Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.O objetivo deste trabalho foi a caracterização dos perfis protéicos expressos durante o desenvolvimento da semente de O. catharinensis, uma espécie recalcitrante, via eletroforese bidimensional. A extração de proteínas foi realizada utilizando tampão uréia/tiouréia seguido de uma etapa de precipitação com TCA 10%. A análise comparativa durante o desenvolvimento da semente mostrou um grande número de proteínas exclusivas em cada estádio do desenvolvimento. O estádio cotiledonar, que representa a fase de transição entre a embriogênese e o começo do metabolismo relacionado a maturação, apresentou o maior número de proteínas estádio-específicas. Proteínas relacionadas ao metabolismo oxidativo e a síntese de reservas foram identificadas via MS/MS. Estes resultados contribuem para o melhor entendimento do metabolismo de proteínas durante o desenvolvimento de sementes recalcitrantes, além de prover informações no estabelecimento de parâmetros para protocolos de embriogênese somática.(FAPESP) São Paulo Research Foundation(CNPq) National Council for Scientific and Technological Developmen

Proteômica comparativa aplicada à cultura de tecidos de plantas

Nas últimas décadas os códigos genéticos de diversos organismos foram completamente seqüenciados, representando um importante avanço do conhecimento na área genômica. No contexto das “ômicas” grande ênfase tem-se dado para a genômica funcional, que visa a identificação da seqüência e função dos genes e proteínas através de estudos do transcriptoma e proteoma dos organimos vivo

WUSCHEL-related genes are expressed during somatic embryogenesis of the basal angiosperm Ocotea catharinensis Mez. (Lauraceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Ocotea catharinensis is a basal angiosperm and an endangered tree species from the Brazilian Atlantic Rain Forest. Despite its economical and ecological importance, mass-propagation of this species is hampered by seldom-produced short-lived seeds, and in vitro propagation is challenged by frequently malformed somatic embryos. Therefore, O. catharinensis somatic embryos are also a good experimental material to study the physiological and molecular mechanisms underlying in vitro morphogenesis. In an ongoing effort to characterize genes expressed during somatic embryogenesis of O. catharinensis we have cloned two Ocotea WUSCHEL-related genes. According to our RT-PCR data, both genes were preferentially expressed in embryogenic cell aggregates. One of them, OcWUS, is a possible ortholog of the Arabidopsis WUSCHEL (WUS) gene, which codes for a homeodomain-containing protein involved in the specification and maintenance of the shoot apical meristem. We analyzed the expression patterns of OcWUS and OcWOX4 by RT-PCR, and OcWUS expression was also assessed by in situ hybridization. The expression patterns of OcWUS were very similar to those described for the Arabidopsis WUS. OcWUS transcripts were generally restricted to a small group of cells in the center of the putative shoot apical meristem of O. catharinensis somatic embryos. Perturbed expression of OcWUS might be related to abnormally formed somatic embryos of O. catharinensis obtained through tissue culture.262493501Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

A gymnosperm homolog of SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE-1 (SERK1) is expressed during somatic embryogenesis

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)The physiological and molecular processes controlling zygotic and somatic embryo development in angiosperms are mediated by a hierarchically organized program of gene expression. Despite the overwhelming information available about the molecular control of the embryogenic processes in angiosperms, little is known about these processes in gymnosperms. Here we describe the cloning and characterization of the expression pattern of the Araucaria angustifolia putative homolog of a SOMATIC EMBRYOGENESIS RECEPTOR-LIKE KINASE (SERK) gene family member, designated as AaSERK1. The Araucaria AaSERK1 gene encodes a leucine-rich repeat receptor-like kinase showing significant similarity to angiosperm homologs of SERK1, known to be involved in early somatic and zygotic embryogenesis. Accordingly, RT-PCR results showed that AaSERK1 is preferentially expressed in Araucaria embryogenic cell cultures. Additionally, in situ hybridization results showed that AaSERK1 transcripts initially accumulate in groups of cells at the periphery of the embryogenic calli and then are restricted to the developing embryo proper. Our results indicate that AaSERK1 might have a role during somatic embryogenesis in Araucaria, suggesting a potentially conserved mechanism, involving SERK-related leucine-rich repeat receptor-like kinases, in the embryogenic processes among all seed plants.10914150Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq

Ethylene and polyamine production patterns during in vitro shoot organogenesis of two passion fruit species as affected by polyamines and their inhibitor

Levels of ethylene and polyamines (PAs) were measured during organogenesis of hypocotyl explants of two species of passion fruit (Passiflora cincinnata Masters and Passiflora edulis Sims f. flavicarpa Degener `FB-100`) to better understand the relationships of these regulators and their influence on cell differentiation and morphogenesis. Moreover, histological investigation of shoot ontogenesis was conducted to characterize the different events involved in cell redifferentiation and regulation of PA and ethylene levels. A delay was observed in morphogenic responses of P. edulis f. flavicarpa as compared to P. cincinnata, and these changes coincided with production of elevated levels of polyamine and ethylene levels. During differentiation, cells showed high rates of expansion and elongation, and high ethylene levels were associated with high PA levels, suggesting that the two biosynthesis pathways were highly regulated. Moreover, their interaction might be an important factor for determining cell differentiation. The addition of PAs to the culture medium did not promote organogenesis; however, the incorporation of the PA inhibitor methylglyoxal bisguanylhydrazone in the culture medium reduced shoot bud differentiation, suggesting the need to maintaining a minimum level of PAs for morphogenic events to take place.Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG)FAPEMIG (Belo Horizonte, MG, Brazil)CNPq (Brasilia, DF, Brazil)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)CAPES (Brasilia, DF, Brazil)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES

Neolignans and sesquiterpenes from leaves and embryogenic cultures of Ocotea Catharinensis (Lauraceae)

The extracts from leaves of Ocotea catharinensis Mez (Lauraceae) were found to contain fourteen neolignans and two sesquiterpenes: nine benzofuran types (including three new compounds 1e, 2f and 4b), one new seco-benzofuran type (3b), two bicyclo[3.2.1]octane types (including the new compound 5c), two new dimers of bicyclo[3.2.1]octane type (7a and 7b) and two sesquiterpenes (including a new humulanol 9). In addition, seven neolignans were also showed to occur in somatic embryos of O. catharinensis including one new bicyclo[3.2.1]octane type (4a).Foram isoladas de extratos de folhas de Ocotea catharinensis Mez (Lauraceae) quatorze neolignanas sendo nove benzofurânicas (incluindo três novas substâncias 1e, 2f e 4b), uma secobenzofurânica inédita (3b), duas biciclo[3.2.1]octânicas (incluindo a nova 5c), dois novos dímeros biciclo[3.2.1]octânicos (7a e 7b) e ainda dois sesquiterpenos (incluindo o novo humulanol 9). Nos embriões somáticos de O. catharinensis foram identificadas sete neolignanas incluindo uma nova neolignana biciclo[3.2.1]octânica (4a).FAPESPCNPqCoordenacao de Aperfeicoamento de Pessoal de Nivel Superior (CAPES)International Foundation for Science (IFS, Sweden