Initiation of Axillary and Floral Meristems in Arabidopsis

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Developmental Biology 218, 341–353 (2000)doi:10.1006/dbio.1999.9572, available online at http://www.idealibrary.com on

Initiation of Axillary and Floral Meristemsin Arabidopsis

Jeff Long* and M. Kathryn Barton*,†,1

†Department of Genetics and *Program in Cell and Molecular Biology, Universityf Wisconsin at Madison, 445 Henry Mall, Madison, Wisconsin 053706

hoot development is reiterative: shoot apical meristems (SAMs) give rise to branches made of repeating leaf and stem unitsith new SAMs in turn formed in the axils of the leaves. Thus, new axes of growth are established on preexisting axes. Heree describe the formation of axillary meristems and floral meristems in Arabidopsis by monitoring the expression of theHOOT MERISTEMLESS and AINTEGUMENTA genes. Expression of these genes is associated with SAMs and organrimordia, respectively. Four stages of axillary meristem development and previously undefined substages of floral meristemevelopment are described. We find parallels between the development of axillary meristems and the development of floraleristems. Although Arabidopsis flowers develop in the apparent absence of a subtending leaf, the expression patterns ofINTEGUMENTA and SHOOT MERISTEMLESS RNAs during flower development suggest the presence of a highly

educed, “cryptic” leaf subtending the flower in Arabidopsis. We hypothesize that the STM-negative region that developsn the flanks of the inflorescence meristem is a bract primordium and that the floral meristem proper develops in the “axil”f this bract primordium. The bract primordium, although initially specified, becomes repressed in itsrowth. © 2000 Academic Press

Key Words: meristem; bud; SHOOT MERISTEMLESS; Arabidopsis; AINTEGUMENTA.

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INTRODUCTION

The shoot apical meristem (SAM) of seed plants is a smallmound of cells located at the tip of the growing shoot. Theprimary SAM forms during embryogenesis with additionalSAMs forming after embryogenesis on the body of the plant.The outgrowth of the resulting buds gives plants theircharacteristic branching growth habit. New SAMs typicallydevelop in the axils of leaves—the axil is the junctionbetween leaf and stem—and for this reason we shall refer tothem as axillary meristems. In vegetatively growing Arabi-dopsis plants, axillary SAMs form in a gradient from thebase of the plant upward with new SAMs forming first inthe axils of the oldest (i.e., earliest formed) leaves (Grbic andBleecker, 1996).

Several observations indicate that the subtending leafplays an important role in the development of the axillarybud. In Arabidopsis, the axillary meristem develops in close

1 To whom correspondence should be addressed at the Depart-ment of Genetics, 445 Henry Mall, University of Wisconsin atMadison, Madison, Wisconsin 53706. Fax: (608) 262-2976. E-mail:
[email protected].
0012-1606/00 $35.00Copyright © 2000 by Academic PressAll rights of reproduction in any form reserved.

hysical association with the adaxial base of the subtendingeaf (Talbert et al., 1995). (The adaxial side of the leaf is theide toward the long axis of the shoot or the “top” of theeaf. The abaxial side is the side away from the shoot or thebottom” of the leaf.) Consistent with this, the axillaryeristem and subtending leaf are clonally related, sharing a

ommon ancestor (Furner and Pumfrey, 1992; Irish andussex, 1992). Moreover, when abaxial leaf fates are trans-ormed into adaxial fates, as in the Arabidopsis phabulosa

utant, ectopic meristems arise on the undersides of leavesMcConnell and Barton, 1998). One explanation for theresence of ectopic meristems in the phb mutant is that

positional cues associated with adaxial, basal leaf fate aresufficient to direct the formation of axillary SAMs. Theimportance of the subtending leaf in the development ofaxillary meristems in other dicot species has been suggestedby surgical experiments in which the subtending leaf wasexcised (Snow and Snow, 1942). In such plants, no axillarySAM developed.

Additional evidence suggests that the adaxial leaf pos-sesses a special competence to form meristems. WhenKNOTTED-like genes are overexpressed in dicots, ectopic
meristems arise but only on the adaxial leaf surface (Sinha
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et al., 1993). More recently, the expression pattern of theArabidopsis PINHEAD (PNH) gene in the adaxial half of theeaf primordium as well as in the SAM indicates that theAM and the adaxial leaf domain comprise a unit of sharedositional identity in the shoot (Lynn et al., 1999). Since theNH gene is required for efficient initiation of SAMs andince it is preferentially expressed in the adaxial portion ofhe developing leaf, we have hypothesized that the PNH

gene confers meristem forming competence to the adaxialleaf domain.

In apparent contradiction to the notion that the adaxialleaf domain plays an important role in directing meristemformation, some meristems form in the absence of a sub-tending leaf. Whereas, in most species, the floral meristemdevelops in the axil of a specialized leaf called a bract, inArabidopsis, as in nearly all members of the Brassicaceae,

oral meristems lack subtending bracts. Instead, the Ara-bidopsis SAM, upon receiving a stimulus to flower, appearsto switch from making leaves to making floral primordia.

The discovery of genes expressed in the meristem and inearly organ primordia has greatly aided the study of meris-tem structure and function. The SHOOT MERISTEMLESS(STM) gene is required for SAM initiation and maintenanceBarton and Poethig, 1993; Clark et al., 1996; Felix et al.,1996; Endrizzi et al., 1996). The STM gene encodes ahomeodomain-containing protein of the KNOTTED classand is expressed in the SAM founder cells in the embryo(Long et al., 1996; Long and Barton, 1998). The STMtranscript remains expressed throughout the SAM duringthe life span of the plant. When cells are specified as leafprimordia, STM expression is extinguished. The STM tran-script is found in all types of SAMs: primary, axillary, andfloral. Thus, STM can be used as a marker of SAM fate, evenarly in the development of the SAM. The AINTEGU-ENTA (ANT) gene has a near-reciprocal expression pat-

FIG. 1. Serial, transverse sections of a vegetative meristem showingSections are 8 mm thick and are ordered from most apical (A) to most bo midpoint of section is given in bottom right-hand corner of each iicrograph of a shoot apical meristem grown under similar condition

ection through summit of SAM. Only a small portion of the meristento this section. The most distal part of P3 is present in this section

eristem. P4 curves over the meristem to about its midpoint. P5 anection. (B) Section 11 mm below summit of SAM. STM-negative regetectable in the meristem at this level. (C) Section 19 mm belowTM-negative regions that are continuous with the SAM. P1 extends2 is also apparent in this section and shows that a cleft has develrimordium (P 21) is expected to form. (D) Section 27 mm below summote, in this and the section shown in F, the difference in the shapeeristem (STM expressing) (white arrows). This boundary is more rou

2). (E) Scanning electron micrograph of a vegetative meristem from and conceal the SAM have been removed. (F) Section 35 mm below su

the SAM the large pith cells that are below the SAM begin to be seen. (Hof STM, indicating development of a stage 3 axillary meristem at the a(white arrow). The STM-negative region associated with P0 extends

Copyright © 2000 by Academic Press. All right

ern in the meristem (Elliott et al., 1996). It is expressed inery young organ primordia throughout the plant.Here we describe the early development of axillary and

oral meristems by monitoring the expression of the STMnd ANT genes. This allows us to describe early steps ineaf, axillary meristem, and floral meristem development.his study provides a baseline of normal developmentgainst which the development of mutants can later beompared. This study also reveals similarities between theevelopment of axillary and that of floral meristems anduggests the presence of a highly reduced leaf subtendinghe floral meristem in Arabidopsis.

MATERIALS AND METHODS

Growth conditions. Plants were grown in short days (8 h light,16 h dark) under fluorescent light at 24°C in Metromix 200 (Grace,Sierra). For experiments in which plants were induced to flower bymoving them to long days, short-day-grown plants were moved to24 h light, 24°C.

Histology. Plants were fixed in 4% paraformaldehyde, dehy-drated in an ethanol series, and embedded in Paraplast Plus.Eight-micrometer-thick sections were cut using a Reichert–Jungmicrotome. In situ hybridizations to antisense probes for the STMand ANT genes was carried out as in Long and Barton (1998).

RESULTS

Early Leaf Development

Wild-type Arabidopsis thaliana plants of ecotype Lands-berg erecta were grown under short-day conditions, fixedbetween 23 and 25 days after germination, and sectioned.The resulting serial sections were hybridized to an anti-sense STM probe. Plants were grown under short days so

RNA accumulation in the meristem and down-regulation in leaves.(H); approximate distance (in micrometers) from summit of meristem. Primordia are labeled 0, 1, 2, etc., in order of increasing age. E is aobserved with the scanning electron microscope for comparison. (A)

me is present in this section (arrow). P0, P1, and P2 do not extend uptypically grows straight upward and does not curve inward over thealso curve over the meristem but extend farther apically than this

hat indicate the presence of leaf primordia P0, P1, and P2 are barelyit of SAM. Both P0 and P1 are evident in the SAM as two small

r laterally from the meristem than does P0. The uppermost region ofto separate P2 from the SAM. Arrow indicates site at which next

f SAM. This section is at the level of the insertion point of P2 (arrow).boundary between cells of the presumptive leaf (STM negative) andin young primordia (e.g., P0) and more linear in older primordia (e.g.,ay-old plant grown in short days. Older leaves that normally overtopt of SAM. (G) Section 43 mm below summit of SAM. At the center ofction 51 mm below summit of SAM. Arrow shows stronger expressionl base of P9. The large pith cells below the SAM are readily observedone section below this. This section is just below the level of the

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343Axillary and Floral Meristems in Arabidopsis

Copyright © 2000 by Academic Press. All rights of reproduction in any form reserved.

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344 Long and Barton

that they would produce leaves over an extended vegetativeperiod. The data were collected from transversely sectionedmaterial because this allows all leaf primordia to be ob-served from the same orientation. Eight of the fifteen plantsprocessed exhibited both good preservation of morphologyand strong, clear signal of the STM probe and were used inthe analysis.

The growing shoot tip consists of a population of self-renewing cells and leaves along a continuum of develop-mental stages. The self-renewing “initial” cells reside inthe central zone of the meristem. Some of the descendantsof these cells are pushed outward to the peripheral zone ofthe meristem where they give rise to leaf primordia. Theexact point at which a leaf should be considered separatefrom the SAM, and therefore the outer limit of the SAM, isdifficult to determine. In this study, the SAM is taken tomean the mound of cells apical to the first morphologically

FIG. 2. Longitudinal sections through vegetative shoot apical merposition at which next leaf primordium (P 21) is expected to arise.STM transcript. P4 curves gently over the SAM reaching almost to iThe arrowhead points to the large pith cells that are located just unf P 21. P0 is morphologically indistinguishable from the SAM. (Cxtends apically just above summit of SAM. It is clearly morpholo

distinct leaf.


The SAMs of short-day-grown Arabidopsis plants formroughly triangle-shaped mound of cells (Fig. 1E). Within

he SAM, STM is expressed in both central and peripheralones (Fig. 1). In the peripheral zone, regions that lackTM expression (referred to as STM-negative regions) are

found at positions expected for new leaves —at an angleof about 137° relative to the next youngest primordium.Given the positions of the STM-negative regions andtheir developmental continuity with older leaf primordia,we conclude that they represent early steps in the devel-opment of leaf primordia. In keeping with the nomencla-ture used by Smith et al. (1992) and Jackson et al. (1994),who have described a similar pattern of expression of thehomologous maize KNOTTED gene, we call the youngestof these primordia P0 and the older P1. While P0 isalways completely contained within the SAM, in somecases P1 is distinguishable as a lateral bulge on the SAM

s showing stages P 21 to P4 of leaf development. (A) Arrow showsis section, there is not yet any indication of down-regulation of thedpoint. The adaxial surface of P4 is in close contact with the SAM.ath the SAM. (B) Arrow shows down-regulation of STM at positionate P1/early P2 primordium bulges laterally from the SAM. (D) P3ly distinct from the SAM. Scale bar, 50 mm.

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The youngest primordium, P0, is found central to andbetween P3 and P5 (Fig. 1). P0 extends apically to near thesummit of the SAM and basally to just below the insertionpoint of P5 (Figs. 1 and 2). In fact, for all leaf primordiaexamined (P0 through P5), the STM-negative region extendsbasally into the stem to just below the insertion point of theleaf five plastochrons older than itself. So, for instance, theSTM-negative region associated with P1 extends basallyinto the stem to just below the insertion point of P6. The P0primordium (as detected by lack of STM transcript) is ovaln cross section in apical regions and more circular in crossection in basal regions (compare P0 in Figs. 1D and 1H).he more basal STM-negative regions are at positionsredicted to encompass the developing vascular traces.P1 extends apically to about the same level as P0 but

xtends farther peripherally, or abaxially, than does P0Figs. 1 and 2). While a morphological separation between1 and the rest of the SAM is not obvious in all samples, inome cases, P1 appears as a slight bulge or “shoulder” off ofhe SAM (Figs. 1F and 2C).

The third youngest primordium, P2, is easily distinguish-ble from the SAM as a cleft has formed between it and theAM. P2 extends apically, or upward, in addition to out-ard (Figs. 1 and 2). Its tip does not reach above the summitf the apex.P3 extends farther apically and reaches 10 to 20 mm above

he summit of the apex (Figs. 1 and 2). A very clear cleft isresent between P3 and the SAM.Finally, P4 overtops and curves to about the midpoint of

he SAM. Its adaxial surface may be in very close contactith the SAM, almost appearing to rest on it (Fig. 2). This

tage is defined as stage 1 of leaf development by Carlandnd McHale (1996).Progressively older primordia are less curved over the

AM and ultimately stand straight up (Carland andcHale’s stages 2 through 3 of leaf development). Finally atarland and McHale’s stage 4 of leaf development, the leafends outward and away from the plant.To summarize, initial growth of the leaf primordium is

utward as a bulge from the meristem. In the P2 and P3rimordia, growth is principally upward. Unequal growth ofhe two sides of the primordium (i.e., the ad- and abaxialides) causes the primordium to first curve over the meri-tem (growth is faster on the abaxial side) and later to curveway from the meristem (growth is faster on the adaxialide).

Morphological differences along the ad/abaxial dimen-ion are not apparent until P2, when the cells on the abaxialide of the primordium are larger than those on the adaxialide (Fig. 1). This differential enlargement is at least partlyesponsible for the shift in primordium growth from pri-arily outward to primarily upward and ultimately to the

urvature of the primordium over the top of the SAM. Theifference in cell size is even more exaggerated in the P3rimordium.In one sample a small region of light, patchy STM

xpression was observed at the position at which the next


eaf primordium, P 21, is expected to form (Fig. 2B). Thisas the earliest the P 21 primordium could be detected by

the criterion of STM down-regulation. In general the bound-ary between STM-expressing and -nonexpressing cells ismore diffuse and rounded in younger primordia than inolder primordia in which it becomes more defined andlinear. For example, the adaxial boundary between STM-expressing and -nonexpressing cells in P1 is less roundedthan in P0 (Fig. 1D) and in P2 the junction betweenSTM-expressing and -nonexpressing cells has resolved intoa nearly linear boundary in cross section.

Development of Axillary Meristems

Axillary meristems develop at the junction between thestem and the leaf base. Although the outwardly directedgrowth of the axillary branch gives it the appearance ofemanating from the stem of the plant, examination of earlydevelopment of the axillary meristem indicates that it

FIG. 3. Development of axillary meristems. All sections arelongitudinal sections through the midpoint of the axil. The leaf isto the left and the meristem (or stem) is to the right. (A) Stage 1 axil.STM expression does not extend above the insertion point of theleaf. (B) Stage 2 axil. STM expression extends above the insertionoint of the leaf but no “bump” has formed. (C) Stage 3 axil. Aorphologically detectable bump has formed but it does not yet

ontain any STM-negative regions. (D) Stage 4 axil. The developingud has two clear STM-negative regions (arrow), indicating theresence of leaf primordia. Scale bar, 50 mm.

develops in close association with the leaf base and indeed


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346 Long and Barton

initially is directed toward the center of the plant (Fig. 3D).We have divided axillary meristem development into fourstages described below.

Stage 1 axils show STM expression only at the base of theaxil itself (Figs. 1, 2, and 3A). This expression is initiallyspread along the entire adaxial surface of the primordium atthe insertion point (e.g., P2 in Fig. 1F) but in later leavesbecomes increasingly focused into a medial area of the leafaxil (e.g., P4 in Fig. 1G).

Stage 2 axils show expression of STM transcript on thedaxial leaf base just above the insertion point of the leafut show no morphological indications of bud formationFig. 3B). The leaf in which stage 2 axils were first detectedanged from P8 to P 10 (Table 1) in the eight meristemsxamined. This stage was usually found in one to twoeaves per rosette and therefore appears to last for about oneo two plastochrons.

Stage 3 axils have morphologically distinguishable

IG. 4. Serial, transverse sections of an inflorescence meristemegulation in floral primordia. Sections are 8 mm thick and are ordicrometers) from summit of meristem to midpoint of section is

abeled 0, 1, 2, etc., in order of increasing age. The plant from whichor 25 days and then under long-day conditions for 7 days prior toimilar conditions and observed with the scanning electron microsarrow). A small portion of FPO, the youngest floral primordium ineen in this section are cauline leaves 2 and 3 (L2 and L3) and floralnd more basal sections, down-regulation associated with two floras observed. We have dubbed these stage 0 primordia. The next flsterisk. (C) Section 19 mm below summit of IM. (D) Section 27 mmhe IM. As in leaf primordia, the boundary between the STM-negaounded and somewhat diffuse in younger FPs (e.g., FP0–2) and becolectron micrograph of an inflorescence meristem grown under cond

ABLE 1tages in Axillary Meristem Development in Relation to Rosette L

Meristema Stage 1b Stage 2c

A P8, P9 P10B P7 P8C P8, P9 P10D P8 P9E P8 P9, P10F P8, P9 P10G P8 P9 (P10 not determH P8, P9 P10

a Letter identifies meristem sampled.b Stage 1 axils exhibit STM expression at insertion point of leaf.c Stage 2 axils exhibit STM expression on adaxial left base, abovd Stage 3 axils exhibit STM expression above insertion point. Axe Stage 4 axillary meristems have begun to produce leaf primordia

f ANT expression.

rom youngest (0) to oldest (9). (F) Section 35 mm below summit of IM. I


umps on the adaxial leaf base that exhibit strong, uniformxpression of STM transcript (Fig. 3C). No STM-negativeegions are found within the stage 3 axillary meristems.tage 3 axillary meristems were first seen in P9 through P11nd were typically seen in two primordia per plant, indicat-ng that this stage lasts about two plastochrons.

Stage 4 axillary meristems appear as morphologicallyetectable bumps that exhibit strong STM expression andlso show STM-negative regions, indicative of the forma-ion of leaf primordia (Figs. 3D and 4K). Stage 4 axillaryeristems were first observed in the axils of leaf primordia

1 through 13 (Table 1).Thus, in vegetatively growing Arabidopsis plants, axil-

ary meristems form in older leaves before they form inounger leaves. However, when the plant receives a floralnduction stimulus, the direction of axillary bud develop-

ent changes such that the youngest leaves are the first toevelop axillary meristems (Hempel and Feldman, 1994).

showing STM RNA accumulation in the meristem and down-from most apical (A) to most basal (L); approximate distance (inin bottom right-hand corner of each image. Floral primordia are

nflorescence was taken had grown first under short-day conditionsion. E is a micrograph of an inflorescence meristem grown underfor comparison. (A) Uppermost section of inflorescence meristemch STM is down-regulated, is just detectable in this section. Alsoordia 8, 10, and 11. (B) Section 11 mm below summit of IM. In this

ordia, FP 0 and FP1, that are not yet distinct from the meristem,primordium (FP 21) will develop at the position marked by theow summit of IM. Stage 2 primordia (FP2–4) bulge outward fromregions associated with the developing FP and the IM is initiallymore linear and defined in older primordia (e.g., FP4). (E) Scannings similar to those shown in other images. Primordia are numbered

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Stage 3d Stage 4e

P11, P12P9, P10 P11P11, P12 P13P10, P11, P12 (P13 not determined)P11, P12 (P13 not determined)P11, P12 P13

) P11 P12P11, P12 P13, P14

ertion point of leaf.meristem is detectable morphologically as a bump.

videnced by down-regulation of STM expression and up-regulation

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the IM and the floral meristem. Despite this morphological boundary, STM expression in these primordia is continuous with theinflorescence meristem. In early stage 2 FPs (e.g., FP5 and 6), sepal primordia (as judged by down-regulation of STM within the floralprimordium proper) are not present. In later stage 2 FPs (e.g., FP7 and 8), sepal primordia can be detected. (G) Section 43 mm below summitof IM. Note STM-negative region of cells in FP5. This region subtends STM-expressing cells in this FP (see D and C). (H) Section 51 mmelow summit of IM. (I) Section 59 mm below summit of IM. (J) Section 67 mm below summit of IM. (K) Section 75 mm below summit ofM. am—axillary meristem in axil of cauline leaf. Note STM-negative regions where the first two leaf primordia are expected to form. (L)

Section 83 mm below summit of IM. Scale bar, 50 mm.


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These leaves, called cauline leaves, are associated with thelong internodes of the inflorescence. Thus, the uppermost,or youngest, cauline leaf develops a bud prior to the two orthree older, more basal, cauline leaves. Axillary meristemsdeveloping in the axils of cauline leaves differ from those inthe axils of rosette leaves in the timing of their developmentrelative to the subtending leaf. For example, in rosetteleaves, a stage 4 axillary meristem was not found until atleast P11. A P11 leaf has well-developed vasculature withclear indication of xylem thickenings (Fig. 3D) as well as arelatively well-developed blade. In contrast stage 4 axillarymeristems are found in the axils of cauline leaves whilethese are still quite undeveloped (Fig. 4K). These leaves aresmall and lack developed vasculature and blades.

Development of Floral Meristems

Like leaf primordia, floral primordia form on the flanks ofthe inflorescence meristem at circa 137° angles from oneanother. For the analysis of floral primordium development,23-day-old short-day-grown plants were shifted to long daysand fixed 7 days later. The resulting inflorescence meris-tems were sectioned transversely and hybridized to anantisense STM probe and the resulting serial sections werexamined to describe the early events in floral meristemormation. We were especially interested in describing thevents that occur in the establishment of STM expression inhe floral meristem. We have previously shown that STMxpression is negatively regulated in regions on the flanks ofhe inflorescence meristem that correspond to early floralrimordia. By the end of stage 2 of flower development, STMxpression is clearly established in the center of the floraleristem (Long et al., 1996; Fig. 5C). We describe here the

teps between the early down-regulation of STM in thenflorescence meristem (the stage “0” flower) and the latetage 2 flower. Our description is based on the observationf greater than 10 serially sectioned inflorescence meri-tems.

The development of the floral primordium has beenivided into several discrete stages (Smyth et al., 1990).

FIG. 5. Longitudinal section through inflorescence meristems shhrough C were probed with an anti-STM probe. D was probed withf STM-negative region (cb) subtending STM-positive region. We intote that the STM-positive region is continuous with the infloresceid-stage 2 flower (arrow) showing intense mass of STM express

howing STM down-regulation within this primordium may indicaig. 4. Stage 0 (0) and 1 (1) floral primordia are also detectableown-regulation of STM in sepal (s) primordia. This primordium isepal primordia. (D) Inflorescence meristem and stage 2 floral meristoints to a region in the IM that may correspond to a stage 0 florrimordia. In mid- to late stage 2 floral primordia, ANT is expressresent in the growth-suppressed cryptic bract.IG. 6. ANT expression in the inflorescence. (A) ANT transcri

primordium is forming in an axillary meristem. Compare to a leaf pri


tage 1 floral primordia appear as outward bulges on thenflorescence meristem (e.g., floral primordia 2 (FP2) andP3 in Fig. 4E). With further growth, a crease forms be-ween the developing primordium and the IM. With theormation of this crease, the floral primordium has enteredtage 2 (e.g., FP6 and FP7 in Fig. 4E). Stage 3 begins whenepal primordia become visible on the floral primordium.The analysis of STM expression allows several substages

o be observed in early flower development. The firstndication of floral primordium formation is the presence ofn STM-negative region on the flanks of the inflorescenceeristem (Figs. 4 and 5). This occurs before the floral

rimordium is recognizable as a bulge on the IM, i.e., beforetage 1. We will refer to these as stage 0 floral primordia.wo stage 0 primordia are typically found in the IM, and weave designated these FP0 and FP1, where FP0, like P0, ishe most recently arisen primordium in the meristem (Figs.

and 5). Similar to young leaf primordia, FP0 and FP1xtend nearly to the summit of the meristem and areompletely contained within the inflorescence meristem,nd the adaxial boundaries between the STM-expressingnd -nonexpressing cells are convex (Fig. 4). The next floralrimordium to form, FP 21, is expected to form central toP4 (see asterisk in Fig. 4B).With entry into stage 1 of floral development comes

utward, or abaxial, growth of the primordium (Figs. 4C–Gnd 5). Typically, three stage 1 primordia are found on thenflorescence axis (Smyth et al., 1990). In Fig. 4, FP2, FP3,nd FP4 are stage 1 primordia.As the primordium grows outward, STM expression ex-

ends continuously from the IM into the developing floralrimordium with the STM-negative region at the leadingdge of growth. So for instance, the STM-negative regionssociated with FP2, FP3, and FP4 protrudes progressivelyarther laterally from the inflorescence meristem (Figs. 4C,D, and 4F). Similar to leaf primordium development, theoundary between STM-expressing and -nonexpressingells changes from convex to linear as the primordiumrows outward. So, for instance, the STM-negative regionssociated with FP2 retains its convex border with the SAM

g different stages of floral meristem development. Sections in Anti-ANT probe. (A) Early stage 2 flower (arrow) showing presencet this STM-negative region as a suppressed leaf or “cryptic bract.”meristem. This primordium is similar in stage to FP5 in Fig. 4. (B)n region above STM-negative region (cb). A small patch of cellsrly sepal development. This primordium is similar to FP6 or 7 inis section. (C) Late stage 2 floral primordium (arrow) showing

lar to FP8 in Fig. 4. Stage 3 floral primordium shows outgrowth ofo which a probe for the ANT transcript has been hybridized. Arrowimordium. Arrowheads indicate ANT expression in stage 1 floral

positions of newly developing sepal primordia and is no longer

found in P0 leaf primordia (arrowhead). In this case, the leaf

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xpression becomes limited. Marginal domains, sites of continued growth, are especially rich in ANT transcript. (B) Section throughummit of inflorescence meristem showing ANT-expressing region consistent with early floral primordium development. (C) More basalection through same inflorescence meristem as in B, showing a stage 1 floral primordium (arrow) expressing ANT transcript in mostbaxial region. This floral primordium is similar to that shown in Fig. 5D.IG. 7. Model for anatomy of floral meristem formation. (A) Dark blue marks regions of STM expression. Initially, just as in leaf primordiumormation, STM is down-regulated and ANT is turned on. Green indicates the region of STM down-regulation and ANT up-regulation. (B) Growthf the floral primordium is primarily directed outward, just as in the leaf primordium, to give a stage 1 floral primordium. (C) As the floralrimordium becomes morphologically distinct from the inflorescence meristem (i.e., enters stage 2), a mass of cells that expresses STM intenselyomes to overlie the subtending region of STM-negative cells. We call this mass the floral meristem proper. This mass of cells is analogous inosition, molecular morphology, and function to a stage 3 axillary meristem while the region that subtends it is analogous in position and initialevelopment to a leaf primordium. (D) In late stage 2 floral primordia, evidence of STM down-regulation in sepal primordia (marked with light
lue) is seen. At this stage the floral primordium is analogous to a stage 4 axillary meristem.

imrtisTP

350 Long and Barton

while the uppermost section that contains FP3 shows astraight boundary between the IM and the STM-negativeregion at the edge of the IM (Figs. 4D and 4F).

Once a crease becomes evident between the FP and theIM, the primordium enters stage 2 of development (Figs. 4and 5). Floral primordia 5 through 9 in Fig. 4 are stage 2floral primordia. Young stage 2 primordia consist of anSTM-negative region that is abaxial to and subtends anSTM-expressing region. The STM-expressing region growsin size to become an intensely staining mass of cells (Figs.4 and 5). As in the case of stage 1 primordia, the STM-expressing region is contiguous with the inflorescencemeristem. In older stage 2 primordia (e.g., FP8 and FP9 inFig. 4), STM-negative regions arise within this dark-stainingmass of cells at the positions expected for the sepal primor-dia (Figs. 4 and 5).

Floral meristems in Arabidopsis and in other members ofthe Brassicaceae lack subtending leaves. Thus, unlike mostother species and unlike axillary meristems, Arabidopsisfloral primordia develop in the apparent absence of a sub-tending leaf. However, the finding that a small region ofSTM-negative cells arises early in the development of thefloral primordium suggests that this STM-negative region isa highly reduced leaf in the axil of which the floral meri-stem develops. To test this hypothesis, we examined theexpression of a gene expressed in leaf and other organprimordia—the AINTEGUMENTA gene—in the developingfloral primordium. Although its function in leaf develop-ment is unknown, ANT is expressed in organ primordiathroughout the plant (Elliott et al., 1996). If the STM-negative region is a reduced leaf, it should express the ANTgene.

Figure 6A shows the expression of the ANT gene inleaves. ANT is initially expressed throughout the young leafprimordium. In a pattern near reciprocal to that of STM,ANT is found in the developing leaf trace that lies below the

TABLE 2Events in Early Leaf Development

Primordium

P 2 1 Leaf trace is distinguishable by PINHEAD mRNAP0 STM transcript is down-regulated. ANT transcript

primordium.a,b

P1 Primordium begins to bulge outward from SAM.P2 Primordium extends apically but not past summi

mRNA localized to adaxial side of primordiumabaxial polarity distinguishable at morphologicthose on the adaxial side.

P3 Primordium extends apically to just beyond summP4 Primordium curves over the top of SAM. Adaxial

(1996) stage 1 leaf.

a Lynn et al. (1998).b Siegfried et al. (1999).

developing leaf primordium (data not shown). As the pri- S


mordium grows ANT becomes progressively localized tocentral and lateral regions of the leaf. ANT is thus localizedto actively developing regions of leaves. Figure 6B shows aregion of ANT expression about 10 mm below the tip of theinflorescence meristem where a developing floral primor-dium is expected to arise (compare to FP0 and FP1 in Fig.4B). Figure 6C shows ANT expression in a stage 1 primor-dium in the area where the STM-negative region is expectedto be (compare to FP2 in Fig. 4D). We conclude that theSTM-negative region associated with young floral primor-dia, like a leaf primordium, expresses the ANT gene.

Figure 5D shows an example of a longitudinal sectionthrough an inflorescence meristem hybridized to a probe forthe ANT transcript. ANT is found in peripheral regions ofstage 0 and 1 floral primordia (and in the developingvascular trace). By stage 2, however, it has vanished fromthe region thought to be the subtending bract (Fig. 5D) andis now associated with the regions of developing sepals.

DISCUSSION

Early Leaf DevelopmentSTM gene expression provides a tool with which to

analyze early events in the development of leaves. Table 2summarizes gene expression patterns and morphologicalevents in early leaf development as described in this paperand in other work. In Arabidopsis, loss of STM expressionn groups of cells in the peripheral zone represents an early

arker of leaf development. By the criterion of STM down-egulation, there are one to two leaf primordia (P0 and P1)hat are physically continuous with and morphologicallyndistinct from the Arabidopsis SAM. Loss of STM expres-ion is, however, not the earliest marker of leaf formation.he vascular trace below the position of P 21 expresses theINHEAD transcript and thus precedes down-regulation of

haracteristics

lization below the SAM.a

resent. PINHEAD and YABBY mRNAs are found throughout the

AM. Cleft forms between SAM and primordium. PINHEADBBY mRNA localized to abaxial side of primordium.b Adaxial/el: cells on the abaxial side of the primordium are larger than

f SAM.of primordium appears to “rest” on SAM. Carland and McHale

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The P0 primordium lacks any morphological or molecu-lar indications of polarity along the ad/abaxial dimension.For instance, the PINHEAD and YABBY transcripts arefound throughout the primordium (Siegfrid et al., 1999;

ynn et al., 1999). However, in the P2 primordium, expres-ion of the PNH gene is localized to the adaxial side (Lynnt al., 1999) and the YABBY family of genes is expressed in

the abaxial leaf domain by the P2 stage (Siegfried et al.,1999). Morphological differences also exist in the P2 leaf,with cells on the abaxial side appearing larger than those onthe adaxial side.

While down-regulation of STM occurs in the bulk of thedeveloping leaf primordium, STM remains expressed ininterprimordial regions. STM may function in the interpri-mordial regions to prevent these cells from proliferating;such a function would prevent organ fusion and promoteorgan separation. During embryogenesis STM is expressedbetween the developing cotyledons and is required to pre-vent fusion of the developing cotyledons (Clark et al., 1996;Long and Barton, 1998; Aida et al., 1999). Evidence thatTM is required for organ separation outside of embryogen-sis has been presented by Felix et al. (1997), who noted thatoral organs were often fused in a weak stm mutant.

“Detached Meristem” vs “de Novo Induction”Models for Axillary Meristem Formation

Two alternative models for axillary meristem formationcan be envisioned. In the “detached meristem” model, asmall number of SAM cells remains associated with the leafaxil as the leaf grows away from the SAM, forming aso-called detached meristem. At some later point, thesecells are released from repression and develop into anaxillary meristem. Alternatively, in the “de novo induc-ion” model, cells lose their identity as SAM cells andifferentiate in part or in total, only to be respecified asAM cells at some later point in the life of the plant.In the former model, the detached meristem cells are

onequivalent to their neighbors in their ability to form aeristem. This means that there is no need to postulate a

ocalized signal to elicit their development as axillaryeristem cells. It also implies that SAM fate is imparted de

ovo only once in the lifetime of a plant—duringmbryogenesis—and that this developmental state is thenassed on to daughter cells. In the alternative, de novonduction model, cells that give rise to axillary meristemsre equivalent to their neighbors in their ability to form aeristem. Thus, one must invoke a localized signal to

xplain the development of these cells and not their neigh-ors as axillary meristem cells. It also implies that SAMate can be conferred on cells even after embryogenesis.

One way to distinguish between these two models woulde to follow the expression of a SAM-specific marker andetermine whether this marker was transiently repressednd then reactivated in regions that develop as axillaryeristems. It was our hope that the STM mRNA would be
marker that would allow us to distinguish the two d

odels. However, the interprimordial expression of STMakes it difficult to distinguish the detached meristemodel for axillary meristem formation from the de novoodel for axillary meristem formation. Since STM mRNA

ersists in the “trough” of the axil, it is possible that therough cells make up the detached meristem and that theirrogeny are subsequently displaced upward into the leafase. Alternatively, the trough cells may function princi-ally in creating separations between leaf primordia whileells located farther up on the leaf base redifferentiate asAM cells.

Similarities between Axillary Meristem and FloralMeristem Formation

Although several lines of evidence suggest that the sub-tending leaf is required for the formation of an axillary bud,floral meristems in Arabidopsis and in most other membersof the Brassicaceae develop in the apparent absence of asubtending leaf. Our analysis of STM (the expression of

hich is associated with shoot apical meristems) and ANTthe expression of which is associated with organ primordia)ranscript accumulation suggests the presence of a highlyeduced leaf subtending the floral bud in Arabidopsis.

Figure 7 summarizes our interpretation of early events inoral primordium formation and highlights similaritiesetween development of the floral primordium and thexillary bud. The earliest indications of leaf development inhe vegetative meristem and of floral primordium develop-ent in the inflorescence meristem are the presence of theNT transcript and the down-regulation of the STM tran-

cript. Initial growth of both the floral primordium and theeaf primordium is outward, away from the meristem. Inhe vegetatively growing plant, the ANT-positive, STM-egative region continues to grow (with ANT transcriptocalized to growing regions of the primordium) and be-omes a leaf. An axillary meristem develops in associationith this leaf at some later time. We interpret the ANT-ositive, STM-negative regions that arise in the inflores-ence meristem as leaf primordia that become growthuppressed. We will refer to these hypothesized leaf primor-ia as “cryptic bracts.” “Cryptic” because they are notbvious unless the appropriate molecular markers are usednd “bracts” because they subtend flowers. ANT transcriptecomes extinguished in the cryptic bract early; this isonsistent with its failure to grow out. The cryptic bractemains STM negative and, as it grows outward from thenflorescence meristem, comes to subtend a ball-shapedegion of cells that exhibits a high level of STM expression.

e interpret this ball-shaped region of cells as the floraleristem proper. Sepal primordia, appearing as STM-

egative regions, appear within the floral meristem properear the end of stage 2 of floral development.Consistent with this work, patches of YABBY gene ex-

ression (which, like ANT expression, is associated with
eveloping leaf primordia) are found in the inflorescence

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352 Long and Barton

meristem in a pattern similar to that of ANT transcriptccumulation (Siegfried et al., 1999).Further consistent with the presence of a cryptic bract

ubtending the developing flower, structures similar totipules, called squamules, have been noted previously inssociation with floral primordia (Levin et al., 1995). Squa-ules develop in pairs and, when present, are found in

ositions similar to where stipules are found. Squamulesre seen rarely in wild-type plants and more commonly innusual floral organ mutants.One difference between the meristem that develops in

he axil of a rosette or cauline leaf and the floral meristemhat develops in the axil of the proposed cryptic bract is theize of the leaf when the meristem develops. In vegetativelyrowing plants, we find the first evidence for axillaryeristem development at the P9 to P10 stage of leaf

evelopment. Upon floral induction, the direction of budormation reverses in the plant; axillary meristems are

ade in top–down order, with the youngest leaf axil mak-ng a bud before older leaf axils do (Hempel and Feldmann,994). Thus, cauline leaves (i.e., those associated with thenflorescence stem) are associated with axillary meristemst a much earlier point in their development than rosetteeaves are. This is even more exaggerated in the case of theoral meristem. The floral meristem proper is obvioushen the cryptic bract is very small. Cauline leaves are

maller than rosette leaves and are associated with longernternodes. The proposed cryptic bract could be interpreteds the most extreme endpoint in this gradient, with floraleristems produced extremely early in the life of the

ubtending leaf and the subtending leaf showing the mostxtreme amount of growth suppression.Consistent with this model, Hempel and Feldman have

rgued that cauline leaves are the leaves that are alreadynitiated at the meristem when the plant is subject to aoral stimulus and that the rapid development of thexillary meristem and the suppression of leaf size is aonsequence of this induction. Furthermore, the same au-hors have shown that the fate of leaf primordia alreadynitiated at the vegetative meristem can be modified inesponse to a floral inductive signal (Hempel and Feldman,994; Hempel et al., 1998). Primordia already present at theegetative meristem (primordia that would have given riseo rosette leaves in a vegetatively growing plant) could beonverted to primordia showing aspects of both vegetativend floral development. In some cases these consisted of aower with a subtending leaf; the subtending leaf was atimes extremely reduced in size.

An experiment in which the floral meristems were ab-ated supports this model and also suggests that the floral

eristem is responsible for inhibiting growth of the sub-ending bract. Nilsson et al. (1998) expressed the gene foriphtheria toxin under the control of the LEAFYromoter—a promoter that acts at highest levels in floralrimordia. When the floral meristem was ablated, a subsetf transgenic plants produced leaves in place of floral
eristems.

Finally, our results suggest a somewhat refined interpre-ation of the leafy mutant. The leafy mutation has beennterpreted as causing a transformation of a floral meristemnto an inflorescence meristem (Weigel et al., 1992). Ouresults suggest that leafy mutations cause a transformationf floral meristem plus associated cryptic bract to axillaryeristem plus associated cauline leaf.

ACKNOWLEDGMENTS

We thank Matthew Evans, Anita Fernandez, Karyn Lynn, andJane McConnell for critically reading the manuscript. The workwas supported by grants from the NSF and the USDA. This is PaperNo. 3524 from the Laboratory of Genetics.

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Received for publication September 28, 1999Revised November 10, 1999

Accepted November 10, 1999


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Initiation of Axillary and Floral Meristems in Arabidopsis