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Inflorescence Architecture: A Developmental Genetics Approach [*].











I. Abstract

We are characterizing a suite of Pisum sativum mutants that alter inflorescence architecture to construct a model for the genetic regulation of inflorescence development in a plant with a compound raceme. Such a model, when compared with those created for Antirrhinum majus and Arabidopsis thaliana, both of which have simple racemes, should provide insight into the evolution of the development of inflorescence architecture. The highly conserved nature of cloned genes that regulate reproductive development in plants and the morphological similarities among our mutants and those identified in A. majus and A. thaliana enhance the probability that a developmental genetics approach will be fruitful. Here we describe six P. sativum mutants that affect morphologically and architecturally distinct aspects of the inflorescence, and we analyze interactions among these genes. Both vegetative and inflorescence growth of the primary axis is affected by UNIFOLIATA, which is necessary for the function of DETERMINATE (DET). DET maintains indeterminacy in the first-order axis. In its absence, the meristem differentiates as a stub covered with epidermal hairs. DET interacts with VEGETATIVE1 (VEG1). VEG1 appears essential for second-order inflorescence ([I.sub.2]) development. veg1 mutants fail to flower or differentiate the [I.sub.2] meristem into a rudimentary stub. det veg1 double mutants produce true terminal flowers with no stubs, indicating that two genes must be eliminated for terminal flower formation in P. sativum, whereas elimination of a single gene accomplishes this in A. thaliana and A. majus. NEPTUNE also affects [I.sub.2] development by limiting to two the number of flowers produced prior to stub formation. Its role is independent of DET, as indicated by the additive nature of the double mutant det nep. UNI, BROC, and PIM all play roles in assigning floral meristem identity to the third-order branch. pim mutants continue to produce inflorescence branches, resulting in a highly complex architecture and aberrant flower s. uni mutants initiate a whorl of sepals, but floral organogenesis is aberrant beyond that developmental point, and the double mutant uni pim lacks identifiable floral organs. A wild-type phenotype is observed in broc plants, but broc enhances the pim phenotype in the double mutant, producing inflorescences that resemble broccoli. Collectively these genes ensure that only the third-order meristem, not higher- or lower-order meristems, generates floral organs, thus precisely regulating the overall architecture of the plant.

II. Introduction

Investigations of the diversity in inflorescence architecture among angiosperms have led to extensive typologies focused on final structure, with more limited emphasis on the development of the inflorescence (Weberling, 1989; Coen & Nugent, 1994; Grimes, 1996; Souer et al., 1998). Developmental studies can contribute substantially to our understanding of evolution (Diggle, 1992; Wray, 1994). For example, Tucker (1997) has devoted her career to comparative floral developmental studies in the Fabaceae (Leguminosae), leading to clear distinctions between parallel and convergent evolution of floral traits. Cladistic analysis in the Fabaceae has been enhanced by the complementation of ontogeny and molecular sequence analysis (Chappill, 1995; Doyle, 1997). Our approach to understanding inflorescence architecture is to analyze developmental mutants. The goal is to understand the genetic regulation of inflorescence development, beginning with the identification of key regulatory genes. Ultimately a comparative analy sis of these genes among species will provide a complementary perspective on the evolution of inflorescence patterns.

The relationship between single gene mutations and evolutionary events is a continuing source of debate. Hilu (1983) surveyed the literature on mutations affecting development in flowering plants and concluded that the predominance of single-gene mutations may affect macroevolution, leading to new taxa. The cloning of the CYCLOIDEA (CYC) gene in Antirrhinum majus (Asteridae) has focused the discussion on the evolution of zygomorphic flowers (Luo et al., 1996). CYC is required for normal development of zygomorphic flowers. Donoghue et al. (1998) conducted an expanded phylogenetic analysis of the Asteridae and concluded that zygomorphic flowers arose at least eight times and that there were at least nine reversals to actinomorphy. Not all reversals were loss-of-function mutations. They suggest that several developmental pathways with different genes (not necessarily CYC loss of function) will be found to account for derived actinomorphy. This illustrates the potential for a genetics approach to distinguish bet ween parallel and convergent evolution, much as Tucker (1997) has done with developmental morphology. Doebley and Lukens (1998), however, emphasize that protein function is highly conserved and that transcriptional regulators play key roles in plant development. Plant form may evolve by alterations in cis-regulatory elements in genes or transcription regulators. They propose that the predominant mechanism in the evolution of form is change in the cis-regulatory elements of transcription regulator genes. Temporal and spatial changes in gene expression can result in substantial morphological change.

Genes affecting reproductive development in Arabidopsis thaliana and A. majus have been shown to be highly conserved among the angiosperms (Frohlich & Meyerowitz, 1997). Floral meristem identity genes affect inflorescence architecture by initiating floral development. The MADS-box floral genes (transcription regulators), which include the A. thaliana floral meristem identity gene APETALAI (API) and its homolog, SQUAMOSA (SQUA), in A. majus, first began to diverge from one another before the origin of seed plants (Mandel et al., 1992; Munster et al., 1997; Purugganan, 1997). LEAFY (LFY), an A. thaliana floral meristem identity gene, is another highly conserved regulatory gene with homologs in the basal angiosperms and Gnetales (Weigel et al., 1992; Frohlich & Meyerowitz, 1997). The LFY/FLO homologs are essential for normal development in plants with cymose inflorescences (the sympodial growth of the Solonaceae) and racemose inflorescences (simple and compound). LFY and its homolog in A. majus, FLORICAULA (FLO ), are expressed primarily, though not exclusively, in floral meristems, while homologs in Pisum (UNI), Nicotiana (NFL), Impatiens, and Petunia (ALF) are expressed in vegetative meristems as well (Kelly et al., 1995; Hofer et al., 1997; Poteau et al., 1997; Souer et al., 1998). A vegetative phenotype, however, is found only in P. sativum uni mutants, in which compound leaves become simple (Hofer et al., 1997). Our work is aimed at understanding how highly conserved genes are used to create diverse inflorescence architectural patterns. We are elucidating and comparing the genetic regulation of inflorescence development in Pisum sativum (garden pea) with A. thaliana and A. majus, two model systems for molecular genetic analysis of flowering (reviewed by Weigel, 1995; Yanofsky, 1995; Amasino, 1996).

P. sativum was selected for several reasons. As a result of the long history of the pea as both a model genetic system and an agriculturally important crop, numerous mutants alter reproductive development. The inflorescence of the pea is one step more complex than the simple racemes of A. thaliana and A. majus. Thus comparative analysis may provide insight into an underlying genetic mechanism that distinguishes simple and compound racemes. Our long-range plan to extend our work to other family members will be facilitated by comparative data on more than 200 taxa within the Fabaceae, which is broader than any other group (Endress, 1994; Tucker & Douglas, 1994). Although P. sativum is a highly derived and domesticated legume, it shares basic inflorescence architectural features with its wild relatives (P. abyssinicum, P. humile, and P. elatius) and with sweet peas (Lathyrus). The indeterminacy of the inflorescence is most likely the basal state in the Fabaceae (Tucker, 1998). Also, some of our mutations appear to "unmask" ancestral traits found in more basal Fabaceae.

III. Overview of Pea Inflorescence Development

The pea is a quantitative, long-day plant with flowering time regulated by both a graft-transmissible floral promoter and inhibitor with meristem sensitivity to the signal(s) controlled by the gene LF (LATE FLOWERING) (Reid et al., 1996; Weller et al., 1997). The transition from vegetative to inflorescence development parallels an increasing complexity in leaf structure. Commitment to inflorescence development precedes the initiation of nodes that will contain second-order inflorescence meristems ([I.sub.2]) meristems) (Ferguson et al., 1991). The first order inflorescence ([I.sub.1]) is comparable to the preceding vegetative ([V.sub.1]) axis, except that [I.sub.2] rather than [V.sub.2] meristems are initiated in each leaf axil (Figs. 1-2). Multiple axillary meristems may be initiated at anode, but in wild-type peas only one develops into a shoot. An [I.sub.1] meristem is indeterminate. Determination for inflorescence development found in cultured meristems provides evidence that the meristem that initiates the [I.sub.1] is in a developmentally distinct state from the [V.sub.1] even though flowers are initiated only on the third-order axis (Ferguson et al., 1991). An [I.sub.2] meristem initiates one or more floral meristems before terminating in a stub covered with epidermal hairs (Hole & Hardwick, 1976; Singer et al., 1990). The result is a compound raceme.

In this article we use a developmental genetics approach to construct a model for regulation of inflorescence architecture in pea (see Table I for a summary of the P. sativum mutants reported here). Descriptive and experimental analysis of the following mutants provides the basis for this model. Vegetative I (veg I) fails to make floral structures or [I.sub.2] stubs (Reid & Murfet, 1984). determinate (det) is ahomeotic mutation that converts an [I.sub.1] meristem to an [I.sub.2] menstem, so that the primary axis of the plant terminates as a stub (Singer et al., 1990; Ferguson et al., 1991). Unlike terminal flower (tfl) in A. thaliana and centroradialus (cen) in A. ma]us, suppression of indeterminacy in the main axis by det does not result in the production of a terminal flower (Shannon & Meeks-Wagner, 1991; Alvarez et al., 1992; Bradley et al., 1996). neptune (nep) increases the number of flowers produced by an [I.sub.2] meristem. PROLIFERATING INFLORESCENCE MERISTEM (PIM), a floral menistem identity gene, i s necessary for the third-order meristem of the inflorescence to develop into a flower. The pim mutant phenotype is more severe in the broccoli (broc) background, much like the interaction reported between cauliflower (cat) and apl in A. thaliana (Kempin et al., 995). UNA the homolog of LEY/FLO, is another floral menistem identity gene, but it also plays a role in leaf development (Hofer et al., 1997). Mutations in these genes alter the branching patterns and floral development of pea inflorescences.

IV. Methods


Seeds heterozygous for det were provided by the late G. A. Marx (New York State Agricultural Experiment Station, Geneva, NY). Heterozygotes were continuously selected and selfed for nine generations prior to the selection of the mutant and wild-type lines used in this study. Marx also provided st (line B88-l81, a mutation causing reduced stipules), multipodded det, and nep seed. pim was a spontaneous mutant isolated in our greenhouse out of an early-flowering line (line C76-505) from Marx. The mutant was outcrossed into a number of different lines to assess genetic background effects on phenotype and was allowed to self-pollinate for at least seven generations to near isogeneity. To determine genetic interactions with other developmental genes, double mutants were costructed using pim as the pollen parent. Seeds heterozygous for vegl were provided by I. C. Murfet (University of Tasmania, Hobart, Australia). uni (line JI 2171 T) was procured from the John Innes Centre (Norwich, England) as a heterozygote. The uni allele, tac, which alters leaf development but not floral development (Marx, 1987), was not used in these studies.


Unless otherwise noted, seeds were sown in a soilless potting mix (Prime-Gro 7, Therm-O-Rock East, New Eagle, PA) in 10cm pots in growth rooms. Long-day conditions (18 hours light/6 hours dark) were maintained with high-pressure sodium vapor and metal halide lights (1:1 ratio) for 8 hours (600-700 [micro]mol photons/[m.sup.2] /[second.sup.1] of photosynthetically active radiation) and low-level incandescent bulb irradiation for 10 hours. Day/night temperatures were 21.5/18.5 C. Plants were fed weekly with 200 ppm of Plantex 20:10:20 fertilizer (Plantex Corp., Brampton, Ontario, Canada).


Plant age was determined by counting the number of nodes acropetally from the first node above the cotyledons to the last unfolded sepal pair. The first node to contain an [I.sub.2] meristem is termed the NFI (node of floral initiation). Typically all nodes initiated after the NFI also have developed [I.sub.2] branches. [V.sub.2] buds are capable of initiating inflorescence axillaries ([I.sub.3] branches in this case), but only after initiating vegetative nodes.


Shoot apices were dissected under a stereoscope and fixed in 2% gluteraldehyde in Millonig's buffer (1961), dehydrated in an acetone series, and critical-point dried with [CO.sub.2] in a Balzers Union CPD020 apparatus. Samples were mounted on stubs and coated with gold in a Hummer Jr. Sputter coater before viewing in a JEOL JSM-350 JEM at 15 kV. Photomicrographs were taken on Polaroid Type 55 film, or images were digitized with slow-scan acquire system from JEOL.


In wild-type plants, the [I.sub.1] stem is indeterminate and has compound leaves with axillary meristems that produce [I.sub.2] inflorescences. [I.sub.2] meristems have suppressed leaf development, produce floral (F) meristems in their axils, and terminate as rudimentary stubs. Early in floral development, third-order floral meristems initiate zygomorphic flowers with an outer whorl of five sepals, a second common whorl of five petals and five stamens, a third whorl of five stamens, and a carpel in the fourth whorl (Tucker, 1989).

Some of our mutations give rise to second-, third-and fourth-order branches that do not fit the description of [I.sub.1], [I.sub.2], or F branches found in the wild type. The classification of mutant stem complexes was based on the presence of floral, infloral, and vegetative traits inclusive to a given axis. Floral traits scored included the presence of floral organs (stamens, carpels, petals, and sepals), whorled phyllotaxy, internode compression, lack of axillary structures, and determinate growth. Infloral traits included nodes with modified or suppressed leaves and termination as a stub. Vegetative structures included complex leaves (or parts thereof), distichous phyllotaxy, internode elongation, and apical meristem indeterminacy, as did first-order inflorescence stems. When the stipules at the seventh node above the NFI unfolded, the apex of the third- and fourth-order branches were categorized according to their developmental fate: termination as a stub; termination as a "flower" (determinate or indet erminate); or indeterminate vegetative stem.

V. Results and Discussion


Analysis of det plants is consistent with the interpretation that DET is necessary to maintain indeterminacy in the [I.sub.1]. In the absence of DET, the terminal (first-order) meristem initiates two or three [I.sub.2] meristems before forming a rudimentary stub (Fig. 3; Singer et al., 1990). The terminal meristem of wild-type plants ceases growth as flowers and fruits develop, but it maintains its meristematic state. This is in contrast to the det terminal meristem (Figs. 4-6). Removal of flowers and fruits results in resumed meristematic growth in wild-type plants but not in det plants (Maki & Singer, unpubl.).

det does not affect the vegetative meristem, which is consistent with the interpretation that [I.sub.1] and [V.sub.1] meristems are developmentally distinct. Murfet (1989) has suggested that a minimum number of vegetative nodes (possibly 10) is required before det apices terminate, even in very early flowering lines. This is consistent with the interpretation that the transition from a [V.sub.1] to an [I.sub.1] meristem represents a phase change. Given that det has no effect on the vegetative phenotype, it should affect the vegl mutant phenotype only if vegl, which never initiates floral organs (Figs. 7-8), has made the transition to an [I.sub.1] meristem. To test this, we constructed det vegl double mutants and obtained a surprising result. The plants flowered, and no stubs formed on any of the axes (Figs. 9-11). Thus, elimination of the function of two genes is necessary for terminal flower formation in P. sativum.

In A. thaliana and A. majus, a single gene (the homologs TFL and CEN, respectively) maintains indeterminacy in the terminal meristem (Shannon & Meeks-Wagner, 1991; Bradley et al., 1997). It is possible that either DET or VEGl is homologous to TFL and CEN; however, since VEGl appears to be responsible for [I.sub.2] stub formation, DET is the more likely candidate. Alternatively, the DET VEGl gene combination that suppresses determinacy may have evolved independently. Stebbins (1974) has argued that a cymose inflorescence was basal in the angiosperms. Suppression of terminal flower formation appears to have occurred before the Fabaceae arose, since racemes are basal in this family (Tucker, 1998). The compound raceme observed in Pisum may be accounted for by the suppression of second-order flower development by a second gene. TFL has been proposed to have a more global effect on shoot architecture by extending both the vegetative and reproductive phases, so that senescence occurs before a terminal flower forms (Ratcliffe et al., 1998). CEN, however, is only expressed in later inflorescence stages. DET function appears to be restricted to the [I.sub.1] and does not alter the number of vegetative nodes before flowering.


Unlike DET, UNI function is necessary for normal vegetative development. Two mutant alleles of UNI have altered leaf morphology-simple leaves or conversion of tendrils to leaflets (Marx, 1987). We have observed a second phenotypic effect of union the first-order axis. After initiating several 12 axillary meristems, uni plants terminate with the formation of a stub, just like det plants (Figs. 12-14). The same phenotype is observed in uni det plants, which are indistinguishable from uni plants. Thus DET probably functions downstream of UNI. This is consistent with our hypothesis that DET and CEN/TFL are homologs. Expression of CEN is dependent on expression of FLO, the homolog of UNI (Bradley et al., 1996; Hofer et al., 1997). Terminal flower suppression results when FLO suppresses CEN. The interactions between FLO and CEN are indirect (Ratcliffe et al., 1998). In the pea, [I.sub.1] identity is regulated by UNI and DET, which appear to function in the same pathway. VEGI may be necessary for the [I.sub.1] meri stem to initiate an [I.sub.2] meristem. It may be essential for stub formation.


[I.sub.2] meristems initiate one or two floral meristems before terminating as a stub. NEP appears to be responsible for limiting the number of flowers initiated by the [I.sub.2] meristem before stub formation. nep-1 mutants can produce as many as five pods on the first inflorescence branch under long-day conditions before stub formation (Fig. 15). We have identified another allele of NEP (nep-2) that has a similar mutant phenotype in the multipodded det line obtained from Marx. The multipodded trait behaves as a recessive single gene. Complementation tests revealed that this multipodded gene is allelic to NEP. Thus we refer to nep as nep-1 and to the multipodded allele as nep-2. These alleles should be particulary useful in separating resource-allocation effects on architecture from basic parameters established genetically. Genetic and environmental effects were distinguished by growing wild-type, nep-1, and nep-2 plants in 10 cm and 15 cm pots (Table II). Wild-type plants always produced two flowers per no de, whereas mutants produced more nodes than did the wild type in both 10 cm and 15 cm pots. Unlike the wild type, both nep-1 and nep-2 plants produced more flowers in 15 cm pots than in 10 cm pots. The larger pot size increases the root mass and, presumably, the resources available for growth. Thus, the nep-1 and nep-2 mutations appear to release the growth of the [I.sub.2] meristem from strict genetic control and allow for plasticity in development based on nutritional factors.

Diggle (1992) suggests that developmental processes themselves are targets of natural selection and provides numerous examples illustrating complex relationships between development and evolution. It is intriguing that NEP plays a role in limiting F meristem production on an [I.sub.2] in P. sativum and most likely its wild relatives; yet the perennial sweet pea, Lathyrus latifolius, produces large numbers of flowers and pods at each reproductive node for an extended period of time. One hypothesis is that the flower- and fruit-induced senescence in Pisum is not present in L. latifolius. Wild-type P. sativum produces flowers over an extended period of time as each new reproductive node expands; however, in nep-1 and nep-2 plants all flowers form and set fruit over a reduced time period. Given the constraint of flower/fruit-induced senescence, an extended period of time for flowering maybe more advantageous than obtaining a few more seeds from the nep plant. Such mutants make possible a more integrated approach to the study of the evolution of plant development.

We investigated the interaction of DET and NEP by creating the double mutant det nep-I (Fig. 16). The phenotype is identical to the multipodded det line (containing nep-2) that we obtained from Marx. The phenotype is additive, with additional flowers forming directly in the axils of the first-order branch before the first-order meristem terminates as a stub. Visually, this illustrates the homeotic conversion of the [I.sub.1] meristem to an [I.sub.2] meristem. The pheno-type of the second-order inflorescence branch is altered by nep-2 but not by det, which also has no effect on the 12 in the single det mutant. Based on these observations, it is clear that [I.sub.1] and [I.sub.2] meristems have distinct, genetically controlled programs.


1. The Effect of pim Mutation on Inflorescence Architecture

In the reproductive phase of P. sativum development, the third-order meristem initiates floral organs. PIM is critical in specifying floral meristem identity. It does not have a direct effect on [I.sub.1] or [I.sub.2] meristems; nor does it alter the node of floral initiation. The pim mutant replaces flowers with extensive inflorescence and vegetative branching (Fig. 17). The [I.sub.2] terminates in a stub after producing one or two nodes (Fig. 18). Curiously, both primary and secondary buds form and develop in these [I.sub.2] nodes. In wild-type P. sativum, secondary axillary buds are present in the vegetative axils, but they seldom develop. We have observed secondary axillary bud formation in wild-type [I.sub.2] nodes, but these buds do not develop. In pim these secondary axillaries develop later as vegetative shoots, perhaps because functional floral signal(s) are no longer present (Fig. 19).

In the third-order branch, single flowers are replaced with elaborate branch systems that exhibit amalgams of floral, infloral, and vegetative character states. The first lateral appendages of the third- and higher-order meristems are paired, opposite foliar structures consistent with Tucker's (1987) definition of bracteoles in other Fabaceae, except that bracteoles tend not to have shoots or buds. Morphologically the subtending, decussate lateral appendages in pim flowers (presepal) seem homologous with bracteoles, as seen in Sophora japonicum (Tucker & Douglas, 1994). After producing one to several sets of paired bracteate nodes, each stem terminates either as a stub or as a flower, or else exhibits indeterminate vegetative growth, reiterating the entire V[right arrow]I[right arrow]"F" pattern. From out of the bracteate nodes grow axillary stems, which, in turn, produce more axillary stems before the growing point pursues one of the three terminal options: stub, carpel, or vegetative shoot (Figs. 20-23). T hese triad structures are distinct from those reported for Petunia, in which the inflorescence meristem bifurcates into an F and an I meristem (Souer et al., 1998). Tucker (1987) has found pseudoracemes that resemble the pim triads in 5 of about 32 papilionaceous tribes. While there is more variability in the fate of the three meristems in the pim triad, it is possible that PIM suppresses this phenotype found in other relatives and may have evolutionary significance for inflorescence architecture. The absence of bracteoles and other leafy outgrowths in the third-order axis of wild-type P. sativum, and their presence in pim plants, is indicative of another aspect of PIM suppression of vegetative growth.

The development and outgrowth of more than one axis from the high-order nodes provides additional complexity to the branching pattern. Generally, these secondary axillaries develop as vegetative shoots. One result is the presence of compound leaves in the inflorescence. Although their development lags significantly behind that of the primary axillaries, they can eventually outgrow the remainder of the inflorescence. Thus, there exists the potential of indefinite proliferation of the pim inflorescence (Figs. 24-25).

Floral development is aberrant in comparison with the carefully documented development of wild-type flowers by Tucker (1989). The petals are most affected, and organ fusions occur regularly (Figs. 26-28). The first initiated petals in P. sativum are those that fuse as the keel. These two petals can be distinguished by color in certain genetic backgrounds and are almost always missing in pim. Petals are lacking to various degrees. If only one petal is present, it is invariably the vexillum. Even when, in the rare instance, all five petals are present, their arrangement is skewed, so that the wild-type imbricate pattern is not followed. There is also a great deal of organ fusion, especially between sepals and anthers, and anthers and carpels. In some cases accessory flowers or inflorescences form in the axil of the sepals, which morphologically take on more leaflike, specifically stipulelike, traits (Fig. 29).

The phenotype of the pim mutant is consistent with Poethig's (1990) contention that juvenile, adult, and reproductive programs are discrete and can overlap. In pim, insufficiencies in the reproductive program may permit the phenotypes of other programs to become more apparent. Alternatively, PIM may be required to suppress vegetative growth. Although reproductive development does not require the termination of a vegetative phase in maize, it may be that the vegetative program prevents normal floral development (Bassiri et al., 1992; Evans & Poethig 1997). Many examples of vegetative development exist in pim inflorescences, from the lack of bract suppression in some [I.sub.2] branches to the presence of compound leaves in later-order inflorescence branches.

The relative activity or level of products of these different programs (vegetative, infloral, and reproductive, in the terminology used in this article) may result in the distinct developmental states reported by McDaniel et al. (1992) in the progression from vegetative to reproductive development. For example, a meristem can be determined for inflorescence development in P. sativum, and this determination is distinct from determination for flower development (Ferguson et al., 1991). In the case of pim, the third- and higher-order branches appear to express components of the vegetative, inflorescence, and floral programs in a nonlinear fashion (Fig.30)

Time of development of specific meristems and primordia appears to be as critical as position in this mutant, in which reversion can occur. Hempel and coworkers (1998) have shown in A. thaliana that developing primordia have a quantitative response to floral induction signals and that the fate of primordia can be modified in situ after initiation. PIM may act coordinately with other floral meristem identity genes to provide floral identity signals, not only in sufficient quantity but also at the correct times.

2. broccoli Enhances the pim Phenotype

We have identified a single gene, the recessive mutation broccoli (broc), which has a normal phenotype in a wild-type background. The double mutant, pim broc, has a more extreme pim phenotype (Figs. 31-32). The branching that occurs after the initiation of the third-order meristem is more extensive, and flower development is minimal. The phenotype resembles a head of broccoli. Thus BROC and PIM may serve partially redundant functions in assigning floral meristem identity.

Morphologically, pim resembles the apetala-1 mutant of A. thaliana, which exhibits a more extreme phenotype when combined with the cauliflower (cal) mutation (Bowman et al., 1993). BROC and CAL are also potential homologs. The triple mutant ap1 cal tfl has the same phenotype as ap1 tfl plants. Thus tfl appears to inhibit the enhancement of the ap1 phenotype by cal. Based on phenotype, we believe that DET in P. sativum, CEN in A. majus, and TFL in A. thaliana have potential functional homologies. The triple mutant pim broc det is additive, unlike the ap1 cal tfl triple mutant, indicating that det does not interfere enhancement of the pim phenotype by broc (Fig. 33). Thus, if homologies exist among these three genes in A. thaliana and P. sativum, the ways they coordinate to effect inflorescence architecture will differ.


UNI is necessary for normal floral development, in addition to its role in vegetative and [I.sub.1] development (Fig. 12). The [I.sub.2] of uni plants is normal and terminates with a stub. The [I.sub.3] begins to initiate what resembles a whorl of sepals, and then a series of flowerlike structures begins to develop within these axils and the axils of later-initiated structures (Fig. 34; Hofer et al., 1997). Primarily sepal-carpel intermediates, carpels, and sepals form. Although we have observed an occasional anther, petals are absent. The phenotype is quite similar to the uni homolog, lfy, in A. thaliana (Weigel et al., 1992; Hofer et al., 1997).

The double mutant pim uni branches like pim plants creating nested triads, although single axillaries initiated are usually suppressed in the wild type and often in pim. In place of flowers there are whorls of leaflike structures at later branch points (Figs. 35-36). uni appears to enhance the outgrowth of [I.sub.2] bracts. If pim is the homolog of apI in A. thaliana, then the pheno-type of pim uni in P. sativum is consistent with the interactions reported between API and LFY in A. thaliana (Bowman et al., 1993; Parcy et al., 1998). The double mutants apI lfy (there are multiple alleles of these A. thaliana genes) are morphologically similar to the pim uni plants. API and LFY are believed to have overlapping and distinct functions. LFY has been shown to induce API expression (Parcy et al., 1998). UNI is expressed in the vegetative phase of P. sativum development and controls more than the floral meristem identity role proposed for PIM. Clearly the additive phenotypic effects seen in the aberrant inflorescenc es of pim uni plants indicate that UNI plays a role in flower development that is at least partially distinct from that of PIM.

VI. Summary

The evolution and development of inflorescence architecture can be fruitfully investigated through analysis of mutants that appear to play a key regulatory role in inflorescence development. Our study of mutants in P. sativum has allowed us to identify genes that regulate key steps in compound raceme inflorescence development and to begin to explore their functions (Fig. 37). Not only are inflorescence and floral meristems developmentally distinct, but in a compound raceme like that of P. sativum, inflorescences of different branch orders are also distinct. What is particularly valuable about this suite of mutants is that they affect several distinct stages of inflorescence development, which allows us to identify [I.sub.1], [I.sub.2], and F meristems in terms of key genes as well as of morphological traits. This has allowed us to explore the effects of changing certain inflorescence components on the overall architecture.

Finding homologies between these genes and those that have been identified in A. majus and A. thaliana, which have simple rather than compound racemes, will further our understanding of the evolution of inflorescence architectures. A second avenue of pursuit is to take advantage of the extensive morphological data on Fabaceae and begin a comparative study to determine how gene function may have been modified, deleted, or added over time within this family.

VII. Acknowledgments

We dedicate this article to the late Dr. Gerry Marx, who generously provided us with the mutants, advice, and gentle encouragement that launched our exploration of developmental regulation of inflorescence architecture in peas. Conversations about pea developmental mutants with Ian Murfet and Scott Taylor (University of Tasmania) and with Julie Hofer and Noel Ellis (John Innes Institute) have been most helpful. We thank the students on the Carleton Greenhouse Crew for excellent plant care. This work was supported by NSF RUI grant 9405799 and USDA grant 9103136 to Susan Singer.

(1.) Department of Biology Carleton College Northfield, MN 55057, U.S.A.

(2.) Department of Horticulture Clemson University Clemson, SC 29634, U.S.A.

(*.) Gene symbols used in this article: For clarity a common symbolization is used for genes of all species discussed in this article. Genes are symbolized with italicized capital letters. Mutant alleles are represented by lowercase, italicized letters. In both cases, the number immediately following the gene symbol differentiates among genes with the same symbol. If there are multiple alleles, a hyphen followed by a number is used to distinguish alleles. Protein products are represented by capital letters without italics.

VIII. Literature Cited

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 Pisum sativum inflorescence architecture genes
Gene Mutant phenotype
VEGI No flowers, no [I.sub.2] stub
DET Converts [I.sub.1] meristem to
 [I.sub.2] meristem terminating as a stub
NEP Multiple pods per inflorescence branch
 in both nep-1 and nep-2 alleles
PIM Floral meristem develops as anflorescence meristem
BROC Enhances pim phenotype; broc alone is wild type
UNI Reduces compound leaves to simple leaves; flowers
 contain mainly leaflike sepals and carpels
Gene Reference
VEGI Reid & Murfet, 1984
DET Singer et al., 1990
NEP Singer, unpubl.
PIM Reid et al., 1996
BROC Singer, unpubl.
UNI Hofer et al., 1997; Marx, 1987
 Effect of genotype and pot size on number of
 flowers produced by second-order
 inflorescence meristems [a]
 Pot size Mean number of flowers
Genotype (inches) [plus or minus] SE (n)
NEP 4 2.0 [plus or minus] 0.0 (15)
 6 2.0 [plus or minus] 0.0 (15)
nep-1 4 2.7 [plus or minus] 0.2 (19) [b]
 6 4.2 [plus or minus] 0.3 (20) [b]
nep-2 4 2.8 [plus or minus] 0.1 (45) [c]
 6 4.0 [plus or minus] 0.1 (28) [c]
(a.)All data are for the first floral node of each plant.
(b.)Two-tailed t-test indicates that means are
different (p [less than].0001).
(c.)Two-tailed t-test indicates that means are
different (p [less than].0001).
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Publication:The Botanical Review
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Date:Oct 1, 1999
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