Isolation of mtpim proves Tnt1 a useful reverse genetics tool in Medicago truncatula and uncovers new aspects of AP1-like functions in legumes

Abstract

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.

Figure 1.

Identification of a Tnt1 insertion in a MADS-box gene. A, Schematic diagram of the Tnt1 element (5.3 kb) with flanking LTRs and a consensus MADS-box gene (approximately 0.7 kb), where conserved regions are shown (MADS-box, intermediary region, K domain, and C-terminal domain). Position and orientation of primers used for screening the population are shown. B, Sequence of the PCR product amplified in pool number 5 with primers LTR6 and MAD2. Sequence in capital letters corresponds to the M. truncatula genome sequence, whereas the lowercase sequence corresponds to the LTR sequence in Tnt1 element. C, PCR amplification products with primers LTR6 and MAD2 in the Tnt1 population DNA pools 1 to 20 (top); and in DNA extracted from individual plants from pool 5 (bottom).

Figure 2.

Sequence analysis of MtPIM. A, ClustalW alignment of the predicted amino acid sequences of MtPIM (accession no. DQ139345), PIM, AP1, and SQUA cDNAs. The MADS domain conserved region is underlined. The prenylation motif (CaaX) is underlined at the carboxy-terminal end of AP1 and SQUA. B, Neighbor-joining tree of the predicted amino acid sequences of genes from the AP1/SQUA subfamily. Arabidopsis: AP1 (CAA64789), CAL (AAA64789), and FUL (AAA97403); Antirrhinum: SQUA (CAA45228); Eucalyptus: EAP1 (AAG24909); Gerbera: GSQUA (CAA08805); Lycopersicon: TM4 (Q40170); Malus: MdAP1 (AAL61543); Nicotiana: NAP1-1 (AAD01422) and NAP1-2 (AAD01422); Petunia: PFG (AAF19721); Pisum: PIM (AJ279089); Silene: SLM4 (CAA56658); Medicago: MtPIM. The tree was rooted with NGL9, a M. sativa MADS protein with homology to members of the PISTILLATA subfamily (AF335473; Zucchero et al., 2001).

Figure 3.

Structure of the Tnt1 insertion in the MtPIM locus. A, Schematic diagram of the MtPIM gene; position of exons (boxes) and introns (lines) is shown. The Tnt1 insertion lies at the end of the first exon. Restriction sites for HindIII are indicated (H). B, Southern blot of HindIII-digested wild-type and mutant genomic DNA hybridized with the complete cDNA of MtPIM. In the wild-type sample, digestion generates two hybridizing fragments of 3.3 (white arrow) and 3.8 kb. The Tnt1 insertion in mtpim generates a new fragment of 1.9 kb (black arrow).

Figure 4.

Expression pattern of MtPIM. A, Northern-blot analysis of MtPIM expression in roots (R), stems (S), leaves (L), and flowers (F) of wild-type M. truncatula plants. B, Northern-blot analysis of MtPIM expression in inflorescence apexes of wild-type and mtpim mutants. Blots in A and B were loaded with samples of 15-μg RNA. A picture of each gel stained with ethidium bromide is shown below. C to H, In situ hybridization of MtPIM RNA in wild-type M. truncatula inflorescences and flowers. C, Longitudinal section of a secondary inflorescence meristem differentiating a floral meristem. MtPIM is strongly expressed in the floral meristem. D, Early stage 2 floral meristem, showing uniform expression of MtPIM. MtPIM RNA can also be detected in the developing bract. E, Floral meristem at late stage 2. MtPIM expression is restricted to the periphery of the meristem. At this stage, no floral organ primordia have been initiated. F, Stage 4 floral meristem, where the common primordia can be observed. Expression is restricted to the region of the common primordia, which will give rise to petals. G, Floral meristem at stage 5, when petals and stamens start differentiating from the common primordia. H, In later stages (stage 6), expression is maintained in sepals and petals. F, Floral meristem; I₂, inflorescence meristem; Br, bract; CP, common primordia; P, petal; S, sepal; St, stamen; C, carpel. Developmental stages were defined according to Benlloch et al. (2002).

Figure 5.

Floral phenotype of the mtpim mutant. A, Cartoon of a wild-type M. truncatula inflorescence. After floral transition, the shoot apical meristem becomes a primary inflorescence meristem (I₁). I₁ gives rise to secondary inflorescence meristems (I₂) in the axils of leaves, which in turn laterally differentiate flowers (F) subtended by bracts before terminating in a residual stub or spike. B, Apical part of the main stem of a M. truncatula plant that has gone through floral transition. The shoot apex and two secondary inflorescences, each of them subtended by a trifoliate leaf, can be distinguished. C, Close-up of a wild-type secondary inflorescence. Br, Bract; Sk, spike. D, Detail of a flower where the subtending bract is clearly visible. E, Close-up of a mtpim secondary inflorescence. The secondary inflorescence meristem produces a bract that subtends a proliferating structure and finally differentiates as a spike. F, Secondary inflorescence of a mtpim plant. After producing several proliferating meristems, an abnormal flower (F*) has finally differentiated. G, A different mtpim secondary inflorescence, found in a more apical position, in which the pattern of symmetrical divisions is clearly observed. After a number of divisions, most of the meristems have differentiated as flowers. H to J, Homeotic transformations in floral organs of the mtpim mutant. H, mtpim flower with transformation of a sepal into a leaf-like organ. I, Mosaic organ of sepal and petal tissues. J, Some of the stamens in the staminal tube develop petaloid extensions. K to S, SEM analysis of epidermal cell types of first-whorl leaf-like organs of mtpim flowers. K, View of a wild-type bract. L, Epidermis of the adaxial side of a wild-type leaf. M, Adaxial side of a leaf-like first-whorl organ of a mtpim flower. N, Adaxial side of a wild-type sepal. O, Close-up of a wild-type bract, showing the epidermal cell types. P, Abaxial side of a wild-type leaf. R, Abaxial side of a transformed first-whorl mtpim floral organ. S, Abaxial side of a wild-type sepal. Scale bar = 600 μm (K), 100 μm (O), 60 μm (L–N and P–S).

Figure 6.

Development of inflorescence meristems in the wild-type and mtpim mutant. A and B, Schematic representation of meristem fates in M. truncatula wild-type (A) and mtpim (B) mutants. V, Vegetative shoot apical meristem; I₁, primary inflorescence meristem; I₂, secondary inflorescence meristem; F, floral meristem; ♦, 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.