ROLES OF THE AF AND TL GENES IN PEA LEAF

Transcription

1 American Journal of Botany 84(10): ROLES OF THE AF AND TL GENES IN PEA LEAF MORPHOGENESIS: CHARACTERIZATION OF THE DOUBLE MUTANT (AFAFTLTL) 1 PHILIP J. VILLANI 2 AND DARLEEN A. DEMASON Department of Botany and Plant Sciences, University of California, Riverside, California The pleiofila phenotype (afaftltl double mutant) of Pisum sativum arises from two single-gene, recessive mutations known to affect the identity of leaf pinnae, afila (af ), and acacia (tl). The wild-type leaf consists of proximal leaflets and distal tendrils, whereas the pleiofila leaf consists of branched pinnae terminating in small leaflets. Using morphological measurements, histology, and SEM, we characterized the variation in leaf form along the plant axis, in leaflet anatomy, and in leaf development in embryonic, early postembryonic, and late postembryonic leaves of aftl and wild-type plants. Leaves on aftl plants increase in complexity more rapidly during shoot ontogeny than those on wild-type plants. Leaflets of aftl plants have identical histology to wild-type leaflets although they have smaller and fewer cells. Pinna initiation is acropetal in early postembryonic leaves of aftl plants and in all leaves of wild-type plants, whereas in late postembryonic leaves of aftl plants pinna initiation is bidirectional. Most phenotypic differences between these genotypes can be attributed to differential timing (heterochrony) of major developmental events. Key words: acacia; afila; Fabaceae; heterochrony; leaf morphogenesis; Pisum sativum; pleiofila. The leaf is the most variable organ of the plant body. This diversity is expressed both from one plant species to another and during the life of a single plant. Among the important developmental questions still unresolved with regard to leaf form diversity are: (1) How are the various regions of the leaf determined?; and (2) How is the existing variability in basic leaf form determined developmentally? Theoretically, this diversity must result from differences in position, timing, and functioning of a small number of leaf meristems. The existence of a number of single-gene mutations that are known to affect leaf form in several plant systems, including Arabidopsis, cotton, maize, tomato, and pea holds considerable promise for studying the genetic regulation of leaf development (for reviews, see Marx, 1983; Smith and Hake, 1992, 1994). Among the most interesting are a suite of mutations in pea (Blixt, 1972; Marx, 1977, 1987; Murfet and Reid, 1993). A typical leaf of pea (Pisum sativum L.) is composed of a basal pair of foliaceous stipules, one or more pairs of proximal leaflets, one or more pairs of distal tendrils, and a single terminal tendril (Fig. 1). There has been considerable interest in two leaf form mutations: acacia (tl) in which leaflets occur at all positions (Vilmorin and Bateson, 1912; White, 1917) and afila (af) in which compound (branched) tendrils occur in proximal positions and simple tendrils occur in distal positions (Kujala, 1 Manuscript received 17 January 1997; revision accepted 26 June The authors thank Dr. Julia Bailey-Serres and Dr. Beth Bray for their critical comments on the manuscript, Janet Giles for her help with the microscopic measurements and data entry, Dr. Manuel Mundo-Ocampo for his technical assistance with the SEM, Dr. Mark Brunell and Dr. Robert Beaver for their assistance with the statistical analysis of the data, and Dr. Todd Cooke for his insightful comments during many discussions. This study is a portion of a Ph.D. dissertation by the first author at the University of California, Riverside. 2 Author for correspondence (FAX: , Villani@citrus.ucr.edu) ; Goldenberg, 1965; Lu et al., 1996). Gould, Cutter, and Young (1986) determined that leaflets of acacia and tendrils of afila are true leaflets and tendrils because they showed no histological differences from those structures in wild-type leaves. The wild-type, acacia, and afila phenotypes exhibit heteroblastic leaf variation (an ontogenic progression in which early or juvenile structures are markedly different from later or adult ones). Leaves at lower nodes have fewer lateral pinna pairs than do leaves at upper nodes (Gould, Cutter, and Young, 1986, 1992; Lu et al., 1996). Recently these pea genotypes have been characterized morphologically and developmentally by various authors (Meicenheimer et al., 1983; Gould, Cutter, and Young 1986, 1989, 1992, 1994; Marx, 1987; Cote et al., 1992; Lu et al., 1996) who have attempted to explain the phenotypic differences observed. Meicenheimer et al. (1983) described the differences in terms of leaf meristematic activities. They observed that leaf primordia of the mutant genotypes diverged in their development from the events in wild-type plants because in af, pinnae exhibit only radial marginal meristems characteristic of tendrils, and in tl, terminal primordia exhibit marginal and adaxial meristems rather than radial, marginal meristems. Their conclusions were that the af allele affects marginal meristem function in the leaflet and the tl allele gives rise to adaxial and marginal meristems where none existed before. Young (1983) published an elegant computer model of pea leaf morphogenesis based on a few, simple assumptions: that phenotypic determination proceeds sequentially by subdivision, giving rise to multiple, independent pinna meristems, and that the developmental fate depends on size at a critical stage, but does not require any information of position or previous developmental history. Thus, a primordium above a critical size threshold becomes a rachis; primordia smaller than the threshold become leaflets and smaller than a second threshold become tendrils. Young defined size as a vague property,

2 1324 AMERICAN JOURNAL OF BOTANY [Vol. 84 Fig. 1. Late postembryonic leaves of wild-type (AfAfTlTlstst) and the aftl double mutant (afaftltlstst) plants. Leaves with five pinna pairs from wild-type (A) and double mutant (B) leaves of garden pea (Pisum sativum). Bar 1 cm. Figure Abbreviations: Fig. 1. L, leaflet; P, petiole; r, rachilla; R, rachis; T, tendril. Fig. 2. E, embryonic leaf, F, flower; MP2 and MP3, mixed pair at pinna position 2 and 3, respectively. Figs A, areole; BE, abaxial epidermis; DE, adaxial epidermis; M, midrib; P, palisade mesophyll; S, spongy mesophyll; ST, stomate; V, vascular bundle. Figs B, branching; D, distal primordium; M, shoot apical meristem; P, primordium of interest; R, rachis; S, stipule; t, tendril formation; 1,2,3, etc., pinna position 1,2,3, etc. which could be volume, mass, cell number, or amount of a chemical signal. Subsequently, Gould, Cutter, and Young (1986) hypothesized that af and tl cause a change in the thresholds precluding the possibilities of forming leaflets or tendrils. Young (1983) likened the af and tl genes to homeotic mutations in Drosophila since the parts are structurally normal, but the arrangement is altered. More recently, Lu et al. (1996) have presented a heterochronic, or timing model to explain the phenotypic differences determined during pea leaf morphogenesis. Using the morphological characteristics of mature leaves in all possible homozygous and heterozygous genotypes, Lu et al. (1996) concluded that positioning, identity, and branching of the pinnae are governed by precise morphological relationships, which are primarily dependent on the relative position along the leaf axis. Thus, they disagreed with the validity of one basic assumption of Young s (1983) model. Further, using the genetic principles basic to the ABC model of homeotic floral mutants (Coen and Meyerowitz, 1991; Weigel and Meyerowitz, 1994), they designated the double recessive condition (aftl) as the ground state (absence of any activity of the genes being examined) in this model (Lu et al., 1996). Using a hypothetical representation of the developmental events leading to the final morphologies of each genotype, they hypothesized that the phenotypes are differentiated by differences in timing of two developmental processes: leaf axis branching and lamina initiation. Lu et al. (1996) regard the Af and Tl genes, then, as gain-offunction genes responsible for regulating the timing of these two processes. Morphological and developmental characterization of the phenotype of the aftl plants (pleiofila) is necessary to test this model. The pleiofila phenotype possesses compound pinnae, which terminate in small leaflet-like structures at all positions (Marx, 1987) (Fig. 1). Since the shape and size of double mutant leaflets are so dissimilar to those of wild-type and acacia leaves, histological analysis is necessary to determine if they are, indeed, true leaflets. Although Gould, Cutter, and Young (1992) described embryonic and early postembryonic leaf variation of aftl plants (nodes 3-8), the features of late postembryonic leaves in this genotype have not been previously characterized. Meicenheimer et al. (1983) briefly described the development of early postembryonic leaves as the result of profuse branching of leaf primordia and ultimate lamina formation due to activities of adaxial and marginal meristems. However, we do not know whether simpler, early postembryonic leaves are representative of more complex, late postembryonic leaves and the details necessary to test the timing hypothesis are not yet available. Also, Lu et al. (1996) observed that position 2 pinnae on late postembryonic leaves with five pinna pairs were more highly branched than those in position 1. They suggested that late postembryonic leaves may have bidirectional pinna initiation, which is inconsistent with observations made in previous developmental studies (Meicenheimer et al., 1983; Gould, Cutter, and Young, 1986). Investigation of the sequence of pinna initiation in late postembryonic leaves of aftl plants is therefore the major goal of this study. Tomato is another compound-leafed species in which a number of single-gene mutations exist. Wild-type plants have a pinnately to bipinnately compound leaf, which is typically characterized as having several pairs of large lateral leaflets, which are often highly lobed, but some of which may be deeply incised (Dengler, 1984). Recently, Hareven et al. (1996) have made tomato transformants that overexpressed the maize Knottedl (Kn1) gene, which in some genotypes resulted in an increase in leaf branching order and produced a highly compound leaf very similar in appearance to the pleiofila phenotype of pea. The implication of these experiments is that Kn1 or Kn1-like genes are involved in compound leaf development. The remarkable morphological similarity between these tomato transformants and the aftl double mutant of pea makes it even more important to adequately characterize the morphology and development of the leaves of this genotype. Our goal in this study is to compare the development and morphology of leaves in aftl plants to those of wild-type plants, to use these data to test the validity of the proposed models of pea leaf morphogenesis, and to understand the roles of the Af and Tl genes. We asked the following questions: (1) What is the entire spectrum of morphological variation in leaf form (from embryonic to late postembryonic) on aftl plants compared to that of wild-type plants? (2) Are there differences between the genotypes in rates of change in leaf form complexity along the shoot? (3) What are the histological characteristics of leaflets of aftl plants? and (4)

3 October 1997] VILLANI AND DEMASON ROLES OF THE AF AND TL GENES IN PEA 1325 What are the morphological events that lead to the formation of both early and late postembryonic leaves of aftl plants and how do they differ in sequence and timing from those leading to the formation of comparable wildtype leaves? MATERIALS AND METHODS Plant material and growth conditions The seeds of two near-isogenic lines of the common pea, Pisum sativum, including wild-type (AfAfTlTlstst) and the aftl double recessive (afaftltlstst) genotypes as described by Marx (1987) were obtained as described previously (Lu et al., 1996). Currently, the Marx collection resides with Dr. Charles Simon, Germplasm Curator, USDA, ARS, Pacific West Area. To simplify morphological measurements and developmental observations on these plants, all were homozygous recessive for the gene reduced stipules (st), which results in dramatic reduction in stipule size without affecting other leaf traits (Meicenheimer, 1983; Marx, 1987). Seeds were soaked in water at 26 C for 24 h, transferred to a commercial fungicide for 15 min, and then planted one seed per pot (3.8 L) in commercial potting mixture. Plants were grown in a full-light greenhouse at the University of California-Riverside, which maintained 23 C year-round. Plants were watered as needed and fertilized with Hoagland s nutrient solution weekly. Lateral structure terminology is used as defined previously (Lu et al., 1996). Briefly these structures are: leaflet, a broad dorsiventral structure with a prominent midvein and numerous lateral veins; tendril, a narrow unbranched cylindrical structure with a central vein; and compound pinna, a branched structure with the lower order branches composed of cylindrical axes with central veins and terminating in small, expanded, dorsiventral leaflets. In wild-type plants a mixed pair is defined as a pinna position at which a leaflet occurs opposite a tendril. Morphological measurements and growth rates Twenty plants of each genotype were grown for the purpose of making morphological measurements on leaves at nodes Leaves were numbered in acropetal order, with the cotyledonary node designated as node 1. Measurements were conducted in two phases: (1) nodes 4-17 were measured as soon as leaf 17 was fully expanded; and (2) nodes were measured as soon as leaf 27 was fully expanded. Leaves at nodes 2 and 3 were not measured because they are scalar leaves without any lateral structures and show no discernible differences in morphology (Gould, Cutter, and Young, 1986). Pinna positions within a leaf were also numbered in acropetal order. The following morphological measurements were recorded: (1) petiole length, the distance from the point of attachment of leaf and stem to the first lateral structure (i.e., leaflet or compound pinna); (2) total leaf length, the distance from the attachment of leaf and stem to the distal-most point of the leaf; (3) the number and length of each pinna pair along the rachis; and (4) in the case of pleiofila, the number of secondary branches and the branch order complexity per pinna position. The lengths of pinna pairs were converted to relative data by dividing the length of a pinna pair by the total length of the leaf to which it was attached. The number of secondary branches per pinna position was defined as the number of primary lateral pairs produced from the central pinna axis. Order of complexity per position was defined as the total number of branch orders occurring at a position (1, 2, 3, etc.). Leaflet areas were determined by photocopying ten wild-type and pleiofila leaflets, cutting the leaf and leaflets out, and weighing them compared to a standard. To simplify the determination of pleiofila leaflet areas, leaflets were separated from rachillae and then photocopied. The percentage differences in leaflet area due to cell size and cell number for pleiofila compared to wild-type leaflets were determined using leaflet area and adaxial epidermal cell surface area. Light microscopy Material for light microscopy was taken from node 8 leaves of 20 wild-type and aftl plants. Approximately 0.5 cm 2 of leaflet tissue was taken consistently from the right and central portion of leaflets from wild-type plants or entire leaflets from aftl plants from a similar location on compound pinnae. Samples were fixed overnight in 2% glutaraldehyde in a 50 mmol/l phosphate buffer at ph 7.2, dehydrated in a graded ethanol series, and embedded in JB-4 plastic (Polysciences). Sections were cut at 3 m and stained with 0.05% toluidine blue O (Certified Index 52040) in benzoate buffer at ph 4.4. Ten leaflet clearings from node 8 of wild-type and aftl plants were prepared by immersion in 100% ethanol until specimens were devoid of chlorophyll, and then 10 mol/l sodium hydroxide for h or until cytoplasmic material was decolorized. Cleared leaflets were then rinsed in distilled water, dehydrated to 70% ethanol, stained with 1.0% chlorazole black E (Certified Index 30235) in 70% ethanol, transferred to xylene, and mounted with Permount. Leaf transverse sections and clearings were photographed on a Zeiss standard brightfield microscope using Kodak Technical Pan 2415 film at ASA 50. Microscopic measurements Measurements were made on a Hewett-Packard 85 computer and graphics tablet to which a Nikon microscope and camera lucida were mounted as described previously (DeMason, 1984). Clearings and sections described above were used to make the following microscopic measurements: adaxial epidermal surface area, palisade cell length, and mesophyll cell and areole surface area. Stomatal frequencies were measured by counting the number of stomata in clearings under the entire field of view of a Nikon compound microscope with 10 oculars and a 40 objective. The area of the field was 115 mm 2. Scanning electron microscopy (SEM) Shoot apices were collected to contain developing stages of leaves 8 and 14 of aftl plants (to represent early and late postembryonic leaves, respectively) and leaves of wild-type plants (to represent late postembryonic leaves of comparable complexity) and were fixed in 2% glutaraldehyde in 50 mmol/l phosphate buffer ph 7.2 overnight, dehydrated in a graded ethanol series, and critical point dried (Samdri PVT-3). Shoot apices where then dissected under a dissecting microscope, mounted on copper stubs, and gold sputter-coated (Hummer V). Specimens were viewed on a JEOL-35C SEM and photographed on Polaroid Type 55 film. The term plastochron was defined as the period of time between the initiation of one leaf primordium and the initiation of the next primordium. The age of a leaf primordium was determined by counting the number of primordia initiated after it plus one. Statistics Excel 5.0 or BMDP Statistical Software package (Dixon, 1990) were used to calculate means, standard errors, and to do pairwise comparisons. RESULTS Leaf morphology The habits of wild-type (AfAfTlTl) and afaftltl plants were very similar. The cotyledons were positioned at the ground level in both genotypes. The leaves at nodes 2 and 3 were small scalar leaves without pinnae. Subsequent leaves produced in both genotypes were compound and exhibited increasing complexity during the ontogeny of the plant. Leaf length in wild-type plants increased steadily until node 19, and subsequent leaves had decreased lengths (Fig. 2). This leaf length decrease corresponded with the onset of flowering, which typically occurred at node 20 and above. Leaves of wild-type plants ranged in complexity from one to six pinna pairs. Embryonic and early postembryonic leaves, which typically occurred at nodes 4-12, were composed of 1-3 pinna pairs (i.e., 1-2 pairs

4 1326 AMERICAN JOURNAL OF BOTANY [Vol. 84 Fig. 2. Heteroblastic progression of leaf length and complexity. Mean total leaf length (solid symbols) and number of pinna pairs per node (open symbols) of wild-type (circles) and aftl (squares) plants from nodes 4 to 27. Asterisks indicate nodes at which means were not significantly different. of leaflets, one pair of tendrils, and a single terminal tendril), whereas late postembryonic leaves at nodes were composed of 4-6 pinna pairs (i.e., 1-3 pairs of leaflets and 3-4 pairs of tendrils and a single terminal tendril). Leaflets were always proximal to tendrils and occasionally leaves would end without a terminal tendril. In wildtype plants the number of pinna pairs per leaf (i.e., leaflets or tendrils) increased slowly and gradually during the ontogeny of the plant from one to six pairs (Fig. 2). Mixed pinna pairs (leaflet opposite a tendril) commonly occurred at two nodes on wild-type plants: (1) at node 14 on leaves that generally had one pair of leaflets, one mixed pair, and one to two pairs of tendrils, and (2) at node 22 on leaves that generally had two pairs of leaflets, one mixed pair, and 1-3 pairs of tendrils. Mixed pairs were not observed on every plant. Leaf length in aftl plants was considerably less than that of wild-type plants at nearly every node (Fig. 2). Leaf length increased steadily until node 23 and then decreased slightly. Again, the decrease in leaf length corresponded to the onset of flowering. Leaves on aftl plants ranged in complexity from one to seven pinna pairs. Leaves with seven pinna pairs were very common in this genotype but were never observed on wild-type plants. The number of pinna pairs per leaf on aftl plants was similar to that on the wild type at early nodes but increased rapidly after node 8 during ontogeny (Fig. 2). Although the mean first node to flower on the wild-type plants was node 20, it was significantly later (node 22) on aftl plants (P 0.01). During ontogeny of wild-type plants, petiole length increased in leaves with 1-4 pinna pairs but stabilized to nearly the same length in leaves with 4-6 pinna pairs. On the other hand petiole length on aftl plants increased Fig. 3. Petiole length and leaf complexity. Mean petiole lengths of leaves with 1-6 pinna pairs of wild-type (circles) and 1-7 pinna pairs of aftl (squares) plants. Data are plotted as the mean 1 SE of replicate leaves. Means plotted without SE bar have standard errors smaller than the symbol. steadily throughout plant ontogeny regardless of the number of pinna pairs (Fig. 3). Measurements of relative pinna lengths in wild-type plants revealed two trends (Table 1). First, relative leaflet lengths decreased from proximal to distal regions within a leaf. When the switch from leaflets to tendrils occurred, there was a corresponding increase in pinna length, but then there was a decrease in relative tendril length, again, distally along the remaining leaf axis. And secondly, leaflet relative length at one particular position decreased in leaves with increasing complexity (i.e., leaves with one, two, three pinna pairs, etc.). On the other hand, relative tendril length at a position increased as leaves increased in complexity. On aftl plants, however, there was a constant decrease in relative pinna length from proximal to distal positions within leaves at each complexity, and as complexity increased, relative pinna length increased at each position (Table 1). Observations on the number of secondary laterals and the order of complexity within leaves of aftl plants revealed three similar trends: (1) as the number of pinna pairs increased on a leaf, the number of secondary branches (Fig. 4) and the order of complexity (Fig. 5) at each position within the leaf increased; (2) for early postembryonic leaves (i.e., two or three pinna pairs), the number of secondary branches and the order of complexity at each position decreased from proximal to distal positions and; (3) for late postembryonic leaves (i.e., 4-7 pinna pairs), both parameters increased between positions 1 and 2 and subsequently decreased distally. Leaflet histology Mean leaflet areas were 4.19 and 0.36 cm 2 for wild-type and aftl plants, respectively. Leaflets of aftl plants were histologically similar to those of wild-type plants. The types and numbers of cell layers from the adaxial to abaxial surface of both genotypes included: one adaxial epidermal layer with stomata, 1-2 palisade layers, 5-6 spongy mesophyll layers, and an abaxial epidermal layer with stomata (Figs. 6, 8). Leaflets

5 October 1997] VILLANI AND DEMASON ROLES OF THE AF AND TL GENES IN PEA 1327 TABLE 1. The relative lengths (%) of all pinna pairs between nodes 4 and 27 (inclusive) with 1 6 or 7 pinna pairs from 20 wild-type and aftl plants. Relative lengths were calculated by dividing the length of the pinna by the total leaf length. Standard errors are in parentheses. Phenotype Pinna pairs per leaf Pinna positions (numbered acropetally) Wild type (7.84) a (2.21) a (2.72) c (0.62) a (2.11) b (1.17) c (0.82) a (2.55) b (0.70) c (0.61) c (0.55) a (0.35) a (1.08) b (0.55) c (0.40) c (0.60) a (0.47) a (1.42) b (0.91) c (0.61) c 8.14 (0.76) c Pleiofila (2.85) (2.93) (3.25) (3.50) (5.43) 9.34 (2.45) (1.48) (1.92) (1.41) 8.43 (0.76) (0.94) (0.91) (0.77) 8.54 (0.48) 6.58 (0.30) (0.67) (0.86) (0.63) (0.42) 4.92 (0.24) 4.56 (0.14) (0.94) (1.02) (0.98) (0.92) 8.74 (0.59) 2.90 (0.27) 3.62 (0.13) a Pinnae at this position were leaflets. b Pinnae at this position were either leaflets, a mixed pair, or tendrils. c Pinnae at this position were tendrils. of wild-type and aftl plants had similar cell shapes: adaxial and abaxial epidermal cells were highly lobed; palisade parenchyma cells were elongate; and spongy mesophyll cells were elliptical and nearly lobed. Adaxial epidermal cell area, palisade cell length, mesophyll cell area, and areole area were significantly smaller for aftl plants than for wild-type plants at the 0.01 level (Table 2). On the other hand, stomatal frequency was greater in aftl than wild-type plants. Leaflet venation pattern in both genotypes was similar in that leaflets contained primary, secondary, tertiary, and quaternary veins and veinlet endings, although secondary veins in pleiofila leaflets were somewhat less defined (Figs. 7, 9). In general, areoles were somewhat irregular in shape and contained few to no veinlet endings within them. Leaf development in wild-type plants Leaf primordia at nodes (late postembryonic leaves) were initiated as lateral outgrowths of the shoot apical meristem in a manner similar to early postembryonic leaves (Lyndon, Fig. 4. Mean number of secondary laterals as a function of pinna position from leaves with 2-7 pinna pairs of aftl plants. A silhouette of a leaf with five pinna pairs is provided to demonstrate the counting scheme used. Lines connecting points reflect pinna positions within leaves with the same number of pinna pairs. Data are plotted as the mean 1 SE of replicate leaves. Means plotted without SE bar have standard errors smaller than the symbol. Means marked with an asterisk are not significantly different within a leaf at the 5% level. Fig. 5. Mean order of complexity per pinna position of leaves with 2 7 pinna pairs on aftl plants. A silhouette of a leaf with five pinna pairs is provided to demonstrate the counting scheme used. Lines connecting points reflect pinna positions within leaves with the same number of pinna pairs. Data are plotted as the mean 1 SE of replicate leaves. Means plotted without SE bar have standard errors smaller than the symbol. Means marked with an asterisk are not significantly different within a leaf at the 5% level.

6 1328 AMERICAN JOURNAL OF BOTANY [Vol. 84 TABLE 2. Microscopic measurements for wild-type and aftl peas. All means differed between wild-type and aftl genotypes at the 1% level. Standard errors are in parentheses. Cell type Wild type Pleiofila Epidermal cell surface area ( m 2 ) 3451 (127.75) 2183 (105.33) Palisade cell length ( m) 82 (0.88) 73 (1.00) Mesophyll cell transverse area ( m 2 ) 3027 (30.04) 2855 (16.98) Areole surface area ( m 2 ) 1916 (64.50) 1184 (108.80) Stomatal frequency a 13 (0.008) 23 (0.014) a Stomatal frequency was the number of stomata occurring in an area of 115 mm 2. Figs Leaflet histology. Transverse sections of leaflets from wild-type (6) and aftl (8) plants and cleared leaflets from wild-type (7) and aftl (9) plants. Bar 50 m for transverse sections and 400 m for clearings.

7 October 1997] VILLANI AND DEMASON ROLES OF THE AF AND TL GENES IN PEA 1329 Figs Development stages of late postembryonic leaves (four or five pinna pairs) on wild-type plants. 10. Initiation of position 2 pinna primordia. 11. Initiation of position 3 pinna primordia. 12. Early stages of lamina initiation in position 1 and Early stages of lamina initiation at position Early stages of tendril elongation. Three leaflet primordia have been removed to allow a better view of developing leaf. 15. Later stages of tendril and rachis elongation. Two leaflet primordia were removed to allow a better view of the developing leaves. Bars 100 m.

8 1330 AMERICAN JOURNAL OF BOTANY [Vol , 1971; Meicenheimer et al., 1983; Gould, Cutter, and Young, 1986). During P 2 (second plastochron), pinnae at positions 1 and 2 were initiated in an acropetal sequence (Fig. 10). Subsequent pinna pairs ( i.e., 3, 4, and 5) were also initiated acropetally in order during P 3 and P 4 (Figs. 11, 12). Lamina initiation started early in P 4 and occurred first at position 1 and proceeded acropetally throughout the positions that form leaflets (Fig. 12). During P 5, pinna positions 1 through 3 had undergone considerable lamina formation before positions 4 and 5 were recognizable as tendrils (i.e., radial structures) (Fig. 13). As tendril primordia began to elongate during later stages of P 5, they curved adaxially (Fig. 14) These two processes proceeded acropetally along the leaf axis and occurred last in the terminal tendril. Elongation of the leaf axis was initiated in later stages of P 5 or early stages of P 6 (Fig. 15). Leaf development in aftl plants Node 8 leaf primordia (early postembryonic) on aftl plants were initiated at the flank of the shoot apical meristem and encircled approximately two-thirds of the apex early in the first plastochron (P 1 ) (Fig. 16). Shortly thereafter, the primordium initiated two pairs of lateral structures: two stipule primordia and two lateral pinna primordia (Fig. 17). During P 3, a second pair of lateral pinna primordia (position 2) was initiated distal to that at position 1 and pinna primordia at position 1 initiated a second round of branching (Fig. 18). The secondary laterals produced on position 1 pinnae were unequal in size such that the primordia produced toward the central axis of the leaf were smaller than those produced toward the outside. Lamina initiation occurred first on the terminal segment during P 4 (Fig. 19). Subsequently the terminal segments of the compound pinnae at each position underwent elongation and lamina initiation before the proximal segments on each (Figs. 19, 20). Therefore, these processes started at the most distal tip of the leaf axis and proceeded basipetally within the leaf as a whole, as well as within each lateral position. During P 5 individual leaflet primordia had distinct marginal ridges (Fig. 21). Elongation of the leaf axis started during P 5 (Fig. 22). The most proximal pinna pairs did not separate by rachis elongation to any extent during the development of the leaf compared to more distal positions. There was considerable variation in the number of pinna pairs, which ranged from one to five pairs, per leaf at node 8 (Figs. 21 vs. 22). Node 14 leaf primordia (late postembryonic) on aftl plants were initiated at the flank of the shoot apical meristem and encircled two-thirds of the apex within P 1 (Fig. 23). The leaf primordium continued to grow in size during P 2. Thereafter, primordia initiated two pairs of structures: stipule primordia and lateral pinna primordia (which correspond to position 2) (Fig. 24). In P 3 the tip of the primordium began to curve over the top of the shoot apical meristem (Fig. 25). During the early stages of P 4 a second pair of pinnae was initiated basipetal to the pair at position 2 and a second round of branching occurred on pinnae on position 2 (Figs. 26, 27). Later in P 4, the third and fourth pinna pairs were initiated distal to position 2 in an acropetal sequence (Fig. 27). Therefore, in leaves at this node, the order of pinna initiation was 2, 1, 3, 4, and 5. By the time the fifth pinna pair was initiated during P 5, several rounds of secondary branching had occurred in an acropetal direction on position 2, and position 1 had undergone the first round of secondary branching (Fig. 28). During later stages of P 5, an additional round of secondary branching occurred in an acropetal direction in position 1 and, subsequently, a third order of branching was produced on the proximal pair of laterals at position 2 (Fig. 29). During very late stages of P 5 or early stages of P 6, lamina initiation was evident on the terminal segment of the leaf and pinna positions 2-5 (Fig. 30). Lamina initiation proceeded in a basipetal fashion on the leaf as a whole and on each pinna position as it did on node 8 (data not shown). DISCUSSION In this study we used two different approaches to compare the diversity in pea leaf form between the wild-type (AfAfTlTl) and the afaftltl genotypes. The first approach was to compare the ontogenetic progression of leaf form changes from node to node along the plant axis, or heteroblastic changes between the genotypes. The other approach was to compare various morphological features of leaves with comparable morphological complexity (i.e., same number of pinna pairs) across the genotypes. Leaf morphology Peas exhibit heteroblastic leaf variation in that leaf length and the number of pinna pairs along the leaf axis increase during ontogeny of the plant. Leaves on wild-type plants are longer than those at the same positions on aftl plants. Two exceptions to this trend are at nodes 4-6, which correspond to embryonic leaves, and at nodes 23-26, which correspond to flowering nodes, where they are not significantly different. The same trend was observed with respect to the average number of pinna pairs as a function of leaf position on wild-type and aftl plants. Both genotypes have similar numbers of pinna pairs in their preformed embryonic leaves, however, on postembryonic leaves from nodes 8 to 24 the number of pinna pairs on leaves of aftl plants increase more rapidly than leaves at similar positions on wild-type plants until late in ontogeny when the number of pinna pairs on both genotypes approach similar values. Lu et al. (1996) saw a similar trend for the acacia and afila genotypes, which exhibit leaf complexity rates more similar to that of wildtype plants. Pleiofila differs from the wild type in that the leaves exhibit an additional complexity (seven pinna pairs), which is never observed on wild-type plants. Using L-system analysis Gould, Cutter, and Young (1992) also showed that leaf complexity was greater in aftl plants than in the other genotypes. Alberch et al. (1979) categorized changes in developmental timing during plant ontogeny by determining whether the reproductive or somatic transitions were being altered. He defined acceleration as an increase in the rate of vegetative development while time to flowering remains unchanged. The rate of increase in the number of pinna pairs relative to nodal position is much greater in aftl plants compared to wild-type plants (i.e., on average, node 12 leaves of aftl plants have five pinna pairs compared to three at the same position in wild-type plants). It appears that vegetative development is accelerated in aftl plants, and the time to flowering is also

9 October 1997] VILLANI AND DEMASON ROLES OF THE AF AND TL GENES IN PEA 1331 Figs Developmental stages of node 8 leaves (early postembryonic) on aftl plants. 16. Initiation of leaf primordium. 17. Initiation of stipule and position 1 pinna primordia. 18. Initiation of secondary branching at position 1 and of position 2 primordia distal to position Early lamina initiation on the terminal primordium. 20. Early lamina initiation at position Early lamina initiation at position 1. One pinna of each pair at positions 1 and 2 was removed to allow a better view of the developing leaf. 22. Early rachis and rachillae elongation. One of the pair of pinnae at positions 1 and 2 was removed to allow a better view of the developing leaf. Bars 50 m (Figs ), 100 m (Figs ), and 1 mm (Fig. 22).

10 1332 AMERICAN JOURNAL OF BOTANY [Vol. 84 Figs Developmental stages of node 14 leaves (late postembryonic) on aftl plants. 23. Initiation of leaf primordium. 24. Initiation of stipule and position 2 pinna primordia. 25. Adaxial curvature of leaf primordium. 26. Initiation of position 1 pinna primordia. 27. Initiation of position 3 pinna primordia and secondary branching of primordia at position Position 2 primordium with four secondary branches and initiation of secondary branching at position Initiation of tertiary branching at position 2 and early lamina initiation on terminal pinna primordium at the distal tip of the primary leaf axis. Bars 100 m.

11 October 1997] VILLANI AND DEMASON ROLES OF THE AF AND TL GENES IN PEA 1333 Fig. 30. Lamina initiation at node 14 of aftl plants. One pinna of each pair at positions 1 and 2 was removed to allow a better view of the developing leaf. Bar 100 m. slightly delayed. Therefore, the Af and Tl genes have minor effects on the rate of vegetative development and time to flowering, in addition to the drastic effects on leaf morphology. Wiltshire, Murfet, and Reid (1994) identified nine other mutations in pea that act via heterochronic mechanisms to produce dramatic morphological changes, ranging from precocious flowering (progenesis) to slower rates of vegetative change (neoteny) as well as other examples. Marx (1987) proposed that the wild-type pea leaf has three compartments, corresponding to stipule, leaflet, and tendril (or base, proximal, and distal, respectively), based on a number of mutations that affect only one specific compartment within the leaf. Lu et al. (1996) used morphological features of wild-type, acacia, afila, and pleiofila leaves to assess whether distinct proximal and distal compartments, or regions, exist in these phenotypes. Based on the characteristics of pinna length, mass, and branching in the wild type, afila, and pleiofila, they demonstrated that proximal and distal regions are an inherent feature of pea leaf blades. Lu et al. (1996) observed that leaves with five pinna pairs on afila and aftl plants had greater branching complexity at position 2 compared to position 1 in the proximal region of the blade. We observed that position 2 pinnae have greater pinnae lengths, numbers of secondary branches, and orders of complexity compared to position 1 on late postembryonic leaves. In addition, with L-system analysis, Gould, Cutter, and Young (1992) also observed a higher order of complexity in position 2 (it required additional transition states compared to position 1). For leaves with six and seven pinna pairs on aftl plants, the greater complexity in position 2 pinnae allows us to distinguish the proximal and distal leaf regions and to determine that the regional boundary is located between pinna positions 2 and 3. Lu et al. (1996) characterized relative pinna positions along the leaf axis, which ultimately results from differential elongation, for wild-type, acacia, afila, and aftl plants and showed, that wild-type plants exhibit an almost perfectly equidistant spacing of pinna pairs, whereas acacia and afila pinna pairs are displaced distally relative to wild-type leaves, resulting in longer petioles. Further, the placement of pinna pairs along the leaf blade axis exhibits crowding in either the distal or proximal regions for the acacia and afila phenotypes, respectively. In aftl plants the displacements are additive in that the leaves have the longest petioles and exhibit crowding of pinna pairs in both proximal and distal regions along the leaf blade axis. In this study we show that petiole lengths of aftl plants increase linearly in leaves of increasing complexity, whereas in wild-type plants petiole lengths increase in early postembryonic leaves but stabilize at near equal lengths in late postembryonic leaves irrespective of pinna pair number. Therefore our observations support the Lu et al. (1996) hypothesis that the Af and/or Tl genes appear to be involved in regulating elongation along the leaf axis, which ultimately affects pinna placement, and this regulation is lost in late postembryonic leaves on aftl plants. Although the wild-type pea leaf is typically symmetrical such that leaflets occur opposite leaflets and tendrils opposite tendrils, asymmetries in the form of mixed pairs also occur. Using L-system analysis, Gould, Cutter, and Young (1992) showed that asymmetric leaves also occur in afila and aftl genotypes. Lu et al. (1996) plotted the position of mixed pinna pairs on wild-type, afila, and pleiofila leaves and found that they occur only at the boundary between the distal and proximal regions of the leaf. Here we report that leaves with mixed pairs occur at specific nodal positions on wild-type plants and mark positions on the shoot where leaf complexity increases such that an additional pair of pinnae is added to the proximal compartment of the leaves. We predict that mixed pairs on afila and aftl plants should occur at shoot positions where the second pinna pair is added to the proximal compartment of these genotypes. Histology Gould, Cutter, and Young (1986) saw no discernible histological differences between proximal wild-type and distal acacia leaflets or between wild-type and afila stem, petioles, rachis, or tendrils. Based on these anatomical similarities they concluded that distal leaflets of acacia leaves and the proximal tendrils of afila leaves are true leaflets and tendrils. We have now shown that the leaflets of aftl plants are histologically equivalent to those of wild-type plants. The only differences between them are cell sizes and leaflet areas, which are smaller in aftl plants. Meicenheimer et al. (1983) have previously shown that total leaf areas at comparable nodes on wildtype and aftl plants do not differ. Individual leaflet area for aftl plants is 11.7 times smaller than it is for wildtype plants. Differences in leaflet area can occur either by differences in cell size, total cell number, or a combination of both. We determined that cells sizes are significantly smaller in pleiofila leaves, which contributes to 40% of the difference in leaflet size between the genotypes. The remaining difference (60%) must be attributable to fewer cells. Similarly, Dengler (1984) found that two tomato leaf form mutants, entire and lanceolate, have smaller leaf areas than wild-type plants, which she attributed to both smaller and fewer cells. Leaf development Several groups have characterized the events during development of embryonic, early post-

12 1334 AMERICAN JOURNAL OF BOTANY [Vol. 84 embryonic (Meicenheimer et al., 1983; Gould, Cutter, and Young, 1986), and late postembryonic (Cote et al., 1992) leaves on wild-type pea plants. Meicenheimer et al. (1983) and Gould, Cutter, and Young (1992) observed slight differences in timing of developmental events, possibly due to background differences. All authors agree that pinnae in position 1 are initiated during late P 1 or P 2 and subsequent pinna pairs are initiated acropetally. Lamina initiation occurs acropetally starting at position 1 during P 3 (Gould, Cutter, and Young, 1986) or P 4 (Meicenheimer et al., 1983). We found that in both early postembryonic and late postembryonic wild-type leaves, pinna pair and lamina initiation are strictly acropetal and occur during P 2 and P 4, respectively. Tendril initiation starts in late P 4 and is also acropetal. These observations confirm those of Cote et al. (1992). Previous observations confirm that the timing of events in the proximal region of early postembryonic and late postembryonic leaves is nearly identical, except for lamina initiation. Gould, Cutter, and Young (1986) observed lamina initiation during P 3, whereas Meicenheimer et al. (1983), Cote et al. (1992), and we observed that in late postembryonic leaves it occurs, instead, during P 4. The additional pinna pairs produced by late postembryonic leaves are produced later and, therefore, late postembryonic leaves initiate pinna primordia over a longer time span. Meicenheimer et al. (1983) were the first to describe leaf development of aftl plants. They observed that the first pinna pair is initiated earlier than on wild-type leaves, that the pleiofila phenotype arises from profuse branching of pinna primordia similar to that of the afila phenotype, and that the terminal primordium of the leaf axis, as well as all branch termini, initiate adaxial and marginal meristems during the sixth plastochron to produce a lamina. These authors stated that they observed leaves produced soon after node 7, which are early postembryonic leaves as we have defined them in this study. The focus of our reinvestigation of early postembryonic leaf development was to more accurately establish the timing of the sequence of events that leads to the formation of these leaves in this genotype. We picked a specific nodal position (node 8) to more accurately evaluate the timing of events. The first pair of pinnae is produced during P 2 and subsequent pinna pairs are initiated in an acropetal sequence. The process of lamina initiation occurs during P 4, starting with the terminal primordium, and proceeds in a basipetal direction throughout the leaf as a whole and within each pinna position. Lu et al. (1996) proposed a hypothetical model to explain the developmental events leading to the acacia, afila, and pleiofila phenotypes. They hypothesized that pleiofila results from successive orders of branching before pinna termini undergo leaflet formation. The above characterization of early postembryonic leaf development in aftl plants confirms the sequence of events that produce the pleiofila phenotype outlined in their model. Early stages in development of late postembryonic leaves on aftl plants show no discernible differences from the events described above in early postembryonic leaves of this genotype. However, during P 2 two significant divergences in development between these leaves occur. Early postembryonic leaves initiate the second pair of pinnae (position 2) distal to those at position 1 and secondary branching on pinnae in position 1 at this stage. In contrast, late postembryonic leaf primordia do not initiate the second pair of pinnae (position 1) until P 4 and it is proximal to the first pair initiated (position 2). Since subsequent pinna pairs are initiated distal to the first initiated position, initiation of pinna pairs in late postembryonic leaves is bidirectional, whereas it is strictly acropetal in early postembryonic leaves. During P 4, early postembryonic leaves undergo elongation and lamina initiation, whereas during this time period late postembryonic leaves initiate position 3 and 4 pinna pairs. During P 5, early postembryonic leaf primordia continue lamina initiation and late postembryonic leaves are initiating position 5 pinnae and secondary and tertiary branching on pinnae in positions 1 and 2, respectively. Elongation and lamina initiation do not occur until approximately two plastochrons later in late postembryonic leaves. The process of lamina initiation in early postembryonic and late postembryonic leaves of aftl plants, although it occurs at different times, is similar. Lamina initiation is initiated first in the most distal position on the leaf or pinna axis and proceeds basipetally throughout the leaf as a whole and within a pinna position. Early postembryonic leaves exhibit precocious development in comparison to late postembryonic leaves of this genotype as they do in wildtype plants. Leaf development of late postembryonic leaves on aftl plants is quite different from previous descriptions, which have been based on early postembryonic leaves (Meicenheimer et al., 1983). The major differences are bidirectional initiation of pinna pairs and timing of branching and maturation events. Lu et al. (1996) suggested the possibility of bidirectional pinna initiation based on the greater morphological complexity of position 2 pinnae on leaves with five pinna pairs. Similar morphological patterns occur on afila plants. It is likely that late postembryonic leaves of this latter genotype may also have bidirectional initiation of pinna pairs, but previous descriptions of leaf development have been based on early postembryonic leaves. If bidirectional pinnae initiation occurs on leaves of afila plants, then we would hypothesize that acropetal initiation is established by Af function. The sequence of leaf development of early postembryonic leaves of wild-type and aftl plants diverges by P 1. Pinnae in position 1 of pleiofila leaves are initiated earlier than those at the same position in wild-type plants, although pinnae at position 2 are initiated during P 4 in both phenotypes. In addition, the secondary branching of pinnae in position 1 of aftl occurs during P 3, which is not observed in wild-type plants. Lamina initiation in both genotypes occurs at approximately comparable times but in different positions and opposite directions: acropetally in wild-type and basipetally in aftl plants. Leaf development in late postembryonic leaves of wild-type and aftl plants are even more dissimilar in the timing and sequence, especially in the proximal region of the blade. In the late postembryonic leaves of aftl plants, the first pinnae are initiated at position 2 as opposed to position 1 on wild-type plants. The next pinna pair is initiated at a distal position one plastochron later in wild-type leaves and proximally two plastochrons later in pleiofila leaves. Subsequent positions (i.e., 3, 4, and 5) are initiated at similar time points in both genotypes.