Monolignol Biosynthetic Pathway Research Articles

Impact of phenology, brown midrib (BMR), seed treatment, and herbivory on epicuticular wax content and fall armyworm (Spodoptera frugiperda) feeding in sorghum-sudangrass (Sorghum xdrummondii)

In crop production, management strategies have historically relied upon chemical control to mitigate insect pest pressures. As resistance to pesticides grows at an alarming rate, and as non-target effects are found more frequently, the search for sustainable, novel pest management strategies has become more important. Plants are equipped with diverse physical and chemical defenses against insect pests. Among these physical defenses, epicuticular waxes serve as an important first line of defense. Yet, how these chemical control methods such as seed treatment affects epicuticular waxes is underexplored. Brown midrib (BMR) is a host-plant trait from a recessive gene that affects the monolignol biosynthetic pathway, reducing lignin content, and presents as a gene of interest for evaluation as a form of pest management. Sorghum-sudangrass, Sorghum x drummondii, is an economically important forage crop with BMR; however, the effects of BMR have primarily been studied in animal agriculture and its effects on insects is largely unknown. In addition, how BMR affects epicuticular waxes is also unknown. This study aims to understand how the quantity of epicuticular wax in Sorghum x drummondii is affected by chemical control (seed treatments), BMR, phenological stages, and how wax affects a polyphagous, destructive pest, fall armyworm, Spodoptera frugiperda (FAW). Through wax quantification and FAW feeding induction and wax added artificial diet experiments, we show that while BMR and seed treatment does not affect wax content, plant phenology affects wax, and wax is induced by FAW feeding and has negative consequences for caterpillar growth.

Integrative analysis of metabolome, proteome, and transcriptome for identifying genes influencing total lignin content in Populus trichocarpa.

Lignin, a component of plant cell walls, possesses significant research potential as a renewable energy source to replace carbon-based products and as a notable pollutant in papermaking processes. The monolignol biosynthetic pathway has been elucidated and it is known that not all monolignol genes influence the total lignin content. However, it remains unclear which monolignol genes are more closely related to the total lignin content and which potential genes influence the total lignin content. In this study, we present a combination of t-test, differential gene expression analysis, correlation analysis, and weighted gene co-expression network analysis to identify genes that regulate the total lignin content by utilizing multi-omics data from transgenic knockdowns of the monolignol genes that includes data related to the transcriptome, proteome, and total lignin content. Firstly, it was discovered that enzymes from the PtrPAL, Ptr4CL, PtrC3H, and PtrC4H gene families are more strongly correlated with the total lignin content. Additionally, the co-downregulation of three genes, PtrC3H3, PtrC4H1, and PtrC4H2, had the greatest impact on the total lignin content. Secondly, GO and KEGG analysis of lignin-related modules revealed that the total lignin content is not only influenced by monolignol genes, but also closely related to genes involved in the "glutathione metabolic process", "cellular modified amino acid metabolic process" and "carbohydrate catabolic process" pathways. Finally, the cinnamyl alcohol dehydrogenase genes CAD1, CADL3, and CADL8 emerged as potential contributors to total lignin content. The genes HYR1 (UDP-glycosyltransferase superfamily protein) and UGT71B1 (UDP-glucosyltransferase), exhibiting a close relationship with coumarin, have the potential to influence total lignin content by regulating coumarin metabolism. Additionally, the monolignol genes PtrC3H3, PtrC4H1, and PtrC4H2, which belong to the cytochrome P450 genes, may have a significant impact on the total lignin content. Overall, this study establishes connections between gene expression levels and total lignin content, effectively identifying genes that have a significant impact on total lignin content and offering novel perspectives for future lignin research endeavours.

Dynamic regulation of phenylpropanoid pathway metabolites in modulating sorghum defense against fall armyworm.

Plants undergo dynamic metabolic changes at the cellular level upon insect infestation to better defend themselves. Phenylpropanoids, a hub of secondary plant metabolites, encompass a wide range of compounds that can contribute to insect resistance. Here, the role of sorghum (Sorghum bicolor) phenylpropanoids in providing defense against the chewing herbivore, fall armyworm (FAW), Spodoptera frugiperda, was explored. We screened a panel of nested association mapping (NAM) founder lines against FAW and identified SC1345 and Ajabsido as most resistant and susceptible lines to FAW, respectively, compared to reference parent, RTx430. Gene expression and metabolomic studies suggested that FAW feeding suppressed the expression level of genes involved in monolignol biosynthetic pathway and their associated phenolic intermediates at 10 days post infestation. Further, SC1345 genotype displayed elevated levels of flavonoid compounds after FAW feeding for 10 days, suggesting a diversion of precursors from lignin biosynthesis to the flavonoid pathway. Additionally, bioassays with sorghum lines having altered levels of flavonoids provided genetic evidence that flavonoids are crucial in providing resistance against FAW. Finally, the application of FAW regurgitant elevated the expression of genes associated with the flavonoid pathway in the FAW-resistant SC1345 genotype. Overall, our study indicates that a dynamic regulation of the phenylpropanoid pathway in sorghum plants imparts resistance against FAW.

Probable Biosynthetic Pathways of Silymarin Precursors

Background: Silymarin is composed of a mixture of flavonolignans derived from secondary plant metabolism. These constituents are present in substantial amounts in milk thistle [Silybum marianum (L.) Gaertn. (Asteraceae)] seeds. Silymarin has antioxidant properties that impact it with protective effects. Because of its chemoprotective effect against liver disease, silymarin is considered a complementary and alternative hepatoprotective medicine.BRMethods and Results: Coniferyl alcohol and taxifolin, the two precursors for silymarin biosyntesis, are derived from phenylpropane and flavonoid units, respectively. Coniferyl alcohol is synthesized via the monolignol biosynthetic pathway, whereas taxifolin is synthesized via the flavonoid pathway. Multiple variables, including related substrates, production, and activating enzymes require consideration to study the biosynthetic pathway of silymarin.BRConclusions: This review is helpful as it summarizes the probable biosynthetic pathways of silymarin and multiple related activating enzymes and substrates found in various plants. A further understanding of silymarin is expected to increase its industrial use value.

Transcriptome of the coralline alga Calliarthron tuberculosum (Corallinales, Rhodophyta) reveals convergent evolution of a partial lignin biosynthesis pathway.

The discovery of lignins in the coralline red alga Calliarthron tuberculosum raised new questions about the deep evolution of lignin biosynthesis. Here we present the transcriptome of C. tuberculosum supported with newly generated genomic data to identify gene candidates from the monolignol biosynthetic pathway using a combination of sequence similarity-based methods. We identified candidates in the monolignol biosynthesis pathway for the genes 4CL, CCR, CAD, CCoAOMT, and CSE but did not identify candidates for PAL, CYP450 (F5H, C3H, C4H), HCT, and COMT. In gene tree analysis, we present evidence that these gene candidates evolved independently from their land plant counterparts, suggesting convergent evolution of a complex multistep lignin biosynthetic pathway in this red algal lineage. Additionally, we provide tools to extract metabolic pathways and genes from the newly generated transcriptomic and genomic datasets. Using these methods, we extracted genes related to sucrose metabolism and calcification. Ultimately, this transcriptome will provide a foundation for further genetic and experimental studies of calcifying red algae.

Conformational Rearrangements in the Redox Cycling of NADPH-Cytochrome P450 Reductase from Sorghum bicolor Explored with FRET and Pressure-Perturbation Spectroscopy.

Simple SummaryNADPH-cytochrome P450 reductase (CPR) enzymes are known to undergo an ample conformational transition between the closed and open states in the process of their redox cycling. To explore the conformational landscape of CPR from the potential biofuel crop Sorghum bicolor (SbCPR), we incorporated a FRET donor/acceptor pair into the enzyme and employed rapid scanning stop-flow and pressure perturbation spectroscopy to characterize the equilibrium between its open and closed states at different stages of the redox cycle. Our results suggest the presence of several open conformational sub-states differing in the system volume change associated with the opening transition (ΔV0). Although the closed conformation always predominates in the conformational landscape, the population of the open conformations increases by order of magnitude upon the two-electron reduction and the formation of the disemiquinone state of the enzyme. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.NADPH-cytochrome P450 reductase (CPR) from Sorghum bicolor (SbCPR) serves as an electron donor for cytochrome P450 essential for monolignol and lignin production in this biofuel crop. The CPR enzymes undergo an ample conformational transition between the closed and open states in their functioning. This transition is triggered by electron transfer between the FAD and FMN and provides access of the partner protein to the electron-donating FMN domain. To characterize the electron transfer mechanisms in the monolignol biosynthetic pathway better, we explore the conformational transitions in SbCPR with rapid scanning stop-flow and pressure-perturbation spectroscopy. We used FRET between a pair of donor and acceptor probes incorporated into the FAD and FMN domains of SbCPR, respectively, to characterize the equilibrium between the open and closed states and explore its modulation in connection with the redox state of the enzyme. We demonstrate that, although the closed conformation always predominates in the conformational landscape, the population of open state increases by order of magnitude upon the formation of the disemiquinone state. Our results are consistent with several open conformation sub-states differing in the volume change (ΔV0) of the opening transition. While the ΔV0 characteristic of the oxidized enzyme is as large as −88 mL/mol, the interaction of the enzyme with the nucleotide cofactor and the formation of the double-semiquinone state of CPR decrease this value to −34 and −18 mL/mol, respectively. This observation suggests that the interdomain electron transfer in CPR increases protein hydration, while promoting more open conformation. In addition to elucidating the functional choreography of plant CPRs, our study demonstrates the high exploratory potential of a combination of the pressure-perturbation approach with the FRET-based monitoring of protein conformational transitions.

Synthesis of hydroxycinnamoyl shikimates and their role in monolignol biosynthesis

Abstract Hydroxycinnamoyl shikimates were reported in 2005 to be intermediates in monolignol biosynthesis. 3-Hydroxylation of p-coumarate, originally thought to occur via coumarate 3-hydroxylase (C3H) from p-coumaric acid or its CoA thioester, was revealed to be via the action of coumaroyl shikimate 3′-hydroxylase (C3′H) utilizing p-coumaroyl shikimate as the substrate, itself derived from p-coumaroyl-CoA via hydroxycinnamoyl-CoA: shikimate hydroxycinnamoyltransferase (HCT). The same HCT was conjectured to convert the product, caffeoyl shikimate, to caffeoyl-CoA to continue on the pathway starting with its 3-O-methylation. At least in some plants, however, a more recently discovered caffeoyl shikimate esterase (CSE) enzyme hydrolyzes caffeoyl shikimate to caffeic acid from which it must again produce its CoA thioester to continue on the monolignol biosynthetic pathway. HCT and CSE are therefore monolignol biosynthetic pathway enzymes that have provided new opportunities to misregulate lignification. To facilitate studies into the action and substrate specificity of C3H/C3′H, HCT, and CSE enzymes, as well as for metabolite authentication and for enzyme characterization, including kinetics, a source of authentic substrates and products was required. A synthetic scheme starting from commercially available shikimic acid and the four key hydroxycinnamic acids (p-coumaric, caffeic, ferulic, and sinapic acid) has been developed to provide this set of hydroxycinnamoyl shikimates for researchers.

Flavonoids naringenin chalcone, naringenin, dihydrotricin, and tricin are lignin monomers in papyrus.

Recent studies demonstrate that several polyphenolic compounds produced from beyond the canonical monolignol biosynthetic pathways can behave as lignin monomers, participating in radical coupling reactions and being incorporated into lignin polymers. Here, we show various classes of flavonoids, the chalconoid naringenin chalcone, the flavanones naringenin and dihydrotricin, and the flavone tricin, incorporated into the lignin polymer of papyrus (Cyperus papyrus L.) rind. These flavonoids were released from the rind lignin by Derivatization Followed by Reductive Cleavage (DFRC), a chemical degradative method that cleaves the β-ether linkages, indicating that at least a fraction of each was integrated into the lignin as β-ether-linked structures. Due to the particular structure of tricin and dihydrotricin, whose C-3ʹ and C-5ʹ positions at their B-rings are occupied by methoxy groups, these compounds can only be incorporated into the lignin through 4ʹ–O–β bonds. However, naringenin chalcone and naringenin have no substituents at these positions and can therefore form additional carbon–carbon linkages, including 3ʹ– or 5ʹ–β linkages that form phenylcoumaran structures not susceptible to cleavage by DFRC. Furthermore, Nuclear Magnetic Resonance analysis indicated that naringenin chalcone can also form additional linkages through its conjugated double bond. The discovery expands the range of flavonoids incorporated into natural lignins, further broadens the traditional definition of lignin, and enhances the premise that any phenolic compound present at the cell wall during lignification could be oxidized and potentially integrated into the lignin structure, depending only on its chemical compatibility. This study indicates that papyrus lignin has a unique structure, as it is the only lignin known to date that integrates such a diversity of phenolic compounds from different classes of flavonoids. This discovery will open up new ways to engineer and design lignins with specific properties and for enhanced value.

De novo transcriptome analysis of white teak (Gmelina arborea Roxb) wood reveals critical genes involved in xylem development and secondary metabolism

BackgroundGmelina arborea Roxb is a fast-growing tree species of commercial importance for tropical countries due to multiple industrial uses of its wood. Wood is primarily composed of thick secondary cell walls of xylem cells which imparts the strength to the wood. Identification of the genes involved in the secondary cell wall biosynthesis as well as their cognate regulators is crucial to understand how the production of wood occurs and serves as a starting point for developing breeding strategies to produce varieties with improved wood quality, better paper pulping or new potential uses such as biofuel production.In order to gain knowledge on the molecular mechanisms and gene regulation related with wood development in white teak, a de novo sequencing and transcriptome assembly approach was used employing secondary cell wall synthesizing cells from young white teak trees.ResultsFor generation of transcriptome, RNA-seq reads were assembled into 110,992 transcripts and 49,364 genes were functionally annotated using plant databases; 5071 GO terms and 25,460 SSR markers were identified within xylem transcripts and 10,256 unigenes were assigned to KEGG database in 130 pathways. Among transcription factor families, C2H2, C3H, bLHLH and MYB were the most represented in xylem. Differential gene expression analysis using leaves as a reference was carried out and a total of 20,954 differentially expressed genes were identified including monolignol biosynthetic pathway genes. The differential expression of selected genes (4CL, COMT, CCoAOMT, CCR and NST1) was validated using qPCR.ConclusionsWe report the very first de novo transcriptome of xylem-related genes in this tropical timber species of commercial importance and constitutes a valuable extension of the publicly available transcriptomic resource aimed at fostering both basic and breeding studies.

Association of gene expression with syringyl to guaiacyl ratio in sugarcane lignin.

A transcriptome analysis reveals the transcripts and alleles differentially expressed in sugarcane genotypes with contrasting lignin composition. Sugarcane bagasse is a highly abundant resource that may be used as a feedstock for the production of biofuels and bioproducts in order to meet increasing demands for renewable replacements for fossil carbon. However, lignin imparts rigidity to the cell wall that impedes the efficient breakdown of the biomass into fermentable sugars. Altering the ratio of the lignin units, syringyl (S) and guaiacyl (G), which comprise the native lignin polymer in sugarcane, may facilitate the processing of bagasse. This study aimed to identify genes and markers associated with S/G ratio in order to accelerate the development of sugarcane bioenergy varieties with modified lignin composition. The transcriptome sequences of 12 sugarcane genotypes that contrasted for S/G ratio were compared and there were 2019 transcripts identified as differentially expressed (DE) between the high and low S/G ratio groups. These included transcripts encoding possible monolignol biosynthetic pathway enzymes, transporters, dirigent proteins and transcriptional and post-translational regulators. Furthermore, the frequencies of single nucleotide polymorphisms (SNPs) were compared between the low and high S/G ratio groups to identify specific alleles expressed with the phenotype. There were 2063 SNP loci across 787 unique transcripts that showed group-specific expression. Overall, the DE transcripts and SNP alleles identified in this study may be valuable for breeding sugarcane varieties with altered S/G ratio that may provide desirable bioenergy traits.

Field Evaluation of Sorghum (Sorghum bicolor) Lines that Overexpress Two Monolignol-Related Genes that Alter Cell Wall Composition

Modifying lignin content and composition of cell walls are key targets for bioenergy feedstock improvement for a range of renewable chemical applications. Sorghum (Sorghum bicolor) is a drought-tolerant C4 grass being developed as a dedicated bioenergy feedstock. The monolignol biosynthetic pathway produces the hydroxycinnamic subunits of lignin, a major target to increase the energy content of sorghum biomass for thermal bioenergy conversion processes. SbMyb60 is a transcription factor linked to the activation of the monolignol biosynthetic pathway and caffeoyl CoA O-methyltransferase (SbCCoAOMT) is a S-adenosyl methionine (SAM)–dependent O-methyltransferase responsible for the methylation of caffeoyl-CoA to generate feruloyl-CoA in this pathway. Overexpression of each gene resulted in increased energy content of biomass and increased levels of several aromatic compounds in previous greenhouse experiments. To assess how SbMyb60 and SbCCoAOMT overexpression lines performed under field conditions, these lines were planted in field trials over two growing seasons, 2017 and 2018, and the effects on lignin composition and content as well as agronomic traits were assessed. Field-grown plants of SbMyb60 and SbCCoAOMT overexpression lines had increased total energy and phenolic compounds. Furthermore, two SbMyb60 transgenic events, SbMyb-10a and SbMyb-15a, with decreased stover yield and seed set were combined with three sorghum lines, N32, N4692, and Tx3118, to investigate whether yield reduction could be rescued. Hybrids improved yield of SbMyb60-15a; however, hybrids were unable to improve performance for SbMyb60-10a. Overall, these results demonstrated that field-grown sorghum overexpressing SbMyb60 or SbCCoAOMT can have biomass with increased phenolic compounds and total energy.

Dwarfism of high-monolignol Arabidopsis plants is rescued by ectopic LACCASE overexpression.

Lignin is a key secondary cell wall chemical constituent, and is both a barrier to biomass utilization and a potential source of bioproducts. The Arabidopsis transcription factors MYB58 and MYB63 have been shown to upregulate gene expression of the general phenylpropanoid and monolignol biosynthetic pathways. The overexpression of these genes also results in dwarfism. The vascular integrity, soluble phenolic profiles, cell wall lignin, and transcriptomes associated with these MYB‐overexpressing lines were characterized. Plants with high expression of MYB58 and MYB63 had increased ectopic lignin and the xylem vessels were regular and open, suggesting that the stunted growth is not associated with loss of vascular conductivity. MYB58 and MYB63 overexpression lines had characteristic soluble phenolic profiles with large amounts of monolignol glucosides and sinapoyl esters, but decreased flavonoids. Because loss of function lac4 lac17 mutants also accumulate monolignol glucosides, we hypothesized that LACCASE overexpression might decrease monolignol glucoside levels in the MYB‐overexpressing plant lines. When laccases related to lignification (LAC4 or LAC17) were co‐overexpressed with MYB63 or MYB58, the dwarf phenotype was rescued. Moreover, the overexpression of either LAC4 or LAC17 led to wild‐type monolignol glucoside levels, as well as wild‐type lignin levels in the rescued plants. Transcriptomes of the rescued double MYB63‐OX/LAC17‐OX overexpression lines showed elevated, but attenuated, expression of the MYB63 gene itself and the direct transcriptional targets of MYB63. Contrasting the dwarfism from overabundant monolignol production with dwarfism from lignin mutants provides insight into some of the proposed mechanisms of lignin modification‐induced dwarfism.

Structural insight of two 4-Coumarate CoA ligase (4CL) isoforms in Leucaena suggests targeted genetic manipulations could lead to better lignin extractability from the pulp.

4-Coumarate: coenzyme A ligase (4CL) is a key enzyme involved in the early steps of the monolignol biosynthetic pathway. It is hypothesized to modulate S and G monolignol content in the plant. Lignin removal is imperative to the paper industry and higher S/G ratio governs better extractability of lignin and economics of the pulping process. This background prompted us to predict 3D structure of two isoforms of 4CL in Leucaena leucocephala and evaluate their substrate preferences. The 3D structure of Ll4CL1 and Ll4CL2 protein were created by homology modeling and further refined by loop refinement. Molecular docking studies suggested differential substrate preferences of both the isoforms. Ll4CL1 preferred sinapic acid (- 4.91kcal/mole), ferulic acid (- 4.84kcal/mole), hydroxyferulic acid (- 4.72kcal/mole), and caffeic acid (- 4.71kcal/mole), in their decreasing order. Similarly, Ll4CL2 preferred caffeic acid (- 6.56kcal/mole, 4 H bonds), hydroxyferulic acid (- 6.56kcal/mole, 3 H bonds), and ferulic acid (- 6.32kcal/mole) and sinapic acid (- 5.00kcal/mole) in their decreasing order. Further, active site residues were identified in both the isoforms and in silico mutation and docking analysis was performed. Our analysis suggested that ASP228, TYR262, and PRO326 for Ll4CL1 and SER165, LYS247 and PRO315 for Ll4CL2 were important for theirfunctional activity. Based on differential substrate preferences of the two isoforms, as a first step towards genetically modified Leuaena having the desired phenotype, it can be proposed that over-expression of Ll4CL1 gene and/or down-regulation of Ll4CL2 gene could yield higher S/G ratio leading to better extractability of lignin.

Identification of enzymatic genes with the potential to reduce biomass recalcitrance through lignin manipulation in Arabidopsis

BackgroundDuring 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.ResultsTo 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.ConclusionsSimple 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.

Natural variation in ZmFBL41 confers banded leaf and sheath blight resistance in maize.

Rhizoctonia solani is a widely distributed phytopathogen that causes banded leaf and sheath blight in maize and sheath blight in rice. Here, we identified an F-box protein (ZmFBL41) that confers resistance to banded leaf and sheath blight through a genome-wide association study in maize. Rice overexpressing ZmFBL41 showed elevated susceptibility to R. solani. Two amino acid substitutions in this allele prevent its interaction with ZmCAD, which encodes the final enzyme in the monolignol biosynthetic pathway, resulting in the inhibition of ZmCAD degradation and, consequently, the accumulation of lignin and restriction of lesion expansion. Knocking out the ZmCAD-homologous gene OsCAD8B in rice enhanced susceptibility to R. solani. The results reveal a susceptibility mechanism in which R. solani targets the host proteasome to modify the secondary metabolism of the plant cell wall for its invasion. More importantly, it provides an opportunity to generate R. solani-resistant varieties of different plant species.

Deployment of SNP (CAPS and KASP) markers for allelic discrimination and easy access to functional variants for brown midrib genes bmr6 and bmr12 in Sorghum bicolor

The brown midrib (bmr) phenotype is a recessive trait of sorghum (Sorghum bicolor L. Moench) that results in overall lignin reduction and is associated with enhanced ruminant digestibility and increased feedstock to ethanol conversion efficiency. The three cloned Bmr loci encode genes involved in the monolignol biosynthetic pathway. There are eight known bmr loci in sorghum, but the two widely known are bmr6 and bmr12. A number of sorghum cultivars are referred to as bmr, but there is limited information as to their specific grouping, and it is important that new bmr lines in the pipeline can be easily genetically characterized. Hence, this study was performed to translate the genetic and DNA sequence information for bmr6 and bmr12, for use in the biomarker technology of single-nucleotide polymorphisms (SNPs). Through an analysis of variation for the two bmr genes, DNA markers in the form of cleave amplified polymorphic sequence (CAPS) and Kompetitive Allelele Specific Polymerase chain reaction (KASP) SNP markers were developed. The utility of the DNA markers for rapid and accurate identification of bmr6 or 12 individuals at the seedling stage were validated in a group of sorghum germplasm and a genetic population through marker-assisted selection. Two CAPS DNA markers (one each for bmr6 and 12) were evaluated and found to positively identify lines that harbor the different bmr genes. Fifteen KASP SNP markers (six for bmr6 and nine for bmr12) were developed and utilized for allelic discrimination and selection of bmr individuals at the seedling stage. The KASP marker bmr6-132 positively identified bmr6-ref allele among materials evaluated and individuals of segregating F2 population at the seedling stage. The KASP DNA marker bmr12-129, positively identified germplasm among materials evaluated and individuals of the F2 population at the seedling stage to select plants that carry the bmr12-ref allele . Results from this study also classified two sorghum germplasm as bmr12 and these lines can be utilized as specific donor of this bmr class using marker-assisted selection. This work demonstrated the successful translation and deployment of molecular information into specific CAPS and KASP DNA markers that are easy to access and can be applied in a breeding program, for use in efficient marker-assisted selection of bmr in sorghum.

The lignin toolbox of the model grass Setaria viridis.

The core set of biosynthetic genes potentially involved in developmental lignification was identified in the model C4 grass Setaria viridis. Lignin has been recognized as a major recalcitrant factor negatively affecting the processing of plant biomass into bioproducts. However, the efficient manipulation of lignin deposition in order to generate optimized crops for the biorefinery requires a fundamental knowledge of several aspects of lignin metabolism, including regulation, biosynthesis and polymerization. The current availability of an annotated genome for the model grass Setaria viridis allows the genome-wide characterization of genes involved in the metabolic pathway leading to the production of monolignols, the main building blocks of lignin. Here we performed a comprehensive study of monolignol biosynthetic genes as an initial step into the characterization of lignin metabolism in S. viridis. A total of 56 genes encoding bona fide enzymes catalyzing the consecutive ten steps of the monolignol biosynthetic pathway were identified in the S. viridis genome. A combination of comparative phylogenetic studies, high-throughput expression analysis and quantitative RT-PCR analysis was further employed to identify the family members potentially involved in developmental lignification. Accordingly, 14 genes clustered with genes from closely related species with a known function in lignification and showed an expression pattern that correlates with lignin deposition. These genes were considered the "core lignin toolbox" responsible for the constitutive, developmental lignification in S. viridis. These results provide the basis for further understanding lignin deposition in C4 grasses and will ultimately allow the validation of biotechnological strategies to produce crops with enhanced processing properties.

Enzyme-Enzyme Interactions in Monolignol Biosynthesis.

The enzymes that comprise the monolignol biosynthetic pathway have been studied intensively for more than half a century. A major interest has been the role of pathway in the biosynthesis of lignin and the role of lignin in the formation of wood. The pathway has been typically conceived as linear steps that convert phenylalanine into three major monolignols or as a network of enzymes in a metabolic grid. Potential interactions of enzymes have been investigated to test models of metabolic channeling or for higher order interactions. Evidence for enzymatic or physical interactions has been fragmentary and limited to a few enzymes studied in different species. Only recently the entire pathway has been studied comprehensively in any single plant species. Support for interactions comes from new studies of enzyme activity, co-immunoprecipitation, chemical crosslinking, bimolecular fluorescence complementation, yeast 2-hybrid functional screening, and cell type–specific gene expression based on light amplification by stimulated emission of radiation capture microdissection. The most extensive experiments have been done on differentiating xylem of Populus trichocarpa, where genomic, biochemical, chemical, and cellular experiments have been carried out. Interactions affect the rate, direction, and specificity of both 3 and 4-hydroxylation in the monolignol biosynthetic pathway. Three monolignol P450 mono-oxygenases form heterodimeric and heterotetrameric protein complexes that activate specific hydroxylation of cinnamic acid derivatives. Other interactions include regulatory kinetic control of 4-coumarate CoA ligases through subunit specificity and interactions between a cinnamyl alcohol dehydrogenase and a cinnamoyl-CoA reductase. Monolignol enzyme interactions with other pathway proteins have been associated with biotic and abiotic stress response. Evidence challenging or supporting metabolic channeling in this pathway will be discussed.

Transcriptional analysis and histochemistry reveal a dominant role for cell wall signaling in mediating Pythium myriotylum resistance in Zingiber zerumbet

Although post infection changes in cell wall constituents are known to induce the immune response in plants against necrotrophs, little is known about the role of the cell wall in mediating resistance in Zingiber zerumbet (L.) Smith (Zingiberaceae) against the soil-borne necrotrophic oomycete Pythium myriotylum Drechsler, which causes soft-rot disease. Using RNA-Seq in combination with custom gene expression microarray we studied the temporal expression profile of 46 wall-associated genes in Z. zerumbet against P. myriotylum inoculation. Many genes that promote cell wall loosening were suppressed. Similarly, the genes involved in the biosynthesis and the signaling of phytohormones and the receptor-like kinases that mediate cell elongation were also suppressed. Several monolignol biosynthetic pathway genes were up-regulated. Histochemistry of the collar region of the aerial stem revealed H2O2 accumulation, increased lignification of the mesophyll cells surrounding vascular bundles in the leaf sheath and the significant increase in total lignin content. Pathogen hyphae were restricted to peripheral leaf sheath and were not extended into the pith through which the principal portion of the connective tissues passes in the aerial stem. Results highlight a coordinated transcriptional reprogramming of cell wall-associated genes to bring about changes in cell wall composition to minimize the cell wall damage caused by pathogen factors and to render the cell wall less amenable to pathogen penetration. The study illustrates a key role for cell wall fortification in preventing pathogen colonization in the vascular tissues, thus ensuring the transport system remains unaffected by the pathogen invasion.

Assessing the impact of the 4CL enzyme complex on the robustness of monolignol biosynthesis using metabolic pathway analysis

Lignin is a polymer present in the secondary cell walls of all vascular plants. It is a known barrier to pulping and the extraction of high-energy sugars from cellulosic biomass. The challenge faced with predicting outcomes of transgenic plants with reduced lignin is due in part to the presence of unique protein-protein interactions that influence the regulation and metabolic flux in the pathway. Yet, it is unclear why certain plants have evolved to create these protein complexes. In this study, we use mathematical models to investigate the role that the protein complex, formed specifically between Ptr4CL3 and Ptr4CL5 enzymes, have on the monolignol biosynthesis pathway. The role of this Ptr4CL3-Ptr4CL5 enzyme complex on the steady state flux distribution was quantified by performing Monte Carlo simulations. The effect of this complex on the robustness and the homeostatic properties of the pathway were identified by performing sensitivity and stability analyses, respectively. Results from these robustness and stability analyses suggest that the monolignol biosynthetic pathway is resilient to mild perturbations in the presence of the Ptr4CL3-Ptr4CL5 complex. Specifically, the presence of Ptr4CL3-Ptr4CL5 complex increased the stability of the pathway by 22%. The robustness in the pathway is maintained due to the presence of multiple enzyme isoforms as well as the presence of alternative pathways resulting from the presence of the Ptr4CL3-Ptr4CL5 complex.