Anatomy and fructan distribution in vegetative organs of Dimerostemma vestitum (Asteraceae) from the campos rupestres.

Among the compounds stored by plants, several functions are assigned to fructans, such as source of energy and protection against drought and extreme temperatures. In the present study we analyzed the anatomy and distribution of fructans in vegetative organs of Dimerostemma vestitum (Asteraceae), an endemic species from the Brazilian campos rupestres. D. vestitumhas amphistomatic and pubescent leaves, with both glandular and non-glandular trichomes. In the basal aerial stem the medulla has two types of parenchyma, which differ from the apical portion. The xylopodium has mixed anatomical origin. Interestingly, although inulin-type fructans with high degree of polymerization were found in all analyzed organs except the leaves, the highest amount and maximum degree of polymerization were detected in the xylopodium. Inulin sphero-crystals were visualized under polarized light in the medulla and in the vascular tissues mainly in the central region of the xylopodium, which has abundant xylem parenchyma. Secretory structures accumulating several compounds but not inulin were identified within all the vegetative organs. The presence of these compounds, in addition to inulin, might be related to the strategies of plants to survive adverse conditions in a semi-arid region, affected seasonally by water restriction and frequently by fire.

Short-term changes of fructans in ryegrass (Lolium multiflorum 'Lema') in response to urban air pollutants and meteorological conditions.

We investigated whether the fructan content, a storage carbohydrate, of Lolium multiflorum 'Lema' plants grown in a subtropical urban environment characterized by typical diurnal profiles of air pollutants and meteorological conditions changed over the course of a day during different seasons. Plants were collected every 2h on the last day of each two-month seasonal field experiment and separated into shoot (stubble or stubble+leaf blades) and roots for carbohydrate analyses and biomass determination. Diurnal contents of total fructose in the stubbles increased with high temperatures. In the roots, fructose accumulation showed a positive relation with hourly variations of both temperature and particulate matter and a negative relation with irradiance and SO2. Seasonal variation in shoot and root biomasses coincided with the seasonal variation of total fructose and were negatively affected by relative humidity and SO2, respectively. We concluded that hourly changes of fructans over the course of a day may increase the ability of L. multiflorum to tolerate short-term oscillations in weather and air pollution commonly observed in the subtropical urban environment, increasing its efficiency in monitoring air quality.

Production, characterization and application of inulinase from fungal endophyte CCMB 328.

Inulinase (ß-2,1-D- fructan fructanohydrolase), EC 3.2.1.7, targets the ß-2,1 linkage of inulin, a polyfructan consisting of linear ß-2,1 linked fructose, and hydrolyzes it into fructose. This use provides an alternative to produce fructose syrup through the hydrolysis of inulin. The objective of this work was to study the production, characterization and applications of inulinases from the fungal endophyte CCMB 328 isolated from the Brazilian semi-arid region. Response Surface Methodology (RSM) was employed to evaluate the effect of variables (concentration of glucose and yeast extract), on secreted inulinase activities detected in the culture medium and also in the inulin hydrolysis. The results showed that the best conditions for inulinase production by CCMB 328 are 9.89 g / L for glucose and 1.09 g / L for yeast extract. The concentration of 0.20 mol/L of NaCl and KCl increased the activity of inulinase from CCMB 328 by approximately 63% and 37%, respectively. The results also showed that the inulinase has potential for inulin hydrolysis, whose conversion yields roughly 72.48 % for an initial concentration of inulin at 1% (w/v).

Biochemical aspects of a serine protease from Caesalpinia echinata Lam. (Brazilwood) seeds: a potential tool to access the mobilization of seed storage proteins.

Several proteins have been isolated from seeds of leguminous, but this is the first report that a protease was obtained from seeds of Caesalpinia echinata Lam., a tree belonging to the Fabaceae family. This enzyme was purified to homogeneity by hydrophobic interaction and anion exchange chromatographies and gel filtration. This 61-kDa serine protease (CeSP) hydrolyses H-D-prolyl-L-phenylalanyl-L-arginine-p-nitroanilide (K(m) 55.7 µM) in an optimum pH of 7.1, and this activity is effectively retained until 50 °C. CeSP remained stable in the presence of kosmotropic anions (PO(4) (3-), SO(4) (2-), and CH(3)COO(-)) or chaotropic cations (K(+) and Na(+)). It is strongly inhibited by TLCK, a serine protease inhibitor, but not by E-64, EDTA or pepstatin A. The characteristics of the purified enzyme allowed us to classify it as a serine protease. The role of CeSP in the seeds cannot be assigned yet but is possible to infer that it is involved in the mobilization of seed storage proteins.

Changes in cell wall composition and ultrastructure related to desiccation during the seed maturation of Paubrasilia echinata (brazilwood).

Paubrasilia echinata (brazilwood) is an endangered native tree from the Brazilian Atlantic Forest whose seeds tolerate maturation drying, but, unlike classic orthodox seeds, they quickly lose viability after shedding. This work analyzed the biochemical and ultrastructural changes during the maturation of brazilwood seeds, with particular attention to the cell walls and organization of the cellular components. The physiological seed maturity was accompanied by increased starch content and decreased soluble sugars. Arabinose increased considerably and was the predominant cell-wall sugar during maturation, suggesting a rise in arabinans that contribute to greater cell wall flexibility. This increase was consistent with the cell wall infolding observed in the hypocotyl axis and cotyledons during the maturation of brazilwood seeds. Ultrastructural analyses showed changes in the number and distribution of protein bodies and amyloplasts and the reorganization of lipid droplets into large drops or masses during seed desiccation. Our findings demonstrate that brazilwood seeds behave like other orthodox seeds during maturation, performing the cell wall and metabolic changes before the major decline in the seed water content. However, the high vacuolization and reorganization of lipid bodies observed at 65 DAA suggest that cell deterioration occurs to some extent at the end of the maturation period and could be responsible for reducing the longevity of the brazilwood dried seeds.

Hydrolase and fructosyltransferase activities implicated in the accumulation of different chain size fructans in three Asteraceae species.

Fructans are widely distributed in Asteraceae from floras with seasonal growth and are thought to be involved in drought and freezing tolerance, in addition to storage function. Reserve organs of Vernonia herbacea and Viguiera discolor, from the cerrado, and of the perennial herb Smallanthus sonchifolius, endemic to Andean region, store over 80% inulin, with different DP (35, 150, and 15, respectively). The fructan pattern in Asteraceae species could be explained by characteristics of their respective 1-FFTs. Hydrolases and fructosyltransferases from S. sonchifolius, V. herbacea and V. discolor were analyzed in plants at the same environmental conditions. The higher 1-FEH activities found in the species with lower DP, S. sonchifolius and V. herbacea reinforce the hypothesis of the involvement of 1-FEH in fructan profile and suggest that the high DP fructan of V. discolor is a consequence of the low affinity of its 1-FEH to the native long chain inulin. Long term incubation with sucrose suggested that the affinity of 1-FFT of V. discolor for 1-kestose is low when compared to that of V. herbacea. Indeed 1-FFT from V. discolor was shown to be an hDP 1-FFT, preferring longer inulins as acceptors. Conversely, 1-FFT from V. herbacea seems to have a higher affinity for short fructo-oligosaccharides, including 1-kestose, as acceptor substrates. Differences in fructan enzymes of the three Asteraceae provide new information towards the understanding of fructan metabolism and control of carbon flow between low and high DP fructans.

Purification and properties of exo-inulinases from Penicillium janczewskii growing on distinct carbon sources.

Penicillium janczewskii, isolated from the rhizosphere of Vernonia herbacea, grows rapidly on media containing either sucrose or inulin, although inulin more than sucrose induced the production of inulinases. Three different extracellular beta-fructofuranosidases (two inulinases and one invertase) were purified from fungal cultures grown on sucrose or inulin, through precipitation with ammonium sulfate, and anion-exchange, hydrophobic interaction and gel filtration chromatographies. The optimum temperature of the three enzymes was approximately 60 C, optimum pH 4-5.5 and apparent molecular mass of 80 kDa. K(m) and V(max) values determined for invertase on sucrose were respectively 3.7 10(-4) M and 7.9 10(-2) micromol/min/mL, and on inulin 6.3 10(-2) M and 2.09 10(-2) micromol/min/mL. The values of k(m) for the two inulinases were 8.11 10(-4) and 2.62 10(-3) M, being lower for inulin when compared to those obtained for sucrose. The inulinases did not produce oligofructans from inulin, indicating they are primarily exoinulinases. The differences found in inulinase induction patterns when inulin or sucrose was used seem to be related to modifications on the enzyme properties, mainly concerning substrate affinity.

Cell-wall structure and composition of Penicillium janczewskii as affected by inulin.

Penicillium janczewskii, a filamentous fungus isolated from the rhizosphere of Vernonia herbacea (Asteraceae), grows rapidly on media containing either sucrose or inulin as carbon sources. Maintenance of P. janczewskii on inulin medium induces secretion of proteins with high inulinase activity but results in a mycelium that easily collapses and breaks. We evaluated the influence of inulin on fungal growth and colony morphology and on cell-wall structure and composition in comparison with growth and wall characteristics on sucrose-containing medium. P. janczewskii grown on Czapek medium with agar containing 1% (w/v) sucrose or inulin showed differences in the color and morphology of the colonies, although growth rates were similar on both carbon sources. Scanning-electron microscopy revealed that the hyphae from fungus grown on inulin-containing medium are much thinner than those from fungus cultivated on sucrose. Ultrastructural analysis of 5 d old cultures using transmission-electron microscopy indicated significant differences in the cell-wall thickness between hyphae grown on inulin or sucrose media. No differences were detected in the overall carbohydrate and protein contents of cell walls isolated from cultures grown on the two carbon sources. Glycosyl composition analyses showed glucose and galactose as the predominant neutral monosaccharides in the walls but showed no differences attributable to the carbon source. Glycosyl linkage composition analyses indicated a predominance of 3-linked glucopyranosyl in the hyphal walls when P. janczewskii was grown on inulin-containing medium. Our results suggest that growth on inulin as the sole carbon source results in structural changes in the mycelia of P. janczewskii that lead to mycelial walls with altered physical and biological properties.

Structural and metabolic changes in rhizophores of the Cerrado species Chrysolaena obovata (Less.) Dematt. as influenced by drought and re-watering.

The high fructan contents in underground organs of Cerrado species, high water solubility, and fast metabolism of these compounds highlight their role as carbon storage and as an adaptive feature in plants under drought. In this study, we showed that anatomical structure, in association with soluble compounds and metabolism of inulin-type fructans were modified in rhizophores of Crysolaena obovata submitted to water suppression and recovery after re-watering. Plants were subjected to daily watering (control), suppression of watering for 22 days (water suppression) and suppression of watering followed by re-watering after 10 days (re-watered). Plants were collected at time 0 and after 3, 7, 10, 12, 17, and 22 days of treatment. In addition to changes in fructan metabolism, high proline content was detected in drought stressed plants, contributing to osmoregulation and recovery after water status reestablishment. Under water suppression, total inulin was reduced from approx. 60 to 40%, mainly due to exohydrolase activity. Concurrently, the activity of fructosyltransferases promoted the production of short chain inulin, which could contribute to the increase in osmotic potential. After re-watering, most parameters analyzed were similar to those of control plants, indicating the resumption of regular metabolism, after water absorption. Inulin sphero-crystals accumulated in parenchymatic cells of the cortex, vascular tissues and pith were reduced under drought and accompanied anatomical changes, starting from day 10. At 22 days of drought, the cortical and vascular tissues were collapsed, and inulin sphero-crystals and inulin content were reduced. The localization of inulin sphero-crystals in vascular tissues of C. obovata, as well as the decrease of total inulin and the increase in oligo:polysaccharide ratio in water stressed plants is consistent with the role of fructans in protecting plants against drought.

Metabolic and structural changes during early maturation of Inga vera seeds are consistent with the lack of a desiccation phase.

Inga vera, native to South America, is an important leguminous species used for ecological restoration of riparian forests and its seeds are among the most recalcitrant ones described up to date. In this work, we analysed the metabolic profile, cell ultrastructure as well as cell wall polysaccharides of I. vera seeds in order to better understand its maturation, which allows embryo germination without a quiescent phase. Increased amounts of citric, glutamic, pyroglutamic, and aspartic acids from stages I to II (120 and 129 days after flowering (DAF)) corroborate the hypothesis of high metabolism, shifting from fermentative to aerobic respiration at seed maturity. This phase was characterized by an extensive vacuolization of embryonic cells, which also indicate high metabolic activity. The proportion of arabinose in the cell walls of embryonic axis (approx. 20%) was lower than those found in some orthodox seeds (nearly 40%), suggesting that arabinose-containing polysaccharides, which are thought to provide more flexibility to the cell wall during natural drying, are less abundant in I. vera seeds. Taken together, our results provide evidence that the major changes occurred during early stages of seed maturation of I. vera, indicating that the rapid temporary metabolic shift observed between stages I and II may be related to the lack of desiccation phase, moving directly to germination.

Using a Caesalpinia echinata Lam. protease inhibitor as a tool for studying the roles of neutrophil elastase, cathepsin G and proteinase 3 in pulmonary edema.

Acute lung injury (ALI) is characterized by neutrophil infiltration and the release of proteases, mainly elastase (NE), cathepsin G (Cat G) and proteinase 3 (PR3), which can be controlled by specific endogenous inhibitors. However, inhibitors of these proteases have been isolated from different sources, including plants. For this study, CeEI, or Caesalpinia echinata elastase inhibitor, was purified from C. echinata (Brazil-wood) seeds after acetone fractionation, followed by ion exchange and reversed phase chromatographic steps. Characterization with SDS-PAGE, stability assays, amino acid sequencing and alignment with other protein sequences confirmed that CeEI is a member of the soybean Kunitz trypsin inhibitor family. Like other members of this family, CeEI is a 20 kDa monomeric protein; it is stable within a large pH and temperature range, with four cysteine residues forming two disulfide bridges, conserved amino acid residues and leucine-isoleucine residues in the reactive site. CeEI was able to inhibit NE and Cat G at a nanomolar range (with K(i)s of 1.9 and 3.6 nM, respectively) and inhibited PR3 within a micromolar range (K(i) 3.7 µM), leading to hydrolysis of specific synthetic substrates. In a lung edema model, CeEI reduced the lung weight and pulmonary artery pressure until 180 min after the injection of zymosan-activated polymorphonuclear neutrophils. In experiments performed in the presence of a Cat G and PR3, but not an NE inhibitor, lung edema was reduced only until 150 min and pulmonary artery pressure was similar to that of the control. These results confirm that NE action is crucial to edema establishment and progression. Additionally, CeEI appears to be a useful tool for studying the physiology of pulmonary edema and provides a template for molecular engineering and drug design for ALI therapy.

Free cyclitol, soluble carbohydrate and protein contents in Vigna unguiculata and Phaseolus vulgaris bean sprouts.

Seeds sprouts have been used as a good source of basic nutrients and nutraceutical compounds. The high nutritional value of seeds derives from the deposition of compounds during development. However some of these molecules are used in metabolic processes like germination, which leads to a considerable variation in their concentrations once these events are completed. In this work, we investigate the levels of inositols (myo-inositol, D-pinitol and ononitol), soluble carbohydrates and proteins in cotyledons of Phaseolus vulgaris and Vigna unguiculata sprouts. Sprouting increased myo-inositol and glucose content and reduction of raffinose and ononitol was observed. The protein levels increased in P. vulgaris and decreased in V. unguiculata sprouting. The level of sucrose was maintained in both sprouts. D-Pinitol was detected only in quiescent seeds. Our results suggested that bean sprout is an important source of proteins, sucrose, glucose and myo-inositol. Additionally, bean sprouts have low levels of raffinose, an antinutritional compound.

Low temperature and defoliation affect fructan-metabolizing enzymes in different regions of the rhizophores of Vernonia herbacea.

In addition to the storage function, fructans in Asteraceae from floras with seasonal growth have been associated with drought and freezing tolerance. Vernonia herbacea, native of the Brazilian Cerrado, bears underground reserve organs, rhizophores, accumulating inulin-type fructans. The rhizophore is a cauline branched system with positive geotropic growth, with the apex (distal region) presenting younger tissues; sprouting of new shoots occurs by development of buds located on the opposite end (proximal region). Plants induced to sprouting by excision of the aerial organs present increased 1-fructan exohydrolase (1-FEH) activity in the proximal region, while plants at the vegetative stage present high 1-sucrose:sucrose fructosyltransferase (1-SST) in the distal region. The aim of the present study was to analyze how low temperature (5 degrees C) could affect fructan-metabolizing enzymes and fructan composition in the different regions of the rhizophores of intact and excised plants. 1-SST and 1-fructan:fructan fructosyltransferase (1-FFT) were higher in the distal region decreasing towards the proximal region in intact plants at the vegetative phase, and were drastically diminished when cold and/or excision were imposed. In contrast, 1-FEH increased in the proximal region of treated plants, mainly in excised plants subjected to cold. The ratio fructo-oligo to fructo-polysaccharides was significantly higher in plants exposed to low temperature (1.17 in intact plants and 1.64 in excised plants) than in plants exposed to natural temperature conditions (0.84 in intact vegetative plants and 0.58 in excised plants), suggesting that oligosaccharides are involved in the tolerance of plants to low temperature via 1-FEH, in addition to 1-FFT. Principal component analysis indicated different response mechanisms in fructan metabolism under defoliation and low temperature, which could be interpreted as part of the strategies to undergo unfavorable environmental conditions prevailing in the Cerrado during winter.

Sub-tropical urban environment affecting content and composition of non-structural carbohydrates of Lolium multiflorum ssp. italicum cv. Lema.

This study analyzed the relationship between environmental factors, especially air pollution and climatic conditions, and non-structural carbohydrates (NSC) in plants of Lolium multiflorum exposed during 10 consecutive periods of 28 days at a polluted site (Congonhas) and at a reference site in São Paulo city (Brazil). After exposure, NSC composition and leaf concentrations of Al, Fe, Cu, Zn, Pb and Cd were measured. The seasonal pattern of NSC accumulation was quite similar in both sites, but plants at Congonhas showed higher concentrations of these compounds, especially fructans of low and medium degree of polymerization. Regression analysis showed that NSC in plants growing at the polluted site were explained by variations on temperature and leaf concentration of Fe (positive effect), as well as relative humidity and particulate material (negative effect). NSC in the standardized grass culture, in addition to heavy metal accumulation, may indicate stressing conditions in a sub-tropical polluted environment.

Reserve carbohydrates and lipids from the seeds of four tropical tree species with different sensitivity to desiccation

Considering the importance of water content for the conservation and storage of seeds, and the involvement of soluble carbohydrates and lipids for embryo development, a comparative study was carried out among the seeds of Inga vera (ingá), Eugenia uniflora (pitanga), both classified as recalcitrant, and Caesalpinia echinata (brazilwood) and Erythrina speciosa (mulungu), considered as orthodox seeds. Low concentrations of cyclitols (0.3-0.5 percent), raffinose family oligosaccharides (ca. 0.05 percent) and unsaturated fatty acids (0-19 percent) were found in the seeds of ingá and pitanga, while larger amounts of cyclitols (2-3 percent) and raffinose (4.6-13 percent) were found in brazilwood and mulungu, respectively. These results, in addition to higher proportions of unsaturated fatty acids (53-71 percent) in orthodox seeds, suggested that sugars and lipids played important role in water movement, protecting the embryo cell membranes against injuries during dehydration.

Os compostos de reserva das sementes, além de suprirem energia para o desenvolvimento embrionário, desempenham importantes funções relacionadas à proteção celular contra secagem e congelamento. Considerando a importância do teor de água para a conservação e para o armazenamento de sementes de espécies arbóreas e o envolvimento dos carboidratos solúveis e de lipídios nesses processos, foi realizada uma análise comparativa desses compostos em sementes de Inga vera (ingá) e Eugenia uniflora (pitanga), consideradas recalcitrantes, e em Caesalpinia echinata (pau-brasil) e Erythrina speciosa (mulungu), com comportamento ortodoxo. Baixas concentrações de ciclitóis (0,3-0,5 por cento), de oligossacarídeos da série da rafinose (0,05 por cento) e de ácidos graxos insaturados (0-19 por cento) foram encontradas em sementes de ingá e pitanga, enquanto maiores quantidades de ciclitóis (2-3 por cento) e de rafinose (4,6-13 por cento) foram encontradas nas sementes de pau-brasil e mulungu, respectivamente. Estes resultados, juntamente com as altas proporções de ácidos graxos insaturados (53-71 por cento) presentes em sementes de pau-brasil e mulungu, sugerem que os açúcares e os lipídios poderiam desempenhar papel importante no movimento de água, protegendo as membranas celulares embrionárias contra injúrias provocadas durante a dessecação.

Maturation of seeds of Caesalpinia echinata Lam. (Brazilwood), an endangered leguminous tree from the Brazilian Atlantic Forest

Sementes de Caesalpinia echinata Lam. têm sido consideradas como de curta longevidade. Contudo, quando lotes são submetidos à seleção prévia ao armazenamento, é possível conservar sua viabilidade por até 18 meses. Considerando a falta de informações conclusivas quanto à melhor época de colheita dessas sementes, o presente trabalho descreve as modificações que ocorrem durante o processo de maturação das sementes. Flores foram etiquetadas no dia de sua antese e os frutos foram colhidos diretamente dos ramos dos 32 aos 65 dias após a antese (DAA). Sementes dispersas naturalmente por período não superior a 24 horas também foram coletadas, sendo designadas sementes recém-dispersas. As características externas e as dimensões (comprimento, largura e espessura) de frutos e sementes foram registradas. A avaliação da qualidade fisiológica das sementes foi baseada no teor de água, no conteúdo de matéria seca e na germinação. Os resultados sugerem que a maturidade fisiológica das sementes de C. echinata ocorreu por volta de 60-65 DAA, imediatamente antes da deiscência, quando as sementes tinham 30-40% de água.

Woronin bodies in Penicillium Janczewskii Zaleski

Penicillium janczewskii Zaleski is an efficient microorganism for the production of extracellular inulinases and grows rapidly on medium containing sucrose or inulin as carbon source. Maintenance of this filamentous fungus on inulin medium induces secretion of large amounts of inulinases, but the resulting mycelium has thinner cell walls that easily collapse and break. Woronin bodies in hyphae of P. janczewskii grown on sucrose and inulin substrates were observed. No significant differences in the number, location, size and shape of Woronin bodies and level of plugging were observed in cultures of the fungus grown on the two carbon sources. The data indicate that the presence of Woronin bodies in P. janczewskii could not be associated with more easily damaged hyphae, although the function of these organelles in pore plugging has been confirmed.

Fructans and water suppression on intact and fragmented rhizophores of Vernonia herbacea

A vegetação herbácea do cerrado brasileiro apresenta sistemas subterrâneos ricos em frutanos e estacionalmente expostos à restrição hídrica. A fim de avaliar o envolvimento dos frutanos na tolerância à dessecação foram conduzidos experimentos utilizando plantas intactas e fragmentos de rizóforos de Vernonia herbacea. O conteúdo de água nos rizóforos de plantas intactas foi mantido por 30 dias, quando as plantas foram molhadas a cada 7 ou 15 dias, sendo que as plantas permaneceram vivas até 60 dias sem água. O conteúdo total de frutanos, a razão oligo/polissacarídeos e a massa molecular média dos polissacarídeos nessas plantas aumentaram, indicando haver ocorrido despolimerização de moléculas com tamanho intermediário das cadeias. Nos fragmentos apicais de rizóforos submetidos à dessecação, os oligossacarídeos aumentaram em relação aos polissacarídeos, um dia após a excisão dos tecidos tratados. Essas alterações foram facilmente detectadas através dos perfis de oligossacarídeos analisados por HPLC, nos quais a proporção de frutose livre e de frutanos com GP 4 - 10 aumentou acentuadamente. Os resultados indicam que o metabolismo de frutanos está envolvido na tolerância à dessecação de Vernonia herbacea.