plant micrornas: key regulators of root architecture and ......coding rnas, post-transcriptional...

Tansley review

Plant microRNAs: key regulators of rootarchitecture and biotic interactions

Authors for correspondence:Jean-Philippe Combier

Tel: +33 (0)5 34 32 38 11Email: [email protected]

Jean-Malo Couzigou

Tel: +44 632 36 54Email: [email protected]

Received: 5 February 2016

Accepted: 8 May 2016

Jean-Malo Couzigou1,2 and Jean-Philippe Combier1

1UMR5546, Laboratoire de Recherche en Sciences V�eg�etales, UPS, CNRS, Universit�e de Toulouse, Castanet-Tolosan 31326, France;

2Present address: Institute of Microbiology, ETH Z€urich, Vladimir-Prelog-Weg 4, Z€urich 8093, Switzerland

Contents

Summary 22

I. Introduction 22

II. The roles of miRNAs in the specification of embryonic roots 24

III. The roles of miRNAs at the post-embryonic rootmeristem (Fig. 2) 25

IV. The roles of miRNAs during lateral and adventitiousroot formation (Fig. 3) 27

V. The roles of miRNAs during root endosymbioses 29

VI. Concluding remarks and future perspectives 10

Acknowledgements 31

References 32

New Phytologist (2016) 212: 22–35doi: 10.1111/nph.14058

Key words: lateral root, microRNAs, non-coding RNAs, post-transcriptional regulation,root apical meristem symbiosis, root systemarchitecture.

Summary

Plants have evolved a remarkable faculty of adaptation to deal with various and changing

environmental conditions. In this context, the roots have taken over nutritional aspects and the

root system architecture can be modulated in response to nutrient availability or biotic

interactionswith soilmicroorganisms. This adaptability requires a fine tuning of gene expression.

Indeed, root specification and development are highly complex processes requiring gene

regulatory networks involved in hormonal regulations and cell identity. Among the different

molecular partners governing root development, microRNAs (miRNAs) are key players for the

fast regulation of gene expression. miRNAs are small RNAs involved in most developmental

processes andare required for thenormal growthof organisms, by thenegative regulationof key

genes, such as transcription factors and hormone receptors. Here, we review the known roles of

miRNAs in root specification anddevelopment, from the embryonic roots to theestablishment of

root symbioses, highlighting the major roles of miRNAs in these processes.

I. Introduction

One of the fascinating properties of land plants is represented by anindeterminate post-embryonic growth. This feature notably relieson particular cellular microenvironments, called the primarymeristems, located at the shoot and root apices (the shoot apicalmeristems and root apicalmeristems, SAMandRAM, respectively;

for reviews, see Scheres, 2007; Stahl& Simon, 2010). The stem cellniches (SCNs) of SAM and RAM fulfill two functions. First, theyare involved in their own self-renewal via the integration of varioussignals for meristem maintenance (for reviews, see Petricka et al.,2012; Drisch& Stahl, 2015). Second, they provide all the cells thatare necessary for organogenesis through division, elongation anddifferentiation activities. In Arabidopsis roots, the SCN is

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composed of a central and rarely dividing quiescent center (QC)surrounded by the stem cells or initials that will divide to producethe different cell types (Fig. 1). Rootward to the SCN, a protectiveroot cap is produced, and shootward, three main regions can bedistinguished: the meristematic, elongation and differentiationzones (Fig. 1). Radially, Arabidopsis root cells are organized inconcentric cylinders of epidermis, cortex, endodermis, pericycleand a central vasculature with xylem and phloem poles.

Being sessile organisms, plants are also characterized by a highdegree of plasticity depending on their environment (for a review,see Gaillochet & Lohmann, 2015). For example, the root systemarchitecture (RSA) can be modulated to cope with the limitingavailability (or excess) of minerals and water, depending on theirbiotic environment (for reviews, see Lugtenberg & Kamilova,2009; Dastidar et al., 2012; Gutjahr & Paszkowski, 2013; Kazan,2013). The modulation of RSA notably relies on the regulation ofprimary root growth through division and elongation activities,and also on the regulation of lateral or adventitious root branching.Understanding how plants integrate external signals to modulateRSA is also particularly important for breeding as it participates inplant fitness (for a review, see Rogers & Benfey, 2015). Anotherstrategy used by plants relies on the interactions with their bioticenvironment at the root level. It is generally assumed that thecapability of land plant ancestors to interact with arbuscularmycorrhizal fungi (AMF) was an important factor for successfulterrestrial colonization in terms of stress tolerance, limiting waterand mineral nutrients in this newly colonized environment (Smith& Read, 2008). In this symbiosis, roots cells are colonizedintracellularly by fungi, characterized by a high degree ofinvagination in colonized cells. This feature obviously increasesthe exchange interface between the roots and the fungus. These

fungal arbuscular cells are connected to an extensive extraradicalhyphal network that also considerably increases the availability ofnutrients for the plant (reviewed in Gutjahr & Parniske, 2013 andGobbato, 2015). This intimate association is widespread andestimated to be found in 80% of land plant species (Smith&Read,2008). Some plants, mainly from the Legume family, also acquirethe ability to host intracellularly soil bacteria collectively known asrhizobia in specialized root-derived organs, symbiotic nodules. Inthis interaction, the plants host symbiotic rhizobia in a favorableecological niche with a carbohydrate supply to the bacteria.Conversely, bacterial nitrogenase allows the conversion of atmo-spheric dinitrogen into ammonium, which is a form of nitrogen(N) that can be assimilated by the plant (for a review, see Suzakiet al., 2015). Given the huge amount of atmospheric dinitrogen(78%of atmospheric gas), this represents an inexhaustible potentialof N fertilizer for legume plants. Interestingly, the mycorrhizationandnodulation,which favor phosphorus (P) uptake andNfixation,respectively, are each inhibited by a high concentration of therespective compound (Thornton, 1936; Gibson & Pagan, 1977;Graham et al., 1981; Balzergue et al., 2011).

Beyond the linear and binary scheme in which a gene containsthe coding information to produce a protein through an interme-diary messenger RNA (mRNA), several processes for the finetuning of this multi-step and compartmentalized biologicalreaction have arisen in recent decades. Among them, the smallregulatory non-codingRNAs are of particular importance and haveconsiderably increased the number of non-coding genetic loci. Twomain classes of small non-coding RNAs can be established based ontheir origin and biogenesis (Axtell, 2013). Among them, themicroRNAs (miRNAs; Box 1) are small regulatory RNAs (20–24nucleotides) that are encoded by genes and participate in numerous

Fig. 1 Schematic representation of the rootsystem and meristem in Arabidopsis thaliana.LR, lateral root; SCN, stem cell niche. The stemcells and quiescent center are surrounded bywhite lines.

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biological processes in plants and animals. In particular, they areinvolved in various stress responses and in many, if not all,developmental processes. In plants (Box 2), miRNAs are generallyorganized in multigenic families that regulate key genes, such astranscription factors. As protein-coding genes, the miRNAs aremainly transcribed by RNA polymerase II, with the requirement ofthe multi-subunit complex Mediator (Kim et al., 2011) and theRNA splicingmachinery (Bielewicz et al., 2013), and they harbor a50-7mGTP-cap and a 30-polyadenylated tail, such as mRNAs (Xieet al., 2005; reviewed in Rogers &Chen, 2013). Another similaritywith coding genes involves the TATA boxes in the cis-elements ofmiRNAs that are recognized for the assembly of the Pol II pre-initiation complex (Xie et al., 2005; Megraw et al., 2006). Thetranscription of miRNA genes and the above-mentioned subse-quent steps lead to the production of a ‘long’ primary transcript ofmiRNA (pri-miRNA) that harbors a typical stem loop structure.The processing of the pri-miRNA into an activematuremiRNA is atwo-step process catalyzed by the same enzyme, acting in concertwith multiple protein partners. First, the stem loop intermediate,the miRNA precursor (pre-miRNA), is excised from the pri-miRNA by DICER-LIKE (DCL) enzymes, RNase III endori-bonucleases. This pre-miRNA is then matured into a -5p/-3pmiRNA duplex, with two nucleotide 30 overhangs. The miRNAsare predominantly processed by theDCL1 enzymewhich processes21-nucleotide-long miRNA, the main miRNA population inplants. Once loaded in a complex that contains an ARGONAUTE(AGO) protein, generally AGO1, the miRNAs negatively regulategene expression based on the complementarity between themiRNA and the target with two possible mechanisms: transcriptcleavage and translational inhibition (Brodersen et al., 2008;reviewed in Rogers & Chen, 2013; Reis et al., 2015).

The number of plant miRNAs deposited in public databases,such as miRbase and PNRD (for Plant Non-coding RNADatabase), has increased considerably in the last two decades (Yiet al., 2015; Zhang & Wang, 2015). Many have been in silicoannotated from the increasing number of available genomicsequences, but also identified from small RNA profiling studiesusing next-generation sequencing technologies. Overall, the latterstudies have highlighted many miRNA families responsive toabiotic and biotic stresses or specific to a developmental process.Notably, they have allowed the discovery of newmiRNAs, but alsoprovide a powerful framework at the spatiotemporal level forfurther functional studies. Although, in most cases, the up-regulation or, conversely, down-regulation of miRNAsmight be offunctional relevance in a given context, their modified expressionmight also represent a secondary side effect. For this reason, theexperimental validation of miRNAs is still a necessary approach.Ideally, the use of a null mutant in a given miRNA represents aclassical approach, but has been little used in plants. Conversely,miRNA overexpression, mainly using the Cauliflower MosaicVirus (CaMV) 35S strong promoter, has been extensively used, buthas the main disadvantage that it may lead to ectopic silencing oftarget genes. Finally, the use of target genes mutated in theirmiRNA binding site, and thus insensitive or less sensitive tomiRNA inhibition (miRresistant), represents a very powerfulapproach to unravel the contribution of miRNAs in the regulationof their targets. In a complementary approach, target mimicryallows the sequestration of miRNAs from the same family and theefficient study of their overall contribution to target inhibition(Franco-Zorrilla et al., 2007).

In this review, we propose to emphasize miRNAs validatedmainly by the latter approaches in the context of root development,the modulation of root architecture and root biotic interactions(Box 3), mainly using Arabidopsis and legume plants as models,because numerous studies presenting an exhaustive list of miRNAprofiles in these biological contexts have been discussed elsewhere.

II. The roles of miRNAs in the specification ofembryonic roots

Plants differ from animals by their continuous post-embryonicgrowth. Likewise, in plants, the embryo is very simple and does notcontain adult organs, such as leaves, branches and lateral roots, butalso lacks the reproductive lineages. Most studies have focused onArabidopsis, in particular because embryo patterning from a singlezygotic cell is easily accessible, and because of the very regular,

Box 3Main features and properties of microRNAs in roots

They participate in:

� Embryo and organ polarity.

� Meristem establishment and maintenance.� Vasculature differentiation.

� Lateral and adventitious root formation.� The regulation of symbioses (nodulation and mycorrhization).

Box2Main features andproperties ofmicroRNAs (miRNAs) in plants

� They often target transcription factors.

� They are mostly found in multigenic families.� They only exert a negative action on their target genes (mainly via

transcript cleavage).

� Some may be sequestered by long non-coding RNAs (lncRNAs) or

ARGONAUTE (AGO) proteins.

� The primary transcripts of miRNAs may also encode for small

regulatory peptides (miPEPs).

Box 1Main features and properties of microRNAs

� Short regulatory RNAs (mainly 21–22 nucleotides).� Produced from primary transcripts after a complex biogenesis.

� Found in several multicellular eukaryotic lineages.

� Participate inmostof thedevelopmental processes and stress responses.

� May act as mobile signals.

� Negatively regulate gene expression.

� Trigger transcript cleavage or translational repression.

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predictable and reproducible cell divisions (for a review, see tenHove et al., 2015). Root specification in the Arabidopsis embryostarts at the globular stage, where the uppermost suspensor cell,called the hypophysis, divides to furnish QC and columellaprecursor cells. The importance of miRNAs for correct embryo-genesis has been highlighted using mutants impaired in miRNAaccumulation, strong alleles in such genes often resulting in embryolethality. Accordingly, c. 400 miRNAs were detected in theArabidopsis embryo (Willmann et al., 2011), suggesting animportant role for post-transcriptional regulation during thisdevelopmental stage.

The C2H2 zinc finger protein SERRATE (SE) is involved inmiRNA processing and various se alleles have been associated withembryo patterning defect or lethality (Prigge & Wagner, 2001;Lobbes et al., 2006; Yang et al., 2006). Among the embryo defectsobserved using a se null mutant, aberrant cell division occurs in thehypophysis and, subsequently, QC precursor cells are not estab-lished and embryonic root development is impaired. At themolecular level, the accumulation of mature miRNAs is largelyaffected in se, in particular for miR165/166 (Grigg et al., 2005;Lobbes et al., 2006; Yang et al., 2006). The miR165/166 familytargets members of the class III homeodomain leucine zipper (HD-ZIP): PHAVULOTA (PHV), PABULOSA (PHB), CORONA(CNA), REVOLUTA (REV) and ARABIDOPSIS THALIANAHOMEOBOX-8 (ATHB8) (Emery et al., 2003; Mallory et al.,2004b). They are involved in numerous developmental processes,such as leaf polarity, vascular differentiation, and RAM and SAMfunctioning (reviewed in Byrne, 2006). During embryogenesis,PHV and PHB are expressed in the central apical domain at theglobular stage (Grigg et al., 2009; Smith & Long, 2010), whereasmiR165/166 are expressed more basally (Miyashima et al., 2013).Interestingly, the basal expression of PHV and PHB in the embryoresults in the conversion of RAM into SAM, showing that theexclusion of PHV/PHB from this domain is necessary for thecorrect specification of root identity (Smith & Long, 2010).Accordingly, miR165/166 have been shown to act non-cell

autonomously to restrict PHV and PHB from the base of theembryo (Miyashima et al., 2013). Another level of complexity hasarisen from recent studies in which AGO10 sequesters miR165/166 and thus represses their activity in the expression domain ofPHB and REV, thus competing with AGO1 which is necessary forclass III HD-ZIP inhibition by miR165/166 (Zhu et al., 2011;Zhou et al., 2015a).

dcl1 mutants have also been associated with defective or lethalembryo formation depending on the mutation (Schwartz et al.,1994; McElver et al., 2001; Nodine & Bartel, 2010; Seefried et al.,2014). Among them, dcl1 null alleles failed to establish correct celldivision at the base of the embryo, a defect resulting in an abnormalhypophysis (Schwartz et al., 1994; Nodine & Bartel, 2010). Theloss of root identity is attested by the absence of the QC markerWUSCHEL HOMEOBOX5 (WOX5) in dcl1-5 globularembryos (Nodine & Bartel, 2010). Numerous miRNA targetedgenes are upregulated in dcl1-5 embryos and mis-regulation ofSQUAMOSA PROMOTER-LIKE 10 (SPL10) and SPL11 bymiR156 could explain part of the dcl1-5 defects (Nodine & Bartel,2010); it has also been proposed that another possible miRNA-target couple might also be involved in embryo patterning defectsof dcl1 (Seefried et al., 2014).

III. The roles of miRNAs at the post-embryonic rootmeristem (Fig. 2)

Distally to the root SCN in the rootward orientation, the root capfulfills twomain functions: the protection of the rootmeristem andSCN when foraging the soil, and the perception of gravitropism.The redundant AUXIN RESPONSE FACTORS 10 (ARF10),ARF16 and ARF17 are targeted by miR160 (Mallory et al., 2005;Wang et al., 2005). Simultaneous loss of function of ARF10 andARF16 or miR160 overexpression leads to a significant decrease inroot elongation and defective root cap formation, thus resulting inagravitropic roots. Interestingly, miR160 overexpression pheno-types are suppressed when expressing miR-resistant versions of

Fig. 2 MicroRNAs (miRNAs) involved in rootapicalmeristemfunctioning. Quiescent center(red) is surrounded bymitotically active initials(green). ARF, AUXIN RESPONSE FACTOR;bHLH, basic helix-loop-helix; GRF,GROWTH-REGULATING FACTOR; HAMs,HAIRY MERISTEMs; HD-ZIP, homeodomainleucine zipper; NFYA, nuclear factor-YA; SCN,stem cell niche.





ARF10 or ARF16, but not ARF17 (Wang et al., 2005). ARF17 isnot involved inmiR160-dependent root cap formation, and acts inan opposite way to that of ARF10 and ARF16 for root elongation(Mallory et al., 2005; Wang et al., 2005).

The root SCN is maintained by several pathways, including theSHORTROOT (SHR) and SCARECOW (SCR) GRAS tran-scription factors initially, and also the PLETHORA (PLT)pathway, which have been extensively reviewed (Petricka et al.,2012;Heyman et al., 2014). After the first divisionof root initials inthe shootward orientation, their progenitor cells enter into rapidand transit-amplifying cell divisions (Scheres, 2007). In Medicagoand Arabidopsis,miR396 overexpression has been shown to reduceroot elongation with possible contradictory effects when monitor-ing the root meristem size of plants either overexpressing orsequestering miR396 (Bazin et al., 2013; Rodriguez et al., 2015).However, in the last two studies, an increased miR396 abundanceresulted in a reduced expression of cell cycle marker genes. TheGROWTH-REGULATING FACTORS (GRFs) are conservedtargets of the miR396 family in plants (Debernardi et al., 2012),and have been shown to promote leaf growth and to be expressed intransit-amplifying cells at the root meristem where they repressPLT gene expression (Debernardi et al., 2014; Rodriguez et al.,2015). Using a combination of elegant experiments, Rodriguezet al. (2015) deciphered anmiR396/GRFs-PLT regulatorymoduleto balance the division activities in the SCN and transit-amplifyingcells. In this model, PLT activates miR396 in the SCN, which, inturn, downregulates GRF in this territory. Stabilized expression ofGRFs (miR-resistant andmiR396mimicry) in the SCNwill lead todistorted QC and columella cells. Accordingly, the exclusion ofGRFs is proposed to be essential for formative periclinal celldivisions to occur in the SCN, as is observed in transit-amplifyingcells when GRFs are downregulated or, conversely, by increasingPLT levels (Galinha et al., 2007). In the transit-amplifying cells,GRFs are necessary for correct spatiotemporal regulation of thismitotically active territory, notably through the inhibition of PLTexpression. In conclusion, themutual repression betweenGRF andPLT, with miR396 as an intermediate partner, is crucial for thecorrect positioning of SCN and transit-amplifying cells, mainly bybalancing cell division activity (Rodriguez et al., 2015).

A recent study nicely highlighted the importance of the LOSTMERISTEMS (LOMs)/HAIRY MERISTEM (HAM) in thecontrol of plant SCNs, notably via interaction with the QCmarkerWOX5, but presumably also in a WOX5-independent manner(Zhou et al., 2015b). HAMs are GRAS transcription factors of theSCR-LIKE family required for both shoot and root indeterminacy(Stuurman et al., 2002; Engstrom et al., 2011). In Arabidopsis,four HAM copies exist, three of which (HAM1, HAM2 andHAM3) are cleaved by the miR171 family (Llave et al., 2002;Engstrom et al., 2011). They participate in a wide variety ofdevelopmental processes, such as the regulation of reproductivetransition, meristem determinacy and trichome patterning invarious species (Stuurman et al., 2002; Schulze et al., 2010; Wanget al., 2010; Engstrom et al., 2011; Curaba et al., 2013; David-Schwartz et al., 2013; Xue et al., 2014). Interestingly, miR171coverexpression reduces the primary root length, as is the case withsimultaneous mutation in the HAM genes (Wang et al., 2010;

Engstrom et al., 2011; Zhou et al., 2015b). Indeed, quadruplemutants for the HAM genes display aberrant QC and columellastem cells, resulting in root growth arrest, according to thedetection of HAM2 transcriptional and translational fusions in theQCand rootmeristem (Zhou et al., 2015b). It remains to be shownhow HAM restriction by the different miR171 members partic-ipates in root SCN maintenance as miR171 is expressed inMedicago and Arabidopsis roots (Wang et al., 2010; Lauressergueset al., 2015).

In addition to their roles during embryogenesis, miR165/166also occupy a central role in the regulation of root development, inparticular for the correct differentiation of the vascular elementsand the regulation of meristem size. The SHR protein moves fromthe stele, its site of production, to the endodermis, where it activatesthe expression of miR165a and miR166b. In turn, miR165/166diffuse in the central stele, thus establishing an opposite gradient tothat of their class III HD-ZIP targets (Carlsbecker et al., 2010).Similar to the shr mutant, the miR-resistant phd-7d allele ischaracterized by the frequent replacement of protoxylem bymetaxylem cells. Conversely, simultaneous mutations of phv, phd,can, rev and athb8 result in protoxylem instead of metaxylem cells.Interestingly, phb-shr double loss-of-function mutants displaynormal vascular patterning, and miR165a under the control of aground-tissue specific promoter was able to rescue vascular defectsof the shrmutant (Carlsbecker et al., 2010). Finally, using the greenfluorescent protein (GFP) sensor system formiR165/166 actions, ithas been confirmed that these miRNAs act non-cell autonomouslyin a dose-dependent manner (Carlsbecker et al., 2010; Miyashimaet al., 2011). Together, the last two studies elegantly demonstratedthat opposite gradients of miR165/166 and class III HD-ZIP arecrucial for correct xylemdifferentiation (highmiR165/166 and lowHD-ZIP) on control of the mobile SHR diffusible signal.Collectively, this provides a case study in which mobile transcrip-tion factors, opposite gradients of miRNAs and targets participatein the establishment of sharp and robust developmental boundaries(Benkovics & Timmermans, 2014). In addition, a mathematicalmodel showed that the integration of auxin, cytokinin (CK), SHR,PHB and miR165/166 can explain the establishment of thebisymmetric pattern in the root of Arabidopsis (Muraro et al.,2014). Recently, mi857 was shown to target LACCASE7 inArabidopsis and, using both overexpression and mutantapproaches, the authors revealed a negative role for miR857 inthe regulation of lignin content and secondary xylem differenti-ation in Arabidopsis (Zhao Y et al., 2015).

phb-1d, another miR-resistant allele of PHB, displays a shortroot phenotype (Hawker & Bowman, 2004), and the rootmeristem size of both phb-1d and phv-1d alleles is reduced,opposite to that observed in phv-phb double mutants (Dello Ioioet al., 2012). As for the embryonic defects of se, phv-phb doublemutations suppress the short root phenotype observed in se (Grigget al., 2009), suggesting an additional and important role for themiR165/166 family in the regulation of root growth andmeristemlongitudinally. CK plays an important role in the control of rootmeristem size (Dello Ioio et al., 2007, 2008), and PHB has beenshown to directly activate a CK biosynthetic gene ISOPENTYLTRANSFERASE7 (IPT7). In turn, the resulting CK inhibits




miR165a transcription, thus building an incoherent feedbackregulatory loop between CK, PHB and miR165a. This circuit hasbeen proposed to regulate the balance between division anddifferentiation, on CK fluctuations in the roots, thus controllingmeristem size (Dello Ioio et al., 2012). Recently, it has beenproposed that IPT3, in addition to IPT7, might contribute to theactivation of CK biosynthesis by the PHB–SHR circuit in thiscontext of meristem size adjustment (Sebastian et al., 2015).

IV. The roles of miRNAs during lateral andadventitious root formation (Fig. 3)

In plants, RSA varies greatly and is highly plastic depending on theenvironment. Lateral and adventitious roots are distinguishedbased on their origin: root tissue or not, respectively (reviewed inAtkinson et al., 2014; Bellini et al., 2014). In dicots, the primaryroot generally arises from the embryonic radicle and branches intolateral roots of several orders and thus contributes to RSAestablishment (taproots or allorhizic systems). In monocots,adventitious roots are of particular importance for root systemestablishment (fibrous roots or homorhizic systems). They derivefrom below-ground or underground shoot nodes, thus formingcrown and brace roots, respectively. In Arabidopsis, adventitiousroots derive from the hypocotyl or at the root–hypocotyl junction.These junction roots can be viewed as a rescue system when theprimary root arrests or is damaged (Lucas et al., 2011). Theformation of lateral and adventitious roots has been particularlystudied in recent decades; it represents a post-embryonic rootspecification process from cells that have already been engaged intoa differentiation route. In Arabidopsis, lateral roots form from thereactivation of pericycle cells facing the xylem poles that undergoanticlinal, periclinal and tangential cell divisions to form aprimordium (reviewed in P�eret et al., 2009). Despite the verysimilar patterning and final anatomy of lateral and adventitiousroots, different molecular players contribute to their establishmentin Arabidopsis (reviewed in Atkinson et al., 2014; Bellini et al.,

2014). Although a series of elegant experiments have led to theformulation of two non-exclusive hypotheses to explain themechanism of the early specification and patterning of lateral rootsin Arabidopsis, their relative contributions and interplay are still amatter of debate (De Smet et al., 2007; Ditengou et al., 2008;Dubrovsky et al., 2008; Moreno-Risueno et al., 2010; Kircher &Schopfer, 2016). The availability of macro- and micro-nutrients isan important factor modulating RSA. Several miRNAs participatein the regulation of these processes from themodulation of nutrientuptake, translocation, sensing and assimilation to an adaptiveresponse via the modulation of the root architecture. As exhaustiveand recent articles have reviewed themiRNAs involved inN, P andpotassium (K) homeostasis (Kulcheski et al., 2015; Nguyen et al.,2015), more attention is paid here to the miRNA-target nodes thatparticipate in the modulation of root architecture. Many factorscontribute to the regulation of lateral root formation, frompreinitiation events tomeristem activation (reviewed in P�eret et al.,2009;Overvoorde et al., 2010; Petricka et al., 2012). Among them,the hormone auxin plays a central role (Lavenus et al., 2013) atthree possible levels: homeostasis, transport and signaling.

Three ARFs are targeted by miR160: ARF10, ARF16 andARF17 (Mallory et al., 2005; Wang et al., 2005). miR-resistantalleles of ARF16 and ARF17 exhibit reduced root branching,whereas double arf10-arf16 loss-of-function mutants andmiR160c-overexpressing plants show the opposite defects (Malloryet al., 2005; Wang et al., 2005). ARF16 and ARF17 downregula-tion by miR160 thus exerts a positive role on lateral rootdevelopment in Arabidopsis. Surprisingly, neither arf10/arf16single loss-of-function mutants nor an miR-resistant ARF10 alleleexhibits an obvious phenotype in terms of lateral root formation(Wang et al., 2005; Liu et al., 2007).

Other members of the ARF family involved in auxin homeosta-sis, namely ARF2, ARF3 and ARF4, are targeted by trans-actingshort interfering RNAs (tasiRNAs; Allen et al., 2005; Williamset al., 2005). These tasiRNAs are produced from miRNA-mediated cleavage products of non-coding TAS transcripts. The

Fig. 3 MicroRNAs (miRNAs) involved in thecontrol of the root system architecture. Redarrows, negative role on the process; greenarrows, positive role on the process. Pleasenote that the numerous and complexconnections between miRNA/target moduleshave been omitted for clarity. Please also notethat these different modules may be requiredat different stages of organ development andin certain environmental conditions (nitrate,auxin, stresses). AFB, AUXIN SIGNALING F-BOX PROTEIN; ARF, AUXIN RESPONSEFACTOR; bHLH, basic helix-loop-helix; GRF,GROWTH-REGULATING FACTOR; HAMs,HAIRY MERISTEMs; HD ZIP, homeodomainleucine zipper; IAR, IAA-Ala RESISTANT;NAC, NAM, ATAF, CUC (NO APICALMERISTEM, ARABIDOPSIS TRANSCRIPTIONACTIVATION FACTOR, CUP-SHAPEDCOTYLEDON); TIR, TRANSPORT INHIBITORRESPONSE.





produced tasiRNAs will, in turn, trans-regulate target genes. Forexample, TAS3 is cleaved by miR390 and the resulting tasiRNAsnegatively regulate ARF2, ARF3 and ARF4 in the context ofpolarity establishment of aerial organs and developmental timing(Fahlgren et al., 2006; Garcia et al., 2006; Hunter et al., 2006). Arole of TAS3/miR390/ARF modules has been revealed in thecontext of lateral root formation (Marin et al., 2010; Yoon et al.,2010). miR390 expression is induced by auxin and a complexinterplay between ARF2, ARF3 and ARF4 (Marin et al., 2010;Yoon et al., 2010). During lateral root initiation, miR390 isexpressed in pericycle cells facing the xylem poles, and, in turn, willlead to tasiARF production that ultimately negatively regulatesARF2, ARF3 and ARF4 in lateral root primordia, thus allowingtheir correct growth (Marin et al., 2010). In summary, Marin et al.(2010) elegantly revealed a complex autoregulatory loop betweenmiR390, tasiARFs, ARF2/3/4 and auxin to quantitatively regulatethe growth of lateral roots in Arabidopsis.

The miR164 family is predicted to target five members of theNAC transcription factor family (for NAM, ATAF, CUC: NOAPICAL MERISTEM, ARABIDOPSIS TRANSCRIPTIONACTIVATION FACTOR, CUP-SHAPED COTYLEDON,respectively; Guo et al., 2005; for a review, see Olsen et al.,2005). In addition to their roles in aerial development (Laufs et al.,2004;Mallory et al., 2004a; for a review, see Blein et al., 2010), onemember of theNAC family,NAC1, positively regulates lateral rootformation, notably via the transduction of the auxin signaldownstream of an F-box auxin receptor, TRANSPORTINHIBITOR RESPONSE1, TIR1 (Ruegger et al., 1998; Xieet al., 2000; Guo et al., 2005). NAC1 is expressed in pericycle cellsduring lateral root initiation (Xie et al., 2000) and miR164 isinduced by auxin, which, in turn, cleaves theNAC1 transcript (Guoet al., 2005). UsingmiR164a andmiR164bmutants, miR-resistantNAC1 and the inducible expression system for both miR164 andNAC1, Guo et al. (2005) revealed an autoregulatory loop betweenauxin, miR164 and NAC1 for lateral root initiation, wheremiR164 acts as a negative regulator. Interestingly, a later studyrevealed a correlation between RSA, miR164 and NAC1 levels inmaize (Li J et al., 2012), and miR164 expression is modified inresponse toN and P deficiencies in lupin andmaize roots and leaves(Zhu et al., 2010). Together, these results might place the auxin/miR164/NAC1 loop as a conserved module participating in theadaptation of root architecture to nutrient availability.

The auxin receptors TIR1, AUXIN SIGNALING F-BOXPROTEIN 2 (AFB2) and AFB3 (also known as TAAR, for TIR1/AFB1 auxin receptors) are targeted by miR393a and miR393b inArabidopsis (Jones-Rhoades & Bartel, 2004; Dharmasiri et al.,2005). AFB1 is partially resistant to miR393 cleavage, a propertywhichmight be a result of themismatch in themiR393 binding siteof AFB1, and is repressed by miR393 at the translational level(Navarro et al., 2006; Parry et al., 2009). Parry et al. (2009) alsodemonstrated that tir1-1 mutants, as miR393a- and miR393b-overexpressing lines, are less responsive to auxin in the stimulationof lateral roots. Interestingly, auxin has long been proposed to beinvolved in the integration of the nitrate signal into themodulationof root development (Forde, 2002; Walch-Liu et al., 2006).miR393 and AFB3 have been shown to be upregulated in

Arabidopsis following nitrate treatment (Vidal et al., 2010). Inparticular, miR393 was induced specifically in roots 2 h followingnitrate treatment. According to this and by contrast with AFB3,only reduced and assimilated (so-called) nitrate, and not nitratetreatment itself, led to miR393 induction. In addition, miR393overexpression, as a null afb3 mutation, suppresses root modifi-cation following nitrate treatment: (1) inhibition of root elongationand (2) stimulation of lateral roots. Interestingly, none of the othermiR393 targets (TIR1, AFB1 and AFB2) are necessary for theestablishment of RSA modulation triggered by exogenous nitratesupply. This apparently incoherent AFB3/miR393 feed-forwardloop might provide a mechanism by which miR393 can be rapidlyand precisely adjusted to regulate its target AFB3. In other words,this may provide a robust mechanism for rapid adaptation of RSAdepending on the fluctuating internal and external nitrate concen-trations (Vidal et al., 2010). pri-miR393b is also induced followingauxin treatment (Chen et al., 2011). Overexpression of a miR393-resistant form of TIR1 significantly increased lateral root numberand, conversely, either tir1 null mutation or miR393a/b overex-pression is associatedwith reduced lateral root number (Chen et al.,2011). miR393b is the predominant source of miR393 in all aerialorgans studied so far (Si-Ammour et al., 2011), and miR393a andmiR393bboth contribute tomiR393production in roots (Windelset al., 2014). In the latter study, it was also shown that AFB2 andAFB3 degradation by miR393 led to the production of secondaryshort interfering RNAs (si-TAARs) using tasiRNA pathways (Si-Ammour et al., 2011).TheTAAR-miR393modules have also beenshown to be required for: (1) adaptive root growth inhibitionpromoted by osmotic stress or ABA treatment (Chen et al., 2012);(2) root acclimation to saline stress via the regulation of redox statusand auxin signaling (Iglesias et al., 2014); and (3) correctestablishment of auxin signaling output (Windels et al., 2014).As nitrate treatment induced the auxin synthetic responsiveconstruct DR5::GUS in Arabidopsis (Vidal et al., 2010), thepossible crosstalk and interference between miR393, its targets,auxin, and internal and external nitrate concentration still need tobe deciphered in detail. Moreover, by combining computationaland experimental data, it was shown that NAC4, which is alsoregulated by the miR164 family, acts downstream of AFB3 for theroot adaptation to nitrate, at least for the modulation of lateral rootformation, in an NRT1.1-dependent manner (Vidal et al., 2013,2014), thus adding another layer of interplay between nitratehomeostasis, TIR1/miR393 and NAC1/miR164 modules in theregulation of root architecture (Xie et al., 2000, 2002).

ARF6 andARF8, the targets ofmiR167, were initially studied inthe context of reproductive stages, as this module regulates ovuleand anther fertility (Nagpal et al., 2005; Ru et al., 2006; Wu et al.,2006). Using cell-specific profiling, miR167/ARF8 have beenproposed to mediate the root adaptive response to nitrate stimulusin Arabidopsis (lateral root initiation stimulated vs lateral rootgrowth inhibited). Accordingly, both themiR167a-overexpressingline and arf8nullmutant exhibited a loss of lateral root growth afternitrate stimulation (Gifford et al., 2008). Later, it was shown thatmiR167, ARF6 and ARF8, together with miR160 and ARF17,regulate adventitious root formation in Arabidopsis (Gutierrezet al., 2009). Using a detailed analysis of promoter-GUS fusions,




genetics, overexpression and quantitative expression analyses, theauthors demonstrated a complex interplay and feedback loopsbetween these three ARFs, their miRNAs, light and adventitiousroot formation. Schematically, ARF6 and ARF8 stimulate adven-titious root formation, whereas ARF17 does the opposite, and theirmutual regulation and post-transcriptional repression by miR167and miR160 are crucial for the correct formation of shoot-borneroots (Gutierrez et al., 2009). A subsequent study demonstrated therequirement of three auxin responsive genesGRETCHENHAGEN3 (GH3) and jasmonate homeostasis downstream of this complexARFs/miR160/miR167 network (Gutierrez et al., 2012).miR167a also targets IAA-Ala RESISTANT 3 (IAR3) on osmoticstress to adapt root architecture, whereas ARF6 and ARF8 cleavageby miR167 appears to be dispensable for this plant adaptiveresponse (Kinoshita et al., 2012). IAR3 is an enzyme responsible forthe conversion of an inactive auxin form, Ala-IAA, into thebioactive auxin IAA (Davies et al., 1999). On high osmotic stress,miR167a and miR167b, and IAR3, are downregulated andupregulated, respectively. According to this, iar3 mutants havereduced IAA levels and lack the root architecture modificationstriggered by osmotic stress. Conversely, overexpression ofmiR167-resistant IAR3 led to increased IAA accumulation and increasedlateral root development. Finally, a positive role for IAR3 indrought stress tolerance was demonstrated, and Kinoshita et al.(2012) proposed a model with specific miR167 regulationdepending on the tissue and stress (roots vs aerial, osmotic vs Nsupply). This last example illustrates the importance of thebiological context in the regulation of different targets by the samemiRNA family.

INDOLE-3-ACETIC ACID INDUCIBLE 28 (IAA28) is anAux/IAA repressor initially identified in a genetic study of theiaa28-1 gain-of-function allele. IAA28 has recently been shown tobe necessary for the transcriptional repression of auxin-induciblegenes required for lateral root promotion (Rogg et al., 2001).IAA28 is post-transcriptionally regulated by miR847, a lowabundant miRNA that is induced on auxin treatment (Wang &Guo, 2015). Accordingly, miR847 overexpression and the iaa28null mutant exhibitedmore lateral roots, whereas overexpression ofthe IAA28-1 gain-of-function allele or miR847-resistant IAA28had a negative impact on the lateral root number (Wang & Guo,2015). Interestingly, post-transcriptional cleavage of IAA28 trig-gered by miR847 is synergistic to the proteasome degradation ofAux/IAAs, and both degradation processes are stimulated by auxin,thus providing a robust degradation mechanism. Using a somaticembryogenesis assay, the authors proposed that IAA28 degradationaffects lateral organ growth via the regulation of cell proliferation.

Many other studies have suggested the potential implication ofmiRNAs in the regulation of lateral roots. For example, miRNAparticipation in the homeostasis of macronutrients (uptake,assimilation, translocation, etc.) might, in fact, modify RSAthrough an indirect mechanism, as the perception of nutrientconcentration and their assimilates has a drastic effect on rootgrowth. Small RNA profiling studies have also suggested theimplication of some miRNAs in the regulation of lateral roots inresponse to nutrient starvation. For example, Liang et al. (2012)proposed that the combined action of miR160-ARF10/16/17,

miR167/ARF6/8 and miR171-HAM1/2/3 modules might beimportant for the modulation of RSA on N starvation. ThemiR171 family and their HAM targetsmight be suspected to play arole in the regulation of lateral root formation, as miR171b isexpressed in lateral root primordia and miR171b overexpressionreduces the lateral root density in Medicago (Lauressergues et al.,2015). In the same line, miR396 detection in Arabidopsis andMedicago lateral root primordia and the reduced root dryweight ofMIM396 plants in Medicago might favor a role for miR396 in theformation of lateral roots (Bazin et al., 2013; Bao et al., 2014). InMedicago, miR166a overexpression led to a reduced lateral rootnumber (Boualem et al., 2008), as is the case in the triple phb-phv-revmutant in Arabidopsis (Hawker & Bowman, 2004). Accordingto this and the increased lateral root density in the miR165/166-resistant rev-10d allele, themiR165/6-HD-ZIPmodule might alsobe suspected to play a role in the regulation of lateral rootformation, in addition to those well established in the longitudinaland radial patterning of RAM in Arabidopsis.

V. The roles of miRNAs during root endosymbioses

Themycorrhization process has been studied for decades because ofits wide distribution in the plant lineage, its ecological importanceand the advantages it provides in terms of nutrition and stresstolerance. This process was first studied in a plant–microbeinteraction perspective, but can also be viewed as a dynamic andreversible developmental process for the plant cell. Indeed, rootcortical cells that are invaded by arbuscules are already engaged in adefined differentiation stage. On P deficiency, these cells can hostfungi based on molecular exchanges between the two partners thatultimately lead to cell specialization. Conversely, arbuscules cancollapse within a few days, leaving cortical cells devoid of branchedarbuscules (Kobae et al., 2016). For this reason, it would not besurprising to unravel an increasing number of developmentalregulators participating in this particular cell differentiationprogram and reversible infection process. Among them, miR396,which is known to regulate GRF transcription factors, basic helix-loop-helix (bHLH) and cell proliferation activity (see above), hasalso been shown to regulate the mycorrhizal colonization rate inMedicago (Bazin et al., 2013). Indeed, Medicago plants overex-pressing or sequesteringmiR396 are characterized bydecreased andincreased colonization rates, respectively, thus defining aninhibitory role for miR396 on the mycorrhization process. Toour knowledge, it remains to be shownwhich of themiR396 targetsparticipate in the regulation of fungal colonization. Low auxinlevels have also been shown to stimulate mycorrhization inMedicago, tomato and rice according to DR5::GUS induction inarbuscules and pre-miR393 repression during mycorrhization(Etemadi et al., 2014). However, the targets of miR393, AFB/TIR1, auxin transport and other signaling elements involvedduring mycorrhization still need to be further investigated. TheGRAS transcription factor, NODULATION SIGNALINGPATHWAY 2 (NSP2), was first isolated on the basis of its essentialrole for the formation of symbiotic nodules in the Legume family(Oldroyd & Long, 2003). Later, it was shown that NSP2 is alsorequired formycorrhizal colonization and is regulated bymiR171h





in Medicago (Devers et al., 2011; Maillet et al., 2011; Lauresser-gues et al., 2012). According to themyc� phenotype of nsp2 plants,miR171h overexpression also impairs fungal colonization. Inter-estingly, the correct post-transcriptional regulation of NSP2 bymiR171h is crucial for the restriction of the fungus, bothquantitatively and spatially, in Medicago roots (Lauressergueset al., 2012) (Fig. 4).

Legume nodules are generally root-derived organs, and althoughthey are formed on stems, their formation is tightly linked to shoot-borne root primordia. Two main classes of nodule can bedistinguished based on the persistence or not of a nodulemeristem:indeterminate nodules (e.g. in Medicago) and determinate round-shaped nodules (e.g. in soybean, bean and Lotus). Despite theanatomical differences between roots and nodules, the nodulationprogram has co-opted part of the root developmental program(Couzigou et al., 2012, 2013; Franssen et al., 2015). The nodu-lation signaling pathway has also been acquired through the co-option of the mycorrhizal signaling pathway. It is interesting tonote here that both mycorrhizal fungi and rhizobia, as theirsymbiotic signals (Myc and Nod factors), trigger lateral rootorganogenesis (Maillet et al., 2011). As for lateral roots, noduleinitiation and organogenesis are tightly controlled processes, inwhichmiRNAs have been reported to participate. Excellent reviewshave described the nodulation signaling program compared withthat of the lateral roots (Desbrosses & Stougaard, 2011; Oldroydet al., 2011). In Medicago, miR166 targets a class III HD ZIPtranscription factor, and has a negative effect on both lateral rootand nodule formation when it is overexpressed (Boualem et al.,2008). Overexpression ofmiR166 also leads to aberrant patterningof vascular bundles in roots (multiplication of xylem poles),according to the well-known functions of the miR166/HD-ZIPmodule in this latter process in Arabidopsis (see earlier). miR169regulates HAP2-1, a transcription factor of the NFY-A family(Combier et al., 2006). The overexpression of miR169 as RNAinterference against its target, HAP2-1, leads to delayed noduleformation and arrested meristem development, thus resulting innon-fixating round-shaped nodules. By contrast, plants expressinga miR169-resistant version ofHAP2-1 develop nodules associatedwith a defect in nodule apical growth, thus resulting in shorternodules, defining an important role for HAP2-1 restriction during

the course of nodule organogenesis. Interestingly, the miR169e/f/g-NFYA2/10 modules have also been shown to regulate rootarchitecture in Arabidopsis (Sorin et al., 2014). Soybean is perhapsthe legumemodel for whichmost examples ofmiRNAs involved inthe regulation of nodulation exist. Indeed, several miRNAsaffecting nodule number have been identified recently (Li et al.,2010; Turner et al., 2013; Wang et al., 2014, 2015; Yan et al.,2015). In these latter studies, overexpression ofmiR167c,miR172c,miR482,miR1512,miR1515 andmiR2606bhas a positive effect onnodule formation in soybean, whereas overexpression of miR156,miR160 and miR4416 has the opposite effect. The miR172c-AP2module has been shown to play a similar role during noduleorganogenesis in Phaseolus vulgaris (Nova-Franco et al., 2015), andrecent evidence suggests a similar situation in Lotus japonicus (Holtet al., 2015). Generally, these studies have combined miRNAoverexpression, miR-resistant target constructs and miRNAmimicry to unravel the miRNA-target module in the symbioticprocess. It should be noted here that an miRNA affecting theindeterminate type of nodule does not necessarily affect determi-nate nodule development, in particular in terms of the regulation ofauxin signaling (Mao et al., 2013) (Fig. 5). During infection by soilnematodes, root cells are also reprogrammed, and some studieshave suggested local and systemic implications of miRNA-targetmodules in the establishment of this interaction (Hewezi et al.,2008; Li X et al., 2012; Zhao W et al., 2015). Finally, small RNAprofiling studies have suggested the implication of miRNA post-transcriptional regulation in the context of infection by rootpathogens (Radwan et al., 2011; Verma et al., 2014). However,

Fig. 4 MicroRNAs (miRNAs) involved in the control of mycorrhization.Photograph of a lateral root colonized by the arbuscular mycorrhizal fungusRhizophagus irregularis. Red arrows indicate a negative role on the process.Note: the targets and the connection betweenmiRNA/target modules havebeen omitted for clarity. The fungal structures (green) have been stainedwith ink (see Lauressergues et al., 2012).

Fig. 5 MicroRNAs (miRNAs) involved in the control of nodulation. Redarrows, negative role on the process; green arrows, positive role on theprocess. Note: the targets and the connection between miRNA/targetmodules have been omitted for clarity.




functional studies still need to be performed to investigate the rolesof these miRNA-target modules in these detrimental bioticinteractions.

VI. Concluding remarks and future perspectives

Since their initial discovery in the early 1990s (Lee et al., 1993;Wightman et al., 1993) and their distinction as a new class ofregulatory molecules in the early 2000s (Pasquinelli et al., 2000;Reinhart et al., 2000, 2002; Lagos-Quintana et al., 2001; Lauet al., 2001; Lee & Ambros, 2001; Llave et al., 2002), miRNAshave emerged as widespread regulatory RNAs in most, if not all,biological processes in plants and animals. Nowadays, they arecrucial actors in the regular patterning of embryo and adultorganisms, and also in the adaptation to fluctuating biotic andabiotic environments. The simple scenario in which an miRNArepresses gene expression via transcript cleavage or translationinhibition seems to be even more complex today with theemergence of additional players in this scheme: activatingmiRNAs, pseudo targets, miRNA sponges, target mimicry,miRNA-encoded peptides (miPEPs) and, finally, many otherclasses of non-coding RNAs, such as tasiRNAs and long non-coding (lnc) RNAs (for reviews see Cech & Steitz, 2014; Guil &Esteller, 2015). In addition, the regulation of miRNA biogenesisand turnover is made up of extremely complex and tightlycontrolled mechanisms. One breakthrough was the discovery ofIPS1 lncRNA, which sequesters miR399 and thus antagonizes thecleavage of the miR399 target, PHO2 (Franco-Zorrilla et al.,2007). Since then, the proposed artificial strategy to sequester anymiRNA isoform via a target mimic has been successfully andwidely used. It appears that other miRNA endogenous targetmimics (eTMs) may also exist in plants; most of the miRNAsdiscussed above might have an eTM in Arabidopsis (Wu et al.,2013). In plants, recent studies have highlighted the importanceof lncRNA regulation (Ariel et al., 2015) and have shown that asignificant amount of lncRNAs (as miRNAs) can be loaded intoribosomes, thus suggesting a possible role in translationalregulation for these so-called non-coding transcripts (Juntawonget al., 2014). In animals, there is also increasing evidence forregulatory peptides derived from lncRNA transcripts, firstthought to be non-coding (Payre & Desplan, 2016). Recently,we have shown that miRNA transcripts can also produce smallregulatory peptides, called miPEPs, which, in turn, favor theaccumulation of their corresponding miRNAs (Lauressergueset al., 2015; Couzigou et al., 2016). This latter finding needs to bestudied in other miRNA families and species to determine theextent of this mechanism. If widespread, these new regulatorypeptides might also serve as tools for the study of separatemiRNAs from the same family because of their apparentspecificity (Couzigou et al., 2015). A major challenge will be todecipher how this miRNA-miPEP couple might be transcrip-tionally regulated and how these peptides might act on theirmiRNA. In the context of plant development, several miRNAshave been shown to act as mobile signals, thus resembling animalmorphogens (Benkovics & Timmermans, 2014). However, theregulatory and selectivity mechanism by which a miRNA moves

or not is still not clear. Indeed, opposite gradients of miRNA andtargets could be required for the correct determination of rootvasculature, SAM and embryogenesis. This non-cell autonomousaction of miRNA has been shown to be tightly controlled by theopposite action of AGO1 and AGO10 in Arabidopsis (Zhu et al.,2011; Zhou et al., 2015a). A similar mechanism in the context ofroot development has not yet been elucidated. Another challengefor miRNAs with dual roles in the regulation of root meristemhomeostasis and lateral roots will be to dissect their contributionat the lateral root level locally, and possibly from their role in themeristem, a region that is close to the site of lateral root priming.A common feature of miRNA-target modules is the presence ofincoherent regulatory loops and the complex interplay betweenthese modules. The existence of incoherent regulatory loops can,however, be interpreted as a possible mechanism to providerobustness and a rapid adaptability to regulatory gene networks(Li et al., 2009; Osella et al., 2011). The global study of thesemiRNA-target modules, their interconnection and their biolog-ical relevance in response to perturbed environments (e.g. singlevs multiple nutrient deprivation) is now possible in the era ofsystems biology and the tools that have been developed. From anevolutionary perspective, it is not surprising to find miRNA-target nodes involved in both shoot and root regulation in thelight of the ‘shoot–root dichotomy hypothesis’, where the rootmorphogenetic program can be thus considered as a co-optedshoot program. Moreover, the conservation of such miRNAs inbasal embryophytes (Cuperus et al., 2011; Jones-Rhoades, 2012)that do not have roots or even vascular elements raises thequestion of their functions in organisms with such differentlifestyles. More extensively, it might be interesting to study themiRNA-target nodes involved in embryogenesis, developmentaltiming and reproductive development in species with an oppositebalance of diploid and haploid phases. The development ofefficient model systems in each of these clades will undoubtedlyallow the study of miRNAs in an evolutionary and develop-mental perspective in future years. The study of the relative rolesof each miRNA isoform and their associated targets present invarious numbers among species also represents an interestingframework for our understanding of miRNA evolution. Afteralmost two decades of intense research in the non-coding RNAfield, it appears that many elements still need to be, and will be,discovered. In other words, and as mentioned in a recent reviewsummarizing 50 yr of Arabidopsis research (Provart et al., 2016),Churchill’s famous sentence about World War II can also applyto the non-coding RNA field: ‘This is not the end. It is not eventhe beginning of the end. But it is, perhaps, the end of thebeginning’.

Acknowledgements

The authors are very grateful to Dr Alexandre Tromas (CCG,UNAM, Mexico) for sharing confocal sections of Arabidopsis andtoDr Pascal Ratet (IPS2, Orsay, France) for the permission to use apicture of Medicago root nodules, both used for drawing thefigures. This work was funded by the French ANR projectmiRcorrhiza (ANR-12-JSV7-0002-01).





References

Allen E, Xie Z, Gustafson AM, Carrington JC. 2005.microRNA-directed phasing

during trans-acting siRNA biogenesis in plants. Cell 121: 207–221.Ariel F, Romero-Barrios N, Jegu T, Benhamed M, Crespi M. 2015. Battles and

hijacks: noncoding transcription in plants. Trends in Plant Science 20: 362–371.Atkinson JA, Rasmussen A, Traini R, Voss U, Sturrock C,Mooney SJ,Wells DM,

Bennett MJ. 2014. Branching out in roots: uncovering form, function, and

regulation. Plant Physiology 166: 538–550.AxtellMJ. 2013.Classification and comparison of small RNAs from plants. AnnualReview of Plant Biology 64: 137–159.

Balzergue C, Puech-Pages V, Becard G, Rochange SF. 2011. The regulation of

arbuscular mycorrhizal symbiosis by phosphate in pea involves early and systemic

signalling events. Journal of Experimental Botany 62: 1049–1060.BaoM, BianH, Zha Y, Li F, Sun Y, Bai B, Chen Z,Wang J, ZhuM,HanN. 2014.

miR396a-mediated basic helix-loop-helix transcription factor bHLH74repression acts as a regulator for root growth in Arabidopsis seedlings. Plant andCell Physiology 55: 1343–1353.

Bazin J, Khan GA, Combier JP, Bustos-Sanmamed P, Debernardi JM, Rodriguez

R, Sorin C, Palatnik J, Hartmann C, Crespi M et al. 2013.miR396 affects

mycorrhization and root meristem activity in the legumeMedicago truncatula.Plant Journal 74: 920–934.

Bellini C, Pacurar DI, Perrone I. 2014. Adventitious roots and lateral roots:

similarities and differences. Annual Review of Plant Biology 65: 639–666.Benkovics AH, Timmermans MC. 2014. Developmental patterning by

gradients of mobile small RNAs. Current Opinion in Genetics & Development27: 83–91.

Bielewicz D, Kalak M, Kalyna M,Windels D, Barta A, Vazquez F, Szweykowska-

KulinskaZ, JarmolowskiA. 2013. Introns of plant pri-miRNAs enhancemiRNA

biogenesis. EMBO Reports 14: 622–628.Blein T, Hasson A, Laufs P. 2010. Leaf development: what it needs to be complex.

Current Opinion in Plant Biology 13: 75–82.Boualem A, Laporte P, Jovanovic M, Laffont C, Plet J, Combier JP, Niebel A,

Crespi M, Frugier F. 2008.MicroRNA166 controls root and nodule

development inMedicago truncatula. Plant Journal 54: 876–887.Brodersen P, Sakvarelidze-Achard L, Bruun-Rasmussen M, Dunoyer P,

Yamamoto YY, Sieburth L, Voinnet O. 2008.Widespread translational

inhibition by plant miRNAs and siRNAs. Science 320: 1185–1190.ByrneME.2006.Shootmeristem function and leaf polarity: the role of class IIIHD-

ZIP genes. PLoS Genetics 2: e89.Carlsbecker A, Lee JY, Roberts CJ, Dettmer J, Lehesranta S, Zhou J, Lindgren O,

Moreno-Risueno MA, Vaten A, Thitamadee S et al. 2010. Cell signalling bymicroRNA165/6 directs gene dose-dependent root cell fate.Nature 465: 316–321.

Cech TR, Steitz JA. 2014. The noncoding RNA revolution—trashing old rules to

forge new ones. Cell 157: 77–94.Chen H, Li Z, Xiong L. 2012. A plant microRNA regulates the adaptation of roots

to drought stress. FEBS Letters 586: 1742–1747.Chen ZH, BaoML, Sun YZ, Yang YJ, Xu XH,Wang JH, Han N, Bian HW, Zhu

MY. 2011.Regulation of auxin response by miR393-targeted transport inhibitor

response protein 1 is involved in normal development in Arabidopsis. PlantMolecular Biology 77: 619–629.

Combier JP, Frugier F, de Billy F, BoualemA, El-Yahyaoui F,Moreau S, VernieT,

Ott T, Gamas P, Crespi M et al. 2006.MtHAP2-1 is a key transcriptional

regulator of symbiotic nodule development regulated by microRNA169 in

Medicago truncatula. Genes & Development 20: 3084–3088.Couzigou JM, Andr�e O, Guillotin B, Alexandre M, Combier JP. 2016. Use of

microRNA-encoded peptide miPEP172c to stimulate nodulation in soybean.

New Phytologist 211: 379–381.Couzigou JM, Lauressergues D, Becard G, Combier JP. 2015.miRNA-encoded

peptides (miPEPs): a new tool to analyze the roles of miRNAs in plant biology.

RNA Biology 12: 1178–1180.Couzigou JM, Mondy S, Sahl L, Gourion B, Ratet P. 2013. To be or noot to be:evolutionary tinkering for symbiotic organ identity.Plant Signaling&Behavior 8:pii: e24969.

Couzigou JM, Zhukov V,Mondy S, Abu elHebaG, CossonV, Ellis TH, Ambrose

M, Wen J, Tadege M, Tikhonovich I et al. 2012. NODULE ROOT and

COCHLEATA maintain nodule development and are legume orthologs of

Arabidopsis BLADE-ON-PETIOLE genes. Plant Cell 24: 4498–4510.Cuperus JT, Fahlgren N, Carrington JC. 2011. Evolution and functional

diversification ofMIRNA genes. Plant Cell 23: 431–442.Curaba J, Talbot M, Li Z, Helliwell C. 2013.Over-expression of microRNA171affects phase transitions and floral meristem determinancy in barley. BMC PlantBiology 13: 1–10.

Dastidar MG, Jouannet V, Maizel A. 2012. Root branching: mechanisms,

robustness, and plasticity.Wiley Interdisciplinary Reviews. Developmental Biology1: 329–343.

David-Schwartz R, Borovsky Y, Zemach H, Paran I. 2013. CaHAM is

autoregulated and regulates CaSTM expression and is required for shoot apical

meristem organization in pepper. Plant Science 203–204: 8–16.Davies RT,GoetzDH, Lasswell J, AndersonMN,Bartel B. 1999. IAR3 encodes anauxin conjugate hydrolase from Arabidopsis. Plant Cell 11: 365–376.

De Smet I, Tetsumura T, De Rybel B, Frei dit Frey N, Laplaze L, Casimiro I,

Swarup R, Naudts M, Vanneste S, Audenaert D et al. 2007. Auxin-dependentregulation of lateral root positioning in the basal meristem of Arabidopsis.

Development 134: 681–690.Debernardi JM,MecchiaMA, Vercruyssen L, Smaczniak C, KaufmannK, InzeD,

Rodriguez RE, Palatnik JF. 2014. Post-transcriptional control of GRF

transcription factors by microRNAmiR396 and GIF co-activator affects leaf size

and longevity. Plant Journal 79: 413–426.Debernardi JM, Rodriguez RE, Mecchia MA, Palatnik JF. 2012. Functional

specialization of the plant miR396 regulatory network through distinct

microRNA–target interactions. PLoS Genetics 8: e1002419.Dello Ioio R, Galinha C, Fletcher AG, Grigg SP,Molnar A,Willemsen V, Scheres

B, Sabatini S, Baulcombe D, Maini PK et al. 2012. A PHABULOSA/cytokinin

feedback loop controls root growth in Arabidopsis. Current Biology 22: 1699–1704.

Dello Ioio R, Linhares FS, Scacchi E, Casamitjana-Martinez E, Heidstra R,

CostantinoP, Sabatini S. 2007.Cytokinins determineArabidopsis root-meristem

size by controlling cell differentiation. Current Biology 17: 678–682.Dello Ioio R, Nakamura K, Moubayidin L, Perilli S, Taniguchi M, Morita MT,

Aoyama T, Costantino P, Sabatini S. 2008. A genetic framework for the control

of cell division and differentiation in the root meristem. Science 322: 1380–1384.Desbrosses GJ, Stougaard J. 2011. Root nodulation: a paradigm for how plant–microbe symbiosis influences host developmental pathways. Cell Host &Microbe10: 348–358.

Devers EA, Branscheid A, May P, Krajinski F. 2011. Stars and symbiosis:

microRNA- andmicroRNA*-mediated transcript cleavage involved in arbuscular

mycorrhizal symbiosis. Plant Physiology 156: 1990–2010.Dharmasiri N, Dharmasiri S, Weijers D, Lechner E, Yamada M, Hobbie L,

Ehrismann JS, Jurgens G, Estelle M. 2005. Plant development is regulated

by a family of auxin receptor F box proteins. Developmental Cell 9: 109–119.

Ditengou FA,TealeWD,Kochersperger P, Flittner KA,Kneuper I, van derGraaff

E, Nziengui H, Pinosa F, Li X, Nitschke R et al. 2008.Mechanical induction of

lateral root initiation in Arabidopsis thaliana. Proceedings of the National Academyof Sciences, USA 105: 18818–18823.

Drisch RC, Stahl Y. 2015. Function and regulation of transcription factors involved in

root apical meristem and stem cell maintenance. Frontiers in Plant Science 6: 505.Dubrovsky JG, Sauer M, Napsucialy-Mendivil S, Ivanchenko MG, Friml J,

Shishkova S, Celenza J, Benkova E. 2008. Auxin acts as a local morphogenetic

trigger to specify lateral root founder cells. Proceedings of the National Academy ofSciences, USA 105: 8790–8794.

Emery JF, Floyd SK,Alvarez J, Eshed Y,HawkerNP, Izhaki A, BaumSF, Bowman

JL. 2003. Radial patterning of Arabidopsis shoots by class III HD-ZIP and

KANADI genes. Current Biology 13: 1768–1774.Engstrom EM, Andersen CM, Gumulak-Smith J, Hu J, Orlova E, Sozzani R,

Bowman JL. 2011. Arabidopsis homologs of the Petunia HAIRY MERISTEMgene are required for maintenance of shoot and root indeterminacy. PlantPhysiology 155: 735–750.

Etemadi M, Gutjahr C, Couzigou JM, Zouine M, Lauressergues D, Timmers A,

Audran C, Bouzayen M, Becard G, Combier JP. 2014. Auxin perception is

required for arbuscule development in arbuscular mycorrhizal symbiosis. PlantPhysiology 166: 281–292.




Fahlgren N, Montgomery TA, Howell MD, Allen E, Dvorak SK, Alexander AL,

Carrington JC. 2006.Regulation ofAUXINRESPONSEFACTOR3 byTAS3 ta-siRNA affects developmental timing and patterning in Arabidopsis. CurrentBiology 16: 939–944.

Forde BG. 2002. Local and long-range signaling pathways regulating plant

responses to nitrate. Annual Review of Plant Biology 53: 203–224.Franco-Zorrilla JM, Valli A, Todesco M, Mateos I, Puga MI, Rubio-Somoza I,

Leyva A,Weigel D, Garcia JA, Paz-Ares J. 2007.Target mimicry provides a new

mechanism for regulation of microRNA activity. Nature Genetics 39:1033–1037.

Franssen HJ, Xiao TT, Kulikova O, Wan X, Bisseling T, Scheres B, Heidstra R.

2015. Root developmental programs shape theMedicago truncatula nodulemeristem. Development 142: 2941–2950.

Gaillochet C, Lohmann JU. 2015. The never-ending story: from pluripotency to

plant developmental plasticity. Development 142: 2237–2249.Galinha C, Hofhuis H, Luijten M,Willemsen V, Blilou I, Heidstra R, Scheres B.

2007. PLETHORA proteins as dose-dependent master regulators of Arabidopsis

root development. Nature 449: 1053–1057.Garcia D, Collier SA, Byrne ME, Martienssen RA. 2006. Specification of leaf

polarity in Arabidopsis via the trans-acting siRNA pathway. Current Biology 16:933–938.

Gibson AH, Pagan JD. 1977. Nitrate effects on the nodulation of legumes

inoculated with nitrate-reductase-deficient mutants of Rhizobium. Planta 134:17–22.

Gifford ML, Dean A, Gutierrez RA, Coruzzi GM, Birnbaum KD. 2008. Cell-

specific nitrogen responses mediate developmental plasticity. Proceedings of theNational Academy of Sciences, USA 105: 803–808.

Gobbato E. 2015. Recent developments in arbuscular mycorrhizal signaling.

Current Opinion in Plant Biology 26: 1–7.Graham JH, Leonard RT, Menge JA. 1981.Membrane-mediated decrease in root

exudation responsible for phosphorus inhibition of vesicular–arbuscularmycorrhiza formation. Plant Physiology 68: 548–552.

Grigg SP, Canales C, Hay A, Tsiantis M. 2005. SERRATE coordinates shoot

meristem function and leaf axial patterning in Arabidopsis. Nature 437: 1022–1026.

Grigg SP, Galinha C, Kornet N, Canales C, Scheres B, Tsiantis M. 2009.

Repression of apical homeobox genes is required for embryonic root development

in Arabidopsis. Current Biology 19: 1485–1490.Guil S, Esteller M. 2015. RNA–RNA interactions in gene regulation: the coding

and noncoding players. Trends in Biochemical Sciences 40: 248–256.GuoHS, XieQ, Fei JF, ChuaNH. 2005.MicroRNAdirectsmRNA cleavage of the

transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral

root development. Plant Cell 17: 1376–1386.Gutierrez L, Bussell JD, Pacurar DI, Schwambach J, Pacurar M, Bellini C. 2009.

Phenotypic plasticity of adventitious rooting in Arabidopsis is controlled bycomplex regulation of AUXIN RESPONSE FACTOR transcripts and

microRNA abundance. Plant Cell 21: 3119–3132.Gutierrez L, Mongelard G, Flokova K, Pacurar DI, Novak O, Staswick P,

Kowalczyk M, Pacurar M, Demailly H, Geiss G et al. 2012. Auxin controls

Arabidopsis adventitious root initiation by regulating jasmonic acid homeostasis.

Plant Cell 24: 2515–2527.Gutjahr C, Parniske M. 2013. Cell and developmental biology of arbuscular

mycorrhiza symbiosis. Annual Review of Cell and Developmental Biology 29: 593–617.

Gutjahr C, Paszkowski U. 2013.Multiple control levels of root system remodeling

in arbuscular mycorrhizal symbiosis. Frontiers in Plant Science 4: 204.HawkerNP, Bowman JL. 2004.Roles for Class IIIHD-Zip andKANADI genes in

Arabidopsis root development. Plant Physiology 135: 2261–2270.Hewezi T, Howe P, Maier TR, Baum TJ. 2008. Arabidopsis small RNAs and their

targets during cyst nematode parasitism.Molecular Plant–Microbe Interactions 21:1622–1634.

Heyman J, Kumpf RP, De Veylder L. 2014. A quiescent path to plant longevity.

Trends in Cell Biology 24: 443–448.Holt DB, Gupta V,Meyer D, Abel NB, Andersen SU, Stougaard J, Markmann K.

2015.microRNA 172 (miR172) signals epidermal infection and is expressed in

cells primed for bacterial invasion in Lotus japonicus roots and nodules. NewPhytologist 208: 241–256.

tenHoveCA, LuKJ,WeijersD. 2015.Building a plant: cell fate specification in the

early Arabidopsis embryo. Development 142: 420–430.Hunter C, Willmann MR, Wu G, Yoshikawa M, de la Luz Gutierrez-Nava M,

Poethig SR. 2006. Trans-acting siRNA-mediated repression of ETTIN and

ARF4 regulates heteroblasty in Arabidopsis. Development 133: 2973–2981.Iglesias MJ, Terrile MC, Windels D, Lombardo MC, Bartoli CG, Vazquez F,

Estelle M, Casalongue CA. 2014.MiR393 regulation of auxin signaling and

redox-related components during acclimation to salinity in Arabidopsis. PLoSONE 9: e107678.

Jones-Rhoades MW. 2012. Conservation and divergence in plant microRNAs.

Plant Molecular Biology 80: 3–16.Jones-Rhoades MW, Bartel DP. 2004. Computational identification of plant

microRNAs and their targets, including a stress-induced miRNA.Molecular Cell14: 787–799.

Juntawong P, Girke T, Bazin J, Bailey-Serres J. 2014. Translational dynamics

revealed by genome-wide profiling of ribosome footprints in Arabidopsis.Proceedings of the National Academy of Sciences, USA 111: E203–E212.

Kazan K. 2013. Auxin and the integration of environmental signals into plant root

development. Annals of Botany 112: 1655–1665.Kim YJ, Zheng B, Yu Y, Won SY, Mo B, Chen X. 2011. The role of Mediator in

small and long noncoding RNA production in Arabidopsis thaliana. EMBOJournal 30: 814–822.

Kinoshita N,Wang H, Kasahara H, Liu J, Macpherson C, Machida Y, Kamiya Y,

Hannah MA, Chua NH. 2012. IAA-Ala Resistant3, an evolutionarily conserved

target of miR167, mediates Arabidopsis root architecture changes during highosmotic stress. Plant Cell 24: 3590–3602.

Kircher S, SchopferP. 2016.Priming andpositioningof lateral roots inArabidopsis.

An approach for an integrating concept. Journal of Experimental Botany67: 1411–1420.

Kobae Y, Ohmori Y, Saito C, Yano K, Ohtomo R, Fujiwara T. 2016. Phosphate

treatment strongly inhibits new arbuscule development but not the maintenance

of arbuscule in mycorrhizal rice roots. Plant Physiology 171: 566–579.Kulcheski FR, Correa R, Gomes IA, de Lima JC, Margis R. 2015. NPK

macronutrients and microRNA homeostasis. Frontiers in Plant Science 6: 451.Lagos-Quintana M, Rauhut R, Lendeckel W, Tuschl T. 2001. Identification of

novel genes coding for small expressed RNAs. Science 294: 853–858.LauNC, LimLP,Weinstein EG,BartelDP. 2001.An abundant class of tinyRNAs

with probable regulatory roles in Caenorhabditis elegans. Science 294: 858–862.Laufs P, Peaucelle A, Morin H, Traas J. 2004.MicroRNA regulation of the CUCgenes is required for boundary size control inArabidopsismeristems.Development131: 4311–4322.

Lauressergues D, Couzigou JM, ClementeHS,Martinez Y, Dunand C, BecardG,

Combier JP. 2015. Primary transcripts of microRNAs encode regulatory

peptides. Nature 520: 90–93.Lauressergues D, Delaux PM, Formey D, Lelandais-Briere C, Fort S, Cottaz S,

Becard G, Niebel A, Roux C, Combier JP. 2012. The microRNA miR171h

modulates arbuscular mycorrhizal colonization ofMedicago truncatula bytargeting NSP2. Plant Journal 72: 512–522.

Lavenus J, Goh T, Roberts I, Guyomarc’h S, Lucas M, De Smet I, Fukaki H,

Beeckman T, Bennett M, Laplaze L. 2013. Lateral root development in

Arabidopsis: fifty shades of auxin. Trends in Plant Science 18: 450–458.Lee RC, Ambros V. 2001. An extensive class of small RNAs in Caenorhabditiselegans. Science 294: 862–864.

Lee RC, Feinbaum RL, Ambros V. 1993. The C. elegans heterochronic gene lin-4encodes small RNAswith antisense complementarity to lin-14.Cell 75: 843–854.

Li H, Deng Y, Wu T, Subramanian S, Yu O. 2010.Misexpression of miR482,miR1512, and miR1515 increases soybean nodulation. Plant Physiology 153:1759–1770.

Li J, Guo G, Guo W, Guo G, Tong D, Ni Z, Sun Q, Yao Y. 2012.miRNA164-

directed cleavage ofZmNAC1 confers lateral root development inmaize (ZeamaysL.). BMC Plant Biology 12: 220.

Li X, Cassidy JJ, Reinke CA, Fischboeck S, Carthew RW. 2009. A microRNA

imparts robustness against environmental fluctuation during development. Cell137: 273–282.

Li X,Wang X, Zhang S, Liu D, Duan Y, DongW. 2012. Identification of soybean

microRNAs involved in soybean cyst nematode infection by deep sequencing.

PLoS ONE 7: e39650.





Liang G, He H, Yu D. 2012. Identification of nitrogen starvation-responsive

microRNAs in Arabidopsis thaliana. PLoS ONE 7: e48951.

Liu PP, Montgomery TA, Fahlgren N, Kasschau KD, Nonogaki H, Carrington

JC. 2007. Repression of AUXIN RESPONSE FACTOR10 by microRNA160 is

critical for seed germination and post-germination stages. Plant Journal 52: 133–146.

Llave C, Xie Z, Kasschau KD, Carrington JC. 2002. Cleavage of Scarecrow-likemRNAtargets directed by a class ofArabidopsismiRNA. Science297: 2053–2056.

LobbesD, Rallapalli G, SchmidtDD,MartinC, Clarke J. 2006. SERRATE: a new

player on the plant microRNA scene. EMBO Reports 7: 1052–1058.LucasM, SwarupR, Paponov IA, SwarupK, Casimiro I, LakeD, P�eret B, Zappala

S, Mairhofer S, Whitworth M et al. 2011. SHORT-ROOT regulates primary,

lateral, and adventitious root development in Arabidopsis. Plant Physiology 155:384–398.

Lugtenberg B, Kamilova F. 2009. Plant-growth-promoting rhizobacteria. AnnualReview of Microbiology 63: 541–556.

Maillet F, Poinsot V, AndreO, Puech-Pages V,Haouy A, GueunierM, Cromer L,

Giraudet D, Formey D, Niebel A et al. 2011. Fungal lipochitooligosaccharidesymbiotic signals in arbuscular mycorrhiza. Nature 469: 58–63.

Mallory AC, Bartel DP, Bartel B. 2005.MicroRNA-directed regulation of

Arabidopsis AUXIN RESPONSE FACTOR17 is essential for proper development

andmodulates expression of early auxin response genes. Plant Cell 17: 1360–1375.Mallory AC, Dugas DV, Bartel DP, Bartel B. 2004a.MicroRNA regulation of

NAC-domain targets is required for proper formation and separation of adjacent

embryonic, vegetative, and floral organs. Current Biology 14: 1035–1046.Mallory AC, Reinhart BJ, Jones-RhoadesMW,TangG, Zamore PD, BartonMK,

Bartel DP. 2004b.MicroRNA control of PHABULOSA in leaf development:

importance of pairing to the microRNA 50 region. EMBO Journal 23: 3356–3364.

Mao G, Turner M, Yu O, Subramanian S. 2013.miR393 and miR164 influence

indeterminate but not determinate nodule development. Plant Signaling &Behavior 8: e26753.

Marin E, Jouannet V, Herz A, Lokerse AS,Weijers D, Vaucheret H, Nussaume L,

Crespi MD, Maizel A. 2010.miR390, Arabidopsis TAS3 tasiRNAs, and their

AUXIN RESPONSE FACTOR targets define an autoregulatory network

quantitatively regulating lateral root growth. Plant Cell 22: 1104–1117.McElver J, Tzafrir I, Aux G, Rogers R, Ashby C, Smith K, Thomas C, Schetter A,

ZhouQ,CushmanMA et al. 2001. Insertional mutagenesis of genes required for

seed development in Arabidopsis thaliana. Genetics 159: 1751–1763.MegrawM,BaevV,RusinovV, JensenST,KalantidisK,HatzigeorgiouAG.2006.

MicroRNA promoter element discovery in Arabidopsis. RNA 12:

1612–1619.MiyashimaS,HondaM,HashimotoK,TatematsuK,HashimotoT, Sato-NaraK,

Okada K, Nakajima K. 2013. A comprehensive expression analysis of the

ArabidopsisMICRORNA165/6 gene family during embryogenesis reveals a

conserved role in meristem specification and a non-cell-autonomous function.

Plant and Cell Physiology 54: 375–384.Miyashima S, Koi S, Hashimoto T, Nakajima K. 2011. Non-cell-autonomous

microRNA165 acts in a dose-dependent manner to regulate multiple

differentiation status in the Arabidopsis root. Development 138: 2303–2313.Moreno-Risueno MA, Van Norman JM, Moreno A, Zhang J, Ahnert SE, Benfey

PN. 2010.Oscillating gene expression determines competence for periodic

Arabidopsis root branching. Science 329: 1306–1311.MuraroD,MellorN, PoundMP,HelpH, LucasM,Chopard J, ByrneHM,Godin

C,HodgmanTC,King JR et al. 2014. Integration of hormonal signaling networks

and mobile microRNAs is required for vascular patterning in Arabidopsis roots.Proceedings of the National Academy of Sciences, USA 111: 857–862.

Nagpal P, Ellis CM, Weber H, Ploense SE, Barkawi LS, Guilfoyle TJ, Hagen G,

Alonso JM, Cohen JD, Farmer EE et al. 2005.Auxin response factors ARF6 andARF8 promote jasmonic acid production and flower maturation. Development132: 4107–4118.

Navarro L, Dunoyer P, Jay F, Arnold B, Dharmasiri N, Estelle M, Voinnet O,

Jones JDG. 2006. A plant miRNA contributes to antibacterial resistance by

repressing auxin signaling. Science 312: 436–439.Nguyen GN, Rothstein SJ, Spangenberg G, Kant S. 2015. Role of microRNAs

involved in plant response to nitrogen and phosphorous limiting conditions.

Frontiers in Plant Science 6: 629.

NodineMD,BartelDP. 2010.MicroRNAs prevent precocious gene expression and

enable pattern formation during plant embryogenesis. Genes & Development 24:2678–2692.

Nova-Franco B, Iniguez LP, Valdes-Lopez O, Alvarado-Affantranger X, Leija A,

Fuentes SI, Ramirez M, Paul S, Reyes JL, Girard L et al. 2015. The micro-

RNA72c-APETALA2-1 node as a key regulator of the common bean–Rhizobiumetli nitrogen fixation symbiosis. Plant Physiology 168: 273–291.

Oldroyd GE, Long SR. 2003. Identification and characterization of Nodulation-Signaling Pathway 2, a gene ofMedicago truncatula involved in Nod actor

signaling. Plant Physiology 131: 1027–1032.Oldroyd GE,Murray JD, Poole PS, Downie JA. 2011.The rules of engagement in

the legume–rhizobial symbiosis. Annual Review of Genetics 45: 119–144.Olsen AN, Ernst HA, Leggio LL, Skriver K. 2005. NAC transcription factors:

structurally distinct, functionally diverse. Trends in Plant Science 10:79–87.

Osella M, Bosia C, Cora D, Caselle M. 2011. The role of incoherent microRNA-

mediated feedforward loops in noise buffering. PLoS Computational Biology 7:e1001101.

Overvoorde P, Fukaki H, Beeckman T. 2010. Auxin control of root development.

Cold Spring Harbor Perspectives in Biology 2: a001537.Parry G, Calderon-Villalobos LI, Prigge M, P�eret B, Dharmasiri S, Itoh H,

Lechner E, Gray WM, Bennett M, Estelle M. 2009. Complex regulation of the

TIR1/AFB family of auxin receptors. Proceedings of the National Academy ofSciences, USA 106: 22540–22545.

Pasquinelli AE, Reinhart BJ, Slack F, Martindale MQ, Kuroda MI, Maller B,

Hayward DC, Ball EE, Degnan B, Muller P et al. 2000. Conservation of the

sequence and temporal expression of let-7 heterochronic regulatory RNA.Nature408: 86–89.

Payre F, Desplan C. 2016. RNA. Small peptides control heart activityScience 351:226–227.

P�eret B, De Rybel B, Casimiro I, Benkova E, Swarup R, Laplaze L, Beeckman T,

Bennett MJ. 2009. Arabidopsis lateral root development: an emerging story.

Trends in Plant Science 14: 399–408.Petricka JJ, Winter CM, Benfey PN. 2012. Control of Arabidopsis root

development. Annual Review of Plant Biology 63: 563–590.Prigge MJ, Wagner DR. 2001. The Arabidopsis SERRATE gene encodes a zinc-

finger protein required for normal shoot development.Plant Cell 13: 1263–1279.ProvartNJ,Alonso J, AssmannSM,BergmannD,Brady SM,Brkljacic J, Browse J,

Chapple C, Colot V, Cutler S et al. 2016. 50 years of Arabidopsis research:highlights and future directions. New Phytologist 209: 921–944.

Radwan O, Liu Y, Clough SJ. 2011. Transcriptional analysis of soybean root

response to Fusarium virguliforme, the causal agent of sudden death syndrome.

Molecular Plant–Microbe Interactions 24: 958–972.Reinhart BJ, Slack FJ, Basson M, Pasquinelli AE, Bettinger JC, Rougvie AE,

Horvitz HR, Ruvkun G. 2000. The 21-nucleotide let-7 RNA regulates

developmental timing in Caenorhabditis elegans. Nature 403: 901–906.Reinhart BJ, Weinstein EG, Rhoades MW, Bartel B, Bartel DP. 2002.

MicroRNAs in plants. Genes & Development 16: 1616–1626.Reis RS, Eamens AL, Waterhouse PM. 2015.Missing pieces in the puzzle of plant

microRNAs. Trends in Plant Science 20: 721–728.Rodriguez RE, ErcoliMF,Debernardi JM,BreakfieldNW,MecchiaMA, Sabatini

M, Cools T, De Veylder L, Benfey PN, Palatnik JF. 2015.MicroRNAmiR396

regulates the switch between stem cells and transit-amplifying cells in Arabidopsis

roots. Plant Cell 27: 3354–3366.Rogers ED, Benfey PN. 2015. Regulation of plant root system architecture:

implications for crop advancement. Current Opinion in Biotechnology 32: 93–98.Rogers K, Chen X. 2013. Biogenesis, turnover, and mode of action of plant

microRNAs. Plant Cell 25: 2383–2399.Rogg LE, Lasswell J, Bartel B. 2001. A gain-of-function mutation in IAA28suppresses lateral root development. Plant Cell 13: 465–480.

Ru P, Xu L,MaH,HuangH. 2006. Plant fertility defects induced by the enhanced

expression of microRNA167. Cell Research 16: 457–465.RueggerM,Dewey E, GrayWM,Hobbie L, Turner J, EstelleM. 1998.The TIR1

protein of Arabidopsis functions in auxin response and is related to human SKP2

and yeast grr1p. Genes & Development 12: 198–207.Scheres B. 2007. Stem-cell niches: nursery rhymes across kingdoms.Nature ReviewsMolecular Cell Biology 8: 345–354.




Schulze S, Schafer BN, Parizotto EA, Voinnet O, Theres K. 2010. LOSTMERISTEMS genes regulate cell differentiation of central zone descendants in

Arabidopsis shoot meristems. Plant Journal 64: 668–678.Schwartz BW, Yeung EC, Meinke DW. 1994. Disruption of morphogenesis and

transformation of the suspensor in abnormal suspensor mutants of Arabidopsis.

Development 120: 3235–3245.Sebastian J, Ryu KH, Zhou J, Tarkowska D, Tarkowski P, Cho YH, Yoo SD, Kim

ES, Lee JY. 2015. PHABULOSA controls the quiescent center-independent root

meristem activities in Arabidopsis thaliana. PLoS Genetics 11: e1004973.Seefried WF, Willmann MR, Clausen RL, Jenik PD. 2014.Global regulation of

embryonic patterning inArabidopsis bymicroRNAs.Plant Physiology165: 670–687.Si-Ammour A, Windels D, Arn-Bouldoires E, Kutter C, Ailhas J, Meins F Jr,

Vazquez F. 2011.miR393 and secondary siRNAs regulate expression of theTIR1/AFB2 auxin receptor clade and auxin-related development of Arabidopsis leaves.

Plant Physiology 157: 683–691.Smith ES, Read JD. 2008.Mycorrhizal symbiosis, 3rd edn. London, UK: Academic

Press.

Smith ZR, Long JA. 2010. Control of Arabidopsis apical–basal embryo polarity by

antagonistic transcription factors. Nature 464: 423–426.SorinC,DeclerckM,ChristA,BleinT,MaL,Lelandais-BriereC,NjoMF,Beeckman

T, Crespi M, Hartmann C. 2014. A miR169 isoform regulates specific NF-YA

targets and root architecture in Arabidopsis.New Phytologist 202: 1197–1211.Stahl Y, Simon R. 2010. Plant primary meristems: shared functions and regulatory

mechanisms. Current Opinion in Plant Biology 13: 53–58.Stuurman J, Jaggi F, Kuhlemeier C. 2002. Shoot meristem maintenance is

controlled by a GRAS-gene mediated signal from differentiating cells. Genes &Development 16: 2213–2218.

Suzaki T, Yoro E, Kawaguchi M. 2015. Leguminous plants: inventors of root

nodules to accommodate symbiotic bacteria. International Review of Cell andMolecular Biology 316: 111–158.

Thornton HG. 1936. The action of sodium nitrate upon the infection of lucerne

root-hairs by nodule bacteria. Proceedings of the Royal Society of London. Series B,Biological Sciences 119: 474–492.

Turner M, Nizampatnam NR, Baron M, Coppin S, Damodaran S, Adhikari S,

Arunachalam SP, Yu O, Subramanian S. 2013. Ectopic expression of miR160

results in auxin hypersensitivity, cytokinin hyposensitivity, and inhibition of

symbiotic nodule development in soybean. Plant Physiology 162: 2042–2055.Verma SS, Rahman MH, Deyholos MK, Basu U, Kav NN. 2014. Differential

expression of miRNAs in Brassica napus root following infection with

Plasmodiophora brassicae. PLoS ONE 9: e86648.

Vidal EA, �Alvarez JM, Guti�errez RA. 2014.Nitrate regulation of AFB3 andNAC4gene expression in Arabidopsis roots depends on NRT1.1 nitrate transport

function. Plant Signaling & Behavior 9: e28501.Vidal EA, Araus V, Lu C, Parry G, Green PJ, Coruzzi GM, Guti�errez RA. 2010.

Nitrate-responsive miR393/AFB3 regulatory module controls root system

architecture in Arabidopsis thaliana. Proceedings of the National Academy ofSciences, USA 107: 4477–4482.

Vidal EA, Moyano TC, Riveras E, Contreras-Lopez O, Guti�errez RA. 2013.

Systems approaches map regulatory networks downstream of the auxin receptor

AFB3 in the nitrate response of Arabidopsis thaliana roots. Proceedings of theNational Academy of Sciences, USA 110: 12840–12845.

Walch-Liu P, Ivanov II, Filleur S, Gan Y, Remans T, Forde BG. 2006. Nitrogen

regulation of root branching. Annals of Botany 97: 875–881.Wang JJ, GuoHS. 2015.Cleavage of INDOLE-3-ACETIC ACID INDUCIBLE28mRNA by microRNA847 upregulates auxin signaling to modulate cell

proliferation and lateral organ growth in Arabidopsis. Plant Cell 27:574–590.

Wang JW,Wang LJ, Mao YB, CaiWJ, Xue HW,Chen XY. 2005.Control of root

cap formation by MicroRNA-targeted auxin response factors in Arabidopsis.

Plant Cell 17: 2204–2216.Wang L, Mai YX, Zhang YC, Luo Q, Yang HQ. 2010.MicroRNA171c-targeted

SCL6-II, SCL6-III, and SCL6-IV genes regulate shoot branching in Arabidopsis.Molecular Plant 3: 794–806.

Wang Y, Li K, Chen L, Zou Y, LiuH, Tian Y, Li D,Wang R, Zhao F, Ferguson BJ

et al. 2015.MicroRNA167-directed regulation of the auxin response factors

GmARF8a and GmARF8b is required for soybean nodulation and lateral root

development. Plant Physiology 168: 984–999.

Wang Y, Wang L, Zou Y, Chen L, Cai Z, Zhang S, Zhao F, Tian Y, Jiang Q,

Ferguson BJ et al. 2014. Soybean miR172c targets the repressive AP2

transcription factor NNC1 to activate ENOD40 expression and regulate nodule

initiation. Plant Cell 26: 4782–4801.Wightman B, Ha I, Ruvkun G. 1993. Posttranscriptional regulation of the

heterochronic gene lin-14 by lin-4mediates temporal pattern formation in

C. elegans. Cell 75: 855–862.WilliamsL,CarlesCC,OsmontKS, Fletcher JC. 2005.Adatabase analysismethod

identifies an endogenous trans-acting short-interfering RNA that targets the

Arabidopsis ARF2,ARF3, andARF4 genes. Proceedings of the National Academy ofSciences, USA 102: 9703–9708.

WillmannMR,Mehalick AJ, Packer RL, Jenik PD. 2011.MicroRNAs regulate the

timing of embryo maturation in Arabidopsis. Plant Physiology 155: 1871–1884.Windels D, Bielewicz D, Ebneter M, Jarmolowski A, Szweykowska-Kulinska Z,

Vazquez F. 2014.miR393 is required for production of proper auxin signalling

outputs. PLoS ONE 9: e95972.

WuHJ,Wang ZM,WangM,Wang XJ. 2013.Widespread long noncoding RNAs

as endogenous target mimics for microRNAs in plants. Plant Physiology 161:1875–1884.

WuMF, Tian Q, Reed JW. 2006. Arabidopsis microRNA167 controls patterns ofARF6 and ARF8 expression, and regulates both female and male reproduction.

Development 133: 4211–4218.Xie Q, Frugis G, Colgan D, Chua NH. 2000. ArabidopsisNAC1 transduces auxin

signal downstream of TIR1 to promote lateral root development. Genes &Development 14: 3024–3036.

Xie Q, Guo HS, Dallman G, Fang S, Weissman AM, Chua NH. 2002. SINAT5

promotes ubiquitin-related degradation of NAC1 to attenuate auxin signals.

Nature 419: 167–170.Xie Z, Allen E, Fahlgren N, Calamar A, Givan SA, Carrington JC. 2005.

Expression of ArabidopsisMIRNA genes. Plant Physiology 138: 2145–2154.Xue XY, Zhao B, Chao LM, Chen DY, Cui WR, Mao YB, Wang LJ, Chen XY.

2014. Interactionbetween two timingmicroRNAs controls trichomedistribution

in Arabidopsis. PLoS Genetics 10: e1004266.YanZ,HossainMS,Arikit S,Valdes-LopezO,Zhai J,Wang J, LibaultM, JiT,Qiu

L, Meyers BC et al. 2015. Identification of microRNAs and their mRNA targets

during soybean nodule development: functional analysis of the role of miR393j-

3p in soybean nodulation. New Phytologist 207: 748–759.Yang L, Liu Z, Lu F, Dong A, Huang H. 2006. SERRATE is a novel nuclear

regulator in primary microRNA processing in Arabidopsis. Plant Journal 47:841–850.

Yi X, Zhang Z, Ling Y, Xu W, Su Z. 2015. PNRD: a plant non-coding RNA

database. Nucleic Acids Research 43(Database issue): 982–989.Yoon EK, Yang JH, Lim J, Kim SH, Kim SK, Lee WS. 2010. Auxin regulation of

the microRNA390-dependent transacting small interfering RNA pathway in

Arabidopsis lateral root development. Nucleic Acids Research 38: 1382–1391.Zhang B,WangQ. 2015.MicroRNA-based biotechnology for plant improvement.

Journal of Cellular Physiology 230: 1–15.Zhao W, Li Z, Fan J, Hu C, Yang R, Qi X, Chen H, Zhao F, Wang S. 2015.

Identification of jasmonic acid-associatedmicroRNAs and characterization of the

regulatory roles of themiR319/TCP4module under root-knot nematode stress in

tomato. Journal of Experimental Botany 66: 4653–4667.Zhao Y, Lin S, Qiu Z, Cao D, Wen J, Deng X, Wang X, Lin J, Li X. 2015.

MicroRNA857 is involved in the regulation of secondary growth of vascular

tissues in Arabidopsis. Plant Physiology 169: 2539–2552.Zhou Y, Honda M, Zhu H, Zhang Z, Guo X, Li T, Li Z, Peng X, Nakajima K,

DuanL et al.2015a.Spatiotemporal sequestration ofmiR165/166byArabidopsisArgonaute10 promotes shoot apical meristem maintenance. Cell Reports 10:1819–1827.

ZhouY, LiuX,EngstromEM,NimchukZL,Pruneda-Paz JL,Tarr PT,YanA,Kay

SA, Meyerowitz EM. 2015b. Control of plant stem cell function by conserved

interacting transcriptional regulators. Nature 517: 377–380.ZhuH,HuF,WangR,ZhouX, Sze SH,LiouLW,BarefootA,DickmanM,Zhang

X. 2011. ArabidopsisArgonaute10 specifically sequesters miR166/165 to regulate

shoot apical meristem development. Cell 145: 242–256.Zhu YY, Zeng HQ, Dong CX, Yin XM, Shen QR, Yang ZM. 2010.microRNA

expression profiles associated with phosphorus deficiency in white lupin (Lupinusalbus L.). Plant Science 178: 23–29.





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