REVIEW / SYNTHÈSE

The shoot apical meristem and development ofvascular architecture1

Nancy G. Dengler

Abstract: The shoot apical meristem (SAM) functions to generate external architecture and internal tissue pattern as wellas to maintain a self-perpetuating population of stem-cell-like cells. The internal three-dimensional architecture of the vas-cular system corresponds closely to the external arrangement of lateral organs, or phyllotaxis. This paper reviews this cor-respondence for dicotyledonous plants in general and in Arabidopsis thaliana (L.) Heynh., specifically. Analysis is partlybased on the expression patterns of the class III homeodomain-leucine zipper transcription factor ARABIDOPSIS THALI-ANA HOMEOBOX GENE 8 (ATHB8), a marker of the procambial and preprocambial stages of vascular development, andon the anatomical criteria for recognizing vascular tissue pattern. The close correspondence between phyllotaxis and vascu-lar pattern present in mature tissues arises at early stages of development, at least by the first plastochron of leaf primor-dium outgrowth. Current literature provides an integrative model in which local variation in auxin concentration regulatesboth primordium formation on the SAM and the first indications of a procambial prepattern in the position of primordiumleaf trace as well as in the elaboration of leaf vein pattern. The prospects for extending this model to the development ofthe complex three-dimensional vascular architecture of the shoot are promising.

Key words: ATHB8, auxin, phyllotaxis, ATPIN1, procambium, vascular development.

Résumé : La fonction du méristème apical de la tige est de générer l’architecture externe et le patron histologique interne,ainsi que de maintenir une population de cellules de nature caulinaire par auto-perpétuation. L’architecture interne tridi-mensionnelle du système vasculaire correspond étroitement à l’arrangement externe des organes latéraux, ou phyllotaxie.L’auteur passe en revue cette correspondance chez les plantes dicotyles en général, et plus particulièrement chez l’Arabi-dopsis thaliana. L’analyse est partiellement basée sur l’expression des patrons de classe III du facteur de transcription del’homéodomaine-leucine-zipper, ARABIDOPSIS THALIANA HOMEOBOX GENE 8 (ATHB8), un marqueur des stadescambial et procambial du développement vasculaire, ainsi que sur des critères anatomiques pour reconnaı̂tre le patron destissus vasculaires. L’étroite correspondance entre la phyllotaxie et le patron des tissus vasculaires, dans les tissus matures,apparaı̂t à un stade précoce du développement, au moins au premier plastochron de l’apparition du primordium foliaire. Lalittérature courante présente un modèle intégrateur dans lequel la variation locale des teneurs en auxines règle à la fois laformation du primordium sur le méristème apical de la tige et les premières indications d’un pré-patron procambial, dansla position de la trace foliaire du primordium, ainsi que dans l’élaboration du patron vasculaire foliaire. La perspectived’étendre ce modèle de développement de l’architecture vasculaire tridimensionnelle complexe de la tige apparaı̂t promet-teuse.

Mots clés : ATHB8, auxine, phyllotaxie, ATP1N1, procambium, développement vasculaire.

[Traduit par la Rédaction]

Introduction

The shoot apical meristem (SAM) functions to generateexternal architecture and internal tissue pattern as well as tomaintain a self-perpetuating population of cells. Knowledgeof the development and behavior of the apical meristems is

prerequisite for understanding plant development as well asthe special properties of plants as organisms with an indeter-minate body plan. Recently, attention has focused on thegeneration of external architecture, specifically the place-ment of lateral organs (e.g., Fleming 2005; Reinhardt 2005),and on the formation and maintenance of the population ofstem-cell-like cells at the core of the SAM (e.g., Baurle andLaux 2003; Carles and Fletcher 2003). Less attention hasbeen given to the generation of the pattern of dermal,ground, and vascular tissues within the shoot. While the pro-toderm (precursor of the dermal tissue system) is derivedfrom the surface layer (L1) of the SAM simply by a restric-tion of division plane to anticlinal, the gradual emergence ofvascular pattern from more homogeneous-appearing precur-

Received 22 February 2006. Published on the NRC ResearchPress Web site at http://canjbot.nrc.ca on 6 February 2007.

N.G. Dengler. Department of Botany, University of Toronto,Toronto, ON M5S 1A1, Canada (e-mail:dengler@botany.utoronto.ca).

1This review is one of a selection of papers published on theSpecial Theme of Shoot Apical Meristems.

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sors derived from the L2 and deeper layers of the SAM isless well understood (Steeves and Sussex 1989). The pro-cambium (vascular tissue precursor) becomes distinct fromsurrounding ground meristem (ground tissue precursor) bydifferential patterns of cellular vacuolation, division, and en-largement (Esau 1965a, 1965b). Procambial pattern can berecognized because the component cells are elongate inshape and less vacuolated than adjacent ground meristemand form continuous strands (Esau 1965a, 1965b; Nelsonand Dengler 1997). As it emerges, the procambial systemcan be seen to form a complex, three-dimensional architec-ture within the shoot that is continuous with more matureparts of the vascular system. Moreover, the complex internalarchitecture of the vascular system corresponds closely tothe external architecture of lateral organ arrangement, orphyllotaxis.

The purpose of this review is to examine the close corre-spondence between phyllotaxis and primary vascular archi-tecture. The literature on this subject extends back foralmost 150 years, since botanists such as Nägeli (1858) andDeBary (1884) noted that the geometric array of developingand mature leaves gives an external clue to the internal ar-rangement of vascular bundles. In past decades, the causalityof this correspondence has been hotly debated, but as em-phasized by Esau (1965a, 1965b), the opposing views thateither (i) new primordia induce the formation of the vascularbundles that supply them or (ii) acropetal development ofvascular bundles induces the formation of primordia areoversimplifications, and it is more likely that both phyllo-taxis and vascular architecture are determined by a commonmechanism. Recent experimental and modeling studies haveprovided strong evidence for such a common mechanism(Fleming 2005; Reinhardt and Kuhlemeier 2002; Reinhardtet al. 2003; Reinhardt 2005; Smith et al. 2006; Jönsson etal. 2006). In this paper, I first review the expression patternof ARABIDOPSIS THALIANA HOMEOBOX GENE8(ATHB8), a putative marker of procambium, and its precur-sors within the SAM region. Second, I review primary vascu-lar architecture of dicotyledonous shoots and itscorrespondence to phyllotaxis and then describe this corre-spondence for A. thaliana based on an analysis of pro-cambium anatomy and the expression pattern of ATHB8(based on the results of Kang et al. 2003). Third, I describedevelopmental aspects of procambium pattern, including thepattern of ATHB8 expression. Finally, I review a currentmodel for regulation of both phyllotaxis and its correspond-ing vascular architecture by active modulation of local auxinconcentration.

ATHB8 as a marker of primary vasculardevelopment

The ARABIDOPSIS THALIANA HOMEOBOX GENE8(ATHB8) is one of five members of a family of class IIIhomeodomain-leucine zipper (HD-Zip) transcription factorsin the Arabidopsis genome that functions in the formationof meristems, in the dorsiventral patterning of lateral or-gans, and in the patterning and differentiation of vasculartissues (McConnell et al. 2001; Emery et al. 2003; Floydand Bowman 2004). Four members of the family (REVO-LUTA, PHABULOSA, PHAVOLUTA, and ATHB15/CO-

RONA) are expressed in the SAM, adaxial domains oflateral organs, and developing vascular tissues, while ex-pression of ATHB8 is restricted to developing vascular tis-sues (Baima et al. 1995; McConnell et al. 2001; Emery etal. 2003; Prigge et al. 2005; Williams et al. 2005). Duringthe development of leaf venation pattern, ATHB8-GUS isexpressed at very early stages in positions where veins arepredicted to appear, but before the diagnostic anatomicalfeatures of procambium are expressed (Kang and Dengler2004; Scarpella et al. 2004). In developing leaves, linearlyadjacent ground tissue cells initiate ATHB8 expression, anddevelopment is continuous and polar in the sense that thefirst cells to express ATHB8-GUS are adjacent to pre-existing procambial strands and that ground cells at theterminus of a developing file are recruited to the ATHB8-GUS-expressing file, extending it across a panel of groundtissue (Kang and Dengler 2004; Scarpella et al. 2004). Thisexpression pattern within the ground meristem presages theanatomical emergence of procambium and has been termed‘‘preprocambium’’ (Mattsson et al. 2003). Following thepreprocambial stage of development, cells in the file ac-quire the distinctive anatomical features of procambium,and ATHB8-GUS expression increases (Kang and Dengler2004; Scarpella et al. 2004). In contrast with the progres-sive appearance of the preprocambial phase, the emer-gence of procambium anatomy appears to occursimultaneously along the file of cells (Scarpella et al.2004). As xylem and phloem cells gradually differentiatefrom procambial tissue, ATHB8-GUS expression becomesrestricted to the residual procambium between the vasculartissues and undifferentiated cells on the adaxial (xylem)side of procambial strands; expression ceases in fully dif-ferentiated veins (Kang and Dengler 2002). Similarly, instem vascular bundles, ATHB8 expression becomes re-stricted to a narrow zone of procambium between the dif-ferentiating xylem and phloem tissues (Baima et al. 1995).Thus, ATHB8 expression provides a uniquely suitablemarker for analysis of vascular architecture, as it definesboth an early prepattern and procambium itself throughoutits development.

Despite this distinctive expression pattern, the specificdevelopmental function of ATHB8 is unknown (Emery etal. 2003; Prigge et al. 2005). Homozygous ATHB8 loss-of-function mutants have no detectable phenotype (Baima etal. 2001), while ectopic expression of ATHB8 results in pro-liferation of xylem precursor cells and subsequent increasein the numbers of mature xylem cells (Baima et al. 2001).In contrast, single loss- or gain-of-function mutations inother members of the class III HD-Zip gene family resultin dramatic conversions of vegetative and floral lateral or-gan dorsiventral symmetry (McConnell et al. 2001; Emeryet al. 2003; Prigge et al. 2005). Mutants of ATHB8 alsohave relatively little effect other than smaller stature in tri-ple, quadruple, and quintuple combinations with other classIII HD-Zip mutants (Prigge et al. 2005). There is some evi-dence, however, that ATHB8 may interact antagonisticallywith the REVOLUTA and ATHB15/CORONA loci as defectsin the differentiation of sclerenchyma fibers from theground tissue in inflorescence stems normally associatedwith these mutants are suppressed in triple mutants (Priggeet al. 2005). Members of the class III HD-Zip gene family

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clearly have overlapping and redundant functions in meris-tem establishment, organ polarity, and vascular develop-ment, making it difficult to establish the functions of asingle member such as ATHB8 through mutant analysis.

Evidence of a function for ATHB8 in vascular patterningand development comes from observations of ATHB8 ex-pression in response to the plant growth hormone auxin.ATHB8-GUS expression is upregulated in response to auxintreatment in wounded tobacco stems (Baima et al. 1995),and ATHB8 mRNA increases in Arabidopsis whole seed-lings or detached leaves incubated with auxin (Baima et al.1995; Mattsson et al. 2003). Additionally, ATHB8 transcriptlevels are reduced when the auxin response factor MONOP-TEROS is impaired or are increased when MONOPTEROS isoverexpressed (Mattsson et al. 2003). GUS expressiondriven by the synthetic auxin response element DR5 marksthe preprocambial stage of leaf vein development (Mattssonet al. 2003), as does ATHB8-GUS (Kang and Dengler 2004;Scarpella et al. 2004). These observations indicate thatATHB8 expression might represent a downstream event inthe process that translates an auxin signal into a procambialstrand. The canalized flow of auxin through shoot tissues ishypothesized to be the specific signal that induces formationof a vascular strand (Sachs 1981, 1991). Support for thismodel comes from experiments demonstrating that an artifi-cial auxin source can induce the formation of a vascularstrand within wounded stem tissue (reviewed in Sachs1981; Lyndon 1990). Numerous physiological experimentshave shown that auxin moves basipetally within intact stems(reviewed in Lomax et al. 1995) and that movement is de-pendent on the cellular localization of a plasma membrane-bound protein ARABIDOPSIS THALIANA PIN-FORMED1 (ATPIN1) (reviewed in Paponov et al. 2005). ATPIN1 isone of eight PIN genes present in the Arabidopsis genome(Paponov et al. 2005). The encoded PIN proteins have beenshown to be required for normal plant embryogenesis, orga-nogenesis, phototropism, and gravitropism and are thoughtto act through a role in polar auxin transport, although it iscurrently unknown whether they act as auxin efflux carriersdirectly or as regulators of polar auxin transport (Paponov etal. 2005). Nevertheless, polar auxin movement is dependenton the polar localization of ATPIN1 protein within the cell(Galweiler et al. 1998; Benková et al. 2003).

The developmental responses to polar auxin movementand signaling are not simple, as a complex series of coordi-nated cell divisions, cell enlargement, and patterned differ-entiation events are required to produce the highlyorganized and functional vascular strand rather than a broadunpatterned zone of vascular cells (Berleth and Mattsson2000; Berleth et al. 2000). A role for ATHB8 in this processis still a putative one, but one that we have exploited for acharacterization of primary vascular architecture in the com-pressed, miniature shoot of Arabidopsis.

Phyllotaxis and shoot vascular architecture

The pattern of initiation of leaf primordia on the flanks ofthe SAM gives rise to one of the most conspicuous featuresof whole-shoot morphology, phyllotaxis. Phyllotactic pat-terns generally are regular (although exceptions occur: Kellyand Cooke 2003; Jeune and Barabé 2004), and individual

species or whole taxonomic groups are characterized by spe-cific patterns, usually helical, distichous, decussate, orwhorled. Developmental shifts in phyllotaxis may occur,such as those associated with shoot phase change from juve-nile to adult or from vegetative to reproductive (Poethig1990; Kwiatkowska 1995). Helical phyllotaxis is the mostcommon pattern among dicotyledons and has received themost attention in terms of analysis of the geometrical pat-terns and modeling pattern generation within the SAM(Richards 1951; Mitchison 1977; Steeves and Sussex 1989;Lyndon 1990, 1998; Jean 1994; Adler et al. 1997; Reinhardtand Kuhlemeier 2002; Smith et al. 2006; Jönsson et al.2006). In species with helical phyllotaxis, leaf primordia areinitiated at a more or less constant divergence angle (ap-proximately 137.58) along a shallow helix, the ontogenetic(or ‘‘genetic’’) helix. Additional helices that are steeperthan the ontogenetic helix, the parastichies, can be recog-nized on the exterior of the shoot and, more readily, intransverse sections of the shoot apex region (Esau 1965a,1965b; Beck et al. 1982; Kirchoff 1984) (Fig. 1A). Leafprimordia that are in direct contact form conspicuous con-tact parastichies, while steeper noncontact parastichies canalso be recognized (Kirchoff 1984). The serial positions ofleaves within a parastichy as well as the numbers of clock-wise and anticlockwise parastichies are integers belonging tothe Fibonacci summation series (Richards 1951; Mitchison1977; Esau 1965a, 1965b). Knowledge of specific parametersof a helical phyllotactic system makes it possible to predictthe position of the next leaf primordium to be initiated onthe flanks of the SAM with considerable accuracy.

Similarly, knowledge of phyllotaxis permits predictionsabout the placement of vascular bundles within the stem(Girolami 1953; Skipworth 1962; Philipson and Balfour1963; Esau 1965a, 1965b; Beck et al. 1982; Kirchoff 1984).The longitudinal vascular bundles of the stem (here referredto as vascular sympodia and most clearly recognizable inimmature portions of the stem) branch at intervals to giverise to the leaf traces, the individual smaller vascular bun-dles that supply the leaves. The number of vascular sympo-dia usually reflects phyllotaxis: for instance, plants withdistichous or decussate phyllotaxis typically have even num-bers of vascular sympodia (e.g., 4, 6), while plants with hel-ical phyllotaxis have a number of sympodia belonging to theFibonacci summation series (e.g., 5, 8, 13: Beck et al. 1982;Kirchoff 1984). In species with a single trace supplying eachleaf (the common condition: Esau 1965a, 1965b; Beck et al.1982), leaves belonging to one parastichy all derive theirtraces from the same vascular sympodium. In species withthree or more traces per leaf, the central traces are suppliedfrom the same vascular sympodium, while the lateral tracesare derived from adjacent sympodia (e.g., Larson 1975). Insome species, there are no interconnections between adja-cent vascular sympodia (an open pattern), so that each vas-cular sympodium extends in a steep helix that mirrors oneparastichy on the exterior of the stem and branches to giverise to leaf traces at regular intervals. This simple, open pat-tern gives rise to the sectoriality observed in some studies oflong-distance transport of water, solutes, and signaling mol-ecules (Marshall 1996; Orians and Jones 2001). In other spe-cies, regular anastomoses between vascular sympodia form aclosed, reticulate pattern in which leaf traces are derived as

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branches of two adjacent vascular sympodia. Interestingly, acorrelation between the relative timing of the onset of cam-bial activity and the nature of the vascular system has beennoted in a modest species sample: secondary vascular devel-opment is initiated early when primary vascular architectureis open, but initiated late and (or) is limited in extent whenprimary vascular architecture is closed (Dormer 1945, 1946;Philipson and Balfour 1963).

Primary vascular architecture in Arabidopsis:mature pattern

Vegetative rosetteBased on analysis of the patterns of ATHB8-GUS expres-

sion and of anatomically defined procambium and (or) vas-cular tissues, vascular architecture of the vegetative rosetteof Arabidopsis is a closed, reticulate pattern that correspondsclosely to phyllotaxis (Figs. 1 and 2) (see Kang et al. 2003).In vegetatively growing shoots with 20 or more leaves, themost conspicuous contact parastichies are those connectingevery third leaf (n + 3) and every fifth leaf (n + 5) of theontogenetic helix (Fig. 1A). These contact parastichies ex-tend up the shoot axis in either a clockwise or a counter-clockwise direction (n + 5 and n + 3, respectively, inFig. 1A), depending on the overall orientation of shoot helicalphyllotaxis (anticlockwise for the shoot illustrated inFig. 1A). Individual rosettes with clockwise or anticlockwisehelical phyllotaxis occur with approximately equal frequen-cies in Arabidopsis (Kang et al. 2003). Generally, three n + 3

contact parastichies (connecting leaves 7, 10, 13, 16, etc.,leaves 5, 8, 11, 14, etc., leaves 6, 9, 12, 15, etc.) and fiven + 5 contact parastichies (connecting leaves 5, 10, 15,etc., leaves 6, 11, 16, etc., leaves 7, 12, 17, etc., leaves 8,13, 18, etc., leaves 9, 14, 19, etc.) are present. In additionto these more obvious contact parastichies, steeper, non-contact parastichies (n + 8, n + 13; Fig. 1A) can be super-imposed on overall shoot helical phyllotaxis. Geometricalproperties such as these intersecting parastichies and otherproperties of leaf arrangement accurately predict the spatialpositioning of vascular bundles within the stem.

The vascular bundles seen in any one cross section of thestem represent either individual leaf traces (4 in Fig. 1B) orthe vascular sympodia that branch and give rise to the indi-vidual leaf traces (e.g., branches from the sympodium la-beled 9 in Fig. 1B give rise to the traces of leaves 9, 14,and 17). The levels of branching of parent sympodia andthe divergence of leaf traces determine the number of vascu-lar strands observed in individual transverse sections, but thenumber in wild-type Arabidopsis is typically eight (Fig. 1B).When individual vacular bundles are traced through succes-sive serial sections, they can be seen to branch and give riseto the traces of leaves that are positionally related in the n +8 and n + 5 parastichies. For instance, the sympodium sup-plying leaf 12 is derived from branches of bundles supplyingleaf 4 (antecedent in n + 8 parastichy) and leaf 7 (antecedentin n + 5 parastichy) (Fig. 2), forming an anastomosing pat-tern and the closed pattern of primary vascular architecturethat characterizes Arabidopsis vegetative growth (Fig. 2).Thus, vascular architecture of the Arabidopsis shoot is a

Fig. 1. Phyllotaxis and primary vascular architecture in a vegetative shoot of Arabidopsis. (A) Cross section at the level of the shoot apicalmeristem. Rosette leaves are numbered according to order of ontogenetic helix. The n + 3 and n + 5 contact parastichies are indicated, asare the steeper n + 8 and n + 13 noncontact parastichies. (B) Cross section at 66 mm below the shoot apical meristem. Vascular bundlesrepresent either individual leaf traces (3, 4, 5, 6, 7) or vascular sympodia that will give rise to multiple leaf traces (8, 9, 10, 11, 12). Solidlines, clockwise parastichies; broken lines, anticlockwise parastichies. Scale bar = 50 mm. (From Kang et al. 2003, reproduced with permis-sion of the New Phytol. 158: 53–64).

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highly integrated system in which the vascular supply toeach leaf is directly connected to antecedent leaves in twodifferent parastichies, providing alternative pathways for thelong-distance movement of water and solutes.

Although the fully expressed primary vascular architec-ture is a closed, reticulate system, the formation of the anas-tomosing procambial strands is not synchronous in that thevascular connection with the n + 5 antecedent leaf lags be-hind the n + 8 connection during development. When a leaftrace is first detectable, it extends as a branch of the trace ofits n + 8 antecedent leaf and terminates just below its corre-sponding primordium within the SAM (see Kang et al.2003). Connections with the n + 5 antecedent leaf trace areformed three to four plastochrons later; thus, on a develop-mental basis, shoot primary vascular architecture is initiallyan open system but quickly becomes modified as a closedreticulum (summarized in Fig. 2).

The closed, reticulate pattern of vascular architecture withregular connections between the sympodia corresponding tothe n + 5 and n + 8 parastichies characterizes the adultphase of shoot ontogeny. During the juvenile phase, how-ever, phyllotaxis is subdecussate, and the traces to leaves 1to 4 arise directly from the vascular cylinder of the hypoco-tyls, as do those of the cotyledons (see Busse and Evert1999; Kang et al. 2003) (Fig. 2). Branches from these tracesanastomose to form the traces of the first-formed adultleaves, and connections are initially between the sympodiacorresponding to the n + 3 and n + 5 parastichies. The for-

mation of leaf 9, which establishes the first n + 8 parastichy,initiates the fully expressed pattern summarized in Fig. 2.

InflorescenceUpon the induction of reproductive growth, the SAM ini-

tiates floral meristems in place of leaves along the ontoge-netic helix of the vegetative rosette (Fig. 3). In Arabidopsisplants grown under long-day inductive conditions, floralmeristems are first formed at position 10, 11, or 12 (12 inFig. 3A and 10 in Fig. 4; see Kang et al. 2003). At first, thevascular architecture of the inflorescence extends the reticu-late pattern of the vegetative rosette, with floral meristemtraces derived from those of the antecedent organs in then + 8 and n + 5 parastichies. For instance, in the inflores-cence illustrated in Fig. 4, the sympodium supplying flower12 is derived from those supplying leaves 4 (n + 8 para-stichy) and 7 (n + 5 parastichy). After formation of the sec-ond flower in each n + 5 parastichy, however, vasculararchitecture switches to an open pattern, with primary con-nections along the n + 5 parastichies (e.g., flower 17,Fig. 4). Although the basic pattern follows that of the vege-tative rosette, the inflorescence pattern differs in two spe-cific ways: (i) the flower trace connects with the trace/sympodium corresponding to the n + 5, not the n + 8 para-stichy, and (ii) a second connection with the adjacent sym-podium was not detected, resulting in a primarily openpattern of inflorescence vascular architecture (summarizedin Fig. 4). ATHB8-GUS expression within new procambial

Fig. 2. Idealized two-dimensional diagram representing primary vascular architecture in vegetative shoots of Arabidopsis. Foliage leavesare represented by squares, numbered according to ontogenetic helix. Squares represent the approximate level of the leaf base. Leaf tracesare derived as branches of vascular sympodia supplying antecedent leaves in the n + 8 (blue) and n + 5 (green) parastichies, a patternestablished only after the second leaf in each n + 8 parastichy. Leaf traces are detectable during P1 (leaf 19) as branches from the antece-dent leaf trace/sympodium in the n + 8 parastichy, while the n + 5 connections are present 3–4 plastochrons later (leaf 16). Branches con-necting sympodia are shown as horizontal, reflecting the vertically compressed architecture of the rosette. Vascular bundles connected toantecedent sympodia are illustrated as double cylinders, although they are anatomical coherent (see Fig. 1B); those connected to one ante-cedent sympodium are illustrated as one. The blue line at the base of the illustration represents the solid cylinder of the hypocotyl vascula-ture, which gives rise directly to cotyledonary and juvenile leaf traces (see Busse and Evert 1999). Not to scale.

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strands is weaker in the inflorescence apex than in vegeta-tive apices, but the timing in relation to primordium forma-tion and the longitudinal pattern of ATHB8-GUS expressionappears to be similar (Kang et al. 2003).

Although inflorescence vascular architecture is initially anopen system, secondary modifications reinstate the closedreticulate nature of the shoot’s primary vascular architecture.Accessory traces form bridges between individual flowerbud traces and the adjacent n + 3 and n + 5 sympodia(Fig. 4). Such accessory traces are formed relatively late(usually 10 or more plastochrons after floral meristem initia-tion), are narrow in diameter relative to the floral meristemtraces, and have a horizontal course.

Axillary budsUpon transition from the vegetative to the reproductive

phase, axillary buds are initiated basipetally, starting withthe cauline leaves (Long and Barton 2000). Axillary budmeristems initially lack detectable vascular bundles, but theappearance of two short procambial strands is coincidentwith formation of the first two leaf primordia on the axillaryshoot (see Kang et al. 2003). These procambial strands arenot isolated but appear to be continuous as branches of ei-ther the leaf trace alone (most rosette leaves) or the adjacentvascular sympodia (cauline leaves) or a combination of thetwo patterns (summarized in Fig. 4; Kang et al. 2003).Thus, the vascular connections for the primary inflorescencebranches associated with the cauline leaves are well inte-grated with at least three vascular sympodia, providing abuilt-in redundancy of vascular pathways supplying theflowers and developing siliques born on those branches ifindividual vascular bundles are damaged.

The stepwise elaboration of the primary vascular systemin vegetative and reproductive shoots of Arabidopsis in-volves the SAM itself as well as older regions of the stem.Formation of the leaf trace (derived from the n + 8 sympo-dium) occurs during the first plastochron (P1), while the

connection with the n + 5 sympodium occurs later (P3 orP4). Vascular connections between axillary buds and adja-cent vascular sympodia develop outside the SAM in oldertissues (depending on growth conditions), as do those thatconnect the traces of developing flower buds with adjacentsympodia. These later developmental events are superim-posed on the initial primary pattern and presumably requirecomparable developmental signals and signaling pathways,although the developmental environment in terms of overalltissue differentiation differs from that within the SAM. Theprogressive elaboration of the primary vascular system al-lows plants to respond appropriately to variation in thegrowth environment in that the pattern may be arrested at asimple phase when plants are short-lived or elaborated whengrowth is prolonged. Under some growth conditions, secon-dary vascular development occurs within the rosette and thebasal portion of the inflorescence axis (Altamura et al. 2001;Chaffey et al. 2002), replacing the primary vascular systemfunctionally.

The basic features of vascular development described forthe rosette plant Arabidopsis on the basis of ATHB8-GUSexpression correspond to descriptions of elongate shoots inother species studied, most notably for Linum usitatissimumL. (Linaceae) (Girolami 1953), Hectorella caespitosaJ.D. Hooker (Portulaceae) (Skipworth 1962), and Populusdeltoides Bartr. ex Marsh. (Saliceae) (Larson 1975). In her-baceous Linum and Hectorella, primary vascular architectureis a closed, reticulate system with single acropetally devel-oping leaf traces connected to vascular sympodia that corre-spond to parastichies. As these shoots mature, the numbersof parastichies (and the numbers of intervening leaves alonga parastichy: n + 13, n + 21, etc.) and vascular sympodia in-crease, just as seen for the juvenile to adult transitions inArabidopsis. In woody Populus, vascular architecture is amore complex, open system with three traces per leaf, yetprocambial strands develop acropetally and precisely ac-cording to phyllotaxis (Larson 1975, 1977). In Populus, vas-

Fig. 3. Phyllotaxis and primary vascular architecture in the inflorescence of Arabidopsis. (A) Cross section at the level of the shoot apicalmeristem. Rosette leaves (5–8), cauline leaves (9–11), and floral meristems (12–21) are numbered in order of formation on the ontogenetichelix. The n + 3 and n + 5 contact parastichies are indicated, as are the steeper n + 8 and n + 13 noncontact parastichies. (B) Cross sectionat 490 mm below the shoot apical meristem. Rosette leaves 5–8 and the trace/sympodia supplying cauline leaves 9–11 and flowers 12–15 arenumbered. Note the axillary buds associated with rosette leaves 5–8 and axillary bud leaf primordia (arrows). Scale bar = 50 mm. (FromKang et al. 2003, reproduced with permission of the New Phytol. 158: 53–64).

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culature may be elaborated secondarily by the formation ofaccessory bundles that provide additional connections be-tween leaf traces and adjacent vascular sympodia (Larson1980a, 1980b).

Development of vascular architecture andexpression of ATHB8

Despite the progressive elaboration of primary vasculararchitecture within rosette and inflorescence stems of Arabi-dopsis, the developmental steps taking place within theSAM itself are similar during both vegetative and reproduc-tive phases. Both leaf primordia and floral meristems aresupplied by a single trace that appears to develop acrope-tally and in continuity with pre-existing vasculature and ispositioned precisely in relation to earlier-formed vascularbundles and to the placement of newly formed lateral organprimordia. Based on ATHB8 expression, procambial strand(or at least preprocambial strand) formation appears to becoincident with the initial formation of an externally detect-able primordium (P1), although detection of primordia fromserial cross sections may have identified only slightly olderprimordia (P2 or P3; Kang et al. 2003). During the vegeta-tive stage of development, procambial strands are continu-ous (based on a combination of anatomical criteria andATHB8 expression), with the trace/sympodium procambialstrand corresponding to the n + 8 parastichy. Although thispattern shifts subtly in the inflorescence (connections corre-spond to the n + 5 parastichy), the formation of new strandsfollows a highly predictable pattern with no indication of astochastic component to linkages between strands. The con-

tinuous, acropetal nature of procambial strand formation ap-pears to be general for the dicotyledons, virtually withoutexception (Esau 1965a, 1965b) and has been previously re-ported for Arabidopsis (Vaughan 1955; Busse and Evert1999).

Although procambial strands within the vegetative and re-productive shoot apical meristems of Arabidopsis appear tobe continuous with antecedent procambial strands, the zoneof ATHB8-GUS expression is initially discontinuous (Kanget al. 2003). The longitudinally oriented narrow files of cellsbelow each new primordium express the ATHB8-GUS con-struct strongly, while ATHB8-GUS activity initially is notdetectable at the basal end where these strands curve to con-nect with the antecedent parent strand. This stage is ephem-eral, as ATHB8-GUS expression becomes establishedthroughout the length of the leaf trace as the procambialstrand grows in diameter. Such a pattern could indicate thatATHB8 expression is induced by a basipetally moving signaland that the discontinuity reflects a moving front that inter-acts with acropetally moving signals, possibly traveling inthe phloem (reviewed in Lough and Lucas 2006). This ideais highly speculative, however, as the function of ATHB8 isstill not established; for instance, ATHB8 expression mightsimply be a marker of acquisition of xylem cell identity andonly presages the discontinuous nature of xylem differentia-tion (Kang et al. 2003).

An auxin model that integrates phyllotaxisand vascular development

The SAM generates regular, predictable patterns of leaves

Fig. 4. Idealized two-dimensional diagram representing primary vascular architecture in the Arabidopsis inflorescence. Foliage leaves arerepresented by squares and flowers by hexagons, numbered according to the ontogenetic helix; symbols represent the approximate level ofthe primordium base. Axillary buds (triangles) are associated with cauline leaves 7–9 and with rosette leaves 3–6. After the second flower ineach n + 5 parastichy, traces are derived as branches of vascular sympodia supplying antecedent flowers in the n + 5 parastichy, forming anopen pattern. Accessory bundles (yellow) connect flower traces with adjacent sympodia after seven or more plastochrons. Axillary bud pro-cambial strands (purple) form as branches of either the leaf trace (leaves 3–6) or adjacent sympodia (9) or a combination of the two (7, 8).The blue line at the base of the illustration represents the solid cylinder of the hypocotyl vasculature. Not to scale.

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and other lateral organs that are generally robust to experi-mental manipulation (e.g., Reinhardt et al. 2003, 2005;Smith et al. 2006). Phyllotaxis and the three-dimensional ar-chitecture of the vasculature supplying the lateral organs arehighly coordinated in their development. Although the na-ture of this coordinated development is not yet fully under-stood, current models point to mechanisms that regulate thepositioning of leaf and floral primordia and of their vascularsupply in an integrated manner. Earlier models of the regu-lation of phyllotaxis fall into two broad categories: in some(i), interactions among primordia on the flanks of the shootapical meristem are thought to determine the placement ofleaves and flowers, while in others (ii), inductive signalsfrom older portions of the shoot are thought to play a role(Larson 1983; Jean 1994; Lyndon 1998; Reinhardt andKuhlemeier 2002; Reinhardt et al. 2003). The nature of in-teractions among primordia is thought to be either biophysi-cal (e.g., Green 1996) or biochemical (e.g., Mitchison 1977;Schwabe 1984) and to display features of a lateral inhibitionsystem in which new primordia are placed at the maximumdistance possible from the ‘‘inhibitory’’ antecedent primor-dia on the flanks of the meristem (Lyndon 1990; Meinhardt1996). Observations that the placement of new primordiamerely reiterates that of older portions of the shoot and thatdeveloping vascular strands sometimes precede the externalappearance of the primordia that they will supply have ledto suggestions that inductive signals move acropetallythrough the vascular system (discussed in Esau 1965a,1965b; Larson 1983; Lyndon 1990). Current evidence,however, strongly supports a model in which the phyllotac-tic pattern is generated within the SAM through biochemi-cal interactions of pre-existing primordia, specificallythrough regulation of local variation in auxin concentration.

The auxin model postulates that auxin concentrations act,not as inhibitors of primordium development, but as activeeffectors of a specific developmental sequence of events(Reinhardt et al. 2003; Reinhardt 2005; Fleming 2004,2005; Heisler et al. 2005; Smith et al. 2006; Jönsson et al.2006). The key features of this model (summarized inFig. 5) are (i) uniform acropetal movement of auxin in thesurface (L1) layer of the apical meristem from regions out-side the SAM, (ii) formation of foci of auxin concentrationin positions that presage the position of primordia throughthe polar localization of ATPIN1 proteins, and (iii) redistrib-ution of ATPIN1 polarity upon the outgrowth of primordiaso that polar auxin movement is directed toward the interiorof the meristem, along a narrow file of cells in the positionof the future midvein procambial strand. Support for thismodel comes from a number of sources. First, when polarauxin transport is inhibited by pharmacological treatments,primordium formation is suppressed (Reinhardt et al. 2000;Stieger et al. 2002; Benková et al. 2003). Second, as shownfor inhibitor-treated tomato shoot tips or pin1 mutants ofArabidopsis, suppression of primordium formation can bereversed by localized auxin treatment (Reinhardt et al.2000). Third, the auxin efflux carrier protein ATPIN1 accu-mulates in the L1 layer of the meristem, specifically on theacropetal side of individual cells, indicating that net move-ment of auxin is toward the center of the SAM (Benkováet al. 2003; Reinhardt et al. 2003; Heisler et al. 2005).Fourth, ATPIN1 protein accumulation is strongest in posi-

tions that anticipate primordium formation by one to threeplastochrons (Reinhardt et al. 2003), as is AUXIN RESIST-ANT 1, a putative auxin influx carrier (Stieger et al. 2002;Benková et al. 2003). Expression of the reporter constructDR5-GFP, thought to reflect the endogenous auxin concen-tration (Benková et al. 2003), also is restricted to the L1layer of the SAM and expression peaks in incipient primor-dia (Smith et al. 2006). The phyllotactic pattern of AT-PIN1 accumulation is disrupted in the mutants pin1,pinoid, and monopteros, indicating that auxin signalingand transport are required for correct PIN1 localization(Reinhardt et al. 2003). Fifth, ATPIN1 protein is dynami-cally redistributed during each plastochron: ATPIN1 polar-ity is first directed toward the center of the presumptiveprimordium (I1 in Fig. 5), but with outgrowth, L1 layerexpression decreases and a narrow file of internal cells be-gins to accumulate ATPIN1 with a basipetal polarity.(Reinhardt et al. 2003; Heisler et al. 2005; P1 in Fig. 5).Live imaging of ATPIN1 distribution within the Arabidop-sis floral meristem also shows that ATPIN1 concentrationis highest in the I3, I2, and I1 presumptive floral meristemsites, when the polarity of surface cells adjacent to older,antecedent primordia is strongly directed toward the centerof the presumptive primordium (Heisler et al. 2005). Withthe outgrowth of the primordium, ATPIN1 becomes local-ized to the basal ends of the internal cells forming a nar-row file (Heisler et al. 2005). Thus, the positive inductionof primordium position and vascular strand position by thelocalized regulation of auxin concentrations could providea single mechanism that regulates both phyllotaxis and vas-cular architecture, much as hypothesized by Esau (1965a).

The auxin model provides a mechanism for the formationof leaf trace procambial strands in a pattern that is coordi-nated with phyllotaxis, but almost all aspects of how the ini-tial pattern of ATPIN1 distribution is translated into thecomplex three-dimensional vascular architecture of the shootare unknown. In many ways, the localization of ATPIN1protein to an internal strand of cells during the early plasto-chrons of primordium development (Reinhardt et al. 2003;Heisler et al. 2005) is comparable with the generation ofthe two-dimensional, yet complex, pattern of leaf veins. Indeveloping leaves, PIN1 is expressed first in the protoderm(derived from the L1 layer) and PIN1-GFP protein is local-ized subcellularly to the acropetal side of protodermal cells,producing a convergence point at the apex (Scarpella et al.2006); thus, the pattern observed for the SAM is preservedduring early stages of leaf development. Internal cells adja-cent to the apical convergence point accumulate PIN1-GFPat the basal side of the cells, and the zone of expressingcells extends towards the base of the leaf, forming the mid-vein prepattern (Scarpella et al. 2006). The looped secon-dary veins are generated similarly, starting with aconvergence point at the leaf margin and with a series ofcells expressing PIN1-GFP extending toward the midvein.These cells accumulate PIN1-GFP on the side toward themidvein, suggesting that auxin moves along the incipientvein from a source at the margin to the sink represented bythe earlier-formed strand (Scarpella et al. 2006).

Strands of preprocambial tissue are polar in their develop-ment and extend unidirectionally from pre-existing strands;if strand extension is arrested, a ‘‘freely ending veinlet’’ is

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formed. During the development of most of the secondaryand higher-order venation, however, strand extension rapidlyconnects with an adjacent strand, thus forming a continuouslink between pre-existing strands (Scarpella et al. 2004,2006). The prepatterns represented by PIN1-GFP, the auxinreporter DR5-GUS, the gene trap marker ET1335-GUS, orATHB8-GUS all appear when the leaf (or field of tissue forhigher-order veins) consists of relatively few cells and is ex-tended by intercalary growth as the leaf enlarges (Mattssonet al. 2003; Kang and Dengler 2004; Scarpella et al. 2004,2006); thus, the stage of discontinuity and unidirectionalgrowth is ephemeral (except for the freely ending veinlets).The cytological features of cells expressing these markersare indistinguishable from those of cells of the ground tissuein which this preprocambial pattern arises; only later do thedistinctive elongate shape and dense cytoplasm of anatomi-cally defined procambial cells emerge (Mattsson et al.2003; Kang and Dengler 2004; Scarpella et al. 2004, 2006).Thus, elements of pattern formation are shared by develop-ment of both two-dimensional leaf vein patterns and three-dimensional shoot vascular pattern: (i) unidirectionalelaboration of the system that may reflect auxin transportfrom localized sources to sinks within the tissue, (ii) forma-tion of major elements of the system, such as leaf primaryand secondary veins or the n + 8 sympodial strands of thestem followed by formation of minor pattern elements, suchas the higher order venation or the n + 5 sympodial strands,and (iii) discontinuities in the accumulation pattern of certainproteins, such as that observed for PIN1 expression in an in-

cipient leaf primordium (Reinhardt et al. 2003; Heisler et al.2005) or in the expression of ATHB8 within leaf traces(Kang et al. 2003). An important distinction between thetwo is that while leaf vein pattern development points to ahighly flexible self-organizing patterning mechanism (Scar-pella et al. 2006), shoot vascular architecture appears to behighly predictable, with almost no stochastic element to theformation of connections between strands (Kang et al. 2003).

Future prospects

Plants are increasingly regarded as supracellular organ-isms in which long-distance transport of, not only water anddissolved nutrients, but also of macromolecules and othersignaling substances depends on the vascular system (Loughand Lucas 2006). Knowledge of the three-dimensional archi-tecture of the vascular system, and particularly how it corre-sponds to leaf position on the shoot, is essential forunderstanding how photosynthate moves through the phloemfrom specific source to specific sink leaves or how chemicaldefense signals might move from herbivore-damaged leavesto particular newly expanding ones (e.g., Larson 1977;Marshall 1996; Orians and Jones 2001). For instance, inpoplar, when 14C label was supplied to a photosynthesizingsource leaf, it was possible to predict where the relativepercentages of the labeled photosynthates would appearbased on knowledge of the connections of central and lat-eral leaf traces with adjacent vascular sympodia and of thetransitions in phyllotaxis appearing during shoot ontogeny

Fig. 5. Idealized two-dimensional diagram representing the spatial pattern of polar auxin flow (based on localization of the ATPIN1 protein:Reinhardt et al. 2003; Heisler et al. 2005) and of ATHB8 expression (based on promoter-GUS expression: Kang et al. 2003) in vegetativeand inflorescence SAMs of Arabidopsis. Arrows, coinciding with localization of ATPIN1, indicate net movement of auxin toward the cen-ters of incipient primordia (I1, I2) and toward internal tissue during early primordium outgrowth (P1, P2). An isolated file of ATHB8-GUS-expressing cells (blue) is present at the P1 and later stages below the base of the primordium, although a connecting strand of anatomicallydefined procambium (pink) is consistently present, connecting it to the antecedent procambial strand associated with a leaf that is eightplastochrons older (vegetative SAM).

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(Larson 1977). Analyses of phloem sap and use of graftingbetween stocks and scions of different genotype have dem-onstrated that numerous macromolecules function in long-distance communication and use phloem tissue as a conduit(reviewed by Lough and Lucas 2006). One example ofthese is the FLOWERING LOCUS T protein, a componentof a phloem-mobile florigenic signal. Induction of gene ex-pression in a source leaf by heat shock results in accumu-lation of FLOWERING LOCUS T mRNA in both thesource leaf and the SAM, indicating that the mRNA (orperhaps the protein) moves along the vascular sympo-dium(a) connecting the source leaf and the primordia clos-est to the SAM (Huang et al. 2005). Although manyenvironmental and developmental stimuli may not be local-ized, so that signaling moves through the entire shoot vas-culature and affects leaves of all parastichies equally, manytriggers will be specific to regions of the shoot or root sys-tem, and vascular architecture will contribute to determin-ing which targets will be reached by those signals.

Knowledge of shoot primary vascular architecture of Ara-bidopsis, specifically, might form the basis for better under-standing aspects of the developmental biology of this modelorganism. The information described herein is especiallyuseful for the interpretation of mutations that affect the vas-cular system, for instance the supernumerary vascular bun-dles that appear in mutants such as REVOLUTA (Zhong etal. 1999) or the effects of mutants in other HD-ZIP familygenes on shoot vascular pattern as well as ground scleren-chyma (Prigge et al. 2005). Expression patterns of genessuch as ATHB8 or DR5 that are restricted to developingprocambial strands might be used in mutant screens toidentify genes that function in the construction of thiscomplex three-dimensional pattern. Although the analysispresented here was based on laborious reconstruction fromserial cross sections (Kang et al. 2003), analysis of clearedwhole shoots of plants carrying these reporter constructs oruse of confocal microscopy and other imaging systemswould allow identification of mutants. Most importantly,knowledge of the primary vascular system of Arabidopsisand how it remains coordinated with changes in phyllotaxisand organ identity through juvenile, vegetative adult, andreproductive phases will contribute to understanding thegenetic framework that underlies shoot development.

In summary, developmental biologists have made dra-matic headway in providing evidence from gene reporterstudies, experimental manipulations, and modeling thatstrongly support the role of the PIN proteins in the regula-tion of localized auxin concentrations, leading to the pat-terned initiation of leaf primordia in the correct phyllotacticsequence on the flanks of the SAM. Tantalizingly for thetopic at hand, the subcellular localization of PIN1 proteinsin Arabidopsis indicates that auxin flows from the center ofthe primordium site inwards along a narrow path, presagingthe location of the leaf midvein and its connecting leaftrace. Thus far, imaging techniques have been limited tosurface layers, so it has not been possible to ‘‘connect thedots’’ and relate this midvein prepattern with the predictedconnection of the leaf trace with the vascular sympodiumcorresponding to the n + 8 parastichy. Detailed analysis ofthe development of leaf vein pattern indicates that localizedconcentrations of auxin also initiate the expression of a pro-

cambial prepattern that connects a source of auxin with tis-sues acting as a sink and that reiterations of the processcreate a complex hierarchical pattern (Scarpella et al.2006). Although such concentrations only have been shownto occur in the L1 layer of the SAM and the protoderm ofdeveloping leaves, a similar process is likely to occur withininternal ground tissues and would be integral to the genera-tion of the three-dimensional vascular pattern of shoots andto its secondary modification during development.

AcknowledgmentsI thank Thomas Berleth, Julie Kang, Bill Remphrey, and

two anonymous reviewers for helpful comments on themanuscript, Janice Wong for illustrations, and the NaturalSciences and Engineering Research Council of Canada forresearch support.

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