Secondary growth as a determinant of plant shape and form

Accepted Manuscript Title: Secondary Growth as a Determinant of Plant Shape and Form Authors: Laura Ragni, Thomas Greb PII: DOI: Reference:

S1084-9521(17)30283-5 http://dx.doi.org/10.1016/j.semcdb.2017.08.050 YSCDB 2359

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Seminars in Cell & Developmental Biology

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Please cite this article as: Ragni Laura, Greb Thomas.Secondary Growth as a Determinant of Plant Shape and Form.Seminars in Cell and Developmental Biology http://dx.doi.org/10.1016/j.semcdb.2017.08.050 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Secondary Growth as a Determinant of Plant Shape and Form

Laura Ragni1*, Thomas Greb2*

1 ZMBP, University of Tübingen, Auf der Morgenstelle 32, 72076 Tübingen, Germany ORCID: 0000-0002-3651-8966

2 Centre for Organismal Studies (COS), Heidelberg University, Im Neuenheimer Feld 230, 69129 Heidelberg ORCID: 0000-0002-6176-646X

*corresponding authors: [email protected] [email protected]

Abstract Plants are the primary producers of biomass on earth. As an almost stereotypic feature, higher plants generate continuously growing bodies mediated by the activity of different groups of stem cells, the meristems. Shoot and root thickening is one of the fundamental growth processes determining form and function of these bodies. Mediated by a group of cylindrical meristems located below organ surfaces, vascular and protective tissues are continuously generated in a highly plastic manner, a competence essential for the survival in an ever changing environment. Acknowledging the fundamental role of this process, which is overall designated as secondary growth, we discuss in this review our current knowledge about the 1

evolution and molecular regulation of the vascular cambium. The cambium is the meristem responsible for the formation of wood and bast, the two types of vascular tissues important for long-distance transport of water and assimilates, respectively. Although regulatory patterns are only beginning to emerge, we show that cambium activity represents a highly rewarding model for studying cell fate decisions, tissue patterning and differentiation, which has experienced an outstanding phylogenetic diversification.

Keywords: secondary growth; cambium; xylem; phloem; meristems

1. Introduction The constant and very plastic mode of organ and tissue production is one basis for the dominance of higher plants in today’s terrestrial ecosystems. In particular, secondary growth is substantial for constant plant growth and the remodeling of body structures. As an important meristem involved, the vascular cambium forms a cylindrical domain below the organ surface producing tissues for long-distance transport and mechanical support: wood (xylem) and bast (phloem). Moreover, the phellogen typically encompasses the expanding vascular tissues as a second likewise cylindrical meristem and produces cork and phelloderm, tissues protecting the growing organ against water loss, pathogen attack and wounding. Both meristem types produce tissues, e.g. xylem and phloem for the cambium, in a strictly bidirectional manner rendering these systems as interesting models for studying cell fate decisions and patterning. In addition, a broad range of quantitative and qualitative differences in tissue production and responsiveness to the environment among species suggest a strong selective pressure on individual aspects of secondary growth during evolution potentially providing tools for accessing distinct cambium features. Motivated by a substantial increase of molecular studies targeting the vascular cambium in the last few years, we discuss in this summary cambium regulation in particular and the evolutionary path of this important tissue type.

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1.1 Evolution of secondary growth Secondary growth is restricted to seed plants in extant taxa. However, the process occurred already in extinct lineages of ferns and lycopods [1] (Figure 1A). The earliest record of secondary growth was recently documented in fossils of three shrubs from the early Devonian (409–394 Mya) [2, 3]. As a characteristic feature, cambial cells of these ancient euphyllophytes divided already both anticlinally (perpendicular to the next organ surface) and periclinally (parallel to the next organ surface) and the generated wood exhibited xylem rays, all features associated with secondary growth in modern plants [2-4]. These findings indicate that the invention of secondary growth may have predated the origin of the stem-leaf-root organography. Prior to this discovery, secondary growth was reported in several lineages of late Devonian (382- 358 Mya) including lycopsids, sphenopsids, and lignophytes [5, 6]. The first evidence of secondary phloem formation comes from a late Devonian fossil of Callixylon, a member of the progymnosperms – an extinct clade of spore-bearing plants among lignophytes (Figure 1A). However, it is not possible to exclude that the lack of phloem in older fossils is due to preservation issues [7]. It appears that the phloem of Callixylon was already quite complex and consisted of fibers, sclereids, rays, axial parenchyma, and putative sieve tubes. [7]. Consistently, the secondary phloem of seed plants in the early Carboniferous (359–347 Mya) was already highly diverse in different taxa. In fact, three possible phloem topologies, based on the presence/absence and spatial distribution of fibers, were identified suggesting that early lignophytes were more advanced than what was previously thought [8]. The anatomy and the structure of the vascular cambium of earlier plant lineages remains an open question since the cambium is rarely preserved in fossils. However, the bifacial cambium of extant seed plants is thought to be homologous to the one present in progymnosperms [1]. Supporting this idea, the PHLOEM INTERCALATED WITH XYLEM/TDIF RECEPTOR (PXY/TDR) signaling network, which regulates cambial proliferation and vascular patterning (see section 3.1) is highly conserved among euphyllophytes (ferns and seed plants) [9]. Although the increase in organ girth driven by a bifacial vascular cambium may be the common ancestral state, the process of secondary growth has been clearly reshaped in different plant lineages [1]. Gymnosperms are predominantly woody 3

plants, whereas angiosperms encompass “primary” woody species, derived herbaceous species and species, which re-acquired the capacity for secondary growth during evolution [10]. Another major difference lays in the wood anatomy, as gymnosperms usually do not develop vessels, water conducting elements typical for angiosperms. Strikingly, also the xylem of the basal angiosperm Amborella trichopoda comprises only tracheids, whereas in other basal angiosperm clades, species with and without primitive vessels co-exist, suggesting that the common ancestor of angiosperms was still vessel-less [11, 12]. Thus, basal angiosperms may be potent models for studying the evolution and function of water conducting tissues. Monocots represent one of the most striking cases in which woody growth was entirely lost during angiosperm evolution. Most likely, this loss was due to the transition of primary vascular bundles to a ‘closed’ anatomy, meaning that no (pro)cambium cells are maintained between xylem and phloem tissues. However, a novel lateral meristem the so-called “monocot cambium” has evolved in some arborescent monocots of the Aspargales [13, 14]. The monocot cambium produces unique secondary bundles with the xylem surrounding the phloem. Recently, the comparison between the transcriptomes of the monocot cambium and the cambium of two tree species revealed a considerable overlap that included key cambial regulators as KNAT class I and class III homeodomain-leucine zipper (HD-Zip III) transcription factors (see section 4.2 and 4.3). This suggests that the reactivation of regulatory principles of cambium regulation may have contributed to the evolution of the monocot cambium [15]. Within eudicots, the degree of variation in woodiness is remarkable, even in closely related species and the so-called cambial variants represent extreme examples. One variant is the presence of concentric domains of successive vascular cambia documented in more than 30 different plant families (see Figure 1B). For instance, the storage root of sugar beet contains parenchyma cells between several cambial layers [16]. Often, these successive cambia are concentric. However, in the mangrove genus Avicennia they are organized in patches, so that the stems develop non-concentric xylem tissue surrounded by internal phloem tissue. In shrubs and trees, the presence of successive cambia correlates with periodically dry and/or saline environments, thus, suggesting an adaptive value in extreme environments [16-18]. 4

Other cambial oddities, present in lianas, are phloem wedges or phloem arcs [19, 20] (see Figure 1C-D). They result from differential cambial activity occurring in four equidistant parts of the cambium. In these regions, xylem production is reduced and phloem formation is enhanced, leading to the production of soft-walled tissues interspersed within thick-walled tissues, probably contributing to the mechanical flexibility of these climbing plants [11, 20].

1.2 The herbaceous Arabidopsis plants as model to study secondary growth The current understanding of the molecular network regulating secondary growth mainly comes from studies using the model plant Arabidopsis. The annual Arabidopsis plant undergoes secondary growth in the stem, in the root and in the hypocotyl. Within the stem and the root a gradient of secondary growth is found with the tips still displaying only primary growth and the region closer to the rosette showing extensive secondary growth [21, 22]. In comparison, in the hypocotyl secondary growth progression is rather uniformly resulting in comparable cambium activity in longitudinal orientation (Figure 2) [23]. The arrangement of the vasculature in the hypocotyl and the root is very similar throughout development: after embryogenesis the vasculature is organized in four equidistant poles: two protoxylem poles and two protophloem poles, whereas during secondary growth a continuous cambial ring produces xylem inward and phloem outward [22-24] (Figure 2). Moreover, in the root and hypocotyl it is possible to distinguish two phases of secondary growth based on the xylem production rate and the presence of fibers. During the first phase, which occurs before the onset of flowering, the xylem comprises only vessels and parenchyma cells, whereas during the so-called xylem expansion phase, xylem fibers appear [23, 25]. Also, it is noteworthy that in the root and hypocotyl a phellogen is formed, which replaces the epidermis as a protective tissue during the increase in girth [22-24]. In contrast to the root and the hypocotyl, stem development is characterized by the formation of vascular bundles with a fascicular cambium located at the center of those bundles, therefore representing an ‘open’ anatomy. Later, a change of cell identity results in the de-novo establishment of cambia between primary bundles, 5

(designated as ‘interfascicular’) connecting the individual bundles and establishing a secondary stem anatomy [21, 26]. Notably, in specific conditions as when the stem is subjected to weight stress, secondary rays and storied cambium occur in the Arabidopsis stem. The presence of uniseriate rays suggests that the cambium comprises two different types of initials: fusiforms and rays as in trees [27]. All together these characteristics render Arabidopsis as an attractive model to study secondary growth. In particular, many regulators found in Arabidopsis have been shown to be conserved in more woody species [28-30].

2. Hormonal control of secondary growth – a potential target of evolution A multitude of phytohormones have been shown to regulate different aspects of secondary growth. Nevertheless, their distinct effects, spatio-temporal arrangements and, in most cases, integrating downstream targets remain elusive making the speculation about the evolutionary history of these pathways challenging. Still, considering their importance it is expected that the role and connectivity of those pathways in cambium regulation are under strong selection in various contexts.

2.1 The promotion of cambium initiation and proliferation The positive effect of auxin on cambial activity has been known for more than 80 years [31]. In fact, exogenous application of auxin to decapitated stems leads to interfascicular cambium formation, whereas decapitated stems alone fail to produce it [32]. Consistently, when the auxin source is removed in explants of pine, cambial cells differentiate into parenchyma cells suggesting that auxin is also required to maintain cambial identity [33]. In trees, differences in auxin levels correlate with topological zones: auxin peaks in the cambium and decreases toward the xylem and the phloem [34-37]. Intermediate auxin concentrations are found in the xylem and phloem expansion zone, whereas low concentrations are measured in the maturation zone [34-37]. Intriguingly, the auxin response markers DR5:GUS or DR5:GFP are not active in cambium stem cells in both Arabidopsis and poplar [38, 39]. This can be explained either by a low auxin 6

sensitivity of those cells or insufficient complexity of the artificial DR5 promoters in these cases, which contain predominantly auxin responsive elements. In line with auxin to promote cambium activity, though, in transgenic trees in which the auxin response was ubiquitously reduced, the number of cambium stem cells was also reduced [40]. The main sources of cambial auxin are shoot apices as auxin biosynthesis is limited in the cambium [41]. Accordingly, PINs (auxin efflux carriers) have been shown to be polarly localized at plasma membranes along the stem. In more detail, a spatiotemporal map of PIN proteins in vascular bundles has been recently published [42]. PIN1 accumulates in the xylem parenchyma and in the cambium, while PIN2 and PIN3 are gathered mainly in xylem parenchyma. Moreover, PIN4 and PIN7 are broadly expressed in young stems and their localization becomes more polar and restricted during development [42]. Consistently, pin1 and pin3 mutants showed reduced interfascicular cambium production [43]. Corroborating the importance of auxin transport, also mutants in other auxin transporter families such as the abcb14 mutant, display defects in secondary growth [44]. Downstream of auxin signaling, the transcription factor WUSCHEL-RELATED HOMEOBOX 4 (WOX4) mediates auxindependent cambium proliferation. In more detail, WOX4 expression is enhanced by auxin, and wox4 mutants fail to initiate the interfascicular cambium even when auxin accumulation is induced [38]. Interestingly, the action of strigolactones (SLs), the branching hormone, is tightly connected to the auxin signaling pathway [45]. SL influences polar auxin transport in the stem, which becomes obvious by the fact that auxin levels and PIN accumulation at the plasma membrane are enhanced in the SL biosynthesis mutant more axillary branches 1 (max1) [46, 47]. However, there is evidence for a role of SLs in cambium regulation downstream of auxin signaling [43]. First of all, max and pin mutants display similar phenotypes, which is a reduction of secondary growth. Secondly, the auxin signaling mutant auxin resistant 1 (axr1) still responds to SL application by a cambium stimulation similar to wild type. Finally, auxin induces the expression of the SL biosynthesis genes MAX3 and MAX4 [43]. Cambial stimulation by SL treatment occurs also in Eucalyptus trees and the ramosus1 mutant in pea (impaired in SL biosynthesis) shows reduced cambial growth suggesting that the positive role of SL in the cambium is conserved among species [43]. 7

In comparison to auxin, which peaks in the cambium, cytokinins (CK) reach the highest concentration in developing phloem and the expression of CK signaling genes reflects this hormone gradient [34, 48, 49]. In both Arabidopsis and poplar, reduced CK levels result in a dramatic reduction of secondary growth [48-50]. For instance, the root of the atipt1;3;5;7 quadruple mutant, deficient for the isopentenyltransferase biosynthesis genes, lacks a cambium in the first place [48]. Consistently, overexpression of PtIPT7 in poplar results in an increased number of cambial cells and biomass overall, without altering cell size and vascular patterning [34]. Furthermore, mutants with impaired CK perception display defects in the procambium and do not form an interfascicular cambium in the Arabidopsis stem [50]. The transcription factor AINTEGUMENTA (ANT, see section 4.1) and the cell cycle regulator CYCLIN D3 (CYCD3) are co-regulated by CK: CK application results in an increase of expression, whereas in plants overexpressing a CK catabolic enzyme, both genes are downregulated [51]. ANT and CYCD3 are expressed in the cambium and single mutants show reduced secondary growth and reduced cambial cell number [51, 52]. In contrast to the ipt quadruple mutant, the ant-9 cycd3;1 double mutant still shows secondary growth, suggesting that other unknown regulators act downstream of CK signaling in cambium formation and proliferation [51]. Collectively, auxin, strigolactones and cytokinin seem to act as general promoters of cambium initiation and activity. Although especially a mutual interaction between auxin and cytokinin signaling cascades exists during primary vascular development [53], such an interaction has not yet been investigated in the context of the vascular cambium. Nevertheless, exploitation of the dual capacities of auxin to act as a selforganizing founder of provascular strands [54] and as a meristem regulator coordinating growth processes over long distances [35], may have been a prerequisite for cambium evolution. Although distinct auxin dependent features of cambium biology are only beginning to emerge (see below), a general association of both hormones has been observed in all species analyzed so far including those displaying anomalous cambial topologies [55]. It is therefore well possible that modulation of the auxin-cytokinin interaction contributed to the generation of the broad diversity in secondary growth during evolution.

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2.2 The production of cambium-derived tissues As a distinct aspect of cambium biology, auxin controls xylem formation. The level of thermospermine (Tspm) in the xylem of both Arabidopsis and poplar is promoted by the auxin-responsive transcription factor MONOPTEROS (MP) and its direct target gene ATHB8 encoding for an HD-Zip III transcription factor [56-58]. Tspm is a recently discovered signaling molecule, controlling xylem differentiation [57, 59]. Tspm is produced by the thermospermine synthase, which is encoded by the ACAULIS5 (ACL5) gene in Arabidopsis [60]. acl5 mutants display reduced height and xylem overproliferation in stems and in leaf veins. In contrast, in the hypocotyl initial xylem over-proliferation is masked in later growth stages by the lack of secondary growth and the resulting lack of xylem fibers [59, 60]. Tspm has been shown to antagonize the function of the bHLH transcription factors TARGET OF MONOPTEROS5 (TMO5), TMO5-LIKE1 (T5L1) and their binding partner LONESOME HIGHWAY (LHW), which induce periclinal division and xylem differentiation during vascular development [61]. At the molecular level, Tspm promotes the translation of SAC51-LIKE (SACL) proteins, which, in turn, bind LHW thereby preventing the formation of TMO5/LHW and TMO5L1/LHW dimers [62-65]. Tspm also affects gene transcription, since the application of Tspm and the Tspm inhibitor xylemin results in a change of expression of auxin biosynthesis and transport genes, and several other secondary growth regulators including PXY/TDR, VASCULAR RELATED NAC-DOMAIN 6 (VND6) and VND7 [56, 57, 66, 67]. Interestingly, MP also directly stimulates TMO5 expression [68] suggesting that auxin induces a whole developmental program promoting and, at the same time, balancing xylem production. Gibberellins (GA) have been shown to have two distinct functions in promoting wood formation: the stimulation of xylogenesis via the promotion of the polar transport of auxin and fiber elongation in the developing xylem [35, 69]. Consistently, trees overproducing GA display enhanced secondary growth and have longer fibers. The expression pattern of GA biosynthetic genes, the profile of GA levels and transgenic trees with altered GA signaling lead to the suggestion that a GA precursor is transported from the phloem toward the xylem [34, 69, 70]. Mobile GA has also been shown to control xylem differentiation in Arabidopsis hypocotyl, where it triggers the 9

“xylem expansion” phase upon flowering (see section 1.3) and fiber formation via the degradation of DELLA proteins [71]. In addition, the competency to respond to GA depends on the homeobox transcription factor BREVIPEDICELLUS/KNAT1 (BP), see section 4.2) [72]. Furthermore, pioneering work on xylogenic Zinnia elegans cell cultures showed that the brassinosteroids (BR) biosynthesis inhibitor unicazol represses xylem differentiation [73]. Consistent with this observation, BR biosynthesis genes and BR precursors highly accumulate during transdifferentiation of Zinnia mesophyll cells into tracheary elements [74]. Moreover, BR signaling and biosynthesis mutants in Arabidopsis show reduced xylem to phloem ratio in the stem vascular bundles [75]. Recently, it has been shown that the BR responsive factor BES1 it is also targeted by the PXY/TDR signaling network, via the BRASSINOSTEROID-INSENSITIVE 2 (BIN2) and other GLYCOGEN SYNTHASE KINASE 3 (GSK3)-like kinases, suggesting a cross-talk between the two pathways in controlling xylem differentiation (see section 3.1) [76]. Remarkably, there is a clear imbalance in the amount of our knowledge when comparing secondary xylem and phloem formation with the short end of the stick on the side of the phloem. However, emerging mechanisms important for phloem formation during primary growth [77] and a possible link to SL signaling [78] may provide novel avenues in this regard. Moreover, information on the role of xylem- or phloem-promoting hormonal control to the phylogenetic history of secondary growth is limited and also challenging to collect. Natural variation of secondary growth present in Arabidopsis [71, 79] harbors, however, a great potential.

2.3 Integrating environmental cues In line with the role of ethylene in plant adaptation to changing environments, ethylene has been shown to promote tension wood formation [80, 81]. Tension wood is produced in leaning stems as a response to mechanical and/or gravitational stress and characterized by increased cambial proliferation and altered anatomy and the chemical composition of cell walls in the xylem. Consistently, pharmacological experiments demonstrated a positive role for ethylene in cambial cell proliferation and xylem cell expansion [80]. Moreover, trees insensitive to ethylene fail to produce normal tension wood upon tilting [80, 82]. In line with these results, several 10

ETHYLENE RESPONSIVE FACTORS (ERFs) are expressed during tension wood formation [83]. Accordingly, the ethylene overproducer 1 (eto1) mutant in Arabidopsis shows enhanced cambial proliferation and erf018 erf109 double mutants display reduced secondary growth [84]. Furthermore, ethylene signaling is upregulated in more lateral growth 1 (mol1) mutants and in pxy mutants as a compensatory mechanism [84, 85]. All together, these results provide evidence for a broad role of ethylene in cambium regulation, possibly as a mediator of mechanical constraints. Jasmonate, one of the major plant defense hormones, regulates also several aspects of plant development. For instance, jasmonate mutants are often early flowering, male sterile and display less adventitious root [86]. Growth and defense against biotic stress are two sides of the same coin that drive plant life. Thus, it is not surprising that also jasmonate affects secondary growth. A positive role for jasmonate in cambium proliferation has been suggested by studies in Arabidopsis stem mutants with impaired jasmonate production or signaling [87]. Consistently, in a transcriptome analysis of cambium formation in the perennial and woody suppressor of overexpression of constans 1 (soc1) fruitfull (ful1) double mutant, jasmonate biosynthesis/signaling genes were upregulated [79, 88]. As it seems vital for a growth process providing mechanical support for the growing plant body, to respond to mechanical stimuli, it is not surprising that the integration of pathways mediating such stimuli in cambium regulation evolved over time.

3. Regulating stem cell attributes by peptides and receptors 3.1 Similar but different - regulation of apical and lateral meristems by CLV1/WUS-like signaling loops Leucine-rich repeat receptor-like kinases (LRR-RLKs) play decisive roles in regulating a broad spectrum of developmental processes and the cambium is no exception in this regard (Figure 3). In fact, the first cambium-specific regulatory pathway discovered in Arabidopsis is mediated by the LRR-RLK PXY/TDR [89, 90]. The receptor belongs to a small group of LRR-RLKs [85] from which CLAVATA1 (CLV1) received most of the attention in the context of the regulation of the shoot 11

apical meristem (SAM) serving as an early paradigm for plant stem cell regulation [91]. CLV1 restricts stem cell attributes by repressing the WUSCHEL (WUS) gene another member of the WOX gene family, which in turn promotes stem cell activity [92, 93]. The regulatory loop has been substantially elaborated since then [94] and non-cell autonomous signaling was identified as an essential feature. The WUS protein is specifically expressed in a small SAM domain called the organizing center (OC) located just below the SAM-specific stem cells [95, 96]. From there, it moves to those stem cells and promotes the expression of the CLV1 peptide ligand CLV3 [96]. CLV3 moves back to the OC where it stimulates CLV1 activity by direct binding, which ultimately leads to the attenuation of the WUS gene [92, 97]. Interestingly, the CLV-WUS concept can be partly transferred to the cambium where PXY/TDR binds the CLV3/EMBRYO SURROUNDING REGION (CLE)-related peptide CLE41 and activates WOX4 and WOX14 genes [98-104] (Figure 3). Similar to WUS in the SAM, especially WOX4 is expressed very locally in a narrow domain of the cambium from where it promotes stem cell attributes also by physically interacting with transcriptional regulators of the HAIRY MERISTEM family [38, 98, 103, 105]. Supporting a fundamental role of the pathway, the CLE41-PXY/TDR loop is well conserved among euphyllophytes [9, 29, 30]. Orthologues of PXY/TDR and CLE41 have been identified not only in gymnosperms but also in ferns and exogenous application of CLE41 repress tracheary element differentiation of procambial cells in these species similarly as in Arabidopsis [9]. Parallels in regulatory pathways between different meristems suggest that existing loops diversified during evolution and adapted to distinct niche requirements. In addition, those parallels highlight striking gaps of knowledge in the case of the cambium. CLE41, together with the redundantly acting CLE44 peptide, is expressed in the phloem from where it is believed to move to PXY/TDR- and WOX4-expressing stem cells [90, 98, 99]. However, it is currently unclear whether the action of WOX4 requires intercellular movement, like WUS and its counterpart WOX5 in the root apical meristem (RAM) [106]. Moreover, how or to which extend the terms ‘organizing center’ and ‘stem cells’ are to be applied to the cambium still must be established. Do PXY/TDR-expressing cells represent a common stem cell pool for both the xylem and the phloem? Does WOX4 expression mark a domain with reduced cell division rates in comparison to more rapidly dividing xylem and phloem mother cells, which would 12

be analogous to the OC or the quiescent center (QC) in the RAM? How is overproliferation of cambium stem cells prevented in light of the fact that the PXY-CLE41 module stimulates WOX4 activity, whereas the CLV pathway represses WUS? In the context of these questions it is interesting that MOL1, another CLV1-like LRR-RLK, is expressed distally to the PXY/TDR expression domain revealing a bipartite organization of the domain of undifferentiated cambium cells [85] (see Figure 3). In fact, although up- and downstream factors still have to be identified, MOL1 represses cambium activity and, in contrast to PXY/TDR, is able to replace CLV1 in the SAM [26, 85]. Therefore, a concerted and spatially highly organized action of several connected and independent LRR-RLK-related pathways seem to be important for balancing the bidirectional tissue production found in the cambium. This expectation is supported by at least two observations. First, prominent overproliferation of clv1 SAMs is only observed in the presence of dominant-negative alleles, whereas true clv1 null mutants show only a mild increase in proliferation [107]. Second, mol1 knock-out mutants show indeed only a very mild increase in cambium proliferation [85]. Therefore, in analogy to the SAM, other LRR-RLKs may support MOL1 in its role as a cambium repressor. LRR-RLKs outside of the CLV1 subgroup associated with cambium regulation are PXY-CORRELATED1 (PXC1) and REDUCED IN LATERAL GROWTH1 (RUL1), which were found to be co-expressed with PXY/TDR during transcriptome analyses [26, 108]. Whereas the only information on RUL1 is that it promotes cambium activity [26], PXC1 stimulates secondary cell wall formation in xylem fibers suggesting that it controls differentiation of cambiumderived cells [108]. In that sense, PXC1 fulfills the opposite function to PXY/TDR which, in addition to promoting stem cell attributes, represses xylem differentiation in a WOX4-independent manner [98, 108]. In this context, the PXY/TDR receptor interacts directly with the glycogen synthase kinase 3 BRASSINOSTEROIDINSENSITIVE2 (BIN2) at the plasma membrane and, thereby, attenuates the brassinosteroid signaling pathway [76] associated with xylem differentiation (see section 2.2) [75].

3.2 The role of non-CLV1-like receptors

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ERECTA (ER) and ER-LIKE1 (ERL1) are two additional non-CLV1-like LRR-RLKs regulating cambium activity [72, 103, 109]. In fact, not in single but in er erl1 double mutants, especially xylem production is strongly increased in the hypocotyl and xylem fiber cells develop ectopically [72]. Because mutations in the ER locus at the same time enhance the patterning defects found in pxy mutants, ER and ERL genes may particularly support PXY/TDR in its role as a repressor of xylem differentiation or specification [72, 103] (Figure 3). Reduced hypocotyl diameter in er erl1 erl2 triple mutants [103] would then mainly be due to disturbed xylem formation and not due to disturbed cambium activity overall. Such a rather specific role of ER and ERL genes in the context of secondary growth contrasts the broadness of their expression [72, 109, 110]. Interestingly, the fiber overproliferation phenotype of er erl1 depends on GA biosynthesis and BP [72]. As ligands for ER and ERL1, secreted cysteine-rich peptides called EPIDERMAL PATTERNING FACTOR LIKE (EPFL) were identified [111]. Different EPFLs show overlapping functions in different contexts [109] and those EPFLs acting in cambium regulation are still to be discovered.

4. Determining cell fate – the role of transcription factors 4.1 AP2/EREBP transcription factors – mediating the radial developmental gradient? Beyond WOX transcription factors and transcription factors directly mediating hormonal signaling, other transcriptional regulators modulate cambium activity or are involved in patterning the cambium area. ANT, a member of the APETALA2/Ethylene Response Element Binding Protein (AP2/EREBP) family of transcription factors, is locally expressed in cambium cells of Arabidopsis and poplar and in ant mutants secondary growth is substantially reduced [30, 51]. As mentioned above, the role of ANT has been associated with cytokinin signaling although ant mutants do not show an altered cytokinin responsiveness with respect to secondary growth [51]. Interestingly, PLETHORA (PLT) genes, encoding for AP2/EREBP transcription factors closely related to ANT, are mediators of RAM zonation in close interaction 14

with auxin signaling [112, 113]. Considering the fundamental role of auxin in cambium regulation, it is therefore tempting to speculate that similar principles determining zones of proliferation, elongation and differentiation are found in both longitudinal and radial plant growth processes.

4.2 KNOX transcription factors – balancing stem cell identity and cell differentiation Likewise gaining attention based on their role in regulating apical meristems, the class I KNOX homeodomain transcription factors SHOOT MERISTEMLESS (STM) and BP [114-116] were investigated in the context of cambium regulation. Because expression and function of both factors are tightly associated with maintaining the undifferentiated state of cells in the SAM [114-116], the notion that they are also expressed along vascular tissues gave rise to the idea that they fulfill similar roles in the cambium [117-120]. However, STM and BP expression is not restricted to undifferentiated cambium cells but also found in developing and mature phloem and xylem [117, 120]. In addition, STM and BP proteins move between cells [121]. Indeed, in stm and bp mutants xylem fiber differentiation is reduced, whereas the total number of cambium-derived cells is only mildly affected [120]. Although difficult to interpret due to the severely affected overall growth habit, the effect is even more pronounced in stm bp double mutants [120]. As in other contexts [122], the class I KNOX gene KNAT6 acts antagonistically to KNAT1 as defects in xylem formation present in bp mutants are suppressed in bp knat6 double mutants [123]. Interestingly, the fiber defects in bp mutants cannot be rescued by GA application [72] suggesting that, in contrast to the ER and ERL genes, STM and BP act downstream of GA signaling. The defects depend, however, on BLADE ON PETIOLE1 (BOP1) and BOP2 genes encoding BTB/POZ domain and ankyrin repeatcontaining proteins [124] and which are thought to be repressed by STM and BP in wild type [120, 123]. In contrast, the mild cambium proliferation defect does not depend on BOP genes demonstrating that promotion of fiber differentiation and of stem cell activity is genetically separable and that the latter role is similar to the role of STM and BP in the SAM [120].

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4.3 Cambium development in a polar environment – HD-ZIP III and KAN In addition to transcription factors promoting stem cell activity, there are other factors predominantly involved in regulating the development of cambium-derived cells. In this regard, factors from the HD-Zip III and KANADI (KAN) families are interesting to look at. Both families determine polarity of organs originating from the SAM, are expressed in complementary domains along the adaxial-abaxial organ axis and mutually repress each other’s activity (reviewed in [125]). Remarkably, their influence on organ polarity includes polarity of vascular tissues. For example, kan1,2,3 triple mutants develop leaves, which tend to be radial instead of flat with an adaxialized outer appearance and vascular bundles in which phloem is surrounded by xylem [126, 127]. Therefore, organ polarity and vascular polarity is connected, and especially adaxially active HD-ZIP III genes promote xylem differentiation in Arabidopsis and poplar (recently reviewed in [128]). This becomes obvious when overexpressing the HD-ZIP III gene ATHB8, which leads to ectopic xylem formation [129] and a reduction of xylem formation in plants with reduced HD-ZIP III activity [130, 131]. As indicated above, ATHB8 directly promotes transcription of the thermospermine biosynthesis genes ACL5 and BUSHY AND DWARF2 (BUD2), which in turn seem to counterbalance xylem differentiation [56]. Moreover, the HDZip III gene REVOLUTA controls interfascicular fiber formation in the stem [132]. Supporting a role of KAN genes in promoting vascular cell differentiation, cambium proliferation is increased in kan1,2,3,4 quadruple mutants and cambium-specific markers disappear in plants with ectopic KAN1 expression [130]. In addition, procambium activity is severely impaired possibly through a negative effect on polar auxin transport along (pro)cambium cells [130]. Although KAN genes do not seem to directly promote phloem formation because even multiple kan mutants still develop phloem [130, 133], restriction of stem cell attributes is, thus, an important role of KAN genes.

5. Concluding remarks As it may have become obvious by the sometimes vague description of phenotypes of cambium-associated mutants, the identification of the primary role of participating genes is still challenging in the context of the cambium. Distinct effects on 16

proliferation, cell specification or patterning are difficult to determine as specific read outs for these features are often missing and short term effects of gene disruption are hardly explored. In the absence of the possibility to look at cambium performance by live cell imaging, usually only long-term effects are used to conclude gene functions. Due to the integrated nature and expected feedback regulations, this approach is naturally prone to misinterpretations. To overcome these fundamental limitations, informative markers and innovative experimental tools like novel cell culture or tissue culture systems [134, 135] have urgently to be established as routine experimental tools. The investigation of other lateral meristems like the phellogen [136] which are located closer to the organ surface or taking advantage of natural variation in radial thickening occurring in Arabidopsis may also be solutions to get an in depth view on secondary plant growth.

Acknowledgments LR is indebted to the Baden-Württemberg Stiftung for the financial support of this research project by the Elite program for Postdocs and to the DFG [RA-2590/1-1]. TG is supported by a Heisenberg professorship from the DFG (GR 2104/5-1) and an ERC Consolidator grant (PLANTSTEMS, #647148).

17

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Figure legends Figure 1. Evolution of secondary growth in land plants A. Today, secondary growth occurs only in seeds plants. However, it was recorded in fossils of progymnosperm (extinct clade of spore-bearing plants) suggesting that lignophytes share a common ancestor regarding secondary growth. Moreover, secondary growth has been documented in extinct taxa of lycophytes and monilophytes. B-E Diversity in the arrangement of the vasculature within eudicots. B The most common arrangement of the vasculatures consists in a ring of cambium (orange) that produces phloem outward (red) and xylem inward (yellow), however other variants exist such as successive cambia (C) or phloem arcs or phloem wedges (D-E).

Figure 2. Secondary growth in Arabidopsis Sketch of an Arabidopsis plants showing the organs that undergo secondary growth. The root and the stem display a gradient of secondary growth progression, whereas secondary growth in the hypocotyl is rather uniform. On the left side, schematic organization of the vasculature arrangement: red=phloem; green=cambium; purple=interfascicular cambium; yellow=xylem; black=epidermis and blue=periderm. On the right side, cross-sections of the stem, hypocotyl and root (scale bar: 50 micrometers).

Figure 3. Many LRR-RLK receptors regulate secondary growth Close up of the cambial area of a hypocotyl cross-section. Several LRR-RLK receptors such as PXY, MOL1, ER and ERL1 regulate cambial activity. However, their expression profiles overlap only marginally. The color of the different regulators represents where they are expressed: yellow=xylem, pale orange=proximal cambium, dark orange=distal cambium, red=phloem. The ligands are displayed as circles, the transcription factors as rectangles, the receptors as sketch of their 3Dstructures.

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