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Switchgrass is a leading dedicated bioenergy feedstock in the United States because it is a native, high-yielding, perennial prairie grass with a broad cultivation range and low agronomic input requirements. Biomass conversion research has developed processes for production of ethanol and other biofuels, but they remain costly primarily because of the intrinsic recalcitrance of biomass. We show here that genetic modification of switchgrass can produce phenotypically normal plants that have reduced thermal-chemical ([less-than or equal to]180 °C), enzymatic, and microbial recalcitrance. Down-regulation of the switchgrass caffeic acid O-methyltransferase gene decreases lignin content modestly, reduces the syringyl:guaiacyl lignin monomer ratio, improves forage quality, and, most importantly, increases the ethanol yield by up to 38% using conventional biomass fermentation processes. The down-regulated lines require less severe pretreatment and 300-400% lower cellulase dosages for equivalent product yields using simultaneous saccharification and fermentation with yeast. Furthermore, fermentation of diluted acid-pretreated transgenic switchgrass using Clostridium thermocellum with no added enzymes showed better product yields than obtained with unmodified switchgrass. Therefore, this apparent reduction in the recalcitrance of transgenic switchgrass has the potential to lower processing costs for biomass fermentation-derived fuels and chemicals significantly. Alternatively, such modified transgenic switchgrass lines should yield significantly more fermentation chemicals per hectare under identical process conditions.

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.

Summary Ferulate 5‐hydroxylase (F5H) catalyses the hydroxylation of coniferyl alcohol and coniferaldehyde for the biosynthesis of syringyl (S) lignin in angiosperms. However, the coordinated effects of F5H with caffeic acid O‐methyltransferase (COMT) on the metabolic flux towards S units are largely unknown. We concomitantly regulated F5H expression in COMT‐down‐regulated transgenic switchgrass (Panicum virgatum L.) lines and studied the coordination of F5H and COMT in lignin biosynthesis. Down‐regulation of F5H in COMT‐RNAi transgenic switchgrass plants further impeded S lignin biosynthesis and, consequently, increased guaiacyl (G) units and reduced 5‐OH G units. Conversely, overexpression of F5H in COMT‐RNAi transgenic plants reduced G units and increased 5‐OH units, whereas the deficiency of S lignin biosynthesis was partially compensated or fully restored, depending on the extent of COMT down‐regulation in switchgrass. Moreover, simultaneous regulation of F5H and COMT expression had different effects on cell wall digestibility of switchgrass without biomass loss. Our results indicate that up‐regulation and down‐regulation of F5H expression, respectively, have antagonistic and synergistic effects on the reduction in S lignin resulting from COMT suppression. The coordinated effects between lignin genes should be taken into account in future studies aimed at cell wall bioengineering.

• The lignin content of feedstock has been proposed as one key agronomic trait impacting biofuel production from lignocellulosic biomass. 4‐Coumarate:coenzyme A ligase (4CL) is one of the key enzymes involved in the monolignol biosynthethic pathway. • Two homologous 4CL genes, Pv4CL1 and Pv4CL2, were identified in switchgrass (Panicum virgatum) through phylogenetic analysis. Gene expression patterns and enzymatic activity assays suggested that Pv4CL1 is involved in monolignol biosynthesis. Stable transgenic plants were obtained with Pv4CL1 down‐regulated. • RNA interference of Pv4CL1 reduced extractable 4CL activity by 80%, leading to a reduction in lignin content with decreased guaiacyl unit composition. Altered lignification patterns in the stems of RNAi transgenic plants were observed with phloroglucinol‐HCl staining. The transgenic plants also had uncompromised biomass yields. After dilute acid pretreatment, the low lignin transgenic biomass had significantly increased cellulose hydrolysis (saccharification) efficiency. • The results demonstrate that Pv4CL1, but not Pv4CL2, is the key 4CL isozyme involved in lignin biosynthesis, and reducing lignin content in switchgrass biomass by silencing Pv4CL1 can remarkably increase the efficiency of fermentable sugar release for biofuel production.

Background Sucrose nonfermenting 1-related protein kinase 2 (SnRK2) proteins constitute a family of plant-specific serine/threonine kinases that play critical roles in mediating abscisic acid (ABA) signaling and responses to abiotic stresses, including drought and salinity. Nevertheless, systematic bioinformatics analysis and expression profiling of the SnRK2 gene family in broomcorn millet ( Panicum miliaceum L.) have not yet been reported. Results A total of 16 PmSnRK2 genes were identified in the broomcorn millet genome, unevenly distributed across 12 chromosomes and phylogenetically classified into three subfamilies (I–III). Gene structure analysis revealed that the majority of PmSnRK2 genes harbor seven to eight introns. Promoter region mining uncovered abundant cis-elements responsive to hormones and abiotic stresses. Synteny analysis detected 12 PmSnRK2–PmSnRK2 paralogous pairs, with duplication events dated between ~ 1.28 and 219.36 million years ago; all Ka/Ks ratios were below 1, consistent with strong purifying selection. Multiple sequence alignment confirmed the presence of the conserved serine/threonine kinase active-site motif, the ATP-binding signature, and two characteristic C-terminal domains (I and II) across all family members. Structural superimposition of four representative PmSnRK2 proteins (PmSnRK2.6.2, PmSnRK2.11.1, PmSnRK2.13.1, PmSnRK2.15.1) onto Arabidopsis AtSnRK2.3 (PDB: 3UC3) yielded RMSD values of 0.922–1.134 Å within the kinase domain, underscoring three-dimensional conservation. Expression profiling using public RNA-seq datasets demonstrated that all 16 genes are transcribed in at least one of eight tissues, with individual members peaking in shoots, leaf blades, stems, inflorescences, roots, or seeds. Under exogenous ABA (50–100 µM), salt (100 mM NaCl), and PEG (20%) treatments, PmSnRK2 genes exhibited distinct induction and repression dynamics, revealing functional divergence in stress-response pathways. Conclusion Our study provides comprehensive insights into the genomic characteristics, evolutionary patterns, and potential functional roles of the PmSnRK2 gene family in broomcorn millet. These results enhance the current understanding of how PmSnRK2 genes may contribute to abiotic stress tolerance, offering a valuable foundation for further functional validation and targeted improvement in broomcorn millet breeding programs.

Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3′-hydroxylase (C3′H) enzymes (PvC3′H1 and PvC3′H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

Drought stress is one of the abiotic stresses and it may alter plant growth, metabolism and yield. The present study aims to analyze the growth, chlorophyll pigments, osmotic adjustment and antioxidative enzymes activity in Panicum sumatrense under drought stress. The control was irrigated daily and treated plants were irrigated at 4-, 7-, 10-, 13-day intervals. Later, they were irrigated at 3-day interval up to 70 DAS. The root and leaf samples were collected on 30 DAS, 50 DAS and 70 DAS and used for analysis. The root length increased gradually in all drought treatments at all growth stages of P. sumatrense . The chlorophyll pigments and plant height showed a reduction in 13 DID treatment when compared to all treatment. Compatible solutes like proline, glycine betaine and free amino acid increased in all drought treatment when compared to control at 70 DAS. Furthermore, stress treatment caused an increase in activity of antioxidant enzymes like superoxide dismutase, catalase and peroxidase. Panicum sumatrense possess many growth and physiological drought tolerance characters which can be used in future breeding program.

Class III peroxidases (CIIIPRX) catalyze the oxidation of monolignols, generate radicals, and ultimately lead to the formation of lignin. In general, CIIIPRX genes encode a large number of isozymes with ranges of in vitro substrate specificities. In order to elucidate the mode of substrate specificity of these enzymes, we characterized one of the CIIIPRXs (PviPRX9) from switchgrass (Panicum virgatum), a strategic plant for second-generation biofuels. The crystal structure, kinetic experiments, molecular docking, as well as expression patterns of PviPRX9 across multiple tissues and treatments, along with its levels of coexpression with the majority of genes in the monolignol biosynthesis pathway, revealed the function of PviPRX9 in lignification. Significantly, our study suggested that PviPRX9 has the ability to oxidize a broad range of phenylpropanoids with rather similar efficiencies, which reflects its role in the fortification of cell walls during normal growth and root development and in response to insect feeding. Based on the observed interactions of phenylpropanoids in the active site and analysis of kinetics, a catalytic mechanism involving two water molecules and residues histidine-42, arginine-38, and serine-71 was proposed. In addition, proline-138 and gluntamine-140 at the 137P-X-P-X140 motif, leucine-66, proline-67, and asparagine-176 may account for the broad substrate specificity of PviPRX9. Taken together, these observations shed new light on the function and catalysis of PviPRX9 and potentially benefit efforts to improve biomass conservation properties in bioenergy and forage crops.

The down-regulation of enzymes of the monolignol pathway results in reduced recalcitrance of biomass for lignocellulosic ethanol production. Cinnamoyl CoA reductase (CCR) catalyzes the first step of the phenylpropanoid pathway specifically dedicated to monolignol biosynthesis. However, plants contain multiple CCR-like genes, complicating the selection of lignin-specific targets. This study was undertaken to understand the complexity of the CCR gene family in tetraploid switchgrass (Panicum virgatum) and to determine the biochemical properties of the encoded proteins. Four switchgrass cDNAs (most with multiple variants) encoding putative CCRs were identified by phylogenetic analysis, heterologously expressed in Escherichia coli, and the corresponding enzymes were characterized biochemically. Two cDNAs, PvCCR1 and PvCCR2, encoded enzymes with CCR activity. They are phylogenetically distinct, differentially expressed, and the corresponding enzymes exhibited different biochemical properties with regard to substrate preference. PvCCR1 has higher specific activity and prefers feruloyl CoA as substrate, whereas PvCCR2 prefers caffeoyl and 4-coumaroyl CoAs. Allelic variants of each cDNA were detected, but the two most diverse variants of PvCCR1 encoded enzymes with similar catalytic activity. Based on its properties and expression pattern, PvCCR1 is probably associated with lignin biosynthesis during plant development (and is therefore a target for the engineering of improved biomass), whereas PvCCR2 may function in defense.

Background: Broomcorn millet (Panicum miliaceum L.), a drought-tolerant C4 crop, is crucial for agricultural resilience in arid regions. Lipoxygenases (LOXs), key enzymes in plant stress responses, have not been studied in broomcorn millet. This study aimed to identify LOX genes in broomcorn millet and elucidate their role in drought tolerance. Methods: We employed bioinformatics and physiological analyses to identify LOX genes in broomcorn millet. Expression profiles were assessed in different organs, and drought stress responses were evaluated in tolerant (HSZ, YXDHM) and sensitive (YS10) varieties. Antioxidant enzyme activities (SOD, POD, CAT) and malondialdehyde (MDA) levels were measured. Results: Twelve LOX genes were identified, classified into three subfamilies, and mapped across seven chromosomes. These genes contained stress-responsive cis-elements and showed organ-specific expression, with PmLOX5 exhibiting no detectable expression. Under drought stress, tolerant varieties showed elevated antioxidant activities and reduced MDA accumulation. PmLOX2, a homolog of Arabidopsis AtLOX1/AtLOX5, was significantly induced in tolerant varieties, correlating with enhanced antioxidant capacity and reduced oxidative damage. Conclusions:PmLOX genes, particularly PmLOX2, play a pivotal role in drought tolerance by modulating ROS scavenging and membrane protection. This study provides a foundation for leveraging LOX genes to improve drought resilience in broomcorn millet and related crops.