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Annals of Botany 88: 225±234, 2001 doi:10.1006/anbo.2001.1448, available online at http://www.idealibrary.com on Leaf and Flower Development in Pea (Pisum sativum L.): Mutants cochleata and unifoliata J E N N I F E R L . YA X L E Y{, W I E SL AW J A B LO N SK I{ and J A M E S B . R E I D *{ {School of Plant Science, University of Tasmania, GPO Box 252-55, Hobart, 7001, Australia and {Central Science Laboratory, University of Tasmania, GPO Box 252-55, Hobart, 7001, Australia Received: 23 September 2000 Returned for revision: 3 November 2000 Accepted: 6 April 2001 The stipule mutant cochleata (coch) and the simple-leaf mutant unifoliata (uni) are utilized to increase understanding of the control of compound leaf and ¯ower development in pea. The phenotype of the coch mutant, which aects the basal stipules of the pea leaf, is described in detail. Mutant coch ¯owers have supernumerary organs, abnormal fusing of ¯ower parts, mosaic organs and partial male and female sterility. The wild-type Coch gene is shown to have a role in in¯orescence development, ¯oral organ identity and in the positioning of leaf parts. Changes in meristem size may be related to changes in leaf morphology. In the coch mutant, stipule primordia are small and their development is retarded in comparison with that of the ®rst lea¯et primordia. The diameter of the shoot apical meristem of the uni mutant is approx. 25 % less than that of its wild-type siblings. This is the ®rst time that a signi®cant dierence in apical meristem size has been observed in a pea leaf mutant. Genetic controls in the basal part of the leaf are illustrated by interactions between coch and other mutants. The mutant coch gene is shown to change stipules into a more `compound leaf-like' identity which is not aected by the stipules reduced mutation. The interaction of coch and tendril-less(tl) genes reveals that the expression of the wild-type Tl gene is reduced at the base of the leaf, supporting # 2001 Annals of Botany Company the theories of gradients of gene action. Key words: Pisum sativum, garden pea, leaf morphogenesis, compound leaf, leaf mutants, ¯ower morphology. I N T RO D U C T I O N The garden pea (Pisum sativum L.), along with tomato (Lycopersicon esculentum Miller), is a model species for understanding compound leaf development. The pea leaf is pinnately compound and consists of basal, foliaceous stipules, proximal lea¯ets and distal tendrils (Fig. 1A). In a major review of pea leaf development mutants, Marx (1987) suggested that various mutants appear to operate in these three dierent `domains' within the leaf (basal, proximal and distal), and that the action of these leaf development genes might help to de®ne these areas. However, studies of pea leaf development have concentrated on the recessive mutant a®la(af) (Kujala, 1953; Goldenberg, 1965), the semidominant mutant tendril-less(tl) (Vilmorin and Bateson, 1911) and the af tl double mutant (Fig. 1D, F and G), which alter lea¯et and tendril pinna identity in the proximal and distal domains (Meicenheimer et al., 1983; Gould et al., 1986, 1994; Cote et al., 1992; Lu et al., 1996; Villani and DeMason, 1997, 1999a, b, 2000). The stipules and basal compartment have received little attention, with only one model of pea leaf development (Gourlay et al., 2000) considering the cochleata (coch) mutant (Wellensiek, 1959), in which the stipules of the middle nodes of the plant are compound (Fig. 1B). This study gives a detailed description of the phenotype of the coch mutant and investigates some of its interactions with other pea leaf genes to increase knowledge of genes controlling the basal domain of the pea leaf. The * For correspondence. Fax 61 3 62 26 2698, e-mail Jim.Reid@utas. edu.au 0305-7364/01/080225+10 $35.00/00 interactions of coch with tl in the basal compartment and of coch with the recessive mutant stipules reduced (st) are described. As the names suggest, st mutants have stipules that are reduced to small, narrow structures (Pellew and Sverdrup, 1923) (Fig. 1C) and the leaves of tl mutants have lea¯ets in place of terminal tendrils (Vilmorin and Bateson, 1911) (Fig. 1D). Interactions between these mutants provide information about domains of gene activity that are not evident in the single mutants. Meristem size has been linked to the size of the structures they produce (Smith and Hake, 1992; Sundberg and Orr, 1996; Lijsebettens and Clarke, 1998; Jackson and Hake, 1999). It has been suggested that dierences in meristem or primordial size could produce dierent pea leaf mutants (Young, 1983; Gould et al., 1992), although no size dierence was found in the meristems of af, tl and af tl mutants (Meicenheimer et al., 1983; Gould et al., 1986; Cote et al., 1992). Here, we examine the early leaf development of coch and unifoliata (uni) (Eriksson, 1929) mutants to investigate the role of meristem size in the development of dierent types of leaf structures in addition to lea¯ets and tendrils. Strong alleles of the uni mutant change the multiple lea¯ets and tendrils of a wild-type leaf into a single leaf blade with basal stipules (Lamprecht, 1933) (Fig. 1E) and seem to merge the proximal and distal compartments into one. The uni gene is the pea homologue of the ¯oral meristem identity genes LEAFY in arabidopsis and FLORICAULA in Antirrhinum (Hofer et al., 1997). Pea shoot apical meristems were examined by environmental scanning electron microscopy (ESEM), since this allows # 2001 Annals of Botany Company 226 Yaxley et al.ÐLeaf and Flower Development in Pea here was generated by J. Weller in Hobart through mutagenesis of the cultivar Torsdag (Hobart line 107). Seeds were treated with 1 % EMS (ethylmethanesulfonate) for 6 h at 18 8C. Among 1100 M2 families, one showed the leaf-like stipules typical of coch mutants. This mutant line (AF99) was crossed to a coch type line, JI 2757, produced by S. Blixt (Blixt, 1972; WL5446) from the variety Parvus and provided by M. Ambrose (John Innes Institute, UK). The F1 plants of this cross had leaves typical of coch plants, including leaf-like stipules in the middle nodes, indicating that our coch mutant line AF99 possessed a mutant allele at the known cochleata locus. AF99 has been backcrossed twice with its progenitor line 107. To generate the coch st double mutant, coch (AF99) (male parent) was crossed with Hobart multiple marker line HL31 ( female parent) which is homozygous recessive for st. To examine the expression of Tl in a coch background, coch (AF99) (male parent) was crossed with Hobart multiple marker line HL111 ( female parent) which is homozygous recessive for the leaf development genes tl, af and st. Identi®cation of coch st double mutant plants F I G . 1. The wild-type pea leaf (A) with basal stipules (S), proximal lea¯ets (L) and distal tendrils (T) and leaves of various leaf mutants: B, cochleata(coch); C, stipules reduced(st); D, tendril-less(tl); E, unifoliata(uni); F, a®la(af); and G, the af tl double mutant. Bars 4 cm. objects to be seen in their natural state (Danilatos, 1993; Jablonski, 1997). Two wild-type F1 heterozygotes from the cross of coch (AF99) (male parent) and st (HL31) ( female parent) were grown; from these plants 40 F2 seeds were sown. It was determined that the cross was successful because the F1 plants had wild-type stipules. However, the identity of the double mutant plants amongst the 40 plants of the F2 generation was not immediately obvious, as both coch and st mutations have the eect of reducing stipules. The coch st double mutants were identi®ed from their ¯oral morphology, and their leaf phenotype was then examined. (Homozygous coch plants have mutant ¯owers while the st mutation does not aect ¯owers.) The F3 progeny of seven F2 plants with reduced stipules (st st) and wild-type ¯owers were grown. Two-thirds of these plants were expected to be heterozygous for the coch mutation (Coch coch); however, only one of the seven F3 families planted showed segregation of ¯ower phenotype. In this family there were ten plants with reduced stipules and wild-type ¯owers (Coch±st st), and also two plants with both reduced stipules and compound stipules and mutant coch-like ¯owers ( putative coch coch st st plants). The F4 progeny from the putative double mutant plants (14 plants), and some of their heterozygous (Coch coch st st) siblings (16 plants) were grown to con®rm the double mutant phenotype, which was clear based on ¯ower morphology in the homozygous st background. M AT E R I A L S A N D M E T H O D S Plant material Identi®cation of coch coch Tl tl plants The lines of Pisum sativum L. used during this work are held in the collection at Hobart, Australia. The uni line JI 2171 ( provided by J. Hofer, John Innes Institute, UK), is descended from the original spontaneous mutant described by Eriksson (1929). This line is maintained as a heterozygote, and in our meristem studies uni plants were compared with their wild-type siblings. The coch line used Four F1 heterozygotes from the cross of coch (AF99) (male parent) and HL111 ( female parent) were grown, and from these plants 64 F2 seeds were sown (16 from each F1 plant). The cross was found to be successful as the F1 plants had wild-type leaves. Leaf forms of the ten F2 plants which were heterozygous for Tl (Tl tl) and had coch leaf-like stipules were observed. Plants heterozygous for Tl can be Yaxley et al.ÐLeaf and Flower Development in Pea distinguished because their leaves have ¯attened tendrils (White, 1917; Villani and DeMason, 1999b). Growing conditions All plants were grown in a 1 : 1 mixture (v/v) of vermiculite and 10 mm dolerite chips topped with 2±3 cm of pasteurized potting mix (1 : 1 mixture (v/v) of coarse river sand and peat moss, with added macro and micro nutrients). Two plants were grown in each 14 cm slimline pot. Before planting the seeds, a small section (about 3 mm2) of testa was removed with a sharp razor blade to facilitate even germination. Seeds were then coated in Thiram 800 fungicide (active ingredient Thiram 800 g kg ÿ1; Agchem, Para®eld Gardens, SA, Australia) to help prevent fungal infection during germination, and planted into wet soil. The pots were watered sparingly for the ®rst 5 d until seedlings began to emerge. Watering was then carried out three times a week for 3 weeks, and then daily thereafter. Plants were provided with nutrient solution [1 g l ÿ1 Aquasol (Hortico, Laverton North, Australia) and 0.05 g l ÿ1 iron chelate (Kendon Chemicals, Thornbury, Australia)] weekly. Vertical strings were used to support the plants once they reached 10 cm in height. The photoperiod was 18 h, consisting of natural daylight extended by mixed incandescent (100 W bulbs) and ¯uorescent (40 W cool white tubes) light, giving approx. 25 mmol m ÿ2 s ÿ1 at the pot surface. Growth of the segregating populations and of plants used to examine in¯orescence structure took place during autumn/winter with mean maximum/minimum day and night temperatures of approx. 22 and 14 8C, respectively. Plants used for microscopic examination and for all other morphological measurements were grown during spring, with mean maximum/minimum day and night temperatures of approx. 25 and 15 8C, respectively. Plants were treated as required with fungicide or pesticide. Scoring of morphological characters Counting of nodes was acropetal, with the cotyledons as node 0, the two subsequent scale leaves as nodes one and two and the ®rst true leaf as node three. Leaves at the apex were numbered using the plastochron index (Lamoreaux et al., 1978; Sylvester et al., 1996) to describe their state of development. The plastochron one (P1) leaf is the youngest leaf which has been initiated on the ¯ank of the apex, the plastochron two leaf (P2) is the next oldest primordium, and so on. Plastochron zero (P0) marks the site where the next leaf will be initiated on the ¯anks of the apical dome. Means were compared using Student's t-test. Environmental scanning electron microscopy Three mutant and three wild-type leaves, for both the coch and uni mutants and their corresponding wild types, were examined for each of nodes 8 to 12. To observe each successive node, groups of plants were harvested every 2± 3 d. Ten plants of each mutant line and the corresponding wild type were examined 4 weeks later to determine the 227 typical mature leaf morphology of each node examined during development. The whole apices of plants were harvested directly before they were to be examined. The meristem was then dissected from the apical leaves under a stereo-microscope with a minimum light intensity and with the sample surrounded by a pool of water to prevent dehydration. The apex was then attached to an aluminium stub with double-adhesive conductive tape and placed on a Peltier-eect thermoelectric cold stage (Omega CN 900A, ElectroScan, Wilmington, Australia) attached to an environmental scanning electron microscope (ESEM 2020, ElectroScan). The specimen chamber was closed and the temperature reduced to 2.5± 2.8 8C before the pressure was reduced. The specimen was localized and the primary focus and video signal level established at a water vapour pressure of 8 Torr. The pressure was then slowly reduced to 6.0±7.5 Torr (to minimize water on the specimen surface). Once an image was acquired (at 2048 2048 resolution), Ilford FP±4 (125) ®lm was used to obtain a hard copy. Using this technique the apices do not require any treatment before microscopy, thus substantially reducing the possibility of artefacts (Crang and Klomparens, 1988). The structural integrity of the specimen is maintained when the internal pressure corresponds to the saturated water pressure at a given temperature. (The surrounding medium also acts as a neutralizing agent to prevent primary electron charge build-up on the surface of the specimen.) Measurements of apical meristem size were made on screen during ESEM observation. R E S ULT S Phenotype of the coch mutant The coch mutation aects only the stipules of the leaf; lea¯ets and tendrils are unchanged. Stipules were mostly absent from the ®rst few nodes (nodes 3-5). Above this (nodes 6 and 7), coch stipules were sometimes small and straplike, sometimes sessile and elliptical, or sometimes spatulate. At higher nodes (nodes 8-11), the stipules were largely compound, with proximal lea¯ets and distal tendrils, that re-iterated the structure of the leaf blade (Fig. 1B). Above node 11, leaf complexity was reduced, and spatulate and sessile-elliptical stipule forms became common; occasionally thread-like forms occurred (Figs 2 and 3). Approximately 25 % of coch leaves had mismatched stipule pairs. For example, an elliptical sessile stipule occurring opposite a spatulate stipule (e.g. node 12, Fig. 2), or a compound stipule opposite a thread-like stipule. In addition, approximately 10 % of compound coch stipules had lea¯et and tendril pairs which were not opposite each other on the stipule rachis (e.g. node 11, Fig. 2). This occurred less often (approximately 2 %) on the main rachis of wild-type leaves. The stipules of coch mutants did not have visible axillary buds. This applied to both compound coch stipules and simple coch stipules. Thus, coch compound stipules appear to re-iterate only the proximal and distal parts of the compound pea leaf (the lea¯ets and tendrils). They do not 228 Yaxley et al.ÐLeaf and Flower Development in Pea F I G . 2. The stipule morphology of a single coch mutant. The pairs of stipules from node 5 (top right) to node 14 (bottom left) are shown. Percentage of stipule forms have stipules or axillary buds at their bases, as occurs in true compound leaves. In addition, almost half of the coch plants with compound stipules had no apparent axillary bud in the main leaf axil. This compares with an approximately 3 % occurrence of `blind' leaf axils in wild-type leaves. The node at which leaves with two pairs of lea¯ets ®rst appeared was similar for coch (11.3 + 0.2) and wild-type (11.8 + 0.2) plants (0.1 4 P 4 0.05, n 24). On average, compound coch stipules also changed to two lea¯et pairs at the same node (10.4 + 0.7) as the main leaf rachis (11.3 + 0.2) (0.4 4 P 4 0.2, n 24). However, the compound stipules exhibited more variability, with some changing to two lea¯et pairs substantially earlier (node 7) than the main leaf rachis, and other compound coch stipules never showing more than three lea¯ets (one lea¯et pair and a mixed lea¯et-tendril pair). Flowering time and ¯owering node were also not signi®cantly aected. Mutant coch plants ¯owered at node 15.5 + 0.9 in 65.2 + 0.9 d, and wild-type plants ¯owered at node 16.0 + 0.3 in 65.4 + 0.6 d (0.8 4 P 4 0.7 for ¯owering node; 0.9 4 P 4 0.8 for ¯owering time, n 24). Wild-type pea ¯owers have ®ve sepals, two fused keel petals, two wing petals and a standard petal ( ®ve petals in all), ten anthers (nine fused into a ®lament tube and one partially free) and a single central carpel (Tucker, 1989; Ferrandiz et al., 1999) (Figs 4 and 5). Flowers of coch mutants ranged from nearly normal in appearance (Fig. 4), to open ¯owers with supernumerary organs in each whorl, abnormal organ fusing and some organs which were a mosaic of dierent organ types (Fig. 5, Table 1). The more severely aected ¯owers occurred at later nodes. The ®rst formed ¯owers on the main stem and on laterals were the most normal in appearance and set the most seed. Mutant coch ¯owers were largely self-sterile and coch plants normally produced no more than ten seeds per plant (compared with an average of 60 seeds per plant for the wild-type progenitor line). Flowers of coch mutants were partially sterile when cross-fertilized with wild-type pollen, and the pollen from coch mutants also showed reduced fertility when it was used to pollinate wild-type ¯owers. Due to the low fertility of coch plants, they produced more lateral branches than the wild type. There was an increase in sepal number in coch ¯owers (Table 1) and mosaic sepal/petal organs appeared in the sepal whorl in ¯owers with severe mutant phenotypes. There was also an increase in the number of wing and keel petals (Table 1), and the keel petals did not pair and fuse properly to make a keel (Figs 4 and 5). Based on petal shape and colour (white or green), some petals appeared to be a mosaic of wing and keel petals. In ¯owers with a weak mutant phenotype, there was no change in the number of standard petals, but in ¯owers with a strong mutant phenotype two or three standard petals were common (Table 1), forming a radially symmetric ¯ower structure (Fig. 5). All coch petals were narrower at the base (Fig. 6) than those of the wild type. Wild-type petals form an enclosing sheath around the 100 STIPULE FORM 75 absent elliptical 50 spatulate strap-like 25 0 compound threadlike 3 4 5 nodes formed in the embryo 6 7 8 9 10 11 12 13 14 15 16 17 Node average node of first flower F I G . 3. Occurrence of dierent stipule forms at dierent nodes of mutant cochleata plants (n 40). Yaxley et al.ÐLeaf and Flower Development in Pea 229 T A B L E 1. Number of ¯oral organs in wild-type and cochleata mutant ¯owers Average number of organs + s.e. Floral organ 6.0 + 0.2b 7.1 + 0.4c Anthers 1.0 + 0a 2.0 + 0a 2.0 + 0a 10.0 + 0a 1.0 + 0a 2.1 + 0.1a 2.8 + 0.1b 11.8 + 0.4b 2.7 + 0.2b 2.9 + 0.3b 2.5 + 0.2b 12.6 + 0.8b Carpels 1.0 + 0a 1.0 + 0a 1.8 + 0.6b Petals Standard Wing Keel F I G . 5. coch ¯owers with strong mutant phenotypes (a wild-type ¯ower is shown on the far left). In addition to the changes seen in Fig. 4, more severe mutant ¯owers may have two (A, B) or three (C) standard petals and two to four gynoecia (E and F: these ¯owers are several days old which has allowed the carpels to grow out and become more visible). Flower A has an additional, less developed, ¯ower in the panicle, and ¯ower B has another small ¯oral structure arising from its pedicel. coch mutant weak coch mutant strong phenotype phenotype (n 20) (n 20) 5.0 + 0a Sepals F I G . 4. coch ¯owers with weak mutant phenotypes (a wild-type ¯ower is shown on the far left). The mutant ¯owers with weak phenotypes have additional wing and keel petals which become disorganized, and additional sepals and anthers. Wild-type (n 50) Means with dierent letters in the same row are signi®cantly dierent (P 5 0.02). anthers and stigma, whereas the narrower petal bases of the coch ¯ower cause the petals to fall open so the anthers and stigma are not enclosed within the keel. This means that coch ¯owers have a more open structure. Their pollen dried quickly and tended not be deposited on the stigma(s), thus contributing to their reduced fertility. The number of anthers was also altered in coch ¯owers (Table 1). Occasionally less than ten anthers were present, but generally ¯owers had more than ten anthers (up to 17 in ¯owers with severe mutant phenotypes). Anthers of coch ¯owers were commonly fused together above the level of the ®lament tube, and also fused to petals above the base (the outer stamens are normally basally adnate to the petals; Tucker, 1989). In coch mutant ¯owers with severe phenotypes, most of the anthers were abnormally fused. The gynoecium remained unchanged in coch ¯owers with weak mutant phenotypes, whereas in highly disturbed ¯owers up to four carpels were present (Fig. 5, Table 1). Wild-type pea ¯owers normally have two ¯owers per panicle. This varies somewhat at dierent nodes, with more single-¯owered panicles at the highest nodes (Hole and Hardwick, 1976; Fig. 7). The number of single-¯owered panicles also varied with planting season; more single¯owered panicles occurred during winter than spring (data not shown). In mutant coch plants, there was a greater occurrence of single-¯owered panicles at all nodes at both planting dates (Fig. 7). This varied with node and season in a similar manner to the wild type, with more single-¯owered panicles occurring at higher nodes and during winter. Approximately 10 % of coch panicles contained one ¯ower plus a partially developed ¯ower or ¯ower bud (Fig. 5). In addition, approximately 10 % of coch pods have a `collar' of leafy bract tissue with crenulate edges surrounding the base of the pedicel. Interaction of coch and st F I G . 6. Mutant coch ¯owers (left) have narrowed petal bases compared with the wild type (right). Whole ¯owers are shown at the top and a single standard petal at the bottom. (Petal bases are narrower in coch mutant ¯owers even when there are no extra petals in the whorl.) The st mutant had stipules reduced to small strap-like structures (Fig. 1C). This morphology is the same at each node, and other leaf parts and ¯owers are normal (Pellew and Sverdrup, 1923). The coch st double mutant had 230 Yaxley et al.ÐLeaf and Flower Development in Pea Single flowers (%) 100 80 60 coch wild-type 40 16 18 Node 20 22 F I G . 7. Occurrence of single-¯owered panicles in mutant coch plants and their wild-type progenitor (`Torsdag') for a winter planting. (Panicles of coch mutants with one ¯ower plus a partially developed bud were considered to be panicles with more than one ¯ower.) n 13. coch-like stipules, indicating that coch is largely epistatic to st in this background. The coch st double mutants showed all the stipule forms typical of coch plants: compound, elliptical and sessile, strap-like, spatulate and ®lamentous. However, the double mutant plants had signi®cantly fewer (P 5 0.001) compound stipules than coch plants. Mutant coch plants had 7 + 0.3 nodes carrying compound stipules and coch st double mutant plants had 4 + 0.5 nodes with compound stipules (n 24). Interaction of coch and tl Leaves of the tl mutant have lea¯ets in distal positions where tendrils are normally present (Fig. 1D). The tl mutation is incompletely dominant, and heterozygotes can be distinguished by the presence of ¯attened, rather than cylindrical, tendrils. Compound coch stipules showed reduced expression of the wild-type Tl gene compared with the main leaf rachis. Leaves of the genotype coch coch Tl tl had ¯attened tendrils typical of Tl tl heterozygotes in the distal region of the leaf, but the compound stipules had only lea¯ets (Fig. 8), like the leaf blades of the homozygous recessive tl mutant. A similar reduction in Tl expression was seen in the compound stipules of coch coch af af Tl tl plants. The leaf blade of coch coch af af Tl tl plants had tendrils like that of an af mutant leaf, with the tendrils somewhat ¯attened at their tips due to the incomplete dominance of the Tl gene. However, the compound stipules of this genotype consisted of branched tendrils ending in small terminal lea¯ets (Fig. 9), similar to the af tl double mutant phenotype (Fig. 1G). Early leaf development of coch and uni In the coch apical meristems examined, stipule primordia were smaller and less developed than the primordia of the ®rst lea¯et pair, from the time they were ®rst visible (Fig. 10B) until late stages of leaf expansion (e.g. P9 , Fig. 11). In contrast, wild-type leaf primordia have early stipule primordia (P2 to P4) that are larger than the primordia F I G . 8. Leaf-like stipules of the genotype coch coch Tl tl have small terminal lea¯ets typical of the homozygous recessive genotype tl tl (arrow). The main leaf rachis has ¯attened tendrils typical of a Tl tl heterozygote. Bar 4 cm. F I G . 9. Leaf-like stipules of the genotype coch coch af af Tl tl have small terminal lea¯ets typical of the double recessive genotype af af tl tl (arrow). The main leaf rachis has the ¯attened tendrils typical of a Tl tl heterozygote. Bar 4 cm. of the ®rst lea¯et pair (compare Fig. 10A and B). During P5 , compound coch stipule primordia initiated lateral pinna. During P6 these ®rst stipule lateral pinna became more dorsiventral and secondary lateral pinna were initiated (Fig. 12). The diameter of the apical dome of coch mutant plants, when P0 is node 11, was similar (190 + 4.7 mm) to that of the corresponding wild type (200 + 5.1 mm) (n 6). The phenotype of the uni mutant has been described previously (Eriksson, 1929; Lamprecht, 1933; Hofer and Ellis, 1996; Hofer et al., 1997). Flowers of the uni mutant have an incomplete sepal whorl, no petals or stamens, and an open gynoecium, with numerous iterations of axillary ¯owers (Hofer et al., 1997). At the nodes examined (7-13), all uni leaves consisted of normal stipules with a single leaf blade. Approximately half of these uni leaves were lobed (Fig. 13), more commonly at the lower nodes examined (nodes 7±9: 70 % lobed leaves). Leaves of uni plants at the higher nodes examined (nodes 11-13) had longer petioles than those leaves at nodes 7±9 which were mostly sessile (80 % sessile or almost sessile; Fig. 13). Leaf primordia of the uni mutant initiated a single large lea¯et primordium and two normal stipule primordia (Fig. 10C). At the same stage, the wild type had smaller lea¯et primordia with two large lateral stipule primordia (Fig. 10A). During P3 , the uni leaf primordium developed a groove on its adaxial side, indicating the incipient midrib. This was more obvious in the P4 uni lea¯et (Fig. 10C), which Yaxley et al.ÐLeaf and Flower Development in Pea 231 development between wild type, coch and uni plants are also seen when the position in which hairs ®rst appear on these leaves is compared; in the wild type they appear on stipules in P3 ±P4 (Fig. 10A), in coch they appear on the ®rst lea¯et pair in P3 ±P4 (Fig. 10B) and in uni they ®rst appear on the leaf blade and stipule primordia in P3 ±P4 (Fig. 10C). The diameter of the apical dome of uni plants, when P0 is node 11, was signi®cantly less (148.5 + 3.3 mm) than that of the corresponding wild-type siblings (193.6 + 3.6 mm) (P 5 0.001, n 7) (see Fig. 10). However, the node and time of ®rst ¯ower initiation were similar in mutants and wild types. Mutant uni plants ¯owered at node 22.6 + 0.4 in 58.9 + 0.4 d in summer, and their wild-type siblings ¯owered at node 21.6 + 0.3 in 57.9 + 0.4 d (both 0.1 4 P 4 0.05, n 20). DISCUSSION Phenotype of the coch mutant F I G . 10. A, A wild-type apex from line JI2171 with the largest leaf subtending node 12. The stipule primordia of the P3 and P4 leaves (S3 and S4) are larger and more developed than the ®rst lea¯et primordia (L3 and L4). The L4 lea¯et primordia are just beginning to develop an adaxial groove. B, A coch apex from line AF99 with the largest leaf subtending node 12. An in¯orescence meristem is also present (IF). The stipule primordia of the P3 and P4 leaves (S3 and S4) are small and undeveloped compared with the primordia of the ®rst lea¯et pairs (L3 and L4). The stipule primordia are also small and undeveloped compared with the wild-type stipule primordia (see Fig. 11B). C, The apex of a uni mutant from line JI2171, with the largest leaf (P4) subtending node 12. The P3 primordium shows a single large leaf blade primordium and stipule primordia (S3). The P3 leaf blade primordium has a central adaxial groove where the midrib is forming. The P4 leaf blade primordium shows a clear central channel and is developing a laterally ¯attened folded lea¯et shape. The P3 and P4 leaf blade primordia also show the development of lobes on their ¯anks (lo). was clearly a laterally ¯attened structure, with two halves folded along a central groove. The wild type lea¯et primordia showed no lateral ¯attening or midrib-groove development until late P4 (Fig. 10A). The dierences in early leaf The stipule forms displayed by the coch mutant vary with node of insertion. The ®rst ®ve to six nodes of a pea plant are formed in the developing embryo (Gould et al., 1987; Villani and DeMason, 1997). Nodes 3-5 usually have no stipules in the coch mutant, so there may be an interaction between the environment of the embryo and the coch mutation which results in the absence of stipules, or the formation of only small stipules. The vegetative nodes of the coch plant which develop after germination show predominantly compound stipule forms. After ¯owering commences (nodes 15-16), the stipule forms found in coch mutants are simpli®ed. Again, it seems likely that the major change in the condition of the shoot apex is responsible for the corresponding change in stipule form, which parallels the increase in leaf blade complexity in the middle nodes, and the decrease in leaf complexity after ¯owering, seen in wild-type plants (Wiltshire et al., 1994; Villani and DeMason, 2000). In experiments in which excised pea leaf primordia were grown in culture (Gould et al., 1994), it has been shown that the two pinnae of a lea¯et or tendril pair are not always determined simultaneously. The tendency for opposite pinnae to develop into dierent structures is increased in coch compound stipules. Since the development of coch stipules is retarded (Gourlay et al., 2000; Figs 10 and 11), it is possible that they remain meristematic for longer, which could lead to their greater developmental ¯exibility. In addition, it was more common for the pinnae of coch compound stipules not to be positioned opposite each other. This is a shoot-like characteristic (Sattler and Rutishauser, 1992; Lacroix and Sattler, 1994), and supports the contention that the coch mutation reduces determinacy in the stipules. It seems likely that the open structure of the coch ¯ower will reduce the viability of pollen and the receptive life of the stigmatic surface. However, while generating crosses in this study, we also found poor seed set of coch ¯owers pollinated by hand with wild-type (Coch) pollen. This is consistent with structural and developmental defects in the gynoecium of coch mutants that reduce seed set; such abnormal development of the gynoecium and of the female gametophyte in putative coch ¯owers was described by 232 Yaxley et al.ÐLeaf and Flower Development in Pea F I G . 11. Stipules of coch mutants remain less developed than wild-type stipules at later stages of leaf growth. A, P7 leaves at node 13 of coch mutant (left) and wild-type (right) plants; B, P8 leaves at node 12 of coch mutant (right) and wild-type (left) plants; C, P9 leaves at node 11 of coch mutant (left) and wild-type (right) plants. (In A and B the apical meristem of the coch mutant has been removed for clarity.) F I G . 12. P6 coch stipule primordia (S6), from node 11, showing three pairs of lateral primordia (l1 , l2 , l3). The primary pair (l1) is becoming ¯attened, indicating that they will become lea¯ets. The P6 lea¯et (L6), and the P5 leaf are also indicated. Molhova et al. (1988). In the putative coch line examined by Molhova there was sometimes no ovule formation and sometimes early formation of the embryo sac was disturbed. Molhova et al. (1988) also described the production of feminized stamens in coch ¯owers, leading to the production of multipistillate ¯owers. While the production of multipistillate ¯owers through an increase in carpel number was observed in this study, no feminization of the stamens was seen. This dierence could be due to the dierent lines examined. Molhova et al. (1988) found a high percentage of sterility in pollen from coch mutants. Pollen from the coch mutant utilized in this study also showed reduced fertility when crossed onto wild-type ¯owers, thus it is also likely to show some abnormalities. It is interesting to note that it is the base of the wing and keel petals that is altered in all coch ¯owers, even in those with the weakest phenotypes. This suggests that there may be some homology between the base of the petals and the base of the leaves (stipules) in the action of the Cochleata gene. The defective petal bases may be partly responsible for the improper fusing of the keel petals and anthers. In F I G . 13. A lobed, almost sessile uni leaf (node 8). pea, the petals and stamens originate from a common primordium (Tucker, 1989; Ferrandiz et al., 1999). Incorrect separation of these organs may result in the fused stamens and petals seen in coch ¯owers. The formation of some ¯oral organs which are a mosaic of dierent organ types indicates that the coch mutation also has an eect on ¯oral organ identity. The reduced formation of two-¯owered panicles seen in coch in¯orescences shows that the early development of the in¯orescence is also altered by the mutation. In pea ¯owers, once the in¯orescence meristem has formed and the meristematic region which will form the ¯ower has become distinct, the remaining region will form another ¯ower initial, or will revert to a vegetative state and form a small stub (Hole and Hardwick, 1976). In coch ¯owers, a second ¯oral initial is much less likely to form (there are more single-¯owered panicles), and in addition, more developmental ¯exibility is seen as half-formed second ¯owers are sometimes seen in coch mutants (Fig. 5), whereas these forms do not appear in the wild type. Thus, the Coch gene has been shown to have pleiotropic eects on leaf and ¯ower development, altering stipule form, ¯oral organ number, shape and identity, fertility and in¯orescence structure. Yaxley et al.ÐLeaf and Flower Development in Pea Interaction of coch and st Double mutant coch st plants resembled coch plants. This is in contrast to the ®ndings of Marx (1987) and Gourlay et al. (2000) who reported that coch st double mutants had no stipules. This may be due to dierences in the genetic background and/or allele-speci®c dierences. In this study, coch is predominantly epistatic to st. It is possible that the coch mutation changes the fate of the stipule cells into more `leaf-like' cells and thus prevents the action of st, which acts only on stipule cells. Epistasis occurs at all nodes and all stipule forms are seen in coch st double mutant plants. The suggestion that coch alters the fate of stipule cells is strongly supported by the fact that coch compound stipules behave genetically as if they were leaf blades in a basal position (Marx, 1987). For example, the coch af double mutant has stipules which consist of tendrils like the leaf blade. Interaction of coch and tl The interaction between coch and tl suggests there is a gradient of wild-type Tendril-less gene action in the leaf, with Tl action being reduced at the base of the leaf. This cannot normally be seen as Tl does not act on the stipules, but in the compound stipules of coch the reduced action of the wild-type Tl gene in the base of the leaf is evident. This supports proposals that gradients of gene action could be involved in determining leaf form in pea (Hofer and Ellis, 1996; Lu et al., 1996). Lu et al. (1996) suggested that a gradient of Tl gene function would be a plausible explanation of pea leaf morphology, with Tl function producing a tendril-inducing or branching-inhibiting morphogen at the leaf tip. Villani and DeMason (1999b) showed, via morphological measurement of a series of mutants with dierent numbers of tl alleles, that Tl acts in both the proximal and distal parts of the leaf, but has a greater eect in the distal region. Early leaf development of coch and uni mutants The small size of coch stipules and their slow growth and development strongly suggests that coch stipules are developmentally retarded compared with lea¯et primordia of the same leaf. Early slow growth of coch stipules was also shown by Gourlay et al. (2000). It is possible that this is the primary consequence of the coch gene mutation, and that because of their developmental retardation and probably more meristematic nature, coch stipules respond inappropriately to normal developmental signals, and so develop into complex compound structures. The formation of compound stipules seems to be due, at least in part, to the expression of Uni seen in coch S3 and S4 stipules, which is not seen in wild-type stipules (Gourlay et al., 2000) (Uni is important in maintaining blastozone activity). However, this ectopic expression of Uni in compound coch stipules is not seen until after morphological changes have occurred in coch primordia (at late P1 or early P2), and it is seen only in the compound coch stipules, not in the other more simple coch forms; thus, this alteration in Uni expression is not likely to be the primary eect of the coch mutation. 233 The eects of the mutations coch and uni seem to be apparent at an earlier stage (late P1 to early P2) of leaf primordia development than the mutants tendril-less and a®la (apparent late P4 ±P6 and late P2 ±P3 , respectively) (Meicenheimer et al., 1983; Gould et al., 1986; Gourlay et al., 2000; Villani and DeMason, 2000), so they may act earlier in the leaf developmental pathway. No dierences in the size of the shoot apical dome have previously been reported in pea leaf mutants. The smaller diameter (reduced by 25 %) of uni meristems may be a consequence or a cause of their altered leaf morphology. It is possible that a reduction in the number of cells in the incipient leaf primordium of the uni plant results in the production of a simpler leaf structure. However, apical size is not always linked to leaf size (Smith and Hake, 1992; Goliber et al., 1999). The wild-type Uni gene is not expressed in the shoot apical meristem (Gourlay et al., 2000), so the reduction in Uni meristem size may not be a primary eect of the mutation. It is possible that cells expressing Uni in the marginal blastozones normally signal to cells in the shoot apical meristem. Recent work has implicated auxin concentration as a determinant of primordial size in tomato, with increasing concentrations of auxin increasing the number of cells recruited into primordia (Reinhardt et al., 2000). CO N C L U S I O N S The most recent model of pea leaf development (Hofer and Ellis, 1998) proposes that Uni, with one or more other genes, acts to maintain a shoot-like fate in the leaf meristem, with the inhibition of Uni by Af and Tl producing determinate lea¯ets and tendrils. Hofer and Ellis (1998) suggest that Coch acts to inhibit Uni at the base of the leaf to distinguish determinate stipule cells from the partiallyindeterminate rachis. Recent molecular evidence upholds this model. Gourlay et al. (2000) show that Uni is expressed in the leaf primordium when leaf primordia are initiated and is down-regulated when they become determined. The loss of Uni gene function results in a prematurely determined leaf primordium which produces a simple leaf form. In keeping with this, there is prolonged Uni expression in the primordia of the complex leaf forms seen in the a®la and a®la tendril-less mutants. This model is largely supported by the evidence from mutant phenotypes reported here. A loss of determinacy is seen in the formation of compound stipules rather than simple ones in coch plants, and some loss of determinacy occurs in ¯owers resulting in the production of supernumerary organs. However, the primary eect of the coch mutation seems to be to retard the development of the stipules; this then appears to allow expression of Uni in some stipule primordia, leading to their development into compound structures. The retarded development of the stipules seems to allow them to behave more like leaf blades. This is shown by the way coch mutant stipules mimic the form of the leaf blade (Marx, 1987), and is supported by the epistasis shown here between coch and st. We also add information to this model by showing that uni mutants have a reduced shoot apical meristem diameter, which could result in their simpler leaf form. 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