Single-cell RNA sequencing reveals developmental trajectories and environmental regulation of callus formation in Arabidopsis

Abstract

Plant cells exhibit an extraordinary regenerative potential, achieving cellular totipotency by dedifferentiating to form new tissues. While significant progress has been made in understanding cell fate mechanisms, the regulatory networks governing callus cell development remain insufficiently explored, particularly regarding cell classification, morphology, and regulatory processes. This study provides a detailed investigation into the developmental dynamics and transcriptomic profiles of callus cells in Arabidopsis at key stages: initiation, proliferation, and greening. Employing single-cell RNA sequencing and UMAP-based clustering, we annotated cell clusters based on highly enriched gene expressions. Developmental trajectories were further mapped through pseudotime analysis, revealing distinct transcription factor networks. Additionally, functional analysis of key regulatory genes was conducted using mutant and overexpression lines, affirming their roles in callus development. Gene Ontology analysis highlighted the involvement of environmental factors—low oxygen and salinity promoted callus formation, while light inhibited it, though essential for greening. These findings shed light on the complex regulatory landscape of plant tissue regeneration and guide future research avenues.

Keywords

Plant regeneration / Callus cells / Dedifferentiation / ScRNA-seq / Developmental trajectories / Environmental factors

References

[1] AlonsoJM, StepanovaAN, SolanoR, WismanE, FerrariS, AusubelFM, EckerJR. Five components of the ethylene-response pathway identified in a screen for weak ethylene-insensitive mutants in Arabidopsis. Proc Natl Acad Sci U S A, 2003, 100(5): 2992-2997.

[2] AngelidisI, SimonLM, FernandezIE, StrunzM, MayrCH, GreiffoFR, TsitsiridisG, AnsariM, GrafE, StromTM, NagendranM, DesaiT, EickelbergO, MannM, TheisFJ, SchillerHB. An atlas of the aging lung mapped by single cell transcriptomics and deep tissue proteomics. Nat Commun, 2019, 101963.

[3] AnzolaJM, SiebererT, OrtbauerM, ButtH, KorbeiB, WeinhoferI, MüllnerAE, LuschnigC. Putative Arabidopsis transcriptional adaptor protein (PROPORZ1) is required to modulate histone acetylation in response to auxin. Proc Natl Acad Sci U S A, 2010, 107(22): 10308-10313.

[4] Asp M, Giacomello S, Larsson L, Wu C, Furth D, Qian X, Wardell E, Custodio J, Reimegard J, Salmen F, Osterholm C, Stahl PL, Sundstrom E, Akesson E, Bergmann O, Bienko M, Mansson-Broberg A, Nilsson M, Sylven C, Lundeberg J (2019) A Spatiotemporal Organ-Wide Gene Expression and Cell Atlas of the Developing Human Heart. Cell 179(7):1647–1660 e1619. https://doi.org/10.1016/j.cell.2019.11.025

[5] AttaR, LaurensL, Boucheron-DubuissonE, Guivarc’hA, CarneroE, Giraudat-PautotV, RechP, ChriquiD. Pluripotency of Arabidopsis xylem pericycle underlies shoot regeneration from root and hypocotyl explants grown in vitro. Plant J, 2009, 57(4): 626-644.

[6] BaccinC, Al-SabahJ, VeltenL, HelblingPM, GrunschlagerF, Hernandez-MalmiercaP, Nombela-ArrietaC, SteinmetzLM, TrumppA, HaasS. Combined single-cell and spatial transcriptomics reveal the molecular, cellular and spatial bone marrow niche organization. Nat Cell Biol, 2020, 22(1): 38-48.

[7] BatistaDS, FelipeSHS, SilvaTD, de CastroKM, Mamedes-RodriguesTC, MirandaNA, Ríos-RíosAM, FariaDV, FortiniEA, ChagasK, Torres-SilvaG, XavierA, ArencibiaAD, OtoniWC. Light quality in plant tissue culture: does it matter?. In Vitro Cellular & Developmental Biology - Plant, 2018, 54(3): 195-215.

[8] BoutilierK, OffringaR, SharmaVK, KieftH, OuelletT, ZhangL, HattoriJ, LiuCM, van LammerenAA, MikiBL, CustersJB, van Lookeren CampagneMM. Ectopic expression of BABY BOOM triggers a conversion from vegetative to embryonic growth. Plant Cell, 2002, 14(8): 1737-1749.

[9] CapiteLD. ACTION OF LIGHT AND TEMPERATURE ON GROWTH OF PLANT TISSUE CULTURES IN VITRO. Am J Bot, 1955, 42: 869-873.

[10] CheP, LallS, HowellSH. Developmental steps in acquiring competence for shoot development in Arabidopsis tissue culture. Planta, 2007, 226(5): 1183-1194.

[11] ChenX. MicroRNA biogenesis and function in plants. FEBS Lett, 2005, 579(26): 5923-5931.

[12] ChenZH, BaoML, SunYZ, YangYJ, XuXH, WangJH, HanN, BianHW, ZhuMY. Regulation of auxin response by miR393-targeted transport inhibitor response protein 1 is involved in normal development in Arabidopsis. Plant Mol Biol, 2011, 77(6): 619-629.

[13] ChenLQ, TongJH, XiaoLT, RuanY, LiuJC, ZengMH, HuangH, WangJW, XuL. YUCCA-mediated auxin biogenesis is required for cell fate transition occurring during de novo root organogenesis in Arabidopsis. J Exp Bot, 2016, 67(14): 4273-4284.

[14] ChenJJ, WangLY, ImmanenJ, NieminenK, SpicerR, HelariuttaY, ZhangJ, HeXQ. Differential regulation of auxin and cytokinin during the secondary vascular tissue regeneration in Populus trees. New Phytol, 2019, 224(1): 188-201.

[15] ChengY, DaiX, ZhaoY. Auxin synthesized by the YUCCA flavin monooxygenases is essential for embryogenesis and leaf formation in Arabidopsis. Plant Cell, 2007, 19(8): 2430-2439.

[16] ChengZJ, ZhuSS, GaoXQ, ZhangXS. Cytokinin and auxin regulates WUS induction and inflorescence regeneration in vitro in Arabidopsis. Plant Cell Rep, 2010, 29(8): 927-933.

[17] CkurshumovaW, SmirnovaT, MarcosD, ZayedY, BerlethT. Irrepressible MONOPTEROS/ARF5 promotes de novo shoot formation. New Phytol, 2014, 204(3): 556-566.

[18] DastidarMG, ScarpaA, MageleI, Ruiz-DuarteP, von BornP, BaldL, JouannetV, MaizelA. ARF5/MONOPTEROS directly regulates miR390 expression in the Arabidopsis thaliana primary root meristem. Plant Direct, 2019, 32. e00116

[19] Denyer T, Ma X, Klesen S, Scacchi E, Nieselt K, Timmermans MCP (2019) Spatiotemporal Developmental Trajectories in the Arabidopsis Root Revealed Using High-Throughput Single-Cell RNA Sequencing. Dev Cell 48(6):840–852 e845. https://doi.org/10.1016/j.devcel.2019.02.022

[20] DewitteW, ScofieldS, AlcasabasAA, MaughanSC, MengesM, BraunN, CollinsC, NieuwlandJ, PrinsenE, SundaresanV, MurrayJA. Arabidopsis CYCD3 D-type cyclins link cell proliferation and endocycles and are rate-limiting for cytokinin responses. Proc Natl Acad Sci U S A, 2007, 104(36): 14537-14542.

[21] DuclercqJ, Sangwan-NorreelB, CatterouM, SangwanRS. De novo shoot organogenesis: from art to science. Trends Plant Sci, 2011, 16(11): 597-606.

[22] EfroniI, MelloA, NawyT, IpPL, RahniR, DelRoseN, PowersA, SatijaR, BirnbaumKD. Root Regeneration Triggers an Embryo-like Sequence Guided by Hormonal Interactions. Cell, 2016, 165(7): 1721-1733.

[23] FanM, XuC, XuK, HuY. LATERAL ORGAN BOUNDARIES DOMAIN transcription factors direct callus formation in Arabidopsis regeneration. Cell Res, 2012, 22(7): 1169-1180.

[24] Fellers JP, Guenzi AC, Taliaferro CM (1995) Factors affecting the establishment and maintenance of embryogenic callus and suspension cultures of wheat (Triticum aestivum L.). Plant Cell Rep 15(3–4):232–237. https://doi.org/10.1007/bf00193726

[25] GaoS, GuoW, FengW, LiuL, SongX, ChenJ, HouW, ZhuH, TangS, HuJ. LTP3 contributes to disease susceptibility in Arabidopsis by enhancing abscisic acid (ABA) biosynthesis. Mol Plant Pathol, 2016, 17(3): 412-426.

[26] GarzonM, EiflerK, FaustA, ScheelH, HofmannK, KonczC, YephremovA, BachmairA. PRT6/At5g02310 encodes an Arabidopsis ubiquitin ligase of the N-end rule pathway with arginine specificity and is not the CER3 locus. FEBS Lett, 2007, 581(17): 3189-3196.

[27] GrafiG, BarakS. Stress induces cell dedifferentiation in plants. Biochim Biophys Acta, 2015, 4: 378-384.

[28] HellmannE, HelariuttaY. Plant Genetics: Advances in Regeneration Pathways. Curr Biol, 2019, 29(14): R702-R704.

[29] HibaraK, TakadaS, TasakaM. CUC1 gene activates the expression of SAM-related genes to induce adventitious shoot formation. Plant J, 2003, 36(5): 687-696.

[30] HoldsworthMJ, VicenteJ, SharmaG, AbbasM, ZubryckaA. The plant N-degron pathways of ubiquitin-mediated proteolysis. J Integr Plant Biol, 2020, 62(1): 70-89.

[31] IkedaY, BannoH, NiuQW, HowellSH, ChuaNH. The ENHANCER OF SHOOT REGENERATION 2 gene in Arabidopsis regulates CUP-SHAPED COTYLEDON 1 at the transcriptional level and controls cotyledon development. Plant Cell Physiol, 2006, 47(11): 1443-1456.

[32] IkeuchiM, SugimotoK, IwaseA. Plant callus: mechanisms of induction and repression. Plant Cell, 2013, 25(9): 3159-3173.

[33] IwaseA, MitsudaN, KoyamaT, HiratsuK, KojimaM, AraiT, InoueY, SekiM, SakakibaraH, SugimotoK, Ohme-TakagiM. The AP2/ERF transcription factor WIND1 controls cell dedifferentiation in Arabidopsis. Curr Biol, 2011, 21(6): 508-514.

[34] IwaseA, HarashimaH, IkeuchiM, RymenB, OhnumaM, KomakiS, MorohashiK, KurataT, NakataM, Ohme-TakagiM, GrotewoldE, SugimotoK. WIND1 Promotes Shoot Regeneration through Transcriptional Activation of ENHANCER OF SHOOT REGENERATION1 in Arabidopsis. Plant Cell, 2017, 29(1): 54-69.

[35] IwaseA, KondoY, LaohavisitA, TakebayashiA, IkeuchiM, MatsuokaK, AsahinaM, MitsudaN, ShirasuK, FukudaH, SugimotoK. WIND transcription factors orchestrate wound-induced callus formation, vascular reconnection and defense response in Arabidopsis. New Phytol, 2021, 232(2): 734-752.

[36] JhaAK, DahleenLS, SuttleJC. Ethylene influences green plant regeneration from barley callus. Plant Cell Rep, 2007, 26(3): 285-290.

[37] JiménezVM. Involvement of Plant Hormones and Plant Growth Regulators on in vitro Somatic Embryogenesis. Plant Growth Regul, 2005, 47(2): 91-110.

[38] KimJ-Y, SymeonidiE, PangTY, DenyerT, WeidauerD, BezrutczykM, MirasM, ZöllnerN, HartwigT, WudickMM, LercherM, ChenL-Q, TimmermansMCP, FrommerWB. Distinct identities of leaf phloem cells revealed by single cell transcriptomics. Plant Cell, 2021, 33(3): 511-530.

[39] LeeHW, KimNY, LeeDJ, KimJ. LBD18/ASL20 regulates lateral root formation in combination with LBD16/ASL18 downstream of ARF7 and ARF19 in Arabidopsis. Plant Physiol, 2009, 151(3): 1377-1389.

[40] LeibfriedA, ToJPC, BuschW, StehlingS, KehleA, DemarM, KieberJJ, LohmannJU. WUSCHEL controls meristem function by direct regulation of cytokinin-inducible response regulators. Nature, 2005, 438(7071): 1172-1175.

[41] LimaJE, BeneditoVA, FigueiraA, PeresLE. Callus, shoot and hairy root formation in vitro as affected by the sensitivity to auxin and ethylene in tomato mutants. Plant Cell Rep, 2009, 28(8): 1169-1177.

[42] LiuZ, DaiX, LiJ, LiuN, LiuX, LiS, XiangF. The Type-B Cytokinin Response Regulator ARR1 Inhibits Shoot Regeneration in an ARR12-Dependent Manner in Arabidopsis. Plant Cell, 2020, 32(7): 2271-2291.

[43] LiuZ, ZhouY, GuoJ, LiJ, TianZ, ZhuZ, WangJ, WuR, ZhangB, HuY, SunY, ShangguanY, LiW, LiT, HuY, GuoC, RochaixJD, MiaoY, SunX. Global Dynamic Molecular Profiling of Stomatal Lineage Cell Development by Single-Cell RNA Sequencing. Mol Plant, 2020, 13(8): 1178-1193.

[44] LuoL, ZengJ, TianZ, ZhaoZ. Plant development: From cells to individuals. Chin Sci Bull, 2016, 61: 3532-3540.

[45] MacoskoEZ, BasuA, SatijaR, NemeshJ, ShekharK, GoldmanM, TiroshI, BialasAR, KamitakiN, MartersteckEM, TrombettaJJ, WeitzDA, SanesJR, ShalekAK, RegevA, McCarrollSA. Highly Parallel Genome-wide Expression Profiling of Individual Cells Using Nanoliter Droplets. Cell, 2015, 161(5): 1202-1214.

[46] Miyashima S, Roszak P, Sevilem I, Toyokura K, Blob B, Heo JO, Mellor N, Help-Rinta-Rahko H, Otero S, Smet W, Boekschoten M, Hooiveld G, Hashimoto K, Smetana O, Siligato R, Wallner ES, Mähönen AP, Kondo Y, Melnyk CW, Greb T, Nakajima K, Sozzani R, Bishopp A, De Rybel B, Helariutta Y (2019) Mobile PEAR transcription factors integrate positional cues to prime cambial growth. Nature 565(7740):490-+. https://doi.org/10.1038/s41586-018-0839-y

[47] MotteH, VerstraetenI, WerbrouckS, GeelenD. CUC2 as an early marker for regeneration competence in Arabidopsis root explants. J Plant Physiol, 2011, 168(13): 1598-1601.

[48] MotteH, VereeckeD, GeelenD, WerbrouckS. The molecular path to in vitro shoot regeneration. Biotechnol Adv, 2014, 32(1): 107-121.

[49] MullerR, BleckmannA, SimonR. The receptor kinase CORYNE of Arabidopsis transmits the stem cell-limiting signal CLAVATA3 independently of CLAVATA1. Plant Cell, 2008, 20(4): 934-946.

[50] ParkDH, LimPO, KimJS, ChoDS, HongSH, NamHG. The Arabidopsis COG1 gene encodes a Dof domain transcription factor and negatively regulates phytochrome signaling. Plant J : for Cell and Molecular Biology, 2003, 34(2): 161-171.

[51] Park JE, Botting RA, Dominguez Conde C, Popescu DM, Lavaert M, Kunz DJ, Goh I, Stephenson E, Ragazzini R, Tuck E, Wilbrey-Clark A, Roberts K, Kedlian VR, Ferdinand JR, He X, Webb S, Maunder D, Vandamme N, Mahbubani KT, Polanski K, Mamanova L, Bolt L, Crossland D, de Rita F, Fuller A, Filby A, Reynolds G, Dixon D, Saeb-Parsy K, Lisgo S, Henderson D, Vento-Tormo R, Bayraktar OA, Barker RA, Meyer KB, Saeys Y, Bonfanti P, Behjati S, Clatworthy MR, Taghon T, Haniffa M, Teichmann SA (2020) A cell atlas of human thymic development defines T cell repertoire formation. Science 367(6480). https://doi.org/10.1126/science.aay3224

[52] ParkSH, JeongYJ, KimS, LeeJ, KimCY, JeongJC. Trichostatin A promotes de novo shoot regeneration from Arabidopsis root explants via a cytokinin related pathway. Sci Reps, 2025, 151978.

[53] QiaoM, XiangF. A set of Arabidopsis thaliana miRNAs involve shoot regeneration in vitro. Plant Signal Behav, 2013, 83. e23479

[54] RosspopoffO, ChelyshevaL, SaffarJ, LecorgneL, GeyD, CaillieuxE, ColotV, RoudierF, HilsonP, BerthoméR, Da CostaM, RechP. Direct conversion of root primordium into shoot meristem relies on timing of stem cell niche development. Development, 2017, 144(7): 1187-1200.

[55] RyuKH, HuangL, KangHM, SchiefelbeinJ. Single-Cell RNA Sequencing Resolves Molecular Relationships Among Individual Plant Cells. Plant Physiol, 2019, 179(4): 1444-1456.

[56] ShangB, XuC, ZhangX, CaoH, XinW, HuY. Very-long-chain fatty acids restrict regeneration capacity by confining pericycle competence for callus formation in Arabidopsis. Proc Natl Acad Sci U S A, 2016, 113(18): 5101-5106.

[57] ShemerO, LandauU, CandelaH, ZemachA, Eshed WilliamsL. Competency for shoot regeneration from Arabidopsis root explants is regulated by DNA methylation. Plant Sci, 2015, 238: 251-261.

[58] SkoogF, MillerCO. Chemical regulation of growth and organ formation in plant tissues cultured in vitro. Symp Soc Exp Biol, 1957, 11: 118-130

[59] SolimanERS, MeyerP. Responsiveness and Adaptation to Salt Stress of the REDOX-RESPONSIVE TRANSCRIPTION FACTOR 1 (RRTF1) Gene are Controlled by its Promoter. Mol Biotechnol, 2019, 61(4): 254-260.

[60] SuYH, LiuYB, ZhangXS. Auxin-cytokinin interaction regulates meristem development. Mol Plant, 2011, 4(4): 616-625.

[61] SugimotoK, TemmanH, KadokuraS, MatsunagaS. To regenerate or not to regenerate: factors that drive plant regeneration. Curr Opin Plant Biol, 2019, 47: 138-150.

[62] TangF, BarbacioruC, WangY, NordmanE, LeeC, XuN, WangX, BodeauJ, TuchBB, SiddiquiA, LaoK, SuraniMA. mRNA-Seq whole-transcriptome analysis of a single cell. Nat Methods, 2009, 6(5): 377-382.

[63] TrapnellC, CacchiarelliD, GrimsbyJ, PokharelP, LiSQ, MorseM, LennonNJ, LivakKJ, MikkelsenTS, RinnJL. The dynamics and regulators of cell fate decisions are revealed by pseudotemporal ordering of single cells. Nat Biotechnol, 2014, 32(4): 381-U251.

[64] TrinhDC, LavenusJ, GohT, BoutteY, DrogueQ, VaissayreV, TellierF, LucasM, VossU, GantetP, FaureJD, DussertS, FukakiH, BennettMJ, LaplazeL, Guyomarc’hS. PUCHI regulates very long chain fatty acid biosynthesis during lateral root and callus formation. Proc Natl Acad Sci U S A, 2019, 116(28): 14325-14330.

[65] WangL, LiuN, WangT, LiJ, WenT, YangX, LindseyK, ZhangX. The GhmiR157a-GhSPL10 regulatory module controls initial cellular dedifferentiation and callus proliferation in cotton by modulating ethylene-mediated flavonoid biosynthesis. J Exp Bot, 2018, 69(5): 1081-1093.

[66] WangJ, NanN, ShiL, LiN, HuangS, ZhangA, LiuY, GuoP, LiuB, XuZY. Arabidopsis BRCA1 represses RRTF1-mediated ROS production and ROS-responsive gene expression under dehydration stress. New Phytol, 2020, 228(5): 1591-1610.

[67] WuG, ParkMY, ConwaySR, WangJW, WeigelD, PoethigRS. The sequential action of miR156 and miR172 regulates developmental timing in Arabidopsis. Cell, 2009, 138(4): 750-759.

[68] XieM, ChenH, HuangL, O’NeilRC, ShokhirevMN, EckerJR. A B-ARR-mediated cytokinin transcriptional network directs hormone cross-regulation and shoot development. Nat Commun, 2018, 911604.

[69] XuL. De novo root regeneration from leaf explants: wounding, auxin, and cell fate transition. Curr Opin Plant Biol, 2018, 41: 39-45.

[70] XuC, CaoH, ZhangQ, WangH, XinW, XuE, ZhangS, YuR, YuD, HuY. Control of auxin-induced callus formation by bZIP59–LBD complex in Arabidopsis regeneration. Nature Plants, 2018, 4(2): 108-115.

[71] ZengJ, LiX, GeQ, DongZ, LuoL, TianZ, ZhaoZ. Endogenous stress-related signal directs shoot stem cell fate in Arabidopsis thaliana. Nat Plants, 2021, 7(9): 1276-1287.

[72] ZhaiN, XuL. Pluripotency acquisition in the middle cell layer of callus is required for organ regeneration. Nat Plants, 2021, 7(11): 1453-1460.

[73] ZhangG, ZhaoF, ChenL, PanY, SunL, BaoN, ZhangT, CuiCX, QiuZ, ZhangY, YangL, XuL. Jasmonate-mediated wound signalling promotes plant regeneration. Nat Plants, 2019, 5(5): 491-497.

[74] ZhangH, RundleC, WinterN, MiricescuA, MooneyBC, BachmairA, GracietE, TheodoulouFL. BIG enhances Arg/N-degron pathway-mediated protein degradation to regulate Arabidopsis hypoxia responses and suberin deposition. Plant Cell, 2024, 36(9): 3177-3200.

Funding

National Key Research and Development Program of China(2022YFD1200300)

RIGHTS & PERMISSIONS

The Author(s)