O estudo do metabolismo de poliaminas (PAS), envolvendo parâmetros fisiológicos, bioquímicos e moleculares, pode gerar uma melhor compreensão do processo de maturação, e criar estratégias importantes para a otimização da embriogênese somática (ES) em cana-de-açúcar. O objetivo deste trabalho foi estudar a relação entre os conteúdos endógenos de PAs, associados à determinação da competência embriogenética e a maturação progressiva de embriões somáticos em cana-de-açúcar. Calos embriogênicos (E) e não-embriogênicos (NE) da variedade SP-803280, foram submetidos a diferentes tratamentos, utilizando agentes de maturação adicionados ao meio MS. Observou-se que as culturas NE não foram capazes de promover a diferenciação de embriões somáticos, devido principalmente, ao alto grau de oxidação das culturas. Os tratamentos suplementados com 0,75 e 1,5 g.L-1 de carvão ativado (CA), foram os que apresentaram as maiores freqüências de formação de embriões somáticos. A partir dos resultados obtidos, foi possível associar a ES em calos de cana-de-açúcar ao aumento do conteúdo endógeno de Spd (Espermidina) e Spm (Espermina), principalmente Spd, acompanhado por uma redução nos níveis de Put (Putrescina). / Somatic embryogenesis (SE) is a highly potential supplier of the material involved in the regeneration and genetic transformation of transgenic plants. The aim was to study correlations between endogenous PA contents, associated to defining embryogenetic competence and the progressive maturation of somatic embryos. Embryogenic (E) and non-embryogenic (NE) calli from the SP-803280 variety, were submitted to different maturation treatments. It was found that the NE cultures submitted to the two maturation experiments, were incapable of promoting somatic embryo differentiation, mainly due to browning. It was noted that in the first experiment, total PA endogenous content of the E callus was higher than in the NE, although, in the control, no significant differences between both callus types were encountered. We can associate the progression of sugarcane somatic embryogenesis to an increase in endogenous content of Spd e Spm, mainly Spd, accompanied for a reduction in Put levels. Thus, it was shown that somatic embryo maturation in sugarcane can be related with PA biosynthesis, thereby indicating the importance of their metabolism in the competence of sugarcane embryogenic cultures.

The aim of this thesis was to develop stable-isotope steady-state metabolic flux analysis (MFA) based on ¹³C labeling to quantify intracellular fluxes of central carbon metabolism in plants. The experiments focus on the analysis of a heterotrophic cell suspension culture of Arabidopsis thaliana (L) Heynh. (ecotype Landsberg erecta). The first objective was to develop a robust methodology based on combining high quality steady-state stable labeling data, metabolic modeling and computational analysis. A comprehensive analysis of the factors that influence the outcome of MFA was undertaken and best practice established. This allowed a critical analysis of the subcellular compartmentation of carbohydrate oxidation in the cell culture. The second objective was to apply the methodology to nutritional perturbations of the cell suspension. A comparison of growth on different nitrogen sources revealed that transfer to an ammonium-free medium: (i) increased flux through the oxidative pentose phosphate pathway (oxPPP) by 10% relative to glucose utilisation; (ii) caused a substantial decrease in entry of carbon into the tricarboxylic acid cycle (TCA); and (iii) increased the carbon conversion efficiency from 55% to 69%. Although growth on nitrate alone might be expected to increase the demand for reductant, the cells responded by decreasing the assimilation of inorganic N. Cells were also grown in media containing different levels of inorganic phosphate (Pi). Comparison of the flux maps showed that decreasing Pi availability: (i) decreased flux through the oxPPP; (ii) increased the proportion of substrate fully oxidised by the TCA cycle; and (iii) decreased carbon conversion efficiency. These changes are consistent with redirection of metabolism away from biosynthesis towards cell maintenance as Pi is depleted. Although published genome-wide transcriptomic and metabolomic studies suggest that Pi starvation leads to the restructuring of carbon and nitrogen metabolism, the current analysis suggests that the impact on metabolic organisation is much less extreme.