JXB Advance Access originally published online on May 21, 2004
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Journal of Experimental Botany, Vol. 55, No. 401, pp. 1391-1399, June 1, 2004
© 2004 Oxford University Press
RESEARCH PAPER |
Ectopic expression of LLAG1, an AGAMOUS homologue from lily (Lilium longiflorum Thunb.) causes floral homeotic modifications in Arabidopsis
Received 11 December 2003; Accepted 19 March 2003
1 Plant Research International, 6700 AA Wageningen, The Netherlands
2 Laboratory of Biochemistry, Wageningen University, 6703 HA Wageningen, The Netherlands
* To whom correspondence should be addressed. Fax: +31 317 41 8094. E-mail: frans.krens{at}wur.nl
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Abstract |
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The ABC model for floral development was proposed more than 10 years ago and since then many studies have been performed on model species, such as Arabidopsis thaliana, Antirrhinum majus, and many other species in order to confirm this hypothesis. This led to additional information on flower development and to more complex molecular models. AGAMOUS (AG) is the only C type gene in Arabidopsis and it is responsible for stamen and carpel development as well as floral determinacy. LLAG1, an AG homologue from lily (Lilium longiflorum Thunb.) was isolated by screening a cDNA library derived from developing floral buds. The deduced amino acid sequence revealed the MIKC structure and a high homology in the MADS-box among AG and other orthologues. Phylogenetic analysis indicated a close relationship between LLAG1 and AG orthologues from monocot species. Spatial expression data showed LLAG1 transcripts exclusively in stamens and carpels, constituting the C domain of the ABC model. Functional analysis was carried out in Arabidopsis by overexpression of LLAG1 driven by the CaMV35S promoter. Transformed plants showed homeotic changes in the two outer floral whorls with some plants presenting the second whorl completely converted into stamens. Altogether, these data strongly indicated the functional homology between LLAG1 and AG.
Key words: ABCDE model, double flower, flower architecture, function analysis, MADS-box genes, phylogenetic study.
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Introduction |
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TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
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Mechanisms of flower development are under the control of a complex genetic system. The formation of organs in the four whorls of a typical eudicotyledonous flower, consisting of sepals, petals, stamens, and carpels, requires many genes for proper organ and tissue development (Blázquez, 2000).
Transcription factors play major roles in the genetic regulation, being responsible for orchestrating the cascade of processes for cellular development, differentiation, and maintenance. Homeotic genes encode transcription factors involved in the specification of organ identity; their loss of function results in the replacement of one type of organ by another.
Several studies with Arabidopsis thaliana and Antirrhinum majus led to the so-called ABC model of flower development (Coen and Meyerowitz, 1991). This model postulates that three classes of homeotic genes control floral organ formation in a combinatorial fashion. The expression of genes from type A alone, A plus B, B plus C, and C alone trigger the formation of sepal, petal, stamen, and carpel, respectively. In addition, A- and C-functions are considered antagonists since expression of one type represses the function of the other (Bowman et al., 1991).
The ABC model of flower development has been fine-tuned in time. Important extensions in the model were the addition of a D function for ovule development (Angenent et al., 1995) and, more recently, the inclusion of an E function of which transcription is active in the inner three whorls of the flower and contributes to protein complex formation in order to trigger organ development (Pelaz et al., 2000, 2001; Honma and Goto, 2001). New proposals for transcriptional protein interaction, among others the ‘quartet model’ which postulates the multimeric complex formation of MADS box proteins and DNA regions for different organ development in the floral whorls, also arose recently due to the aforementioned discoveries on the ABC model, and hence called the ABCDE model (Theissen and Saedler, 2001; Honma and Goto, 2001).
All the genes involved in the ABCDE model are MADS-box genes, with the exception of the type A AP2 gene. The MADS-box genes constitute a superfamily of transcription factors found in very simple organisms, for example, yeast, as well as in complex species such as plants and animals (Schwarz-Sommer et al., 1990; Shore and Sharrocks, 1995; Ng and Yanofsky, 2001). In plants, they are involved in many developmental processes, especially in the reproductive organs (Ng and Yanofsky, 2001), but also in roots, leaves, and other organs (Alvarez-Buylla et al., 2000; Causier et al., 2002). Plant type II MADS-box proteins carry a conserved organization called MIKC, which is characterized by the highly conserved motif of 56 amino acids called MADS-box (M), which has a DNA-binding domain. The weakly conserved intervening (I) region is thought to be the determinant factor of selective dimerization. The moderately conserved K-domain, with its keratin-like coiled-coil structure is involved in protein–protein interactions and is exclusively found in plant species. Finally, the variable carboxy(C)-terminal domain may be responsible for transcriptional activation and protein interaction stabilization (Shore and Sharrocks, 1995).
AGAMOUS (AG) is the only C-functional gene found in Arabidopsis, whilst in other species redundant or complementary AG paralogues have been found, such as PLENA (PLE) and FARINELLI (FAR) in Antirrhinum majus (Davies et al., 1999), FLORAL BINDING PROTEIN 6 (FBP6) and pMADS3 in Petunia hybrida (Kater et al., 1998). In Arabidopsis, recessive mutants for AG show petals instead of stamens and a new flower in the place of carpels, giving rise to indeterminacy of the flower as an additional effect of AG absence. Constitutive overexpression of AG induces homeotic changes in the flower: carpels instead of sepals and stamens in the place of petals, presenting the characteristics of apetala2 (ap2) mutants (Yanofsky et al., 1990). This ap2 loss-of-function-like phenotype induced by overexpression of AG orthologues in Arabidopsis or tobacco is being used as a heterologous system for testing the functional homology of genes from diverse species such as hazelnut (Rigola et al., 2001), grapevine (Boss et al., 2001), hyacinth (Li et al., 2002), and the conifer black spruce (Rutledge et al., 1998).
Knowledge obtained with MADS-box genes in model species may provide tools for potential applications in important commercial crops. Among them, ornamental species are the most obvious candidates for floral morphology manipulations, in order to create novel varieties with high market values. Petunia, which has become another model species for studying MADS-box genes (Angenent et al., 1992, 1995; Kater et al., 1998), Antirrhinum, which has been used as a model species since the start of the ABC model (Coen and Meyerowitz, 1991), rose (Kitahara and Matsumoto, 2000; Kitahara et al., 2001), carnation (Baudinette et al., 2000), gerbera (Yu et al., 1999; Kotilainen et al., 2000), lisianthus (Tzeng et al., 2002), primula (Webster and Gilmartin, 2003), orchids (Lu et al., 1993; Yu and Goh, 2000, 2001; Hsu and Yang, 2002), hyacinth (Li et al., 2002), and lily (Tzeng and Yang, 2001; Tzeng et al., 2002) are among the ornamental crops in which ABCDE model genes have been under study.
Lily (Lilium longiflorum Thunb.) is one of the most important ornamental species in the world. Tzeng and Yang (2001) and Tzeng et al. (2002) recently described its flower structure and development. Importantly, organs of the two outermost whorls of lily flower are very similar, generating a perianth of tepals, instead of sepals and petals. It has been supposed that this similarity would be due to a modification in the ABC model, leading to an extension of B function towards the first whorl in Liliaceae species (Theissen et al., 2000).
Lily double flowers are highly appreciated in the market and this phenotype is thought to involve the C function. The results of these investigations on the molecular characterization of flower development in lily are presented here. In order to understand more about floral homeotic genes in this species, the MADS-box gene LLAG1 has been isolated and characterized. Sequence analysis, expression patterns, and functional characterization in the heterologous species Arabidopsis thaliana led to the conclusion that LLAG1 is the functional AG orthologue in lily.
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Materials and methods |
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Plant materials
Lily (Lilium longiflorum Thunb. cv. Snow Queen) plants used in this study were grown in the greenhouse at 18–25 °C /14–18 °C day/night with a natural light regime during the growth season, in Wageningen, the Netherlands. Arabidopsis thaliana Columbia (Col) ecotype plants were grown in the greenhouse under a long-day regime (22 °C, 14/10 h light/dark) after breaking the dormancy of the seeds 3 d at 4 °C.
RNA extraction
Total RNA was extracted from lily floral buds (1.0–3.5 cm), leaves, and mature floral organs (tepal, stamens, and pistil) according to Zhou et al. (1999). Arabidopsis total RNA from flowers was isolated using the RNeasy Plant Mini kit (Qiagen, GmbH, Hilden, Germany).
cDNA library preparation and screening
cDNA was synthesized using the ZAP-cDNA Gigapack III Gold Cloning Kit (Stratagene, La Jolla, CA, USA) with 5 µg of a poly(A)+ RNA pool from 1.0–3.5 cm lily buds purified through the Poly(A) Quick mRNA isolation column (Stratagene). cDNA fractions containing 1–1.5 kb fragments were unidirectionally inserted between the EcoRI and XhoI sites of the Uni-ZAP XR phage vector.
Approximately 200 000 pfu were screened using a 760 bp fragment of LRAG, a putative AG homologue from Lilium regale (Theissen et al., 2000), without the MADS-box, labelled with [32P] dATP using the RadPrime DNA Labeling System (Invitrogen, Carlsbad, CA, USA). Hybridization procedures were carried out at night at 56 °C on nylon membranes and washes reached the stringency of 0.5x SSC, 0.1% SDS. Blots were exposed to X-ray films for 24 h and positive clones were collected from plates for in vivo excision procedures.
Sequence analysis
cDNA clone sequencing was performed in both directions with T3 (5'-AAT TAA CCC TCA CTA AAG GG) and T7 (5'-GCC CTA TAG TGA GTC GTA TTA C) primers using rhodamine dye (Applied Biosystems, Foster City, CA, USA). Sequence analyses were carried out with DNASTAR software package.
Multiple protein sequence alignment was performed using the 185 domain (185 amino acids starting from the MADS-box) (Rigola et al., 1998). The accession numbers of the protein sequences used in this study are as followed: AG (CAA37642 [GenBank] ; AGL11 (AAC49080 [GenBank] ; BAG1 (AAA32985 [GenBank] ; CaMADS1 (AAD03486 [GenBank] ; CUM1 (AAC08528 [GenBank] ; CUM10 (AAC08529 [GenBank] ; CUS1 (CAA66388 [GenBank] ; DAL2 (CAA55867 [GenBank] ; FAR (CAB42988 [GenBank] ; FBP6 (CAA48635 [GenBank] ; FBP7 (CAA57311 [GenBank] ; FBP11 (CAA57445 [GenBank] ; pMADS3 (CAA51417 [GenBank] ; GAG1 (CAA86585 [GenBank] ; GAGA1 (CAA08800 [GenBank] ; GAGA2 (CAA08801 [GenBank] ; GGM3 (CAB44449 [GenBank] ; HAG1 (AAD19360 [GenBank] ; HaAG (AAN47198 [GenBank] ; HvAG1 (AAL93196 [GenBank] ; HvAG2 (AAL93197 [GenBank] ; LAG (AAD38119 [GenBank] ; LMADS2 (Tzeng et al., 2002); MdMADS15 (CAC80858 [GenBank] ; NAG1 (AAA17033 [GenBank] ; OsMADS3 (AAA99964 [GenBank] ; OsMADS13 (AAF13594 [GenBank] ; PeMADS1 (AAL76415 [GenBank] ; PLE (AAB25101 [GenBank] ; PTAG1 (AAC06237 [GenBank] ; PTAG2 (AAC06238 [GenBank] ; RAG1 (AAD00025 [GenBank] ; RAP1 (CAA61480 [GenBank] ; SAG1 (AAC97157 [GenBank] ; SLM1 (CAA56655 [GenBank] ; STAG1 (AAD45814 [GenBank] ; TAG1 (AAA34197 [GenBank] ; VvMADS1 (AAK58564 [GenBank] ; WAG (BAC22939 [GenBank] ; ZAG1 (AAA02933 [GenBank] , and ZMM1 (considered here equivalent to ZAG2, CAA56504 [GenBank] . The sequence of the putative AG homologue from Lilium regale (LRAG) (Theissen et al., 2000) was not included in the alignment because the 185 domain sequence data for the entry was not made publicly available.
Expression analysis
RT-PCR analysis was performed using the SuperScript First-Strand Synthesis System for RT-PCR (Invitrogen). Five micrograms of total RNA extracted from floral organs and leaves were used to synthesize the first-strand cDNA using an oligo-dT primer. For amplification of LLAG1 cDNA, a 2 µl aliquot of the first-strand RT reaction were used in a 50 µl PCR reaction with LLAG1 specific primers (5'-GAT TGC TGA AAA TGA GAG G and 5'-AAA GTC ACA AAA TAA TAC AGC as forward and reverse primers, respectively). PCR was performed with a 10 min 95 °C denaturation step, followed by 35 cycles of 1 min at 95 °C, 1 min annealing at 54 °C and 1 min extension at 72 °C, and a final extension period of 10 min. Twelve microlitres of the RT-PCR reaction were run in an agarose gel and photographed under UV light. As a control, 2 µl of the first-strand RT reaction was used for amplification of the constitutive glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA using degenerated primers (5'-GTK GAR TCN ACY GGY GTC TTC ACT and 5'-GTR TGR AGT TGM CAN GAR ACA TC as forward and reverse primers, respectively) conceived from the alignment of GAPDH sequences of several monocot species available in the GenBank. The PCR conditions for GAPDH were as described for LLAG1.
Northern blot of transgenic Arabidopsis was prepared with 5 µg total RNA from flowers of first generation transformants. Probes were prepared as described for cDNA screening, using LLAG1 without the MADS-box and, as constitutive expression control, a 1.2 kb actin cDNA fragment, obtained by RT-PCR amplification of leaf RNA with specific primers (5'-GCG GTT TTC CCC AGT GTT GTT G and 5'-TGC CTG GAC CTG CTT CAT CAT ACT as forward and reverse primers, respectively). Hybridization was carried out at 60 °C and washes reached 0.5x SSC, 0.1% SDS at the same hybridization temperature in both cases. After LLAG1 hybridization and development, the membrane was stripped with 0.1x SSC, 0.1% SDS at boiling temperature for 20 min. The membrane was exposed for 4 h to a Phosphor Imaging Plate (Fuji Photo Film Co., Tokyo, Japan) and developed in a related scanner system. Autoradiographic signals were processed using the TINA 2.10 software (Raytest, Straubenhardt, Germany).
Binary vector construction and Arabidopsis transformation
A pBINPLUS-derived binary vector (van Engelen et al., 1995) with a multiple cloning site between the CaMV35S promoter and the NOS terminator was used for sense-oriented LLAG1 insertion and overexpression in Arabidopsis. LLAG1 was excised from the pBluescript SK+ vector using XbaI and XhoI restriction enzymes and inserted into the binary vector treated with the same enzymes. After confirmation of the sequence, Agrobacterium tumefaciens strain C58C1 competent cells were prepared and transformed by electroporation according to Mattanovich et al. (1989).
Arabidopsis thaliana ecotype Columbia (Col) plants were transformed using the floral dip method (Clough and Bent, 1998). T1 seeds were placed onto agar plates containing 0.5x MS medium (Murashige and Skoog, 1962) with 50 µg ml–1 kanamycin as selection agent. In order to break the dormancy, plates were placed at 4 °C for 3 d and then at 23 °C in a growth chamber with long-day conditions (14/10 h light/dark). T1 seedlings were transferred to soil and kept in the same temperature and light regime. The self-pollinated T2 population was sown directly in soil and grown in identical conditions.
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Results |
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Isolation and sequence analysis of LLAG1 cDNA from lily.
In order to isolate the AG homologue from Lilium longiflorum, a cDNA library derived from developing flowers was screened, using as probe a fragment of 760 bp without the MADS-box from LRAG, a putative AG homologue from Lilium regale (Theissen et al., 2000). Five positive clones were selected out of about 200 000 pfu, of which four clones were identical and designated as LLAG1.
LLAG1 cDNA is 1171 bp long with a 5' leader region of 73 bp and a 3' untranslated region of 366 bp upstream of the poly(A) tail. Deduced protein sequence analysis of LLAG1 revealed a 244 amino acid product.
Amino acid sequence alignment of LLAG1 and AG homologues from monocot and dicot species is displayed in Fig. 1. A high sequence conservation in the 56 amino acids of the MADS-box is evident, and also, to a lesser extent, in the K-box, revealing the MIKC structure, typical of type II plant MADS box proteins. An N-terminal extension preceding the MADS-box, commonly present in AG homologues, was not found in LLAG1.
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Within the MADS-box, LLAG1 shares 100% (56/56) amino acid similarity with AG from Arabidopsis thaliana, HAG from Hyacinthus orientalis, PeMADS1 from Phalaenopsis equestris, OsMADS3 from Oryza sativa, WAG from Triticum aestivum, pMADS3 from Petunia hybrida, and CaMADS1 from Corylus avellana while there is only one non-conserved amino acid substitution in PLE from Antirrhinum majus, leading to 98% similarity. The C-terminus is the least conserved portion of the AG homologues. LLAG1 shares 81% (58/72) amino acid similarity with HAG1, 92% (67/73) with PeMADS1, 59% (47/80) with OsMADS3, 82% (64/78) with WAG, 93% (65/70) with CaMADS1, 89% (62/70) with pMADS3, 84% (59/70) with PLE, and 60% (48/80) with AG. As a whole, the similarities in the predicted primary structure of LLAG1 range from 89% with HAG1 (202/228) to 67% with AG (191/284) among the sequences shown in Fig. 1.
Phylogenetic analyses indicate that MADS-box subfamilies representing monophyletic clades tend to show similar sequences, expression patterns, and related functions (Purugganan, 1997). Multiple alignment with LLAG1 and other members of the monophyletic AG clade is presented as a phylogram in Fig. 2. It shows that LLAG1 is closely related to the monocot AG orthologues, specially HAG1 and PeMADS1, the latter being from an orchid species.
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LLAG1 expression pattern
The spatial expression pattern of LLAG1 in floral organs of lily was investigated by RT-PCR using gene-specific primers designed to amplify the 3' portion, which is its least conserved section, in order to avoid cross-annealing with other MADS-box genes. Amplification of a GAPDH fragment was used as a constitutive control. A fragment of approximately 500 bp corresponding to LLAG1 transcripts could only be detected in stamens and carpels while it was not detectable in tepals or leaves (Fig. 3).
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This expression pattern suggests that this gene is involved in the development of reproductive floral organs since it is expressed in stamens and carpels, but remains inactive in the perianth and vegetative tissues. These findings are consistent with the hypothesis that LLAG1 has a similar function as AG in lily floral development.
Ectopic expression of LLAG1 in Arabidopsis
Functional analysis of LLAG1 cDNA was investigated by ectopic expression in Arabidopsis to understand whether the sequence and expression similarities between LLAG1 and AG also point to a functional relationship. A binary vector carrying 35S::LLAG1 and a kanamycin-resistance gene was introduced into Arabidopsis via Agrobacterium transformation and the phenotypic alterations of the transformed plants were analysed in the T1 and T2 generations.
According to the ABC model of flower development, transgenic plants overexpressing AG are expected to show homeotic modifications in the first and second whorls of the flower. This results in the formation of carpelloid organs in the first whorl and petals replaced by organs with a staminoid identity, acquiring characteristics of the ap2 mutant phenotype (Bowman et al., 1991), due to the negative interaction between A- and C-functions.
Out of 60 independent kanamycin-resistant plants analysed in the T1, 26 exhibited phenotypic alterations. Plants showing homeotic changes were divided into strong and weak ap2-like phenotypes. In general, strong ap2-like plants displayed reduced height, small and curled leaves, loss of inflorescence indeterminacy, bumpy siliques, and they also flowered earlier than wild-type plants (Fig. 4), whereas the weak ap2-like group showed normal vegetative growth with less pronounced floral homeotic modifications.
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Flowers derived from plants showing the strong ap2-like phenotype had evident homeotic modifications in the first two whorls, with distinct stigmatic papillae at the apex of the modified sepals (data not shown) and, occasionally, complete homeotic changes of petals into stamens. The most important features of the C-function overexpression are the homeotic mutations in the organs of the two outer floral whorls when expressed in these places, and in Fig. 4C, a close-up is shown of an Arabidopsis flower with a genuine stamen in the place of a petal. This was certified by its position in the flower, i.e. the second whorl which is by definition between two sepals in a more internal adjacent whorl. The basis of this stamen between the two sepals and the absence of petals is clear in Fig. 4C. The overall weak phenotype of a strong ap2-like individual is demonstrated in Fig. 4F showing small and curled leaves.
Plants with a weak phenotype often presented partly or completely developed petals.
T2 progeny analysis was carried out with six self-pollinated strong ap2-like T1 plants. Offspring plants revealed a clear segregation of the wild type and strong ap2-like phenotypes. No weak ap2-like phenotype was found in the T2 progeny analysed. Northern blots confirmed the expression of LLAG1 in the flowers of T1 transformed Arabidopsis (Fig. 5).
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Discussion |
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Since lily (Lilium sp.) is one of the major ornamental crops in the world and ABCDE model genes are involved in the morphology of the flower, research on those genes can be of great commercial interest. Evolutionary and developmental biology may also take advantage from these studies since not many floral homeotic genes from monocot species have been investigated in detail so far.
LLAG1, a Lilium longiflorum MADS-box gene isolated from a cDNA library from developing flowers, is specifically expressed in the stamens and carpels, constituting the C domain of the ABCDE model for floral development.
The primary structure of the LLAG1 protein was shown to be highly homologous to AGAMOUS from Arabidopsis and other known orthologues. As expected, the phylogenetic dendrogram revealed a close relationship to AG orthologues from monocot species.
Although no specific function has been given so far to the N-terminal extension of the MADS-box, since AG with a truncated N-terminus was still shown to be functionally active in vitro (Pollock and Treisman, 1991; Huang et al., 1993), it was suggested that all functional AG homologues would contain this extension (Kater et al., 1998). However, it was found that the AG homologues HAG1 from hyacinth (Hyacinthus orientalis), OsMADS3 from rice (Oryza sativa) and CUM10 from cucumber (Cucumis sativus) have their presumed start codon at the MADS-box. In addition, besides showing a putative N-terminal portion before the MADS-box, PeMADS1 from Phalaenopsis equestris and WAG from wheat (Triticum aestivum) contain an ATG codon just in front of their MADS-box, which could be their actual start codon. LLAG1 does not carry an N-terminal domain preceding the MADS-box, indicating that this extension might have been abolished during plant evolution, possibly due to its lack of functionality.
Based on RT-PCR, the spatial expression of LLAG1 in mature flowers corresponded with the AG expression in Arabidopsis (Yanofsky et al., 1990), being expressed in the third and fourth whorls of the flower in accordance to the ABC model.
Due to the very low transformation efficiency of lily, functional studies of LLAG1 were undertaken in the model species Arabidopsis. Constitutive overexpression of LLAG1 led to homeotic changes of floral organs. The modifications observed were entirely in accordance with reports on AG overexpression in Arabidopsis (Mizukami and Ma, 1992) and the functional AG orthologues from hazelnut (Rigola et al., 2001), hyacinth (Li et al., 2002), and spruce (Picea mariana; Rutledge et al., 1998).
These results provided additional evidence of the capability for in vivo cross-interaction of proteins belonging to the ABC model from different species, even with those distantly related, like Arabidopsis and lily. It can also reiterate the evolutionary importance of AG function in flowering plants due to the preservation of in vivo functionality of protein–protein interaction among MADS-box transcription factors from diverse species. Molecular evidence with cross-interactions among several MADS-box proteins from Arabidopsis and Petunia (Immink and Angenent, 2002) and also between Petunia and rice (Favaro et al., 2002) were provided in recent studies. However, there are indications that MADS-box protein dimerization may occur in a different way in monocot species (Winter et al., 2002).
There are several sequences from the ABCDE class of genes from bulbous crops, such as the AG orthologue HAG1 from hyacinth (Li et al. 2002), the B-type genes LMADS1 from L. longiflorum (Tzeng and Yang, 2001) and LRDEF, LRGLOA, and LRGLOB from L. regale (Winter et al., 2002), and a partial sequence of PeMADS1 from an orchid (Phalaenopsis equestris) available in the public gene database. Recently, LMADS2, a new MADS-box gene from L. longiflorum was described as a D-functional gene (Tzeng et al., 2002). However, functional analysis of LMADS2 in Arabidopsis induced the same characteristics found when overexpressing the C type AG or its orthologues instead of promoting ectopic ovule formation as it was observed with other D functional genes, such as FBP11 from petunia (Colombo et al., 1995).
Among the oriental hybrids of Lilium sp. currently in the market, there are double flower varieties that resemble the AG loss of function, having tepals instead of stamens and a new flower instead of carpels with loss of flower determinacy. This variant pattern arises naturally and occurs for many species, being of great interest to flower breeders for creating novel characteristics. Nevertheless, the phenomenon is very rare and not completely understood yet. It can be due to mutations in the AG orthologue directly or even caused by abnormal interactions with trans-regulatory elements (Roeder and Yanofsky, 2001; Franks et al., 2002). Knowledge about the mechanisms involved in this process would allow crops, such as lily, to be modified, in order to create new varieties with the double flower phenotype.
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Acknowledgements |
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The authors are grateful to Antonio Chalfun Jr, Jurriaan Mes, Santie de Villiers, and Anna Shchennikova for valuable contributions and discussions for this study. Akira Kanno and Günter Theissen are specially thanked for providing the Lilium regale probe.
This work was supported by CNPq, the Science and Technology Development Organ of the Brazilian Government (200851/98-5 GDE) and by the Dutch Ministry of Agriculture, Nature Management and Fisheries (DWK 364).
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References |
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|
TopAbstractIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
Alvarez-Buylla ER, Liljegren SJ, Pelaz S, Gold SE, Burgeff C, Ditta GS, Vergara-Silva F, Yanofsky MF. 2000. MADS-box gene evolution beyond flowers: expression in pollen, endosperm, guard cells, roots and trichomes. The Plant Journal 24, 457–466.[CrossRef][Web of Science][Medline]
Angenent GC, Busscher M, Franken J, Mol JNM, van Tunen AJ. 1992. Differential expression of two MADS box genes in wild-type and mutant petunia flowers. The Plant Cell 4, 983–993.
Angenent GC, Franken J, Busscher M, van Dijken A, van Went JL, Dons HJM, van Tunen AJ. 1995. A novel class of MADS box genes is involved in ovule development in Petunia. The Plant Cell 7, 1569–1582.[Abstract]
Baudinette SC, Stevenson TW, Savin KW. 2000. Isolation and characterization of the carnation floral-specific MADS box gene, CMB2. Plant Science 155, 123–131.[Medline]
Blázquez MA. 2000. Flower development pathways. Journal of Cell Science 113, 3547–3548.[Web of Science][Medline]
Boss P, Vivier M, Matsumoto S, Dry IB, Thomas MR. 2001. A cDNA from grapevine (Vitis vinifera L.), which shows homology to AGAMOUS and SHATTERPROOF, is not only expressed in flowers but also throughout berry development. Plant Molecular Biology 45, 541–553.[CrossRef][Web of Science][Medline]
Bowman JL, Smyth DR, Meyerowitz EM. 1991. Genetic interactions among floral homeotic genes of Arabidopsis. Development 112, 1–20.[Abstract]
Causier B, Kieffer M, Davies B. 2002. MADS-box genes reach maturity. Science 296, 275–276.
Clough SJ, Bent AF. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal 16, 735–743.[CrossRef][Web of Science][Medline]
Coen ES, Meyerowitz EM. 1991. The war of the whorls: genetic interactions controlling flower development. Nature 353, 31–37.[CrossRef][Medline]
Colombo L, Franken J, Koetje E, van Went J, Dons HJ, Angenent GC, van Tunen AJ. 1995. The Petunia MADS-box gene FBP11 determines ovule identity. The Plant Cell 7, 1859–1868.[Abstract]
Davies B, Motte P, Keck E, Saedler H, Sommer H, Schwarz-Sommer Z. 1999. PLENA and FARINELLI: redundancy and regulatory interactions between two Antirrhinum MADS-box factors controlling flower development. EMBO Journal 18, 4023–4034.[CrossRef][Web of Science][Medline]
Favaro R, Immink RGH, Ferioli V, Bernasconi B, Byzova M, Angenent GC, Kater M, Colombo L. 2002. Ovule-specific MADS-box proteins have conserved protein–protein interactions in monocot and dicot plants. Molecular Genetics and Genomics 268, 152–159.[CrossRef][Web of Science][Medline]
Franks R, Wang C, Levin JZ, Liu Z. 2002. SEUSS, a member of a novel family of plant regulatory proteins, represses floral homeotic gene expression with LEUNIG. Development 129, 253–263.
Honma T, Goto K. 2001. Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409, 525–529.[CrossRef][Medline]
Hsu HF, Yang CH. 2002. An orchid (Oncidium Gower Ramsey) AP3-like MADS gene regulates floral formation and initiation. Plant and Cell Physiology 43, 1198–1209.
Huang H, Mizukami Y, Hu Y, Ma H. 1993. Isolation and characterization of the binding sequences for the product of the Arabidopsis floral homeotic gene AGAMOUS. Nucleic Acids Research 21, 4769–4776.
Immink RGH, Angenent GC. 2002. Transcription factors do it together: the hows and whys of studying protein-protein interactions. Trends in Plant Science 7, 531–534.[CrossRef][Web of Science][Medline]
Kater MM, Colombo L, Franken J, Busscher M, Masiero S, Campagne MML, Angenent GC. 1998. Multiple AGAMOUS homologs from cucumber and petunia differ in their ability to induce reproductive organ fate. The Plant Cell 10, 171–182.
Kitahara K, Hirai S, Fukui H, Matsumoto S. 2001. Rose MADS-box genes ‘MASAKO BP and B3’ homologous to class B floral identity genes. Plant Science 161, 549–557.
Kitahara K, Matsumoto S. 2000. Rose MADS-box genes ‘MASAKO C1 and D1’ homologous to class C floral identity genes. Plant Science 151, 121–134.[Medline]
Kotilainen M, Elomaa P, Uimari A, Albert VA, Yu D, Teeri TH. 2000. GRCD1, and AGL2-like MADS box gene, participates in the C function during stamen development in Gerbera hybrida. The Plant Cell 12, 1893–1902.
Li QZ, Li XG, Bai SN, Lu WL, Zhang XS. 2002. Isolation of HAG1 and its regulation by plant hormones during in vitro floral organogenesis in Hyacinthus orientalis L. Planta 215, 533–540.[CrossRef][Web of Science][Medline]
Lu ZX, Wu M, Loh CS, Yeong CY, Goh CJ. 1993. Nucleotide sequence of a flower-specific MADS box cDNA clone from orchid. Plant Molecular Biology 23, 901–904.[CrossRef][Web of Science][Medline]
Mattanovich D, Rüker F, Machado A C, Laimer M, Regner F, Steinkellner H, Himmler G, Katinger H. 1989. Efficient transformation of Agrobacterium spp. by electroporation. Nucleic Acids Research 17, 6747.
Mizukami Y, Ma H. 1992. Ectopic expression of the floral homeotic gene AGAMOUS in transgenic Arabidopsis plants alters floral organ identity. Cell 71, 119–131.[CrossRef][Web of Science][Medline]
Murashige T, Skoog F. 1962. A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiologia Plantarum 15, 473–479.[CrossRef]
Ng M, Yanofsky MF. 2001. Function and evolution of the plant MADS-box gene family. Nature Review Genetics 2, 186–195.[CrossRef][Web of Science][Medline]
Page RDM. 1996. TREEVIEW: an application to display phylogenetic trees on personal computers. Computational Applied Bioscience 12, 357–358.
Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF. 2000. B and C floral organ identity require SEPALLATA MADS-box genes. Nature 405, 200–203.[CrossRef][Medline]
Pelaz S, Tapia-López R, Alvarez-Buylla ER, Yanofsky MF. 2001. Conversion of leaves into petals in Arabidopsis. Current Biology 11, 182–184.[CrossRef][Web of Science][Medline]
Pollock R, Treisman R. 1991. Human SRF-related proteins: DNA-binding properties and potential regulatory targets. Genes and Development 5, 2327–2341.
Purugganan MD. 1997. The MADS-box floral homeotic gene lineages predate the origin of seed plants: phylogenetic and molecular clock estimates. Journal of Molecular Evolution 45, 392–396.[CrossRef][Web of Science][Medline]
Rigola D, Pè ME, Fabrizio C, Mè G, Sari-Gorla M. 1998. CaMADS1, a MADS box gene expressed in the carpel of hazelnut. Plant Molecular Biology 38, 1147–1160.[CrossRef][Web of Science][Medline]
Rigola D, Pè ME, Mizzi L, Ciampolini F, Sari-Gorla M. 2001. CaMADS1, an AGAMOUS homologue from hazelnut, produces floral homeotic conversion when expressed in Arabidopsis. Sexual Plant Reproduction 13, 185–191.[CrossRef]
Roeder AHL, Yanofsky MF. 2001. Unraveling the mystery of double flowers. Developmental Cell 1, 4–6.[CrossRef][Web of Science][Medline]
Rutledge R, Regan S, Nicolas O, Fobert P, Côté C, Bosnich W, Kauffeldt C, Sunohara G, Séguin A, Stewart D. 1998. Characterization of an AGAMOUS homologue from the conifer black spruce (Picea mariana) that produces floral homeotic conversions when expressed in Arabidopsis. The Plant Journal 15, 625–634.[CrossRef][Web of Science][Medline]
Schwarz-Sommer Z, Huijser P, Nacken W, Saedler H, Sommer H. 1990. Genetic control of flower development by homeotic genes in Antirrhinum majus. Science 250, 931–936.
Shore P, Sharrocks AD. 1995. The MADS-box family of transcription factors. European Journal of Biochemistry 229, 1–13.[Web of Science][Medline]
Theissen G, Becker A, Rosa AD, Kanno A, Kim JT, Münster T, Winter K-U, Saedler H. 2000. A short history of MADS-box genes in plants. Plant Molecular Biology 42, 115–149.[CrossRef][Web of Science][Medline]
Theissen G, Saedler H. 2001. Floral quartets. Nature 409, 469–471.[CrossRef][Medline]
Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F, Higgins DG. 1997. The ClustalX windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. Nucleic Acids Research 24, 4876–4882.
Tzeng TS, Chen HY, Yang CH. 2002. Ectopic expression of carpel-specific MADS box genes from lily and lisianthus causes similar homeotic conversion of sepal and petal in Arabidopsis. Plant Physiology 130, 1827–1836.
Tzeng TS, Yang CH. 2001. A MADS-box gene from lily (Lilium longiflorum) is sufficient to generate dominant negative mutation by interacting with PISTILLATA (PI) in Arabidopsis thaliana. Plant and Cell Physiology 42, 1156–1168.
van Engelen FA, Molthoff JW, Conner AJ, Nap JP, Pereira A, Stiekema WJ. 1995. pBINPLUS: an improved plant transformation vector based on pBIN19. Transgenic Research 4, 288–290.[CrossRef][Web of Science][Medline]
Webster MA, Gilmartin PM. 2003. A comparison of early floral ontogeny in wild-type and floral homeotic mutant phenotypes of Primula. Planta 216, 903–917.[Web of Science][Medline]
Winter K-U, Weiser C, Kaufmann K, Bohne A, Kirchner C, Kanno A, Saedler H, Theissen G. 2002. Evolution of class B floral homeotic proteins: obligate heterodimerization originated from homodimerization. Molecular Biology and Evolution 19, 587–596.
Yanofsky MF, Ma H, Bowman JL, Drews GN, Feldmann, KA, Meyerowitz EM. 1990. The protein encoded by the Arabidopsis homeotic gene agamous resembles transcription factors. Nature 346, 35–39.[CrossRef][Medline]
Yu D, Kotilainen M, Pöllänen E, Mehto M, Elomaa P, Helariutta Y, Albert VA, Teeri TH. 1999. Organ identity genes and modified patterns of flower development in Gerbera hybrida (Asteraceae). The Plant Journal 17, 51–62.[CrossRef][Web of Science][Medline]
Yu H, Goh CJ. 2000. Identification and characterization of three orchid MADS-box genes of the AP1/AGL9 subfamily during floral transition. Plant Physiology 123, 1325–1336.
Yu H, Goh CJ. 2001. Molecular genetics of reproductive biology in orchids. Plant Physiology 127, 1390–1393.
Zhou J, Pesacreta TC, Brown RC. 1999. RNA isolation without gel formation from oligosaccharide-rich onion epidermis. Plant Molecular Biology Reports 17, 397–407.[CrossRef]
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