Profiling of Essential Oils from the Leaves of Pistacia lentiscus Collected in the Algerian Region of Tizi-Ouzou: Evidence of Chemical Variations Associated with Climatic Contrasts between Littoral and Mountain Samples.

Leaves of Pistacia lentiscus were collected from two Algerian sites in the mountains and the littoral of the Tizi-Ouzou region. The harvest was conducted in four consecutive seasons on the same selected set of trees. Essential oils (EOs) were extracted by hydrodistillation; then, they were analyzed by gas chromatography coupled mass spectrometry (GC-MS). Forty-seven constituents could be detected and quantified, including α-pinene (2-13%), ß-caryophyllene (8-25%), ß-myrcene (0.3-19%), bornyl acetate (0.8-7%), δ-cadinene (3-8%), bisabolol (1-9%), ß-pinene (0.9-7%), caryophyllene oxide (4-9%), and α-cadinol (3-11%). Antioxidant (AOx) activities of the EOs were assessed by ferric reducing antioxidant power (FRAP), 2,2-diphenyl-1-picrylhydrazyl (DPPH), and 2,2'-azino-bis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS) assays. Significant differences in EO composition and AOx activities appeared dependent on the season and the site. Variations of AOx activities were significant for the FRAP and ABTS tests but not for DPPH. Characterization of the leaf fatty acyl (FA) profiles was performed by GC-MS. Variability appeared according to season and altitude. Polyunsaturated fatty acids levels were high (27-55%) at the coldest date and place. The levels of linolenic acyl in the leaves were significantly correlated with bisabolol levels in the EOs (Spearman's correlation coefficient: 0.818). Such results will be useful for the sustainable local valorization of wild P. lentiscus. These data also open new routes for further studies on terpenoid biosynthesis using correlation networks and fluxomic approaches.

Origin and Function of Structural Diversity in the Plant Specialized Metabolome.

The plant specialized metabolome consists of a multitude of structurally and functionally diverse metabolites, variable from species to species. The specialized metabolites play roles in the response to environmental changes and abiotic or biotic stresses, as well as in plant growth and development. At its basis, the specialized metabolism is built of four major pathways, each starting from a few distinct primary metabolism precursors, and leading to distinct basic carbon skeleton core structures: polyketides and fatty acid derivatives, terpenoids, alkaloids, and phenolics. Structural diversity in specialized metabolism, however, expands exponentially with each subsequent modification. We review here the major sources of structural variety and question if a specific role can be attributed to each distinct structure. We focus on the influences that various core structures and modifications have on flavonoid antioxidant activity and on the diversity generated by oxidative coupling reactions. We suggest that many oxidative coupling products, triggered by initial radical scavenging, may not have a function in se, but could potentially be enzymatically recycled to effective antioxidants. We further discuss the wide structural variety created by multiple decorations (glycosylations, acylations, prenylations), the formation of high-molecular weight conjugates and polyesters, and the plasticity of the specialized metabolism. We draw attention to the need for untargeted methods to identify the complex, multiply decorated and conjugated compounds, in order to study the functioning of the plant specialized metabolome.

Maize specialized metabolome networks reveal organ-preferential mixed glycosides.

Despite the scientific and economic importance of maize, little is known about its specialized metabolism. Here, five maize organs were profiled using different reversed-phase liquid chromatography-mass spectrometry methods. The resulting spectral metadata, combined with candidate substrate-product pair (CSPP) networks, allowed the structural characterization of 427 of the 5,420 profiled compounds, including phenylpropanoids, flavonoids, benzoxazinoids, and auxin-related compounds, among others. Only 75 of the 427 compounds were already described in maize. Analysis of the CSPP networks showed that phenylpropanoids are present in all organs, whereas other metabolic classes are rather organ-enriched. Frequently occurring CSPP mass differences often corresponded with glycosyl- and acyltransferase reactions. The interplay of glycosylations and acylations yields a wide variety of mixed glycosides, bearing substructures corresponding to the different biochemical classes. For example, in the tassel, many phenylpropanoid and flavonoid-bearing glycosides also contain auxin-derived moieties. The characterized compounds and mass differences are an important step forward in metabolic pathway discovery and systems biology research. The spectral metadata of the 5,420 compounds is publicly available (DynLib spectral database, https://bioit3.irc.ugent.be/dynlib/).

Development of a Pteris vittata L. compound database by widely targeted metabolomics profiling.

The objective of this work was the development of a detailed, extensive and reliable database of the metabolomes of P. vittata. Using an ultra-performance liquid chromatography-triple quadrupole tandem mass spectrometry system (UPLC-QqQ-MS/MS) and based on the knowledge of retention time and mass spectral characteristics of an in-house collection of authentic standards, we screened for the presence of a large collection of natural compounds. The database represents 359 authenticated metabolites, comprising 220 primary and 139 secondary metabolites (70 flavonoids, 16 phenylpropanoic acid derivatives, five coumarins, two stilbenoids, 14 benzoic acids, nine phenols, 20 alkaloids and three terpenoids). Comparison of the accumulation of these compounds in two tissues showed that the aerial parts were enriched in flavonols, whereas the subterranean parts were enriched in anthocyanins. The comprehensive database developed here will be beneficial in improving the understanding of the chemical basis of plant therapeutic profile using multivariate analysis, with a particular example of antioxidant activity.

An untargeted liquid chromatography-mass spectrometry-based workflow for the structural characterization of plant polyesters.

Cell wall localized heterogeneous polyesters are widespread in land plants. The composition of these polyesters, such as cutin, suberin, or more plant-specific forms such as the flax seed coat lignan macromolecule, can be determined after total hydrolysis of the ester linkages. The main bottleneck in the structural characterization of these macromolecules, however, resides in the determination of the higher order monomer sequences. Partial hydrolysates of the polyesters release a complex mixture of fragments of different lengths, each present in low abundance and therefore are challenging to structurally characterize. Here, a method is presented by which liquid chromatography-mass spectrometry (LC-MS) profiles of such partial hydrolysates are searched for pairs of related fragments. LC-MS peaks that show a mass difference corresponding to the addition of one or more macromolecule monomers were connected in a network. Starting from the lowest molecular weight peaks in the network, the annotation of the connections as the addition of one or more polyester monomers allows the prediction of consecutive and increasingly complex adjacent peaks. Multi-stage MS (MSn) experiments further helped to reject, corroborate, and sometimes refine the structures predicted by the network. As a proof of concept, this procedure was applied to partial hydrolysates of the flax seed coat lignan macromolecule, and allowed to characterize 120 distinct oligo-esters, consisting of up to six monomers, and containing monomers and linkages for which incorporation in the lignan macromolecule had not been described before. These results showed the capacity of the approach to advance the structural elucidation of complex plant polyesters.

Metabolic markers for the yield of lipophilic indole alkaloids in dried woad leaves (Isatis tinctoria L.).

The pharmacologically active dichloromethane extracts of dried woad leaves (Isatis tinctoria L.), and the methanol extracts of comparable fresh leaves of the same plants, were analyzed by LC-MSn. The fresh leaf metabolite profile revealed a complex pattern of indolic compounds. Besides the known indigo precursors, isatan A, isatan B and indican, seven previously unreported indole derivatives were characterized: acetylindican, malonylindican, two dioxindole glucosides, dioxindole malonylglucoside, 6-hydroxyindole-3-carboxylic acid 6-O-glucoside and 6-hydroxyindole-3-carboxylic acid glucose ester. The integration of 122 compounds in fresh leaves and of five selected compounds (indoxyl, isatin, indigo, indirubin, and tryptanthrin) in dried leaves, formed the input data for a stepwise modelling procedure generating five predictive linear models. The structure of the predictive models and a cross validation provide evidence that the models could predict well or moderately well the accumulation of the selected lipophilic compounds, and were simple enough to be used in a woad cultivation program. PLS regression models relating each of the five selected dry leaf indolics to the fresh leaf metabolome were then fitted in order to deduct potential precursors and mechanisms leading to the formation of these lipophilic indolics in drying woad leaves. The models suggested glucobrassicin, isatan A and isatan B as the main candidate precursors of these compounds, besides a minor contribution of other fresh leaf indolics, including malonylindican, actylindican and dioxindole malonylglucoside. Dioxindole malonylglucoside was identified here as isatan C. The models further suggested that the accumulation of phenylpropanoid antioxidants in woad leaves has a negative impact on the formation of indoxyl, isatin, indigo, indirubin and tryptanthrin.

Assessing the between-background stability of metabolic effects arising from lignin-related transgenic modifications, in two Populus hybrids using non-targeted metabolomics.

The advances in 'high-throughput' biology have significantly expanded our fundamental understanding of complex biological processes inherent to tree growth and development. Relative to the significant achievements attained with whole genome re-sequencing and transcriptomics efforts, the development and power of post-transcriptional tools such as proteomics and metabolomics continue to lag behind in tree biology. However, the inclusion of these powerful functional genomics platforms should substantially enable systems biology assessments of tree development, physiology and response(s) to biotic and abiotic stresses. Herein, we employ a non-targeted metabolomics platform to elucidate the metabolic plasticity of xylem lignification in distinct hybrid poplar genetic backgrounds, as well as in transgenic trees in these backgrounds expressing two common constructs: the first construct (C4H::F5H) augments monolignol content (syringyl:guaiacyl (S:G) ratio), while the second construct (C3'H-RNAi) reduces cell wall lignification. The results clearly show that genotype-specific metabolism exists, and provide an appropriate foundation for properly comparing the influence of background on the relationships between metabolic and specific phenotypic traits. Moreover, it was apparent that transgene-induced phenotypic gradients in cell wall chemical wood can be associated with global metabolism of secondary xylem biosynthesis, however in a genotype-specific manner. This result implies that the same may be true for phenotypic gradients arising through natural genetic variation, intensive breeding or environmental factors. It is also apparent that while distinct, at a global level the wood-forming metabolisms of different poplar hybrids can, to some extent, respond similarly to the influences of genetic manipulation of lignin-related genes. This further implies that with the correct approach, it may be possible to associate the emergence of specific wood traits from different genetic backgrounds-be they transgene-induced or otherwise-with stable metabolic signatures.

Phenylpropanoid profiling reveals a class of hydroxycinnamoyl glucaric acid conjugates in Isatis tinctoria leaves.

The brassicaceous herb, Isatis tinctoria, is an ancient medicinal plant whose rosette leaf extracts have anti-inflammatory and anti-allergic activity. Brassicaceae are known to accumulate a variety of phenylpropanoids in their rosette leaves acting as antioxidants and a UV-B shield, and these compounds often have pharmacological potential. Nevertheless, knowledge about the phenylpropanoid content of I. tinctoria leaves remains limited to the characterization of a number of flavonoids. In this research, we profiled the methanol extracts of I. tinctoria fresh leaf extracts by liquid chromatography - mass spectrometry (LC-MS) and focused on the phenylpropanoid derivatives. We report the structural characterization of 99 compounds including 18 flavonoids, 21 mono- or oligolignols, 2 benzenoids, and a wide spectrum of 58 hydroxycinnamic acid esters. Besides the sinapate esters of malate, glucose and gentiobiose, which are typical of brassicaceous plants, these conjugates comprised a large variety of glucaric acid esters that have not previously been reported in plants. Feeding with 13C6-glucaric acid showed that glucaric acid is an acyl acceptor of an as yet unknown acyltransferase activity in I. tinctoria rosette leaves. The large amount of hydroxycinnamic acid derivatives changes radically our view of the woad metabolite profile and potentially contributes to the pharmacological activity of I. tinctoria leaf extracts.

Poplar trees reconfigure the transcriptome and metabolome in response to drought in a genotype- and time-of-day-dependent manner.

BACKGROUND: Drought has a major impact on tree growth and survival. Understanding tree responses to this stress can have important application in both conservation of forest health, and in production forestry. Trees of the genus Populus provide an excellent opportunity to explore the mechanistic underpinnings of forest tree drought responses, given the growing molecular resources that are available for this taxon. Here, foliar tissue of six water-deficit stressed P. balsamifera genotypes was analysed for variation in the metabolome in response to drought and time of day by using an untargeted metabolite profiling technique, gas chromatography/mass-spectrometry (GC/MS). RESULTS: Significant variation in the metabolome was observed in response the imposition of water-deficit stress. Notably, organic acid intermediates such as succinic and malic acid had lower concentrations in leaves exposed to drought, whereas galactinol and raffinose were found in increased concentrations. A number of metabolites with significant difference in accumulation under water-deficit conditions exhibited intraspecific variation in metabolite accumulation. Large magnitude fold-change accumulation was observed in three of the six genotypes. In order to understand the interaction between the transcriptome and metabolome, an integrated analysis of the drought-responsive transcriptome and the metabolome was performed. One P. balsamifera genotype, AP-1006, demonstrated a lack of congruence between the magnitude of the drought transcriptome response and the magnitude of the metabolome response. More specifically, metabolite profiles in AP-1006 demonstrated the smallest changes in response to water-deficit conditions. CONCLUSIONS: Pathway analysis of the transcriptome and metabolome revealed specific genotypic responses with respect to primary sugar accumulation, citric acid metabolism, and raffinose family oligosaccharide biosynthesis. The intraspecific variation in the molecular strategies that underpin the responses to drought among genotypes may have an important role in the maintenance of forest health and productivity.

Unravelling the architecture and dynamics of tropane alkaloid biosynthesis pathways using metabolite correlation networks.

The tropane alkaloid spectrum in Solanaceae is highly variable within and between species. Little is known about the topology and the coordination of the biosynthetic pathways leading to the variety of tropine and pseudotropine derived esters in the alkaloid spectrum, or about the metabolic dynamics induced by tropane alkaloid biosynthesis stimulating conditions. A good understanding of the metabolism, including all ramifications, is however necessary for the development of strategies to increase the abundance of pharmacologically interesting compounds such as hyoscyamine and scopolamine. The present study explores the tropane alkaloid metabolic pathways in an untargeted approach involving a correlation-based network analysis. Using GC-MS metabolite profiling, the variation and co-variation among tropane alkaloids and primary metabolites was monitored in 60 Datura innoxia Mill. individuals, of which half were exposed to tropane alkaloid biosynthesis stimulating conditions by co-culture with Agrobacterium rhizogenes. Considerable variation was evident in the relative proportions of the tropane alkaloids. Remodeling of the tropane alkaloid spectrum under co-culture with A. rhizogenes involved a specific and strong increase of hyoscyamine production and revealed that the accumulation of hyoscyamine, 3-tigloyloxy-6,7-epoxytropane, and 3-methylbutyryloxytropane was controlled independently of the majority of tropane alkaloids. Based on correlations between metabolites, we propose a biosynthetic origin of hygrine, the order of esterification of certain di-oxygenated tropanes, and that the rate of acetoxylation contributes to control of hyoscyamine production. Overall, this study shows that the biosynthesis of tropane alkaloids may be far more complex and finely controlled than previously expected.

Kinetics of glucosylated and non-glucosylated aryltetralin lignans in Linum hairy root cultures.

Due to their pronounced cytotoxic activity, a number of aryltetralin lignans (ATLs), such as podophyllotoxin (PTOX), are used as antitumor compounds. The production of such molecules from entire plants or plant cell-tissue-organ cultures is thus of interest to the pharmaceutical industry. Hairy root cultures constitute a good tool not only for phytochemical production but also for investigating plant secondary metabolism. This work reports on the growth and ATL biosynthesis in two hairy root cultures of Linum album Kotschy ex Boiss. and Linum flavum. The kinetics of accumulation of the intermediates of MPTOX biosynthesis and of their glucosylated forms are described over a 21-day period of growth. An accumulation of non-glucosylated forms of the ATLs during the exponential phase of the cultures is followed by an accumulation of the glucosylated forms during the stationary phase. Our results show a strong coordination of the biosynthetic paths derived from deoxypodophyllotoxin via deoxypodophyllotoxin 6-hydroxylase and deoxypodophyllotoxin 7-hydroxylase, and a coordinated glucosylation of podophyllotoxin, methoxypodophyllotoxin, and 5'-demethoxymethoxypodophyllotoxin. Furthermore, our results suggest an important role of ß-peltatin-6-glucoside formation in the control of ATL accumulation in Linum hairy root cultures.

Ectopic lignification in the flax lignified bast fiber1 mutant stem is associated with tissue-specific modifications in gene expression and cell wall composition.

Histochemical screening of a flax ethyl methanesulfonate population led to the identification of 93 independent M2 mutant families showing ectopic lignification in the secondary cell wall of stem bast fibers. We named this core collection the Linum usitatissimum (flax) lbf mutants for lignified bast fibers and believe that this population represents a novel biological resource for investigating how bast fiber plants regulate lignin biosynthesis. As a proof of concept, we characterized the lbf1 mutant and showed that the lignin content increased by 350% in outer stem tissues containing bast fibers but was unchanged in inner stem tissues containing xylem. Chemical and NMR analyses indicated that bast fiber ectopic lignin was highly condensed and rich in G-units. Liquid chromatography-mass spectrometry profiling showed large modifications in the oligolignol pool of lbf1 inner- and outer-stem tissues that could be related to ectopic lignification. Immunological and chemical analyses revealed that lbf1 mutants also showed changes to other cell wall polymers. Whole-genome transcriptomics suggested that ectopic lignification of flax bast fibers could be caused by increased transcript accumulation of (1) the cinnamoyl-CoA reductase, cinnamyl alcohol dehydrogenase, and caffeic acid O-methyltransferase monolignol biosynthesis genes, (2) several lignin-associated peroxidase genes, and (3) genes coding for respiratory burst oxidase homolog NADPH-oxidases necessary to increase H2O2 supply.

Investigating the drought-stress response of hybrid poplar genotypes by metabolite profiling.

Drought stress is perhaps the most commonly encountered abiotic stress plants experience in the natural environment, and it is one of the most important factors limiting plant productivity. Here, we employed untargeted metabolite profiling to examine four drought-stressed hybrid poplar (Populus spp.) genotypes for their metabolite content, using gas chromatography coupled to mass spectrometry. The primary objective of these analyses was to characterize the metabolite profile of poplar trees to assess relative drought resistance and to investigate the underlying biochemical mechanisms employed by the genotypes to combat drought. Metabolite profiling identified key metabolites that increased or decreased in relative abundance upon exposure to drought stress. Overall, amino acids, the antioxidant phenolic compounds catechin and kaempferol, as well as the osmolytes raffinose and galactinol exhibited increased abundance under drought stress, whereas metabolites involved in photorespiration, redox regulation and carbon fixation showed decreased abundance under drought stress. One clone in particular, Okanese, displayed unique responses to the imposed drought conditions. This clone was found to have higher leaf water potential, but lower growth rate relative to the other clones tested. Okanese also had lower accumulation of osmolytes such as raffinose, galactinol and proline, but higher overall levels of antioxidants such as catechin and dehydroascorbic acid. As such, it was proposed that osmotic adjustment as a mechanism for drought avoidance in this clone is not as well developed in comparison with the other clones investigated in this study, and that a possible alternative mechanism for the enhanced drought avoidance displayed by Okanese may be due to differential allocation of resources or better retention of water.

Metabolic dynamics during autumn cold acclimation within and among populations of Sitka spruce (Picea sitchensis).

• Autumnal cold acclimation in conifers is a complex process, the timing and extent of which vary widely along latitudinal gradients for many tree species and reflect local adaptation to climate. Although previous studies have detailed some aspects of the metabolic remodelling that accompanies cold acclimation in conifers, little is known about global metabolic dynamics, or how these changes vary among phenotypically divergent populations. • Using untargeted GC-MS metabolite profiling, we monitored metabolic dynamics during autumnal cold acclimation in three populations of Sitka spruce from the southern, central, and northern portions of the species range, which differ in both the timing and extent of cold acclimation. • Latitudinal variation was evident in the nature, intensity, and timing of metabolic events. Early development of strong freezing tolerance in the northern population was associated with a transient accumulation of amino acids. By late autumn, metabolic profiles were highly similar between the northern and central populations, whereas profiles for the southern population were relatively distinct. • Our results provide insight into the metabolic architecture of latitudinal adaptive variation in autumn acclimation and show that different mechanisms are the basis of early October cold hardiness and autumn-acclimated cold hardiness.

Mass spectrometry-based sequencing of lignin oligomers.

Although the primary structure of proteins, nucleic acids, and carbohydrates can be readily determined, no sequencing method has been described yet for the second most abundant biopolymer on earth (i.e. lignin). Within secondary-thickened plant cell walls, lignin forms an aromatic mesh arising from the combinatorial radical-radical coupling of monolignols and many other less abundant monomers. This polymerization process leads to a plethora of units and linkage types that affect the physicochemical characteristics of the cell wall. Current methods to analyze the lignin structure focus only on the frequency of the major monomeric units and interunit linkage types but do not provide information on the presence of less abundant unknown units and linkage types, nor on how linkages affect the formation of neighboring linkages. Such information can only be obtained using a sequencing approach. Here, we describe, to our knowledge for the first time, a sequencing strategy for lignin oligomers using mass spectrometry. This strategy was then evaluated on the oligomers extracted from wild-type poplar (Populus tremula x Populus tremuloides) xylem. In total, 134 lignin trimers to hexamers were observed, of which 36 could be completely sequenced. Interestingly, based on molecular mass data of the unknown monomeric and dimeric substructures, at least 10 unknown monomeric units or interunit linkage types were observed, one of which was identified as an arylglycerol end unit.

Predicting the regenerative capacity of conifer somatic embryogenic cultures by metabolomics.

Somatic embryogenesis in gymnosperms is an effective approach to clonally propagating germplasm. However, embryogenic cultures frequently lose regenerative capacity. The interactions between metabolic composition, physiological state, genotype and embryogenic capacity in Pinus taeda (loblolly pine) somatic embryogenic cultures were explored using metabolomics. A stepwise modelling procedure, using the Bayesian information criterion, generated a 47 metabolite predictive model that could explain culture productivity. The model performed extremely well in cross-validation, achieving a correlation coefficient of 0.98 between actual and predicted mature embryo production. The metabolic composition and structure of the model implied that variation in culture regenerative capacity was closely linked to the physiological transition of cultures from the proliferation phase to the maturation phase of development. The propensity of cultures to advance into this transition appears to relate to nutrient uptake and allocation in vivo, and to be associated with the tolerance and response of cultures to stress, during the proliferation phase.

Downregulation of cinnamoyl-coenzyme A reductase in poplar: multiple-level phenotyping reveals effects on cell wall polymer metabolism and structure.

Cinnamoyl-CoA reductase (CCR) catalyzes the penultimate step in monolignol biosynthesis. We show that downregulation of CCR in transgenic poplar (Populus tremula x Populus alba) was associated with up to 50% reduced lignin content and an orange-brown, often patchy, coloration of the outer xylem. Thioacidolysis, nuclear magnetic resonance (NMR), immunocytochemistry of lignin epitopes, and oligolignol profiling indicated that lignin was relatively more reduced in syringyl than in guaiacyl units. The cohesion of the walls was affected, particularly at sites that are generally richer in syringyl units in wild-type poplar. Ferulic acid was incorporated into the lignin via ether bonds, as evidenced independently by thioacidolysis and by NMR. A synthetic lignin incorporating ferulic acid had a red-brown coloration, suggesting that the xylem coloration was due to the presence of ferulic acid during lignification. Elevated ferulic acid levels were also observed in the form of esters. Transcript and metabolite profiling were used as comprehensive phenotyping tools to investigate how CCR downregulation impacted metabolism and the biosynthesis of other cell wall polymers. Both methods suggested reduced biosynthesis and increased breakdown or remodeling of noncellulosic cell wall polymers, which was further supported by Fourier transform infrared spectroscopy and wet chemistry analysis. The reduced levels of lignin and hemicellulose were associated with an increased proportion of cellulose. Furthermore, the transcript and metabolite profiling data pointed toward a stress response induced by the altered cell wall structure. Finally, chemical pulping of wood derived from 5-year-old, field-grown transgenic lines revealed improved pulping characteristics, but growth was affected in all transgenic lines tested.

Molecular phenotyping of lignin-modified tobacco reveals associated changes in cell-wall metabolism, primary metabolism, stress metabolism and photorespiration.

Lignin is an important component of secondarily thickened cell walls. Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) are two key enzymes that catalyse the penultimate and last steps in the biosynthesis of the monolignols. Downregulation of CCR in tobacco (Nicotiana tabacum) has been shown to reduce lignin content, whereas lignin in tobacco downregulated for CAD incorporates more aldehydes. We show that altering the expression of either or both genes in tobacco has far-reaching consequences on the transcriptome and metabolome. cDNA-amplified fragment length polymorphism-based transcript profiling, combined with HPLC and GC-MS-based metabolite profiling, revealed differential transcripts and metabolites within monolignol biosynthesis, as well as a substantial network of interactions between monolignol and other metabolic pathways. In general, in all transgenic lines, the phenylpropanoid biosynthetic pathway was downregulated, whereas starch mobilization was upregulated. CCR-downregulated lines were characterized by changes at the level of detoxification and carbohydrate metabolism, whereas the molecular phenotype of CAD-downregulated tobacco was enriched in transcript of light- and cell-wall-related genes. In addition, the transcript and metabolite data suggested photo-oxidative stress and increased photorespiration, mainly in the CCR-downregulated lines. These predicted effects on the photosynthetic apparatus were subsequently confirmed physiologically by fluorescence and gas-exchange measurements. Our data provide a molecular picture of a plant's response to altered monolignol biosynthesis.