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. 2013 Sep;64(12):3869-84.
doi: 10.1093/jxb/ert218.

Virus-induced Gene Silencing Unravels Multiple Transcription Factors Involved in Floral Growth and Development in Phalaenopsis Orchids

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Free PMC article

Virus-induced Gene Silencing Unravels Multiple Transcription Factors Involved in Floral Growth and Development in Phalaenopsis Orchids

Ming-Hsien Hsieh et al. J Exp Bot. .
Free PMC article

Abstract

Orchidaceae, one of the largest angiosperm families, has significant commercial value. Isolation of genes involved in orchid floral development and morphogenesis, scent production, and colouration will advance knowledge of orchid flower formation and facilitate breeding new varieties to increase the commercial value. With high-throughput virus-induced gene silencing (VIGS), this study identified five transcription factors involved in various aspects of flower morphogenesis in the orchid Phalaenopsis equestris. These genes are PeMADS1, PeMADS7, PeHB, PebHLH, and PeZIP. Silencing PeMADS1 and PebHLH resulted in reduced flower size together with a pelaloid column containing petal-like epidermal cells and alterations of epidermal cell arrangement in lip lateral lobes, respectively. Silencing PeMADS7, PeHB, and PeZIP alone resulted in abortion of the first three fully developed flower buds of an inflorescence, which indicates the roles of the genes in late flower development. Furthermore, double silencing PeMADS1 and PeMADS6, C- and B-class MADS-box genes, respectively, produced a combinatorial phenotype with two genes cloned in separate vectors. Both PeMADS1 and PeMADS6 are required to ensure the normal development of the lip and column as well as the cuticle formation on the floral epidermal cell surface. Thus, VIGS allows for unravelling the interaction between two classes of MADS transcription factors for dictating orchid floral morphogenesis.

Keywords: Cymbidium mosaic virus; PeMADS1; PeMADS6; PebHLH; Phalaenopsis; virus-induced gene silencing..

Figures

Fig. 1.
Fig. 1.
VIGS phenotypes of PebHLH-silenced flowers. (A) Front view of mock-treated (left) and PebHLH-silenced flowers (right) in Doritaenopsis I-Shin Sunrise Cinderella ‘OX1357’; solid white arrows indicate the lateral lobe (LL) and midlobe (ML); solid black arrows indicate the lateral lobes of the lip bowed outward. (B) Top-down view of the lateral lobes of the mock-treated (left) and PebHLH-silenced flowers (right); solid black arrows indicate the lateral lobes of the lip bowed outward; white bars, 1cm. (C) Diameter of flowers in mock-treated and PebHLH-silenced plants; a total of four flowers (the 3rd flower at blooming stage) from four plants were examined for each treatment; data are mean ± SD (n = 4); significance was accepted (one-tailed t-test) if P < 0.01 (*). (D) Relative level of PebHLH in mock-treated and PebHLH-silenced flowers. mRNA was extracted from the 3rd floral bud at 15 d post inoculation; data are mean ± SD (n = 4); significance was accepted (one-tailed t-test) if P < 0.01 (*). (E–H) SEM of the adaxial surface cells of lateral lobes in lips from mock-treated (E and F) and PebHLH-silenced flowers (G and H) at the flower-blooming stage; black arrowheads indicate node-like cuticular folds in the central fields of cells; white arrowheads indicate parallel cuticular folds in the anticline fields of cells; dotted black arrows indicate irregular cuticular folds in the central fields of cells; dotted black arrows indicate epicuticular wax crystals in the anticline field. Bars, 30 μm (this figure is available in colour at JXB online).
Fig. 2.
Fig. 2.
Silencing of PebHLH and PeMADS1 affects cell size and number in the floral organ. (A–F) SEM of the sepal and petal adaxial surface cells at the flower-blooming stage in mock-treated (A, B), PebHLH-silenced (C, D), and PeMADS1-silenced flowers (E, F). (G–J) Cell size and cell number in the distal adaxial surface of sepals (G, I) and petals (H, J) in mock-treated (M), PebHLH-silenced (1), and PeMADS1-silenced flowers (2); data are mean ± SD (n = 6); the same letters above the bars indicate that there is no statistical difference by Duncan’s multiple range test (P < 0.05) Bar, 30 μm. Materials were Dtps. I-Hsin Sunrise Cinderella ‘OX 1357’.
Fig. 3.
Fig. 3.
VIGS phenotypes of PeMADS1-silenced flowers. (A) Front view of mock-treated (left) and PeMADS1-silenced (right) flowers; solid black arrow indicates a longer and wider column. (B) Front and back view of columns in mock-treated (left) and PeMADS1-silenced flowers (right); solid white arrows indicate the anther cap (ac), rostellum (r), and stigmatic cavity (sc); white arrowheads indicate that the opposite site of stigmatic cavity in the column became longer and curved; black arrowheads indicate that the rostellum became longer. (C) Diameter of flowers in mock-treated (M) and PeMADS1-silenced plants (S); a total of six flowers (the 7th flower at blooming stage) from six plants were examined for each treatment; data are mean ± SD (n = 6); significance was accepted (one-tailed t-test) if P < 0.01 (*). (D) Relative mRNA level of PeMADS1 in M and S flowers; mRNA was extracted from the 7th floral bud on 35 d post inoculation; data are mean ± SD (n = 6); significance was accepted (one-tailed t-test) if P < 0.01 (*). (E) The 5th to 8th blooming flowers in a PeMADS1-silenced inflorescence and (F) columns of the 5th to 8th flowers; solid black arrows indicate that the column became longer and wider. Bars, 1cm.
Fig. 4.
Fig. 4.
Cyro-SEM micrographs of top views of epidermal cells of floral organs of the 7th blooming flowers of mock-treated (A–G), PeMADS1-silenced (H–N), PeMADS6-silenced (O–U), and double-silenced floral organs (V–AB). (A, H, O, V) Sepal adaxial epidermal cells had polygonal, polygonal, irregular, and polygonal forms. (B, I, P, W) Sepal abaxial epidermal cells all had irregular forms. (C, J, Q, X) Petal adaxial epidermal cells had polygonal, polygonal, irregular, and polygonal forms. (D, K, R, Y) Petal abaxial epidermal cells all had irregular forms. (E, L, S, Z) Lip adaxial epidermal cells had polygonal, polygonal, irregular, and irregular forms. (F, M, T, AA) Lip abaxial epidermal cells had elongated polygonal, elongated polygonal, irregular, and irregular forms. (G, N, U, AB) Column epidermal cells had polygonal, polygonal, polygonal, and elongated polygonal forms. Solid white arrows indicate a protuberance on the top of the cell; solid black arrow indicates epicuticular wax crystals; white arrowheads indicate irregular cuticular folds in the central field; black arrowheads indicate parallel cuticular folds in the central field; dashed white arrows indicate irregular cuticular folds in the anticline field; dashed black arrows indicate parallel cuticular folds in the central field. Bars, 25 μm. Materials were Dtps. OX Red Shoe ‘OX1407’.
Fig. 5.
Fig. 5.
Concurrent silencing of both PeMADS1 and PeMADS6 with a pCymMV-Gateway vector. (A–D) Mock-treated plants: top view (A), back view (B) of the flower and front view of lip (C) and column (D); solid white arrows indicate the lateral lobe (LL) and midlobe (ML); solid black arrows indicate the anther cap (ac), rostellum (r), stigmatic cavity (sc), and cirrus (C). (E–H) 1st blooming flower of double-silenced plants: top view (E), back view (F), and front view of lip (G) and column (H); black arrowheads indicate discoloured characteristics of sepals, petals, and lips; white arrowheads indicate dark green colouration (including leaf-like characteristics) of sepals, petals, and lips. (I–L) 7th blooming flower of double-silenced plants: top view (I), back view (J), and front view of lip (K) and column (L); dashed white arrow indicates that no cirrus developed in the tip of the midlobe; dashed black arrow indicates that the lateral lobes and midlobe were fused into a petal-like lip; dotted white arrow indicates that the base of the column became wider; dotted black arrows indicate that the side of the column developed an extra petal-like organ; dash-dotted white arrow indicates that the anther cap became wider and the appearance of more than one rostellum-like organ; dash-dotted black arrow indicates that the stigmatic cavity did not develop normally. White bars and black bars, 1cm.
Fig. 6.
Fig. 6.
Cyro-SEM micrograph of side views of epidermal cells of floral organs of the 7th blooming flowers of mock-treated (A–G), PeMADS1-silenced (H–N), PeMADS6-silenced (O–U), and double-silenced floral organs (V–AB). (A, H, O, V) Sepal adaxial epidermal cells had papilla, papilla, convex, and papilla shapes. (B, I, P, W) Sepal abaxial epidermal cells all had convex shape; St., stomata. (C, J, Q, X) Petal adaxial epidermal cells had papilla, papilla, papilla/hair papilla, and conical/papilla shapes. (D, K, R, Y) Petal abaxial epidermal cells all had convex shapes. (E, L, S, Z) Lip adaxial epidermal cells had convex, convex, conical, and hemispherical shapes. (F, M, T, AA) Lip abaxial epidermal cells had convex, convex, hemispherical, and convex shapes. (G, N, U, AB) Column epidermal cells had conical, conical, conical, and different shapes (including convex, hemispherical cupola, conical, and papilla). Solid white arrows indicate a protuberance on the top of the cell; solid black arrow indicates epicuticular wax crystals; white arrowheads indicate irregular cuticular folds in the central field; black arrowheads indicate parallel cuticular folds in the central field; dashed white arrows indicate irregular cuticular folds in the anticline field; dashed black arrows indicate parallel cuticular folds in the central field. Bars, 25 μm. Materials were Dtps. OX Red Shoe ‘OX1407’.
Fig. 7.
Fig. 7.
Severity of floral organ morphological changes in ordered blooming flowers in PeMADS6- (dotted bars), PeMADS1- (slash bars), and double-silenced plants (black bars). (A) Sepal morphological changes graded by estimating the percentage of greenish area on the abaxial surface. (B) Petal morphological changes graded by estimating the percentage of greenish and discoloured areas on the adaxial surface. (C–E) Lip morphological changes graded by estimating the percentage of greenish and discoloured areas on the abaxial surface of the midlobe and lateral lobes (C, G&D, greenish and discoloured), calculating the percentage of flowers that did not develop lateral lobes (D, NL, no lateral lobes), and he percentage of flowers that did not develop cirrus as well as the lateral lobes (E, NL&NC, no lateral lobes and no cirrus). (F–H) Column morphological changes were graded by calculating the percentage change in the length of the column (F, EL, elongated length of column; the formula is (length of VIGS column – length of mock column)/(length of mock column) ×100, calculating the percentage of flowers that developed an abnormal stigmatic cavity (G, ASC), and the percentage of flowers that developed an abnormal stigmatic cavity as well as an extra petal-like organ (H, ASC&EPL). Significance was accepted (one-tailed t-test) if P < 0.01 (*).
Fig. 8.
Fig. 8.
Expression of PeMADS6 in PeMADS1-, PeMADS6-, and double-silenced plants according to flower blooming order (1st, 3rd, 5th, 7th, and 9th) in inflorescences of mock-treated plants (Mock) and PeMADS1-, PeMADS6-, and double-silenced plants (Pe1-silenced, Pe6-silenced, and Pe1&Pe6-silenced) in the (A) sepal, (B) petal, (C) lip, and (D) column. mRNA was extracted at flower blooming date in Dtps. I-Hsin Sunrise Cinderella ‘OX 1357’. Data are mean ± SD from three plants for each experiment (n = 3); the same letters above the bars indicate that there is no statistical difference by Duncan’s multiple range test (P < 0.05).
Fig. 9.
Fig. 9.
Expression of PeMADS1 in PeMADS1-, PeMADS6-, and double-silenced plants according to flower blooming order (1st, 3rd, 5th, 7th, and 9th) in inflorescences of mock-treated plants (Mock) and PeMADS1-, PeMADS6-, and double-silenced plants (Pe1-silenced, Pe6-silenced, and Pe1&Pe6-silenced) in the (A) sepal, (B) petal, (C) lip, and (D) column. mRNA was extracted at flower blooming date in Dtps. I-Hsin Sunrise Cinderella ‘OX 1357’. Data are mean ± SD from three plants for each experiment (n = 3); the same letters above the bars indicate that there is no statistical difference by Duncan’s multiple range test (P < 0.05).

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