Phenolic components and degradability of cell walls of grass and legume species

Cell walls separated from the aerial parts of Lolium multiflorum, Lolium perenne and Phleum pratense contained bound cis and trans ferulic and p-coumaric acids and diferulic acid which were released from the walls by treatment with sodium hydroxide. The total content of these acids in L. multiflorum ranged from 5 to 16.8 mg/g of wall, the trans-ferulic acid content varying between 2.8 and 8.9 mg/g of wall. In addition, small amounts of p-hydroxybenzoic acid were released from senescent leaf blade plus sheath parts. Cell walls from legume species gave much smaller amounts of the acids, the total content of aerial parts of Trifolium pratense being <0.8 mg/g of wall. The degra dability of the cell walls with a commercial cellulase preparation was determined and the water-soluble phenolic compounds released were estimated by UV absorption spectroscopy.     

cis-Jasmone induces accumulation of defence compounds in wheat, Triticum aestivum

Liquid phase extraction (LPE) and vapor phase extraction (VPE) methodologies were used to evaluate the impact of the plant activator, cis-jasmone, on the secondary metabolism of wheat, Triticum aestivum, var. Solstice. LPE allowed the measurement of benzoxazinoids, i.e. 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA), 2-hydroxy-7-methoxy-1,4-benzoxazin-3-one (HMBOA) and 6-methoxy-benzoxazolin-2-one (MBOA), and phenolic acids such as trans-p-coumaric acid, syringic acid, p-hydroxybenzoic acid, vanillic acid and cis- and trans-ferulic acid. Using LPE, a significantly higher level of DIMBOA was found in aerial parts and roots of T. aestivum following treatment with cis-jasmone, when compared with untreated plants. Similar results were obtained for phenolic acids, such as trans-ferulic acid and vanillic acid in roots. Using VPE, it was possible to measure levels of 2-hydroxy-7-methoxy-(2H)-1,4-benzoxazin-3(4H)-one (HBOA), benzoxazolin-2(3H)-one (BOA), ferulic acid, syringic acid and coumaric acid. The levels of HBOA in aerial parts and roots were significantly greater in cis-jasmone treated plants compared to untreated plants. cis-Jasmone is known to be a plant activator in terms of production of defence-related volatile semiochemicals that repel aphids and increase the foraging activity of aphid parasitoids. These results show, for the first time, that cis-jasmone also induces selective production of secondary metabolites that are capable of directly reducing development of pests, diseases and weeds.     

Cell wall-bound trans- and cis-ferulic acids in growing maize roots

The levels of cell wall-bound trans - and cis -ferulic acids in roots of dark grown Zea mays cv. LG11 plants were measured. They were quantified after alkaline hydrolysis of purified cell walls by reversed phase HPLC using trans -cinnamic acid as internal standard. The total amount of ferulic acid ( trans - and cis -ferulic acid) in the root base was 3–4 times higher than in the root tip. Cis -ferulic acid represented between 2% (tip) and 18% (base) of the total ferulic acid content. The total content of trans - and cis -ferulic acids was approximately the same in the stele and the cortex, but the level of cis -ferulic acid in the stele was 5–6 times higher than in the cortex. Trans - and cis -ferulic acid levels as well as the percentage of cis -ferulic acid in the elongation zone were steady between 48 and 96 h after the beginning of germination. Slowly growing roots contained more wall-bound ferulic acids, particularly cis -ferulic acid, than fast growing roots. This relationship was found in the differentiation zone but not in the elongation zone. The importance of cell wall-bound trans - and cis -ferulic acids is discussed in the context of root growth and differentiation.     

Carbohydrates and carbohydrate esters of ferulic acid released from cell walls of Lolium multiflorum by treatment with cellulolytic enzymes

41% of the cell walls from mature leaf blades of Lolium multiflorum were digested by treatment during 14 days with C₁ enzyme (cellulase) which had been purified by gel filtration and ion-exchange chromatography. Cellobiose was the main sugar released from the walls, together with some glucose and higher oligosaccharides. Considerable amounts of carbohydrate esters of ferulic and p-coumaric acids were also released. When the C₁ enzyme was further purified by isoelectric focusing, only 8% of the cell walls were digested. Purified C_x (CM-cellulase) containing β-glucosidase digested 51% of the cell walls in 16 hours: the major component detected in the soluble products was glucose together with some β (1 → 4)-xylobiose, xylose and arabinose. Higher oligosaccharides and carbohydrate esters of ferulic and p-coumaric acids were also present. It was shown that these acids were present in the cell walls mainly in the trans-configuration.     

Microbiologically Catalyzed Enantio- and Diastereoselective Oxidation of Chrysanthemol Stereoisomers to Chrysanthemic Acids

The diastereo- and enantioselective microbial oxidation of a mixture of racemic cis/trans-chrysanthemols to the corresponding stereoisomeric chrysanthemic acids by Aspergillus species is described. Of the three microorganisms which were found capable of oxidizing racemic cis/trans-chrysanthemols, A. ochraceus ATCC 18500 showed complete enantioselectivity for (+)-stereoisomers [(+)-trans-chrysanthemol and (+)-cis-chrysanthemol), whereas A. flavipes ATCC 1030 and ATCC 11013 showed complete enantioselectivity for the (+)-cis-chrysanthemol but a time-dependent enantioselectivity during oxidation of trans-chrysanthemol [oxidation of (+)-trans-chrysanthemol prior to (−)-trans-chrysanthemol]. The diastereoselectivity of all three microorganisms was time dependent, in that the trans-stereoisomers were oxidized prior to the cis-isomers.     

Microbial Resolution of (±)-1 and 2-Decalols

By microorganisms or esterase they produce, (±)-1 and 2-decalyl acetates were asymmetrically hydrolyzed to (?)-1-(R)-trans,cis-1-decalol (IIa), (+)-1-(S)-cis,cis-1-decalol (IIIb), (+)-1-(R)-cis,trans-1-decalol (IVa) and (+)-1-(S)-trans,trans-2-decalol (VIIb), (?)-cis,cis-2-decalol (IXb) with the acetates of their antipodes, whereas the axial acetates of (±)-decalols were scarecely hydrolyzed.     

Involvement of cell wall-bound phenolic acids in decrease in cell wall susceptibility to expansins during the cessation of rapid growth in internodes of floating rice

The cell walls in the elongating zone of submerged floating rice internodes show high susceptibility to expansins. When internode sections corresponding to such an elongation zone were incubated for 24 h under osmotic stress conditions produced by treatment with 100 mM polyethylene glycol 4000 (PEG), the cell wall susceptibility to expansins remained at its initial level, while the susceptibility of internode sections incubated under unstressed conditions decreased considerably during the same period. The contents of polysaccharides and phenolic acids as ferulic, diferulic and p-coumaric acids in the cell walls of internode sections increased substantially under unstressed conditions, but the increases were almost completely prevented by osmotic stress. Ferulic acid applied to internode sections under osmotic stress reduced the susceptibility of the cell walls to expansins and increased the levels of ferulic and diferulic acids in the cell walls, with little effect on the accumulation of polysaccharides. In contrast, applied p-coumaric acid increased the level of p-coumaric acid in the cell walls without a change in the levels of ferulic and diferulic acids but did not reduce the susceptibility to expansins. These results suggest that the deposition of ferulic and diferulic acids is a primary determinant in regulating the reduction of the susceptibility of cell walls to expansins in floating rice internodes.     

Influence of Plant Phenolic Acids on Growth and Cellulolytic Activity of Rumen Bacteria

Isolated rumen bacteria were examined for growth and, where appropriate, for their ability to degrade cellulose in the presence of the hydroxycinnamic acids trans-p-coumaric acid and trans-ferulic acid and the hydroxybenzoic acids vanillic acid and 4-hydroxybenzoic acid. Ferulic and p-coumaric acids proved to be the most toxic of the acids examined and suppressed the growth of the cellulolytic strains Ruminococcus albus, Ruminococcus flavefaciens, and Bacteroides succinogenes when included in a simple sugars medium at concentrations of >5 mM. The extent of cellulose digestion by R. flavefaciens and B. succinogenes but not R. albus was also substantially reduced. Examination of rumen fluid from sheep maintained on dried grass containing 0.51% phenolic acids showed the presence of phloretic acid (0.1 mM) and 3-methoxyphloretic acid (trace) produced by hydrogenation of the 2-propenoic side chain of p-coumaric and ferulic acids, respectively. The parent acids were found in trace amounts only, although they represented the major phenolic acids ingested. Phloretic and 3-methoxyphloretic acids proved to be considerably less toxic than their parent acids. All of the cellulolytic strains (and Streptococcus bovis) showed at least a limited ability to hydrogenate hydroxycinnamic acids, with Ruminococcus spp. proving the most effective. No further modification of hydroxycinnamic acids was produced by the single strains of bacteria examined. However, a considerable shortfall in the recovery of added phenolic acids was noted in media inoculated with rumen fluid. It is suggested that hydrogenation may serve to protect cellulolytic strains from hydroxycinnamic acids.     

Preparation of methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d4

An eight-step synthesis is described which gives an overall yield of ～30% methyl cis-9,cis-12-octadecadienoate-16,16,17,17-d₄. The preparation utilizes easily obtainable starting materials. Tris(triphenylphosphine)chlororhodium (I) catalyst is used for incorporation of the deuterium isotopes. The double bond in the 9 position is created by the Wittig coupling of 1-non-3-enyl-d₄-triphenylphosphonium bromide to methyl 8-formyloctanoate. Various methods for preparation of the intermediate and final products are discussed. Partial argentation resin chromatography was used to remove the ～9% trans/cis, cis/trans, and trans/trans isomers also produced. Analysis of the final product by mass spectrometry (MS) indicated 96%-d₄.     

Flavonoids from Eustoma grandiflorum flower petals

The major flavonoids responsible for flower colours of Eustoma grandiflorum were characterized by TLC, HPLC, spectral and chemical analyses. Anthocyanins were delphinidin 3-rhamnosylgalactoside-5-glucoside and delphinidin 3-galactoside-5-glucoside, each acylated with p-coumaric acid, from the purple cultivar ‘Murasaki no Homare’ and the pelargonidin analogues, each acylated with either p-coumaric or ferulic acids, from the pink cultivar ‘Momo no Mine’. The major flavonol copigments were the 3-rhamnosylgalactoside-7-rhamnoside of myricetin, kaempferol and isorhamnetin and the 3-rhamnosylglucoside-7-rhamnoside of kaempferol and isorhamnetin. Flavonols present acylated with p-coumaric acid were myricetin 3-rhamnosylgalactoside-7-rhamnoside and robinin in both cis and trans forms, and isorhamnetin 3-rhamnosylgalactoside-7-rhamnoside. Robinin also was present acylated with caffeic or ferulic acids. Simulated in vitro colours obtained from the flavonoids present in this germplasm indicated that good blue colours were not attainable. Good blue colours were formed with delphinidin 3-p-coumaroyl-rhamnosylgalactoside-5-glucoside and C-glycosylflavone copigments such as swertisin and isoorientin. These copigments are readily available in other members of the Gentianaceae and this suggests the possibility of genetical engineering endeavours for increasing the colour range of this important new ornamental plant.     

5-hydroxyferulic acid in Zea mays and Hordeum vulgare cell walls

Considerable amounts of esterified E-5-hydroxyferulic acid and very small amounts of esterified E-sinapic acid were detected and identified in cell walls of young Zea mays and Hordeum vulgare, in addition to known E-p-coumaric and ferulic acids. Their relative amounts were determined by peak areas using GC. The ratios of E-p-coumaric-5-hydroxyferulic-sinapic-ferulic acid were 440:46:2:100 in corn, and 37:26:3:100 in barley, respectively.     

The phenolics and a hydrolysable tannin polyphenol oxidase of Medinilla magnifica

The production of viable meristem cultures of Medinilla magnifica has proved to be very difficult. This may be due, in part, to a pronounced ‘browning’ response of the tissues on cutting. For this reason the phenolic compounds and the hydrolysable-tannin polyphenol oxidase from Medinilla were studied. The distribution of the compounds was: simple phenols 19% , flavonoids 5% , hydrolysable tannins 69% , condensed tannins 7%. Amongst the simple phenols and phenolic acids, the following were identified: phloroglucinol, p-hydroxybenzoic acid, vanillic acid, protocatechuic acid, gallic acid (both in free and bound form the most abundant simple phenol), syringic acid, trans-p-coumaric acid, trans-ferulic acid and trans-caffeic acid. No kaempferol or quercetin or their derivatives were detected but condensed tannins are present. Methods for the extraction, fractionation and quantitative determination of phloroglucinol and the phenolic acids, as well as correction factors for losses during the extraction, alkali treatment and derivatization, are presented in a supplementary publication. With regard to the hydrolysable tannin polyphenol oxidase activity of Medinilla stems, the enzyme(s) is rather specific since at neither of its two pH optima (6 and 7) could a classical polyphenol oxidase activity be detected. The enzyme was strongly inhibited by 2-mercaptoethanol. Preliminary experiments have further shown that in addition to the hydrolysable tannins of the tissue, the ferrous ions of the medium, and oxygen together with the hydrolysable tannin polyphenol oxidase could play a role in the browning response. Ways to overcome this difficulty have been suggested.     

Long chain esters of Virola species

The esters of n-fatty acids and ω-hydroxy n-fatty acids of β-sitosterol, D-glucose and ferulic acid (trans and cis) as well as β-sitosterol, fatty acids and β-sitosteryl-β-D-glucoside were isolated from three Virola species and identified by optical data and chemical reactions. A novel series of acidic esters derived from C₂₂C₂₉ ω-hydroxy fatty acids and cis- and trans-ferulic acid is reported for the first time. These compounds also occurred as the corresponding diester 1-monoglycerides whereas the ω-hydroxy acids themselves were also present as the corresponding glucosyl esters.     

Time-course tracer studies on the metabolism of cinnamic acid in Cestrum poeppigii

Time-course tracer studies were performed on the metabolism of trans-cinnamic acid-[3-¹⁴C] and trans-p-coumaric acid-[2-¹⁴C] in the     

Stereochemistry of phytoene

Samples of phytoene (7,8,11,12,7′,8′,11′,12′-octahydro-ψ,ψ-carotene) isolated from higher plant sources, from Neurospora crassa and strains of Phycomyces blakesleeanus and from diphenylamine-inhibited cultures of Rhodospirillum rubrum have been examined by a number of physical methods. All the organisms accumulate predominantly 15-cis phytoene while only traces of all-trans phytoene are normally present. A comparison with synthetic model compounds has shown that the predominant isomer has a trans,cis,trans triene chromophore.     

Symmetry and Cell Division in Raphid Diatoms

The development and final morphology of the valve in raphe-bearingdiatoms exhibit a cryptic lateral polarity, and hence two typesof frustule can be distinguished. In the cis type both valveshave the same orientation; in the trans type they have oppositeorientations. Examination of a variety of taxa suggests thatin all dividing raphid diatoms, both new valves have the sameorientation and so only three types of division are possible:cis; cis + cis, cis trans + trans, trans; cis+trans. The possessionof different combinations of these explains the observed ratiosof cis: trans in different taxa, viz all cis; 1: 2 cis: trans;and roughly 1: 1 cis: trans. The implications of the resultsfor diatom systematics are examined, with special referenceto Navicula Bory. Diatom systematic, diatom valve morphogenesis, cell symmetry, raphe structure     

Langaside,a novel secoiridolactone glycoside derivative from Tachiadenus longiflorus Griseb. (Gentianaceae) formed by a [2+2] cycloaddition reaction

Langaside (1), a secoiridoid lactone glucoside possessing a novel skeleton formed by a [2 + 2] cycloaddition reaction between the secoiridolactone glucoside, 1,9-trans-9,5-cis-sweroside, and p-coumaric acid was isolated from the fruits and flowers of the Malagasy Tachiadenus longiflorus Griseb. (Gentianaceae), alongside another seven known compounds. The structure of langaside was established using HRESIMS, IR and NMR spectroscopy and comparison of experimental and calculated electronic circular dichroism (ECD) spectra. Langaside was screened for its neuritogenic activity against SHSY-5Y cells and anticancer activity against the NCI59 human tumour cell panel but not found to be active.     

The phenolic profile of maize primary cell wall changes in cellulose-deficient cell cultures

Cultured maize cells habituated to grow in the presence of the cellulose synthesis inhibitor dichlobenil (DCB) have a modified cell wall in which the amounts of cellulose are reduced and the amounts of arabinoxylan increased. This paper examines the contribution of cell wall-esterified hydroxycinnamates to the mechanism of DCB habituation. For this purpose, differences in the phenolic composition of DCB-habituated and non-habituated cell walls, throughout the cell culture cycle and the habituation process were characterized by HPLC. DCB habituation was accompanied by a net enrichment in cell wall phenolics irrespective of the cell culture phase. The amount of monomeric phenolics was 2-fold higher in habituated cell walls. Moreover, habituated cell walls were notably enriched in p-coumaric acid. Dehydrodimers were 5–6-fold enhanced as a result of DCB habituation and the steep increase in 8,5′-diferulic acid in habituated cell walls would suggest that this dehydrodimer plays a role in DCB habituation. In summary, the results obtained indicate that cell wall phenolics increased as a consequence of DCB habituation, and suggest that they would play a role in maintaining the functionality of a cellulose impoverished cell wall.     

Effects of Dichloroethene Isomers on the Induction and Activity of Butane Monooxygenase in the Alkane-Oxidizing Bacterium “Pseudomonas butanovora”

We examined cooxidation of three different dichloroethenes (1,1-DCE, 1,2-trans DCE, and 1,2-cis DCE) by butane monooxygenase (BMO) in the butane-utilizing bacterium “Pseudomonas butanovora.” Different organic acids were tested as exogenous reductant sources for this process. In addition, we determined if DCEs could serve as surrogate inducers of BMO gene expression. Lactic acid supported greater rates of oxidation of the three DCEs than the other organic acids tested. The impacts of lactic acid-supported DCE oxidation on BMO activity differed among the isomers. In intact cells, 50% of BMO activity was irreversibly lost after consumption of ~20 nmol mg protein⁻¹ of 1,1-DCE and 1,2-trans DCE in 0.5 and 5 min, respectively. In contrast, a comparable loss of activity required the oxidation of 120 nmol 1,2-cis DCE mg protein⁻¹. Oxidation of similar amounts of each DCE isomer (~20 nmol mg protein⁻¹) produced different negative effects on lactic acid-dependent respiration. Despite 1,1-DCE being consumed 10 times faster than 1,2,-trans DCE, respiration declined at similar rates, suggesting that the product(s) of oxidation of 1,2-trans DCE was more toxic to respiration than 1,1-DCE. Lactate-grown “P. butanovora” did not express BMO activity but gained activity after exposure to butane, ethene, 1,2-cis DCE, or 1,2-trans DCE. The products of BMO activity, ethene oxide and 1-butanol, induced lacZ in a reporter strain containing lacZ fused to the BMO promoter, whereas butane, ethene, and 1,2-cis DCE did not. 1,2-trans DCE was unique among the BMO substrates tested in its ability to induce lacZ expression.     

Effect of ammonium ions on spiramycin biosynthesis in Streptomyces ambofaciens

The physiological significance of trans unsaturated fatty acids, which are constituents of membrane lipids of the phenol-degrading bacterium Pseudomonas putita P8, was studied. The addition of phenol or phenol derivatives to the cells induced the formation of trans unsaturated fatty acids, yielding an overall maximal amount of 41.3% of total fatty acids. The inhibition of de-novo lipid synthesis by cerulenin prevented the change in the degree of saturation in the lipids. However, the cells could still respond to phenols with an amplified conversion of cis into trans unsaturated fatty acids, which is apparently a post-synthesis mechanism of isomerization of the double bond. The cis/trans conversion correlated with growth inhibition induced by toxic concentrations of 4-chlorophenol, whereas only growing cells were able to change the degree of saturation. In cells that were protected against phenol by immobilization in calcium alginate, the conversion of cis into trans fatty acids occurred at higher toxin concentrations compared with free cells. Cells entering the stationary growth phase increased the prodortion of saturated to unsaturated fatty acids but maintained a constant trans/cis ratio.P. putida P8 reacted to an increase or decrease in the growth temperature with an appropriate change in the ratio of saturated to unsaturated fatty acids and in cells inhibited by cerulenin with a change in the trans/cis ratio. This study shows that the physiological role of the cis/trans conversion is probably the regulation of membrane fluidity when the most important mechanism for this, the modification of the degree of saturation, cannot by used by the cells due to inhibition of growth and lipid biosynthesis. Correspondence to: H. Keweloh