A small proportion of the thousands of pesticides on the market today are associated with a disproportionately high incidence of severe acute pesticide poisoning and suicide. Paraquat stands out as one of the most lethal pesticides in common use, frequently involved in fatal incidents due to suicides or accidental exposure. Even though paraquat has been banned in over 67 countries, it is still widely used in many others, particularly in Asia and Latin America. Based on a literature review and consultations, this paper identifies options for replacing paraquat and distils practical lessons from numerous successes around the world. Our aim is to support regulators, policymakers, agronomists and the supply chain sector with practical information related to phasing out paraquat. Production data consistently failed to show any negative effects of banning paraquat on agricultural productivity. A wide range of alternative approaches to weed management and crop defoliation are available, many of which do not rely on herbicides. Over 1.25 million farmers in low- and middle-income countries (LMICs) successfully produce a range of crops for private voluntary standards (PVS) in food and fiber supply chains which prohibit paraquat use. We conclude from the findings of this study that eliminating paraquat will save lives without reducing agricultural productivity. Less hazardous and more sustainable alternatives exist. To enhance successful adoption and uptake of these methods on a wide scale, farmers require training and support within an enabling policy environment.

Ecological engineering using linear flower strips is proposed as an alternative to insecticide-based rice pest management. However, its success depends on farmers’ appreciations of related interventions as part of an ecosystem restoration process. We examined agronomic and pest management responses to flower strips among 305 farmers surveyed at 12 villages in the Mekong Delta Region (MDR) of Vietnam. Practices by conventional farmers at the same villages were used as a baseline. The ecological engineering farmers mainly integrated flower strips with pest management practices by reducing insecticide applications before 40 days after rice crop establishment (ca 38% of farmers; 9% more than on conventional farms). Flower strips were also associated with less frequent and irregular insecticide applications or with insecticide-free rice (i.e., possibly IPM: ca 19% of ecological engineering farmers). Otherwise, farmers (ca 43% of ecological engineering farmers) continued to apply insecticides prophylactically and, in some cases, applied more insecticides than their conventional neighbors. Flower strips were not associated with reductions in any other pesticides. Reported yields were not directly affected by flower strips or pesticide inputs. Our results suggest that ecological engineering was not widely regarded by participating farmers as an ecosystem restoration practice, but rather, as a pest management action. Further promotion of flower strips as a component of ecosystem restoration is required to break the lock-in to pesticide use at ecologically engineered rice farms in the MDR.

Introduction Suicide is a major public health problem in Pakistan, accounting to approximately 19,331 deaths every year. Many are due to consumption of acutely toxic pesticides; however, there is a lack of national suicide data, limiting knowledge and potential for intervention. In this paper, we aimed to review the literature on pesticide self-poisoning in Pakistan to identify the most problematic pesticides in relation to national pesticide regulations. Methods Information on the currently registered and banned pesticides was obtained from Ministry of National Food Security and Research while data on pesticide import and use was extracted from FAOSTAT. We searched the following sources for articles and research papers on poisoning in Pakistan: Cumulative Index to Nursing and Allied Health (CINAHL), Google Scholar, Applied Social Sciences Index and Abstracts (ASSIA), Excerpta Medica (EMBASE), National Library of Medicine’s MEDLINE (PUBMED), PS102YCHINFO and Pakmedinet.com using the search terms ‘self-poisoning’, ‘deliberate self-harm’, ‘suicide’, ‘methods and means of suicide’, ‘organophosphate’, ‘wheat pill’, ‘aluminium phosphide’, ‘acute poisoning’, OR ‘pesticides’, AND ‘Pakistan’. Results As of May 2021, 382 pesticide active ingredients (substances) were registered in Pakistan, of which five were WHO hazard class Ia (extremely hazardous) and 17 WHO hazard class Ib (highly hazardous). Twenty-six pesticides, four formulations, and seven non-registered pesticides had been banned, of which two were WHO class Ia and five Ib. We identified 106 hospital-level studies of poisoning conducted in Pakistan, of which 23 did not mention self-poisoning cases and one reported no suicidal poisoning cases. We found no community or forensic medicine studies. Of 52,323 poisoning cases identified in these papers, 24,546 [47%] were due to pesticides. The most commonly identified pesticide classes were organophosphorus (OP) insecticides (13,816 cases, 56%) and the fumigant aluminium phosphide (3 g 56% tablets, often termed ‘wheat pills’; 686 cases, 2.7%). Few studies identified the particular pesticides involved or the resulting case fatality. Conclusion We found pesticide poisoning to be a major cause of poisoning in Pakistan, with OP insecticides and the fumigant aluminium phosphide the main pesticides identified. Withdrawal of Class I pesticides (as proposed to occur nationally in 2022) and high concentration aluminium phosphide tablets should rapidly reduce suicidal deaths by reducing the case fatality for low-intention poisoning cases. National cause of death data and forensic toxicology laboratory data identifying the pesticides responsible for deaths will be important to assess impacts of the proposed national ban.

Background Removing highly hazardous pesticides from agricultural practice in low- and middle-income countries is crucial to ensuring community and environmental health and occupational safety of farmers. However, the approach has been challenged as threatening food production, despite evidence from Asian countries that curbing agricultural use of highly hazardous pesticides does not affect crop yields. In 2011, the state of Kerala, India, banned 14 highly hazardous pesticides resulting in a marked reduction in deaths from pesticide poisoning. Objective We aimed to determine whether the Kerala pesticide bans impacted agricultural yields. Methods We collected data on agricultural production, area under cultivation, and rainfall, published by the Kerala state agricultural department from 2004 to 2018 for eight key crops that had been treated with the banned pesticides. Trends in crop yields (total production/area under cultivation) and rainfall across 14 districts in Kerala were aggregated and analysed using joinpoint regression. These trends were evaluated to ascertain possible associations with the pesticide bans. Results The joinpoint regression analyses showed no evidence for any change in yield trends for any of the eight crops in the year of the pesticide bans (2011), or the subsequent year (2012), suggesting a negligible impact of the bans on crop yields. Steady trends of predominately reductions in overall rainfall, without any change around the time of the pesticide bans, was observed in Kerala throughout the period. No evidence of district-level changes in rainfall that might have offset any potential adverse impacts of the pesticide bans on crop yields was noted. Fluctuations in yield until 2018 could be explained by variation in rainfall, changes in land use, and agricultural policies. Conclusion We found no evidence of an adverse effect on agricultural yields in Kerala that could be attributed to bans of highly hazardous pesticides. This work provides further evidence that such pesticides can be withdrawn from agricultural use without affecting yields. Further studies are required for the whole of India after the national bans of 12 pesticides in 2018 to identify state-level effects of the bans.

OBJECTIVE:Although pesticide poisonings occur worldwide, most high-income countries have not been severely affected. Japan is a key exception, with pesticide suicides becoming a major public health concern in the 1980s. We here report the epidemiology of lethal pesticide poisoning in Japan in relation to its pesticide regulation and agricultural output. METHODS:We obtained data on pesticide registration and sales from the Japan Plant Protection Association's annual Pesticide Handbook, National Food and Agricultural Materials Inspection Center, and Food and Agriculture Organization of the United Nations. Data on deaths due to pesticide poisoning and population were obtained from Vital Statistics of Japan. We reviewed the registration status and hazard classification of pesticides in Japan and analysed the relationships between the use/sales, pesticides fatalities, and agricultural output. RESULTS:Five hundred and twenty-nine pesticide active ingredients are currently registered in Japan, including four WHO hazard class IB organophosphorus and carbamate insecticides. Paraquat was registered in 1962 as a liquid SL20 formulation. In 1986, restrictions were imposed on its sale/use and a 4.3% paraquat ion/4.1% diquat ion combination product registered by ICI. There were 221 pesticide poisoning fatalities in 2019, down from 2648 in 1986, a 92% reduction over 33 years. Self-poisoning was responsible for most pesticide deaths in both 1985 (2013/2476, 81.3%) and 2019 (146/221, 66.1%). Pesticide poisoning made up 8.6% of all suicides in 1985, down to 0.7% in 2019. Unintentional pesticide poisoning deaths also all fell by 83.8%, from 463 to 75. These reductions were associated with reduced sales of both OP/carbamate insecticides and paraquat/diquat but no apparent change in agricultural output across a broad range of crops. CONCLUSIONS:Reduced use of highly hazardous pesticides and lowered concentration formulations in Japan were associated with major decreases in all deaths from pesticide poisoning and the proportion of all suicides due to pesticide ingestion.

To achieve a bio‐based economy, it is necessary to consider variability within a feedstock population. We must understand the range of key phenotypic characteristics when selecting economically advantageous genotypes for domestication in an optimized supply chain. In this analysis we measured cell‐wall composition traits in a large natural variant population of Populus trichocarpa . The results were combined with agronomic growth data from the matching genotype to conduct various techno‐economic analyses, evaluating the impacts of physical and compositional variability and determining the ultimate phenotypic drivers for yield and economic metrics. Here we show that, although ethanol yield per land area per year and minimum fuel selling price were most strongly impacted by tree size, when considering the largest 25% of trees, size and carbohydrate content were nearly identical influences on minimal fuel selling price, highlighting the need to focus on both size and carbohydrate content in selecting economically optimal feedstocks. © 2020 The Authors. Biofuels, Bioproducts, and Biorefining published by Society of Chemical Industry and John Wiley & Sons, Ltd

Background Multiple analytical methods have been developed to determine the ratios of aromatic lignin units, particularly the syringyl/guaiacyl (S/G) ratio, of lignin biopolymers in plant cell walls. Chemical degradation methods such as thioacidolysis produce aromatic lignin units that are released from certain linkages and may induce chemical changes rendering it difficult to distinguish and determine the source of specific aromatic lignin units released, as is the case with nitrobenzene oxidation methodology. NMR methods provide powerful tools used to analyze cell walls for lignin composition and linkage information. Pyrolysis-mass spectrometry methods are also widely used, particularly as high-throughput methodologies. However, the different techniques used to analyze aromatic lignin unit ratios frequently yield different results within and across particular studies, making it difficult to interpret and compare results. This also makes it difficult to obtain meaningful insights relating these measurements to other characteristics of plant cell walls that may impact biomass sustainability and conversion metrics for the production of bio-derived fuels and chemicals. Results The authors compared the S/G lignin unit ratios obtained from thioacidolysis, pyrolysis-molecular beam mass spectrometry (py-MBMS), HSQC liquid-state NMR and solid-state (ss) NMR methodologies of pine, several genotypes of poplar, and corn stover biomass. An underutilized approach to deconvolute ssNMR spectra was implemented to derive S/G ratios. The S/G ratios obtained for the samples did not agree across the different methods, but trends were similar with the most agreement among the py-MBMS, HSQC NMR and deconvoluted ssNMR methods. The relationship between S/G, thioacidolysis yields, and linkage analysis determined by HSQC is also addressed. Conclusions This work demonstrates that different methods using chemical, thermal, and nondestructive NMR techniques to determine native lignin S/G ratios in plant cell walls may yield different results depending on species and linkage abundances. Spectral deconvolution can be applied to many hardwoods with lignin dominated by S and G units, but the results may not be reliable for some woody and grassy species of more diverse lignin composition. HSQC may be a better method for analyzing lignin in those species given the wealth of information provided on additional aromatic moieties and bond linkages. Additionally, trends or correlations in lignin characteristics such as S/G ratios and lignin linkages within the same species such as poplar may not necessarily exhibit the same trends or correlations made across different biomass types. Careful consideration is required when choosing a method to measure S/G ratios and the benefits and shortcomings of each method discussed here are summarized.

Background Pyrolysis-molecular beam mass spectrometry (py-MBMS) analysis of a pedigree of Populus trichocarpa was performed to study the phenotypic plasticity and heritability of lignin content and lignin monomer composition. Instrumental and microspatial environmental variability were observed in the spectral features and corrected to reveal underlying genetic variance of biomass composition. Results Lignin-derived ions (including m/z 124, 154, 168, 194, 210 and others) were highly impacted by microspatial environmental variation which demonstrates phenotypic plasticity of lignin composition in Populus trichocarpa biomass. Broad-sense heritability of lignin composition after correcting for microspatial and instrumental variation was determined to be H 2 = 0.56 based on py-MBMS ions known to derive from lignin. Heritability of lignin monomeric syringyl/guaiacyl ratio ( S / G ) was H 2 = 0.81. Broad-sense heritability was also high (up to H 2 = 0.79) for ions derived from other components of the biomass including phenolics (e.g., salicylates) and C5 sugars (e.g., xylose). Lignin and phenolic ion abundances were primarily driven by maternal effects, and paternal effects were either similar or stronger for the most heritable carbohydrate-derived ions. Conclusions We have shown that many biopolymer-derived ions from py-MBMS show substantial phenotypic plasticity in response to microenvironmental variation in plantations. Nevertheless, broad-sense heritability for biomass composition can be quite high after correcting for spatial environmental variation. This work outlines the importance in accounting for instrumental and microspatial environmental variation in biomass composition data for applications in heritability measurements and genomic selection for breeding poplar for renewable fuels and materials.

Development of commercially relevant bioenergy switchgrass cultivars requires reducing recalcitrance for bioprocessing without compromising biomass yield. Low-lignin transgenic switchgrass has been produced via down-regulation of caffeic acid O-methyltransferase (COMT), a lignin biosynthetic enzyme, or by over-expression of the MYB4 transcription factor, a repressor of the lignin biosynthetic pathway. The aim of this study was to evaluate parents and selected hybrids obtained from COMT and MYB4 hybrid families under field conditions for agronomic performance and biomass quality. Plant height, width, number of tillers, dry weight, cell wall composition including lignin content, and sugar release were measured after the establishment year (2014) and the second growing season (2015). For COMT hybrids, biomass yield of the transgenic hybrids was similar to or greater than the wild-type parents selected for high biomass. Lignin content of COMT transgenic hybrids was reduced by 10%, S/G ratio decreased by 27%, and sugar release increased between 20% and 44% compared to their wild-type parents. These results indicate that hybridization of COMT with a high-yielding locally selected genotype resulted in both improved agronomic performance and enhanced biomass quality in the offspring. On the other hand, the MYB transgenic hybrid showed a 10% reduction in biomass yield compared with its wild-type parent in year 1, but not in year 2. The lignin S/G ratio was not reduced in MYB transgenic hybrids, nor was sugar release increased. These data indicate that the MYB transgene may not be suitable for an agronomic setting. More testing is needed of transgenic and wild-type, high-biomass selections for use as breeding parents. These results show that combining low-lignin transgenic switchgrass with a breeding and selection program for biomass yield will allow for the deployment of effective transgenes in high-yielding genetic backgrounds.

Switchgrass (Panicum virgatum L.) is a lignocellulosic perennial grass with great potential in bioenergy field. Lignocellulosic bioenergy crops are mostly resistant to cell wall deconstruction, and therefore yield suboptimal levels of biofuel. The one-carbon pathway (also known as C1 metabolism) is critical for polymer methylation, including that of lignin and hemicelluloses in cell walls. Folylpolyglutamate synthetase (FPGS) catalyzes a biochemical reaction that leads to the formation of folylpolyglutamate, an important cofactor for many enzymes in the C1 pathway. In this study, the putatively novel switchgrass PvFPGS1 gene was identified and its functional role in cell wall composition and biofuel production was examined by RNAi knockdown analysis. The PvFPGS1-downregulated plants were analyzed in the field over three growing seasons. Transgenic plants with the highest reduction in PvFPGS1 expression grew slower and produced lower end-of-season biomass. Transgenic plants with low-to-moderate reduction in PvFPGS1 transcript levels produced equivalent biomass as controls. There were no significant differences observed for lignin content and syringyl/guaiacyl lignin monomer ratio in the low-to-moderately reduced PvFPGS1 transgenic lines compared with the controls. Similarly, sugar release efficiency was also not significantly different in these transgenic lines compared with the control lines. However, transgenic plants produced up to 18% more ethanol while maintaining congruent growth and biomass as non-transgenic controls. Severity of rust disease among transgenic and control lines were not different during the time course of the field experiments. Altogether, the unchanged lignin content and composition in the low-to-moderate PvFPGS1-downregulated lines may suggest that partial downregulation of PvFPGS1 expression did not impact lignin biosynthesis in switchgrass. In conclusion, the manipulation of PvFPGS1 expression in bioenergy crops may be useful to increase biofuel potential with no growth penalty or increased susceptibility to rust in feedstock.

Cellulose synthase A genes (CesAs) are responsible for cellulose biosynthesis in plant cell walls. In this study, functions of secondary wall cellulose synthases PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B were characterized during wood formation in Populus trichocarpa (Torr. & Gray). CesA RNAi knockdown transgenic plants exhibited stunted growth, narrow leaves, early necrosis, reduced stature, collapsed vessels, thinner fiber cell walls and extended fiber lumen diameters. In the RNAi knockdown transgenics, stems exhibited reduced mechanical strength, with reduced modulus of rupture (MOR) and modulus of elasticity (MOE). The reduced mechanical strength may be due to thinner fiber cell walls. Vessels in the xylem of the transgenics were collapsed, indicating that water transport in xylem may be affected and thus causing early necrosis in leaves. A dramatic decrease in cellulose content was observed in the RNAi knockdown transgenics. Compared with wildtype, the cellulose content was significantly decreased in the PtrCesA4, PtrCesA7 and PtrCesA8 RNAi knockdown transgenics. As a result, lignin and xylem contents were proportionally increased. The wood composition changes were confirmed by solid-state NMR, two-dimensional solution-state NMR and sum-frequency-generation vibration (SFG) analyses. Both solid-state nuclear magnetic resonance (NMR) and SFG analyses demonstrated that knockdown of PtrCesAs did not affect cellulose crystallinity index. Our results provided the evidence for the involvement of PtrCesA4, PtrCesA7-A/B and PtrCesA8-A/B in secondary cell wall formation in wood and demonstrated the pleiotropic effects of their perturbations on wood formation.

In plants, the phenylpropanoid pathway is responsible for the synthesis of a diverse array of secondary metabolites that include lignin monomers, flavonoids, and coumarins, many of which are essential for plant structure, biomass recalcitrance, stress defense, and nutritional quality. Our previous studies have demonstrated that Populus trichocarpa PtrEPSP-TF, an isoform of 5-enolpyruvylshikimate 3-phosphate (EPSP) synthase, has transcriptional activity and regulates phenylpropanoid biosynthesis in Populus. In this study, we report the identification of single nucleotide polymorphism (SNP) of PtrEPSP-TF that defines its functionality. Populus natural variants carrying this SNP were shown to have reduced lignin content. Here, we demonstrated that the SNP-induced substitution of 142nd amino acid (PtrEPSP-TFD142E) dramatically impairs the DNA-binding and transcriptional activity of PtrEPSP-TF. When introduced to a monocot species rice (Oryza sativa) in which an EPSP synthase isoform with the DNA-binding helix-turn-helix (HTH) motif is absent, the PtrEPSP-TF, but not PtrEPSP-TFD142E, activated genes in the phenylpropanoid pathway. More importantly, heterologous expression of PtrEPSP-TF uncovered five new transcriptional regulators of phenylpropanoid biosynthesis in rice. Collectively, this study identifies the key amino acid required for PtrEPSP-TF functionality and provides a strategy to uncover new transcriptional regulators in phenylpropanoid biosynthesis.

The lignin biosynthetic pathway is highly conserved in angiosperms, yet pathway manipulations give rise to a variety of taxon-specific outcomes. Knockout of lignin-associated 4-coumarate:CoA ligases (4CLs) in herbaceous species mainly reduces guaiacyl (G) lignin and enhances cell wall saccharification. Here we show that CRISPR-knockout of 4CL1 in poplar (Populus tremula × alba) preferentially reduced syringyl (S) lignin, with negligible effects on biomass recalcitrance. Concordant with reduced S-lignin was downregulation of ferulate 5-hydroxylases (F5Hs). Lignification was largely sustained by 4CL5, a low-affinity paralog of 4CL1 typically with only minor xylem expression or activity. Levels of caffeate, the preferred substrate of 4CL5, increased in line with significant upregulation of caffeoyl shikimate esterase1 Upregulation of caffeoyl-CoA O-methyltransferase1 and downregulation of F5Hs are consistent with preferential funneling of 4CL5 products toward G-lignin biosynthesis at the expense of S-lignin. Thus, transcriptional and metabolic adaptations to 4CL1-knockout appear to have enabled 4CL5 catalysis at a level sufficient to sustain lignification. Finally, genes involved in sulfur assimilation, the glutathione-ascorbate cycle, and various antioxidant systems were upregulated in the mutants, suggesting cascading responses to perturbed thioesterification in lignin biosynthesis.

Fungal pretreatment of Populus trichocarpa wood genetically modified to reduce lignin and alter lignin chemistry is investigated for its effectiveness as an alternative to common pretreatment methods. The goal of this work is to improve biomass utilization for biofuel and biochemical applications by increasing sugar release. Sugar release after enzymatic hydrolysis was measured after various biomass pretreatments (including wood-rot fungus, hot water, and dilute acid). In the wildtype, and in constructs downregulated in PAL, 4CL, and C3H, the fungal pretreatment resulted in substantial improvements in sugar yields, up to 2.4-fold increase in glucose yield and 6-fold increase in xylose yield after enzymatic hydrolysis compared to the unpretreated control. However, the effects of fungal pretreatment were inconsistent, and in genetic lines down-regulated in 4CL, CCoAOMT, CAld5H, and C3H, fungal pretreatment yielded similar or decreased sugar release after enzymatic hydrolysis.

Wood processing is often performed at elevated temperatures under moisture-saturated conditions; therefore, it is important to understand the impact of the lignin content and lignin chemical structure on the thermo-mechanical properties of wood. In this study, genetically modified Populus trichocarpa wood specimens with down-regulated cinnamyl alcohol dehydrogenase, cinnamate 3-hydroxylase, and cinnamate 4-hydroxylase with altered lignin contents and/or lignin structures were utilized to probe the relationship between the lignin content, lignin monomer composition, and thermo-mechanical properties of solid wood. The thermo-mechanical properties of these unique samples were measured using dynamic mechanical analysis and the nuclear magnetic resonance (NMR) spin-spin relaxation time. The results showed that the transgenic P. trichocarpa samples had decreased storage and loss moduli compared with the wildtype. The solid-state NMR revealed increased lignin molecular mobility in the reduced-lignin transgenic lines. Also, noticeably reduced glass transition temperatures (Tg) were observed in the transgenic lines with reduced lignin contents and altered lignin monomer compositions compared with the wildtype. The increased lignin molecular mobility and reduced Tg in these samples can probably contribute to wood utilization and processing, such as lignin removal for pulp and paper and biofuels production, as well as particle consolidation during wood composite manufacturing.

Background Populus trichocarpa is an important forest tree species for the generation of lignocellulosic ethanol. Understanding the genomic basis of biomass production and chemical composition of wood is fundamental in supporting genetic improvement programs. Considerable variation has been observed in this species for complex traits related to growth, phenology, ecophysiology and wood chemistry. Those traits are influenced by both polygenic control and environmental effects, and their genome architecture and regulation are only partially understood. Genome wide association studies (GWAS) represent an approach to advance that aim using thousands of single nucleotide polymorphisms (SNPs). Genotyping using exome capture methodologies represent an efficient approach to identify specific functional regions of genomes underlying phenotypic variation. Results We identified 813 K SNPs, which were utilized for genotyping 461 P. trichocarpa clones, representing 101 provenances collected from Oregon and Washington, and established in California. A GWAS performed on 20 traits, considering single SNP-marker tests identified a variable number of significant SNPs ( p -value < 6.1479E-8) in association with diameter, height, leaf carbon and nitrogen contents, and δ 15 N. The number of significant SNPs ranged from 2 to 220 per trait. Additionally, multiple-marker analyses by sliding-windows tests detected between 6 and 192 significant windows for the analyzed traits. The significant SNPs resided within genes that encode proteins belonging to different functional classes as such protein synthesis, energy/metabolism and DNA/RNA metabolism, among others. Conclusions SNP-markers within genes associated with traits of importance for biomass production were detected. They contribute to characterize the genomic architecture of P. trichocarpa biomass required to support the development and application of marker breeding technologies.

Abstract Background The cellular machinery for cell wall synthesis and metabolism is encoded by members of large multi-gene families. Maize is both a genetic model for grass species and a potential source of lignocellulosic biomass from crop residues. Genetic improvement of maize for its utility as a bioenergy feedstock depends on identification of the specific gene family members expressed during secondary wall development in stems. Results High-throughput sequencing of transcripts expressed in developing rind tissues of stem internodes provided a comprehensive inventory of cell wall-related genes in maize (Zea mays, cultivar B73). Of 1239 of these genes, 854 were expressed among the internodes at ≥95 reads per 20 M, and 693 of them at ≥500 reads per 20 M. Grasses have cell wall compositions distinct from non-commelinid species; only one-quarter of maize cell wall-related genes expressed in stems were putatively orthologous with those of the eudicot Arabidopsis. Using a slope-metric algorithm, five distinct patterns for sub-sets of co-expressed genes were defined across a time course of stem development. For the subset of genes associated with secondary wall formation, fifteen sequence motifs were found in promoter regions. The same members of gene families were often expressed in two maize inbreds, B73 and Mo17, but levels of gene expression between them varied, with 30% of all genes exhibiting at least a 5-fold difference at any stage. Although presence-absence and copy-number variation might account for much of these differences, fold-changes of expression of a CADa and a FLA11 gene were attributed to polymorphisms in promoter response elements. Conclusions Large genetic variation in maize as a species precludes the extrapolation of cell wall-related gene expression networks even from one common inbred line to another. Elucidation of genotype-specific expression patterns and their regulatory controls will be needed for association panels of inbreds and landraces to fully exploit genetic variation in maize and other bioenergy grass species.

Following publication of the original article [1], the authors reported that the omission of author name.

Engineered switchgrass and poplar are better feedstocks for biofuel synthesis and yield more biomass in multi-year field trials. Cell walls in crops and trees have been engineered for production of biofuels and commodity chemicals, but engineered varieties often fail multi-year field trials and are not commercialized. We engineered reduced expression of a pectin biosynthesis gene (Galacturonosyltransferase 4, GAUT4) in switchgrass and poplar, and find that this improves biomass yields and sugar release from biomass processing. Both traits were maintained in a 3-year field trial of GAUT4-knockdown switchgrass, with up to sevenfold increased saccharification and ethanol production and sixfold increased biomass yield compared with control plants. We show that GAUT4 is an α-1,4-galacturonosyltransferase that synthesizes homogalacturonan (HG). Downregulation of GAUT4 reduces HG and rhamnogalacturonan II (RGII), reduces wall calcium and boron, and increases extractability of cell wall sugars. Decreased recalcitrance in biomass processing and increased growth are likely due to reduced HG and RGII cross-linking in the cell wall.