In the search for renewable energy sources, genetic engineering is a promising strategy to improve plant cell wall composition for biofuel and bioproducts generation. Lignin is a major factor determining saccharification efficiency and, therefore, is a prime target to engineer. Here, lignin content and composition were modified in poplar (Populus tremula × Populus alba) by specifically down-regulating CINNAMYL ALCOHOL DEHYDROGENASE1 (CAD1) by a hairpin-RNA-mediated silencing approach, which resulted in only 5% residual CAD1 transcript abundance. These transgenic lines showed no biomass penalty despite a 10% reduction in Klason lignin content and severe shifts in lignin composition. Nuclear magnetic resonance spectroscopy and thioacidolysis revealed a strong increase (up to 20-fold) in sinapaldehyde incorporation into lignin, whereas coniferaldehyde was not increased markedly. Accordingly, ultra-high-performance liquid chromatography-mass spectrometry-based phenolic profiling revealed a more than 24,000-fold accumulation of a newly identified compound made from 8-8 coupling of two sinapaldehyde radicals. However, no additional cinnamaldehyde coupling products could be detected in the CAD1-deficient poplars. Instead, the transgenic lines accumulated a range of hydroxycinnamate-derived metabolites, of which the most prominent accumulation (over 8,500-fold) was observed for a compound that was identified by purification and nuclear magnetic resonance as syringyl lactic acid hexoside. Our data suggest that, upon down-regulation of CAD1, coniferaldehyde is converted into ferulic acid and derivatives, whereas sinapaldehyde is either oxidatively coupled into S′(8-8)S′ and lignin or converted to sinapic acid and derivatives. The most prominent sink of the increased flux to hydroxycinnamates is syringyl lactic acid hexoside. Furthermore, low-extent saccharification assays, under different pretreatment conditions, showed strongly increased glucose (up to +81%) and xylose (up to +153%) release, suggesting that down-regulating CAD1 is a promising strategy for improving lignocellulosic biomass for the sugar platform industry.

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.

Lignin is the main factor limiting the enzymatic conversion of lignocellulosic biomass into fermentable sugars. To reduce the recalcitrance engendered by the lignin polymer, the coumarin scopoletin was incorporated into the lignin polymer through the simultaneous expression of FERULOYL-CoA 6′-HYDROXYLASE 1 ( F6′H1 ) and COUMARIN SYNTHASE ( COSY ) in lignifying cells in Arabidopsis . The transgenic lines overproduced scopoletin and incorporated it into the lignin polymer, without adversely affecting plant growth. About 3.3% of the lignin units in the transgenic lines were derived from scopoletin, thereby exceeding the levels of the traditional p- hydroxyphenyl units. Saccharification efficiency of alkali-pretreated scopoletin-overproducing lines was 40% higher than for wild type.

Lignin is a phenolic polymer in plants that rigidifies the cell walls of water-conducting tracheary elements and support-providing fibers and stone cells. Different mechanisms have been suggested for the transport of lignin precursors to the site of lignification in the cell wall. Extracellular vesicle (EV)-enriched samples isolated from a lignin-forming cell suspension culture of Norway spruce (Picea abies L. Karst.) contained both phenolic metabolites and enzymes related to lignin biosynthesis. Metabolomic analysis revealed mono-, di- and oligolignols in the EV isolates, as well as carbohydrates and amino acids. In addition, salicylic acid (SA) and some proteins involved in SA signaling were detected in the EV-enriched samples. A proteomic analysis detected several laccases, peroxidases, β-glucosidases, putative dirigent proteins, and cell wall-modifying enzymes such as glycosyl hydrolases, transglucosylase/hydrolases, and expansins in EVs. Our findings suggest that EVs are involved in transporting enzymes required for lignin polymerization in Norway spruce, and that radical coupling of monolignols can occur in these vesicles.

The phenylpropanoid cinnamic acid (CA) is a plant metabolite that can occur under a trans‐ or cis‐form. In contrast to the proven bioactivity of the cis‐form (c‐CA), the activity of trans‐CA (t‐CA) is still a matter of debate. We tested both compounds using a submerged rice coleoptile assay and demonstrated that they have opposite effects on cell elongation. Notably, in the tip of rice coleoptile t‐CA showed an inhibiting and c‐CA a stimulating activity. By combining transcriptomics and (untargeted) metabolomics with activity assays and genetic and pharmacological experiments, we aimed to explain the underlying mechanistic processes. We propose a model in which c‐CA treatment activates proton pumps and stimulates acidification of the apoplast, which in turn leads to the loosening of the cell wall, necessary for elongation. We hypothesize that c‐CA also inactivates auxin efflux transporters, which might cause a local auxin accumulation in the tip of the coleoptile. For t‐CA, the phenotype can partially be explained by a stimulation of cell wall polysaccharide feruloylation, leading to a more rigid cell wall. Metabolite profiling also demonstrated that salicylic acid (SA) derivatives are increased upon t‐CA treatment. As SA is a known antagonist of auxin, the shift in SA homeostasis provides an additional explanation of the observed t‐CA‐mediated restriction on cell growth.