Abstract
Key message
NtRAV4 is a nucleus-localised protein and no self-activation effect. ntrav4 mutants maintain the steady state of the ROS system under drought stress by enhancing antioxidant capacity and defence system.
Abstract
The APETALA2/ethylene response factor (AP2/ERF) transcription factor (TF) family plays an important role in plant responses to environmental stresses. In this study, we identified a novel NtRAV4 TF, a member of RAV subfamily among AP2/ERF gene family, which have AP2 and B3 domain in its N- and C-terminus, respectively. Subcellular localisation and self-activation activity analysis revealed that NtRAV4 localised in the nucleus and had no self-activation effect. The overexpression and gene editing vectors of NtRAV4 were constructed by homologous recombination and CRISPR/Cas9 gene editing methods, and transformed into tobacco by agrobacterium-mediated method. ntrav4 led to the appearance of termination codon in advance and lacked the unique B3 domain of RAV subfamily protein. Further analysis displayed that knockout of the NtRAV4 in tobacco increased drought tolerance with high relative water content, accompanied by reduced stomatal aperture, density, and stomatal opening ratio compared to overexpression lines and WT. Moreover, ntrav4 knockout plants also exhibited increased osmotic tolerance with low malondialdehyde (MDA) and ion leakage (EL), less accumulation of O2•− and H2O2, and high enzymatic antioxidant (SOD, POD, CAT) activities, non-enzymatic antioxidant (AsA-GSH cycle) contents and hormone (IAA, ABA, GA3, and ZR) levels under drought stress. Furthermore, ntrav4 mutants in tobacco improved the expression levels of ROS-related proline synthesis and stress-responsive genes under osmotic stress. Our results indicate that NtRAV4 negatively regulates plant tolerance to drought stress by reducing water loss and activating the antioxidant system and stress-related gene expression to maintain the steady state of the ROS system.
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Abbreviations
- ABA:
-
Abscisic acid
- MeJA:
-
Methyl Jasmonate
- EL:
-
Electrolyte leakage
- GFP:
-
Green fluorescent protein
- GR:
-
Glutathione reductase
- IAA:
-
Indole acetic acid
- NBT:
-
Nitro blue tetrazolium
- NLS:
-
Nuclear localisation signal
- ROS:
-
Reactive oxygen species
- RWC:
-
Relative water content
- SD:
-
Standard deviation
- SOD:
-
Superoxide dismutase
- TF:
-
Transcription factor
- GA3:
-
Gibberellin A3
- ZR:
-
Zeatin riboside
References
Amara I, Odena A, Oliveira E et al (2012) Insights into Maize LEA proteins: from proteomics to functional approaches. Plant Cell Physiol 53:312–329. https://doi.org/10.1093/pcp/pcr183
Bartels D, Sunkar R (2005) Drought and salt tolerance in plants. Crit Rev Plant Sci 24:23–58. https://doi.org/10.1080/07352680590910410
Dahro B, Wang F, Peng T et al (2016) PtrA/NINV, an alkaline/neutral invertase gene of Poncirus trifoliata, confers enhanced tolerance to multiple abiotic stresses by modulating ROS levels and maintaining photosynthetic efficiency. BMC Plant Biol 16:76. https://doi.org/10.1186/s12870-016-0761-0
Dat J, Vandenabeele S, Vranova E et al (2000) Dual action of the active oxygen species during plant stress responses. Cell Mol Life Sci 57:779–795. https://doi.org/10.1007/s000180050041
Fu M, Kang HK, Son SH et al (2014) A subset of RAV transcription factors modulates drought and salt stress responses ABA-independently in Arabidopsis. Plant Cell Physiol 55:1892–1904. https://doi.org/10.1093/pcp/pcu118
Gao Y, Han D, Jia W et al (2020) Molecular characterization and systematic analysis of NtAP2/ERF in tobacco and functional determination of NtRAV-4 under drought stress. Plant Physiol Biochem 156:420–435. https://doi.org/10.1016/j.plaphy.2020.09.027
Giraudat J, Hauge BM, Valon C et al (1992) Isolation of the Arabidopsis ABI3 gene by positional cloning. Plant Cell 4:1251–1261. https://doi.org/10.1105/tpc.4.10.1251
Hu YX, Wang YH, Liu XF et al (2004) Arabidopsis RAV1 is down-egulated by brassinosteroid and may act as a negative regulator during plant development. Cell Res 14:8–15. https://doi.org/10.1038/sj.cr.7290197
Hu W, Huang C, Deng X et al (2013) TaASR1, a transcription factor gene in wheat, confers drought stress tolerance in transgenic tobacco. Plant Cell Environ 36:1449–1464. https://doi.org/10.1111/pce.12074
Jezek M, Blatt MR (2017) The membrane transport system of the guard cell and its integration for stomatal dynamics. Plant Physiol 174:487–519. https://doi.org/10.1104/pp.16.01949
Jha RK, Mishra A (2022) Introgression of SbERD4 gene encodes an early-responsive dehydration-stress protein that confers tolerance against different types of abiotic stresses in transgenic tobacco. Cells 11:62. https://doi.org/10.3390/cells11010062
Kagaya Y, Ohmiya K, Hattori T (1999) RAV1, a novel DNA-binding protein, binds to bipartite recognition sequence through two distinct DNA-binding domains uniquely found in higher plants. Nucleic Acids Res 27:470–478. https://doi.org/10.1093/nar/27.2.470
Lee SC, Luan S (2012) ABA signal transduction at the crossroad of biotic and abiotic stress responses. Plant Cell Environ 35:53–60. https://doi.org/10.1111/j.1365-3040.2011.02426.x
Li CW, Su RC, Cheng CP et al (2011) Tomato RAV transcription factor is a pivotal modulator involved in the AP2EREBP-mediated defense pathway. Plant Physiol 156:213–227. https://doi.org/10.1104/pp.lll.l74268
Li XJ, Li M, Zhou Y et al (2015) Overexpression of cotton RAV1 gene in Arabidopsis confers transgenic plants high salinity and drought sensitivity. PLoS ONE 10:e0118056. https://doi.org/10.1371/journal.pone.0118056
Li WT, Zhu ZW, Chern M et al (2017) A natural allele of a transcription factor in rice confers broad-spectrum blast resistance. Cell 170:114–126. https://doi.org/10.1016/j.cell.2017.06.008
Lim CW, Baek W, Jung J et al (2015) Function of ABA in stomatal defense against biotic and drought stresses. Int J Mol Sci 16(7):15251–15270. https://doi.org/10.3390/ijms160715251
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Lu Q, Zhao L, Li D et al (2014) A GmRAV ortholog is involved in photoperiod and sucrose control of flowering time in soybean. PLoS ONE 9:e89145. https://doi.org/10.1371/journal.pone.0089145
Matias-Hernandez L, Aguilar-Jaramillo AE, Marin-Gonzalez E et al (2014) RAV genes: regulation of floral induction and beyond. Ann Bot 114:1459–1470. https://doi.org/10.1093/aob/mcu069
Mizoi J, Shinozaki K, Yamaguchi-Shinozaki K (2012) AP2/ERF family transcription factors in plant abiotic stress responses. Biochim Biophys Acta 1819:86–96. https://doi.org/10.1016/j.bbagrm.2011.08.004
Osnato M, Castillejo C, Matias-Hernandez L et al (2012) TEMPRANILLO genes link photoperiod and gibberellin pathways to control flowering in Arabidopsis. Nat Commun 3:808. https://doi.org/10.1038/ncomms1810
Osnato M, Cereijo U, Sala J et al (2021) The floral repressors TEMPRANILLO1 and 2 modulate salt tolerance by regulating hormonal components and photo-protection in Arabidopsis. Plant J 105(1):7–21. https://doi.org/10.1111/tpj.15048
Park JM, Park C, Lee S et al (2001) Overexpression of the tobacco Tsi1 gene encoding an EREBPAP2-type transcription factor enhances resistance against pathogen attack and osmotic stress in tobacco. Plant Cell 13:1035–1046. https://doi.org/10.1105/tpc.13.5.1035
Raghavendra AS, Gonugunta VK, Christmann A et al (2010) ABA perception and signalling. Trends Plant Sci 15:395–401. https://doi.org/10.1016/j.tplants.2010.04.006
Schroeder JI, Allen GJ, Hugouvieux V et al (2001) Guard cell signal transduction. Annu Rev Plant Physiol Plant Mol Biol 52:627–658. https://doi.org/10.1146/annurev.arplant.52.1.627
Shigeoka S, Ishikawa T, Tamoi M et al (2002) Regulation and function of ascorbate peroxidase isoenzymes. J Exp Bot 53:1305–1319. https://doi.org/10.1093/jexbot/53.372.1305
Skopelitis DS, Paranychianakis NV, Paschalidis KA et al (2006) Abiotic stress generates ROS that signal expression of anionic glutamate dehydrogenases to form glutamate for proline synthesis in tobacco and grapevine. Plant Cell 18:2767–2781. https://doi.org/10.1105/tpc.105.038323
Song Z, Li Y, Jia Y et al (2021) An endoplasmic reticulum-localized NtHSP70–8 confers drought tolerance in tobacco by regulating water loss and antioxidant capacity. Environ Exp Bot 188:104519. https://doi.org/10.1016/j.envexpbot.2021.104519
Stone SL, Williams LA, Farmer LM et al (2006) KEEP ON GOING, a RING E3 ligase essential for Arabidopsis growth and development, is involved in abscisic acid signaling. Plant Cell 18:3415–3428. https://doi.org/10.1105/tpc.106.046532
Swaminathan K, Peterson K, Jack T (2008) The plant B3 superfamily. Trends Plant Sci 13:647–655. https://doi.org/10.1016/j.tplants.2008.09.006
Xie H, Bai G, Lu P et al (2022) Exogenous citric acid enhances drought tolerance in tobacco (Nicotiana tabacum). Plant Biol (stuttg) 24(2):333–343. https://doi.org/10.1111/plb.13371
Yamasaki K, Kigawa T, Seki M et al (2013) DNA-binding domains of plant-specific transcription factors: structure, function, and evolution. Trends Plant Sci 18(5):267–276. https://doi.org/10.1016/j.tplants.2012.09.001
Yang C, Zhang ZS, Gao HY et al (2014) The mechanism by which NaCl treatment alleviates PSI photoinhibition under chilling-light treatment. J Photoch Photobio B 140:286–291. https://doi.org/10.1016/j.jphotobiol.2014.08.012
Yao M, Ge W, Zhou Q et al (2021) Exogenous glutathione alleviates chilling injury in postharvest bell pepper by modulating the ascorbate-glutathione (AsA-GSH) cycle. Food Chem 352:129458. https://doi.org/10.1016/j.foodchem.2021.129458
Yuan L, Xie GZ, Zhang S et al (2021) GmLCLs negatively regulate ABA perception and signalling genes in soybean leaf dehydration response. Plant Cell Environ 44:412–424. https://doi.org/10.1111/pce.13931
Zhang X, Zhang Z, Chen J et al (2005) Expressing TERF1 in tobacco enhances drought tolerance and abscisic acid sensitivity during seedling development. Planta 222:494–501. https://doi.org/10.1007/s00425-005-1564-y
Zhao L, Luo Q, Yang C et al (2008) A RAV-like transcription factor controls photosynthesis and senescence in soybean. Planta 227:1389–1399. https://doi.org/10.1007/s00425-008-0711-7
Zhao Q, Hu RS, Liu D et al (2020) The AP2 transcription factor NtERF172 confers drought resistance by modifying NtCAT. Plant Biotechnol J 18:2444–2455. https://doi.org/10.1111/pbi.13419
Zoulias N, Harrison EL, Casson SA et al (2018) Molecular control of stomatal development. Biochem J 475:441–454. https://doi.org/10.1042/BCJ20170413
Acknowledgements
This work was supported by the National Natural Science Foundation of China (Grant No. 32102230), Key Scientific Research Foundation of the Higher Education Institutions of Henan Province (Grant No. 22A210019), and Science and Technology Research Project of China Tobacco Sichuan Industrial Co., Ltd (Grant No. 2020510000340393).
Funding
This study was funded by a grant (No. 32102230) of the National Natural Science Foundation of China, a grant (No. 22A210019) of the Key Scientific Research Foundation of the Higher Education Institutions of Henan Province, and a grant (No. 2020510000340393) of Science and Technology Research Project funded by China Tobacco Sichuan Industrial Co., Ltd.
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Gao, Y., Yang, J., Duan, W. et al. NtRAV4 negatively regulates drought tolerance in Nicotiana tabacum by enhancing antioxidant capacity and defence system. Plant Cell Rep 41, 1775–1788 (2022). https://doi.org/10.1007/s00299-022-02896-5
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DOI: https://doi.org/10.1007/s00299-022-02896-5