Structure of arabinogalactan oligosaccharides derived from arabinogalactan-protein of Coffea arabica instant coffee powder

Arabinogalactan-protein, previously isolated from instant coffee powder of Coffea arabica, has been subjected to partial mild acidic and enzymatic hydrolyses. Separation of obtained mixtures by size exclusion and HPLC chromatographies afforded series of oligosaccharides, structure of which were studied by NMR spectroscopy. Mild acidic hydrolysis afforded oligosaccharides without any αAraf substituent while after enzymatic hydrolysis αAraf was found in di-, tri-, and tetrasaccharides. In all cases αAraf was a terminal substituent linked separately to O3, O6, and to both, O3 and O6, of βGal residues. Identification of di-, tri-, and tetrasaccharides containing α-Araf enabled to distinguish in the ¹H NMR spectra αAraf signals linked to O6 and O3 of neighboring βGal unit. Composition of polymeric residues after enzymatic and mild acidic hydrolyses was also analyzed.     

Ribonucleic acid from coffee beans

A method of extraction of RNA from coffee based on phenol treatment is described. Effectsf of various agents and pH of the extracting buffer on the efficiency of extraction were studied. The best extracting solution is 0·2 M Tris-HCl buffer at pH 7·4 with 1% sodium dodecyl sulphate and 0·05% EDTA. RNA (5–6%) is lost in the tissue residue and 4·6% in the interphase layer. No significant deviation of the spectral characteristics of the RNA solutions obtained from three samples of coffee from that for purified yeast RNA is observed. The purine-pyrimidine ratio for the RNA has been found to be in the range of 1·25–1·38.     

Four new immunomodulating steroidal glycosides from the stems of Stephanotis mucronata

Four new C-21 steroidal glycosides, mucronatosides E (1), F (2), G (3), and H (4), were isolated from the stems of Stephanotis mucronata. Two of them had the rare aglycone with a double bond between C-6 and C-7. Their structures were elucidated on the basis of spectroscopic data and chemical evidence. These isolated compounds were assayed for their immunological activities in vitro against concanavalin A (Con A)- and lipopolysaccharide (LPS)-induced proliferation of mice splenocytes. Compounds 2 and 4 showed immunosuppressive activities in a dose-dependent manner, while compounds 1 and 3 possessed immunoenhancing activities.     

Properties of family 79 beta-glucuronidases that hydrolyze beta-glucuronosyl and 4-O-methyl-beta-glucuronosyl residues of arabinogalactan-protein

The carbohydrate moieties of arabinogalactan-proteins (AGPs), which are mainly composed of Gal, L-Ara, GlcA, and 4-Me-GlcA residues, are essential for the physiological functions of these proteoglycans in higher plants. For this study, we have identified two genes encoding family 79 beta-glucuronidases, designated AnGlcAase and NcGlcAase, in Aspergillus niger and Neurospora crassa, respectively, based on the amino acid sequence of a native beta-glucuronidase purified from a commercial pectolytic enzyme preparation from A. niger. Although the deduced protein sequences of AnGlcAase and NcGlcAase were highly similar, the recombinant enzymes expressed in Pichia pastoris exhibited distinct substrate specificity toward 4-Me-GlcA residues of AGPs: recombinant AnGlcAase (rAnGlcAase) substantially liberated both GlcA and 4-Me-GlcA residues from radish AGPs, whereas recombinant NcGlcAase (rNcGlcAase) activity on the 4-Me-GlcA residues of AGPs was very low. Maximum activity of rAnGlcAase hydrolyzing PNP beta-GlcA occurred at pH 3.0-4.0, whereas the maximum rNcGlcAase activity was at pH 6.0. The apparent Km values of rAnGlcAase were 30.4 microM for PNP beta-GlcA and 422 microM for beta-GlcA-(1-->6)-Gal, and those of rNcGlcAase were 38.3 microM and 378 microM, respectively. Similar to the native enzyme, rAnGlcAase was able to catalyze the transglycosylation of GlcA residues from PNP beta-GlcA to various monosaccharide acceptors such as Glc, Gal, and Xyl. We propose that both AnGlcAase and NcGlcAase are instances of a novel type of beta-glucuronidase with the capacity to hydrolyze beta-GlcA and 4-Me-beta-GlcA residues of AGPs, although they differ significantly in their preferences.     

An immunomodulating pectic polymer from Glinus oppositifolius

An immunomodulating pectic polymer, GOA1, obtained from the aerial parts of the Malian medicinal plant Glinus oppositifolius (L.) Aug. DC. (Aizoaceae) has previously been reported to consist of arabinogalactans type I and II, probably linked to a rhamnogalacturonan backbone. To further elucidate the structure of the polymer GOA1, enzymatic degradation studies and weak acid hydrolysis were performed. Five different glycosidases were used, endo-alpha-D-(1-->4)-polygalacturonase, exo-alpha-L-arabinofuranosidase, endo-alpha-L-(1-->5)-arabinanase, endo-beta-D-(1-->4)-galactanase and exo-beta-D-galactosidase. It appears that GOA1 may contain a structural moiety consisting of a 1,3-linked galactopyranosyl (Galp) main chain with 1,6-linked Galp side chains attached to position 6 of the main chain. The 1,6-linked Galp side chain may be branched in position 3 with arabinofuranosyl (Araf) side chains. A 1,4-linked Galp backbone which might carry side chains or glycosyl units attached to position 3 is also a structural element in the polymer. We further show that GOA1 induce proliferation of B cells and the secretion of IL-1beta by macrophages, in addition to a marked increase of mRNA for IFN-gamma in NK-cells. To elucidate structure-activity relations the native polymer and the digested fractions were tested for complement fixing activity and intestinal immune stimulating activity. The partial removal of Araf residues after enzymatic degradations did not affect the bioactivities, while the acid hydrolysed fraction showed reduced complement fixing activity. A decrease in Araf units, 1,3,6-linked Galp units and a partial hydrolysed rhamnogalacturonan backbone, in addition to a reduction in molecular weight are factors that might have contributed to reduced bioactivity.     

Antitussive activity of an extracellular Rhodella grisea proteoglycan on the mechanically induced cough reflex

A mucilagineous extracellular proteoglycan (EPG) composed of xylose and its 3-O-and 4-O-methyl-derivates (55%), glucuronic acids (17%), rhamnose (14%), galactose (8%), glucose (4%) and minor amounts of other sugars (∼2%) has been isolated from culture medium of Rhodella grisea. A white fluffy algal biopolymer of molecular mass over 8.1 × 10⁵ contained protein (13%), methoxyl (6%), acetyl and succinyl groups. EPG was tested in vivo on mechanically induced cough in non-anaesthetized cats as a test system. The biopolymer showed a cough suppressing effect on laryngopharyngeal type of cough while the cough from tracheobronchial mucous area was slightly or not affected. Further, the intensity of maximal cough efforts from laryngopharyngeal and tracheobronchial parts in expirium and inspirium were influenced slightly only indicating that the expectoration effect was not suppressed by biopolymer application.     

Allantoin has a limited role as nitrogen source in cultured coffee cells

In plants the ureides allantoin (ALN) and allantoic acid (ALA) are formed in purine metabolism, and in some legumes both compounds play an important role as nitrogen (N) sources. In coffee plants, ALN and ALA are catabolites of caffeine degradation. Caffeine is found throughout the coffee plant and in some parts this alkaloid can accumulate up to 4% dry basis. Therefore, caffeine degradation via ureides may make an important contribution to N metabolism of the plant. Using coffee cell suspension as a model we investigated the contribution of ALN as a source of N in coffee. ALN was incorporated in the liquid medium and after 20 d of cultivation, cell mass, NO(3), NH(4), amino acids, soluble proteins, ALN and caffeine were determined in the cells. The activity of glutamine synthetase was also studied. The results showed that despite being taken up by cells ALN does not contribute significantly as a source of N in coffee cells. Compared with mineral N sources, cells grown with ALN-N accumulated much less mass. The inclusion of ALN in the medium caused significant alterations in the content of some N compounds indicating a stress condition.     

Aluminum induces changes in oxidative burst scavenging enzymes in Coffea arabica L. suspension cells with differential Al tolerance

The accumulation of reactive oxygen species (ROS) and concomitant oxidative stress have been considered deleterious consequences of aluminum toxicity. However, several lines of evidence suggest that ROS can function as important signaling molecules in the plant defense system for protection from abiotic stress and the acquisition of tolerance. The role of ROS-scavenging enzymes was assayed in two different coffee cell suspension lines. We treated L2 (Al-sensitive) and LAMt (Al-tolerant) Coffea arabica suspension cells with 100 μM AlCl₃ and observed significant differences in catalase activity between the two cell lines. However, we did not observe any differences in superoxide dismutase or glutathione reductase activity in either cell line following Al treatment. ROS production was diminished in the LAMt cell line. Taken together, these results indicate that aluminum treatment may impair the oxidative stress response in L2 cells but not in LAMt cells. We suggest a possible role for Al-induced oxidative bursts in the signaling pathways that lead to Al resistance and protection from Al toxicity.     

Cytochemistry and immunolocalisation of polysaccharides and proteoglycans in the endosperm of green Arabica coffee beans

Summary. The major noncellulosic polysaccharides and proteoglycans in the coffee bean (Coffea arabica) cell wall are (galacto)mannans and arabinogalactan proteins. Immunological and chemical probes demonstrated that the mannans and arabinogalactan proteins were located continuously across the width of the cell wall, but that the concentration of different structural epitopes within these polysaccharide types showed considerable spatial variation. For the mannans this was implied by the striated pattern demonstrated by fluctuation of the affinity between the mannan monoclonal antibody BGM C6 and (galacto)mannan. The arabinogalactan proteins labelled by the Yariv reagent and the arabinogalactan protein-specific antibody LM2 appeared to be located in all regions of the wall except the middle lamella, but showed some differences in intensity of labelling. However, the LM6 antibody, specific for (15)--arabinan epitopes, was located only as a compact region adjacent to the cell lumen in the body of the endosperm; though, it did label throughout the wall of epidermal cells. This implied that either some of the more highly arabinosylated arabinogalactan proteins contained contiguous 5-arabinosyl residues or that a rhamnogalacturonan which contained 5-arabinosyl residues as side chains existed in the cell wall. In either case the polymers were very restricted in their distribution. A second category of pectin, a homogalacturonan detected by JIM7, was located only in the middle lamella region. The architecture of the wall, as revealed by resin etching, appeared to reflect the chemical heterogeneity, with three distinct physical zones identifiable in a cross section across a single wall.Correspondence and reprints: Nestlé Research Center, Nestec Ltd., Vers-chez-les-Blanc, P.O. Box 44, 1000 Lausanne 26, Switzerland     

Complementary ecosystem services provided by pest predators and pollinators increase quantity and quality of coffee yields

Wild animals substantially support crop production by providing ecosystem services, such as pollination and natural pest control. However, the strengths of synergies between ecosystem services and their dependencies on land-use management are largely unknown. Here, we took an experimental approach to test the impact of land-use intensification on both individual and combined pollination and pest control services in coffee production systems at Mount Kilimanjaro. We established a full-factorial pollinator and vertebrate exclosure experiment along a land-use gradient from traditional homegardens (agroforestry systems), shaded coffee plantations to sun coffee plantations (total sample size = 180 coffee bushes). The exclusion of vertebrates led to a reduction in fruit set of ca 9%. Pollinators did not affect fruit set, but significantly increased fruit weight of coffee by an average of 7.4%. We found no significant decline of these ecosystem services along the land-use gradient. Pest control and pollination service were thus complementary, contributing to coffee production by affecting the quantity and quality of a major tropical cash crop across different coffee production systems at Mount Kilimanjaro.     

Aluminium induces changes in organic acids metabolism in Coffea arabica suspension cells with differential Al-tolerance

The primary Al-tolerance mechanism in plants involves exudation and/or accumulation of specific organic acid species, which form non-phytotoxic complexes with Al³⁺ under physiological conditions. An evaluation was done of the role of organic acids in the tolerance mechanism of a cell suspension line of coffee Coffea arabica that exhibits Al-tolerance (LAMt) but for which the metabolic tolerance mechanism remains unknown. Significant differences existed in malate dehydrogenase and citrate synthase activities (key enzymes in organic acids metabolism) between protein extracts (day 7 of culture cycle) of the L2 (Al-sensitive) and LAMt (Al-tolerant) cells when cell suspensions were treated with 100 μM AlCl₃. HPLC analysis showed that the suspension cells of both lines exudate malate when incubated in a minimal solution but that exudation was not enhanced by treatment with AlCl₃ (100 μM). This is the first study demonstrating that plant Al-tolerance may be associated with down-regulation of malate dehydrogenase and citrate synthase activities.     

Endogenous IAA levels and development of coffee flower buds from dormancy to anthesis

Dormant coffee (Coffea arabica L.) flower buds require water stress to stimulate regrowth. A xylem specific water-soluble dye, azosulfamide, was used to quantify water uptake of buds after their release from dormancy by water stress. In non-stressed flower buds, the rate of water uptake was generally slower and variable compared to stressed flower buds, where the rate of uptake tripled from 1 to 3 days after rewatering and preceded the doubling of fresh and dry weight of buds. Free, ester and amide IAA levels of developing flower buds were measured by gas chromatography-mass spectrometry-selective ion monitoring using an isotope dilution technique with [¹³C₆]IAA as an internal standard. Throughout development, the majority of IAA was present as amide IAA. The proportions of amide and free IAA increased one day after plants were released from water stress, and preceded the doubling of fresh and dry weight. Free and conjugated IAA content per bud remained stable during the period of rapid flower growth until one day before anthesis.Abbreviations FW fresh weight - IAA indole 3-acetic acid - HPLC high performance liquid chromatography - GC-MS-SIM gas chromatography-mass spectrometry selected ion monitoring - NAA naphthalene acetic acid - IBA indole butyric acid     

Boron translocation in coffee trees

Boron deficiency in coffee trees (Coffea arabica) is widespread, however, responses to B fertilizer have been erratic, depending on the year, method, and time of application. A better understanding of B uptake, distribution, and remobilization within the plant is important in developing a rational fertilization program. Field and greenhouse experiments were conducted to study B distribution and remobilization in coffee trees. Boron was provided either in the nutrient solution or sprayed on the leaves of trees grown under adequate or transient B deficiency. There was clear evidence for B translocation via symplast (remobilization) to coffee grains, even in well-nourished plants. When ¹⁰B was present in the nutrient solution during most part of fruit filling, from 33 to 40% of the B found in coffee fruits was absorbed during this period, depending on the timing and duration of the B deficiency treatment. In the field, when B was sprayed once on the leaves, around 4% of the fruit B was derived from the foliar fertilizer. Boron remobilization within coffee trees is limited in well nourished plants, but it can be significant during periods of temporary B deficiency in plants otherwise well nourished with B. The implications of these findings for B fertilization practice, are discussed.     

Aluminium-induced phospholipid signal transduction pathway in Coffea arabica suspension cells and its amelioration by silicic acid

Coffee (Coffea arabica L.) is of economic importance worldwide. Its growth in organic-rich acidic soils is influenced by aluminium such that coffee yield may be impaired. Herein we have used the Al-sensitive C. arabica suspension cell line L2 to analyse the effect of two different Al species on the phosphoinositide signal transduction pathway. Our results have shown that the association of Al with coffee cells was affected by the pH and the form of Al in media. More Al was associated with cells at pH 4.3 than 5.8, whereas when Al was present as hydroxyaluminosilicates (HAS) the association was halved at pH 4.3 and unchanged at pH 5.8. Two signal transduction elements were also evaluated; phospholipase C (PLC) activity and phosphatidic acid (PA) formation. PLC was inhibited ( approximately 50%) when cells were incubated for 2 h in the presence of either AlCl(3) or Al in the form of HAS. PA formation was tested as a short-term response to Al. By way of contrast to what was found for PLC, incubation of cells for 15 min in the presence of AlCl(3) decreased the formation of PA whereas the same concentration of Al as HAS produced no effect upon its formation. These results suggest that Al is capable to exert its effects upon signal transduction as Al((aq))(3+) acting upon a mechanism linked to the phosphoinositide signal transduction pathway.     

Purification and immunomodulating activity of C-phycocyanin from Spirulina platensis cultured using power plant flue gas

In this study, flue gas from a power plant smokestack was applied to culture Spirulina platensis microalgae. Our results will not only achieve the fixation of carbon from the emissions, products can also be produced from the algal biomass that possess physiological activities which could be beneficial to human health. An improved one-step process of chromatography was used to produce high-purity C-phycocyanin with a PC ratios >3.5. Adding different concentrations of ammonium sulfate produced different amounts of C-phycocyanin, with 40% generating the highest yield, followed by 35% and 30% concentrations. Immunomodulating activities were evaluated in the murine macrophage cell line J774A.1. We found that C-phycocyanin had the capability to induce secretion of inflammatory cytokines, including TNF-α, IL-1β, and IL-6, and that these results were not due to contamination with LPS. Treatment with C-phycocyanin also increased proIL-1β and COX-2 protein expression dose-dependently. Furthermore, C-phycocyanin rapidly stimulated phosphorylation of inflammatory-related signaling molecules, including ERK, JNK, p38 and IκB. In addition, although C-phycocyanin decreased production of LPS-induced ROS, it did not inhibit LPS-induced inflammatory cytokines in J774A.1 cells. This is the first report to show that C-phycocyanin exhibited a detailed molecular mechanism of bioactivity by boosting immunomodulation performance.     

Polyphenoloxidase activity in coffee leaves and its role in resistance against the coffee leaf miner and coffee leaf rust

In plants, PPO has been related to defense mechanism against pathogens and insects and this role was investigated in coffee trees regarding resistance against a leaf miner and coffee leaf rust disease. PPO activity was evaluated in different genotypes and in relation to methyl-jasmonate (Meja) treatment and mechanical damage. Evaluations were also performed using compatible and incompatible interactions of coffee with the fungus Hemileia vastatrix (causal agent of the leaf orange rust disease) and the insect Leucoptera coffeella (coffee leaf miner). The constitutive level of PPO activity observed for the 15 genotypes ranged from 3.8 to 88 units of activity/mg protein. However, no direct relationship was found with resistance of coffee to the fungus or insect. Chlorogenic acid (5-caffeoylquinic acid), the best substrate for coffee leaf PPO, was not related to resistance, suggesting that oxidation of other phenolics by PPO might play a role, as indicated by HPLC profiles. Mechanical damage, Meja treatment, H. vastatrix fungus inoculation and L. coffeella infestation caused different responses in PPO activity. These results suggest that coffee resistance may be related to the oxidative potential of the tissue regarding the phenolic composition rather than simply to a higher PPO activity.     

Gibberellic acid causes earlier flowering and synchronizes fruit ripening of coffee

The effect of 100 mgl^–1 gibberellic acid (GA₃) on flowering and fruit ripening synchrony, fruit set, fruit fresh weight, and vegetative growth were studied for different size classes of coffee (Coffea arabica L. cv. Guatemalan) flower buds. Flower buds that were > 4 mm, but not developed to the candle stage at the time of GA₃ treatment, reached anthesis 20 days earlier than the controls, and their development was independent of precipitation, unlike the controls. Fruit from buds that were treated with GA₃ at the candle stage showed earlier and more synchronous ripening than the control, although no differences in flowering were found during anthesis. Buds that were smaller than 4 mm at the time of treatment did not respond to GA₃ applications. Treatment with GA₃ did not affect fruit set, fresh weight of fruits, or vegetative shoot growth.