A semente de café: desenvolvimento e perspectivas genômicas

Abstract

Apesar da importância econômica do café, pouco se sabe sobre os mecanismos moleculares que atuam durante a formação de sementes de cafeeiros. Estas fases são essenciais no estabelecimento de todas as características da semente madura e rendimento final da lavoura. O estabelecimento da era genômica vem transformando os estudos de expressão gênica em biologia de plantas, em função de tecnologias de alto desempenho hoje existentes em transcriptoma ('chips de DNA') e proteoma (eletroforese bidimensional e espectrometria de massa). Estudos nestes níveis apresentam grande potencial e certamente contribuirão para o desenvolvimento da produção cafeeira como um todo, sendo essencial que o biologista leve em consideração o posicionamento preciso do evento considerado ao longo do desenvolvimento. O objetivo do presente trabalho foi estabelecer um modelo funcional preliminar de desenvolvimento de sementes de cafeeiro (Coffea arábica L.)como pré requisito para as análises de expressão gênica. Utilizou-se microscopia eletrônica na análise estrutural dos tecidos; citometria na análise de síntese e ploidia do DNA; "western blotting" no acúmulo de b-tubulina; e microscopia de imuno-fluorescência nas configurações do citoesqueleto microtubular. O estudo possibilitou o esclarecimento conclusivo da origem dos diferentes tecidos, definir com precisão a organogênese, as fases do desenvolvimento, a ocorrência de eventos fisiológicos e a anatomia da semente. O modelo funcional descreve a complexidade do desenvolvimento da semente do cafeeiro, compreendendo um pré requisito fundamental em estudos de desenvolvimento e implementação de análises de expressão gênica em escala genômica (transcriptoma e proteoma) e de função dos genes na era pós-genômica (metaboloma).

Although coffee has great world wide economic importance, little is known about the factors that act during flowering and fruit and seed formation in coffee plants. These developmental phases are essential for the establishement of all characteristics of the mature seed and final yield of the crop. It is fundamental a better understanding of the molecular mechanisms and regulatory programs the determine seed quality as propagule and as commodity. Besides, it is relevant to consider the fact that seeds are the main form of survival of the coffee as species, despite the fact that they usually have short longevity. The establishment of the genomic era is transforming the studies of gene expression in plant biology and promisses to revolutionize the way that cells and cell processes are studied. This transformation is due to the existence nowadays of high throughput (bio)tecnologies in transcriptomics (DNA microarrays) and proteomics (2D-electrophoresis and mass spectrometry). Studies at this level show great potential and will certainly be essential also for the development of the coffee production as a whole. However, the success in these types of studies depend not only on the proper use of the technologies, but also on the precise positioning of the event considered in developmental studies. It is presented here a funcional model of development of coffee (Coffea arabica L.) seeds, which allows a greater precision on the identification of the different stages and occurrence of physiological events during development, contributing therefore, for the success of the gene expression analysis to be considered in a subsequent phase. The stablishment of the model considered the functional analysis of cell cycle and organogenesis events in relation to physiological events during seed development, taking as reference the number of ‘days after pollination’ (dap), the size and color of fruits. Scanning electron microscopy was utilized for the structural analysis of differences in the seed tissues. Cell cycle and organogenesis events were analysed in relation to the processes of tissue differentiation by cell division, and growth by cell expansion. Synthesis and duplication of DNA, as well as DNA ploidy, were analysed by means of citometry; the accumulation of ß-tubulin by eletrophoresis and western blotting, and the citological analysis the microtubular cytoskeleton configurations by immuno-fluorescence microscopy. The accumulation of dry matter, water content, and germinability and desiccation tolerance tests were considered for the evaluation of the physiological state of the seeds. The relation among DNA synthesis, B-tubulin accumulation and configurations of the microtubular cytoskeleton allowed a precise difinition of the phases of development. The phase of differentiation of seed tissues and embryogenesis (60 to 150 DAP) is marked by a decreasing activity of DNA synthesis, as well as of tubulin accumulation and of the mitotic configurations of the microtubular cytoskeleton. The results of the analysis of ploidy by citometry and of struture by electron microscopy, it is verified that the initial development of the fruit is due to the growth of the diploid maternal nucellar tissue ("false seed"), which is subsequently partially degraded giving up space for the development of the triploid endosperm and diploid embryo ("true seed"). By the end of this phase (150 DAP), the differentiated embryos are shown to be viable, when isolated and incubated in vitro. However, the seeds do not germinate when evaluated in germination test. Both isolated embryos, as seeds, do not show desiccation tolerance in this phase. In the maturation phase (150 to 225 DAP), it is predominantly observed complementary morphogenetic events as cell expasion growth, since mitotic cytoskeleton is no longer observed, existing only cortical cytoskeleton, and increasing accumulation of dry matter. With the progress of maturation and desiccation, it is observed the complete degradation of the remaining cortical cytosketon and tubulin, leading to the inactivation of the cell cycle events and induction of embryo and seed quiescence. The acquisition of germinability by the seed is only initiated when fruits reach the larger-green stage (210 DAP), becoming maximum in the cherry stage (225 DAP), when desiccation tolerance also becomes maximum. It was possible, by means of the present study, to conclusively clarify the origin of the different tissues, to define with precision the organogenesis, the different phases of development, the occurrence of specific physiological events and the seed anatomy. Therefore, it is established a funcional model that describes the complexity of the development of the coffee fruit and seed, and that complies an essential prerequisite for the success on the implementation of gene expression studies at the genomic level (transcriptomics and proteomics) and of gene function at the post-genomic level (metabolomics).