Characterization of C- and D-Class MADS-Box Genes in Orchids

Abstract

Orchids (members of the Orchidaceae family) possess unique flower morphology and adaptive reproduction strategies. Although the mechanisms underlying their perianth development have been intensively studied, the molecular basis of reproductive organ development in orchids remains largely unknown. Here, we report the identification and functional characterization of two AGAMOUS (AG)-like MADS-box genes, Dendrobium 'Orchid' AG1 (DOAG1) and DOAG2, which are putative C- and D-class genes, respectively, from the orchid Dendrobium 'Chao Praya Smile'. Both DOAG1 and DOAG2 are highly expressed in the reproductive organ, known as the column, compared to perianth organs, while DOAG2 expression gradually increases in pace with pollination-induced ovule development and is localized in ovule primordia. Ectopic expression of DOAG1, but not DOAG2, rescues floral defects in the Arabidopsis (Arabidopsis thaliana) ag-4 mutant, including reiteration of stamenoid perianth organs in inner whorls and complete loss of carpels. Downregulation of DOAG1 and DOAG2 in orchids by artificial microRNA interference using l-Met sulfoximine selection-based gene transformation systems shows that both genes are essential for specifying reproductive organ identity, yet they, exert different roles in mediating floral meristem determinacy and ovule development, respectively, in Dendrobium spp. orchids. Notably, knockdown of DOAG1 and DOAG2 also affects perianth organ development in orchids. Our findings suggest that DOAG1 and DOAG2 not only act as evolutionarily conserved C- and D-class genes, respectively, in determining reproductive organ identity, but also play hitherto unknown roles in mediating perianth organ development in orchids.

Figure 1.

Quantitative analyses of DOAG1 and DOAG2 expression in Dendrobium ‘Chao Praya Smile’. A, A Dendrobium ‘Chao Praya Smile’ plant with inflorescences and flowers. FB, Floral bud; IA, inflorescence apex; Lf, leaf; OF, open flower; Rt:, root; Sm, stem. B, Close-up view of the inflorescence bearing open flowers of Dendrobium ‘Chao Praya Smile’. C, A Dendrobium ‘Chao Praya Smile’ flower comprising three sepals (Se), two petals (Pe), one lip (Li), and the reproductive organ column (Co). D, Close-up view of the column showing the anther cap (Ac), stigmatic cavity (Sc), and ovary (Ov). E, Top view of the anther cap. F, Close-up view of two pollinia. G, Seed pods of Dendrobium ‘Chao Praya Smile’ at different DAP. Scale bars = 1 cm (A–D and G) and 500 μm (E and F)..H and I, Expression of DOAG1 (H) and DOAG2 (I) in various tissues. J and K, Expression of DOAG1 (J) and DOAG2 (K) in different flower organs. The anther cap and pollinia from the column were collected as male reproductive organs (indicated as Po), whereas the remaining tissue of the column surrounding the stigmatic cavity and the ovary were collected as female reproductive organs. L and M, Expression of DOAG1 (L) and DOAG2 (M) in developing seed pods at different DAP, as shown in G. In H to M, expression of DOAG1 and DOAG2 was examined by qPCR analysis of three independent biological samples. The levels of DOAG1 and DOAG2 expression were normalized to the expression of the orchid polyubiquitin gene DOUbi. Error bars indicate the mean ± sd.

Figure 2.

In situ localization of DOAG1 and DOAG2 transcripts in Dendrobium ‘Chao Praya Smile’. Sections in this figure were hybridized with DOAG1 and DOAG2 gene-specific antisense RNA probes or the corresponding sense probes, which served as negative controls (Supplemental Fig. S5). A to D, DOAG1 mRNA localization in an inflorescence apex (A), young floral buds (B and C), and an old floral bud (D). E to H, DOAG2 mRNA localization in an inflorescence apex (E), young floral buds (F and G), and an old floral bud (H). I to K, DOAG2 mRNA localization in ovules at different developmental stages. DOAG2 is barely detectable in sections of ovaries at 0 DAP (I) or 6 DAP (J), but is expressed in ovule primordia at 16 DAP (K). Red arrows indicate ovule primordia. The inset in K shows an enlarged view of the ovule primordium in the dotted square. Scale bars = 200 μm. Ac, Anther cap; Co, column; FP, floral primordium; IM, inflorescence meristem; Li, lip; Pe, petal; Po, pollinia; Ro, rostellum; Se, sepal.

Figure 3.

Phenotypic analyses of transgenic Arabidopsis plants overexpressing DOAG1. A, A 35S:DOAG1 plant displays early flowering and curled leaves compared to a wild-type (WT) plant. B, Close-up view of a wild-type Arabidopsis flower consisting of four whorls of floral organs, including sepals, petals, stamens, and carpels. C, Inflorescence apex of a 35S:DOAG1 plant. D, A 35S:DOAG1 flower showing stamenoid petals. E, Flowering time distribution of wild-type and 35S:DOAG1 plants under long-day conditions. F, ag-4 35S:DOAG1 flowered earlier with fewer rosette leaves than ag-4. G and H, Close-up views of ag-4 (G) and ag-4 35S:DOAG1 (H) flowers. I, Quantitative analysis of DOAG1 expression in representative 35S:DOAG1 transgenic plants and ag-4 35S:DOAG1. Results are normalized against the expression levels of TUB2 and shown as relative values to the highest level set to 100%. Error bars indicate the mean ± sd. Asterisks indicate significant differences in DOAG1 expression levels between 35S:DOAG1 and wild-type plants or between ag-4 35S:DOAG1 and ag-4 using two-tailed paired Student's t test (*P < 0.05). Scale bars = 1 cm (A and F) and 0.5 mm (B–D, G, and H).

Figure 4.

Phenotypic analyses of transgenic Arabidopsis plants overexpressing DOAG2. A, A 35S:DOAG2 plant displays early flowering and curled leaves compared to a wild-type (WT) plant. B, Close-up view of a 35S:DOAG2 seedling. C, Close-up view of a wild-type Arabidopsis flower. D, Inflorescence apex of a 35S:DOAG2 plant. E, Close-up view of a 35S:DOAG2 flower. Scale bars = 1 cm (A), 0.5 cm (B), and 0.5 mm (C–E). F, Flowering time distribution of wild-type and 35S:DOAG2 plants under long-day conditions. G, Quantitative analysis of DOAG2 expression in representative 35S:DOAG2 transgenic plants. Results are normalized against the expression levels of TUB2 and shown as relative values to the highest level set to 100%. Error bars indicate the mean ± sd. Asterisks indicate significant differences in DOAG2 expression levels between 35S:DOAG2 and wild-type plants using two-tailed paired Student's t test (*P < 0.05).

Figure 5.

Identification of AmiR-doag1 and AmiR-doag2 transgenic orchids. A, PCR genotyping of putative AmiR-doag1 (top) and AmiR-doag2 (bottom) transgenic orchids using the 35S promoter primer and the universal primer G4369 listed in Supplemental Table S1. The transformation plasmids and genomic DNA from wild-type orchid (WT) were included as positive controls (C+) and negative controls, respectively. B, Southern blot analysis of genomic DNAs from representative AmiR-doag1 and AmiR-doag2 transgenic orchids. The DNA gel blot containing 20 μg of genomic DNA digested with EcoRI was hybridized with the specific probe for the bar gene in transgenic plants. Genomic DNAs from wild-type orchids and the pGreen-0229 plasmid containing the bar gene (P) served as negative and positive controls, respectively.

Figure 6.

DOAG1 affects floral meristem determinacy and floral organ development in Dendrobium ‘Chao Praya Smile’. A, Front view of a wild-type (WT) open flower shows three sepals (S1–S3), two petals (P1 and P2), and one lip. B, Front view of an AmiR-doag1 (line 1) open flower shows five petals (P1 to P5), two pieces of gynostemium (G1 and G2), and one lip. C, Back view of an AmiR-doag1 (line 1) open flower shows five sepals (S1 to S5) supported by one bract (B). D, A wild-type pollinarium consists of two normal pollinia. E, An AmiR-doag1 (line 1) pollinarium consists of two underdeveloped pollinia. F, Front view of an AmiR-doag1 (line 3) open flower shows multiple whorls of green bract-like structures. Scale bars = 0.5 cm (A–C and F) and 0.5 mm (D and E). G and H, Quantitative analysis of DOAG1 (G) and DOAG2 (H) expression in wild-type and representative AmiR-doag1 transgenic orchids. Total RNA extracted from open flowers and inflorescence apex was used for expression analysis. Results are normalized against the expression levels of DOUbi and shown as relative values to the wild-type level set at 100% (G) or 1.0 (H). Error bars indicate the mean ± sd. Asterisks indicate significant differences in expression levels of DOAG1 (G) and DOAG2 (H) between specified transgenic lines and wild-type plants using two-tailed paired Student's t test (*P < 0.05).

Figure 7.

Downregulation of DOAG2 affects perianth and gynostemium development in Dendrobium ‘Chao Praya Smile’. A, A wild-type (WT) open flower. B and C, AmiR-doag2 transgenic orchids, lines 1 (B) and 2 (C), develop only two perianth organs without the gynostemium. D to F, AmiR-doag2 transgenic orchids, lines 3 (D), 4 (E), and 5 (F), develop three perianth organs without the gynostemium. Scale bars = 0.5 cm. G and H, Quantitative analysis of DOAG2 (G) and DOAG1 (H) expression in wild-type and representative AmiR-doag2 transgenic orchids. Total RNA extracted from open flowers and inflorescence apex was used for expression analysis. Results are normalized against the expression levels of DOUbi and shown as relative values to the wild-type level set at 100% (G) or 1.0 (H). Error bars indicate the mean ± sd. Asterisks indicate significant differences in expression levels of DOAG2 (G) and DOAG1 (H) between specified transgenic lines and wild-type plants using two-tailed paired Student's t test (*P < 0.05).

Figure 8.

Schematic representation of DOAG1 and DOAG2 function in Dendrobium ‘Chao Praya Smile’. A, Schematic representation of a wild-type Dendrobium ‘Chao Praya Smile’ flower showing perianth organs, including sepals (Se), petals (Pe), lip (Li), and the reproductive organ column (Co), which is a fused structure of stigmatic cavity (Sc), ovary (Ov), and pollinia (Po) covered by the anther cap (Ac). B, Relative expression of DOAG1 and DOAG2 in different floral organs in a wild-type Dendrobium ‘Chao Praya Smile’ flower. C, Summary of floral phenotypes of DOAG1 and DOAG2 knockdown lines. Weak knockdown of DOAG1 produces indeterminate floral meristems, resulting in more floral organs than wild-type flowers, while strong knockdown of DOAG1 results in leaf-like reiterated structures (green leaves). Knockdown of DOAG2 abolishes the generation of reproductive organs and produces greenish chimeric perianth organs (light green leaves).