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Two euAGAMOUS genes control C-function in Medicago truncatula.

Serwatowska J, Roque E, Gómez-Mena C, Constantin GD, Wen J, Mysore KS, Lund OS, Johansen E, Beltrán JP, Cañas LA - PLoS ONE (2014)

Bottom Line: This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily.Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern.In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV). Ciudad Politécnica de la Innovación, Valencia, Spain.

ABSTRACT
C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.

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Overexpression of MtAGa and MtAGb in Arabidopsis thaliana: floral phenotypes.Wild-type Arabidopsis flower (A). Floral phenotypes observed in Arabidopsis plants overexpressing MtAGa or MtAGb genes have been classified into mild (B), medium (C) and strong (D). Scanning electron micrographs showing the characteristic cellular types of wild-type sepals (E), petals (F), stamens (G) and carpels (H). Scanning electron micrographs of sepals from overexpression lines with mild phenotype (I) showing wax accumulation (arrow). Narrow petals from overexpressing lines (J) showing staminoid cellular types (K). Wax accumulation in sepals (L) of plants with medium and strong phenotypes. Near complete homeotic conversion of petal into stamen (M) showing characteristic cellular types from anther (N) and filament (O). Flower with strong phenotype showing ectopic ovules and stigmatic tissue (P) in the first whorl. Relative proportion of phenotypes observed in plants overexpressing MtAGa or MtAGb genes (Q). Bars indicate: 1mm in A, B, C, and D.
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pone-0103770-g005: Overexpression of MtAGa and MtAGb in Arabidopsis thaliana: floral phenotypes.Wild-type Arabidopsis flower (A). Floral phenotypes observed in Arabidopsis plants overexpressing MtAGa or MtAGb genes have been classified into mild (B), medium (C) and strong (D). Scanning electron micrographs showing the characteristic cellular types of wild-type sepals (E), petals (F), stamens (G) and carpels (H). Scanning electron micrographs of sepals from overexpression lines with mild phenotype (I) showing wax accumulation (arrow). Narrow petals from overexpressing lines (J) showing staminoid cellular types (K). Wax accumulation in sepals (L) of plants with medium and strong phenotypes. Near complete homeotic conversion of petal into stamen (M) showing characteristic cellular types from anther (N) and filament (O). Flower with strong phenotype showing ectopic ovules and stigmatic tissue (P) in the first whorl. Relative proportion of phenotypes observed in plants overexpressing MtAGa or MtAGb genes (Q). Bars indicate: 1mm in A, B, C, and D.

Mentions: Ectopic expression of AG results in the homeotic conversion of sepals and petals into carpels and stamens, respectively [55], [56]. Other reported phenotypes include early flowering and curling of leaves. To investigate whether MtAGa and MtAGb proteins differ in their ability to induce reproductive organ fate we overexpressed these two genes in Arabidopsis. The coding sequence of these genes was located under the control of the CaMV 35S constitutive promoter. We analysed 75 independent primary transgenic (T1) lines for every construct (35S::MtAGa and 35S::MtAGb plants) and found plants with different floral phenotypes that we classified as mild, medium and strong phenotype (Figure 5B-D). Transgenic plants with mild phenotype showed wax accumulations on the surface of the sepals (Figure 5I) that are characteristic of the carpel (Figure 5H). The petals are narrow and asymmetric (Figure 5B and 5J) with partial homeotic conversions to stamens (Figure 5K). Flowers with medium phenotypes showed curved sepals (Figure 5C) with abundant accumulation of wax (Figure 5L). T1 plants with medium and strong phenotypes showed near complete homeotic transformations of sepals to stamens (Figure 5M and 5N). Finally, flowers with strong phenotype showed thickened and curved sepals (Figure 5D) that contain carpelloid structures with ectopic ovules and stigmatic papillae (Figure 5P). Although these three phenotypical classes were observed for both constructs, we notice a higher proportion of plants with medium/strong phenotypes (80.3%) in the MtAGb overexpressing lines (Figure 5Q). In contrast, 59.5% of MtAGa overexpressing lines showed mild homeotic transformations.


Two euAGAMOUS genes control C-function in Medicago truncatula.

Serwatowska J, Roque E, Gómez-Mena C, Constantin GD, Wen J, Mysore KS, Lund OS, Johansen E, Beltrán JP, Cañas LA - PLoS ONE (2014)

Overexpression of MtAGa and MtAGb in Arabidopsis thaliana: floral phenotypes.Wild-type Arabidopsis flower (A). Floral phenotypes observed in Arabidopsis plants overexpressing MtAGa or MtAGb genes have been classified into mild (B), medium (C) and strong (D). Scanning electron micrographs showing the characteristic cellular types of wild-type sepals (E), petals (F), stamens (G) and carpels (H). Scanning electron micrographs of sepals from overexpression lines with mild phenotype (I) showing wax accumulation (arrow). Narrow petals from overexpressing lines (J) showing staminoid cellular types (K). Wax accumulation in sepals (L) of plants with medium and strong phenotypes. Near complete homeotic conversion of petal into stamen (M) showing characteristic cellular types from anther (N) and filament (O). Flower with strong phenotype showing ectopic ovules and stigmatic tissue (P) in the first whorl. Relative proportion of phenotypes observed in plants overexpressing MtAGa or MtAGb genes (Q). Bars indicate: 1mm in A, B, C, and D.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4126672&req=5

pone-0103770-g005: Overexpression of MtAGa and MtAGb in Arabidopsis thaliana: floral phenotypes.Wild-type Arabidopsis flower (A). Floral phenotypes observed in Arabidopsis plants overexpressing MtAGa or MtAGb genes have been classified into mild (B), medium (C) and strong (D). Scanning electron micrographs showing the characteristic cellular types of wild-type sepals (E), petals (F), stamens (G) and carpels (H). Scanning electron micrographs of sepals from overexpression lines with mild phenotype (I) showing wax accumulation (arrow). Narrow petals from overexpressing lines (J) showing staminoid cellular types (K). Wax accumulation in sepals (L) of plants with medium and strong phenotypes. Near complete homeotic conversion of petal into stamen (M) showing characteristic cellular types from anther (N) and filament (O). Flower with strong phenotype showing ectopic ovules and stigmatic tissue (P) in the first whorl. Relative proportion of phenotypes observed in plants overexpressing MtAGa or MtAGb genes (Q). Bars indicate: 1mm in A, B, C, and D.
Mentions: Ectopic expression of AG results in the homeotic conversion of sepals and petals into carpels and stamens, respectively [55], [56]. Other reported phenotypes include early flowering and curling of leaves. To investigate whether MtAGa and MtAGb proteins differ in their ability to induce reproductive organ fate we overexpressed these two genes in Arabidopsis. The coding sequence of these genes was located under the control of the CaMV 35S constitutive promoter. We analysed 75 independent primary transgenic (T1) lines for every construct (35S::MtAGa and 35S::MtAGb plants) and found plants with different floral phenotypes that we classified as mild, medium and strong phenotype (Figure 5B-D). Transgenic plants with mild phenotype showed wax accumulations on the surface of the sepals (Figure 5I) that are characteristic of the carpel (Figure 5H). The petals are narrow and asymmetric (Figure 5B and 5J) with partial homeotic conversions to stamens (Figure 5K). Flowers with medium phenotypes showed curved sepals (Figure 5C) with abundant accumulation of wax (Figure 5L). T1 plants with medium and strong phenotypes showed near complete homeotic transformations of sepals to stamens (Figure 5M and 5N). Finally, flowers with strong phenotype showed thickened and curved sepals (Figure 5D) that contain carpelloid structures with ectopic ovules and stigmatic papillae (Figure 5P). Although these three phenotypical classes were observed for both constructs, we notice a higher proportion of plants with medium/strong phenotypes (80.3%) in the MtAGb overexpressing lines (Figure 5Q). In contrast, 59.5% of MtAGa overexpressing lines showed mild homeotic transformations.

Bottom Line: This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily.Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern.In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function.

View Article: PubMed Central - PubMed

Affiliation: Instituto de Biología Molecular y Celular de Plantas (CSIC-UPV). Ciudad Politécnica de la Innovación, Valencia, Spain.

ABSTRACT
C-function MADS-box transcription factors belong to the AGAMOUS (AG) lineage and specify both stamen and carpel identity and floral meristem determinacy. In core eudicots, the AG lineage is further divided into two branches, the euAG and PLE lineages. Functional analyses across flowering plants strongly support the idea that duplicated AG lineage genes have different degrees of subfunctionalization of the C-function. The legume Medicago truncatula contains three C-lineage genes in its genome: two euAG genes (MtAGa and MtAGb) and one PLENA-like gene (MtSHP). This species is therefore a good experimental system to study the effects of gene duplication within the AG subfamily. We have studied the respective functions of each euAG genes in M. truncatula employing expression analyses and reverse genetic approaches. Our results show that the M. truncatula euAG- and PLENA-like genes are an example of subfunctionalization as a result of a change in expression pattern. MtAGa and MtAGb are the only genes showing a full C-function activity, concomitant with their ancestral expression profile, early in the floral meristem, and in the third and fourth floral whorls during floral development. In contrast, MtSHP expression appears late during floral development suggesting it does not contribute significantly to the C-function. Furthermore, the redundant MtAGa and MtAGb paralogs have been retained which provides the overall dosage required to specify the C-function in M. truncatula.

Show MeSH
Related in: MedlinePlus