The roles of microRNAs in regulating root formation and growth in plants

YAN Xiao-xiao,LIU Xiang-yang,CUI Hong,ZHAO Ming-qin

College of Tobacco Science,Henan Agricultural University/National Tobacco Cultivation and Physiology and Biochemistry Research Center/Key Laboratory for Tobacco Cultivation of Tobacco Industry,Zhengzhou 450002,P.R.China

Abstract MicroRNAs (miRNAs) are small (ca.20-24 nucleotides) non-coding RNAs that have recently been recognized as key post-transcriptional modulators of gene expression;and they are involved in many biological processes in plants,such as root growth and development.The miRNAs regulate root elongation,lateral root (LR) formation and adventitious root (AR)development in response to hormone signaling,nutrient uptake and biotic/abiotic stress.This review provides multiple perspectives on the involvement of miRNAs in regulating root growth and development in plants.We also discuss several crucial mechanisms of miRNAs,their relationships with transcription factors and the target gene-mediated hormone signaling interactions in the regulation of root growth and development.

Keywords:microRNAs,adventitious root,lateral root,primary root

1.Introduction

The root system is an important facilitator for the colonization of land by plants and for water and mineral nutrient uptake.Optimal root morphology is therefore the foundation of normal growth and development in plants.Root systems are different between dicotyledons and monocotyledons,such asArabidopsisthalianaandOrazasativa(rice) (Fig.1).The primary root (PR) or seminal root (SR),generally are derived from the embryonic radicle,while the lateral root (LR) and adventitious root (AR) are derived post-embryonically.LRs are branches of PRs and are derived from the reactivation of pericycle cells through anticlinal,periclinal and tangential cell division to form the primordium (Peretet al.2009).The AR always develops from cells neighboring vascular tissues and is used to increase transport capacities from soil to the aerial parts of the plant (Lakehal and Bellini 2019).In dicotyledons,AR is produced from the junction of the shoot and root (Coudertet al.2010;Lucaset al.2011).The AR is initiated directly from hypocotyl pericycle-like cells ofArabidopsisfrom stem tissues or vascular tissues of the secondary growth (Goldfarbet al.1998;Bustillo-Avendanoet al.2018),and is similar to LR formation (Rasmussenet al.2012).In monocotyledons,two main developmental pathways can lead to AR formation,and the AR is named crown roots (CR) or shoot-borne roots in cereals.The AR/CR differentiates from the ground meristem that is assimilated to a stem pericycle-like tissue in rice plant(Coudertet al.2013).The AR primordia are also initiated from cells close to the vascular system of older seedling stems in woody species such as poplar and apple,relevant to the adult phase of plants (Xuet al.2017;Goninet al.2019).The modulation of root system architecture mainly relies on the regulation of PR growth through cell division and elongation activities,and also on the regulation of LR or AR branching.

Fig.1 Sketch map of rice and Arabidopsis roots.AR,adventitious root;LR,lateral root;SR,seminal root;PR,primary root.

However,plant growth sometimes must be achieved under conditions that provide minimal water and nutrients.More rapid and more extensive root growth and development are important for plant growth under such adverse environmental conditions (Giriet al.2018).The rhizosphere composition elements affecting rootformation and growth are usually thought of as external environmental conditions,such as soil nutrient or water supply (Sunet al.2014,2016;Giriet al.2018),biotic interactions including soil fungi and neighboring plants,and intrinsic factors such as phytohormones,mRNAs,proteins,and small RNAs,which are energetically involved in the regulation of plant root growth and development (Morriset al.2017;Vissenberget al.2020).Root system architecture formation is a highly complex process that requires a fine-tuning of gene expression involved in the hormonal regulation of cell division and differentiation.The radial tissue organization in the root is highly conserved with a central vascular cylinder,which is regulated by crosstalk between the vascular cylinder and the surrounding endodermis mediated by cell-to-cell movement of the transcription factors and microRNAs (miRNAs)(Carlsbeckeret al.2010;Couzigou and Combier 2016).

1.1.Auxin

The phytohormones crosstalk,with auxin as a hub,and this occurs in response to environmental signals in order to tightly and spatiotemporally control the root development.Auxin is synthesized mainly in the shoot apex and young leaves byYUCCAgenes,and has been described as one essential positive regulator during plant root growth dependent on its localized synthesis,secretion and modification of the root hair tip cell wall,and it is the major growth-promoting hormone for AR initiation (Zhao 2012;Schoenaerset al.2018).The auxin effects on root growth are supplemented with an identified non-transcriptionalTIR1-dependent signaling branch mediated by pH changes and calcium signaling (Fendrychet al.2018),as well as by extracellular reactive oxygen species (ROS).Auxin carriers andAUXINRESPONSEFACTOR(ARF) play important roles in regulating root growth and formation(Shenet al.2013;Huanget al.2016;Giriet al.2018).As the important regulatory inhibitor,miRNAs regulate root system architecture by forming a feedback loop withARFs,such as miR160-ARF10/16/17(Wanget al.2005;Huanget al.2016),miR167-ARF12(Qiet al.2012),and miR390-ARF2/3/4(Marinet al.2010;Yoonet al.2010).The local auxin maximum controlled by polar auxin transport (PAT)through auxin efflux carriers has a key role in AR architecture formation in rice (Lin and Sauter 2019).A basic helix-loophelix (bHLH) transcription factor,root hair defective-like 4(RLS4),is regarded to be the first auxin responsive gene in the timeline of root hair morphogenesis (Yiet al.2010),while genes functioning upstream ofRLS4have nothing to do with auxin signaling (Vissenberget al.2020),suggesting that auxin is involved in the LR development.

1.2.Cytokinins (CKs)

In 2015,Antoniadiet al.(2015) revealed the presence of a CKs gradient within theArabidopsisroot tip,with a concentration maximum in the lateral root cap.A C2H2 zinc finger protein,ZINC FINGER PROTEIN 5 (ZFP5) induced by CKs,has been proved to be a key regulator of root hair initiation and morphogenesis inArabidopsis,whereZFP5mediates CKs and ethylene effects on the formation and growth of root hairs (Anet al.2012).A CKs biosynthetic gene,ISOPENTYLTRANSFERASE7(IPT7),is activated by Class IIIHOMEODOMAIN-LEUCINEZIPPER(HD-ZIP IIIs) transcripts,which inhibits the expression of miR165 in root meristem (Dello Ioioet al.2012).miR165-SHR(SHORT ROOT) is also known to involved in a cell signaling process meditated by auxin in roots (Carlsbeckeret al.2010).Therefore,CKs and auxin interweave into a complex network to regulate the root system architecture.The heterogeneous distribution of CKs can be controlled to maintainArabidopsisand rice root meristem size and development alone with auxin,and the complexity in auxin,CKs and ethylene crosstalk requires a combined experimental and systematic modeling approach (Peterssonet al.2009;Gaoet al.2014;Liuet al.2017).A rice AP2/ERF protein,ERF3,is essential for CR development and acts in auxin-and CKs-responsive gene expression,and functions cooperatively withWOX11to regulate CR elongation and development (Antoniadiet al.2015;Zhaoet al.2015;Dominik and Jan 2019).

1.3.Abscisic acid (ABA)

ABA inhibits root elongation synergistically with ethylene(Maet al.2014).ABA is also a negative regulator of AR development in tomato (Belliniet al.2014).ABA and ethylene cascades share some common features in terms of the mediation of root growth:low concentrations promote root growth and high concentrations inhibit root growth (Joshi-Sahaet al.2011;Arcet al.2013).miR159 is induced by ABA in germinating seedlings ofArabidopsis,and its targetMYB33was previously verified to be expressed in the root,which shows that an ABA-miR159-MYB33module plays an important role in root development (Reyes and Chua 2007).Moreover,ABA induces PR growth inhibition through miR393 and a secondary interfering RNA (si-TAAR) (Chenet al.2012).ABA regulates root growth by acting upstream of ethylene and by directly affecting auxin accumulation and/or auxin signaling (Luoet al.2014).

1.4.Ethylene

Ethylene promotes root hair growth and also mediates the effects of different signals that stimulate hair cell development.Ethylene inhibits root growth or formation through auxin action by modulating its biosynthesis,transport and/or signaling (Pacheco-Villaloboset al.2013;Liet al.2015;Duboiset al.2018).The regular pairs of ethylene-activated transcription factorETHYLENEINSENSITIVE3(EIN3)/EIN3-Like1(EIL1) andROOT HAIRDEFECTIVE6(RHD6)/RDH6-Like1(RSL1) act as a well-documented positive regulator of hair cells,and provide a molecular framework for the control of root hair initiation and elongation (Fenget al.2017).Ethylene regulates the distribution of AR initiation sites on the apical part of the hypocotylviathe changes inPIN-FORMED(PIN) localization (Velocciaet al.2016).Ethylene is a positive regulator of root hair development while auxin also regulates the epidermal cell fate determination pathway.The auxin response occurring in the elongation zone mediates a substantial part of the ethylene effect on root growth(Vanstraelen and Benkov 2012).Data integration reveals a patterning in root development consisting of ethylene and CK pathways,which act through a phosphorelay mechanism meditated by both transcription factors and miRNA,such as miR393a-MYB33(Zhanget al.2016;Liuet al.2017).Ethylene and ABA synergistically inhibit root elongation (Maet al.2014;Chenet al.2018).

1.5.Brassinosteroids (BRs)

BRs are not only involved in root cell elongation but are also involved in many aspects of root development,such as the maintenance of meristem size,root hair formation and LR initiation (Wei and Li 2016).Xia Ket al.(2015) showed thatOsCYCP51G3,a cytochrome P enzyme gene mediating BRs biosynthesis,is inhibited by miR1848 in rice,which affects plant root growth and responses to external stress such as salinity.Studies show that the BRs-responsive transcription factors could regulate a subset of growthpromoting genesviaconserved bipartitecis-regulatory elements together with an auxin-regulated transcription factor.AnARF5-BES1/BEH4transcriptional module acts to promote hypocotyl growth by modulation of a diverse set of growth-associated genes (Lachowiecet al.2018;Galstyan and Nemhauser 2019).BRs and auxin counteract each otherviaBRs-activated transcription factorBZR1during root cell elongation (Wei and Li 2016).The opposite effects have been shown to antagonistically control root elongation between BRs and auxin (Chaiwanon and Wang 2015).

1.6.Strigolactones (SLs)

SLs synthesized mainly in the root and some parts of the stem have been identified as one novel phytohormones that regulate root development (Kapulniket al.2011;Mayzlish-Gatiet al.2012;Rasmussenet al.2012;De Cuperet al.2015).Liuet al.(2011) found that two GRAS transcription factors meditated by miR171 regulate the expression ofDWARF27,a gene essential for SLs biosynthesis,to affect the LR density in rice.SLs and the targetMAX2F-box have a positive effect on root-hair elongation and repress LR initiation (Kapulniket al.2011).SLs are known to have an interfering function on the development of root-cell length against auxin inArabidopsisand rice (Rasmussenet al.2012;Sunet al.2016),and SLs spatially influence LR development through the auxin and CKs signaling network (Jianget al.2016).SLs and CKs appear to act independently to suppress adventitious rooting,while SLs and auxin hormonal pathways may convergeviathe ethylene pathway for the regulation of root hair elongation,in which SLs and additive-epistatic ethylene regulate root hair elongation (Kapulniket al.2011;Rasmussenet al.2012).

1.7.Gibberellins (GAs)

A growing body of evidence suggests that GAs have an inhibitory effect on LR and AR formation but a stimulatory effect on root elongation.GAs appear to be involved in the hypoxia response and AR development inhibition in barley (Moriconiet al.2019).GAs inhibit AR formation by stabilizingPINand perturbing the establishment of thePATin auxin (Lombardi-Crestanaet al.2012).High levels of GAs negatively regulate the early initiation step of root formation in conjunction with auxin (El-Sharkawyet al.2012;Niuet al.2013).GAs are also involved in the reduction of cell division in the LR meristems,resulting in a shorter LR length through the accumulation of growth-inhibitory DELLA proteins(Hetheringtonet al.2021).The relationship between GAs and auxin appears to be both complex and context specific.The function of auxin promotion of root growth is built on enhancing the GA-induced destabilization (Niuet al.2013).Furthermore,the interwoven GAs,ethylene and ABA signaling pathways regulate the root system architecture meditated by miR159 (Zhanget al.2008).

1.8.Jasmonate (JA)

JA,being an inhibitor of adventitious rooting,is modulated byCOI1,miRNA,ARFand auxin-inducibleGretchenHagen3(GH3) protein to control the inducible adventitious rooting inArabidopsis(Gutierrezet al.2012;Lakehal and Bellini 2019).Both of the transgenicJAR1andMYC2lines in which the JA signaling pathways are upregulated have significantly fewer ARs than the wild type (WT),and those mutants such as thejar1-12,coi1-16andmyc2lines with a downregulated JA signaling pathway all had more ARs.Exogenous JA or JA-Ile treatments yielded significantly fewer ARs than the controls (Lischweskiet al.2015).The core repressorJAZ1has been verified to the determinant of the root traits under stress.Overexpression ofOsJAZ1/EG2induces longer and more ARs (Liet al.2017).Studies also found that submicromolar amounts of methyl jasmonate promoted AR development from thin cell layers of tobacco and culturedArabidopsisthin cell layers (Fattoriniet al.2009;Gutierrezet al.2012).Jasmonate elevates local auxin accumulation and transportation in the root,and it modifies the expression of auxin transport genes and regulates LR formation in anANTHRANILATESYNTHASEα1(ASA1)-dependent manner(Sunet al.2009).

1.9.Salicylic acid (SA)

SA is involved in various biotic and abiotic stress responses and promotes AR organogenesis and formation (Yanget al.2013).Exogenous SA,GAs and JA modulate the activity of miR475b and its targets to participate in plant growth and responses to abiotic stress inPopulussuaveolens(Niuet al.2016).SA is also possibly a positive regulator of adventitious rooting inArabidopsis(Gutierrezet al.2012).SA also enhances indoleacetic acid (IAA) decarboxylation and inhibits IAA-induced AR formation in apple microcuttings(De Klerket al.2011).

1.10.MicroRNAs

Among the different molecular partners governing root development,miRNAs are a kind of non-negligible regulators.They are small (ca.20-24 nucleotides)non-coding RNAs that act as key negative regulators by binding to mRNA complementary sequences for mRNA destabilisation and translational inhibition with the formation of an RNA-induced silencing complex (RISCs) in several plants species (Bartel 2004;Kawamata and Tomari 2010;Rogers and Chen 2013;Jean-Malo and Jean-Philippe 2016).In addition,miRNAs were shown to participate in the embryonic root and vasculature differentiation,and LR or AR formation in response to biotic and abiotic stresses,nutrient supply and hormonal stimulation (Couzigou and Combier 2016).

A number of plant miRNAs have been deposited in the public databases,such as miRbase and PNRD (for Plant Non-coding RNA Database) (Yiet al.2015;Zhang and Wang 2015).A large number of miRNAs have been identified and reported in various terrestrial plants such asArabidopsis(Rajagopalanet al.2006),Populus(poplar) (Barakatet al.2007),rice (Zhuet al.2008),Zeamays(maize) (Zhanget al.2009),Arachishypogaea(peanut) (Zhaoet al.2010),Vitisvinifera(grape) (Pantaleoet al.2010),Citrusreticulata(orange) (Xuet al.2010),Glycinemax(soybean) (Songet al.2011),Maluspumila(apple) (Xiaet al.2012;Xuet al.2017),Piceaabies(Norway Spruce) (Xia Ret al.2015),Medicagosativa(alfalfa) (Aunget al.2017),andHordeum vulgare(barley) (Baiet al.2017).

As a phylogenetically conserved family of endogenous small RNAs,miRNAs show species-specificity,reflecting their fast evolving and functionally diverging natures.The miRNAs are thought to regulate more than 60% of human protein-coding genes,fine-tuning a diverse array of biological processes (Friedmanet al.2009;Kimet al.2009;Ghildiyal and Zamore 2009).Also,miRNAs are essential for plant growth and play important regulatory roles in response to biotic or abiotic stresses (Kantaret al.2010,2011;Budak and Akpinar 2011;Budaket al.2015;Alptekinet al.2017;Brant and Budak 2018),and nutrients (Huet al.2011;Chenet al.2012;Yanet al.2014;Liet al.2016) by affecting root growth.For example,miR393 and a si-TAAR are involved in root growth inhibition following osmotic stress and ABA treatment (Chenet al.2012).miR167 regulates LR development triggered by osmotic stress and nitrogen(N) supply (Couzigou and Combier 2016).Several miRNAs participate in regulating the availability of macro-and micronutrients through the modulation of the root architecture.miR160,miR164,miR167,miR171,and miR393 might be important for root system architecture under N starvation(Giffordet al.2008;Vidalet al.2010;2013,2014;Zhuet al.2010;Lianget al.2012).The expression of miR164 is also modified in response to phosphate (Pi) deficiencies during maize root growth (Zhuet al.2010).Besides,many miRNAs are known to regulate root growth and development by targeting different transcription factors or genes,either in monocotyledons or dicotyledons (Huet al.2011;Yanet al.2014;Huanget al.2016;Gautamet al.2017).For example,root hair patterning in apple is regulated by miR828-targetedS15MYBs,which is a eudicot-specific regulatory function(Xiaet al.2012).Meanwhile,miR529 and its targetSQUAMOSAPROMOTERBINDINGPROTEIN-LIKE(SPL)genes are present in mosses and monocots but absent in most eudicots (Cuperuset al.2011;Jeonget al.2011).Moreover,the modes of miR160 interactions with its target genesARF10andARF16are required for root tip formation inArabidopsis(Wanget al.2005).miR393 plays a key role in the regulation of ARs growth and formation in rice (Bianet al.2012).In addition,the miRNAs regulatory network is intertwined with various hormone pathways.For example,the miR164 level in roots is involved in LR formation and growth by down-regulating auxin signaling (Guoet al.2005;Gutierrezet al.2009;Liet al.2012).The miR390-TAS3-ARFmodule plays a critical role in auxin signaling and is involved in the regulation of LRs growth (Marinet al.2010).miR159 and its target transcription factors,such as GAMYB,HD-ZIP,and ARF,modify auxin,CKs,ABA,ethylene,and GAs biosynthesis leading to variations in PR,LR and AR morphology (Zhanget al.2008;Xueet al.2017).A spatiotemporal expression pattern meditated by miR165/166 and their targets decreases root lengthviathe complex crosstalk of auxin,CKs,ABA,GAs,JA,and SA (Singhet al.2017).This review highlights the recent advances made in deciphering the role of miRNAs in the regulation of root growth and development in plants.

2.miRNAs are involved in root elongation

Several lines of evidence have suggested that miRNAs regulate root elongation inArabidopsis(Fig.2) (Carlsbeckeret al.2010;Wanget al.2010;Zhouet al.2015;Singhet al.2017).miR156 and its targetSPLgenes play the crucial roles in regulating the root length.A miR156-SPL10module inArabidopsisregulates PR growth and length by altering root meristem activity mostly by modulating the auxin and CKs responses (Gaoet al.2018;Barrera-Rojaset al.2020).The root length ofmiR156overexpression plants is shorter than WTLotus japonicasplants with significant SLs changes,although it remains to be determined whether SLs are impacted by miR156 and theSPLregulatory system (Wanget al.2015).Aunget al.(2017) showed that overexpression of MsmiR156 inhibits the auxin signaling pathway in order to enhance root initiation in alfalfa,which also indicated an extra effect on the CKs,ABA,GAs,and ethylene signaling pathways.miR159 acts as an important inhibitor of PR growth through its target geneMYB65and regulates the activity of the meristem in root tips through auxin and CKs responses (Xueet al.2017).miR160 and its targetsARF10/16regulate root elongation inArabidopsisby affecting cell division and differentiation in the root tip.miR160c overexpression plants andarf10arf16double mutants show uncontrolled cell division and blocked cell differentiation (Wanget al.2005),which suggests that a miR160-ARF10/16axis plays the important role in root elongation and development.Other studies have shown more functional miRNAs and more relationships with plant root growth and development.Light repression ofPXMT1is tenable in the complementation lines expressingprimiR163gene in themir163mutant background,and is restored to wild-type levels;whilemir163mutants orPXMT1overexpression lines show shorter PR lengths,which proves that miR163 and its targetPXMT1modulate root architecture during early development ofArabidopsisseedlings (Chunget al.2016).miR163 overexpression limits the PR elongation by inhibiting targetPXMT1expression,but rescues the defective PR elongation of thehy5mutant,which provides insight into the further understanding the post-transcriptional regulation of root photomorphogenesis meditated by theHY5-miR163-PXMT1axis (Liet al.2021).miR165/166 and their targets communicate radial positional information between cells of the root meristem.A cell signaling mediated by miR165/166,SHRandSCR(SCARECROW) is involved in controlling xylem patterning,in whichSHRmoves from the vascular cylinder into the endodermis and activatesSCR,which activates both miR165a and miR166b (Carlsbeckeret al.2010).The miR165/166 with target genesHD-ZIPIIIsinhibits root elongation and development by affecting the above cell signaling (Carlsbeckeret al.2010;Singhet al.2017),and regulating xylem patterning (Fanet al.2021).A spatio-temporal expression pattern of miR165/166,with targetHD-ZIPIIIsandKANADIgenes,decreases root lengthviaphytohormonal crosstalk,such as auxin,CKs,GAs,ABA,JA,and SA (Singhet al.2017).PHB,which belongs to theHD-ZIPIIIsubfamily,directly activates the CKs biosynthetic geneIPT7,while CKs inhibit the expression of miR165a.Then,an incoherent feedback regulatory loop between CKs,PHBand miR165a is constructed to regulate the mechanism of root meristem size adjustment and differentiation(Dello Ioioet al.2012).Together,a mathematical model showed that the integration of auxin,CKs,SHR,PHB,and miR165/166 can explain the root system establishment of the bisymmetric pattern (Muraroet al.2014).miR169defg and targetNF-YA2/YA10work in the inhibition of PR growth inArabidopsis(Sorinet al.2014).miR171 has been reported to have an important effect on root development (Liet al.2019).miR171 regulates PR elongation by affecting the quiescent centre and root growth by inhibitingHAM,and miR171-targetedSCARECROW-LIKE6-II(SCL6-II),SCL6-IIIandSCL6-IVinfluence the PR elongation (Wanget al.2010;Zhouet al.2015).miR319b and targetMYB33are involved in the phosphorylation progress ofCBP20regulation by ethylene (Zhanget al.2016).miR393 inhibits the root elongation in response to toxic aluminium stress mediated by the auxin signaling pathway in barley (Baiet al.2017).miR393 with a si-TAAR inhibits PR growth involved in the ABA signaling pathway (Chenet al.2012),and miR393 with its targetAFB3suppress root elongation under nitrate treatment (Vidalet al.2010).miR396 also decreases the expression ofGROWTHRESPONSEFACTORS(GRFs)to impede PR growth through regulating cell division inArabidopsisandMedicagotruncatula,which shows a potential link between BRs and miR396 (Bazinet al.2013;Rodriguezet al.2015).All of the above results suggest that many miRNAs play indispensable roles in the regulation of plant root elongation and development through their target genes in response to the complex rhizosphere environment.

Fig.2 Roles of miRNAs in regulating root elongation.Solid arrows denote regulatory pathways;the terminal arrow indicates facilitation,and the terminal dash indicates inhibition.CKs,cytokinins;ABA,abscisic acid;GAs,gibberellins;SLs,strigolactones;JA,jasmonate;SA,salicylic acid;BRs,brassinosteroids.-,unknown.

In addition toArabidopsis,many miRNAs are involved in root elongation in response to nutrient uptake and abiotic stress in crops,such as rice and barley (Fig.2).The miR156-SPLregulatory module has an important role in rice root development through the auxin signaling pathway(Liuet al.2009;Curabaet al.2014).Some studies have verified that the OsmiR160 target geneOsARF18is an orthologue ofArabidopsisARF16which leads to abnormal growth and rice root elongation by affecting auxin signaling,which is similar toOsARF10,OsARF16andOsARF17in rice (Huanget al.2016).Mutants harbouringARF18have shorter root lengths than those of WT,and the application of exogenous naphthylacetic acid (NAA) has also been shown to cause a similar and significant inhibition of root elongation and growth in WT andosarf18mutants (Huanget al.2016).miR167d in rice also inhibits theOsARF12regulation of root elongation,and shorter root lengths ofosarf12mutants are observed with lower auxin concentrations (Qiet al.2012).A longer length of PR is observed in miR393a/b transgenic barley than in WT,andOsTIR1andOsAFB2are confirmed to be negatively regulated by miR393 in rice (Bianet al.2012),which indicates the fundamental importance of miR393-regulated root growth by auxin signal transduction regulation in both rice and barley.These results indicate that most miRNAs are closely associated with auxin or auxin signaling in regulating root elongation in plants.In addition,theARFs,the auxin response factors,play important roles in this process,though few miRNAs are involved in root development induced by other hormones such as CKs,ABA,ethylene,GAs,and JA.Moreover,miR399 in rice and its downstream geneLTN1(PHO2),as a crucial Pi starvation signaling component,are involved in promoting root length under Pi deficiency (Huet al.2011).miR444 is one unique miRNA in monocots,and miR444a in rice promotes PR and AR elongationviathe NO3-and NH4+signaling pathways.Studies have proven that miR444 regulates four genes that are homologous to a MADS-box transcription factorANR1,which is a major component in the NO3-signaling pathway (Yanet al.2014).Furthermore,NH4+promotes BRs biosynthesis through miR444 with its target five homologous MADS-boxes,such asOsMADS57,to regulate rice root growth (Jiaoet al.2020).

3.The roles of miRNAs in the regulation of lateral root development

LRs attached to PRs or ARs are the major determinants of water and nutrient uptake from the soil.Studies have shown that miRNA regulates LR formation through various hormone signals in response to rhizosphere environmental stress in plants (Fig.3) (Guoet al.2005;Menget al.2009;Yanet al.2014;Chunget al.2016;Zhanget al.2018).miR156 and its targetSPLgenes (SPL3,SPL9andSPL10) are responsive to auxin signaling and have been demonstrated to regulate LR formation and growth inArabidopsis(Yuet al.2015;Zhenget al.2019;Barrera-Rojaset al.2020).A miR156-SPL10-AGL79module has been proven to affect LR growth inArabidopsis(Gaoet al.2018).miR160 regulates LR formation by downstream target genesARF16andARF17inArabidopsis(Wanget al.2005).The root morphology of increased number of LR frommir163mutants orPXMT1overexpression lines under long-day conditions illustrates that light-inducible miR163 and targetPXMT1transcripts must regulate LR development inArabidopsisthrough as yet unknown hormonal mechanisms (Chunget al.2016).miR164-mediated auxin signaling is required for LR formation inArabidopsis.mir164a/bmutants promote LR formation and show a decrease of miR164 and an increase ofNAC1mRNA,as well as NAA induction of miR164 with the increase ofNAC1mRNA,which is not observed in the auxin-insensitiveArabidopsismutants,such asauxin resistant1(axr1),axr2andtransportinhibitorresponse1.These results suggest that miR164 and targetNAC1are involved in transducing the auxin signal for LR formation(Guoet al.2005).Moreover,NAC4,another target gene of miR164,acts as the downstream gene ofAFB3to participate in the LR formation and root adaptation to nitrate treatment(Vidalet al.2013,2014).In potato plants,Stu-miR164 inhibits the downstream target geneStNAC262to alter LR formation and elongation in response to osmotic stress,which has the opposite relationship under polyethylene glycol (PEG) treatment.Stu-miR164 transgenic potato lines show a smaller number of LR relative to WT plants,whileStNAC262transgenic lines show more but shorter LR(Zhanget al.2018).LR density is increased and LR lengths are shortened by decreasing the length of the meristem zone in miR169defg transgenicArabidopsislines compared with WT,while the miR169defg targetNF-YA2/YA10inhibits PR growth (Sorinet al.2014).Moreover,miR171 participates in the regulation of LR formation inArabidopsis(Chenet al.2020) andMedicagotruncatula(Lauressergueset al.2015).A mechanism of miR390-TAS3-ARFsis involved in auxin signaling and affects LR formation inArabidopsis(Pekkeret al.2005;Marinet al.2010;Yoonet al.2010).Hobeckeret al.(2017) suggested that the miR390-TAS3regulatory module also promotes LR elongation inMedicagotruncatula.Another possible mechanism of miR393-regulated LR formation inArabidopsisviainhibition of the auxin signaling consists of downstream targets of the F-box auxin receptors transport inhibitor response 1 (TIR1) and auxin signaling F-box proteins 2/3 (AFB2/3) (Xieet al.2000;Navarroet al.2006).In 2010,Vidal pointed out that miR393 and its targetAFB3promote LR formation under nitrate treatment.Liu(2012) demonstrated that miR393 and its targetTIR1-like(F-box) gene in maize may participate in LR development.The proposed model of the miR393-TIR1-NAC1interacting with the miR164-NAC1pathway is involved in the LR formation meditated by auxin and CKs inArabidopsis.miR408 and miR528 targetCUPREDOXINand regulate LR formation in maize (Liuet al.2012).miR847 is also an important gene involved in LR development,and miR847 with the downregulation geneINDOLEACETICACID28(IAA28) promotes LR development (Wang and Guo 2015).These results showed that miRNAs regulate LR formation mainly by transcription factors and the target gene-mediated auxin signaling pathway.

Fig.3 Roles of miRNAs in regulating lateral root (LR) formation.Solid arrows denote regulatory pathways;the terminal arrow indicates facilitation,and the terminal dash indicates inhibition.CKs,cytokinins;ABA,abscisic acid;GAs,gibberellins;SLs,strigolactones.-,unknown.

In rice plants,studies show that the numbers of LRs are similar betweenosarf16,osarf18mutants and WT plants under normal growth conditions.The number of LRs significantly decreases inosarf16andosarf18mutants while it increases in WT plant after the treatment with IAA/NAA,which suggests that miR160 could be involved in auxin-regulated LR development byOsARF16/18-mediated auxin signaling in rice (Shenet al.2013;Huanget al.2016).Based on the results of microarray data and Northern blots,the higher expression levels of OsmiR164 family genes in an auxin-insensitive mutantosaxrmay be a legitimate explanation for the LR defect (Menget al.2009),which is consistent with the above results inArabidopsisand potato (Guoet al.2005;Zhanget al.2018),but needs more experimental validation.miR444a overexpression rice lines show reduced LR elongation under NO3--rich treatment (Yanet al.2014).Moreover,a total of 153 known miRNAs and 119 predicted novel miRNAs are shown to be involved in increases of AR and LR numbers in grapevine after root restriction cultivation (Liet al.2020).However,compared withArabidopsis,the mechanisms underlying miRNA-regulated LR formation in monocotyledons remain unclear and require further study.

4.miRNAs regulate adventitious root formation

Most published studies about AR development regulation by miRNAs also make a parallel with the data available for in the involvement of hormone signaling crosstalk in LR development.AR formation in dicotyledons and monocotyledons is different but shares several similar regulatory mechanisms on the basis of current research(Fig.4).Several studies have identified regulatory genes common to AR growth and development (Hochholdinger and Zimmermann 2008;Goninet al.2019).Therefore,the function of miRNAs in regulating AR formation is crucial.The adventitious rooting ability is necessary during the juvenile-to-adult phase change that is mediated by the miR156 in many species.The reduction of miR156 activity inhibits AR production in the hypocotyl,implying that its target SPL proteins inhibit AR production (Curabaet al.2014;Xuet al.2016),just as they inhibit LR production in the PR processviathe auxin signaling pathway (Yuet al.2015).AR production increases inTeopod/Corngrassmutants of maize (Poethig 1988) and tobacco (Fenget al.2016) overexpressed with miR156,which show the lower expression levels ofSPLgenes.Higher expression of miR156 and lower expression ofMxSPL26and someMxARFsare shown in juvenile phase trees with a higher adventitious rooting ability compared to those from adult phase trees in the presence of the synthetic auxin indole-3-butyric acid (IBA).Overexpressing the miR156-resistantMxrSPLgenes in tobacco confirms the involvement ofMxSPL20,MxSPL21/22andMxSPL26in AR formation,manifesting the necessity of miR156-MxSPL26for auxininduced AR formation in apple (Xuet al.2017).In 2017,Aung verified that MsmiR156 overexpression increases root regeneration capacity,but has little effect on root biomass at the early stages of root growth and development in alfalfa.In 2009,Gutierrezet al.(2009) suggested that auxin-associated miRNAs,such as miR160 and miR167,regulate AR development through complex processes.The regulation of AR by the miR167-ARFsmodules has been observed inArabidopsisand maize,the miR160-target geneARF17is a negative regulator of AR development through the integration of auxin and light signalling pathways,and miR167-target genesARF6andARF8have been demonstrated to be positive regulators of AR formation,and theseARFs interact genetically and regulate mutual expression at the transcriptional or posttranscriptional levels by modulating miR160 and miR167 availability (Gutierrezet al.2009;Liuet al.2012).Moreover,GH3,JA homeostasis and the COI1 signaling pathway are required downstream of the complex miR160/miR167-ARFsnetwork involved in AR development (Gutierrezet al.2012).These results show that miR160 and miR167 play key roles in plant root developmentviatheARFgenes within the auxin and JA signaling pathway.

Fig.4 Roles of miRNAs in regulating adventitious root (AR) formation.Solid arrows denote regulatory pathways and the dashed arrow represents a possible pathway;the terminal arrow indicates facilitation,and the terminal dash indicates inhibition.JA,jasmonate.

Several studies on the regulation of AR/CR by miRNAs have focused on plant species other than rice (Baiet al.2017;Chenet al.2020).The expression of miR167 family is downregulated inosaxrmutants,resulting in decreased numbers of ARs (Menget al.2009).Unlike inArabidopsis,miR167 acts as a negative regulator of AR through its downstream target genes in rice,which are likely homologs ofARF6andARF8inArabidopsis(Gutierrezet al.2009).vvi-miPEP171d1,a functional,small peptide encoded byprimiR171din grapevine,promotes LR and AR development by activating the expression of its targetsVvSCL15andVvSCL27(Chenet al.2020).Moreover,a miR393-TIR1/AFB2-IAA1module has been verified to regulate AR/CR formation in rice and barley associated with altered auxin signaling.Transgenic miR393 rice plants had longer PRs and produced fewer CRs than in WT (Bianet al.2012),similar to the results of AR suppression in barley (Baiet al.2017).

5.Conclusion and future developments

Several miRNAs are needed to fine-tune the root system architecture via many different target genes in plants.Most of them areAUXIN RESPONSE FACTOR(ARF) or involved in the auxin signaling pathway (Fig.5).Among them,miR156,miR160,miR171,and miR393 can participate in PR,LR,and AR growth and development simultaneously.Taking into consideration the above recent advances in deciphering the genetic elements controlling root development,miRNAs are involved primarily as the reinforcers,and their activities which are coherent with transcriptional patterns have sharpened plant root developmental transitions and entrenched root cellular identities (Ebert and Sharp 2012).Specialized and integrated rooting systems are necessary for the evolution of increasingly larger and more sophisticated plants that are capable of exploiting a broad range of habitats (Kenrick 2013).The evidence for microRNAs controlling phenotypic variability is a significant step forward in understanding the molecular mechanisms regulating roots,so it is very possible that miRNAs buffer fluctuations in gene expression and more faithfully signal outcomes in the context of certain regulatory networks.

Fig.5 Summary schematic diagram of miRNAs in root system architecture.▲,auxin;?,cytokinins (CKs);?,abscisic acid (ABA)?,ethylene;?,brassinosteroids (BRs);☆,strigolactones (SLs);?,gibberellins (GAs);★,jasmonate (JA);?,salicylic acid (SA),and the short line represents the unknown signaling pathway.

Acknowledgements

This work was funded by the Science and Technology Department of Henan Province,China (212102110046),the State Tobacco Monopoly Administration of China(110202101005 (JY-05)),the Science and Technology Project of China National Tobacco Corporation Henan Tobacco Company,China (2018410000270095),and the Undergraduate Innovation and Entrepreneurship Project of Henan Province,China (202110466042).

Declaration of competing interest

The authors declare that they have no conflict of interest.