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The Plant Cell, Vol. 4, 901-913, August 1992 0 1992 American society of Plant Physiologists

LEAFY lnteracts with Floral Homeotic Genes to Regulate Arabidopsis Floral Development

Eva Huala' and lan M. Sussex Department of Plant Biology, University of California, Berkeley, California 94720

In the leafy mutant of Arabidopsis, most of the lateral meristems that are fated to develop as flowers in a wild-type plant develop as inflorescence branches, whereas a few develop as abnormal flowers consisting of whorls of sepals and carpels. We have isolated several new alleles of leafy and constructed a series of double mutants with leafy and other homeotic mutants affecting floral development to determine how these genes interact to specify the developmental fate of lateral meristems. We found that leafy is completely epistatic to pistillata and interacts additively with agamous in early floral whorls, whereas in later whorls leafy is epistatic to agamous. Double mutants with leafy and either apetalal or apetala2 showed a complete loss of the whorled phyllotaxy, shortened internodes, and suppression of axillary buds typical of flowers. Our results suggest that the products of LEAFY, APETALAl, and APETALAP together control the differentiation of lateral meristems as flowers rather than as inflorescence branches.

INTRODUCTION

The angiosperm flower is a modified branch that differs in sev- era1 essential ways from a vegetative shoot. One obvious distinguishing feature of a flower is the presence of floral organs (sepals, petals, stamens, and -1s) rather than leaves. Other differences include a determinate pattern of growth, the absence of lateral meristems in the axils of the floral organs, and the lack of internode elongation. A common but not invar- iant feature of flowers is the initiation of floral organ primordia in a whorled rather than a helical phylldaxy. In Arabidopsis, the conversion from vegetative to reproductive growth is marked by bolting and production of several cauline leaves with axillary buds, followed by flowers that lack bracts. The apical meristem progresses from the vegetative stage, which initiates rosette leaf primordia, to an early inflorescence stage, which initiates cauline leaf (bract) primordia, and finally to a late inflorescence stage, which initiates an indefinite number of floral primordia in the form of an open raceme. The lateral meristems present in the axils of the cauline leaves and up per rosette leaves may develop into additional inflorescences consisting of one or more cauline leaves followed by an indeterminate number of flowers.

Mutations at the LEAFY locus cause cauline leaves with axillary buds to develop in many of the positions that would be occupied by flowers in a wild-type plant (Haughn and Somerville, 1988; Meyerowitz et al., 1991; Schultz and Haughn, 1991). These axillary buds develop into inflorescence branches that repeat the abnormal pattern of the primary inflorescence axis, resulting in a highly branched vegetative plant. Although

To whom correspondence should be addressed.

many of these extra inflorescence branches appear relatively normal except for a tendency to terminate in carpels, others exhibit characteristics of both branches and flowers or develop as nearly normal flowers consisting of whorls of sepals and carpels. The /e@ mutation is proposed to block the transition of the inflorescence meristem from the early inflorescence stage, which initiates cauline leaves with axillary buds, to the late inflorescence stage, which initiates flowers (Schultz and Haughn, 1991). A similar mutation, floricaula, has been stud- ied in Antirrhinum (Coen et al., 1990).

To investigate the genetic basis for the switch from development of inflorescence branch primordia to development of flower primordia, we have examined the relationship between LEAFYand several other genes of Arabidopsis that participate in the differentiation of primordia as flowers rather than inflorescence branches. The apetala7-7 (ap7-7) mutant fails to suppress development of axillary buds in the flower, resulting in the appearance of secondary flowers in the axils of the leaflike first whorl organs of the primary flower. These secondary flowers in turn initiate flower primordia in first whorl axillary positions, resulting in a multiply branched flower. Some elongation of the internode between the media1 and lateral first whorl organs also occurs (Irish and Sussex, 1990). Although ap7-7 flowers show some of the characteristics of branches, the third and fourth whorls are not affected by the mutation and the flower remains a determinate structure, terminating in two carpels as in wild-type flowers. When the ap7-7 mutation is combined with apetala2-7 (ap2-7), a mutation that causes homeotic transformations of organs in the first and second whorls, the whorled phyllotaxy of ap7-7 and ap2-7 flowers is replaced by a helical phyllotaxy. Up to 30 lateral structures, each having

902 The Plant Cell

two or more carpels sometimes accompanied by stamens andleaflike organs, are produced in a helical pattern by each flo-ral meristem of the double homozygote. The first few floralmeristems of this double mutant develop into indeterminatestructures, but later-formed flowers have a more determinatepattern of growth and terminate in fused carpels.

Flowers of agamous, another mutant of Arabidopsis, havelost the determinate growth pattern of a wild-type flower butretain the whorled phyllotaxy, absence of lateral buds, and abil-ity to initiate floral organs characteristic of flowers, agamousflowers consist of a normal first and second whorl followed bya third whorl in which stamens have been replaced by petals(Bowman etal., 1989). In place of a fourth whorl, a new flowerprimordium is formed that repeats the pattern of the originalflower. Although no internode elongation occurs when aga-mous is present in the Landsberg erecfa ecotype, someinternode elongation does occur between the third whorl andthe new first whorl for agamous in the Columbia ecotype, whichdoes not carry the erecta mutation (Bowman et al., 1991).

We have isolated and characterized several new alleles ofleafy. In addition, through the construction and analysis of dou-ble mutants we have investigated the interaction of leafy withother genes that control floral development. Our results sug-gest that the products of LEAFY, APETALA1, and APETALA2together control the differentiation of lateral meristems asflowers rather than as inflorescence branches.

RESULTS

Isolation of New Alleles of leafy

The previously described /eary-7 (lfy-1) mutation results in thetransformation of most flowers into inflorescence branches orstructures intermediate between inflorescence branches andflowers, whereas the remainder develop as abnormal flowersconsisting only of sepals and carpels (Haughn and Somerville,

Figure 1. leafy Allelic Series.Plants were grown for 5 to 6 weeks under 16-hr photoperiods. The arrow indicates the position of the earliest flower. For wild type, lfy-13, andlfy-14, the transition from inflorescence branches (below arrow) to flowers (above arrow) is relatively sharp, whereas this is not the case for lfy-1and lfy-12. x3.1.(A) Wild-type Col-0. Two cauline leaves subtend inflorescence branches, and a third cauline leaf subtends an arrested axillary bud.(B) lfy-13. A series of 11 flowers is visible above the arrow, followed by several structures intermediate between flowers and inflorescence branches.(C) lfy-14. At least eight flowers in a series are visible above the arrow.(D) lfy-1. The earliest flower is followed by a mixture of flowers and inflorescence branches.(E) lfy-12. No flowers are visible.

LEAFY and Floral Development in Arabidopsis 903

Figure 2. Range of leafy Lateral Structures Showing Some Floral Characteristics.

Plants were grown under 16-hr photoperiods. Bars = 500 |im.(A) lfy-12. A lateral structure in which internode elongation, axillary buds (a), and stigmatic tissue (s) are visible.(B) lfy-12. A lateral structure with decreased internode elongation.(C) lfy-13. A whorled flowerlike structure. A carpelloid stamen is visible.(D) Wild type (one petal removed).

1988; Schultz and Haughn, 1991). We have isolated three newrecessive alleles at the LEAFY locus, as shown in Figure 1.lfy-12 is a strong allele that closely resembles the previouslydescribed lfy-1 (Schultz and Haughn, 1991), whereas lfy-13 andlfy-14 are less severe. {Alleles were assigned numbers in con-sultation with D. Weigel and E. Meyerowitz to avoid duplicationof allele numbers with other laboratories.) Both lfy-1 and lfy-12carry the same point mutation that generates a premature stopcodon near the start of the coding region (D. Weigel and E.Meyerowitz, personal communication). The majority of lateralmeristems initiated by a lfy-12 plant form inflorescence

branches or intermediates between branches and flowers. Al-though the early development of these inflorescence branchesis normal, the more distal leaves are increasingly carpelloidand the branches eventually terminate in several carpels. Laterinflorescence branches become increasingly flowerlike, withshorter internodes and fewer axillary buds.

Figure 2 illustrates the range of leafy lateral structures hav-ing some floral characteristics. Some of the lateral structuresinitiated in the upper third of the bolt have the whorled phyl-lotaxy and general appearance of flowers, but, unlike wild-typeflowers, these generally consist of five to 11 sepals and sepaloid

904 The Plant Cell

Table 1. Number of Flowers for leafy Allelic Seriesa

Flowers + Flowersl Branchesl

Allele Plant Plantb O/, Flowers

LFY 27 30 90 Ify-1 (13) 11 42 26 lfy-12 (25) 2.3 33 6.3 4 - 1 3 (18) 20 41 48 IW-14 (13) 17 40 43

a Plants were grown in 16-hr days. lntermediate structures were counted as branches. Number of plants examined is shown for each allele.

carpels located in two to three irregular whorls that enclose an additional whorl of two to fioe partly to fully fused carpels. These flowers are sometimes female fertile, although the number of seeds produced by outcrossing is quite low. After the initiation of a few relatively normal flowers, severa1 more intermediate structures with elongated internodes are produced before growth of the primary inflorescence meristem terminates in carpels.

The development of the milder lfy-73 and lfy-74 alleles fol- lows the same general pattern described above, except that a much larger number of flowers and fewer intermediate structures are produced and the flowers appear more normal. The relative numbers of flowers and inflorescence branches for each allele of lfy are compared in Table 1, whereas Table 2 shows the effect of different alleles on organ identity. The flowers of lfy-73 mutants have a first whorl consisting of four sepals and a second whorl that generally contains four smaller sepals or mosaic organs consisting of both sepal and peta1 tissue. The remaining organs are difficult to assign to a par- ticular whorl, and often include a stamen and two to three fused carpels along with an occasional carpelloid sepal. In addition, flowers of lfy-73 rarely have axillary buds, although these frequently occur in even the most flowerlike structures of lfy-7 and lfy-72 mutants (Table 2). lfy-73 flowers are generally female fertile and occasionally self-fertile.

Phenotype of leafy 1s Affected by Daylength

Arabidopsis is a facultative long-day plant in which flower initiation is greatly accelerated by long photoperiods (Napp-Zinn, 1985). Therefore, genes controlling flower initiation and development are likely to be regulated in part by photoperiod length. The Columbia ecotype flowers after about 3 weeks when grown under long days (16 hr of lighff8 hr of dark), whereas growth under short-day conditions (8 hr of lighff16 hr of dark) delays bolting and flowering by about 2 months. As a result of the delay in flowering, plants grown under short days develop larger rosettes and larger inflorescences (Shannon and Meeks-Wagner, 1991).

Figure 3 shows the phenotypes of wild-type and leafy plants grown under 8hr days. Wild-type plants grown under long days produced an average of 3.2 inflorescence branches in our growth conditions, whereas plants grown in short days produced an average of 6.7 inflorescence branches, as shown in Table 3. Although more inflorescence branches are produced in wild-type plants grown in short days, these plants also produce more flowers, so that the ratio of flowers to the total number of inflorescence nodes remains about the same. In contrast, lfy-73 plants grown in short days have about the same number of inflorescence nodes as those grown in long days, but produce many more inflorescence branches and fewer flowers (Table 3). The effect of short days on the leafy phenotype is more pronounced on the milder alleles (lfy-73 and lfy-74) but also affects the more severe alleles (lfy-7 and lfy-72), causing inflorescence branches to be produced in all positions nor- mally occupied by flowers.

Effect of leafy on the lnflorescence Meristem and Early Primordia

We compared the inflorescence meristems of wild type, lfy-7, and lfy-73 grown under 16-hr days to determine whether there were any obvious morphological differences. Figure 4 shows the primary inflorescence meristem and lateral meristems of lfy-73 during early and late stages of growth. When the early development of wild-type and leafy lateral meristems was

Table 2. Floral Oraan Number for leafy Allelic Series

Allele Sepals Petalsa Stamensb Carpels Se/CaC Buds

LFY (lO)d 4 4 6 2 O O

lfy-12 (10) 4.5 O O 3.5 3.2 1.3

lfy-14 (10) 5.5 O 0.1 3.3 2.8 0.1

l fy-1 (9) 3.6 O O 3.9 3.3 0.56

lfy-73 (10) 7.8 0.2 1.2 2.5 0.5 0.4

a lncludes petaloid sepals. b lncludes sepaloid and carpelloid stamens. c Se/Ca, sepaloid carpels. d Number of flowers examined is shown for each allele.


Figure 3. Effect of Growth under Short Days on leafy Phenotype.

Allelic series of leafy mutants grown for 16 weeks under 8-hr photoperiods. No flowers have developed. x3.1.(A) Wild-type Col-0.(B) lfy-13.(Q lfy-14.(D) lfy-1.(E) lfy-12.

compared, several differences were immediately obvious. Incontrast to wild-type inflorescence meristems, which initiateflowers without subtending bracts, bracts are present at allstages of leafy inflorescence development. Bract primordiawere visible early in the development of the leafy lateralmeristems, appearing as a ridge on the abaxial side of the

lateral meristems (Figure 4). In young inflorescences, the de-velopment of the bract was relatively slow and the developmentof the adjacent lateral meristem was rapid. The relative ratesof development of the bract and lateral meristem were reversedin older inflorescences, resulting in a prominent bract primor-dium with a small lateral meristem visible in its axil.

Table 3. Effect of Daylength on Wild-Type and lfy-13 Inflorescences

Total Inflorescence NodesNumber of BranchesNumber of Flowers% Flowers

Wild Type8 hr

50 ± 12°6.7 ± 2.743 ± 1286 ± 7

16 hr

30 ± 93.2 ± 1.227 ± 889 ± 3

lfy-13

8 hr

45 ± 1542 ± 153.4 ± 47.4 ± 9.2

16 hr

41 ± 721 ± 420 ± 548 ± 6

JSD.

906 The Plant Cell

Figure 4. Wild-Type and leafy Inflorescence Meristems.Primary inflorescence meristems from lfy-13 grown under 16-hr photoperiods. Bars = 100 urn.(A) Wild-type Col-0.(B) lfy-13 early in inflorescence growth. Bracts (b) are barely visible, and lateral meristems (Im) are prominent.(C) lfy-13 at an intermediate stage of inflorescence growth. Bracts (b) are enlarged relative to lateral meristems (Im).(D) lfy-13 late in inflorescence growth. The inflorescence meristem is closely associated with bract primordia (b).

As others have reported, development of the lateral meristemitself also differs in wild-type and leafy inflorescences (Schultzand Haughn, 1991). Whereas wild-type floral meristems initi-ate two pairs of sepals in a cruciform pattern, many of the lateralmeristems of leafy initiated organs in a helical phyllotaxy typi-cal of an inflorescence meristem. These lateral meristemsprobably develop into the lateral structures of leafy that havemostly branchlike characteristics. Other lateral meristems ofleafy initiated organs in the cruciform pattern of a wild-typefloral meristem. These are more frequent on mid and late stage

inflorescences and probably correspond to the mature flower-like structures that occur in mid and upper parts of theinflorescence (Figure 1).

The transition from cauline leaf and inflorescence branchproduction to flower production is rapid in the wild type butoccurs much more slowly in leafy. Unlike the wild type, leafyplants have a transitional zone where intermediate structuresdevelop with a mixture of floral and branchlike characteristics.Figure 5 shows young lateral structures of a single lfy-1 in-dividual that were initiated before, during, and after the


transition from initiation of inflorescence branches to initiationof flowers. In the most branchlike structure (Figure 5A), theaxillary buds are prominent, the early bracts are leaf I ike, andthe later bracts are small and filamentous. The bracts in thesecond (transitional) structure shown in Figure 5B are well de-veloped and show the epidermal features of sepals on the outerbracts and carpels on the inner bracts, but secondary flowerbuds are still present and no central gynoecium of fused car-pels exists. In the most flowerlike structure (Figure 5C), relativelynormal sepals enclose the inner whorls of sepaloid carpelsand fused carpels with an occasional secondary flower bud.

Interaction between leafy and Floral OrganIdentity Genes

A leafy mutant, in addition to producing many branchlike lateralstructures, also produces a few flowerlike structures consist-ing of whorls of sepals, sepaloid carpels, and carpels, withpetals and stamens reduced in number or entirely absent. Be-cause leafy can alter not only floral organ identity but also thestructure of a flower, leafy is likely to act at a stage of develop-ment that precedes the determination of floral organ identity.We have constructed Ify pi-1 (pistillata-1), Ify ag-1 (agamous-1),Ify ap1-1, and Ify ap2-1 double mutants using two different al-leles of Ify to study the interaction of Ify with other genescontrolling floral organ identity. The lateral structures formedin the upper portion of the bolt were examined for the effectsof the second mutation because flowerlike lateral structuresappear most frequently in this part of a Ify plant. The segrega-tion ratios obtained for each double mutant are listed in Table 4.

Hy-12 pi-1

The pistillata mutation causes the conversion of second whorlorgans (petals) into first whorl organs (sepals), and third whorlorgans (stamens) into fourth whorl organs (carpels) (Bowmanet al., 1989). Both the flowers of pi-1 and the most flowerlikestructures of lfy-1 and Hy-12 lack petals and stamens and con-sist entirely of sepals and carpels. However, whereas the sepaland carpel organ types are distinct and confined to separatewhorls in pistillata flowers, in leafy flowers only the organs ofthe first whorl are consistently normal and the organs in laterwhorls very often have characteristics of both sepals and car-pels, becoming progressively more carpelloid toward the centerof the flower.

To investigate the relationship between pistillata and leafy,we have constructed pi-1 lfy-12 double homozygotes. Becauseboth leafy and pistillata are male sterile, a lfy-12 heterozygotewas used as the male parent in a cross with a plant homozy-gous for pi-1, a strong allele of pistillata (Bowman et al., 1991).Only the progeny of selfed individuals from the F, generationthat segregated for both pi-1 and lfy-12 were subjected to fur-ther analysis. The F2 progeny from this cross were eitherphenotypically wild type or indistinguishable from one of the

Figure 5. Transition from Inflorescence Branches to Flowers in lfy-1.

Lateral meristems from lfy-1 grown under 16-hr photoperiods. Bars =100 \im.(A) Branchlike structure. Axillary buds (a) are prominent; bracts (b)vary from leaflike to filamentous.(B) Transitional structure. The outer whorl has developed as sepals,but several axillary buds have formed and no fused gynoecium is pres-ent. Stigmatic tissue (s) is visible on an organ of an inner whorl.(C) Flowerlike structure. The developing flower is enclosed by twowhorls of sepals.

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I

Table 4. Segregation Ratios foi Single and Double Mutant Phenotypes

Cross % Wild Type % leafy Parenta1 Type % New Phenotype Total Scoreda

Expectedb 56 19 19 6

ag-1 x Ify-1 61 16 17 6.6 183

apl-1 x l fy-7 58 24 13 5.0 160 apl-1 x lfy-13 59 23 15 3.2 185

ap2-1 x lfy-13 62 12 17 8.3 346

% Second

- pi-1 x lfy-12 56 26 19 O 534

ag-1 x lfy-13 58 20 14 7.7 220

ap2-1 x &-1 53 24 17 6.3 222

a Number of F2 plants scored.

gation ratio for phenotypically wild type to leafy to the second parental type to the double mutants is 9:3:3:1. The PI, AG, AP1, and AP2 loci are unlinked to LEAFY (Koornneef et al., 1983; Schultz and Haughn, 1991), and, therefore, the expected segre-

parental types, indicating that one of these mutations is completely epistatic to the other. We examined the phenotypes of 534 F2 plants and obtained a 9:4:3 segregation ratio of 298 wild-type, 137 leafy, and 99pistiIIafa plants (Table 4). This suggests that leafy is epistatic to pisfillata.

/fy-7 ag7 and lfy-73 ag7

The agamous-7 mutation causes the conversion of third whorl organs (stamens) into second whorl organs (petals) and the replacement of the fourth whorl organs (carpels) with a new flower primordium, resulting in a flower that indefinitely repeats a pattern of one whorl of sepals followed by two whorls of petals (Bowman et al., 1989). The flowers of Ify-7, ag-1, and the lfy-7 ag-1 double mutant are shown in Figures 6A to 6C. Flowers of the lfy-7 ag-1 homozygotes exhibited characteristics of both the lfy-1 and ag-7 phenotypes. The identity of the first whorl organs was unaffected by either mutation, and these organs developed as sepals. Second whorl organs are converted from petals to sepals in lfy-7 and are unaffected in an ag-7 mutant. The combined effect of both mutations produced a second whorl of four sepals in the lfy-7 ag-7 double mutant. The ag-7 mutation causes the identity of second and third whorl organs to be the same, resulting in petals in the third whorl of the ag-7 parent and sepals in the third whorl of the lfy-7 ag-7 double mutant. The organs of subsequent whorls of Ify-7 ag-7 flowers were more irregular in number and position and became less sepal-like and increasingly carpelloid in the inner whorls, with ovules appearing along the margins and stigmatic tissue at the tips. These carpelloid leaves frequently had axillary buds that, like the primary flower, consisted of many whorls of sepals and carpelloid leaves with additional axillary buds. A similar result was obtained with lfy-13 ag-1 double mutants, except that some organs with both sepal and petal tissue were ObSeNed in the third whorl of the double mutant flowers.

Elongation of the later internodes and a return to a branchlike mode of growth was frequently observed in both lfy-7 ag-7

and lfy-73 ag-7 flowers, particularly in those double mutant individuals that were not homozygous for erecta, which suppresses internode elongation between the successive nested flowers of the ag parent (Bowman et al., 1991). No fusion of the carpelloid leaves into a gynoecium was ever observed, and organ initiation appeared to proceed continu- ously, as organs in the earliest stages of development were always present in the center of the flower even when older flowers with senescing outer whorls were examined. These results suggest both additive and synergistic interactions between leafy and agamous in the determination of floral organ identity.

lfy-7 ap7-7 and lfy-73 ap7-7

The ap7-7 mutation causes the conversion of first whorl organs (sepals) into cauline leaves with axillary floral buds (Figure 6D). Second whorl organs (petals) are absent or are mosaics of leaf, stamen, and petal tissue, and the third and fourth whorls are unaffected (Irish and Sussex, 1990). The flowers of ap7-7 and the lfy-7 ap7-1 double mutant are shown in Figures 6D and 6E. All lateral structures of the lfy-7 ap7-7 and lfy-73 ap7-7 double mutants had elongated internodes and leaflike organs with axillary buds arranged in a helical phyllotaxy, as shown in Fig- ure 7A. These double homozygotes failed to produce any flowers with suppressed axillary buds, shortened internodes, and a whorled phyllotaxy. As with the leafy parent, the lateral structures and the primary inflorescence produced by the double mutant eventually terminated in carpels (Figure 7C). Many of the more dista1 organs borne on these lateral structures showed some characteristics of carpels, including stigmatic tissue at the tips, occasional ovules along the margin, and a lack of trichomes. The increased branchlike characteristics of the lateral structures produced by the lfy-7 ap7-7 and lfy-73 apl-1 double mutants suggest that the lfy and ap7 mutations interact synergistically with regard to flower structure.

Figure 6. Flowers of Single and Double Mutants.

Plants were grown under 16-hr photoperiods. Bars = 1 mm.(A) tty-1.(B) ag-1.(C) ag-1 tty-1.(D) apT-7.(E) apr-7 lfy-1.(F) ap2-r.(G) ap2-7 lfy-1.

910 The Plant Cell

Figure 7. Lateral Structures of lfy-1 ap1-t and lfy-1 ap2-l.

Bars = 100 urn.(A) ap1-1 lfy-1. Young lateral structure showing helical phyllotaxy and prominent bracts (b) with axillary buds (a).(B) ap2-1 lfy-1. Young lateral structure showing helical phyllotaxy and prominent bracts (b) with many stellate trichomes and an axillary bud (a).(C) ap1-1 lfy-1. Older lateral structure with stigmatic tissue (s) at distal ends of bracts.(D) ap2-1 lfy-1. Older lateral structure lacking stigmatic tissue at distal ends of bracts (b).

lty-1 ap2-1 and I1y-13 ap2-1

Mutations in AP2 cause the conversion of first whorl organs(sepals) into fourth whorl organs (carpels), leaves, or carpel-loid leaves and second whorl organs (petals) to third whorlorgans (stamens) (Komaki et al., 1988; Bowman et al., 1989;Kunstetal., 1989). The AP2 gene product represses AGAMOUSexpression in the first and second whorls, and induces genesrequired for the normal development of sepals and petals inthese whorls. The ap2-1 allele is postulated to be defectivein the function required for sepal and petal development butretains the ability to prevent expression of AG in the first andsecond whorls, resulting in the transformation of the first whorlorgans from sepals to leaves, rather than carpels as with the

more severe alleles ap2-2 and ap2-6 (Bowman et al., 1991;Drews et al., 1991). In a previous study in which lfy-1 was com-bined with ap2-6, no significant difference between the lfy-1parent and the double mutant was observed (Schultz andHaughn, 1991).

We constructed lfy-1 ap2-1 and lfy-13 ap2-1 double homo-zygotes. The flowers of ap2-1 and the lfy-1 ap2-1 double mutantare shown in Figures 6F and 6G. Like the lfy-1 apl-1 and lfy-13ap1-1 double homozygotes, these showed a much greater de-gree of transformation of lateral structures from flowers tobranches than was seen with leafy alone (Figure 7B). In addi-tion, the frequency with which the primary inflorescence andthe inflorescence branches terminated in carpels was greatlyreduced (Figure 7D). As a result, the transformation of lateral

LEAFY and Floral Development in Arabidopsis 91 1

structures from flowers to inflorescence branches was complete. No floral characteristics were visible in the lateral structures produced by these double mutants, which had the elongated internodes, helical phyllotaxy, leaves with stellate trichomes and axillary buds, and indeterminate growth pattern of wild-type inflorescence branches. No phenotypic difference was observed between lfy-7 ap2-7 and lfy-73 ap2-7. These data suggest that LFY and AP2, like LFY and AP7, interact synergistically to produce the structural features typical of flowers.

DlSCUSSlON

Effect of Daylength on Ieafy lnflorescence Development

Growth in short days produced a more severe Ieafy phenotype in all alleles of leafy that we tested. Because growth under short days affects the phenotype of the severe alleles of leafy as well as the mild alleles, it seems likely that some product other than LEAFY also participates in the initiation of floral meristems and is regulated in response to daylength. Alter- natively, a small amount of LEAFY activity could be responsible for the effect of daylength on the leafy phenotype if the expression or activity of LEAFY is decreased under short days.

Growth in short days has a similar effect on apl-7 mutants, causing them to produce a large number of inflorescence branches and some transitional flowers, with very long pedicels and extensive internode elongation between successive first whorl organs, before producing typical apl-7 flowers (E. Huala and I. M. Sussex, unpublished observations). Growth in short days also causes the development of secondary flowers in the axils of first whorl organs in ap2 flowers (Komaki et al., 1988). Another mutant, ffl-7, also shows a dramatic difference in phenotype in response to a change in daylength (Shannon and Meeks-Wagner, 1991). It is perhaps significant that in each of the mutants cited above, growth in short days favors initiation of inflorescence meristems (&, terminalflower[fflj) or shifts the development of floral meristems toward that of inflorescence meristems (lfy, ap7, ap2), whereas growth in long days has the opposite effect. The similarity of the effect of growth in short days on each of these mutants suggests that some factor that is necessary for either the expression or the activity of these genes is regulated in response to daylength.

Genetic lnteractions of leafy with Homeotic Floral Genes

Ify and pi

The P l and AP3 gene products are required to specify the identity of organs in whorls 2 and 3 as sepals and petals (Bowman et al., 1989). The most flowerlike structures produced by &-7, like the flowers of pi-7 or ap3-7, show a conversion of petals

to sepals and stamens to carpels. The similar effect that mutations in LFY; Pl, and AP3 have on organ identity in the second and third whorls suggests that LFY is required along with PI and AP3 for the development of petals and stamens. One pos- sible role for LFY in the development of these whorls is as a positive regulator of Pl and/or AP3. However, lfy-7 has additional effects on the second and third whorls that are not found in pi-l or ap3-7 flowers. Flowers of lfy-7, unlike those of pi-7 or ap3-7, show a gradual transition in organ identity from sepals in the outer whorls to carpels in the inner whorls, with organs intermediate between sepals and carpels present in the second and third whorls. Therefore, the role of LFY in establishing the identity of organs in the second and third whorls is probably not confined to positive regulation of PlorAP3 expression.

Ify and ag

The first severa1 whorls of the Ify-7 ag-7 double mutant flowers resembled the previously described ap3-7 ag-7 double mutant flowers that produce only whorls of sepals (Bowman et al., 1989). However, later whorls of the Ify-7 ag-7 double mutant appear increasingly branchlike, with carpelloid leaves, axillary buds, a helical phyllotaxy, internode elongation, and an indeterminate mode of growth. The appearance of some carpelloid characteristics in the flowers of lfy-7 ag-7 in the absence of AG activity demonstrates that AG is not absolutely required for some aspects of carpel development such as the formation of ovules and stigmatic tissue. Other functions associated with AG, such as carpel fusion and determinate growth, do not appear in the lfy ag double mutants and may require AG.

The current model for the control of floral organ identity suggests that the sharp boundary between the second and third whorls is maintained through negative regulation of AG expression byAP2 and vice versa (Bowman et al., 1989,1991; Drews et al., 1991). Because this sharp boundary fails to appear in a lfy mutant, LFY must play a role in establishing this boundary, perhaps through some effect on the expression or function of AP2 or AG. One possibility consistent with the data is that the LFY gene product participates in both the repression of AG in the first and second whorls, and the activation of AG in the third and fourth whorls.

Ify and apl-1, Ify and ap2-1

When lfywas combined with either ap7-7 or ap2-7, the double mutants showed a complete conversion of all whorled flowers into inflorescence branches rather than the partia1 conversion seen in lfy-7 or lfy-73 alone. The loss in the double mutants af the few whorled flowers produced by the /fy parent indicates that AP7, AP2, and LFY act together to produce the whorled phyllotaxy, short internodes, and suppression of axillary buds found in a flower. The synergistic interaction of Ify with both ap7-7 and ap2-7 is reminiscent of an ap7-1 ap2-1 double homozygote (Irish and Sussex, 1990) and suggests some

912 The Plant Cell

degree of redundancy in the roles of these three genes in bring- ing about the whorled phyllotaxy, shortened internodes, and suppressed axillary buds characteristic of flowers.

The Ify ap7-7 double mutants differed in one respect from Ify ap2-7. The branchlike structures and the primary inflorescence of the Ify ap7-7 plants, like those of the Ify-7 and lfy-73 parents, eventually terminated in carpels, whereas those of the Ify ap2-7 double mutants did not. In addition, the leaves of the lfy ap7-7 branchlike structures frequently exhibited carpelloid characteristics such as aborted ovules along their margins and stigmatic tissue at their tips, and these characteristics appeared more frequently on the most dista1 organs. Although AG is not required for the formation of carpelloid leaves (Bowman et al., 1991), it may be essential for the termi- nation of growth after formation of carpels, suggesting that some late AG activity may be present in the branchlike structures and the primary inflorescence meristem of the Ify ap7-7 mutants.

Although the formation of carpelloid leaves and terminal carpels was entirely lost in the Ifyap2-7 double mutants, different results have been reported for a double mutant that was constructed using Ify-7 and ap2-6, a strong allele of ap2 (Schultz and Haughn, 1991). In the Ify-7 ap2-6 double mutant, some carpels did develop. Because the ap2-7 allele used in the construction of our My ap2-7 double mutants is not a null allele and may still retain the ability to suppress AG (Drews et al., 1991), the loss of all carpelloid traits in the Ify-7 ap2-7 and lfy-73 ap2-7 double mutants could be due to the negative regulation of AG expression by ap2-7. In this case, a triple mutant carrying strong alleles of Ify, ap2, and ag will show a similarly complete transformation of all flowers into inflorescence branches. Alternatively, the presence of carpelloid leaves in the lfy ap7-7 double mutants, but not in the Ify ap2-7 double mutants, could be due to the negative regulation of an AG-like gene by the ap2-7 gene product that was proposed to account for a similar effect when triple mutants carrying either ap2-7 or ap2-2 were compared (Bowman et al., 1991). Although mutations at the AP2 locus affect organ identity in only the first two whorls, the loss of carpels in the lfyap2-7 double mutant implies a role for AP2 in later whorls that could be masked by the presence of the wild-type LFYgene product. The effect of strong mutations in AP2 on organ number and position in the third and fourth whorls (Bowman et al., 1991) also sup- ports the possibility of a function for AP2 in later whorls.

Function of LFY

The predominant effect of mutations at the LEAFY locus is to cause a large proportion of the lateral structures that ordinar- ily develop as flowers to be converted into inflorescence branches, or intermediate structures with characteristics of both flowers and branches. The failure of even the severe alleles lfy-7 and Ify-72 to entirely block floral development suggests that, although the LEAFYgene plays an important role in determining the developmental fate of a floral meristem, this function of LEAFY may be duplicated in part byAP7 and AP2.

In addition to its role in floral meristems, LEAFY may also play a role in maintenance of inflorescence meristems. In all mutant alleles of LEAFY examined here, the primary inflorescence meristem and inflorescence branches eventually terminated in one or more carpels. The eventual conversion of all inflorescence branches and even the primary inflorescence into determinate structures that end in carpels demonstrates that the distinction between floral meristems, which develop in adeterminate way, and inflorescence meristems, which develop in an indeterminate way, is not maintained in a/fy mutant. The conversions of floral meristems into inflorescence meristems in young Ify plants and of inflorescence meristems into floral meristems in older Ify plants seem to require contradic- tory roles for LFY at different times or in different types of meristems.

One way around this apparent contradiction is offered by the possibility that, rather than being itself the determining factor that distinguishes a floral meristem from an inflorescence meristem, LFY might interact with such determining factors in both the floral meristem and the inflorescence meristem and, as a consequence of this interaction, activate genes appropri- ate for the development of each type of meristem. This is analogous to the role we have postulated for LFY in the determination of sepal and carpel identity within the flower. The effect of mutations in LEAFYon the inflorescence meristem suggests that the expression pattern of LFY may include the inflorescence meristem. Alternatively, LFY could exert its effect on the inflorescence meristem indirectly through generation of a factor in young flower primordia that is translocated to the inflorescence meristem.

METHODS

Plants were grown in a greenhouse with supplemental lights (16-hr day/8-hr night, 18 to 24T) unless othewise indicated. Plants grown in short days were maintained in a growth chamber under 8-hr days and 16-hr nights at 22T. Light intensities were 100 pmol m-2 sec-I for the growth chamber and ranged from 250 to 1200 pmol m-2 sec-I for the greenhouse, depending on the intensity of the ambient light.

Mutagenesis was carried out in the Columbia ecotype by soaking seeds in 03% ethyl methanesulfonate for 16 hr. lfy-72, lfy-73, and lfy-74 were isolated by screening the progeny of 860 M1 plants as individual families.

lfy-7 was a gift from George Haughn (University of Saskatchewan, Saskatoon, Canada). ag-7, pi-7, ap7-7, and ap2-7 were obtained from the AIS seed bank (A. R. Kranz, Botanisches Institut, J. W. Goethe Universitat, Frankfurt, Germany).

The LEAFY locus has been mapped to chromosome 5 between CER-3 (at 81.9 cM) and Yl (at 87.4 cM) by Schultz and Haughn (1991).

Because leafy is male sterile but somewhat female fertile, crosses with homozygous ap7-7 and ap2-7 were carried out using lfy-7 or lfy-73 homozygous mutants as the female parent. Crosses with ag-7 and pí-7 were carried out using plants heterozygous for these mutations as the pollen donor, and F2 families that segregated for both mutations were chosen for the double mutant analysis. Scanning electron microscopy was carried out on tissue fixed in 3.7% formaldehyde, 50% ethanol, 5% acetic acid for 30 to 60 min, dehydrated in a graded etha- no1 series, and critical point dried in liquid C02. Samples were


sputter-coated with palladium and examined in an ISI DS-130 scanning electron microscope with an accelerating voltage of 10 kV.

ACKNOWLEDGMENTS

We thank Keith Allen, Mark Carrier, Vivian Irish, Chad Nusbaum, and Gary Peter for a critical reading of the manuscript. We are grateful to Elizabeth Schultz and George Haughn for providing 4 - 7 . E.H. was supported by a postdoctoral fellowship from the National Science Foundation.

Received March 19, 1992; accepted June 5, 1992.

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DOI 10.1105/tpc.4.8.901 1992;4;901-913Plant Cell

E. Huala and I. M. SussexLEAFY Interacts with Floral Homeotic Genes to Regulate Arabidopsis Floral Development.

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