PISTILLATA-like Gene in Phalaenopsis

Plant Cell Physiol. 46(7): 1125–1139 (2005) doi:10.1093/pcp/pci125, available online at www.pcp.oupjournals.org JSPP © 2005

PeMADS6, a GLOBOSA/PISTILLATA-like Gene in Phalaenopsis equestris Involved in Petaloid Formation, and Correlated with Flower Longevity and Ovary Development Wen-Chieh Tsai 1, Pei-Fang Lee 2, Hong-Ie Chen 2, Yu-Yun Hsiao 1, Wan-Ju Wei 2, Zhao-Jun Pan 1, Ming-Hsiang Chuang 1, Chang-Sheng Kuoh 1, Wen-Huei Chen 3 and Hong-Hwa Chen 1, 4, * 1

Department of Life Sciences, National Cheng Kung University, Tainan 701, Taiwan Department of Biotechnology, Fooyin University, Kaohsiung County 831, Taiwan 3 Department of Life Sciences, National University of Kaohsiung, Kaohsiung 811, Taiwan 4 Institute of Biotechnology, National Cheng Kung University, Tainan 701, Taiwan 2

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In this study, we isolated and characterized the function of a GLOBOSA/PISTILLATA-like gene, PeMADS6, from a native Phalaenopsis species, P. equestris. Southern blot analysis showed PeMADS6 as a single copy in the Phalaenopsis genome. Results of the determination of temporal and spatial expression showed that PeMADS6 was expressed and thus participated in the development of the sepals, petals, labellum and column in Phalaenopsis. Further confirmation of the expression pattern of PeMADS6 was carried out with in situ hybridization. Repressed expression of PeMADS6 in the orchid ovary was found to be pollination regulated, which suggests that the gene may have an inhibitory effect on the development of the ovary or ovule. In addition, auxin acted as the candidate signal to regulate the repression of PeMADS6 expression in the ovary. Furthermore, the flowers of transgenic Arabidopsis plants ectopically overexpressing PeMADS6 showed the morphology of petaloid sepals, with a 3- to 4-fold increase in flower longevity. Concomitantly, delayed fruit maturation was also observed in the transgenic Arabidopsis, which is consistent with the inhibitory effect of PeMADS6 on the development of the ovary. Thus, as a B-function gene, PeMADS6, not only specifies floral organ identity but has functions in flower longevity and ovary development in orchids. Keywords: Flower longevity — Floral organ identity — GLOBOSA/PISTILLATA-like gene — Ovary/ovule development — Overexpression — Phalaenopsis. Abbreviations: ACC, 1-aminocyclopropane-1-carboxylic acid; AVG, aminoethoxyvinylglycine; CaMV, cauliflower mosaic virus; DAF, days after flowering; EST, expressed sequence tag; NAA, naphthaleneacetic acid; ORF, open reading frame; PeMADS, Phalaenopsis equestris MADS gene; PeMADS, Phalaenopsis equestris MADS protein; 5′-RACE, 5′-rapid amplification of cDNA ends; UTR, untranslated region. The nucleotide sequence reported in this paper has been submitted to NCBI under accession number AY678299 (PeMADS6).

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Introduction Members of the MADS domain family of transcriptional regulators participate in a variety of developmental processes in plants, including reproductive (flower, seed and fruit) and vegetative (root and leaf) development (Ng and Yanofsky 2001). Based on phenotypic and genetic analyses of Arabidopsis and snapdragon homeotic mutants, the development of the four types of floral organs is governed by overlapping activities of three classes of regulatory genes (Weigel and Meyerowitz 1994). In the well-known ‘ABC model’, the expression of A alone specifies sepal formation. The combination of AB determines the development of petals, and that of BC stamen formation. Expression of the C function controls formation of carpels (Weigel and Meyerowitz 1994, Theissen et al. 2000). Recently, it was shown that the Arabidopsis B- and C-function genes, which control petal, stamen and carpel development, are functionally dependent on three highly similar MADS-box genes, SEP1, SEP2 and SEP3 (Pelaz et al. 2000). Most well characterized plant MADS-box genes belong to a monophyletic gene superclade with a conserved structural organization, the MIKC-type domain structure, including a MADS (M-), intervening (I-), keratin-like (K-) and C-terminal (C-) domain (Becker et al. 2000, Theissen et al. 2000, Parenicov et al. 2003). All B-function genes belong to the family of MADS-box genes. They fall into either one of two different clades, namely DEFICIENS (DEF)/APETALA3 (AP3)or GLOBOSA (GLO)/PISTILLATA (PI)-like genes (Theissen et al. 1996, Theissen et al. 2000). DEF/AP3- and GLO/PI-like genes are closely related within the MADS-box gene family, because these two clades together also represent a wellsupported gene clade (Theissen et al. 1996, Theissen et al. 2000). Mutation in AP3 or PI caused flowers with the sepal structure in two outer whorls and the carpels in two inner whorls (Bowman et al. 1989, Jack et al. 1992, Jack et al. 1994). In Antirrhinum majus, similar mutant phenotypes are described, which are due to a mutation in the gene DEF or GLO (Sommer et al. 1990, Tröbner et al. 1992), homologs to AP3 and PI,

Corresponding author: E-mail, [email protected]; Fax, +886-6-235-6211. 1125

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A GLO-like PeMADS6 gene in Phalaenopsis orchid

Fig. 1 Structure of Phalaenopsis equestris flower. (A) Diagram of the front view of the Phalaenopsis showing flower organs. Inset: the column showing the stigmatic cavity. (B) Diagram of the back view of the same flower showing the inferior ovary. (C) The post-pollination syndrome of flowers at 4 DAP. The swelling ovary and senescent perianth. Inset: the swelling of the stigma enclosing the pollinia. (D) Morphological changes at day 4 of treatment with auxin (NAA). The swelling ovary and senescent perianth. Inset: the enclosing stigma. (E) Senescence-related degradative changes in all organs of flower at day 4 of treatment with ACC. Whole flower and ovary senescence was observed.

respectively. In Petunia, duplicated B-function genes control the meristem fate of whorls 2 and 3 (van der Krol et al. 1993, Vandenbussche et al. 2004). The GLO/PI-like gene, MdPI, identified from apple, had not only a floral organ identity function but also a role in parthenocarpic apple fruit production (Yao et al. 2001). Cloning of the ABC genes also allows for validation of the ABC model using gain-of-function experiments with transgenic plants. For example, overexpression of both AP3 and PI leads to the formation of flowers in which the first whorl is occupied by petals and the fourth whorl carpels are replaced by stamens (Krizek and Meyerowitz 1996). Transgenic rice that ectopically expressed OSMADS16 displayed an innermost whorl occupied by multiple stamen-like organs that also fit the ABC model (Lee et al. 2003). Ectopic expression of B-function genes in heterogous systems is also used to prove their functions. For example, transgenic Nicotiana tabacum ectopically expressing the B-function genes from A. majus revealed two outer whorls of petals (Davies et al. 1996). Over-

expression of GGM2 from gymnosperm Gnetum in Arabidopsis suggests that GGM2 is related to class B floral organ identity genes of angiosperm (Winter et al. 2002). Ectopic expression of LMADS1 truncated with the MADS domain in Arabidopsis generated the ap3-like dominant negative mutation, supporting that LMADS1 is the functional counterpart of the AP3 gene in lily (Tzeng and Yang 2001). In Oncidium, OMADS3, an AP3-like gene, regulates floral formation and initiation (Hsu and Yang 2002). Outside the higher eudicots, B-function genes have been most intensively studied in cereal grasses (Poaceae), mainly rice and maize (Chung et al. 1995, Kang et al. 1998, Moon et al. 1999, Ambrose et al. 2000, Kyozuka et al. 2000, Münster et al. 2001, Lee et al. 2003, Nagasawa et al. 2003). The phenotypes of the mutant and transgenic plants and the expression patterns support the view that DEF/AP3- and GLO/PI-like genes from rice and maize are similar in function to the floral homeotic B-function genes in eudicots. However, Liliaceae, whose flowers superficially look very different from the flow-


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Fig. 2 Alignment of the deduced amino acid sequences of PeMADS genes and other GLO/PI- and DEF/AP3-like genes. The multiple alignment was generated by the computer program PILEUP and displayed by PRETTYBOX. Identity with the consensus is denoted by a black box. Similarity with the consensus is denoted by the color gray; differences are indicated by white, gaps in the alignment are indicated by points, and positions that are not occupied by an amino acid by a ‘∼’. The MADS-, I-, K- and C-domains are indicated on top of the columns. The PI motif is indicated by a black line. OSMADS4 (AAC05723) and OSMADS2 (AAB52709) are from Oryza sativa; ZMM16 (CAC33848), ZMM18 (CAC33849) and ZMM29 (CAC33850) are from Zea mays; DEF (S12378) and GLO (CAA48725) are from Antirrhium majus; PI (BAA06465) and AP3 (A42059) are from Arabidopsis thaliana; and PeMADS6 is from Phalaenopsis equestris.

ers of grasses, for example, have a perianth composed of two whorls of organs, each containing three petaloid tepals. Floral structures like these could be easily explained by a modified ABC model in that the expression of the B-function genes has expanded to whorl 1 (van Tunen et al. 1993). This modified ABC model was strongly supported by expression and gel retardation assay analyses of B-function genes in tulip (Kanno et al. 2003). Both unique floral morphology and reproductive biology have made orchids attractive models for botanists. Phalaenopsis is a member of the Orchidaceae, one of the largest families of flowering plants. The Phalaenopsis flower is highly evolved, with a gynostemium or column (a fusion of the male and female reproductive organs) and a highly modified petal, the labellum or lip. The sepals and petals are usually more or less similar in size, shape and coloration. Modifications of the perianth and fusion of androecium and gynoecium are the bases for variations in orchid floral morphology (Fig. 1A). In

Phalaenopsis, most flowers are long lasting, usually remaining in good condition for at least 2 weeks, but the perianth withers following pollination (Christenson 2001). The perianth is persistent and remains attached to the apex of the ovary as small, shriveled structures during fruit development. The ovary of Phalaenopsis, which eventually develops into a fruit, is termed inferior. The ovary, defined by the region with ovules and/or seeds, internally tapers at the base to a sterile segment, the pedicel (Fig. 1B). These regions are usually visually undifferentiated prior to pollination (Christenson 2001). Pollination of flowers is a key regulatory event in plant reproduction. In Phalaenopsis, post-pollination development is precisely and completely triggered by pollination. It includes perianth senescence, pigmentation changes, swelling of the column leading to enclosure of the pollinia within the stigmatic camber, and induction and coordination of ovary and ovule development in preparation for fertilization (Zhang and O’Neill 1993). Ethylene, with auxin, is required for ovary development

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and ovule differentiation following pollination (Zhang and O’Neill 1993). In this report, we isolated a GLO/PI-like MADS-box gene, PeMADS6, from Phalaenopsis and established its floral organ identity function. In addition, we found that the role of

PeMADS6 was closely correlated with the extension of flower longevity and post-pollination development of the ovary. These results provide the basis for an expanded model of floral and ovary development in orchids.


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tion of the ORF yielded a protein of 210 amino acids. Multiple sequence alignments with other MADS-box proteins showed PeMADS6 to have a typical MIKC-type domain structure (Fig. 2). Almost all of the C-terminal regions of GLO-like proteins have the PI motif MPFxFRVQPxQPNLQE (Kramer et al. 1998). The sequence of PeMADS6 contained MPMTFRVQPIQPNLQG and showed 87.5% similarity in this region (Fig. 2).

Fig. 4 Southern blot analysis of the PeMADS6 gene in the P. equestris genome. DNA gel blots containing 10 µg of genomic DNA digested with BglII (lane 1), HindIII (lane 2) and EcoRI (lane 3) were hybridized under stringent conditions with use of probes derived from the 3′-specific region of the PeMADS6 gene. The sizes of DNA markers are shown at the left margin (kb).

Results Identification of GLO/PI-like MADS-box genes in P. equestris Five sequences with high similarity to GLO/PI-like MADS-box proteins were identified in the established floral expressed sequence tag (EST) database of P. equestris (Tsai et al. 2004). The sequences were assembled into one contig and determined to be a partial cDNA with an incomplete open reading frame (ORF). The full-length sequence of the cDNA was obtained by use of 5′-rapid amplification of cDNA ends (5′-RACE). Nucleotide sequences of the 10 cloned RACE products of the individual MADS-box genes were determined, assembled with the contig of the GLO/PI-like EST. A complete sequence of a 919 bp GLO/PI-like cDNA was identified and named PeMADS6 (the P. equestris MADS6 gene). Conceptual transla-

Phylogenetic relationship of PeMADS6 with other B-function genes To determine the phylogenetic relationship of PeMADS6 and other MADS-box genes, the phylogeny of known B-function genes was reconstructed with the use of the conceptual amino acid sequences of the respective genes as input data. The topology of the phylogenetic tree confirmed that PeMADS6 fell well into the clade of the GLO/PI-like genes (Fig. 3). Sequences of B-function genes from monocots formed a monophyletic subclade (Fig. 3). Consistent with this result, only one GLO/PI-like gene existed in the last common ancestor of monocots and eudicots, about 160–200 million years ago (Goremykin et al. 1997). Within the clade of monocot GLO/PIlike genes, PeMADS6 is more closely related to the ORCPI gene (accession No. BAC22579) in Orchis italica, an orchid GLO/PI-like gene. These results together strongly suggest that the PeMADS6 gene belongs to the GLO/PI-like gene family. Genomic organization of the PeMADS6 gene To investigate the genomic organization of PeMADS6 in the orchid genome, Southern blot analysis was carried out with a probe containing sequences of the partial I-region and the entire K-box region of PeMADS6. Results showed a single band in all four enzyme digestions, including BglII, HindIII, EcoRV and EcoRI (data not shown). To confirm this result further, we used a probe containing the C-terminal region and 3′untranslated region (UTR) of PeMADS6. Again it revealed only one band for each digestion (Fig. 4, lanes 1–3). These results demonstrate that the probes were gene specific for PeMADS6 and suggest the existence of only one GLO/PI-like gene in the Phalaenopsis orchid genome. Spatial and temporal expression patterns of PeMADS6 The expression of B-function genes in Arabidopsis and Antirrhinum is known to be flower specific (Jack et al. 1992,

Fig. 3 Phylogenetic tree of B-function MADS-box genes in the DEF, GLO and GGM2 subfamilies. Published plant MADS-box protein sequences were retrieved from the GenBank database [DAL11 (AAF18372), DAL13 (AAF18376), GGM2 (CAB44448), OSMADS2 (AAB52709), ZMM16 (CAC33848), ZMM18 (CAC33849), ZMM29 (CAC33850), OSMADS4 (AAC05723), HPI2 (AAD22494), HPI1 (AAD22493), ORCPI (BAC22579), LRGLOA (BAB91551), LRGLOB (BAB91552), TGGLO (BAC75972), SMPI (AAF73941), NTGLO (CAA48142), FBP1 (AAA33731), GLO (S28062), PMADS2 (CAA49568), GGLO1 (CAA08804), SLM2 (CAA56656), EGM2 (AAC78283), PI (BAA06465), PeMADS2 (AY378149), PeMADS3 (AY378150), PeMADS4 (AY378147), PeMADS5 (AY378148), DEF (S12378), AP3 (A42095)]. Phylogeny was conducted with the neighbor-joining algorithm. The tree was rooted by use of DAL11, DAL13 and GGM2, members of the sister clade of the B proteins, as the outgroup (Becker et al. 2000). Bootstrap values from 1,000 replicates are indicated on most major nodes. Genus names of the species from which the respective genes were isolated are given in parentheses following the corresponding protein names. PeMADS6 is highlighted by open boxes. The monophyletic floral homeotic gene groups are labeled by brackets at the right margin.

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Fig. 5 (A) Expression of the PeMADS6 gene during the development of the flower buds and in different orchid tissues. Each lane contained 10 µg of total RNA from: stage 1–4 flower buds (lanes 1–4), immature ovaries (lane 5), shoots (lane 6), leaves (lane 7) and roots (lane 8). (B, C) Northern blot analysis of the PeMADS6 gene in different floral organs of the wild-type plant (B) and its peloric mutant (C). The blots in each lane contained 10 µg of total RNA and were hybridized with the 3′-specific region probe described above. The rRNA indicated the same amount of total RNA loaded in each lane. RNA sources were sepal (B, C, lane 1), petal (B, lane 2), lip-like petal (lane 2), lip (B, C, lane 3), pollinium (B, lane 4) and column (B, lane 5; C, lane 4) as indicated.

Schwarz-Sommer et al. 1992, Tröbner et al. 1992, Goto and Meyerowitz 1994). To investigate whether the expression of PeMADS6 is also flower specific, results of Northern blot analysis with use of total RNA isolated from different developmental stages of flower buds and several vegetative tissues was performed. Results showed that expression of PeMADS6 was detected in all the developmental stages of flower buds (stage 1,