Identifying transcription factors that reduce wood recalcitrance and improve enzymatic degradation of xylem cell wall in Populus
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ABSTRACT:
SUBMITTER: Hori C
PROVIDER: S-EPMC7744511 | biostudies-literature | 2020 Jan
REPOSITORIES: biostudies-literature
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Project description:Tension wood (TW) is a specialized xylem tissue developed under mechanical/tension stress in angiosperm trees. TW development involves transregulation of secondary cell wall genes, which leads to altered wood properties for stress adaptation. We induced TW in the stems of black cottonwood (Populus trichocarpa, Nisqually-1) and identified two significantly repressed transcription factor (TF) genes: class B3 heat-shock TF (HSFB3-1) and MYB092. Transcriptomic analysis and chromatin immunoprecipitation (ChIP) were used to identify direct TF-DNA interactions in P. trichocarpa xylem protoplasts overexpressing the TFs. This analysis established a transcriptional regulatory network in which PtrHSFB3-1 and PtrMYB092 directly activate 8 and 11 monolignol genes, respectively. The TF-DNA interactions were verified for their specificity and transactivator roles in 35 independent CRISPR-based biallelic mutants and overexpression transgenic lines of PtrHSFB3-1 and PtrMYB092 in P. trichocarpa. The gene-edited trees (mimicking the repressed PtrHSFB3-1 and PtrMYB092 under tension stress) have stem wood composition resembling that of TW during normal growth and under tension stress (i.e., low lignin and high cellulose), whereas the overexpressors showed an opposite effect (high lignin and low cellulose). Individual overexpression of the TFs impeded lignin reduction under tension stress and restored high levels of lignin biosynthesis in the TW. This study offers biological insights to further uncover how metabolism, growth, and stress adaptation are coordinately regulated in trees.
| S-EPMC8154086 | biostudies-literature
Project description:UnlabelledBackgroundThe recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.ResultsA pot trial was designed to test if the 'RW response' varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.ConclusionsOur findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a 'RW response') indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.
| S-EPMC3541151 | biostudies-literature
Project description:Plant lignocellulosic biomass, i.e. secondary cell walls of plants, is a vital alternative source for bioenergy. However, the acetylation of xylan in secondary cell walls impedes the conversion of biomass to biofuels. Previous studies have shown that REDUCED WALL ACETYLATION (RWA) proteins are directly involved in the acetylation of xylan but the regulatory mechanism of RWAs is not fully understood. In this study, we demonstrate that overexpression of a Populus trichocarpa PtRWA-C gene increases the level of xylan acetylation and increases the lignin content and S/G ratio, ultimately yielding poplar woody biomass with reduced saccharification efficiency. Furthermore, through gene coexpression network and expression quantitative trait loci (eQTL) analysis, we found that PtRWA-C was regulated not only by the secondary cell wall hierarchical regulatory network but also by an AP2 family transcription factor HARDY (HRD). Specifically, HRD activates PtRWA-C expression by directly binding to the PtRWA-C promoter, which is also the cis-eQTL for PtRWA-C. Taken together, our findings provide insights into the functional roles of PtRWA-C in xylan acetylation and consequently saccharification and shed light on synthetic biology approaches to manipulate this gene and alter cell wall properties. These findings have substantial implications for genetic engineering of woody species, which could be used as a sustainable source of biofuels, valuable biochemicals, and biomaterials.
| S-EPMC10762510 | biostudies-literature
Project description:Regulation of gene expression, as determined by the genetics of the tree species, is a major factor in determining wood quality. Therefore, the identification of genes that play a role in xylogenesis is extremely important for understanding the mechanisms shaping the plant phenotype. Efforts to develop new varieties characterized by higher yield and better wood quality will greatly benefit from recognizing and understanding the complex transcriptional network underlying wood development. The present study provides a detailed comparative description of the changes that occur in genes transcription and the biosynthesis of cell-wall-related compounds during xylogenesis in Populus trichocarpa pioneer roots and stems. Even though results of microarray analysis indicated that only approximately 10% of the differentially expressed genes were common to both organs, many fundamental mechanisms were similar; e.g. the pattern of expression of genes involved in the biosynthesis of cell wall proteins, polysaccharides, and lignins. Gas chromatography time-of-flight mass spectrometry (GC-TOF-MS) shows that the composition of monosaccharides was also very similar, with an increasing amount of xylose building secondary cell wall hemicellulose and pectins, especially in the stems. While hemicellulose degradation was typical for stems, possibly due to the intensive level of cell wall lignification. Notably, the main component of lignins in roots were guiacyl units, while syringyl units were dominant in stems, where fibers are especially needed for support. Our study is the first comprehensive analysis, at the structural and molecular level, of xylogenesis in under- and aboveground tree parts, and clearly reveals the great complexity of molecular mechanisms underlying cell wall formation and modification during xylogenesis in different plant organs.
| S-EPMC6861220 | biostudies-literature
Project description:Background:During the chemical and biochemical decomposition of lignocellulosic biomasses, lignin is highly recalcitrant. Genetic transformation of plants to qualitatively and/or quantitatively modify lignin may reduce these recalcitrant properties. Efficient discovery of genes to achieve lignin manipulation is thus required. Results:To screen for new genes to reduce lignin recalcitrance, we heterologously expressed 50 enzymatic genes under the control of a cinnamate 4-hydroxylase (C4H) gene promoter, derived from a hybrid aspen, which is preferentially active in tissues with lignified cell walls in Arabidopsis plants. These genes encode enzymes that act on metabolites in shikimate, general phenylpropanoid, flavonoid, or monolignol biosynthetic pathways. Among these genes, 30, 18, and 2 originated from plants, bacteria, and fungi, respectively. In our first screening step, 296 independent transgenic plants (T1 generation) harboring single or multiple transgenes were generated from pools of seven Agrobacterium strains used for conventional floral-dip transformation. Wiesner and Mäule staining patterns in the stems of the resultant plants revealed seven and nine plants with apparent abnormalities in the two respective staining analyses. According to genomic PCR and subsequent direct sequencing, each of these 16 plants possessed a gene encoding either coniferaldehyde dehydrogenase (calB), feruloyl-CoA 6'-hydroxylase (F6H1), hydroxycinnamoyl-CoA hydratase/lyase (couA), or ferulate 5-hydroxylase (F5H), with one transgenic plant carrying both calB and F6H1. The effects of these genes on lignin manipulation were confirmed in individually re-created T1 transgenic Arabidopsis plants. While no difference in lignin content was detected in the transgenic lines compared with the wild type, lignin monomeric composition was changed in the transgenic lines. The observed compositional change in the transgenic plants carrying calB, couA, and F5H led to improved sugar release from cell walls after alkaline pretreatment. Conclusions:Simple colorimetric characterization of stem lignin is useful for simultaneous screening of many genes with the potential to reduce lignin recalcitrance. In addition to F5H, the positive control, we identified three enzyme-coding genes that can function as genetic tools for lignin manipulation. Two of these genes (calB and couA) accelerate sugar release from transgenic lignocelluloses.
| S-EPMC7260809 | biostudies-literature
Project description:Plant cell wall associated hydroxyproline-rich glycoproteins (HRGPs) are involved in several aspects of plant growth and development, including wood formation in trees. HRGPs such as arabinogalactan-proteins (AGPs), extensins (EXTs), and proline rich proteins (PRPs) are important for the development and architecture of plant cell walls. Analysis of publicly available gene expression data revealed that many HRGP encoding genes show tight spatio-temporal expression patterns in the developing wood of Populus that are indicative of specific functions during wood formation. Similar results were obtained for the expression of glycosyl transferases putatively involved in HRGP glycosylation. In situ immunolabelling of transverse wood sections using AGP and EXT antibodies revealed the cell type specificity of different epitopes. In mature wood AGP epitopes were located in xylem ray cell walls, whereas EXT epitopes were specifically observed between neighboring xylem vessels, and on the ray cell side of the vessel walls, likely in association with pits. Molecular mass and glycan analysis of AGPs and EXTs in phloem/cambium, developing xylem, and mature xylem revealed clear differences in glycan structures and size between the tissues. Separation of AGPs by agarose gel electrophoresis and staining with β-D-glucosyl Yariv confirmed the presence of different AGP populations in phloem/cambium and xylem. These results reveal the diverse changes in HRGP-related processes that occur during wood formation at the gene expression and HRGP glycan biosynthesis levels, and relate HRGPs and glycosylation processes to the developmental processes of wood formation.
| S-EPMC7768015 | biostudies-literature
Project description:Wood represents a promising source of sugars to produce bio-based renewables, including biofuels. However, breaking down lignocellulose requires costly pretreatments because lignocellulose is recalcitrant to enzymatic saccharification. Increasing saccharification potential would greatly contribute to make wood a competitive alternative to petroleum, but this requires improving wood properties. To identify wood biomass traits associated with saccharification, we analyzed a total of 65 traits related to wood chemistry, anatomy and structure, biomass production and saccharification in 40 genetically engineered Populus tree lines. These lines exhibited broad variation in quantitative traits, allowing for multivariate analyses and mathematical modeling. Modeling revealed that seven wood biomass traits associated in a predictive manner with saccharification of glucose after pretreatment. Four of these seven traits were also negatively associated with biomass production, suggesting a trade-off between saccharification potential and total biomass, which has previously been observed to offset the overall sugar yield from whole trees. We therefore estimated the "total-wood glucose yield" (TWG) from whole trees and found 22 biomass traits predictive of TWG after pretreatment. Both saccharification and TWG were associated with low abundant, often overlooked matrix polysaccharides such as arabinose and rhamnose which possibly represent new markers for improved Populus feedstocks.
| S-EPMC5693926 | biostudies-literature
Project description:BackgroundAflatoxins are carcinogenic compounds produced by certain species of Aspergillus fungi. The consumption of crops contaminated with this toxin cause serious detrimental health effects, including death, in both livestock and humans. As a consequence, both the detection and quantification of this toxin in food/feed items is tightly regulated with crops exceeding the allowed limits eliminated from food chains. Globally, this toxin causes massive agricultural and economic losses each year.ResultsIn this paper we investigate the feasibility of using an aflatoxin-degrading enzyme strategy to reduce/eliminate aflatoxin loads in developing maize kernels. We used an endoplasmic reticulum (ER) targeted sub-cellular compartmentalization stabilizing strategy to accumulate an aflatoxin-degrading enzyme isolated from the edible Honey mushroom Armillariella tabescens and expressed it in embryo tissue in developing maize kernels. Three transgenic maize lines that were determined to be expressing the aflatoxin-degrading enzyme both at the RNA and protein level, were challenged with the aflatoxin-producing strain Aspergillus flavus AF13 and shown to accumulate non-detectable levels of aflatoxin at 14-days post-infection and significantly reduced levels of aflatoxin at 30-days post-infection compared to nontransgenic control Aspergillus-challenged samples.ConclusionsThe expression of an aflatoxin-degrading enzyme in developing maize kernels was shown to be an effective means to control aflatoxin in maize in pre-harvest conditions. This aflatoxin-degradation strategy could play a significant role in the enhancement of both US and global food security and sustainability.
| S-EPMC8684248 | biostudies-literature
Project description:Current research and development in cellulosic ethanol production has been focused mainly on agricultural residues and dedicated energy crops such as corn stover and switchgrass; however, woody biomass remains a very important feedstock for ethanol production. The precise composition of hemicellulose in the wood is strongly dependent on the plant species, therefore different types of enzymes are needed based on hemicellulose complexity and type of pretreatment. In general, hardwood species have much lower recalcitrance to enzymes than softwood. For hardwood, xylanases, beta-xylosidases and xyloglucanases are the main hemicellulases involved in degradation of the hemicellulose backbone, while for softwood the effect of mannanases and beta-mannosidases is more relevant. Furthermore, there are different key accessory enzymes involved in removing the hemicellulosic fraction and increasing accessibility of cellulases to the cellulose fibres improving the hydrolysis process. A diversity of enzymatic cocktails has been tested using from low to high densities of biomass (2-20% total solids) and a broad range of results has been obtained. The performance of recently developed commercial cocktails on hardwoods and softwoods will enable a further step for the commercialization of fuel ethanol from wood.
| S-EPMC4767290 | biostudies-other
Project description:Wood, the most abundant biomass on Earth, is composed of secondary xylem differentiated from vascular cambium. However, the underlying molecular mechanisms of wood formation remain largely unclear. To gain insight into wood formation, we performed a series of wood-forming tissue-specific transcriptome analyses from a hybrid poplar (Populus alba × P. glandulosa, clone BH) using RNA-seq. Together with shoot apex and leaf tissue, cambium and xylem tissues were isolated from vertical stem segments representing a gradient of secondary growth developmental stages (i.e., immature, intermediate, and mature stem). In a comparative transcriptome analysis of the 'developing xylem' and 'leaf' tissue, we could identify critical players catalyzing each biosynthetic step of secondary wall components (e.g., cellulose, xylan, and lignin). Several candidate genes involved in the initiation of vascular cambium formation were found via a co-expression network analysis using abundantly expressed genes in the 'intermediate stem-derived cambium' tissue. We found that transgenic Arabidopsis plants overexpressing the PtrHAM4-1, a GRAS family transcription factor, resulted in a significant increase of vascular cambium development. This phenotype was successfully reproduced in the transgenic poplars overexpressing the PtrHAM4-1. Taken together, our results may serve as a springboard for further research to unravel the molecular mechanism of wood formation, one of the most important biological processes on this planet.
| S-EPMC6770981 | biostudies-literature