A Mutational Analysis of Leaf Morphogenesis in Arabidopsis thaliana

Corresponding author: José Luis Micol, División de Genética, Universidad Miguel Hernández, Campus de San Juan, 03550 Alicante, Spain., jlmicol{at}umh.es (E-mail)

Communicating editor: J. CHORY

	ABSTRACT

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

As a contribution to a better understanding of the developmental processes that are specific to plants, we have begun a genetic analysis of leaf ontogeny in the model system Arabidopsis thaliana by performing a large-scale screening for mutants with abnormal leaves. After screening 46,159 M₂ individuals, arising from 5770 M₁ parental seeds exposed to EMS, we isolated 1926 M₂ putative leaf mutants, 853 of which yielded viable M₃ inbred progeny. Mutant phenotypes were transmitted with complete penetrance and small variations in expressivity in 255 lines. Most of them were inherited as recessive monogenic traits, belonging to 94 complementation groups, which suggests that we did not reach saturation of the genome. We discuss the nature of the processes presumably perturbed in the phenotypic classes defined among our mutants.

THE development of dicotyledonous plant leaves has received little attention in the way of causal analysis (reviewed in TELFER and POETHIG 1994 ; TSUKAYA 1995 ; HALL and LANGDALE 1996 ; POETHIG 1997 ; BRUTNELL and LANGDALE 1998 ; VAN LIJSEBETTENS and CLARKE 1998 ). In fact, the identification of genes that act as developmental controls in leaf ontogeny is problematic for two reasons. On the one hand, the inexistence of analogous phenomena in animals forces us to discard strategies based on gene cloning by homology. On the other hand, two important processes that take place in the leaf, photosynthesis and the exchange of gases with the environment, both require the participation of a large number of gene products, many of them presumably absent or poorly represented in other plant organs. Hence, a search for genes involved in the control of leaf morphogenesis by attempting to isolate gene products spatially restricted to this organ would identify those related to the execution of leaf functions rather than to the developmental mechanisms responsible for leaf architecture. As a result of the above, the spectrum of possible experimental approaches that can be used to identify the genes that control leaf morphogenesis is almost inevitably restricted to the identification and study of mutants.

Saturation mutagenesis is the term given to experiments intended to isolate mutant alleles in every gene affecting a particular developmental phenomenon (WILKINS 1993 ). Such a genetic approach to the study of ontogeny was pioneered by WADDINGTON 1940 to analyze wing morphogenesis in Drosophila melanogaster and later allowed the understanding of the process of segmentation in this insect (NUSSLEIN-VOLHARD and WIESCHAUS 1980 ). The method continues to show its usefulness, since it has been successfully applied to animals and plants, in analyzing embryonic development of both the fish Danio rerio (HAFFTER et al. 1996 ) and the crucifer Arabidopsis thaliana (JURGENS et al. 1991 ).

We are interested in the study of leaf ontogeny as a model for answering general questions on pattern formation. To this end, we have isolated and analyzed A. thaliana variants showing abnormal venation pattern, marginal configuration, shape, or size of their vegetative leaves. Such alternatives in leaf architecture have been identified among wild-type strains, usually termed ecotypes (CANDELA et al. 1999 ; J. SERRANO-CARTAGENA, J. M. PÉREZ-PÉREZ and J. L. MICOL, unpublished results), mutants from existing collections (SERRANO-CARTAGENA et al. 1999 ; J. SERRANO-CARTAGENA, H. CANDELA, P. ROBLES and J. L. MICOL, unpublished results), and new mutants (this work; P. ROBLES and J. L. MICOL, unpublished results). As a contribution to the causal analysis of plant leaf development, we first performed a large-scale screen for EMS-induced mutants with aberrantly shaped or sized leaves. Our intention was to isolate as many mutants as possible showing abnormal leaves (1) to define the spectrum of possible perturbations in leaf morphogenesis, (2) to determine the number of genes whose mutations disturb the process, and (3) to choose for further studies those mutants whose phenotype and/or genetic interactions with others would suggest they are involved in the control of leaf development.

	MATERIALS AND METHODS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Plant materials and growth:
The M₂ A. thaliana (L.) Heyhn. seeds used for mutant screens were derived from mutagenesis of the ecotype Landsberg erecta with ethyl methanesulfonate (EMS; to the 0.2% v/v during 12 hr at 23°), carried out by a commercial supplier, Lehle Seeds (catalog number M2E-4-2). Wild-type seeds were also provided by Lehle Seeds. Both sterile (in Petri dishes) and nonsterile (in pots containing a 1:1:1 mixture of perlite, vermiculite, and sphagnum moss) cultures were performed at 20 ± 1° under continuous illumination of 7000 lux as described in PONCE et al. 1998 .

Conditions of the mutant screening:
Seeds were sown by Pasteur pipette in a water suspension, in 150-mm Petri dishes containing 100 ml of solid culture medium, at a density of 100 regularly spaced seeds per plate. Growth was at 20 ± 1° and 60–70% relative humidity in Conviron (Winnipeg, Canada) TC16 tissue culture chambers under constant fluorescent light (7000 lux). These growth conditions allowed us to observe, 15 days after sowing, the architecture of the rosette and of its leaves as well as to identify putative mutants on the basis of their morphological differences from the wild-type individuals. The selection of mutants was carried out after observation of the M₂ plants, eliminating, 20 days after sowing, all those plantlets showing no clear differences from the ecotype Ler. All putative mutants were transplanted to soil between 3 and 5 wk after sowing.

	RESULTS

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

Isolation of putative mutants:
M₂ seeds were sown and mutants sought that showed macroscopic alterations in the architecture of vegetative leaves and/or in their arrangement on the stem. Putative mutants were assigned to phenotypic classes, the number of new classes being determined after each screening experiment (Table 1). The two right-hand columns of Table 1 show that the number of phenotypic classes from each of the sowings varied from 30 to 54 and that the number of new phenotypic classes dropped from 30 in the first screening experiment to 0 in the eighth, suggesting that we were near saturation.

We sowed a total of 46,159 M₂ seeds, which originated from 5770 M₁ parental lines mutagenized with EMS. Of the M₂ seeds sown, 23.5% did not germinate or presented abortive germination against a 2.7% rate of wild-type Ler control sowings. Together with this high percentage of early lethals, the appearance of a high number of albino plants (fifth column from the left in Table 1) indicated the efficiency of the mutagenesis.

Phenotypic classification of putative mutants:
The strategy followed for the isolation and characterization of mutants is summarized in Figure 1. First, we phenotypically classified the 1926 isolated putative leaf mutants (4.2% of M₂ plants) that survived at least 3 wk after germination into three broad categories, corresponding to alterations (1) in the proportions or size of the lamina, (2) in the form of the margin, and (3) in the structure of the basal rosette, which is dependent upon the length of the petiole of the leaves and their phyllotaxis. Each group was subdivided into other more specific groups, as presented in Table 2, that were further subdivided into several phenotypic classes, whose total number reached 85. We provisionally assigned the putative mutants to only one class according to their most conspicuous trait, although many of them presented more than one alteration.

View larger version (43K): In this window In a new window Download PPT slide   Figure 1. Steps followed for the isolation and genetic analysis of mutants. The symbols and indicate, respectively, growth and selfing. The time scale refers to each single mutant line. Tasks indicated in italics were not performed by the authors (EMS mutagenesis accomplished by Lehle Seeds) or were initiated in this work but remain to be completed (isolation of double mutants and linkage analysis).

Study of the heritability of the mutant phenotypes:
More than half of the putative mutants bore lethal-effect mutations, dying before completing their life cycle, or were sterile. They were only studied to obtain photographs of their phenotypes. About 30 M₃ inbred seeds were sown from each of 853 putative mutants yielding viable progeny to ascertain the heritability of their phenotypes. Only in 333 lines was transmission of the phenotype unequivocally Mendelian, without variations in expressivity or incomplete penetrance. Among the remaining 520 families, which received no further attention, 135 showed only subtle phenotypes that were considerably weaker than those of the M₂ parents, variable expressivity was observed in 109 cases, and no mutant phenotype was observed in the M₃ progeny of the remaining 276 lines.

Determination of the inheritance patterns of mutant phenotypes:
Among the 333 M₂ mutant lines that presented a stable phenotype, 81 displayed alterations in the structure of the basal rosette. These strains were not studied further, since it was considered that their mutations disturbed leaf phyllotaxis, leaf primordium initiation, or petiole elongation, aspects of leaf development beyond the scope of this work. One or more M₃ individuals from each of the remaining 252 lines were testcrossed to wild type to determine the inheritance pattern in the F₁ progeny. About 30 F₁ individuals were analyzed from each testcross. We found 245 cases of recessive pattern of inheritance, 6 of semidominance (all F₁ individuals displayed a mutant phenotype weaker than their M₃ parental), and only 1 of complete dominance (all F₁ individuals were indistinguishable from their M₃ parental; Table 3).

Identification of lines displaying monogenic mutant phenotypes:
About 100 seeds of each F₂ family were sown to study the segregation of the mutant phenotype (Table 3). Two or more different mutant phenotypes were found in some F₂ populations, which demonstrated the existence of several mutations in their M₂ ancestor. Thirteen new mutations affecting leaf morphology were found in this way, 3 of which were studied, thus increasing to 255 the number of lines under study. Finally, three F₂ individuals displaying a phenotype identical to that of their M₂ and M₃ ancestors were chosen from each mutant line, with the aim of studying 100 of their F₃ inbred descendants. We chose a total of 153 lines with a fully heritable mutant phenotype and similar M₂, M₃, and F₃ individuals for subsequent studies. The phenotypes of 122 of these mutant lines were monogenic and recessive, 6 being semidominant and 1 completely dominant (Table 3). Of the remaining lines, 23 displayed unequivocally recessive mutant phenotypes but had F₂ phenotypic proportions that deviated significantly from the 3:1 ratio. However, we decided to include these lines in further studies, because the underrepresentation of the putative homozygous recessive individuals in the F₂ progeny might have been due to certation (KOORNNEEF 1994 ), to the poor viability of their embryos compared with the heterozygous and wild-type homozygous, or to an insufficiently large sample size. The 15:1 and 9:7 phenotypic segregations observed in some F₂ populations might also be explained as a consequence of epistasis: in the first case the concurrence of two mutations, with no phenotypic effect in themselves, being responsible for the mutant phenotype; in the second the presence of at least one wild-type allele at each of two genes being essential for wild-type leaf morphology.

Complementation analysis:
To simplify allelism testing, we defined a series of phenotypic classes (Table 4 and Figure 2), limiting the crosses to lines within a class. This decision was based on the assumption that most mutant alleles of a gene cause similar phenotypes, which can sometimes be put into a series, especially when they correspond to hypomorphic mutations that cause different degrees of loss of function. Consequently, it is reasonable to assume that mutants with similar phenotypes are candidates to be damaged in the same gene. However, the possibility that two mutations in the same gene determine phenotypes that are so different as to be assigned to different phenotypic classes cannot be excluded.

View larger version (65K): In this window In a new window Download PPT slide   Figure 2. Diagram showing representative individuals of the phenotypic classes defined for the complementation analysis of mutants with abnormal leaves. Images were obtained from pictures taken 21 days after sowing. Bars, 5 mm.

Figure 2 shows representative examples of the phenotypes of the 19 classes, obtained by grouping several of the 70 fertile classes initially defined (Table 2). The names given to these phenotypic classes are new, with the exception of Asymmetric leaves, which was first used by REDEI and HIRONO 1964 . For the remaining names we elected Latin words, following STEARN 1995 as closely as possible. In cases involving dominant or semidominant mutations, the complementation criterion was the appearance of wild-type individuals in the F₂ generation.

The 153 mutant lines studied, grouped in 19 phenotypic classes, were assigned to 94 complementation groups (Table 5), 76 of which had a single allele, while 11, 6, and 1 groups had 2, 3, and 4 alleles, respectively. The number of genes affected in those mutants would be <94 if any class included different phenotypic manifestations of damages in a single gene. On the other hand, the possibility that the phenotype of some of the mutants subjected to complementation analysis was due to the concurrent action of mutations in at least two different genes cannot be excluded.

Allelism tests with existing mutants:
Some previously described mutants were included in the complementation tests: two nonallelic asymmetric leaves mutants, as1-1 (NASC NW146; REDEI and HIRONO 1964 ) and as2-1 (ABRC CS3117; FABRI and SCHAFFNER 1994 ), and five nonallelic mutants displaying involute leaves, the phenotype that we have called Incurvata (Table 5; GOODRICH et al. 1997 ; KIM et al. 1998A ; TELFER and POETHIG 1998 ; J. SERRANO-CARTAGENA, H. CANDELA, P. ROBLES and J. L. MICOL, unpublished results), three of them recessive, clf-18, icu2, and hst-5, one semidominant, icu4-2, and one dominant, icu5 (NASC N345, N329, N314, N401, and N379, respectively; BURGER 1971 ).

Several other already described lines, which resembled some of our mutants, were tested for allelism: revoluta (rev; TALBERT et al. 1995 ), displaying large rosette and cauline leaves; cab underexpressed1 (cue1; LI et al. 1995 ), differential development of vascular associated cells (dov1; NASC N557; KINSMAN and PYKE 1998 ), and reticulata-1 (re-1; NASC NW129; REDEI and HIRONO 1964 ) showing reticulate leaves with veins greener than their interveinal regions; angustifolia (an; TSUGE et al. 1996 ) with narrow leaves; and rotundifolia3 (rot3; TSUGE et al. 1996 ), with rounded leaves. Crosses were performed involving rev and elo1-elo4 mutants, while cue1-5, dov1, and re-1 were crossed by ven1-ven6. In addition, an was crossed by ang1-ang4 and elo1-elo4, as well as rot3-1 by ron1-ron3. Complementation was found in 32 of these 33 crosses, the only exception being the cross involving ven2 and re-1, which were found to be allelic.

	DISCUSSION

TOP ABSTRACT MATERIALS AND METHODS RESULTS DISCUSSION LITERATURE CITED

To answer the question of how plant leaves are constructed, the most obvious approach involves the identification, characterization, and manipulation of genes that are candidates for controlling leaf development in a model system such as A. thaliana. Previous authors have isolated mutants with abnormal leaves in Arabidopsis as well as in other plant species, but only some have subjected their findings to detailed study, leading in a few cases to the molecular characterization of the genes involved in leaf development. This is shown in Table 6, where Arabidopsis genes known to affect leaf morphology are listed, most of them still to be cloned. The situation is similar in other plant systems, such as pea, tobacco, and tomato (see MARX 1987 ; MCHALE 1993 ; KEDDIE et al. 1996 ; HOFER et al. 1997 ).

The monocot Zea mays is, alongside Arabidopsis, the plant species that has received most attention regarding the genetic and molecular analyses of leaf development. The KNOTTED1 (KN1) gene, the first homeobox gene found in plants, plays an indirect role in leaf development, maintaining the undifferentiated meristematic state (VOLLBRECHT et al. 1991 ; SINHA et al. 1993 ). The LIGULELESS (LG) genes code for nuclear proteins involved in the differentiation of the ligule, a concrete region of the leaf that appears between the blade and the sheath (MORENO et al. 1997 ; MUEHLBAUER et al. 1997 ; WALSH et al. 1997 ). A gene specifically affecting epidermal differentiation is CRINKLY4 (CR4), whose coded protein, a TNFR-like receptor kinase, might be part of a signal transduction chain (BECRAFT et al. 1996 ).

Usefulness of a screen for Arabidopsis mutants with abnormal leaves:
Mutations in genes that control leaf development might have phenotypic effects that vary from lethality to the absence of visible alterations. The lethal alleles would include the most hypomorphic and null mutations, affecting genes with housekeeping functions or those required for initiating the leaf, an essential organ for the plant, or those with products shared by other developmental pathways, such as embryogenesis. A second class would be represented by mutations in genes of redundant functions, which would not cause phenotypes distinguishable from the wild type. After considering these two extreme cases, it is reasonable to assume that mutations in the genes involved in the control of leaf ontogeny might determine viable phenotypes, characterized by alterations in leaf shape or size. Such mutants might express morphological abnormalities in the leaves alone or in other parts of the plant, too, depending on whether or not the damaged morphogenetic controls were leaf specific. If mutations in some of the genes crucial to leaf development cause viable phenotypes, distinguishable from the wild type by leaf architectural traits, a search for such mutants merely based on the observation of plants derived from mutagenesis might be considered worth attempting. This is what we have done in this work. Such mutant screening has the advantage of not necessitating manipulation of the individuals under study, although it is a very slow process, since plants should be studied one by one, under conditions that do not limit or prevent the growth of wild-type individuals. Another disadvantage is that lethal mutations and those mutations affecting genes of redundant functions would be lost in the search for viable and visible mutants.

The criterion used in our search was to distinguish between mutant and wild-type plants simply by observation of structural alterations. Mutants obtained through this procedure should show disturbances in the structure, spatial arrangement and/or division or expansion patterns of their leaf cells. One objection that could be made to this screen procedure is that it might lose all those mutations that, while altering some of the parameters of cell proliferation or differentiation, do not determine any obvious modification in leaf structure. In fact, mutations modifying cell division patterns without disturbing morphogenesis are known. Such is the case of the mutant alleles of TANGLED-1 (TAN-1) in maize (SMITH et al. 1996 ), whose deviations in the direction of cell division do not affect leaf shape, and the ton (tonneau) mutants, in Arabidopsis, in which the planes of cell division are altered while the correct position of all the plant organs is maintained (TRAAS et al. 1995 ). Therefore, it is reasonable to suppose that leaf morphology does not depend exclusively on cell division patterns and that intercellular communication is one of the processes responsible for the final shape of the organ.

Degree of saturation of the Arabidopsis genome reached in the mutant screening:
In a saturation mutagenesis experiment, the number of mutants obtained should be high enough to render negligible the probability of having found no representative alleles for any of the genes involved in the process under study. Assuming that all the genes are equally mutable, the occurrence of mutations in a mutagenesis should follow Poisson's distribution. Consequently, the proportion of genes undetected in a mutant search will be smaller than 5% when the average number of allelic mutations is 3 (JURGENS et al. 1991 ). To achieve a proportion of undetected genes of less than 1%, the average number of alleles obtained must be 5, which can be possible only in organisms that allow screening of large progenies of mutagenized individuals, such as Drosophila, Arabidopsis, or the zebra fish.

We subjected a total of 153 mutant lines to complementation analysis, assigning them to 94 complementation groups. Hence, the average number of alleles obtained per gene is 153/94 = 1.6276. It follows that the class of genes not represented among our mutants would represent almost one-fifth (19.64%) of those that can yield phenotypically abnormal leaves, since m = 1.6276, f(0) = e^-m = e^-1.6276 = 0.1964 = 19.64%. On the other hand, we obtained in several cases 2 or more noncomplementing mutant lines with quite similar phenotypes from the same parental group. If such lines represented the isolation of individuals with identical genotypes, the number of different mutations would decrease from 153 to 120, so the average number of alleles per gene would be 1.275, and we would not have identified 27.94% of the genes that we were looking for. In the previous calculations we have assumed that all the studied mutant lines display monogenic phenotypes, including those whose phenotypic segregation in F₂ is significantly different from 3:1 (at least 23 of the 153) and in some instances (7 lines) is very acceptably close to the 15:1 ratio. Nonetheless, we believe that we have covered a substantial part of the Arabidopsis genome with our collection of mutants. The results obtained indicate that the proportion of genes not represented among our mutants might be up to one-third of those that give rise to phenotypes characterized by an abnormal leaf morphology. A more accurate approach to a saturation state of the genome would probably require additional sowings, although the relation between benefit and effort would be slight.

Speculations regarding the function of the genes identified by isolating mutants:
The genetic operations at work in organogenesis remain poorly understood in most biological systems. Such an assertion is particularly true when it comes to the study of leaf organogenesis, a field where many questions still await answers. Up to now, leaf development has not been analyzed following a mutational approach involving a large-scale screen as starting point, aimed to define as far as possible the spectrum of genes participating in the process. On the other hand, molecular analyses, until now, have not identified genes controlling the process, with only three possible exceptions: the MYB superfamily member PHANTASTICA (PHAN; WAITES et al. 1998 ), in Antirrhinum, whose participation in the establishment of the dorsoventral asymmetry seems to be required mostly in the leaves, bracts, and petals (WAITES and HUDSON 1995 ); ARGONAUTE1 (AGO1; BOHMERT et al. 1998 ) in Arabidopsis, whose product, a protein without homologs encoded by unicellular organisms, such as yeasts or bacteria, is required in the entire plant body for correct cell proliferation, its mutation resulting in a pleiotropic phenotype mainly characterized by the absence of lateral expansion in leaves and floral organs; and ROTUNDIFOLIA3 (ROT3; KIM et al. 1998B ), which encodes for a cytochrome P-450 required for the polar elongation of cells in the longitudinal axis, its mutant alleles determining short petioles and rounded leaves.

When trying to genetically dissect leaf ontogeny in Arabidopsis, we have employed logic similar to that applied by JURGENS et al. 1991 with respect to pattern formation in the embryo. These authors attempted to identify most of the genes generating the body plan of the Arabidopsis embryo by means of a large-scale screening for seedlings with an abnormal morphology. Such an approach was based on the supposition that, as occurs in D. melanogaster (see, for example, NUSSLEIN-VOLHARD and WIESCHAUS 1980 ), most of the mutations that cause embryonic lethality would affect genes with housekeeping cell functions, while mutations in many of the genes responsible for embryonic morphogenesis would not prevent completion of embryogenesis, despite causing strong morphological alterations that would be expressed in the seedling. The assumption of Jürgens et al. with respect to the embryo can also be applied to the leaf, since mutants in ubiquitous and essential metabolic processes should determine lethality. Viable mutants with aberrant leaves should correspond to dysfunctions in morphogenetic controls or in the synthesis, spatial distribution, or assembly of the structural or metabolic elements required for the proliferation, spatial arrangement, and differentiation of the cells of the organ.

A large number of new mutants with abnormal leaves are presented in this work. It cannot be established, a priori, how many of them are affected in genes that control the morphogenesis of this organ and how many represent genes that participate in the process by merely providing, locating, or assembling cell structural elements. It cannot be ruled out that some, or even most, of our mutants correspond to partial loss-of-function damage in genes participating in metabolic pathways whose alteration causes the modification of leaf form. The most obvious candidates to be included in this group are the mutants that show an anomalous pigmentation, which are probably associated with a generalized (in the mutants of the Exigua, Orbiculata, and Angulata classes) or localized (Venosa) abnormal chloroplast development as occurs in the cue1 (LI et al. 1995 ), dov1 (KINSMAN and PYKE 1998 ), and pac (pale cress; REITER et al. 1994 ) mutants. Other mutants, the most extreme examples of which are those of the Exigua class, show anomalies in size, which is inferior to that of the wild type. This might reflect disorders in the general processes that control cell division or expansion, as occurs in the diminuto (dim; TAKAHASHI et al. 1995 ) mutant and its allele dwarf1 (dwf1; KAUSCHMANN et al. 1996 ) or in the constitutive triple response 1 (ctr1; KIEBER et al. 1993 ) mutant, which displays constitutive ethylene response. The phenotypic classes Rugosa and Scabra include members that display irregularities on the surface of the lamina that might be due to uncoordinated division and/or expansion of epidermal and/or mesophyll cells, as in the crinkly-4 mutant of maize (BECRAFT et al. 1996 ). Another explanation for the rough surface of these mutants could be the existence of local processes of cell death affecting the epidermis or mesophyll. In any case, it is difficult to determine on only a gross phenotype basis if these mutants are affected in tissue patterning or in morphogenesis.

It has been proposed that at least two independent controls contribute to the final size of the cells of the expanding Arabidopsis leaf (TSUGE et al. 1996 ), which would determine either cell width or cell length. Evidence for the existence of such polar controls is provided by some mutant phenotypes: Rotundifolia, with short rounded leaves (TSUGE et al. 1996 ), and Angustifolia, with narrow thick leaves (REDEI 1962 ; TSUGE et al. 1996 ). Such a model is applicable to some of our mutants, which might be affected in the polar controls of cell expansion: the lateral (expansion in width; the Angusta, Asymmetric leaves, Rotunda and Erosa classes) and the longitudinal or proximo-distal (in length; Elongata). The final morphology of all these mutants, however, might also be due to perturbations in the patterns of cell division.

The phenotypic classes whose members are characterized by different types of indentations in the margin constitute a sizeable part of the obtained mutants, which may be due to localized cell death or to local disturbances of the division and expansion patterns. On the other hand, it has been demonstrated that hydathodes, guttation organs connected to the vasculature, are associated with prominences in the leaf margin (TSUKAYA and UCHIMIYA 1997 ; VAN LIJSEBETTENS and CLARKE 1998 ). A direct correlation exists between the number of hydathodes and the number of prominences in the leaf margin, both increasing in parallel in successive vegetative leaves of the ecotype Ler. In fact, a mutant, extrahydathodes, that presents an increased number of hydathodes, and, consequently, of prominences in the leaf margin was obtained in our laboratory (CANDELA et al. 1999 ). In accordance with the above hypothesis, we may assume that our mutants that show alterations in leaf marginal configuration represent disturbances in the distribution of such elements of the vascular pattern, which are externally expressed in the prominences appearing in the leaf margin, or in the differentiation of adjacent tissues or the expansion or division of their cells. It is striking that the three phenotypic classes that are characterized by alterations in the marginal configuration of the leaf are among those showing a higher number of complementation groups: The first is Denticulata with 17, the second is Angulata with 12, and the fourth is Apiculata with 7.

Since cell migration does not play any role in plant morphogenesis, the body pattern of an adult plant is a consequence of mechanisms that control the number and direction of cell divisions, the direction of cell expansion, and the local differentiation of the cells (MEYEROWITZ 1994 ) and, eventually, of the existence of programmed cell death (VAN LIJSEBETTENS and CLARKE 1998 ). On the other hand, it is generally assumed that developmental genetic decisions of individual cells are subordinated to coordination systems among groups of cells, which are mediated by intercellular communication mechanisms. The leaf of Arabidopsis is a fundamentally flat structure, which consists of only a few cell layers (PYKE et al. 1991 ; TSUGE et al. 1996 ), its final form resulting from cell division and expansion. The growth of the adaxial and abaxial surfaces, which are equivalent in area, must be coordinated by some unknown mechanism. It is reasonable to suppose that perturbations in such a coordination mechanism would lead to differences between the dorsal and ventral cell division and/or expansion patterns. This would determine differences between the adaxial and abaxial surfaces, which, in turn, would cause alterations in the morphology of the leaf reflected by its curling up or downward. We obtained some mutants whose leaves display different kinds of deviation from planarity, their lamina being curled upward (the Incurvata phenotype), spirally rolled downward (Ultracurvata), or obliquely folded (Transcurvata). Since all these mutants are candidates to be affected in genes involved in coordinating the growth of the dorsal and ventral surfaces of the leaf, their characterization might provide information on the coordination mechanisms between proliferating cells during development. Alternatively, the above-mentioned phenotypes might arise from hormonal imbalances. In fact, it is likely that some of our mutants have suffered damage in the synthesis, transport, or perception of phytohormones as well as in the related responses. Abnormal size and/or morphology have been observed in mutants impaired in such processes with regard to auxin, gibberellins, brassinosteroids, and ethylene (see, for reviews, HOBBIE 1998 ; MCGRATH and ECKER 1998 ; PHILLIPS 1998 ; SZEKERES and KONCZ 1998 ).

Pleiotropy of the mutant phenotypes:
Some of our mutants seem to be affected in processes mainly or exclusively required in leaf ontogeny, whereas others seem to have suffered damage in functions that participate in the construction of several organs. We have verified that 60 of the 153 mutants studied show anomalies visible only in the vegetative leaves, while another 15 also express alterations in the cauline leaves although not in the rest of the plant. Not infrequently (a total of 78 mutant lines) morphological abnormalities were observed in other organs, mainly the flowers. This is not surprising, since leaves represent the basal state of floral organs, which they preceded in the evolutionary history of flowering plants. In fact, it is generally assumed that floral organs are modified leaves, which have acquired specific functions throughout evolution. Furthermore, the elimination by mutation of all major A, B, and C morphogenetic functions has led to the homeotic transformation of floral organs into leaves (COEN and MEYEROWITZ 1991 ). All of the above lends plausibility to the fact that mutations that disturb the morphogenetic controls shared by leaves and floral organs might perturb the morphology of both flowers and leaves.

Speculations regarding the molecular nature of the genes identified by isolating mutants:
Little can be said about the molecular nature of the genes affected in our mutants. Nevertheless, those termed incurvata1 deserve commentary, since we have found them to be allelic to the CURLY LEAF (CLF) gene. The phenotype associated with such mutant alleles consists of curvature of the leaf margin and disturbances in the size and the spatial arrangement of some floral organs. The cloning of CLF (GOODRICH et al. 1997 ) has revealed that it is structurally related to members of the Polycomb and trithorax groups of D. melanogaster (KENNISON 1993 ; SIMON 1995 ), which include regulatory genes required for the maintenance of the expression of homeotic genes such as those belonging to the Bithorax and Antennapedia complexes. It has been demonstrated that the leaf phenotype of clf mutants is due to the ectopic derepression of some genes required for flower morphogenesis, but inactive in wild-type leaves, such as AGAMOUS (AG). Since CLF represses AG in leaves as well as in some floral organs, its loss of function leads to the ectopic derepression of AG, which causes leaf curling. On the other hand, mutant phenotypes such as those of our Incurvata class have been observed in Arabidopsis plants transformed with an antisense construction of the METI gene, whose product is a methyltransferase (FINNEGAN et al. 1996 ). These plants suffer severe hypomethylation of their DNA, one of the consequences of which is the ectopic expression of the AGAMOUS and APETALA3 genes in the leaf. According to the above data, some of our incurvata, transcurvata, or ultracurvata mutants might be regarded as suffering the ectopic derepression in the leaf of genes whose normal realm of action is the flower. We hypothesize that some of such mutants owe their phenotype to mutations in genes whose function is to repress floral genes in the leaf. Such genes would have appeared in order to safeguard the identity of leaves as organs, distinguishing them from floral organs. The Incurvata phenotype can be explained as a consequence of the ectopic expression in the leaf of some floral organ identity gene that participates in the control of cell division patterns and probably contributes to building partially concave organs in the flower, such as sepals and petals, or fundamentally cylindrical ones, such as the stamens and carpels. In the leaf, however, a flat structure is desirable and the curvature observed in some incurvata mutants would be a consequence of the imposition on this organ of the typical division patterns of floral organs.

Given the nature of the chemical mutagen that we have employed, EMS, it is likely that most of the alleles obtained are hypomorphic. However, the semidominance of six of them and the complete dominance of another constitute singular cases that could correspond to hypermorphic or antimorphic mutations or null alleles of haploinsufficient loci. The semidominant ven3-2 and ven3-4 mutations probably deserve attention, since they are members of an allelic series including a further two alleles that are recessive. The same can be said of the ucu1 alleles, two of which are semidominant (ucu1-1 and ucu1-2), and the third recessive. Finally, the icu6 mutation is the only dominant one that we have found, its mutant phenotype in icu6/ICU6 heterozygous being more extreme than that of most of the homozygotes for recessive mutations in the remaining ICU loci.

Perspectives for the genetic and molecular analyses of mutants obtained:
Histological analyses of the mutants presented in this work will be necessary for establishing the nature of the underlying perturbations in their phenotypes at the cellular level. Other information to be determined is their eventual allelism with mutants already isolated by previous authors. We have performed some of such allelism tests, finding that at least four of the genes that we have identified are alleles of already known Arabidopsis genes: AS1, AS2, CLF, and RE. Although additional intercrosses with old mutations could be made to test for allelism, we decided to restrict this approach to existing mutants with phenotypes very similar to those obtained in this work. The reason such a choice was made is that the sheer volume of both old and new mutations affecting leaf morphology is quite large. Take, as an example, the AIS collection of form mutants (BURGER 1971 ; KRANZ and KIRCHHEIM 1987 , KRANZ and KIRCHHEIM 1990 ; ANDERSON 1993 ), which includes >150 old mutant strains, all of which are candidates to be intercrossed with representative individuals of our 94 complementation groups. The genetic analysis of 77 lines of the AIS collection of form mutants revealed that they represent 47 different genes (J. SERRANO-CARTAGENA, H. CANDELA, P. ROBLES and J. L. MICOL, unpublished results; SERRANO-CARTAGENA et al. 1999 ). To choose further candidates for allelism tests, proximity between genetic map positions might be a better criterion than a mere resemblance between phenotypes. Hence, another aspect in the study of our mutants that would constitute a natural continuation of the work presented here is their genetic mapping. Crosses to reach this objective to obtain the F₂ plants that will be employed for simple sequence length polymorphism (SSLP) linkage analysis have been accomplished (BELL and ECKER 1994 ; PONCE et al. 1999 ).

An obvious avenue that opens up with the availability of the mutants described in this work is an analysis of their interactions in double mutant combinations. A study of their phenotypes will help to discriminate mutations that disturb morphogenetic controls from those affecting other processes. By analyzing the interactions that take place between the genes that we have identified, we will be able to propose models for the genetic control of leaf development. Advantage will be taken of the progeny obtained from the crosses made for the complementation analysis. Since some of the mutants that we have obtained display flower abnormalities, it is possible that they may correspond to any of the numerous mutations already known in genes affecting floral morphogenesis in Arabidopsis. Finally, we emphasize that the collection of mutants that we have obtained provides the scientific community with new tools for studying leaf morphogenesis in A. thaliana.

	ACKNOWLEDGMENTS

We are grateful to H. Candela, A. Martínez-Laborda, J. M. Pérez-Pérez, P. Piqueras, M. R. Ponce, V. Quesada, and A. Vera for comments on the manuscript, to the Instituto de Neurociencias of the Universidad Miguel Hernández for the use of its facilities, to S. Gerber, J. M. Serrano, and J. C. Pastor for their expert technical assistance, and to the Nottingham Arabidopsis Stock Centre and Arabidopsis Biological Resource Center for providing seeds. We especially thank Drs. L. Comai, J. Chory, and H. Tsukaya for kindly providing seeds of rev-1, cue1-5, and rot3-1, respectively. Critical reading of the manuscript and helpful suggestions by A. García-Bellido, J. Chory, and two anonymous referees are most appreciated. This work was supported by grants from the Dirección General de Enseñanza Superior of Spain (PB91-0749, APC95-0191, and PB95-0685). G. Berná and P. Robles were supported by fellowships from the Dirección General de Enseñanza Superior of Spain.

Manuscript received February 5, 1999; Accepted for publication March 10, 1999.

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