect plant water status. Plant J 45:523–539. doi:10.1111/j.1365-313X.2005.02593.x
Weigel D, Glazebrook J (2002) Arabidopsis: a laboratory manual. Cold Spring Harbor Laboratory Press, NY, USA
Xie Z, Zhang ZL, Zou XL, Huang J, Ruas P, Thompson D, Shen QJ (2005) Annotations and functional analyses of the rice WRKY gene superfamily reveal positive and negative regulators of abscisic acid signaling in aleurone cells. Plant Physiol 137:176–189. doi:10.1104/pp.104.054312
Xiong L, Lee H, Ishitani M, Zhu J-K (2002a) Regulation of osmotic stress responsive gene expression by LOS6/ABA1 locus in Arabidopsis. J Biol Chem 277:8588–8596. doi:10.1074/jbc.M109275200
Xiong L, Schumaker K, Zhu J-K (2002b) Cell signaling during cold, dehydration, and salt stress. Plant Cell 14:S165–S183. doi:10.1105/tpc.010278
Xu YH, Wang JW, Wang S, Wang JY, Chen XY (2004a) Characterization of GaWRKY1, a cotton transcription factor that regulates the sesquiterpene synthase gene (+)-delta-cadinene synthase-A. Plant Physiol 135:507–515. doi:10.1104/pp.104.038612
Xu Z, Escamilla-Trevino L, Zeng L, Lalgondar M, Bevan D, Winkel B, Mohamed A, Cheng CL, Shih MC, Poulton J, Esen A (2004b) Functional genomic analysis of Arabidopsis thaliana glycoside hydrolase family 1. Plant Mol Biol 55:343–367. doi:10.1007/s11103-004-0790-1
Yamaguchi-Shinozaki K, Shinozaki K (1994) A novel cis-acting element in an Arabidopsis gene is involved in responsiveness to dehydration, low-temperature, or high-salt stress. Plant Cell 6:251–264
Yamaguchi-Shinozaki K, Shinozaki K (2006) Transcriptional regulatory networks in cellular responses and tolerance to dehydration and cold stresses. Annu Rev Plant Biol 57:781–803. doi:10.1146/annurev.arplant.57.032905.105444
Yang LX, Wang RY, Ren F, Liu J, Cheng J, Lu YT (2005) AtGLB1 enhances the tolerance of Arabidopsis to hydrogen peroxide stress. Plant Cell Physiol 46:1309–1316. doi:10.1093/pcp/pci140
Yoo SY, Bomblies K, Yoo SK, Yang JW, Choi MS, Lee JS, Weigel D, Ahn JH (2005) The 35S promoter used in a selectable marker gene of a plant transformation vector affects the expression of the transgene. Planta 221:523–530. doi:10.1007/s00425-004-1466-4
Zhang JZ (2003) Overexpression analysis of plant transcription factors. Curr Opin Plant Biol 6:430–440. doi:10.1016/S1369-5266(03)00081-5
Zhang ZL, Xie Z, Zou X, Casaretto J, Ho TD, Shen QJ (2004) A rice WRKY gene encodes a transcriptional repressor of the gibberellin signaling pathway in aleurone cells. Plant Physiol 134:1500–1513. doi:10.1104/pp.103.034967
Zheng Z, Qamar SA, Chen Z, Mengiste T (2006) Arabidopsis WRKY33 transcription factor is required for resistance to necrotrophic fungal pathogens. Plant J 48:592–605. doi:10.1111/j.1365-313X.2006.02901.x
Zheng Z, Mosher SL, Fan B, Klessig DF, Chen Z (2007) Functional analysis of Arabidopsis WRKY25 transcription factor in plant defense against Pseudomonas syringae. BMC Plant Biol 7:2. doi:10.1186/1471-2229-7-2
Zhou QY, Tian AG, Zou HF, Xie ZM, Lei G, Huang J, Wang CM, Wang HW, Zhang JS, Chen SY (2008) Soybean WRKY-type transcription factor genes, GmWRKY13, GmWRKY21, and GmWRKY54, confer differential tolerance to abiotic stresses in transgenic Arabidopsis plants. Plant Biotechnol J 6:486–503. doi:10.1111/j.1467-7652.2008.00336.x
Zhu J-K (2001) Cell signaling under salt, water and cold stresses. Curr Opin Plant Biol 4:401–406. doi:10.1016/S1369-5266(00)00192-8
Zimmermann P, Hirsch-Hoffmann M, Hennig L, Gruissem W (2004) GENEVESTIGATOR: Arabidopsis microarray database and analysis toolbox. Plant Physiol 136:2621–2632. doi:10.1104/pp.104.046367
Zou X, Seemann JR, Neuman D, Shen QJ (2004) A WRKY gene from creosote bush encodes an activator of the abscisic acid signaling pathway. J Biol Chem 279:55770–55779. doi:10.1074/jbc.M408536200
Acknowledgments
We thank the ABRC for providing mutant seeds and Mohsen Mohammadi for help in statistical analyses. The project was funded by a NSERC (Natural Sciences and Engineering Research Council) Discovery grant to M.K.D.
Author information
Authors and Affiliations
Corresponding author
Electronic Supplementary Material
Rights and permissions
About this article
Cite this article
Jiang, Y., Deyholos, M.K. Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol 69, 91–105 (2009). https://doi.org/10.1007/s11103-008-9408-3
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11103-008-9408-3