A Genomics Approach to Deciphering Lignin Biosynthesis in Switchgrass

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.     

Time Course Field Analysis of COMT-Downregulated Switchgrass: Lignification,Recalcitrance, and Rust Susceptibility

Modifying plant cell walls by manipulating lignin biosynthesis can improve biofuel yields from lignocellulosic crops. For example, transgenic switchgrass lines with downregulated expression of caffeic acid O-methyltransferase, a lignin biosynthetic enzyme, produce up to 38 % more ethanol than controls. The aim of the present study was to understand cell wall lignification over the second and third growing seasons of COMT-downregulated field-grown switchgrass. COMT gene expression, lignification, and cell wall recalcitrance were assayed for two independent transgenic lines at monthly intervals. Switchgrass rust (Puccinia emaculata) incidence was also tracked across the seasons. Trends in lignification over time differed between the 2 years. In 2012, sampling was initiated in mid-growing season on reproductive-stage plants and there was little variation in the lignin content of all lines (COMT-downregulated and control) over time. COMT-downregulated lines maintained 11–16 % less lignin, 33–40 % lower S/G (syringyl-to-guaiacyl) ratios, and 15–42 % higher sugar release relative to controls for all time points. In 2013, sampling was initiated earlier in the season on elongation-stage plants and the lignin content of all lines steadily increased over time, while sugar release expectedly decreased. S/G ratios increased in non-transgenic control plants as biomass accumulated over the season, while remaining relatively stable across the season in the COMT-downregulated lines. Differences in cell wall chemistry between transgenic and non-transgenic lines were not apparent until plants transitioned to reproductive growth in mid-season, after which the cell walls of COMT-downregulated plants exhibited phenotypes consistent with what was observed in 2012. There were no differences in rust damage between transgenics and controls at any time point. These results provide relevant fundamental insights into the process of lignification in a maturing field-grown biofuel feedstock with downregulated lignin biosynthesis.     

Silencing of 4-coumarate:coenzyme A ligase in switchgrass leads to reduced lignin content and improved fermentable sugar yields for biofuel production

? 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.     

Simultaneous regulation of F5H in COMT‐RNAi transgenic switchgrass alters effects of COMT suppression on syringyl lignin biosynthesis

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.     

Developmental Control of Lignification in Stems of Lowland Switchgrass Variety Alamo and the Effects on Saccharification Efficiency

The switchgrass variety Alamo has been chosen for genome sequencing, genetic breeding, and genetic engineering by the US Department of Energy Joint Genome Institute (JGI) and the US Department of Energy BioEnergy Science Center. Lignin has been considered as a major obstacle for cellulosic biofuel production from switchgrass biomass. The purpose of this study was to provide baseline information on cell wall development in different parts of developing internodes of tillers of switchgrass cultivar Alamo and evaluate the effect of cell wall properties on biomass saccharification. Cell wall structure, soluble and wall-bound phenolics, and lignin content were analyzed from the top, middle, and bottom parts of internodes at different developmental stages using ultraviolet autofluorescence microscopy, histological staining methods, and high-performance liquid chromatography (HPLC). The examination of different parts of the developing internodes revealed differences in the stem structure during development, in the levels of free and well-bound phenolic compounds and lignin content, and in lignin pathway-related gene expression, indicating that the monolignol biosynthetic pathway in switchgrass is under complex spatial and temporal control. Our data clearly show that there was a strong negative correlation between overall lignin content and biomass saccharification efficiency. The ester-linked p-CA/FA ratio showed a positive correlation with lignin content and a negative correlation with sugar release. Our data provide baseline information to facilitate genetic modification of switchgrass recalcitrance traits for biofuel production.     

Cloning, characterization and impact of up- and down-regulating subabul cinnamyl alcohol dehydrogenase (CAD) gene on plant growth and lignin profiles in transgenic tobacco

Both cDNA including 5′UTR and 3′UTR and genomic clones of cinnamyl alcohol dehydrogenase (CAD) were isolated and characterized from a pulp-yielding leguminous tree Leucaena leucocephala (LlCAD1). The deduced amino acid sequence shared high identity with orthologous sequences of Acacia mangium?×?Acacia auriculiformis (83%), Medicago sativa (83%), Nicotiana tabaccum (83%) and Aralia cordata (81%). Full length cDNA contained 78 bases of 5′UTR and 283 bases of 3′UTR, while the genomic clone contained 5 exons and 4 introns. Western blot analysis revealed elevated expression of LlCAD1 in seedling roots and shoots compared to leaves. Sense and antisense CAD tobacco transgenics showed increased and reduced CAD activity accompanied by a change in monomeric lignin composition. Histochemical staining of lignin in down-regulated plants suggested an increase in aldehyde units and a decrease in S/G ratio. Down-regulation of CAD resulted in accumulation of syringic, ferulic, p-coumaric and sinapic acids compared to untransformed controls. These observations were validated by anatomical studies of down-regulated transgenic stems which showed thin walled, elongated phloem and xylem fibres, accompanied by a reduction in the density of vessel elements and amount of secondary xylem when compared to untransformed plants. Furthermore, Klason lignin analysis of CAD antisense transgenics showed 7–32% reduced lignin and normal phenotype as compared to untransformed plants. Such a reduction was not noticed in up-regulated transgenics. These results demonstrate a unique opportunity to explore the significant role that down-regulation of CAD gene plays in reducing lignin content thereby offering potential benefits to the pulp and paper industry.     

LtuCAD1 Is a Cinnamyl Alcohol Dehydrogenase Ortholog Involved in Lignin Biosynthesis in Liriodendron tulipifera L., a Basal Angiosperm Timber Species

Cinnamyl alcohol dehydrogenase (CAD) is a key enzyme in lignin biosynthesis and catalyzes the final step in the synthesis of monolignols. Seven CAD homologs (LtuCAD1 to LtuCAD7) have been previously identified from a basal angiosperm species Liriodendron tulipifera L., which is an important timber tree species with significant ecological and economic values. The phylogenetic analysis indicates that LtuCAD1 is the only Liriodendron CAD grouped with the bona fide CADs, the primary CAD genes involved in lignification. In this study, the predicted protein sequence of LtuCAD1 was found to have conserved domains and the same key determinant site with the bona fide CADs in other plant species. Additionally, LtuCAD1 had the highest expression level in xylem as revealed by quantitative RT-PCR analysis. The expression of beta-glucuronidase (GUS) driven by the LtuCAD1 promoter was largely localized in vascular tissues in Arabidopsis. In stem cross sections, GUS staining was found exclusively in xylem and phloem. When expressed in the Arabidopsis cad4 cad5 double mutant, LtuCAD1 was able to restore the total lignin content and decrease the S/G lignin ratio. Our data indicate that LtuCAD1 is a CAD ortholog involved in lignin biosynthesis in Liriodendron.     

Improving Saccharification Efficiency of Alfalfa Stems Through Modification of the Terminal Stages of Monolignol Biosynthesis

A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50–60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.     

Overexpression of Poplar Xylem Sucrose Synthase in Tobacco Leads to a Thickened Cell Wall and Increased Height

Sucrose synthase (SuSy) is considered the first key enzyme for secondary growth because it is a highly regulated cytosolic enzyme that catalyzes the reversible conversion of sucrose and UDP into UDP-glucose and fructose. Although SuSy enzymes preferentially functions in the direction of sucrose cleavage at most cellular condition, they also catalyze the synthetic reaction. We isolated a gene that encodes a SuSy from Populus simonii×Populus nigra and named it PsnSuSy2 because it shares high similarity to SuSy2 in Populus trichocarpa. RT-PCR revealed that PsnSuSy2 was highly expressed in xylem, but lowly expressed in young leaves. To characterize its functions in secondary growth, multiple tobacco overexpression transgenic lines of PnsSuSy2 were generated via Agrobacterium-mediated transformation. The PsnSuSy2 expression levels and altered wood properties in stem segments from the different transgenic lines were carefully characterized. The results demonstrated that the levels of PsnSuSy2 enzyme activity, chlorophyll content, total soluble sugars, fructose and glucose increased significantly, while the sucrose level decreased significantly. Consequently, the cellulose content and fiber length increased, whereas the lignin content decreased, suggesting that PsnSuSy2 plays a significant role in cleaving sucrose into UDP-glucose and fructose to facilitate cellulose biosynthesis and that promotion of cellulose biosynthesis suppresses lignin biosynthesis. Additionally, the noticeable increase in the lodging resistance in transgenic tobacco stem suggested that the cell wall characteristics were altered by PsnSuSy2 overexpression. Scanning electron microscopy was performed to study the cell wall morphology of stem, and surprisingly, we found that the secondary cell wall was significantly thicker in transgenic tobacco. However, the thickened secondary cell wall did not negatively affect the height of the plants because the PsnSuSy2- overexpressing lines grew taller than the wildtype plants. This systematic analysis demonstrated that PsnSuSy2 plays an important role in cleaving sucrose coupled with cellulose biosynthesis in wood tissue.     

Identification and functional analysis of the Pm4CL1 gene in transgenic tobacco plant as the basis for regulating lignin biosynthesis in forest trees

Reducing the lignin content of trees could provide both economic and environmental benefits. To this end, the coumarate:coenzyme A ligase 1 gene (4CL1) was isolated from Pinus massoniana Lamb (Pm4CL1). The sequence of the full-length Pm4CL1 cDNA (accession no. FJ810495) contained an entire open reading frame (ORF) of 1,614 bp, which encoded a polypeptide of 537 amino acid residues. Tobacco (Nicotiana tabacum L.) as a model plant was used for functional characterization of the Pm4CL1 gene in transgenic plants. Results revealed that 4CL1 enzyme activity and lignin content in most antisense Pm4CL1 transgenic tobacco lines were decreased as compared to wild-type; the average 4CL1 enzyme activity was decreased by 48.75% and lignin content was decreased by 24.5%. In contrast, in the sense Pm4CL1 transgenic tobacco lines, average 4CL1 enzyme activity was increased by 72.3% and lignin content was increased by 27.6%. These results suggest that the Pm4CL1 gene from P. massoniana could be applied to regulate lignin biosynthesis in transgenic trees.     

Impact of Harvest Time and Switchgrass Cultivar on Sugar Release Through Enzymatic Hydrolysis

Switchgrass (Panicum virgatum L.) is a native North American prairie grass being developed for bioenergy production in the central and eastern USA. The objective of this study was to identify the impacts of harvest time and switchgrass cultivar had on sugar release variables determined through enzymatic hydrolysis. Previously, we reported that delaying harvest of switchgrass until after frost and until after winter resulted in decreased yields of switchgrass but it reduced the amount of ash and nutrients in the biomass. The current study used near-infrared reflectance spectroscopy (NIRS) to broaden an existing set of calibration equations designed to predict composition and sugar release variables of switchgrass. These updated calibrations were then applied to the full set of samples from a multi-year and multi-location switchgrass harvest-management study. Composition and processor sugar yields were significantly affected by location, year, cultivar, and harvest time, of which the time of harvest was the most important. Delaying the time of harvest until after frost or post-winter increased the concentration of structural carbohydrates from 500 to over 570 g kg^?1 in the biomass and lignin content from 160 to over 200 g kg^?1. Conversely, delaying harvest time lowered the amounts of ash and soluble sugars. The later harvest times also yielded more sugars following processing with yields increasing over 20% from the first harvest. Increased sugar yields are attributable to both increased concentration of sugars in the biomass upon harvest and reduced biomass recalcitrance. Based upon processed sugar yields, it is estimated that a biorefinery producing 76 million liters of ethanol per year would require 229–373 km² of land cultivated with switchgrass.