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, 123 (4), 1325-36

Identification and Characterization of Three Orchid MADS-box Genes of the AP1/AGL9 Subfamily During Floral Transition

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Identification and Characterization of Three Orchid MADS-box Genes of the AP1/AGL9 Subfamily During Floral Transition

H Yu et al. Plant Physiol.

Abstract

Gene expressions associated with in vitro floral transition in an orchid hybrid (Dendrobium grex Madame Thong-In) were investigated by differential display. One clone, orchid transitional growth related gene 7 (otg7), encoding a new MADS-box gene, was identified to be specifically expressed in the transitional shoot apical meristem (TSAM). Using this clone as a probe, three orchid MADS-box genes, DOMADS1, DOMADS2, and DOMADS3, were subsequently isolated from the TSAM cDNA library. Phylogenetic analyses show that DOMADS1 and DOMADS2 are new members of the AGL2 subfamily and SQUA subfamily, respectively. DOMADS3 contains the signature amino acids as with the members in the independent OSMADS1 subfamily separated from the AGL2 subfamily. All three of the DOMADS genes were expressed in the TSAM during floral transition and later in mature flowers. DOMADS1 RNA was uniformly expressed in both of the inflorescence meristem and the floral primordium and later localized in all of the floral organs. DOMADS2 showed a novel expression pattern that has not been previously characterized for any other MADS-box genes. DOMADS2 transcript was expressed early in the 6-week-old vegetative shoot apical meristem in which the obvious morphological change to floral development had yet to occur. It was expressed throughout the process of floral transition and later in the columns of mature flowers. The onset of DOMADS3 transcription was in the early TSAM at the stage before the differentiation of the first flower primordium. Later, DOMADS3 transcript was only detectable in the pedicel tissues. Our results suggest that the DOMADS genes play important roles in the process of floral transition.

Figures

Figure 1
Figure 1
mRNA differential display. Total RNA from VSAM (V) and TSAM (T) were treated with RNase-free DNase I and analyzed by differential display using T4 and P9 primers. Amplified products were separated on 5% (w/v) denaturing polyacrylamide gels under thermostatic conditions. The arrow indicates the gene (otg7) differentially expressed in the TSAM.
Figure 2
Figure 2
Alignment of the deduced amino acid sequences of DOMADS1, DOMADS2, and DOMADS3. Black box, MADS domain; gray box, K-box region. Identical residues to the DOMADS1 reference sequence are indicated by dots, and gaps introduced to maximize the alignment are shown by dashes. The positions of amino acids are shown on the right.
Figure 3
Figure 3
Phylogenetic tree of plant MADS-box genes in the AGL2, OSMADS1, and SQUA subfamilies. Orchid MADS-box proteins are indicated by asterisks. Genus names of respective species are given in the parentheses behind the corresponding protein names. Subfamilies generally representing monophyletic gene clades (Theissen et al., 1996) are indicated by brackets at the right margin. The subfamily of OSMADS1-like genes appears here for the first time as an independent clade. The horizontal branch length is proportional to the estimated number of base substitutions. The numbers next to the nodes indicate bootstrap values (>50%) in 100 replicates.
Figure 4
Figure 4
Comparison of MADS-box domains between the genes in the AGL2 and OSMADS1 subfamilies. A consensus sequence (amino acids most frequently encountered at each particular position) is displayed, and only the deviations from consensus are listed in the individual sequence. The identical amino acids with consensus sequence are represented by dashes, and the missing data are indicated by blanks. The α-helix and two β-strands in the MADS-box domain are indicated respectively by the square dots and the lines above the consensus sequence. The characteristic amino acids of the OSMADS1 subfamily, which is distinguished from the AGL2 subfamily, are in black boxes.
Figure 5
Figure 5
Genomic DNA-blot analysis of DOMADS genes. DNA gel blots containing 15 μg of genomic DNA digested with EcoRl, EcoRV, and Xbal were hybridized at high stringency with digoxigenin-labeled probes that were derived from the 3′-specific region of DOMADS genes. The size of the DNA markers is given on right in kb.
Figure 6
Figure 6
Expression of DOMADS genes during the development of the orchid. The temporal scheme of main events during the orchid development is outlined above the northern results. The horizontal double arrows above the temporal scheme indicate the different developmental phases of the orchid. From left to right, total RNA (30 μg per lane) was successively prepared from thin sections of protocorms (0-week length = 1 mm), PLBs (3-week length = 4–5 mm), VSAMs including the youngest leaf primordium (6-week length = 1.5 mm), TSAMs including bracts and the youngest leaf primoridium (9- and 12-week length = 2 mm), inflorescence meristems including bracts and the youngest leaf primordium (15-week length = 3 mm), and floral buds (18-week length = 2–4 mm). Blots were hybridized with the specific digoxigenin-labeled probes described in “Genomic DNA-Blot Analysis.” The ribosomal RNAs stained by methylene blue indicate the amount of total RNA loaded in each lane.
Figure 7
Figure 7
A schematic median vertical section of the orchid flower bud showing the different floral parts. c, Column (fused structure of stigmas, styles, and stamens indicated by shaded region); o, ovary; p, petal; pd, pedicel; pl, pollinarium; r, rostellum; s, sepal; st, stigma.
Figure 8
Figure 8
Northern analysis of DOMADS genes in different orchid tissues (A) and in different floral organs (B). All of the blots were hybridized with the specific digoxigenin-labeled probes described in “Genomic DNA-Blot Analysis.” The ribosomal RNAs stained by methylene blue indicate the amount of total RNA loaded in each lane. A, The blots contain 25 μg of total RNA extracted from different tissues in each lane. B, The blots contain 15 μg of total RNA extracted from different mature floral organs in each lane.
Figure 9
Figure 9
In situ localization of DOMADS1 expression in longitudinal sections of SA and developing floral buds. Sections hybridized with the DOMADS1-specific antisense RNA probe (a, b, d, f, and h) or the DOMADS1 sense RNA probe (c, e, g, and i) are shown. Hybridization signals were visualized using a blue filter in bright-field illumination (a–e, h, and i) or dark-field illumination (f and g). Expression of DOMADS1 in: a, the SA of 12-week-old culture in the apical region of the inflorescence meristem and the first floral primordium (magnification, ×100). b and c, The SA of 15-week-old culture in the inflorescence meristem and the developing floral primordium (magnification, ×80). d and e, The young developing floral bud of 17-week-old culture in all of the floral organ primordia and the basal floral meristem (magnification, ×60). f and g, The floral bud of 19-week-old culture mainly in the maturing pollinarium, the rostellum, and the column (arrowheads) (magnification, ×50). h and i, The mature flower of 23-week-old culture in the rostellum located below the pollinarium (magnification, ×20). ac, Anther cap; am, apical meristem; b, bract; c, column; fm, floral meristem; fp, floral primordium; im, inflorescence meristem; lp, leaf primordium; p, petal; pl, pollinarium; r, rostellum; s, sepal.
Figure 10
Figure 10
In situ localization of DOMADS2 and DOMADS3 transcripts in longitudinal sections of SA and developing floral buds. Sections hybridized with the DOMADS2-specific antisense RNA probe (a, b, d, and e) and the DOMADS3-specific antisense RNA probe (f–h) or the DOMADS2 sense RNA probe (c) are shown. Hybridization signals were visualized using a blue filter in bright-field illumination (a, d, f, and g) or dark-field illumination (b, c, e, and h). Accumulation of DOMADS2 transcripts in: a, the SA of 11-week-old culture in the TSAM, both of the bract primordia and the last leaf primordium (magnification, ×100). b and c, The first floral primordium of 16-week-old culture in the central zone of the floral meristem (magnification, ×80). d and e, The floral bud of 18-week-old culture in the column primordium located at the bottom of the floral bud (magnification, ×30). Accumulation of DOMADS3 transcripts in: f, the SA of 11-week-old culture in the central zone of the TSAM and the region where the floral primordium would initiate (arrowhead; magnification, ×110). g and h, The SA of 16-week-old culture in the region below the floral meristem and the area flanking the procambium tissues below the inflorescence meristem (arrowheads; magnification, ×80). am, Apical meristem; b, bract; c, column; fm, floral meristem; im, inflorescence meristem; lp, leaf primordium; p, petal; pl, pollinarium; s, sepal.

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