Abstract
Salinity affects plant growth, alters physiology and causes changes in DNA methylation. However, complete understanding of DNA methylation mechanism in response to salt stress remains unclear. Kenaf seedlings were pre-treated with 0, 50, 100, 150, and 200 µM of the DNA methylation inhibitor 5-azacytidine (5-azaC) for 7 days, and exposed to uniform NaCl concentration (150 mM) in nutrient solution for 9 days. Morpho-physiological, hormonal, and ultrastructural changes of leaves and DNA methylation sequencing were investigated, and function of DNA demethylase gene HcROS1 was verified by virusinduced gene silencing (VIGS) technology. Pretreatment with 5-azaC alleviated salt stress in kenaf, significantly increased the seedlings biomass, antioxidant enzyme activities, and contents of chlorophyll and carbohydrates while, reduced ROS production. Furthermore, 150 µM 5-azaC pretreatment relieved salt stress damage to cell ultrastructure, particularly chloroplast structure, whose lamellar structure was neatly stacked. Methylome analysis showed that 5-azaC pretreatment significantly reduced genomic DNA methylation, and a total of 441 differentially methylated genes (DMGs) were detected in 5-azaC150, of which 186 and 255 were characterized as up- and down- regulated DMGs, respectively. DMGs HcMDH, pyruvate kinase, triosephosphate isomerase, G6PDH, NADPH, and Hsps are mainly involved in carbon metabolism, amino acid biosynthesis, and fatty acid metabolism were characterized as differentially expressed genes (DEGs). Moreover, silencing HcROS1 could significantly increased the sensitivity of kenaf seedlings to salt. 5-azaC pretreatment altered physiological indexes, reduced DNA methylation levels, and improved kenaf salt tolerance. Furthermore, HcROS1 can positively regulate kenaf’s response to salt stress.
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Highlight
• Pretreatment with 5-azaC activates the antioxidant system, reducing reactive oxygen species (ROS) and alleviating salt stress in kenaf.
• 5-azaC pretreatment can increase the content of chlorophyll, starch, soluble sugars, and adenosine triphosphate (ATP) in kenaf seedlings under salt stress.
• MethylRAD methylome analysis showed 5-azaC pretreatment significantly decreased genome-wide DNA methylation level under salt stress.
• 5-azaC pretreatment regulated differentially methylated genes (DMGs) and metabolic pathways played essential roles in kenaf salt tolerance.
• VIGS silencing of DNA demethylase gene, of repressor of silencing 1 (HcROS1), reduced kenaf salt tolerance capacity.
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Data Availability
All the raw sequence data were submitted to the Sequence Read Archive (SRA, https://www.ncbi.nlm.nih.gov/sra/) of NCBI (accession numbers: SRR11743656-SRR11743661). Data will be made available on request.
References
Auriga A, Wrobel J (2018) Effect of effective micro-organisms on the proline and mda contents in herb plant material of Ocimum basilicum L. var. Piccolino. Fresen Environ Bull 27:7409–7415. https://doi.org/10.3390/genes13081476
Cao YB, Zhang M, Liang XY, Li FR, Shi YL, Yang XH, Jiang CF (2020) Natural variation of an EF-hand Ca-binding-protein coding gene confers saline-alkaline tolerance in maize. Nat Commun 11:186. https://doi.org/10.1038/s41467-019-14027-y
Cao Q, Huang L, Li JM, Qu P, Tao P, Crabbe MJC, Zhang TC, Qiao Q (2022) Integrated transcriptome and methylome analyses reveal the molecular regulation of drought stress in wild strawberry. Bmc Plant Biol 22. https://doi.org/10.1186/s12870-022-04006-9
Chen P, Li ZQ, Luo DJ, Jia RX, Lu H, Tang MQ, Hu YL, Yue J, Huang Z (2021) Comparative transcriptomic analysis reveals key genes and pathways in two different cadmium tolerance kenaf (Hibiscus cannabinus L.) cultivars. Chemosphere 263:128211. https://doi.org/10.1016/j.chemosphere.2020.128211
Cheng Y, Zhao Y (2007) A role for Auxin in Flower Development. J Integr Plant Biol 49:99–104. https://doi.org/10.1111/j.1744-7909.2006.00412.x
Chiavarina B, Whitaker-Menezes D, Martinez-Outschoorn UE (2011) Pyruvate kinase expression (PKM1 and PKM2) in cancer-associated fibroblasts drives stromal nutrient production and tumor growth. Cancer Biol Ther 12:1101–1113. https://doi.org/10.4161/cbt.12.12.18703
Chodavarapu RK, Feng SH, Bernatavichute YV, Chen PY, Stroud H, Yu YC, Hetzel JA, Kuo F, Kim J, Cokus SJ, Casero D, Bernal M, Huijser P, Clark AT, Krämer U, Merchant SS, Zhang XY, Jacobsen SE, Pellegrini M (2010) Relationship between nucleosome positioning and DNA methylation. Nature 466:388–392. https://doi.org/10.1038/nature09147
Cingolani P, Platts A, Wang LL, Coon M, Nguyen T, Wang L, Land SJ, Lu XY, Ruden DM (2012) A program for annotating and predicting the effects of single nucleotide polymorphisms, SnpEff: SNPs in the genome of Drosophila melanogaster strain w1118; iso-2; iso-3. https://doi.org/10.4161/fly.19695
Cramer GR, Urano K, Delrot S, Pezzotti M, Shinozaki K (2011) Effects of abiotic stress on plants: a systems biology perspective. Bmc Plant Biol 11. https://doi.org/10.1186/1471-2229-11-163
Dar AA, Choudhury AR, Kancharla PK, Arumugam N (2017) The gene in plants: occurrence, regulation, and role. Front Plant Sci 8. https://doi.org/10.3389/fpls.2017.01789
De Santis A, Landi P, Genchi G (1999) Changes of mitochondrial properties in maize seedlings associated with selection for germination at low temperature. Fatty acid composition, cytochrome oxidase, and adenine nucleotide translocase activities. Plant Physiol 119:743–754. https://doi.org/10.1104/pp.119.2.743
Feng SH, Cokus SJ, Zhang XY, Chen PY, Bostick M, Goll MG, Hetzel J, Jain J, Strauss SH, Halpern ME, Ukomadu C, Sadler KC, Pradhan S, Pellegrini M, Jacobsen SE (2010) Conservation and divergence of methylation patterning in plants and animals. P Natl Acad Sci USA 107:8689–8694. https://doi.org/10.1073/pnas.1002720107
Garg R, Narayana Chevala V, Shankar R, Jain M (2015) Divergent DNA methylation patterns associated with gene expression in rice cultivars with contrasting drought and salinity stress response. Rep 5:14922. https://doi.org/10.1038/srep14922
Gill SS, Tuteja N (2010) Reactive oxygen species and antioxidant machinery in abiotic stress tolerance in crop plants. Plant Physiol Bioch 48:909–930. https://doi.org/10.1016/j.plaphy.2010.08.016
Gong ZH, Morales-Ruiz T, Ariza RR, Roldán-Arjona T, David L, Zhu JK (2002) ROS1, a repressor of transcriptional gene silencing in Arabidopsis, encodes a DNA glycosylase/lyase. Cell 111:803–814. https://doi.org/10.1016/S0092-8674(02)01133-9
Griffin PT, Niederhuth CE, Schmitz RJ (2016) A comparative analysis of 5-Azacytidine- and Zebularine-Induced DNA demethylation. G3-Genes Genom Genet. 6:2773–2780. https://doi.org/10.1534/g3.116.030262
Grigorova B, Vaseva I, Demirevska K, Feller U (2011) Combined drought and heat stress in wheat: changes in some heat shock proteins. Biol Plant 55:105–111. https://doi.org/10.1007/s10535-011-0014-x
Hu YL, Yue J, Nie JZ, Luo DJ, Cao S, Wang CJ, Pan J, Chen CN, Zhang H, Wu QJ, TanYQ, Li R, Chen P (2023a) Salicylic acid alleviates the salt toxicity in kenaf by activating antioxidant system and regulating crucial pathways and genes. Ind Crop Prod 199. https://doi.org/10.1016/j.indcrop.2023.116691
Hu YL, Li ZQ, Tang MQ, Luo DJ, Kashif MH, Cao S, Wang CJ, Yue J, Huang Z, Pan J, Wu X, Wu QJ, Zhang H, Li R, Chen P (2023b) Comparative physiological and transcriptomic analysis provide new insights of crucial pathways and genes regulating kenaf salt tolerance. J Plant Growth Regul 42:3582–3605. https://doi.org/10.1007/s00344-022-10822-y
Huang ZK, Xiao QZ, Yu F, Gan Y, Lu CK, Peng WZ, Zhang YF, Luo X, Chen N, You WW, Ke CH (2021) Comparative transcriptome and DNA methylation analysis of phenotypic plasticity in the pacific abalone. Front Physiol 12:994. https://doi.org/10.3389/fphys.2021.683499
Im YJ, Han O, Chung GC, Cho BH (2002) Antisense expression of an Arabidopsis ω-3 fatty acid desaturase gene reduces salt/drought tolerance in transgenic tobacco plants. Mol Cells 13:264–271. https://doi.org/10.1046/j.1432-1327.1999.00382.x
Kashif MH, Tang DF, Li ZQ, Wei F, Liang ZC, Chen P (2020a) Comparative cytological and gene expression analysis reveals potential metabolic pathways and target genes responsive to salt stress in kenaf (Hibiscus cannabinus L). J Plant Growth Regul 39:1245–1260. https://doi.org/10.1007/s00344-019-10062-7
Kashif MH, Wei F, Tang DF, Tang MQ, Luo DJ, Hai L, Li R, Chen P (2020b) iTRAQ-based comparative proteomic response analysis reveals regulatory pathways and divergent protein targets associated with salt-stress tolerance in kenaf (Hibiscus cannabinus L). Ind Crop Prod 153. https://doi.org/10.1016/j.indcrop.2020.112566
Kim JS, Lim JY, Shin H, Kim BG, Yoo SD, Kim WT, Huh JH (2019) ROS1-dependent DNA demethylation is required for ABA-inducible expression. Plant Physiol 179:1810–1821. https://doi.org/10.1104/pp.18.01471
Kochian LV, Piñeros MA, Hoekenga OA (2005) The physiology, genetics and molecular biology of plant aluminum resistance and toxicity. Plant Soil 274:175–195. https://doi.org/10.1007/s11104-004-1158-7
Kornicka K, Marycz K, Maredziak M, Tomaszewski KA, Nicpon J (2017) The effects of the DNA methyltranfserases inhibitor 5-Azacitidine on ageing, oxidative stress and DNA methylation of adipose derived stem cells. J Cell Mol Med 21:387–401. https://doi.org/10.1111/jcmm.12972
Langmead B, Salzberg SL (2012) Fast gapped-read alignment with Bowtie 2. Nat Methods 9:357–U354. https://doi.org/10.1038/Nmeth.1923
Lee JH, Van Montagu M, Verbruggen N (1999) A highly conserved kinase is an essential component for stress tolerance in yeast and plant cells. P Natl Acad Sci USA 96:5873–5877. https://doi.org/10.1073/pnas.96.10.5873
Li ZQ, Hu YL, Chang MM, Kashif MH, Tang MQ, Luo DJ, Cao S, Lu H, Zhang WX, Huang Z, Yue J, Chen P (2021) 5-azacytidine pre-treatment alters DNA methylation levels and induces genes responsive to salt stress in kenaf (Hibiscus cannabinus L). Chemosphere 271. https://doi.org/10.1016/j.chemosphere.2021.129562
Liang CW, Zhang XW, Chi XY, Guan XY, Li YX, Qin S, Shao HB (2011) Serine/threonine protein kinase SpkG is a candidate for high salt resistance in the unicellular cyanobacterium synechocystis sp. PCC 6803. PLoS ONE 6:e18718. https://doi.org/10.1371/journal.pone.0018718
Liu XS, Wu H, Ji X, Stelzer Y, Wu X, Czauderna S, Shu J, Dadon D, Young RA, Jaenisch R (2016) Editing DNA methylation in the mammalian genome. Cell 167:233–247. e17
Liu CT, Mao BG, Yuan DY, Chu CC, Duan MJ (2022) Salt tolerance in rice: physiological responses and molecular mechanisms. Crop J 10:13–25. https://doi.org/10.1016/j.cj.2021.02.010
Luo DJ, Cao S, Li ZQ, Tang MQ, Wang CJ, Mackon E, Huang Z, Pan J, Wu X, Wu QJ, Zhang H, Li RX, Li X, Li R, Chen P (2022) Methyl-sensitive amplification polymorphism (MSAP) analysis provides insights into the DNA methylation underlying heterosis in kenaf drought tolerance. J Nat Fibers 19:13665–13680. https://doi.org/10.1080/15440478.2022.2103610
Luo DJ, Lu H, Wang CJ, Mubeen S, Cao S, Yue J, Pan J, Wu X, Wu QJ, Zhang H, Chen CN, Rehman M, Li R, Chen P (2023a) Physiological and DNA methylation analysis provides epigenetic insights into kenaf cadmium tolerance heterosis. Plant Sci 331. https://doi.org/10.1016/j.plantsci.2023.111663
Luo DJ, Wang CJ, Cao S, Mubeen S, Mackon E, Yue J, Rehman M, Pan J, Wu X, Wu QJ, Zhang H, Chen T, Li R, Chen P (2023b) Physiological and transcriptome analysis reveals key genes and molecular basis into heterosis of kenaf (Hibiscus cannabinus L.) under drought stress. Environ Exp Bot 209. https://doi.org/10.1016/j.envexpbot.2023.105293
Ma J, Lv CF, Xu ML, Chen GX, Lv CE, Gao ZP (2016) Photosynthesis performance, antioxidant enzymes, and ultrastructural analyses of rice seedlings under chromium stress. Environ Sci Pollut R 23:1768–1778. https://doi.org/10.1007/s11356-015-5439-x
Malatrasi M, Corradi M, Svensson JT, Close TJ, Gulli M, Marmiroli N (2006) A branched-chain amino acid aminotransferase gene isolated from is differentially regulated by drought stress. Theor Appl Genet 113:965–976. https://doi.org/10.1007/s00122-006-0339-6
Marconi G, Pace R, Traini A, Raggi L, Lutts S, Chiusano M, Guiducci M, Falcinelli M, Benincasa P, Albertini E (2013) Use of MSAP markers to analyse the effects of salt stress on DNA methylation in rapeseed (Brassica napus var. oleifera). PLoS ONE 8. https://doi.org/10.1371/journal.pone.0075597
Mirouze M, Paszkowski J (2011) Epigenetic contribution to stress adaptation in plants. Curr Opin Plant Biol 14:267–274. https://doi.org/10.1016/j.pbi.2011.03.004
Mittler R (2006) Abiotic stress, the field environment and stress combination. Trends Plant Sci 11:15–19. https://doi.org/10.1016/j.tplants.2005.11.002
Munns R, Tester M (2008) Mechanisms of salinity tolerance. Annu Rev Plant Biol 59:651–681. https://doi.org/10.1146/annurev.arplant.59.032607.092911
Murelli C, Rizza F, Marinone Albini F, Dulio A, Terzi V, Cattivelli L (2010) Metabolic changes associated with cold-acclimation in contrasting cultivars of barley. Physiol Plant 94:87–93. https://doi.org/10.1111/j.1399-3054.1995.tb00788.x
Nemoto Y, Sasakuma T (2000) Specific expression of glucose-6-phosphate dehydrogenase (G6PDH) gene by salt stress in wheat (Triticum aestivum L). Plant Sci 158:53–60. https://doi.org/10.1016/S0168-9452(00)00305-8
Ortega-Galisteo AP, Morales-Ruiz T, Ariza RR, Roldán-Arjona T (2008) Arabidopsis DEMETER-LIKE proteins DML2 and DML3 are required for appropriate distribution of DNA methylation marks. Plant Mol Biol 67:671–681. https://doi.org/10.1007/s11103-008-9346-0
Pandey G, Yadav CB, Sahu PP, Muthamilarasan M, Prasad M (2017) Salinity induced differential methylation patterns in contrasting cultivars of foxtail millet. Plant Cell Rep 36:759–772. https://doi.org/10.1007/s00299-016-2093-9
Petridis A, Therios I, Samouris G, Tananaki C (2012) Salinity-induced changes in phenolic compounds in leaves and roots of four olive cultivars and their relationship to antioxidant activity. Environ Exp Bot 79:37–43. https://doi.org/10.1016/j.envexpbot.2012.01.007
Qian YX, Hu WJ, Liao JY, Zhang J, Ren QY (2019) The dynamics of DNA methylation in the maize inbred line B73 response to heat stress at the seedling stage. Biochem Bioph Res Co 512:742–749. https://doi.org/10.1016/j.bbrc.2019.03.150
Quinlan AR, Hall IM (2010) BEDTools: a flexible suite of utilities for comparing genomic features. Bioinformatics 26:841–842. https://doi.org/10.1093/bioinformatics/btq033
Rudrabhatla P, Reddy MM, Rajasekharan R (2006) Genome-wide analysis and experimentation of plant serine/threonine/tyrosine-specific protein kinases. Plant Mol Biol 60:293–319. https://doi.org/10.1007/s11103-005-4109-7
Salekdeh GH, Siopongco J, Wade LJ, Ghareyazie B, Bennett J (2002) A proteomic approach to analyzing drought- and salt-responsiveness in rice. Field Crop Res 76, 199–219. https://doi.org/10.1016/S0378-4290(02)00040-0
Shukla S, Singh K, Patil RV, Kadam S, Bharti S, Prasad P, Singh NK, Khanna-Chopra R (2015) Genomic regions associated with grain yield under drought stress in wheat. Euphytica 203:449–467. https://doi.org/10.1007/s10681-014-1314-y
Tang K, Lang ZB, Zhang H, Zhu JK (2016) The DNA demethylase ROS1 targets genomic regions with distinct chromatin modifications. Nat Plants 2. https://doi.org/10.1038/nplants.2016.169
Tang DF, Wei F, Kashif MH, Munsif F, Zhou RY (2019) Identification and analysis of RNA editing sites in chloroplast transcripts of kenaf. https://doi.org/10.1007/s13205-019-1893-3. 3 Biotech 9
Tang M, Li R, Chen P (2023) Exogenous glutathione can alleviate chromium toxicity in kenaf by activating antioxidant system and regulating DNA methylation. https://doi.org/10.1016/j.2023.139305. Chemosphere.139305
Tesfaye M, Temple SJ, Allan DL, Vance CP, Samac DA (2001) Overexpression of malate dehydrogenase in transgenic alfalfa enhances organic acid synthesis and confers tolerance to aluminum. Plant Physiol 127:1836–1844. https://doi.org/10.1104/pp.010376
Van Zelm E, Zhang YX, Testerink C (2020) Salt tolerance mechanisms of plants. Annu Rev Plant Biol 71:403–433. https://doi.org/10.1146/annurev-arplant-050718-100005
Verhoeven KJF, Jansen JJ, van Dijk PJ, Biere A (2010) Stress-induced DNA methylation changes and their heritability in asexual dandelions. New Phytol 185:1108–1118. https://doi.org/10.1111/j.1469-8137.2009.03121.x
Wang M, Qin LM, Xie C, Li W, Yuan JR, Kong LN, Yu WL, Xia GM, Liu SW (2014) Induced and constitutive DNA methylation in a salinity-tolerant wheat introgression line. Plant Cell Physiol 55:1354–1365. https://doi.org/10.1093/pcp/pcu059
Wang S, Lv J, Zhang LL, Dou JZ, Sun Y, Li X, Fu XT, Dou HQ, Mao JX, Hu XL, Bao ZM (2015) MethylRAD: a simple and scalable method for genome-wide DNA methylation profiling using methylation-dependent restriction enzymes. Open Biol 5. https://doi.org/10.1098/rsob.150130
Yao YX, Dong QL, Zhai H, You CX, Hao YJ (2011) The functions of an apple gene in growth and tolerance to cold and salt stresses. Plant Physiol Bioch 49:257–264. https://doi.org/10.1016/j.plaphy.2010.12.009
Zemach A, McDaniel IE, Silva P, Zilberman D (2010) Genome-wide evolutionary analysis of eukaryotic DNA methylation. Science 328:916–919. https://doi.org/10.1126/science.1186366
Zhang B, Tieman DM, Jiao C, Xu YM, Chen KS, Fei ZJ, Giovannoni JJ, Klee HJ (2016) Chilling-induced tomato flavor loss is associated with altered volatile synthesis and transient changes in DNA methylation. P Natl Acad Sci USA 113:E8007–E8007. https://doi.org/10.1073/pnas.1618362113
Zhang HM, Lang ZB, Zhu JK (2018) Dynamics and function of DNA methylation in plants. Nat Rev Mol Cell Bio 19:489–506. https://doi.org/10.1038/s41580-018-0016-z
Zhang LW, Xu Y, Zhang XT, Ma XK, Zhang LL, Liao ZY, Zhang Q, Wan XB, Cheng Y, Zhang JS, Li DX, Zhang LM, Xu JT, Tao AF, Lin LH, Fang PP, Chen S, Qi R, Xu XM, Qi JM, Ming R (2020) The genome of kenaf provides insights into bast fibre and leaf shape biogenesis. Plant Biotechnol J 18:1796–1809. https://doi.org/10.1111/pbi.13341
Zheng GQ, Dong XY, Wei JP, Liu ZG, Aslam A, Cui JM, Li H, Wang Y, Tian HY, Cao XD (2022) Integrated methylome and transcriptome analysis unravel the cold tolerance mechanism in winter rapeseed (Brassica napus L). Bmc Plant Biol 22:1–18. https://doi.org/10.1186/s12870-022-03797-1
Zhong L, Xu YH, Wang JB (2010) The effect of 5-azacytidine on wheat seedlings responses to NaCl stress. Biol Plant 54:753–756. https://doi.org/10.1007/s10535-010-0135-7
Zhu JK (2016) Abiotic stress signaling and responses in plants. Cell 167:313–324. https://doi.org/10.1016/j.cell.2016.08.029
Zhu N, Cheng SF, Liu XY, Du H, Dai MQ, Zhou DX, Yang WJ, Zhao Y (2015) The R2R3-type MYB gene has a function in coordinating plant growth and salt stress tolerance in rice. Plant Sci 236:146–156. https://doi.org/10.1016/j.plantsci.2015.03.023
Zhu WL, Yang CL, Liu QY, Peng M, Li QY, Wang HL, Chen XL, Zhang B, Feng PF, Chen TC, Zeng DG, Zhao YZ (2023) Integrated analysis of DNA methylome and transcriptome reveals epigenetic regulation of cold tolerance in Litopenaeus vannamei. Int J Mol Sci 24. https://doi.org/10.3390/ijms241411573
Zorina AA, Bedbenov VS, Novikova GV, Panichkin VB, Los DA (2014) Involvement of serine/threonine protein kinases in the cold stress response in the cyanobacterium Synechocystis Sp PCC 6803: functional characterization of SpkE protein kinase. Mol Biol 48:390–398. https://doi.org/10.1134/S0026893314030212
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This research work was supported by the National Natural Science Foundation of China (Grant No. 31960368).
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PC conceived the project; ZL and DL performed the experiment and wrote the manuscript; SM, TC and RL revised the manuscript; MR constructed VIGS vector; GJ, WZ and CW performed RT-qPCR analysis; SC assisted in plant materials management. All the authors have read and approved the manuscript.
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Li, Z., Luo, D., Cao, S. et al. DNA Methylome Provide New Insights into the Physiological-Molecular Regulation of Salt Stress in Kenaf Using 5-azaC Pretreatment. J Soil Sci Plant Nutr (2024). https://doi.org/10.1007/s42729-024-01807-9
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DOI: https://doi.org/10.1007/s42729-024-01807-9