AINTEGUMENTA-LIKE proteins-hubs in a plethora of networks

Feature Review

AINTEGUMENTA-LIKE proteins: hubsin a plethora of networksAnneke Horstman1, Viola Willemsen2, Kim Boutilier1, and Renze Heidstra2

1 Plant Research International, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen,

The Netherlands2 Plant Developmental Biology, Wageningen University and Research Centre, Droevendaalsesteeg 1, 6708 PB Wageningen,

The Netherlands

Review

Members of the AINTEGUMENTA-LIKE (AIL) family ofAPETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF)domain transcription factors are expressed in all dividingtissues in the plant, where they have central roles indevelopmental processes such as embryogenesis, stemcell niche specification, meristem maintenance, organpositioning, and growth. When overexpressed, AIL pro-teins induce adventitious growth, including somaticembryogenesis and ectopic organ formation. The Arabi-dopsis (Arabidopsis thaliana) genome contains eight AILgenes, including AINTEGUMENTA, BABY BOOM, andthe PLETHORA genes. Studies on these transcriptionfactors have revealed their intricate relationship withauxin as well as their involvement in an increasingnumber of gene regulatory networks, in which extensivecrosstalk and feedback loops have a major role.

The AIL transcription factor family in Arabidopsis

The eight AINTEGUMENTA-LIKE (AIL) transcriptionfactor genes within the Arabidopsis (Arabidopsis thaliana)genome [1] include AINTEGUMENTA (ANT) [2,3], BABYBOOM (BBM) [4], and the PLETHORA (PLT) genes [5,6](Box 1; Figure 1), which are all expressed in young, divid-ing tissues in the plant. They have overlapping roles in theestablishment and maintenance of meristems, as well asorgan initiation and growth (Table 1). A wealth of geneticstudies have shown that AIL proteins are master regula-tors of these developmental processes. Loss-of-functioncombinations and gain-of-function mutants of this genefamily show spectacular phenotypes in which meristems orcomplete organs are missing or arise at ectopic positions.The central role of this family in meristem and organdevelopment extends as far back as mosses [7–9]. In thisreview, we provide an overview of recent AIL research inthe model plant Arabidopsis and point out future chal-lenges in AIL research.

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� 2013 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tplants.2013.10.010

Corresponding author: Heidstra, R. ([email protected]).Keywords: AINTEGUMENTA-LIKE; development; embryogenesis; meristemmaintenance.

146 Trends in Plant Science, March 2014, Vol. 19, No. 3

AIL function during embryogenesisPLT1, PLT2, AIL6/PLT3, and BBM (collectively calledPLT/BBM) genes have a major role in basal patterning ofthe embryo (Figure 2A). PLT1 and PLT2 gene expressionhas been described from the octant stage onwards, in thelower tier of the embryo proper [5]. Early embryonic AIL6/PLT3 and BBM expression has not been reported. Later inembryogenesis, PLT1 expression becomes restricted to thequiescent centre (QC) and surrounding stem cells, where-as the PLT2, AIL6/PLT3, and BBM expression domainsare slightly expanded to include the ground tissue andprovascular cells. Post-embryonically, the AIL6/PLT3 ex-pression maximum is in the columella stem cells, in con-trast to the QC peak expression observed for PLT1, PLT2,and BBM [5,6]. Only combinations of plt1, plt2, ail6/plt3,and bbm mutants show embryonic abnormalities [5,6].Subtle defects in plt1;plt2 embryos include enlargedmis-specified QC progenitor cells, and plt1;plt2 seedlingsshow defective root development, confirming that PLT1and PLT2 are required for stem cell niche specification [5].plt1;plt2;ail6/plt3 triple mutant embryos show aberrantorganization of the embryonic root pole and seedlings arerootless. plt2;bbm double mutants fail to develop past theearly embryo stage, indicating the importance of PLT2and BBM for embryogenesis [6].

BBM or AIL5/PLT5 overexpression induces the ectopicformation of embryos on the meristem, cotyledons, andfirst leaves of seedlings [4,10]. Overexpression of PLT1 orPLT2 during embryogenesis ectopically induces root stemcell niches and, in the most extreme cases, can lead to acomplete transformation of the embryo towards root iden-tity [5]. In line with this, induced overexpression of PLT2 inseedlings can also produce roots from the shoot apex [6].Together with the mutant phenotypes, these results sug-gest that BBM, AIL5/PLT5, PLT1, and PLT2 genes act asmaster regulators for early embryo and root development.This might reflect a specific function for these genes inembryo initiation or the maintenance of cell potency.

In most plant species, the initial phase of embryo cellproliferation and morphogenesis is followed by the matu-ration phase, in which cell division stops, storage reservesaccumulate, and the seed becomes desiccation tolerant anddormant. In Arabidopsis, the onset of seed maturation ischaracterized by an increase in the level of the planthormone abscisic acid (ABA) and by increased expression

Box 1. AILs form a subgroup within the large AP2/ERF family

The AINTEGUMENTA-LIKE (AIL) proteins are members of the

APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain family

of transcription factors that are found in a phylogenetically wide

group of plants, including moss, algae, gymnosperms, and angios-

perms, and comprise the second largest group of transcription factors

in plants, with up to 200 members in a single genome [1,121,126–133].

The AP2/ERF domain, a DNA binding domain of approximately 60 to

70 amino acids [134], was identified initially in the Arabidopsis AP2

protein [135], and shortly thereafter in four tobacco (Nicotiana

tabacum) ERFs [136]. The AP2/ERF domain was thought to be plant-

specific until the discovery that the same domain exists in HNH (His-

Asp-His) homing endonucleases from viruses, cyanobacteria, ciliates,

and parasitic protists [55,137,138]. Homing endonucleases are

invasive DNA sequences that are usually found in self-splicing introns

or inteins, which can transpose and duplicate themselves within and

between species [139].

The AIL proteins fall under the AP2 subfamily of proteins, which

contain two AP2/ERF domains separated by a linker region [126]. This

AP2 subfamily is divided into the euAP2 lineage, which is characterized

by a miR172 binding motif that lies C-terminal to the AP2/ERF domains,

and the AINTEGUMENTA (ANT) lineage, which is characterized by

signature amino acid insertions in the first and second AP2/ERF

domain. The ANT lineage is further divided into the basalANT and

euANT/AIL lineages (see Figure 1 in main text) on the basis of a number

of conserved amino acid insertions in the first AP2/ERF domain

(euANT1) and the N-terminal region (euANT2–4) of the euANT/AIL

proteins [127,140]. These motifs are generally well conserved among

AILs, both within and between species [127,140,141].

The 3D structure of AP2/ERF proteins has only been resolved for the

single AP2 domain-containing protein ERF1. The 3D solution structure

of ERF1 showed that its AP2 domain forms a three-stranded anti-

parallel b-sheet that lies mostly parallel to a conserved a-helix [142].

Modelling and DNA-binding experiments suggest that the anti-

parallel b-sheet is responsible for the DNA-binding properties of

ERF1 [142], which has since been confirmed for other single AP2

domain proteins in the large ERF1 subfamily [113,143]. Homology

modelling of Arabidopsis ANT using the ERF1 3D structure as a

template suggests that each AP2 repeat of ANT forms an a-helix,

similarly to ERF1. However, unlike ERF1, the first AP2 repeat of ANT is

predicted to contain two b-sheets, at different positions from those in

ERF1, whereas the second AP2/ERF repeat does not seem to form b-

sheets [109]. Both AP2 domains of ANT are required for DNA binding,

and each domain is thought to use different amino acids to contact

the DNA [109,110]. It was proposed that the first AP2 repeat binds to

the 50 part of the target sequence and the second AP2 repeat to the 30

part of the target sequence, with the ANT linker region serving as a

bridge [110]. The importance of the linker region between the AP2/

ERF domains is illustrated by the high conservation of this region

within the AIL gene family and by the observation that mutations in

the linker abolish DNA binding in vitro. These observations led to the

suggestion that the linker region directly binds DNA or serves to

position the AP2 repeats on the DNA [109].

AP2/ERF domain proteins regulate two major processes in plants:

response to stress and control of growth and development. Notably,

AIL proteins have been shown to function exclusively in pathways

related to development.

Review Trends in Plant Science March 2014, Vol. 19, No. 3

of an interwoven network of transcription factors, includ-ing ABSCISIC ACID-INSENSITIVE 3 (ABI3), that togeth-er regulate expression of maturation phase genes.(reviewed in [11,12]). Recently, a heterologous systembased on activation of the Phaseolus vulgaris ABI3 tran-scription factor PvALF and expression of its target genePHASEOLIN (PHAS) identified AIL5/PLT5 as being co-expressed with PHAS [13]. AIL5/PLT5 is bound by PvALFin the presence of ABA and is required for expression ofendogenous seed storage genes in Arabidopsis [13]. It is notknown whether AIL5/PLT5 directly binds to seed storagegenes to activate their expression or whether it acts up-stream of other maturation phase transcription factors,thereby regulating the final phase of embryo development.

AIL–auxin feedback loop

The phytohormone auxin has been shown to play an im-portant part in the formation of the apical–basal axis of theembryo. Auxin binding by its receptors TRANSPORT IN-HIBITOR RESISTANT 1 (TIR1) and TIR1-related proteinspromotes degradation of the AUXIN/INDOLE ACETICACID (Aux/IAA) family proteins that bind to AUXIN RE-SPONSE FACTOR (ARF) family transcription factors,inhibiting ARF transcriptional activity on auxin targetgenes (reviewed in [14]).

The expression of PLT1 and PLT2 is dependent on theredundant action of ARF5/MONOPTEROS (MP) and that ofits close homologue NONPHOTOTROPIC HYPOCOTYL 4(NPH4/ARF7) [5,15]. Although the dynamics of auxin-in-duced PLT1 and PLT2 transcription implies that they are(late) auxin response genes, exogenous application of auxinfails to rescue plt1;plt2 mutants, indicating that auxincannot bypass the requirement for PLT1 and PLT2 [5].Expression of the auxin efflux facilitator PIN-FORMED 4(PIN4) is largely absent in plt1;plt2 embryos, showing that

PIN4 is downstream of PLT1 and PLT2 in the transcrip-tional network. Reduced transcription of PIN1 and PIN3 isalso observed in plt1;plt2;ail6/plt3 triple mutant embryos[6,16]. By contrast, the PLT1 expression domain is expandedto the whole embryo in pin2;pin3;pin4;pin7 mutants.Explanted pin2;pin3;pin4;pin7 embryos show impaired cot-yledon development, and root hairs emerge at apical posi-tions on the seedling. These findings suggest a feedback loopin which PIN proteins, by directing auxin transport andaccumulation, restrict PLT1 and PLT2 expression to thebasal embryo domain to initiate embryonic root specifica-tion. In turn, PLT activity regulates PIN transcription tostabilize the position of the root primordium [16] (Figure 3).

Defects in QC patterning are observed in RHO guanyl-nucleotide exchange factor 7 (RopGEF7) RNA interferencemutants during embryogenesis that correlate with thereduced expression of PIN1, PLT1, and PLT2 [17]. Rop-GEF7, which encodes a RAC/ROP GTPase activator, isexpressed in the same domain as PLT1 and PLT2, and itsexpression is unaffected in plt1;plt2 double mutants [17]. Ithas been suggested that RopGEF7, via RAC/ROP GTPases[18], regulates correct PIN endocytosis, thereby directlyaffecting local auxin concentrations and indirectly affect-ing PLT1 and PLT2 expression to mediate root meristempatterning [17].

Reduced expression of PLT1, PLT2, BBM, and severalPIN genes is observed in JAGGED LATERAL ORGAN(JLO) mutant embryos [19]. Strong jlo mutants are embryolethal, but plants carrying the hypomorphic jlo2 allele pro-duce viable embryos in addition to those resembling mp andbdl mutant embryos [19,20]. JLO might control embryopatterning, either alone or together with its interactionpartner ASYMMETRIC LEAVES 2 (AS2) [21], throughthe auxin-dependent MP–BDL pathway, or by directly reg-ulating PLT/BBM gene expression (Figure 3).

147

98

100

100

100

98

88

41

3151

75

95

975834

630.05

ADAP

AIL1

PLT1

euANT

basalANT

PLT2

PLT7

AIL6/PLT3

ANT

AIL5/PLT5

BBM

Wrinkled

Other

Selaginella

Selaginella

Selaginella

Physcomitrella

Physcomitrella

98

81

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Figure 1. Phylogenetic tree for the AINTEGUMENTA-LIKE (AIL) family of transcription factors. Neighbour-joining tree of the euANT and basalANT family using Molecular

Evolutionary Genetics Analysis (MEGA) version 5 [144]. A total of 251 sequences were analysed using the APETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domain

region (240 positions). Numbers at the nodes indicate bootstrap support calculated using 100 replicates. Only bootstrap values over 30% are indicated. The ‘Other’

basalANT clade corresponds to genes with no apparent orthologue in Arabidopsis. Selaginella refers to Selaginella moellendorffii and Physcomitrella refers to

Physcomitrella patens. Abbreviations: ADAP, ARIA-INTERACTING DOUBLE AP2 DOMAIN PROTEIN is given as the representative member of a clade similar to Wrinkled;

ANT, AINTEGUMENTA; BBM, BABY BOOM; PLT, PLETHORA.

Review Trends in Plant Science March 2014, Vol. 19, No. 3

Together, these studies highlight the importance of AILfunction and its relation to auxin for embryo development.

Embryonic AIL regulatory networks

PLT1 and PLT2 are directly regulated by the TOPLESS(TPL) transcriptional co-repressor [22]. tpl loss-of-function

Table 1. Arabidopsis AIL genes and their functionsa

AGI numberb Gene names Function

At4g37750 ANT Shoot and flower meristem maintenance

ovule development, floral organ identity

At1g72570 AIL1

At5g17430 AIL2/BBM/PLT4 Embryogenesis, root SCN patterning an

At3g20840 AIL3/PLT1 Embryogenesis, root SCN patterning an

At1g51190 AIL4/PLT2 Embryogenesis, root SCN patterning an

At5g57390 AIL5/CHO1/

EMK/PLT5

germination, phyllotaxy, rhizotaxy, cell p

At5g10510 AIL6/PLT3 Shoot and flower meristem maintenance

identity, embryogenesis, root SCN patte

phyllotaxy, rhizotaxy, cell proliferation

At5g65510 AIL7/PLT7 Shoot meristem maintenance, phyllotax

aIndividual AIL members are given different names. We refer to the genes by their mo

bAbbreviations: AGI, Arabidopsis Genome Initiative; AIL, AINTEGUMENTA-LIKE; ANT, AIN

PLETHORA; SCN, stem cell niche.

148

mutants display ectopic PLT1 and PLT2 expression andform a secondary root pole [22,23]. A mutation in themiR165/166 binding site of PHABULOSA (PHB) sup-presses the formation of the tpl double root. PHB, likeREVOLUTA (REV), is a member of the miR165/166-regu-lated class III homeodomain-leucine zipper (HD-ZIP III)

Refs

, organ size and polarity, flower initiation,

, cell proliferation

[2,3,51,78,83,84,92,93,97,

103,104]

d meristem maintenance, cell proliferation [4,6]

d meristem maintenance, cell proliferation [5,6]

d meristem maintenance, cell proliferation [5,6]

roliferation, seed maturation [10,13,50,57,59,79,85]

, organ size, flower initiation, floral organ

rning and meristem maintenance,

[6,50,51,78,79,85,93,95]

y, rhizotaxy, cell proliferation [50,78,79,85]

st commonly used name (in bold).

TEGUMENTA; BBM, BABY BOOM; CHO1, CHOTTO 1; EMK, EMBRYOMAKER; PLT,

(A)

(F)(E)

(D)

(B)

(C) Peri

Stemcells

Col

Stele EnCor

EpiLrc

Vasc Gt

Stage I Stage II

AIL6/PLT3,AIL5/PLT5,PLT7

AIL6/PLT3,AIL5/PLT5,PLT7

ANT

ANT

ANT

AIL6/PLT3

AIL6/PLT3

ANTAIL6/PLT3

PLT7

PLT1,PLT2,AIL6/PLT3,BBM

PLT1,PLT2,AIL6/PLT3,BBM

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137°

Figure 2. Sites of AINTEGUMENTA-LIKE (AIL) function during plant development. (A) Embryogenesis. During embryo development, the apical–basal body axis is

established by an asymmetric division of the zygote. The apical cell gives rise to most of the embryo, including the cotyledons, shoot apical meristem (SAM), hypocotyl, and

root stem cells. The basal cell forms the hypophyseal cell, which gives rise to the columella and the quiescent centre (QC), which is specified at early heart stage [145,146].

Together the QC and surrounding stem cells form the stem cell niche. (B) Root development. The root meristem provides new cells for tissues of the growing root: stele,

ground tissue, epidermis, lateral root cap, and columella. The QC is essential for maintenance of the surrounding stem cells [147]. Daughter cells produced by the stem cells

traverse the meristematic zone, where they divide to generate a pool of cells that exit the cell cycle when they reach the transition zone, and increase in length in the

elongation zone before acquiring their specific characteristics in the differentiation zone. (C) Rhizotaxis. In Arabidopsis, lateral roots arise from two files of pericycle cells

that lie adjacent to the protoxylem within the differentiating root [86,148]. A subset of these cells, called founder cells, is stimulated by a local accumulation of auxin to

divide and form a lateral root primordium (LRP) [87,149–151]. LRP spacing correlates with the root curvature [86,87]. (D–F) Shoot development. The SAM gives rise to the

leaves. The transition to reproductive growth is marked by the conversion of the vegetative meristem to an inflorescence meristem, which produces floral meristems that

differentiate into flowers containing different types of organs with distinct sizes and shapes. Organ size is determined by cell number and cell size. (D) Organ polarity.

Leaves and floral organs are polarized along their adaxial–abaxial axis, and different cell types exist on either side of this axis. The main players controlling organ polarity in

Arabidopsis correspond to three classes of transcription factors: the adaxial-specifying Class III homeodomain-leucine zipper (HD-ZIP III) proteins, and the abaxial-

specifying KANADI (KAN) and YABBY (YAB) proteins [152]. The arrow in (D) represents the role of AINTEGUMENTA (ANT) and AIL6/PLETHORA 3 (PLT3) in lateral organ

growth. (E) Phyllotaxis. In Arabidopsis seedlings, the cotyledons and the first pair of leaves are formed in a decussate pattern (opposite to each other), and subsequent

leaves develop in a spiral pattern with an angle close to the ‘golden angle’ of 137.58. This spiral pattern of organ initiation is maintained in the inflorescence meristem

(reviewed in [153]). (F) Floral organ identity and ovule development. Floral organs are specified by the combined activity of the so-called A, B, C, and E classes of organ

identity genes, which are expressed in overlapping domains. In addition, class A and C activities inhibit each other (reviewed in [49]). During gynoecium development, two

carpel margin meristems (CMMs) form on the adaxial (inner) portion of the medial domain of the gynoecium, and ultimately give rise to the ovules and other organs of the

carpel. Abbreviation: BBM, BABY BOOM.

Review Trends in Plant Science March 2014, Vol. 19, No. 3

gene family of transcription factors that promote apicalfate during early embryogenesis [24]. Class III homeodo-main-leucine zipper (HD-ZIP III) gene expression is absentin the apical region of tpl mutants, but a miR165/166sensor still accumulates, indicating a mechanism for con-trol of HD-ZIP III gene expression that is independent ofmiR165/166 action. Apical expression of PHB and REV isrestored in tpl;plt1;plt2 triple mutants, suggesting thatPLT1 and PLT2 act as repressors of HD-ZIP III expressionduring embryogenesis [22]. In turn, genetic- and gain-of-function studies have shown that HD-ZIP III proteinsrepress the PLT1–PLT2 pathway. Thus, the antagonisticaction of the HD-ZIP III and PLT1–PLT2 proteins mightfacilitate proper apical–basal patterning [22] (Figure 3).

Ectopic root formation was also observed at the apicalregion of ANGUSTIFOLIA 3/GRF INTERACTING FAC-TOR 1 (AN3/GIF1) and HANABA TARANU (HAN) doubleloss-of-function mutant embryos (an3/gif;han) [25]. AN3/GIF1 encodes a putative transcriptional co-activator regu-lating various aspects of shoot development [26–29]. HANencodes a GATA-type transcription factor required to po-sition the proembryo boundary in the early Arabidopsisembryo [30,31]. PLT1 expression is expanded to the apicalregion of the globular embryo in an3/gif1;han doublemutants, and ectopic root formation was suppressed bythe plt1 mutation. These results suggest that AN3/GIF1and HAN repress PLT1, possibly via TPL, to establishapical identity [25].

149

OBE1;OBE2

PLT1;PLT2 PLT3/AIL6ANT

MP;NPH4

Auxin

YUCCAPIN

AN3/GIF1

JLO(-AS2)

BBM

TPL HD-ZIP III

PLT5/AIL5PLT7

ARF7;ARF19

TPST

RGF/GLV

AB14

GA

JA

MYC2

GCN5ADA2b

PKLAG

AP3

SLR/IAA14

ARGOS

ADF9

HAN

ABA

RopGEF7

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Figure 3. AINTEGUMENTA-LIKE (AIL) gene regulatory networks. Genes in purple, green or orange are directly associated with the jasmonate (JA), auxin, or abscisic acid

(ABA) pathways, respectively. Unbroken black lines represent direct interactions, whereas broken lines show possible interactions. Abbreviations: ABI4, ABA INSENSITIVE

4; ADF9, ACTIN DEPOLYMERIZING FACTOR 9; AG, AGAMOUS; AN3/GIF1, ANGUSTIFOLIA 3/GRF INTERACTING FACTOR 1; ANT, AINTEGUMENTA; AP3, APETALA 3; ARF,

AUXIN RESPONSE FACTOR; ARGOS, AUXIN-REGULATED GENE INVOLVED IN ORGAN SIZE; BBM, BABY BOOM; GA, gibberellic acid; GCN5, GENERAL CONTROL NON-

REPRESSIBLE 5; GLV, GOLVEN; HAN, HANABA TARANU; JLO, JAGGED LATERAL ORGAN; MP, MONOPTEROS; NPH4, NONPHOTOTROPIC HYPOCOTYL 4; OBE, OBERON;

PIN, PIN-FORMED; PKL, PICKLE; PLT, PLETHORA; RGF, ROOT GROWTH FACTOR; RopGEF7, RHO guanyl-nucleotide exchange factor 7; SLR, SOLITARY ROOT: TPL,

TOPLESS; TPST, TYROSYLPROTEIN SULFOTRANSFERASE.

Review Trends in Plant Science March 2014, Vol. 19, No. 3

Root development and meristem maintenancePLT/BBM expression patterns set up during embryogene-sis are maintained during post-embryonic root develop-ment (Figure 2B), where they show partly overlappinggradients of protein accumulation, with the highest pro-teins levels in the stem cell niche [5,6]. Concomitantly, theSHORT ROOT (SHR) transcription factor is expressed inthe stele and moves to the surrounding cells, including theQC, where it activates SCARECROW (SCR) [32,33].plt1;plt2, shr and scr mutants display loss of (different)QC identity markers and premature termination of rootgrowth [5,34]. PLT1 transcription is not affected in shr andscr mutants, and PLT1–PLT2 activity is not required forSHR or SCR expression. Thus, QC specification requiresthe parallel action of PLT1–PLT2 and SHR–SCR path-ways [5].

Additive activities of AIL genes

The QC is the site of the root auxin maximum [35–37],which is generated by local biosynthesis [38–42] andthrough the activity of directional auxin transporters suchas PIN proteins [16,43–45]. The PIN-mediated, root-direct-ed auxin transport in the vasculature and the shoot-direct-ed transport in the lateral root cap and epidermis arethought to generate an auxin gradient in the root that iscrucial for root meristem size and maintenance[16,43,46,47]. The proposed root auxin gradient correlateswith an expression gradient of PLT/BBM proteins that actin a largely additive fashion [5,6]. First, stepwise loweringof PLT/BBM gene dosage results in increasingly enhancedroot phenotypes. Second, complementing plt1;plt2 mutantswith a shortened gradient of PLT2 expression results inseverely decreased root and meristem sizes, but rescuesthe stem cell niche. Third, expression of PLT2 in thetransition zone of the plt1;plt2 mutant meristem prolongs

150

meristem activity but fails to maintain the stem cell niche.Finally, inducible PLT2 overexpression results in continu-ous growth of the meristematic zone while inhibiting cellexpansion at the elongation zone [6]. Simultaneously low-ering RETINOBLASTOMA-RELATED (RBR) levels, as anindependent stem cell input [48], and inducing overexpres-sion of PLT2 increases stem cell numbers and activity [6].Thus, high PLT/BBM levels in the QC are required tospecify and maintain the root stem cell niche, intermediatelevels are required for the proliferation of root meristemcells, and exit from the meristem to differentiation requiresPLT/BBM levels to drop below a certain threshold [6].Together, the auxin–PLT/BBM gradient can provide aconcentration-based mechanism for specification andmaintenance of root stem cells, as well as for regulatingthe proliferation, elongation, and differentiation of cells.

Translating auxin to AIL expression

Several studies implicate ARFs as general upstream med-iators of the auxin–AIL pathway during embryogenesis(MP and NPH4), flower primordia initiation and ovuledevelopment (MP), and lateral root formation (ARF7 andARF19) [5,49–51] (see below).

Auxin also positively regulates TYROSYLPROTEINSULFOTRANSFERASE (TPST) and several tyrosine-sul-fated peptide encoding ROOT GROWTH FACTOR (RGF)/GOLVEN (GLV) genes that are highly expressed in the rootstem cell niche [52–54]. tpst mutants have pleiotropicphenotypes, including short roots, additional QC cells,and differentiation of columella stem cells. The observedroot defects correlate with impaired expression of auxinbiosynthesis genes and PLT1 and PLT2 transcript andprotein levels. tpst root defects were partially restored inthe presence of RGF1 peptide. Induced overexpression ofPLT2 also partially rescued tpst mutant root defects,

Review Trends in Plant Science March 2014, Vol. 19, No. 3

whereas plt1;plt2 mutant roots could not be complementedby addition of RGF1 peptide [52,53]. In addition, RGF1-treated roots show an expanded PLT2 protein expressiondomain without associated changes in transcription [52].Together these results suggest TPST-sulfated RGF pep-tides link the auxin pathway to transcriptional and post-transcriptional regulation of PLT/BBM in root stem cellmaintenance (Figure 3).

AIL genes integrate multiple hormonal inputs

Germination results in emergence of the radicle (hypocotyl +embryonic root) from the seed through cell enlargement andactivation of the embryonic root meristem [55]. This processis regulated by environmental factors, such as nutrients,light, and temperature, as well as by the endogenous planthormones ABA and gibberellic acid (GA) [56]. The ail5/plt5mutant was first identified (and named chotto1; Table 1) as amutant showing reduced sensitivity to ABA during germi-nation [57]. Increased AIL5/PLT5 expression in the radicleof imbibed seeds requires the transcription factor ABAINSENSITIVE 4 (ABI4), a key component of ABA-mediatedsignalling in seeds [58], and genetic analyses confirms thatAIL5/PLT5 acts downstream of ABI4 [59] (Figure 3). Fresh-ly harvested ail5/plt5 mutant seeds show reduced primaryseed dormancy, which was recently also shown for abi4 [59–61]. However, neither ABA nor GA seems to regulate AIL5/PLT5 expression [59,61]. Genetic analysis further showsthat the ail5/plt5 mutant background partially restores GAbiosynthesis and the associated delayed germination phe-notype in an ABA over-accumulating mutant. However,ail5/plt5 is not able to restore germination in a GA-deficientmutant [61]. Finally, ail5/plt5 seedlings display partialinsensitivity to excess nitrate independent of ABI4. Theseresults indicate that AIL5/PLT5 integrates multiple andindependent inputs towards regulation of germination andseedling growth.

Jasmonate (JA) negatively affects root growth in a dose-dependent manner resulting in reduced root cell sizes, areduced meristem size and activity, and QC divisionsfollowed by columella stem cell differentiation [62]. TheJA growth inhibitory effect is mediated via its receptorCOI1 [63,64]. The downstream MYC2 transcription factor[65,66] acts, at least in part, through direct transcriptionaldownregulation of the PLT1 and PLT2 genes, independentof the auxin pathway [62] (Figure 3). The effects of JAapplication are only partly reduced in plt1;plt2 mutant andPLT2 overexpression roots, suggesting that other PLT/BBM members also play a part in mediating JA effects [62].

Regulation of PLT expression at the chromatin level

Chromatin factors affect key regulators of cell proliferationand stem cell maintenance, such as WUS, WOX5, PLT1,and PLT2 genes [67–72]. The histone acetyltransferaseGENERAL CONTROL NON-REPRESSIBLE 5 (GCN5)and its associated factor ADA2b were found to regulatePLT1 and PLT2 gene expression levels and genetic analy-sis confirmed that GCN5 and ADA2b act in the PLT1–PLT2 pathway [71] (Figure 3). In addition, the root meri-stem of gcn5 mutants, which is gradually lost owing tocompromised stem cell niche maintenance, is partiallyrescued by induced overexpression of PLT2 [71].

Mutation of the chromatin remodelling factor PICKLE(PKL) causes short primary roots with a reduced meristemsize due to impaired stem cell niche activity. By contrast,mutation of the Polycomb-group (PcG) gene CURLY LEAF(CLF) results in longer roots with an increased meristemsize, which is associated with increased stem cell activity[67]. These mutants do not show altered auxin accumula-tion. Rather, their phenotypes correlated with, and wereshown genetically to be caused by, decreased PLT1–PLT2expression or increased WOX5 expression, respectively.Decreased expression levels of root stem cell and meristemmarker genes in pkl correlated with increased levels of(PcG-mediated) trimethylation of histone H3 on lysine 27(H3K27me3) at their promoter regions, indicating that rootstem cell niche activity is regulated by the antagonisticactivities of PcG proteins and PKL [67]. The similar phe-notypes of gcn5 and pkl suggest linked gene activities.PKL-mediated remodelling might allow the recruitmentof a GCN5–ADA2b-containing histone acetyltransferasecomplex, thereby generating a suitable chromatin statefor the accurate interpretation of auxin-dependent PLTsignals and reinforcing stem cell fate [67]. Such a stem cell-specific chromatin state would be analogous to that ob-served in animal systems [73,74].

The OBERON 1 (OBE1) and OBE2 genes encode planthomeodomain (PHD)-finger proteins, so-called epigeneticreaders that recognize and bind to both methylated andunmodified histone H3 tails [75,76]. The OBE proteins actredundantly in MP-dependent embryonic root initiation[76,77]. obe1;obe2 double mutants resemble mp mutantsand lack PLT1 and PLT2 expression, whereas MP expres-sion is unaffected [76]. Because PLT1 and PLT2 expressionis dependent on MP [5], OBE1 and OBE2 might act tocontrol embryonic root meristem formation downstream orat the level of MP, and in this way control PLT1 and PLT2expression [76] (Figure 3).

Shoot and flower meristem maintenanceANT, AIL6/PLT3, and PLT7 are expressed in distinct butoverlapping domains in the inflorescence and vegetativeshoot meristems. AIL6/PLT3 and PLT7 are expressedthroughout the meristem, but their expression is increasedin the peripheral zones and in the central zone, respective-ly [78,79]. By contrast, ANT is expressed exclusively in theperipheral zones and marks the cryptic bract region of thefloral meristem [80,81]. The ant;ail6/plt3;plt7 triple mu-tant shoot stops growing after the production of a fewleaves owing to reduced cell divisions in the meristemand differentiation of the meristematic cells, showing thatANT, AIL6/PLT3, and PLT7 genes are required for shootapical meristem (SAM) maintenance [78] (Figure 2D). Theant and ail6/plt3 mutations enhance wus and shoot mer-istemless (stm) phenotypes, whereas plt7 partially rescuesthese phenotypes, indicating that ANT, AIL6/PLT3, andPLT7 do not function in a strictly redundant fashion [78].

Both the ant and ap2 single mutants have fewer cells intheir floral meristems (Figure 2F), resulting in a reducednumber of floral organs [82,83]. When ap2 and ant muta-tions are combined, sepal, petal, and stamen formation iscompletely abolished [2]. This organ loss is likely to be aresult of reduced proliferation owing to ectopic expression

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of the growth-repressing class C MADS-box gene AGA-MOUS (AG) [2,84].

Organ developmentPhyllotaxy and rhizotaxy

Cotyledons and the first pair of leaves are formed oppositeto each other in Arabidopsis, and subsequent leaves devel-op in a spiral pattern (Figure 2E). Unlike AIL6/PLT3 andPLT7, AIL5/PLT5 is expressed in a largely uniform man-ner throughout the entire SAM [78,79]. In the ail5/plt5;ail6/plt3;plt7 triple mutant, the switch to the spiralphyllotactic pattern is delayed by a few leaf pairs, withdouble mutant combinations showing lower penetrance[79]. The spiral pattern in the inflorescence meristem(Figure 2E) is also affected in this triple mutant, whichshows a tendency to develop successive siliques at angles of�908 or �1808 instead of at 137.58 in wild-type Arabidop-sis. Reducing PIN1 levels leads to the same defects in thephyllotactic pattern as reduced AIL/PLT expression [79].It was shown recently by ail5/plt5;ail6/plt3;plt7 comple-mentation experiments that AIL/PLT-induced auxin bio-synthesis genes, YUCCA 1 (YUC1) and YUC4, mediatehigh auxin levels in the central zone that are required forthe correct spiral phyllotactic pattern [85].

Lateral root primordia (LRPs) are distributed along theArabidopsis root, and their spacing correlates with the rootcurvature [86,87] (Figure 2C). ARF7 and ARF19, togetherwith the gene encoding SOLITARY ROOT (SLR)/IAA14,which represses these ARFs, are all expressed in the vascu-lar tissue and lateral root initiation sites [88–91]. arf7;arf19double mutant roots are defective in the first asymmetricdivision that leads to LRP formation and almost completelylack lateral roots. AIL6/PLT3, AIL5/PLT5, and PLT7 areexpressed prior to the first founder cell division, and theirexpression is absent in arf7;arf19 mutants [50]. Outgrowthof LRP is severely impaired in ail6/plt3;plt7 double andail6/plt3;ail5/plt5;plt7 triple mutants, and LRPs are fre-quently grouped in longitudinal or radial clusters. Comple-mentation experiments reveal that expression of AIL6/PLT3, AIL5/PLT5, or PLT7 from their endogenous promo-ters rescues LRP emergence more readily than the cluster-ing phenotype, suggesting distinct targets and/or dosagedependency reminiscent of PLT/BBM function in the pri-mary root [6,50]. Overexpression of AIL6/PLT3, AIL5/PLT5, or PLT7 in arf7;arf19 can overcome the block inLRP formation. These results indicate that the AIL6/PLT3, AIL5/PLT5, and PLT7 genes are downstream com-ponents of the ARF7- and ARF19-mediated auxin responsemodule [50] (Figure 3), although they do not seem to bedirect targets of ARF7 and ARF19 [89,91].

Despite the differences in the processes of shoot and rootprimordia initiation, the involvement of the same threeAIL proteins in both processes suggests that similar mech-anisms regulate plant architecture in different organs.

Organ size

Loss-of-function ant mutants have smaller leaves andflowers as a result of having fewer cells, whereas over-expression of ANT prolongs the cell proliferation period,leading to larger leaves and flowers with more and/orlarger cells [83,92] (Figure 2D). The reduced cell prolifera-

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tion observed in ant is enhanced in the ant;ail6/plt3 doublemutant, which has even smaller leaves [93]. Overexpres-sion of the auxin-inducible gene AUXIN-REGULATEDGENE INVOLVED IN ORGAN SIZE (ARGOS) prolongsANT expression and leads to the formation of larger organs[94]. This effect was lost in the ant mutant, suggesting thatARGOS functions downstream of auxin and upstream ofANT in organ size control [94] (Figure 3).

An increase in floral organ size due to increased cellnumber was also observed in AIL5/PLT5 and AIL6/PLT3overexpression lines [1,95]. However, high expressionlevels of AIL6/PLT3 block cellular differentiation, result-ing in floral organs with altered morphology [95]. The verysmall and aberrantly shaped sepals of these AIL6/PLT3overexpression lines are unable to cover the entire flowerbud, a phenotype that was also reported in BBM over-expression lines [4,95]. Together with the sustained meri-stem cell proliferation observed on PLT2 overexpression[6], these results indicate that AIL transcription factorsregulate the balance between cell proliferation and differ-entiation.

Organ polarity

Double and triple mutant combinations of ant with loss-of-function alleles of the polarity genes FILAMENTOUSFLOWER (FIL) and YABBY 3 produce dwarfed seedlingsthat develop small, narrow leaves with loss of both adaxialand abaxial epidermal cell characteristics, which are notobserved in single mutants [96,97]. These defects are alsopresent in some floral organs. The expression of the adaxialspecifying HD-ZIP III gene PHB is reduced in fil;antflowers, suggesting that ANT and FIL act together topromote organ polarity (Figure 2D) by activating PHB[97] (Figure 3).

Flower and floral organ developmentFloral initiation

LEAFY (LFY) encodes a transcriptional regulator thatpromotes the transition to flowering and also specifiesfloral meristem identity [98–101]. Auxin treatmentinduces higher LFY gene expression and results in preco-cious flower formation. Auxin response elements in thepromoter of LFY are bound by the MP and BDL proteins[51]. Initiation of flower primordia from the inflorescencemeristem (Figure 2F) is known to rely on MP; mp mutantsform naked inflorescence pins lacking flowers, phenocopy-ing loss-of-function mutants in the PIN1 auxin effluxcarrier [45,102]. MP and LFY expression overlaps withthat of the ANT and AIL6/PLT3 genes, and MP was shownto bind to the promoters and activate expression of ANTand AIL6/PLT3 [51]. The pin-like inflorescences formed inthe ant;ail6/plt3;lfy triple mutant and the additive abilityof ANT and LFY overexpression to rescue flower primordi-um initiation in the hypomorphic mp-S319 mutant indi-cate that ANT and LFY function in parallel pathwaysdownstream of MP in the initiation of flower primordia[51] (Figure 3). As with AIL proteins, LFY feeds backpositively into the auxin pathway [51,79,85]. MP-depen-dent upregulation of LFY, ANT, and AIL6/PLT3, togetherwith their positive feedback to the auxin pathway, mightcommit cells to flower primordium formation [51].

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Floral organ identity

The petals of the ant single mutant develop stomata, whichare absent in wild-type petals, indicating partial loss ofpetal identity in this mutant [84]. A more severe phenotypein which the petals become partially stamenoid was ob-served in the hypomorphic ap2-1;ant double mutant [84].Expression of AG was extended to the second whorl in theap2-1;ant mutant, where it caused the partial homeotictransformation [84]. Single ail6/plt3 mutants develop nor-mally, but flowers of the ant;ail6/plt3 double mutant havereduced petal, stamen, and carpel identities and consistprimarily of small sepals, filamentous organs, and unfusedcarpel valves, showing that ANT and AIL6/PLT3 functionredundantly in determining floral organ identity [93](Figure 2F). This phenotype correlates with altered expres-sion of the floral organ identity genes APETALA 3 (AP3, B-class) and AG (C-class) [93], and suggests their regulationby ANT and AIL6/PLT3 (Figure 3).

Ovule development

The number of ovule primordia in the ant mutant isreduced owing to carpel margin meristem (CMM) defects.Also, the integuments of ant ovules do not initiate properlyand female gametophyte development is disrupted, lead-ing to female sterility [2,3,103,104]. The CUP SHAPEDCOTYLEDON 1 (CUC1) and CUC2 genes act in parallel toANT in controlling the number of developing ovule primor-dia [49]. ANT, CUC1, and CUC2 are downregulated inhypomorphic mp-S319 mutant pistils, which do not devel-op ovules owing to the absence of carpel margin tissue.Binding of MP to the ANT, CUC1, and CUC2 genomicregions was confirmed in this tissue [49]. Thus, MP acts asan upstream regulator of ANT during ovule development(Figure 3), reminiscent of its role in regulating AIL geneexpression during embryogenesis and floral meristem ini-tiation [5,49,51].

HUELLENLOS (HLL) and ANT have overlapping rolesin ovule primordium outgrowth [105]. The transcriptionalco-regulators SEUSS (SEU) and LEUNIG (LUG) showpartial redundancy with ANT in the control of medialdomain development [106,107], and these defects are as-sociated with reduced expression levels of the HD-ZIP IIIgenes REV and PHB [106]. Double mutant ant;rev gynoeciaexhibit loss of CMM-derived tissue, which is correlatedwith lower expression of PHB [108]. These studies showthat ANT has a central role in ovule development(Figure 2F) through the activation of HD-ZIP III genes(Figure 3).

AIL target genesAlthough many genetic interactions have been described forAIL genes, our knowledge about target genes that aredirectly regulated by AIL binding remains limited. BothAPETALA 2/ETHYLENE RESPONSE FACTOR (AP2/ERF) domains of ANT are required for DNA binding, andeach domain is thought to use different amino acids tocontact the DNA [109,110] (Box 1). An ANT DNA bindingmotif was determined using SELEX, an in vitro oligonucle-otide selection method, which yielded the consensus se-quence gCAC(A/G)N(A/T)TcCC(a/g)ANG(c/t) [110]. AIL5/PLT5 also binds to the ANT consensus sequence in vitro

[61], suggesting that the observed overlapping functions ofAIL proteins is realized through a set of common targetgenes. The ANT DNA binding motif is different from that ofother single AP2/ERF proteins, which bind to short GC-richboxes [111–113].

Currently, only the targets of BBM have been identified.Using a microarray approach in which the BBM proteinwas inducibly activated in the presence of the translationalinhibitor cycloheximide to identify direct targets, it wasshown that the vast majority of target genes are upregu-lated on DNA binding, suggesting that BBM acts mainly asa transcriptional activator [114]. BBM was reported tointeract with TPL-related (TPR) corepressors in yeast[115], suggesting that BBM also acts as a transcriptionalrepressor. AP2 has also been shown to form complexes withTOPLESS (TPL) to repress the floral organ identity genesAG, AP3, PI, and SEP3 [116], suggesting that similarprotein–protein complexes are co-opted by different groupsof AP2-family proteins.

The functional diversity of BBM targets suggests thatthis protein functions in a wide range of developmentalpathways, which is consistent with the large number ofmutant studies in which BBM and other AIL genes wereshown to be regulated incorrectly. One of the few function-ally characterized BBM targets is RGF8/GLV6, whichencodes a homologue of the RGF1 signalling peptide thathas been shown to regulate PLT1 and PLT2 expression[52]. This suggests a positive feedback loop between RGF/GLV and PLT/BBM genes (Figure 3) that ensures suffi-ciently high PLT/BBM levels to preserve the meristem.Another gene that is directly upregulated by BBM isACTIN DEPOLYMERIZING FACTOR 9 (ADF9) [114],which encodes an ADF/cofilin cytoskeletal protein[117,118]. ADF9 is required for hormone-mediated cellproliferation in vitro [119]. Notably, only a few minutesof inducible BBM overexpression is sufficient to stimulatereorganization of the actin cytoskeleton. ADF9 thereforeprovides a potential link between the actin cytoskeletonand BBM-mediated meristematic growth. Finally, it wasshown that BBM also stimulates its own expression, sug-gesting it functions in a positive auto-regulatory feedbackloop to control its own activity [114]. BBM targets supporta role for AIL proteins in the regulation of cell proliferationand differentiation.

Concluding remarks and future prospectsAlmost 20 years of AIL research in Arabidopsis has shownthat these transcription factors are crucial for stem cellniche specification, meristem maintenance, organ initia-tion, and growth, and that they function as master reg-ulators of embryogenesis and root formation. At present,functions have been ascribed to all Arabidopsis AIL genesexcept AIL1. Research on AIL expression and function inother species is still at an early stage. Analysis of theexpression patterns of ANT-like genes from different spe-cies suggest that they are [120–122] or are not [120,123]conserved with the Arabidopsis ANT gene. Nevertheless,functional studies on AIL proteins from different speciesindicate that they also regulate stem cell specification andplant developmental processes, as in Arabidopsis. Forexample, in poplar (Populus spp.), ANT-like AILs have

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been shown to control meristem activity during adventi-tious rooting [9] and to directly feed into the cell cyclemachinery to establish growth cessation on exposure toshort days [8]. Similarly, in the moss Physcomitrellapatens, four AIL genes are indispensable for the formationof one type of moss stem cell, the apical gametophore cell[7].

AIL proteins interact with auxin pathways throughoutplant development and at multiple levels, including viaARFs and through PIN feedback loops (Figure 3). Futurestudies will undoubtedly reveal additional auxin–AILrelationships, as well as new relationships with otherhormones, signalling pathways and chromatin-levelchanges. Factors involved in regulating AIL expressionduring the earliest stages of embryogenesis have not beenidentified, even though at least two AILs, BBM and PLT2,are required for progression through early embryogenesis.

Specific AIL family members seem to be expressed andfunction in either the root or shoot meristem. This divisioncould reflect protein subfunctionalization, which is alsosuggested by the different AIL–HD-ZIP III genetic inter-actions that appear as a general module in the regulationof several developmental processes (Figure 3). Alternative-ly, the regional expression and interaction of AIL proteinswith locally expressed transcriptional co-activators orrepressors might be responsible for this differential effect.Identification of the protein complexes in which AIL pro-teins act might shed light on this matter. In addition,promoter swapping to express root AILs in the shootand vice versa may reveal how much of this apparentsubfunctionalization is due to differences in protein char-acteristics versus gene expression patterns.

One aspect of AIL function is their dosage-dependentregulation of developmental processes, as observed duringroot development. This dosage-dependency could be me-diated by quantitative differences in the activation oftarget genes containing AIL-specific DNA binding motifs.Alternatively, AIL proteins could exert their dosage-de-pendent regulation via differences in binding affinity forDNA motifs that are present in common target genepromoters. The fact that both ANT and AIL5/PLT5 canbind the same DNA motif suggests a common set of down-stream targets. Elucidating the targets and DNA bindingsites of additional AIL proteins might clarify either of theabove scenarios.

A poorly understood aspect of AIL function is theirability to induce totipotency and pluripotency when over-expressed. This property offers new possibilities toimprove the efficiency of plant regeneration for a rangeof biotechnology applications. For example, the ability ofBBM to induce somatic embryogenesis was used toimprove Chinese white poplar and sweet pepper (Capsi-cum anuum) transformation [124,125]. More importantly,a better understanding of how AIL proteins function willprovide a framework for understanding how regenerationis regulated in vitro and during normal plant development.

AcknowledgementsWe thank Gabino Sanchez-Perez for constructing an updated phyloge-netic tree for the AIL family, and Ben Scheres, Gerco Angenent, andStephen Grigg for valuable comments on the manuscript. A.H. was

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supported by a Technology Top Institute Green Genetics project. R.H.was supported by a Netherlands Genomics Initiative Horizon grant (no.93516054) from The Netherlands Organization for Scientific Research.

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