Pea Compound Leaf Architecture Is Regulated by Interactions Among the Genes UNIFOLIATA, COCHLEATA, AFILA, and TENDRIL-LESS

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 activity 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 primordia (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 development occurred early after leaf primordium initiation (Figure 2C). Pea Leaf Development The blastozone emerged laterally on the SAM, and stipule primordia (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). and trifoliate. The semidominant tl mutation (de Vilmorin and Bateson, 1911) replaces distal and terminal tendrils with leaflets (Figure 1C), whereas the recessive af mutation Analysis of Wild-Type, tl, af, and af tl Compound (Kujala, 1953; Goldenberg, 1965) replaces leaflets with Leaf Development branching rachides bearing tendrils (Figure 1D). In the af tl double mutant, branching rachides at proximal, distal, and Leaf primordium initiation and early development in wild- terminal positions terminate in small leaflets (Figure 1E). type, tl, af, and af tl pea leaves have been described in detail

Pea Compound Leaf Architecture 1281

previously by several authors (Meicenheimer et al., 1983; Gould et al., 1986; Villani and DeMason, 1997, 1999a). A brief SEM analysis of near-isogenic lines of these genotypes—JI 1194, JI 1197, JI 1195, and JI 1199, respectively—is presented in Figure 3 to facilitate the identification of those structures labeled in subsequent in situ hybridization sections. All plants included in the SEM analysis were 2 weeks old and had five fully expanded leaves; the leaf primordium being initiated from the SAM on these plants at the time it was dissected corresponded to node 13 on a mature plant, counting the first scale leaf as node 1. Stipule primordium initiation and development in all four lines appeared similar to that described for wild-type JI 2171 above. No differences in proximal organ development were observed over the first two plastochrons, in agreement with a previous report (Meicenheimer et al., 1983). On wild-type and tl mutant samples, primordia initiated during P2 formed a proximal pair of leaflets that during P3, expanded and began to bend inward toward the SAM (Figures 3A and 3C). Expansion continued during P4, and a central fold in the leaflets became clearly visible (Figures 3B and 3D). Late in P3, the second pair of organ primordia began to emerge from the marginal blastozone of the wild type and tl mutants (Figures 3B and 3D). On 2-week-old plants, these organ primordia typically formed tendrils in wild-type plants and leaflets in tl mutants. At P4, the blastozone did not usually initiate any further lateral organ primordia and became determinate, forming a terminal tendril or a terminal leaflet on wild-type and tl leaves, respectively. Leaf development on af and af tl mutant shoots was similar to that of wild-type shoots during P1 and P2; however, differences in lateral primordium development became apparent during P3, as has been reported previously (Meicenheimer et al., 1983; Villani and DeMason, 1999a). Lateral primordia initiated during P2 did not form determinate organs as in wild-type and tl leaves. Instead, they initiated further primordia during P3 in a manner similar to that of the primary leaf marginal blastozone (Figures 3E and 3G) and are hereafter denoted as secondary marginal blastozones. This observed increase in organogenic potential in af and af tl leaves compared with the wild type and tl showed that AF function in the suppression of secondary blastozone activity was first manifested in lateral primordia at P3. During

Figure 1. Morphology of Wild-Type and Mutant Pea Leaves. (D) An af (JI 1195) mutant leaf with a basal pair of stipules and all po- (A) A wild-type (JI 1194) compound leaf showing a basal pair of sitions on the leaf blade occupied by branching rachides bearing stipules, a petiole and a blade comprising a pair of proximal leaflets, tendrils. two distal pairs of tendrils, and a terminal tendril, all borne on a ra- (E) An af tl (JI 1199) double mutant leaf with a basal pair of stipules chis. and all positions on the leaf blade occupied by branching rachides (B) A uni (JI 2171) mutant leaf with a basal pair of stipules and the terminating in small leaflets. leaf blade reduced to a unifoliate form. (F) A coch (JI 2165) mutant leaf showing that each structure occupy- (C) A tl (JI 1197) mutant leaf with a basal pair of stipules and leaflets ing the position of a stipule mimicks the organization of the leaf at all positions on the leaf blade. blade with a pair of leaflets, pairs of tendrils, and a terminal tendril. 1282 The Plant Cell

P3, both primary and secondary blastozones of af and af tl mutants initiated primordia acropetally. The rate of acropetal organ initiation observed on af and af tl primary blastozones appeared similar to that observed on wild-type and tl plants in that all were initiating their second pair of lateral organs at P3 (cf. Figures 3B and 3G). During P4, the primary blastozones of af and af tl leaves developed an additional distal pair of lateral primordia not seen on wild-type and tl plants. This showed that the function of AF in the suppression of primary blastozone activity was first manifested at P4. Differences in the development of af and af tl leaves were observed during P4. On af leaves, lateral primordia initiated from the primary and secondary blastozones during P3 and P4 did not branch further, were seen to elongate during P5, and formed tendrils during P6 (Figure 3F). In contrast, primordia arising from similar positions on af tl mutant leaves remained organogenic and formed additional branches, which were denoted as tertiary blastozones (Figure 3H). This showed that the function of TL in the suppression of tertiary blastozone activity in af secondary blastozones was first manifested during P5. The quaternary branching that results from tertiary blastozone activity in af tl leaves accounts for the greater complexity of these leaves than the af leaves when both are fully developed (cf. the number of organs in Figures 1D and 1E). The quaternary primordia of af tl leaves developed into small leaflets (Figure 1E), which started to form late in P5 and were clearly visible as folded laminae at P6 (Figure 3H). In summary, our SEM analysis showed that in a wild-type leaf, the function of UNI in the maintenance of the marginal blastozone was first evident during P2, whereas the function of AF in the suppression of blastozone activity in lateral primordia and in the primary blastozone was first evident at P3 and P4, respectively. The function of TL in the suppression of blastozone activity in the tertiary lateral primordia of af mutant leaves was first apparent during P5. Our observations of changes during leaf ontogeny were in broad agreement with those made in earlier studies that compared wild-type and mutant leaf development (Meicenheimer et al., 1983; Gould et al., 1986; Villani and DeMason, 1997, 1999a). Because previous reports had provided genetic evidence to suggest that UNI, AF, and TL interact to pattern the pea leaf (Marx, 1987; Hofer and Ellis, 1996, 1998), we examined UNI expression in the wild type, tl, af, and af tl mutants to deter-

(A) and (B) Two-week-old wild-type (JI 2171) vegetative shoot apices. (C) and (D) Two-week-old sibling uni mutant (JI 2171) vegetative shoot apices. The white arrow in (C) indicates a central crease marking the beginning of lamina folding of a single, terminal leaflet. A, shoot apex; P1 to P4, plastochron 1 to plastochron 4 of leaf development; S1 to S3, stipule primordia present on P1 to P3 marginal blastozones; L2 to L4, proximal leaflet primordia present on P2 to P4 Figure 2. SEM Showing the Effects of the uni Mutation on Leaf Pri- marginal blastozones; T3 and T4, tendril primordia present on P3 mordium Initiation and Early Development. and P4 marginal blastozones. Bars in (A) to (D) ϭ 100 ␮m. Pea Compound Leaf Architecture 1283

mine whether there were any differences in UNI transcript accumulation in these different genetic backgrounds.

UNI Expression in Vegetative Apices

Figure 4 shows frontal longitudinal sections of vegetative apices hybridized in situ to a digoxigenin-labeled UNI RNA probe. In all four lines, UNI was expressed in marginal blastozones but not in the main shoot axis or SAM. In wild-type leaves, transcripts were first detected at P1, had accumulated by P2, and then accumulated distally in the marginal blastozone up to P4. At P5, UNI expression appeared to be downregulated, and transcripts were not detected above background (Figure 4A). No signal was detected in control sections probed with UNI sense RNA probes (Figure 4B). The duration of UNI expression in tl mutant leaves appeared similar to that in the wild type. Transcripts were first detected at P1, accumulated distally up to P4, and were downregulated at P5 (Figure 4C). In af mutant sections, UNI expression was detected distally in the primary blastozone up to P5, one plastochron longer than expression in wild- type and tl primordia (Figure 4D). The leaves of the af tl double mutant showed even more prolonged UNI expression in the primary blastozone, up to P6 (Figures 4E and 4F); by P7, however, expression was not detectable above background in the primary blastozone (Figure 4G). At P6 and P7, UNI was still strongly expressed in secondary and tertiary blastozones and developing leaflets (Figures 4F and 4G). In contrast to the pattern of UNI expression, HISTONE H3 (HH3) transcripts were detected in a widespread pattern in af tl sections (Figure 4H). Because HH3 transcription is likely to be more abundant in actively dividing cells (Robertson et al., 1997), this probe provides a control to verify that signal above background from the UNI antisense RNA probe is not a consequence of high cell density in areas of active division. The differences in expression patterns observed between these lines in longitudinal sections showed that interactions

ces. White arrowhead in (E) indicates a secondary blastozone, initiating tertiary primordia acropetally. These tertiary primordia will develop into tendrils, indicated by a white arrowhead in (F). A, vege- Figure 3. SEM Showing the Effects of the tl, af, and af tl Mutations tative shoot apex; P3 to P6, plastochron 3 to plastochron 6 of leaf on Early Leaf Development. development; S4 and S5, stipule primordia present on P4 and P5 (A) and (B) Two-week-old wild-type (JI 1194) vegetative shoot api- primary marginal blastozones. ces. White arrowheads indicate leaflet primordia. A, vegetative (G) and (H) Two-week-old af tl double mutant (JI 1199) vegetative shoot apex; P1 to P4, plastochron 1 to plastochron 4 of leaf devel- shoot apices. White arrowhead in (G) indicates a secondary blasto- opment; S2 to S4, stipule primordia present on P2 to P4 marginal zone initiating tertiary blastozones acropetally. The distal tip of a blastozones. secondary blastozone, marked by the P6 label in (H), has been re- (C) and (D) Two-week-old tl mutant (JI 1197) vegetative shoot api- moved. Quaternary leaflet primordia, derived from tertiary blasto- ces. White arrowheads indicate leaflet primordia. A, vegetative zones, are indicated with white arrowheads in (H). A, vegetative shoot apex; P1 to P4, plastochron 1 to plastochron 4 of leaf devel- shoot apex; P2 to P6, plastochron 2 to plastochron 6 of leaf development; S3 and S4, stipule primordia present on P3 and P4 mar- opment; S3 and S5, stipule primordia present on P3 and P5 primary ginal blastozones. marginal blastozones. (E) and (F) Two-week-old af mutant (JI 1195) vegetative shoot api- Bars in (A) to (H) ϭ 100 ␮m. 1284 The Plant Cell

between the UNI, AF, and TL genes do occur. The prolongation of UNI expression to P5 in af mutant sections suggested that UNI gene expression is suppressed by AF late in P4 and during P5 in wild-type primary marginal blastozones. The prolongation of UNI expression to P6 in the primary blastozone of af tl primordia suggested that UNI is suppressed by TL in af primary blastozones late in P5 and during P6. The similar patterns of expression seen in wild-type and tl mutant sections, however, suggested that TL does not regulate UNI transcript accumulation in primary blastozones during P1 to P4 in a wild-type background. UNI expression in primary blastozones was most clearly visible in the frontal longitudinal sections examined above. UNI transcripts were detected in the secondary blastozones of af and af tl leaves but not in the determinate lateral organ primordia of wild-type and tl leaves. Clear zones, in which UNI transcripts were less abundant, were visible on wild- type and tl P4 blastozones. These demarcated the position of lateral primordia (Figures 4A and 4C). To show UNI expression patterns in lateral primordia more clearly, we used transverse sections, as seen in Figure 5. The sections were ␮m 70ف ␮m below (Figures 5A to 5C and 5E) and 40ف taken above (Figures 5D and 5F) the shoot apex. No marked differences in UNI expression were detected between wild-type and tl mutant samples. Transcripts were detected up to P4 and accumulated distally in the marginal blastozone, as shown in the longitudinal sections (cf. Fig- ures 4A and 4C with Figures 5A and 5B). UNI expression was not detected in lateral organ primordia or in the SAM of these samples (Figures 5A and 5B). Differences in UNI expression were more clearly apparent between wild-type and af single or af tl double mutant samples in transverse sections. In af mutants during P2, transcripts accumulated distally in the primary blastozone but

Sections were taken from 2-week-old seedlings. (A) and (B) Wild type (JI 1194). (C) tl mutant (JI 1197). (D) af mutant (JI 1195). (E) to (H) af tl double mutant (JI 1199). Sections shown in (A) and (C) to (G) were hybridized with an antisense digoxigenin-labeled UNI probe, section shown in (B) was hybridized with a sense digoxigenin-labeled UNI probe, and section shown in (H) was hybridized with an antisense digoxigenin-labeled HH3 probe. The plastochronic ages indicated in (B) apply to sections (A) to (E) and (H), where (A) and (E) are in midplastochron, (C) and (D) are very early in a plastochron, and (B) and (H) are late in a plastochron. White arrowheads in (A) and (C) indicate the positions of lateral organs that emerged in front of the plane of section. The section in (D) is lateral to P1 on the shoot apex, reducing the view of Figure 4. Localization of UNI Transcripts in Wild-Type, tl, af, and af the P2 and P3 blastozones. A, vegetative shoot apex; L, leaflet; P1 tl Mutant Samples by RNA in Situ Hybridization on Frontal Longitu- to P7, plastochronic age of leaf primordia; PR, primary marginal dinal Sections. blastozone; S, secondary blastozone; T, tertiary blastozone. Pea Compound Leaf Architecture 1285

were barely detectable in the first-initiated lateral primordia (Figure 5C). This expression pattern was similar to that of the wild type at this stage. SEM analysis demonstrated that P2 wild-type and af marginal blastozones are similar in ap- pearance, but during P3 the first-initiated lateral primordia of af mutant leaves behave differently, like blastozones, because they initiate further organ primordia instead of be- coming determinate organs. In accordance with their morphological similarity to the primary marginal blastozone, these secondary blastozones expressed UNI at P3 (Figure 5C). By P4, the af primordium shown had initiated a second pair of secondary blastozones, and these also showed UNI expression (Figure 5D). Signal was detected faintly in the primary and secondary blastozones of af mutants at P5, but expression was downregulated in the lateral organ primordia that arose from them (Figure 5D). As was demonstrated by SEM and discussed above (Figure 3F), these lateral primordia initiated from the primary and secondary blastozones are determinate and develop into tendrils during subsequent plastochrons. In summary so far, UNI expression was detected in the secondary blastozone primordia of an af mutant but was not detected in lateral organ primordia at the same position on wild-type leaves. This result, in combination with SEM analysis, suggested that both UNI transcription and blastozone activity were suppressed by AF in the lateral organ primordia of wild-type leaves after P2 (during P3 to P5). These transverse sections also confirmed results obtained from longitudinal sections, which showed that UNI was expressed in the primary marginal blastozone at P5 in af mutant primordia compared with expression at P4 in the wild- type primordia. Thus, these results suggest that UNI expression is also suppressed by AF in the primary blastozone of wild-type leaves after P4 (during P5). The UNI expression pattern appeared similar in transverse sections of af single mutants and af tl double mutants up to P4. At P3 and P4, UNI expression was observed in the primary and secondary marginal blastozones of af tl leaf primordia (Figure 5E). At P5, UNI expression was detected in the primary, secondary, and tertiary blastozones of af tl leaf primordia (Figure 5F), whereas UNI expression was not clearly detectable in the tertiary organ primordia of af mutant leaves at P5 (Figure 5D). This suggested that UNI expression Figure 5. Localization of UNI Transcripts in Wild-Type, tl, af, and af is suppressed by TL in the lateral tendril primordia of af mu- tl Mutant Samples by RNA in Situ Hybridization on Transverse Sec- tant leaves during P5. As was seen in longitudinal section, tions. Sections taken from 2-week-old seedlings hybridized with an antisense digoxigenin-labeled UNI probe. (A) Wild type (JI 1194). (B) tl mutant (JI 1197). dium in which UNI expression was downregulated compared with (C) and (D) af mutant (JI 1195). the expression in an adjacent secondary blastozone (marked with a (E) and (F) af tl double mutant (JI 1199). black arrowhead). Black arrowheads in (F) indicate tertiary blasto- White arrowheads in (A) and (B) indicate lateral primordia on P3 and zones where UNI transcripts were detected. (A) to (C) and (E) were 70ف ␮m below the shoot apex; (D) and (F) were taken 40ف P4 marginal blastozones where transcripts were not detected. The taken black arrowheads in (C) to (E) indicate secondary lateral blasto- ␮m above the shoot apex. A, vegetative shoot apex; P2 to P5, plas- zones on P3, P4, and P5 primary blastozones where UNI transcripts tochronic age of leaf primordia; S3 to S6, stipule primordia present were detected. White arrowhead in (D) indicates a tertiary primor- on P3 to P6 marginal blastozones. 1286 The Plant Cell

UNI expression was prolonged in an af tl primary blastozone compared with that in an af primary blastozone, suggesting suppression by TL after P5 in an af mutant background. Fi- nally, these data suggest that AF suppresses UNI in the lateral organ primordia of tl mutant leaves, because UNI expression was prolonged in lateral primordia of the af tl double mutant.

Stipule Development and UNI Expression in coch Mutant Plants

Our results from SEM combined with longitudinal and transverse sections showed that UNI expression was correlated with blastozone activity. To test this correlation further, we performed in situ hybridization with coch plants to deter- mine whether UNI expression could be detected in the stipule primordia at a time when they exhibited blastozone activity and gave rise to compound stipule architectures. Previous work has shown that compound stipules form in plants homozygous for the coch-5137 allele between nodes 8 and 15 (Blixt, 1967; Gourlay, 1999). To demonstrate this and to aid in identification of the structures in section, we performed SEM analysis of stipule development in 2-week- old coch-5137 plants (JI 2165). Leaflets and tendrils in the coch mutant samples were initiated in a manner similar to that of their corresponding wild type, variety Weitor (Blixt, 1967). Figure 6A shows that one pair of leaflets, two pairs of tendrils, and a terminal tendril had initiated normally on P4 coch leaves. Early stipule development is also shown (Figure 6A). Stipule primordia (labeled S1 and S2) emerged during P1 and remained indistinguishable from their corresponding wild-type primordia at P2. Wild-type stipules broadened and flattened during P3, P4, and P5, whereas the coch stipule primordia observed did not; they appeared to expand at a markedly slower rate. For example, P3 stipule primordia of all wild-type lines examined to date, including variety Weitor, were comparable in size to their adjacent P3 leaflet primordia (see Figures 2A and 3A; Gourlay, 1999); however, coch stipule primordia at P3 (labeled S3 in Figure 6A) were much smaller than their adjacent P3 leaflet primordia. These observations showed Figure 6. SEM Showing Effects of the coch Mutation on Early that COCH promotes normal stipule growth and develop- Stipule Development and Localization of UNI Transcripts by RNA in ment and that this activity was first evident at P3. Later Situ Hybridization. stages of stipule development on coch mutant plants are (A) and (B) Two-week-old coch mutant (JI 2165) vegetative shoot shown in Figure 6B, when compound stipules were ob- apices. Black arrow in (B) indicates a compound stipule (S5) with served. One compound stipule with two pairs of lateral or- two pairs of lateral organ primordia. gan primordia (S5 in Figure 6B) is visible on a P5 marginal (C) and (D) Transverse sections taken from a 2-week-old coch seed- blastozone. Note that the other member of the pair of S5 ling hybridized with an antisense digoxigenin-labeled UNI probe. (C) was taken 40 ␮m below the shoot apex, and (D) was taken 100 ف ف stipules does not appear to be compound; therefore, the ␮m below the shoot apex, at a slightly oblique angle. stipules at this position would be predicted to resemble A, vegetative shoot apex; P1 to P6, plastochronic age of leaf primor- those examined by in situ hybridization in Figure 6C. dia; S1 to S6, stipule primordia present on P1 to P6 marginal blasto- In coch leaves, UNI expression was detected in primary zones. Bars in (A) and (B) ϭ 100 ␮m. marginal blastozones up to P4 (Figure 6C), as was previously seen in wild-type (JI 1194) leaves (Figures 4A and 5A). This part of the expression pattern was in accordance with Pea Compound Leaf Architecture 1287

the fact that patterning in the leaf blade is not altered by the coch mutation; only stipule development is affected. Unlike the case in wild-type plants, UNI expression was detected in the developing stipule primordia of coch mutant plants; in the example shown, transcripts accumulated on one side only, in S3 and S4 (Figure 6D). This represented ectopic UNI expression, because transcripts were not detected in wild- type S3 and S4 stipules (data not shown for the corresponding wild-type variety Weitor; a comparable wild-type expression pattern is shown in Figure 5A). No signal was detected in S1 and S2 coch mutant stipule primordia or in those older than S4. The detection of ectopic UNI expression in the stipule primordia of 2-week-old JI 2165 plants coincides with a time when compound architectures arise at this position. At other points in ontogeny, such as from node 16 on- ward, when compound stipules are not formed on coch- 5137 plants (Gourlay, 1999), UNI expression was not detected in stipule primordia (data not shown).

Gene Expression in uni Mutants

The presence of a rachis and additional pairs of leaflets or tendrils on some double and triple uni mutant combinations indicated that UNI gene function was not always correlated with extended marginal blastozone activity. It was described previously that uni af and uni af tl leaves can be pentafoliate and thus more complex than uni leaves, which are trifoliate at their maximum complexity (Hofer and Ellis, 1998). An example of a uni af tl triple mutant leaf with a rachis and two pairs of leaflets is shown in Figure 7A. The complexity of this leaf suggests that in the absence of AF and UNI gene activities, a redundant UNI-like function permits the formation of a rachis bearing more than one pair of leaflets. This has been referred to previously as a function of “gene X,” and candidates for the identity of this gene have been discussed (Hofer and Ellis, 1998). Among those considered was KNOTTED1 (KN1), which is expressed in the vegetative shoot apex of maize but is downregulated at sites of leaf Figure 7. Morphology of a uni af tl Triple Mutant and Localization of initiation (Smith et al., 1992; Jackson et al., 1994). KNOX PSKN1 Transcripts by RNA in Situ Hybridization. homologs are expressed in tomato compound leaf primor- (A) Nodes 5 and above of a uni af tl plant before flowering. The white dia, and overexpression of KNOX genes has been shown to arrow indicates a leaf at node 12 with two pairs of leaflets, borne on result in more ramified leaves, suggesting that KNOX genes a rachis (obscured from view). can influence pattern formation in leaves and shoots (B) Frontal longitudinal section of a 3-week-old uni af tl (XM 7175) (Hareven et al., 1996; Parnis et al., 1997; Janssen et al., shoot apex hybridized to an antisense PSKN1 probe. 1998). It therefore seemed possible that in the absence of (C) Frontal longitudinal section of a 3-week-old uni (JI 2171) shoot UNI, ectopic expression of a pea KNOX homolog in pea leaf apex hybridized to an antisense PSKN1 probe. (D) Frontal longitudinal section of a 3-week-old wild-type (JI 1194) primordia might result in the formation of additional pairs of shoot apex hybridized to an antisense PSKN1 probe. The black ar- lateral organs. rowhead indicates an inflorescence meristem developing in the axil To investigate this possibility, we isolated a pea class I of the P2 marginal blastozone. KNOX homolog (Hofer et al., 2000) and examined its expres- A, shoot apex; LS, lateral shoot; P1 to P7, plastochron 1 to plastochron sion pattern in uni af tl triple mutant leaves by RNA in situ hy- 7 of leaf development; V, developing vasculature of the main stem. bridization. The expression patterns of PSKN1 in uni af tl (Figure 7B), uni (Figure 7C), and wild-type shoot apices (Fig- ure 7D) were similar. In all cases, PSKN1 gene expression was confined to the shoot apex and developing vasculature 1288 The Plant Cell

in the main stem and was downregulated in leaf primordia, primordium becomes determined over four plastochrons of in a pattern similar to that observed in maize (Smith et al., growth (Gould et al., 1994). Our SEM analysis on uni plants 1992; Jackson et al., 1994). No signal was obtained when a demonstrated that they had a reduction in the number of sense PSKN1 probe was used as a control (data not shown). plastochrons, from four to two, over which the marginal Given these gene expression patterns, we concluded that blastozone is maintained (Figures 2A to 2D). This reduction PSKN1 was unlikely to confer a redundant UNI-like function in organogenic potential was accompanied by the early ex- in pea marginal blastozones. pression of processes associated with determinate growth and differentiation such as lamina expansion and folding and epidermal hair formation (Figures 2B and 2D). This suggests that UNI promotes blastozone activity and inhibits DISCUSSION lamina-forming processes in the wild-type compound leaf primordium. In accordance with this role, UNI transcripts were found to accumulate in the distal region of the marginal The term blastozone was proposed to designate regions of blastozone of wild-type leaves over the first four plasto- the shoot competent for organogenesis. Leaves are derived chrons of development (Figure 4A) but were not detected in from marginal blastozones; shoot axes are derived from api- determinate organ primordia, that is, in stipule, leaflet, and cal blastozones (Hagemann and Gleissberg, 1996). In this tendril primordia (Figure 5A). Presumably, therefore, sup- article, we focus on UNI function in the pea leaf, in the con- pression of UNI transcription in lateral primordia is an impor- text of a marginal blastozone, in which it may perform a role tant event in ensuring formation of determinate organs. different from that proposed for LFY in the transition to flow- A recently proposed model for compound leaf develop- ering (Blázquez and Weigel, 2000). A wild-type pea com- ment in pea is based on the phenotypes of double and tri- pound leaf primordium exhibits a prolonged period as a ple mutants at the loci uni, af, and tl (Hofer and Ellis, 1998). blastozone, compared with a primordium initiated at an In this model, the compound leaf is divided into four do- equivalent node on a uni mutant plant, because the wild- mains, defined by interactions between UNI, AF, TL, and type primordium possesses a greater growth potential and COCH. The model also suggests that UNI may be inhibited capacity for organogenesis. Previously, we described the by COCH in domain 1 (stipule and petiole), by AF in do- wild-type pea leaf as developing from a transiently indeter- main 2 (proximal leaflets and rachis), by AF and TL in do- minate primordium (Hofer et al., 1997), although the term in- main 3 (distal tendril pairs and rachis), and by TL in domain determinacy is usually used to refer to the quality of 4 (a trefoil of tendrils), thus preventing stipule-, leaflet-, and prolonged growth and organogenesis that is observed in a tendril-fated cells from adopting a central rachis, or blasto- shoot (Steeves and Sussex, 1989). We use the term mar- zone, fate. The molecular evidence we present here is ginal blastozone in this paper to help avoid any possible am- summarized in Figure 8 and is in general agreement with biguity in descriptions of compound leaves as opposed to this model. shoots. We showed that UNI expression is negatively regulated by COCH, AF, and TL and that UNI expression in pea leaf primordia is correlated with maintenance of marginal Interactions between UNI and COCH blastozones. We suggest that these interactions are fundamental in establishing a wild-type pea leaf architecture UNI expression was detected in the stipule primordia of through the regulation of marginal blastozone activity. coch mutant plants at a time when compound stipule architectures were predicted to form (Figures 6B and 6D). This represents ectopic expression, because wild-type stipules UNI Maintains the Wild-Type Leaf as a do not accumulate UNI transcripts. Ectopic UNI expression Marginal Blastozone was also observed in compound stipule primordia of coch af double mutants at a time when these plants were develop- Blastozone activity is maintained in the distal portion of a ing compound stipules bearing unbranched tendrils (Gourlay, developing wild-type pea leaf and is lost in lateral primordia 1999). We hypothesize that ectopic UNI expression confers as the cells are organized, or become determined, into a blastozone identity on stipule primordia, allowing com- stipule, leaflet, or tendril pathways. Cells at the apex of the pound architectures to form. The timing of UNI expression is marginal blastozone also eventually lose their organogenic critical to this hypothesis, however, and causality is difficult capacity and form a terminal tendril (Meicenheimer et al., to establish in the absence of appropriate transgenic stud- 1983). Our SEM analysis confirmed that in 2-week-old wild- ies. If ectopic UNI expression causes a change in stipule pri- type plants, all compound leaf organ primordia were initi- mordium fate, then UNI gene expression would be expected ated during the first three plastochrons and that the mar- to precede detectable changes in morphology. However, we ginal blastozone became determined during the fourth were able to detect ectopic expression only at P3, when plastochron (Figures 3A and 3B). This is also in agreement morphological changes were already apparent. Therefore, with tissue culture experiments suggesting that a pea leaf the misexpression of UNI may be a consequence of the Pea Compound Leaf Architecture 1289

change in morphology, not its cause. The uni mutant phenotype provides indirect support in favor of our hypothesis. By demonstrating that UNI function is required to maintain the marginal blastozone, we have inferred that ectopic UNI function in stipule primordia maintains them as blastozones (as in coch) and lack of expression fails to maintain the primordia as blastozones (as in the wild type). Further evidence to support our hypothesis comes from the fact that compound stipules are not found on coch uni double mutant plants. Although the full description of this double mutant phenotype is beyond the scope of this paper, Figure 9A shows five nodes on a representative coch uni plant that began to flower at node 15. Maximum leaf complexity in pea is usually found at or just below the first node of flowering (Makasheva, 1983), and compound stipules are formed between nodes 8 and 15 on coch (JI 2165) plants. The stipules at nodes 11 to 15 of the coch uni plant shown were all simple laminae (Figure 9A). This epistasis with respect to stipule development further indicates that UNI function is required for the development of compound stipule architectures. The detection of ectopic UNI transcripts in coch mutant stipule primordia at the time when compound architectures form and the lack of compound stipules in coch uni double mutant plants suggest that COCH normally inhibits UNI in wild-type stipule primordia. There is no evidence for a direct interaction, but this inhibition would prevent a blastozone fate in stipule primordia, as discussed above, and is in agreement with the domain 1 interaction suggested by

(top center), but during P3, both the morphology and the expression patterns changed. In the far left-hand column, UNI expression was detected in the primary marginal blastozones of wild-type and tl mutant seedlings, up to and during P4. Lateral primordia that arose from these showed no signal and formed determinate organs (stipules, leaflets, and tendrils). In the second column from the left, UNI transcripts accumulated in primary blastozones and in the secondary blastozones that arose during P3 in both af and af tl mutant leaves. Both of these mutants initiated tertiary branches late in P3 and during P4 (second and third columns). In af mutant leaves, the tertiary branches were determinate tendril primordia showing no detectable UNI expression (second column), whereas af tl tertiary blas- Figure 8. Summary Diagram Showing UNI Expression in Vegetative tozones showed UNI signal, remained indeterminate, and exhibited Shoot Apices of Wild-Type, tl, af, af tl, and coch Mutant Seedlings. quaternary branching during P5 (third column). In af tl mutant leaves, Representations of transverse sections through the shoot apex are UNI transcripts were detected in the primary marginal blastozone up followed through the early stages of leaf development (arrows). The to P6. In the far right-hand column, UNI expression was detected in main shoot axis is shown as a large green circle, blastozones in the primary marginal blastozones of coch mutant seedlings, up to which UNI expression was detected are shown as red ovals, and or- and during P4, but not in the lateral leaflet or tendril organ primordia gans in which UNI expression was not detected are shown as blue that arose from these. During P3 and P4, UNI expression was de- ovals. Small black double-headed arrows represent the rachis and tected in stipule primordia that behaved like blastozones and initi- rachides. The plastochronic age of each marginal blastozone is ated lateral leaflet and tendril organ primordia (these lateral shown at left (P1 to P6). During P1 and P2, the morphology and pat- primordia are not shown). After this time, no further signal was de- tern of UNI expression in all the genotypes studied was the same tected. 1290 The Plant Cell

volved in expansion of the stipule (Pellew and Sverdrup, 1923), may be an additional factor that suppresses UNI in stipule primordia. We have observed compound stipule architectures at some nodes on st af tl (JI 1201) triple mutant plants, whereas we have never seen compound stipules on af tl double mutant plants (see Figure 1E). Figure 9B shows an example of an st af tl triple mutant plant with a compound stipule opposite a simple stipule at node 18. If a simple model proposes that COCH and ST both act redundantly to repress UNI, and hence blastozone activity, in stipule-fated cells, then the stipules of coch st double mutants would be predicted to be compound. However, such a simple model does not hold, because coch st plants fail to form stipule laminae at all (Blixt, 1967; Marx, 1987; Gourlay, 1999). This novel phenotype suggests that COCH and ST act in the same pathway. Further investigation is required to ascertain whether ST repression of blastozone activity in stipules is also dependent on the absence of AF or TL.

Interactions between UNI and AF

An antagonistic relationship between AF and TL has been suggested previously (Marx, 1987), such that AF inhibits TL in the proximal part of the leaf to allow leaflet formation and that TL inhibits AF in the distal part of the leaf, leading to the formation of distal and terminal tendrils. This model predicts that the loss of TL function would lead to leaflet formation at distal and terminal positions; that is, a tl mutant leaf, and loss of AF function would allow tendrils to form at proximal positions. It does not, however, account for the branching rachides found at proximal positions on af mutant leaves, nor does it provide a satisfactory explanation for the supercompound architecture of the leaf of an af tl double mutant. The UNI expression data presented here and summarized in Figure 8 provide an explanation for the tl, af, and af tl mutant leaf architectures and suggest an alternative model for the development of the pea leaf. Organ primordia initiated from the marginal blastozone during P2 on af single mutant and af tl double mutant leaves are morphologically indistinguishable from those of wild- Figure 9. Stipule Morphology in Mutant Backgrounds. type plants and tl mutants at this stage (Meicenheimer et al., 1983; Gould et al., 1986; Villani and DeMason, 1997, 1999). (A) A coch uni double mutant. Stipules at five nodes are indicated Differences between these four genotypes first occur during with white arrows. Node 11 is at the bottom of the panel; node 15, with an axial flower, is at the top. P3, when these first-initiated organ primordia (which would (B) An st af tl (JI 1201) plant with a compound stipule opposite a form determinate leaflets in wild-type and tl mutant leaves) simple stipule at node 18. The compound stipule is indicated with a behave like blastozones in af and af tl leaves (Figures 3E and white arrow. 3G) and initiate tertiary primordia. At this time, UNI expression is detectable in both af and af tl rachide primordia but not in the determinate leaflet primordia of wild-type or tl plants (cf. P3 in Figures 5A and 5B with P3 in Figures 5C Hofer and Ellis (1998). However, because neither ectopic and 5E). Therefore, in the absence of AF function, ectopic UNI expression nor compound stipule architectures are UNI expression in the first-initiated organ primordia of af and observed at every node on coch mutant plants, UNI appar- af tl mutant leaves was associated with a change in devel- ently is suppressed in stipule primordia by factors other than opmental fate, from a determinate leaflet primordium to that COCH. The STIPULES REDUCED (ST) gene, which is in- of a blastozone. This suggests that AF normally inhibits UNI Pea Compound Leaf Architecture 1291

in the proximal leaflet primordia of wild-type and tl leaves. 1998), we examined the expression pattern of a class I Despite the lack of evidence for a direct interaction, this pro- KNOX homolog. We thought it possible that misexpression posed inhibition is in agreement with the domain 2 interac- of a KNOX gene in uni af marginal blastozones could result tion in the model suggested by Hofer and Ellis (1998). in a more complex leaf, given that overexpression of genes Previous authors have suggested that the AF gene either of this class confers ramification on tomato compound directs (Gould et al., 1994) or promotes (Lu et al., 1996; Villani leaves (Hareven et al., 1996; Parnis et al., 1997; Janssen et and DeMason, 1997, 1999a; Hofer and Ellis, 1998) lamina al., 1998). This possibility was also considered in light of formation in a wild-type pea leaf. Clearly, AF is not required dominant knox mutants in maize with distal leaf tissue trans- for leaflet formation per se, because leaflets form on af tl, af formed into more proximal tissue types (Freeling, 1992). uni (Marx, 1987), and af tl uni plants (Hofer and Ellis, 1996). Continuation of blastozone activity in uni af and uni af tl However, AF clearly is required for leaflets to form at proxi- leaves could be accounted for as a proximal transformation, mal positions on a wild-type leaf (with a functional UNI gene) with the shoot meristem viewed as the ultimate proximal cell because on af and af tl mutants these are replaced by group (Muehlbauer et al., 1997). In a previous model, we branching rachides. We propose that AF normally sup- suggested that such a transformation could occur as a re- presses UNI expression in proximal primordia positions and sult of an additional activity (“gene X”; Hofer and Ellis, 1998). that the branched af and af tl mutant phenotypes occur as a Our in situ hybridization analysis (Figure 9) showed no dif- result of ectopic UNI expression, which confers blastozone ferences in PSKN1 expression between wild-type, uni, and identity on proximal leaflet primordia. Again, the timing of uni af tl leaf primordia. We concluded that it was unlikely UNI gene expression in the leaflet primordia is critical in de- that ectopic PSKN1 gene expression corresponded to gene termining whether it causes, or is a consequence of, X activity in uni af tl marginal blastozones. However, the uni changed primordial fates. We were unable to detect differ- af tl leaf primordia at the particular nodes we examined ences in UNI expression in wild-type and af blastozones could have been constrained in their complexity and there- during P2, before any morphological differences were ap- fore might not have reflected the expression pattern in leaf parent. Therefore, these in situ hybridization studies alone primordia that would go on to develop a rachis. The possi- do not support the hypothesis that UNI gene activity confers bility also exists that PSKN1 influences patterning in uni af tl a blastozone fate. The uni mutant phenotype does, how- blastozones from a distance, given the demonstration that ever, support this hypothesis indirectly. We have inferred KN1 protein is mobile (Lucas et al., 1995). Finally, other pea from the uni mutant phenotype that UNI expression not only KNOX homologs may be expressed ectopically in the ab- is associated with a blastozone fate but also is part of its sence of UNI; therefore, we cannot rule out KNOX homologs cause. This hypothesis remains to be confirmed by UNI as candidates for gene X. overexpression studies. Further support comes from the uni af double mutant leaf, which has a variable phenotype ranging from unifoliate to Interactions between UNI and TL pentafoliate (Hofer and Ellis, 1998). The first-initiated lateral organs may be tendrils or leaflets, but ramified rachides The epistasis of uni over tl (Marx, 1987) suggests that these have never been observed at this position. Similarly, uni af tl two genes act in the same pathway; however, the role of the triple mutant leaves can be pentafoliate at their maximum wild-type TL gene in the determination of leaf pattern is complexity, but the lateral organs are always simple leaflets, uncertain because the tl mutation is semidominant. On a never secondary blastozones. This epistasis of uni over af at heterozygous plant (TL/tl), the tendrils are straplike and con- lateral leaflet positions supports the hypothesis that UNI is sidered to be intermediate between leaflets and tendrils required for blastozone activity in the lateral primordia of af (Marx, 1987; Villani and DeMason, 1999b). Perhaps the wild- leaves. When another aspect of lateral organ development— type TL gene plays no role in lamina or tendril formation, but leaflet shape—is considered, a novel phenotype clearly is the altered activity of the gene in the tl mutant allows it to associated with uni af and uni af tl leaflets that is absent in have an influence on leaf patterning that it would not nor- uni leaves. Most uni af and uni af tl leaflets are not planar; mally exert. In attempting to understand the function of the their midveins are curved in the dorsoventral plane (as can TL gene, we can consider legume leaf form more broadly. be seen at lower nodes in Figure 7A). This novel phenotype The wild-type tendrilled pea leaf is a relatively rare form that occurs after leaflet identity has been established and sug- may have arisen after a loss or gain of function in the leaf gests that UNI and AF act nonindependently later in leaflet relative to other legumes. Therefore, one interpretation of development. the tl mutation is that it represents the restoration of a typi- cal legume leaf function. The effects of TL on UNI expression were seen in the af tl Class I KNOX Gene Expression double mutant. Our expression data showed that UNI expression was extended to P6 in the primary blastozone of an To address the surprising finding that uni af and uni af tl af tl double mutant leaf primordium, compared with P5 in an leaves can be more complex than uni leaves (Hofer and Ellis, af mutant leaf primordium. Furthermore, UNI expression in 1292 The Plant Cell

the tertiary primordia of af leaves that would go on to form METHODS tendrils (Figure 3F) was not above background values at P5 (Figure 5D), whereas the tertiary blastozones of af tl mutant Plant Material leaves expressed UNI at P5 (Figure 5F) and would remain indeterminate to initiate quaternary primordia (Figure 3H). In All plant lines were obtained from the John Innes germ plasm collec- this proposed role as a repressor of UNI expression, TL tion. The lines JI 1195 (af/af), JI 1197 (tl/tl), JI 1199 (af/af, tl/tl), and JI would be functionally redundant with AF, which would ex- 1201 (st/st, af/af, tl/tl) are part of a near-isogenic series with JI 1194 plain why UNI expression was not observed to extend later- as the corresponding wild-type line (Marx, 1974, 1987). JI 2165 car- ally or distally in a tl mutant leaf compared with wild-type ries the coch mutation and was generated by ethyl methanesulfonate expression. Perhaps TL suppression of UNI transcription is mutagenesis of the Weitor (JI 379) variety (Blixt, 1967). JI 2171 is the so weak in a wild-type background, and AF suppression of type line for the uni mutation, and XM 7175 carries a deletion allele of UNI is sufficiently more important, that differences in UNI uni in an af tl genetic background (Hofer et al., 1997). Plants were expression between wild-type and tl plants are not detect- potted individually in John Innes No. 1 potting mix plus 30% grit and grown in greenhouses under a 16-hr photoperiod supplemented with able by in situ hybridization. 360-W sodium lamps. af tl Leaflets Have a Novel Phenotype In Situ Hybridization

The af tl leaf is more ramified at its extremities than the af Sense and antisense digoxigenin-labeled UNI, HH3, and PSKN1 leaf, and this ramification appears to be more than a simple probes were prepared from full-length cDNA clones transcribed by additive phenotype. For example, there are between 20 and using T3 or T7 RNA polymerase. Probes were hybridized to 8-␮m 30 tendrils on the af leaf shown in Figure 1D. The replace- longitudinal or transverse sections cut from wax-embedded samples, as previously described (Coen et al., 1990). Sections were ment of tendrils by leaflets on an equivalent af tl leaf would counterstained with 0.1% (w/v) calcofluor white fluorescent bright- .leaflets if the phenotype were ener and viewed by light microscopy with epifluorescent illumination 30ف be expected to result in additive, but as Figure 1E shows, Ͼ60 leaflets can be counted. This novel, supercompound phenotype suggests that AF and TL can act together in the suppression of blas- Scanning Electron Microscopy tozone activity. We showed that UNI expression was correlated with blas- Samples were dissected to reveal the shoot apical meristem (SAM), tozone activity in compound stipules and rachides. A con- frozen in liquid nitrogen, sublimated under vacuum at Ϫ95ЊC for 2 min, and sputter-coated in platinum at 10 mA for 2 min. Samples tradictory result was the detection of UNI transcripts in the were viewed under vacuum in a 3-kV electron beam by using a field miniature leaflets of the developing af tl leaf. UNI expression emission gun (FEG) scanning electron microscope (model XL 30; was observed even though the leaflets had ceased organo- Philips Electronics N.V., Eindhoven, The Netherlands). genesis and had begun to differentiate, which demonstrates that UNI expression alone is insufficient to confer blastozone activity. With respect to UNI expression, these miniature leaflets ACKNOWLEDGMENTS appeared to differ from the larger leaflets found on wild-type and tl leaves. Regulation of wax deposition in af tl leaflets has already been noted to be distinctive compared with that We thank Mike Ambrose, the curator of the John Innes Pisum germ in the wild type and tl (Marx, 1987). The wachslos (wlo) mu- plasm collection, Richard Gould and Miriam Balcam for horticultural services, Andrew Davis for photography, and Kim Findlay for advice tation normally suppresses wax deposition on the adaxial on electron microscopy. We thank members of the laboratory for surfaces of leaflets. In af genetic backgrounds, such as af tl stimulating discussions and Tony Michael (Institute of Food wlo, the miniature leaflets have waxy adaxial surfaces, Research, Norwich, UK) for the gift of the HH3 probe. C.W.G. was which suggests that the af mutation counteracts the effect supported by a John Innes Foundation studentship. J.M.I.H was of the wlo mutation (Marx, 1987). An alternative interpreta- supported by Grant No. AR0125 from the Ministry of Agriculture, tion is that laminae in an af mutant background may not be Fisheries and Food. equivalent to wild-type leaflets and therefore may not be subject to the same regulation (Murfet and Reid, 1993). In our model of leaf pattern determination in pea, we would Received February 14, 2000; accepted May 31, 2000. need to invoke a UNI transcript-independent mechanism in af tl leaflets to explain the determination and differentiation REFERENCES of these laminae despite the maintenance of UNI expression. Further refinement of the model and a deeper under- standing of these complex genetic interactions await Angiosperm Phylogeny Group. (1998). An ordinal classification for molecular characterization of the AF and TL genes. the families of flowering plants. Ann. Mo. Bot. Gard. 85, 531–553. Pea Compound Leaf Architecture 1293

Anthony, R.G., James, P.E., and Jordan, B.R. (1993). Cloning and Hofer, J., Gourlay, C., Michael, A., and Ellis, T.H.N. (2000). sequence analysis of a flo/lfy homologue isolated from cauliflower Expression of a class 1 knotted1-like homeobox gene is downreg- (Brassica oleracea L. var. botrytis). Plant Mol. Biol. 22, 1163–1166. ulated in pea compound leaf primordia. Plant Mol. Biol. (in press). Blázquez, M.A., and Weigel, D. (2000). Integration of floral induc- Jackson, D., Veit, B., and Hake, S. (1994). Expression of maize tive signals in Arabidopsis. Nature 404, 889–892. KNOTTED1-related homeobox genes in the shoot apical meristem predicts patterns of morphogenesis in the vegetative shoot. Blázquez, M.A., Soowal, L.N., Lee, I., and Weigel, D. (1997). Development 120, 405–413. LEAFY expression and flower initiation in Arabidopsis. Develop- ment 124, 3835–3844. Janssen, B.J., Lund, L., and Sinha, N. (1998). Overexpression of a homeobox gene, LeT6, reveals indeterminate features in the Blixt, S. (1967). Linkage studies in Pisum VII. The manifestation of tomato compound leaf. Plant Physiol. 117, 771–786. the genes cri, and coch, and the double-recessive in Pisum. Agric. Hort. Genet. 25, 131–144. Kelly, A.J., Bonnlander, M.B., and Meeks-Wagner, D.R. (1995). NFL, the tobacco homolog of FLORICAULA and LEAFY, is tran- Coen, E.S., Romero, J.M., Doyle, S., Elliot, R., Murphy, G., and scriptionally expressed in both vegetative and floral meristems. Carpenter, R. (1990). floricaula: A homeotic gene required for Plant Cell 7, 225–234. flower development in Antirrhinum majus. Cell 63, 1311–1322. Kujala, V. (1953). Felderbse beiwelcher die ganze Blattspreite in Colombo, L., Marziani, G., Masiero, S., Wittich, P.E., Schmidt, Ranken umgewandelt ist. Arch. Soc. Zool. Bot. Fenn. 8, 44–55. R.J., Sari Gorla, M., and Enrico Pé, M. (1998). BRANCHED SILKLESS mediates the transition from spikelet to floral meristem Kyozuka, J., Konishi, S., Nemoto, K., Izawa, T., and Shimamoto, during Zea mays ear development. Plant J. 16, 355–363. K. (1998). Down-regulation of RFL, the FLO/LFY homolog of rice, accompanied with panicle branch initiation. Proc. Natl. Acad. Sci. Dengler, N.G. (1984). Comparison of leaf development in normal (ϩ/ϩ), USA 95, 1979–1982. entire (e/e), and lanceolate (La/ϩ) plants of tomato, Lycopersicon esculentum ‘Ailsa Craig’. Bot. Gaz. 145, 66–77. Lu, B., Villani, P.J., Watson, J.C., DeMason, D.A., and Cooke, T.J. (1996). The control of pinna morphology in wildtype and mutant de Vilmorin, P., and Bateson, W. (1911). A case of gametic cou- leaves of the garden pea (Pisum sativum L.). Int. J. Plant Sci. 157, pling in Pisum. Proc. R. Soc. Lond. Ser. B 84, 9–11. 659–673. Eriksson, G. (1929). Erbkomplexe des Rotklees und der Erbsen. Z. Lucas, W.J., Bouché-Pillon, S., Jackson, D.P., Nguyen, L., Baker, Pflanzen. 84, 735–744. L., Ding, B., and Hake, S. (1995). Selective trafficking of Freeling, M. (1992). A conceptual framework for maize leaf develop- KNOTTED1 homeodomain protein and its mRNA through plas- ment. Dev. Biol. 153, 44–58. modesmata. Science 270, 1980–1983. Frohlich, M.W., and Meyerowitz, E.M. (1997). The search for flower Makasheva, R.K. (1983). Morphology and classification of culti- homeotic gene homologs in basal angiosperms and gnetales: A vated peas. In The Pea, V.S. Kothekar, ed, B.K. Sharma, trans potential new source of data on the evolutionary origin of flowers. (New Delhi: Oxonian Press), pp. 20–77. Int. J. Plant Sci. 158, S131–S142. Marx, G.A. (1974). Stocks available. Pisum Genet. 6, 60. Goldenberg, J.B. (1965). afila, a new mutation in pea (Pisum sati- Marx, G.A. (1986). Tendrilled acacia (tac): An allele at the Uni locus. vum L.). Biol. Genet. 1, 27–31. Pisum Genet. 18, 49–52. Gould, K.S., Cutter, E.G., and Young, J.P.W. (1986). Morphogene- Marx, G.A. (1987). A suite of mutants that modify pattern formation sis of the compound leaf in three genotypes of the pea, Pisum in pea leaves. Plant Mol. Biol. Rep. 5, 311–335. sativum. Can. J. Bot. 64, 1268–1276. Meicenheimer, R.D., Muehlbauer, F.J., Hindman, J.L., and Gritton, Gould, K.S., Cutter, E.G., and Young, J.P.W. (1994). The determina- E.T. (1983). Meristem characteristics of genetically modified pea tion of pea leaves, leaflets, and tendrils. Am. J. Bot. 81, 352–360. (Pisum sativum) leaf primordia. Can. J. Bot. 61, 3430–3437. Gourlay, C.W. (1999). An Analysis of Genes Involved in Pea Com- Molinero-Rosales, N., Jamilena, M., Zurita, S., Gómez, P., Capel, pound Leaf Development. Ph.D. Dissertation (Norwich, UK: Uni- J., and Lozano, R. (1999). FALSIFLORA, the tomato orthologue versity of East Anglia). of FLORICAULA and LEAFY, controls flowering time and floral meristem identity. Plant J. 20, 685–693. Hagemann, W., and Gleissberg, S. (1996). Organogenetic capacity of leaves: The significance of marginal blastozones in angio- Mouradov, A., Glassick, T., Hamdorf, B., Murphy, L., Fowler, B., sperms. Plant Syst. Evol. 199, 121–152. Marla, S., and Teasdale, R.D. (1998). NEEDLY, a Pinus radiata ortholog of FLORICAULA/LEAFY genes, expressed in both repro- Hareven, D., Gutfinger, T., Parnis, A., Eshed, Y., and Lifschitz, E. ductive and vegetative meristems. Proc. Natl. Acad. Sci. USA 95, (1996). The making of a compound leaf: Genetic manipulation of 6537–6542. leaf architecture in tomato. Cell 84, 735–744. Muehlbauer, G.J., Fowler, J.E., and Freeling, M. (1997). Sectors Hofer, J.M.I., and Ellis, T.H.N. (1996). The effect of Uni on leaf expressing the homeobox gene liguleless3 implicate a time- shape. Pisum Genet. 28, 21–23. dependent mechanism for cell fate acquisition along the proxi- Hofer, J.M.I., and Ellis, T.H.N. (1998). The genetic control of pat- mal–distal axis of the maize leaf. Development 124, 5097–5106. terning in pea leaves. Trends Plant Sci. 3, 439–444. Murfet, I.C., and Reid, J.B. (1993). Developmental mutants. In Hofer, J., Turner, L., Hellens, R., Ambrose, M., Matthews, P., Peas: Genetics, Molecular Biology and Biotechnology, R. Casey Michael, A., and Ellis, N. (1997). UNIFOLIATA regulates leaf and and D.R. Davies, eds (Wallingford, UK: CAB International), pp. flower morphogenesis in pea. Curr. Biol. 7, 581–587. 165–216. 1294 The Plant Cell

Parnis, A., Cohen, O., Gutfinger, T., Hareven, D., Zamir, D., and ectopic expression in leaf cells with altered fates. Development Lifschitz, E. (1997). The dominant development mutants of 116, 21–30. tomato, mouse-ear and curl, are associated with distinct modes Souer, E., van der Krol, A., Kloos, D., Spelt, C., Bliek, M., Mol, J., of abnormal transcriptional regulation of a knotted gene. Plant and Koes, R. (1998). Genetic control of branching pattern and flo- Cell 9, 2143–2158. ral identity during Petunia inflorescence development. Develop- Pellew, C., and Sverdrup, A. (1923). New observations on the ment 125, 733–742. genetics of peas. J. Genet. 13, 125–131. Steeves, T.A., and Sussex, I.M. (1989). Organogenesis in the Pnueli, L., Carmel-Goren, L., Hareven, D., Gutfinger, T., Alvarez, shoot: Later stages of leaf development. In Patterns in Plant J., Ganal, M., Zamir, D., and Lifschitz, E. (1998). The SELF- Development, 2nd ed (Cambridge, UK: Cambridge University PRUNING gene of tomato regulates vegetative to reproductive Press), pp. 147–175. switching of sympodial meristems and is the ortholog of CEN and Villani, P.J., and DeMason, D.A. (1997). Roles of the af and tl genes TFL1. Development 125, 1979–1989. in pea leaf morphogenesis: Characterisation of the double mutant Pouteau, S., Nicholls, D., Tooke, F., Coen, E., and Battey, N. (af/af,tl/tl). Am. J. Bot. 84, 1323–1336. (1997). The induction and maintenance of flowering in Impatiens. Villani, P.J., and DeMason, D.A. (1999a). The Af gene regulates Development 124, 3343–3351. timing and direction of major developmental events during leaf Robertson, A.J., Kapros, T., and Waterborg, J. (1997). A cell morphogenesis in garden pea (Pisum sativum). Ann. Bot. 83, cycle–regulated Histone H3 gene of alfalfa with an atypical pro- 117–128. moter structure. DNA Sequence 7, 209–216. Villani, P.J., and DeMason, D.A. (1999b). Roles of the Af and Tl Rottman, W.H., Boes, T.K., and Strauss, S.H. (1993). Structure genes in pea leaf morphogenesis: Leaf morphology and pinna and expression of a LEAFY homologue from Populus. Cell Bio- anatomy of the heterozygotes. Can. J. Bot. 77, 611–622. chem. Suppl. 17B, 23. Weigel, D., Alvarez, J., Smyth, D.R., Yanofsky, M.F., and Sharma, B. (1981). Genetic pathway of foliage development in Meyerowitz, E.M. (1992). LEAFY controls floral meristem identity Pisum sativum. Pulse Crop Newsl. 1, 56–57. in Arabidopsis. Cell 69, 843–859. Smith, L.G., Greene, B., Veit, B., and Hake, S. (1992). A dominant Wellensiek, S.J. (1959). Neutronic mutations in peas. Euphytica 8, mutation in the maize homeobox gene, Knotted-1, causes its 209–215. Pea Compound Leaf Architecture Is Regulated by Interactions among the Genes UNIFOLIATA, COCHLEATA, AFILA, and TENDRIL-LESS Campbell W. Gourlay, Julie M. I. Hofer and T. H. Noel Ellis Plant Cell 2000;12;1279-1294 DOI 10.1105/tpc.12.8.1279

This information is current as of May 2, 2019

References This article cites 41 articles, 14 of which can be accessed free at: /content/12/8/1279.full.html#ref-list-1 Permissions https://www.copyright.com/ccc/openurl.do?sid=pd_hw1532298X&issn=1532298X&WT.mc_id=pd_hw1532298X eTOCs Sign up for eTOCs at: http://www.plantcell.org/cgi/alerts/ctmain CiteTrack Alerts Sign up for CiteTrack Alerts at: http://www.plantcell.org/cgi/alerts/ctmain Subscription Information Subscription Information for The Plant Cell and Plant Physiology is available at: http://www.aspb.org/publications/subscriptions.cfm