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An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF

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Abstract

Main conclusion

Overexpression of BnaWGR1 causes ROS accumulation and promotes leaf senescence. BnaWGR1 binds to promoters of RbohD and RbohF and regulates their expression.

Manipulation of leaf senescence process affects agricultural traits of crop plants, including biomass, seed yield and stress resistance. Since delayed leaf senescence usually enhances tolerance to multiple stresses, we analyzed the function of specific MAPK–WRKY cascades in abiotic and biotic stress tolerance as well as leaf senescence in oilseed rape (Brassica napus L.), one of the important oil crops. In the present study, we showed that expression of one WRKY gene from oilseed rape, BnaWGR1, induced an accumulation of reactive oxygen species (ROS), cell death and precocious leaf senescence both in Nicotiana benthamiana and transgenic Arabidopsis (Arabidopsis thaliana). BnaWGR1 regulates the transcription of two genes encoding key enzymes implicated in production of ROS, that is, respiratory burst oxidase homolog (Rboh) D and RbohF. A dual-luciferase reporter assay confirmed the transcriptional regulation of RbohD and RbohF by BnaWGR1. In vitro electrophoresis mobility shift assay (EMSA) showed that BnaWGR1 could bind to W-box cis-elements within promoters of RbohD and RbohF. Moreover, RbohD and RbohF were significantly upregulated in transgenic Arabidopsis overexpressing BnaWGR1. In summary, these results suggest that BnaWGR1 could positively regulate leaf senescence through regulating the expression of RbohD and RbohF genes.

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Abbreviations

CaMV:

Cauliflower mosaic virus

EMSA:

Electrophoretic mobility shift assay

GUS:

β-Glucuronidase

NBT:

Nitroblue tetrazolium

ROS:

Reactive oxygen species

SAG:

Senescence-associated gene

References

  • Adachi H, Nakano T, Miyagawa N, Ishihama N, Yoshioka M, Katou Y, Yaeno T, Shirasu K, Yoshioka H (2015) WRKY transcription factors phosphorylated by MAPK regulate a plant immune NADPH oxidase in Nicotiana benthamiana. Plant Cell 27:2645–2663

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Apel K, Hirt H (2004) Reactive oxygen species: metabolism, oxidative stress, and signal transduction. Annu Rev Plant Biol 55:373–399

    Article  CAS  PubMed  Google Scholar 

  • Bakshi M, Oelmuller R (2014) WRKY transcription factors: Jack of many trades in plants. Plant Signal Behav 9:e27700

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Balazadeh S, Riano-Pachon DM, Mueller-Roeber B (2008) Transcription factors regulating leaf senescence in Arabidopsis thaliana. Plant Biol (Stuttg) 10(Suppl 1):63–75

    Article  Google Scholar 

  • Baxter A, Mittler R, Suzuki N (2014) ROS as key players in plant stress signalling. J Exp Bot 65:1229–1240

    Article  CAS  PubMed  Google Scholar 

  • Besseau S, Li J, Palva ET (2012) WRKY54 and WRKY70 co-operate as negative regulators of leaf senescence in Arabidopsis thaliana. J Exp Bot 63:2667–2679

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bhattarai KK, Atamian HS, Kaloshian I, Eulgem T (2010) WRKY72-type transcription factors contribute to basal immunity in tomato and Arabidopsis as well as gene-for-gene resistance mediated by the tomato R gene Mi-1. Plant J 63:229–240

    Article  CAS  PubMed  Google Scholar 

  • Birkenbihl RP, Diezel C, Somssich IE (2012) Arabidopsis WRKY33 is a key transcriptional regulator of hormonal and metabolic responses toward Botrytis cinerea infection. Plant Physiol 159:266–285

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Bustin SA (2000) Absolute quantification of mRNA using real-time reverse transcription polymerase chain reaction assays. J Mol Endocrinol 25:169–193

    Article  CAS  PubMed  Google Scholar 

  • Chen L, Zhang L, Yu D (2010) Wounding-induced WRKY8 is involved in basal defense in Arabidopsis. Mol Plant Microbe Interact 23:558–565

    Article  CAS  PubMed  Google Scholar 

  • Ciolkowski I, Wanke D, Birkenbihl RP, Somssich IE (2008) Studies on DNA-binding selectivity of WRKY transcription factors lend structural clues into WRKY-domain function. Plant Mol Biol 68:81–92

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Clough SJ, Bent AF (1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J 16:735–743

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Daudi A, Cheng Z, O’Brien JA, Mammarella N, Khan S, Ausubel FM, Bolwell GP (2012) The apoplastic oxidative burst peroxidase in Arabidopsis is a major component of pattern-triggered immunity. Plant Cell 24:275–287

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Eulgem T, Rushton PJ, Robatzek S, Somssich IE (2000) The WRKY superfamily of plant transcription factors. Trends Plant Sci 5:199–206

    Article  CAS  PubMed  Google Scholar 

  • Gao S, Gao J, Zhu X, Song Y, Li Z, Ren G, Zhou X, Kuai B (2016) ABF2, ABF3, and ABF4 promote ABA-mediated chlorophyll degradation and leaf senescence by transcriptional activation of chlorophyll catabolic genes and senescence-associated genes in Arabidopsis. Mol Plant 9:1272–1285

    Article  CAS  PubMed  Google Scholar 

  • Gepstein S, Sabehi G, Carp MJ, Hajouj T, Nesher MF, Yariv I, Dor C, Bassani M (2003) Large-scale identification of leaf senescence-associated genes. Plant J 36:629–642

    Article  CAS  PubMed  Google Scholar 

  • Hellens RP, Allan AC, Friel EN, Bolitho K, Grafton K, Templeton MD, Karunairetnam S, Gleave AP, Laing WA (2005) Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods 1:13

    Article  PubMed  PubMed Central  Google Scholar 

  • Hinderhofer K, Zentgraf U (2001) Identification of a transcription factor specifically expressed at the onset of leaf senescence. Planta 213:469–473

    Article  CAS  PubMed  Google Scholar 

  • Huang W, Miao M, Kud J, Niu X, Ouyang B, Zhang J, Ye Z, Kuhl JC, Liu Y, Xiao F (2013) SlNAC1, a stress-related transcription factor, is fine-tuned on both the transcriptional and the post-translational level. New Phytol 197:1214–1224

    Article  CAS  PubMed  Google Scholar 

  • Jambunathan N (2010) Determination and detection of reactive oxygen species (ROS), lipid peroxidation, and electrolyte leakage in plants. Methods Mol Biol 639:292

    PubMed  Google Scholar 

  • Jiang Y, Deyholos MK (2009) Functional characterization of Arabidopsis NaCl-inducible WRKY25 and WRKY33 transcription factors in abiotic stresses. Plant Mol Biol 69:91–105

    Article  CAS  PubMed  Google Scholar 

  • Jiang Y, Liang G, Yang S, Yu D (2014) Arabidopsis WRKY57 functions as a node of convergence for jasmonic acid- and auxin-mediated signaling in jasmonic acid-induced leaf senescence. Plant Cell 26:230–245

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Kadota Y, Sklenar J, Derbyshire P, Stransfeld L, Asai S, Ntoukakis V, Jones JD, Shirasu K, Menke F, Jones A, Zipfel C (2014) Direct regulation of the NADPH oxidase RBOHD by the PRR-associated kinase BIK1 during plant immunity. Mol Cell 54:43–55

    Article  CAS  PubMed  Google Scholar 

  • Kim K, Franceschi V, Davin L, Lewis N (2006) β-Glucuronidase as reporter gene. Advantages and limitations. In: Salinas J, Sanchez-Serrano JJ (eds) Arabidopsis protocols. Humana Press Inc, Totowa, pp 263–273

    Chapter  Google Scholar 

  • Knoth C, Ringler J, Dangl JL, Eulgem T (2007) Arabidopsis WRKY70 is required for full RPP4-mediated disease resistance and basal defense against Hyaloperonospora parasitica. Mol Plant Microbe Interact 20:120–128

    Article  CAS  PubMed  Google Scholar 

  • Kwak JM, Mori IC, Pei ZM, Leonhardt N, Torres MA, Dangl JL, Bloom RE, Bodde S, Jones JD, Schroeder JI (2003) NADPH oxidase AtrbohD and AtrbohF genes function in ROS-dependent ABA signaling in Arabidopsis. EMBO J 22:2623–2633

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lee S, Seo PJ, Lee HJ, Park CM (2012) A NAC transcription factor NTL4 promotes reactive oxygen species production during drought-induced leaf senescence in Arabidopsis. Plant J 70:831–844

    Article  CAS  PubMed  Google Scholar 

  • Li L, Ye C, Zhao R, Li X, Liu WZ, Wu F, Yan J, Jiang YQ, Yang B (2015) Mitogen-activated protein kinase kinase kinase (MAPKKK) 4 from rapeseed (Brassica napus L.) is a novel member inducing ROS accumulation and cell death. Biochem Biophys Res Commun 467:792–797

    Article  CAS  PubMed  Google Scholar 

  • Liang W, Yang B, Yu B, Zhou Z, Li C, Sun Y, Zhang Y, Jia M, Wu F, Zhang H, Wang B, Deyholos M, Jiang Y-Q (2013) Identification and analysis of MKK and MPK gene families in canola (Brassica napus L.). BMC Genom 14:392

    Article  CAS  Google Scholar 

  • Liang C, Wang Y, Zhu Y, Tang J, Hu B, Liu L, Ou S, Wu H, Sun X, Chu J, Chu C (2014) OsNAP connects abscisic acid and leaf senescence by fine-tuning abscisic acid biosynthesis and directly targeting senescence-associated genes in rice. Proc Natl Acad Sci USA 111:10013–10018

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Lim PO, Nam HG (2005) The molecular and genetic control of leaf senescence and longevity in Arabidopsis. Curr Top Dev Biol 67:49–83

    Article  CAS  PubMed  Google Scholar 

  • Lim PO, Kim HJ, Nam HG (2007) Leaf senescence. Annu Rev Plant Biol 58:115–136

    Article  CAS  PubMed  Google Scholar 

  • Liu D, Shi L, Han C, Yu J, Li D, Zhang Y (2012) Validation of reference genes for gene expression studies in virus-infected Nicotiana benthamiana using quantitative real-time PCR. PLoS ONE 7:e46451

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Marino D, Dunand C, Puppo A, Pauly N (2012) A burst of plant NADPH oxidases. Trends Plant Sci 17:9–15

    Article  CAS  PubMed  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

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

    Article  CAS  PubMed  Google Scholar 

  • Navabpour S, Morris K, Allen R, Harrison E, Mackerness SAH, Buchanan-Wollaston V (2003) Expression of senescence-enhanced genes in response to oxidative stress. J Exp Bot 54:2285–2292

    Article  CAS  PubMed  Google Scholar 

  • Niu F, Wang C, Yan J, Guo X, Wu F, Yang B, Deyholos MK, Jiang YQ (2016) Functional characterization of NAC55 transcription factor from oilseed rape (Brassica napus L.) as a novel transcriptional activator modulating reactive oxygen species accumulation and cell death. Plant Mol Biol 92:89–104

    Article  CAS  PubMed  Google Scholar 

  • Oh CS, Martin GB (2011) Tomato 14-3-3 protein TFT7 interacts with a MAP kinase kinase to regulate immunity-associated programmed cell death mediated by diverse disease resistance proteins. J Biol Chem 286:14129–14136

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Pfaffl MW (2001) A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 29:e45

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rivero RM, Kojima M, Gepstein A, Sakakibara H, Mittler R, Gepstein S, Blumwald E (2007) Delayed leaf senescence induces extreme drought tolerance in a flowering plant. Proc Natl Acad Sci USA 104:19631–19636

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Robatzek S, Somssich IE (2002) Targets of AtWRKY6 regulation during plant senescence and pathogen defense. Genes Dev 16:1139–1149

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Rogers H, Munne-Bosch S (2016) Production and scavenging of reactive oxygen species and redox signaling during leaf and flower senescence: similar but different. Plant Physiol 171:1560–1568

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Torres MA, Dangl JL, Jones JD (2002) Arabidopsis gp91phox homologues AtrbohD and AtrbohF are required for accumulation of reactive oxygen intermediates in the plant defense response. Proc Natl Acad Sci USA 99:517–522

    Article  CAS  PubMed  Google Scholar 

  • Yang B, Jiang Y, Rahman MH, Deyholos MK, Kav NN (2009) Identification and expression analysis of WRKY transcription factor genes in canola (Brassica napus L.) in response to fungal pathogens and hormone treatments. BMC Plant Biol 9:68

    Article  PubMed  PubMed Central  Google Scholar 

  • Yang J, Worley E, Udvardi M (2014) A NAP-AAO3 regulatory module promotes chlorophyll degradation via ABA biosynthesis in Arabidopsis leaves. Plant Cell 26:4862–4874

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Ye Z, Rodriguez R, Tran A, Hoang H, de los Santos D, Brown S, Vellanoweth RL (2000) The developmental transition to flowering represses ascorbate peroxidase activity and induces enzymatic lipid peroxidation in leaf tissue in Arabidopsis thaliana. Plant Sci 158:115–127

    Article  CAS  PubMed  Google Scholar 

  • Yoshioka H, Numata N, Nakajima K, Katou S, Kawakita K, Rowland O, Jones JD, Doke N (2003) Nicotiana benthamiana gp91phox homologs NbrbohA and NbrbohB participate in H2O2 accumulation and resistance to Phytophthora infestans. Plant Cell 15:706–718

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • 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

    Article  CAS  PubMed  Google Scholar 

  • Zhou X, Jiang Y, Yu D (2011) WRKY22 transcription factor mediates dark-induced leaf senescence in Arabidopsis. Mol Cells 31:303–313

    Article  CAS  PubMed  PubMed Central  Google Scholar 

  • Zimmermann P, Heinlein C, Orendi G, Zentgraf U (2006) Senescence-specific regulation of catalases in Arabidopsis thaliana (L.) Heynh. Plant Cell Environ 29:1049–1060

    Article  CAS  PubMed  Google Scholar 

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Acknowledgements

This work was supported by the National Natural Science Foundation of China [No. 31301648 to BY and 31270293 to Y-QJ]. Natural Science Foundation of Shaanxi Province [no. 2014JQ3102 to BY]. The Fundamental Research Funds for the Central Universities of China [no. 2109021137, nos. 2452015413, 2452016009, to BY]. We thank Profs. Jian-Ye Chen (South China Agricultural University) and Benke Kuai (Fudan University) for technical assistance.

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Authors and Affiliations

  1. State Key Laboratory of Crop Stress Biology for Arid Areas, College of Life Sciences, Northwest A&F University, Yangling, 712100, Shaanxi, China

    Liu Yang, Chaofei Ye, Yuting Zhao, Xiaolin Cheng, Yiqiao Wang, Yuan-Qing Jiang & Bo Yang

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  1. Liu Yang
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  2. Chaofei Ye
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Correspondence to Yuan-Qing Jiang or Bo Yang.

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Yang, L., Ye, C., Zhao, Y. et al. An oilseed rape WRKY-type transcription factor regulates ROS accumulation and leaf senescence in Nicotiana benthamiana and Arabidopsis through modulating transcription of RbohD and RbohF. Planta 247, 1323–1338 (2018). https://doi.org/10.1007/s00425-018-2868-z

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