B-class MADS-box Genes Research Articles

Isolation and Characterization of Three MADS-box Genes from Alpinia hainanensis (Zingiberaceae)

Three complementary DNA clones, AhMADS5, AhMADS6, and AhMADS8 were isolated from the young inflorescences of Alpinia hainanensis. Sequence comparisons and phylogenetic analyses indicated that AhMADS5 and AhMADS8 are highly homologous to AP3 and PI family, respectively, and AhMADS6 is an AG homologue. In situ hybridization analyses showed that AhMADS5 and AhMADS8 genes are expressed in the second and third whorls, whereas, the AhMADS6 signal is present in the third and fourth whorls. The expression patterns of AhMADS5, AhMADS8, and AhMADS6 genes are very similar to those of AP3-, PI-, and AG-like genes in other plant species, respectively. These results suggest that while AhMADS5 and AhMADS8 belong to the B-class MADS-box gene family, AhMADS6 belongs to a different category, the C-class MADS-box gene family. Present studies strongly support the hypothesis that the labellum of A. hainanensis originated from stamens.

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.

Functional Diversification of B MADS-Box Homeotic Regulators of Flower Development: Adaptive Evolution in Protein–Protein Interaction Domains after Major Gene Duplication Events

B-class MADS-box genes have been shown to be the key regulators of petal and stamen specification in several eudicot model species such as Arabidopsis thaliana, Antirrhinum majus, and Petunia hybrida. Orthologs of these genes have been found across angiosperms and gymnosperms, and it is thought that the basic regulatory function of B proteins is conserved in seed plant lineages. The evolution of B genes is characterized by numerous duplications that might represent key elements fostering the functional diversification of duplicates with a deep impact on their role in the evolution of the floral developmental program. To evaluate this, we performed a rigorous statistical analysis with B gene sequences. Using maximum likelihood and Bayesian methods, we estimated molecular substitution rates and determined the selective regimes operating at each residue of B proteins. We implemented tests that rely on phylogenetic hypotheses and codon substitution models to detect significant differences in substitution rates (DSRs) and sites under positive adaptive selection (PS) in specific lineages before and after duplication events. With these methods, we identified several protein residues fixed by PS shortly after the origin of PISTILLATA-like and APETALA3-like lineages in angiosperms and shortly after the origin of the euAP3-like lineage in core eudicots, the 2 main B gene duplications. The residues inferred to have been fixed by positive selection lie mostly within the K domain of the protein, which is key to promote heterodimerization. Additionally, we used a likelihood method that accommodates DSRs among lineages to estimate duplication dates for AP3-PI and euAP3-TM6, calibrating with data from the fossil record. The dates obtained are consistent with angiosperm origins and diversification of core eudicots. Our results strongly suggest that novel multimer formation with other MADS proteins could have been crucial for the functional divergence of B MADS-box genes. We thus propose a mechanism of functional diversification and persistence of gene duplicates by the appearance of novel multimerization capabilities after duplications. Multimer formation in different combinations of regulatory proteins can be a mechanistic basis for the origin of novel regulatory functions and a gene regulatory mechanism for the appearance of morphological innovations.

Expression of a DEFICIENS-like gene correlates with the differentiation between sepal and petal in the orchid, Habenaria radiata (Orchidaceae)

The B-class MADS-box genes play an important role in controlling petal development in dicots, and orthologs have been isolated from many plants. The Orchidaceae includes many species with petaloid sepals in whorl 1, while there are also several genera with two whorls of perianth, petals and greenish sepals. Since the morphological differentiation between sepals and petals is observed in different orchid groups such as Cypripedium and Habenaria, this characteristic would be evolved independently in Orchidaceae. Habenaria radiata, which is an ornamental plant, has green sepals and white petals in its perianth. Among the cultivars of H. radiata, one floral homeotic mutant has undergone an alteration from sepals to petaloid organs in whorl 1. In order to investigate the molecular mechanism of the morphological differentiation between sepals and petals in Orchidaceae, it is very interesting to analyze the B-class gene expression in wild type and petaloid-sepal mutant of H. radiata. In this study, we isolated and characterized three B-class genes, HrGLO1, HrGLO2 and HrDEF, from this species. Northern hybridization and RT-PCR analyses revealed that HrGLO1 and HrGLO2 were expressed in sepals, petals and columns, whereas HrDEF expression was detected only in the petals and columns, but not in the first whorl, sepals. In the petaloid-sepal mutant, all three B-class genes were expressed in petaloid-sepals, petals and columns. These results suggest that the distinctive expression of HrDEF plays a role in differentiation between sepal and petal in H. radiata.

Phylogeny and divergence of basal angiosperms inferred from APETALA3- and PISTILLATA-like MADS-box genes.

The B-class MADS-box genes composed of APETALA3 ( AP3) and PISTILLATA ( PI) lineages play an important role in petal and stamen identity in previously studied flowering plants. We investigated the diversification of the AP3-like and PI-like MADS-box genes of eight species in five basal angiosperm families: Amborella trichopoda (Amborellaceae); Brasenia schreberi and Cabomba caroliniana (Cabombaceae); Euryale ferox, Nuphar japonicum, and Nymphaea tetragona (Nymphaeaceae); Illicium anisatum (Illiciaceae); and Kadsura japonica (Schisandraceae). Sequence analysis showed that a four amino acid deletion in the K domain, which was found in all previously reported angiosperm PI genes, exists in a PI homologue of Schisandraceae, but not in six PI homologues of the Amborellaceae, Cabombaceae, and Nymphaeaceae, suggesting that the Amborellaceae, Cabombaceae, and Nymphaeaceae are basalmost lineages in angiosperms. The results of molecular phylogenetic analyses were not inconsistent with this hypothesis. The AP3 and PI homologues from Amborella share a sequence of five amino acids in the 5' region of exon 7. Using the linearized tree and likelihood methods, the divergence time between the AP3 and PI lineages was estimated as somewhere between immediately after to several tens of millions of years after the split between angiosperms and extant gymnosperms. Estimates of the age of the most recent common ancestor of all extant angiosperms range from approximately 140-210 Ma, depending on the trees used and assumptions made.

Genes from the APETALA3 and PISTILLATA lineages are expressed in developing vascular bundles of the tuberous rhizome, flowering stem and flower Primordia of Eranthis hyemalis.

In Arabidopsis thaliana expression of the B-class MADS-box genes APETALA3 (AP3) and PISTILLATA (PI) is confined to petals and stamens but in other plant species these genes are also transcribed in non-flower tissues; in Solanum tuberosum they are transcribed specifically in vascular bundles leading to petals and stamens. Transcription analysis of B-class genes in Eranthis hyemalis using reverse transcribed in situ PCR revealed that both AP3 and PI are expressed in developing vascular bundles in the tuberous rhizome, flowering stem and floral primordia. In addition, AP3 and PI transcripts are also found in stems and leaves. These results suggest a more complex role of B-class genes in Eranthis and possible involvement in the development of vascular tissue.