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Title: Diversification of MADS-box genes in flowering plants.
Other Titles: Diversificatie van MADS-box genen in bloemplanten.
Authors: Vekemans, Dries
Issue Date: 24-Sep-2012
Abstract: People are fascinated by the bewildering floral diversity displayed among flowering plants. Yet, the developmental mechanisms and evolutionary events that have created this diversity still remain largely unidentified. Since members of the MIKCc-type MADS-box transcription factor family are generally accepted to play prominent roles in flower development, the central theme of this thesis is dedicated to the study of these genes. In particular, we investigate how MIKCc-genes are involved in the origin and development of diverse floral morphologies, thereby contributing to floral diversity and, in general, angiosperm biodiversity. In chapter two and three, we focus on ‘transference of function’, an important evolutionary mechanism shaping floral diversity. More specifically, in Davidia involucrata and Impatiens walleriana transference of function is displayed by the transfer of petaloidy towards respectively extrafloral bracts and a nectariferous, spurred sepal. In both cases, we question whether and how transference of function might be associated with the remodelling of the floral organ identity program.Concerning Davidia, two creamy white bracts subtend a remarkable reproductive structure, of which the exact nature has been controversial in the past. While this structure resembles a single flower, our detailed ontogeny illustrates it is in fact an inflorescence comprising perianthless male and bisexual flowers, though the latter may be sometimes absent. Despite their high level of reduction, the expression patterns of floral organ identity genes in both flower types are consistent with those observed in model plants, as indicated by qRT-PCR and in situ hybridization experiments. The bracts, however, express a subset of genes that could rather be expected to establish stamen identity than petal identity early in development, but this expression is not maintained at later stages. We also performed yeast-two-hybrid assays that are in close agreement with data acquired for model plant species. Together, our data suggest that the transfer of petaloidy towards the bracts in Davidia was associated in evolution mainly with spatial changes in expression patterns, rather than with changes in protein interaction specificity. In the case of Impatiens, a different mechanism, compared to Davidia, seems to be responsible for the observed heterotopic petaloidy. While both semi-quantitative and quantitative RT-PCR did not detect expression of any of the genes found in Davidia’s bracts among several other floral organ identity genes, we show that SEP3 is heterotopically expressed in the nectariferous, spurred sepal, suggesting a possible correlation. Somewhat unexpectedly, these same experiments reveal that CRC, proposed to play a key role in the nectaries of core eudicots, does not seem to fulfil this role in Impatiens, as both extrafloral and floral nectaries lack CRC expression.To also functionally characterize genes putatively involved in the regulation of the petaloid sepal, we set up a technique called Virus Induced Gene Silencing (VIGS).After several optimization rounds, we demonstrated that this viral based gene silencing method is capable of eliciting an IwPDS silenced phenotype in Impatiens leaves. However, subsequent attempts at silencing floral developmental genes were not successful and failed at exhibiting floral phenotypes, suggesting that at this point, TRV-based VIGS is not sufficient to assay floral gene function in Impatiens walleriana.Following these two chapters, we deal with an ancient polyploidy event, called gamma, in chapter four. In general, these events are considered to have contributed to the origin of morphological novelties and diversification of some of the largest groups in the plant kingdom. In order to evaluate the evolutionary impact of such a whole genome duplication, it is of crucial importance to accurately place this event both in absolute time and relative to other speciation events in the phylogeny. In this study, detailed phylogenies of subfamilies of MADS-box genes suggest that the gamma triplication has occurred before the divergence of Gunnerales but after the divergence of Buxales and Trochodendrales. Large-scale phylogenetic and KS-based approaches on the inflorescence transcriptomes of Gunnera manicata (Gunnerales) and Pachysandra terminalis (Buxales) provide further support for this placement, enabling us to position the gamma triplication in the stem lineage of the core eudicots. This triplication likely initiated the functional diversification of key regulators of reproductive development in the core eudicots, comprising 75% of flowering plants. While it is possible that the gamma event triggered early core eudicot diversification, our dating estimates suggest that the event occurred early in the stem-lineage, well before the rapid speciation of the earliest core eudicot lineages. The evolutionary significance of this paleopolyploidy event may thus rather lie in establishing a species lineage that was resilient to extinction but with the genomic potential for later diversification.In a fifth chapter, we continue to assess the impact of duplication events on the increase in morphological complexity. We use a combination of phylogenetic and collinearity analyses to reconstruct the evolutionary history of the AGL9-lineage, known to be essential for flower development. In particular, we try to position a previously unplaced duplication that seems to have been involved in the acquisition of novel floral morphologies in the Asteraceae. By conducting targeted cloning efforts and BLAST searches in public databases we expanded our original dataset with 86 sequences, of which 39 are newly isolated. This enabled us to trace back the AGL9-duplication to the base of the euasteridsII, suggesting that this event might have contributed to novel floral morphologies in much more taxonomic lineages than previously proposed.
Publication status: published
KU Leuven publication type: TH
Appears in Collections:Ecology, Evolution and Biodiversity Conservation Section
Plant Systematics and Ecology Section - miscellaneous
Molecular Physiology of Plants and Micro-organisms Section - miscellaneous

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