RsERF40 contributes to cold stress tolerance and cell expansion of taproot in radish (Raphanus sativus L.)

Abstract

The growth and development of taproots are inhibited by cold stress in radish (Raphanus sativus L.). Ethylene-responsive element binding factors (ERF) are key participators in the cold stress response and growth regulation of plants. However, the function of ERF genes in cold tolerance and root development in radish remains elusive. Here, we showed that the secondary growth of radish taproots was inhibited by cold stress. Comparative transcriptome analysis demonstrated that the RsERF40 gene is an important regulator of the cold stress response and root growth regulation. The cold tolerance of transgenic Arabidopsis plants overexpressing the RsERF40 gene was significantly improved. Overexpressing RsERF40 in the cold-sensitive radish genotype and silencing RsERF40 in the cold-tolerant radish genotype indicated that RsERF40 was beneficial for alleviating oxidative damage under cold stress in radish. Transgenic Arabidopsis seedlings showed an increase in the elongation and radial growth of dark-grown roots. RT-qPCR analysis showed that the expression of the cold-related genes (CORs) RsCOR78 and RsCOR413PM1 and the cell wall strengthening-related genes RsCESA6 and RsEXPB3 was upregulated in transgenic Arabidopsis seedlings. Yeast one-hybrid (Y1H) and dual-luciferase reporter assays (DLA) revealed that RsERF40 directly regulates RsCOR78, RsCOR413PM1, RsCESA6 and RsEXPB3 expression, illustrating that RsERF40 enhances cold tolerance and taproot growth by modulating osmotic adjustment and cell wall mechanical strength in radish. In this study, the RsERF40-regulon was firstly found to be a new cold response pathway independent of the CBF-COR pathway conferring cold stress tolerance with increasing radish taproot growth. These results provided novel insight into the molecular mechanism underlying cold stress response and would facilitate the genetic improvement of cold tolerance in radish and other root vegetable crops.

Acknowledgements

This work was funded by grants from the National Key Technology R&D Program of China (2018YFD1000800), National Natural Science Foundation of China (32172579), Jiangsu Seed Industry Revitalization Project[JBGS(2021)071], the earmarked fund for Jiangsu Agricultural Industry Technology System [JATS(2022)463], Jiangsu Agricultural Science and Technology Innovation Fund (CX (21)2020) and the Project Founded by the Priority Academic Program Development of Jiangsu Higher Education Institutions (PAPD).

Author contributions

C.L. and L.L. conceived and designed the research. C.L. and B.M. performed the data analysis and wrote the paper. C.L., K.W., and L.F. performed experiments. L.X., Y.W., and Y.M. contributed powerful analytical tools. L.L., L.W., and Y.L. critically reviewed the manuscript. All authors read and approved the final paper.

Data availability statement

The raw data of RNA-seq was deposited in the Genome Sequence Archive (GSA) database of the National Genomics Data Center (NGDC) with BioProject ID PRJCA011633.

Conflict of interest

The authors declare that they have no conflict of interest.

References

[1] Ding Y, Yang S . Surviving and thriving: how plants perceive and respond to temperature stress. Dev Cell. 2022; 57: 947-58.

[2] Gong Z, Xiong L, Shi H et al. Plant abiotic stress response and nutrient use efficiency. Sci China Life Sci. 2020; 63: 635-74.

[3] Kidokoro S, Shinozaki K, Yamaguchi-Shinozaki K . Transcriptional regulatory network of plant cold-stress responses. Trends Plant Sci. 2022; 27: 922-35.

[4] Barah P, Jayavelu ND, Rasmussen S et al. Genome-scale cold stress response regulatory networks in ten Arabidopsis thaliana ecotypes . BMC Genomics. 2013; 14: 722.

[5] Shen C, Li D, He R et al. Comparative transcriptome analysis of RNA-seq data for cold-tolerant and cold-sensitive rice genotypes under cold stress. J Plant Biol. 2014; 57: 337-48.

[6] Ma L, Coulter JA, Liu L et al. Transcriptome analysis reveals key cold-stress-responsive genes in winter rapeseed (Brassica rapa L.). Int J Mol Sci. 2019; 20: 1071.

[7] Ritonga FN, Ngatia JN, Wang Y et al. AP2/ERF, an important cold stress-related transcription factor family in plants: a review. Physiol Mol Biol Plants. 2021; 27: 1953-68.

[8] Shi Y, Ding Y, Yang S . Molecular regulation of CBF signaling in cold acclimation. Trends Plant Sci. 2018; 23: 623-37.

[9] Ding Y, Shi Y, Yang S . Advances and challenges in uncovering cold tolerance regulatory mechanisms in plants. New Phytol. 2019; 222: 1690-704.

[10] Illgen S, Zintl S, Zuther E et al. Characterisation of the ERF102 to ERF105 genes of Arabidopsis thaliana and their role in the response to cold stress . Plant Mol Biol. 2020; 103: 303-20.

[11] Lv K, Li J, Zhao K et al. Overexpression of an AP2/ERF family gene, BpERF13, in birch enhances cold tolerance through upregulating CBF genes and mitigating reactive oxygen species . Plant Sci. 2020; 292: 110375.

[12] Wang Y, Jiang H, Mao Z et al. Ethylene increases the cold tolerance of apple via the MdERF1B-MdCIbHLH1 regulatory module. Plant J. 2021; 106: 379-93.

[13] Wei S, Li X, Lu Z et al. A transcriptional regulator that boosts grain yields and shortens the growth duration of rice. Science. 2022; 377: eabi8455.

[14] Chiab N, Kammoun M, Charfeddine S et al. Impact of the overexpression of the StDREB1 transcription factor on growth parameters, yields, and chemical composition of tubers from greenhouse and field grown potato plants . J Plant Res. 2021; 134: 249-59.

[15] Lee SY, Hwang EY, Seok HY et al. Arabidopsis AtERF71/HRE2 functions as transcriptional activator via cis-acting GCC box or DRE/CRT element and is involved in root development through regulation of root cell expansion . Plant Cell Rep. 2015; 34: 223-31.

[16] An D, Ma Q, Yan W et al. Divergent regulation of CBF regulon on cold tolerance and plant phenotype in cassava overexpressing Arabidopsis CBF3 gene . Front Plant Sci. 2016; 7: 1866.

[17] Xie Y, Xu L, Wang Y et al. Comparative proteomic analysis provides insight into a complex regulatory network of taproot formation in radish (Raphanus sativus L.). Hortic Res. 2018; 5: 51.

[18] Hoang NV, Choe G, Zheng Y et al. Identification of conserved gene-regulatory networks that integrate environmental sensing and growth in the root cambium. Curr Biol. 2020; 30: 2887-2900.e7.

[19] Wilson K, Long D, Swinburne J et al. A dissociation insertion causes a semidominant mutation that increases expression of TINY, an Arabidopsis gene related to APETALA2 . Plant Cell. 1996; 8: 659-71.

[20] Wang G, Xu X, Wang H et al. A tomato transcription factor, SlDREB3 enhances the tolerance to chilling in transgenic tomato. Plant Physiol Biochem. 2019; 142: 254-62.

[21] Hamant O, Traas J . The mechanics behind plant development. New Phytol. 2010; 185: 369-85.

[22] Desprez T, Vernhettes S, Fagard M et al. Resistance against herbicide isoxaben and cellulose deficiency caused by distinct mutations in same cellulose synthase isoform CESA6. Plant Physiol. 2002; 128: 482-90.

[23] Hu H, Zhang R, Feng S et al. Three AtCesA6-like members enhance biomass production by distinctively promoting cell growth in Arabidopsis. Plant Biotechnol J. 2018; 16: 976-88.

[24] Sakamoto S, Somssich M, Nakata MT et al. Complete substitution of a secondary cell wall with a primary cell wall in Arabidopsis. Nat Plants. 2018; 4: 777-83.

[25] Tenhaken R. Cell wall remodeling under abiotic stress. Front Plant Sci. 2015; 5: 771.

[26] Qiao G, Liu M, Song K et al. Phenotypic and comparative transcriptome analysis of different ploidy plants in Dendrocalamus latiflorus Munro. Front Plant Sci. 2017; 8: 1371.

[27] Takahashi H, Yamauchi T, Rajhi I et al. Transcript profiles in cortical cells of maize primary root during ethylene-induced lysigenous aerenchyma formation under aerobic conditions. Ann Bot. 2015; 115: 879-94.

[28] Su C, Chen K, Ding Q et al. Proteomic analysis of the function of a novel cold-regulated multispanning transmembrane protein COR413-PM1 in Arabidopsis. Int J Mol Sci. 2018; 19: 2572.

[29] Jia H, Zhang S, Ruan M et al. Analysis and application of RD29 genes in abiotic stress response . Acta Physiol Plant. 2012; 34: 1239-50.

[30] Canella D, Gilmour SJ, Kuhn LA et al. DNA binding by the Arabidopsis CBF1 transcription factor requires the PKKP/RAGRxK-FxETRHP signature sequence . Biochim Biophys Acta. 2010; 1799: 454-62.

[31] Liu J, Shi Y, Yang S . Insights into the regulation of C-repeat binding factors in plant cold signaling. J Integr Plant Biol. 2018; 60: 780-95.

[32] Stefanowska M, Kuraś M, Kacperska A . Low temperature-induced modifications in cell ultrastructure and localization of phenolics in winter oilseed rape (Brassica napus L. var. oleifera L.) leaves . Ann Bot. 2002; 90: 637-45.

[33] Heidari P, Ahmadizadeh M, Izanlo F et al. In silico study of the CESA and CSL gene family in Arabidopsis thaliana and Oryza sativa: focus on post-translation modifications . Plant Gene. 2019; 19: 100189.

[34] Zhang SS, Sun L, Dong X et al. Cellulose synthesis genes CESA6 and CSI1 are important for salt stress tolerance in Arabidopsis. J Integr Plant Biol. 2016; 58: 623-6.

[35] Feng X, Xu Y, Peng L et al. TaEXPB7-B, a β-expansin gene involved in low-temperature stress and abscisic acid responses, promotes growth and cold resistance in Arabidopsis thaliana. J Plant Physiol. 2019; 240: 153004.

[36] Yamauchi Y, Ogawa M, Kuwahara A et al. Activation of gibberellin biosynthesis and response pathways by low temperature during imbibition of Arabidopsis thaliana seeds . Plant Cell. 2004; 16: 367-78.

[37] Tang M, Xu L, Wang Y et al. Melatonin-induced DNA demethylation of metal transporters and antioxidant genes alleviates lead stress in radish plants. Hortic Res. 2021; 8: 124.

[38] Dhindsa RS, Pulmb-Dhindsa P, Thorpe TA . Leaf senescence: correlated with increased levels of membrane permeability and lipid peroxidation and decreased levels of superoxide dismutase and catalase. J Exp Bot. 1981; 32: 93-101.

[39] Jiménez-Bremont JF, Becerra-Flora A, Hernandéz-Lucero E et al. Proline accumulation in two bean cultivars under salt stress and the effect of polyamines and ornithine. Biol Plant. 2006; 50: 763-6.

[40] Updegraff DM . Semimicro determination of cellulose in biological materials. Anal Biochem. 1969; 32: 420-4.

[41] Janshekar H, Brown C, Fiechter A . Determination of biodegraded lignin by ultraviolet spectrophotometry. Anal Chim Acta. 1981; 130: 81-91.

[42] Jang G, Lee JH, Rastogi K et al. Cytokinin-dependent secondary growth determines root biomass in radish (Raphanus sativus L.). J Exp Bot. 2015; 66: 4607-19.

[43] Moreno-Sanz P, D’Amato E, Nebish A et al. An optimized histological proceeding to study the female gametophyte development in grapevine. Plant Methods. 2020; 16: 61.

[44] Kim D, Langmead B, Salzberg SL . HISAT: a fast spliced aligner with low memory requirements. Nat Methods. 2015; 12: 357-60.

[45] Love MI, Huber W, Anders S . Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 2014; 15: 550.

[46] Powers RK, Goodspeed A, Pielke-Lombardo H et al. GSEA-InContext: identifying novel and common patterns in expression experiments. Bioinformatics. 2018; 34: i555-64.

[47] Kumar S, Stecher G, Li M et al. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol. 2018; 35: 1547-9.

[48] Robert X, Gouet P . Deciphering key features in protein structures with the new ENDscript server. Nucleic Acids Res. 2014; 42: W320-4.

[49] Clough SJ, Bent AF . Floral dip: a simplified method for agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 1998; 16: 735-43.

[50] Wang Z, Chen CB, Xu YY et al. A practical vector for efficient knockdown of gene expression in rice (Oryza sativa L.). Plant Mol Biol Rep. 2004; 22: 409-17.

[51] Sparkes IA, Runions J, Kearns A et al. Rapid, transient expression of fluorescent fusion proteins in tobacco plants and generation of stably transformed plants. Nat Protoc. 2006; 1: 2019-25.

[52] Fan L, Wang Y, Xu L et al. A genome-wide association study uncovers a critical role of the RsPAP2 gene in red-skinned Raphanus sativus L. Hortic Res. 2020; 7: 164.

[53] Bournonville CF, Díaz-Ricci JC . Quantitative determination of superoxide in plant leaves using a modified NBT staining method. Phytochem Anal. 2011; 22: 268-71.

[54] Daudi A, O’Brien JA . Detection of hydrogen peroxide by DAB staining in Arabidopsis leaves . Bio Protoc. 2012; 2: e263.

[55] Liu C, Ou S, Mao B et al. Early selection of bZIP73 facilitated adaptation of japonica rice to cold climates . Nat Commun. 2018; 9: 3302.

[56] Hellens RP, Allan AC, Friel EN et al. Transient expression vectors for functional genomics, quantification of promoter activity and RNA silencing in plants. Plant Methods. 2005; 1: 13.

[57] Lin R, Ding L, Casola C et al. Transposase-derived transcription factors regulate light signaling in Arabidopsis. Science. 2007; 318: 1302-5.

[58] Wang HW, Zhang B, Hao YJ et al. The soybean Dof-type transcription factor genes, GmDof4 and GmDof11, enhance lipid content in the seeds of transgenic Arabidopsis plants . Plant J. 2007; 52: 716-29.

[59] Xu Y, Zhu X, Gong Y et al. Evaluation of reference genes for gene expression studies in radish (Raphanus sativus L.) using quantitative real-time PCR . Biochem Biophys Res Commun. 2012; 424: 398-403.

[60] Livak KJ, Schmittgen TD . Analysis of relative gene expression data using real-time quantitative PCR and the 2 ^−ΔΔCT method . Methods. 2001; 25: 402-8.