Regulatory role of MicroRNA164 in heat and salinity stress responses via candidate target genes during seed germination in petunia

Abstract

MicroRNA164 (miR164) is a highly conserved miRNA that targets NAC transcription factors in plants. This study explores its role in regulating stress responses in petunia by generating MIR164 overexpression (MIR164-ox) and miR164 suppression (miR164-STTM) lines. Under heat stress, MIR164-ox lines exhibited enhanced tolerance, while miR164-STTM lines showed reduced tolerance with growth reduction in both shoots and roots. Conversely, under salinity stress, miR164-STTM lines displayed improved tolerance, while MIR164-ox lines were more sensitive, suggesting that miR164 may have a differential regulatory effect on heat and salinity stress responses. To further investigate the molecular mechanisms underlying these differential stress responses mediated by miR164, we conducted a bioinformatic analysis of the NAC transcription factor family in Petunia, the primary targets of miR164. A genome-wide analysis of the NAC gene family in Petunia identified 80 NAC genes in P. axillaris and 91 in P. inflata. Phylogenetic analysis revealed nine distinct clades, indicating both conserved and divergent functions of NAC proteins. Expression analysis of miR164-targeted NAC genes showed tissue- and stress-specific patterns. Protein–protein interaction (PPI) network and Gene Ontology (GO) enrichment analyses provided insights into the potential functions of NAC proteins in petunia, particularly in secondary cell wall biogenesis and xylem development. Real-time qRT-PCR analysis revealed differential expression of specific NAC genes in miR164 transgenic lines under heat and salinity stress, with NAC06 and NAC39/NAC54 showing the most pronounced changes, suggesting their potential involvement in heat and salinity stress responses, respectively. Taken together, our findings suggest that miR164 plays a crucial role in regulating NAC gene expression and stress responses in petunia, providing a foundation for further functional characterization of miR164-NAC regulatory modules in plant stress adaptation.

Keywords

MicroRNA164 / NAC transcription factors / Petunia / Heat and salinity stress / Genome-wide analysis / Biological Sciences / Biochemistry and Cell Biology / Genetics

References

[1] Addo-QuayeC, EshooTW, BartelDP, AxtellMJ. Endogenous siRNA and miRNA targets identified by sequencing of the Arabidopsis degradome. Curr Biol, 2008, 18: 758-762.

[2] AminiZ, SalehiH, ChehraziM, EtemadiM, XiangM. miRNAs and their target genes play a critical role in response to heat stress in Cynodon dactylon (L.) Pers. Mol Biotechnol., 2023, 65: 2004-17.

[3] BaileyTL, BodenM, BuskeFA, FrithM, GrantCE, ClementiL, et al. . MEME SUITE: tools for motif discovery and searching. Nucleic Acids Res, 2009, 37: W202-W208.

[4] ChenC, ChenH, ZhangY, ThomasHR, FrankMH, HeY, et al. . TBtools: an integrative toolkit developed for interactive analyses of big biological data. Mol Plant, 2020, 13: 1194-1202.

[5] ChibaY, MinetaK, HiraiMY, SuzukiY, KanayaS, TakahashiH, et al. . Changes in mRNA stability associated with cold stress in Arabidopsis cells. Plant Cell Physiol, 2013, 54: 180-194.

[6] CuiJ, SunZ, LiJ, ChengD, LuoC, DaiC. Characterization of miRNA160/164 and their targets expression of beet (Beta vulgaris) seedlings under the salt tolerance. Plant Mol Biol Rep, 2018, 36: 790-799.

[7] DiaoW, SnyderJC, WangS, LiuJ, PanB, GuoG, et al. . Genome-wide analyses of the NAC transcription factor gene family in pepper (Capsicum annuum L.): chromosome location, phylogeny, structure, expression patterns, cis-elements in the promoter, and interaction network. Int J Mol Sci., 2018, 19: 1028.

[8] DuanX, TianD, GaoP, SunY, PengX, WenJ, et al. . Transcriptome-wide identification of dark-and salt-induced senescence-related NAC gene family members in alfalfa. Int J Mol Sci, 2024, 25: 8908.

[9] FangY, XieK, XiongL. Conserved miR164-targeted NAC genes negatively regulate drought resistance in rice. J Exp Bot, 2014, 65: 2119-2135.

[10] FuX, ZhuL, YuX, YangQ, YuanF, JinH. Identification of NAC transcription factors in suaeda glauca and their responses to salt stress. Curr Issues Mol Biol, 2024, 46: 8741-8751.

[11] GeSX, JungD, YaoR. ShinyGO: a graphical gene-set enrichment tool for animals and plants. Bioinformatics, 2020, 36: 2628-2629.

[12] GermanMA, PillayM, JeongDH, HetawalA, LuoS, JanardhananP, et al. . Global identification of microRNA–target RNA pairs by parallel analysis of RNA ends. Nat Biotechnol, 2008, 26: 941-946.

[13] GuerinC, RocheJ, AllardV, RavelC, MouzeyarS, BouzidiMF. Genome-wide analysis, expansion and expression of the NAC family under drought and heat stresses in bread wheat (T. aestivum L.). PLoS One., 2019, 14: e0213390.

[14] GuoHS, XieQ, FeiJF, ChuaNH. MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development. Plant Cell, 2005, 17: 1376-1386.

[15] GuoZ, KuangZ, ZhaoY, DengY, HeH, WanM, et al. . PmiREN2. 0: from data annotation to functional exploration of plant microRNAs. Nucleic Acids Res., 2022, 50: D1475-82.

[16] HernandezY, GoswamiK, Sanan-MishraN. Stress induced dynamic adjustment of conserved miR164: NAC module. Plant Environ Interact, 2020, 1: 134-151.

[17] IqbalA, BocianJ, HameedA, OrczykW, Nadolska-OrczykA. Cis-regulation by NACs: a promising frontier in wheat crop improvement. Int J Mol Sci, 2022, 23: 15431.

[18] JiangT, LiuF, RodriguezK, RabanalN, LianZ, LinM, et al. . Genome-wide analysis of the SPL gene family in lettuce and its role in miR156/SPL module mediated plant development and regeneration. Hortic Plant J, 2025.

[19] Jin Y, Chen Z, Liu X, Zhou X. Evaluating the microRNA targeting sites by luciferase reporter gene assay. Ying SY, editors. Methods Mol Biol. 2013:117–27. https://doi.org/10.1007/978-1-62703-083-0_10.

[20] KamiuchiY, YamamotoK, FurutaniM, TasakaM, AidaM. The CUC1 and CUC2 genes promote carpel margin meristem formation during Arabidopsis gynoecium development. Front Plant Sci, 2014, 5: 165.

[21] KimHJ, NamHG, LimPO. Regulatory network of NAC transcription factors in leaf senescence. Curr Opin Plant Biol, 2016, 33: 48-56.

[22] KozomaraA, BirgaoanuM, Griffiths-JonesS. miRBase: from microRNA sequences to function. Nucleic Acids Res, 2019, 47: D155-D162.

[23] KumarS, StecherG, LiM, KnyazC, TamuraK. MEGA X: molecular evolutionary genetics analysis across computing platforms. Mol Biol Evol, 2018, 35: 1547-1549.

[24] LeeMH, JeonHS, KimHG, ParkOK. An Arabidopsis NAC transcription factor NAC4 promotes pathogen-induced cell death under negative regulation by microRNA164. New Phytol, 2017, 214: 343-360.

[25] LescotM, DéhaisP, ThijsG, MarchalK, MoreauY, Van de PeerY, et al. . PlantCARE, a database of plant cis-acting regulatory elements and a portal to tools for in silico analysis of promoter sequences. Nucleic Acids Res, 2002, 30: 325-327.

[26] LetunicI, BorkP. Interactive tree of life (iTOL) v6: recent updates to the phylogenetic tree display and annotation tool. Nucleic Acids Res, 2024, 52: W78-82.

[27] LiJ, GuoG, GuoW, GuoG, TongD, NiZ, et al. . miRNA164-directed cleavage of ZmNAC1 confers lateral root development in maize (Zea mays L.). BMC Plant Biol., 2012, 12: 220.

[28] LiQ, ZhangH, YangY, TangK, YangY, OuyangW, et al. . Genome-wide identification of NAC family genes and their expression analyses in response to osmotic stress in cannabis sativa L. Int J Mol Sci, 2024, 25: 9466.

[29] LiuQ, WangF, AxtellMJ. Analysis of complementarity requirements for plant microRNA targeting using a Nicotiana benthamiana quantitative transient assay. Plant Cell, 2014, 26: 741-753.

[30] LivakKJ, SchmittgenTD. Analysis of relative gene expression data using real-time quantitative PCR and the 2− ΔΔCT method. Methods, 2001, 25: 402-408.

[31] LuX, DunH, LianC, ZhangX, YinW, XiaX. The role of peu-miR164 and its target PeNAC genes in response to abiotic stress in Populus euphratica. Plant Physiol Biochem, 2017, 115: 418-438.

[32] MallonaI, LischewskiS, WeissJ, HauseB, Egea-CortinesM. Validation of reference genes for quantitative real-time PCR during leaf and flower development in Petunia hybrida. BMC Plant Biol, 2010, 10: 4.

[33] MaoX, ZhangH, QianX, LiA, ZhaoG, JingR. TaNAC2, a NAC-type wheat transcription factor conferring enhanced multiple abiotic stress tolerances in Arabidopsis. J Exp Bot, 2012, 63: 2933-2946.

[34] MasoodJ, ZhuW, FuY, LiZ, ZhouY, ZhangD, et al. . Scaffold protein RACK1A positively regulates leaf senescence by coordinating the EIN3-miR164-ORE1 transcriptional cascade in Arabidopsis. J Integr Plant Biol, 2023, 65: 1703-1716.

[35] MazalmazraeiT, NejadsadeghiL, Mehdi KhanlouK, AhmadiDN. Comparative analysis of differentially expressed miRNAs in leaves of three sugarcanes (Saacharum officinarum L.) cultivars during salinity stress. Mol Biol Rep., 2023, 50: 485-92.

[36] NakashimaK, TranLSP, Van NguyenD, FujitaM, MaruyamaK, TodakaD, et al. . Functional analysis of a NAC-type transcription factor OsNAC6 involved in abiotic and biotic stress-responsive gene expression in rice. Plant J, 2007, 51: 617-630.

[37] NguyenDQ, NguyenNL, NguyenVT, TranTHG, NguyenTH, NguyenTKL, et al. . Comparative analysis of microRNA expression profiles in shoot and root tissues of contrasting rice cultivars (Oryza sativa L.) with different salt stress tolerance. Plos One., 2023, 18: e0286140.

[38] Oda-YamamizoC, MitsudaN, SakamotoS, OgawaD, Ohme-TakagiM, OhmiyaA. The NAC transcription factor ANAC046 is a positive regulator of chlorophyll degradation and senescence in Arabidopsis leaves. Sci Rep, 2016, 6: 23609.

[39] PuranikS, SahuPP, SrivastavaPS, PrasadM. NAC proteins: regulation and role in stress tolerance. Trends Plant Sci., 2012, 17: 369-81.

[40] QingC, WenM, Ruo-yuN, Bao-weiW, Li-jianG, MengM, et al. . The miR164-TaNAC14 module regulates root development and abiotic-stress tolerance in wheat seedlings. J Integr Agric, 2023, 22: 981-998.

[41] RamanS, GrebT, PeaucelleA, BleinT, LaufsP, TheresK. Interplay of miR164, CUP-SHAPED COTYLEDON genes and LATERAL SUPPRESSOR controls axillary meristem formation in Arabidopsis thaliana. Plant J, 2008, 55: 65-76.

[42] RhoadesMW, ReinhartBJ, LimLP, BurgeCB, BartelB, BartelDP. Prediction of plant microRNA targets. Cell, 2002, 110: 513-520.

[43] Rodríguez-CazorlaE, AndújarA, RipollJJ, BaileyLJ, Martínez-LabordaA, YanofskyMF, et al. . 3’rapid amplification of cDNA ends (3’RACE) using Arabidopsis samples. Bio Protoc., 2015, 5: e1604.

[44] FrohmanMA. 5′ end cDNA amplification using classic RACE. CSH Protoc., 2006, 2006: pdb.prot4131.

[45] ShanT, FuR, XieY, ChenQ, WangY, LiZ, et al. . Regulatory mechanism of maize (Zea mays L.) miR164 in salt stress response. Russ J Genet., 2020, 56: 835-42.

[46] Sieber P, Wellmer F, Gheyselinck J, Riechmann JL, Meyerowitz EM. Redundancy and specialization among plant microRNAs: role of the MIR164 family in developmental robustness. 2007;134:1051–60 . https://doi.org/10.1242/dev.02817.

[47] SinumpornS. A drought senescence NAC gene, OMTN4, confers seed development defects in rice. RMUTI Journal, 2020, 13: 150-161

[48] SmootME, OnoK, RuscheinskiJ, WangPL, IdekerT. Cytoscape 2.8: new features for data integration and network visualization. Bioinformatics., 2011, 27: 431-2.

[49] SongSY, ChenY, ChenJ, DaiXY, ZhangWH. Physiological mechanisms underlying OsNAC5-dependent tolerance of rice plants to abiotic stress. Planta, 2011, 234: 331-345.

[50] SunkarR, LiYF, JagadeeswaranG. Functions of microRNAs in plant stress responses. Trends Plant Sci., 2012, 17: 196-203.

[51] SzklarczykD, GableAL, LyonD, JungeA, WyderS, Huerta-CepasJ, et al. . STRING v11: protein–protein association networks with increased coverage, supporting functional discovery in genome-wide experimental datasets. Nucleic Acids Res, 2019, 47: D607-D613.

[52] TangG, YanJ, GuY, QiaoM, FanR, MaoY, et al. . Construction of short tandem target mimic (STTM) to block the functions of plant and animal microRNAs. Methods, 2012, 58: 118-125.

[53] TsaiWA, SungPH, KuoYW, ChenMC, JengST, LinJS. Involvement of microRNA164 in responses to heat stress in Arabidopsis. Plant Sci, 2023, 329: 111598.

[54] VillarinoGH, HuQ, ScanlonMJ, MuellerL, BombarelyA, MattsonNS. Dissecting tissue-specific transcriptomic responses from leaf and roots under salt stress in petunia hybrida Mitchell. Genes (Basel), 2017, 8: 195.

[55] WanX, WangZ, DuanW, HuangT, SongH, XuX. Knockdown of sly-miR164a enhanced plant salt tolerance and improved preharvest and postharvest fruit nutrition of tomato. Int J Mol Sci, 2023, 24: 4639.

[56] WangA, HuJ, GaoC, ChenG, WangB, LinC, et al. . Genome-wide analysis of long non-coding RNAs unveils the regulatory roles in the heat tolerance of Chinese cabbage (Brassica rapa ssp. chinensis). Sci Rep., 2019, 9: 5002.

[57] WangQ, ZhouL, YuanM, PengF, ZhuX, WangY. Genome-wide identification of NAC gene family members of tree peony (Paeonia suffruticosa Andrews) and their expression under heat and waterlogging stress. Int J Mol Sci., 2024, 25: 9312.

[58] XueGP, WayHM, RichardsonT, DrenthJ, JoycePA, McIntyreCL. Overexpression of TaNAC69 leads to enhanced transcript levels of stress up-regulated genes and dehydration tolerance in bread wheat. Mol Plant, 2011, 4: 697-712.

[59] YueY, JiangH, DuJ, ShiL, BinQ, YangX, et al. . Variations in physiological response and expression profiles of proline metabolism-related genes and heat shock transcription factor genes in petunia subjected to heat stress. Sci Hortic, 2019, 258: 108811.

[60] ZhuJK. Abiotic stress signaling and responses in plants. Cell, 2016, 167: 313-324.

[61] ZhuoX, ZhengT, ZhangZ, ZhangY, JiangL, AhmadS, et al. . Genome-wide analysis of the NAC transcription factor gene family reveals differential expression patterns and cold-stress responses in the woody plant Prunus mume. Genes (Basel), 2018, 9: 494.

Funding

National Institute of Food and Agriculture(2019-67013-29236)

RIGHTS & PERMISSIONS

The Author(s)