Lignin engineering in field-grown poplar trees affects the endosphere bacterial microbiome

Cinnamoyl-CoA reductase (CCR), an enzyme central to the lignin bio-synthetic pathway, represents a promising biotechnological target to reduce lignin levels and to improve the commercial viability of lignocellulosic biomass. However, silencing of the CCR gene results in considerable flux changes of the general and monolignol-specific lignin pathways, ultimately leading to the accumulation of various extractable phenolic compounds in the xylem. Here, we evaluated host genotype-dependent effects of field-grown, CCR-down-regulated poplar trees (Populus tremula x Populus alba) on the bacterial rhizosphere microbiome and the endosphere microbiome, namely the microbiota present in roots, stems, and leaves. Plant-associated bacteria were isolated from all plant compartments by selective isolation and enrichment techniques with specific phenolic carbon sources (such as ferulic acid) that are up-regulated in CCR-deficient poplar trees. The bacterial microbiomes present in the endosphere were highly responsive to the CCR-deficient poplar genotype with remarkably different metabolic capacities and associated community structures compared with the WT trees. In contrast, the rhizosphere microbiome of CCR-deficient and WT poplar trees featured highly overlapping bacterial community structures and metabolic capacities. We demonstrate the host genotype modulation of the plant microbiome by minute genetic variations in the plant genome. Hence, these interactions need to be taken into consideration to understand the full consequences of plant metabolic pathway engineering and its relation with the environment and the intended genetic improvement

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)

Lignin biosynthesis perturbations affect secondary cell wall composition and saccharification yield in Arabidopsis thaliana.

BACKGROUND: Second-generation biofuels are generally produced from the polysaccharides in the lignocellulosic plant biomass, mainly cellulose. However, because cellulose is embedded in a matrix of other polysaccharides and lignin, its hydrolysis into the fermentable glucose is hampered. The senesced inflorescence stems of a set of 20 Arabidopsis thaliana mutants in 10 different genes of the lignin biosynthetic pathway were analyzed for cell wall composition and saccharification yield. Saccharification models were built to elucidate which cell wall parameters played a role in cell wall recalcitrance. RESULTS: Although lignin is a key polymer providing the strength necessary for the plant's ability to grow upward, a reduction in lignin content down to 64% of the wild-type level in Arabidopsis was tolerated without any obvious growth penalty. In contrast to common perception, we found that a reduction in lignin was not compensated for by an increase in cellulose, but rather by an increase in matrix polysaccharides. In most lignin mutants, the saccharification yield was improved by up to 88% cellulose conversion for the cinnamoyl-coenzyme A reductase1 mutants under pretreatment conditions, whereas the wild-type cellulose conversion only reached 18%. The saccharification models and Pearson correlation matrix revealed that the lignin content was the main factor determining the saccharification yield. However, also lignin composition, matrix polysaccharide content and composition, and, especially, the xylose, galactose, and arabinose contents influenced the saccharification yield. Strikingly, cellulose content did not significantly affect saccharification yield. CONCLUSIONS: Although the lignin content had the main effect on saccharification, also other cell wall factors could be engineered to potentially increase the cell wall processability, such as the galactose content. Our results contribute to a better understanding of the effect of lignin perturbations on plant cell wall composition and its influence on saccharification yield, and provide new potential targets for genetic improvement.RIGHTS : This article is licensed under the BioMed Central licence at http://www.biomedcentral.com/about/license which is similar to the 'Creative Commons Attribution Licence'. In brief you may : copy, distribute, and display the work; make derivative works; or make commercial use of the work - under the following conditions: the original author must be given credit; for any reuse or distribution, it must be made clear to others what the license terms of this work are

Stacking of a low-lignin trait with an increased guaiacyl and 5-hydroxyguaiacyl unit trait leads to additive and synergistic effects on saccharification efficiency in Arabidopsis thaliana

Background: Lignocellulosic biomass, such as wood and straw, is an interesting feedstock for the production of fer-mentable sugars. However, mainly due to the presence of lignin, this type of biomass is recalcitrant to saccharification. In Arabidopsis, lignocellulosic biomass with a lower lignin content or with lignin with an increased fraction of guaiacyl (G) and 5-hydroxyguaiacyl (5H) units shows an increased saccharification efficiency. Here, we stacked these two traits and studied the effect on the saccharification efficiency and biomass yield, by combining either transaldolase (tra2), cinnamate 4-hydroxylase (c4h-3), or 4-coumarate:CoA ligase (4cl1-1) with caffeic acid O-methyltransferase (comt-7 or comt-4) mutants. Results: The three double mutants (tra2 comt-1, c4h-3 comt-4, and 4cl1-7 comt-4) had a decreased lignin amount and an increase in G and 5H units in the lignin polymer compared to wild-type (WT) plants. The tra2 comt-1 double mutant had a better saccharification efficiency compared to the parental lines when an acid or alkaline pretreatment was used. For the double mutants, c4h-3 comt-4 and 4cl1-1 comt-4, the saccharification efficiency was significantly higher compared to WT and its parental lines, independent of the pretreatment used. When no pretreatment was used, the saccharification efficiency increased even synergistically for these mutants. Conclusion: Our results show that saccharification efficiency can be improved by combining two different mutant lignin traits, leading to plants with an even higher saccharification efficiency, without having a yield reduction of the primary inflorescence stem. This approach can help improve saccharification efficiency in bio-energy crops

Plant cell wall profiling by fast maximum likelihood reconstruction (FMLR) and region-of-interest (ROI) segmentation of solution-state 2D 1H–13C NMR spectra

BACKGROUND: Interest in the detailed lignin and polysaccharide composition of plant cell walls has surged within the past decade partly as a result of biotechnology research aimed at converting biomass to biofuels. High-resolution, solution-state 2D (1)H–(13)C HSQC NMR spectroscopy has proven to be an effective tool for rapid and reproducible fingerprinting of the numerous polysaccharides and lignin components in unfractionated plant cell wall materials, and is therefore a powerful tool for cell wall profiling based on our ability to simultaneously identify and comparatively quantify numerous components within spectra generated in a relatively short time. However, assigning peaks in new spectra, integrating them to provide relative component distributions, and producing color-assigned spectra, are all current bottlenecks to the routine use of such NMR profiling methods. RESULTS: We have assembled a high-throughput software platform for plant cell wall profiling that uses spectral deconvolution by Fast Maximum Likelihood Reconstruction (FMLR) to construct a mathematical model of the signals present in a set of related NMR spectra. Combined with a simple region of interest (ROI) table that maps spectral regions to NMR chemical shift assignments of chemical entities, the reconstructions can provide rapid and reproducible fingerprinting of numerous polysaccharide and lignin components in unfractionated cell wall material, including derivation of lignin monomer unit (S:G:H) ratios or the so-called SGH profile. Evidence is presented that ROI-based amplitudes derived from FMLR provide a robust feature set for subsequent multivariate analysis. The utility of this approach is demonstrated on a large transgenic study of Arabidopsis requiring concerted analysis of 91 ROIs (including both assigned and unassigned regions) in the lignin and polysaccharide regions of almost 100 related 2D (1)H–(13)C HSQC spectra. CONCLUSIONS: We show that when a suitable number of replicates are obtained per sample group, the correlated patterns of enriched and depleted cell wall components can be reliably and objectively detected even prior to multivariate analysis. The analysis methodology has been implemented in a publicly-available, cross-platform (Windows/Mac/Linux), web-enabled software application that enables researchers to view and publish detailed annotated spectra in addition to summary reports in simple spreadsheet data formats. The analysis methodology is not limited to studies of plant cell walls but is amenable to any NMR study where ROI segmentation techniques generate meaningful results. Please see Research Article: http://www.biotechnologyforbiofuels.com/content/6/1/46/

Polyploidy affects plant growth and alters cell wall composition

Polyploidization has played a key role in plant breeding and crop improvement. Although its potential to increase biomass yield is well described, the effect of polyploidization on biomass composition has largely remained unexplored. Here, we generated a series of Arabidopsis (Arabidopsis thaliana) plants with different somatic ploidy levels (2n, 4n, 6n, and 8n) and performed rigorous phenotypic characterization. Kinematic analysis showed that polyploids developed slower compared to diploids; however, tetra- and hexaploids, but not octaploids, generated larger rosettes due to delayed flowering. In addition, morphometric analysis of leaves showed that polyploidy affected epidermal pavement cells, with increased cell size and reduced cell number per leaf blade with incrementing ploidy. However, the inflorescence stem dry weight was highest in tetraploids. Cell wall characterization revealed that the basic somatic ploidy level negatively correlated with lignin and cellulose content, and positively correlated with matrix polysaccharide content (i.e. hemicellulose and pectin) in the stem tissue. In addition, higher ploidy plants displayed altered sugar composition. Such effects were linked to the delayed development of polyploids. Moreover, the changes in polyploid cell wall composition promoted saccharification yield. The results of this study indicate that induction of polyploidy is a promising breeding strategy to further tailor crops for biomass production

Rewired phenolic metabolism and improved saccharification efficiency of a Zea mays cinnamyl alcohol dehydrogenase 2 (zmcad2) mutant

Lignocellulosic biomass is an abundant byproduct from cereal crops that can potentially be valorized as a feedstock to produce biomaterials. Zea mays CINNAMYL ALCOHOL DEHYDROGENASE 2 (ZmCAD2) is involved in lignification, and is a promising target to improve the cellulose‐to‐glucose conversion of maize stover. Here, we analyzed a field‐grown zmcad2 Mutator transposon insertional mutant. Zmcad2 mutant plants had an 18% lower Klason lignin content, whereas their cellulose content was similar to that of control lines. The lignin in zmcad2 mutants contained increased levels of hydroxycinnamaldehydes, i.e. the substrates of ZmCAD2, ferulic acid and tricin. Ferulates decorating hemicelluloses were not altered. Phenolic profiling further revealed that hydroxycinnamaldehydes are partly converted into (dihydro)ferulic acid and sinapic acid and their derivatives in zmcad2 mutants. Syringyl lactic acid hexoside, a metabolic sink in CAD‐deficient dicot trees, appeared not to be a sink in zmcad2 maize. The enzymatic cellulose‐to‐glucose conversion efficiency was determined after 10 different thermochemical pre‐treatments. Zmcad2 yielded significantly higher conversions compared with controls for almost every pre‐treatment. However, the relative increase in glucose yields after alkaline pre‐treatment was not higher than the relative increase when no pre‐treatment was applied, suggesting that the positive effect of the incorporation of hydroxycinnamaldehydes was leveled off by the negative effect of reduced p‐coumarate levels in the cell wall. Taken together, our results reveal how phenolic metabolism is affected in CAD‐deficient maize, and further support mutating CAD genes in cereal crops as a promising strategy to improve lignocellulosic biomass for sugar‐platform biorefineries

Different routes for conifer- and sinapaldehyde and higher saccharification upon deficiency in the dehydrogenase CAD1

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 3 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