Control of compound leaf development by FLORICAULA/LEAFY ortholog SINGLE LEAFLET1 in Medicago truncatula

Abstract

Molecular genetic studies suggest that FLORICAULA (FLO)/LEAFY (LFY) orthologs function to control compound leaf development in some legume species. However, loss-of-function mutations in the FLO/LFY orthologs result in reduction of leaf complexity to different degrees in Pisum sativum and Lotus japonicus. To further understand the role of FLO/LFY orthologs in compound leaf development in legumes, we studied compound leaf developmental processes and characterized a leaf development mutant, single leaflet1 (sgl1), from the model legume Medicago truncatula. The sgl1 mutants exhibited strong defects in compound leaf development; all adult leaves in sgl1 mutants are simple due to failure in initiating lateral leaflet primordia. In addition, the sgl1 mutants are also defective in floral development, producing inflorescence-like structures. Molecular cloning of SGL1 revealed that it encodes the M. truncatula FLO/LFY ortholog. When properly expressed, LFY rescued both floral and compound leaf defects of sgl1 mutants, indicating that LFY can functionally substitute SGL1 in compound leaf and floral organ development in M. truncatula. We show that SGL1 and LFY differed in their promoter activities. Although the SGL1 genomic sequence completely rescued floral defects of lfy mutants, it failed to alter the simple leaf structure of the Arabidopsis thaliana plants. Collectively, our data strongly suggest that initiation of lateral leaflet primordia required for compound leaf development involves regulatory processes mediated by the SGL1 function in M. truncatula.

Figure 1.

The ontogeny of compound leaf development in wild-type M. truncatula. A, Morphology of M. truncatula ‘Jemalong’ A17. Shown in inset were juvenile (left) and adult (right) leaves. B to M, SEM analysis of compound leaf development. B, Sites of incipient leaf primordia were specified at the periphery of the SAM at S0, albeit no morphological changes were visible at this stage. At S1, a common leaf primordium was initiated as a strip of cells outgrown along the periphery of SAM (asterisk). C, A pair of stipule primordia (ST) was initiated from the proximal end of the common leaf primordium at S2. D, At S3, a pair of lateral leaflet primordia (LL) emerged between the stipule and common leaf primordia. E, Boundaries (arrow) between the stipule and lateral leaflet primordia formed, and the common leaf primordium differentiated into a terminal leaflet primordium (TL) as indicated by development of trichomes from the abaxial surface at S4. F, At S5, boundaries (arrows) formed between the lateral and terminal leaflet primordia. G, While trichomes developed from the abaxial surface of both stipule and lateral leaflet primordia, the terminal leaflet primordium folded as a result of outgrowth of the abaxial surface at S6. H, At S7, a petiole primordium (Pet) formed between the stipule and lateral leaflet primordia. I, A close-up view of H. J, The lateral and terminal leaflet primordia folded due to outgrowth of the abaxial surface at S8. Trichomes developed from the adaxial surface of the petiole primordium at this stage. K, A close-up view of J. L, A rachis primordium (Rac) formed between the lateral and terminal leaflet primordia and trichomes developed from its adaxial surface at S9. M, A close-up view of L. Scale bars, 50 or 200 μm as indicated.

Figure 2.

Phenotypes of M. truncatula sgl1 mutants. Three-week-old wild-type M. truncatula (ecotype R108; A) and sgl1-1 mutant (B) exhibited compound and simple leaf forms, respectively. C, Close-up views of adult leaves of wild type R108 (left) and sgl1-1 mutant (right). In S2 and S4 (D), S3 and S5 (E), and S4 and S6 (F) leaf primordia of sgl1-1 mutant, lateral leaflet primordia did not form at the proximal end of common leaf primordia. G, Morphology of a mature sgl1-1 mutant plant, exhibiting simple leaf and floral homeotic phenotypes. H, Petiole length was significantly reduced in sgl1-1 mutant plants compared to the wild-type R108 plants, but no significant differences in petiole length were found in newly developed leaves of wild-type and sgl1-1 mutant (node 10). SL, Single leaflet; ST, stipule. Scale bars, 10 mm (A–C); 50 μm; or 100 μm as indicated (D–F). [See online article for color version of this figure.]

Figure 3.

Flower phenotypes of M. truncatula sgl1 mutants. Two mature flowers developed on a single spike in wild type (R108). The bilateral symmetry along the dorsal-ventral axis was shown. B, Defective flowers developed on a single spike in sgl1-1 mutant. C, A close-up view of a single flower of the sgl1-1 mutant. D to I, Flower development in wild type (R108). D, Three FMs were initiated from I₂. E, In S3, the abaxial (Sab) and two lateral (Sl) sepal primordia were initiated. F, In S4, two adaxial sepal primordia (Sad) were initiated. The carpel primordium (C) and the abaxial (CPab) and two lateral (CPl) common primordia formed. G, In S5, four common primordia differentiated into petal and stamen primordia along the abaxial to adaxial axis. Subsequently, the adaxial common primordia produced one inner antepetal (Stp) and two outer antesepal (Sts) stamens and the standard primordium (Vexillum or Vx). H, At subsequent stages, all floral organ primordia formed. A, Alae petals; K, keel petals. I, Stamen primordia differentiated anthers at S8 (asterisk). Sg, Stigma. J to O, Flower development in sgl1 mutants. J, Multiple FM developed from a single I₂ inflorescence meristem in sgl1-1 mutant. K, A close-up view of an I₂ with multiple FM. L and M, An S5 FM in sgl1-1 mutant initiated three to six common primordia (CP) between sepal (S) and carpel (C) primordia. The carpel primordium was occasionally missing from the center of the FMs (asterisks in K and M). M, Secondary FM were initiated from the second whorl of FMs in sgl1-1 mutant. N, An S7 FM in sgl1-1 mutant with its carpel primordium (C) started to fold. Secondary FM started to differentiate. O, Secondary floral-like meristems developed between the carpel and sepal primordia and gave rise to proliferating structures with elongated sepals (S). Scale bars as indicated. [See online article for color version of this figure.]

Figure 4.

Molecular cloning of SGL1 gene and functional complementation of sgl1-1 mutant. A, PCR amplification of SGL1gene from wild-type M. truncatula (ecotype R108), sgl1-1, and sgl1-2 mutants. A single insertion of tobacco Tnt1 retrotransposon was detected as a shift in molecular weights of the amplified SGL1gene from each sgl1 mutant allele. B, The intron and exon structure of SGL1 and positions and orientation of Tnt1 insertions in sgl1 mutants. Tnt1 was inserted in the first exon at positions 198 bp, 314 bp, and 333 bp, and in the third exon at the position 831 bp downstream from the translation initiation codon of SGL1 in sgl1-1 to sgl1-4 mutants, respectively. C, A representative sgl1-1 transgenic line transformed with the SGL1 genomic sequence (SGL1:SGL1) exhibited completely rescued wild type-like leaves. D, Representative flowers of three independent sgl1 SGL1:SGL1 transgenic lines, showing completely rescued (line 1), partially rescued (line 2), and nonrescued (line 3) flowers, albeit leaves from all three lines were rescued. E, RT-PCR analysis of SGL1 expression level in developing flowers of three independent sgl1 SGL1:SGL1 transgenic lines (lanes 1–3) compared with that of the sgl1-1 mutant (lane 4) and wild type (lane 5). An Actin gene was used as an internal control (bottom). F, Phylogenetic analysis of SGL1 and its putative orthologs: FLO of snapdragon, LFY of Arabidopsis, NFL of tobacco, UNI of pea, ALF of petunia, Imp-FLO of impatiens, FA of tomato, and PFM of L. japonicus. Bootstrap supports above 50% from 1,000 replicates were shown. [See online article for color version of this figure.]

Figure 5.

Expression pattern of SGL1 gene. A to C, RNA in situ hybridization analysis of SGL1 gene expression. A, SGL1 gene expression was detected in SAM, developing leaf primordia (S2 and S4), and I₂. B, SGL1 gene expression was detected in developing floral organs in S7 flowers of wild-type plants. P, Petal; S, sepal; St; stamen. C, SGL1 sense probes were used as a negative control, no hybridization signal was detected in SAM and leaf primordia (S2, S4, and S6). D to G, SGL1:GUS histochemical staining pattern. D, GUS staining was restricted to the SAM (inset), developing leaf primordia at early stages (inset), vascular tissues of petioles, and the basal regions of leaflets at late stages. E and F, Close-up views of GUS staining patterns at the basal region of leaflets. G, GUS staining in mature leaves.

Figure 6.

Genetic complementation of sgl1 mutants by LFY. A, Homozygous sgl1-1 mutant plants transformed with SGL1:LFY, all adult leaves exhibiting wild-type compound leaf morphology. B, Wild-type-like flowers were developed from secondary inflorescence from axils of compound leaves of sgl1 SGL1:LFY transgenic lines. C, PCR-based genotyping of sgl1 SGL1:LFY transgenic lines indicated that three independent transgenic lines were homozygous for Tnt1 insertion in SGL1 gene (lanes 1–3) in contrast to the wild-type M. truncatula (ecotype R108), where no Tnt1 insertion was detected in SGL1 gene (lane 4). Top segment for detecting Tnt1 inserts; bottom for detecting Tnt1 insertion in SGL1. D, RT-PCR analysis of expression of LFY gene in sgl1 SGL1:LFY transgenic lines. Shown were LFY expression in two independent sgl1 SGL1:LFY transgenic lines (lanes 1 and 2), sgl1-1 mutant (lane 3), and wild-type M. truncatula (lane 4). Expression of an Actin gene was used as an internal loading control (bottom). E, A typical mature flower of wild-type M. truncatula (ecotype R108), exhibiting bilateral symmetric zygomorphic morphology. F to I, Floral organs of wild-type M. truncatula were dissected. Shown were a top view of vexillum (F), top (G), and side (H) views of keel and alae, the central carpel enclosed by the staminal tube, and a top view of dissected sepals (I). J, A representative mature flower of sgl1 SGL1:LFY transgenic lines, exhibiting wild-type-like morphology. K to N, Floral organs of sgl1 SGL1:LFY transgenic lines were dissected. Shown were a top view of vexillum (K), top (L) and side (M) views of keel and alae, the central carpel, and a top view of dissected sepals (N).

Figure 7.

Genetic complementation of lfy mutants by SGL1 and comparison of expression patterns of LFY:GUS and SGL1:GUS in Arabidopsis. A, Inflorescence of homozygous lfy (left) and lfy transformed with LFY:SGL1 (right). B to D, Two-week-old wild-type (B), lfy LFY:SGL1 (C), and lfy SGL1:SGL1 (D) seedlings; insets, mature flowers. E, Morphology of lfy mutant flowers. F, RT-PCR analysis of SGL1 expression in wild-type M. truncatula (lane 1), lfy (lane 2), and three independent transgenic lfy lines transformed with LFY:SGL1 (lanes 3–5). G, RT-PCR analysis of SGL1 expression in M. truncatula (lane 1), Arabidopsis (lane 2), and two independent transgenic lfy lines transformed with the SGL1 genomic sequence (SGL1:SGL1; lanes 3 and 4). An Actin gene was used as an internal loading control (F and G; bottom). H and L, Histochemical staining of LFY:GUS in mature and young seedlings. Strong GUS staining was restricted to the base of flowers, vascular tissues of inflorescence stems, and the shoot apex but not of pedicels and leaves. M to Q, Histochemical staining of SGL1:GUS in mature and young seedlings. Strong GUS staining was detected in style, but not at the base of flowers, and in vascular tissues of inflorescence stems, pedicels, sepals, carpel, cauline, and rosette leaves, and the shoot apex. In cauline and rosette leaves, a high level of GUS staining was localized to the distal margins (arrows).