The underground reserve organs of yacon (Polymnia sonchifolia Poep. Endl.), similarly to other economically important Asteraceae, accumulate more than 60%, on a DW basis, of inulin type β(2‐1) fructans, mainly oligomers (GF2–GF16). Although sucrose:sucrose 1‐fructosyl transferase (1‐SST), fructan:fructan 1‐fructosyl transferase (1‐FFT) and fructan 1‐exohydrolase (1‐FEH) were properly described and characterized from a number of plant species, detailed information about their activities in different organs during development are rather scarce in the literature. In the present work 1‐SST, 1‐FFT and 1‐FEH activities were measured monthly in rhizophores and tuberous roots of yacon plants during their complete growth cycle under field conditions. Results showed that 1‐SST activity in rhizophores was always higher than 1‐FFT activity and increased up to 8 months of cultivation, decreasing to initial values at the end of the growth period. In the tuberous roots 1‐SST activity was also higher than 1‐FFT but varied differently. The higher values were found at the beginning of tuberization (3‐month‐old plants) and at the flowering phase (7‐month‐old plants). Results also showed that synthesizing activities in yacon plants were always higher in rhizophores than in the tuberous roots, while hydrolysing activity predominated in the latter, mainly when 1‐kestose and nystose were used as substrates. 1‐FEH from yacon plants showed low efficiency when commercial inulin from Helianthus tuberosus was utilized as substrate. The analysis of the enzymatic activities performed during growth of yacon clearly indicated the most appropriate source organ and phase of development to obtain the highest enzymatic activities for purification purposes and for the production of fructo‐oligosaccharides (FOS). Furthermore, the results suggested that the relative levels of activities of 1‐SST, 1‐FFT and 1‐FEH could be involved in the chain length distribution of the fructan molecules found in rhizophores and in tuberous roots of this species.

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Sucrose:sucrose fructosyltransferase (SST) and fructan:fructan fructosyl-transferase (FFT) activities from crude extracts of tuberous roots of Viguiera discolor growing in a preserved area of cerrado were analyzed in 1995-1996. SST activity was characterized by the synthesis of 1-kestose from sucrose and FFT activity by the production of nystose from 1-kestose. The highest fructan-synthesizing activity was observed during early dormancy (autumn), when both (SST and FFT) activities were high. The increase in synthetic activity seemed to start during the fruiting phase in the summer, when SST activity was higher than in spring. During winter and at the beginning of sprouting, both activities declined. The in vitro synthesis of high molecular mass fructans from sucrose by enzymatic preparations from tuberous roots collected in summer showed that long incubations of up to 288 h produced consistently longer polymers which resembled those found in vivo with respect to chromatographic profiles.

In order to evaluate the involvement of fructans in drought tolerance

SUMMARYTuberous roots of Viguiera discolor accumulate up to 80% of their dry mass as fructans. The distribution pattern of oligomers suggests the predominance of anabolic reactions at the beginning of dormancy, when a continuous series of fructans between sucrose and higher polymers is present. This paper describes the synthesis in vitro of fructans of high molecular mass by enzyme extracts prepared from growing tuberous roots of V. discolor at the beginning of dormancy. Sucrose: sucrose fructosyl transferase activity was characterized by the synthesis of 1-kestose from sucrose and fructan: fructan fructosyl transferase activity by the production of nystose from 1-kestose. The optimal temperature for both activities was 30 °C and the optimal pH range was between 5-5 and 6-0. The apparent K^ for sucrose with respect to 1-kestose synthesis was 173 mol m^^, whereas the K^.^ for nystose synthesis from 1-kestose was much higher (592 mol m"^^). Long incubation periods (up to 264 h) with sucrose, 1-kestose or nystose produced fructans of high molecular mass in vitro that resembled those found in vivo. Our data show that the higher the molecular mass of the substrate, the higher the average molecular mass of the product that u'as formed.

-(Fructan degradation and hydrolytic activity in tuberous roots of Viguiera discolor Baker (Asteraceae), a herbaceous species from the cerrado). Fructans of the inulin type are the major reserve carbohydrates in tuberous roots of Viguiera discolor, a perennial herb native to the cerrado. Changes in molecular mass of the polymer, followed by releasing free fructose suggested that hydrolysis could be related to the sprouting of the buds after the dormant period, when aerial parts of the plant are naturally absent. Excision of aerial parts resulted in the increase of fructan 1-exohydrolase (1-FEH) activity in tuberous roots after sprouting. 1-FEH was partially purified from this material by binding to ConA-Sepharose and the highest activity was detected at pH 5.4 and between 20 and 40 °C. Values of K m for V. discolor inulin could not be determined since no saturation was observed up to 10%. The study of the kinetics of the 1-FEH activity showed that it does not follow MichaelisMenten and apparently presents allosteric behaviour, as data fits in the Hill equation. The 1-FEH from V. discolor is a glycoprotein, more active on low molecular mass fructans than on high molecular mass inulin from the same species.Key words -Asteraceae, cerrado, fructan 1-exohydrolase, inulin RESUMO -(Degradação de frutanos e atividade hidrolítica em raízes tuberosas de Viguiera discolor Baker, uma espécie herbácea de Asteraceae do cerrado). Frutano do tipo inulina é o principal carboidrato de reserva das raízes tuberosas de Viguiera discolor, uma espécie herbácea nativa do cerrado. Variações na massa molecular do polímero, acompanhadas de altas proporções de frutose livre sugeriram que a hidrólise do mesmo poderia estar associada à brotação das plantas, após o período de dormência, quando toda a parte aérea é naturalmente eliminada. Após indução experimental de brotação e estímulo da atividade hidrolítica, por eliminação da parte aérea, o extrato enzimático foi parcialmente purificado por adsorção em Sepharose ConA, sendo a maior atividade detectada em pH 5,4 e entre 20 e 40 °C. Não foi possível determinar os valores de K m para a inulina, uma vez que até a concentração de 10% não houve a saturação do substrato. O estudo da cinética da atividade de frutano 1-exohidrolase mostrou que a mesma não corresponde à cinética de Michaelis-Menten, apresentando um comportamento aparentemente alostérico, uma vez que os dados se enquadram na equação de Hill. A 1-FEH de V. discolor é uma glicoproteína, mais ativa sobre oligofrutanos do que sobre inulina de alta massa molecular, como a encontrada em outras Asteráceas, e principalmente na própria espécie.Palavras-chave -Asteraceae, cerrado, frutano 1-hexohidrolase, inulina

Fructans in calli of Smallanthus sonchifolius (Poepp.) H. Rob.). Smallanthus sonchifolius is an Asteraceae native from the Andes Mountains that accumulates inulin-type fructans in the underground reserve organs, the tuberous roots and the rhizophores. Yellow callus was obtained from tuberous roots, giving origin, spontaneously to red callus. Fructans and their metabolizing enzymes were analyzed in calli of both types, showing similar fructan contents in both lineages. However, synthesizing activity was higher in red callus, while hydrolyzing activity was higher in yellow callus. HPAEC/ PAD analyses revealed the presence of fructo-oligosaccharides in both calli with degrees of polymerization lower than those found in plants cultured ex vitro, indicating that this characteristic was maintained in undifferentiated cells. The presence of fructans and the differences between the activities of fructosyltransferases and hydrolases found in red and yellow calli indicate this material as suitable for studies of fructan metabolism under controlled conditions. Key words: fructan, in vitro conditions, inulin, yacon RESUMO -(Frutanos em calos de Smallanthus sonchifolius (Poepp.) H. Rob.). Smallanthus sonchifolius, Asteraceae originária dos Andes, possui rizóforos e raízes tuberosas que armazenam frutanos do tipo inulina. Explantes de raízes tuberosas dão origem a calos de linhagem amarela que, espontaneamente, originaram a linhagem vermelha. O conteúdo de frutanos foi semelhante nas duas linhagens, enquanto a atividade de síntese foi mais elevada na linhagem vermelha e a de hidrólise foi mais elevada na amarela. Análises por HPAEC/PAD demonstraram a presença, em ambos os calos, de frutooligossacarídeos com grau de polimerização inferior ao encontrado em plantas cultivadas sob condições naturais, indicando que esta característica é mantida em células indiferenciadas. A presença de frutanos e as diferenças entre as atividades de frutosiltransferases e hidrolases em calos vermelhos e amarelos possibilitam a aplicação desse material em estudos de regulação do metabolismo desses compostos em condições controladas. Palavras-chave: cultivo in vitro, frutanos, inulina, yacon

RESUMO -(Produção de frutanos em calos e plântulas clonadas in vitro de Viguiera discolor Baker (Asteraceae)). Viguiera discolor Baker é uma espécie nativa do cerrado, cuja sobrevivência está ameaçada pela invasão de espécies exóticas. Considerando sua elevada produção e a vasta aplicação de frutanos, o presente trabalho foi conduzido visando à obtenção in vitro dessa espécie e à prospecção desses carboidratos nessas condições. Sementes foram germinadas in vitro, em meio MS modificado, e após cinco semanas de incubação, nós caulinares foram isolados e incubados no mesmo meio adicionado de 0,5 mg L -1 de ANA, regenerando plantas uniformes, raízesnão espessadas, raízes tuberosas e estruturas semelhantes a calo (calo tipo1), formadas na região caulinar basal. Análise desse material evidenciou a presença de frutanos do tipo inulina nas raízes tuberosas e nos calos tipo 1. Na presença de 2,4-D obteve-se a formação de calos friáveis (calo tipo 2), nos quais também foram detectados frutanos e suas enzimas de síntese sacarose: sacarose 1-frutosiltransferase (SST) e frutano: frutano 1-frutosiltransferase (FFT). Embora em concentrações menores às observadas nas plantas cultivadas sob condições naturais, o material produzido in vitro apresentou frutanos do mesmo tipo e razão SST/FFT menor do que um. Em meio de cultura sem hormônios, foi verificada a regeneração de 50% de plantas a partir dos nós caulinares. A propagação de V. discolor in vitro pode viabilizar a multiplicação e a preservação da espécie, bem como a produção de frutanos nessas condições. Palavras-chave: cerrado, inulina, carboidratos de reserva, raízes tuberosas ABSTRACT -(Fructan production in callus and in vitro cloned seedlings of Viguiera discolor Baker (Asteraceae)). Viguiera discolorBaker is a herbaceous species, native to cerrado and its survival has been threatened by the invasion of exotic species. Considering its high production and the wide application of fructans, the present work has aimed to establish in vitro culture of this species and to investigate the presence of fructans under these conditions. Seeds were germinated in vitro on modified MS medium and after plant growth, stem nodes were isolated and incubated on the previous culture medium, supplemented with 0.5 mg L -1 NAA, which allowed the production of uniform plants, and formation of non thickened roots, tuberous roots and callus-like structures (callus type 1). Analysis of these materials showed the presence of inulin-type fructans. Incubation of stem nodes in the presence of 2,4-D induced growth of friable callus (callus type 2), in which fructans and their synthesis enzymes sucrose: sucrose 1-fructosyltransferase (SST) and fructan: fructan 1-fructosyltransferase (FFT) were detected. Although in lower concentrations, the ratio SST/FFT activities and their fructans were similar to values found in plants cultivated under natural conditions. Stem nodes incubation on hormone-free medium resulted in regeneration of 50% plantlets. In vitro propagation, of V. discolor, may allow large-scale...