Plant Genomics
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Our group is interested in the mechanisms underlying sexual reproduction and early embryogenesis with a particular focus on the role of the male gametes. Contrary to previous assumptions recent studies have shown that male gametes both in the plant and animal kingdom carry complex sets of RNA molecules, including not only mRNAs but also small RNAs. We are particularly interested in the role of these two RNA classes before, during and after double fertilization as it occurs in higher plants. Using Arabidopsis thaliana as our primary experimental model we are addressing specific questions like: (1) What are the functions of small RNA and DNA methylation pathways in sperm cells? (2) Do sperm cell derived RNAs play a role after fertilization? (3) Do conserved core sets of genetic modules underlie common characteristics of male gametes across kingdoms?. In a second research line we are analyzing the symbiotic transcriptome of the model legume Medicago truncatula, both for nodule and mycorrhizal symbiosis, leading to the creation of a gene expression database that will allow the in-depth analysis of the symbiontic genetic programme.
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Funding
Fundação para a Ciência e a Tecnologia
(FCT) Research Grant (PTDC/AGR-GPL/103778/2008)
Collaborators
Rob Martienssen, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY, USA
Keith Slotkin, Ohio State University, Columbus, OH, USA
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- The nodulin VfENOD18 is an ATP-binding protein in infected cells of Vicia faba L. nodulesPublication . Becker, J.D.; Moreira, L.M.; Kapp, D.; Frosch S.C.; Pühler A.; Perlick, A.M.Recently we described the novel nodulin gene VfENOD18, whose corresponding transcripts were restricted to the nitrogen-fixing zone III of broad bean root nodules. To characterize VfENOD18 on the protein level, polyclonal antibodies were generated using the purified recombinant VfENOD18 protein produced in Escherichia coli by employing the pMAL-c expression system. These antibodies recognized immunoreactive proteins isolated from indeterminate nodules of different leguminous plants, but also from non-symbiotic tissues of Glycine max and from tissues of Arabidopsis thaliana and Zea mays. Using immunogold labelling the nodulin VfENOD18 was localized to the cytoplasm of infected cells in the nitrogen-fixing zone of broad bean nodules. Due to the homology of the VfENOD18 sequence to that of the ATP-binding protein MJ0577 from the hyperthermophile Methanococcus jannaschii the recombinant VfENOD18 protein was tested for ATP-binding. Using the biotin photoaffinity ATP analogue 8N3ATP[gamma]biotin it could be demonstrated that VfENOD18 is an ATP-binding protein. PCR experiments revealed that the amino acid sequences of the putative C-terminal ATP-binding sites of the VfENOD 18 homologues from Lens culinaris, Vicia hirsuta, Vicia sativa and Vicia villosa were conserved. We propose that VfENOD18 is a member of a novel family of ATP-binding proteins in plants.
- Transcriptional profiling of arabidopsis tissues reveals the Unique Characteristics of the pollen transcriptomePublication . Becker, J.D.; Boavida, L.C.; Carneiro, J.; Haury, M.; Feijó, J.A.Pollen tubes are a good model for the study of cell growth and morphogenesis because of their extreme elongation without cell division. Yet, knowledge about the genetic basis of pollen germination and tube growth is still lagging behind advances in pollen physiology and biochemistry. In an effort to reduce this gap, we have developed a new method to obtain highly purified, hydrated pollen grains of Arabidopsis through flowcytometric sorting, and we used GeneChips (Affymetrix, Santa Clara, CA; representing approximately 8,200 genes) to compare the transcriptional profile of sorted pollen with those of four vegetative tissues (seedlings, leaves, roots, and siliques). We present a new graphical tool allowing genomic scale visualization of the unique transcriptional profile of pollen. The 1,584 genes expressed in pollen showed a 90% overlap with genes expressed in these vegetative tissues, whereas one-third of the genes constitutively expressed in the vegetative tissues were not expressed in pollen. Among the 469 genes enriched in pollen, 162 were selectively expressed, and most of these had not been associated previously with pollen. Their functional classification reveals several new candidate genes, mainly in the categories of signal transduction and cell wall biosynthesis and regulation. Thus, the results presented improve our knowledge of the molecular mechanisms underlying pollen germination and tube growth and provide new directions for deciphering their genetic basis. Because pollen expresses about one-third of the number of genes expressed on average in other organs, it may constitute an ideal system to study fundamental mechanisms of cell biology and, by omission, of cell division.
- Signalling by tipsPublication . Feijó, J.A.; Costa, S.S.; Prado, A.M.; Becker, J.D.; Certal, A.C.New molecules, including protein kinases, lipids and molecules that have neurotransmitter activities in animals have emerged as important players in tip-growing cells. Transcriptomics analysis reveals that the largest single class of genes expressed in pollen tubes encode signal transducers, reflecting the necessity to decode complex and diverse pathways that are associated with tip growth. Many of these pathways may use common intracellular second messengers, with ions and reactive oxygen species emerging as two major common denominators in many of the processes involved in tip growth. These second messengers might influence the actin cytoskeleton through known interactions with actin-binding proteins. In turn, changes in the dynamic properties of the cytoskeleton would define the basic polarity events needed to shape and modify tip-growing cells.
- The making of gametes in higher plantsPublication . Boavida, L.; Becker, J.D.; Feijó, J.A.Higher plants have evolved to be one of the predominant life forms on this planet. A great deal of this evolutionary success relies in a very short gametophytic phase which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower. In most species the processes of gametogenesis, pollination, syngamy and embryogenesis are sequentially coordinated to give rise to a functional seed in a matter of few weeks. Any of these processes is so intricately complex and precisely regulated that it becomes no wonder that each involves more specific genes and cellular processes than any other function in the plant life cycle. While variability generation - the evolutionary output of the sexual cycle - is the same as in any other Kingdom, plants do it using a completely original set of mechanisms, many of which are not yet comprehended. In this paper, we cover the fundamental features of male and female gametogenesis. While the physiological and cellular bases of these processes have been continuously described since the early nineteen century, recent usage of Arabidopsis and other species as central models has brought about a great deal of specific information regarding their genetic regulation. Transcriptomics has recently enlarged the repertoire and pollen became the first gametophyte to have a fully described transcriptome in plants. We thus place special emphasis on the way this newly accumulated genetic and transcriptional information impacts our current understanding of the mechanisms of gametogenesis.
- Gametophyte interaction and sexual reproduction: how plants make a zygotePublication . Boavida, L.C.; Vieira, A.M.; Becker, J.D.; Feijó, J.A.The evolutionary success of higher plants relies on a very short gametophytic phase, which underlies the sexual reproduction cycle. Sexual plant reproduction takes place in special organs of the flower: pollen, the male gametophyte, is released from the anthers and then adheres, grows and interacts along various tissues of the female organs, collectively known as the pistil. Finally, it fertilizes the female gametophyte, the embryo sac. Pollen is released as bi or tricellular, highly de-hydrated and presumably containing all the biochemical components and transcripts to germinate. Upon hydration on the female tissues, it develops a cytoplasmic extension, the pollen tube, which is one of the fastest growing cells in nature. Pollen is completely "ready-to-go", but despite this seemingly simple reaction, very complex interactions take place with the female tissues. In higher animals, genetic mechanisms for sex determination establish striking developmental differences between males and females. In contrast, most higher plant species develop both male and female structures within the same flower, allowing self-fertilization. Outcrossing is ensured by self-incompatibility mechanisms, which evolved under precise genetic control, controlling self-recognition and cell-to-cell interaction. Equally important is pollen selection along the female tissues, where interactions between different cell types with inherent signalling properties correspond to check-points to ensure fertilization. Last but not least, pollen-pistil interaction occurs in a way that enables the correct targeting of the pollen tubes to the receptive ovules. In this review, we cover the basic mechanisms underlying sexual plant reproduction, from the structural and cellular determinants, to the most recent genetic advances.
- Cyclooxygenase-2 (COX-2) expression by in vitro produced bovine embryos. Preliminary results = Expressão da ciclo-oxigenase-2 (COX-2) em embriões bovinos produzidos in vitro. Resultados preliminaresPublication . Marques, C.C.; Horta, A.E.; Vasques, M.I.; Baptista, M.C.; Becker, J.D.; Pimenta, J.; Pereira, R.M.Expression of the two isoforms of cyclooxygenase enzyme, cyclooxygenase-1 (COX-1) and cyclooxygenase-2 (COX-2), by in vitro produced bovine embryos was examined. Cumulus-oocyte complexes were recovered from ovaries of slaughtered animals and subsequently in vitro matured and in vitro fertilized. Presumptive zygotes were cultured in serum containing medium (TCM 199+10% bovine superovulated oestrus serum) on a granulosa cell monolayer for 12 days.
- Gene family analysis of the Arabidopsis pollen transcriptome reveals biological implications for cell growth, division control and gene expression regulationPublication . Pina, C.; Pinto, F.; Feijó, J.A.; Becker, J.D.Upon germination, pollen forms a tube that elongates dramatically through female tissues to reach and fertilize ovules. While essential for the life cycle of higher plants, the genetic basis underlying most of the process is not well understood. We previously used a combination of flow cytometry sorting of viable hydrated pollen grains and GeneChip array analysis of onethird of the Arabidopsis (Arabidopsis thaliana) genome to define a first overview of the pollen transcriptome. We now extend that study to approximately 80% of the genome of Arabidopsis by using Affymetrix Arabidopsis ATH1 arrays and perform comparative analysis of gene family and gene ontology representation in the transcriptome of pollen and vegetative tissues. Pollen grains have a smaller and overall unique transcriptome (6,587 genes expressed) with greater proportions of selectively expressed (11%) and enriched (26%) genes than any vegetative tissue. Relative gene ontology category representations in pollen and vegetative tissues reveal a functional skew of the pollen transcriptome toward signaling, vesicle transport, and the cytoskeleton, suggestive of a commitment to germination and tube growth. Cell cycle analysis reveals an accumulation of G2/Massociated factors that may play a role in the first mitotic division of the zygote. Despite the relative underrepresentation of transcription-associated transcripts, nonclassical MADS box genes emerge as a class with putative unique roles in pollen. The singularity of gene expression control in mature pollen grains is further highlighted by the apparent absence of small RNA pathway components.
- BOLITA, an Arabidopsis AP2/ERF-like transcription factor that affects cell expansion and proliferation/differentiation pathwaysPublication . Marsch-Martinez, Nayelli; Greco, Raffaella; Becker, Jorg D.; Dixit, Shital; Bergervoet, Jan H. W.; Karaba, Aarati Aarati; de Folter, Stefan; Pereira, AndyThe BOLITA (BOL) gene, an AP2/ERF transcription factor, was characterized with the help of an activation tag mutant and overexpression lines in Arabidopsis and tobacco. The leaf size of plants overexpressing BOL was smaller than wild type plants due to a reduction in both cell size and cell number. Moreover, severe overexpressors showed ectopic callus formation in roots. Accordingly, global gene expression analysis using the overexpression mutant reflected the alterations in cell proliferation, differentiation and growth through expression changes in RBR, CYCD, and TCP genes, as well as genes involved in cell expansion (i.e. expansins and the actin remodeling factor ADF5). Furthermore, the expression of hormone signaling (i.e. auxin and cytokinin), biosynthesis (i.e. ethylene and jasmonic acid) and regulatory genes was found to be perturbed in bol-D mutant leaves
- Genetic subtraction profiling identifies genes essential for Arabidopsis reproduction and reveals interaction between the female ganetophyte and the maternal sporophytePublication . Johnston, A.J.; Meier, P.; Gheyselinck, J.; Federer, M.; Wuest, A.E.J.; Schlagenhauf, E.; Becker, J.D.; Grossnikalus, U.The embryo sac contains the haploid maternal cell types necessary for double fertilization and subsequent seed development in plants. Large-scale identification of genes expressed in the embryo sac remains cumbersome because of its inherent microscopic and inaccessible nature. We used genetic subtraction and comparative profiling by microarray between the Arabidopsis thaliana wild-type and a sporophytic mutant lacking an embryo sac in order to identify embryo sac expressed genes in this model organism. The influences of the embryo sac on the surrounding sporophytic tissues were previously thought to be negligible or nonexistent; we investigated the extent of these interactions by transcriptome analysis.
- How many genes are needed to make a pollen tube? lessons from transcriptomicsPublication . Becker, Jorg D.; Feijó, José ABackground Pollen is the male gametophyte of higher plants. Upon pollination, it germinates and develops into a fast-growing cytoplasmic extension, the pollen tube, which ultimately delivers the sperm into the ovary. The biological relevance of its role, and the uniqueness of this kind of cellular organization, have made pollen the focus of many approaches, and it stands today as one of the best-known models in plant cell biology. In contrast, the genetic background of its development has been until recently largely unknown. Some genes involved have been described and a few functional mutants have been characterized, but only to a limited extent and allowing only a limited understanding of the regulatory mechanisms. Yet, being a relatively simple organ (2 or 3 cells), pollen stands as an excellent target for molecular-biology-based approaches. Recent studies on Arabidopsis thaliana have characterized the transcriptional profile of pollen grains and microgametogenesis in comparison to sporophytic tissues. They underline the unique characteristics of pollen, not only in terms of a strongly reduced set of genes being expressed, but also in terms of the functions of the proteins encoded and the pathways they are involved in. These approaches have expanded the number of genes with known expression in pollen from a few hundred to nearly eight thousand. While for the first time allowing systems and/or gene-family approaches, this information also expands dramatically the possibility of hypothesis-driven experimentation based on specific gene function predictions. Recent studies reveal this to be the case in, for example, transcriptional regulation, cell-cycle progression and gene-silencing mechanisms in mature pollen