Increased expression of fructan 1-exohydrolase in rhizophores of Vernonia herbacea during sprouting and exposure to low temperature

Rhizophores of Vernonia herbacea, an Asteraceae found in the Brazilian Cerrado, store high amounts of fructans that vary in composition over the phenological cycle. Fructan 1-exohydrolase (1-FEH) activity is detectable during the sprouting phase, mainly in the proximal regions of rhizophores, of plants induced to sprout by defoliation and/or cold storage. We found an increase in 1-FEH gene expression during natural and induced sprouting and further enhancement through low-temperature treatment. Furthermore, a comparative analysis of 1-FEH gene expression in different regions of the rhizophores during the transition from dormancy to sprouting is presented. Transcripts were detected mainly in the proximal region, coinciding with high 1-FEH activity and a high concentration of free fructose. Low temperature promoted the accumulation of fructans of a low degree of polymerization (DP) and enhanced 1-FEH activity and gene expression. It is hypothesized that a set of 1-FEH proteins acts in two different ways during fructan mobilization: (1) by hydrolyzing fructo-oligosaccharides and -polysaccharides in sprouting plants (naturally or induced) for carbon supply and (2) by hydrolyzing preferably fructo-polysaccharides under low temperature to maintain the oligosaccharide pool for plant cold acclimation. (C) 2010 Elsevier GmbH. All rights reserved.

Cloning, characterization and functional analysis of a 1-FEH cDNA from Vernonia herbacea (Vell.) Rusby

Variations in the inulin contents have been detected in rhizophores of Vernonia herbacea during the phenological cycle. These variations indicate the occurrence of active inulin synthesis and depolymerization throughout the cycle and a role for this carbohydrate as a reserve compound. 1-Fructan exohydrolase (1-FEH) is the enzyme responsible for inulin depolymerization, and its activity has been detected in rhizophores of sprouting plants. Defoliation and low temperature are enhancer conditions of this 1-FEH activity. The aim of the present work was the cloning of this enzyme. Rhizophores were collected from plants induced to sprout, followed by storage at 5C. A full length 1-FEH cDNA sequence was obtained by PCR and inverse PCR techniques, and expressed in Pichia pastoris. Cold storage enhances FEH gene expression. Vh1-FEH was shown to be a functional 1-FEH, hydrolyzing predominantly -2,1 linkages, sharing high identity with chicory FEH sequences, and its activity was inhibited by 81 in the presence of 10 mM sucrose. In V. herbacea, low temperature and sucrose play a role in the control of fructan degradation. This is the first study concerning the cloning and functional analysis of a 1-FEH cDNA of a native species from the Brazilian Cerrado. Results will contribute to understanding the role of fructans in the establishment of a very successful fructan flora of the Brazilian Cerrado, subjected to water limitation and low temperature during winter.

Soluções nutritivas para cultivo e produção de frutanos em plantas de Vernonia herbacea

O crescimento limitado de rizóforos de Vernonia herbacea (Asteraceae) em solução de Hoagland levou à necessidade de estabelecer uma solução nutritiva para o cultivo dessa planta, visando ao incremento da biomassa de seus rizóforos ricos em frutanos. Essa solução (denominada Vernonia), constituída de Ca(NO3)2.4H2O 2,5 mmol L-1, KNO3 2,3 mmol L-1 , KH2PO4 0,52 mmol L-1, Mg(NO3)2.6H2O 1,7 mmol L-1 e Na2SO4 1,3 mmol L-1, foi comparada com a de Hoagland nas forças iônicas de 50%, 100% e 200%. Foram realizadas duas avaliações para análise de crescimento e conteúdo de frutanos. As plantas não sobreviveram até os dois meses na solução de Hoagland 200%. A solução Vernonia diluída duas vezes (50%) foi a mais eficiente para o incremento de massa seca dos rizóforos e produção de frutanos por planta. Maior crescimento da parte aérea foi verificado nas soluções de Hoagland e Vernonia 100%. Em comparação com a solução de Hoagland, a solução Vernonia é mais pobre em macronutrientes, confirmando a hipótese de que plantas adaptadas a solos oligotróficos são menos exigentes em nutrientes minerais.

Inulin production by Vernonia herbacea as influenced by mineral fertilization and time of harvest

The underground organs of Vernonia herbacea (Vell.) Rusby, known as rhizophores, acumulate 80% of their dry mass as fructans of the inulin type. In view of the growing industrial use of fructans as dietetic and general food products, and of their medical application, the present investigation aimed at evaluating the effect of mineral fertilization and period of cultivation on the production of these carbohydrates in field trials. Plants used in the experiments were obtained by vegetative propagation from rhizophores collected from plants growing in natural areas of the cerrado, and cultivated for two years. Fertilization consisted of N:P2O5:K2O (80:200:150 kg.ha-1) plus 80 kg.ha-1 nitrogen as dressing. Soil fertilization did not stimulate biomass or inulin production, but in the second year of cultivation a dramatic gain in biomass and inulin was detected in both treated and control plants. Inulin production varied from 113 to 674 kg.ha-1 which corresponds to 43% of the rhizophore dry mass. The composition of fructans was not altered by fertilization, although treated plants had a higher proportion of sucrose and fructans with degree of polymerization 3-8 in the second year of cultivation. The results identify this species as a fructan source similar to other commercial crops and recommend further agronomic studies, aimed at increasing the production of this polysaccharide.

Fructan variation in the rhizophores of Vernonia herbacea (Vell.) Rusby, as influenced by temperature

The influence of climatic variations on fructan content in tropical regions is not well known. The present study deals with the effects of temperature on fructan contents in rhizophores of plants of Vernonia herbacea, a native species from the Brazilian cerrado vegetation. Intact plants and fragmented rhizophores were subjected to different temperatures under natural and controlled environmental conditions. Rhizophores of plants in pre-dormant stage (aerial parts showing some yellowish leaves) presented higher fructan content at 5oC than those kept at 25oC, whereas in dormant plants (aerial parts absent) temperature treatments did not affect fructan contents. Fragmented rhizophores obtained from dormant plants presented higher levels of fructo-polysaccharides at the end of the experiment than at the beginning of the treatment, regardless of the temperature they were stored, whereas fragments obtained from vegetative plants showed a decrease in fructan content under the same treatments. It was concluded that variations observed in fructan contents are related to the phenological state of the plants prior to the treatment rather than to extraneous temperatures they are subjected to during this stage.

Crescimento e conteúdo de frutanos em plantas de Vernonia herbacea (Vell.) Rusby submetidas à adubação nitrogenada

Vernonia herbacea acumula altas concentrações de frutanos do tipo inulina em seus órgãos subterrâneos de reserva, denominados rizóforos. Foi estudada a influência da adubação nitrogenada no crescimento dessa espécie e na produção de frutanos. Plantas obtidas a partir de fragmentos de rizóforos foram cultivadas em área de cerrado onde receberam três doses de nitrogênio: 6, 12 e 24 kg N.ha-1 na forma de (NH4)2SO4. Plantas jovens, com seis meses de idade, mostraram aumento em altura, área foliar e massa seca da parte aérea e dos rizóforos em resposta ao aumento da dose de nitrogênio. Por outro lado, o teor de frutanos decresceu com o aumento da concentração do nitrogênio. Após doze meses de cultivo, o crescimento das plantas foi estimulado apenas nas doses entre 6 e 12 kg.ha-1 de nitrogênio. Os resultados mostraram, também, que o teor de frutanos em plantas jovens está correlacionado negativamente tanto ao crescimento quanto às concentrações crescentes de nitrogênio. Já as plantas tratadas com 24 kg N.ha-1 chegaram a acumular 6,0 g de frutanos por planta, enquanto nas plantas controle, que não receberam N suplementar, esse valor foi de 3,5 g. Assim, aplicação de 24 kg N.ha-1 é recomendada para uma colheita antecipada (12 meses) visando à produção de frutanos.

Fructan production in Vernonia herbacea (Vell.) Rusby is related to adequate nitrogen supply and period of cultivation

Previous studies showed that plants of Vernonia herbacea grown for one year under a limited nitrogen supply presented reduced growth and higher fructan content than plants treated with sufficient nitrogen supply. However, the total fructan production was similar in both plant groups due to the higher biomass of the underground reserve organ in nitrogen-sufficient (N-sufficient) plants. In the present study we aimed to evaluate if a stress growing condition under nitrogen-limited (N-limited) supply, following cultivation under N-sufficient supply would have a positive effect on fructan production. Plants cultivated during one year under N-sufficient supply (10.7 mmol L-1 N-NO3-) were separated in two groups. During the following six months, one group continued to receive the same treatment (control) while the other received an N-limited supply (1.3 mmol L-1 N-NO3-). Growth, photosynthesis and soluble carbohydrates were measured at days 0, 30, 60, 90 and 180. At day 30, plants transferred to N-limited supply showed a significant increase in growth and a decrease in fructan concentration, as a response to the stressing condition. However, in the following period growth was reduced and fructan concentration was increased, confirming the inverse relationship between nitrogen concentration and fructan content. After 180 days, although the fructan concentration in N-limited was significantly higher, with a fructan production of 6.0 g plant¹, the higher gain in rhizophore biomass after 18 months of cultivation in N-sufficient solution led to a fructan production of 8.3 g plant¹, thus surpassing the higher fructan concentration of N-limited plants.

Morfoanatomia comparada dos frutos em desenvolvimento de Vernonia brevifolia Less. e V. herbacea (Vell.) Rusby (Asteraceae)

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Underground systems of Asteraceae species from the Brazilian Cerrado

Underground systems of Asteraceac species from the Brazilian Cerrado. The aim or this study was to describe the underground systems structure, the origin of the shoot buds, and to identify the storage reserve of seven Asteraceae species in order to understand the adaptive strategies of these species in burned Cerrado areas and their higher frequency in the floristic Surveys for herbaceous and undershrub layers of this biome. The subterranean systems types-diffuse underground system, rhizophore, tuberous roots, and xylopodium-varied among the studied species, but all of them have high shoot bud-forming potential. Fructans of the inulin type were detected as storage substances. The presence of these characteristics on the underground systems Could explain the frequency of these species in the floristic surveys from the Brazilian Cerrado, in which Fire and seasonal drought are frequent. It is possible that the belowground bud banks in the Cerrado biome are as important as demonstrated in North American prairies where fire is also frequent. However, in the Cerrado, as demonstrated here, there are types of underground systems other than rhizomes that are usually described in the prairie. Understanding the anatomical features of these plants is one of the steps to appropriate conservation management of these species in the Cerrado.

Produção de concentrados de frutose por inulinases de Penicillium janczewskii e atividade sobre o nível de glicose plasmática em ratos diabéticos

A frutose é utilizada atualmente como adoçante para diabéticos, sendo produzida comercialmente por hidrólise do amido, sob um processo de alto custo que envolve três etapas enzimáticas usando alfa-amilase, amiloglicosidase e glicose isomerase. Uma alternativa para a produção de concentrados de frutose é a hidrólise enzimática da inulina, polímero de frutose encontrado em Asteráceas, incluindo espécies nativas do cerrado. Nesse caso, através de uma única etapa enzimática obtêm-se concentrados com até 95% de frutose. Embora baixos níveis desse açúcar possam ser metabolizados na ausência de insulina, seu efeito sobre a redução do nível de glicose plasmática ainda não está completamente esclarecido. No presente trabalho foi avaliada a ação da frutose produzida por hidrólise da inulina de Vernonia herbacea (Asteraceae) por inulinases de Penicillium janczewskii no nível de glicose plasmática de ratos diabéticos. Dentre os animais diabéticos tratados não foi verificada mortalidade, havendo redução de 46% em média (p<1% no teste de Tukey) dos níveis de glicose plasmática quando comparados aos controles. A considerável produção de inulina em V. herbacea, a alta atividade inulinásica de P. janczewskii e a ausência de mortalidade dos animais tratados demonstram que a frutose obtida por essa via pode ser uma alternativa viável para a produção comercial desse açúcar.

Morfoanatomia comparada dos frutos em desenvolvimento de Vernonia brevifolia Less. e V. herbacea (Vell.) Rusby (Asteraceae)

Neste trabalho, o pericarpo e pápus de Vernonia brevifolia e V. herbacea são descritos morfoanatômica e ontogeneticamente. As espécies são muito similares entre si, possuindo ovário ínfero, bicarpelar, sincárpico e unilocular. Na formação do pericarpo, nenhuma das regiões é multiplicativa. O exocarpo é unisseriado e recoberto por fina cutícula. Observam-se tricomas tectores longos, multicelulares e bisseriados, que persistem até a maturidade; os tricomas glandulares capitados não se mantêm na maturidade. O mesocarpo externo é composto por duas ou três camadas de fibras em V. herbacea e apenas uma em V. brevifolia, acumulando cristais prismáticos. O mesocarpo interno é similar nas espécies, de natureza parenquimática. Feixes vasculares colaterais ocorrem imersos entre o mesocarpo externo e o interno. O endocarpo é unisseriado, havendo duas a três camadas apenas nas regiões de fusão dos carpelos. Na porção apical do pericarpo, observa-se uma protuberância onde se insere o pápus duplo, composto por células lignificadas, algumas projetadas perifericamente. Na base do fruto, ocorre o carpopódio que, em V. herbacea, é mais amplo e abriga drusas e estilóides; em V. brevifolia, o carpopódio é reduzido e não ocorrem cristais. Na maturidade, o pericarpo de ambas as espécies é desidratado, de modo que a maioria das camadas fica colapsada, distinguindo-se apenas algumas células do exocarpo e tricomas tectores, as fibras e cristais mesocárpicos externos e o xilema dos feixes vasculares.

Morfoanatomia da raiz tuberosa de Vernonia oxylepis Sch. Bip. in Mart. ex Baker - Asteraceae

Several native herbaceous and subshrub species native to the Cerrado in Brazil are geophytes, that is, they survive the unfavorable dry season and low temperatures, that sometimes coincide with fire, with only the underground system intact. Vernonia oxylepis is one of these species and the aim of this study was to describe the morpho-anatomy of the tuberous root and bud formation on this structure. The main axis of this root is perpendicular to the soil surface, and from which aerial shoots arise periodically throughout the life cycle. On the upper portion of the root, self-grafting of the shoots occurs. The root stores lipids and fructans, exhibits contraction and produces reparatory buds; adventitious buds arise from proliferated pericycle. These characteristics may be related to adaptation of this species to conditions in the Cerrado.

Cellular origin of chlorinated diketopiperazines in the dictyoceratid sponge Dysidea herbacea (Keller)

The tropical marine sponge Dysidea herbacea (Keller) contains the filamentous unicellular cyanobacterium Oscillatoria spongeliae (Schulze) Hauck as an endosymbiont, plus numerous bacteria, both intracellular and extracellular. Archaeocytes and choanocytes are the major sponge cell types present. Density gradient centrifugation of glutaraldehyde-fixed cells with Percoll as the support medium has been used to separate the cyanobacterial symbiont from the sponge cells on the basis of their differing densities. The protocol also has the advantage of separating broken from intact cells of O. spongeliae. The lighter cell preparations contain archaeocytes and choanocytes together with damaged cyanobacterial cells, whereas heavier cell preparations contain intact cyanobacterial cells, with less than 1% contamination by sponge cells. Gas chromatography/mass spectrometry analysis has revealed that the terpene spirodysin is concentrated in preparations containing archaeocytes and choanocytes, whereas nuclear magnetic resonance analysis of the symbiont cell preparations has shown that they usually contain the chlorinated diketopiperazines, dihydrodysamide C and didechlorodihydrodysamide C, which are the characteristic metabolites of the sponge/symbiont association. However, one symbiont preparation, partitioned by a second Percoll gradient, has been found to be devoid of chlorinated diketopiperazines. The capability to synthesize secondary metabolites may depend on the physiological state of the symbiont; alternatively, there may be two closely related cyanobacterial strains within the sponge tissue.

Sesquiterpene lactones from Vernonia

Some informations about the sesquiterpene lactones isolated from Brazilian species of Vernonia are described, as well the results of tests developed with such compounds with respect to their anti-feedant, molluscicide, antimicrobial and analgesic properties.