The Shoot Apical Meristem and Development of Vascular Architecture1

1660

REVIEW / SYNTHE` SE

The shoot apical meristem and development of vascular architecture1

Nancy G. Dengler

Abstract: The shoot apical meristem (SAM) functions to generate external architecture and internal tissue pattern as well as to maintain a self-perpetuating population of stem-cell-like cells. The internal three-dimensional architecture of the vascular system corresponds closely to the external arrangement of lateral organs, or phyllotaxis. This paper reviews this correspondence for dicotyledonous plants in general and in Arabidopsis thaliana (L.) Heynh., specifically. Analysis is partly based 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, and on the anatomical criteria for recognizing vascular tissue pattern. The close correspondence between phyllotaxis and vascular pattern present in mature tissues arises at early stages of development, at least by the first plastochron of leaf primordium outgrowth. Current literature provides an integrative model in which local variation in auxin concentration regulates both primordium formation on the SAM and the first indications of a procambial prepattern in the position of primordium leaf trace as well as in the elaboration of leaf vein pattern. The prospects for extending this model to the development of the complex three-dimensional vascular architecture of the shoot are promising. Key words: ATHB8, auxin, phyllotaxis, ATPIN1, procambium, vascular development. Re´sume´ : La fonction du me´riste`me apical de la tige est de geńe´rer l’architecture externe et le patron histologique interne, ainsi que de maintenir une population de cellules de nature caulinaire par auto-perpe´tuation. L’architecture interne tridimensionnelle du syste`me vasculaire correspond e´troitement a` l’arrangement externe des organes late´raux, ou phyllotaxie. L’auteur passe en revue cette correspondance chez les plantes dicotyles en geńe´ral, et plus particulie`rement chez l’Arabi- dopsis thaliana. L’analyse est partiellement baseé sur l’expression des patrons de classe III du facteur de transcription de l’homeódomaine-leucine-zipper, ARABIDOPSIS THALIANA HOMEOBOX GENE 8 (ATHB8), un marqueur des stades cambial et procambial du de´veloppement vasculaire, ainsi que sur des crite`res anatomiques pour reconnaıˆtre le patron des tissus vasculaires. L’e´troite correspondance entre la phyllotaxie et le patron des tissus vasculaires, dans les tissus matures, apparaıˆta` un stade prećoce du de´veloppement, au moins au premier plastochron de l’apparition du primordium foliaire. La litte´rature courante pre´sente un mode`le inte´grateur dans lequel la variation locale des teneurs en auxines re`gle a` la fois la formation du primordium sur le me´riste`me apical de la tige et les premie`res indications d’un pre´-patron procambial, dans la position de la trace foliaire du primordium, ainsi que dans l’e´laboration du patron vasculaire foliaire. La perspective d’e´tendre ce mode`le de de´veloppement de l’architecture vasculaire tridimensionnelle complexe de la tige apparaıˆt promet- teuse. Mots cle´s : ATHB8, auxine, phyllotaxie, ATP1N1, procambium, de´veloppement vasculaire. [Traduit par la Re´daction]

Introduction prerequisite for understanding plant development as well as the special properties of plants as organisms with an indeter- The shoot apical meristem (SAM) functions to generate minate body plan. Recently, attention has focused on the external architecture and internal tissue pattern as well as to generation of external architecture, specifically the place- maintain a self-perpetuating population of cells. Knowledge ment of lateral organs (e.g., Fleming 2005; Reinhardt 2005), of the development and behavior of the apical meristems is and on the formation and maintenance of the population of stem-cell-like cells at the core of the SAM (e.g., Baurle and Received 22 February 2006. Published on the NRC Research Press Web site at http://canjbot.nrc.ca on 6 February 2007. Laux 2003; Carles and Fletcher 2003). Less attention has been given to the generation of the pattern of dermal, N.G. Dengler. Department of Botany, University of Toronto, ground, and vascular tissues within the shoot. While the pro- Toronto, ON M5S 1A1, Canada (e-mail: toderm (precursor of the dermal tissue system) is derived [email protected]). from the surface layer (L1) of the SAM simply by a restric- 1This review is one of a selection of papers published on the tion of division plane to anticlinal, the gradual emergence of Special Theme of Shoot Apical Meristems. vascular pattern from more homogeneous-appearing precur-

Can. J. Bot. 84: 1660–1671 (2006) doi:10.1139/B06-126 # 2006 NRC Canada Dengler 1661 sors derived from the L2 and deeper layers of the SAM is RONA) are expressed in the SAM, adaxial domains of less well understood (Steeves and Sussex 1989). The pro- lateral organs, and developing vascular tissues, while ex- cambium (vascular tissue precursor) becomes distinct from pression of ATHB8 is restricted to developing vascular tis- surrounding ground meristem (ground tissue precursor) by sues (Baima et al. 1995; McConnell et al. 2001; Emery et differential patterns of cellular vacuolation, division, and en- al. 2003; Prigge et al. 2005; Williams et al. 2005). During largement (Esau 1965a, 1965b). Procambial pattern can be the development of leaf venation pattern, ATHB8-GUS is recognized because the component cells are elongate in expressed at very early stages in positions where veins are shape and less vacuolated than adjacent ground meristem predicted to appear, but before the diagnostic anatomical and form continuous strands (Esau 1965a, 1965b; Nelson features of procambium are expressed (Kang and Dengler and Dengler 1997). As it emerges, the procambial system 2004; Scarpella et al. 2004). In developing leaves, linearly can be seen to form a complex, three-dimensional architec- adjacent ground tissue cells initiate ATHB8 expression, and ture within the shoot that is continuous with more mature development is continuous and polar in the sense that the parts of the vascular system. Moreover, the complex internal first cells to express ATHB8-GUS are adjacent to pre- architecture of the vascular system corresponds closely to existing procambial strands and that ground cells at the the external architecture of lateral organ arrangement, or terminus of a developing file are recruited to the ATHB8- phyllotaxis. GUS-expressing file, extending it across a panel of ground The purpose of this review is to examine the close corre- tissue (Kang and Dengler 2004; Scarpella et al. 2004). This spondence between phyllotaxis and primary vascular archi- expression pattern within the ground meristem presages the tecture. The literature on this subject extends back for anatomical emergence of procambium and has been termed almost 150 years, since botanists such as Na¨geli (1858) and ‘‘preprocambium’’ (Mattsson et al. 2003). Following the DeBary (1884) noted that the geometric array of developing preprocambial stage of development, cells in the file ac- and mature leaves gives an external clue to the internal ar- quire the distinctive anatomical features of procambium, rangement of vascular bundles. In past decades, the causality and ATHB8-GUS expression increases (Kang and Dengler of this correspondence has been hotly debated, but as em- 2004; Scarpella et al. 2004). In contrast with the progres- phasized by Esau (1965a, 1965b), the opposing views that sive appearance of the preprocambial phase, the emer- either (i) new primordia induce the formation of the vascular gence of procambium anatomy appears to occur bundles that supply them or (ii) acropetal development of simultaneously along the file of cells (Scarpella et al. vascular bundles induces the formation of primordia are 2004). As xylem and phloem cells gradually differentiate oversimplifications, and it is more likely that both phyllo- from procambial tissue, ATHB8-GUS expression becomes taxis and vascular architecture are determined by a common restricted to the residual procambium between the vascular mechanism. Recent experimental and modeling studies have tissues and undifferentiated cells on the adaxial (xylem) provided strong evidence for such a common mechanism side of procambial strands; expression ceases in fully dif- (Fleming 2005; Reinhardt and Kuhlemeier 2002; Reinhardt ferentiated veins (Kang and Dengler 2002). Similarly, in et al. 2003; Reinhardt 2005; Smith et al. 2006; Jo¨nsson et stem vascular bundles, ATHB8 expression becomes re- al. 2006). In this paper, I first review the expression pattern stricted to a narrow zone of procambium between the dif- of ARABIDOPSIS THALIANA HOMEOBOX GENE8 ferentiating xylem and phloem tissues (Baima et al. 1995). (ATHB8), a putative marker of procambium, and its precur- Thus, ATHB8 expression provides a uniquely suitable sors within the SAM region. Second, I review primary vascu- marker for analysis of vascular architecture, as it defines lar architecture of dicotyledonous shoots and its both an early prepattern and procambium itself throughout correspondence to phyllotaxis and then describe this corre- its development. spondence for A. thaliana based on an analysis of pro- Despite this distinctive expression pattern, the specific cambium anatomy and the expression pattern of ATHB8 developmental function of ATHB8 is unknown (Emery et (based on the results of Kang et al. 2003). Third, I describe al. 2003; Prigge et al. 2005). Homozygous ATHB8 loss-of- developmental aspects of procambium pattern, including the function mutants have no detectable phenotype (Baima et pattern of ATHB8 expression. Finally, I review a current al. 2001), while ectopic expression of ATHB8 results in pro- model for regulation of both phyllotaxis and its correspond- liferation of xylem precursor cells and subsequent increase ing vascular architecture by active modulation of local auxin in the numbers of mature xylem cells (Baima et al. 2001). concentration. In contrast, single loss- or gain-of-function mutations in other members of the class III HD-Zip gene family result ATHB8 as a marker of primary vascular in dramatic conversions of vegetative and floral lateral or- development gan dorsiventral symmetry (McConnell et al. 2001; Emery et al. 2003; Prigge et al. 2005). Mutants of ATHB8 also The ARABIDOPSIS THALIANA HOMEOBOX GENE8 have relatively little effect other than smaller stature in tri- (ATHB8) is one of five members of a family of class III ple, quadruple, and quintuple combinations with other class homeodomain-leucine zipper (HD-Zip) transcription factors III HD-Zip mutants (Prigge et al. 2005). There is some evi- in the Arabidopsis genome that functions in the formation dence, however, that ATHB8 may interact antagonistically of meristems, in the dorsiventral patterning of lateral or- with the REVOLUTA and ATHB15/CORONA loci as defects gans, and in the patterning and differentiation of vascular in the differentiation of sclerenchyma fibers from the tissues (McConnell et al. 2001; Emery et al. 2003; Floyd ground tissue in inflorescence stems normally associated and Bowman 2004). Four members of the family (REVO- with these mutants are suppressed in triple mutants (Prigge LUTA, PHABULOSA, PHAVOLUTA, and ATHB15/CO- et al. 2005). Members of the class III HD-Zip gene family

# 2006 NRC Canada 1662 Can. J. Bot. Vol. 84, 2006 clearly have overlapping and redundant functions in meris- species or whole taxonomic groups are characterized by spe- tem establishment, organ polarity, and vascular develop- cific patterns, usually helical, distichous, decussate, or ment, making it difficult to establish the functions of a whorled. Developmental shifts in phyllotaxis may occur, single member such as ATHB8 through mutant analysis. such as those associated with shoot phase change from juve- Evidence of a function for ATHB8 in vascular patterning nile to adult or from vegetative to reproductive (Poethig and development comes from observations of ATHB8 ex- 1990; Kwiatkowska 1995). Helical phyllotaxis is the most pression in response to the plant growth hormone auxin. common pattern among dicotyledons and has received the ATHB8-GUS expression is upregulated in response to auxin most attention in terms of analysis of the geometrical pat- treatment in wounded tobacco stems (Baima et al. 1995), terns and modeling pattern generation within the SAM and ATHB8 mRNA increases in Arabidopsis whole seed- (Richards 1951; Mitchison 1977; Steeves and Sussex 1989; lings or detached leaves incubated with auxin (Baima et al. Lyndon 1990, 1998; Jean 1994; Adler et al. 1997; Reinhardt 1995; Mattsson et al. 2003). Additionally, ATHB8 transcript and Kuhlemeier 2002; Smith et al. 2006; Jo¨nsson et al. levels are reduced when the auxin response factor MONOP- 2006). In species with helical phyllotaxis, leaf primordia are TEROS is impaired or are increased when MONOPTEROS is initiated at a more or less constant divergence angle (ap- overexpressed (Mattsson et al. 2003). GUS expression proximately 137.58) along a shallow helix, the ontogenetic driven by the synthetic auxin response element DR5 marks (or ‘‘genetic’’) helix. Additional helices that are steeper the preprocambial stage of leaf vein development (Mattsson than the ontogenetic helix, the parastichies, can be recog- et al. 2003), as does ATHB8-GUS (Kang and Dengler 2004; nized on the exterior of the shoot and, more readily, in Scarpella et al. 2004). These observations indicate that transverse sections of the shoot apex region (Esau 1965a, ATHB8 expression might represent a downstream event in 1965b; Beck et al. 1982; Kirchoff 1984) (Fig. 1A). Leaf the process that translates an auxin signal into a procambial primordia that are in direct contact form conspicuous con- strand. The canalized flow of auxin through shoot tissues is tact parastichies, while steeper noncontact parastichies can hypothesized to be the specific signal that induces formation also be recognized (Kirchoff 1984). The serial positions of of a vascular strand (Sachs 1981, 1991). Support for this leaves within a parastichy as well as the numbers of clock- model comes from experiments demonstrating that an artifi- wise and anticlockwise parastichies are integers belonging to cial auxin source can induce the formation of a vascular the Fibonacci summation series (Richards 1951; Mitchison strand within wounded stem tissue (reviewed in Sachs 1977; Esau 1965a, 1965b). Knowledge of specific parameters 1981; Lyndon 1990). Numerous physiological experiments of a helical phyllotactic system makes it possible to predict have shown that auxin moves basipetally within intact stems the position of the next leaf primordium to be initiated on (reviewed in Lomax et al. 1995) and that movement is de- the flanks of the SAM with considerable accuracy. pendent on the cellular localization of a plasma membrane- Similarly, knowledge of phyllotaxis permits predictions bound protein ARABIDOPSIS THALIANA PIN-FORMED about the placement of vascular bundles within the stem 1 (ATPIN1) (reviewed in Paponov et al. 2005). ATPIN1 is (Girolami 1953; Skipworth 1962; Philipson and Balfour one of eight PIN genes present in the Arabidopsis genome 1963; Esau 1965a, 1965b; Beck et al. 1982; Kirchoff 1984). (Paponov et al. 2005). The encoded PIN proteins have been The longitudinal vascular bundles of the stem (here referred shown to be required for normal plant embryogenesis, orga- to as vascular sympodia and most clearly recognizable in nogenesis, phototropism, and gravitropism and are thought immature portions of the stem) branch at intervals to give to act through a role in polar auxin transport, although it is rise to the leaf traces, the individual smaller vascular bun- currently unknown whether they act as auxin efflux carriers dles that supply the leaves. The number of vascular sympo- directly or as regulators of polar auxin transport (Paponov et dia usually reflects phyllotaxis: for instance, plants with al. 2005). Nevertheless, polar auxin movement is dependent distichous or decussate phyllotaxis typically have even num- on the polar localization of ATPIN1 protein within the cell bers of vascular sympodia (e.g., 4, 6), while plants with hel- (Galweiler et al. 1998; Benkova´ et al. 2003). ical phyllotaxis have a number of sympodia belonging to the The developmental responses to polar auxin movement Fibonacci summation series (e.g., 5, 8, 13: Beck et al. 1982; and signaling are not simple, as a complex series of coordi- Kirchoff 1984). In species with a single trace supplying each nated cell divisions, cell enlargement, and patterned differ- leaf (the common condition: Esau 1965a, 1965b; Beck et al. entiation events are required to produce the highly 1982), leaves belonging to one parastichy all derive their organized and functional vascular strand rather than a broad traces from the same vascular sympodium. In species with unpatterned zone of vascular cells (Berleth and Mattsson three or more traces per leaf, the central traces are supplied 2000; Berleth et al. 2000). A role for ATHB8 in this process from the same vascular sympodium, while the lateral traces is still a putative one, but one that we have exploited for a are derived from adjacent sympodia (e.g., Larson 1975). In characterization of primary vascular architecture in the com- some species, there are no interconnections between adja- pressed, miniature shoot of Arabidopsis. cent vascular sympodia (an open pattern), so that each vascular sympodium extends in a steep helix that mirrors one Phyllotaxis and shoot vascular architecture parastichy on the exterior of the stem and branches to give rise to leaf traces at regular intervals. This simple, open pat- The pattern of initiation of leaf primordia on the flanks of tern gives rise to the sectoriality observed in some studies of the SAM gives rise to one of the most conspicuous features long-distance transport of water, solutes, and signaling mol- of whole-shoot morphology, phyllotaxis. Phyllotactic pat- ecules (Marshall 1996; Orians and Jones 2001). In other spe- terns generally are regular (although exceptions occur: Kelly cies, regular anastomoses between vascular sympodia form a and Cooke 2003; Jeune and Barabe´ 2004), and individual closed, reticulate pattern in which leaf traces are derived as

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Fig. 1. Phyllotaxis and primary vascular architecture in a vegetative shoot of Arabidopsis. (A) Cross section at the level of the shoot apical meristem. Rosette leaves are numbered according to order of ontogenetic helix. The n + 3 and n + 5 contact parastichies are indicated, as are the steeper n + 8 and n + 13 noncontact parastichies. (B) Cross section at 66 mm below the shoot apical meristem. Vascular bundles represent 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). Solid lines, clockwise parastichies; broken lines, anticlockwise parastichies. Scale bar = 50 mm. (From Kang et al. 2003, reproduced with permission of the New Phytol. 158: 53–64).

branches of two adjacent vascular sympodia. Interestingly, a contact parastichies (connecting leaves 7, 10, 13, 16, etc., correlation between the relative timing of the onset of cam- leaves 5, 8, 11, 14, etc., leaves 6, 9, 12, 15, etc.) and five bial activity and the nature of the vascular system has been n + 5 contact parastichies (connecting leaves 5, 10, 15, noted in a modest species sample: secondary vascular devel- etc., leaves 6, 11, 16, etc., leaves 7, 12, 17, etc., leaves 8, opment is initiated early when primary vascular architecture 13, 18, etc., leaves 9, 14, 19, etc.) are present. In addition is open, but initiated late and (or) is limited in extent when to these more obvious contact parastichies, steeper, non- primary vascular architecture is closed (Dormer 1945, 1946; contact parastichies (n +8,n + 13; Fig. 1A) can be super- Philipson and Balfour 1963). imposed on overall shoot helical phyllotaxis. Geometrical properties such as these intersecting parastichies and other Primary vascular architecture in Arabidopsis: properties of leaf arrangement accurately predict the spatial positioning of vascular bundles within the stem. mature pattern The vascular bundles seen in any one cross section of the stem represent either individual leaf traces (4 in Fig. 1B) or Vegetative rosette the vascular sympodia that branch and give rise to the indi- Based on analysis of the patterns of ATHB8-GUS expres- vidual leaf traces (e.g., branches from the sympodium la- sion and of anatomically defined procambium and (or) vas- beled 9 in Fig. 1B give rise to the traces of leaves 9, 14, cular tissues, vascular architecture of the vegetative rosette and 17). The levels of branching of parent sympodia and of Arabidopsis is a closed, reticulate pattern that corresponds the divergence of leaf traces determine the number of vascu- closely to phyllotaxis (Figs. 1 and 2) (see Kang et al. 2003). lar strands observed in individual transverse sections, but the In vegetatively growing shoots with 20 or more leaves, the number in wild-type Arabidopsis is typically eight (Fig. 1B). most conspicuous contact parastichies are those connecting When individual vacular bundles are traced through succes- every third leaf (n + 3) and every fifth leaf (n +5)ofthe sive serial sections, they can be seen to branch and give rise ontogenetic helix (Fig. 1A). These contact parastichies ex- to the traces of leaves that are positionally related in the n + tend up the shoot axis in either a clockwise or a counter- 8 and n + 5 parastichies. For instance, the sympodium sup- clockwise direction (n + 5 and n + 3, respectively, in plying leaf 12 is derived from branches of bundles supplying Fig. 1A), depending on the overall orientation of shoot helical leaf 4 (antecedent in n + 8 parastichy) and leaf 7 (antecedent phyllotaxis (anticlockwise for the shoot illustrated in in n + 5 parastichy) (Fig. 2), forming an anastomosing pat- Fig. 1A). Individual rosettes with clockwise or anticlockwise tern and the closed pattern of primary vascular architecture helical phyllotaxis occur with approximately equal frequen- that characterizes Arabidopsis vegetative growth (Fig. 2). cies in Arabidopsis (Kang et al. 2003). Generally, three n +3 Thus, vascular architecture of the Arabidopsis shoot is a

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

highly integrated system in which the vascular supply to mation of leaf 9, which establishes the first n + 8 parastichy, each leaf is directly connected to antecedent leaves in two initiates the fully expressed pattern summarized in Fig. 2. different parastichies, providing alternative pathways for the long-distance movement of water and solutes. Inflorescence Although the fully expressed primary vascular architec- Upon the induction of reproductive growth, the SAM ini- ture is a closed, reticulate system, the formation of the anas- tiates floral meristems in place of leaves along the ontoge- tomosing procambial strands is not synchronous in that the netic helix of the vegetative rosette (Fig. 3). In Arabidopsis vascular connection with the n + 5 antecedent leaf lags be- plants grown under long-day inductive conditions, floral hind the n + 8 connection during development. When a leaf meristems are first formed at position 10, 11, or 12 (12 in trace is first detectable, it extends as a branch of the trace of Fig. 3A and 10 in Fig. 4; see Kang et al. 2003). At first, the its n + 8 antecedent leaf and terminates just below its corre- vascular architecture of the inflorescence extends the reticu- sponding primordium within the SAM (see Kang et al. late pattern of the vegetative rosette, with floral meristem 2003). Connections with the n + 5 antecedent leaf trace are traces derived from those of the antecedent organs in the formed three to four plastochrons later; thus, on a develop- n + 8 and n + 5 parastichies. For instance, in the inflores- mental basis, shoot primary vascular architecture is initially cence illustrated in Fig. 4, the sympodium supplying flower an open system but quickly becomes modified as a closed 12 is derived from those supplying leaves 4 (n + 8 para- reticulum (summarized in Fig. 2). stichy) and 7 (n + 5 parastichy). After formation of the sec- The closed, reticulate pattern of vascular architecture with ond flower in each n + 5 parastichy, however, vascular regular connections between the sympodia corresponding to architecture switches to an open pattern, with primary con- the n + 5 and n + 8 parastichies characterizes the adult nections along the n + 5 parastichies (e.g., flower 17, phase of shoot ontogeny. During the juvenile phase, how- Fig. 4). Although the basic pattern follows that of the vege- ever, phyllotaxis is subdecussate, and the traces to leaves 1 tative rosette, the inflorescence pattern differs in two spe- to 4 arise directly from the vascular cylinder of the hypoco- cific ways: (i) the flower trace connects with the trace/ tyls, as do those of the cotyledons (see Busse and Evert sympodium corresponding to the n + 5, not the n + 8 para- 1999; Kang et al. 2003) (Fig. 2). Branches from these traces stichy, and (ii) a second connection with the adjacent sym- anastomose to form the traces of the first-formed adult podium was not detected, resulting in a primarily open leaves, and connections are initially between the sympodia pattern of inflorescence vascular architecture (summarized corresponding to the n + 3 and n + 5 parastichies. The for- in Fig. 4). ATHB8-GUS expression within new procambial

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

strands is weaker in the inflorescence apex than in vegeta- connection with the n + 5 sympodium occurs later (P3 or tive apices, but the timing in relation to primordium forma- P4). Vascular connections between axillary buds and adja- tion and the longitudinal pattern of ATHB8-GUS expression cent vascular sympodia develop outside the SAM in older appears to be similar (Kang et al. 2003). tissues (depending on growth conditions), as do those that Although inflorescence vascular architecture is initially an connect the traces of developing flower buds with adjacent open system, secondary modifications reinstate the closed sympodia. These later developmental events are superim- reticulate nature of the shoot’s primary vascular architecture. posed on the initial primary pattern and presumably require Accessory traces form bridges between individual flower comparable developmental signals and signaling pathways, bud traces and the adjacent n + 3 and n + 5 sympodia although the developmental environment in terms of overall (Fig. 4). Such accessory traces are formed relatively late tissue differentiation differs from that within the SAM. The (usually 10 or more plastochrons after floral meristem initia- progressive elaboration of the primary vascular system al- tion), are narrow in diameter relative to the floral meristem lows plants to respond appropriately to variation in the traces, and have a horizontal course. growth environment in that the pattern may be arrested at a simple phase when plants are short-lived or elaborated when Axillary buds growth is prolonged. Under some growth conditions, secon- Upon transition from the vegetative to the reproductive dary vascular development occurs within the rosette and the phase, axillary buds are initiated basipetally, starting with basal portion of the inflorescence axis (Altamura et al. 2001; the cauline leaves (Long and Barton 2000). Axillary bud Chaffey et al. 2002), replacing the primary vascular system meristems initially lack detectable vascular bundles, but the functionally. appearance of two short procambial strands is coincident The basic features of vascular development described for with formation of the first two leaf primordia on the axillary the rosette plant Arabidopsis on the basis of ATHB8-GUS shoot (see Kang et al. 2003). These procambial strands are expression correspond to descriptions of elongate shoots in not isolated but appear to be continuous as branches of ei- other species studied, most notably for Linum usitatissimum ther the leaf trace alone (most rosette leaves) or the adjacent L. (Linaceae) (Girolami 1953), Hectorella caespitosa vascular sympodia (cauline leaves) or a combination of the J.D. Hooker (Portulaceae) (Skipworth 1962), and Populus two patterns (summarized in Fig. 4; Kang et al. 2003). deltoides Bartr. ex Marsh. (Saliceae) (Larson 1975). In her- Thus, the vascular connections for the primary inflorescence baceous Linum and Hectorella, primary vascular architecture branches associated with the cauline leaves are well inte- is a closed, reticulate system with single acropetally devel- grated with at least three vascular sympodia, providing a oping leaf traces connected to vascular sympodia that corre- built-in redundancy of vascular pathways supplying the spond to parastichies. As these shoots mature, the numbers flowers and developing siliques born on those branches if of parastichies (and the numbers of intervening leaves along individual vascular bundles are damaged. a parastichy: n + 13, n + 21, etc.) and vascular sympodia in- The stepwise elaboration of the primary vascular system crease, just as seen for the juvenile to adult transitions in in vegetative and reproductive shoots of Arabidopsis in- Arabidopsis. In woody Populus, vascular architecture is a volves the SAM itself as well as older regions of the stem. more complex, open system with three traces per leaf, yet Formation of the leaf trace (derived from the n + 8 sympo- procambial strands develop acropetally and precisely ac- dium) occurs during the first plastochron (P1), while the cording to phyllotaxis (Larson 1975, 1977). In Populus, vas-

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Fig. 4. Idealized two-dimensional diagram representing primary vascular architecture in the Arabidopsis inflorescence. Foliage leaves are represented by squares and flowers by hexagons, numbered according to the ontogenetic helix; symbols represent the approximate level of the primordium base. Axillary buds (triangles) are associated with cauline leaves 7–9 and with rosette leaves 3–6. After the second flower in each n + 5 parastichy, traces are derived as branches of vascular sympodia supplying antecedent flowers in the n + 5 parastichy, forming an open pattern. Accessory bundles (yellow) connect flower traces with adjacent sympodia after seven or more plastochrons. Axillary bud procambial 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.

culature may be elaborated secondarily by the formation of tinuous, acropetal nature of procambial strand formation ap- accessory bundles that provide additional connections be- pears to be general for the dicotyledons, virtually without tween leaf traces and adjacent vascular sympodia (Larson exception (Esau 1965a, 1965b) and has been previously re- 1980a, 1980b). ported for Arabidopsis (Vaughan 1955; Busse and Evert 1999). Development of vascular architecture and Although procambial strands within the vegetative and re- expression of ATHB8 productive shoot apical meristems of Arabidopsis appear to be continuous with antecedent procambial strands, the zone Despite the progressive elaboration of primary vascular of ATHB8-GUS expression is initially discontinuous (Kang architecture within rosette and inflorescence stems of Arabi- et al. 2003). The longitudinally oriented narrow files of cells dopsis, the developmental steps taking place within the below each new primordium express the ATHB8-GUS con- SAM itself are similar during both vegetative and reproduc- struct strongly, while ATHB8-GUS activity initially is not tive phases. Both leaf primordia and floral meristems are detectable at the basal end where these strands curve to con- supplied by a single trace that appears to develop acrope- nect with the antecedent parent strand. This stage is ephem- tally and in continuity with pre-existing vasculature and is eral, as ATHB8-GUS expression becomes established positioned precisely in relation to earlier-formed vascular throughout the length of the leaf trace as the procambial bundles and to the placement of newly formed lateral organ strand grows in diameter. Such a pattern could indicate that primordia. Based on ATHB8 expression, procambial strand ATHB8 expression is induced by a basipetally moving signal (or at least preprocambial strand) formation appears to be and that the discontinuity reflects a moving front that inter- coincident with the initial formation of an externally detect- acts with acropetally moving signals, possibly traveling in able primordium (P1), although detection of primordia from the phloem (reviewed in Lough and Lucas 2006). This idea serial cross sections may have identified only slightly older is highly speculative, however, as the function of ATHB8 is primordia (P2 or P3; Kang et al. 2003). During the vegeta- still not established; for instance, ATHB8 expression might tive stage of development, procambial strands are continu- simply be a marker of acquisition of xylem cell identity and ous (based on a combination of anatomical criteria and only presages the discontinuous nature of xylem differentia- ATHB8 expression), with the trace/sympodium procambial tion (Kang et al. 2003). strand corresponding to the n + 8 parastichy. Although this pattern shifts subtly in the inflorescence (connections corre- An auxin model that integrates phyllotaxis spond to the n + 5 parastichy), the formation of new strands follows a highly predictable pattern with no indication of a and vascular development stochastic component to linkages between strands. The con- The SAM generates regular, predictable patterns of leaves

# 2006 NRC Canada Dengler 1667 and other lateral organs that are generally robust to experi- tions that anticipate primordium formation by one to three mental manipulation (e.g., Reinhardt et al. 2003, 2005; plastochrons (Reinhardt et al. 2003), as is AUXIN RESIST- Smith et al. 2006). Phyllotaxis and the three-dimensional ar- ANT 1, a putative auxin influx carrier (Stieger et al. 2002; chitecture of the vasculature supplying the lateral organs are Benkova´ et al. 2003). Expression of the reporter construct highly coordinated in their development. Although the na- DR5-GFP, thought to reflect the endogenous auxin concen- ture of this coordinated development is not yet fully under- tration (Benkova´ et al. 2003), also is restricted to the L1 stood, current models point to mechanisms that regulate the layer of the SAM and expression peaks in incipient primor- positioning of leaf and floral primordia and of their vascular dia (Smith et al. 2006). The phyllotactic pattern of AT- supply in an integrated manner. Earlier models of the regu- PIN1 accumulation is disrupted in the mutants pin1, lation of phyllotaxis fall into two broad categories: in some pinoid, and monopteros, indicating that auxin signaling (i), interactions among primordia on the flanks of the shoot and transport are required for correct PIN1 localization apical meristem are thought to determine the placement of (Reinhardt et al. 2003). Fifth, ATPIN1 protein is dynami- leaves and flowers, while in others (ii), inductive signals cally redistributed during each plastochron: ATPIN1 polar- from older portions of the shoot are thought to play a role ity is first directed toward the center of the presumptive (Larson 1983; Jean 1994; Lyndon 1998; Reinhardt and primordium (I1 in Fig. 5), but with outgrowth, L1 layer Kuhlemeier 2002; Reinhardt et al. 2003). The nature of in- expression decreases and a narrow file of internal cells be- teractions among primordia is thought to be either biophysi- gins to accumulate ATPIN1 with a basipetal polarity. cal (e.g., Green 1996) or biochemical (e.g., Mitchison 1977; (Reinhardt et al. 2003; Heisler et al. 2005; P1 in Fig. 5). Schwabe 1984) and to display features of a lateral inhibition Live imaging of ATPIN1 distribution within the Arabidop- system in which new primordia are placed at the maximum sis floral meristem also shows that ATPIN1 concentration distance possible from the ‘‘inhibitory’’ antecedent primor- is highest in the I3, I2, and I1 presumptive floral meristem dia on the flanks of the meristem (Lyndon 1990; Meinhardt sites, when the polarity of surface cells adjacent to older, 1996). Observations that the placement of new primordia antecedent primordia is strongly directed toward the center merely reiterates that of older portions of the shoot and that of the presumptive primordium (Heisler et al. 2005). With developing vascular strands sometimes precede the external the outgrowth of the primordium, ATPIN1 becomes local- appearance of the primordia that they will supply have led ized to the basal ends of the internal cells forming a nar- to suggestions that inductive signals move acropetally row file (Heisler et al. 2005). Thus, the positive induction through the vascular system (discussed in Esau 1965a, of primordium position and vascular strand position by the 1965b; Larson 1983; Lyndon 1990). Current evidence, localized regulation of auxin concentrations could provide however, strongly supports a model in which the phyllotac- a single mechanism that regulates both phyllotaxis and vas- tic pattern is generated within the SAM through biochemi- cular architecture, much as hypothesized by Esau (1965a). cal interactions of pre-existing primordia, specifically The auxin model provides a mechanism for the formation through regulation of local variation in auxin concentration. of leaf trace procambial strands in a pattern that is coordi- The auxin model postulates that auxin concentrations act, nated with phyllotaxis, but almost all aspects of how the ini- not as inhibitors of primordium development, but as active tial pattern of ATPIN1 distribution is translated into the effectors of a specific developmental sequence of events complex three-dimensional vascular architecture of the shoot (Reinhardt et al. 2003; Reinhardt 2005; Fleming 2004, are unknown. In many ways, the localization of ATPIN1 2005; Heisler et al. 2005; Smith et al. 2006; Jo¨nsson et al. protein to an internal strand of cells during the early plasto- 2006). The key features of this model (summarized in chrons of primordium development (Reinhardt et al. 2003; Fig. 5) are (i) uniform acropetal movement of auxin in the Heisler et al. 2005) is comparable with the generation of surface (L1) layer of the apical meristem from regions out- the two-dimensional, yet complex, pattern of leaf veins. In side the SAM, (ii) formation of foci of auxin concentration developing leaves, PIN1 is expressed first in the protoderm in positions that presage the position of primordia through (derived from the L1 layer) and PIN1-GFP protein is local- the polar localization of ATPIN1 proteins, and (iii) redistrib- ized subcellularly to the acropetal side of protodermal cells, ution of ATPIN1 polarity upon the outgrowth of primordia producing a convergence point at the apex (Scarpella et al. so that polar auxin movement is directed toward the interior 2006); thus, the pattern observed for the SAM is preserved of the meristem, along a narrow file of cells in the position during early stages of leaf development. Internal cells adja- of the future midvein procambial strand. Support for this cent to the apical convergence point accumulate PIN1-GFP model comes from a number of sources. First, when polar at the basal side of the cells, and the zone of expressing auxin transport is inhibited by pharmacological treatments, cells extends towards the base of the leaf, forming the mid- primordium formation is suppressed (Reinhardt et al. 2000; vein prepattern (Scarpella et al. 2006). The looped secon- Stieger et al. 2002; Benkova´ et al. 2003). Second, as shown dary veins are generated similarly, starting with a for inhibitor-treated tomato shoot tips or pin1 mutants of convergence point at the leaf margin and with a series of Arabidopsis, suppression of primordium formation can be cells expressing PIN1-GFP extending toward the midvein. reversed by localized auxin treatment (Reinhardt et al. These cells accumulate PIN1-GFP on the side toward the 2000). Third, the auxin efflux carrier protein ATPIN1 accu- midvein, suggesting that auxin moves along the incipient mulates in the L1 layer of the meristem, specifically on the vein from a source at the margin to the sink represented by acropetal side of individual cells, indicating that net move- the earlier-formed strand (Scarpella et al. 2006). ment of auxin is toward the center of the SAM (Benkova´ Strands of preprocambial tissue are polar in their develop- et al. 2003; Reinhardt et al. 2003; Heisler et al. 2005). ment and extend unidirectionally from pre-existing strands; Fourth, ATPIN1 protein accumulation is strongest in posi- if strand extension is arrested, a ‘‘freely ending veinlet’’ is

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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 vegetative and 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 anatomically defined procambium (pink) is consistently present, connecting it to the antecedent procambial strand associated with a leaf that is eight plastochrons older (vegetative SAM).

formed. During the development of most of the secondary cipient leaf primordium (Reinhardt et al. 2003; Heisler et al. and higher-order venation, however, strand extension rapidly 2005) or in the expression of ATHB8 within leaf traces connects with an adjacent strand, thus forming a continuous (Kang et al. 2003). An important distinction between the link between pre-existing strands (Scarpella et al. 2004, two is that while leaf vein pattern development points to a 2006). The prepatterns represented by PIN1-GFP, the auxin highly flexible self-organizing patterning mechanism (Scar- reporter DR5-GUS, the gene trap marker ET1335-GUS, or pella et al. 2006), shoot vascular architecture appears to be ATHB8-GUS all appear when the leaf (or field of tissue for highly predictable, with almost no stochastic element to the higher-order veins) consists of relatively few cells and is ex- formation of connections between strands (Kang et al. 2003). tended by intercalary growth as the leaf enlarges (Mattsson et al. 2003; Kang and Dengler 2004; Scarpella et al. 2004, Future prospects 2006); thus, the stage of discontinuity and unidirectional growth is ephemeral (except for the freely ending veinlets). Plants are increasingly regarded as supracellular organ- The cytological features of cells expressing these markers isms in which long-distance transport of, not only water and are indistinguishable from those of cells of the ground tissue dissolved nutrients, but also of macromolecules and other in which this preprocambial pattern arises; only later do the signaling substances depends on the vascular system (Lough distinctive elongate shape and dense cytoplasm of anatomi- and Lucas 2006). Knowledge of the three-dimensional archi- cally defined procambial cells emerge (Mattsson et al. tecture of the vascular system, and particularly how it corre- 2003; Kang and Dengler 2004; Scarpella et al. 2004, 2006). sponds to leaf position on the shoot, is essential for Thus, elements of pattern formation are shared by develop- understanding how photosynthate moves through the phloem ment of both two-dimensional leaf vein patterns and three- from specific source to specific sink leaves or how chemical dimensional shoot vascular pattern: (i) unidirectional defense signals might move from herbivore-damaged leaves elaboration of the system that may reflect auxin transport to particular newly expanding ones (e.g., Larson 1977; from localized sources to sinks within the tissue, (ii)forma- Marshall 1996; Orians and Jones 2001). For instance, in tion of major elements of the system, such as leaf primary poplar, when 14C label was supplied to a photosynthesizing and secondary veins or the n + 8 sympodial strands of the source leaf, it was possible to predict where the relative stem followed by formation of minor pattern elements, such percentages of the labeled photosynthates would appear as the higher order venation or the n + 5 sympodial strands, based on knowledge of the connections of central and lat- and (iii) discontinuities in the accumulation pattern of certain eral leaf traces with adjacent vascular sympodia and of the proteins, such as that observed for PIN1 expression in an in- transitions in phyllotaxis appearing during shoot ontogeny

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(Larson 1977). Analyses of phloem sap and use of grafting cambial prepattern that connects a source of auxin with tis- between stocks and scions of different genotype have dem- sues acting as a sink and that reiterations of the process onstrated that numerous macromolecules function in long- create a complex hierarchical pattern (Scarpella et al. distance communication and use phloem tissue as a conduit 2006). Although such concentrations only have been shown (reviewed by Lough and Lucas 2006). One example of to occur in the L1 layer of the SAM and the protoderm of these is the FLOWERING LOCUS T protein, a component developing leaves, a similar process is likely to occur within of a phloem-mobile florigenic signal. Induction of gene ex- internal ground tissues and would be integral to the genera- pression in a source leaf by heat shock results in accumu- tion of the three-dimensional vascular pattern of shoots and lation of FLOWERING LOCUS T mRNA in both the to its secondary modification during development. source leaf and the SAM, indicating that the mRNA (or perhaps the protein) moves along the vascular sympo- Acknowledgments dium(a) connecting the source leaf and the primordia clos- est to the SAM (Huang et al. 2005). Although many I thank Thomas Berleth, Julie Kang, Bill Remphrey, and environmental and developmental stimuli may not be local- two anonymous reviewers for helpful comments on the ized, so that signaling moves through the entire shoot vas- manuscript, Janice Wong for illustrations, and the Natural culature and affects leaves of all parastichies equally, many Sciences and Engineering Research Council of Canada for triggers will be specific to regions of the shoot or root sys- research support. tem, and vascular architecture will contribute to determining which targets will be reached by those signals. References Knowledge of shoot primary vascular architecture of Ara- Adler, I., Barabe´, D., and Jean, R.V. 1997. A history of the study bidopsis, specifically, might form the basis for better under- of phyllotaxis. Ann. Bot. (Lond.), 80: 231–244. standing aspects of the developmental biology of this model Altamura, M.M., Possenti, M., Matteuci, A., Baima, S., Ruberti, I., organism. The information described herein is especially and Morelli, G. 2001. Development of the vascular system in the useful for the interpretation of mutations that affect the vas- inflorescence stem of Arabidopsis. New Phytol. 151: 381–389. cular system, for instance the supernumerary vascular bun- doi:10.1046/j.0028-646x.2001.00188.x. dles that appear in mutants such as REVOLUTA (Zhong et Baima, S., Nobili, F., Sessa, G., Lucchette, S., Ruberti, I., and Mor- al. 1999) or the effects of mutants in other HD-ZIP family elli, G. 1995. The expression of the Athb-8 homeobox gene is genes on shoot vascular pattern as well as ground scleren- restricted to provascular cells in Arabidopsis thaliana. Develop- chyma (Prigge et al. 2005). Expression patterns of genes ment, 121: 4171–4182. PMID:8575317. such as ATHB8 or DR5 that are restricted to developing Baima, S., Possenti, M., Matteucci, A., Wisman, E., Altamura, M.M., Ruberti, I., and Morelli, G. 2001. 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