The life cycle of all land plants is characterized by the alternation between two multicellular generations: haploid gametophytes and diploid sporophytes. Whereas meiotic recombination and fertilization via sexual reproduction is known to confer adaptive evolutionary benefits, some plant species can reproduce asexually bypassing meiosis and fertilization yet still undergo alternation of generations. In ferns, the two asexual reproductive pathways are termed apogamy and apospory, i.e. the asexual reproduction of sporophytes and gametophytes, respectively. Although understanding the process of asexual reproduction in flowering plants would be of considerable agricultural value, the molecular mechanisms underlying this process have yet to be identified. Similarly, the genes in ferns that underlie apogamy and apospory are unknown. Knowledge of such genes will provide invaluable information not only for the evolutionary study of asexual reproduction in land plants, but also insight into the developmental program for each generation. My PhD research focuses on the investigation of candidate genes involved in asexual reproduction in the model fern Ceratopteris richardii. Though the molecular mechanisms underlying asexual reproduction are not understood, it is hypothesized that asexual reproduction is the result of altered regulation of certain sexual reproduction-related genes. Therefore, I selected candidate genes for asexual reproduction in C. richardii, CrEMS1, CrKNOX3 and CrANT, based on the functions of their respective homologs during sexual reproduction in two other model systems: Arabidopsis thaliana and Physcomitrella patens. I showed that, in C. richardii, CrEMS1 is expressed during key events of sexual reproduction, including sporogenesis, gametogenesis and embryo development; CrKNOX3 expression is specific for the mature egg cells and CrANT expression is restricted to sperm cells. The presence of CrEMS1 during early embryo development made it a suitable candidate for an embryonic marker for future research. To study the function of the candidate genes, I overexpressed CrKNOX3 and BnBBM, a flowering plant BBM gene that has similar expression patterns in Brassica to CrANT. The overexpression of CrKNOX3 results in unique cell outgrowth from the antheridia, and overexpression of BnBBM results in spontaneous apogamy induction. Whole mount in situ also showed CrEMS1 expression, marking embryonic identity, in pre-apogamous cells undergoing sugar-induced and overexpression of BnBBM-induced apogamy. The results of my PhD research provides, for the first time, insight into the molecular mechanisms underlying asexual reproduction in the model fern C. richardii, and invaluable tools, transient and stable Agrobacterium-mediated transformation protocols, for functional genomics research in this fern. The spontaneous apogamy induction on BnBBM-expressing gametophytes and the presence of CrEMS1 in pre-apogamous cells confirm the hypothesis that asexual reproduction is the result of the alteration in expression of sexual-related genes, verifying that the two pathways share a common set of genes.

Orientadores: Sandra Maria Carmello-Guerreiro, Simone de Padua Teixeira / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-15T16:53:33Z (GMT). No. of bitstreams: 1 Caetano_AnaPauladeSouza_M.pdf: 5552395 bytes, checksum: ddd2d3795d5da107ef405fe68e7a02a3 (MD5) Previous issue date: 2010 / Resumo: Em Miconia fallax, uma espécie apomítica, e Miconia pepericarpa, uma espécie sexuada, os processos de esporogênese e gametogênese apresentam diversas características semelhantes, muitas delas compartilhadas com outros membros da família Melastomataceae. Entretanto, apesar das similaridades, algumas diferenças foram observadas. Em relação à antera, o formato da célula generativa no grão de pólen maduro é fusiforme em M. pepericarpa e esférico em M. fallax. Além disso, a quantidade de grãos de pólen viáveis produzidos é marcadamente menor na espécie apomítica. Quanto ao óvulo, as diferenças encontradas são ainda mais marcantes. Em M. fallax, células denominadas iniciais apospóricas se diferenciam juntamente com as células-mãe de megásporos, levando à formação de mais de um saco embrionário. Assim, enquanto M. fallax apresenta até três sacos embrionários no óvulo maduro, sendo um sexuado e até dois apospóricos, M. pepericarpa exibe apenas um saco embrionário. Além disso, na espécie apomítica M. fallax, constatou-se a formação de embriões adventícios em sementes jovens. Provavelmente, o desenvolvimento de embriões a partir dos sacos embrionários apospóricos juntamente com a formação do embrião zigótico e adventício levam à poliembrionia em M. fallax, com formação de até quatro embriões em uma mesma semente. Das diferenças encontradas entre as duas espécies, apenas o formato da célula generativa não foi relacionado à apomixia, sendo um caráter específico. Já as demais diferenças, são características exclusivas de M. fallax, e estão diretamente relacionadas à apomixia, sendo elas: 1) formação de uma proporção menor de grãos de pólen viáveis; 2) diferenciação de células iniciais apospóricas; 3) formação de múltiplos sacos embrionários; 4) desenvolvimento de embriões apomíticos; 5) poliembrionia. A espécie apomítica M. fallax é poliplóide e, neste caso, a viabilidade polínica baixa pode ser explicada pela ocorrência de alterações na meiose, comuns em espécies poliplóides. A poliembrionia, também tem sido bastante relacionada à apomixia, sendo esta relação confirmada pelos dados obtidos neste trabalho. Além disso, com os resultados alcançados, foi possível reconhecer que M. fallax é uma apomítica facultativa, uma vez que, além da possibilidade de produção de embriões apomíticos, a espécie ainda preserva os mecanismos de formação de grãos de pólen e sacos embrionários sexuados, com possibilidade de produção de embriões zigóticos. / Abstract: In Miconia fallax, an apomictic species, and Miconia pepericarpa, a sexual species, the processes of gametogenesis and sporogenesis have a number of similar characteristics, many of which are shared with other members of the family Melastomataceae. However, despite the similarities, some differences were observed. Concerning the anther, the generative cell in mature pollen grain is spindle shaped in M. pepericarpa and spherical in M. fallax. Furthermore, the quantity of viable pollen grains produced is markedly lower in apomictic species. Regarding the ovule, differences are even more striking. In M. fallax, cells called aposporous initials differentiate themselves along with the megaspore mother cell, leading to the formation of more than one embryo sac. Thus, while M. fallax exhibit up to three embryo sacs in mature ovule, one being sexed and a maximum of two aposporous, M. pepericarpa displays only one embryo sac. Moreover, in apomictic species M. fallax, we found the formation of adventitive embryos in young seeds. Probably the development of embryos from the aposporous embryo sac parallel to the formation of zygotic and adventitious embryos lead to polyembryony in M. fallax, with formation of up to four embryos in a single seed. About differences found between this two species, only the shape of the generative cell was not related to apomixis, as it is a specific character. The other differences are unique features of M. fallax and are directly related to apomixis, namely: 1) the formation of a smaller proportion of viable pollen grains, 2) the differentiation of aposporous initial cells 3) the formation of multiple embryo sacs, 4) the development of apomictic embryos (adventitious or aposporic), and 5) polyembryony. The apomictic species M. fallax is polyploid, and, in this case, the low pollen viability can be explained by the occurrence of changes in meiosis, common in polyploid species. The polyembryony, has also been frequently related to apomixis, and this relationship is confirmed by data obtained in this work. Furthermore, with these results, it was possible to recognize that M. fallax is a facultative apomictic, since in addition to the possibility of apomictic embryo production the species still preserves the mechanisms of the formation of pollen grain and sexual embryo sac, with the possibility of producing zygotic embryos. / Mestrado / Mestre em Biologia Vegetal