JXB Advance Access originally published online on March 5, 2007
Journal of Experimental Botany 2007 58(6):1245-1259; doi:10.1093/jxb/erl263
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
RESEARCH PAPER |
Functional characterization of MADS box genes involved in the determination of oil palm flower structure
IRD/CIRAD Palm Group, UMR 1098, Centre IRD Montpellier, 911 avenue Agropolis, 34394 Montpellier, France
* To whom correspondence should be addresssed. E-mail: tregear{at}mpl.ird.fr
Received 14 June 2006; Revised 7 November 2006 Accepted 9 November 2006
 |
Abstract |
|---|
 Top Abstract IntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
In order to study the molecular regulation of flower development in the monoecious species oil palm (Elaeis guineensis), cDNAs of 12 MADS box genes from this plant belonging to seven distinct subfamilies were previously isolated and characterized. Here studies carried out on five of these genes, each likely to be involved in floral morphogenesis: EgSQUA1 (SQUAMOSA subfamily); EgAGL2-1 (AGL2 subfamily); EgGLO2 (GLOBOSA subfamily); EgDEF1 (DEFICIENS subfamily); and EgAG2 (AGAMOUS subfamily), are described. In order to determine where and when in the plant these genes are likely to function, their spatial and temporal patterns of expression were studied during the development of male and female inflorescences, either of normal phenotype or displaying a homeotic flowering abnormality known as mantled. In parallel, the phenotypic effects of ectopically expressing these genes in transgenic Arabidopsis thaliana plants were analysed. The data suggest a broad conservation of floral homeotic gene functions between oil palm and previously described model species, although a few minor variations in the zones of activity of certain genes cannot be excluded. The data also indicate distinct molecular identities for the morphologically similar floral organs of whorls 1 and 2. They also reveal reduced expression of putative B, C/D, and E class genes in mantled flowers, which undergo a homeotic transformation comparable to B class mutants of model species.
Key words: ABCDE model, flower, homeotic, MADS box, mantled, oil palm
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
The mechanisms which govern the morphology of flowers in higher plants have been extensively studied in recent years. In the generic hermaphrodite angiosperm flower, floral organs are arranged in a series of concentric whorls containing sepals, petals, stamens, and carpels. Many variations to this basic structure exist; however, a large body of data has revealed that the regulation of flower morphology works along similar lines in a wide range of higher plant species. Floral organ identity determination can be explained by the ABCDE model (Coen and Meyerowitz, 1991; Colombo et al., 1995; Pelaz et al., 2000; Honma and Goto, 2001) with variations to the basic scheme applying to specific plant groups (Theissen et al., 2000). According to the generic ABCDE model, the expression of class A genes specifies sepal formation in the calyx. The combination of class A, B, and E gene activities specifies the formation of petals in the corolla. In a similar way, class B, C, and E gene expression specifies stamen formation in the androecium, while class C and E gene activities determine the formation of carpels in the gynoecium. Additionally, class C, D, and E genes determine ovule identity. With the exception of some class A genes such as APETALA2 from Arabidopsis thaliana, all floral organ identity genes characterized to date code for proteins of the MADS box transcription factor family displaying a typical MIKC modular structure. Molecular studies have revealed interesting parallels between the phylogeny of the MADS box gene family and the evolution of land plants (Purugganan et al., 1995; Theissen et al., 1996, 2000; Alvarez-Buylla et al., 2000; Lamb and Irish, 2003). The known A, B, C, D, and E class genes fall into separate clades on the basis of their sequence similarities, namely those containing the SQUAMOSA (class A), DEFICIENS (class B), GLOBOSA (class B), AGAMOUS (classes C and D), and AGL2-like (class E) genes, as defined by Becker and Theissen (2003).
Since its conception, the ABCDE model has been shown to be widely applicable amongst dicot species (Pnueli et al., 1994; Kater et al., 1998, 2001; Berbel et al., 2001; Immink et al., 2003). In more recent years, homologues of the dicot ABCDE MADS box genes have also been identified in a range of monocot species (Mena et al., 1995; Ambrose et al., 2000; Jeon et al., 2000; Fornara et al., 2003; Xiao et al., 2003). Overall, less is known about the function of floral MADS box genes in monocots. It should also be noted that the majority of functional MADS box gene studies in monocots to date concern the grass family (Poaceae). Whilst some MADS box gene studies have been described in the literature for species of the Asparagales, Liliales, and Commelinales groups, none has as yet been reported for the other monocot orders. One of the most notable gaps concerns the palm family (Arecaceae), which forms the order Arecales, a group of great economic significance, particularly in tropical countries, consisting of 187 recognized genera (Dransfield et al., 2005). One of the most agriculturally important species of this group is the African oil palm (Elaeis guineensis), a plant cultivated extensively in inter-tropical regions of Asia, Africa, and Latin America. Here the first reported study of floral MADS box gene function in this species is presented, which should serve as a useful basis for understanding how the flower structure determination of palms fits in with those of other monocot groups.
The inflorescences of oil palm are compound panicles consisting of a central rachis which gives rise to branches known as rachillae. The oil palm normally produces in alternation male and female inflorescences carrying unisexual flowers (Fig. 1A, B). The staminate flower consists of a perianth surrounding an androecium of six stamens (in two concentric whorls of three) which themselves enclose a rudimentary gynoecium (Fig. 1C). The perianth consists of a calyx and a corolla of three sepals and three petals, respectively, sometimes collectively referred to as tepals. The female inflorescence produces floral triads each consisting of two accompanying male flowers flanking a single pistillate flower. Unlike functional staminate flowers, the accompanying male flowers do not develop sufficiently to produce pollen. The structure of pistillate flowers is similar to that of staminate flowers except that the perianth encloses a rudimentary androecium of usually six staminodes, themselves surrounding a tricarpellate gynoecium (Fig. 1D). In the staminate flower, petals and sepals display some subtle differences in their appearance whereas in pistillate flowers, which are considered to be homochlamydeous, they show a close resemblance.
|
For the purpose of understanding the molecular processes which determine flower structure in oil palm, a previously described homeotic epimutant, known as mantled (Corley et al., 1986), is of particular interest. Mantled palms exhibit a transformation of stamens and staminodes into carpel-like structures in staminate and pistillate flowers, respectively (Fig. 1E, F; Adam et al., 2005). In the mantled staminate flower, the transformation of the stamens into pseudocarpels results in sterility, whereas in the mantled pistillate flower characteristic fertile or parthenocarpic fruits may be produced in less severe cases (Fig. 1G, H). The mantled phenotype is observed in oil palms regenerated from tissue culture (Corley et al., 1986) and may be transmitted by genetic crossing (Rao and Donough, 1990); however, reversion to wild type is observed in the field in some but not all individuals (Durand-Gasselin et al., 1990), indicating an epigenetic origin. It has been proposed that the mechanism involved might involve differential genomic DNA methylation (Jaligot et al., 2000).
A structural characterization of oil palm MADS box cDNAs and their encoded proteins was carried out previously (Adam et al., 2006). Twelve oil palm cDNAs identified in this way were revealed by phylogenetic reconstructions to belong to five different MADS box gene subfamilies of known importance in flower structure determination, namely the SQUAMOSA (SQUA), DEFICIENS (DEF), GLOBOSA (GLO), AGAMOUS (AG), and AGL2 groups described by Becker and Theissen (2003). The broad aim of the work described herein was to identify putative functions for the genes in question. The investigation of floral gene function in oil palm is complicated by its large size and long life cycle, the production of functional flowers starting only 3 years after germination. On the basis of their sequence similarities and expression profiles, five different oil palm MADS box genes likely to be involved in floral morphogenesis were selected for detailed analyses. A study of the temporal and spatial expression of these five MADS box genes in oil palm and an investigation of their phenotypic effects when ectopically expressed in transgenic A. thaliana plants are described here. In parallel with the transgenic approach, a comparative study of mantled and wild-type material was performed, which helped provide an additional insight into the molecular regulation of petal and stamen/staminode development. Taken together, the results allow a model of floral organ identity control for oil palm to be proposed, which resembles the generic ABCDE dicot model, with the possibility of some minor divergences.
|
Materials and methods |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
Plant material
Inflorescence material of seed-derived (DelixLa Mé origin) and regenerant (FC166 clonal lines) oil palms used for the reverse transcription-polymerase chain reaction (RT-PCR) study was harvested at the Tun Razak plantation, Malaysia, with the kind permission and help of FELDA Agricultural Services Sdn Bhd. Root and leaf material were harvested from seed-derived greenhouse plants grown in Montpellier.
Plant material used for in situ hybridization (DelixAvros origin) was harvested at the Coto plantation, Costa Rica, with the kind permission and help of the ASD company. Additional material was harvested in Malaysia as described above.
RT-PCR
Total RNA was isolated from different tissues of oil palm according to Corre et al. (1996). First-strand cDNA was prepared using an oligo(dT) primer and 2 µg of RNA in conjunction with a SuperScript first strand kit (Invitrogen). The primer pairs used and conditions for RT-PCR amplification are shown in Supplementary Table S1 at JXB online. The primers of the control elongation factor gene EgEF1-
1 (accession no. AY550990) were designed around an intron sequence to test for the presence of genomic DNA contamination in the reverse transcription reaction. Amplified cDNAs were cloned and sequenced to check the specificity of the amplification products.
Total RNA of A. thaliana flowers was extracted using the RNeasy kit (Qiagen). First-strand cDNA was prepared using oligo(dT) as primer and 0.7 µg of RNA in conjunction with a SuperScript first strand kit (Invitrogen). The primer pairs used and conditions for RT-PCR amplification are shown in Supplementary Table S2 at JXB online.
RNA in situ hybridization
DNA substrates for riboprobe synthesis were PCR products obtained with a 5' primer and a 3' primer, containing sequences detailed in Supplementary Table S1 at JXB online, plus the T7 RNA polymerase initiation site. Primers used are shown in Supplementary Table S3 at JXB online. The resulting fragments were used as templates for synthesizing antisense digoxigenin-labelled riboprobes employing UTP-DIG (Roche) and a T7 Maxi Script kit (Ambion). Each amplification product was tested by Southern blotting to check the specificity of the probe (data not shown).
Flower material was fixed for 8 h at room temperature in fixation buffer (4% paraformaldehyde, 0.1 M phosphate buffer pH 7), dehydrated through a graded series of ethanol and butanol dilutions, embedded in Paraplast plus (Paraplast X-Tra, Oxford Labware) and sectioned to 7 µm with a microtome. Tissue sections were de-paraffinized with Safesol (LaboNord, France), rehydrated through an ethanol series, and then pre-treated with proteinase K (0.1 U ml–1) in TRIS-HCl pH 7.5 at 37 °C for 15 min. Digestion was stopped by washing with 0.2x phosphate-buffered saline (PBS)/glycine and then twice with PBS for 2 min each. After dehydrating in ethanol baths, hybridization was performed at 42 °C overnight with 0.2 µg ml–1 of the digoxigenin-labelled RNA probe in hybridization solution (50% formamide, 2x SSC, 4x Denhardt, 20% dextran, 2 mg ml–1 tRNA). After hybridization, slides were washed in 2x SSC at 50 °C for 45 min and twice in 1x NTE (TRIS-HCl 1 mM, NaCl 0.5 M, EDTA 1 mM, pH 7.5) at 37 °C for 2 min each. An RNase A digestion (20 µg ml–1 in 1x NTE) was carried out for 30 min at 37 °C and stopped by washing with 1x NTE at 37 °C. Final washes were conducted in 2x SSC, 1x SSC, and 0.5x SSC for 15 min each at 50 °C prior to rinsing in PBS. For signal detection, samples were incubated in blocking reagent [Roche; 10% (w/v) in PBS] for 1 h and afterwards for 30 min in blocking reagent containing anti-digoxigenin alkaline phosphatase-conjugated Fab fragment antibody (Roche) diluted at 1:500. After three washes for 10 min in PBS, tissues were equilibrated in detection buffer (100 mM TRIS-HCl pH 9.5, 100 mM NaCl, 50 mM MgCl2) for 15 min prior to incubating in the same buffer supplemented with 0.2 mM nitroblue tetrazolium (NBT) and 0.2 mM bromochloroindolyl phosphate (BCIP) substrates.
Plant transformation and transgenic plant analysis
The complete open reading frames of EgSQUA1, EgDEF1, EgGLO2, EgAGL2-1, and EgAG2 were each generated by PCR using a 5' primer containing a restriction enzyme site and a reverse primer designed after the stop codon with a restriction site. The PCR fragment was placed downstream of the cauliflower mosaic virus 35S promoter in the 35S-2 plasmid (see http://www.pGreen.ac.uk) and the expression cassette was subsequently cloned into the pGreen binary vectors (Hellens et al., 2000).
Arabidopsis thaliana plants of the ecotype Columbia were transformed using the floral dip method (Clough and Bent, 1998), and the Agrobacterium tumefaciens C58C1 strain was used for the transformation. Transformants that survived in the medium containing phosphinotricine were checked for transgene insertions by PCR. The plants were grown at 22 °C under short day conditions.
|
Results |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
Temporal and spatial expression patterns of oil palm MADS box genes during wild-type flower development
A structural characterization of 12 oil palm MADS box cDNAs belonging to the SQUAMOSA (SQUA), DEFICIENS (DEF), GLOBOSA (GLO), AGAMOUS (AG), and AGL2 subfamilies was described previously (Adam et al., 2006). In the study presented here, a detailed analysis of the expression of five of these genes, chosen on the basis of their sequences and expression patterns, was carried out. The five genes were: EgSQUA1 (SQUA subfamily); EgGLO2 (GLO subfamily); EgDEF1 (DEF subfamily); EgAG2 (AG subfamily); and EgAGL2-1 (AGL2 subfamily) (see Supplementary Fig. S1 at JXB online). The expression pattern of each MADS box gene was characterized by gene-specific semi-quantitative RT–PCR using rachillae of normal and mantled oil palm inflorescences at five different developmental stages plus leaves and roots (Fig. 2). The key features of each developmental stage studied are described in detail elsewhere (Adam et al., 2005) and reference microscopic sections are shown in Supplementary Fig. S2 at JXB online. The spatial expression pattern of each of the five chosen genes was studied by in situ hybridization on longitudinal sections of pistillate and staminate flowers (Fig. 3). In situ hybridization was also performed in the same way on floral triads of mantled female inflorescences and on the rachilla meristem for some of the genes characterized (Fig. 3).
|
|
SQUA subfamily:
The EgSQUA1, EgSQUA2, and EgSQUA3 genes belong to the SQUAMOSA subfamily and display similar expression patterns (see Supplementary Fig. S3 at JXB online). The focus in the present study was on EgSQUA1 which is more related to FRUITFULL-like genes of the SQUA subfamily (Adam et al., 2006; see Supplementary Fig. S1 at JXB online). RT-PCR analysis revealed the presence of EgSQUA1 transcripts in the inflorescence, rachilla, and floral meristems, but not, in contrast to EgSQUA2 and EgSQUA3, in the vegetative shoot meristem (see Supplementary Fig. S3 at JXB online). Thus, its function appears to be restricted to reproductive development. EgSQUA1 transcript abundance is relatively constant during the development of pistillate and staminate flowers; however, no signal was detected in either leaves or roots (Fig. 2).
The histo-cytological localization of EgSQUA1 transcripts was studied on several different types of material by in situ hybridization (Fig. 3). In longitudinal sections of the developing rachis, a signal was detected in cells of the outer layers of the rachilla meristem (Fig. 3A). In situ hybridization performed on pistillate and staminate flowers at different stages of development revealed a localization of EgSQUA1 transcripts concentrated in the central dividing zone of floral primordia in the case of the pistillate flower (Fig. 3B). At stage 3 of development in the staminate flower, EgSQUA1 was observed to be expressed in the cells of stamen primordia and weakly in sepals and petals (Fig. 3C). Overall, it appears that the oil palm EgSQUA1 gene is expressed at a generally constant level during inflorescence development, with no noticeable changes occurring at the stages associated with floral organ initiation and differentiation. The RT-PCR and in situ hybridization data obtained suggest that this gene might play a role in determining the meristematic character of cells during inflorescence and flower development, with a possible additional function in the later phases of development of sepal and petals.
AGL2 subfamily:
For the detailed studies reported here, it was decided to focus attention on EgAGL2-1, which belongs to the gene clade represented by the A. thaliana SEPALLATA3 (SEP3) E function gene (Pelaz et al., 2000; Honma and Goto, 2001; see Supplementary Fig. S1 at JXB online). RT-PCR analysis revealed that EgAGL2-1 is expressed during the late stages of floral development, transcripts being detected from stage 4 onwards in the female inflorescence and from stage 3 onwards in the male inflorescence (Fig. 2).
In situ hybridization studies performed on pistillate flowers revealed a weak accumulation of EgAGL2-1 transcripts principally at the base of petals at stage 3 (Fig. 3D). Once all organs of pistillate flowers were in place, expression was observed to be concentrated in the cells of petals and in ovule primordia (Fig. 3E). In stage 3 staminate flowers, EgAGL2-1 transcripts were seen to be localized in petals and in the primordia of stamens (Fig. 3F).
In conclusion, the spatial pattern of expression revealed for EgAGL2-1 suggests that this gene might be involved in the development of petals and ovule primordia in pistillate flowers, and in stamen and perianth development in the staminate flower.
DEF subfamily:
One gene belonging to the DEFICIENS subfamily, named EgDEF1, was identified and characterized in oil palm. EgDEF1 transcripts were not detected at the floral meristem stage in female and male inflorescences (Fig. 2), but were found to be present in similar quantities at all subsequent stages studied. EgDEF1 transcripts were not detected in leaves or roots.
In situ hybridization revealed a weak signal in staminodal cells and at the centre of the carpel (Fig. 3H). In the pistillate flower displaying developed reproductive organs, EgDEF1 transcripts were detected mostly in staminodes and in the distal regions of petals (Fig. 3I). Accompanying male flowers also displayed an accumulation of EgDEF1 transcripts in petal cells and in stamens. In stage 3 staminate flowers, an intense signal was observed in the primordia of stamens (Fig. 3J). Overall, the transcript accumulation pattern of EgDEF1 in pistillate and staminate flowers suggests a role for this gene in the determination of the identity of the inner perianth whorl and stamens as seen for genes belonging to the DEF subfamily in other species.
GLO subfamily:
Two closely related genes belonging to the GLOBOSA subfamily, named EgGLO1 and EgGLO2, were identified in oil palm. Their closely related sequences (Adam et al., 2006) and qualitatively identical expression patterns (see Supplementary Fig. S3 at JXB online) suggest that they are young paralogues. In the present study attention was focused on the expression and function of EgGLO2. A progressive accumulation of EgGLO2 transcripts was observed during the development of the female and male inflorescences. Transcripts were only barely detected at stage 1 (Fig. 2), a weak signal also being observed in roots.
In situ hybridization studies revealed that EgGLO2 transcripts were mostly localized in sepals, petals, and in the floral meristem in stage 3 pistillate flowers (Fig. 3L). In stage 4 pistillate flowers, EgGLO2 transcripts were only weakly detected, principally in whorls 1 and 2 (Fig. 3M). In staminate flowers, EgGLO2 transcripts were found to be localized in the organs of all whorls at stage 3 (Fig. 3N).
Overall, the expression of EgGLO2 seems to be mainly localized in sepals, petals, and staminodes or stamens of pistillate and staminate flowers, respectively, at early stages of development, whereas at later stages, transcripts are detected mostly in the perianth. It is doubtful that the presence of EgGLO2 transcripts in sepals signifies a homeotic activity for this gene in these organs, since this would require the production of not only a functional EgGLO2 protein, but also its putative heterodimeric partner EgDEF1. Given the apparent lack of expression of EgDEF1 in sepals, this scenario seems unlikely. On the basis of currently available data, the most plausible role for EgGLO2 appears to be, like many of its homologues, in the determination of the identity of the inner perianth and androecium. Nevertheless, it is interesting to note that GLO gene transcript accumulation in whorl 1 has already been reported for other monocot species such as tulip (Liliales; Kanno et al., 2003) and Tradescantia reflexa (Commelinales; Ochiai et al., 2004), although in the former case this was to be expected since in tulip flowers the outer perianth whorl is petaloid, with DEF gene expression also being detected.
AG subfamily:
The EgAG1 and EgAG2 genes belong to the AGAMOUS subfamily and are similar in both their sequence and expression pattern (see Supplementary Figs S1 and S3 at JXB online), suggesting that, like EgGLO1 and EgGLO2, they are young paralogues. Attention was focused on EgAG2, for which transcripts were weakly detected in roots and leaves, progressively increasing amounts being observed during female and male inflorescence development (Fig. 2).
In situ hybridization experiments showed that EgAG2 transcripts were localized mostly in the cells of carpel primordia at stage 3 of pistillate flower development (Fig. 3P). Subsequently at stage 4, EgAG2 transcripts were most strongly detected in cells of ovule primordia (Fig. 3Q). In the staminate flower at stage 3, EgAG2 transcripts were observed to accumulate in each of the four whorls (Fig. 3R). Overall, the spatial expression pattern of EgAG2 suggests a function comparable with that of C or D class genes as observed for genes of this subfamily in other species.
Analysis of gene expression patterns associated with the mantled phenotype
RT-PCR analysis revealed that the expression of several oil palm MADS box genes was perturbed in mantled palms (Fig. 2). The mantled phenotype may be identified morphologically from stage 4 (initiation of androecium and gynoecium organs) onwards in pistillate and staminate flowers. No difference in the accumulation of EgSQUA1 transcripts was observed between normal and mantled material. In contrast, transcripts of EgAGL2-1 were found to be accumulated in smaller quantities in male and female inflorescences displaying the mantled phenotype. The most marked case of perturbed expression in mantled palms was that of EgDEF1, transcripts of this gene accumulating in smaller amounts at all stages studied for the female inflorescence. However, in the mantled male inflorescence, EgDEF1 transcripts were less abundant only at stages 2 and 3 compared with the same stage of development in the wild type. For EgGLO2, a pattern of reduced expression in mantled palms was observed, at stage 4 for the female inflorescence and at stages 2 and 3 for the mantled male inflorescence. In the case of EgAG2, a mantled-dependent profile of expression was observed only in the female inflorescence, for which reduced transcript abundance was seen at stages 3 and 4.
In order to characterize the spatial pattern of EgAGL2-1, EgDEF1, EgGLO2, and EgAG2 expression in mantled plants, in situ hybridization was performed on longitudinal sections of mantled floral triads at stage 4, which display the formation of pseudocarpels in the androecium of both pistillate and accompanying male flowers (Fig. 3; see Supplementary Fig. S4 at JXB online). The question of whether the mantled phenotype also involves a transformation of petals into sepals as seen in class B mutants was unresolved at this point, owing to the close physical resemblance of petals and sepals in oil palm. In experiments performed with EgAGL2-1 and EgDEF1 probes, no hybridization signal was observed either in petals or in the androecium, in contrast to the situation seen in normal staminate and pistillate flowers (Fig. 3G, K; see Supplementary Fig. S4 at JXB online). Similar experiments performed with an EgGLO2 probe enabled the identification of a hybridization signal localized mostly in whorls 1 and 2 of mantled pistillate flowers (Fig. 3O; see Supplementary Fig. S3 at JXB online). Finally, in situ hybridization analysis of EgAG2 expression revealed a concentration of transcripts of this gene, as for wild-type pistillate flowers, in the ovule primordia of whorl 4. No significant signal was seen in the pseudocarpels, which do not form ovules (see Supplementary Fig. S3 at JXB online).
Taken together, these data indicate that in mantled plants, the accumulation of transcripts of several genes belonging to the DEF, GLO, AGL2, and AG subfamilies is reduced. This suggests that these genes might be involved in the determination of organ identity in the androecium and also possibly in the inner perianth. Indeed the in situ hybridization data obtained revealed differences in gene expression between normal and mantled flowers, not only for the androecium but also for whorl 2 organs, suggesting a homeotic transformation of both as seen in the class B mutants of model plants. For the genes belonging to DEF and GLO subfamilies, transcript accumulation is perturbed in distinctly different developmental patterns in male and female inflorescences of the mantled phenotype, suggesting that the temporal regulation of these genes is different in pistillate and staminate flowers.
Analysis of oil palm MADS box gene function by ectopic expression in A. thaliana
To investigate the biological functions of the proteins encoded by the oil palm MADS box genes, transgenic plants ectopically expressing EgSQUA1, EgDEF1, EgAG2, EgGLO2, and EgAGL2-1 were generated. For the genes EgAG2 and EgDEF1, a total of 44 and 61 independent transgenic lines, respectively, were obtained. However, none of the transformants obtained in these two cases was observed to display phenotypic alterations compared with wild-type plants, either in flowers or in any other organs or tissues of the plant. In contrast, the results obtained with the transgenic lines overexpressing EgSQUA1, EgGLO2, and EgAGL2-1 revealed changes in phenotype.
Heterologous expression of EgSQUA1 in A. thaliana results in a tall phenotype:
Previous functional studies of SQUAMOSA-like genes showed that when overexpressed, they cause an early flowering phenotype (Mandel and Yanofsky, 1995; Berbel et al., 2001; Shchennikova et al., 2004). To investigate whether EgSQUA1 has a similar biological effect, 25 independent transgenic lines overexpressing this gene were obtained. No significant effect on flowering time was observed; however, 17 plants were found to display a greater height than wild-type plants. Four independent transgenic lines were studied, and for each line, five T2 plants were analysed (Table 1). All plants displayed the same tall phenotype. Various measurements were performed on the transgenic plants at the same defined time points. Measurements of the transgenic plants showed that they were on average 17 cm taller than the wild type (Table 1; Fig. 4A). This elongation was found to be due to a greater number of internodes produced during the reproductive phase of the plant. Internode length was found to be unaffected. Transgenic plants carrying the 35S::EgSQUA1 construction also displayed longer pedicels and shorter siliques in comparison with those of the wild type (Fig. 4B, C).
|
|
Overall, it can be seen that the heterologous expression of EgSQUA1 in transgenic A. thaliana did not affect floral structure but rather the architecture of the plants, probably due to a modification of the meristematic activity of the plants during their reproductive phase.
Ectopic expression of EgGLO2 in A. thaliana results in the same phenotype as 35S::PI plant lines:
Twenty-six independent transgenic lines that overexpressed EgGLO2 were obtained. Six of these lines (31%) displayed homeotic transformation of floral organs, of the same type in each case. This involved a partial conversion of sepals to petals in whorl 1 (Fig. 4D). In the wild-type plant, the adaxial (inner) and abaxial (outer) epidermis of sepals were seen to contain elongated cells (Fig. 4E, F) whereas petals displayed rounded cells with prominent cuticular thickenings on their abaxial surface and ovoid cells on their adaxial surface (Fig. 4K). Examination of the flowers of 35S::EgGLO2 lines revealed that whorl 1 contained mosaic organs with both sepal-like and petal-like tissues (Fig. 4G, H). The peripheral regions of the first whorl organs were found to be characteristic of petals, while cells in their central region appeared to be characteristic of sepals. The same type of homeotic conversion was observed in the case of ectopic expression of PI in A. thaliana (Krizek and Meyerowitz, 1996), suggesting that the EgGLO2 and PI proteins have similar biochemical specificities.
Ectopic expression of EgAGL2-1 in A. thaliana results in modifications to the epidermis of sepals and petals and the production of a flower in the place of the ovule:
Twenty-nine independent transgenic lines that overexpressed EgAGL2-1 were obtained. Six of these lines (20%) displayed the same floral alterations, namely homeotic transformation of sepals and petals, shorter stamens, and gynoecium malformation (Fig. 4I). The gynoecium was often seen to be supported by an elongated gynophore, to display malformed carpels, to lack a style, and to possess only a short stigma. Upon microscopic examination, floral whorls 1 and 2 of 35S::EgAGL2-1 plants were found to be transformed into leaf-like organs. The abaxial epidermal cells of the sepals of transgenic flowers appeared to have a mixture of both leaf- and sepal-like characteristics (Fig. 4J). Whereas wild-type petals displayed an abundance of rounded cells with prominent cellular thickenings (Fig. 4K), the abaxial surface of petals of 35S::EgAGL2-1 transgenic lines were found to consist almost entirely of irregular puzzle-shaped cells typical of those normally found on leaves (Fig. 4L). The adaxial surface of petals did not show homeotic transformation, although the cells displayed less cuticular thickening (Fig. 4M). Whereas the sepals from flowers of wild-type plants had simple unbranched trichomes on the abaxial surface and no trichomes were formed on the pistil, flowers of 35S::EgAGL2-1 transgenic lines possessed sepals and carpels which displayed some branched stellate trichomes (Fig. 4I, 4N). In severe cases, the carpels of 35S::EgAGL2-1 transgenic lines were completely malformed (Fig. 4N). Moreover, the epidermis of carpels displayed some elongated rectangular cells instead of smaller epidermal cells (Fig. 4O), although the puzzle-shaped cells characteristic of leaves were not seen. The development of the ovules of 35S::EgAGL2-1 transgenic lines was studied. It was observed that some ovules were malformed with an undeveloped funiculus (data not shown). In several cases, flowers of 35S::EgAGL2-1 plants displayed a loss of determinacy in their innermost whorl, witnessed by the production of a flower within a flower (Fig. 4P). The additional flower was found to be formed in the place of an ovule. The new flower was found to be composed of perianth organs, stamens, and organs similar to the stigma (Fig. 4P). In order to assess whether A. thaliana floral organ identity genes were affected by the ectopic expression of the oil palm EgAGL2-1 gene, RT-PCR was performed on wild-type and transgenic flowers at two stages of development. The expression of the endogenous A. thaliana genes AP1, SEP1, SEP2, SEP3, SEP4, AP3, PI, and AG was found to be unaffected, in terms of transcript accumulation, in transgenic flowers (see Supplementary Fig. S5 at JXB online). It is interesting to note that the modified floral phenotype observed in 35S::EgAGL2-1 plants has common features with that of the sep1sep2sep3sep4 quadruple mutant of A. thaliana (Ditta et al., 2004), including the production of a flower within the carpel.
Overall, these results suggest that the EgAGL2-1 protein may act in a dominant negative manner to perturb the SEP regulatory pathway in transgenic A. thaliana plants.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
An in-depth characterization is described of five florally expressed MADS box genes in oil palm belonging to five different MADS box subfamilies, namely the SQUA, DEF, GLO, AGL2, and AG groups (Adam et al., 2006). The study included a detailed analysis of their expression during flower development in relation to sex and floral phenotype, and a functional analysis by means of genetic transformation of A. thaliana. Genetic transformation has been achieved for oil palm (Parveez et al., 2000); however, studies of floral gene function in transgenic oil palm would require many years and a large amount of space in order to yield exploitable results. On the other hand, by using ectopic expression in A. thaliana as a functional tool, it was possible to obtain information on the conservation of MADS box protein function between oil palm and its dicot counterpart. On the basis of the available data, a working model of homeotic gene activities regulating oil palm flower structure is proposed here (Fig. 5).
|
Meristem identity and A function
The main function of SQUA-like genes in higher plants appears to be the determination of the identity of the floral meristem. In A. thaliana, three genes belonging to the SQUA subfamily act in redundancy in the determination of inflorescence architecture (AP1, FUL, and CAL; Ferrandiz et al., 2000). The AP1 gene also acts to specify the identity of sepals and petals, thus performing an A function (Mandel et al., 1992). Despite evidence that a rice SQUA gene might exert a dominant negative effect on floral organ identity when heterologously expressed in Arabidopsis (Fornara et al., 2004), there has been no clear demonstration to date of an A function for SQUA genes in monocots (Kater et al., 2006). Indeed, as illustrated by the wheat VRN1 gene involved in vernalization, SQUA-like gene function may have diverged in various species (Yan et al., 2003).
EgSQUA1 transcripts accumulate in cells that retain meristematic characteristics in the rachilla and floral meristems and also to a lesser extent throughout most regions of the staminate and pistillate flower. The specific expression of EgSQUA1 in inflorescence and floral meristems is a pattern commonly observed amongst members of the SQUA subfamily of MADS box genes (Huijser et al., 1992; Mandel et al., 1992; Mena et al., 1995; Moon et al., 1999a; Kyozuka et al., 2000). The functional studies of EgSQUA1 in A. thaliana revealed that its ectopic expression increases the number of internodes during the reproductive phase of the plant. Floral morphogenesis and floral induction timing are not affected, in contrast to Arabidopsis plants which overexpress AP1 (Mandel and Yanofsky, 1995). When interpreting the phenotype of 35S::EgSQUA1 plants, it is of interest to note that like the Arabidopsis ful mutant, reduced fertility is observed (Gu et al., 1998). Thus the hypothesis of a dominant negative effect cannot be ruled out. Whatever the original cause of the reduced fertility of 35S::EgSQUA1 plants, it is a possibility that this phenotypic effect might lead to changes in resource allocation which could, in turn, affect inflorescence growth.
B function
The archetypal B function observed in the model dicot A. thaliana involves the activity of APETALA3 and PISTILLATA, genes belonging, respectively, to the DEF and GLO subfamilies. The corresponding proteins act in a multimeric complex. In monocots, the situation appears to be similar, but often more complex, multiple GLO and DEF genes have been observed in a number of cases. In the case of maize, a DEF-like gene (SILKY1) and three GLO-like genes (ZMM16, ZMM18, and ZMM29) have been identified. Expression patterns and functional data support the idea that these genes do indeed exert a B function (Ambrose et al., 2000; Münster et al., 2001). In the case of rice, similarly based on expression patterns in flowers and functional analyses, two GLO orthologues (OsMADS2 and OsMADS4), and a DEF orthologue (OsMADS16 or SUPERWOMAN1, SPW1) with B class activity have been identified (Kang et al., 1998; Moon et al., 1999b; Kyozuka et al., 2000; Nagasawa et al., 2003). In oil palm, three genes of putative B function have been identified: EgGLO1 and EgGLO2 belonging to the GLO subfamily; and EgDEF1 belonging to the DEF subfamily. In situ hybridization analysis revealed that unlike their model dicot counterparts, EgDEF1 and EgGLO2 do not display the same spatial pattern of expression in the flower. EgDEF1 expression is essentially restricted to the inner perianth and stamens/staminodes in staminate/pistillate flowers, respectively. This result indicates a distinct molecular identity for whorl 2 organs compared with those of whorl 1, despite their similar appearance in oil palm. It is relevant to note in this context that most palm species produce distinguishable petals and sepals (Dransfield and Uhl, 1998). In contrast to EgDEF1, EgGLO2 expression is mainly detected in sepals and petals in nearly mature oil palm pistillate flowers, whereas in staminate flowers a strong signal is also seen in the stamens. The question of homeotic identity in perianth organs has been addressed for a number of other monocot species, including some which produce morphologically similar tepals rather than distinct petals and sepals. In the case of tulip, which produces tepals of a petaloid nature, DEF and GLO gene expression is observed in both whorls of the perianth (Kanno et al., 2003). In contrast, Asparagus, which also has tepal organs resembling petals, exhibits expression of DEF and GLO genes in the inner tepals and in stamens, but not in the outer tepals (Park et al., 2003, 2004). It is also interesting to compare the oil palm data with results obtained for Tradescantia reflexa and Commelina communis (Ochiai et al., 2004), which produce distinctly different sepals and petals. The expression of the DEF gene homologues was found to be restricted to the inner perianth and androecium. In contrast, one of the GLO genes isolated was found to be expressed in whorl 1, as observed for EgGLO2 in oil palm.
Consistent with their similarities to known B class genes in respect of sequence and spatial expression in the flower, EgDEF1 and EgGLO2 both display reduced expression in mantled palms, which resemble B class mutants in that they show transformation of stamens or staminodes into carpeloid structures. The additional question remains as to whether the mantled abnormality involves, like the def and glo mutants of Antirrhinum, a transformation of petals into sepals. This appears to be the case, on the basis of the perturbation of expression of EgDEF1 in the inner perianth of mantled palm flowers. Thus the phenotype of mantled palms may be considered to be similar in nature to previously characterized B mutants. In the case of EgGLO2, although a general quantitative reduction in transcript amounts is observed in mantled flowers at specific stages, spatial changes observed by in situ hybridization are less marked.
Additional indications concerning the conservation of B function in oil palm can be drawn from the A. thaliana ectopic expression data. Ectopic expression of EgGLO2 in transgenic A. thaliana results in conversion of sepals into petals as observed with PI (Krizek and Meyerowitz, 1996), suggesting that EgGLO2 has common biochemical specificities with its A. thaliana homologue which is necessary for petal and stamen formation. In the case of EgDEF1, it might be considered surprising that the ectopic expression of this gene does not result in homeotic transformation in A. thaliana flowers. However, a similar result was obtained by Tzeng and Yang (2001) for a lily AP3 homologue LMADS1. If the A. thaliana AP3 gene is ectopically expressed in its native species, a homeotic conversion of carpels to stamens in whorl 4 is observed to be induced. This involves the induction of the expression of PI (Jack et al., 1994), which is, in turn, maintained through activation of the PI promoter by the PI–AP3 complex (Honma and Goto, 2000). One possible explanation for the absence of homeotic transformation in A. thaliana flowers ectopically expressing EgDEF1 (or LMADS1) is that the two monocot DEF proteins mentioned might not have the specificities necessary for binding to their protein partner homologues in A. thaliana and/or interaction with the PI promoter.
Overall, the spatial expression data indicate that the two perianth organs of oil palm have a different homeotic identity despite being morphologically similar and that the oil palm DEF and GLO genes described here might have a B function. On the basis of the available data, EgDEF1 is likely to be involved in petal determination, and in stamen/staminode initiation and functional stamen development. Similarly, EgGLO2 might play a role in stamen and petal determination. The additional detection of EgGLO2 expression in sepals might be taken to suggest a role for this gene in whorl 1; however, it should be stressed that no studies to date have as yet demonstrated a DEF-independent biological function for GLO proteins.
C and D functions
Two oil palm genes belong to the AG subfamily, which is known to be involved, in model plants, in the determination of stamens and carpels in the case of C class genes and in ovule development in the case of C and D class genes. Phylogenetic studies indicate that the two oil palm AG genes do not belong to the monophyletic D class group (Kramer et al., 2004; Adam et al., 2006). EgAG2 expression was found to be localized principally in stamens and carpel primordia in staminate and pistillate flowers, respectively. In mature flowers, transcripts are present in the precursor cells of ovules and in the microspores of stamens. RT-PCR analysis of EgAG2 transcript accumulation shows that the expression of this gene is perturbed in mantled plants, suggesting a role in the determination of androecium identity as for other C class genes. In model species, C class genes are expressed in stamens and carpels while D class genes show specific expression in ovules. However, it has been shown that, in the A. thaliana mature flower, the expression of AG occurs in restrictive cellular regions of stamens and carpels (Bowman et al., 1991). Moreover, it was shown that AG controls the microsporogenesis process leading to pollen formation via the regulation of SPOROCYTELESS (Ito et al., 2004). It has been shown that ovule identity in A. thaliana is promoted by three redundant genes closely related to AG, namely SHATTERPROOF1 (SHP1), SHATTERPROOF2 (SHP2), and SEEDSTICK (STK), which act in concert with AG (Favaro et al., 2003; Pinyopich et al., 2003). Functional analyses indicate that in A. thaliana a C class gene may be involved in the development of male and female gametes and that some genes may have a specific D function and others a mixed C/D function.
Based on these observations and on the expression pattern of EgAG2, it is possible to hypothesize that this oil palm gene exhibits a mixed C/D function involving the control of organ identity in the androecium and the gynoecium and that it might also play a role in male and female gamete development. This situation is similar to that proposed for the SHP1 and SHP2 genes of A. thaliana. Nevertheless, ectopic expression of EgAG2 in A. thaliana does not result in either a homeotic floral phenotype, or in any of the other developmental alterations associated with AG gene heterologous expression, such as altered leaf morphology or early flowering (Mizukami and Ma, 1992; Kater et al., 1998; Rutledge et al., 1998). These observations suggest that the specificities of the EgAG2 protein are divergent from those of the A. thaliana homologues and/or that EgAG2 is not sufficient on its own to provide a C or C/D function in A. thaliana. A number of studies in various different species have revealed that the C/D function is often shared by two or more closely related genes as a result of gene duplication, and that partial redundancy in the control of reproductive organ identity is common amongst angiosperms (Ferrario et al., 2004). Interestingly, in rice, the functions regulated by AG have been partially partitioned between two paralogous genes, OsMADS3 and OsMADS58, which display subfunctionalization in terms of their activity in different whorls of the flower (Yamaguchi et al., 2006).
On the basis of the present data, the possibility that a specific D function controlling ovule identity and development exists in oil palm cannot be excluded. Ovules are initiated from the remaining meristematic region located inside the three carpels of the gynoecium (Adam et al., 2005). The hypothesis of a specific D function in oil palm is compatible with the mantled phenotype in which carpels are produced instead of stamens or staminodes in the androecium. These carpels do not form ovules, possibly implying that in oil palm the development of the ovule requires additional homeotic gene functions compared with those necessary for carpel development.
E function
EgAGL2-1, belonging to the AGL2/SEP subfamily, shows late expression in pistillate and staminate flowers from the stage of reproductive organ initiation, and this expression is affected in mantled plants. In situ hybridization shows that EgAGL2-1 is expressed in petals and ovules in pistillate flowers and in petals and stamens in staminate flowers. EgAGL2-1 might therefore play a role in petal development and in ovule and stamen development in pistillate and staminate flowers, respectively, like previously described E class genes.
The ectopic expression of EgAGL2-1 in A. thaliana results in modifications in different whorls of the flower. The overall phenotype has similarities to that of the sep1sep2sep3sep4 mutant (Ditta et al., 2004), although, in the latter case, the additional flower produced within the carpel was found to originate from the floral meristem and not from the conversion of an ovule as seen in 35S::EgAGL2-1 plants. Interestingly, no A. thaliana floral organ identity genes were found to be affected in their transcript accumulation in 35S::EgAGL2-1 plants. A possible explanation of these data is that the protein encoded by EgAGL2-1 might interact with native A. thaliana proteins to form a biologically inactive complex in a dominant negative manner. All floral whorls display modifications when EgAGL2-1 is introduced, suggesting that EgAGL2-1 is able to interact within each of the different native protein complexes necessary for correct whorl identity in A. thaliana. On the other hand, the fact that 35S::EgAGL2-1 plants display a sep mutant phenotype suggests that the functional specificities of EgAGL2-1 and Arabidopsis SEP proteins have diverged significantly during evolution. Clearly it would be of interest to assess whether EgAGL2-1 is able to complement the Arabidopsis sep mutants genetically. Overall, the data support the idea that EgAGL2-1 acts as an E class protein involved in petal, androecium, and gynoecium development in oil palm flowers and that it may be involved in protein complexes which are slightly different from those of A. thaliana. The complement of E class genes functioning in monocots appears to be different since SEP1/2 and SEP4-like genes do not seem to be present, in contrast to the SEP3-like genes. Moreover SEP-related genes specific to monocots have been described, notably the LHS1 and the OsMADS34 clades (Kramer et al., 2004; Adam et al., 2006). LHS1-like genes play a role in determining the identity of the paleas and lemmas, in inflorescence meristem identity, and in specifying the upper floret in certain species (Malcomber and Kellogg, 2004). In rice, five SEP-related genes are present: two from the SEP3 clade, two from the LHS1 clade, and one from the OsMADS34 clade (Adam et al., 2006). All of them show specific expression patterns during flower development (Kang and An, 1997; Prasad et al., 2001; Pelucchi et al., 2002). Within the second group, an E-type mutant phenotype was recently demonstrated for the OsMADS1 gene (Kumar Agrawal et al., 2005).
|
Conclusion |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
The working model of flower structure determination in oil palm implicates different combinations of gene activities necessary for correct organ identity in each whorl (Fig. 5). The scheme in Fig. 5 shows a strong overall resemblance to the generic dicot ABCDE model and contrasts with the ‘modified ABC model’ proposed for members of the Liliales (Tzeng et al., 2001; Kanno et al., 2003) and Asparagales (Nakamura et al., 2005) displaying petal identity in floral whorl 1. Overall, the oil palm data indicate a closer similarity to the situation observed in species of the Commelinales (Ochiai et al., 2004), which show a clear distinction of sepals and petals and which are more closely related to palms (with which they form the Commelinid group) than to the Liliales or Asparagales. Further studies will now be required in order to obtain a deeper understanding of the diverse roles played by MADS box genes in the determination and evolution of flower form both within the palm family and throughout angiosperms as a whole.
|
Supplementary data |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
The following supplementary data are available at JXB online.
Fig. S1. Relationships between MADS box proteins from Elaeis guineensis and their relatives from other angiosperms.
Fig. S2. Histological definition of developmental stages in staminate and pistillate flowers of oil palm.
Fig. S3. Expression pattern analyses of oil palm MADS box genes.
Fig. S4. In situ hybridization studies to investigate the accumulation of transcripts of the oil palm genes EgAGL2-1, EgDEF1, EgGLO2 and EgAG2 in mantled flowers (stage 4) of the male oil palm inflorescence.
Fig. S5. Endogenous gene expression levels in flowers of wild type and transgenic 35S::EgAGL2-1 Arabidopsis plants.
Table S1. List of primers and RT-PCR conditions used for the expression analysis of oil palm MADS box genes.
Table S2. List of primers and RT-PCR conditions used for the expression analysis of A. thaliana MADS box genes.
Table S3. List of primers used for the synthesis of in situ hybridization probes.
|
Note added in Proof |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
Syed Alwee et al. (Plant Cell Tissue and Organ Culture 85, 331–344; 2006) recently described the identification of oil palm MADS box genes including EgMADS16, a paralogue of EgGL02 (identical to EgGL01; Adam et al., Journal of Molecular Evolution 62, 15–31; 2006) which complements the pistillate mutant.
|
Acknowledgements |
|---|
We would like to thank Jean-Luc Verdeil for helpful discussions and technical advice for in situ hybridization, Eric Tailliez and Zahia Mammasse for technical assistance in A. thaliana transgenic line analyses, and Phil Mullineaux for providing pGREEN plasmids. We thank Alain Rival for fixation of mantled flowers and Axel Labeyrie for photographs. We also thank Tim Tranbarger and Patrick Laufs for critical reading of the manuscript. The authors acknowledge the generous support of colleagues at CNRA La Mé Experimental Station in Côte d'Ivoire, FELDA Agricultural Services in Malaysia, and ASD Coto in Costa Rica for providing plant material. This work was supported by an MESR PhD grant to HA and institutional funding from IRD and CIRAD.
|
Abbreviations |
|---|
PBS, phosphate-buffered saline; RT-PCR, reverse transcription-polymerase chain reaction.
|
References |
|---|
|
TopAbstractIntroductionMaterials and methodsResultsDiscussionConclusionSupplementary dataNote added in ProofReferences |
|---|
Adam H, Jouannic S, Escoute J, Verdeil JL, Duval Y, Tregear JW. (2005) Reproductive developmental complexity in the African oil palm (Elaeis guineensis). American Journal of Botany 92 1836–1852.
Adam H, Jouannic S, Morcillo F, Richaud F, Duval Y, Tregear JW. (2006) MADS box genes in oil palm (Elaeis guineensis): patterns within the evolution of the SQUAMOSA, DEFICIENS, GLOBOSA, AGAMOUS and SEPALLATA subfamilies. Journal of Molecular Evolution 62 15–31.[CrossRef][Web of Science][Medline]
Alvarez-Buylla ER, Pelaz S, Liljegren SJ, Gold SE, Burgeff C, Ditta GS, Ribas de Pouplana L, Martinez-Castilla L, Yanofsky MF. (2000) An ancestral MADS-box gene duplication occurred before the divergence of plants and animals. Proceedings of the National Academy of Sciences, USA 97 5328–5333.
Ambrose BA, Lerner DR, Ciceri P, Padilla CM, Yanofsky MF, Schmidt RJ. (2000) Molecular and genetic analyses of the silky1 gene reveal conservation in floral organ specification between eudicots and monocots. Molecular Cell 5 569–579.[CrossRef][Web of Science][Medline]
Becker A and Theissen G. (2003) The major clades of MADS-box genes and their role in the development and evolution of flowering plants. Molecular Phylogenetics and Evolution 29 464–489.[CrossRef][Web of Science][Medline]
Berbel A, Navarro C, Ferrandiz C, Canaz KA, Madueno F, Beltran J-P. (2001) Analysis of PEAM4, the pea AP1 functional homologue, supports a model for AP1-like genes controlling both floral meristem and floral organ identity in different plant species. The Plant Journal 25 441–451.[CrossRef][Web of Science][Medline]
Bowman JL, Drews GN, Meyerowitz EM. (1991) Expression of the Arabidopsis floral homeotic gene AGAMOUS is restricted to specific cell types late in flower development. The Plant Cell 3 749–758.
Clough SJ and 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 and 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]
Corley RHV, Lee CH, Law LH, Wong CY. (1986) Abnormal flower development in oil palm clones. Planter, Kuala Lumpur 62 233–240.
Corre F, Henry Y, Rode A, Hartmann C. (1996) Em gene expression during somatic embryogenesis in the monocot Triticum aestivum L. Plant Science 117 139–149.
Ditta G, Pinyopich A, Robles P, Pelaz S, Yanofsky MF. (2004) The SEP4 gene of Arabidopsis thaliana functions in floral organ and meristem identity. Current Biology 14 1935–1940.[CrossRef][Web of Science][Medline]
Dransfield J and Uhl NW. (1998) Palmae. In Kubitzi K (Ed.). Families and genera of vascular plants. IV. Flowering plants: monocotyledonsBerlin Springer-Verlag pp. 306–389.
Dransfield J, Uhl NW, Asmussen CB, Baker WJ, Harley MM, Lewis C. (2005) A new phylogenetic classification of the palm family, Arecaceae. Kew Bulletin 60 559–569.
Durand-Gasselin T, Le Guen VL, Konan E, Duval Y. (1990) Oil palm (Elaeis guineensis Jacq.) plantations in Côte d'Ivoire obtained through in vitro culture—first results. Oléagineux 45 1–11.[Web of Science]
Favaro R, Pinyopich A, Battaglia R, Kooiker M, Borghi L, Ditta G, Yanofsky MF, Kater MM, Colombo L. (2003) MADS-box protein complexes control carpel and ovule development in Arabidopsis. The Plant Cell 15 2603–2611.
Ferrandiz C, Gu Q, Martienssen R, Yanofsky MF. (2000) Redundant regulation of meristem identity and plant architecture by FRUITFULL, APETALA1 and CAULIFLOWER. Development 127 725–734.[Abstract]
Ferrario S, Immink RGH, Angenent GC. (2004) Conservation and diversity in flower land. Current Opinion in Plant Biology 7 84–91.[CrossRef][Web of Science][Medline]
Fornara F, Marziani G, Mizz L, Kater M, Colombo L. (2003) MADS box genes controlling flower development in rice. Plant Biology 5 16–22.[Medline]
Fornara F, Parenicova L, Falasca G, Pelucchi N, Masiero S, Ciannamea S, Lopez-Dee Z, Altamura MM, Colombo L, Kater M. (2004) Functional characterization of OsMADS18, a member of the AP1/SQUA subfamily of MADS box genes. Plant Physiology 135 2207–2219.
Gu Q, Ferrandiz C, Yanofsky MF, Martienssen R. (1998) The FRUITFULL MADS-box gene mediates cell differentiation during Arabidopsis fruit development. Development 125 1509–1517.[Abstract]
Hellens RP, Edwards A, Leyland NR, Beab S, Mullineaux PN. (2000) pGreen: a versatile and flexible binary Ti vectors. Trends in Plant Science 5 446–551.[CrossRef][Web of Science][Medline]
Honma T and Goto K. (2000) The Arabidopsis floral homeotic gene PISTILLATA is regulated by discrete cis-elements responsive to induction and maintenance signals. Development 127 2021–2030.[Abstract]
Honma T and Goto K. (2001) Complexes of MADS-box proteins are sufficient to convert leaves into floral organs. Nature 409 525–529.[CrossRef][Medline]
Huijser P, Klein J, Lonnig WE, Meijer H, Saedler H, Sommer H. (1992) Bracteomania, an inflorescence anomaly, is caused by the loss of function of the MADS-box gene SQUAMOSA in Antirrhinum majus. EMBO Journal 11 1239–1249.[Web of Science][Medline]
Immink RG, Ferrario S, Busscher-Lange J, Kooiker M, Busscher M, Angenent GC. (2003) Analysis of the petunia MADS-box transcription factor family. Molecular Genetics and Genomics 268 598–606.[Web of Science][Medline]
Ito T, Wellmer F, Yu H, Das P, Ito N, Alves-Ferreira M, Riechmann JL, Meyerowitz EM. (2004) The homeotic protein AGAMOUS controls microsporogenesis by regulation of SPOROCYTELESS. Nature 430 356–360.[CrossRef][Medline]
Jack T, Fox G, Meyerowitz EM. (1994) Arabidopsis homeotic gene APETALA3 ectopic expression: transcriptional and post-transcriptional regulation determine floral organ identity. Cell 76 703–716.[CrossRef][Web of Science][Medline]
Jaligot E, Rival A, Beulé T, Dussert S, Verdeil JL. (2000) Somaclonal variation in oil palm (Elaeis guineensis Jacq.): the DNA methylation hypothesis. Plant Cell Reports 7 684–690.[CrossRef]
Jeon JS, Jang S, Lee S, Nam J, Kim C, Lee SH, Chung YY, Kim SR, Lee YH, Cho YG, An G. (2000) leafy hull sterile1 is a homeotic mutation in a rice MADS box gene affecting rice flower development. The Plant Cell 12 871–884.
Kang HG and An G. (1997) Isolation and characterization of a rice MADS box gene belonging to the AGL2 gene family. Molecular Cell 71 45–51.
Kang HG, Jeon JS, Lee S, An G. (1998) Identification of class B and class C floral organ identity genes from rice plants. Plant Molecular Biology 38 1021–1029.[CrossRef][Web of Science][Medline]
Kanno A, Seaki H, Kameya T, Saedler H, Theissen G. (2003) Heteropic expression of class B floral homeotic genes supports a modified ABC model for tulip (Tulipa gesneriana). Plant Molecular Biology 52 831–841.[CrossRef][Web of Science][Medline]
Kater MM, Colombo L, Franken J, Busscher M, Masiero S, Van Lookeren Campagne MM, 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.
Kater MM, Franken J, Carney KJ, Colombo L, Angenent GC. (2001) Sex determination in the monoecious species cucumber is confined to specific floral whorls. The Plant Cell 13 481–493.
Kater MM, Dreni L, Colombo L. (2006) Functional conservation of MADS-box factors controlling floral organ identity in rice and Arabidopsis. Journal of Experimental Botany 57 3433–3444.
Kramer EM, Jaramillo MA, Di Stilio VS. (2004) Patterns of gene duplication and functional evolution during the diversification of the AGAMOUS subfamily of MADS box genes in Angiosperms. Genetics 166 1011–1023.
Krizek BA and Meyerowitz EM. (1996) The Arabidopsis homeotic genes APETALA3 and PISTILLATA are sufficient to provide the B class organ identity function. Development 122 11–22.[Abstract]
Kumar Agrawal G, Abe K, Yamazaki M, Miyao A, Hirochika H. (2005) Conservation of the E-function for floral organ identity in rice revealed by the analysis of tissue culture-induced loss-of-function mutants of the OsMADS1 gene. Plant Molecular Biology 59 125–135.[CrossRef][Web of Science][Medline]
Kyozuka J, Kobayashi T, Morita M, Shimamoto K. (2000) Spatially and temporally regulated expression of rice MADS box genes with similarity to Arabidopsis class A, B and C genes. Plant and Cell Physiology 41 710–718.
Lamb RS and Irish VF. (2003) Functional divergence within the APETALA3/PISTILLATA floral homeotic gene lineages. Proceedings of the National Academy of Sciences, USA 100 6558–6563.
Malcomber ST and Kellog EA. (2004) Heterogeneous expression patterns and separate roles of the SEPALLATA gene LEAFY HULL STERILE1 in grasses. The Plant Cell 16 1692–1706.
Mandel MA, Gustafson-Brown C, Savidge B, Yanofsky MF. (1992) Molecular characterization of the Arabidopsis floral homeotic gene APETALA1. Nature 360 273–277.[CrossRef][Medline]
Mandel MA and Yanofsky MF. (1995) The Arabidopsis AGL8 MADS box gene is expressed in inflorescence meristems and is negatively regulated by APETALA1. The Plant Cell 7 1763–1771.[Abstract]
Mizukami Y and 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]
Mena M, Mandel MA, Lerner DR, Yanofsky MF, Schmidt RJ. (1995) A characterization of the MADS-box gene family in maize. The Plant Journal 8 845–854.[CrossRef][Web of Science][Medline]
Moon YH, Jung JY, Kang HG, An G. (1999b) Identification of a rice APETALA3 homologue by yeast two-hybrid screening. Plant Molecular Biology 40 167–177.[CrossRef][Web of Science][Medline]
Moon YH, Kang HG, Jung JY, Jeon JS, Sung SK, An G. (1999a) Determination of the motif responsible for interaction between the rice APETALA1/AGAMOUS-LIKE9 family proteins using a yeast two-hybrid system. Plant Physiology 120 1193–1204.
Münster T, Wingen LU, Faigl W, Werth S, Saedler H, Theissen G. (2001) Characterization of three GLOBOSA-like MADS-box genes from maize: evidence for ancient paralogy in one class of floral homeotic B-function genes of grasses. Gene 262 1–13.[CrossRef][Web of Science][Medline]
Nagasawa N, Miyoshi M, Sano Y, Satoh H, Hirano H, Sakai H, Nagato Y. (2003) SUPERWOMAN1 and DROOPING LEAF genes control floral organ identity in rice. Development 130 705–718.
Nakamura T, Fukuda T, Nakano M, Hasebe M, Kameya T, Kanno A. (2005) The modified ABC model explains the development of the petaloid perianth of Agapanthus praecox ssp. orientalis (Agapanthaceae) flowers. Plant Molecular Biology 58 435–445.[CrossRef][Web of Science][Medline]
Ochiai T, Nakamura T, Mashiko Y, Fukuda T, Yokoyama J, Kanno A, Kameya T. (2004) The differentiation of sepal and petal morphologies in Commelinaceae. Gene 343 253–262.[CrossRef][Web of Science][Medline]
Park JH, Ishikawa Y, Yoshida R, Kanno A, Kameya T. (2003) Expression of AODEF, a B-functional MADS-box gene, in stamens and inner tepals of the dioecious species Asparagus officinalis L. Plant Molecular Biology 51 867–875.[CrossRef][Web of Science][Medline]
Park JH, Ishikawa Y, Ochiai T, Kanno A, Kameya T. (2004) Two GLOBOSA-like genes are expressed in second and third whorls of homochlamydeous flowers in Asparagus officinalis L. Plant and Cell Physiology 45 325–332.
Parveez GK, Masri MM, Zainal A, Majid NA, Yunus AM, Fadilah HH, Rasid O, Cheah SC. (2000) Transgenic oil palm: production and projection. Biochemical Society Transactions 28 969–972.[CrossRef][Web of Science][Medline]
Pelaz S, Ditta GS, Baumann E, Wisman E, Yanofsky MF. (2000) B and C floral organ identity functions require SEPALLATA MADS-box genes. Nature 405 200–203.[CrossRef][Medline]
Pelucchi N, Fornara F, Favalli C, Masiero S, Lago C, Pe ME, Colombo L, Kater MM. (2002) Comparative analysis of rice MADS-box genes expressed during flower development. Sexual Plant Reproduction 15 113–122.[CrossRef][Web of Science]
Pinyopich A, Ditta GS, Savidge B, Liljegren SJ, Baumann E, Wisman E, Yanofsky MF. (2003) Assessing the redundancy of MADS-box genes during carpel and ovule development. Nature 424 85–88.[CrossRef][Medline]
Pnueli L, Hareven D, Broday L, Hurwitz C, Lifschitz E. (1994) The TM5 MADS box gene mediates organ differentiation in the three inner whorls of tomato flowers. The Plant Cell 6 175–186.[Abstract]
Prasad K, Sriram P, Kumar CS, Kushalappa K, Vijayraghavan U. (2001) Ectopic expression of rice OsMADS1 reveals a role in specifying the lemma and palea, grass floral organs analogous to sepals. Development, Genes and Evolution 211 281–290.[CrossRef][Web of Science][Medline]
Purugganan MD, Rounsley SD, Schmidt RJ, Yanofsky MF. (1995) Molecular evolution of flower development: diversification of the plant MADS-box regulatory gene family. Genetics 140 345–356.[Abstract]
Rao V and Donough CR. (1990) Preliminary evidence of a genetic cause for the floral abnormalities in some oil palm ramets. Elaeis 2 199–207.
Rutledge R, Regan S, Nicolas O, Fobert P, Cote C, Bosnich W, Kauffeldt C, Sunohara G, Seguin 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]
Shchennikova AV, Shulga OA, Immink R, Skryabin KG, Angenent GC. (2004) Identification and characterization of four chrysanthemum MADS-box genes, belonging to the APETALA1/FRUITFULL and SEPALLATA3 subfamilies. Plant Physiology 134 1632–1641.
Theissen G, Becker A, Di Rosa A, Kanno A, Kim JT, Munster T, Winter KU, 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, Kim JT, Saedler H. (1996) Classification and phylogeny of the MADS-box multigene family suggest defined roles of MADS-box gene subfamilies in the morphological evolution of eukaryotes. Journal of Molecular Evolution 43 484–516.[Web of Science][Medline]
Tomlinson PB. (1990) The structural biology of palms Oxford Science Publications.
Tzeng TY and 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.
Xiao H, Wang Y, Liu D, Wang W, Li X, Zhao X, Xu J, Zhai W, Zhu L. (2003) Functional analysis of the rice AP3 homologue OsMADS16 by RNA interference. Plant Molecular Biology 52 957–966.[CrossRef][Web of Science][Medline]
Yamaguchi T, Lee DY, Miyao A, Hirochika H, An G, Hirano HY. (2006) Functional diversification of the two-C-class MADS box genes OSMADS3 and OSMADS58 in Oryza sativa. The Plant Cell 18 15–28.
Yan L, Loukoianov A, Tranquilli G, Helguera M, Fahima T, Dubcovsky J. (2003) Positional cloning of the wheat vernalization gene VRN1. Proceedings of the National Academy of Sciences, USA 100 6263–6268.
CiteULike 
Connotea 
Del.icio.us What's this?
This article has been cited by other articles:
|
|
 |
|
  Y.-Y. Chang, N.-H. Kao, J.-Y. Li, W.-H. Hsu, Y.-L. Liang, J.-W. Wu, and C.-H. Yang Characterization of the Possible Roles for B Class MADS Box Genes in Regulation of Perianth Formation in Orchid Plant Physiology, February 1, 2010; 152(2): 837 - 853. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J. C. Preston, A. Christensen, S. T. Malcomber, and E. A. Kellogg MADS-box gene expression and implications for developmental origins of the grass spikelet Am. J. Botany, August 1, 2009; 96(8): 1419 - 1429. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. C. Hileman and V. F. Irish More is better: the uses of developmental genetic data to reconstruct perianth evolution Am. J. Botany, January 1, 2009; 96(1): 83 - 95. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 Compiled by, F. Tooke, T. Chiurugwi, and N. Battey Flowering Newsletter bibliography for 2007 J. Exp. Bot., July 18, 2008; (2008) ern109v1. [Full Text] [PDF]
|
|
|
|
 |
|
 H. Adam, F. Ouellet, N. A. Kane, Z. Agharbaoui, G. Major, Y. Tominaga, and F. Sarhan Overexpression of TaVRN1 in Arabidopsis Promotes Early Flowering and Alters Development Plant Cell Physiol., August 1, 2007; 48(8): 1192 - 1206. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||