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American Society of Plant Biologists The early phase change Gene in MaizePlant Science Institute, Biology Department, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6018 3 To whom correspondences should be addressed. E-mail spoethig{at}sas.upenn.edu; fax 215-898-8780
Recessive mutations of the early phase change (epc) gene in maize affect several aspects of plant development. These mutations were identified initially because of their striking effect on vegetative phase change. In certain genetic backgrounds, epc mutations reduce the duration of the juvenile vegetative phase of development and cause early flowering, but they have little or no effect on the number of adult leaves. Except for a transient delay in leaf production during germination, mutant plants initiate leaves at a normal rate both during and after embryogenesis. Thus, the early flowering phenotype of epc mutations is explained completely by their effect on the expression of the juvenile phase. The observation that epc mutations block the rejuvenation of leaf primordia in excised shoot apices supports the conclusion that epc is required for the expression of juvenile traits. This phenotype suggests that epc functions normally to promote the expression of the juvenile phase of shoot development and to suppress the expression of the adult phase and that floral induction is initiated by the transition to the adult phase. epc mutations are epistatic to the gibberellin-deficient mutation dwarf1 and interact additively with the dominant gain-of-function mutations Teopod1, Teopod2, and Teopod3. Genetic backgrounds that enhance the mutant phenotype of epc demonstrate that, in addition to its role in phase change, epc is required for the maintenance of the shoot apical meristem, leaf initiation, and root initiation.
Growth of the plant shoot can be divided into several discrete phases based on the character of the organs produced during these phases and the capacity of the shoot for reproductive development (Hackett, 1985  ; Zimmerman et al., 1985; Poethig, 1990). Genetic analyses of phase change in herbaceous plants have focused primarily on floral induction—that is, on the transition from an adult vegetative phase of shoot development to a reproductive phase in which the shoot produces flowers and flowering branches (Simpson et al., 1999). The regulation of earlier developmental transitions is poorly understood. We are particularly interested in the regulation of the transition from the juvenile to the adult phase of vegetative growth. Traditionally, this transition has been defined as a change in the reproductive competence of the shoot, but it also is marked by species-specific changes in a variety of vegetative traits, including leaf shape, leaf anatomy, root production, disease resistance, and the synthesis of a number of secondary compounds (Hackett, 1985; Kerstetter and Poethig, 1998). Mutations affecting this vegetative transition have been identified in several species, including pea (Wiltshire et al., 1994), Arabidopsis (Telfer and Poethig, 1998; Clarke et al., 1999; Hamada et al., 2000; Berardini et al., 2001; Prigge and Wagner, 2001), and maize (Freeling et al., 1992; Lawson and Poethig, 1995), and are beginning to provide some insight into the mechanism of this important but neglected aspect of plant development.
In maize, the juvenile and adult phases of vegetative development are distinguished primarily by features of the epidermis of the leaf blade, the most obvious of which are the presence of epicuticular wax (a juvenile trait) and epidermal hairs (an adult trait) (Freeling and Lane, 1994
Two other dominant mutations, Hairy sheath frayed-O (Bertrand-Garcia and Freeling, 1991 Although genetic analyses of vegetative phase change in maize have revealed some of the major features of this process, one of the major limitations of these studies is that they have largely used dominant gain-of-function mutations. Dosage studies have provided some information about the nature of these mutations, but their effect on gene function still is unknown. As noted above, gene function can be inferred accurately only from loss-of-function mutations, and only a few such mutations have been identified in maize. Here, we describe a new gene, early phase change (epc), that is required for the expression of the juvenile phase and that plays a critical role in several other aspects of shoot and root development.
Most of the work presented here was conducted with two alleles of epc, one that arose spontaneously in a W23 genetic background (epc-W23) and an allele (epc-1s2p) present in the inbred background 1s2p. Several other alleles have been identified in our laboratory and by others (see Methods), but these were not characterized in detail in this study. The phenotype of all of these alleles is influenced strongly by genetic background and undefined environmental and/or developmental conditions. In a W23 background, epc-W23 and epc-1s2p primarily affect the number of juvenile and transition leaves, although sometimes this phenotype is poorly penetrant and variably expressed in this inbred background. This is illustrated for epc-W23 (W23) in Figure 1A and Table 1, rows 3 to 9, where each row represents the progeny of a single self-pollinated plant from the row above it. Even in this highly inbred background, some families have only a few affected progeny with an extremely mild phenotype, whereas others have a large number of affected plants with a wide range of phenotypes. This phenomenon also is characteristic of epc-1s2p in a W23 background (Table 2) as well as epc-W23/epc-1s2p (W23/1s2p) hybrids (Figure 1B, Table 1, rows 11 and 12). In other genetic backgrounds, the phenotype of epc-W23, epc-1s2p, and other epc alleles is much more severe and is fully penetrant (Figure 2B , Table 2). In the Oh43 and A632 backgrounds, for example, mutant shoots either fail to emerge above soil level or produce only one or two rudimentary leaves. epc mutations have no obvious effect on endosperm morphology.
epc was mapped by testing linkage to simple sequence repeat (SSR) markers using F2 families from self-pollinated epc-W23/+ (W23/Oh43) plants. Only mutant progeny were genotyped to avoid misscoring as a result of the poor penetrance of this mutation. Data from 133 mutant progeny place epc on the long arm of chromosome 8 in the following position: epc—0.4 centimorgan (cM)—bnlg 2082, bnlg 1067—10.9 cM—bnlg 162—1.5 cM—bnlg 666.
Effect of epc on Vegetative Identity
To determine if epc regulates mesophyll identity, we examined its effect on the expression of Ragged leaves (Rg), a dominant mutation that causes adult leaves to develop necrotic lesions as a result of cytolysis of mesophyll cells (Mericle, 1950 ; Evans et al., 1994; Bongard-Pierce et al., 1996), and on Rolled leaf (Rld), a dominant mutation that affects leaf polarity and causes leaf curling (Hay et al., 2000). We found that leaf necrosis occurred two leaves earlier in epc Rg/+ double mutants than in Rg/+ plants and that this effect was correlated with the first leaf expressing adult-specific epidermal traits (i.e., the first transition leaf). Rg/+ plants produced their first transition leaf at node 5.7 ± 0.2 and their first ragged leaf at node 5.8 ± 0.2, whereas epc Rg/+ double mutants produced their first transition leaf at node 3.2 ± 0.1 and their first ragged leaf at node 3.5 ± 0.3. A similar result was obtained with Rld. Rld/+ plants produced their first transition leaf and their first rolled leaf at node 5.6 ± 0.2, whereas epc Rld/+ plants produced their first transition leaf and their first rolled leaf at nodes 3.3 ± 0.2 and 3.9 ± 0.1, respectively. This result is consistent with the effects of epc on leaf morphology and leaf anatomy and suggests that epc affects all aspects of phase-specific leaf identity. To quantify the effect of epc mutations on the duration of juvenile and adult development, we examined their effects on the two most obvious phase-specific traits, epicuticular wax (a juvenile trait) and epidermal hairs (an adult trait) (Table 3). The data demonstrate that epc reduces the number of juvenile leaves but has only a minor effect on adult leaf number. In a W23-Rnj background, epc-W23 had approximately one juvenile leaf, one transition leaf, and 10 adult leaves, compared with wild-type plants, which usually had five juvenile leaves, two transition leaves, and nine adult leaves. epc-W23/epc-1s2p (W23/1s2p) plants had one or two juvenile leaves, one or two transition leaves, and 10 adult leaves, whereas +/epc-1s2p (W23/1s2p) plants produced five juvenile leaves, three transition leaves, and 10 or 11 adult leaves. The reduced leaf number of mutant plants therefore is accounted for entirely by a reduction in the number of juvenile and transition leaves.
Effect of epc on Shoot Growth epc mutants often germinated slightly later than wild-type seedlings and usually had fewer expanded leaves than wild-type plants throughout shoot growth. The severity of this effect usually was correlated with the severity of the phase change phenotype of mutant seedlings, raising the possibility that the primary function of epc may be to regulate leaf production and shoot growth rather than the developmental identity of the shoot. If epc mutations delayed leaf production without affecting the timing of vegetative maturation, mutant plants would produce fewer juvenile leaves. To test this hypothesis, we studied the effect of epc mutations on the rate of leaf initiation before and after germination. epc had no effect on the rate of leaf initiation early in embryogenesis (Figure 5) , but it had a significant effect on leaf and meristem growth late in embryogenesis. By 30 days after pollination, leaves 3, 4, and 5 were significantly smaller in mutant embryos than in wild-type embryos (Table 4, Figure 6) , and mutant shoot apical meristems (SAMs) were morphologically aberrant (Figure 6A). The dimensions of the SAM in 30-day-old mutant and wild-type embryos revealed that the epc SAM is significantly (P < 0.01 for each dimension) broader at the base, narrower at the tip, and taller than the wild-type SAM (Figure 6A).
Mutant seedlings exhibited a transient delay in leaf initiation during germination, but they initiated leaves at the same rate as wild-type plants throughout the remainder of shoot growth and produced a tassel 3 days earlier than normal (Figure 5). This effect on tassel initiation was reflected in the timing of anthesis, which also occurred 3 days earlier in mutant than in wild-type plants (49.7 versus 53.3 days after planting). The reduced leaf number of mutant plants therefore is attributable to both an early pause in leaf production and early flowering. In the families used for this study, the majority of mutant plants produced their first transition leaf at node 2 or 3 and their first completely adult leaf at node 3 or 4 (Table 1, row 11, and Table 3). Most wild-type plants produced their first transition leaf at node 6 and their first completely adult leaf at node 8 (Table 1, row 10, and Table 3). Thus, mutant plants produce their first adult leaves during embryogenesis, whereas wild-type plants do not make this transition until after germination. We conclude that the effect of epc mutations on leaf identity does not result from a delay in the timing of leaf initiation because mutant plants actually initiate adult leaves earlier than wild-type plants.
epc Prevents Rejuvenation in Vitro
Effect of epc on Shoot Viability and Root Growth As noted above, the phenotypes of epc-W23 and epc-1s2p are particularly strong in the Oh43 and A632 inbred backgrounds. In both of these backgrounds, the shoots of most mutant plants either fail to emerge above soil level or produce only one or two rudimentary expanded leaves (Figure 2B). To determine the basis of this phenotype, five mutant and five wild-type F2 seedlings from a self-pollinated epc-W23/Oh43 plant were harvested 5 days after planting and analyzed histologically. At this stage, leaf 1 had just emerged from the coleoptile. Mutant seedlings could be distinguished from wild-type seedlings by their unfused coleoptiles and short mesocotyls; confirmation that these abnormal seedlings were homozygous for epc-W23 was obtained by genotyping these seedlings for the SSR marker bnlg 2082, located 0.4 cM from epc. The shoot apices of mutant seedlings had six leaf primordia, the youngest of which (leaves 4, 5, and 6) were significantly smaller than the corresponding wild-type leaves (Table 4). There was no obvious sign of cell death in leaf 1, but other leaves in the shoot were either partially or completely necrotic (Figure 8) . Mutant seedlings also had an unusually large SAM compared with wild-type SAMs and had a lower rate of cell division. Wild-type SAMs had 56 ± 6 cells in a median longitudinal section, whereas mutant plants had 139 ± 18. Wild-type plants had a mitotic index of 0.015 ± 0.003 in the SAM, whereas the mitotic index in epc was 0.004 ± 0.003. The cells in mutant SAMs also had an unusually large amount of starch or, in the case of the most severely affected SAMs, were highly vacuolated. This phenotype is consistent with the morphology of mutant SAMs in viable seedlings and suggests that shoot abortion in mutant seedlings occurs either late in seed development or during germination—that is, at approximately the same time as the transient delay in leaf initiation observed in less severely affected plants.
epc mutations also affect root development. As shown in Figure 2A, epc-W23 plants produce few lateral roots and produce no roots at the coleoptile node by 12 days after planting. In contrast, wild-type plants produce an extensively branched root system and usually have three or four roots at the coleoptile node by this stage. Adventitious roots usually develop some time later at the coleoptile node and other basal nodes of mutant plants, but the number and extent of these roots usually is reduced significantly compared with wild-type plants. The most severely affected plants have such a rudimentary root system that they have dif-ficulty remaining upright. The effect of epc mutations on lateral root development is more severe in genetic backgrounds in which these mutations cause shoot abortion (Figure 2B). Although this phenotype may be an indirect result of the effect of these mutations on the growth of the shoot, it is true that adventitious root production is a juvenile trait in maize (Poethig, 1988 ), as it is in many other species (Hackett, 1985). Therefore, the reduction in root production in mutant plants may either result from the effect of this mutation on phase change or reflect a more direct role for this gene in root initiation.
Genetic Interactions
epc and d1 In maize, GA promotes vegetative and reproductive maturation (Evans and Poethig, 1995 ). In addition to reducing stem and leaf expansion, this mutation has been shown to prolong the expression of juvenile traits and to increase total leaf number (Evans and Poethig, 1995). To test the hypothesis that epc promotes adult development by increasing the endogenous level of GA or the sensitivity of cells to this hormone, we examined the interaction between epc-1s2p and the GA-deficient d1 mutation. The phenotypes of single and double mutant plants were compared in F2 families segregating both mutations to minimize the effects of genetic background. Wild-type, d1, epc, and d1 epc-1s2p double mutant plants could be identified readily in these families by their unique phenotypes. Double mutant plants were identical to epc single mutants in terms of leaf identity and leaf number, but they had the dwarf phenotype of d1 (Figures 9B and 10A)
. Thus, the epc mutation is completely epistatic to d1 with respect to phase-specific traits but does not correct the leaf and stem expansion phenotype of d1. We conclude that the wild-type allele of epc is required for the effect of the d1 mutation on both vegetative and reproductive phase change and that epc acts downstream of GA.
epc and the Teopod Mutations Tp1, Tp2, and Tp3 are dominant gain-of-function mutations that prolong the expression of juvenile vegetative traits (Poethig, 1988 ). In contrast to the epistasis displayed by epc in combination with d1 and id, epc mutations interacted with all three of these dominant mutations in an additive fashion. In families segregating epc and any one of the Tp1, Tp2, or Tp3 mutations, plants with a wild-type and a Tp mutant phenotype occurred at the expected ratio of 1:1 (data not shown). However, approximately half of these Tp plants produced adult traits at the same, or earlier, leaf position than wild-type plants and had the same, or fewer, total leaves than wild-type plants. Because Tp1, Tp2, and Tp3 always produce more juvenile leaves and more total leaves than wild-type plants (Poethig, 1988), these plants were assumed to be epc Tp double mutants. In all three cases, the number of juvenile and transition leaves in putative epc Tp double mutants was, on average, intermediate between epc and Tp/+ plants (Figures 9C to 9E). Furthermore, although the overall morphology of these putative double mutants was "Tp-like," this phenotype clearly was less severe than that of Tp single mutants (Figures 10B and 10C). In particular, putative double mutants had broader leaves, fewer tillers, and, in the case of epc Tp2, a more highly branched tassel than single mutant plants. Thus, epc mutations partially suppress the dominant gain-of-function phenotype of these mutations, suggesting that wild-type epc function normally is required for this phenotype.
Recessive mutant alleles of epc affect many aspects of the vegetative phase identity of the shoot and, in some genetic backgrounds, also affect root production, seedling morphology, and the viability of the shoot apex. We were particularly interested in the effect of these mutations on phase change because they have a more dramatic effect on this process than any mutations described previously in maize. Recessive mutations of gl15 are phenotypically similar to epc mutations in accelerating the expression of adult traits, but gl15 mutations affect only a subset of phase-specific epidermal traits and have no obvious effect on the development of the first two juvenile leaves or on flowering time (Evans et al., 1994 ; Moose and Sisco, 1994). The dominant gain-of-function mutations Tp1, Tp2, Tp3, and Cg have a phenotype opposite to that of epc mutations in that they prolong the expression of many juvenile traits. However, these mutations do not significantly affect the onset of the adult vegetative phase (Poethig, 1988) and, at least in the case of Tp1 and Tp2, do not affect the duration of shoot growth or reproductive competence (Bassiri et al., 1992). vp8 (Evans and Poethig, 1997) and GA-deficient mutations (Evans and Poethig, 1995) delay both vegetative and reproductive phase change, but they have relatively subtle effects on these transitions compared with epc mutations. Therefore, the dramatic effect of epc mutations on vegetative and reproductive phase change is unusual, and suggests that this gene plays a crucial role in the regulation of shoot maturation.
Role of epc in Phase Change
The relationship between vegetative maturation and reproductive maturation is a major unsolved problem in plant biology. Although the vegetative morphology of a shoot generally is correlated with its reproductive competence, it is unknown if these processes are regulated independently or by a single developmental program. This question has been addressed by investigators studying woody plants (Greenwood and Hutchinson, 1993
Other genes involved in phase change fall into three classes with respect to their effect on vegetative and reproductive development. Genes that play a role in both vegetative phase change and floral induction are defined by mutations/transgenes that simultaneously affect the duration of both the juvenile and adult phases of vegetative development. We are not aware of any mutations with this phenotype in maize, but several such mutations and transgenes have been described in Arabidopsis (Martinez-Zapater et al., 1995
The observation that epc mutations affect the duration of the juvenile phase but not the adult phase is significant for several reasons. First, this phenotype demonstrates that the vegetative development of the shoot is divided into at least two discrete developmental phases. Although this has been accepted widely by investigators studying woody plants, investigators studying herbaceous plants tend to think of vegetative shoot maturation as a gradual, quantitative process (Schultz and Haughn, 1993
Genetic Interactions The functional relationship between epc and the tp genes is less clear, largely because the tp genes are defined by gain-of-function mutations. Thus, although the additive interaction between epc and the Tp mutations could mean that epc and the tp genes operate in parallel pathways, it is conceivable that these genes act in the same pathway. For example, if epc is an upstream activator of tp2, epc-W23 could partially correct the Tp2 mutant phenotype by reducing the expression level of this neomorphic mutation.
Function of epc This severe phenotype and the pleiotropic effects of epc mutations on shoot and root development in backgrounds in which these mutations are partially suppressed indicate that epc is involved in a number of different processes in maize. In addition to regulating vegetative phase change and leaf initiation, epc plays a role in leaf expansion (mutant leaves are slightly uprolled) and root development. This does not, of course, make its role in phase change any less interesting. Most major regulatory factors have diverse roles in growth and development; obvious examples include all major phytohormones and photoreceptors. Defining the functions of such molecules by mutational analysis can be difficult because loss-of-function mutations often have pleiotropic phenotypes that obscure the role of these factors in specific developmental or physiological processes. In such cases, weak alleles or conditions that suppress some aspects of a mutant phenotype provide critical information about gene function. Thus, the phenotype of epc mutations in W23 reveals a role for this gene in the regulation of phase change that is not obvious from its more severe phenotype in other genetic backgrounds. The genes responsible for this difference in expressivity clearly are of interest and could be identified by analyzing crosses between mutant lines that display suppressed and more severe phenotypes.
Penetrance and Expressivity of epc The paucity of mutations in maize that have the precocious adult phenotype of epc mutations is surprising because mutations with this phenotype block epicuticular wax production. Many mutations that reduce or eliminate epicuticular wax have been identified in maize because the mutants have an obvious "glossy" appearance. However, we identified only one phase change gene, gl15, in the existing collection of glossy mutations at the Maize Stock Center (Urbana, IL). It is possible that genes involved in promoting juvenile development have an aborted shoot phenotype like that of epc, in which case they would not exist in collections of glossy mutations. The other obvious possibility is that many genes that regulate juvenile development in maize are functionally redundant and therefore have no obvious loss-of-function phenotype. This conclusion is supported by the observation that an ethyl methanesulfonate–induced revertant of the dominant Tp1 mutation and a Mutator-induced revertant of the dominant Tp2 mutation both have homozygous wild-type phenotypes (R.S. Poethig, unpublished results). At present, epc is the only gene known to be essential for the expression of the juvenile phase in maize. Therefore, it is an important entry point into the mechanism of phase change in this species.
Genetic Stocks and Alleles epc-W23 was identified in an inbred maize (Zea mays) W23 line obtained from E.H. Coe, Jr. (University of Missouri, Columbia). The epc-1s2p allele was identified in a 1s2p stock obtained from M. Freeling (University of California, Berkeley); the 1s2p inbred line was found subsequently to be homozygous for this mutation. For the sake of clarity, the 1s2p line is referred to as epc-1s2p (1s2p) in this article. Two other mutant alleles of epc have been identified in our laboratory. epc-mu was identified in a targeted screen for Mutator-induced alleles of epc; it was the only allele identified in a screen of  100,000 F1 plants produced by crossing epc-W23 to lines containing Robertson's Mu. epc-Mo was preexisting in a Mu stock obtained from Guri Johal (University of Missouri, Columbia). epc-mu and epc-Mo mutations are unlikely to represent reisolates of epc-W23 and epc-1s2p because the stocks in which they arose possess unique alleles of one or more SSR markers (bnlg 2082, bnlg 1067, bnlg 667) located 0.4 centimorgan (cM) from epc. The aborted shoot 3 gene, which was identified by D. Laudencia-Chingcuanco (Plant Gene Expression Center, Albany, CA), and the narrow leaf 4 gene, which was identified by R.F. Baker and M. Freeling (University of California, Berkeley), also have been shown to be allelic to epc. The epc-W23 (W23) stocks used in this study were generated by crossing epc-W23 six times to an ACR-nj W23 stock (W23-Rnj) obtained from J. Beckett (University of Missouri, Columbia) that does not contain this mutation. epc-W23 (OH43) was generated by crossing epc-W23 six times to Oh43. F1 progeny produced by crossing epc-1s2p (1s2p) to the original epc-W23 (W23) stock are vigorous and display a strong phase change phenotype; they were used for many of the experiments described in this article. The wild-type controls for these mutants were the progeny of a cross between epc-1s2p (1s2p) and W23-Rnj. Families segregating epc-W23 and the dominant Ragged leaves1 (Rg1) were constructed by crossing epc-W23 as males onto Rg1/+ and then crossing F1 Rg1 plants as females by epc. The first leaves with any small chlorotic regions on the blade were scored as the first leaves to express Rg1. Families segregating epc-1s2p and Rld were constructed in a similar fashion. Families segregating indeterminate growth (id) and epc were generated by crossing +/id females by epc-1s2p and self-crossing the F1. Mutant id plants were identified as those that had not flowered by the end of the summer field season. Families segregating epc and Tp1, Tp2, or Tp3 were generated by crossing Tp/+ (W23-Rnj) females by epc-W23 (W23) and then backcrossing F1 Tp plants by epc-W23 (W23). Families segregating d1 and epc were generated by crossing epc-1s2p females by d1 and self-pollinating the F1.
In all of these crosses, double mutants were identified as plants that expressed a combination of traits not found in either single mutant. Double mutants between epc and mutations that prolong the expression of juvenile traits (d1, Tp1, Tp2, Tp3) were identified as plants that had the distinctive morphology of these mutants and produced glossy leaves with epidermal hairs (adult traits) earlier than the wild-type plants segregating in the same family. We assumed that these plants were homozygous for epc because d1, Tp1, Tp2, and Tp3 single mutants always produce these adult traits later than wild-type plants (Poethig, 1988
Phenotypic Analysis Leaf dimensions were measured on plants growing in the greenhouse in 6-inch pots in Metromix 200 (Scotts, Hope, AR). Leaf width was measured at the widest part of the leaf blade, and leaf length was measured from the tip of the leaf blade to the ligule. To study the basis for the effect of epc mutations on shoot viability, seed from a self-pollinated plant heterozygous for epc-W23 (OH43) were planted on filter paper in a growth chamber (25°C, 16-hr-light/8-hr-dark) and harvested 5 days later. The shoots of germinated seedlings were fixed and processed for histological examination as described below. To distinguish mutant from wild-type seedlings, each specimen was genotyped for the SSR marker bngl 2082 before embedding using root tissue harvested at the time of fixation. The size of the shoot apical meristem (SAM) was determined by counting the number of cells in the region of the shoot apex above the last leaf primordium in a median longitudinal section of five mutant and five wild-type specimens. The mitotic index was calculated as the ratio of the number of mitotic figures (metaphase through anaphase) to the total number of cells in the SAM in this longitudinal section.
Leaf Initiation The rate of leaf initiation after germination was determined using plants from these same crosses. Plants were grown in a growth chamber (16-hr-light/8-hr-dark, 25°C), six to 10 plants of each genotype were dissected every few days with the aid of a stereomicroscope, and the total number of leaves and visible leaf primordia was counted. Some of these plants were transferred to the greenhouse after 3 weeks to measure the effect of epc on flowering time.
Shoot Tip Culture
Histological Analysis The effect of epc-W23/epc-1s2p on the anatomy of the shoot apex was studied using shoot apices fixed in formalin–acetic acid–ethanol or CRAFIII and embedded in paraffin. Sections (10 µm) were deparaf-finized, stained with periodic acid–Schiff's reagent–Weigert's hematoxylin, mounted in Permount (Fisher Scientific), and photographed with Kodak Ektachrome 160 film or with a digital camera. Images were adjusted and composed subsequently using Photoshop 5.5 (Adobe Systems, Mountain View, CA).
SSR Analysis
We are grateful to Doris Wagner and Christine Hunter for helpful comments on the manuscript and to Jennifer Schumacher for technical assistance. This research was supported by a National Institutes of Health training grant to S.H.V. and by grants from the National Science Foundation and the National Institutes of Health to R.S.P.
Article, publication date, and citation information can be found at www.plantcell.org/cgi/doi/10.1105/tpc.010406.
1 Current address: Sungene GmbH, Corrensstrasse 3, 06466 Gatersleben, Germany.
2 Current address: Biology Department, Villanova University, Villanova, PA 19085. Received July 17, 2001; accepted October 11, 2001.
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INTRODUCTION
