Protoplasma DOI 10.

1007/s00709-010-0211-z

REVIEW ARTICLE

The vascular cambium: molecular control of cellular structure

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 new organs in plants require a constant supply of new cells. The majority of these cells are produced in mitotic regions called meristems. For primary or tip growth of the roots and shoots, the meristems are located in the apices. These apical meristems have been shown to function as developmentally regulated and environmentally responsive stem cell niches. The principle requirements to maintain a functioning meristem in a dynamic system are a balance of cell division and differentiation and the regulation of the planes of cell division and expansion. Woody plants also have secondary indeterminate mitotic regions towards the exterior of roots, stems and branches that produce the cells for continued growth in girth. The chief secondary meristem is the vascular cambium (VC). As its name implies, cells produced in the VC contribute to the growth in girth via the production of secondary vascular elements. Although we know a considerable amount about the cellular and
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. Jones FAFNR, University of Sydney, Sydney 2006, Australia Y. Sun : B. J. Jones (*) Ume Plant Science Centre, Department of Plant Physiology, Ume Universitet, 901 83 Ume, Sweden e-mail: brian.jones@sydney.edu.au Y. Sun Hangzhou Normal University, College of Life and Environmental University, Hangzhou 310036, China

molecular basis of the apical meristems, our knowledge of the cellular basis and molecular functioning of the VC has been rudimentary. This is now changing as a growing body of research shows that the primary and secondary meristems share some common fundamental regulatory mechanisms. In this review, we outline recent research that is leading to a better understanding of the molecular forces that 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 the importance of wood-based products to industry, it is perhaps surprising that we know so little about the cellular and molecular basis of wood production in trees. Growth and biomass accumulation in plants rely on the interdependent processes of cell proliferation, expansion and differentiation. These processes begin in stem cell niches at the heart of indeterminate mitotic regions called meristems (Iqbal 1990; reviewed in Scheres 2007). Meristems provide the microenvironment necessary to protect a stem cell population from differentiation signals, while at the same time acting as central control points for growth and development, receiving, integrating, responding to and broadcasting growthregulating 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 cells for primary or tip growth. Secondary growth, or the postgermination expansion in girth of stems, branches and roots that occurs in woody dicotyledon and gymnosperm species, results from the activities of the secondary, lateral meristems,

J. P. Matte Risopatron et al.

the vascular cambium (VC) (Larson 1994) and the cork cambium (phellogen; Fig. 1; Li et al. 2006). The VC provides the cells for continued vascular development. Plant vasculature is comprised of two main elements: xylem and phloem. The xylem stream transports water and dissolved mineral nutrients taken up by the roots, while the phloem is the primary transport route for photoassimilates, signalling molecules and some mineral ions throughout the plant (Vanbel 1990; Plomion et al. 2001). In most higher plants, both the primary and secondary vasculatures are bifacial, where the xylem and phloem differentiate from either side of an intervening layer of relatively slowly dividing, pluripotent stem cells (Larson

Bark Periderm

1994). The production of VC-derived secondary vasculature ensures that as woody plants grow, the translocation of water and nutrients is adequately maintained. Of equal importance, the secondary xylem provides the essential structural support for the growing shoot. Following advances in our understanding of the structure and function of the apical meristems (Barton 2010; Moubayidin et al. 2010), significant progress has been made recently in unravelling the cellular and molecular basis of VC formation and function (see below and Supplemental Table 1). Most of the recent insights into VC function have come from work conducted in two plant models: Arabidopsis thaliana and Poplar (Populus spp.). Although Arabidopsis is an herbaceous annual, it forms secondary vasculature (Fig. 2) in the root (Dolan et al. 1993; Lev-Yadun 1994; Dolan and Roberts 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 inflorescence stem (Lev-Yadun 1994; Altamura et al. 2001; Baima et al. 2001; Oh et al. 2003; Lev-Yadun et al. 2004; Liu et al.

Ray cell Fusiform cell

a.

Phloem Cambial Zone Xylem

b.
Phloem Xylem Pith Parenchyma
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Matte J.P.

Gro

c.
wth

Cork Phellogen Phloem Ray Primary Phloem Secondary Phloem

Cambial Zone Xylem Ray Secondary Xylem Primary Xylem Pith 150 mm

Fig. 1 Internal structure of a woody plant stem. The vascular cambium consists of a centrifugal layer of fusiform secondary phloem and a centripetal layer of secondary xylem cells surrounding a central zone comprising phloem and xylem transit amplifying cells with a central uniseriate layer of cambial stem cells. Most angiosperm and gynomsperm tree species also contain radial files of near isodiametric ray cells that play a role in nutrient transport and storage

Epidermis Endodermis Cortex Pericycle

Primary Phloem Cambial Zone

Primary Xylem

Fig. 2 Arabidopsis plant with transverse sections of three tissues used for the analysis of cambial development (a) inflorescence stem showing primary vascular bundles (b), hypocotyl (c) and upper section of main root (c). Bar =200 m

Protoplasma vascular cambium review

2008). As each tissue offers specific advantages, studies from all three have contributed valuable insights. Whereas the rapid life cycle of Arabidopsis provides considerable advantages for forward and reverse molecular genetic studies, Poplar has numerous complementary advantages as a model for cambium function and wood production. The purpose of this review is to highlight a number of research areas where recent results have made significant inroads into an understanding of the cellular structure and molecular function of the VC stem cell niche.

Apical meristem structure Plant meristems are complex tissues that for long-term stability require a tight, yet flexible control of the balance of the stem cell renewal and cell differentiation processes. In most higher plants, the shoot apical meristem (SAM) is the ultimate progenitor of all organs above ground. A wellordered structure and integrated molecular regulation set up during embryogenesis (Moller and Weijers 2009) allow for iterative cell division and differentiation processes that lead to genetically pre-determined and environmentally responsive (Maughan et al. 2006) shoot growth and development. In plants, cell fate and the mature pattern of differentiated tissues and organs are determined primarily by positional information rather than by cell lineage (Costa and Shaw 2006). The lack of cell migration in plants and the ordered structure of meristems mean, however, that cell fates can to some extent be reliably predicted from some cell lineages. In dicots, the SAM is ordered into distinctive zones and cell layers (Fig. 3). Growth and development of the shoot proceed as stem cells at the apex of the central zone divide symmetrically, producing daughter cells that are eventually

displaced into the peripheral zones away from factors inhibiting differentiation and towards those promoting specific cell fates (Fig. 3; Grandjean et al. 2004). In the root apical meristem (RAM), depending on their position, stem cells act as progenitors for cell layers that differentiate to form the epidermal, ground or vascular tissues (Figs. 2c and 4a). In Arabidopsis, stem cells at the heart of the RAM surround the organising centre (quiescent centre (QC)) cells in a uniseriate layer (Fig. 5). In asymmetric RAM stem cell divisions, the daughter cell adjacent to the QC retains stem cell identity. The nonadjacent daughter, or progenitor cell, will generally act as a transit amplifying (TA) cell, dividing several more times before attaining the characteristics of a mature, fully differentiated cell type (Moubayidin et al. 2010).

Cambium structure In the shoot apex, the primary stem vasculature is formed from procambial strands, narrow, densely cytoplasmic cells which form from the ground tissue in the early stages of stem development (Fig. 4c, d; Xia and Steeves 1999). The procambial strands develop in turn into mediolaterally organised vascular bundles with centripetal xylem and centrifugal phloem layers surrounding an intervening layer of relatively slowly dividing, pluripotent stem cells (Larson 1994). Asymmetric, periclinal stem cell divisions produce progenitor, TA cells that contribute to continued xylem and phloem cell production. As the stem ages, a layer of extraxylary ground tissue cells regains mitotic capacity, forming an interfascicular cambium that integrates with the fascicular cambia to form a cylindrical VC (Esau 1943). A considerable range of cell-specific markers has been developed for cell types in the root and shoot apical meristems (Birnbaum et al. 2005; Aggarwal et al. 2010). However, despite a long progression of studies that has clearly identified several aspects of VC structure and function (Larson 1994), very little is known about what cell types comprise the VC stem cell niche and how the production of cells destined for secondary vascular stem cell fates is regulated. The processes of ordered cell division and differentiation, and the determination of the plane of division and axes of elongation are critical elements in the maintenance of structure and function in constantly evolving meristems. Evidence based primarily on histological examination of wood structure indicates that the cambial stem cell population exists as a uniseriate, cylindrical layer of infrequently dividing cells (Larson 1994). In dicotyledon and gymnosperm tree species, two types of stem cells, fusiform and ray, exist within this cambial layer (Fig. 1). The fusiform stem cells are the ultimate progenitors of all classes of xylem and phloem

Central Zone CZ Organizing Centre OC Rib Zone RZ Peripheral Zone PZ Cell Layer L1 Cell Layer L2 Cell Layer L3

Fig. 3 Schematic representation of shoot apical meristem (SAM) layers and domains. Central zone (ZN), organising centre (OC), rib zone (RZ), peripheral zone (PZ), cell layer L1, cell layer L2 and cell layer L3. The SAM stem cell population is localised in layers 13 of the central zone. WUS is expressed in OC cells that subtend the stem cell population

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J. P. Matte Risopatron et al. Fig. 4 Transverse sections of Arabidopsis thaliana showing cellular organisation according at its use in the study of secondary growth. a Base of hypocotyl of 10-day-old seedling, an advantage of this tissue is that it is possible to obtain a clear and detailed picture of the cellular organization and any perturbations. b Hypocotyls at 8 weeks showing phase I and II of secondary xylem production, with this tissue it is possible to get similar structures as a woody plant (although without ray files). c Schematic representation of inflorescence stem showing structure of primary vascular bundles. d Base of inflorescence stem at 6 weeks, where it is possible to study the growth of the fascicular and interfascicular cambia, giving a clear model for the characterization of the initiation of secondary growth. Structure in a will develop into structure in b and structure in c will develop into structure in d

a.

b.

10 mm Cortex Endodermis Pericycle Phloem Procambium Metaxylem Protoxylem Phloem Cambial Zone Vessels

100 mm Xylem Phase II Xylem Phase I

c.

d.

100 mm Primary Phloem Procambium Primary Xylem Phloem Cap Phloem Epidermis Cortex Pith Parenchyma Fascicular Cambium Interfascicular Cambium Xylem Sclerenchyma Cortex / Starch Sheath

Matte J.P.

cells. The ray stem cells initiate the ray files, the radial strands of cuboidal cells that fulfil a nutrient transport and storage role in developing stems (Vanbel 1990). Ray files extend from the cortex to the interior of the tree (Fig. 1), allowing the transport of nutrients throughout the living vascular elements. Daughter cells from ordered asymmetric periclinal divisions of the fusiform stem cells can retain stem cell characteristics or differentiate, becoming xylem or phloem progenitors. These progenitors will, depending on both genetic and environmental conditions, divide several more times before losing their mitotic capacity. The number of cells within a radial cell file that are in the mitotic, cambial zone can vary greatly, depending on genotype and environmental conditions (Davis and Evert 1968; Fahn et al. 1968; Waisel et al. 1970; Ghouse and Hashmi 1979; Larson 1994; Fuchs et al. 2010). The factors determining which of the daughter cells proceeds down a path to differentiation are not fully known; however, this cell fate determination process is both genetically and environmen-

tally determined (Deslauriers et al. 2009; Li et al. 2009). In general, more xylem is produced than phloem, as a result of a preferential differentiation of the centripetal daughter and because xylem progenitors generally undergo a greater number of secondary periclinal divisions before losing their mitotic capacity than do phloem TA cells (Liphschitz et al. 1981; Mizukami and Fischer 2000; Zhao et al. 2005). As the production of xylem increases the radius of the stem and the circumference of the VC, new radial files of fusiform cells are formed in order to maintain an unbroken cambial cylinder. These files originate almost exclusively through symmetric anticlinal divisions of the stem cells (Schrader et al. 2004). While these aspects of cambium structure have been determined, many aspects remain unresolved. For example, in the root and shoot apical meristems, the maintenance of stem cell identity depends on short range signalling between the stem cells and adjacent organising centre cells (Birnbaum et al. 2005; Williams and Fletcher 2005). Whereas these cells have been clearly identified in the

Protoplasma vascular cambium review

Stele

Vasculature Stem Cells Vasculature Pericycle Stem Cells Pericycle Columella Columella Stem Cells Lateral Root Cap Epidermis Quiescent Centre (QC) Cortex Endodermis Epidermis/Lateral Root Cap Stem Cells Cortex/Endodermis Stem and Doughter Cells

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

apical meristems, cells that fulfil an organising centre role in the 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 developing organs in order to provide continuity with the water, nutrients and signalling components flowing through existing structures. The plant hormone auxin plays a determining role in many plant developmental programs and environmental response adaptations (Grunewald and Friml 2010; Jaillais and Chory 2010). In the SAM, auxin maxima and minima play central roles in the regulation of structure and the ordered, iterative initiation and development of organs and tissues (Busch et al. 2010; Ha et al. 2010; Vernoux et al. 2010). The PIN-FORMED (PIN) family of auxin efflux carrier proteins is critical for cell-to-cell auxin transport and for its polar distribution throughout the plant (Galweiler et al. 1998; Petrasek et al. 2006; Feraru and Friml 2008). At the SAM surface, PIN1-directed auxin polarisation regulates the initiation and development of new organs 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 establishment of 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 procambial strands from isodiametric preprocambial cells in the ground tissue of the leaf primordium (Mattsson et al. 2003; Kang and Dengler 2004; Scarpella et al. 2006) has been proposed to be driven by a self-reinforcing canalisation of auxin flow. The canalisation theory proposes that auxin exerts a positive feedback on the rate and polarity of its own transport (Sachs 1981, 1991; Petrasek and Friml 2009). Polarisation of PIN1 amplifies and stabilises small cellular fluxes of the hormone, reinforcing its directional movement and ultimately the establishment of laterally restricted channels of auxin flow (Mattsson et al. 2003; Scarpella et al. 2006). The expression domains of PIN1 and an auxin response element, MONOPTEROS (MP), are essential components in the process, indicating that primary vascular development is regulated by changes in both concentration and responsiveness to hormone (Wenzel et al. 2007). During primary vascular development, PIN1 and MP expression domains overlap and become increasing restricted along with the zone of elevated auxin transport to specific cell files (Kramer 2004; Wenzel et al. 2007). The onset of expression of the auxin responsive ATHB8 gene in these cell files signals the acquisition of a preprocambial cell fate (Scarpella et al. 2004; Sawchuk et al. 2007). ATHB8 encodes a member of the Class III HOMEODOMAIN LEUCINE ZIPPER (HD-ZIP III) family of transcriptional regulators (Baima et al. 1995, 2001). Its presence in preprocambial cells stabilises the cells against perturbations in auxin transport and synchronises leaf procambial cell fate acquisition (Donner et al. 2009). Auxin derived from young, developing aerial tissues has long been thought to be important for the establishment and maintenance of the VC (Digby and Wareing 1966; Sundberg et al. 2000). Considerable evidence has accumulated in support of a role for polar auxin transport (PAT) in cambial cell division. Early physiological experiments such as those by Digby and Wareing (1966) and others (Avery et al. 1937; Reinders-Gouwentak 1965) showed that the application of auxin to decapitated non-dormant stems stimulates VC cell proliferation. Auxin transported basipetally by the PAT stream and presumably that produced in situ in the cambium (see Table 1 for VC expression of auxin biosynthetic genes) contribute to a steep radial auxin gradient extending across the VC (Tuominen et al. 1997). The peak of the gradient coincides roughly with the cambial mitotic zone (Tuominen et al. 1997; Uggla et al. 1998; Sundberg et al. 2000), suggesting that this gradient provides the positional information required for the development and maintenance of VC structure and function (Uggla et al. 1996). A recent study of auxin responsive transcript accumulation in Poplar stems showed that many known cambial expressed genes are auxin responsive (Nilsson et al. 2008). These authors also showed that there

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J. P. Matte Risopatron et al. Table 1 Candidate molecular regulators of vascular cambium structure and function
Gene CYP83B1 SUR2 NIT1 NIT2 ATHB15/CNA/ICU4 AUX1 BP/KNAT1 PDC2 AT-E1_ALPHA STM TSB1 TRP2 TSB2 ATR1 F23N19.18 REV/IFL1 ATR2 MAB1 COV1 LCV1 ATPAO2 CYP83A1 CYP79B2 CYP79B3 BAM1 SUR1 RPL GN IAR4 IGS-like CLE41 TGG1 TGG2 PHV ASA1 PDH-E1_ALPHA TSA1 MAX1/CYP711A1 AAO1 ARR5 F4I10.4 ATHB8 AAO4 T14P4.22 ASB1 ASB1-like A ASB1-like B ASB1-like C ASB1-like D ASB2 AHK3 CLV1 PDH-E1_BETA WOL ACL5 AGI AT4G31500 AT3G44310 AT3G44300 AT1G52150 AT2G38120 AT4G08150 AT5G54960 AT1G59900 AT1G62360 AT5G54810 AT4G27070 AT4G24520 AT1G62810 AT5G60690 AT4G30210 AT5G50850 AT2G20120 AT2G20130 AT2G43020 AT4G13770 AT4G39950 AT2G22330 AT5G65700 AT2G20610 AT5G02030 AT1G13980 AT1G24180 AT2G04400 AT3G24770 AT5G26000 AT5G25980 AT1G30490 AT5G05730 AT1G01090 AT3G54640 AT2G26170 AT5G20960 AT3G48100 AT4G33070 AT4G32880 AT1G04580 AT1G02590 AT1G25220 AT1G25155 AT1G24807 AT1G24909 AT1G25083 AT5G57890 AT1G27320 AT1G75820 AT1G30120 AT2G01830 AT5G19530 AtH 4.19 4.02 4.02 3.98 3.93 3.88 3.85 3.83 3.83 3.80 3.80 3.77 3.73 3.73 3.67 3.67 3.65 3.65 3.62 3.61 3.60 3.60 3.59 3.57 3.56 3.52 3.51 3.51 3.51 3.50 3.50 3.50 3.50 3.49 3.48 3.48 3.47 3.43 3.43 3.41 3.41 3.41 3.40 3.40 3.40 3.40 3.40 3.40 3.38 3.38 3.36 3.35 3.32 AtX 3.93 3.86 3.86 4.26 4.14 3.90 3.88 3.92 3.91 3.54 3.54 3.79 3.85 4.04 3.60 3.68 3.67 3.67 3.72 2.82 2.96 2.81 3.16 3.35 3.52 3.53 3.47 3.44 2.47 2.67 2.67 3.77 3.24 3.43 3.29 3.72 3.44 3.29 3.58 3.68 3.11 3.11 3.29 3.29 3.29 3.29 3.29 3.29 3.42 2.97 3.30 3.49 3.63 Pop. Ort. NCBI-Gene ID POPTRDRAFT_346721 POPTRDRAFT_782522 POPTRDRAFT_782522 POPTRDRAFT_797557 POPTRDRAFT_653103 POPTRDRAFT_710537 POPTRDRAFT_835585 POPTRDRAFT_657555 POPTRDRAFT_811717 POPTRDRAFT_888009 POPTRDRAFT_888009 POPTRDRAFT_818445 POPTRDRAFT_744008 POPTRDRAFT_741921 POPTRDRAFT_825890 POPTRDRAFT_829373 POPTRDRAFT_669317 POPTRDRAFT_669317 POPTRDRAFT_831582 POPTRDRAFT_346721 POPTRDRAFT_555694 POPTRDRAFT_584081 POPTRDRAFT_717990 POPTRDRAFT_836654 POPTRDRAFT_218986 POPTRDRAFT_578433 POPTRDRAFT_1090032 POPTRDRAFT_834976 POPTRDRAFT_569594 POPTRDRAFT_800515 POPTRDRAFT_800515 POPTRDRAFT_815792 POPTRDRAFT_227100 POPTRDRAFT_755473 POPTRDRAFT_818693 POPTRDRAFT_352660 POPTRDRAFT_767106 POPTRDRAFT_824636 POPTRDRAFT_835585 POPTRDRAFT_832118 POPTRDRAFT_767106 POPTRDRAFT_588889 POPTRDRAFT_566399 POPTRDRAFT_419417 POPTRDRAFT_419417 POPTRDRAFT_419417 POPTRDRAFT_419417 POPTRDRAFT_566399 POPTRDRAFT_554773 POPTRDRAFT_583546 POPTRDRAFT_668506 POPTRDRAFT_766213 POPTRDRAFT_717791 1.11 4.18 1.13 0.69 0.21 0.12 0.48 0.77 1.69 0.11 0.96 0.02 0.05 0.68 0.32 0.08 0.30 0.03 0.02 0.15 0.08 0.03 2.03 0.01 0.11 0.93 0.15 0.02 2.42 0.23 0.19 1.06 0.32 0.42 4.99 0.38 0.69 2.35 0.45 2.42 0.17 1.30 0.00 0.21 0.07 0.37 0.09 0.41 0.30 0.56 0.10 0.47 0.11 0.82 0.06 0.30 0.14 0.21 0.09 0.01 0.08 0.10 0.19 0.04 0.32 0.16 0.15 0.16 0.24 0.12 0.12 0.18 1.20 1.48 1.37 0.69 0.25 0.17 0.50 0.60 1.13 0.06 0.28 0.03 0.09 0.62 0.05 0.12 0.03 0.04 0.47 0.28 0.19 0.62 1.12 0.28 0.08 0.42 0.20 0.12 0.03 0.02 0.38 0.13 0.17 0.38 0.19 0.11 0.48 0.02 0.23 0.07 0.41 0.10 0.16 2.46 0.02 0.15 0.37 0.16 0.16 0.19 0.21 0.00 0.20 0.20 0.74 0.08 0.20 0.04 0.04 0.35 0.02 0.27 0.12 0.12 0.11 0.10 0.45 0.15 0.15 0.29 0.03 0.09 0.14 0.14 0.04 0.11 0.57 0.21 0.21 0.58 0.35 0.08 0.34 0.34 0.06 0.36 0.33 0.06 0.06 0.96 0.54 0.69 0.04 0.65 0.45 0.57 0.45 0.17 0.06 0.02 0.00 0.18 0.13 0.07 0.37 0.42 0.09 0.09 0.48 0.30 0.15 0.79 0.10 0.07 1.00 0.11 0.03 A1 B4 A2a A3a B6 A4 B7 A5 B8

Protoplasma vascular cambium review Table 1 (continued)

Gene PIN1 ARR7 BP/KNAT6 IRX3 TDR/PXY BRL3 MAX3/CCD7 BRL1 KAPP WES1 PIN3 IAR1 AMI1 AT2G34590 PHB IAR3 ILL5 MAX4/CCD8 BP/KNAT2 TSA-like AO2 NIT4 KAN3 MAX2 ARR6 ELF5A-1 BRL2 APL PAT1/TRP1 LAX3 F17M19.7 HPA1 ILR1 PDC3 T10O8.30 F11A3.11 IAGLU AAO3 MP CLV2 PAI1 PAI2 PAI3 AHK2 KAN2 GH3.6/DFL1 T24P13.11 ATR1-like T22N19.10 T22N19.20 AT3G25660 BAM2 CLE44 AGI AT1G73590 AT1G19050 AT1G23380 AT5G17420 AT5G61480 AT3G13380 AT2G44990 AT1G55610 AT5G19280 AT4G27260 AT1G70940 AT1G68100 AT1G08980 AT2G34590 AT2G34710 AT1G51760 AT1G51780 AT4G32810 AT1G70510 AT4G02610 AT3G43600 AT5G22300 AT4G17695 AT2G42620 AT5G62920 AT1G13950 AT2G01950 AT1G79430 AT5G17990 AT1G77690 AT1G71920 AT5G10330 AT3G02875 AT5G01330 AT5G01320 AT2G20340 AT4G15550 AT2G27150 AT1G19850 AT1G65380 AT1G07780 AT5G05590 AT1G29410 AT5G35750 AT1G32240 AT5G54510 AT1G26730 AT3G02280 AT5G13360 AT5G13370 AT3G25660 AT3G49670 AT4G13195 AtH 3.32 3.32 3.31 3.30 3.29 3.27 3.27 3.23 3.22 3.20 3.19 3.18 3.18 3.18 3.15 3.14 3.14 3.13 3.12 3.09 3.07 3.05 3.03 3.02 3.01 3.01 3.00 2.97 2.95 2.90 2.89 2.89 2.89 2.89 2.89 2.87 2.85 2.85 2.83 2.82 2.82 2.82 2.82 2.80 2.78 2.78 2.77 2.77 2.74 2.74 2.74 2.71 2.65 AtX 3.56 3.42 3.29 3.68 3.56 3.15 3.57 3.18 3.24 3.11 3.17 3.22 2.74 2.96 3.40 3.08 3.08 3.33 3.02 3.10 3.12 2.66 2.65 2.96 2.89 2.88 2.96 2.00 2.91 2.57 2.79 2.79 2.86 3.00 3.00 2.93 2.64 2.83 2.77 2.88 2.73 2.73 2.73 2.72 1.86 2.65 2.75 2.79 2.63 2.63 2.66 2.53 2.25 Pop. Ort. NCBI-Gene ID POPTRDRAFT_728847 POPTRDRAFT_824636 POPTRDRAFT_658310 POPTRDRAFT_717644 POPTRDRAFT_1073831 POPTRDRAFT_672125 POPTRDRAFT_781295 POPTRDRAFT_844734 POPTRDRAFT_805045 POPTRDRAFT_571444 POPTRDRAFT_803601 POPTRDRAFT_720768 POPTRDRAFT_662772 POPTRDRAFT_668506 POPTRDRAFT_815792 POPTRDRAFT_711792 POPTRDRAFT_711792 POPTRDRAFT_561749 POPTRDRAFT_658310 POPTRDRAFT_818693 POPTRDRAFT_767106 POPTRDRAFT_782522 POPTRDRAFT_590054 POPTRDRAFT_1099491 POPTRDRAFT_824636 POPTRDRAFT_717121 POPTRDRAFT_657034 POPTRDRAFT_765696 POPTRDRAFT_423398 POPTRDRAFT_643656 POPTRDRAFT_287731 POPTRDRAFT_287731 POPTRDRAFT_763045 POPTRDRAFT_835585 POPTRDRAFT_835585 POPTRDRAFT_816969 POPTRDRAFT_645347 POPTRDRAFT_767106 POPTRDRAFT_652033 POPTRDRAFT_596988 POPTRDRAFT_564037 POPTRDRAFT_564037 POPTRDRAFT_564037 POPTRDRAFT_775135 POPTRDRAFT_590054 POPTRDRAFT_571444 POPTRDRAFT_765774 POPTRDRAFT_714013 POPTRDRAFT_750995 POPTRDRAFT_750995 POPTRDRAFT_558478 POPTRDRAFT_717990 POPTRDRAFT_569594 0.47 0.62 0.08 0.12 0.38 0.38 0.48 0.07 0.16 0.10 0.30 0.73 0.19 0.11 0.21 0.04 0.08 0.35 0.03 0.04 0.03 0.39 0.14 0.28 0.24 1.06 0.77 0.69 0.69 0.26 0.45 0.45 0.18 0.17 0.17 0.21 0.00 0.00 0.09 0.07 0.07 0.21 0.09 0.09 0.01 0.30 0.30 0.03 0.10 0.10 0.02 0.11 0.11 0.64 0.09 0.09 0.27 0.27 0.08 0.08 0.09 0.09 0.13 0.13 0.04 0.04 0.10 0.10 0.38 0.38 0.05 0.05 0.45 0.56 0.21 0.44 0.29 0.64 0.24 0.01 0.06 0.16 0.24 0.00 0.11 0.31 0.17 0.08 0.11 0.13 0.25 0.10 0.57 0.21 0.17 0.09 0.19 0.13 1.22 0.33 0.30 0.28 0.21 0.02 0.01 0.03 0.10 0.41 0.04 0.21 0.16 0.42 0.16 0.09 0.12 0.73 0.18 0.02 1.20 0.34 0.34 0.32 1.48 0.00 0.00 0.03 1.37 0.49 0.49 0.08 0.69 0.06 0.06 0.01 0.25 0.23 0.23 0.15 0.17 0.35 0.35 0.23 0.50 0.77 0.77 0.32 0.60 0.50 0.50 0.38 1.13 0.00 0.00 0.33 0.21 0.28 0.12 0.02 0.22 0.03 0.09 0.41 0.16 0.21 0.18 0.42 0.20 0.09 0.12 0.73 0.84 A1 1.08 B4 1.17 A2a 0.34 A3a 0.06 B6 0.31 A4 0.49 B7 0.62 A5 0.11 B8 0.01

J. P. Matte Risopatron et al. Table 1 (continued)

Gene SHR ANAC012 LAS PIN4 Dof5.6/HCA2 ARR15 SCR VND7 PLT1 ROP1 KAN4/ATS PXL2 ATIPT8 CLE6 KAN1 WUS ATIPT1 ANAC101 PLT2 ZPR3 ATIPT14 CLE40 AG CKI1 CLV3 ATIPT6 AHP6 ACR4 miR156C miR165A miR165A miR165B miR165B miR166F miR172A WOX4 AGI AT4G37650 AT1G32770 AT1G55580 AT2G01420 AT5G62940 AT1G74890 AT3G54220 AT1G71930 AT3G20840 AT3G51300 AT5G42630 AT4G28650 AT3G19160 AT2G31085 AT5G16560 AT2G17950 AT1G68460 AT5G62380 AT1G51190 AT3G52770 AT4G24650 AT5G12990 AT4G18960 AT2G47430 AT2G27250 AT1G25410 AT1G80100 AT3G59420 AT4G31877 AT1G01183 AT1G01183 AT4G00885 AT4G00885 AT5G43603 AT2G28056 AT1G46480 AtH 2.61 2.60 2.53 2.53 2.49 2.44 2.41 2.36 2.31 2.25 2.25 2.18 2.13 2.03 1.99 1.92 1.90 1.88 1.83 1.83 1.78 1.76 1.73 1.69 1.43 1.33 1.24 1.03 AtX 2.64 2.94 2.47 2.26 1.90 2.46 2.14 2.60 2.31 2.35 2.50 2.16 2.16 1.67 1.16 1.89 2.00 2.11 1.89 2.11 1.78 1.76 1.63 1.70 1.26 1.44 1.19 1.01 Pop. Ort. NCBI-Gene ID POPTRDRAFT_586010 POPTRDRAFT_569285 POPTRDRAFT_550683 POPTRDRAFT_803601 POPTRDRAFT_570095 POPTRDRAFT_824636 POPTRDRAFT_589585 POPTRDRAFT_592235 POPTRDRAFT_758079 POPTRDRAFT_827046 POPTRDRAFT_917686 POPTRDRAFT_553299 POPTRDRAFT_564538 POPTRDRAFT_574352 POPTRDRAFT_755990 POPTRDRAFT_827060 POPTRDRAFT_564538 POPTRDRAFT_773505 POPTRDRAFT_758079 POPTRDRAFT_560740 POPTRDRAFT_564538 POPTRDRAFT_750766 POPTRDRAFT_758707 POPTRDRAFT_898811 POPTRDRAFT_821595 POPTRDRAFT_564538 POPTRDRAFT_815256 POPTRDRAFT_562238 POPTRDRAFT_646571 POPTRDRAFT_778882 POPTRDRAFT_832118 POPTRDRAFT_778882 POPTRDRAFT_778882 POPTRDRAFT_768574 POPTRDRAFT_743690 POPTRDRAFT_836454 0.03 2.25 2.35 2.25 2.25 0.00 0.38 1.95 0.89 2.48 2.42 2.48 2.48 0.14 0.54 2.39 0.00 1.17 1.30 1.17 1.17 0.19 0.25 1.73 0.16 0.03 0.21 0.03 0.03 0.18 0.24 0.16 0.28 0.05 0.37 0.05 0.05 0.09 0.05 0.23 0.04 0.39 0.41 0.39 0.39 0.17 0.01 0.45 0.28 0.41 0.56 0.41 0.41 0.56 0.21 0.43 0.15 0.48 0.47 0.48 0.48 0.56 0.20 0.78 1.35 0.48 0.82 0.48 0.48 0.32 1.13 0.54 0.13 0.22 0.02 0.06 1.20 0.08 0.90 0.05 0.87 0.11 0.22 0.12 0.16 0.27 0.03 0.37 0.09 0.36 A1 B4 A2a A3a B6 A4 B7 A5 B8

Arabidopsis genes and their Poplar (Populus trichocarpa) homologs (Pop. Ort.; UniProt NCBI-Gene ID). Arabidopsis Gene AGI number with the expression in log2 in Arabidopsis hypocotyls (AtH), xylem (AtX) obtained from Genivestigator (Hruz et al. 2008). Poplar expression data derived from Popgenie (Sjdin 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. 1
a

A2 and A3 are the most likely to contain the cambial stem cells according to Schrader et al. (2004)

was a limited correlation between the expression patterns of many classes of auxin-responsive genes and the auxin concentration gradient across the cambium, indicating that as with primary vascular development, auxin responses in the cambium are regulated both by hormone levels and the capacity to respond to the hormone. Global expression profiling experiments such as this one have identified a great number of genes that are likely to play important roles in cambium function (Table 1; Supplemental Table 1; Ko and Han 2004; Schrader et al. 2004). The roles of only a

limited number of these genes have been studied in any detail. Research in a few areas, however, is making significant inroads.

Class III HD-Zip and KANADI regulation of the VC Given the role identified for the ATHB8 gene product in primary vascular development in Arabidopsis (Baima et al. 1995, 2001), the expression of a close homolog, PttHB8, in

Protoplasma vascular cambium review

the cambial zone in Poplar stems (Schrader et al. 2004) and ATHB8 expression in the VC of Arabidopsis inflorescence stems (Pineau et al. 2005) suggest a similar mechanism operates in the establishment of the secondary vasculature. Although the loss of ATHB8 function in Arabidopsis mutants does not affect vascular development, potentially because of functional redundancies, ectopic expression of the gene, driven by the constitutive 35SCaMV promoter, enhances cambial cell proliferation and xylem differentiation in the inflorescence stem VC (Baima et al. 2001; Ohashi-Ito et al. 2005). Whereas this suggests a role for ATHB8 in the cambium, on its own, 35SCaMV-driven expression data is insufficient to determine the role of the endogenous gene. In addition to ATHB8, there are four other Class III HD-Zip genes in Arabidopsis (REVOLUTA/ INTERFASCICULAR FIBERLESS (REV/IFL1), PHABULOSA (PHB), PHAVOLUTA (PHV) and ATHB15/CORONA/INCURVATA4 (ATHB15); Sessa et al. 1998; Prigge et al. 2005). All have vascular expression, primarily in the procambium, cambium and differentiating xylem (Prigge et al. 2005; Ilegems et al. 2010); all except for PHB have been shown to be regulated by auxin, and all are capable of influencing vascular development (Baima et al. 1995; Zhou et al. 2007). ATHB15 and REV/IFL1 are the only members of the gene family for which single gene loss-of-function mutations cause significant vascular phenotypes (Talbert et al. 1995; Zhong et al. 1997; Zhong and Ye 1999; Prigge et al. 2005). A loss of ATHB15 function results in inflorescence stems with an altered pattern of cell lignification and uneven distribution of primary vascular bundles (Prigge et al. 2005). A more dramatic phenotype is seen in single gene loss-of-function rev/ifl1 alleles. The rev/ifl1 mutants are unable to form interfascicular xylem in inflorescence stems, and this is associated with a reduction in PAT in the stems and hypocotyls and a diminished expression of the auxin efflux carrier genes, PIN3 and PIN4 (Zhong and Ye 2001). As the rev/ifl1 mutant phenotype can be phenocopied in wild-type plants 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 and function of the interfascicular cambium (Zhong and Ye 2001). Combining loss-of-function alleles of the other Class III HD-Zip genes with the loss of REV/IFL1 revealed overlapping but divergent functions for gene family members and a complex relationship in terms of their roles in vascular development. The rev phb and rev phv double mutants both enhanced the rev/ifl1 single gene mutant vascular phenotype, whereas the triple mutant combination, rev athb15 athb8, partially suppressed it (Prigge et al. 2005). Carlsbecker et al. (2010) recently showed that in the roots, many multiple Class III HD-Zip mutant combinations result 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 Class III HD-Zip genes in a quadruple mutant led to both a reduction in differentiated xylem and to an increase in vascular-associated cell proliferation (Carlsbecker et al. 2010). This suggests that ATHB8, PHB, PHV and ATHB15 function redundantly to regulate the allied processes of xylem cell fate determination and cambial cell proliferation. Interestingly, the loss of all five genes resulted in roots with no xylem (Carlsbecker et al. 2010). Together with a loss of interfascicular cambium formation in single gene rev/ifl1 mutants, the data indicate that REV/IFL1 plays a central role in procambial cell development. Transcripts for all five Class III HD-Zip genes contain a recognition sequence for miRNA-directed cleavage via the microRNAs, miR165 and miR166 (Yao et al. 2009). Disruption of the miRNA165/6 recognition sequence causes the ectopic accumulation of Class III HD-Zip mRNA and semidominant gain-of-function-related phenotypes (Baima et al. 2001; Emery et al. 2003; Zhong and Ye 2004; OhashiIto et al. 2005). In contrast to the loss-of-function rev/ifl1 alleles, gain-of-function REV/IFL1 mutants have increased fascicular and interfascicular cambial activity in inflorescence stems (Emery et al. 2003; Ohashi-Ito et al. 2005). Given the association between a loss of REV/IFL1 and a perturbation in auxin transport, it is possible that there is increased and/or ectopic auxin accumulation in the gain-offunction plants. The establishment and maintenance of a precise radial pattern of stem cell divisions and differentiation of daughter cells indicate the involvement of radial patterning genes. Much of the research related to the Class III HD-Zips has centred on their roles in dorsiventral patterning (Emery et al. 2003). In leaf abaxial-adaxial polarisation, REV/IFL1, PHB and PHV mRNA are initially present throughout incipient leaf promordia, but become restricted to the adaxial side as the primordia develop (Emery et al. 2003). The accumulation of REV/IFL1, PHB and PHV mRNA in the abaxial domain of developing leaves in gain-of-function mutants results in the global adaxialisation of mutant leaves. In contrast to the Class III HD-Zips, the KANADI family of dorsiventral patterning genes encodes transcriptional regulators that promote abaxial identity (Kerstetter et al. 2001; Eshed et al. 2004). Whereas the Class III HD-Zip genes are expressed in procambium, cambium and xylem tissues (Prigge et al. 2005; Ilegems et al. 2010), the KANADI genes (KAN1, KAN2, KAN3 and KAN4) are expressed on the phloem side of the cambium (Kerstetter et al. 2001; Emery et al. 2003; Eshed et al. 2004). The two gene families therefore have opposite, non-overlapping vascular expression (Zhong and Ye 2001; Izhaki and Bowman 2007; Ilegems et al. 2010). Several lines of evidence suggest that the disjunct in their expression profiles is important in establishing and/or maintaining the

J. P. Matte Risopatron et al.

structure and function of the VC. Similarly to many of the single gene loss-of-function Class III HD-Zip mutant lines, single gene loss-of-function KANADI mutants have no observable vascular phenotype (Eshed et al. 2001; Kerstetter et al. 2001). In order to characterise the relationships between the Class III HD-Zip and KANADI genes in terms of the VC, Ilegems et al. (2010) created a number of mutant and transgenic lines with altered gene expression. A loss of all four KANADI genes in a kan1 kan2 kan3 kan4 quadruple mutant resulted in an increased proliferation of presumptive cambial cells in the hypocotyls (Ilegems et al. 2010). As the quadruple kan1,2,3,4 mutant retained the capacity to produce secondary phloem in the hypocotyl (Ilegems et al. 2010), the data suggest that the primary role of KAN proteins is to suppress cambial cell division, rather than to promote phloem cell fate. In stark contrast to the quadruple mutant, the hypocotyls of transgenic plants carrying a misexpressed KAN1 gene, driven by the procambium-specific, ATHB15 promoter (ATHB15::KAN1) completely lacked vascular tissues. The ectopic expression of KAN1 behind the 35SCaMV or lateral organ-specific ASYMMETRIC LEAVES1 (AS1) promoters has also been shown to have this effect in Arabidopsis shoots (Eshed et al. 2001). The ectopic ATHB15::KAN1 expression correlated with a lack of expression of PIN1 and of the auxin responsive marker, DR5rev::GFP (Ilegems et al. 2010), suggesting that the KANADI proteins suppress cambial cell division by altering PAT and suppressing auxin levels on the phloem side of the cambium. In support of this hypothesis, 35SCaMV-driven expression of PIN1 in the ATHB15::KAN1 line gradually restored vascular development (Ilegems et al. 2010). In contrast to the ATHB15::KAN1 line, ectopically expressing miRNA165 in ATHB15 expressing cells ( ATHB15:: miRNA165) suppressed the levels of some Class III HDZip mRNA and led to an increased accumulation of presumptive cambial stem cells in which the ATHB15, ATHB8 and the synthetic auxin responsive DR5 promoters were activated (Ilegems et al. 2010). Together, the data indicate that Class III HD-Zip proteins promote xylem differentiation and that either directly or indirectly both KANADI and Class III HD-Zip proteins suppress cellular indeterminancy and cell division, from opposite sides of the cambium and at least partially by suppressing auxin levels as cells emerge from the central cambial zone. This also suggests that the KANADI and Class III HD-Zip proteins are involved in regulating the radial auxin concentration gradient in the VC. In wild-type Arabidopsis roots, PHB mRNA are normally restricted to the xylem precursor and cambial cells (Carlsbecker et al. 2010). A point mutation in the PHB miRNA165/6 recognition sequence caused the ectopic accumulation of PHB mRNA throughout and beyond the root central vascular cylinder (stele; Fig. 5), indicating that

similarly to leaf dorsiventral patterning, miRNA165/6 cleavage is important in delimiting the domain of PHB mRNA accumulation (Carlsbecker et al. 2010). The PHB gain-of-function mutant also produced ectopic metaxylem in the roots. The authors showed that miRNA165/6 gene expression in the roots, and hence the delimitation of PHB mRNA and the regulation of xylem differentiation, was activated by the transcriptional regulator, SHORT-ROOT (SHR). SHR has previously been shown to be essential for QC and stem cell maintenance in the RAM (Hauser et al. 1995; Helariutta et al. 2000). Homologs of each of the elements in this mechanism, the miRNA165/6 encoding genes, SHR and the HD-Zips, are expressed in the cambium of Poplar stems, indicating that a similar mechanism may operate to delimit Class III HD-Zip expression in the VC (Schrader et al. 2004). Some evidence has emerged to support this hypothesis. Levels of mRNA for the Poplar ( Populus tremula Populus alba ) homologs of REV (PtaHB1) and microRNA166 (Pta-miR166) have been shown to be inversely correlated in cambial tissue (Ko et al. 2006). Similarly, in Nicotiana sylvestris, a gain-offunction mutation in the miRNA cleavage site of a PHV homolog causes the ectopic accumulation of NsPHAV transcripts in the phloem side of the cambium and secondary vascular malformation in the stems (McHale and Koning 2004). Whereas there is evidence for a role for SHR in VC formation and function: the Arabidopsis shr mutants lack an interfascicular cambium in inflorescence stems and a functional cambium in mature hypocotyls (authors' unpublished data), further research is necessary to determine if SHR regulates the expression of the miRNA165/6 genes and Class III HD-Zip mRNA cleavage in a manner similar to that observed by Carlsbecker et al. (2010) in the roots. Control of the balance between stem cell division and the differentiation of derivatives is important in both the apical meristems and the cambium. Tree stems infected by the crown gall tumour-inducing bacteria, Agrobacterium tumefaciens, for example, provide an easily identified example of the loss of cell division control in the VC (Kado 1976). Together, the data presented here strongly suggest an interactive role for Class III HDZip and KANADI gene products in establishing and maintaining the balance of cell division and differentiation in the VC. Considerable work remains if we are to understand the precise mechanisms by which they act.

WUS/CLV3 and the CLEs One of the earliest discoveries in the molecular regulation of meristem structure and function was the identification of the importance of cell-to-cell signalling in the SAM (reviewed in Lehesranta et al. 2009). The WUSCHEL/

Protoplasma vascular cambium review

CLAVATA (WUS/CLV) feedback loop mechanism acts to maintain the dynamic balance between stem cell division and differentiation (for review, see Wang and Fiers 2010). Basically, WUS, a homeodomain transcription factor, is expressed in a group of cells in the central zone known as the organising centre (OC; Fig. 3) that subtend the apical stem cells (Tax and Durbak 2006). Cells expressing WUS produce a signal that suppresses differentiation in the overlying cells (van den Berg et al. 1997; Mayer et al. 1998). In response, the L1L3 layers of the stem cells produce CLV3, a 96 amino acid protein (Fletcher et al. 1999) that is processed to an active 13 amino acid secreted glycoprotein (Ohyama et al. 2009). The CLV3 glycoprotein diffuses basipetally and interacts with the extracellular leucine-rich repeat (LRR) domains of the receptor kinase, CLAVATA1 (CLV1) and its homolog, CLAVATA2 (CLV2; Kayes and Clark 1998). CLV1 is expressed in the central zone in the L3 layer and below and overlaps with both CLV3 and WUS expression domains (Clark et al. 1997; Tax and Durbak 2006). CLV3-CLV1/CLV2 receptor-ligand binding results in the repression of WUS expression (Brand et al. 2000; Schoof et al. 2000; Ogawa et al. 2008; Ikeda et al. 2009). This feedback regulatory loop provides a dynamic mechanism for maintaining a stem cell population that reflects the requirement for cells at specific developmental stages or under changing environmental conditions (Geier et al. 2008). A related negative feedback loop mechanism has recently been identified in the root for the control of distal stem cell identity. A WUS homolog, WUSRELATED HOMEOBOX 5 (WOX5), is expressed in the RAM organising centre (QC) cells (Haecker et al. 2004; Gonzali et al. 2005; Stahl et al. 2009). Like WUS, WOX5 acts non-cell autonomously to maintain an undifferentiated state in the adjacent stem cells, in this case, the columella precursor, distal stem cells (Sarkar et al. 2007). Similarly to the loss of stem cell identity in the SAM of wus mutants (van den Berg et al. 1997; Mayer et al. 1998), the loss of WOX5 expression leads to the terminal differentiation of the distal stem cells (Sarkar et al. 2007). Conversely, loss of a CLV3 homolog, CLAVATA3/ENDOSPERM SURROUNDING REGION 40 (CLE40), in cle40 null mutant roots leads to an expansion of the WOX5 expression domain into the lateral stem cells surrounding the QC and to the production of supernumerary columella stem cells (Sarkar et al. 2007). The CLE40 ligand interacts in the RAM not with a CLV1like 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 ACR4 and WOX5 in a WOX/CLV-like feedback loop to control distal stem cell proliferation (Chaudhuri et al. 2009). While the CLV3 signal emanates from the SAM stem cells and the related RAM CLE40 signal originates from differentiating

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

CLEs and VC cell proliferation In Zinnia elegans and Arabidopsis, mesophyll cells can be coaxed into undergoing de-differentiation and redifferentiation into tracheary (xylem vessel) elements (TEs) in liquid cell culture medium (Ito et al. 2006; Pesquet et al. 2010). A potent inhibitor of this xylogenesis process, Tracheary Element Differentiation Inhibitory Factor (TDIF), was isolated in Zinnia by Ito et al. (2006) and identified as being a CLE family peptide. TDIF was shown to have the dual functions of inhibiting xylogenesis and enhancing cell division in Zinnia cell cultures (Ito et al. 2006; Hirakawa et al. 2010a). In Arabidopsis, the CLE41 and CLE44 genes encode proteins with C-terminal sequences that share the same 12 amino acid sequence as TDIR (Ito et al. 2006; Strabala et al. 2006), and another, the CLE42 gene, produces a highly similar sequence (Ito et al. 2006; Strabala et al. 2006). All three Arabidopsis gene products performed similarly to TDIF in the Zinnia xylogenesis system, suggesting a conservation of function (Ito et al. 2006). In all, there is predicted to be up to 83 CLE genes in Arabidopsis (Etchells and Turner 2010). Whitford et al. (2008) classified Arabidopsis CLEs into two classes, A-type and B-type, based on the ability of their corresponding synthetic peptides to inhibit growth (Oelkers et al. 2008). Both A-type and B-type CLE peptides are expressed in the VC in Poplar stems (Table 1; Schrader et al. 2004). In Arabidopsis, CLV3 and other A-type peptides suppress the production of stem cells, while B-type peptides such as CLE41 have been shown to have the dual roles of inhibiting differentiation and promoting cell division (Whitford et al. 2008). Whereas this indicates that the two CLE classes act antagonistically to control stem cell proliferation, the simultaneous application of synthetic Btype CLE41 and A-type, CLE6 peptides led to a dramatic increase in ATHB8-expressing presumptive cambial stem cells in Arabidopsis hypocotyls (Whitford et al. 2008). A similar phenotype was observed with a number of other CLE41/A-type CLE combinations and in transgenic plants simultaneously over-expressing the CLE41 and CLE6

J. P. Matte Risopatron et al.

genes (Whitford et al. 2008). These data suggest that rather than a simple antagonistic relationship, A-type CLEs potentiate the action of the B-type CLEs. The synergistic effect was enhanced when the plants were treated at the same time with auxin and was reduced in plants treated with the auxin transport inhibitor, NPA (Whitford et al. 2008). One possible interpretation of these results is that cambial stem and TA cells are specified by a confluence of overlapping domains of A-type and B-type CLEs and a high concentration and/or capacity to respond to auxin. The simultaneous application or overexpression of Atype and B-type CLEs also perturbed the orientation of the plane of cell division, resulting in disorganised vascular patterning (Whitford et al. 2008). This would be expected if an anisotropic apoplastic gradient of CLE peptides from source cells to the sites of perception played a role in cambial vascular patterning. The CLV1-like gene, PHLOEM INTERCALATED WITH XYLEM (PXY), was originally isolated in a screen designed to identify genes involved in secondary vascular cell polarity determination (Fisher and Turner 2007). Fisher and Turner (2007) found that in pxy mutants, in place of the usual bifacial cambium, the secondary xylem, phloem and cambial cells were interspersed in inflorescence stem vascular bundles. Hirakawa et al. (2008) subsequently isolated a pxy allele, TDR (putative TDIF receptor), and showed that it specifically bound the TDIF ligand. In Arabidopsis hypocotyls, the TDIF-like CLE41 and CLE44 peptides are produced in cells on the phloem side of the cambium, whereas the plasma membrane-bound TDR/PXY receptor has been localised to a non-overlapping domain in cambial stem cells (Hirakawa et al. 2008). This suggests that the ordered structure of the VC requires relies on spatial cues provided by phloem produced CLE41/44/TDIF ligands binding to the PXY/TDF receptor in cambial stem cells. Evidence to support this theory came from the misexpression of CLE41 and CLE42 in transgenic plants (Etchells and Turner 2010). Ectopic expression of CLE41 or CLE42 behind either the ubiquitous 35SCaMV or xylem-specific, IRX3 promoters severely disrupts the orientation of cambial cell divisions and both the mediolateral and apical-basal order of phloem and xylem in the hypocotyls (Etchells and Turner 2010). In contrast, ectopic expression behind the phloem-specific, SUC2 promoter failed to alter the orientation of divisions or the bifacial structure of the cambium (Etchells and Turner 2010). Loss-of-function tdr/pxy mutants also exhibited a disorganised cambial cell division phenotype, indicating that both elements, the location of ligand production and the position of the receptor, are important in determining the orientation of cambial cell divisions and the structure of the cambium (Etchells and Turner 2010). Somewhat surprisingly, 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 ectopic expression of CLE41 (IRX3:CLE41), vasculature structure was once again disrupted (Etchells and Turner 2010). The authors hypothesised that in the double 35SCaMV line (35SCaMV:TDR/PXY 35SCaMV:CLE41), a gradient of CLE41 would still be present from the endogenous production and secretion of the ligand. Together, these results indicate the importance of a centripetal apoplastic concentration gradient of the CLE41 ligand for the orientation of cambial cell division and the ordered structure of the VC. Commensurate with a role for CLE41/44/TDIF-PXY/ TDF ligand-receptor binding in specifying cambial stem cell identity, loss-of-function tdr/pxy (Hirakawa et al. 2008, 2010b) or cle41 (Hirakawa et al. 2010a) mutants display reduced cambial cell proliferation. This is in contrast to mutants for the CLV1 and ACR4 ligand receptors in the SAM and RAM. Both clv1 and acr4 mutants have an increased proliferation of stem cells, suggesting that they negatively regulate stem cell fate (Clark et al. 1993; De Smet et al. 2008). As CLV3 and CLE40 are A-type CLEs that act to suppress stem cell proliferation and CLE41 is a B-type that activates it, the contrasting loss-of-function phenotypes may reflect fundamental differences between the 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 role of TDR/PXY-CLE41/44/TDIR receptor ligand binding in promoting VC cambial stem cell identity and cell division. Whereas the vascular structure in the 35SCaMV:TDR/PXY 35SCaMV:CLE41 line was relatively normal, the combined ectopic expression led to a dramatic increase in the number of cells produced in both the fascicular and interfascicular cambia (Etchells and Turner 2010). Combining 35SCaMV: TDR/PXY and 35SCaMV:CLE42 led to a similar outcome (Etchells and Turner 2010). Both fascicular and in interfascicular cambial cell divisions were also activated in the SUC2:CLE41, although to a lesser extent than in the 35SCaMV:TDR/PXY 35SCaMV:CLE41/2 plants (Etchells and Turner 2010). The 35SCaMV promoter drives strong and ubiquitous gene expression in plants, and yet in the 35SCaMV:TDR/PXY 35SCaMV:CLE41/2 lines, the VC is almost wild type. This suggests that it is the increased expression of TDR/PXY and CLE41/42 at the position of the cambial stem cells that is important for the augmentation of cell division. This is reinforced by results in the SUC2: CLE41 line where VC structure is also preserved and cambial cell division increased. The effect is diminished in this line, however, commensurate with only one of the two

Protoplasma vascular cambium review

components, CLE41, being upregulated. Clearly, high levels of TDR/PXY and CLE41/42 expression promote cambial cell division, but the data suggests that they do this only in combination with other factors that determine the precise radial position of the fascicular and interfascicular cambia. The data presented here indicate clearly that CLE ligandreceptor production and binding are important in VC function. If the mechanism is closely related to those operating in the root and shoot apical meristems, however, a WOX homeobox gene family member should act together with CLE41/44/TDIF and TDR/PXY to regulate the cambial stem cell population. In Arabidopsis, the closest homologs to WUS are the RAM regulator, WOX5, and the WOX4 gene. In hypocotyls, WOX4 has been shown to be specifically expressed in the cambial zone (Hirakawa et al. 2010b). Ji et al. (2010) recently found that reducing the expression of WOX4 in Arabidopsis through an RNAi strategy resulted in a strong inhibition of differentiated phloem and xylem cells. They also found that it resulted in delayed and reduced the expression of markers driven by the phloem-specific ALTERED PHLOEM1 (APL1), and cambium and protoxylem-specific ATHB8 promoters (Ji et al. 2010). This suggested that WOX4 promotes cambial cell identity and proliferation. However, it remained unclear whether it acts in conjunction with CLE41/44/TDIF and TDR/PXY. Hirakawa et al. (2010b) recently provided strong evidence for an integrated CLE41/44/TDIF-TDR/ PXY-WOX4 mechanism. They were able to show that whereas the CLE41/44/TDIF peptide activates both periclinal and anticlinal cambial divisions in wild-type hypocotyls, it is not able to do this in wox4-1 loss-of-function mutants. As expected, given the previous evidence that CLE41/44/TDIF acts through TDF/PXY to promote cambial cell proliferation, the ligand was also unable to induce cambial divisions in the hypocotyls of tdr-1 and the tdr-1 wox4-1 double mutant (Hirakawa et al. 2010b). Together with the previous data, these results indicate that cambial cell 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 WOX4 expression in a TDR/PXY-dependent manner. It is possible that cambial cell-specific WOX4 expression is one of the essential elements that determine the capacity of cells to respond to the upregulation of TDR/PXY and CLE41/44/ TDIR in the 35SCaMV:TDR/PXY 35SCaMV:CLE41/2 lines. 35SCaMV overexpression of WOX4 had no effect on cambial cell proliferation on its own, suggesting that although WOX4 is an important element in the regulation of cambial cell proliferation, TDR/PXY and CLE41/44/ TDIR are the key limiting factors. In Poplar, close homologs of WOX4 (PtHB3) and TDR/PXY (PtRLK3) have

been shown to be similarly and strongly expressed on the xylem side of the cambial zone in the stem (Schrader et al. 2004). Further research is needed to determine if these genes are functionally similar to their Arabidopsis homologs and how they fit into the regulation of cambial activity in Poplar and other tree stems. Both intrinsic and extrinsic signals contribute to the function of the cambium and the determination of the fate of cambial stem cell derivatives (Savidge 1988, 2001; Werner and Schmulling 2009). The results presented here are not only important for what they tell us about how cambial cell proliferation is regulated and VC structure is maintained, they are also important because they provide the basis for a more detailed understanding of the structure and function of the VC. Are there specific OC-like cells in the VC? How are the Class III HD-Zip-KANADI and WOX-CLV systems linked in regulating VC structure and function? What are the principle mechanisms that integrate VC activity with whole plant environmental and developmental cues? It is clear from the evidence that mechanisms that operate in the primary meristems also regulate the formation and function of the VC (Ko and Han 2004; Schrader et al. 2004; Hirakawa et al. 2008). There are clear differences between, for example, the WOX/CLV mechanisms in the apices and the VC, but there are also strong similarities. Continuing the strategy of referring to mechanisms that have been well described in other tissues, organs and species will undoubtedly lead to the above questions being answered and eventually to a holistic understanding of the molecular regulation of VC structure and function.
Acknowledgements This work was supported in part by a Ph.D. scholarship for Matte J.P. by Advanced Human Capital Program of the National Commission for Scientific and Technological Research (CONICYT) Bicentennial Becas-Chile Scholarship and the Swedish Research Council FORMAS centre of excellence program, FUNCFIBER. Thanks to Gran Sandberg for the opportunity and inspiration. Conflict of interest The authors declare that they have no conflict of interest.

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