Functional characterization of MADS box genes involved in the determination of oil palm flower structure

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.     

B-class MADS-box genes in trioecious papaya: two paleoAP3 paralogs, CpTM6-1 and CpTM6-2, and a PI ortholog CpPI

In the ABC model of flower development, B function organ-identity genes act in the second and third whorls of the flower to control petal and stamen identity. The trioecious papaya has male, female, and hermaphrodite flowers and is an ideal system for testing the B-class gene expression patterns in trioecious plants. We cloned papaya B-class genes, CpTM6-1, CpTM6-2, and CpPI, using MADS box gene specific degenerate primers followed by cDNA library screening and sequencing of positive clones. While phylogenetic analyses show that CpPI is the ortholog of the Arabidopsis gene PI, the CpTM6-1 and CpTM6-2 loci are representatives of the paralogous TM6 lineage that contain paleoAP3 motifs unlike the euAP3 gene observed in Arabidopsis. These two paralogs appeared to have originated from a tandem duplication occurred approximately 13.4 million year ago (mya) (bootstrap range 13.36 ± 2.42). In-situ hybridization and RT-PCR showed that the papaya B-class genes were highly expressed in young flowers across all floral organ primordia. As the flower organs developed, all three B-class genes were highly expressed in petals of all three-sex types and in stamens of hermaphrodite and male flowers. CpTM6-1 expressed at low levels in sepals and carpels, whereas CpTM6-2 expressed at a low level in sepals and at a high level in leaves. Our results showed that B-class gene homologs could function as predicted by the ABC model in trioecous flowers but differential expressions of CpTM6-1, and CpTM6-2, and CpPI suggested the diversification of their functions after the duplication events. Christine M. Ackerman, Qingyi Yu contributed equally to this work.     

Determination of Flower Structure in Elaeis guineensis: Do Palms use the Same Homeotic Genes as Other Species?

AIMS: In this article a review is made of data recently obtained on the structural diversity and possible functions of MADS box genes in the determination of flower structure in the African oil palm (Elaeis guineensis). MADS box genes play a dominant role in the ABC model established to explain how floral organ identity is determined in model dicotyledon species such as Arabidopsis thaliana and Antirrhinum majus. In the monocotyledons, although there appears to be a broad general conservation of ABC gene functions, the model itself needs to be adapted in some cases, notably for certain species which produce flowers with sepals and petals of similar appearance. For the moment, ABC genes remain unstudied in a number of key monocot clades, so only a partial picture is available for the Liliopsida as a whole. The aim of this article is to summarize data recently obtained for the African oil palm Elaeis guineensis, a member of the family Arecaceae (Arecales), and to discuss their significance with respect to knowledge gained from other Angiosperm groups, particularly within the monocotyledons. SCOPE: The essential details of reproductive development in oil palm are discussed and an overview is provided of the structural and functional characterization of MADS box genes likely to play a homeotic role in flower development in this species. CONCLUSIONS: The structural and functional data provide evidence for a general conservation of the generic 'ABC' model in oil palm, rather than the 'modified ABC model' proposed for some other monocot species which produce homochlamydeous flowers (i.e. with morphologically similar organs in both perianth whorls), such as members of the Liliales. Our oil palm data therefore follow a similar pattern to those obtained for other Commelinid species in the orders Commelinales and Poales. The significance of these findings is discussed.     

Ectopic expression of carpel-specific MADS box genes from lily and lisianthus causes similar homeotic conversion of sepal and petal in Arabidopsis

Two MADS box genes, Lily MADS Box Gene 2 (LMADS2) and Eustoma grandiflorum MADS Box Gene 1 (EgMADS1), with an extensive similarity to the petunia (Petunia hybrida) FLORAL BINDING PROTEIN 7/11 and Arabidopsis AGL11, were characterized from the lily (Lilium longiflorum) and lisianthus (Eustoma grandiflorum). The expression of LMADS2 and EgMADS1 mRNA was restricted to the carpel and was absent in the other flower organs or vegetative leaves. LMADS2 mRNA was detected mainly in ovules and weakly in style tissues of the carpel, whereas EgMADS1 mRNA was only expressed in the ovules. Transgenic Arabidopsis plants ectopically expressing LMADS2 or EgMADS1 showed similar novel phenotypes resembling 35S::AGAMOUS plants by significantly reducing plant size, flowering early, and losing inflorescence indeterminacy. Ectopic expression of these two genes also generated similar ap2-like flowers by inducing homeotic conversion of the sepals into carpel-like structures in which stigmatic papillae and ovules were observed. In addition, the petals were converted into stamen-like structures in the second whorl of 35S::LMADS2 and 35S::EgMADS1 transgenic Arabidopsis. Our data indicated that LMADS2 and EgMADS1 are putative D functional MADS box genes in lily and lisianthus with a function similar to C functional genes once ectopically expressed in Arabidopsis.     

Functional analysis of three lily (Lilium longiflorum) APETALA1-like MADS box genes in regulating floral transition and formation

Three cDNAs showing a high degree of homology to the SQUA subfamily of MADS box genes were isolated and characterized from the lily (Lilium longiflorum). Lily MADS Box Gene 5 (LMADS5) showed high sequence identity to oil palm (Elaeis guineensis) SQUAMOSA3 (EgSQUA3). LMADS6 is closely related to LMADS5 whereas LMADS7 is more related to DOMADS2, an orchid (Dendrobium) gene in the SQUA subfamily. The expression pattern for these three genes was similar and their RNAs were detected in vegetative stem and inflorescence meristem. LMADS5 and 6 were highly expressed in vegetative leaves and carpel, whereas LMADS7 expression was absent. Ectopic expression of LMADS5, 6 or 7 in transgenic Arabidopsis plants showed novel phenotypes by flowering early and producing terminal flowers. Homeotic conversions of sepals to carpelloid structures and of petal to stamen-like structures were also observed in 35S::LMADS5, 6 or 7 flowers. Ectopic expression of LMADS6 or LMADS7 was able to complement the ap1 flower defect in transgenic Arabidopsis ap1 mutant plants. These results strongly indicated that the function of these three lily genes was involved in flower formation as well as in floral induction. Furthermore, the ability of lily LMADS6 and 7 to complement the Arabidopsis ap1 mutant provided further evidence to show that the conserved motifs (paleoAP1 or euAP1) in the C-terminus of the SQUA/AP1 subfamily of MADS box genes is not strictly necessary for their function.     

Male and female flowers of the dioecious plant sorrel show different patterns of MADS box gene expression.

Male and female flowers of the dioecious plant sorrel (Rumex acetosa) each produce three whorls of developed floral organs: two similar whorls of three perianth segments and either six stamens (in the male) or a gynoecium consisting of a fertile carpel and two sterile carpels (in the female). In the developing male flower, there is no significant proliferation of cells in the center of the flower, in the position normally occupied by the carpels of a hermaphrodite plant. In the female flower, small stamen primordia are formed. To determine whether the organ differences are associated with differences in the expression of organ identity genes, cDNA clones representing the putative homologs of B and C function MADS box genes were isolated and used in an in situ hybridization analysis. The expression of RAD1 and RAD2 (two different DEFICIENS homologs) in males and females was confined to the stamen whorl; the lack of expression in the second, inner perianth whorl correlated with the sepaloid nature of the inner whorl of perianth segments. Expression of RAP1 (a PLENA homolog) occurred in the carpel and stamen whorls in very young flower primordia from both males and females. However, as soon as the inappropriate set of organs ceased to develop, RAP1 expression became undetectable in those organs. The absence of expression of RAP1 may be the cause of the arrest in organ development or may be a consequence.     

Expression of AODEF,a B-functional MADS-box gene,in stamens and inner tepals of the dioecious species Asparagus officinalis L.

Garden asparagus (Asparagus officinalis L.) is a dioecious species with male and female flowers on separate unisexual individuals. Since B- and C-functional MADS-box genes specify male and female reproductive organs, it is important to characterize these genes to clarify the mechanism of sex determination in monoecious and dioecious species. In this study, we isolated and characterized AODEF gene, a B-functional gene in the development of male and female flowers of A. officinalis. Southern hybridization identified a single copy of AODEF gene in asparagus genome. Northern blot analysis showed that this gene was specifically expressed in flower buds and not in vegetative tissues. In situ hybridization showed that during early hermaphrodite stages, AODEFgene was expressed in the inner tepal and stamen whorls (whorls 2 and 3, respectively), but not in the outer tepals (whorl 1), in both male and female flowers. In late unisexual developmental stages, the expression of AODEF gene was still detected in the inner tepals and stamens of male flowers, but the expression was reduced in whorls 2 and 3 of female flowers. Our results suggest that AODEF gene is probably not involved in tepal development in asparagus and that the expression of AODEF gene is probably controlled directly or indirectly by sex determination gene in the late developmental stages.     

Characterization of <Emphasis Type="Italic">HoMADS 1</Emphasis> and its induction by plant hormones during <Emphasis Type="Italic">in vitro</Emphasis> ovule development in <Emphasis Type="Italic">Hyacinthus orientalis</Emphasis> L.

To understand the molecular mechanism of ovule development, a MADS box gene,HoMADS 1, has been isolated from the ovule tissues of Hyacinthus. Sequence comparison showed that HoMADS 1 is highly homologous to both class C and D genes. Furthermore, phylogenetic analysis suggests that HoMADS 1 is most likely a class D MADS box gene. RNA hybridization revealed that HoMADS 1 was exclusively expressed in the ovules. Over-expressing HoMADS 1 in transgenic Arabidopsis plants produced ectopic carpelloid structures, including ovules, indicating that HoMADS 1 is involved in the determination of carpel and ovule identities. Interestingly, during in vitro flowering, no HoMADS 1 mRNA was detected in the floral tissues at high level hormones in the media. However, HoMADS 1 mRNA accumulated in the floral tissues when the regenerated flowers were transferred to the media containing low level hormones which could induce in vitro ovule formation. Our data suggest that the induction of HoMADS 1 by plant hormones may play important roles during ovule initiation and development in the regenerated flower. Whether HoMADS 1 expression is also regulated by cytokinin and auxin during ovule development in planta remains to be investigated.     

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

An AGAMOUS/SHATTERPROOF homologue (Vvmads1) was isolated from grapevine by differential display between berry and leaf mRNA. The predicted protein sequence of the full-length clone shows a high degree of homology to PLENA (77% identity) and to SHP1 and SHP2 (75% and 74% identity respectively), and is grouped with AGAMOUS/PLENA homologues when the conserved MADS and K domains are compared. Vvmads1 is expressed only in the later stages of flower development and throughout berry development, although expression is reduced after ripening commenced. When Vvmads1 was over-expressed in tobacco, the resulting plants display altered morphologies in the outer two floral whorls. In the most extreme cases, the inner whorls were surrounded by a carpelloid structure created by the modified sepals. Within these sepals were petals which had been split into sections and which were attached at the base of the flower by structures with the appearance of filaments. The results of this study suggest that Vvmads1 has a regulatory role in flower development before fertilisation and a role in fruit development after fertilisation.     

Expression of ASK1-like genes in arrested stamens of female Silene latifolia plants

Silene latifolia is a dioecious plant in which sex is determined by heteromorphic sex chromosomes. In female plants, stamen development is arrested before microspore mother cells are formed. In this study, we isolated four cDNAs (SlSKP1-1 to 4) encoding ASK1-like protein as expression markers to reveal when expression levels are reduced in arrested stamens of female flowers. Expression patterns of the SlSKP1 genes were analyzed by in-situ hybridization. We use the flower development classification of Grant et al. (in Plant J 6:471–480, 1994). SlSKP1 genes were highly expressed in primary parietal cells and primary sporogenous cells that develop into microspore mother cells in male flowers. Expression levels started to be reduced in the external stamens of the female flowers when stamen development was arrested at stage 7. Although microspore mother cells could not be developed in female flowers and SlSKP1 expression may be unnecessary in arrested stamens, SlSKP1 genes were still expressed in sporogenous cells of degenerated stamens at stage 8. Parietal cells stopped differentiating earlier than sporogenous cells in arrested stamens. These results suggest that not all types of cell are arrested simultaneously at a particular stage of stamen development during stamen suppression of S. latifolia.     

<Emphasis Type="Italic">AOM3</Emphasis> and<Emphasis Type="Italic"> AOM4</Emphasis>: two MADS box genes expressed in reproductive structures of<Emphasis Type="Italic"> Asparagus officinalis</Emphasis>

The MADS box genes participate in different steps of vegetative and reproductive plant development, including the most important phases of the reproductive process. Here we describe the isolation and characterisation of two Asparagus officinalis MADS box genes, AOM3 and AOM4. The deduced AOM3 protein shows the highest degree of similarity with ZAG3 and ZAG5 of maize, OsMADS6 of rice and AGL6 of Arabidopsis thaliana. The deduced AOM4 protein shows the highest degree of similarity with AOM1 of asparagus, the SEP proteins of Arabidopsis and the rice proteins OsMADS8, OsMADS45 and OsMADS7. The high level of identity between AOM1 and AOM4 made impossible the preparation of probes specific for one single gene, so the hybridisation signal previously described for AOM1 is probably due to the expression of both genes. The expression profile of AOM3 and AOM1/AOM4 during flower development is identical, and similar to that of the SEP genes. Asparagus genes, however, are expressed not only in flower organs, but also in the different meristem present on the apical region of the shoot during the flowering season: the apical meristem and the three lateral meristems emerging from the leaf axillary region that will give rise to flowers and lateral inflorescences during flowering season, and to phylloclades and branches during the subsequent vegetative phase. The expression of AOM3 and AOM1/AOM4 in these meristems appears to be correlated with the reproductive function of the apex as the hybridisation signal disappears when the apex switches to vegetative function.     

GRCD1, an AGL2-like MADS box gene, participates in the C function during stamen development in Gerbera hybrida

Despite the differences in flower form, the underlying mechanism in determining the identity of floral organs is largely conserved among different angiosperms, but the details of how the functions of A, B, and C are specified varies greatly among plant species. Here, we report functional analysis of a Gerbera MADS box gene, GRCD1, which is orthologous to AGL2-like MADS box genes. Members of this group of genes are being reported in various species in growing numbers, but their functions remained largely unsettled. GRCD1 expression is detected in all four whorls, but the strongest signal is seen in the developing stamen and carpel. Downregulating GRCD1 expression by antisense transformation revealed that lack of GRCD1 caused homeotic changes in one whorl only: sterile staminodes, which normally develop in whorl 3 of marginal female florets, were changed into petals. This indicates that the GRCD1 gene product is active in determining stamen identity. Transgenic downregulation of GRCD1 causes a homeotic change similar to that in the downregulation of the Gerbera C function genes GAGA1 and GAGA2, but one that is limited to whorl 3. Downregulation of GRCD1 expression does not reduce expression of GAGA1 or GAGA2, or vice versa; and in yeast two-hybrid analysis, GRCD1 is able to interact with GAGA1 and GAGA2. We propose that a heterodimer between the GRCD1 and GAGA1/2 gene products is needed to fulfill the C function in whorl 3 in Gerbera.     

A novel role of BELL1-like homeobox genes, PENNYWISE and POUND-FOOLISH, in floral patterning

Flowers are determinate shoots comprised of perianth and reproductive organs displayed in a whorled phyllotactic pattern. Floral organ identity genes display region-specific expression patterns in the developing flower. In Arabidopsis, floral organ identity genes are activated by LEAFY (LFY), which functions with region-specific co-regulators, UNUSUAL FLORAL ORGANS (UFO) and WUSCHEL (WUS), to up-regulate homeotic genes in specific whorls of the flower. PENNYWISE (PNY) and POUND-FOOLISH (PNF) are redundant functioning BELL1-like homeodomain proteins that are expressed in shoot and floral meristems. During flower development, PNY functions with a co-repressor complex to down-regulate the homeotic gene, AGAMOUS (AG), in the outer whorls of the flower. However, the function of PNY as well as PNF in regulating floral organ identity in the central whorls of the flower is not known. In this report, we show that combining mutations in PNY and PNF enhance the floral patterning phenotypes of weak and strong alleles of lfy, indicating that these BELL1-like homeodomain proteins play a role in the specification of petals, stamens and carpels during flower development. Expression studies show that PNY and PNF positively regulate the homeotic genes, APETALA3 and AG, in the inner whorls of the flower. Moreover, PNY and PNF function in parallel with LFY, UFO and WUS to regulate homeotic gene expression. Since PNY and PNF interact with the KNOTTED1-like homeodomain proteins, SHOOTMERISTEMLESS (STM) and KNOTTED-LIKE from ARABIDOPSIS THALIANA2 (KNAT2) that regulate floral development, we propose that PNY/PNF-STM and PNY/PNF-KNAT2 complexes function in the inner whorls to regulate flower patterning events.