The Plant Cell, Vol. 12, 1279–1294, August 2000, www.plantcell.org © 2000 American Society of Plant Physiologists Pea Compound Leaf Architecture Is Regulated by Interactions among the Genes UNIFOLIATA, COCHLEATA, AFILA, and TENDRIL-LESS Campbell W. Gourlay, Julie M. I. Hofer,1 and T. H. Noel Ellis Department of Applied Genetics, John Innes Centre, Colney Lane, Norwich NR4 7UH, United Kingdom The compound leaf primordium of pea represents a marginal blastozone that initiates organ primordia, in an acropetal manner, from its growing distal region. The UNIFOLIATA (UNI) gene is important in marginal blastozone maintenance because loss or reduction of its function results in uni mutant leaves of reduced complexity. In this study, we show that UNI is expressed in the leaf blastozone over the period in which organ primordia are initiated and is downregulated at the time of leaf primordium determination. Prolonged UNI expression was associated with increased blastozone activ- ity in the complex leaves of afila (af), cochleata (coch), and afila tendril-less (af tl) mutant plants. Our analysis suggests that UNI expression is negatively regulated by COCH in stipule primordia, by AF in proximal leaflet primordia, and by AF and TL in distal and terminal tendril primordia. We propose that the control of UNI expression by AF, TL, and COCH is important in the regulation of blastozone activity and pattern formation in the compound leaf primordium of the pea. INTRODUCTION The genetic analysis of compound leaf development has flowers into proliferating floral structures of mainly sepalloid concentrated on the model organisms pea and tomato. and carpelloid organs (Hofer et al., 1997). The pleiotropic ef- These two species are distantly related, pea (Fabales) being fects of the uni mutation suggest that UNI plays an impor- in the eurosid I group of eudicots and tomato (Solanales) in tant role in patterning both leaves and flowers in pea. In the euasterid I group (Angiosperm Phylogeny Group, 1998). tomato, the mutation that probably corresponds to uni has The leaves of these two species, however, undergo different recently been identified as falsiflora (fa), which is also pleio- morphogeneses: pea leaves initiate organs acropetally tropic in effect. The inflorescences of fa plants are converted (Meicenheimer et al., 1983), whereas tomato leaves do so in into ramified, leafy structures without flowers, and the mu- a basipetal fashion (Dengler, 1984). Recently, genes have tant leaves have fewer small, lateral leaflets than do wild- been identified that influence indeterminacy in tomato and type leaves (Molinero-Rosales et al., 1999). pea leaf primordia and thus control aspects of their leaf ar- The UNI and FA genes are homologs of the floral mer- chitecture. In tomato, two class 1 KNOTTED1-like homeo- istem identity genes FLORICAULA (FLO; Coen et al., 1990) box (KNOX) genes, TKN1 and TKN2, members of a gene and LEAFY (LFY; Weigel et al., 1992) from Antirrhinum and family important in shoot apical meristem (SAM) mainte- Arabidopsis, respectively (Hofer et al., 1997; Molinero- nance and function, promote more ramified leaf forms when Rosales et al., 1999). Other potential homologs of FLO/LFY overexpressed in transgenic plants (Hareven et al., 1996; have been identified in monocotyledonous (Colombo et al., Parnis et al., 1997; Janssen et al., 1998). In pea, the gene 1998; Kyozuka et al., 1998) and dicotyledonous (Anthony et UNIFOLIATA (UNI; Eriksson, 1929) is important in regulating al., 1993; Rottman et al., 1993; Kelly et al., 1995; Pouteau et compound leaf architecture such that the leaves of uni al., 1997; Souer et al., 1998; Molinero-Rosales et al., 1999) plants are reduced to a more simplified form. UNI is thought angiosperm species, in basal angiosperms and gnetales to promote compound architectures by maintaining a period (Frohlich and Meyerowitz, 1997), and in the gymnosperm of indeterminacy in a developing leaf primordium (Hofer et pine (Mouradov et al., 1998). Of the dicots studied to date, al., 1997). In addition to its effect on leaves, the uni mutation FLO/LFY transcripts were detected in the leaf primordia of also perturbs floral development, transforming wild-type tobacco, Arabidopsis, pea, Impatiens, tomato, and petunia (Kelly et al., 1995; Blázquez et al., 1997; Hofer et al., 1997; Pouteau et al., 1997; Pnueli et al., 1998; Souer et al., 1998; Molinero-Rosales et al., 1999), but mutant leaf phenotypes 1 To whom correspondence should be addressed. E-mail hofer@ have been described only for those species with compound bbsrc.ac.uk; fax 44-1603-456844. leaves: pea and tomato. Hofer et al. (1997) proposed a 1280 The Plant Cell common function for UNI in regulating indeterminacy during The coch mutation (Wellensiek, 1959) can increase the both leaf and flower development. An opposite role in spec- complexity of pea leaves at the stipule position. On plants ho- ifying lateral organ determinacy was suggested for the to- mozygous for the coch-5137 allele, stipules between nodes 8 bacco homolog NICOTIANA FLO/LFY (NFL; Kelly et al., and 15 often mimic the morphology of the blade, with fully or- 1995), although no loss-of-function tobacco mutant has ganized leaflets and tendrils (Figure 1F). We refer to these lat- been identified to support this role in leaf development. eral structures as compound stipules because they arise at There are no reported morphological variations in the leaves the stipule position, but note that they lack stipules them- of lfy or the corresponding petunia mutant, aberrant leaf and selves. Between nodes 8 and 15, both stipules may be com- flower (alf), and neither LFY nor ALF is thought to play a pos- pound, both may be reduced to a simple petiolate form, or itive role in leaf development (Weigel et al., 1992; Souer et the pair can be a combination of one compound and one re- al., 1998). Vegetative tissues that accumulated LFY in Arabi- duced stipule (Blixt, 1967; Gourlay, 1999). dopsis were viewed as primordia with the potential to adopt an alternative floral fate. It was suggested that when LFY reached a critical value, these lateral primordia would be- Effects of the uni Mutation on Leaf Development come competent to respond to floral initiation signals (Blázquez et al., 1997). To examine the effects of the uni mutation on leaf primor- Here, we examine the role of UNI in pea leaf development dium initiation and early development, scanning electron mi- by studying its expression in several mutant backgrounds. croscopy (SEM) analysis was performed on the dissected Previous genetic evidence has shown that UNI interacts with apices of 2-week-old wild-type and uni mutant JI 2171 the AFILA (AF) and TENDRIL-LESS (TL) genes to control as- plants, as shown in Figure 2. Leaf primordia arose laterally 180Њ to each other in a sequential mannerف pects of pea leaf architecture (Sharma, 1981; Marx, 1986, from the SAM at 1987; Hofer and Ellis, 1996, 1998). The reduction in overall leaf on wild-type plants (Figure 2A). By the end of plastochron 1 complexity seen in tl, af, and af tl mutant plants when com- (P1), a pair of stipule primordia (S1) had emerged and were bined with the uni mutation (Marx, 1987; Hofer and Ellis, 1996) visible as small bumps on either side of the leaf primordium, suggests that interactions between UNI, AF, and TL are im- or marginal blastozone (Figure 2B). The proximal leaflet pri- portant in regulating the branching potential of a pea com- mordia were next to emerge and could be seen clearly at P2 pound leaf. In this article, we demonstrate that the AF, TL, and (Figure 2A). During P3, the distal tendril primordia were initi- COCHLEATA (COCH) genes negatively regulate UNI expres- ated. The proximal leaflet and stipule primordia had flat- sion. We propose that these interactions influence the organo- tened, were beginning to grow in toward the apex, and had genic potential of the primordium and are fundamental in begun to initiate epidermal hairs (Figure 2A). Whereas the determining the compound leaf architecture of the pea. leaflet primordia had begun to fold during P3, the stipule pri- mordia (S3) had not. No more organs were initiated from the blastozone, which became determined during P4 and formed a terminal tendril (Figure 2B). RESULTS In the JI 2171 uni mutant, disruption to normal leaf develop- ment occurred early after leaf primordium initiation (Figure 2C). Pea Leaf Development The blastozone emerged laterally on the SAM, and stipule pri- mordia (S1) emerged late in P1, as seen in wild-type plants The mature wild-type pea leaf shown in Figure 1A is com- (Figure 2B). The stipules (S1 to S3) appeared to develop nor- pound pinnate, consisting of a basal pair of foliaceous sti- mally and at a similar rate as those of the wild type, but no fur- pules, a pair of proximal leaflets, two pairs of distal tendrils, ther pairs of leaflet or tendril primordia were initiated (Figures and a terminal tendril. Stipule, leaflet, and tendril primordia 2C and 2D). At P2, the blastozone showed signs of differentia- are initiated in an acropetal manner on the compound leaf tion into a terminal, unifoliate leaflet with a central crease primordium (Meicenheimer et al., 1983), which is termed a marking the beginning of lamina folding (Figure 2C). This re- marginal blastozone (Hagemann and Gleissberg, 1996). The duction of organogenic potential indicated that in plants of this recessive uni mutation (Eriksson, 1929) reduces the com- age, UNI was required during P2 to maintain the developing plexity of the pea compound leaf. The single laminate form wild-type marginal blastozone. During P3 and P4, the terminal for which the mutation is named is shown in Figure 1B; how- unifoliate leaflet expanded, began to fold, and initiated epider- ever, leaves on a uni mutant plant may also be lobed, bi-, mal hairs at its tip (Figure 2D).