REVIEW ARTICLE

The vascular cambium: molecular control of cellularstructure

Juan Pablo Matte Risopatron & Yuqiang Sun &

Brian Joseph Jones

Received: 2 September 2010 /Accepted: 9 September 2010# Springer-Verlag 2010

Abstract Indeterminate growth and the production of neworgans in plants require a constant supply of new cells. Themajority of these cells are produced in mitotic regionscalled meristems. For primary or tip growth of the roots andshoots, the meristems are located in the apices. These apicalmeristems have been shown to function as developmentallyregulated and environmentally responsive stem cell niches.The principle requirements to maintain a functioningmeristem in a dynamic system are a balance of cell divisionand differentiation and the regulation of the planes of celldivision and expansion. Woody plants also have secondaryindeterminate mitotic regions towards the exterior of roots,stems and branches that produce the cells for continuedgrowth in girth. The chief secondary meristem is thevascular cambium (VC). As its name implies, cellsproduced in the VC contribute to the growth in girth viathe production of secondary vascular elements. Althoughwe know a considerable amount about the cellular and

molecular basis of the apical meristems, our knowledge ofthe cellular basis and molecular functioning of the VC hasbeen rudimentary. This is now changing as a growing bodyof research shows that the primary and secondary mer-istems share some common fundamental regulatory mech-anisms. In this review, we outline recent research that isleading to a better understanding of the molecular forcesthat shape the cellular structure and function of the VC.

Keywords Vascular cambium . Secondary growth .

Stem cell .WOX . CLE . Class III HD-Zip . KANADI

Introduction

Given the role that forests play in carbon sequestration and theimportance of wood-based products to industry, it is perhapssurprising that we know so little about the cellular andmolecular basis of wood production in trees. Growth andbiomass accumulation in plants rely on the interdependentprocesses of cell proliferation, expansion and differentiation.These processes begin in stem cell niches at the heart ofindeterminate mitotic regions called meristems (Iqbal 1990;reviewed in Scheres 2007). Meristems provide the microen-vironment necessary to protect a stem cell population fromdifferentiation signals, while at the same time acting ascentral control points for growth and development, receiving,integrating, responding to and broadcasting growth-regulating signals (Grieneisen et al. 2007; Petersson et al.2009; Hohm et al. 2010; Uyttewaal et al. 2010; Zhao et al.2010). Meristems in the root and shoot apices provide cellsfor primary or tip growth. Secondary growth, or the post-germination expansion in girth of stems, branches and rootsthat occurs in woody dicotyledon and gymnosperm species,results from the activities of the secondary, lateral meristems,

Handling Editor: David Robinson

Electronic supplementary material The online version of this article(doi:10.1007/s00709-010-0211-z) contains supplementary material,which is available to authorized users.

J. P. Matte Risopatron :B. J. JonesFAFNR, University of Sydney,Sydney 2006, Australia

Y. Sun : B. J. Jones (*)Umeå Plant Science Centre,Department of Plant Physiology, Umeå Universitet,901 83 Umeå, Swedene-mail: brian.jones@sydney.edu.au

Y. SunHangzhou Normal University,College of Life and Environmental University,Hangzhou 310036, China

ProtoplasmaDOI 10.1007/s00709-010-0211-z

the vascular cambium (VC) (Larson 1994) and the corkcambium (phellogen; Fig. 1; Li et al. 2006).

The VC provides the cells for continued vasculardevelopment. Plant vasculature is comprised of two mainelements: xylem and phloem. The xylem stream transportswater and dissolved mineral nutrients taken up by the roots,while the phloem is the primary transport route forphotoassimilates, signalling molecules and some mineralions throughout the plant (Vanbel 1990; Plomion et al.2001). In most higher plants, both the primary andsecondary vasculatures are bifacial, where the xylem andphloem differentiate from either side of an intervening layerof relatively slowly dividing, pluripotent stem cells (Larson

1994). The production of VC-derived secondary vascula-ture ensures that as woody plants grow, the translocation ofwater and nutrients is adequately maintained. Of equalimportance, the secondary xylem provides the essentialstructural support for the growing shoot. Following advan-ces in our understanding of the structure and function of theapical meristems (Barton 2010; Moubayidin et al. 2010),significant progress has been made recently in unravellingthe cellular and molecular basis of VC formation andfunction (see below and Supplemental Table 1). Most of therecent insights into VC function have come from workconducted in two plant models: Arabidopsis thaliana andPoplar (Populus spp.). Although Arabidopsis is an herba-ceous annual, it forms secondary vasculature (Fig. 2) in theroot (Dolan et al. 1993; Lev-Yadun 1994; Dolan andRoberts 1995; Zhao et al. 2005), hypocotyl (Gendreau et al.1997; Busse and Evert 1999; Zhao et al. 2000, 2005, 2008;Chaffey et al. 2002; Sibout et al. 2008) and inflorescencestem (Lev-Yadun 1994; Altamura et al. 2001; Baima et al.2001; Oh et al. 2003; Lev-Yadun et al. 2004; Liu et al.

CorkPhellogenPhloem RayPrimary PhloemSecondary Phloem

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Fig. 1 Internal structure of a woody plant stem. The vascularcambium consists of a centrifugal layer of fusiform secondary phloemand a centripetal layer of secondary xylem cells surrounding a centralzone comprising phloem and xylem transit amplifying cells with acentral uniseriate layer of cambial stem cells. Most angiosperm andgynomsperm tree species also contain radial files of near isodiametricray cells that play a role in nutrient transport and storage

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Fig. 2 Arabidopsis plant with transverse sections of three tissues usedfor the analysis of cambial development (a) inflorescence stemshowing primary vascular bundles (b), hypocotyl (c) and uppersection of main root (c). Bar=200 μm

J. P. Matte Risopatron et al.

2008). As each tissue offers specific advantages, studiesfrom all three have contributed valuable insights. Whereasthe rapid life cycle of Arabidopsis provides considerableadvantages for forward and reverse molecular geneticstudies, Poplar has numerous complementary advantagesas a model for cambium function and wood production.The purpose of this review is to highlight a number ofresearch areas where recent results have made significantinroads into an understanding of the cellular structure andmolecular function of the VC stem cell niche.

Apical meristem structure

Plant meristems are complex tissues that for long-termstability require a tight, yet flexible control of the balanceof the stem cell renewal and cell differentiation processes.In most higher plants, the shoot apical meristem (SAM) isthe ultimate progenitor of all organs above ground. A well-ordered structure and integrated molecular regulation set upduring embryogenesis (Moller and Weijers 2009) allow foriterative cell division and differentiation processes that leadto genetically pre-determined and environmentally respon-sive (Maughan et al. 2006) shoot growth and development.In plants, cell fate and the mature pattern of differentiatedtissues and organs are determined primarily by positionalinformation rather than by cell lineage (Costa and Shaw2006). The lack of cell migration in plants and the orderedstructure of meristems mean, however, that cell fates can tosome extent be reliably predicted from some cell lineages.In dicots, the SAM is ordered into distinctive zones and celllayers (Fig. 3). Growth and development of the shootproceed as stem cells at the apex of the central zone dividesymmetrically, producing daughter cells that are eventually

displaced into the peripheral zones away from factorsinhibiting differentiation and towards those promotingspecific cell fates (Fig. 3; Grandjean et al. 2004).

In the root apical meristem (RAM), depending on theirposition, stem cells act as progenitors for cell layers thatdifferentiate to form the epidermal, ground or vasculartissues (Figs. 2c and 4a). In Arabidopsis, stem cells at theheart of the RAM surround the organising centre (quiescentcentre (QC)) cells in a uniseriate layer (Fig. 5). Inasymmetric RAM stem cell divisions, the daughter celladjacent to the QC retains stem cell identity. Thenonadjacent daughter, or progenitor cell, will generally actas a transit amplifying (TA) cell, dividing several moretimes before attaining the characteristics of a mature, fullydifferentiated cell type (Moubayidin et al. 2010).

Cambium structure

In the shoot apex, the primary stem vasculature is formedfrom procambial strands, narrow, densely cytoplasmic cellswhich form from the ground tissue in the early stages ofstem development (Fig. 4c, d; Xia and Steeves 1999). Theprocambial strands develop in turn into mediolaterallyorganised vascular bundles with centripetal xylem andcentrifugal phloem layers surrounding an intervening layerof relatively slowly dividing, pluripotent stem cells (Larson1994). Asymmetric, periclinal stem cell divisions produceprogenitor, TA cells that contribute to continued xylem andphloem cell production. As the stem ages, a layer ofextraxylary ground tissue cells regains mitotic capacity,forming an interfascicular cambium that integrates with thefascicular cambia to form a cylindrical VC (Esau 1943). Aconsiderable range of cell-specific markers has beendeveloped for cell types in the root and shoot apicalmeristems (Birnbaum et al. 2005; Aggarwal et al. 2010).However, despite a long progression of studies that hasclearly identified several aspects of VC structure andfunction (Larson 1994), very little is known about whatcell types comprise the VC stem cell niche and how theproduction of cells destined for secondary vascular stemcell fates is regulated. The processes of ordered celldivision and differentiation, and the determination of theplane of division and axes of elongation are criticalelements in the maintenance of structure and function inconstantly evolving meristems. Evidence based primarilyon histological examination of wood structure indicates thatthe cambial stem cell population exists as a uniseriate,cylindrical layer of infrequently dividing cells (Larson1994). In dicotyledon and gymnosperm tree species, twotypes of stem cells, fusiform and ray, exist within thiscambial layer (Fig. 1). The fusiform stem cells are theultimate progenitors of all classes of xylem and phloem

Central Zone CZOrganizing Centre OCRib Zone RZPeripheral Zone PZCell Layer L1Cell Layer L2Cell Layer L3 M

atte

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Fig. 3 Schematic representation of shoot apical meristem (SAM)layers and domains. Central zone (ZN), organising centre (OC), ribzone (RZ), peripheral zone (PZ), cell layer L1, cell layer L2 and celllayer L3. The SAM stem cell population is localised in layers 1–3 ofthe central zone. WUS is expressed in OC cells that subtend the stemcell population

Protoplasma vascular cambium review

cells. The ray stem cells initiate the ray files, the radialstrands of cuboidal cells that fulfil a nutrient transport andstorage role in developing stems (Vanbel 1990). Ray filesextend from the cortex to the interior of the tree (Fig. 1),allowing the transport of nutrients throughout the livingvascular elements. Daughter cells from ordered asymmetricpericlinal divisions of the fusiform stem cells can retainstem cell characteristics or differentiate, becoming xylem orphloem progenitors. These progenitors will, depending onboth genetic and environmental conditions, divide severalmore times before losing their mitotic capacity. The numberof cells within a radial cell file that are in the mitotic,‘cambial zone’ can vary greatly, depending on genotypeand environmental conditions (Davis and Evert 1968; Fahnet al. 1968; Waisel et al. 1970; Ghouse and Hashmi 1979;Larson 1994; Fuchs et al. 2010). The factors determiningwhich of the daughter cells proceeds down a path todifferentiation are not fully known; however, this cell fatedetermination process is both genetically and environmen-

tally determined (Deslauriers et al. 2009; Li et al. 2009). Ingeneral, more xylem is produced than phloem, as a result ofa preferential differentiation of the centripetal daughter andbecause xylem progenitors generally undergo a greaternumber of secondary periclinal divisions before losing theirmitotic capacity than do phloem TA cells (Liphschitz et al.1981; Mizukami and Fischer 2000; Zhao et al. 2005). Asthe production of xylem increases the radius of the stemand the circumference of the VC, new radial files offusiform cells are formed in order to maintain an unbrokencambial cylinder. These files originate almost exclusivelythrough symmetric anticlinal divisions of the stem cells(Schrader et al. 2004). While these aspects of cambiumstructure have been determined, many aspects remain unre-solved. For example, in the root and shoot apical meristems,the maintenance of stem cell identity depends on short rangesignalling between the stem cells and adjacent organisingcentre cells (Birnbaum et al. 2005; Williams and Fletcher2005). Whereas these cells have been clearly identified in the

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Interfascicular Cambium

Fig. 4 Transverse sections ofArabidopsis thaliana showingcellular organisation accordingat its use in the study of sec-ondary growth. a Base of hypo-cotyl of 10-day-old seedling, anadvantage of this tissue is that itis possible to obtain a clear anddetailed picture of the cellularorganization and any perturba-tions. b Hypocotyls at 8 weeksshowing phase I and II ofsecondary xylem production,with this tissue it is possible toget similar structures as a woodyplant (although without rayfiles). c Schematic representa-tion of inflorescence stemshowing structure of primaryvascular bundles. d Base ofinflorescence stem at 6 weeks,where it is possible to study thegrowth of the fascicular andinterfascicular cambia, giving aclear model for the character-ization of the initiation ofsecondary growth. Structure in‘a’ will develop into structure in‘b’ and structure in ‘c’ willdevelop into structure in ‘d’

J. P. Matte Risopatron et al.

apical meristems, cells that fulfil an organising centre role inthe vicinity of the cambial stem cells are yet to be defined.

The basics of vascular development in plants:connecting the growing leaves and stem

Primary vascular connections emerge early in developingorgans in order to provide continuity with the water,nutrients and signalling components flowing through exist-ing structures. The plant hormone auxin plays a determin-ing role in many plant developmental programs andenvironmental response adaptations (Grunewald and Friml2010; Jaillais and Chory 2010). In the SAM, auxin maximaand minima play central roles in the regulation of structureand the ordered, iterative initiation and development oforgans and tissues (Busch et al. 2010; Ha et al. 2010;Vernoux et al. 2010). The PIN-FORMED (PIN) family ofauxin efflux carrier proteins is critical for cell-to-cell auxintransport and for its polar distribution throughout the plant(Galweiler et al. 1998; Petrasek et al. 2006; Feraru andFriml 2008). At the SAM surface, PIN1-directed auxinpolarisation regulates the initiation and development of neworgans through the establishment of polar auxin gradients(Reinhardt et al. 2000; Heisler et al. 2005; Borghi et al.2007). Auxin polarisation is also vital for the establishmentof the primary vascular structures (Vernoux et al. 2000;Donner et al. 2010).

The vascularisation of developing leaves is a good,readily accessible example of the role played by auxin in

vascular development. The establishment of procambialstrands from isodiametric preprocambial cells in the groundtissue of the leaf primordium (Mattsson et al. 2003; Kangand Dengler 2004; Scarpella et al. 2006) has been proposedto be driven by a self-reinforcing canalisation of auxin flow.The canalisation theory proposes that auxin exerts apositive feedback on the rate and polarity of its owntransport (Sachs 1981, 1991; Petrasek and Friml 2009).Polarisation of PIN1 amplifies and stabilises small cellularfluxes of the hormone, reinforcing its directional movementand ultimately the establishment of laterally restrictedchannels of auxin flow (Mattsson et al. 2003; Scarpella etal. 2006). The expression domains of PIN1 and an auxinresponse element, MONOPTEROS (MP), are essentialcomponents in the process, indicating that primary vasculardevelopment is regulated by changes in both concentrationand responsiveness to hormone (Wenzel et al. 2007).During primary vascular development, PIN1 and MPexpression domains overlap and become increasing restrict-ed along with the zone of elevated auxin transport tospecific cell files (Kramer 2004; Wenzel et al. 2007). Theonset of expression of the auxin responsive ATHB8 gene inthese cell files signals the acquisition of a preprocambialcell fate (Scarpella et al. 2004; Sawchuk et al. 2007).ATHB8 encodes a member of the Class III HOMEODO-MAIN LEUCINE ZIPPER (HD-ZIP III) family of tran-scriptional regulators (Baima et al. 1995, 2001). Itspresence in preprocambial cells stabilises the cells againstperturbations in auxin transport and synchronises leafprocambial cell fate acquisition (Donner et al. 2009).

Auxin derived from young, developing aerial tissues haslong been thought to be important for the establishment andmaintenance of the VC (Digby and Wareing 1966;Sundberg et al. 2000). Considerable evidence has accumu-lated in support of a role for polar auxin transport (PAT) incambial cell division. Early physiological experiments suchas those by Digby and Wareing (1966) and others (Avery etal. 1937; Reinders-Gouwentak 1965) showed that theapplication of auxin to decapitated non-dormant stemsstimulates VC cell proliferation. Auxin transported basipe-tally by the PAT stream and presumably that produced insitu in the cambium (see Table 1 for VC expression ofauxin biosynthetic genes) contribute to a steep radial auxingradient extending across the VC (Tuominen et al. 1997).The peak of the gradient coincides roughly with thecambial mitotic zone (Tuominen et al. 1997; Uggla et al.1998; Sundberg et al. 2000), suggesting that this gradientprovides the positional information required for the devel-opment and maintenance of VC structure and function(Uggla et al. 1996). A recent study of auxin responsivetranscript accumulation in Poplar stems showed that manyknown cambial expressed genes are auxin responsive(Nilsson et al. 2008). These authors also showed that there

Quiescent Centre (QC)

Cortex/Endodermis Stem and Doughter CellsEpidermis/Lateral Root Cap Stem Cells

Columella Stem CellsPericycle Stem CellsVasculature Stem Cells

ColumellaLateral Root CapEpidermis

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Fig. 5 Diagram of a radial longitudinal section through root apicalmeristem (RAM), showing the cellular organization of the tissue. Themain cell types are the: quiescent centre (QC); epidermis, cortex,endodermis pericycle, provascular and columella cell files and theirstem cell progenitors

Protoplasma vascular cambium review

Table 1 Candidate molecular regulators of vascular cambium structure and function

Gene AGI AtH AtX Pop. Ort. NCBI-Gene ID A1 B4 A2a A3a B6 A4 B7 A5 B8

CYP83B1 SUR2 AT4G31500 4.19 3.93 POPTRDRAFT_346721

NIT1 AT3G44310 4.02 3.86 POPTRDRAFT_782522

NIT2 AT3G44300 4.02 3.86 POPTRDRAFT_782522

ATHB15/CNA/ICU4 AT1G52150 3.98 4.26 POPTRDRAFT_797557

AUX1 AT2G38120 3.93 4.14 POPTRDRAFT_653103

BP/KNAT1 AT4G08150 3.88 3.90 POPTRDRAFT_710537 0.54 0.65 0.45 0.02 −0.13 −0.42 −0.48 −0.79 −1.00PDC2 AT5G54960 3.85 3.88 POPTRDRAFT_835585 −0.69 −0.45 −0.17 0.00 0.07 0.09 0.30 0.10 0.11

AT-E1_ALPHA AT1G59900 3.83 3.92 POPTRDRAFT_657555 −0.04 −0.57 0.06 −0.18 −0.37 −0.09 −0.15 0.07 0.03

STM AT1G62360 3.83 3.91 POPTRDRAFT_811717

TSB1 TRP2 AT5G54810 3.80 3.54 POPTRDRAFT_888009

TSB2 AT4G27070 3.80 3.54 POPTRDRAFT_888009

ATR1 AT4G24520 3.77 3.79 POPTRDRAFT_818445

F23N19.18 AT1G62810 3.73 3.85 POPTRDRAFT_744008

REV/IFL1 AT5G60690 3.73 4.04 POPTRDRAFT_741921

ATR2 AT4G30210 3.67 3.60 POPTRDRAFT_825890 0.15 0.21 0.08 0.02 0.10 −0.03 −0.11 −0.35 −0.36MAB1 AT5G50850 3.67 3.68 POPTRDRAFT_829373 0.37 0.00 0.20 0.27 −0.45 0.09 −0.57 0.08 −0.33COV1 AT2G20120 3.65 3.67 POPTRDRAFT_669317 −0.16 0.20 0.04 0.12 0.15 0.14 −0.21 0.34 0.06

LCV1 AT2G20130 3.65 3.67 POPTRDRAFT_669317 −0.16 0.20 0.04 0.12 0.15 0.14 −0.21 0.34 0.06

ATPAO2 AT2G43020 3.62 3.72 POPTRDRAFT_831582 0.19 0.74 0.35 0.11 −0.29 0.04 −0.58 −0.06 −0.96CYP83A1 AT4G13770 3.61 2.82 POPTRDRAFT_346721

CYP79B2 AT4G39950 3.60 2.96 POPTRDRAFT_555694

CYP79B3 AT2G22330 3.60 2.81 POPTRDRAFT_584081

BAM1 AT5G65700 3.59 3.16 POPTRDRAFT_717990 −0.47 −0.62 0.08 0.12 0.38 0.38 0.48 0.07 −0.16SUR1 AT2G20610 3.57 3.35 POPTRDRAFT_836654 0.28 1.12 0.42 −0.03 −0.13 −0.19 −0.02 −0.41 −2.46RPL AT5G02030 3.56 3.52 POPTRDRAFT_218986 0.19 0.28 0.20 −0.02 0.17 0.11 0.23 −0.10 0.02

GN AT1G13980 3.52 3.53 POPTRDRAFT_578433

IAR4 AT1G24180 3.51 3.47 POPTRDRAFT_1090032

IGS-like AT2G04400 3.51 3.44 POPTRDRAFT_834976 −0.06 0.28 0.03 0.09 0.62 −0.05 0.12 0.03 0.04

CLE41 AT3G24770 3.51 2.47 POPTRDRAFT_569594

TGG1 AT5G26000 3.50 2.67 POPTRDRAFT_800515

TGG2 AT5G25980 3.50 2.67 POPTRDRAFT_800515

PHV AT1G30490 3.50 3.77 POPTRDRAFT_815792 −1.20 −1.48 −1.37 −0.69 0.25 0.17 0.50 0.60 1.13

ASA1 AT5G05730 3.50 3.24 POPTRDRAFT_227100

PDH-E1_ALPHA AT1G01090 3.49 3.43 POPTRDRAFT_755473 0.06 0.14 0.09 −0.08 −0.19 −0.32 −0.15 −0.24 0.12

TSA1 AT3G54640 3.48 3.29 POPTRDRAFT_818693 0.30 −0.21 −0.01 0.10 0.04 −0.16 −0.16 0.12 0.18

MAX1/CYP711A1 AT2G26170 3.48 3.72 POPTRDRAFT_352660

AAO1 AT5G20960 3.47 3.44 POPTRDRAFT_767106

ARR5 AT3G48100 3.43 3.29 POPTRDRAFT_824636

F4I10.4 AT4G33070 3.43 3.58 POPTRDRAFT_835585 −0.69 −0.45 −0.17 0.00 0.07 0.09 0.30 0.10 0.11

ATHB8 AT4G32880 3.41 3.68 POPTRDRAFT_832118 −2.35 −2.42 −1.30 −0.21 0.37 0.41 0.56 0.47 0.82

AAO4 AT1G04580 3.41 3.11 POPTRDRAFT_767106

T14P4.22 AT1G02590 3.41 3.11 POPTRDRAFT_588889

ASB1 AT1G25220 3.40 3.29 POPTRDRAFT_566399

ASB1-like A AT1G25155 3.40 3.29 POPTRDRAFT_419417

ASB1-like B AT1G24807 3.40 3.29 POPTRDRAFT_419417

ASB1-like C AT1G24909 3.40 3.29 POPTRDRAFT_419417

ASB1-like D AT1G25083 3.40 3.29 POPTRDRAFT_419417

ASB2 AT5G57890 3.40 3.29 POPTRDRAFT_566399

AHK3 AT1G27320 3.38 3.42 POPTRDRAFT_554773 0.11 −0.05 −0.08 0.02 0.03 −0.11 −0.02 −0.19 −0.42CLV1 AT1G75820 3.38 2.97 POPTRDRAFT_583546 0.96 0.68 0.30 −0.15 −2.03 −0.93 −2.42 −1.06 −4.99PDH-E1_BETA AT1G30120 3.36 3.30 POPTRDRAFT_668506 0.02 0.32 −0.03 −0.08 0.01 −0.15 −0.23 −0.32 −0.38WOL AT2G01830 3.35 3.49 POPTRDRAFT_766213

ACL5 AT5G19530 3.32 3.63 POPTRDRAFT_717791 −1.11 −4.18 −1.13 −0.69 −0.21 0.12 0.48 0.77 1.69

J. P. Matte Risopatron et al.

Table 1 (continued)

Gene AGI AtH AtX Pop. Ort. NCBI-Gene ID A1 B4 A2a A3a B6 A4 B7 A5 B8

PIN1 AT1G73590 3.32 3.56 POPTRDRAFT_728847 −1.08 −1.17 −0.34 0.06 0.31 0.49 0.62 0.11 −0.01ARR7 AT1G19050 3.32 3.42 POPTRDRAFT_824636

BP/KNAT6 AT1G23380 3.31 3.29 POPTRDRAFT_658310 0.33 0.28 −0.02 −0.03 −0.41 −0.21 −0.42 −0.09 −0.73IRX3 AT5G17420 3.30 3.68 POPTRDRAFT_717644 −0.21 −0.12 −0.22 −0.09 0.16 −0.18 0.20 0.12 0.84

TDR/PXY AT5G61480 3.29 3.56 POPTRDRAFT_1073831

BRL3 AT3G13380 3.27 3.15 POPTRDRAFT_672125

MAX3/CCD7 AT2G44990 3.27 3.57 POPTRDRAFT_781295

BRL1 AT1G55610 3.23 3.18 POPTRDRAFT_844734

KAPP AT5G19280 3.22 3.24 POPTRDRAFT_805045

WES1 AT4G27260 3.20 3.11 POPTRDRAFT_571444

PIN3 AT1G70940 3.19 3.17 POPTRDRAFT_803601

IAR1 AT1G68100 3.18 3.22 POPTRDRAFT_720768

AMI1 AT1G08980 3.18 2.74 POPTRDRAFT_662772

AT2G34590 AT2G34590 3.18 2.96 POPTRDRAFT_668506 0.02 0.32 −0.03 −0.08 0.01 −0.15 −0.23 −0.32 −0.38PHB AT2G34710 3.15 3.40 POPTRDRAFT_815792 −1.20 −1.48 −1.37 −0.69 0.25 0.17 0.50 0.60 1.13

IAR3 AT1G51760 3.14 3.08 POPTRDRAFT_711792 0.34 0.00 0.49 0.06 −0.23 −0.35 −0.77 −0.50 0.00

ILL5 AT1G51780 3.14 3.08 POPTRDRAFT_711792 0.34 0.00 0.49 0.06 −0.23 −0.35 −0.77 −0.50 0.00

MAX4/CCD8 AT4G32810 3.13 3.33 POPTRDRAFT_561749

BP/KNAT2 AT1G70510 3.12 3.02 POPTRDRAFT_658310 0.33 0.28 −0.02 −0.03 −0.41 −0.21 −0.42 −0.09 −0.73TSA-like AT4G02610 3.09 3.10 POPTRDRAFT_818693 0.30 −0.21 −0.01 0.10 0.04 −0.16 −0.16 0.12 0.18

AO2 AT3G43600 3.07 3.12 POPTRDRAFT_767106

NIT4 AT5G22300 3.05 2.66 POPTRDRAFT_782522

KAN3 AT4G17695 3.03 2.65 POPTRDRAFT_590054

MAX2 AT2G42620 3.02 2.96 POPTRDRAFT_1099491

ARR6 AT5G62920 3.01 2.89 POPTRDRAFT_824636

ELF5A-1 AT1G13950 3.01 2.88 POPTRDRAFT_717121 0.45 0.44 0.24 0.16 0.11 −0.08 −0.25 −0.21 −0.19BRL2 AT2G01950 3.00 2.96 POPTRDRAFT_657034 −0.56 −0.29 0.01 0.24 0.31 0.11 0.10 0.17 −0.13APL AT1G79430 2.97 2.00 POPTRDRAFT_765696 0.21 0.64 −0.06 0.00 −0.17 −0.13 −0.57 −0.09 −1.22PAT1/TRP1 AT5G17990 2.95 2.91 POPTRDRAFT_423398

LAX3 AT1G77690 2.90 2.57 POPTRDRAFT_643656

F17M19.7 AT1G71920 2.89 2.79 POPTRDRAFT_287731 −0.09 −0.27 0.08 −0.09 0.13 0.04 0.10 0.38 −0.05HPA1 AT5G10330 2.89 2.79 POPTRDRAFT_287731 −0.09 −0.27 0.08 −0.09 0.13 0.04 0.10 0.38 −0.05ILR1 AT3G02875 2.89 2.86 POPTRDRAFT_763045

PDC3 AT5G01330 2.89 3.00 POPTRDRAFT_835585 −0.69 −0.45 −0.17 0.00 0.07 0.09 0.30 0.10 0.11

T10O8.30 AT5G01320 2.89 3.00 POPTRDRAFT_835585 −0.69 −0.45 −0.17 0.00 0.07 0.09 0.30 0.10 0.11

F11A3.11 AT2G20340 2.87 2.93 POPTRDRAFT_816969 −0.26 −0.18 −0.21 0.09 0.21 −0.01 0.03 0.02 0.64

IAGLU AT4G15550 2.85 2.64 POPTRDRAFT_645347

AAO3 AT2G27150 2.85 2.83 POPTRDRAFT_767106

MP AT1G19850 2.83 2.77 POPTRDRAFT_652033 −0.10 −0.73 0.11 −0.04 0.35 0.04 0.39 0.28 1.06

CLV2 AT1G65380 2.82 2.88 POPTRDRAFT_596988 0.30 −0.19 0.21 −0.08 0.03 0.03 0.14 −0.24 0.77

PAI1 AT1G07780 2.82 2.73 POPTRDRAFT_564037

PAI2 AT5G05590 2.82 2.73 POPTRDRAFT_564037

PAI3 AT1G29410 2.82 2.73 POPTRDRAFT_564037

AHK2 AT5G35750 2.80 2.72 POPTRDRAFT_775135

KAN2 AT1G32240 2.78 1.86 POPTRDRAFT_590054

GH3.6/DFL1 AT5G54510 2.78 2.65 POPTRDRAFT_571444

T24P13.11 AT1G26730 2.77 2.75 POPTRDRAFT_765774

ATR1-like AT3G02280 2.77 2.79 POPTRDRAFT_714013

T22N19.10 AT5G13360 2.74 2.63 POPTRDRAFT_750995

T22N19.20 AT5G13370 2.74 2.63 POPTRDRAFT_750995

AT3G25660 AT3G25660 2.74 2.66 POPTRDRAFT_558478

BAM2 AT3G49670 2.71 2.53 POPTRDRAFT_717990 −0.47 −0.62 0.08 0.12 0.38 0.38 0.48 0.07 −0.16CLE44 AT4G13195 2.65 2.25 POPTRDRAFT_569594

Protoplasma vascular cambium review

was a limited correlation between the expression patterns ofmany classes of auxin-responsive genes and the auxinconcentration gradient across the cambium, indicating thatas with primary vascular development, auxin responses inthe cambium are regulated both by hormone levels and thecapacity to respond to the hormone. Global expressionprofiling experiments such as this one have identified agreat number of genes that are likely to play important rolesin cambium function (Table 1; Supplemental Table 1; Koand Han 2004; Schrader et al. 2004). The roles of only a

limited number of these genes have been studied in anydetail. Research in a few areas, however, is makingsignificant inroads.

Class III HD-Zip and KANADI regulation of the VC

Given the role identified for the ATHB8 gene product inprimary vascular development in Arabidopsis (Baima et al.1995, 2001), the expression of a close homolog, PttHB8, in

Table 1 (continued)

Gene AGI AtH AtX Pop. Ort. NCBI-Gene ID A1 B4 A2a A3a B6 A4 B7 A5 B8

SHR AT4G37650 2.61 2.64 POPTRDRAFT_586010

ANAC012 AT1G32770 2.60 2.94 POPTRDRAFT_569285

LAS AT1G55580 2.53 2.47 POPTRDRAFT_550683

PIN4 AT2G01420 2.53 2.26 POPTRDRAFT_803601

Dof5.6/HCA2 AT5G62940 2.49 1.90 POPTRDRAFT_570095

ARR15 AT1G74890 2.44 2.46 POPTRDRAFT_824636

SCR AT3G54220 2.41 2.14 POPTRDRAFT_589585

VND7 AT1G71930 2.36 2.60 POPTRDRAFT_592235

PLT1 AT3G20840 2.31 2.31 POPTRDRAFT_758079

ROP1 AT3G51300 2.25 2.35 POPTRDRAFT_827046 −0.11 −0.22 0.12 −0.16 −0.27 −0.03 −0.37 −0.09 −0.36KAN4/ATS AT5G42630 2.25 2.50 POPTRDRAFT_917686

PXL2 AT4G28650 2.18 2.16 POPTRDRAFT_553299

ATIPT8 AT3G19160 2.13 2.16 POPTRDRAFT_564538

CLE6 AT2G31085 2.03 1.67 POPTRDRAFT_574352

KAN1 AT5G16560 1.99 1.16 POPTRDRAFT_755990

WUS AT2G17950 1.92 1.89 POPTRDRAFT_827060

ATIPT1 AT1G68460 1.90 2.00 POPTRDRAFT_564538

ANAC101 AT5G62380 1.88 2.11 POPTRDRAFT_773505

PLT2 AT1G51190 1.83 1.89 POPTRDRAFT_758079

ZPR3 AT3G52770 1.83 2.11 POPTRDRAFT_560740

ATIPT14 AT4G24650 1.78 1.78 POPTRDRAFT_564538

CLE40 AT5G12990 1.76 1.76 POPTRDRAFT_750766

AG AT4G18960 1.73 1.63 POPTRDRAFT_758707

CKI1 AT2G47430 1.69 1.70 POPTRDRAFT_898811

CLV3 AT2G27250 1.43 1.26 POPTRDRAFT_821595

ATIPT6 AT1G25410 1.33 1.44 POPTRDRAFT_564538

AHP6 AT1G80100 1.24 1.19 POPTRDRAFT_815256 0.13 0.22 0.02 0.06 1.20 −0.08 0.90 0.05 0.87

ACR4 AT3G59420 1.03 1.01 POPTRDRAFT_562238

miR156C AT4G31877 POPTRDRAFT_646571 0.03 0.89 0.00 0.16 0.28 −0.04 0.28 −0.15 −1.35miR165A AT1G01183 POPTRDRAFT_778882 −2.25 −2.48 −1.17 −0.03 0.05 0.39 0.41 0.48 0.48

miR165A AT1G01183 POPTRDRAFT_832118 −2.35 −2.42 −1.30 −0.21 0.37 0.41 0.56 0.47 0.82

miR165B AT4G00885 POPTRDRAFT_778882 −2.25 −2.48 −1.17 −0.03 0.05 0.39 0.41 0.48 0.48

miR165B AT4G00885 POPTRDRAFT_778882 −2.25 −2.48 −1.17 −0.03 0.05 0.39 0.41 0.48 0.48

miR166F AT5G43603 POPTRDRAFT_768574 0.00 0.14 0.19 0.18 0.09 0.17 0.56 0.56 0.32

miR172A AT2G28056 POPTRDRAFT_743690 0.38 0.54 0.25 0.24 −0.05 −0.01 −0.21 −0.20 −1.13WOX4 AT1G46480 POPTRDRAFT_836454 −1.95 −2.39 −1.73 −0.16 0.23 0.45 0.43 0.78 0.54

Arabidopsis genes and their Poplar (Populus trichocarpa) homologs (Pop. Ort.; UniProt NCBI-Gene ID). Arabidopsis Gene AGI number with theexpression in log2 in Arabidopsis hypocotyls (AtH), xylem (AtX) obtained from Genivestigator (Hruz et al. 2008). Poplar expression data derivedfrom Popgenie (Sjödin et al. 2009). Poplar expression domain: A1, B4, phloem; A2, phloem cambial transition; A3, B6, A4, B7, cambial zone; A5,cambial zone xylem transition zone; and B8, xylem as showed in Supplemental Fig. 1a A2 and A3 are the most likely to contain the cambial stem cells according to Schrader et al. (2004)

J. P. Matte Risopatron et al.

the cambial zone in Poplar stems (Schrader et al. 2004) andATHB8 expression in the VC of Arabidopsis inflorescencestems (Pineau et al. 2005) suggest a similar mechanismoperates in the establishment of the secondary vasculature.Although the loss of ATHB8 function in Arabidopsismutants does not affect vascular development, potentiallybecause of functional redundancies, ectopic expression ofthe gene, driven by the constitutive 35SCaMV promoter,enhances cambial cell proliferation and xylem differentia-tion in the inflorescence stem VC (Baima et al. 2001;Ohashi-Ito et al. 2005). Whereas this suggests a role forATHB8 in the cambium, on its own, 35SCaMV-drivenexpression data is insufficient to determine the role of theendogenous gene. In addition to ATHB8, there are fourother Class III HD-Zip genes in Arabidopsis (REVOLUTA/INTERFASCICULAR FIBERLESS (REV/IFL1), PHABU-LOSA (PHB), PHAVOLUTA (PHV) and ATHB15/CORO-NA/INCURVATA4 (ATHB15); Sessa et al. 1998; Prigge etal. 2005). All have vascular expression, primarily in theprocambium, cambium and differentiating xylem (Prigge etal. 2005; Ilegems et al. 2010); all except for PHB have beenshown to be regulated by auxin, and all are capable ofinfluencing vascular development (Baima et al. 1995; Zhouet al. 2007). ATHB15 and REV/IFL1 are the only membersof the gene family for which single gene loss-of-functionmutations cause significant vascular phenotypes (Talbert etal. 1995; Zhong et al. 1997; Zhong and Ye 1999; Prigge etal. 2005). A loss of ATHB15 function results in inflores-cence stems with an altered pattern of cell lignification anduneven distribution of primary vascular bundles (Prigge etal. 2005). A more dramatic phenotype is seen in single geneloss-of-function rev/ifl1 alleles. The rev/ifl1 mutants areunable to form interfascicular xylem in inflorescence stems,and this is associated with a reduction in PAT in the stems andhypocotyls and a diminished expression of the auxin effluxcarrier genes, PIN3 and PIN4 (Zhong and Ye 2001). As therev/ifl1 mutant phenotype can be phenocopied in wild-typeplants by growing them on 1-N-naphthylphthalamic acid(NPA), an auxin transport inhibitor, it seems clear that REV/IFL1 regulation of PAT is important for the establishment andfunction of the interfascicular cambium (Zhong and Ye 2001).

Combining loss-of-function alleles of the other Class IIIHD-Zip genes with the loss of REV/IFL1 revealed over-lapping but divergent functions for gene family membersand a complex relationship in terms of their roles invascular development. The rev phb and rev phv doublemutants both enhanced the rev/ifl1 single gene mutantvascular phenotype, whereas the triple mutant combination,rev athb15 athb8, partially suppressed it (Prigge et al.2005). Carlsbecker et al. (2010) recently showed that in theroots, many multiple Class III HD-Zip mutant combinationsresult in changes in the type of xylem present in the root.Importantly, they also showed that in the presence of the

wild-type REV/IFL1 gene, mutation of the other four ClassIII HD-Zip genes in a quadruple mutant led to both areduction in differentiated xylem and to an increase invascular-associated cell proliferation (Carlsbecker et al.2010). This suggests that ATHB8, PHB, PHV and ATHB15function redundantly to regulate the allied processes ofxylem cell fate determination and cambial cell proliferation.

Interestingly, the loss of all five genes resulted in rootswith no xylem (Carlsbecker et al. 2010). Together with aloss of interfascicular cambium formation in single generev/ifl1 mutants, the data indicate that REV/IFL1 plays acentral role in procambial cell development. Transcripts forall five Class III HD-Zip genes contain a recognitionsequence for miRNA-directed cleavage via the microRNAs,miR165 and miR166 (Yao et al. 2009). Disruption of themiRNA165/6 recognition sequence causes the ectopicaccumulation of Class III HD-Zip mRNA and semi-dominant gain-of-function-related phenotypes (Baima etal. 2001; Emery et al. 2003; Zhong and Ye 2004; Ohashi-Ito et al. 2005). In contrast to the loss-of-function rev/ifl1alleles, gain-of-function REV/IFL1 mutants have increasedfascicular and interfascicular cambial activity in inflores-cence stems (Emery et al. 2003; Ohashi-Ito et al. 2005).Given the association between a loss of REV/IFL1 and aperturbation in auxin transport, it is possible that there isincreased and/or ectopic auxin accumulation in the gain-of-function plants.

The establishment and maintenance of a precise radialpattern of stem cell divisions and differentiation of daughtercells indicate the involvement of radial patterning genes.Much of the research related to the Class III HD-Zips hascentred on their roles in dorsiventral patterning (Emery etal. 2003). In leaf abaxial-adaxial polarisation, REV/IFL1,PHB and PHV mRNA are initially present throughoutincipient leaf promordia, but become restricted to theadaxial side as the primordia develop (Emery et al. 2003).The accumulation of REV/IFL1, PHB and PHV mRNA inthe abaxial domain of developing leaves in gain-of-functionmutants results in the global adaxialisation of mutantleaves. In contrast to the Class III HD-Zips, the KANADIfamily of dorsiventral patterning genes encodes transcrip-tional regulators that promote abaxial identity (Kerstetter etal. 2001; Eshed et al. 2004). Whereas the Class III HD-Zipgenes are expressed in procambium, cambium and xylemtissues (Prigge et al. 2005; Ilegems et al. 2010), theKANADI genes (KAN1, KAN2, KAN3 and KAN4) areexpressed on the phloem side of the cambium (Kerstetteret al. 2001; Emery et al. 2003; Eshed et al. 2004). The twogene families therefore have opposite, non-overlappingvascular expression (Zhong and Ye 2001; Izhaki andBowman 2007; Ilegems et al. 2010). Several lines ofevidence suggest that the disjunct in their expressionprofiles is important in establishing and/or maintaining the

Protoplasma vascular cambium review

structure and function of the VC. Similarly to many of thesingle gene loss-of-function Class III HD-Zip mutant lines,single gene loss-of-function KANADI mutants have noobservable vascular phenotype (Eshed et al. 2001; Kerstetteret al. 2001). In order to characterise the relationshipsbetween the Class III HD-Zip and KANADI genes in termsof the VC, Ilegems et al. (2010) created a number of mutantand transgenic lines with altered gene expression. A loss ofall four KANADI genes in a kan1 kan2 kan3 kan4 quadruplemutant resulted in an increased proliferation of presumptivecambial cells in the hypocotyls (Ilegems et al. 2010). As thequadruple kan1,2,3,4 mutant retained the capacity to producesecondary phloem in the hypocotyl (Ilegems et al. 2010), thedata suggest that the primary role of KAN proteins is tosuppress cambial cell division, rather than to promotephloem cell fate. In stark contrast to the quadruple mutant,the hypocotyls of transgenic plants carrying a misexpressedKAN1 gene, driven by the procambium-specific, ATHB15promoter (ATHB15::KAN1) completely lacked vasculartissues. The ectopic expression of KAN1 behind the35SCaMV or lateral organ-specific ASYMMETRIC LEAVES1(AS1) promoters has also been shown to have this effect inArabidopsis shoots (Eshed et al. 2001). The ectopicATHB15::KAN1 expression correlated with a lack ofexpression of PIN1 and of the auxin responsive marker,DR5rev::GFP (Ilegems et al. 2010), suggesting that theKANADI proteins suppress cambial cell division by alteringPAT and suppressing auxin levels on the phloem side of thecambium. In support of this hypothesis, 35SCaMV-drivenexpression of PIN1 in the ATHB15::KAN1 line graduallyrestored vascular development (Ilegems et al. 2010). Incontrast to the ATHB15::KAN1 line, ectopically expressingmiRNA165 in ATHB15 expressing cells (ATHB15::miRNA165) suppressed the levels of some Class III HD-Zip mRNA and led to an increased accumulation ofpresumptive cambial stem cells in which the ATHB15,ATHB8 and the synthetic auxin responsive DR5 promoterswere activated (Ilegems et al. 2010). Together, the dataindicate that Class III HD-Zip proteins promote xylemdifferentiation and that either directly or indirectly bothKANADI and Class III HD-Zip proteins suppress cellularindeterminancy and cell division, from opposite sides of thecambium and at least partially by suppressing auxin levels ascells emerge from the central cambial zone. This alsosuggests that the KANADI and Class III HD-Zip proteinsare involved in regulating the radial auxin concentrationgradient in the VC.

In wild-type Arabidopsis roots, PHB mRNA are nor-mally restricted to the xylem precursor and cambial cells(Carlsbecker et al. 2010). A point mutation in the PHBmiRNA165/6 recognition sequence caused the ectopicaccumulation of PHB mRNA throughout and beyond theroot central vascular cylinder (stele; Fig. 5), indicating that

similarly to leaf dorsiventral patterning, miRNA165/6cleavage is important in delimiting the domain of PHBmRNA accumulation (Carlsbecker et al. 2010). The PHBgain-of-function mutant also produced ectopic metaxylemin the roots. The authors showed that miRNA165/6 geneexpression in the roots, and hence the delimitation of PHBmRNA and the regulation of xylem differentiation, wasactivated by the transcriptional regulator, SHORT-ROOT(SHR). SHR has previously been shown to be essential forQC and stem cell maintenance in the RAM (Hauser et al.1995; Helariutta et al. 2000). Homologs of each of theelements in this mechanism, the miRNA165/6 encodinggenes, SHR and the HD-Zips, are expressed in the cambiumof Poplar stems, indicating that a similar mechanism mayoperate to delimit Class III HD-Zip expression in the VC(Schrader et al. 2004). Some evidence has emerged tosupport this hypothesis. Levels of mRNA for the Poplar(Populus tremula×Populus alba) homologs of REV(PtaHB1) and microRNA166 (Pta-miR166) have beenshown to be inversely correlated in cambial tissue (Ko etal. 2006). Similarly, in Nicotiana sylvestris, a gain-of-function mutation in the miRNA cleavage site of a PHVhomolog causes the ectopic accumulation of NsPHAVtranscripts in the phloem side of the cambium andsecondary vascular malformation in the stems (McHaleand Koning 2004). Whereas there is evidence for a role forSHR in VC formation and function: the Arabidopsis shrmutants lack an interfascicular cambium in inflorescencestems and a functional cambium in mature hypocotyls(authors' unpublished data), further research is necessary todetermine if SHR regulates the expression of themiRNA165/6 genes and Class III HD-Zip mRNA cleavagein a manner similar to that observed by Carlsbecker et al.(2010) in the roots. Control of the balance between stemcell division and the differentiation of derivatives isimportant in both the apical meristems and the cambium.Tree stems infected by the crown gall tumour-inducingbacteria, Agrobacterium tumefaciens, for example, providean easily identified example of the loss of cell divisioncontrol in the VC (Kado 1976). Together, the data presentedhere strongly suggest an interactive role for Class III HD-Zip and KANADI gene products in establishing andmaintaining the balance of cell division and differentiationin the VC. Considerable work remains if we are tounderstand the precise mechanisms by which they act.

WUS/CLV3 and the CLEs

One of the earliest discoveries in the molecular regulationof meristem structure and function was the identification ofthe importance of cell-to-cell signalling in the SAM(reviewed in Lehesranta et al. 2009). The WUSCHEL/

J. P. Matte Risopatron et al.

CLAVATA (WUS/CLV) feedback loop mechanism acts tomaintain the dynamic balance between stem cell divisionand differentiation (for review, see Wang and Fiers 2010).Basically, WUS, a homeodomain transcription factor, isexpressed in a group of cells in the central zone known asthe organising centre (OC; Fig. 3) that subtend the apicalstem cells (Tax and Durbak 2006). Cells expressing WUSproduce a signal that suppresses differentiation in theoverlying cells (van den Berg et al. 1997; Mayer et al.1998). In response, the L1–L3 layers of the stem cellsproduce CLV3, a 96 amino acid protein (Fletcher et al.1999) that is processed to an active 13 amino acid secretedglycoprotein (Ohyama et al. 2009). The CLV3 glycoproteindiffuses basipetally and interacts with the extracellularleucine-rich repeat (LRR) domains of the receptor kinase,CLAVATA1 (CLV1) and its homolog, CLAVATA2 (CLV2;Kayes and Clark 1998). CLV1 is expressed in the centralzone in the L3 layer and below and overlaps with bothCLV3 and WUS expression domains (Clark et al. 1997; Taxand Durbak 2006). CLV3-CLV1/CLV2 receptor-ligandbinding results in the repression of WUS expression (Brandet al. 2000; Schoof et al. 2000; Ogawa et al. 2008; Ikeda etal. 2009). This feedback regulatory loop provides adynamic mechanism for maintaining a stem cell populationthat reflects the requirement for cells at specific develop-mental stages or under changing environmental conditions(Geier et al. 2008). A related negative feedback loopmechanism has recently been identified in the root for thecontrol of distal stem cell identity. A WUS homolog, WUS-RELATED HOMEOBOX 5 (WOX5), is expressed in theRAM organising centre (QC) cells (Haecker et al. 2004;Gonzali et al. 2005; Stahl et al. 2009). Like WUS, WOX5acts non-cell autonomously to maintain an undifferentiatedstate in the adjacent stem cells, in this case, the columellaprecursor, distal stem cells (Sarkar et al. 2007). Similarly tothe loss of stem cell identity in the SAM of wus mutants(van den Berg et al. 1997; Mayer et al. 1998), the loss ofWOX5 expression leads to the terminal differentiation of thedistal stem cells (Sarkar et al. 2007). Conversely, loss of aCLV3 homolog, CLAVATA3/ENDOSPERM SURROUND-ING REGION 40 (CLE40), in cle40 null mutant roots leadsto an expansion of the WOX5 expression domain into thelateral stem cells surrounding the QC and to the productionof supernumerary columella stem cells (Sarkar et al. 2007).The CLE40 ligand interacts in the RAM not with a CLV1-like LRR receptor-like kinase (RLK) but with the RLK,ARABIDOPSIS CRINKLY4 (ACR4; Stahl et al. 2009).ACR4 is expressed in columella stem cells and their derivatives(De Smet et al. 2008). CLE40, therefore, operates with ACR4and WOX5 in a WOX/CLV-like feedback loop to controldistal stem cell proliferation (Chaudhuri et al. 2009). Whilethe CLV3 signal emanates from the SAM stem cells and therelated RAM CLE40 signal originates from differentiating

columella cells, the two systems are sufficiently similar toindicate that a common mechanism has been adapted for theregulation of both meristems. Importantly, in both the SAMand RAM, CLV peptide ligands suppress the WUS homeo-domain promotion of stem cell identity. Similarly important,the directed WUS signal acts non-cell autonomously,emanating from organising centre cells (OC in the SAM andQC in the RAM) adjacent to the stem cell population (Figs. 3and 5). The adoption of a WOX/CLV-like feedback loop inboth apical meristems raises the question, does a WOX/CLV-like feedback loop operate in the VC meristem?

CLEs and VC cell proliferation

In Zinnia elegans and Arabidopsis, mesophyll cells can becoaxed into undergoing de-differentiation and re-differentiation into tracheary (xylem vessel) elements(TEs) in liquid cell culture medium (Ito et al. 2006; Pesquetet al. 2010). A potent inhibitor of this xylogenesis process,Tracheary Element Differentiation Inhibitory Factor(TDIF), was isolated in Zinnia by Ito et al. (2006) andidentified as being a CLE family peptide. TDIF was shownto have the dual functions of inhibiting xylogenesis andenhancing cell division in Zinnia cell cultures (Ito et al.2006; Hirakawa et al. 2010a). In Arabidopsis, the CLE41and CLE44 genes encode proteins with C-terminal sequen-ces that share the same 12 amino acid sequence as TDIR(Ito et al. 2006; Strabala et al. 2006), and another, theCLE42 gene, produces a highly similar sequence (Ito et al.2006; Strabala et al. 2006). All three Arabidopsis geneproducts performed similarly to TDIF in the Zinnia xylo-genesis system, suggesting a conservation of function (Itoet al. 2006). In all, there is predicted to be up to 83 CLEgenes in Arabidopsis (Etchells and Turner 2010). Whitfordet al. (2008) classified Arabidopsis CLEs into two classes,A-type and B-type, based on the ability of theircorresponding synthetic peptides to inhibit growth (Oelkerset al. 2008). Both A-type and B-type CLE peptides areexpressed in the VC in Poplar stems (Table 1; Schrader etal. 2004). In Arabidopsis, CLV3 and other A-type peptidessuppress the production of stem cells, while B-typepeptides such as CLE41 have been shown to have the dualroles of inhibiting differentiation and promoting celldivision (Whitford et al. 2008). Whereas this indicates thatthe two CLE classes act antagonistically to control stem cellproliferation, the simultaneous application of synthetic B-type CLE41 and A-type, CLE6 peptides led to a dramaticincrease in ATHB8-expressing presumptive cambial stemcells in Arabidopsis hypocotyls (Whitford et al. 2008). Asimilar phenotype was observed with a number of otherCLE41/A-type CLE combinations and in transgenic plantssimultaneously over-expressing the CLE41 and CLE6

Protoplasma vascular cambium review

genes (Whitford et al. 2008). These data suggest thatrather than a simple antagonistic relationship, A-typeCLEs potentiate the action of the B-type CLEs. Thesynergistic effect was enhanced when the plants weretreated at the same time with auxin and was reduced inplants treated with the auxin transport inhibitor, NPA(Whitford et al. 2008). One possible interpretation of theseresults is that cambial stem and TA cells are specified by aconfluence of overlapping domains of A-type and B-typeCLEs and a high concentration and/or capacity to respondto auxin.

The simultaneous application or overexpression of A-type and B-type CLEs also perturbed the orientation of theplane of cell division, resulting in disorganised vascularpatterning (Whitford et al. 2008). This would be expected ifan anisotropic apoplastic gradient of CLE peptides fromsource cells to the sites of perception played a role incambial vascular patterning. The CLV1-like gene, PHLO-EM INTERCALATED WITH XYLEM (PXY), was originallyisolated in a screen designed to identify genes involved insecondary vascular cell polarity determination (Fisher andTurner 2007). Fisher and Turner (2007) found that in pxymutants, in place of the usual bifacial cambium, thesecondary xylem, phloem and cambial cells were inter-spersed in inflorescence stem vascular bundles. Hirakawa etal. (2008) subsequently isolated a pxy allele, TDR (putativeTDIF receptor), and showed that it specifically bound theTDIF ligand. In Arabidopsis hypocotyls, the TDIF-likeCLE41 and CLE44 peptides are produced in cells on thephloem side of the cambium, whereas the plasmamembrane-bound TDR/PXY receptor has been localisedto a non-overlapping domain in cambial stem cells(Hirakawa et al. 2008). This suggests that the orderedstructure of the VC requires relies on spatial cues providedby phloem produced CLE41/44/TDIF ligands binding tothe PXY/TDF receptor in cambial stem cells. Evidence tosupport this theory came from the misexpression of CLE41and CLE42 in transgenic plants (Etchells and Turner 2010).Ectopic expression of CLE41 or CLE42 behind either theubiquitous 35SCaMV or xylem-specific, IRX3 promotersseverely disrupts the orientation of cambial cell divisionsand both the mediolateral and apical-basal order of phloemand xylem in the hypocotyls (Etchells and Turner 2010). Incontrast, ectopic expression behind the phloem-specific,SUC2 promoter failed to alter the orientation of divisions orthe bifacial structure of the cambium (Etchells and Turner2010). Loss-of-function tdr/pxy mutants also exhibited adisorganised cambial cell division phenotype, indicatingthat both elements, the location of ligand production andthe position of the receptor, are important in determiningthe orientation of cambial cell divisions and the structure ofthe cambium (Etchells and Turner 2010). Somewhatsurprisingly, the authors (Etchells and Turner 2010) found

that combining 35SCaMV ectopic expression of both TDR/PXY (35SCaMV:TDR/PXY) and CLE41 (35SCaMV:CLE41)resulted in plants with relatively normal cell orientations.However, when the ubiquitous expression of 35SCaMV:TDR/PXY was combined with xylem specific ectopicexpression of CLE41 (IRX3:CLE41), vasculature structurewas once again disrupted (Etchells and Turner 2010). Theauthors hypothesised that in the double 35SCaMV line(35SCaMV:TDR/PXY 35SCaMV:CLE41), a gradient ofCLE41 would still be present from the endogenousproduction and secretion of the ligand. Together, theseresults indicate the importance of a centripetal apoplasticconcentration gradient of the CLE41 ligand for theorientation of cambial cell division and the orderedstructure of the VC.

Commensurate with a role for CLE41/44/TDIF-PXY/TDF ligand-receptor binding in specifying cambial stemcell identity, loss-of-function tdr/pxy (Hirakawa et al. 2008,2010b) or cle41 (Hirakawa et al. 2010a) mutants displayreduced cambial cell proliferation. This is in contrast tomutants for the CLV1 and ACR4 ligand receptors in theSAM and RAM. Both clv1 and acr4 mutants have anincreased proliferation of stem cells, suggesting that theynegatively regulate stem cell fate (Clark et al. 1993; DeSmet et al. 2008). As CLV3 and CLE40 are A-type CLEsthat act to suppress stem cell proliferation and CLE41 is aB-type that activates it, the contrasting loss-of-functionphenotypes may reflect fundamental differences betweenthe activities of the apical and VC systems (Whitford et al.2008). The recent work of Etchells and Turner (2010)presented additional evidence for the importance of the roleof TDR/PXY-CLE41/44/TDIR receptor ligand binding inpromoting VC cambial stem cell identity and cell division.Whereas the vascular structure in the 35SCaMV:TDR/PXY35SCaMV:CLE41 line was relatively normal, the combinedectopic expression led to a dramatic increase in the numberof cells produced in both the fascicular and interfascicularcambia (Etchells and Turner 2010). Combining 35SCaMV:TDR/PXY and 35SCaMV:CLE42 led to a similar outcome(Etchells and Turner 2010). Both fascicular and ininterfascicular cambial cell divisions were also activatedin the SUC2:CLE41, although to a lesser extent than in the35SCaMV:TDR/PXY 35SCaMV:CLE41/2 plants (Etchellsand Turner 2010). The 35SCaMV promoter drives strongand ubiquitous gene expression in plants, and yet in the35SCaMV:TDR/PXY 35SCaMV:CLE41/2 lines, the VC isalmost wild type. This suggests that it is the increasedexpression of TDR/PXY and CLE41/42 at the position of thecambial stem cells that is important for the augmentation ofcell division. This is reinforced by results in the SUC2:CLE41 line where VC structure is also preserved andcambial cell division increased. The effect is diminished inthis line, however, commensurate with only one of the two

J. P. Matte Risopatron et al.

components, CLE41, being upregulated. Clearly, highlevels of TDR/PXY and CLE41/42 expression promotecambial cell division, but the data suggests that they dothis only in combination with other factors that determinethe precise radial position of the fascicular and interfascic-ular cambia.

The data presented here indicate clearly that CLE ligand-receptor production and binding are important in VCfunction. If the mechanism is closely related to thoseoperating in the root and shoot apical meristems, however,a WOX homeobox gene family member should act togetherwith CLE41/44/TDIF and TDR/PXY to regulate thecambial stem cell population. In Arabidopsis, the closesthomologs to WUS are the RAM regulator, WOX5, and theWOX4 gene. In hypocotyls, WOX4 has been shown to bespecifically expressed in the cambial zone (Hirakawa et al.2010b). Ji et al. (2010) recently found that reducing theexpression of WOX4 in Arabidopsis through an RNAistrategy resulted in a strong inhibition of differentiatedphloem and xylem cells. They also found that it resulted indelayed and reduced the expression of markers driven bythe phloem-specific ALTERED PHLOEM1 (APL1), andcambium and protoxylem-specific ATHB8 promoters (Ji etal. 2010). This suggested that WOX4 promotes cambial cellidentity and proliferation. However, it remained unclearwhether it acts in conjunction with CLE41/44/TDIF andTDR/PXY. Hirakawa et al. (2010b) recently providedstrong evidence for an integrated CLE41/44/TDIF-TDR/PXY-WOX4 mechanism. They were able to show thatwhereas the CLE41/44/TDIF peptide activates both peri-clinal and anticlinal cambial divisions in wild-type hypo-cotyls, it is not able to do this in wox4-1 loss-of-functionmutants. As expected, given the previous evidence thatCLE41/44/TDIF acts through TDF/PXY to promote cam-bial cell proliferation, the ligand was also unable to inducecambial divisions in the hypocotyls of tdr-1 and the tdr-1wox4-1 double mutant (Hirakawa et al. 2010b). Togetherwith the previous data, these results indicate that cambialcell proliferation is regulated by a CLE41/44/TDIF-TDF/PXY-WOX4-mediated pathway. Hirakawa et al. (2010b)also showed that the exogenous application of the CLE41/44/TDIF peptide was capable of rapidly inducing WOX4expression in a TDR/PXY-dependent manner. It is possiblethat cambial cell-specific WOX4 expression is one of theessential elements that determine the capacity of cells torespond to the upregulation of TDR/PXY and CLE41/44/TDIR in the 35SCaMV:TDR/PXY 35SCaMV:CLE41/2lines. 35SCaMV overexpression of WOX4 had no effect oncambial cell proliferation on its own, suggesting thatalthough WOX4 is an important element in the regulationof cambial cell proliferation, TDR/PXY and CLE41/44/TDIR are the key limiting factors. In Poplar, closehomologs of WOX4 (PtHB3) and TDR/PXY (PtRLK3) have

been shown to be similarly and strongly expressed on thexylem side of the cambial zone in the stem (Schrader et al.2004). Further research is needed to determine if thesegenes are functionally similar to their Arabidopsis homo-logs and how they fit into the regulation of cambial activityin Poplar and other tree stems.

Both intrinsic and extrinsic signals contribute to thefunction of the cambium and the determination of the fateof cambial stem cell derivatives (Savidge 1988, 2001;Werner and Schmulling 2009). The results presented hereare not only important for what they tell us about howcambial cell proliferation is regulated and VC structure ismaintained, they are also important because they providethe basis for a more detailed understanding of the structureand function of the VC. Are there specific OC-like cells inthe VC? How are the Class III HD-Zip-KANADI andWOX-CLV systems linked in regulating VC structure andfunction? What are the principle mechanisms that integrateVC activity with whole plant environmental and develop-mental cues? It is clear from the evidence that mechanismsthat operate in the primary meristems also regulate theformation and function of the VC (Ko and Han 2004;Schrader et al. 2004; Hirakawa et al. 2008). There are cleardifferences between, for example, the WOX/CLV mecha-nisms in the apices and the VC, but there are also strongsimilarities. Continuing the strategy of referring to mech-anisms that have been well described in other tissues,organs and species will undoubtedly lead to the abovequestions being answered and eventually to a holisticunderstanding of the molecular regulation of VC structureand function.

Acknowledgements This work was supported in part by a Ph.D.scholarship for Matte J.P. by Advanced Human Capital Program ofthe National Commission for Scientific and Technological Re-search (CONICYT) Bicentennial Becas-Chile Scholarship and theSwedish Research Council FORMAS centre of excellence program,FUNCFIBER. Thanks to Göran Sandberg for the opportunity andinspiration.

Conflict of interest The authors declare that they have no conflict ofinterest.

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Protoplasma vascular cambium review