Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon.

Brachypodium distachyon (Brachypodium) has been proposed as a model for grasses, but there is limited knowledge regarding its lignins and no data on lignin-related mutants. The cinnamyl alcohol dehydrogenase (CAD) genes involved in lignification are promising targets to improve the cellulose-to-ethanol conversion process. Down-regulation of CAD often induces a reddish coloration of lignified tissues. Based on this observation, we screened a chemically induced population of Brachypodium mutants (Bd21-3 background) for red culm coloration. We identified two mutants (Bd4179 and Bd7591), with mutations in the BdCAD1 gene. The mature stems of these mutants displayed reduced CAD activity and lower lignin content. Their lignins were enriched in 8-O-4- and 4-O-5-coupled sinapaldehyde units, as well as resistant inter-unit bonds and free phenolic groups. By contrast, there was no increase in coniferaldehyde end groups. Moreover, the amount of sinapic acid ester-linked to cell walls was measured for the first time in a lignin-related CAD grass mutant. Functional complementation of the Bd4179 mutant with the wild-type BdCAD1 allele restored the wild-type phenotype and lignification. Saccharification assays revealed that Bd4179 and Bd7591 lines were more susceptible to enzymatic hydrolysis than wild-type plants. Here, we have demonstrated that BdCAD1 is involved in lignification of Brachypodium. We have shown that a single nucleotide change in BdCAD1 reduces the lignin level and increases the degree of branching of lignins through incorporation of sinapaldehyde. These changes make saccharification of cells walls pre-treated with alkaline easier without compromising plant growth.

Arabidopsis wat1 (walls are thin1)-mediated resistance to the bacterial vascular pathogen, Ralstonia solanacearum, is accompanied by cross-regulation of salicylic acid and tryptophan metabolism.

Inactivation of Arabidopsis WAT1 (Walls Are Thin1), a gene required for secondary cell-wall deposition, conferred broad-spectrum resistance to vascular pathogens, including the bacteria Ralstonia solanacearum and Xanthomonas campestris pv. campestris, and the fungi Verticillium dahliae and Verticillium albo-atrum. Introduction of NahG, the bacterial salicylic acid (SA)-degrading salicylate hydroxylase gene, into the wat1 mutant restored full susceptibility to both R. solanacearum and X. campestris pv. campestris. Moreover, SA content was constitutively higher in wat1 roots, further supporting a role for SA in wat1-mediated resistance to vascular pathogens. By combining transcriptomic and metabolomic data, we demonstrated a general repression of indole metabolism in wat1-1 roots as shown by constitutive down-regulation of several genes encoding proteins of the indole glucosinolate biosynthetic pathway and reduced amounts of tryptophan (Trp), indole-3-acetic acid and neoglucobrassicin, the major form of indole glucosinolate in roots. Furthermore, the susceptibility of the wat1 mutant to R. solanacearum was partially restored when crossed with either the trp5 mutant, an over-accumulator of Trp, or Pro35S:AFB1-myc, in which indole-3-acetic acid signaling is constitutively activated. Our original hypothesis placed cell-wall modifications at the heart of the wat1 resistance phenotype. However, the results presented here suggest a mechanism involving root-localized metabolic channeling away from indole metabolites to SA as a central feature of wat1 resistance to R. solanacearum.

Disruption of LACCASE4 and 17 results in tissue-specific alterations to lignification of Arabidopsis thaliana stems.

Peroxidases have been shown to be involved in the polymerization of lignin precursors, but it remains unclear whether laccases (EC 1.10.3.2) participate in constitutive lignification. We addressed this issue by studying laccase T-DNA insertion mutants in Arabidopsis thaliana. We identified two genes, LAC4 and LAC17, which are strongly expressed in stems. LAC17 was mainly expressed in the interfascicular fibers, whereas LAC4 was expressed in vascular bundles and interfascicular fibers. We produced two double mutants by crossing the LAC17 (lac17) mutant with two LAC4 mutants (lac4-1 and lac4-2). The single and double mutants grew normally in greenhouse conditions. The single mutants had moderately low lignin levels, whereas the stems of lac4-1 lac17 and lac4-2 lac17 mutants had lignin contents that were 20 and 40% lower than those of the control, respectively. These lower lignin levels resulted in higher saccharification yields. Thioacidolysis revealed that disrupting LAC17 principally affected the deposition of G lignin units in the interfascicular fibers and that complementation of lac17 with LAC17 restored a normal lignin profile. This study provides evidence that both LAC4 and LAC17 contribute to the constitutive lignification of Arabidopsis stems and that LAC17 is involved in the deposition of G lignin units in fibers.

Impact of the absence of stem-specific ß-glucosidases on lignin and monolignols.

Monolignol glucosides are thought to be implicated in the lignin biosynthesis pathway as storage and/or transportation forms of cinnamyl alcohols between the cytosol and the lignifying cell walls. The hydrolysis of these monolignol glucosides would involve ß-glucosidase activities. In Arabidopsis (Arabidopsis thaliana), in vitro studies have shown the affinity of ß-GLUCOSIDASE45 (BGLU45) and BGLU46 for monolignol glucosides. BGLU45 and BGLU46 genes are expressed in stems. Immunolocalization experiments showed that BGLU45 and BGLU46 proteins are mainly located in the interfascicular fibers and in the protoxylem, respectively. Knockout mutants for BGLU45 or BGLU46 do not have a lignin-deficient phenotype. Coniferin and syringin could be detected by ultra-performance liquid chromatography-mass spectrometry in Arabidopsis stems. Stems from BGLU45 and BGLU46 mutant lines displayed a significant increase in coniferin content without any change in coniferyl alcohol, whereas no change in syringin content was observed. Other glucosylated compounds of the phenylpropanoid pathway were also deregulated in these mutants, but to a lower extent. By contrast, BGLU47, which is closely related to BGLU45 and BGLU46, is not implicated in either the general phenylpropanoid pathway or in the lignification of stems and roots. These results confirm that the major in vivo substrate of BGLU45 and BGLU46 is coniferin and suggest that monolignol glucosides are the storage form of monolignols in Arabidopsis, but not the direct precursors of lignin.

Imbalanced lignin biosynthesis promotes the sexual reproduction of homothallic oomycete pathogens.

Lignin is incorporated into plant cell walls to maintain plant architecture and to ensure long-distance water transport. Lignin composition affects the industrial value of plant material for forage, wood and paper production, and biofuel technologies. Industrial demands have resulted in an increase in the use of genetic engineering to modify lignified plant cell wall composition. However, the interaction of the resulting plants with the environment must be analyzed carefully to ensure that there are no undesirable side effects of lignin modification. We show here that Arabidopsis thaliana mutants with impaired 5-hydroxyguaiacyl O-methyltransferase (known as caffeate O-methyltransferase; COMT) function were more susceptible to various bacterial and fungal pathogens. Unexpectedly, asexual sporulation of the downy mildew pathogen, Hyaloperonospora arabidopsidis, was impaired on these mutants. Enhanced resistance to downy mildew was not correlated with increased plant defense responses in comt1 mutants but coincided with a higher frequency of oomycete sexual reproduction within mutant tissues. Comt1 mutants but not wild-type Arabidopsis accumulated soluble 2-O-5-hydroxyferuloyl-L-malate. The compound weakened mycelium vigor and promoted sexual oomycete reproduction when applied to a homothallic oomycete in vitro. These findings suggested that the accumulation of 2-O-5-hydroxyferuloyl-L-malate accounted for the observed comt1 mutant phenotypes during the interaction with H. arabidopsidis. Taken together, our study shows that an artificial downregulation of COMT can drastically alter the interaction of a plant with the biotic environment.

Sequencing around 5-hydroxyconiferyl alcohol-derived units in caffeic acid O-methyltransferase-deficient poplar lignins.

Caffeic acid O-methyltransferase (COMT) is a bifunctional enzyme that methylates the 5- and 3-hydroxyl positions on the aromatic ring of monolignol precursors, with a preference for 5-hydroxyconiferaldehyde, on the way to producing sinapyl alcohol. Lignins in COMT-deficient plants contain benzodioxane substructures due to the incorporation of 5-hydroxyconiferyl alcohol (5-OH-CA), as a monomer, into the lignin polymer. The derivatization followed by reductive cleavage method can be used to detect and determine benzodioxane structures because of their total survival under this degradation method. Moreover, partial sequencing information for 5-OH-CA incorporation into lignin can be derived from detection or isolation and structural analysis of the resulting benzodioxane products. Results from a modified derivatization followed by reductive cleavage analysis of COMT-deficient lignins provide evidence that 5-OH-CA cross couples (at its beta-position) with syringyl and guaiacyl units (at their O-4-positions) in the growing lignin polymer and then either coniferyl or sinapyl alcohol, or another 5-hydroxyconiferyl monomer, adds to the resulting 5-hydroxyguaiacyl terminus, producing the benzodioxane. This new terminus may also become etherified by coupling with further monolignols, incorporating the 5-OH-CA integrally into the lignin structure.

Transcriptomic analysis of Arabidopsis developing stems: a close-up on cell wall genes.

BACKGROUND: Different strategies (genetics, biochemistry, and proteomics) can be used to study proteins involved in cell biogenesis. The availability of the complete sequences of several plant genomes allowed the development of transcriptomic studies. Although the expression patterns of some Arabidopsis thaliana genes involved in cell wall biogenesis were identified at different physiological stages, detailed microarray analysis of plant cell wall genes has not been performed on any plant tissues. Using transcriptomic and bioinformatic tools, we studied the regulation of cell wall genes in Arabidopsis stems, i.e. genes encoding proteins involved in cell wall biogenesis and genes encoding secreted proteins. RESULTS: Transcriptomic analyses of stems were performed at three different developmental stages, i.e., young stems, intermediate stage, and mature stems. Many genes involved in the synthesis of cell wall components such as polysaccharides and monolignols were identified. A total of 345 genes encoding predicted secreted proteins with moderate or high level of transcripts were analyzed in details. The encoded proteins were distributed into 8 classes, based on the presence of predicted functional domains. Proteins acting on carbohydrates and proteins of unknown function constituted the two most abundant classes. Other proteins were proteases, oxido-reductases, proteins with interacting domains, proteins involved in signalling, and structural proteins. Particularly high levels of expression were established for genes encoding pectin methylesterases, germin-like proteins, arabinogalactan proteins, fasciclin-like arabinogalactan proteins, and structural proteins. Finally, the results of this transcriptomic analyses were compared with those obtained through a cell wall proteomic analysis from the same material. Only a small proportion of genes identified by previous proteomic analyses were identified by transcriptomics. Conversely, only a few proteins encoded by genes having moderate or high level of transcripts were identified by proteomics. CONCLUSION: Analysis of the genes predicted to encode cell wall proteins revealed that about 345 genes had moderate or high levels of transcripts. Among them, we identified many new genes possibly involved in cell wall biogenesis. The discrepancies observed between results of this transcriptomic study and a previous proteomic study on the same material revealed post-transcriptional mechanisms of regulation of expression of genes encoding cell wall proteins.

Impact of CCR1 silencing on the assembly of lignified secondary walls in Arabidopsis thaliana.

A cinnamoyl-CoA reductase 1 knockout mutant in Arabidopsis thaliana was investigated for the consequences of lignin synthesis perturbation on the assembly of the cell walls. The mutant displayed a dwarf phenotype and a strong collapse of its xylem vessels corresponding to lower lignin content and a loss of lignin units of the noncondensed type. Transmission electron microscopy revealed that the transformation considerably impaired the capacity of interfascicular fibers and vascular bundles to complete the assembly of cellulose microfibrils in the S(2) layer, the S(1) layer remaining unaltered. Such disorder in cellulose was correlated with X-ray diffraction showing altered organization. Semi-quantitative immunolabeling of lignins showed that the patterns of distribution were differentially affected in interfascicular fibers and vascular bundles, pointing to the importance of noncondensed lignin structures for the assembly of a coherent secondary wall. The use of laser capture microdissection combined with the microanalysis of lignins and polysaccharides allowed these polymers to be characterized into specific cell types. Wild-type A. thaliana displayed a two-fold higher syringyl to guaiacyl ratio in interfascicular fibers compared with vascular bundles, whereas this difference was less marked in the cinnamoyl-CoA reductase 1 knockout mutant.

Purification, cloning and functional characterization of an endogenous beta-glucuronidase in Arabidopsis thaliana.

Beta-glucuronidase (GUS) activities have been extensively characterized in bacteria, fungi, and animals, and the bacterial enzyme GUSA from Escherichia coli is commonly used as a reporter for gene expression studies in plants. Although endogenous GUS activity has been observed in plants, the nature and function of the enzymes involved remain elusive. Here we report on tissue-specific localization, partial purification and identification of AtGUS2, a GUS active under acidic conditions from Arabidopsis thaliana. This enzyme belongs to the GH79 family in the Carbohydrate-Active Enzymes database, which also includes mammalian heparanases that degrade the carbohydrate moieties of cell surface proteoglycans, and fungal enzymes active on arabinogalactan proteins (AGPs). We characterized a knockout insertion line (atgus2-1) and transgenic lines overexpressing AtGUS2 (Pro(35S):AtGUS2). Endogenous GUS activity assayed histochemically and biochemically was absent in atgus2-1 tissues and four times higher in Pro(35S):AtGUS2 lines. AGPs purified from atgus2-1 and Pro(35S):AtGUS2 seedlings showed higher and markedly lower glucuronic acid content, respectively. Our results suggest that endogenous GUS activity influences the sugar composition of the complex polysaccharide chains of AGPs. We also show that transgenics display hypocotyl and root growth defects compared to wild-type plants. Hypocotyl and root lengths are increased in Pro(35S):AtGUS2 seedlings, whereas hypocoyl length is reduced in atgus2-1 seedlings. These data are consistent with a role for the carbohydrate moieties of AGPs in cell growth.

Field and pulping performances of transgenic trees with altered lignification.

The agronomic and pulping performance of transgenic trees with altered lignin has been evaluated in duplicated, long-term field trials. Poplars expressing cinnamyl alcohol dehydrogenase (CAD) or caffeate/5-hydroxy-ferulate O-methyltransferase (COMT) antisense transgenes were grown for four years at two sites, in France and England. The trees remained healthy throughout the trial. Growth indicators and interactions with insects were normal. No changes in soil microbial communities were detected beneath the transgenic trees. The expected modifications to lignin were maintained in the transgenics over four years, at both sites. Kraft pulping of tree trunks showed that the reduced-CAD lines had improved characteristics, allowing easier delignification, using smaller amounts of chemicals, while yielding more high-quality pulp. This work highlights the potential of engineering wood quality for more environmentally benign papermaking without interfering with tree growth or fitness.

Comparing the impact of conventional pesticide and use of a transgenic pest-resistant crop on the beneficial carabid beetle Pterostichus melanarius.

The potential impact of a chemical pesticide control method has been compared with that of transgenic plants expressing a protease inhibitor conferring insect resistance by utilising a tritrophic system comprising the crop plant Brassica napus (L.) (Oilseed rape), the pest mollusc Deroceras reticulatum (Müller) and the predatory carabid beetle Pterostichus melanarius (Illiger). Cypermethrin, as the most widely used pesticide in UK oilseed rape (OSR) cultivation, was selected as the conventional treatment. OSR expressing a cysteine protease inhibitor, oryzacystatin-1 (OC-1), was the transgenic comparator. In feeding trials, D. reticulatum showed no significant long-term effects on measured life history parameters (survival, weight gain, food consumption) as a result of exposure to either the cypermethrin or OC-1 treatment. However, D. reticulatum was able to respond to the presence of the dietary inhibitor by producing two novel proteases following exposure to OC-1-expressing OSR. Similarly, P. melanarius showed no detectable alterations in mortality, weight gain or food consumption when feeding on D. reticulatum previously fed either pesticide-contaminated or GM plant material. Furthermore, as with the slug, a novel form of protease, approximately M(r) 27 kDa, was induced in the carabid in response to feeding on slugs fed OC-1-expressing OSR.

Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins.

A series of transgenic poplars down-regulated for cinnamyl alcohol dehydrogenase (CAD) was analyzed by thioacidolysis. Among the lignin-derived monomers, the indene compounds that were recently shown to originate from sinapaldehyde incorporated into lignins through 8-O-4-cross-coupling, were found to increase as a function of CAD deficiency level. While these syringyl markers were recovered in substantial amounts in the most severely depressed lines, the markers for coniferaldehyde incorporation were recovered in only low amounts. In conjunction with these additional sinapaldehyde units and relative to the control samples, lignins in CAD-deficient poplar lines had less conventional syringyl-units and beta-O-4-bonds and more free phenolic groups. We found that almost half of the polymers in the most deficient lines could be solubilized in alkali and at room temperature. This unusual behavior suggests that lignins in CAD-deficient poplars occur as small, alkali-leachable lignin domains. That mainly sinapaldehyde incorporates into the lignins of CAD-deficient poplars suggests that the recently identified sinapyl alcohol dehydrogenase (SAD), which is structurally distinct from the CAD enzyme targeted herein, does not play any substantial role in constitutive lignification in poplar.

Lignification in poplar plantlets fed with deuterium-labelled lignin precursors.

Lignification was investigated in wild-type (WT) and in transgenic poplar plantlets with a reduced caffeic acid O-methyl-transferase (COMT) activity. Coniferin and syringin, deuterated at their methoxyl, were incorporated into the culture medium of microcuttings. The gas chromatography-mass spectrometry (GC-MS) analysis of the thioacidolysis guaiacyl (G) and syringyl (S) lignin-derived monomers revealed that COMT deficiency altered stem lignification. GC-MS analysis proved that the deuterated precursors were incorporated into root lignins and, to a lower extent, in stem lignins without major effect on growth and lignification. Deuterium from coniferin was recovered in G and S lignin units, whereas deuterium from syringin was only found in S units, which further establishes that the conversion of G to S lignin precursors may occur at the level of p-OH cinnamyl alcohols.

TL -DNA transformation decreases ABA level.

The endogenous levels of ABA were measured in Agrobacterium rhizogenes A4 Tl -DNA transformed oilseed rape (Brassica napus L. var. oleifera cv. Brutor and cv. Drakkar), cabbage (Brassica oleracea). A4 transformed tobacco (Nicotiana tabacum cv. Xanthi) and their normal counterparts, using high performance liquid chromatography and enzyme-liked immunosorbent assay. Measurements were made on different plant tissues (i. e. floral stem, terminal bud, young leaf, mature leaf, root and root tips) and ABA levels were compared in unstressed and osmotically stressed oilseed rape plants (cv. Brutor). In unstressed Plants. in each of the 5 independent transformation events studied, a significant reduction (about 65% of control) in ABA concentration was observed in all transformed plants. When subjected to an osmotic stress, TL transformed Brutor plants showed a higher ABA accumulation than untransformed plants. The change in ABA content as a consequence of TL -DNA transformation is discussed with regard to phenotype, drought resistance and adaptability.

A TILLING Platform for Functional Genomics in Brachypodium distachyon.

The new model plant for temperate grasses, Brachypodium distachyon offers great potential as a tool for functional genomics. We have established a sodium azide-induced mutant collection and a TILLING platform, called "BRACHYTIL", for the inbred line Bd21-3. The TILLING collection consists of DNA isolated from 5530 different families. Phenotypes were reported and organized in a phenotypic tree that is freely available online. The tilling platform was validated by the isolation of mutants for seven genes belonging to multigene families of the lignin biosynthesis pathway. In particular, a large allelic series for BdCOMT6, a caffeic acid O-methyl transferase was identified. Some mutants show lower lignin content when compared to wild-type plants as well as a typical decrease of syringyl units, a hallmark of COMT-deficient plants. The mutation rate was estimated at one mutation per 396 kb, or an average of 680 mutations per line. The collection was also used to assess the Genetically Effective Cell Number that was shown to be at least equal to 4 cells in Brachypodium distachyon. The mutant population and the TILLING platform should greatly facilitate functional genomics approaches in this model organism.

The simultaneous repression of CCR and CAD, two enzymes of the lignin biosynthetic pathway, results in sterility and dwarfism in Arabidopsis thaliana.

Cinnamoyl CoA reductase (CCR) and cinnamyl alcohol dehydrogenase (CAD) catalyze the last steps of monolignol biosynthesis. In Arabidopsis, one CCR gene (CCR1, At1g15950) and two CAD genes (CAD C At3g19450 and CAD D At4g34230) are involved in this pathway. A triple cad c cad d ccr1 mutant, named ccc, was obtained. This mutant displays a severe dwarf phenotype and male sterility. The lignin content in ccc mature stems is reduced to 50% of the wild-type level. In addition, stem lignin structure is severely affected, as shown by the dramatic enrichment in resistant inter-unit bonds and incorporation into the polymer of monolignol precursors such as coniferaldehyde, sinapaldehyde, and ferulic acid. Male sterility is due to the lack of lignification in the anther endothecium, which causes the failure of anther dehiscence and of pollen release. The ccc hypolignified stems accumulate higher amounts of flavonol glycosides, sinapoyl malate and feruloyl malate, which suggests a redirection of the phenolic pathway. Therefore, the absence of CAD and CCR, key enzymes of the monolignol pathway, has more severe consequences on the phenotype than the individual absence of each of them. Induction of another CCR (CCR2, At1g80820) and another CAD (CAD1, At4g39330) does not compensate the absence of the main CCR and CAD activities. This lack of CCR and CAD activities not only impacts lignification, but also severely affects the development of the plants. These consequences must be carefully considered when trying to reduce the lignin content of plants in order to facilitate the lignocellulose-to-bioethanol conversion process.

Arabidopsis peroxidase-catalyzed copolymerization of coniferyl and sinapyl alcohols: kinetics of an endwise process.

In order to determine the mechanism of the earlier copolymerization steps of two main lignin precursors, sinapyl (S) alcohol and coniferyl (G) alcohol, microscale in vitro oxidations were carried out with a PRX34 Arabidopsis thaliana peroxidase in the presence of H(2)O(2). This plant peroxidase was found to have an in vitro polymerization activity similar to the commonly used horseradish peroxidase. The selected polymerization conditions lead to a bulk polymerization mechanism when G alcohol was the only phenolic substrate available. In the same conditions, the presence of S alcohol at a 50/50 S/G molar ratio turned this bulk mechanism into an endwise one. A kinetics monitoring (size-exclusion chromatography and liquid chromatography-mass spectrometry) of the different species formed during the first 24h oxidation of the S/G mixture allowed sequencing the bondings responsible for oligomerization. Whereas G homodimers and GS heterodimers exhibit low reactivity, the SS pinoresinol structure act as a nucleating site of the polymerization through an endwise process. This study is particularly relevant to understand the impact of S units on lignin structure in plants and to identify the key step at which this structure is programmed.

Cinnamyl alcohol dehydrogenases-C and D, key enzymes in lignin biosynthesis, play an essential role in disease resistance in Arabidopsis.

The deposition of lignin during plant-pathogen interactions is thought to play a role in plant defence. However, the function of lignification genes in plant disease resistance is poorly understood. In this article, we provide genetic evidence that the primary genes involved in lignin biosynthesis in Arabidopsis, CAD-C and CAD-D, act as essential components of defence to virulent and avirulent strains of the bacterial pathogen Pseudomonas syringae pv. tomato, possibly through the salicylic acid defence pathway. Thus, in contrast with cellulose synthesis, whose alteration leads to an increase in disease resistance, alteration of the cell wall lignin content leads directly or indirectly to defects in some defence components.

Characterisation of adult green lacewing (Chrysoperla carnea) digestive physiology: impact of a cysteine protease inhibitor and a synthetic pyrethroid.

BACKGROUND: In spite of concern regarding potential non-target effects of GM crops, few studies have compared GM pest control with conventional methods. The impacts of cypermethrin and oilseed rape expressing oryzacystatin-1 (OC-1) were compared in this study on the predator Chrysoperla carnea (Stephens). RESULTS: Adults fed purified rOC-1 showed a subtle shift in digestive protease profile, with an increasing reliance on serine proteases (chymotrypsin), increase in aspartic proteases and a slight reduction in elastase activity. Although there were no effects on mortality, onset of oviposition was delayed; however, once egg production commenced, egg laying and hatching success rates were comparable with those of controls. Oryzacystatin-1 expressed in pollen showed no detrimental effects. Cypermethrin had no effect on mortality owing to high levels of non-specific esterase activity resulting in partial breakdown of the insecticide. In spite of this, there was a significant delay in onset of oviposition and a significant reduction in egg production and viability. CONCLUSION: This study demonstrates the potential for pest management to impact on predators, but importantly it highlights the ability of the predator to detoxify/respond to treatments with different modes of action. In this case, exposure to an insecticide carried a greater fitness cost than exposure to a protease inhibitor expressed in transgenic crops.

Redirection of the phenylpropanoid pathway to feruloyl malate in Arabidopsis mutants deficient for cinnamoyl-CoA reductase 1.

Cinnamoyl-CoA reductase 1 (CCR1, gene At1g15950) is the main CCR isoform implied in the constitutive lignification of Arabidopsis thaliana. In this work, we have identified and characterized two new knockout mutants for CCR1. Both have a dwarf phenotype and a delayed senescence. At complete maturity, their inflorescence stems display a 25-35% decreased lignin level, some alterations in lignin structure with a higher frequency of resistant interunit bonds and a higher content in cell wall-bound ferulic esters. Ferulic acid-coniferyl alcohol ether dimers were found for the first time in dicot cell walls and in similar levels in wild-type and mutant plants. The expression of CCR2, a CCR gene usually involved in plant defense, was increased in the mutants and could account for the biosynthesis of lignins in the CCR1-knockout plants. Mutant plantlets have three to four-times less sinapoyl malate (SM) than controls and accumulate some feruloyl malate. The same compositional changes occurred in the rosette leaves of greenhouse-grown plants. By contrast and relative to the control, their stems accumulated unusually high levels of both SM and feruloyl malate as well as more kaempferol glycosides. These findings suggest that, in their hypolignified stems, the mutant plants would avoid the feruloyl-CoA accumulation by its redirection to cell wall-bound ferulate esters, to feruloyl malate and to SM. The formation of feruloyl malate to an extent far exceeding the levels reported so far indicates that ferulic acid is a potential substrate for the enzymes involved in SM biosynthesis and emphasizes the remarkable plasticity of Arabidopsis phenylpropanoid metabolism.