Topochemical distribution of lignin and hydroxycinnamic acids in sugar-cane cell walls and its correlation with the enzymatic hydrolysis of polysaccharides

Background: Lignin and hemicelluloses are the major components limiting enzyme infiltration into cell walls. Determination of the topochemical distribution of lignin and aromatics in sugar cane might provide important data on the recalcitrance of specific cells. We used cellular ultraviolet (UV) microspectrophotometry (UMSP) to topochemically detect lignin and hydroxycinnamic acids in individual fiber, vessel and parenchyma cell walls of untreated and chlorite-treated sugar cane. Internodes, presenting typical vascular bundles and sucrose-storing parenchyma cells, were divided into rind and pith fractions. Results: Vascular bundles were more abundant in the rind, whereas parenchyma cells predominated in the pith region. UV measurements of untreated fiber cell walls gave absorbance spectra typical of grass lignin, with a band at 278 nm and a pronounced shoulder at 315 nm, assigned to the presence of hydroxycinnamic acids linked to lignin and/or to arabino-methylglucurono-xylans. The cell walls of vessels had the highest level of lignification, followed by those of fibers and parenchyma. Pith parenchyma cell walls were characterized by very low absorbance values at 278 nm; however, a distinct peak at 315 nm indicated that pith parenchyma cells are not extensively lignified, but contain significant amounts of hydroxycinnamic acids. Cellular UV image profiles scanned with an absorbance intensity maximum of 278 nm identified the pattern of lignin distribution in the individual cell walls, with the highest concentration occurring in the middle lamella and cell corners. Chlorite treatment caused a rapid removal of hydroxycinnamic acids from parenchyma cell walls, whereas the thicker fiber cell walls were delignified only after a long treatment duration (4 hours). Untreated pith samples were promptly hydrolyzed by cellulases, reaching 63% of cellulose conversion after 72 hours of hydrolysis, whereas untreated rind samples achieved only 20% hydrolyzation. Conclusion: The low recalcitrance of pith cells correlated with the low UV-absorbance values seen in parenchyma cells. Chlorite treatment of pith cells did not enhance cellulose conversion. By contrast, application of the same treatment to rind cells led to significant removal of hydroxycinnamic acids and lignin, resulting in marked enhancement of cellulose conversion by cellulases.

Phenolics and Antioxidant Properties of Fruit Pulp and Cell Wall Fractions of Postharvest Banana (Musa acuminata Juss.) Cultivars

Banana fruits are important foods, but there have been very few studies evaluating the phenolics associated with their cell walls. In the present study, (+) catechin, gallocatechin, and (-) epicatechin, as well as condensed tannins, were detected in the soluble extract of the fruit pulp; neither soluble anthocyanidins nor anthocyanins were present. In the soluble cell wall fraction, two hydroxycinnamic acid derivatives were predominant, whereas in the insoluble cell wall fraction, the anthocyanidin delphinidin, which is reported in banana cell walls for the first time, was predominant. Cell wall fractions showed remarkable antioxidant capacity, especially after acid and enzymatic hydrolysis, which was correlated with the total phenolic content released after the hydrolysis of the water-insoluble polymer, but not for the posthydrolysis water-soluble polymer. The acid hydrolysis released various monosaccharides, whereas enzymatic hydrolysis released one peak of oligosaccharides. These results indicate that banana cell walls could be a suitable source of natural antioxidants and that they could be bioaccessible in the human gut.

Seed Cell Wall Storage Polysaccharides: Models to Understand Cell Wall Biosynthesis and Degradation

Cell wall storage polysaccharides (CWSPs) are found as the principal storage compounds in seeds of many taxonomically important groups of plants. These groups developed extremely efficient biochemical mechanisms to disassemble cell walls and use the products of hydrolysis for growth. To accumulate these storage polymers, developing seeds also contain relatively high activities of noncellulosic polysaccharide synthases and thus are interesting models to seek the discovery of genes and enzymes related to polysaccharide biosynthesis. CWSP systems offer opportunities to understand phenomena ranging from polysaccharide deposition during seed maturation to the control of source-sink relationship in developing seedlings. By studying polysaccharide biosynthesis and degradation and the consequences for cell and physiological behavior, we can use these models to develop future biotechnological applications.

Cell wall polysaccharides from cell suspension cultures of the Atlantic Forest tree Rudgea jasminoides (Rubiaceae)

Rudgea jasminoides (Rubiaceae) is a tropical tree species native of the Atlantic Forest in the south of Brazil. Previous studies with leaf cell walls of R. jasminoides showed a different proportion of cross-linked glycans compared to what is usually reported for eudicots. However, due to the difficulties of working with whole plant organs, cell suspensions of R. jasminoides, consisting of predominantly undifferentiated cells with mainly primary cell walls, were used to examine cell walls and extracellular soluble polysaccharides (EP) released into the culture medium. Sugar composition and linkage analysis showed homogalacturonans, xylogalacturonans and arabinogalactans to be the predominant EP. In the cell wall, homogalacturonans and arabinogalactans are the major pectins, and xyloglucans and xylans are the major cross-linking glycans. The presence of xylogalacturonans in the R. jasminoides cell cultures seems to be related to the occurrence of a homogeneous cell suspension with loosely attached cells. Although all alkali extractions from the cell walls yielded amounts of xyloglucan that exceed those of the xylans, the latter was found in a proportion that is higher than what has been usually reported for primary cell walls of most eudicots. The xyloglucan from cell walls of cell suspension cultures of R. jasminoides has low fucosylation levels and high proportion of galactosyl residues, a branching pattern commonly found in storage cell-wall xyloglucans.

Influence of palm fatty acid distillate on rumen degradability and protozoa population in buffaloes

Oil and fat as energy sources at low cost are relevant in ruminant nutrition. The aim of this study was to evaluate the effects of palm fatty acid distillate (PFAD) on the degradability and ciliate protozoa population in buffalo. Four rumen fistulated buffaloes were fed a basal diet in a Latin square (4x4) design trial. Treatments were designed with four of different levels of PFAD added directly into the rumen: 0; 200; 420 and 500 g/animal/d. High levels of PFAD (420 and 500 g/d) promoted higher degradation of the soluble fraction and lower in potentially degradable fraction of dry matter (DM) and neutral detergent fibre (NDF) with lower values of potential and effective degradability in two evaluated grasses, bermudagrass and brachiariagrass. Significant decreases in the total number of protozoa/mL of rumen content, Entodinium and ciliates belonging to subfamily Diplodiniinae were observed at higher level of PFDA addition in the rumen. Also, Epidinium and Holotrich ciliates disappeared from the rumen. Significant correlations were observed of the ciliate concentration and composition as a function of dietary lipids content. Entodinium composition increased from 68.0% to 99.6% and Diplodiniinae reduced from 30.4% to 0.4% with increasing PFAD level indicating higher fat toxicity effect on the Diplodiniinae ciliates than Entodinium species and direct action of the larger ciliates on the fibre degradation.

Utilization of pineapple stem juice to enhance enzyme-hydrolytic efficiency for sugarcane bagasse after an optimized pre-treatment with alkaline peroxide

The enzymatic hydrolysis of sugarcane bagasse was investigated by treating a peroxide-alkaline bagasse with a pineapple stem juice, xylanase and cellulase. Pre-treatment procedures of sugarcane bagasse with alkaline hydrogen peroxide were evaluated and compared. Analyses were performed using 2(4) factorial designs, with pre-treatment time, temperature, magnesium sulfate and hydrogen peroxide concentration as factors. The responses evaluated were the yield of cellobiose and glucose released from pretreated bagasse after enzymatic hydrolysis. The results show that the highest enzymatic conversion was obtained for bagasse using 2% hydrogen peroxide at 60 degrees C for 16 h in the presence of 0.5% magnesium sulfate. Bagasse (5%) was treated with pineapple stem extract, which contains mixtures of protease and esterase, in combination with xylanase and cellulase. It was observed that the amount of glucose and cellobiose released from bagasse increased with the mixture of enzymes. It is believed that the enzymes present in pineapple extracts are capable of hydrolyze specific linkages that would facilitate the action of digesting plant cell walls enzymes. This increases the amount of glucose and other hexoses that are released during the enzymatic treatment and also reduces the amount of cellulase necessary in a typical hydrolysis. (C) 2010 Elsevier Ltd. All rights reserved.

Chitinolytic activity of endophytic Streptomyces and potential for biocontrol

Biological sources for the control of plant pathogenic fungi remain an important objective for sustainable agricultural practices. Actinomycetes are used extensively in the pharmaceutical industry and agriculture owing to their great diversity in enzyme production. In the present study, therefore, we evaluated chitinase production by endophytic actinomycetes and the potential of this for control of phytopathogenic fungi. Endophytic Streptomyces were grown on minimum medium supplemented with chitin, and chitinase production was quantified. The strains were screened for any activity towards phytopathogenic fungi and oomycetes by a dual-culture in vitro assay. The correlation between chitinase production and pathogen inhibition was calculated and further confirmed on Colletotrichum sublineolum cell walls by scanning electron microscopy. This paper reports a genetic correlation between chitinase production and the biocontrol potential of endophytic actinomycetes in an antagonistic interaction with different phytopathogens, suggesting that this control could occur inside the host plant. A genetic correlation between chitinase production and pathogen inhibition was demonstrated. Our results provide an enhanced understanding of endophytic Streptomyces and its potential as a biocontrol agent. The implications and applications of these data for biocontrol are discussed.

SAGE transcript profiling of the juvenile cambial region of Eucalyptus grandis

Despite the importance of Eucalyptus spp. in the pulp and paper industry, functional genomic approaches have only recently been applied to understand wood formation in this genus. We attempted to establish a global view of gene expression in the juvenile cambial region of Eucalyptus grandis Hill ex Maiden. The expression profile was obtained from serial analysis of gene expression (SAGE) library data produced from 3- and 6-year-old trees. Fourteen-base expressed sequence tags (ESTs) were searched against public Eucalyptus ESTs and annotated with GenBank. Altogether 43,304 tags were generated producing 3066 unigenes with three or more copies each, 445 with a putative identity, 215 with unknown function and 2406 without an EST match. The expression profile of the juvenile cambial region revealed the presence of highly frequent transcripts related to general metabolism and energy metabolism, cellular processes, transport, structural components and information pathways. We made a quantitative analysis of a large number of genes involved in the biosynthesis of cellulose, pectin, hemicellulose and lignin. Our findings provide insight into the expression of functionally related genes involved in juvenile wood formation in young fast-growing E. grandis trees.

Adsorption of Ni(2+), Zn(2+) and Pb(2+) onto dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris. I. Single metal systems

Adsorption of Ni(2+), Zn(2+) or Pb(2+) by dry biomass of Arthrospira (Spirulina) platensis and Chlorella vulgaris was studied as a function of contact time and initial metal concentration. The zero point of charge calculated for these biosorbents (pH(zpc) 4.0 and 3.4, respectively) and additional pH tests suggested the use of pH in the range 5.0-5.5 for the experiments. The equilibrium isotherms were evaluated in terms of maximum sorption capacity and sorption affinity. The pseudo first and second order kinetic models were considered to interpret the experimental data, and the latter best described the adsorption system. Both the Freundlich and Langmuir models were shown to well describe the sorption isotherms, thus suggesting an intermediate mono/multilayer sorption mechanism. Compared to A. platensis (q(e) = 0.354, 0.495 and 0.508 mmol g(-1) for Ni(2+), Pb(2)+ and Zn(2+), respectively), C. vulgaris behaved as a better biosorbent because of higher equilibrium sorption capacity (q(e) = 0.499, 0.634 and 0.664 mmol g(-1), respectively). The removal efficiency decreased with increasing metal concentration, pointing out a passive adsorption process involving the active sites on the surface of the biomasses. The FT-IR spectroscopy evidenced that ions removal occurred mainly by interaction between metal and carboxylate groups present on both the cell walls. (C) 2011 Elsevier B.V. All rights reserved.

Novel reports of glands in Neotropical species of Indigofera L. (Leguminosae, Papilionoideae)

By considering controversial discussions in the literature with regard to gland denomination in Indigofera species, as well as the taxonomic value of secretory structures in Leguminosae, we aim to morphologically detail glands that had been previously observed in I. microcarpa and I. sabulicola, and to investigate the occurrence of glands in vegetative and reproductive organs of other six Neotropical species that belong to the genus. Glands analyzed through scanning electronic microscopy (SEM) in combination with anatomic analyses correspond to secretory trichomes that Lire classified into seven types. Main variations in relation to types occurred with regard to head shape and peduncle size. Trichome heads were multicellular, with a thin cuticle. Hollow heads with conspicuous inner space characterized only one type (type I); the other trichome types had massive heads. Peduncles, which varied from biseriate to multiseriate, had thick, pecto-cellulosic cell walls. Trichomes were found on sterns, stipules, petioles, rachis, petiolules, leaflets, bracteoles, sepals, standards and fruits, more commonly along the margins. Each of the eight Indigofera species analyzed had at least two different trichome types out of the seven types that occurred in reproductive and vegetative organs of these taxa. Various types of secretory trichomes were found in I. campestris, I. lespedezioides, I. microcarpa, I. spicata. I. Suffruticosa and I. truxillensis. Stems and rachis were the vegetative organs in which a greater variety of trichomes occurred, and sepals were parts of reproductive organs with the same status. Five out of the seven secretory trichome types occurred on both vegetative and reproductive organs. Distribution and gland types differed between species and these gland distribution patterns can be used as diagnostic characters. Reports of glands in Indigofera campestris, I. hirsuta, I. lepedezioides, I. suffruticosa, I. spicata and I. truxillensis, their recognition as secretory trichomes. and the morphological variety of types found for such trichomes are novel data for Indigofera. (C) 2008 Elsevier GmbH. All rights reserved.

Effect of a highly concentrated lipopeptide extract of Bacillus subtilis on fungal and bacterial cells

Lipopeptides produced by Bacillus subtilis are known for their high antifungal activity. The aim of this paper is to show that at high concentration they can damage the surface ultra-structure of bacterial cells. A lipopeptide extract containing iturin and surfactin (5 mg mL-1) was prepared after isolation from B. subtilis (strain OG) by solid phase extraction. Analysis by atomic force microscope (AFM) showed that upon evaporation, lipopeptides form large aggregates (0.1-0.2 mu m2) on the substrates silicon and mica. When the same solution is incubated with fungi and bacteria and the system is allowed to evaporate, dramatic changes are observed on the cells. AFM micrographs show disintegration of the hyphae of Phomopsis phaseoli and the cell walls of Xanthomonas campestris and X. axonopodis. Collapses to fungal and bacterial cells may be a result of formation of pores triggered by micelles and lamellar structures, which are formed above the critical micelar concentration of lipopeptides. As observed for P. phaseoli, the process involves binding, solubilization, and formation of novel structures in which cell wall components are solubilized within lipopeptide vesicles. This is the first report presenting evidences that vesicles of uncharged and negatively charged lipopeptides can alter the morphology of gram-negative bacteria.

Use of lignin extracted from different plant sources as standards in the spectrophotometric acetyl bromide lignin method

A nongravimetric acetyl bromide lignin (ABL) method was evaluated to quantify lignin concentration in a variety of plant materials. The traditional approach to lignin quantification required extraction of lignin with acidic dioxane and its isolation from each plant sample to construct a standard curve via spectrophotometric analysis. Lignin concentration was then measured in pre-extracted plant cell walls. However, this presented a methodological complexity because extraction and isolation procedures are lengthy and tedious, particularly if there are many samples involved. This work was targeted to simplify lignin quantification. Our hypothesis was that any lignin, regardless of its botanical origin, could be used to construct a standard curve for the purpose of determining lignin concentration in a variety of plants. To test our hypothesis, lignins were isolated from a range of diverse plants and, along with three commercial lignins, standard curves were built and compared among them. Slopes and intercepts derived from these standard curves were close enough to allow utilization of a mean extinction coefficient in the regression equation to estimate : lignin concentration in any plant, independent of its botanical origin. Lignin quantification by use of a common regression equation obviates the steps of lignin extraction, isolation, and standard curve construction, which substantially expedites the ABL method. Acetyl bromide lignin method is a fast, convenient analytical procedure that may routinely be used to quantify lignin.

Morphology and anatomy of inflorescence and inflorescence axis in Paepalanthus sect. Diphyomene Ruhland (Eriocaulaceae, Poales) and its taxonomic implications

Paepalanthus sect. Diphyomene has inflorescences arranged in umbels. The underlying bauplan seems however to be more complex and composed of several distinct subunits. Despite appearing superficially very similar, the morphology and anatomy of the inflorescences can supply useful information for the understanding of the phylogeny and taxonomy of the group. Inflorescences of Paepalanthus erectifolius, Paepalanthus flaccidus, Paepalanthus giganteus, and Paepalanthus polycladus were analyzed in regard to branching pattern and anatomy. In P. erectifolius, P. giganteus and P. polycladus the structure is a tribotryum, with terminal dibotryum, and with pherophylls bearing lateral dibotrya. In P. flaccidus, the inflorescence is a pleiobotryum, with terminal subunit, and without pherophylls. Secondary inflorescences may occur in all species without regular pattern. Especially when grown in sites without a pronounced seasonality, the distinction between enrichment zone (part of the same inflorescence) and new inflorescences may be obscured. The main anatomical features supplying diagnostic and phylogenetic information are as follows: (a) in the elongated axis, the thickness of the epidermal cell walls and the cortex size; (b) in the bracts, the quantity of parenchyma cells (c) in the scapes, the shape and the presence of a pith tissue. Therefore, P. sect. Diphyomene can be divided in two groups; group A is represented by P. erectifolius, P. giganteus and P. polycladus, and group B is represented by P. flaccidus. The differentiation is based in both, inflorescence structure and anatomy. Group A presents a life cycle and anatomical features similar to species of Actinocephalus. Molecular trees also point that these two groups are closely related. However, inflorescence morphology and blooming sequence are different. Species of group B present an inflorescence structure and anatomical features shared with many genera and species in Eriocaulaceae. The available molecular and morphology based phylogenies still do not allow a precise allocation of the group in the bulk of basal species of Paepalanthus collocated in P. sect. Variabiles. The characters described and used here supply however important information towards this goal. (C) 2009 Elsevier GmbH. All rights reserved.

Anatomical and biochemical changes in the composition of developing seed coats of annatto (Bixa orellana L.)

Seeds of Bixa orellana (L.) have a sclerified palisade cell layer, which constitutes a natural barrier to water uptake. In fact, newly fully developed B. orellana seeds are highly impermeable to water and thereby dormant. The purpose of this work is to investigate, from a developmental point of view, the histochemical and physical changes in the cell walls of the seed coat that are associated with the water impermeability. Seed coat samples were analyzed by histochemical and polarization microscopy techniques, as well as by fractionation/HPAEC-PAD. For histochemical analysis the tissue samples were fixed, dehydrated, embedded in paraffin and the slides were dewaxed and tested with appropriate stains for different cell wall components. Throughout the development of B. orellana seeds, there was a gradual thickening of the seed coat at the palisade region. This thickening was due to the deposition of cellulose and hemicelluloses in the palisade layer cell walls, which resulted in a highly water impermeable seed coat. The carbohydrate composition of the cell walls changed dramatically at the late developmental stages due to the intense deposition of hemicelluloses. Hemicelluloses were mainly deposited in the outer region of the palisade layer cell walls and altered the birefringent pattern of the walls. Xylans were by far the most abundant hemicellulosic component of the cell walls. Deposition of cellulose and hemicelluloses, especially xylans, could be responsible for the impermeability to water observed in fully developed B. orellana seeds.

Melatonin inhibits LPS-induced NO production in rat endothelial cells

Endothelial cells produce NO by activation of constitutive nitric oxide synthase (NOS) and transcription of inducible NOS (iNOS). We have previously shown that melatonin, in the nanomolar range, inhibits activation of constitutive NOS, and in the present paper, we evaluated whether it could interfere with the expression of iNOS, which is activated by lipopolysaccharide (LPS), a major component of gram-negative bacteria cell walls. Primary cultures of rat endothelial cells were loaded with fluorescent probe for NO detection. Nuclear factor kappa B (NF-kappa B) translocation in endothelial cells elicited by LPS was measured by electromobility shift assay, and the vasodilation of aortic rings was accessed by recording isometric contraction. Melatonin in a micromolar but not in a nanomolar range inhibits the NO production induced by LPS. This effect is not dependent on the activation of G protein-coupled melatonin receptors. The nuclear NF-kappa B translocation is a process necessary for iNOS transcription, and melatonin also inhibits its translocation. LPS induced vasodilation only in endothelium-intact aortic rings, and melatonin (10 mu m) inhibits the vasodilation. Here, we show that concentrations compatible with nocturnal melatonin surge (nm) did not interfere with the activity of iNOS. Considering that micromolar melatonin concentrations could be locally achieved through production by activated immune competent cells, extra-pineal melatonin could have a protective effect against tissue injury. We propose that melatonin blocked the LPS-induced vasodilation by inhibiting the NF-kappa B pathway. Finally, we propose that the effect of melatonin on vascular reactivity is one of the mechanisms that underlies the protective effect of this indolamine against LPS.