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ScienceDirect How boundaries control plant development Petra Zˇa´dnı´kova´ and Ru¨diger Simon Continuous growth and organ development from the shoot apical meristem (SAM) requires a precise coordination of stem cell proliferation, commitment of stem cell descendants to diverse differentiation pathways and establishment of morphological meristem-to-organ boundaries. These complex biological processes require extensive integration of several components of cell-to-cell signaling and gene regulatory networks whose coordinated actions have an impact on cell division and growth.Here we review the current knowledge of gene networks involved in organogenesis from the SAM in higher plants. We focus on recent advances to show how the interaction between transcriptional regulators, hormonal crosstalk and physical stress regulates the establishment and maintenance of meristem-to-organ boundaries.Continuous growth and organ development from the shoot apical meristem (SAM) requires a precise coordination of stem cell proliferation, commitment of stem cell descendants to diverse differentiation pathways and establishment of morphological meristem-to-organ boundaries. These complex biological processes require extensive integration of several components of cell-to-cell signaling and gene regulatory networks whose coordinated actions have an impact on cell division and growth.Here we review the current knowledge of gene networks involved in organogenesis from the SAM in higher plants. We focus on recent advances to show how the interaction between transcriptional regulators, hormonal crosstalk and physical stress regulates the establishment and maintenance of meristem-to-organ boundaries. Addresses Institute of Developmental Genetics, Cluster of Excellence on Plant Sciences (CEPLAS), Heinrich-Heine-University, Universita¨tsstrasse 1, D-40225 Du¨sseldorf, Germany Corresponding author: Simon, Ru¨diger ([email protected])

Current Opinion in Plant Biology 2014, 17:116–125 This review comes from a themed issue on Growth and development Edited by David R Smyth and Jo Ann Banks

S1369-5266/$ – see front matter, Published by Elsevier Ltd. http://dx.doi.org/10.1016/j.pbi.2013.11.013

Introduction Plant organogenesis is assured by pools of dividing, pluripotent cells that reside in the meristems, the plant’s stem cell niches. The shoot apical meristem (SAM), a group of cells at the growing tip of a plant, generates all the aboveground tissues of the plant. The SAM is organized into a central zone, composed of slowly dividing stem cells and a Current Opinion in Plant Biology 2014, 17:116–125

peripheral zone, containing more rapidly dividing cells that can become incorporated into the organ primordia (Figure 1a and b). The initiation of organs from the peripheral zone requires the creation of meristem-to-organ boundaries that separate these two cell groups with very distinct gene expression programs and morphologies. The boundary itself expresses a unique set of transcription factors that play an important role to locally repress cell proliferation, which is a prerequisite for the development of physically separate organs. Loss-of-function mutants often cause organ fusion, but also defects in organ development and altered phyllotactic patterning. This indicates that the meristem-to-organ boundaries also operate as organizing centers that provide information to adjacent cells to control their developmental programs (Figure 1a–c). Recent studies have uncovered how a combination of mechanical forces, transcriptional regulators and phytohormonal inputs interact to establish and maintain functional boundaries at the meristem (Figure 1d).

Role of mechanical stress in meristem patterning and organ initiation Cells at different positions within the dome-shaped apical meristem experience different growth and expansion rates and therefore are subjected to mechanical stress. In response to this local mechanical stresses, they modify their growth rate and anisotropy accordingly. Growth rate and anisotropy depend on the cell wall properties, since cell growth is driven by internal turgor pressure, which is limited by the stiffness of the wall. The rigid cellulose microfibrils provide the necessary reinforcements that counteract cellular turgor. However, not all cells in the meristem are subjected to the same mechanical stress, indicated by the observation that the outer cell wall is approximately five-times stiffer at the tip of the meristem than on its flanks [1]. Anisotropic growth is mediated by the parallel alignment of cellulose microfibrils in plant cell walls, which is controlled by cortical microtubules. These drive the local insertion and processing trajectory of the cellulose synthase complex (CESA) at the plasma membrane [2,3]. An intimate connection between microtubules and the cellulose synthase complex is essential for organ phyllotaxis: absence of the linker protein CESA INTERACTIVE PROTEIN 1 (CSI1) that connects CESA with cortical microtubules [4,5] cause twisting of the rapidly elongating part of the stem after primordia have arisen, and subsequently affecting phyllotaxy [6]. This revealed that the overall mechanical properties and local resistance to stress within meristem can affect organ positioning [1,6]. Local growth heterogeneity during organ initiation requires a differential response of neighboring cells. In www.sciencedirect.com

How boundaries control plant development Simon and Zˇa´dnı´kova´ 117

a parallel orientation of the microtubules [9], reviewed in [10]. Microtubules quickly reorient upon wounding of the SAM, which locally alters the mechanical forces. Interestingly, relocalization of the auxin efflux carrier protein PINFORMED1 (PIN1) was shown to parallel microtubule reorientation to the direction of the largest stress thereby affecting auxin distribution in the tissue. However, PIN1 was still stably localized after microtubule depolymerization through oryzalin treatment, indicating that microtubules are not causing the PIN1 localization in a direct manner.

Figure 1

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In boundaries, microtubules are oriented along the major axis of cells coinciding with PIN1 localization at anticlinal walls. This orientation of PIN1 will then result in a depletion of auxin from the boundary domain and export towards the organ initial and the remainder of the meristem. Importantly, auxin itself affects the mechanical properties of the cell wall controlling the expression of cell-wall remodeling enzymes such as expansins, and auxin maxima allow primordia to grow out. Thus, a positive feedback loop is created whereby auxin transport is affected by tissue mechanics, which is in turn controlled by auxin through its effect on cell wall characteristics [9].

brassinosteroids Current Opinion in Plant Biology

Transcriptional regulation of boundary functions

The inflorescence meristem (IM) of Arabidopsis thaliana. The shoot apical meristem (SAM) is responsible for the production of rosette leaves and, after transition to the inflorescence meristem (IM), for the production of the stem, cauline leaves, lateral meristems and flowers of the inflorescence, which arise in the axils of cryptic bracts. (a)–(c) Reconstructed views of the SAM expressing membrane-localized PIN1::PIN1-GFP in the epidermis (a, top view; b, optical section; c, detailed view of the top, focused on different stages of meristem-toorgan boundary formation). The localization of PIN1::PIN1-GFP indicates auxin transport towards young flower bud primordia (named P1-P6 from the youngest to the oldest organ primordium). The functional zones are represented and highlighted in different colors. At the meristem summit the central zone (CZ, yellow) comprises the stem cells, primordia (P, green) are initiated in the peripheral zone (PZ, red). The zone between the peripheral zone and the primordium represents the meristem-toorgan boundary (B, blue). (D) Schematic representation of the likely distribution of auxin and brassinosteroids in the SAM.

Organ initiation is closely related to auxin accumulation in organ founder cells due to PIN1-mediated directional auxin transport. Subsequently, when primordia start to grow, PIN1 polarity reverses to form a new auxin maximum at a distant position. The shifts in the auxin transport direction are temporally and spatially correlated with the establishment of the boundary between the new primordium and the meristem, as well as with auxin depletion from boundaries [9].

the SAM, cortical microtubules continuously reorganize to remain parallel to the direction of maximal stress, and the ability to reorient these cortical microtubules in response to mechanical forces is crucial to allow differential growth. This is achieved through proteins like KATANIN that fragment microtubules. In katanin mutants, neighboring cells have the tendency to grow more frequently in the same direction, and the characteristic dome-like shape of the shoot tip inverts [7], reviewed in [8].

CUP-SHAPED COTYLEDON (CUC1,2,3) genes in Arabidopsis and the NO APICAL MERISTEM (MtNAM) gene in Medicago truncatula [11] encode NAC transcription factors that, together with the homeobox gene SHOOTMERISTEM LESS (STM), regulate the formation of shoot meristems. Later in development, CUC genes also control the specification of organ boundaries. Typical for genes regulating boundary functions, cuc1 cuc2 double mutants display organ fusions and growth arrest [12], reviewed in [13].

Cells in boundaries exhibit a very low growth rate and high anisotropy, compared to the neighboring meristem and primordium, which correlates with stiff cell walls and www.sciencedirect.com

The complex interplay of regulatory gene networks involved in boundary establishment and maintenance will now be discussed (see Table 1 for a list of genes involved). The role of some of these genes, and the phytohormone auxin, is illustrated in Figure 2.

Earlier studies showed that there is interdependence between auxin-dependent organ initiation and CUC gene expression. Mutations in PIN1 produce naked inflorescence stems resulting from the ectopic expression of Current Opinion in Plant Biology 2014, 17:116–125

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Table 1 Hormones and genes controlling boundary function Hormones regulating boundary functions Hormone Auxin Brassinosteroids

Meristem effects

Levels and function in meristem-to-organ boundary

Commitment of organ founder cells Regulation of organ spacing (phyllotaxis) Control of plant stature and growth

Low levels in boundaries correlates with meristem-to-organ boundary formation and negatively regulate CUC genes Low levels in boundaries allow boundary specific gene expression (CUC1-3) and to restrict growth

Genes controlling boundary functions Gene

Accession number

Protein family

Meristem-specific genes CLV3 AT2G27250 CLE WUS AT2G17950 HOMEODOMAIN STM AT1G62360 Class I KNOX BP/KNAT1 AT4G08150 Class I KNOX KNAT2 AT1G70510 Class I KNOX Meristem and meristem-to-organ boundary-specific genes LBD15 AT2G40470 LBD Meristem-to-organ boundary and organ primordia-specific genes BOP1 AT3G57130 BTB/POZ BOP2 AT2G41370 BTB/POZ Meristem-to-organ boundary-specific genes CUC1 AT3G15170 NAC CUC2 AT5G53950 NAC CUC3 AT1G76420 NAC JLO AT4G00220 LBD KNAT6 AT1G23380 Class I KNOX LOB AT5G63090 LBD LOF1 AT1G26780 MYB LOF2 AT1G69560 MYB OBO1/LSH3 AT2G31160 ALOG LSH4 AT3G23290 ALOG SUP AT3G23130 C2H2 TFIIIA DPA4 AT5G06250 Putative RAV Organ primordia-specific genes AS1 AT2G37630 MYB AS2 AT1G65620 LBD HIRA AT3G44530 WD repeat HIR1 CLF AT2G23380 Polycomb-group SWN AT4G02020 Polycomb-group PTL AT5G03680 TRIHELIX RBE AT5G06070 C2H2 TFIIIA JAG AT1G68480 C2H2 TFIIIA Genes involved in auxin transport PIN1 AT1G73590 PIN PID AT2G34650 SER/THR KINASE ENP AT4G31820 NPY ABCB19 AT3G28860 ABC Other genes with diverse expression patterns miRNA164 AT2G47585 – EEP1 AT5G27807 – TCP3 AT1G53230 bHLH

Function Controls the size of the stem cell domain by repressing WUS Maintains stem cells in an undifferentiated state Required for SAM formation (embryogenesis) and later for SAM function Meristem maintenance Meristem maintenance Maintains the stem cell pool through upregulation of WUS Regulation of organ cell fate, promotes boundary specification Regulation of organ cell fate, promotes boundary specification Required for SAM formation (embryogenesis) and boundary specification Required for SAM formation (embryogenesis) and boundary specification Required for SAM formation (embryogenesis) and boundary specification Involved in boundary specification Important for SAM formation and organ separation Involved in boundary specification and lateral organ development Functions in boundary specification, meristem initiation and maintenance Functions in boundary specification, meristem initiation and maintenance Involved in meristem growth and floral whorl development Involved in meristem growth and floral whorl development Negative regulation of growth at the stamen-to-carpel boundary Involved in meristem maintenance and lateral organ development Involved in organ primordia development and determination of organ polarity Involved in organ primordia development and determination of organ polarity Chromatin re-organization Chromatin re-organization Chromatin re-organization Role in organ (sepal) separation, development of petals Involved in petal development and inter-sepal boundary maintenance Required for separation of petals Controls directed auxin efflux Positive regulation of auxin efflux by controlling PIN1 localization Positive regulation of auxin efflux Role in auxin efflux Negative regulator of CUC genes Negative regulator of CUC genes Negative regulator of CUC genes through upregulation of miRNA164

CUC2 at a ring-like domain, which is characterized by the expression of primordia-specific genes. During embryogenesis, localization and function of PINs are regulated by PINOID (PID) and ENHANCER OF PINOID (ENP) to promote cotyledon initiation by preventing the expansion of CUC1 and CUC2 expression into the cotyledon primordium. Moreover, in leaves, CUC2 contributes to the generation of PIN-dependent auxin maxima, while Current Opinion in Plant Biology 2014, 17:116–125

auxin represses CUC2 expression in a regulatory loop, reviewed in [13,14]. Interestingly, another auxin transporter ABCB19 (ATP-binding cassette/multi-drug resistance/P-glycoprotein) plays an important role in boundary formation through the positive regulation of CUC2 and also contributes to the depletion of auxin from the boundary region [15]. Altogether, these observations depict interdependence between auxin, auxin transport www.sciencedirect.com

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Figure 2

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Gene networks underlying IM patterning and the transcriptional regulation of the boundary domain in Arabidopsis. Arrows and inhibition lines represent positive (black) and negative interactions (dark red), respectively. Auxin (red) is highlighted. Genes presented by large capital letters are expressed in the relevant zone of SAM, while gene names inidcated in small letters are repressed in the corresponding zone. Diagram represents a schematic drawing of organization of the SAM. Undifferentiated cells in the meristem are represented by grey color, organ primordia by dark grey and boundary cells which separate meristem and primordia from each other are represented by yellow color. In Arabidopsis, the stem cell populations in the meristem are positively regulated by class I KNOX genes and by the WUS-CLV negative feedback loop. In addition, expression of WUS is positively regulated by the transcription factor LBD15 expressed in the SAM and meristem-to-organ boundaries. The formation of meristem-to-organ boundary requires the inhibition of cell division, which is ensured by a combination of boundary-specific transcription factors, including CUC1-3, JLO, LOB, LOF1, BOP1, BOP2 and OBO1. CUC positively regulates class I KNOX genes and LOF1, another boundary-specific gene. Furthermore, CUC genes promote the generation of auxin maxima, while auxin and miR164 repress CUC gene expression in a regulatory feedback loop. Expression of LOB has been shown to be regulated by brassinosteroids (Figure 3) and BOP1/2. JLO acts independently, or in a multimeric complex with AS1/AS2 to suppress KNOX gene expression and induce expression of PIN-famliy auxin efflux carriers. Organ primordia initiation is closely connected to and dependent on the accumulation of auxin, driven by PIN-mediated auxin transport, and with the repression of class I KNOX genes. BOP1/2 transcription factors positively regulate expression of AS1/AS2 complex components. AS1/AS2 then interact with chromatin remodeling complexes (HIRA and PRC2) to ensure KNOX genes repression. For further detail, see text.

and the regulation of CUC expression in the process of organ patterning and boundary establishment (Figure 2). The expression of CUC2 is post-transcriptionally downregulated by members of the miRNA164 family, among them EEP1 (EXTRA EARLY PETALS1) coding for miR164c, and by the transcriptional repressor DPA4 (DEVELOPMENT-RELATED PCG TARGET IN THE APEX 4), encoding a putative RAV (RELATED TO ABI3/VP1). Both genes regulate the spatial restriction of CUC gene expression [16,17,18]. Other factors downregulating CUC genes are TCP (CINCINNATA-like TEOSINTE BRANCHED1, CYCLOIDEA, and PCF) transcription factors, which directly activate EEP1 expression [16,19]. In flowers, CUCs and PTL (PETAL LOSS), a trihelix transcription factor, maintain the inter-sepal boundary, but through independent pathways. While CUCs prevent www.sciencedirect.com

the inter-sepal boundary from differentiating into sepal tissue, PTL controls cell proliferation within boundaries and is required to establish auxin maxima at the presumptive petal initiation sites. Interestingly, PTL acts non-cell-autonomously to influence petal development from the sepal whorl, since PTL is not expressed at sites of petal initiation. Thus, PTL could generate a mobile petal initiation signal, or the PTL protein itself may be transported to petal initiation sites [18,20]. PTL itself is directly repressed by JAG (JAGGED), which is expressed in the distal petal domain and regulates organ development [21]. Another gene, RBE (RABBIT EARS), encoding a zinc finger transcriptional repressor, is expressed in petal primordia and is also required for proper petal development and inter-sepal boundary maintenance. RBE represses miRNA164c expression from the EEP1 locus, Current Opinion in Plant Biology 2014, 17:116–125

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thus allowing the expression of CUC genes. Loss of RBE function results in miR164c upregulation and consequent reduction of CUC1 and CUC2 expression. Therefore RBE acts to fine-tune CUC expression and, ultimately, cell proliferation [22]. The transcriptional repressor SUP (SUPERMAN) is expressed in the inner part of stamen primordia, adjacent to carpels, from where it non-cell-autonomously maintains the boundaries between these two organs. SUP was shown to regulate cell proliferation via interaction with auxin and cytokinin signaling pathways [23]. The aforementioned transcriptional network involving PTL, RBE and SUP reveals the presence of strong feedback control, and consequent interdependency between the establishment of boundaries and organ development. A novel downstream target of CUC genes was recently identified. CUC genes directly activate the expression of LIGHT-DEPENDENT SHORT HYPOCOTYLS 4 (LSH4) and its homolog LSH3, both members of the ALOG (Arabidopsis LSH1 and Oryza G1) family, in shoot organ boundary cells. LSH3 and LSH4 appear to suppress organ initiation in the boundary region and to promote boundary formation [24]. In Arabidopsis thaliana, meristems are maintained as undifferentiated cell pools through the activity of transcription factors of the KNOTTED-LIKE HOMEODOMAIN (KNOX) gene family (Figure 2). As soon as cells are recruited into an organ, these meristematic identity genes are switched off and maintained in a repressed state by organ-specific regulators like the MYB transcription factor AS1 (ASYMMETRIC LEAVES 1), the LBD (LATERAL ORGAN BOUNDARY DOMAIN) transcription factor AS2 (ASYMMETRIC LEAVES 2) [25] and genes encoding proteins with a BTB/POZ domain, such as BOP1, 2 (BLADE ON PETIOLE1) [26,27,28], reviewed in [29]. Permanent repression of KNOX genes involves an epigenetic regulation mechanism: a current model suggests that AS1/AS2 complexes bind to two distinct sites of the BP promoter, thus creating a DNA loop between the two binding sites, and recruit the chromatin-remodeling protein HIRA to maintain the chromatin in a stably repressed state, reviewed in [29]. The proteins POLYCOMB REPRESSIVE COMPLEX 2 (PRC2), CURLY LEAF (CLF) and SWINGER (SWN), which serve to maintain the repressed state of KNOX genes in leaves by catalyzing the dispersed trimethylation of histone H3 at lysine 27 (H3K27) and subsequently inducing chromatin compression and inhibition of transcription, were recently shown to be also recruited by AS1/AS2 complexes and thus to direct PRC2 to repress KNOX genes [30] (Figure 2 and Table 1). Another boundary-specific gene JAGGED LATERAL ORGANS (JLO), a transcription factor of the LATERAL Current Opinion in Plant Biology 2014, 17:116–125

ORGAN BOUNDARY DOMAIN (LBD) gene family, has been recently identified. During organ initiation and development, the expression of JLO in the SAM is highly dynamic and transient. In the peripheral zone of the SAM, JLO is expressed at organ initiation sites. The JLO protein alone, or in a multimeric complex with AS2 and AS1, suppresses KNOX gene expression, thereby allowing the meristematic cells to exit the stem cell state [31]. Once organs are initiated, JLO expression in the SAM is limited exclusively to the meristem-to-organ boundaries. Importantly, expression of KNOX genes in differentiating organs remains repressed, although JLO is no longer expressed in these cells, most probably due to the continuous presence of AS1-AS2 complexes [31]. Furthermore, JLO was shown to upregulate PIN gene expression in the SAM, thereby contributing to the auxin buildup at the organ initiation sites and later to the depletion of auxin from the boundary region [32] (Figure 2). Recently, further transcriptional regulators were identified that act in boundaries to repress cell divisions and growth, e.g. BOP1, BOP2, LOF1, LBD15 and OBO1 (Table 1). BOP1 (BLADE ON PETIOLE 1) and BOP2 genes encode BTB/POZ domain proteins that are expressed in the meristem-to-organ boundary but also at the base of lateral organs. BOP1 and BOP2 genes were shown to directly induce AS2 expression in leaf primordia. In addition, they repress class I KNOX gene (BP, KNAT2 and KNAT6) expression via an AS2-independent pathway [26]. Further, BOP1 and BOP2 induce expression of the boundary genes LOB and LBD36 and upregulate adaxial, while repressing abaxial factors [28,33] (Figure 2). (Additional roles for BOP genes are addressed below.) LOF1 (LATERAL ORGAN FUSION1) and LOF2 encode MYB-domain transcription factors that are expressed in organ boundaries. lof1 and lof2 mutants display defects in organ separation as a result of abnormal cell division and expansion during early boundary formation. LOF1 and LOF2 were also found to play role in meristem initiation by inducing STM expression [34]. Some members of the LBD family were previously shown to play an important role in lateral organ development of plants (e.g. JLO and LOB [31], reviewed in [35]) including lateral root formation (e.g. LBD14, LBD16, LBD18 and LBD33 [36,37]) and vasculature development, together with a control of secondary growth (e.g. LBD1, LBD4 and LBD18 [38,39]). Interestingly, LBD genes could also play important roles in sustaining cell division activity of meristematic cells. For example, LBD15, which is expressed in the SAM and meristemto-organ boundaries, was found to upregulate expression of the homeodomain transcription factor WUS, which is, www.sciencedirect.com

How boundaries control plant development Simon and Zˇa´dnı´kova´ 121

together with class I KNOX genes, required for the maintenance of stem cell populations in shoot and flower meristems [40]. The boundary-specific gene ORGAN BOUNDARY1 (OBO1/LSH3) belongs to a novel gene family (ALOG) consisting of 10 plant-specific members, which encode a single small domain of unknown function. OBO1 overexpression causes petal-stamen fusions, while genetic ablation of cells expressing OBO1 result in a loss of SAM and lateral organs, suggesting that OBO1 plays an important (while not yet fully understood) role in meristem maintenance and organogenesis of Arabidopsis [41].

A novel role for brassinosteroids in boundary formation Auxin has long been known to be important for SAM patterning, and its depletion from the boundary between newly forming organ and the remainder of the meristem correlates with meristem-to-organ boundary formation in this region. Recently, it was shown that another class of plant hormones, brassinosteroids (BRs), may also play a crucial role in boundary formation (Figures 1d and 3). BRs

are known to stimulate growth by promoting cell division and elongation. During lateral organ development, BR signaling contributes to boundary formation at the cellular and molecular levels. High BRs levels cause defects in organ separation, while reduced levels in the boundary domain result in restricted growth and consequently groove formation between the meristem and the new organ. Additionally, BRs inhibit the expression of boundary genes (CUC1, CUC2, CUC3 and LOF1) and thus the expression of CUCs and LOF1 in the meristem-to-organ boundary could result from their de-repression due to the lower BR signaling in this domain [42]. The gene BAS1 (PHYB ACTIVATION TAGGED SUPRESSOR1) encoding a BR-inactivating enzyme was identified as direct target of the boundary-specific transcription factor LOB (LATERAL ORGAN BOUNDARY). Expressing BAS1 under the LOB promoter rescued the organ fusion defect of the lob mutant. This showed that LOB could modulate BR responses partly through activation of BAS1. Based on these findings, the following model could be suggested (Figure 3). The LOB gene, expressed in the meristem-to-organ boundary, represses BR signaling, which subsequently

Figure 3

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Gene networks underlying brassinosteroid signaling during IM patterning in Arabidopsis. Arrows and inhibition lines represent positive (black) and negative interactions (dark red), respectively. The plant hormone brassinosteroid is highlighted in blue. Genes presented with large letters are expressed in the relevant zone of SAM, while gene names indicated with smaller letters are repressed in the respective area. The diagram represents a schematic drawing of SAM tissue organization. Central zone in grey, an organ primordium in dark grey, and the boundary zone in yellow. LOB expression in the meristem-to-organ boundaries is activated by the expression of BOP1 and BOP2 in the primordium and the boundary domain and by the activity of BRZ1 transcription factor, whose expression depends on brassinosteroids. LOB represses brassinosteroid signaling, in particular through the activation of the brassinosteroid-inactivating enzyme BAS1. Consequently, low levels of brassinosteroids release the repression of CUC and LOF and inhibit growth, thus generating the boundary domain. There is cross-talk between brassinosteroids (this figure) and auxin (Figure 2), and their resulting effect on boundary gene’ expression contributes to the patterning of the meristem-to-organ boundaries. www.sciencedirect.com

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restricts cell growth and division in the boundary domain. In turn, BRs signaling activates LOB expression. Presumambly a low concentration of BRs in the boundary is sufficient to activate first LOB expression, thus creating a negative feedback loop. Furthermore, expression of LOB is also induced by BOP1/ 2, which are expressed in the layer of neighboring organ cells and therefore likely act non-cell autonomously [43], and reviewed in [35] (Figure 3). To date, several studies have shown that BRs and auxin play crucial roles in establishing and maintaining boundary functions. The formation of local minima of these two hormones is a prerequisite for boundary establishment (Figure 1D). Furthermore, both hormones share downstream target genes, indicating significant hormonal crosstalk during the formation of the boundary domain.

Boundaries allow organs to let go Boundaries between meristem and organ are established early in development and serve to separate newly forming organs from the meristem. At later stages, when meristems lose their dividing activity, meristem-to-organ boundaries can apparently be differentiated into abscission zones (AZs), the position where organ abscission occurs. Abscission in plants is a crucial process to shed organs such as leaves, flowers and fruits when they are senescent, damaged or mature, reviewed in [44]. The best described organ abscission so far is the release of outer floral organs after pollination. After AZ differentiation, the abscission process is initiated, whereby cell-wall modifying enzymes orchestrate the organ separation process. In Arabidopsis, floral organ abscission is regulated by a receptor ligand module including the signaling peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and the receptor-like kinases HAESA (HAE) and HAESA-LIKE2 (HSL2) [reviewed in 44]. The integration of multiple developmental, hormonal and environmental signals triggers organ abscission. Some components of this signaling pathway are common to both the abscission process and the specification of the boundary domain during plant development. For example, BOP1/2 (BLADE ON PETIOLE) not only activate LOB [43] and reviewed in [35], but are also important for the differentiation of AZs cells. Another example is BP/KNAT1, which was identified as a downstream factor of the boundary specific CUC genes [16], while during the abscission process BP/ KNAT1 functions as a negative downstream component of the IDA signaling pathway. BP/KNAT1 regulates abscission zone size and represses the expression of KNAT2 and KNAT6 [45,46], which are both required for the abscission process to occur. The recently identified tomato MADS-box transcription factors MACROCALYX (MC) and JOINTLESS are involved in regulating the Current Opinion in Plant Biology 2014, 17:116–125

development of tomato pedicel AZs, but were also found to upregulate the expression of CUCs [47]. These recent data suggest a tight connection between organ-boundary formation and organ abscission, two processes that occur at the same cell sites and share several signaling pathway components.

Role of the meristem-to-organ boundaries in grasses (Poaceae) In this review, we mostly focussed on Arabidopsis, in which most of the recent findings have been made. In Poaceae, the SAM produces the primary branches of the inflorescence, thereby controlling inflorescence architecture. In most species, leaves are generated in a distichous pattern, and inflorescence branches are produced in a spiral [48]. In maize (Zea mays), boundaries within phytomers separate three components: an internode, the leaf and the axillary meristem. The SBP-box transcription factor TASSELSHEATH4 (TSH4) plays an essential role in establishing these boundaries within the inflorescence. tsh4 mutants display altered phyllotaxy, fewer lateral meristems and ectopic leaves that grow at the expense of the meristem. TSH4 is expressed in a boundary adjacent to all lateral meristems and was found to be negatively regulated by mi156RNA, which is showing a meristemspecific pattern complementary to that of TSH4. Thus TSH4 and its negative regulator miRNA156 contribute to establishment of lateral meristems and the repression of leaf initiation, thereby playing a major role in defining meristem-to-leaf boundaries [49]. In rice, LAX PANICLE1 (LAX1) function is required for the generation of axillary meristem (AM). In lax1 mutants, the proliferation of meristematic cells is initiated but fails to progress for the formation of AM. LAX1 mRNA accumulates in the boundary region between the initiating AM and the SAM. Its site of action differs from the expression pattern, suggesting that LAX1 acts non-cell — autonomously [50]. Mutations in lax2, a rice gene encoding a nuclear protein that physically interacts with LAX1, phenotypically resemble lax1 mutants. Double mutants synergistically enhance each others phenotype and display reduced branching. Thus, both proteins act at least partially in the same pathway controlling axillary meristems [51].

Conclusion and perspectives In recent years, it was shown that boundaries not only separate the meristem from the organ primordium but also function as important regulatory domains for both meristematic and lateral organ cells, which is highlighted by the expression of specific regulatory genes. Our understanding of the molecular mechanisms that control meristem-toorgan boundary formation, their maintenance and function has greatly advanced. Nevertheless, several aspects of the www.sciencedirect.com

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regulatory pathways remain elusive and further studies are required. In the future, significant progress is expected to come from the use of live-imaging approaches to study the dynamics of boundary formation in vivo, together with approaches to study protein-protein interactions and mobility. A key future challenge will be to combine live-imaging and computational modeling to improve our knowledge of cell behavior and morphogenesis during boundary development. A role for mechanical forces in shaping meristems and coordinating developmental process has been (re)discovered. The first steps towards understanding how gene expression (and predominantly the role of several families of transcription factors), phytohormone transport and signaling, and the physics of the meristem are coordinated have been made, and we expect that further interdependencies between these contributing factors will be uncovered in the near future. We also envisage a strong role for modeling approaches and computer simulations not solely for data analysis, but also to aid us in comprehending this complex and multifactorial interplay.

Acknowledgements Research in RS’ lab is supported through the DFG (IRTG 1525, EXC 1028, EVOREP and individual grants), and the BMELV

References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as:  of special interest  of special interest Milani P, Gholamirad M, Traas J, Arne´odo A, Boudaoud A, Argoul F, Hamant O: In vivo analysis of local wall stiffness at the shoot apical meristem in Arabidopsis using atomic force microscopy. Plant J 2011, 67:1116-1123. An atomic force microscopy approach was designed to investigate the elastic modulus of the outer cell wall in living shoot apical meristems (SAM). By combining modeling and experimental data, the authors designed a protocol to measure local properties of living meristematic cells. The authors identified three levels of complexity at the meristem surface, with significant heterogeneity in stiffness at regional, cellular and subcellular levels. Furthermore, they found that the outer cell wall was approximately five-times stiffer at the tip of the meristem than on its flanks, which correlates with previously proposed mechanical models and can explain regional differences in growth rates.

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The authors studied the relevance of a close connection between microtubules and the cellulose synthase complex (CESA). Loss of the linker protein CESA INTERACTIVE PROTEIN 1 (CSI1) that connects CESA with cortical microtubules cause twisting of the rapidly elongating part of the stem, and thus affecting phyllotaxy of already emerged primordia. 7. 

Uyttewaal M, Burian A, Alim K, Landrein B, Borowska-Wykre˛t D, Dedieu A, Peaucelle A, Ludynia M, Traas J, Boudaoud A et al.: Mechanical stress acts via katanin to amplify differences in growth rate between adjacent cells in Arabidopsis. Cell 2012, 149:439-451. In the SAM, continuous reorganization of cortical microtubules in response to mechanical forces is guiding differential growth. This microtubule reorganization is achieved through proteins like KATANIN that fragment microtubules. In katanin mutants, neighboring cells tend to grow more frequently in the same direction, and the characteristic dome-like shape of the shoot tip inverts.

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28. Khan M, Xu M, Murmu J, Tabb P, Liu Y, Storey K, McKim SM, Douglas CJ, Hepworth SR: Antagonistic interaction of BLADE ON-PETIOLE1 and 2 with BREVIPEDICELLUS and PENNYWISE regulates Arabidopsis inflorescence architecture. Plant Physiol 2012, 158:946-960. The article addresses internode patterning, which relies in part on the activities of two homeodomain transcription factors: BP (BREVIPEDICELLUS) and PNY (PENNYWISE). The authors examined the genetic interactions between BP and PNY, whose expression is upregulated in stems, and the lateral organ boundary genes BOP 1 (BLADE-ONPETIOLE1) and BOP2. They show that bp and pny inflorescence defects are caused by BOP1/2 gain-of-function in stems and pedicels, and inactivation of BOP1/2 can rescue these defects. They further indicate that BOP1/2 are positive regulators of KNAT6 expression and that growth restriction in BOP1/2 gain-of-function plants requires KNAT6 29. Hamant O, Pautot V: Plant development: a TALE story. CR Biol 2010, 333:371-381. 30. Lodha M, Marco CF, Timmermans MCP: The ASYMMETRIC  LEAVES complex maintains repression of KNOX homeobox genes via direct recruitment of Polycomb-repressive complex2. Genes Dev 2013, 27:596-601. Previous studies found that Polycomb repressive complexes (PRCs) are important for epigenetic spatio temporal gene regulation. In this article, the authors describe a mechanism by which PRCs (namely CURLY LEAF, CLF and SWINGER, SWN) complexes are recruited to specific targets. The AS1-AS2 (ASYMMETRIC LEAVES2) complex physically interacts with PRC2 and recruits this complex to KNOX genes (KNAT1/BP and KNAT2) to silence them in newly forming leaves. 31. Rast MI, Simon R: Arabidopsis JAGGED LATERAL ORGANS  acts with ASYMMETRIC LEAVES2 to coordinate KNOX and PIN expression in shoot and root meristems. Plant Cell 2012, 24:2917-2933. This article shows that JLO (JAGGED LATERAL ORGANS), a member of the LBD (LATERAL ORGAN BOUNDARY DOMAIN) gene family, is required for coordinated organ development in shoot and floral meristems. JLO was found to restrict KNOX gene expression. Further, JLO acts in a trimeric protein complex with AS2 and AS1 to suppress BP expression in lateral organs. In addition, AS2 (ASYMMETRIC LEAVES2) together with JLO also regulate PIN (PINFORMED) expression and auxin transport from embryogenesis onwards. 32. Bureau M, Rast MI, Illmer J, Simon R: JAGGED LATERAL ORGAN (JLO) controls auxin dependent patterning during development of the Arabidopsis embryo and root. Plant Mol Biol 2012, 74:479-491. 33. Jun JH, Ha CM, Fletcher JC: BLADE-ON-PETIOLE1 coordinates organ determinacy and axial polarity in Arabidopsis by directly  activating ASYMMETRIC LEAVES2. Plant Cell 2010, 22:62-76. This article found that the BTB/POZ domain proteins BOP1 and BOP2 can dimerize and act as transcriptional coactivators. Both are negative regulators of BP, KNAT2 and KNAT6 in leaf primordia. In double mutants of bop1 bop2 ectopic blade tissue grows on petioles, indicating that they serve a role in proximo distal patterning. In leaves, BOP1 and BOP2 upregulate AS2, LOB and LBD36 together with other adaxial factors, while repressing abaxial factors. 34. Lee D-K, Geisler M, Springer PS: LATERAL ORGAN FUSION1 and LATERAL ORGAN FUSION2 function in lateral organ separation and axillary meristem formation in Arabidopsis. Development 2009, 136:2423-2432. 35. Arnaud N, Laufs P: Plant development: brassinosteroids go out of bounds. Curr Biol 2013, 23:152-154. 36. Berckmans B, Vassileva V, Schmid SPC, Maes S, Parizot B, Naramoto S, Magyar Z, Alvim Kamei CL, Koncz C, Bo¨gre L et al.: Auxin-dependent cell cycle reactivation through transcriptional regulation of Arabidopsis E2Fa by lateral organ boundary proteins. Plant Cell 2011, 23:3671-3683. 37. Kim J, Lee HW: Direct activation of EXPANSIN14 by LBD18 in the gene regulatory network of lateral root formation in Arabidopsis. Plant Signal Behav 2013, 8:e22979.

26. Ha CM, Jun JH, Fletcher JC: Control of Arabidopsis leaf morphogenesis through regulation of the YABBY and KNOX families of transcription factors. Genetics 2010, 186:197-206.

38. Yordanov YS, Busov V: Boundary genes in regulation and evolutionof secondary growth. Plant Signal Behav2011, 6:688-690.

27. Khan M, Tabb P, Hepworth SR: BLADE-ON-PETIOLE1 and 2 regulate Arabidopsis inflorescence architecture in conjunction with homeobox genes KNAT6 and ATH1. Plant Signal Behav 2012, 7:788-792.

39. Yordanov YS, Regan S, Busov V: Members of the LATERAL ORGAN BOUNDARIES DOMAIN transcription factor family are involved in the regulation of secondary growth in Populus. Plant Cell 2010, 22:3662-3677.

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How boundaries control plant development Simon and Zˇa´dnı´kova´ 125

40. Sun X, Feng Z, Meng L, Zhu J, Geitmann A: Arabidopsis ASL11/ LBD15 is involved in shoot apical meristem development and regulates WUS expression. Planta 2013, 237:1367-1378. 41. Cho E, Zambryski PC: Organ boundary1 defines a gene expressed at the junction between the shoot apical meristem and lateral organs. Proc Natl Acad Sci U S A 2011, 108:21542159. 42. Gendron JM, Liu J-S, Fan M, Bai M-Y, Wenkel S, Springer PS,  Barton MK, Wang Z-Y: Brassinosteroids regulate organ boundary formation in the shoot apical meristem of Arabidopsis. Proc Natl Acad Sci U S A 2012, 109:21152-21157. In this study, the authors uncovered a key role for brassinosteroids (BRs) in organ boundary formation. High levels of BRs in boundaries induce organ fusions, while BR deficient mutants have larger or even ectopic organ boundaries that can result in the formation of extra (flower) organs. BRs were shown to promote expression of the transcription factor BRZ1 which regulates expression of the boundary-specific genes CUC (CUPSHAPED COTYLEDON) and LOF (LATERAL ORGAN FUSION). BRZ1 binds to the promoter of CUC genes and represses their expression, resulting also in a downregulation of the CUC-target gene LOF1. 43. Bell EM, Lin W, Husbands AY, Yu L, Jaganatha V, Jablonska B,  Mangeon A, Neff MM, Girke T, Springer PS: Arabidopsis lateral organ boundaries negatively regulates brassinosteroid accumulation to limit growth in organ boundaries. Proc Natl Acad Sci U S A 2012, 109:21146-21151. This article reveals, for the first time, that mutants for LOB (LATERAL ORGAN BOUNDARIES) display a fusion of axillary branches with adjacent cauline leaves. The altered expression of two boundary-specific genes (LATERAL ORGAN FUSION1 and ORGAN BOUNDARY1) in lob mutant revealed an expansion of the boundary domain, suggesting that LOB is required to limit the size of the boundary region. Ectopic expression of LOB results in reduced BR responses, suggesting that LOB acts as a negative regulator of BR accumulation in organ boundaries. In contrast, LOB expression itself is regulated by BRs, indicating that LOB and BRs form a feedback loop to modulate local BR accumulation in organ boundaries, thereby limiting growth in the boundary domain. Furthermore, one of the target genes identified by microarray, BAS1, encoding a BR-inactivating enzyme, is a directly transcriptionally activated by LOB. The organ fusion phenotype of lob mutants can be rescued by BAS1 expression from the LOB promoter, indicating that increased accumulation of BR contributes to the lob mutant phenotype.

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44. Liljegren SJ: Organ abscission: exit strategies require signals and moving traffic. Curr Opin Plant Biol 2012, 15:670-676. 45. Shi C-L, Stenvik G-E, Vie AK, Bones AM, Pautot V, Proveniers M,  Aalen RB, Butenko MA: Arabidopsis class I KNOTTED-like homeobox proteins act downstream in the IDA-HAE/HSL2 floral abscission signaling pathway. Plant Cell 2011, 23:25532567. Earlier studies had shown that in Arabidopsis, floral organ abscission is regulated by a receptor–ligand module involving the signaling peptide INFLORESCENCE DEFICIENT IN ABSCISSION (IDA) and two receptorlike kinases HAESA (HAE) and HAESA-LIKE2 (HSL2). Here, a suppressor screen for mutations that restore ida floral organ abscission identified BP/ KNAT1 as downstream component of the IDA signaling pathway. BP controls cell size in the abscission zone and represses expression of KNAT2 and KNAT6 genes, consistent with the hypothesis that IDA signaling positively regulates KNAT2 and KNAT6 expression. 46. Butenko MA, Shi C-L, Aalen RB: KNAT1, KNAT2 and KNAT6 act downstream in the IDA-HAE/HSL2 signaling pathway to regulate floral organ abscission. Plant Signal Behav 2012, 7:135-138. 47. Nakano T, Kimbara J, Fujisawa M, Kitagawa M, Ihashi N, Maeda H, Kasumi T, Ito Y: MACROCALYX and JOINTLESS interact in the transcriptional regulation of tomato fruit abscission zone development. Plant Physiol 2012, 158:439-450. 48. Kellogg EA, Camara PEAS, Rudall PJ, Ladd P, Malcomber ST, Whipple CJ, Doust AN: Early inflorescence development in the grasses (Poaceae). Front Plant Sci 2013, 4:250. 49. Chuck G, Whipple C, Jackson D, Hake S: The maize SBP-box transcription factor encoded by tasselsheath4 regulates bract development and the establishment of meristem boundaries. Development 2010, 137:1243-1250. 50. Oikawa T, Kyozuka J: Two-step regulation of LAX PANICLE1 protein accumulation in axillary meristem formation in rice. Plant Cell 2009, 21:1095-1108. 51. Tabuchi H, Zhang Y, Hattori S, Omae M, Shimizu-Sato S, Oikawa T, Qian Q, Nishimura M, Kitano H, Xie H et al.: LAX PANICLE2 of rice encodes a novel nuclear protein and regulates the formation of axillary meristems. Plant Cell 2011, 23:3276-3287.

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