Maria Manuela Ribeiro Costa

Evolution of regulatory interactions controlling floral asymmetry

A key challenge in evolutionary biology is to understand how new morphologies can arise through changes in gene regulatory networks. For example, floral asymmetry is thought to have evolved many times independently from a radially symmetrical ancestral condition, yet the molecular changes underlying this innovation are unknown. Here, we address this problem by investigating the action of a key regulator of floral asymmetry, CYCLOIDEA (CYC), in species with asymmetric and symmetric flowers. We show that CYC encodes a DNA-binding protein that recognises sites in a downstream target gene RADIALIS (RAD) in Antirrhinum. The interaction between CYC and RAD can be reconstituted in Arabidopsis, which has radially symmetrical flowers. Overexpression of CYC in Arabidopsis modifies petal and leaf development, through changes in cell proliferation and expansion at various stages of development. This indicates that developmental target processes are influenced by CYC in Arabidopsis, similar to the situation in Antirrhinum. However, endogenous RAD-like genes are not activated by CYC in Arabidopsis, suggesting that co-option of RAD may have occurred specifically in the Antirrhinum lineage. Taken together, our results indicate that floral asymmetry may have arisen through evolutionary tinkering with the strengths and pattern of connections at several points in a gene regulatory network.

A subcellular tug of war involving three MYB-like proteins underlies a molecular antagonism in Antirrhinum flower asymmetry

The establishment of meristematic domains with different transcriptional activity is essential for many developmental processes. The asymmetry of the Antirrhinum majus flower is established by transcription factors with an asymmetric pattern of activity. To understand how this asymmetrical pattern is established, we studied the molecular mechanism through which the dorsal MYB protein RADIALIS (RAD) restricts the activity of the MYB transcription factor DIVARICATA (DIV) to the ventral region of the flower meristem. We show that RAD competes with DIV for binding with other MYB-like proteins, termed DRIF1 and DRIF2 (DIV- and-RAD-interacting-factors). DRIF1 and DIV interact to form a protein complex that binds to the DIV-DNA consensus region, suggesting that the DRIFs act as co-regulators of DIV transcriptional activity. In the presence of RAD, the interaction between DRIF1 and DIV bound to DNA is disrupted. Moreover, the DRIFs are sequestered in the cytoplasm by RAD, thus, preventing or reducing the formation of DRIF-DIV heterodimers in the nuclei. Our results suggest that in the dorsal region of the Antirrhinum flower meristem the dorsal protein RAD antagonises the activity of the ventral identity protein DIV in a subcellular competition for a DRIF protein promoting the establishment of the asymmetric pattern of gene activity in the Antirrhinum flower.

Crystal structure of the MYB domain of the RAD transcription factor from Antirrhinum majus

Introduction. The RAD gene of Antirrhinum majus encodes a single-repeat MYB transcription factor that is implicated in the control of floral asymmetry. Herein we present the crystal structure of the MYB domain of the RAD protein determined using the anomalous signal of iodide and refined to a resolution of 1.9 Å. This constitutes the first X-ray structure of a single-repeat MYB protein. The structure conforms to the three-helix bundle fold of the canonical MYB domain, although the third helix is notably longer. Several developmental genes have been identified in Antirrhinum majus (garden snapdragon) that are implicated in the control of asymmetric floral architecture. Sequence analysis suggests that two of these genes, RADIALIS (RAD) and DIVARICATA (DIV), encode putative MYB transcription factors, this being consistent with their proposed signaling roles. MYB transcription factors are ubiquitous in eukaryotes, most notably in plants, and contain one or more copies of the so-called MYB DNAbinding domain. MYB domains are approximately 50 residues in length, highly variable in sequence, but are often characterized by three regularly spaced Trp residues. MYB proteins can be split into three subfamilies depending on whether they have one, two or three imperfect repeats of the MYB domain. Most MYB proteins from vertebrates consist of three imperfect repeats (R1, R2, and R3), but plants contain large numbers of two repeat (R2 and R3) MYBs. In some cases, however, only one MYB-like domain is present. The DIV protein contains two MYB-like domains, whilst the RAD protein contains only one. The latter is closely related in sequence to the N-terminal MYB domain of DIV, suggesting that RAD may have evolved through a C-terminal deletion of a DIV-like ancestral protein. RAD promotes dorsal identity of the flower, whereas DIV promotes ventral identity. It is thought that RAD is activated in the dorsal region of the developing flower, which then inhibits the activity of DIV, by either competitively binding to DIV’s target DNA sequences or to proteins that interact with DIV. So far, DNA binding functions for both RAD and DIV have yet to be demonstrated experimentally, and the possibility exists that they only bind to DNA as complexes with other proteins. To date, there are relatively few structures of MYB proteins in the Protein Data Bank (PDB), despite their prevalence in nature, possibly reflecting inherent problems in over expressing and crystallizing this family of proteins. Moreover, the majority of known structures have been determined using NMR rather than X-ray crystallography. In this article, we report the crystal structure determination of an 8 kDa fragment of the 10.8 kDa RAD protein from Antirrhinum majus that encompasses the entire MYB domain and was determined using the anomalous signal of iodide from X-ray data collected in-house.

A comprehensive assessment of the transcriptome of cork oak (Quercus suber) through EST sequencing

BackgroundCork oak (Quercus suber) is one of the rare trees with the ability to produce cork, a material widely used to make wine bottle stoppers, flooring and insulation materials, among many other uses. The molecular mechanisms of cork formation are still poorly understood, in great part due to the difficulty in studying a species with a long life-cycle and for which there is scarce molecular/genomic information. Cork oak forests are of great ecological importance and represent a major economic and social resource in Southern Europe and Northern Africa. However, global warming is threatening the cork oak forests by imposing thermal, hydric and many types of novel biotic stresses. Despite the economic and social value of the Q. suber species, few genomic resources have been developed, useful for biotechnological applications and improved forest management.ResultsWe generated in excess of 7 million sequence reads, by pyrosequencing 21 normalized cDNA libraries derived from multiple Q. suber tissues and organs, developmental stages and physiological conditions. We deployed a stringent sequence processing and assembly pipeline that resulted in the identification of ~159,000 unigenes. These were annotated according to their similarity to known plant genes, to known Interpro domains, GO classes and E.C. numbers. The phylogenetic extent of this ESTs set was investigated, and we found that cork oak revealed a significant new gene space that is not covered by other model species or EST sequencing projects. The raw data, as well as the full annotated assembly, are now available to the community in a dedicated web portal at http://www.corkoakdb.org.ConclusionsThis genomic resource represents the first trancriptome study in a cork producing species. It can be explored to develop new tools and approaches to understand stress responses and developmental processes in forest trees, as well as the molecular cascades underlying cork differentiation and disease response.

Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber.

Monoecious species provide a comprehensive system to study the developmental programs underlying the establishment of female and male organs in unisexual flowers. However, molecular resources for most monoecious non-model species are limited, hampering our ability to study the molecular mechanisms involved in flower development of these species. The objective of this study was to identify differentially expressed genes during the development of male and female flowers of the monoecious species Quercus suber, an economically important Mediterranean tree. Total RNA was extracted from different developmental stages of Q. suber flowers. Non-normalized cDNA libraries of male and female flowers were generated using 454 pyrosequencing technology producing a total of 962,172 high-quality reads with an average length of 264 nucleotides. The assembly of the reads resulted in 14,488 contigs for female libraries and 10,438 contigs for male libraries. Comparative analysis of the transcriptomes revealed genes differentially expressed in early and late stages of development of female and male flowers, some of which have been shown to be involved in pollen development, in ovule formation and in flower development of other species with a monoecious, dioecious, or hermaphroditic sexual system. Moreover, we found differentially expressed genes that have not yet been characterized and others that have not been previously shown to be implicated in flower development. This transcriptomic analysis constitutes a major step toward the characterization of the molecular mechanisms involved in flower development in a monoecious tree with a potential contribution toward the knowledge of conserved developmental mechanisms in other species.

Flower development and sex specification in wild grapevine

BackgroundWild plants of Vitis closely related to the cultivated grapevine (V. v. vinifera) are believed to have been first domesticated 10,000 years BC around the Caspian Sea. V. v. vinifera is hermaphrodite whereas V. v. sylvestris is a dioecious species. Male flowers show a reduced pistil without style or stigma and female flowers present reflexed stamens with infertile pollen. V. vinifera produce perfect flowers with all functional structures. The mechanism for flower sex determination and specification in grapevine is still unknown.ResultsTo understand which genes are involved during the establishment of male, female and complete flowers, we analysed and compared the transcription profiles of four developmental stages of the three genders. We showed that sex determination is a late event during flower development and that the expression of genes from the ABCDE model is not directly correlated with the establishment of sexual dimorphism. We propose a temporal comprehensive model in which two mutations in two linked genes could be players in sex determination and indirectly establish the Vitis domestication process. Additionally, we also found clusters of genes differentially expressed between genders and between developmental stages that suggest a role involved in sex differentiation. Also, the detection of differentially transcribed regions that extended existing gene models (intergenic regions) between sexes suggests that they may account for some of the variation between the subspecies.ConclusionsThere is no evidence of differences of expression levels in genes from the ABCDE model that could explain the shift from hermaphroditism to dioecy. We propose that sex specification occurs after floral organ identity has been established and therefore, sex determination genes might be having an effect downstream of the ABCDE model genes.For the first time a full transcriptomic analysis was performed in different flower developmental stages in the same individual. Our experimental approach enabled us to create a comprehensive catalogue of transcribed genes across developmental stages and genders that will contribute for future work in sex determination in seed plants.

Evaluation of the presence of arabinogalactan proteins and pectins during Quercus suber male gametogenesis

BACKGROUND AND AIMS Quercus suber (cork oak) is a dominant tree of the Fagaceae in forests of the south-west Iberian Peninsula. It is monoecious with a long progamic phase that provides a comprehensive system for comparative studies in development and sexual reproduction. In this study the distribution of arabinogalactan protein (AGPs) and pectin epitopes in anthers of Q. suber was assessed to map these hydroxyproline-rich glycoproteins and the galacturonate-rich acidic polysaccharides during pollen development. Methods Immunolocalization in male flowers was performed with a set of monoclonal antibodies directed against the carbohydrate moiety that recognizes AGPs and pectins. To identify AGP genes involved in cork oak male flower development, a search was conducted for annotated AGP genes in the available transcriptome data of the Cork Oak EST Consortium database (www.corkoakdb.org). KEY RESULTS Ubiquitous labelling in all cell types was obtained with anti-homogalacturan antibodies for methyl-esterified pectins. In contrast, the antibody that labelled non-methyl-esterified homogalacturans had a preferential presence in microsporocyte cells walls at the beginning of pollen development. Intense labelling was obtained with anti-AGP antibodies both in the tapetum and in the intine wall near the pollen apertures and later in the generative cell wall and vegetative cell. Evaluation of the putative AGPs highly expressed in the male gametophyte was achieved by quantitative RT-PCR analysis in male and female cork oak flowers. CONCLUSIONS Four putative AGP genes were identified that are preferentially expressed in the male flower compared with the female flower. The putative Arabidopsis thaliana orthologues of these genes are associated with preferential expression in pollen, suggesting that the AGPs probably play a significant role in cork oak reproduction.

Arabinogalactan proteins and pectin distribution during female gametogenesis in Quercus suber L.

BACKGROUND AND AIMS Quercus suber L. (cork oak) is one of the most important monoecious tree species in semi-arid regions of Southern Europe, with a high ecological value and economic potential. However, as a result of its long reproductive cycle, complex reproductive biology and recalcitrant seeds, conventional breeding is demanding. In its complex reproductive biology, little is known about the most important changes that occur during female gametogenesis. Arabinogalactan proteins (AGPs) and pectins are the main components of plant cell walls and have been reported to perform common functions in cell differentiation and organogenesis of reproductive plant structures. AGPs have been shown to serve as important molecules in several steps of the reproductive process in plants, working as signalling molecules, associated with the sporophyte-gametophyte transition, and pectins have been implicated in pollen-pistil interactions before double fertilization. In this study, the distribution of AGP and pectin epitopes was assessed during female gametogenesis. METHODS Immunofluorescence labelling of female flower cells was performed with a set of monoclonal antibodies (mAbs) directed to the carbohydrate moiety of AGPs (JIM8 and JIM13) and pectic homogalacturonans (HGs) (mAbs JIM5 and JIM7). KEY RESULTS The selective labelling obtained with AGP and pectin mAbs JIM8, JIM13, JIM5 and JIM7 during Q. suber female gametogenesis shows that AGPs and pectic HG can work as markers for mapping gametophytic cell differentiation in this species. Pectic HG showed different distribution patterns, depending on their levels of methyl esterification. Methyl-esterified HGs showed a uniform distribution in the overall female flower cells before fertilization and a more specific pattern after fertilization. A low methyl-ester pectin distribution pattern during the different developmental stages appears to be related to the pathway that pollen tubes follow to reach the embryo sac. AGPs showed a more sparse distribution in early stages of development, but specific labelling is shown in the synergids and their filiform apparatus. CONCLUSIONS The labelling obtained with anti-AGP and anti-pectin mAbs in Q. suber female flower cells showed a dynamic distribution of AGPs and pectic HGs, which may render these molecules useful molecular markers during female gametogenesis. Changes occurring during development will be determined in order to help describe cork oak ovule structural properties before and after fertilization, providing new insight to better understand Q. suber female gametogenesis.

The Quest for Molecular Regulation Underlying Unisexual Flower Development

The understanding of the molecular mechanisms responsible for the making of a unisexual flower has been a long-standing quest in plant biology. Plants with male and female flowers can be divided mainly into two categories: dioecious and monoecious, and both sexual systems co-exist in nature in ca of 10% of the angiosperms. The establishment of male and female traits has been extensively described in a hermaphroditic flower and requires the interplay of networks, directly and indirectly related to the floral organ identity genes including hormonal regulators, transcription factors, microRNAs, and chromatin-modifying proteins. Recent transcriptomic studies have been uncovering the molecular processes underlying the establishment of unisexual flowers and there are many parallelisms between monoecious, dioecious, and hermaphroditic individuals. Here, we review the paper entitled “Comparative transcriptomic analysis of male and female flowers of monoecious Quercus suber” published in 2014 in the Frontiers of Plant Science (volume 5 |Article 599) and discussed it in the context of recent studies with other dioecious and monoecious plants that utilized high-throughput platforms to obtain transcriptomic profiles of male and female unisexual flowers. In some unisexual flowers, the developmental programs that control organ initiation fail and male or female organs do not form, whereas in other species, organ initiation and development occur but they abort or arrest during different species-specific stages of differentiation. Therefore, a direct comparison of the pathways responsible for the establishment of unisexual flowers in different species are likely to reveal conserved modules of gene regulatory hubs involved in stamen or carpel development, as well as differences that reflect the different stages of development in which male and/or female organ arrest or loss-of-function occurs.

Deep analysis of wild Vitis flower transcriptome reveals unexplored genome regions associated with sex specification

Key messageRNA-seq of Vitis during early stages of bud development, in male, female and hermaphrodite flowers, identified new loci outside of annotated gene models, suggesting their involvement in sex establishment.AbstractThe molecular mechanisms responsible for flower sex specification remain unclear for most plant species. In the case of V. vinifera ssp. vinifera, it is not fully understood what determines hermaphroditism in the domesticated subspecies and male or female flowers in wild dioecious relatives (Vitis vinifera ssp. sylvestris). Here, we describe a de novo assembly of the transcriptome of three flower developmental stages from the three Vitis vinifera flower types. The validation of de novo assembly showed a correlation of 0.825. The main goals of this work were the identification of V. v. sylvestris exclusive transcripts and the characterization of differential gene expression during flower development. RNA from several flower developmental stages was used previously to generate Illumina sequence reads. Through a sequential de novo assembly strategy one comprehensive transcriptome comprising 95,516 non-redundant transcripts was assembled. From this dataset 81,064 transcripts were annotated to V. v. vinifera reference transcriptome and 11,084 were annotated against V. v. vinifera reference genome. Moreover, we found 3368 transcripts that could not be mapped to Vitis reference genome. From all the non-redundant transcripts that were assembled, bioinformatics analysis identified 133 specific of V. v. sylvestris and 516 transcripts differentially expressed among the three flower types. The detection of transcription from areas of the genome not currently annotated suggests active transcription of previously unannotated genomic loci during early stages of bud development.