Shikimate hydroxycinnamoyl transferase (HCT) activity assays in Populus nigra

Lignin is a complex phenolic polymer deposited in secondarily-thickened plant cell walls. The polymer is mainly derived from the three primary monolignols: p-coumaryl, coniferyl and sinapyl alcohol which give rise to p-hydroxyphenyl, guaiacyl and syringyl units (H, G and S units, respectively) when coupled into the polymer. The building blocks differ in their degree of methoxylation and their biosynthetic pathway is catalyzed by more than 10 enzymes. HCT plays a crucial role by channeling the phenylpropanoids towards the production of coniferyl and sinapyl alcohols. Interestingly, HCT has been reported to be implicated in the pathway both upstream and downstream of the 3-hydroxylation of the aromatic ring of p-coumaroyl shikimate (Figure 1) (Hoffmann et al., 2003; Hoffmann et al., 2004; Vanholme et al., 2013b). These features highlight the importance of developing an assay to reliably measure HCT activity in planta. Here, we describe a UPLC-MS-based method for the analysis of HCT activity in xylem total protein extracts of Populus nigra, which can be adapted to other woody and herbaceous plant species. The protocol was initially described in Vanholme et al. (2013a)

Chemical genetics uncovers novel inhibitors of lignification, including p-iodobenzoic acid targeting CINNAMATE-4-HYDROXYLASE

Plant secondary-thickened cell walls are characterized by the presence of lignin, a recalcitrant and hydrophobic polymer that provides mechanical strength and ensures long-distance water transport. Exactly the recalcitrance and hydrophobicity of lignin put a burden on the industrial processing efficiency of lignocellulosic biomass. Both forward and reverse genetic strategies have been used intensively to unravel the molecular mechanism of lignin deposition. As an alternative strategy, we introduce here a forward chemical genetic approach to find candidate inhibitors of lignification. A high-throughput assay to assess lignification in Arabidopsis (Arabidopsis thaliana) seedlings was developed and used to screen a 10-k library of structurally diverse, synthetic molecules. Of the 73 compounds that reduced lignin deposition, 39 that had a major impact were retained and classified into five clusters based on the shift they induced in the phenolic profile of Arabidopsis seedlings. One representative compound of each cluster was selected for further lignin-specific assays, leading to the identification of an aromatic compound that is processed in the plant into two fragments, both having inhibitory activity against lignification. One fragment, p-iodobenzoic acid, was further characterized as a new inhibitor of CINNAMATE 4-HYDROXYLASE, a key enzyme of the phenylpropanoid pathway synthesizing the building blocks of the lignin polymer. As such, we provide proof of concept of this chemical biology approach to screen for inhibitors of lignification and present a broad array of putative inhibitors of lignin deposition for further characterization

Mutation of the inducible ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE2 alters lignin composition and improves saccharification

ARABIDOPSIS THALIANA CYTOCHROME P450 REDUCTASE1 (ATR1) and ATR2 provide electrons from NADPH to a large number of CYTOCHROME P450 (CYP450) enzymes in Arabidopsis (Arabidopsis thaliana). Whereas ATR1 is constitutively expressed, the expression of ATR2 appears to be induced during lignin biosynthesis and upon stresses. Therefore, ATR2 was hypothesized to be preferentially involved in providing electrons to the three CYP450s involved in lignin biosynthesis: CINNAMATE 4-HYDROXYLASE (C4H), p-COUMARATE 3-HYDROXYLASE1 (C3H1), and FERULATE 5-HYDROXYLASE1 (F5H1). Here, we show that the atr2 mutation resulted in a 6% reduction in total lignin amount in the main inflorescence stem and a compositional shift of the remaining lignin to a 10-fold higher fraction of p-hydroxyphenyl units at the expense of syringyl units. Phenolic profiling revealed shifts in lignin-related phenolic metabolites, in particular with the substrates of C4H, C3H1 and F5H1 accumulating in atr2 mutants. Glucosinolate and flavonol glycoside biosynthesis, both of which also rely on CYP450 activities, appeared less affected. The cellulose in the atr2 inflorescence stems was more susceptible to enzymatic hydrolysis after alkaline pretreatment, making ATR2 a potential target for engineering plant cell walls for biofuel production

Saccharification protocol for small-scale lignocellulosic biomass samples to test processing of cellulose into glucose

Second generation biofuels are derived from inedible lignocellulosic biomass of food and non-food crops. Lignocellulosic biomass is mainly composed of cell walls that contain a large proportion of cellulosic and hemicellulosic polysaccharides. An interesting route to generate biofuels and bio-based materials is via enzymatic hydrolysis of cell wall polysaccharides into fermentable sugars, a process called saccharification. The released sugars can then be fermented to fuels, e.g. by use of yeast. To test the saccharification efficiency of lignocellulosic biomass on a lab-scale, a manual saccharification protocol was established that uses only small amounts of biomass and a low concentration of enzyme. This protocol can be used for different plant species like Arabidopsis thaliana, tobacco, maize and poplar. The low enzyme concentrations make it possible to detect subtle improvements in saccharification yield and to analyze the speed of hydrolysis. Although a specific acid and alkali pretreatment were included, the saccharification step can be preceded by any other pretreatment. Because no advanced equipment is necessary, this protocol can be carried out in many laboratories to analyze saccharification yield. The protocol was initially described in Van Acker et al. (2013)

The role of the secondary cell wall in plant resistance to pathogens

Plant resistance to pathogens relies on a complex network of constitutive and inducible defensive barriers. The plant cell wall is one of the barriers that pathogens need to overcome to successfully colonize plant tissues. The traditional view of the plant cell wall as a passive barrier has evolved to a concept that considers the wall as a dynamic structure that regulates both constitutive and inducible defense mechanisms, and as a source of signaling molecules that trigger immune responses. The secondary cell walls of plants also represent a carbon-neutral feedstock (lignocellulosic biomass) for the production of biofuels and biomaterials. Therefore, engineering plants with improved secondary cell wall characteristics is an interesting strategy to ease the processing of lignocellulosic biomass in the biorefinery. However, modification of the integrity of the cell wall by impairment of proteins required for its biosynthesis or remodeling may impact the plants resistance to pathogens. This review summarizes our understanding of the role of the plant cell wall in pathogen resistance with a focus on the contribution of lignin to this biological process

Overexpression of GA20-OXIDASE1 impacts plant height, biomass allocation and saccharification efficiency in maize

Increased biomass yield and quality are of great importance for the improvement of feedstock for the biorefinery. For the production of bioethanol, both stem biomass yield and the conversion efficiency of the polysaccharides in the cell wall to fermentable sugars are of relevance. Increasing the endogenous levels of gibberellic acid (GA) by ectopic expression of GA20-OXIDASE1 (GA20-OX1), the rate-limiting step in GA biosynthesis, is known to affect cell division and cell expansion, resulting in larger plants and organs in several plant species. In this study, we examined biomass yield and quality traits of maize plants overexpressing GA20-OX1 (GA20-OX1). GA20-OX1 plants accumulated more vegetative biomass than control plants in greenhouse experiments, but not consistently over two years of field trials. The stems of these plants were longer but also more slender. Investigation of GA20-OX1 biomass quality using biochemical analyses showed the presence of more cellulose, lignin and cell wall residue. Cell wall analysis as well as expression analysis of lignin biosynthetic genes in developing stems revealed that cellulose and lignin were deposited earlier in development. Pretreatment of GA20-OX1 biomass with NaOH resulted in a higher saccharification efficiency per unit of dry weight, in agreement with the higher cellulose content. On the other hand, the cellulose-to-glucose conversion was slower upon HCl or hot-water pretreatment, presumably due to the higher lignin content. This study showed that biomass yield and quality traits can be interconnected, which is important for the development of future breeding strategies to improve lignocellulosic feedstock for bioethanol production

Breeding for improved digestibility and processing of lignocellulosic biomass in Zea mays

Forage maize is a versatile crop extensively utilized for animal nutrition in agriculture and holds promise as a valuable resource for the production of fermentable sugars in the biorefinery sector. Within this context, the carbohydrate fraction of the lignocellulosic biomass undergoes deconstruction during ruminal digestion and the saccharification process. However, the cell wall’s natural resistance towards enzymatic degradation poses a significant challenge during both processes. This so-called biomass recalcitrance is primarily attributed to the presence of lignin and ferulates in the cell walls. Consequently, maize varieties with a reduced lignin or ferulate content or an altered lignin composition can have important beneficial effects on cell wall digestibility. Considerable efforts in genetic improvement have been dedicated towards enhancing cell wall digestibility, benefiting agriculture, the biorefinery sector and the environment. In part I of this paper, we review conventional and advanced breeding methods used in the genetic improvement of maize germplasm. In part II, we zoom in on maize mutants with altered lignin for improved digestibility and biomass processing

COSY catalyses trans-cis isomerization and lactonization in the biosynthesis of coumarins

Coumarins, also known as 1,2-benzopyrones, comprise a large class of secondary metabolites that are ubiquitously found throughout the plant kingdom. In many plant species, coumarins are particularly important for iron acquisition and plant defence. Here, we show that COUMARIN SYNTHASE (COSY) is a key enzyme in the biosynthesis of coumarins. Arabidopsis thaliana cosy mutants have strongly reduced levels of coumarin and accumulate o-hydroxyphenylpropanoids instead. Accordingly, cosymutants have reduced iron content and show growth defects when grown under conditions in which there is a limited availabil-ity of iron. Recombinant COSY is able to produce umbelliferone, esculetin and scopoletin from their respective o-hydroxycin-namoyl-CoA thioesters by two reaction steps—a trans–cis isomerization followed by a lactonization. This conversion happens partially spontaneously and is catalysed by light, which explains why the need for an enzyme for this conversion has been overlooked. The combined results show that COSY has an essential function in the biosynthesis of coumarins in organs that are shielded from light, such as roots. These findings provide routes to improving coumarin production in crops or by microbial fermentation

Mass spectrometry-based fragmentation as an identification tool in lignomics

The ensemble of all phenolics for which the biosynthesis is coregulated with lignin biosynthesis, i.e., metabolites from the general phenylpropanoid, monolignol, and (neo)-lignan biosynthetic pathways and their derivatives, as well as the lignin oligomers, is coined the lignome. In lignifying tissues, the lignome comprises a significant portion of the metabolome. However, as is true for metabolomics in general, the structural elucidation of unknowns represents the biggest challenge in characterizing the lignome. To minimize the necessity to purify unknowns for NMR analysis, it would be desirable to be able to extract structural information from liquid chromatography-mass spectrometry data directly. However, mass spectral libraries for metabolomics are scarce, and no libraries exist for the lignome. Therefore, elucidating the gas-phase fragmentation behavior of the major bonding types encountered in lignome-associated molecules would considerably advance the systematic characterization of the lignome. By comparative MS" analysis of a series of molecules belonging to the beta-aryl ether, benzodioxane, phenylcoumaran, and resinol groups, we succeeded in annotating typical fragmentations for each of these bonding structures as well as fragmentations that enabled the identification of the aromatic units involved in each bonding structure. Consequently, this work lays the foundation for a detailed characterization of the lignome in different plant species, mutants, and transgenics and for the MS-based sequencing of lignin oligomers and (neo)lignans