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First published online September 8, 2006; 10.1104/pp.106.083543 Plant Physiology 142:972-983 (2006) © 2006 American Society of Plant Biologists
Isolation of mtpim Proves Tnt1 a Useful Reverse Genetics Tool in Medicago truncatula and Uncovers New Aspects of AP1-Like Functions in Legumes1Instituto de Biología Molecular y Celular de Plantas, Consejo Superior de Investigaciones Científicas-Universidad Politécnica de Valencia, 46022 Valencia, Spain (R.B., C.F., J.P.B., L.A.C., F.M.); and Institut des Sciences du Végétal, Centre National de la Recherche Scientifique, 91198 Gif sur Yvette cedex, France (I.d'E., V.C., A.K., P.R.)
Comparative studies help shed light on how the huge diversity in plant forms found in nature has been produced. We use legume species to study developmental differences in inflorescence architecture and flower ontogeny with classical models such as Arabidopsis thaliana or Antirrhinum majus. Whereas genetic control of these processes has been analyzed mostly in pea (Pisum sativum), Medicago truncatula is emerging as a promising alternative system for these studies due to the availability of a range of genetic tools. To assess the use of the retrotransposon Tnt1 for reverse genetics in M. truncatula, we screened a small Tnt1-mutagenized population using degenerate primers for MADS-box genes, known controllers of plant development. We describe here the characterization of mtpim, a new mutant caused by the insertion of Tnt1 in a homolog to the PROLIFERATING INFLORESCENCE MERISTEM (PIM)/APETALA1 (AP1)/SQUAMOSA genes. mtpim shows flower-to-inflorescence conversion and altered flowers with sepals transformed into leaves, indicating that MtPIM controls floral meristem identity and flower development. Although more extreme, this phenotype resembles the pea pim mutants, supporting the idea that M. truncatula could be used to complement analysis of reproductive development already initiated in pea. In fact, our study reveals aspects not shown by analysis of pea mutants: that the mutation in the AP1 homolog interferes with the specification of floral organs from common primordia and causes conversion of sepals into leaves, in addition to true conversion of flowers into inflorescences. The isolation of mtpim represents a proof of concept demonstrating that Tnt1 populations can be efficiently used in reverse genetics screenings in M. truncatula.
Recent studies have underlined the importance of comparative studies when trying to understand the evolution and divergence of different plant forms and shapes in nature (Vandenbussche et al., 2003a  ; Irish and Litt, 2005). Legumes are one of the largest plant families on earth and are second only to grasses in economic importance. One important agronomic trait is their ability to fix atmospheric nitrogen in symbiosis with bacteria of the genera Rhizobium, Sinorhizobium, and Azorhizobium (Schultze and Kondorosi, 1998). Development in many species of this family presents general and interesting characteristics that cannot be studied in other model species, such as Arabidopsis (Arabidopsis thaliana). Examples of such traits are leaf complexity, complex inflorescence architecture, and very different floral ontogeny and morphology (Hofer et al., 1997; Ferrándiz et al., 1999; Singer et al., 1999; Benlloch et al., 2002; Tucker, 2003).
We are interested in the study of flower and inflorescence architecture in legume species. Flower development has been subjected to detailed analysis in species such as Arabidopsis or Antirrhinum majus, but many questions can only be answered by genetic analysis of the process in species with different floral and inflorescence architectures. The inflorescence of legume species, such as garden pea (Pisum sativum) or M. truncatula, is more complex than that seen in Arabidopsis and Antirrhinum. Moreover, previous comparative studies revealed that the development of legume flowers also dramatically differs from these model species (Ferrándiz et al., 1999
These species differences suggest the existence of new regulatory genes in legumes or the modification of the function and/or regulation of the legume homologs of genes that control inflorescence and flower development in the model species. Clear examples of the acquisition of new functions by legume genes are UNIFOLIATA (UNI) and STAMINA PISTILLOIDA (STP), which are the pea homologs of LEAFY/FLORICAULA and UNUSUAL FLORAL ORGANS/FIMBRIATA, two key regulators of flower development in both Arabidopsis and Antirrhinum, respectively. Whereas these genes only regulate flower development in Arabidopsis and Antirrhinum, in pea they not only regulate flower development, but also compound leaf development (Hofer et al., 1997
Many of the key regulators of inflorescence and flower development belong to the family of MADS-box genes; this has been widely demonstrated not only for model plants, but also for many other species (Theissen et al., 2000
In the last few years, M. truncatula and Lotus japonicus have emerged as model species for the study of legume biology because of their small diploid genomes, short generation times, self fertility, and relative ease of genetic transformation (Cook, 1999
Reverse Genetics Screening of a Tnt1 Insertion Population of M. truncatula
To isolate Tnt1 insertions in MADS-box genes, we screened a population of 200 M. truncatula plants containing random insertions of the Tnt1 retrotransposon. These plants had been generated by transforming the M. truncatula R108 line (d'Erfurth et al., 2003
To check whether the Tnt1 insertion caused any alteration affecting plant development, T2 plants from the tnk148 line were grown in the greenhouse and their phenotype was analyzed. Around one-fourth of this population of tnk148 T2 plants exhibited a mutant phenotype that affected inflorescence architecture and flower development. In the mutant plants, flowers were replaced by a proliferation of meristems that eventually gave rise to abnormal flowers (see below).
Sequence similarity and the phenotype observed in the progeny suggested that the Tnt1 element was inserted in a gene homologous to PIM/AP1. To better characterize the Tnt1 insertion detected in the tnk148 line, we used a fragment of the PIM gene corresponding to the C-terminal region of the polypeptide as a probe to screen a cDNA library from flowers and inflorescences of M. truncatula. Four cDNA clones corresponding to a gene that we named MtPIM were isolated. The longest cDNA clone was 1,161 bp long and contained a 720-bp-long open reading frame encoding for a 240-amino acid protein (accession no. DQ139345).
The protein encoded by the MtPIM cDNA (Fig. 2A
) showed high similarity to PIM (95% amino acid identity), SQUAMOSA (SQUA; Huijser et al., 1992
In addition, we identified one bacterial artificial chromosome clone (AC144726; M. truncatula clone mth2-7k13) in the M. truncatula genomic sequence, available in the public database, containing the complete sequence of the MtPIM gene. The genomic sequence indicated that the MtPIM gene is organized in eight exons and seven introns. The comparison of this sequence with that of the PCR product amplified from line tnk148 indicated that Tnt1 was inserted 128 bp downstream of the ATG start codon (Fig. 3A ), at the end of the first exon, in the MADS-box region. In addition, it indicated that the insertion of the Tnt1 element created the expected 5-bp (GAAGT) duplication at the site of insertion (d'Erfurth et al., 2003 ).
To further confirm the Tnt1 insertion in the MtPIM locus, Southern-blot analysis was performed with genomic DNA from wild-type plants and tnk148 plants exhibiting the mutant phenotype, using the complete MtPIM cDNA as a probe. According to the sequence of the bacterial artificial chromosome clone, in a wild-type background, a HindIII digestion probed with the entire MtPIM cDNA should generate two bands of 3.3 and 3.8 kb, respectively, whereas in mutant plants, the band of 3.3 kb should be shifted to 1.9 kb due to the Tnt1 insertion (Fig. 3A). The results of this Southern-blot experiment confirmed that, in the mutant plants of line tnk148, the Tnt1 transposable element was inserted in the MtPIM gene (Fig. 3B).
To test whether the mutant phenotype cosegregated with the Tnt1 insertion in the MtPIM gene, tnk148 plants with the mutant phenotype were backcrossed with the wild-type R108 line and 111 F2 plants originating from three independent backcrosses were analyzed. About one-fourth of these F2 plants (28/111 plants) exhibited the floral phenotype previously observed in T2 plants. No other phenotype associated with this group of floral mutant plants could be observed. A sample of 83 plants from this population, including the 28 floral mutant plants, was tested by PCR for the presence or absence of the Tnt1 insertion in the MtPIM locus. Plants with a wild-type phenotype either did not contain the Tnt1 insertion in the MtPIM locus (19 plants) or were heterozygous for the insertion (36 plants), whereas all plants exhibiting the mutant phenotype were homozygous for the Tnt1 insertion (data not shown). These data, along with the results of the Southern-blot analysis, strongly support the idea that the mutant floral phenotype in the M. truncatula tnk148 line was caused by the insertion of the Tnt1 retrotransposon in the MtPIM gene.
Northern-blot analysis performed on RNA from different plant organs showed that MtPIM was specifically expressed in floral tissues, although a faint signal was also detected in stems (Fig. 4A ). No expression of MtPIM was detected in root or leaf tissue. Expression of MtPIM was not detected in floral apices of the mtpim mutant (Fig. 4B), indicating that the mutation caused by Tnt1 likely represents a null allele of the gene.
To detail the MtPIM expression pattern, in situ hybridization on wild-type inflorescence apices was performed (Fig. 4, C–H). During flower development, expression of MtPIM was first detected when the secondary inflorescence meristems started producing floral meristems (Fig. 4C). At stage 1 of floral meristem development (developmental stages as defined by Benlloch et al., 2002 ), the expression was uniform in the entire floral meristem. At this stage, expression of MtPIM could also be observed in the subtending bract (Fig. 4D). As the floral meristem developed, expression was soon restricted to the peripheral region of the meristem from which whorls 1 and 2 would form (Fig. 4E). At stage 4, when common primordia are first observed, MtPIM was expressed in sepal and common primordia. Interestingly, from the beginning, expression in common primordia was restricted to the region that would give rise to the petals (Fig. 4F). The expression of MtPIM was maintained through development of the flower in sepals and petals (Fig. 4G), although the signal became fainter in flowers at later developmental stages (Fig. 4H).
The insertion of Tnt1 in the MtPIM gene causes dramatic phenotypical changes affecting inflorescence and flower architecture (Figs. 5 and 6 ). The mutation seems to affect only reproductive stages of development because no effect on vegetative organ development could be observed in mutant plants.
In wild-type M. truncatula plants, flowers are produced by secondary inflorescence meristems (I2) formed in the axils of the leaves produced by the primary inflorescence (I1; Fig. 5, A and B). Each I2 laterally produces one to three floral meristems on its flanks (usually one in M. truncatula R108), each subtended by a bract, and then the I2 meristem differentiates into a residual stub or spike (Fig. 5, A and C). The architecture of the mtpim mutant inflorescence was dramatically altered (Fig. 5, E–G). Each floral position, subtended by the corresponding bract, was occupied by a highly branched structure. The degree of ramification of these structures increased acropetally along the main stem, the apical structures appearing in upper nodes being more branched than the basal ones. Scanning electron microscopy (SEM) analysis of the inflorescences of the mtpim mutant revealed that I2 meristems were normally produced by I1. As in the wild type, each I2 laterally differentiated new meristems, subtended by bracts, and then terminated as a spike (Fig. 6, C and D). However, these lateral meristems, which in the wild type gave rise to floral meristems, behaved as I2 meristems in the mutant: Rather than developing as flowers, they again laterally produced two new meristems subtended by bracts and differentiated into a spike. The two new meristems reiterated this symmetrical division pattern, giving rise to the proliferating structures observed in the mutant (Fig. 6, D and F–H). Eventually, aberrant flowers were able to differentiate from these proliferating meristems (Fig. 6I). The frequency with which this occurred increased acropetally (Fig. 5G). The number and nature of the organs in mtpim flowers were highly variable (Table I ). Sepals were typically reduced in number and transformed into organs similar to leaves both in shape and epidermal cell types (Fig. 5, H and K–S). Second-whorl organs were reduced in number and/or presented an altered morphology. The adaxial petal was frequently the only petal formed and, occasionally, mosaic organs of sepal and petal tissues were found (Fig. 5I). Third- and fourth-whorl organs were frequently absent or reduced in number. Stamens often had patches of petaloid tissue (petaloid stamens; Fig. 5J).
SEM analysis of the development of wild-type and mutant floral meristems showed that, in the mutant, the number of floral organ primordia initiated was reduced and their production did not follow the highly structured pattern observed in wild-type floral meristems (Fig. 6, J and K). This observation agrees with the variable number and nature of floral organs observed in the second and third whorls of the mutant flowers. Whereas the mtpim mutation affected organ number in all floral whorls, phenotypic alterations were particularly evident in the patterning of the common primordia. In wild-type flowers, four common primordia arise between the sepal and carpel whorls and subsequently divide in a fixed and precise pattern to give rise to five petal and 10 stamen primordia (Benlloch et al., 2002 ). In mtpim mutant flowers, common primordia were formed, but did not follow the typical pattern of divisions, producing floral organs that were reduced in number and developed in abnormal positions.
We have screened a Tnt1-tagged population of M. truncatula for insertions in MADS-box genes with the double aim of (1) evaluating the use of this kind of population as an efficient reverse genetics tool in this species; and (2) identifying genes involved in the control of M. truncatula reproductive development. We describe here the isolation and characterization of the mtpim mutant. Our results represent a proof of concept supporting the previous proposal that Tnt1-tagged populations could work efficiently for reverse genetics in M. truncatula. We show in this work that the mutation is caused by an insertion of Tnt1 in the MtPIM gene, a homolog of AP1/SQUA/PIM. Our analyses indicate that this gene is a key controller in M. truncatula of both floral meristem identity and floral development, regulating sepal identity and the development of floral organs from common primordia.
Our results indicate that the floral mutant phenotype observed in the M. truncatula tnk148 line corresponds to an insertion in MtPIM. Because the tnk148 line contains several Tnt1 insertions, the possibility of a second mutation contributing to the observed phenotype has to be taken into account. However, the analysis of a large number of mtpim individuals from an F2 population derived from a backcross of line tnk148 into the wild-type parental line showed that the mutant phenotypes were quite homogeneous and cosegregated perfectly with the Tnt1 insertion in MtPIM. This would imply that, if a second gene was mutated, it would have to be tightly linked to MtPIM. Moreover, as discussed below, the phenotypes of mtpim and pim are, in essence, rather similar, except that the phenotype of the M. truncatula mutant seems more severe than that of the pea mutant. This would mean that the second mutation would have to be affecting a gene playing a role similar to mtpim/pim. Therefore, although a second mutation cannot be formally discarded, we do not consider this as a likely possibility.
MtPIM is very similar to the pea gene PIM, a likely ortholog of the AP1 and SQUA genes from Arabidopsis and Antirrhinum, respectively. The MtPIM polypeptide lacks the C-terminal prenylation motif present in other members of the AP1 family, such as AP1, CAL, or SQUA (Yalovsky et al., 2000
The MtPIM expression pattern is similar to that described for AP1 and other AP1-like genes and agrees with the dual role that these genes have been proposed to play, first in specifying floral meristem identity and later in floral organ identity (Bowman et al., 1993
In mtpim plants, the flowers are replaced by complex structures with inflorescence characteristics indicating that MtPIM is required for specification of floral meristem identity. In this view, mtpim flowers are replaced by shoots and the mtpim phenotype is equivalent to that of ap1, squa, or pim mutants from Arabidopsis, Antirrhinum, and pea, respectively (Huijser et al., 1992
Whereas the Arabidopsis ap1 mutants also exhibit flower-to-inflorescence transformations, in these mutants this transformation is not complete because flowers are actually formed. ap1 flowers have normal stamens and carpels, but petals are absent and secondary floral meristems develop in the axils of the first-whorl organs (Bowman et al., 1993
A second AP1 homolog, PsMADS9, exists in the pea genome (N. Carrasquilla, A. Berbel, J.P. Beltrán, and F. Madueño, unpublished data; cited by Litt and Irish, 2003
MtPIM plays a major role in the specification of floral meristem identity, but mtpim plants are able to eventually produce flowers. Initiation of these flowers is likely due to the activity of other meristem identity genes, partially redundant with MtPIM, such as the homologs to the genes LEAFY or FRUITFULL, which have already been identified in the sequence of the M. truncatula genome (Weigel et al., 1992
Organs from the second and third whorls of the M. truncatula and pea flowers derive from common primordia. These are four short-lived meristems that form between sepal and carpel primordia. Each of these meristems subsequently divides in a characteristic pattern to produce the petal and stamen primordia (Ferrándiz et al., 1999 Interestingly, the mtpim mutation also affects the normal development of organs arising from the common primordia (i.e. petals and stamens), which were often abnormal, reduced in number, and chimeric in nature. Sepal/petal and petal/stamen chimeras are also frequently observed in the pim mutant (J. Hofer, personal communication; A. Berbel, C. Ferrandiz, and F. Madueño, unpublished data). These mosaic organs seem to appear more frequently in the legume mutants than in ap1 or squa, possibly reflecting the common origin of these organs in pea and Medicago. Moreover, SEM analysis of developing mtpim flowers indicated that common primordia divided abnormally. The onset of MtPIM expression in common primordia occurs at early stages of development, prior to any morphological sign of petal and stamen initiation, and very precisely marks the boundaries between the regions of the common primordia that will give rise to petals and stamens. This, together with the mutant phenotype, suggests a possible role of MtPIM in common primordia patterning, maybe providing positional cues for correct organ initiation. In summary, the mtpim mutation affects floral organ identity, as shown by the sepal-to-leaf transformations, but also seems to affect the specification of second- and third-whorl organs from common primordia, possibly by interfering with the establishment of boundaries during common primordia division.
For species such as M. truncatula or L. japonicus to work as useful model legumes, it is necessary that efficient reverse genetics tools are available for them. Collections of lines derived from insertional mutagenesis are a very important resource for the development of this kind of tool. T-DNA and transposon-tagged populations have been very useful for the genetic dissection of developmental processes in different plants (Carpenter and Coen, 1990
Retrotransposons present advantages in comparison with T-DNAs or other transposable elements because of their random insertion in the genome and their low frequency of rearrangements or aberrant or incomplete insertions, which can make isolation of tagged mutants difficult. In L. japonicus, the use of LORE1, an endogenous retrotransposon recently identified, is being explored as a tool for insertional mutagenesis (Madsen et al., 2005
The isolation of MtPIM represents proof of the concept that shows that, in fact, Tnt1 works efficiently for gene tagging in M. truncatula and that Tnt1 populations constitute a useful resource for reverse genetics. Our analysis confirmed that the Tnt1 insertion causing the mtpim mutation showed no rearrangement and that the mutant phenotype is stably inherited and shows Mendelian segregation. Whereas detection of the mtpim mutation in a starting population of only 200 Tnt1-tagged lines might look fortunate, it should be considered that they were independent lines with an average of 15 to 20 insertions per line, therefore representing up to 4,000 Tnt1 inserts. Assuming that one-third of the inserts are in genes (d'Erfurth et al., 2003
While this work confirms Tnt1 populations as a useful resource for reverse genetics in M. truncatula, other mutant resources for reverse genetics are being developed in legumes. A TILLING platform is available for L. japonicus (Perry et al., 2003 What seems clear is that a variety of complementary strategies for mutant isolation through reverse genetics will be efficiently working in model legumes very soon. This will greatly ease the dissection of genetic networks controlling traits of interest in these species and, in general, will allow rapid progress in the understanding of the biology of this family of plants.
Plant Material and Growth Conditions
Medicago truncatula (ecotype R108) was grown in the greenhouse at 22°C (day) and 18°C (night); 16-h photoperiods were maintained with supplementary lighting [400-W Phillips HDK/400 HPI (R) (N)]. Plants were grown in a mixture of soil:sand (3:1) and were irrigated with Hoagland no. 1 solution supplemented with oligoelements (Hewitt, 1966
The M. truncatula population used for the PCR-based screening of mutants was described in detail (d'Erfurth et al., 2003 Primer sequences were as follows: MAD1, 5'-ATGGGRAGRGGAAARATTGARATMAARAGGAT-3'; MAD2, 5'-ATGGGRAGRGGAAGAGTGSARTTGAARAGGAT-3'; MAD3, 5'-ATGGGNMGNGGNAARATHGA-3'; DAM1, 5'-ATCCTYTTKATYTCAATYTTTCCYCTYCCCAT-3'; DAM2, 5'-ATCCTYTTCAAYTSCACTCTTCCYCTYCCCAT-3'; LTR6, 5'-GCTACCAACCAAACCAAGTCAA-3'; and LTR31, 5'-CTCCTCTCGGGGTCGTGGTT-3'.
A cDNA library of M. truncatula (ecotype Jemalong A17) flowers and inflorescence apexes was generated (HybriZAP-2.1 XR Library; Stratagene) and screened at high stringency (65°C, 0.1 x SSC/0.1% SDS) with a 767-bp 3' fragment of PEAM4 cDNA (Berbel et al., 2001
Plant genomic DNA was extracted from leaves as described (Dellaporta et al., 1983
PCR reactions were performed using genomic DNA as a template 20 ng from each F2 individual plant (30 cycles; 58°C annealing temp). The presence of the Tnt1 insertion in the MtPIM locus was tested by the amplification of a 301-bp band with the oligonucleotides Mtm42, 5'-AGGATAGAAAACAAGATCAATCG-3' (nucleotides 28–51 of the coding sequence of MtPIM cDNA) and LTR51, 5'-AAAGCTTCACCCTCTAAAGCCT-3' (nucleotides 178–200 of the LTR of Tnt1), and the absence of the Tnt1 insertion was tested by the amplification of a 156-bp band with the oligonucleotides Mtm42 and Mtm460, 5'-AGAATCAGTTGCATATTCAAAGAG-3' (nucleotides 160–183 of the coding sequence of MtPIM cDNA).
Total RNA was isolated by phenol-chloroform extraction and precipitated with 3 M lithium chloride. RNA was electrophoresed in formaldehyde-agarose gels, transferred to Hybond N+ membranes (Amersham Biosciences), and hybridized with 32P under standard conditions. The probe was the 434-bp-long fragment of the C-terminal region of MtPIM cDNA amplified with the oligonucleotides AP1insF, 5'-ATAGCGGACTGAAGGCAAAG-3' and MtAP1insR, 5'-GCATCCAAGATGGCAGGTAT-3'.
RNA in situ hybridization with digoxigenin-labeled probes was performed on 8-µm longitudinal paraffin sections of M. truncatula inflorescences as described in Ferrándiz et al. (2000)
Light photographs of wild-type and mtpim mutant flowers were obtained using a dissection microscope (Leica MZ8). Specimens were freshly harvested and dissected using a forceps and scalpel. For SEM, samples were harvested, dehydrated, dried, and analyzed as described in Benlloch et al. (2002) Sequence data from this article can be found in the GenBank/EMBL data libraries under accession number DQ139345.
We thank D. Bradley and F. Parcy for critical reading of the manuscript and N. Carrasquilla for helping to generate the M. truncatula cDNA library. We also thank M.T. Caballero for excellent technical assistance and acknowledge the collaboration of R. Martínez Pardo and A. Millán in the greenhouse. Received May 18, 2006; accepted August 7, 2006; published September 8, 2006.
1 This work was supported by the Secretaría General del Plan Nacional de Investigación Científica y Desarrollo Tecnológico (Spain; grant no. BIO2000–0940), the Generalitat Valenciana (grant no. GV–03–66), and the European Union Grain Legumes Integrated Project (grant no. FP6–2002–FOOD–1–506223). R.B. was supported by a fellowship of the Ministerio de Educación y Ciencia (Spain); I.d'E. was supported by a French Ministère de l'Education Nationale, de la Recherche et de la Technologie fellowship and the European Union Medicago Project (grant no. QLG2–CT–2000–00676). 
2 Present address: Laboratories Physiologie Cellulaire Végétale, UMR CNRS 5168-CEA-INRA1200-UJF, 17 Av des Martyrs, bât.c2, 38054 Grenoble cedex, France. The author responsible for distribution of materials integral to the findings presented in this article in accordance with the policy described in the Instructions for Authors (www.plantphysiol.org) is: Francisco Madueño (madueno{at}ibmcp.upv.es). www.plantphysiol.org/cgi/doi/10.1104/pp.106.083543 * Corresponding author; e-mail madueno{at}ibmcp.upv.es; fax 34–963877859.
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ABSTRACT
RESULTS
, spike. C and E, SEM micrographs of wild-type inflorescence apexes. C, The primary inflorescence meristem (I1, central) is differentiating a leaf (L), with the corresponding flanking stipules (Stp), and an axillary secondary inflorescence meristem (I2). At the top left corner, a developing secondary inflorescence shows a flower (F) subtended by a bract (Br), produced by the I2 meristem. E, Similar structures to those shown in C are indicated. The secondary inflorescence in the top left corner shows a flower (F) in a later stage of development, where floral organ primordia are clearly visible. The I2 has already differentiated the terminal spike (Sk). D and F, SEM micrographs of mtpim inflorescence apexes. D, The primary inflorescence meristem (I1), at the right side, is differentiating a leaf and a secondary inflorescence meristem (I2). The secondary inflorescence at the left shows noticeable phenotypic alterations. Whereas I2 is already differentiating as a spike (Sk), in the axil of the bract (Br), instead of floral meristems, a second-order I2 meristem (below the bract) has produced two new lateral structures, which resemble third-order I2 meristems (I2**). F, View of a different mtpim inflorescence apex. In the secondary inflorescence developing at the right side end, second- and third-order I2 are easily distinguished (I2*, I2**). In the top left corner, a highly branched structure has formed. G, Close-up view of the structure formed in place of a floral meristem in a mtpim mutant, showing the symmetrical pattern of meristem division. H, Highly branched structure formed in a secondary inflorescence of a mtpim mutant. An extreme proliferation of bracts with associated meristems is observed. I, mtpim secondary inflorescence in which an aberrant flower has developed (top left). J and K, Flowers of wild type (J) and the mtpim mutant (K). J, In wild type, common primordia have already formed and divided between the sepals (Sp) and the carpel (C) to differentiate five petal and 10 stamen primordia. K, In this mtpim flower, common primordia have divided abnormally to give rise to a total number of seven floral organ primordia.