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Mutational analysis of pistil structure and development of Arabidopsis thaliana
Cell Differentiation
and Development,
21
28 (1989) 27-38
Elsevier Scientific Publishers Ireland. Ltd. CELDIF 00609
Mutational analysis of pistil structure and development of Arabidopsis thaliana Kiyotaka
Okada ‘, Masako
K. Komaki
’ and Yoshiro
Shimura
I*2
’ Division of Gene Expression and Regulation, National Institute for Basic Biology, Okazaki 444, Japan and 2 Department
of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan
(Accepted 21 May 1989)
The developmental and morphogenetic process of pistil formation was examined by analysing flowers of wild type and six flower mutants of Arubidopsis flraliana, a small crucifer. The wild type is suggested to originate from two ‘pistil-forming units’ (carpels) arranged laterally against the axis of the inflorescence at a pistil primordium. Aberrant structures of the pistils of mutants indicate that a set of genes regulate each step of pistil development and morphogenesis, namely arrangement of the units at the pistil primordia, fusion of the units, growth of primordia, formation of the septum in the ovary, and formation of the stigma.
Arubidopsis thuhimu; Flower mutant; Pistil structure; Floral development; Carpel theory; Pistil organogenesis
Introduction
The flower is a reproductive organ in angiosperms, consisting of several floral organs (sepals, petals, stamens and pistil) arranged in whorls or concentric circles. Flower development is a complex process, involving the temporally and spacially controlled initiation of primordia and their coordinated development into mature floral organs. A number of mutants of Arubidopsis thaliana, in which these processes are defective, have been isolated and characterized (Haughn and Somerville, 1988; Komaki et al., 1988; Bowman et al., 1989), suggesting that flowering in this plant is regulated by a number of genes. A. thuliunu, a
Correspondence address: K. Okada, Division of Gene Expression and Regulation, Institute for Basic Biology, Okazaki 444, Japan.
member of the Brassicaceae, has various properties that make it suitable for classical and molecular genetic studies (Redei, 1975; Meyerowitz and Pruitt, 1985; Estelle and Somerville, 1986; Meyerowitz, 1987,1989; Fink, 1988) and the analysis of the flower mutants is expected to be a useful approach in pursuing an understanding of the genetic control of floral development and morphogenesis. Genetic analysis of these mutants has revealed that a single, recessive mutation may result in aberrant arrangement and/or structure of one or several floral organs (Komaki et al., 1988). In the course of systematic morphological analysis of the mutant flowers, we encountered some intriguing problems which led us to examine existing theories concerning morphogenesis in higher plants. For instance, pistil development. and morphogenesis posed an interesting problem in some mutant flowers. The pistil is a complex organ with apparently unique developmental
0922-3371/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland, Ltd.
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processes. On the basis of morphological studies, several hypotheses have been proposed on the origin of the pistil of Brassicaceae, which remain totally unsettled. One of the hypotheses is the ‘ two-lateral-carpel hypothesis’ which assumes that pistils are made by fusion of two carpels located in lateral positions against the inflorescence axis (Eichler, 1865; Eggers, 1935; Allexander, 1952; Esau, 1977). On the other hand, according to a second hypothesis, the ‘four-carpel hypothesis’, pistils are considered to be made of four carpels; two of them form the ovary wall, and the other two form the placentae (Saunders, 1925, 1929; Eames and Wilson, 1928, 1930; Pm-i, 1941, 1945; Merxmtiller and Leins, 1967; Eigner, 1973). The latter hypothesis was based mainly on the arrangement of vascular bundles and the stigma structure (for a review see Lawrence, 1951). There are also other hypotheses claiming that pistils are formed by two carpels positioned on the median plane (Spratt, 1932) or by six carpels (Yen, 1959). Studies on the pistil formation of Brassicaceae flowers have mostly involved microscopic analysis of developing flower buds (Sattler, 1973). In this paper, we present our view on pistil formation based on detailed morphological analyses of several flower mutants as well as evidence indicating that a set of genes participates in pistil formation in A. thaliana. Materials and Methods
Plant lines An A. thaliana wild-type strain, Landsberg (erecta), and a mutant line M7 (apetala 1, clavata I) were obtained from the Arabidopsis Information Service (J.W. Goethe-Universitat, Frankfurtam-Main, F.R.G.). Another mutant line, pistillata I, was a gift from Dr. E.M. Meyerowitz (Caltech, Pasadena, CA). Mutant lines of Fl-series were isolated in our laboratory. Isolation and genetic analyses of the mutants (Fl-40, Fl-82 and Fl-89) were described previously (Komaki et al., 1988). Fl-165 is a newly isolated mutant. Cultivation of plants Plants were grown in accordance with the procedures of Dr. C.R. Somerville (Michigan State
University, personal communication) tinuous illumination (24 L) at 22 o C.
under con-
Thin section analysis Thin sections of flowers were prepared and stained by Azure B, as described by Komaki et al. (1988).
Results and Discussion
Pistil structure and development of Arabidopsis thaliana wild type The floral structure of A. thaliana wild type is typical of the Brassicaceae (Fig. 1A). The pistil is a green cylinder about 2.5 mm long and 0.4 mm wide in mature flowers (Figs. lB, 2D). A long ovary is surmounted by a short style and a clump of stigmatic papillae. Transverse sections show that the ovary is separated into two rooms (biloculous) by a septum (Fig. 2A). The septum may be histologically divided into three regions; central region and two side regions (placental tissues). The central region (the transmitting tissue) consists of loosely-packed cells that are stained pink by Azure B. The tissues of the side regions are indistinguishable from the tissue of the inner layer of the ovary wall. Ovules are attached to the placenta located in the region where the septum merges with the ovary wall. A total of about 30 ovules are lined up in four rows in a wild type pistil. There are four major vascular bundles (two lateral and two median bundles) running parallel to the longitudinal axis of the ovary (Figs. lB, 2A). Small axillary bundles derived from the major bundles appear when the pistil is fertilized, and these develop into pods. Although the presence of an extra vascular bundle in which the positions of the xylem and phloem are reversed was reported in other species of Brassicaceae (Eames and Wilson, 1928, 1930; Puri, 1941, 1945), we could not detect such bundles in the ovary of A. thaliana. The process of the pistil development from undifferentiated meristem to mature flowers may be separated into four stages which are shown schematically in Fig. 3A-D. The pistil originates from a relatively large primordium which appears at the center of the floral meristem. At a very
29
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Fig. 1. Schematic drawings of Arabidopsis rholima flowers and the hypothetical unit structure. Panels A and E and the upper figures in B, C, and D are transverse sections; the lower figures in B, C, and D are side views: cb, central vascular bundle; fmb, fused marginal vascular bundle; ia, infloresoence axis; 1, lateral vascular bundle; m, median vascular bundle; mb, marginal vascular bundle; o, ovule; ov, ovary; p, petal; pfu, pistil-forming unit; pi, pistil; s, sepal; sp, stigmatic papillae; st, septum tissue; sy, style. A, a wild type flower; B, a normal pistil of the wild type; C, an ‘open pistil’ of the FL40 mutant; D, the proposed hypothetical ‘pistil-forming unit’; E, a hypothetical structure of the pistils of Brassicaceae. Two ‘pistil-forming units’ are arranged laterally to the axis of inflorescence.
early stage when the cylindrical wall of the pistil is formed (stage I), two swellings develop from opposite sides of the wall on the median plane (Figs. 2C, 3A). Ovule primordia develop at the side of the swellings (stage II, Fig. 3B) and these swellings begin to fuse when the young ovules appear (stage III, Figs. 2B, 3C). The loosely packed pink-staining tissue in the centre of the septum develops in the last stage just before flowering (Figs. 2A, 3D). Structural changes are also observed at the top of the pistil primordia. At early stages (stages I-II) the top of the cylindrical pistil primordia is open. As development proceeds, the opening is closed to form a rounded top (stages II-III, Fig. 2L), and then stigmatic papillae develop (stage IV). To investigate the genetic regulation of pistil development and morphogenesis, we analyzed the structure and development of some mutant flowers of A. thaliana. Pistil unit hypothesis Pistil development and structure were examined in more than nine flowers of each of the six mutant lines and the mutant phenotypes were
found to be quite stable. The mutants showed a variety of abnormalities in pistil structure and in the course of the analyses it was found that the abnormalities could be best explained if we hypothesised the existence of a ‘pistil-forming unit’. This hypothesis is explained by reference to some of the floral mutants. The Fl-40 mutant has a homeotic conversion in which the sepals are replaced by leaf-like structures bearing ovules along their edges (converted sepal; Fig. 5B, H) (komaki et al., 1988). In other homeotic mutants of A. pistillata 1 and apetela 2, petals are converted to sepals in the former and in the latter sepals are converted to leaves and petals are converted into stamens (Haughn and Somerville, 1988; Bowman et al., 1989). In these cases there is an entire conversion of one floral organ to another and if this is also true of Fl-40, the structure replacing the sepals in this mutant may be interpreted as a developmental ‘pistil-forming unit’. The structure of the putative unit is a leaf-like structure curving inward having three major vascular bundles, one central and two marginal bundles with the placentae located at the margins
30
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Fig. 3. Schematic drawings of pistils of A. fMiuna wild type at different developmental stages. Redrawn from transverse sections. Sixes are ca. 75 gm (A), 100 pm (B), 200 pm (C) and 400 pm (D) in diameter. The position of the axis of the inflorescence is shown relative to the orientation of the pistil: ia, inflorescence axis; 1, lateral vascular bundle; m, median vascular bundle; o, ovule; op, ovule primordium; s, septum; yo, young ode. A, a very young pistil (stage I). A pistil of this stage is shown in Fig. 2C. B, a young pistil which shows swellings corresponding to ovule primordia. Four vascular bundles begin to appear (stage II). C, a young pistil. The septum tissues grow and meet at the center. Young ovules develop integuments (stage III). A pistil of this stage is shown in Fig. 2B. D, a mature pistil (stage IV), A pistil of this stage is shown in Fig. 2A.
(Fig. 1D). The wild type pistil may then be seen to consist of two units which are laterally arranged against the axis of inflorescence as shown in Fig. 1E. The marginal vascular bundles of the units are shared and appear as the median bundles in the developing pistil. Although the structural unit hypothesis described above is based on the anatomical and genetic analyses of mutant flowers of a single species, A. thaliana, and not on phylogenic studies on several evolutionarily related species, the structure of the proposed unit is almost identical to the hypothetical carpel structure deduced from phylogenic analyses (Eames, 1931), except
A
Fig. 4. Carpel hypotheses explaining pistil structure of Brassicaceae. Arrows and dotted lines indicate the hypothetical border of the carpels. The postion of the axis of inflorescence (ia) is shown relative to the orientation of the pistil. A, the twolateral-carpel hypothesis. B, the four-carpel hypothesis. Carpels 1 and 2 (in the median position) are fertile carpels which carry ovules and lost locules. Carpels 3 and 4 (in the lateral position) are sterile carpels forming an ovary wall. C, the two-mediancarpel hypothesis, which is the only one proposing that ovules are generated in the central region.
for the presence of septum tissue which represents a specific characteristic of A. thaliana and possibly of the Brassicaceae. This interpretation strongly supports the twolateral-carpel hypothesis (Fig. 4A: for references, see Introduction). According to the other carpel hypotheses, there are considerable difficulties in interpreting the pistil structure of the mutant flowers of A. thaliana. For example, the FL40 mutant flowers show a variable feature of having a longitudinal split in the pistil in the median position, exposing the ovules. According to the fourcarpel hypothesis (Fig. 4B), the structure of the split pistil of the FL40 mutant flower (open pistil, Figs. lC, 5A, C, E) should be interpreted as follows. One of the fertile carpels which forms the
Fig. 2. Flowers of Arubidopsis thnlium wild type and mutants. Bar =l mm in panels D, E, F; bar =lOO pm in other panels. A, transverse section of a pistil of wild type; 1, lateral vascular bundle; m, median vascular bundle; o, ovule; st, septum tissue (pink-staining tissue). B, transverse section of a young pistil of wild type. C, transverse section of a young bud of wild type: pi, pistil. D, a pistil of a wild type flower. E, a pistil of the Fl-89 mutant; h, horn-shaped projection; sp, stigmatic papillae. F, a pistil of the Fl-82 mutant. G, transverse section of a Fl-82 mutant flower. H, a schematic drawing of panel G. The ovules and the pink-staining tissue are painted in green and purple, respectively. Black spots indicate the position of the major vascular bundles. Dotted lines indicate the border of the hypothetical ‘pistil-forming units’. I, transverse section of a young bud of the FL-82 mutant: pi, pistil primordium. J, transverse section of a pistil of the Fl-165 mutant. A small panel at the bottom left is an enlargement of a portion of panel J (indicated by 5 dots). Arrows show the separated median vascular bundle. K, transverse section of a pistil of the Fl-89 mutant. L, young pistil primordium of wild type. M, young pistil primordium of the Fl-89 mutant.
32
33
placentae in the median position (carpel 2 of Fig. 4B) is split into two halves and the ‘half-carpels’ remain attached at the margins of the sterile lateral carpels (carpels 3 and 4 of Fig. 4B). The converted sepal of the Fl-40 mutant (Fig. 5B, H) should also be regarded as a complex structure composed of one sterile carpel accompanied by two half-carpels on each side. Since other homeotic conversions in this organism have all been of one whole floral organ to another, we believe that the converted sepal of Fl-40 is not such a complex structure, but more likely represents a single carpel. The aberrant structures of other floral mutants are also best explained in terms of the ‘pistil-forming unit’ hypothesis as described below. Mutational defects in the development and morphogenesis of pistils We have analyzed six flower mutants having aberrant pistil structures and the results are summarized in Table I. The mutants have been divided into three groups on the basis of the developmental process of pistil formation where genetic defects seem to occur. Control of the number and arrangement of ‘pistil units’ One group of the mutants is thought to have mutational defects in an early stage(s) of pistil formation where pistil primordia are formed from two units laterally arranged at the center of a flower bud. The analysis of four mutants (Fl-40, Fl-82, clavata I and pistillata 1) seems to indicate that the number and arrangement of the units are genetically determined. The clavata I (&I) mutant has club-like pistils (Fig. 6A) (McKelvie, 1962). Ovaries have four locules separated by crossed septa (Fig. 6C). As shown in the schematic drawing (Fig. 6D), there are eight major vascular bundles whose positions relative to the placentae are identical with those of wild-type pistils; four bundles are observed at the
TABLE
I
Pistil structure
and fertility
of the mutant
lines and fertility
Mutant line
Mutation
Pistil structure
Fl-40
single recessive on chr. 4
Some flowers have pistils split on one side (open pistils). Open pistils are sterile. Sepals are homeotically converted to carpel-like structures.
Fl-82
recessive nuclear
Pistils are large and usually consist of three or more ‘pistil-forming units’. Pistils are semi-sterile.
Fl-89
single recessive on chr. 4
Pistils have two clumps of stigmatic papillae and two horn-like projections on the stigma. In the ovary, the septum is not fused. Upper part of ovary is sometimes split. Pistils are mostly sterile.
Fl-165
recessive nuclear
Pistils have thin ovaries and a long style. In the ovary, the septum is not fused. Median vascular bundles are often divided into two separate bundles. Pistils are mostly sterile.
clauata 1
single recessive on chr. 1
Club-shaped pistils. Ovaries have four locules separated by a cross-shaped septum. Pistils are fertile.
single recessive on chr. 5
Ovaries have multiple locules separated by an irregularlyformed septum. Pistils are fertile.
pistillata
I
base of the septa near the placentae at comparable positions to the median bundles of the wild type, and the other four bundles are located within the
Fig. 5. Flowers of A. thaliana mutants. In panels F and G, the ovules are painted in green. Black spots indicate the position of the major vascular bundles. Bar = 500 pm in panels A-C; bar = 100 pm in panels D, E, H. A, A Fl-40 mutant flower with an open pistil: cs, converted sepal; op, open pistil. B, a converted sepal of the Fl-40 mutant flower: o, ovule; sp, stigmatic papillae. C, an open pistil of the Fl-40 mutant flower cut on the transverse plane to show inside: o, ovule. D, transverse section of a young bud of the Fl-40 mutant: cs, primordium of the converted sepal; o, ovule primordium; op, primordium of the open pistil. An arrowhead shows the splitting site of the open pistil. E, transverse section of a mature open pistil shown in panel C: o, ovule. Arrowheads show the major vascular bundles. F, a schematic drawing of panel E. G, a schematic drawing of panel H. H, transverse section of the converted sepal: o, ovule; sp, stigmatic papillae. Arrowheads show the major vascular bundles.
34
D
1 G
35
ovary wall between the two bundles near the placentae. The loosely packed pink-staining tissue is observed at the intersection of the crossed septa. At an early stage of flower development, four swellings are observed in the young pistil primordium (Fig. 6B). In the context of the pistil unit hypothesis, it could be assumed, therefore, that the pistil of the clul mutant consists of four pistil units which are arranged in a symmetrically ordered fashion and grow coordinately. The number of the units forming a pistil was always four in the 12 flowers examined. This mutant strain has a single, recessive mutation mapped on chromosome 1 (Koornneef et al., 1983). The large pistil of the Fl-82 mutant consisting of several folded structures with ovules and the pink-staining tissue (Fig. 2G) could be interpreted as a four-unit structure, in which the units show incomplete marginal fusion as illustrated schematically in Fig. 2H. However, unlike those of the clul mutant, the units appear to be arranged in a disordered fashion. The disordered pistil primordium is also observed at an early stage which corresponds to stage I of the wild type (Fig. 21). The number of units varies from 3 to 8 (average 4) in the 12 flowers examined. The Fl-82 mutant has recessive, nuclear mutation(s) which are.non-allelic with the Fl-40, &I or pi1 mutations (Komaki et al., 1988). The pistillata 1 (pil) mutant has several structural abnormalities in the flowers; homeotic conversion of petals into sepals, abnormal stamens and pistils (Haughn and Somerville, 1988; Bowman et al., 1989). The mutation is single and recessive, and has been mapped on chromosome 5 (Koornneef et al., 1983). The shape of the pistil is not a simple cylinder as in the wild type, rather the pistils are partly swollen as if small domes were attached on the cylindrical surface (Fig. 6E).
Development of stigmatic papillae is usually poor. Microscopic analyses of the continuous transverse sections show that the septum is formed irregularly. In some pistils, the epidermal tissue of the ovary wall is not fused at the upper part of the ovary (Fig. 6F), but it is fused in the lower part of the same pistil (Fig. 6H). The pink-staining tissue is not fused at the upper part, although it is fused in the lower part of the ovary. As illustrated schematically (Fig. 6G), ovules are attached to the base of the septum. Although the inner structure of the ovary is aberrant, the pistil of the pi2 mutant is fertile. The number of units per pistil was 3 or 4 in the 9 flowers examined. The pi1 mutation results in an abnormal number and somewhat disordered arrangement of the ‘pistil units’ in the pistil primordia. Section analysis of young pistil primordia also indicates aberrantly arranged units (data not shown). Although the mature pistils are closed, the septa pattern is irregular, possibly because the units in the pistil primordium are not exactly in a symmetrical position and/or the coordinate growth of the units is disturbed. As described above, some flowers of the Fl-40 mutant have open pistils (Fig. 5A, C). Examination of young Fl-40 pistils has shown that they are split in the median position in the early stages of development, indicating that the defect occurs in the organization of the primordium and is not due to splitting of a phenotypically normal pistil later (Fig. 5D). The mutation responsible for the Fl-40 phenotype is single, recessive, allelic to the apetala 2 mutation and has been mapped on chromosome 4 (Komaki et al., 1988). Growth of pistil primordia At an intermediary stage of wild type pistil development and morphogenesis, coordinate growth of the joined pistil units occurs. During development, ovule primordia
Fig. 6. Flowers of A. thaliana mutants. In panels D and G, the ovules and the pink tissue are painted in green and purple, respectively. Black spots indicate the position of the major vascular bundles. Dotted lines indicate the border of the hypothetical ‘pistil-forming units’. Bar = 500 pm in panels A, E; bar =lOO pm in panels B, C, F, H. A, a pistil of the clauara I mutant. B, transverse section of a young bud of the clauata I mutant: pi, pistil primordium. C, transverse section of a mature pistil of the clauata I mutant. D, a schematic drawing of panel C. E, a pistil of the pistillata 1 mutant. F, transverse section of a pistil of the pisrilfara I mutant. The place where the ovary wall splits and the space protruding into the pink tissue are indicated by a horizontal arrow and a vertical arrow, respectively. G, a schematic drawing of panel F. H, transverse section of a mutant pistil of pistillata I, sectioned 200 pm below the section shown in panel F.
36
and four major vascular bundles begin to appear (Fig. 3B); two median bundles are formed as the fused form of the marginal bundles of the units, and two lateral bundles corresponding to the central bundle of the units. The Fl-165 mutant seems to have a mutational defect at this stage, since the median vascular bundle is often separated into two bundles (Fig. 25). In addition, the septum is poorly developed and not fused. The pink-staining tissue is not developed at all. Nor is the epidermal tissue of the ovary wall fully developed at the placentae (Fig. 25). These abnormalities could be ascribed to the uncoordinated growth of the two units in the pistil primordium. This mutation is recessive and nuclear. Most of the flowers are sterile due to a functional defect in the pistils, although the pollen grains are fertile. Maturation of pistil At the third and last stage, maturation of the pistils occurs. A stigma having a clump of papillae is formed at the top of the pistils, and the ovules mature. The septum tissues generated on both sides meet and fuse at the center and the transmitting tissue develops at the center of the septum. Genetic defects at this stage were observed in the Fl-89 and FL165 mutants. As reported previously (Komaki et al., 1988), the stigma and the septum of the pistils are abnormal in the Fl-89 mutant. There are two horn-like projections and two clumps of the papillae on the stigma (Fig. 2E). At an early stage of pistil development, when the round top is formed in the wild type, four swellings are observed at the top of the mutant pistil primordia which develop two horns and two stigmatic papillae (Fig. 2M). Transverse sections show that the septa are not fused (Fig. 2K) but growth of the septum tissue is better than that in the FL165 mutant. The defects of this mutant appear in the formation of the stigma and septum. The Fl-89 mutation was shown to be single, recessive and nuclear (Komaki et al., 1988). The Fl-89 and the FL165 mutations have been shown to be non-allelic. An analysis of various mutant flowers has given new insight into the developmental origin of the pistils of Brassicaceae flowers, and the evidence strongly supports the two-carpel hypothesis. It is considered that many genes are responsible for the
regulation of pistil development and morphogenesis. The genes responsible for the six flower mutants described above represent, in all likelihood, part of the group of genes that play particularly important roles in the process. It is of great interest to determine whether the genes are expressed and if they function in a sequential manner as discussed above. Identification and isolation of the genes is in progress in our laboratory. When the genes are identified, their structure and expression will provide clues for understanding the mechanism of pistil morphogenesis and development. A genetic approach using A. thaliana appears to be very promising for elucidating basic molecular mechanisms in higher plants.
Acknowledgements
We thank Drs. K.R. Krantz (Arabidopsis Information Service, J.W. Goethe-Universitat, Frankfurt-am-Main, F.R.G.) and E.M. Meyerowitz (Caltech, Pasadena, CA) for providing seeds, Dr. C.R. Somerville (Michigan State University) for the procedures of mutagenesis, cultivation and artificial pollination and Dr. C.J. Bell (NIBB) for critical reading of the manuscript. This work was supported, in part, by grants from the Ministry of Education, Science and Culture and by funds from the CIBA-GEIGY Foundation for the Promotion of Science and from the Naito Memorial Foundation for the Promotion of Science.
References Allexander, I.: Entwicklungsstudien an Bltiten von Cruciferen und Papaveraceen. Planta 40, 125-144 (1952). Bowman, J.L., D.R. Smyth and E.M. Meyerowitz: Genes directing flower development in Arubidopsis. Plant Cell 1, 37-52 (1989). Eames, A.J.: The vascular anatomy of the flower with refutation of the theory of carpel polymorphism. Am. J. Bot. 18, 147-188 (1931). Eames, A.J. and C.L. Wilson: Carpel morphology in the Cruciferae. Am. J. Bot. 15, 251-270 (1928). Fames, A.J. and C.L. Wilson: Crucifer carpels. Am. J. Bot. 17, 638-656 (1930). Eggers, 0.: iiber die morphologische Bedeutung des Leitbtindelverlaufes in den Bltiten der Rhoeadalen und
37 tiber das Diagramrn der Cruciferen und Capparidaceen. Planta 24, 14-58 (1935). EichIer, A.W.: Uber den Bltithenbau der Fumariaceen, Cruciferen und einiger Capparideen. Flora 48, 433-444, 449-460, 497-511, 513-521, 545-559 (1865). Eigner, von V.: Zur Stempel- und Fruchtentwickhtng ausgew&her Brassicaceae ( = Cruciferae) unter neueren Gesichtspunkten der Bltitenmorphologie und der Systematik. Bietr. Biol. Pflanzen. 49, 359-429 (1973). Esau, K.: The flower: structure and development. In: Anatomy of Seed Plants, 2nd ed., Chap 20, (John Wiley & Sons, New York) pp. 375-401 (1977). Estelle, M.A. and CR. Somerville: The mutants of Arubidopsis. Trends Genet. 2, 89-93 (1986). Fink, G.R.: Notes of a bigamous biologist. Genetics 118, 549-550 (1988). Haughn, G.W. and CR. Somerville: Genetic control of morphogenesis in Arubidopsis. Dev. Genet. 9, 73-85 (1988). Komaki, M.K., K. Okada, E. Nishino and Y. Shimura: Isolation and characterization of novel mutants of Arabidopsis thakmu defective in flower development. Development 104, 195-203 (1988). Koomneef, M., J. Van Eden, C.J. Hanhart, P. Stam, F.J. Braaksma and W.J. Feenstra: Linkage map of Arubidopsis thuliunu. J. Hered. 74, 265-272 (1983). Lawrence, G.H.: Taxonomy of Vascular Plants (Macmillan, New York and London) pp. 520-524 (1951). McKelvie, A.D.: A list of mutant genes in Arubidopsis thuliunu (L.) Heynh. Radiat. Bot. 1, 233-241 (1962).
Merxmtiller, H. and P. Leins: Die Verwandtschaftsbeziehungen der Kreuzbltitler und Mobngew;ichse. Bot. Jahrb. 86, 113-129 (1967). Meyerowitz, E.M.: Arabidopsis thaliunu. Annu. Rev. Genet. 21, 93-111 (1987). Meyerowitz, E.M.: Arabidopsis, a useful weed. Cell 56,263-269 (1989). Meyerowitz, E.M. and R.E. Pruitt: Arubidopsis thaliunu and plant molecular genetics. Science 229, 1214-1218 (1985). Puri, V.: Studies in floral anatomy. I. Gynoecium constitution in the Cruciferae. Proc. Ind. Acad. Sci. 14, 166-187 (1941). Puri, V.: Studies in floral anatomy. III. Gn the origin and orientation of placental strains. Proc. Nat. Acad. Sci. India 15, 74-91 (1945). Redei, G.P.: Arabidopsis as a genetic tool. Annu. Rev. Genet. 9, 111-127 (1975). Sattler, R.: Organogenesis of Flowers. A Photographic TextAtlas. (Univ. Toronto Press, Toronto and Buffalo) pp. 68-71 (1973). Saunders, E.R.: On carpel polymorphism: I. Ann. Bot. 39, 123-167 (1925). Saunders, E.R.: Gn a new view of the nature of the median carpels in the Cruciferae. Am. J. Bot. 16, 122-137 (1929). Spratt, E.R.: The gynoecium of the family Cruciferae. J. Bot. 70, 308-314 (1932). Yen, C.: On a new view of carpel morphology. Acta Bot. Sinica. 8, 271-280 (1959).
and Development,
21
28 (1989) 27-38
Elsevier Scientific Publishers Ireland. Ltd. CELDIF 00609
Mutational analysis of pistil structure and development of Arabidopsis thaliana Kiyotaka
Okada ‘, Masako
K. Komaki
’ and Yoshiro
Shimura
I*2
’ Division of Gene Expression and Regulation, National Institute for Basic Biology, Okazaki 444, Japan and 2 Department
of Biophysics, Faculty of Science, Kyoto University, Kyoto 606, Japan
(Accepted 21 May 1989)
The developmental and morphogenetic process of pistil formation was examined by analysing flowers of wild type and six flower mutants of Arubidopsis flraliana, a small crucifer. The wild type is suggested to originate from two ‘pistil-forming units’ (carpels) arranged laterally against the axis of the inflorescence at a pistil primordium. Aberrant structures of the pistils of mutants indicate that a set of genes regulate each step of pistil development and morphogenesis, namely arrangement of the units at the pistil primordia, fusion of the units, growth of primordia, formation of the septum in the ovary, and formation of the stigma.
Arubidopsis thuhimu; Flower mutant; Pistil structure; Floral development; Carpel theory; Pistil organogenesis
Introduction
The flower is a reproductive organ in angiosperms, consisting of several floral organs (sepals, petals, stamens and pistil) arranged in whorls or concentric circles. Flower development is a complex process, involving the temporally and spacially controlled initiation of primordia and their coordinated development into mature floral organs. A number of mutants of Arubidopsis thaliana, in which these processes are defective, have been isolated and characterized (Haughn and Somerville, 1988; Komaki et al., 1988; Bowman et al., 1989), suggesting that flowering in this plant is regulated by a number of genes. A. thuliunu, a
Correspondence address: K. Okada, Division of Gene Expression and Regulation, Institute for Basic Biology, Okazaki 444, Japan.
member of the Brassicaceae, has various properties that make it suitable for classical and molecular genetic studies (Redei, 1975; Meyerowitz and Pruitt, 1985; Estelle and Somerville, 1986; Meyerowitz, 1987,1989; Fink, 1988) and the analysis of the flower mutants is expected to be a useful approach in pursuing an understanding of the genetic control of floral development and morphogenesis. Genetic analysis of these mutants has revealed that a single, recessive mutation may result in aberrant arrangement and/or structure of one or several floral organs (Komaki et al., 1988). In the course of systematic morphological analysis of the mutant flowers, we encountered some intriguing problems which led us to examine existing theories concerning morphogenesis in higher plants. For instance, pistil development. and morphogenesis posed an interesting problem in some mutant flowers. The pistil is a complex organ with apparently unique developmental
0922-3371/89/$03.50 0 1989 Elsevier Scientific Publishers Ireland, Ltd.
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processes. On the basis of morphological studies, several hypotheses have been proposed on the origin of the pistil of Brassicaceae, which remain totally unsettled. One of the hypotheses is the ‘ two-lateral-carpel hypothesis’ which assumes that pistils are made by fusion of two carpels located in lateral positions against the inflorescence axis (Eichler, 1865; Eggers, 1935; Allexander, 1952; Esau, 1977). On the other hand, according to a second hypothesis, the ‘four-carpel hypothesis’, pistils are considered to be made of four carpels; two of them form the ovary wall, and the other two form the placentae (Saunders, 1925, 1929; Eames and Wilson, 1928, 1930; Pm-i, 1941, 1945; Merxmtiller and Leins, 1967; Eigner, 1973). The latter hypothesis was based mainly on the arrangement of vascular bundles and the stigma structure (for a review see Lawrence, 1951). There are also other hypotheses claiming that pistils are formed by two carpels positioned on the median plane (Spratt, 1932) or by six carpels (Yen, 1959). Studies on the pistil formation of Brassicaceae flowers have mostly involved microscopic analysis of developing flower buds (Sattler, 1973). In this paper, we present our view on pistil formation based on detailed morphological analyses of several flower mutants as well as evidence indicating that a set of genes participates in pistil formation in A. thaliana. Materials and Methods
Plant lines An A. thaliana wild-type strain, Landsberg (erecta), and a mutant line M7 (apetala 1, clavata I) were obtained from the Arabidopsis Information Service (J.W. Goethe-Universitat, Frankfurtam-Main, F.R.G.). Another mutant line, pistillata I, was a gift from Dr. E.M. Meyerowitz (Caltech, Pasadena, CA). Mutant lines of Fl-series were isolated in our laboratory. Isolation and genetic analyses of the mutants (Fl-40, Fl-82 and Fl-89) were described previously (Komaki et al., 1988). Fl-165 is a newly isolated mutant. Cultivation of plants Plants were grown in accordance with the procedures of Dr. C.R. Somerville (Michigan State
University, personal communication) tinuous illumination (24 L) at 22 o C.
under con-
Thin section analysis Thin sections of flowers were prepared and stained by Azure B, as described by Komaki et al. (1988).
Results and Discussion
Pistil structure and development of Arabidopsis thaliana wild type The floral structure of A. thaliana wild type is typical of the Brassicaceae (Fig. 1A). The pistil is a green cylinder about 2.5 mm long and 0.4 mm wide in mature flowers (Figs. lB, 2D). A long ovary is surmounted by a short style and a clump of stigmatic papillae. Transverse sections show that the ovary is separated into two rooms (biloculous) by a septum (Fig. 2A). The septum may be histologically divided into three regions; central region and two side regions (placental tissues). The central region (the transmitting tissue) consists of loosely-packed cells that are stained pink by Azure B. The tissues of the side regions are indistinguishable from the tissue of the inner layer of the ovary wall. Ovules are attached to the placenta located in the region where the septum merges with the ovary wall. A total of about 30 ovules are lined up in four rows in a wild type pistil. There are four major vascular bundles (two lateral and two median bundles) running parallel to the longitudinal axis of the ovary (Figs. lB, 2A). Small axillary bundles derived from the major bundles appear when the pistil is fertilized, and these develop into pods. Although the presence of an extra vascular bundle in which the positions of the xylem and phloem are reversed was reported in other species of Brassicaceae (Eames and Wilson, 1928, 1930; Puri, 1941, 1945), we could not detect such bundles in the ovary of A. thaliana. The process of the pistil development from undifferentiated meristem to mature flowers may be separated into four stages which are shown schematically in Fig. 3A-D. The pistil originates from a relatively large primordium which appears at the center of the floral meristem. At a very
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Fig. 1. Schematic drawings of Arabidopsis rholima flowers and the hypothetical unit structure. Panels A and E and the upper figures in B, C, and D are transverse sections; the lower figures in B, C, and D are side views: cb, central vascular bundle; fmb, fused marginal vascular bundle; ia, infloresoence axis; 1, lateral vascular bundle; m, median vascular bundle; mb, marginal vascular bundle; o, ovule; ov, ovary; p, petal; pfu, pistil-forming unit; pi, pistil; s, sepal; sp, stigmatic papillae; st, septum tissue; sy, style. A, a wild type flower; B, a normal pistil of the wild type; C, an ‘open pistil’ of the FL40 mutant; D, the proposed hypothetical ‘pistil-forming unit’; E, a hypothetical structure of the pistils of Brassicaceae. Two ‘pistil-forming units’ are arranged laterally to the axis of inflorescence.
early stage when the cylindrical wall of the pistil is formed (stage I), two swellings develop from opposite sides of the wall on the median plane (Figs. 2C, 3A). Ovule primordia develop at the side of the swellings (stage II, Fig. 3B) and these swellings begin to fuse when the young ovules appear (stage III, Figs. 2B, 3C). The loosely packed pink-staining tissue in the centre of the septum develops in the last stage just before flowering (Figs. 2A, 3D). Structural changes are also observed at the top of the pistil primordia. At early stages (stages I-II) the top of the cylindrical pistil primordia is open. As development proceeds, the opening is closed to form a rounded top (stages II-III, Fig. 2L), and then stigmatic papillae develop (stage IV). To investigate the genetic regulation of pistil development and morphogenesis, we analyzed the structure and development of some mutant flowers of A. thaliana. Pistil unit hypothesis Pistil development and structure were examined in more than nine flowers of each of the six mutant lines and the mutant phenotypes were
found to be quite stable. The mutants showed a variety of abnormalities in pistil structure and in the course of the analyses it was found that the abnormalities could be best explained if we hypothesised the existence of a ‘pistil-forming unit’. This hypothesis is explained by reference to some of the floral mutants. The Fl-40 mutant has a homeotic conversion in which the sepals are replaced by leaf-like structures bearing ovules along their edges (converted sepal; Fig. 5B, H) (komaki et al., 1988). In other homeotic mutants of A. pistillata 1 and apetela 2, petals are converted to sepals in the former and in the latter sepals are converted to leaves and petals are converted into stamens (Haughn and Somerville, 1988; Bowman et al., 1989). In these cases there is an entire conversion of one floral organ to another and if this is also true of Fl-40, the structure replacing the sepals in this mutant may be interpreted as a developmental ‘pistil-forming unit’. The structure of the putative unit is a leaf-like structure curving inward having three major vascular bundles, one central and two marginal bundles with the placentae located at the margins
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Fig. 3. Schematic drawings of pistils of A. fMiuna wild type at different developmental stages. Redrawn from transverse sections. Sixes are ca. 75 gm (A), 100 pm (B), 200 pm (C) and 400 pm (D) in diameter. The position of the axis of the inflorescence is shown relative to the orientation of the pistil: ia, inflorescence axis; 1, lateral vascular bundle; m, median vascular bundle; o, ovule; op, ovule primordium; s, septum; yo, young ode. A, a very young pistil (stage I). A pistil of this stage is shown in Fig. 2C. B, a young pistil which shows swellings corresponding to ovule primordia. Four vascular bundles begin to appear (stage II). C, a young pistil. The septum tissues grow and meet at the center. Young ovules develop integuments (stage III). A pistil of this stage is shown in Fig. 2B. D, a mature pistil (stage IV), A pistil of this stage is shown in Fig. 2A.
(Fig. 1D). The wild type pistil may then be seen to consist of two units which are laterally arranged against the axis of inflorescence as shown in Fig. 1E. The marginal vascular bundles of the units are shared and appear as the median bundles in the developing pistil. Although the structural unit hypothesis described above is based on the anatomical and genetic analyses of mutant flowers of a single species, A. thaliana, and not on phylogenic studies on several evolutionarily related species, the structure of the proposed unit is almost identical to the hypothetical carpel structure deduced from phylogenic analyses (Eames, 1931), except
A
Fig. 4. Carpel hypotheses explaining pistil structure of Brassicaceae. Arrows and dotted lines indicate the hypothetical border of the carpels. The postion of the axis of inflorescence (ia) is shown relative to the orientation of the pistil. A, the twolateral-carpel hypothesis. B, the four-carpel hypothesis. Carpels 1 and 2 (in the median position) are fertile carpels which carry ovules and lost locules. Carpels 3 and 4 (in the lateral position) are sterile carpels forming an ovary wall. C, the two-mediancarpel hypothesis, which is the only one proposing that ovules are generated in the central region.
for the presence of septum tissue which represents a specific characteristic of A. thaliana and possibly of the Brassicaceae. This interpretation strongly supports the twolateral-carpel hypothesis (Fig. 4A: for references, see Introduction). According to the other carpel hypotheses, there are considerable difficulties in interpreting the pistil structure of the mutant flowers of A. thaliana. For example, the FL40 mutant flowers show a variable feature of having a longitudinal split in the pistil in the median position, exposing the ovules. According to the fourcarpel hypothesis (Fig. 4B), the structure of the split pistil of the FL40 mutant flower (open pistil, Figs. lC, 5A, C, E) should be interpreted as follows. One of the fertile carpels which forms the
Fig. 2. Flowers of Arubidopsis thnlium wild type and mutants. Bar =l mm in panels D, E, F; bar =lOO pm in other panels. A, transverse section of a pistil of wild type; 1, lateral vascular bundle; m, median vascular bundle; o, ovule; st, septum tissue (pink-staining tissue). B, transverse section of a young pistil of wild type. C, transverse section of a young bud of wild type: pi, pistil. D, a pistil of a wild type flower. E, a pistil of the Fl-89 mutant; h, horn-shaped projection; sp, stigmatic papillae. F, a pistil of the Fl-82 mutant. G, transverse section of a Fl-82 mutant flower. H, a schematic drawing of panel G. The ovules and the pink-staining tissue are painted in green and purple, respectively. Black spots indicate the position of the major vascular bundles. Dotted lines indicate the border of the hypothetical ‘pistil-forming units’. I, transverse section of a young bud of the FL-82 mutant: pi, pistil primordium. J, transverse section of a pistil of the Fl-165 mutant. A small panel at the bottom left is an enlargement of a portion of panel J (indicated by 5 dots). Arrows show the separated median vascular bundle. K, transverse section of a pistil of the Fl-89 mutant. L, young pistil primordium of wild type. M, young pistil primordium of the Fl-89 mutant.
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placentae in the median position (carpel 2 of Fig. 4B) is split into two halves and the ‘half-carpels’ remain attached at the margins of the sterile lateral carpels (carpels 3 and 4 of Fig. 4B). The converted sepal of the Fl-40 mutant (Fig. 5B, H) should also be regarded as a complex structure composed of one sterile carpel accompanied by two half-carpels on each side. Since other homeotic conversions in this organism have all been of one whole floral organ to another, we believe that the converted sepal of Fl-40 is not such a complex structure, but more likely represents a single carpel. The aberrant structures of other floral mutants are also best explained in terms of the ‘pistil-forming unit’ hypothesis as described below. Mutational defects in the development and morphogenesis of pistils We have analyzed six flower mutants having aberrant pistil structures and the results are summarized in Table I. The mutants have been divided into three groups on the basis of the developmental process of pistil formation where genetic defects seem to occur. Control of the number and arrangement of ‘pistil units’ One group of the mutants is thought to have mutational defects in an early stage(s) of pistil formation where pistil primordia are formed from two units laterally arranged at the center of a flower bud. The analysis of four mutants (Fl-40, Fl-82, clavata I and pistillata 1) seems to indicate that the number and arrangement of the units are genetically determined. The clavata I (&I) mutant has club-like pistils (Fig. 6A) (McKelvie, 1962). Ovaries have four locules separated by crossed septa (Fig. 6C). As shown in the schematic drawing (Fig. 6D), there are eight major vascular bundles whose positions relative to the placentae are identical with those of wild-type pistils; four bundles are observed at the
TABLE
I
Pistil structure
and fertility
of the mutant
lines and fertility
Mutant line
Mutation
Pistil structure
Fl-40
single recessive on chr. 4
Some flowers have pistils split on one side (open pistils). Open pistils are sterile. Sepals are homeotically converted to carpel-like structures.
Fl-82
recessive nuclear
Pistils are large and usually consist of three or more ‘pistil-forming units’. Pistils are semi-sterile.
Fl-89
single recessive on chr. 4
Pistils have two clumps of stigmatic papillae and two horn-like projections on the stigma. In the ovary, the septum is not fused. Upper part of ovary is sometimes split. Pistils are mostly sterile.
Fl-165
recessive nuclear
Pistils have thin ovaries and a long style. In the ovary, the septum is not fused. Median vascular bundles are often divided into two separate bundles. Pistils are mostly sterile.
clauata 1
single recessive on chr. 1
Club-shaped pistils. Ovaries have four locules separated by a cross-shaped septum. Pistils are fertile.
single recessive on chr. 5
Ovaries have multiple locules separated by an irregularlyformed septum. Pistils are fertile.
pistillata
I
base of the septa near the placentae at comparable positions to the median bundles of the wild type, and the other four bundles are located within the
Fig. 5. Flowers of A. thaliana mutants. In panels F and G, the ovules are painted in green. Black spots indicate the position of the major vascular bundles. Bar = 500 pm in panels A-C; bar = 100 pm in panels D, E, H. A, A Fl-40 mutant flower with an open pistil: cs, converted sepal; op, open pistil. B, a converted sepal of the Fl-40 mutant flower: o, ovule; sp, stigmatic papillae. C, an open pistil of the Fl-40 mutant flower cut on the transverse plane to show inside: o, ovule. D, transverse section of a young bud of the Fl-40 mutant: cs, primordium of the converted sepal; o, ovule primordium; op, primordium of the open pistil. An arrowhead shows the splitting site of the open pistil. E, transverse section of a mature open pistil shown in panel C: o, ovule. Arrowheads show the major vascular bundles. F, a schematic drawing of panel E. G, a schematic drawing of panel H. H, transverse section of the converted sepal: o, ovule; sp, stigmatic papillae. Arrowheads show the major vascular bundles.
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D
1 G
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ovary wall between the two bundles near the placentae. The loosely packed pink-staining tissue is observed at the intersection of the crossed septa. At an early stage of flower development, four swellings are observed in the young pistil primordium (Fig. 6B). In the context of the pistil unit hypothesis, it could be assumed, therefore, that the pistil of the clul mutant consists of four pistil units which are arranged in a symmetrically ordered fashion and grow coordinately. The number of the units forming a pistil was always four in the 12 flowers examined. This mutant strain has a single, recessive mutation mapped on chromosome 1 (Koornneef et al., 1983). The large pistil of the Fl-82 mutant consisting of several folded structures with ovules and the pink-staining tissue (Fig. 2G) could be interpreted as a four-unit structure, in which the units show incomplete marginal fusion as illustrated schematically in Fig. 2H. However, unlike those of the clul mutant, the units appear to be arranged in a disordered fashion. The disordered pistil primordium is also observed at an early stage which corresponds to stage I of the wild type (Fig. 21). The number of units varies from 3 to 8 (average 4) in the 12 flowers examined. The Fl-82 mutant has recessive, nuclear mutation(s) which are.non-allelic with the Fl-40, &I or pi1 mutations (Komaki et al., 1988). The pistillata 1 (pil) mutant has several structural abnormalities in the flowers; homeotic conversion of petals into sepals, abnormal stamens and pistils (Haughn and Somerville, 1988; Bowman et al., 1989). The mutation is single and recessive, and has been mapped on chromosome 5 (Koornneef et al., 1983). The shape of the pistil is not a simple cylinder as in the wild type, rather the pistils are partly swollen as if small domes were attached on the cylindrical surface (Fig. 6E).
Development of stigmatic papillae is usually poor. Microscopic analyses of the continuous transverse sections show that the septum is formed irregularly. In some pistils, the epidermal tissue of the ovary wall is not fused at the upper part of the ovary (Fig. 6F), but it is fused in the lower part of the same pistil (Fig. 6H). The pink-staining tissue is not fused at the upper part, although it is fused in the lower part of the ovary. As illustrated schematically (Fig. 6G), ovules are attached to the base of the septum. Although the inner structure of the ovary is aberrant, the pistil of the pi2 mutant is fertile. The number of units per pistil was 3 or 4 in the 9 flowers examined. The pi1 mutation results in an abnormal number and somewhat disordered arrangement of the ‘pistil units’ in the pistil primordia. Section analysis of young pistil primordia also indicates aberrantly arranged units (data not shown). Although the mature pistils are closed, the septa pattern is irregular, possibly because the units in the pistil primordium are not exactly in a symmetrical position and/or the coordinate growth of the units is disturbed. As described above, some flowers of the Fl-40 mutant have open pistils (Fig. 5A, C). Examination of young Fl-40 pistils has shown that they are split in the median position in the early stages of development, indicating that the defect occurs in the organization of the primordium and is not due to splitting of a phenotypically normal pistil later (Fig. 5D). The mutation responsible for the Fl-40 phenotype is single, recessive, allelic to the apetala 2 mutation and has been mapped on chromosome 4 (Komaki et al., 1988). Growth of pistil primordia At an intermediary stage of wild type pistil development and morphogenesis, coordinate growth of the joined pistil units occurs. During development, ovule primordia
Fig. 6. Flowers of A. thaliana mutants. In panels D and G, the ovules and the pink tissue are painted in green and purple, respectively. Black spots indicate the position of the major vascular bundles. Dotted lines indicate the border of the hypothetical ‘pistil-forming units’. Bar = 500 pm in panels A, E; bar =lOO pm in panels B, C, F, H. A, a pistil of the clauara I mutant. B, transverse section of a young bud of the clauata I mutant: pi, pistil primordium. C, transverse section of a mature pistil of the clauata I mutant. D, a schematic drawing of panel C. E, a pistil of the pistillata 1 mutant. F, transverse section of a pistil of the pisrilfara I mutant. The place where the ovary wall splits and the space protruding into the pink tissue are indicated by a horizontal arrow and a vertical arrow, respectively. G, a schematic drawing of panel F. H, transverse section of a mutant pistil of pistillata I, sectioned 200 pm below the section shown in panel F.
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and four major vascular bundles begin to appear (Fig. 3B); two median bundles are formed as the fused form of the marginal bundles of the units, and two lateral bundles corresponding to the central bundle of the units. The Fl-165 mutant seems to have a mutational defect at this stage, since the median vascular bundle is often separated into two bundles (Fig. 25). In addition, the septum is poorly developed and not fused. The pink-staining tissue is not developed at all. Nor is the epidermal tissue of the ovary wall fully developed at the placentae (Fig. 25). These abnormalities could be ascribed to the uncoordinated growth of the two units in the pistil primordium. This mutation is recessive and nuclear. Most of the flowers are sterile due to a functional defect in the pistils, although the pollen grains are fertile. Maturation of pistil At the third and last stage, maturation of the pistils occurs. A stigma having a clump of papillae is formed at the top of the pistils, and the ovules mature. The septum tissues generated on both sides meet and fuse at the center and the transmitting tissue develops at the center of the septum. Genetic defects at this stage were observed in the Fl-89 and FL165 mutants. As reported previously (Komaki et al., 1988), the stigma and the septum of the pistils are abnormal in the Fl-89 mutant. There are two horn-like projections and two clumps of the papillae on the stigma (Fig. 2E). At an early stage of pistil development, when the round top is formed in the wild type, four swellings are observed at the top of the mutant pistil primordia which develop two horns and two stigmatic papillae (Fig. 2M). Transverse sections show that the septa are not fused (Fig. 2K) but growth of the septum tissue is better than that in the FL165 mutant. The defects of this mutant appear in the formation of the stigma and septum. The Fl-89 mutation was shown to be single, recessive and nuclear (Komaki et al., 1988). The Fl-89 and the FL165 mutations have been shown to be non-allelic. An analysis of various mutant flowers has given new insight into the developmental origin of the pistils of Brassicaceae flowers, and the evidence strongly supports the two-carpel hypothesis. It is considered that many genes are responsible for the
regulation of pistil development and morphogenesis. The genes responsible for the six flower mutants described above represent, in all likelihood, part of the group of genes that play particularly important roles in the process. It is of great interest to determine whether the genes are expressed and if they function in a sequential manner as discussed above. Identification and isolation of the genes is in progress in our laboratory. When the genes are identified, their structure and expression will provide clues for understanding the mechanism of pistil morphogenesis and development. A genetic approach using A. thaliana appears to be very promising for elucidating basic molecular mechanisms in higher plants.
Acknowledgements
We thank Drs. K.R. Krantz (Arabidopsis Information Service, J.W. Goethe-Universitat, Frankfurt-am-Main, F.R.G.) and E.M. Meyerowitz (Caltech, Pasadena, CA) for providing seeds, Dr. C.R. Somerville (Michigan State University) for the procedures of mutagenesis, cultivation and artificial pollination and Dr. C.J. Bell (NIBB) for critical reading of the manuscript. This work was supported, in part, by grants from the Ministry of Education, Science and Culture and by funds from the CIBA-GEIGY Foundation for the Promotion of Science and from the Naito Memorial Foundation for the Promotion of Science.
References Allexander, I.: Entwicklungsstudien an Bltiten von Cruciferen und Papaveraceen. Planta 40, 125-144 (1952). Bowman, J.L., D.R. Smyth and E.M. Meyerowitz: Genes directing flower development in Arubidopsis. Plant Cell 1, 37-52 (1989). Eames, A.J.: The vascular anatomy of the flower with refutation of the theory of carpel polymorphism. Am. J. Bot. 18, 147-188 (1931). Eames, A.J. and C.L. Wilson: Carpel morphology in the Cruciferae. Am. J. Bot. 15, 251-270 (1928). Fames, A.J. and C.L. Wilson: Crucifer carpels. Am. J. Bot. 17, 638-656 (1930). Eggers, 0.: iiber die morphologische Bedeutung des Leitbtindelverlaufes in den Bltiten der Rhoeadalen und
37 tiber das Diagramrn der Cruciferen und Capparidaceen. Planta 24, 14-58 (1935). EichIer, A.W.: Uber den Bltithenbau der Fumariaceen, Cruciferen und einiger Capparideen. Flora 48, 433-444, 449-460, 497-511, 513-521, 545-559 (1865). Eigner, von V.: Zur Stempel- und Fruchtentwickhtng ausgew&her Brassicaceae ( = Cruciferae) unter neueren Gesichtspunkten der Bltitenmorphologie und der Systematik. Bietr. Biol. Pflanzen. 49, 359-429 (1973). Esau, K.: The flower: structure and development. In: Anatomy of Seed Plants, 2nd ed., Chap 20, (John Wiley & Sons, New York) pp. 375-401 (1977). Estelle, M.A. and CR. Somerville: The mutants of Arubidopsis. Trends Genet. 2, 89-93 (1986). Fink, G.R.: Notes of a bigamous biologist. Genetics 118, 549-550 (1988). Haughn, G.W. and CR. Somerville: Genetic control of morphogenesis in Arubidopsis. Dev. Genet. 9, 73-85 (1988). Komaki, M.K., K. Okada, E. Nishino and Y. Shimura: Isolation and characterization of novel mutants of Arabidopsis thakmu defective in flower development. Development 104, 195-203 (1988). Koomneef, M., J. Van Eden, C.J. Hanhart, P. Stam, F.J. Braaksma and W.J. Feenstra: Linkage map of Arubidopsis thuliunu. J. Hered. 74, 265-272 (1983). Lawrence, G.H.: Taxonomy of Vascular Plants (Macmillan, New York and London) pp. 520-524 (1951). McKelvie, A.D.: A list of mutant genes in Arubidopsis thuliunu (L.) Heynh. Radiat. Bot. 1, 233-241 (1962).
Merxmtiller, H. and P. Leins: Die Verwandtschaftsbeziehungen der Kreuzbltitler und Mobngew;ichse. Bot. Jahrb. 86, 113-129 (1967). Meyerowitz, E.M.: Arabidopsis thaliunu. Annu. Rev. Genet. 21, 93-111 (1987). Meyerowitz, E.M.: Arabidopsis, a useful weed. Cell 56,263-269 (1989). Meyerowitz, E.M. and R.E. Pruitt: Arubidopsis thaliunu and plant molecular genetics. Science 229, 1214-1218 (1985). Puri, V.: Studies in floral anatomy. I. Gynoecium constitution in the Cruciferae. Proc. Ind. Acad. Sci. 14, 166-187 (1941). Puri, V.: Studies in floral anatomy. III. Gn the origin and orientation of placental strains. Proc. Nat. Acad. Sci. India 15, 74-91 (1945). Redei, G.P.: Arabidopsis as a genetic tool. Annu. Rev. Genet. 9, 111-127 (1975). Sattler, R.: Organogenesis of Flowers. A Photographic TextAtlas. (Univ. Toronto Press, Toronto and Buffalo) pp. 68-71 (1973). Saunders, E.R.: On carpel polymorphism: I. Ann. Bot. 39, 123-167 (1925). Saunders, E.R.: Gn a new view of the nature of the median carpels in the Cruciferae. Am. J. Bot. 16, 122-137 (1929). Spratt, E.R.: The gynoecium of the family Cruciferae. J. Bot. 70, 308-314 (1932). Yen, C.: On a new view of carpel morphology. Acta Bot. Sinica. 8, 271-280 (1959).