Characterization of the possible roles for B class MADS box genes in regulation of perianth formation in orchid

Abstract

To investigate sepal/petal/lip formation in Oncidium Gower Ramsey, three paleoAPETALA3 genes, O. Gower Ramsey MADS box gene5 (OMADS5; clade 1), OMADS3 (clade 2), and OMADS9 (clade 3), and one PISTILLATA gene, OMADS8, were characterized. The OMADS8 and OMADS3 mRNAs were expressed in all four floral organs as well as in vegetative leaves. The OMADS9 mRNA was only strongly detected in petals and lips. The mRNA for OMADS5 was only strongly detected in sepals and petals and was significantly down-regulated in lip-like petals and lip-like sepals of peloric mutant flowers. This result revealed a possible negative role for OMADS5 in regulating lip formation. Yeast two-hybrid analysis indicated that OMADS5 formed homodimers and heterodimers with OMADS3 and OMADS9. OMADS8 only formed heterodimers with OMADS3, whereas OMADS3 and OMADS9 formed homodimers and heterodimers with each other. We proposed that sepal/petal/lip formation needs the presence of OMADS3/8 and/or OMADS9. The determination of the final organ identity for the sepal/petal/lip likely depended on the presence or absence of OMADS5. The presence of OMADS5 caused short sepal/petal formation. When OMADS5 was absent, cells could proliferate, resulting in the possible formation of large lips and the conversion of the sepal/petal into lips in peloric mutants. Further analysis indicated that only ectopic expression of OMADS8 but not OMADS5/9 caused the conversion of the sepal into an expanded petal-like structure in transgenic Arabidopsis (Arabidopsis thaliana) plants.

Figure 1.

Sequence comparison of OMADS3, OMADS5, OMADS8, and OMADS9 and the related B class MADS domain proteins. The functional MADS box proteins include PeMADS2, PeMADS3, PeMADS4, PeMADS5, and PeMADS6 (P. equestris), LMADS1 (L. longiflorum), OsMADS16 and OsMADS4 (O. sativa), CsAP3A (Crocus sativus), DcOPA3B (Dendrobium crumenatum), TGDEFA (T. gesneriana), and AODEF (Asparagus officinalis). The first and second underlined regions represent the MADS domain and the K domain, respectively. The three blocks in the C-terminal region represent the three motifs conserved among B class MADS box proteins. The paleoAP3 and PI-derived motifs are the two highly conserved motifs for paleoAP3 proteins of monocots. The PI motif is a highly conserved motif for PI orthologs. Amino acid residues identical to OMADS5 are indicated as dots. To improve the alignment, dashes were introduced into the sequence. The names of the OMADS3, OMADS5, OMADS8, and OMADS9 proteins are underlined. This sequence alignment was generated by the ClustalW Multiple Sequence Alignment Program at the DNA Data Bank of Japan (http://clustalw.ddbj.nig.ac.jp/top-e.html).

Figure 2.

Phylogenetic analysis of B class MADS domain proteins. Based on the amino acid sequence of the full-length protein, OMADS8 was closely related to PeMADS6 and OsMADS4 in the PI group of MADS box genes in monocots. OMADS3, OMADS5, and OMADS9 were closely related to genes in the paleoAP3 lineage of monocots. OMADS5 belongs to clade 1, OMADS9 belongs to clade 3, and OMADS3 belongs to clade 2 of paleoAP3 genes of orchids. The names of the OMADS3, OMADS5, OMADS8, and OMADS9 proteins are shown in boldface and underlined. The names of the plant species for each MADS box gene are listed behind the protein names. Amino acid sequences of B class MADS box genes were retrieved via the National Center for Biotechnology Information server (http://www.ncbi.nlm.nih.gov/). The phylogenetic tree was generated using Bayesian analysis as described in “Materials and Methods.”

Figure 3.

Detection of expression of OMADS3, OMADS5, OMADS8, OMADS9, and OMADS1 in O. Gower Ramsey. A, An O. Gower Ramsey mature flower bud (10 mm) consisting of three sepals (S), two petals (P), a lip (Lp) with red-brown around the callus (cal), and a reproductive organs column (col). Sepals and petals are yellow with red-brown bars and blotches toward the base of the segment. ac, Anther cap. Bar = 2 mm. B, Closeup of the column. The anther cap, which covers the reproductive organs column (col in A), was removed, revealing the pollinarium (male reproductive organ), which consists of two pollinia (po), a stalk (sk), and the brown viscidium (arrowed). Bar = 0.5 mm. C, Closeup of the pollinarium. po, Pollinia; sk, stalk; v, viscidium. Bar = 0.2 mm. D, Total RNAs isolated from leaves (L), roots (R), and the flower organs sepal (S), petal (P), lip (Lp), stamen (St), and carpel (C) of 10-mm-long floral buds (F) were used as templates to detect the expression of OMADS3, OMADS5, OMADS8, and OMADS9 by RT-PCR. In this study, two pollinia, a stalk of pollinarium, and the viscidium from the column were isolated as male reproductive organs (indicated as stamen). The remaining tissues of the column were used as female reproductive organs (indicated as carpel). The results indicated that OMADS8 and OMADS3 were expressed in all four floral organs as well as in vegetative leaves and roots. The mRNA for OMADS5 was only strongly detected in sepals and petals, whereas OMADS9 was only strongly detected in petals and lips. The AGL6-like gene OMADS1 was only expressed in lips and carpels. Each experiment was repeated twice with similar results. A fragment of the α-tubulin gene was amplified as an internal control.

Figure 4.

Detection of expression of OMADS3, OMADS5, OMADS8, and OMADS9 in sepal/petal/lip during early flower development in O. Gower Ramsey. A, Flower buds of O. Gower Ramsey at different developmental stages (2, 3, 5, and 8 mm long). Bar = 2 mm. B, Closeup of the young flower buds on the top of the inflorescence in O. Gower Ramsey. The positions of the 2- and 3-mm-long flower buds are indicated by the numbers. Bar = 2 mm. C, A 2-mm-long O. Gower Ramsey flower bud consisting of three sepals (s), two petals (p), and a lip (lp). In this stage, the lip is small and morphologically similar to the petals. Bar = 1 mm. D to G, Total RNAs isolated from the sepal (s), petal (p), and lip (lp) of 2-, 3-, and 5-mm-long young flower buds from A were used as templates to detect the expression of OMADS8 (D), OMADS3 (E), OMADS5 (F), and OMADS9 (G) by real-time PCR. The results indicated that OMADS8 mRNA was consistently detected in sepals, petals, and lips of all three early flower buds. OMADS3 mRNA was also consistently detected in sepals, petals, and lips of all three early flower buds with a relatively lower expression in lips than in sepal/petal. OMADS5 mRNA was consistently expressed only in sepals and petals and was absent in lips, whereas OMADS9 mRNA was only expressed in petals and lips and was not detected in the sepals of all three early flower buds. In quantitative real-time PCR, the columns represent the relative expression of these genes. Transcript levels of these genes were determined using two to three replicates and were normalized using α-tubulin. Error bars represent sd. Each experiment was repeated three times with similar results.

Figure 5.

Gene duplications and expression patterns of the B class genes in O. Gower Ramsey. A major duplication event from an ancestral B gene generated the paleoAP3 and PI lineages. In orchid O. Gower Ramsey, there is only one PI gene, OMADS8, and two more duplications occurred in the paleoAP3 gene that generated at least three paleoAP3-like genes, OMADS5 (clade 1), OMADS3 (clade 2), and OMADS9 (clade 3). OMADS8 and OMADS3 were expressed in the sepal/petal/lip, OMADS5 was expressed in the sepal/petal, while OMADS9 was expressed in the petal/lip. The AGL6-like gene OMADS1 was expressed in lip and carpel. [See online article for color version of this figure.]

Figure 6.

Detection of expression of OMADS1, OMADS3, OMADS5, OMADS8, and OMADS9 in peloric mutants of O. Gower Ramsey. A, Flower phenotypes of wild-type plants (WT), peloric mutants with two petals converted into lips (PL), and peloric mutants with two lateral sepals converted into lips (SL) of O. Gower Ramsey. The top row represents the front side and the bottom row represents the back side of the flower. ds, Dorsal sepal; ls, lateral sepal; p, petal; lp, lip; pl, lip-like petal; lsl, lip-like lateral sepal. Bar = 10 mm. B to F, Total RNAs isolated from the dorsal sepal (ds), lateral sepal (ls), petal (p), and lip (lp) of mature flowers from A, the wild type (WT), peloric mutants with petals converted into lips (PL), and peloric mutants with lateral sepals converted into lips (SL), were used as templates to detect the expression of OMADS5 (B), OMADS9 (C), OMADS8 (D), OMADS3 (E), and OMADS1 (F) by real-time PCR. The level of gene expression in lip-like petals is marked with red stars, and expression in the lip-like lateral sepal (lsl) is marked with blue stars. The results indicated that OMADS5 was significantly down-regulated in both lip-like petals and lip-like lateral sepals in peloric mutants. OMADS9 was significantly up-regulated in both lip-like lateral sepals and normal dorsal sepals in peloric mutants (A, right). OMADS1 was clearly up-regulated in both lip-like petals and lip-like lateral sepals in peloric mutants. In quantitative real-time PCR, the columns represent the relative expression of these genes. Transcript levels of these genes were determined using two to three replicates and were normalized using α-tubulin. Error bars represent sd. Each experiment was repeated three times with similar results.

Figure 7.

Confocal laser scanning microscopy of various floral organs in O. Gower Ramsey. A, A lip (L), two petals (P), and three sepals (S) of a 10-mm O. Gower Ramsey floral bud. The sepals and petals look the same. Bar = 5 mm. B, A lip (L), two petals (P), and three sepals (S) of an O. Gower Ramsey mature flower. The sepals and petals look the same. Bar = 10 mm. C, Irregularly shaped cells in the epidermis of sepals in the wild-type floral bud in A. Bar = 50 μm. D, Irregularly shaped cells in the epidermis of petals in the wild-type floral bud in A. Bar = 50 μm. E, Small cobblestone-like cells in the epidermis of lips in the wild-type floral bud in A. Bar = 50 μm. F to H, Irregularly shaped cells in the epidermis of sepals in the wild-type mature flower in B. Bar = 50 μm. I to K, Irregularly shaped cells in the epidermis of petals in the wild-type mature flower in B. Bar = 50 μm. L to N, Cobblestone-like cells in the epidermis of lips in the wild-type mature flower in B. Bar = 50 μm. O, Small cobblestone-like cells in the epidermis of lip-like petals in a peloric mutant floral bud (Fig. 3A, middle). Bar = 50 μm. P to R, Cobblestone-like cells in the epidermis of lip-like petals in a peloric mutant mature flower. Bar = 50 μm. The cell wall was counterstained with propidium iodide. C to F, I, L, O, and P are single slices to indicate the cell outline; G, J, M, and Q are Z-stacks to present the cell morphology; and H, K, N, and R are three-dimensional rotated images from the Z-stack images using the Fluoview 1000 software.

Figure 8.

Protein interactions among B class proteins of O. Gower Ramsey in a yeast two-hybrid assay. A, β-Galactosidase activity in yeast cells transformed with combinations of truncated OMADS3, OMADS5, OMADS8, and OMADS9 in either the binding domain plasmid (Bait) or the activation domain plasmid (Prey) calculated according to Miller (1992). Yeast cells transformed with OMADS3, OMADS5, OMADS8, and OMADS9 in the binding domain plasmid or the activation domain plasmid alone were used as controls for background activity. B, A summary of the strength of the β-galactosidase activity obtained in A. The number of + signs indicates the relative strength of the activity detected in each reaction. The – sign indicates that no activity was detected. AD, Activation domain in plasmid pGADT7; BK, binding domain in plasmid pGBKT7. C, A summary of the homodimers and heterodimers among the four B class proteins of O. Gower Ramsey obtained in A. [See online article for color version of this figure.]

Figure 9.

Phenotypic analysis of transgenic Arabidopsis plants ectopically expressing OMADS8 or OMADS8-ΔMADS. A, A wild-type inflorescence contained flower buds and mature flowers with normal sepals (s) and petals (p). B, A mature wild-type Arabidopsis flower consisted of four whorls of organs, including four sepals (s), four elongated petals (p), six stamens (arrow), and two fused carpels. The sepals had not opened in this stage. C, A 35S∷OMADS8 inflorescence contained flower buds (fb) with green/white sepals in the first whorl, fully opened mature flowers with white elongated petal-like sepals (ps) in the first whorl, and normal petals (p) in the second whorl of the flower. D and E, Closeup views of the 35S∷OMADS8 transgenic Arabidopsis flowers. White elongated petal-like sepals (ps) and normal petals (p) were produced in the first and second whorls of the flowers, respectively. F, Scanning electron micrograph of the surface cells of the epidermis of the first whorl petal-like sepal of a 35S∷OMADS8 flower was similar to the mature wild-type petal epidermis in G. Bar = 10 μm. G, Scanning electron micrograph of the surface cells of the epidermis of a mature wild-type petal. Bar = 10 μm. H, Scanning electron micrograph of the surface cells with irregular shapes in the epidermis of wild-type sepals. Bar = 10 μm. I, Total RNA isolated from two severe (T5 and T6) and four less severe (T1–T4) 14-d-old 35S∷OMADS8 transgenic Arabidopsis plants and from one untransformed wild-type plant (WT) used as a template. The results indicated that OMADS8 (OM8) was clearly expressed higher in the T5 and T6 than in the T1 to T4 transgenic plants. A fragment of the ACTIN (ACT) gene was amplified as an internal control.

Figure 10.

Possible evolutionary relationships between B class genes in the regulation of sepal/petal/lip formation for O. Gower Ramsey. B group genes in monocots are thought to have been produced by a major duplication event from an ancestral gene that generated the paleoAP3 and PI lineages. In orchid O. Gower Ramsey, there is only one PI gene, OMADS8, and at least two more duplications occurred in the paleoAP3 gene OMADS3 that generated the paleoAP3-like genes OMADS5 and OMADS9. The ancestral complex containing OMADS3/8 and other A/E proteins (X) is sufficient to convert the first and second whorl organs into well-expanded lip-like sepals/petals (in yellow). After gene duplication, OMADS9 may retain the ancestral B gene function of possibly promoting cell proliferation, whereas OMADS5 might have been altered in relation to this function. This caused the possible suppression of cell proliferation for any complex that includes OMADS5 (e.g. OMADS3/8/5/9/X) and might result in the formation of six short sepals/petals (in red) in the orchid flowers. During evolution, one of the short sepals/petals was converted into a well-expanded lip structure (in yellow) due to the loss of OMADS5 expression, resulting in the formation of one expanded lip (in yellow; OMADS3/8/9/X) and five short sepals/petals (in red; OMADS3/8/5/9/X), as seen in the modern O. Gower Ramsey. Unlike O. Gower Ramsey, there is only one paleoAP3 ortholog, LMADS1, and one PI ortholog, LMADS8, in lily. The complex containing LMADS1/8 and other A/E proteins (Y) may have the ability, similar to OMADS3/8/X, to convert the first and second whorl organs into well-expanded tepals (in white) in lily. The production of short tepals by suppressing cell proliferation has not been seen in lily, since no other paleoAP3 gene, such as OMADS5, has been identified in lily. [See online article for color version of this figure.]