Benchtop NMR Coupled with Chemometrics: A Workflow for Unveiling Hidden Drug Ingredients in Honey-Based Supplements.

Recently, benchtop nuclear magnetic resonance (NMR) spectrometers utilizing permanent magnets have emerged as versatile tools with applications across various fields, including food and pharmaceuticals. Their efficacy is further enhanced when coupled with chemometric methods. This study presents an innovative approach to leveraging a compact benchtop NMR spectrometer coupled with chemometrics for screening honey-based food supplements adulterated with active pharmaceutical ingredients. Initially, fifty samples seized by French customs were analyzed using a 60 MHz benchtop spectrometer. The investigation unveiled the presence of tadalafil in 37 samples, sildenafil in 5 samples, and a combination of flibanserin with tadalafil in 1 sample. After conducting comprehensive qualitative and quantitative characterization of the samples, we propose a chemometric workflow to provide an efficient screening of honey samples using the NMR dataset. This pipeline, utilizing partial least squares discriminant analysis (PLS-DA) models, enables the classification of samples as either adulterated or non-adulterated, as well as the identification of the presence of tadalafil or sildenafil. Additionally, PLS regression models are employed to predict the quantitative content of these adulterants. Through blind analysis, this workflow allows for the detection and quantification of adulterants in these honey supplements.

Characterization of fatty acid forms using benchtop NMR in omega-3 oil supplements.

Omega-3 fatty acid supplements, such as fish oil and plant-based oils, have gained popularity because of their potential health benefits. However, the quality and composition of these supplements can vary widely, particularly in terms of the two main forms of omega-3 fatty acids: triacylglycerols (TAGs) and ethyl esters (EEs). TAGs are the natural form found in fish oil but are prone to oxidation, whereas EEs are more stable but less well absorbed by the body. Differentiating between these forms is crucial for assessing the efficacy and tolerance of omega-3 supplements. This article describes a novel approach to differentiate between TAG and EE forms of omega-3 fatty acids in dietary supplements, utilizing a 60-MHz benchtop nuclear magnetic resonance (NMR) spectrometer. The proposed method using 1H and 1H-1H COSY NMR provides a quick and accurate approach to screen the forms of omega-3 fatty acids and evaluate their ratios. The presence of diacylglycerol (DAGs) in some supplements was also highlighted by this method and adds some information about the process used (i.e., esterification/enrichment). The affordability and user-friendliness of benchtop NMR equipment make this method feasible for food processing companies or quality control laboratories. In this study, 24 oil supplements were analyzed using NMR analysis in order to demonstrate the potential of this method for the differentiation of TAG and EE forms in omega-3 supplements.

Discovery of new diaryl ether inhibitors against Mycobacterium tuberculosis targeting the minor portal of InhA.

Tuberculosis (TB) caused by Mycobacterium tuberculosis (Mtb) affects 10 million people each year and the emergence of resistant TB augurs for a growing incidence. In the last 60 years, only three new drugs were approved for TB treatment, for which resistances are already emerging. Therefore, there is a crucial need for new chemotherapeutic agents capable of eradicating TB. Enzymes belonging to the type II fatty acid synthase system (FAS-II) are involved in the biosynthesis of mycolic acids, cell envelope components essential for mycobacterial survival. Among them, InhA is the primary target of isoniazid (INH), one of the most effective compounds to treat TB. INH acts as a prodrug requiring activation by the catalase-peroxidase KatG, whose mutations are the major cause for INH resistance. Herein, a new series of direct InhA inhibitors were designed based on a molecular hybridization approach. They exhibit potent inhibitory activities of InhA and, for some of them, good antitubercular activities. Moreover, they display a low toxicity on human cells. A study of the mechanism of action of the most effective molecules shows that they inhibit the biosynthesis of mycolic acids. The X-ray structures of two InhA/NAD+/inhibitor complexes have been obtained showing a binding mode of a part of the molecule in the minor portal, rarely seen in the InhA structures reported so far.

The POWER saga from 2007 to 2022: An example of a sexual enhancement dietary supplement tainted by different adulterants and still on the market.

Ten POWER dietary supplements, chronologically called tabs, pills then caps, and advertised as 100% natural aphrodisiacs, were analyzed by 1H NMR from 2007 to 2022. They were all tainted by PDE-5 inhibitors. Eight different adulterants were identified (sildenafil (1), sildenafil analogues (6), and vardenafil analogue (1)). Their amounts ranged from 15 to 145 mg/capsule. Four supplements contained at least 100 mg/capsule of PDE-5 inhibitor or analogue, the maximal recommended dose of sildenafil. The nature of the adulterant has changed over time, probably to evade its detection by regulatory agencies routine screening tests. Despite several warnings and/or seizures from several European food and/or health authorities, the dietary supplement POWER is still on sale on the Internet, thus demonstrating the impossibility of controlling this market. Faced with this situation, the consumer should be better informed by establishing at the European level a public database of tainted dietary supplements on the model of that of the US Food and Drug Administration. It should indicate the product name, its photo, the adulterant name, and be easily accessible to everyone.

Quality evaluation of berberine food supplements with high-field and compact 1H NMR spectrometers.

Due to their health benefits, including regulating blood sugar and lowering cholesterol, berberine food supplements (FS) are widely used by consumers. This study aims to evaluate the quality of such products by proposing a new analytical methodology based on low-field NMR. Eighteen berberine FS were analyzed with both conventional (500 MHz) and benchtop (60 MHz) NMR spectrometers. Three quantitative 60 MHz 1H NMR methodologies were performed to determine berberine contents that were compared to those obtained at 500 MHz considered as reference measurements. To make the recording time of the spectra acquired at low field compatible with the requirements of a routine control, the quantification was carried out using a calibration curve established under conditions of incomplete relaxation of BrB protons. This methodology, applied to a test sample of 15 mg of FS, allowed to accurately measure a minimum quantity of berberine of ≈ 10 mg/capsule or tablet in 15 min. Regarding the FS, their labels are often unclear and/or incomplete for the consumer. Moreover, only 56 % of the FS analyzed actually contain the claimed quantity of berberine. The amounts of active they supply per day are extremely variable with only 39 % of the FS delivering a sufficient dose to achieve a hypoglycemic or hypolipidemic effect (1000-1500 mg/day based on literature data). These results show that health authorities should institute much stricter control and regulation over the production, labeling and marketing of berberine-based FS.

Quality assessment of Curcuma dietary supplements: Complementary data from LC-MS and 1H NMR.

Due to the numerous potential health benefits of Curcuma, turmeric dietary supplements (DS) are among the top selling products. To assess the quality of these formulations, thirty Curcuma DS along with five standard Curcuma rhizomes were analyzed with UHPLC-MS and 1H NMR. The chemometric treatment of the UHPLC-MS spectra showed a significant variability of their chemical composition that was confirmed by 1H NMR which allowed the absolute quantification of the Curcuma major bioactive components, i.e. curcuminoids (curcumin, demethoxycurcumin and bisdemethoxycurcumin), and turmerones (aryl-, α- and ß-) as well as piperine, a commonly associated curcumin bioavailability enhancer: respectively 3.5-556, 0-8.6, 0.18-8.1 mg/capsule or tablet. The comparison of the actual and claimed quantities of curcuminoids and piperine showed that 58% of the DS contained the expected amounts of actives.

Synthetic cannabinoids in e-liquids: A proton and fluorine NMR analysis from a conventional spectrometer to a compact one.

The 1H NMR profiles of 13 samples of e-liquids supplied by French customs were obtained with high-field and low-field NMR. The high-field 1H NMR spectra allowed the detection of matrix signals, synthetic cannabinoids, and flavouring compounds. Quantitative results were obtained for the five synthetic cannabinoids detected: JWH-210, 5F-MDMB-PICA, 5F-ADB, 5F-AKB48, and ADB-FUBINACA. Conventional GC-MS analysis was used to confirm compound identification. Fluorine-19 NMR was proposed for the quantification of fluorinated synthetic cannabinoids and was successfully implemented on both 400 MHz and 60 MHz NMR spectrometers. This study based on few examples explored the potentiality of low-field NMR for quantitative and quantitative analysis of synthetic cannabinoids in e-liquids.

Chemometric Analysis of Low-field 1H NMR Spectra for Unveiling Adulteration of Slimming Dietary Supplements by Pharmaceutical Compounds.

The recent introduction of compact or low-field (LF) NMR spectrometers that use permanent magnets, giving rise to proton (1H) NMR frequencies between 40 and 80 MHz, have opened up new areas of application. The two main limitations of the technique are its insensitivity and poor spectral resolution. However, this study demonstrates that the chemometric treatment of LF 1H NMR spectral data is suitable for unveiling medicines as adulterants of slimming dietary supplements (DS). To this aim, 66 DS were analyzed with LF 1H NMR after quick and easy sample preparation. A first PLS-DA model built with the LF 1H NMR spectra from forty DS belonging to two classes of weight-loss DS (non-adulterated, and sibutramine or phenolphthalein-adulterated) led to the classification of 13 newly purchased test samples as natural, adulterated or borderline. This classification was further refined when the model was made from the same 40 DS now considered as representing three classes of DS (non-adulterated, sibutramine-adulterated, and phenolphthalein-adulterated). The adulterant (sibutramine or phenolphthalein) was correctly predicted as confirmed by the examination of the 1H NMR spectra. A limitation of the chemometric approach is discussed with the example of two atypical weight-loss DS containing fluoxetine or raspberry ketone.

Lipo-chitooligosaccharides promote lateral root formation and modify auxin homeostasis in Brachypodium distachyon.

Lipo-chitooligosaccharides (LCOs) are microbial symbiotic signals that also influence root growth. In Medicago truncatula, LCOs stimulate lateral root formation (LRF) synergistically with auxin. However, the molecular mechanisms of this phenomenon and whether it is restricted to legume plants are not known. We have addressed the capacity of the model monocot Brachypodium distachyon (Brachypodium) to respond to LCOs and auxin for LRF. For this, we used a combination of root phenotyping assays, live-imaging and auxin quantification, and analysed the regulation of auxin homeostasis genes. We show that LCOs and a low dose of the auxin precursor indole-3-butyric acid (IBA) stimulated LRF in Brachypodium, while a combination of LCOs and IBA led to different regulations. Both LCO and IBA treatments locally increased endogenous indole-3-acetic acid (IAA) content, whereas the combination of LCO and IBA locally increased the endogenous concentration of a conjugated form of IAA (IAA-Ala). LCOs, IBA and the combination differentially controlled expression of auxin homeostasis genes. These results demonstrate that LCOs are active on Brachypodium roots and stimulate LRF probably through regulation of auxin homeostasis. The interaction between LCO and auxin treatments observed in Brachypodium on root architecture opens interesting avenues regarding their possible combined effects during the arbuscular mycorrhizal symbiosis.

Carotenoid accumulation during tomato fruit ripening is modulated by the auxin-ethylene balance.

BACKGROUND: Tomato fruit ripening is controlled by ethylene and is characterized by a shift in color from green to red, a strong accumulation of lycopene, and a decrease in ß-xanthophylls and chlorophylls. The role of other hormones, such as auxin, has been less studied. Auxin is retarding the fruit ripening. In tomato, there is no study of the carotenoid content and related transcript after treatment with auxin. RESULTS: We followed the effects of application of various hormone-like substances to "Mature-Green" fruits. Application of an ethylene precursor (ACC) or of an auxin antagonist (PCIB) to tomato fruits accelerated the color shift, the accumulation of lycopene, α-, ß-, and δ-carotenes and the disappearance of ß-xanthophylls and chlorophyll b. By contrast, application of auxin (IAA) delayed the color shift, the lycopene accumulation and the decrease of chlorophyll a. Combined application of IAA + ACC led to an intermediate phenotype. The levels of transcripts coding for carotenoid biosynthesis enzymes, for the ripening regulator Rin, for chlorophyllase, and the levels of ethylene and abscisic acid (ABA) were monitored in the treated fruits. Correlation network analyses suggest that ABA, may also be a key regulator of several responses to auxin and ethylene treatments. CONCLUSIONS: The results suggest that IAA retards tomato ripening by affecting a set of (i) key regulators, such as Rin, ethylene and ABA, and (ii) key effectors, such as genes for lycopene and ß-xanthophyll biosynthesis and for chlorophyll degradation.

Transcriptional reprogramming and phenotypical changes associated with growth of Xanthomonas campestris pv. campestris in cabbage xylem sap.

Xylem sap (XS) is the first environment that xylem phytopathogens meet in planta during the early infection steps. Xanthomonas campestris pv. campestris (Xcc), the causative agent of Brassicaceae black rot, colonizes the plant xylem vessels to ensure its multiplication and dissemination. Besides suppression of plant immunity, Xcc has to adapt its metabolism to exploit plant-derived nutrients present in XS. To study Xcc behaviour in the early infection steps, we used cabbage XS to analyse bacterial growth. Mineral and organic composition of XS were determined. Significant growth of Xcc in XS was allowed by the rapid catabolism of amino acids, sugars and organic acids, and it was accompanied by the formation of biofilm-like structures. Transcriptome analysis of Xcc cultivated in XS using cDNA microarrays revealed a XS-specific transcriptional reprogramming compared to minimal or rich media. More specifically, up-regulation of genes encoding transporters such as TonB-dependent transporters (TBDTs), that could be associated with nutrient acquisition and detoxification, was observed. In agreement with the aggregation phenotype, expression of genes important for twitching motility and adhesion was up-regulated in XS. Taken together, our data show specific responses of Xcc to colonization of cabbage XS that could be important for the pathogenesis process and establish XS as a model medium to study mechanisms important for the early infection events.

Aphanomyces euteiches cell wall fractions containing novel glucan-chitosaccharides induce defense genes and nuclear calcium oscillations in the plant host Medicago truncatula.

N-acetylglucosamine-based saccharides (chitosaccharides) are components of microbial cell walls and act as molecular signals during host-microbe interactions. In the legume plant Medicago truncatula, the perception of lipochitooligosaccharide signals produced by symbiotic rhizobia and arbuscular mycorrhizal fungi involves the Nod Factor Perception (NFP) lysin motif receptor-like protein and leads to the activation of the so-called common symbiotic pathway. In rice and Arabidopsis, lysin motif receptors are involved in the perception of chitooligosaccharides released by pathogenic fungi, resulting in the activation of plant immunity. Here we report the structural characterization of atypical chitosaccharides from the oomycete pathogen Aphanomyces euteiches, and their biological activity on the host Medicago truncatula. Using a combination of biochemical and biophysical approaches, we show that these chitosaccharides are linked to ß-1,6-glucans, and contain a ß-(1,3;1,4)-glucan backbone whose ß-1,3-linked glucose units are substituted on their C-6 carbon by either glucose or N-acetylglucosamine residues. This is the first description of this type of structural motif in eukaryotic cell walls. Glucan-chitosaccharide fractions of A. euteiches induced the expression of defense marker genes in Medicago truncatula seedlings independently from the presence of a functional Nod Factor Perception protein. Furthermore, one of the glucan-chitosaccharide fractions elicited calcium oscillations in the nucleus of root cells. In contrast to the asymmetric oscillatory calcium spiking induced by symbiotic lipochitooligosaccharides, this response depends neither on the Nod Factor Perception protein nor on the common symbiotic pathway. These findings open new perspectives in oomycete cell wall biology and elicitor recognition and signaling in legumes.

Cryptogein, a fungal elicitor, remodels the phenylpropanoid metabolism of tobacco cell suspension cultures in a calcium-dependent manner.

Plant cells use calcium-based signalling pathways to transduce biotic and/or abiotic stimuli into adaptive responses. However, little is known about the coupling between calcium signalling, transcriptional regulation and the downstream biochemical processes. To understand these relationships better, we challenged tobacco BY-2 cells with cryptogein and evaluated how calcium transients (monitored through the calcium sensor aequorin) impact (1) transcript levels of phenylpropanoid genes (assessed by RT-qPCR); and (2) derived-phenolic compounds (analysed by mass spectrometry). Most genes of the phenylpropanoid pathway were up-regulated by cryptogein and cell wall-bound phenolic compounds accumulated (mainly 5-hydroxyferulic acid). The accumulation of both transcripts and phenolics was calcium-dependent. The transcriptional regulation of phenylpropanoid genes was correlated in a non-linear manner with stimulus intensity and with components of the cryptogein-induced calcium signature. In addition, calmodulin inhibitors increased the sensitivity of cells to low concentrations of cryptogein. These results led us to propose a model of coupling between the cryptogein signal, calcium signalling and the transcriptional response, exerting control of transcription through the coordinated action of two decoding modules exerting opposite effects.

Strigolactone inhibition of shoot branching.

A carotenoid-derived hormonal signal that inhibits shoot branching in plants has long escaped identification. Strigolactones are compounds thought to be derived from carotenoids and are known to trigger the germination of parasitic plant seeds and stimulate symbiotic fungi. Here we present evidence that carotenoid cleavage dioxygenase 8 shoot branching mutants of pea are strigolactone deficient and that strigolactone application restores the wild-type branching phenotype to ccd8 mutants. Moreover, we show that other branching mutants previously characterized as lacking a response to the branching inhibition signal also lack strigolactone response, and are not deficient in strigolactones. These responses are conserved in Arabidopsis. In agreement with the expected properties of the hormonal signal, exogenous strigolactone can be transported in shoots and act at low concentrations. We suggest that endogenous strigolactones or related compounds inhibit shoot branching in plants. Furthermore, ccd8 mutants demonstrate the diverse effects of strigolactones in shoot branching, mycorrhizal symbiosis and parasitic weed interaction.

Modification of hemicellulose content by antisense down-regulation of UDP-glucuronate decarboxylase in tobacco and its consequences for cellulose extractability.

Extractability and recovery of cellulose from cell walls influences many industrial processes and also the utilisation of biomass for energy purposes. The utility of genetic manipulation of lignin has proven potential for optimising such processes and is also advantageous for the environment. Hemicelluloses, particularly secondary wall xylans, also influence the extractability of cellulose. UDP-glucuronate decarboxylase produces UDP-xylose, the precursor for xylans and the effect of its down-regulation on cell wall structure and cellulose extractability in transgenic tobacco has been investigated. Since there are a number of potential UDP-glucuronate decarboxylase genes, a 490bp sequence of high similarity between members of the family, was chosen for general alteration of the expression of the gene family. Sense and antisense transgenic lines were analysed for enzyme activity using a modified and optimised electrophoretic assay, for enzyme levels by western blotting and for secondary cell wall composition. Some of the down-regulated antisense plants showed high glucose to xylose ratios in xylem walls due to less xylose-containing polymers, while arabinose and uronic acid contents, which could also have been affected by any change in UDP-xylose provision, were unchanged. The overall morphology and stem lignin content of the modified lines remained little changed compared with wild-type. However, there were some changes in vascular organisation and reduction of xylans in the secondary walls was confirmed by immunocytochemistry. Pulping analysis showed a decreased pulp yield and a higher Kappa number in some lines compared with controls, indicating that they were less delignified, although the level of residual alkali was reduced. Such traits probably indicate that lignin was less available for removal in a reduced background of xylans. However, the viscosity was higher in most antisense lines, meaning that the cellulose was less broken-down during the pulping process. This is one of the first studies of a directed manipulation of hemicellulose content on cellulose extractability and shows both positive and negative outcomes.

Introduction of sense constructs of cinnamate 4-hydroxylase (CYP73A24) in transgenic tomato plants shows opposite effects on flux into stem lignin and fruit flavonoids.

Understanding regulation of phenolic metabolism underpins attempts to engineer plants for diverse properties such as increased levels of antioxidant flavonoids for dietary improvements or reduction of lignin for improvements to fibre resources for industrial use. Previous attempts to alter phenolic metabolism at the level of the second enzyme of the pathway, cinnamate 4-hydroxylase have employed antisense expression of heterologous sequences in tobacco. The present study describes the consequences of homologous sense expression of tomato CYP73A24 on the lignin content of stems and the flavonoid content of fruits. An extensive number of lines were produced and displayed four developmental variants besides a normal phenotype. These aberrant phenotypes were classified as dwarf plants, plants with distorted (curly) leaves, plants with long internodes and plants with thickened waxy leaves. Nevertheless, some of the lines showed the desired increase in the level of rutin and naringenin in fruit in a normal phenotype background. However this could not be correlated directly to increased levels of PAL and C4H expression as other lines showed less accumulation, although all lines tested showed increases in leaf chlorogenic acid which is typical of Solanaceous plants when engineered in the phenylpropanoid pathway. Almost all transgenic lines analysed showed a considerable reduction in stem lignin and in the lines that were specifically examined, this was correlated with partial sense suppression of C4H. Although not the primary purpose of the study, these reductions in lignin were amongst the greatest seen in plants modified for lignin by manipulation of structural genes. The lignin showed higher syringyl to coniferyl monomeric content contrary to that previously seen in tobacco engineered for downregulation of cinnamate 4-hydroxylase. These outcomes are consistent with placing CYP73A24 more in the lignin pathway and having a role in flux control, while more complex regulatory processes are likely to be involved in flavonoid and chlorogenic acid accumulation.

Down-regulation of cinnamoyl-CoA reductase in tomato (Solanum lycopersicum L.) induces dramatic changes in soluble phenolic pools.

Health-beneficial properties of many secondary plant metabolites have created much interest into the control of their biosynthesis in crop species. Phenolic compounds, including flavonoids, hydroxycinnamates, and tannins, make up an important group of such phytonutrients. They are formed via the phenylpropanoid pathway and share common precursors with lignin, an insoluble cell wall-associated polymer. In this study, the aim was to reduce lignin biosynthesis so as to enhance the availability of these precursors and, thereby, stimulate the production of soluble, potentially health-promoting, phenolic compounds in tomato (Solanum lycopersicum L.). First two tomato genes encoding cinnamoyl-CoA reductase (CCR), a key enzyme in the formation of lignin monomers, were identified and characterized. Transgenic plants exhibiting a reduced lignin content were subsequently obtained through an RNAi strategy targeting one of these genes. As anticipated, the total level of soluble phenolics was higher in stems and leaves of the transformants as compared with control plants. This was correlated with an increased antioxidant capacity of the corresponding plant extracts. Analysis of the soluble phenolic fraction by HPLC-MS revealed that vegetative organs of CCR down-regulated plants contained higher amounts of chlorogenic acid and rutin, and accumulated new metabolites undetectable in the wild type, such as N-caffeoyl putrescine and kaempferol rutinoside. In fruits, CCR down-regulation triggered the moderate accumulation of two new compounds in the flesh, but the total phenolic content was not affected. Although the prospects of exploiting such a strategy for crop improvement are limited, the results provide further insight into the control of the phenylpropanoid pathway in the Solanaceae.

Metabolite profiling reveals a role for atypical cinnamyl alcohol dehydrogenase CAD1 in the synthesis of coniferyl alcohol in tobacco xylem.

In angiosperms, lignin is built from two main monomers, coniferyl and sinapyl alcohol, which are incorporated respectively as G and S units in the polymer. The last step of their synthesis has so far been considered to be performed by a family of dimeric cinnamyl alcohol dehydrogenases (CAD2). However, previous studies on Eucalyptus gunnii xylem showed the presence of an additional, structurally unrelated, monomeric CAD form named CAD1. This form reduces coniferaldehyde to coniferyl alcohol, but is inactive on sinapaldehyde. In this paper, we report the functional characterization of CAD1 in tobacco (Nicotiana tabacum L.). Transgenic tobacco plants with reduced CAD1 expression were obtained through an RNAi strategy. These plants displayed normal growth and development, and detailed biochemical studies were needed to reveal a role for CAD1. Lignin analyses showed that CAD1 down-regulation does not affect Klason lignin content, and has a moderate impact on G unit content of the non-condensed lignin fraction. However, comparative metabolic profiling of the methanol-soluble phenolic fraction from basal xylem revealed significant differences between CAD1 down-regulated and wild-type plants. Eight compounds were less abundant in CAD1 down-regulated lines, five of which were identified as dimers or trimers of monolignols, each containing at least one moiety derived from coniferyl alcohol. In addition, 3-trans-caffeoyl quinic acid accumulated in the transgenic plants. Together, our results support a significant contribution of CAD1 to the synthesis of coniferyl alcohol in planta, along with the previously characterized CAD2 enzymes.

Laccase down-regulation causes alterations in phenolic metabolism and cell wall structure in poplar.

Laccases are encoded by multigene families in plants. Previously, we reported the cloning and characterization of five divergent laccase genes from poplar (Populus trichocarpa) xylem. To investigate the role of individual laccase genes in plant development, and more particularly in lignification, three independent populations of antisense poplar plants, lac3AS, lac90AS, and lac110AS with significantly reduced levels of laccase expression were generated. A repression of laccase gene expression had no effect on overall growth and development. Moreover, neither lignin content nor composition was significantly altered as a result of laccase suppression. However, one of the transgenic populations, lac3AS, exhibited a 2- to 3-fold increase in total soluble phenolic content. As indicated by toluidine blue staining, these phenolics preferentially accumulate in xylem ray parenchyma cells. In addition, light and electron microscopic observations of lac3AS stems indicated that lac3 gene suppression led to a dramatic alteration of xylem fiber cell walls. Individual fiber cells were severely deformed, exhibiting modifications in fluorescence emission at the primary wall/middle lamella region and frequent sites of cell wall detachment. Although a direct correlation between laccase gene expression and lignification could not be assigned, we show that the gene product of lac3 is essential for normal cell wall structure and integrity in xylem fibers. lac3AS plants provide a unique opportunity to explore laccase function in plants.