Reduced expression of chlorophyllide a oxygenase (CAO) decreases the metabolic flux for chlorophyll synthesis and downregulates photosynthesis in tobacco plants.

Chlorophyll b is synthesized from chlorophyllide a, catalyzed by chlorophyllide a oxygenase (CAO). To examine whether reduced chlorophyll b content regulates chlorophyll (Chl) synthesis and photosynthesis, we raised CAO transgenic tobacco plants with antisense CAO expression, which had lower chlorophyll b content and, thus, higher Chl a/b ratio. Further, these plants had (i) lower chlorophyll b and total Chl content, whether they were grown under low or high light; (ii) decreased steady-state levels of chlorophyll biosynthetic intermediates, due, perhaps, to a feedback-controlled reduction in enzyme expressions/activities; (iii) reduced electron transport rates in their intact leaves, and reduced Photosystem (PS) I, PS II and whole chain electron transport activities in their isolated thylakoids; (iv) decreased carbon assimilation in plants grown under low or high light. We suggest that reduced synthesis of chlorophyll b by antisense expression of CAO, acting at the end of Chl biosynthesis pathway, downregulates the chlorophyll b biosynthesis, resulting in decreased Chl b, total chlorophylls and increased Chl a/b. We have previously shown that the controlled up-regulation of chlorophyll b biosynthesis and decreased Chl a/b ratio by over expression of CAO enhance the rates of electron transport and CO2 assimilation in tobacco. Conversely, our data, presented here, demonstrate that-antisense expression of CAO in tobacco, which decreases Chl b biosynthesis and increases Chl a/b ratio, leads to reduced photosynthetic electron transport and carbon assimilation rates, both under low and high light. We conclude that Chl b modulates photosynthesis; its controlled down regulation/ up regulation decreases/ increases light-harvesting, rates of electron transport, and carbon assimilation. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01395-5.

Heterologous expression of Arabidopsis laccase2, laccase4 and peroxidase52 driven under developing xylem specific promoter DX15 improves saccharification in populus.

BACKGROUND: Secondary cell wall holds considerable potential as it has gained immense momentum to replace the lignocellulosic feedstock into fuels. Lignin one of the components of secondary cell wall tightly holds the polysaccharides thereby enhancing the recalcitrance and complexity in the biomass. Laccases (LAC) and peroxidases (PRX) are the major phenyl-oxidases playing key functions during the polymerization of monolignols into lignin. Yet, the functions of laccase and peroxidases gene families remained largely unknown. Hence, the objective of this conducted study is to understand the role of specific LAC and PRX in Populus wood formation and to further investigate how the altered Lac and Prx expression affects biomass recalcitrance and plant growth. This study of heterologous expression of Arabidopsis Lac and Prx genes was conducted in poplar to avoid any otherwise occurring co-suppression mechanism during the homologous overexpression of highly expressed native genes. In the pursuit of optimizing lignocellulosic biomass for biofuel production, the present study focuses on harnessing the enzymatic potential of Arabidopsis thaliana Laccase2, Laccase4, and Peroxidase52 through heterologous expression. RESULTS: We overexpressed selected Arabidopsis laccase2 (AtLac2), laccase4 (AtLac4), and peroxidase52 (AtPrx52) genes, based on their high transcript expression respective to the differentiating xylem tissues in the stem, in hybrid poplar (cv. 717) expressed under the developing xylem tissue-specific promoter, DX15 characterized the transgenic populus for the investigation of growth phenotypes and recalcitrance efficiency. Bioinformatics analyses conducted on AtLac2 and AtLac4 and AtPrx52, revealed the evolutionary relationship between the laccase gene and peroxidase gene homologs, respectively. Transgenic poplar plant lines overexpressing the AtLac2 gene (AtLac2-OE) showed an increase in plant height without a change in biomass yield as compared to the controls; whereas, AtLac4-OE and AtPrx52-OE transgenic lines did not show any such observable growth phenotypes compared to their respective controls. The changes in the levels of lignin content and S/G ratios in the transgenic poplar resulted in a significant increase in the saccharification efficiency as compared to the control plants. CONCLUSIONS: Overall, saccharification efficiency was increased by 35-50%, 21-42%, and 8-39% in AtLac2-OE, AtLac4-OE, and AtPrx52-OE transgenic poplar lines, respectively, as compared to their controls. Moreover, the bioengineered plants maintained normal growth and development, underscoring the feasibility of this approach for biomass improvement without compromising overall plant fitness. This study also sheds light on the potential of exploiting regulatory elements of DX15 to drive targeted expression of lignin-modifying enzymes, thereby providing a promising avenue for tailoring biomass for improved biofuel production. These findings contribute to the growing body of knowledge in synthetic biology and plant biotechnology, offering a sustainable solution to address the challenges associated with lignocellulosic biomass recalcitrance.

Composition and yield of non-cellulosic and cellulosic sugars in soluble and particulate fractions during consolidated bioprocessing of poplar biomass by Clostridium thermocellum.

BACKGROUND: Terrestrial plant biomass is the primary renewable carbon feedstock for enabling transition to a sustainable bioeconomy. Consolidated bioprocessing (CBP) by the cellulolytic thermophile Clostridium thermocellum offers a single step microbial platform for production of biofuels and biochemicals via simultaneous solubilization of carbohydrates from lignocellulosic biomass and conversion to products. Here, solubilization of cell wall cellulosic, hemicellulosic, and pectic polysaccharides in the liquor and solid residues generated during CBP of poplar biomass by C. thermocellum was analyzed. RESULTS: The total amount of biomass solubilized in the C. thermocellum DSM1313 fermentation platform was 5.8, 10.3, and 13.7% of milled non-pretreated poplar after 24, 48, and 120 h, respectively. These results demonstrate solubilization of 24% cellulose and 17% non-cellulosic sugars after 120 h, consistent with prior reports. The net solubilization of non-cellulosic sugars by C. thermocellum (after correcting for the uninoculated control fermentations) was 13 to 36% of arabinose (Ara), xylose (Xyl), galactose (Gal), mannose (Man), and glucose (Glc); and 15% and 3% of fucose and glucuronic acid, respectively. No rhamnose was solubilized and 71% of the galacturonic acid (GalA) was solubilized. These results indicate that C. thermocellum may be selective for the types and/or rate of solubilization of the non-cellulosic wall polymers. Xyl, Man, and Glc were found to accumulate in the fermentation liquor at levels greater than in uninoculated control fermentations, whereas Ara and Gal did not accumulate, suggesting that C. thermocellum solubilizes both hemicelluloses and pectins but utilizes them differently. After five days of fermentation, the relative amount of Rha in the solid residues increased 21% indicating that the Rha-containing polymer rhamnogalacturonan I (RG-I) was not effectively solubilized by C. thermocellum CBP, a result confirmed by immunoassays. Comparison of the sugars in the liquor versus solid residue showed that C. thermocellum solubilized hemicellulosic xylan and mannan, but did not fully utilize them, solubilized and appeared to utilize pectic homogalacturonan, and did not solubilize RG-I. CONCLUSIONS: The significant relative increase in RG-I in poplar solid residues following CBP indicates that C. thermocellum did not solubilize RG-I. These results support the hypothesis that this pectic glycan may be one barrier for efficient solubilization of poplar by C. thermocellum.

Genetic Modification of KNAT7 Transcription Factor Expression Enhances Saccharification and Reduces Recalcitrance of Woody Biomass in Poplars.

The precise role of KNAT7 transcription factors (TFs) in regulating secondary cell wall (SCW) biosynthesis in poplars has remained unknown, while our understanding of KNAT7 functions in other plants is continuously evolving. To study the impact of genetic modifications of homologous and heterologous KNAT7 gene expression on SCW formation in transgenic poplars, we prepared poplar KNAT7 (PtKNAT7) overexpression (PtKNAT7-OE) and antisense suppression (PtKNAT7-AS) vector constructs for the generation of transgenic poplar lines via Agrobacterium-mediated transformation. Since the overexpression of homologous genes can sometimes result in co-suppression, we also overexpressed Arabidopsis KNAT7 (AtKNAT7-OE) in transgenic poplars. In all these constructs, the expression of KNAT7 transgenes was driven by developing xylem (DX)-specific promoter, DX15. Compared to wild-type (WT) controls, many SCW biosynthesis genes downstream of KNAT7 were highly expressed in poplar PtKNAT7-OE and AtKNAT7-OE lines. Yet, no significant increase in lignin content of woody biomass of these transgenic lines was observed. PtKNAT7-AS lines, however, showed reduced expression of many SCW biosynthesis genes downstream of KNAT7 accompanied by a reduction in lignin content of wood compared to WT controls. Syringyl to Guaiacyl lignin (S/G) ratios were significantly increased in all three KNAT7 knockdown and overexpression transgenic lines than WT controls. These transgenic lines were essentially indistinguishable from WT controls in terms of their growth phenotype. Saccharification efficiency of woody biomass was significantly increased in all transgenic lines than WT controls. Overall, our results demonstrated that developing xylem-specific alteration of KNAT7 expression affects the expression of SCW biosynthesis genes, impacting at least the lignification process and improving saccharification efficiency, hence providing one of the powerful tools for improving bioethanol production from woody biomass of bioenergy crops and trees.

Novel Mutant Alleles Reveal a Role of the Extra-Large G Protein in Rice Grain Filling, Panicle Architecture, Plant Growth, and Disease Resistance.

Plant growth and grain filling are the key agronomical traits for grain weight and yield of rice. The continuous improvement in rice yield is required for a future sustainable global economy and food security. The heterotrimeric G protein complex containing a canonical α subunit (RGA1) couples extracellular signals perceived by receptors to modulate cell function including plant development and grain weight. We hypothesized that, besides RGA1, three atypical, extra-large GTP-binding protein (XLG) subunits also regulate panicle architecture, plant growth, development, grain weight, and disease resistance. Here, we identified a role of XLGs in agronomic traits and stress tolerance by genetically ablating all three rice XLGs individually and in combination using the CRISPR/Cas9 genome editing in rice. For this study, eight (three single, two double, and three triple) null mutants were selected. Three XLG proteins combinatorically regulate seed filling, because loss confers a decrease in grain weight from 14% with loss of one XLG and loss of three to 32% decrease in grain weight. Null mutations in XLG2 and XLG4 increase grain size. The mutants showed significantly reduced panicle length and number per plant including lesser number of grains per panicle compared to the controls. Loss-of-function of all individual XLGs contributed to 9% more aerial biomass compared to wild type (WT). The double mutant showed improved salinity tolerance. Moreover, loss of the XLG gene family confers hypersensitivity to pathogens. Our findings suggest that the non-canonical XLGs play important roles in regulating rice plant growth, grain filling, panicle phenotype, stress tolerance, and disease resistance. Genetic manipulation of XLGs has the potential to improve agronomic properties in rice.

Novel derivatives of plant monomeric phenolics: act as inhibitors of bacterial cell-to-cell communication.

The aim of the present study was to synthesize novel active Anti-Quorum sensing derivatives from secondary metabolites viz. Gallic acid, Protocatechuic acid and Vanillic acid present in the plant Bergenia ciliata. Efficacy of all synthesized derivatives have been evaluated on the formation of bacterial biofilm and inhibition of cell-to-cell communication. Anti-Quorum Sensing activity and biofilm formation of all synthesized compounds was measured on biomonitor strain Chrobacterium violaceum, ATCC 12472 using standard paper disk-diffusion assay and quantification of violacein pigment. Among all derivatives, five derivatives 3,4,5-Trihydroxy-benzoic acid methyl ester (9a), 3,4-Dihydroxy-benzoic acid methyl ester (10a), 3,4,5-Tris-(2,4-dichloro-benzyloxy)-benzoic acid methyl ester (12), 3,4,5-Tris-(2,5-dichloro-benzyloxy)-benzoic acid methyl ester (13) and 4-(2,4-Dichloro-benzyloxy)-3-methoxy-benzoic acid methyl ester (15) has shown Anti-Quorum Sensing activity by inhibiting violacein pigment production and biofilm formation without interfering with its growth. The inhibitory effects in violacein pigment production were: positive control (C-30) 72%, (9a), (10a) 47.2%, (12) 27.3%, (13) 40.1% and (15) 22.7% at the concentration of 1 mg/mL and biofilm percent inhibition were found (C-30) 64% (9a) 46.2%, (10a) 40.3%, (12) 18.4%, (13) 35.2%, and (15) 17.3% when compared with the untreated control. Results reveal that synthesized derivatives seem to be good compounds for inhibition and formation of biofilm and AHL-mediated Quorum-sensing mechanism. The present article highlights the importance of derivatives derived from secondary metabolites as potent drug for biofilm formation and inhibition of cell-to-cell communication.

Multitrait genome-wide association analysis of Populus trichocarpa identifies key polymorphisms controlling morphological and physiological traits.

Genome-wide association studies (GWAS) have great promise for identifying the loci that contribute to adaptive variation, but the complex genetic architecture of many quantitative traits presents a substantial challenge. We measured 14 morphological and physiological traits and identified single nucleotide polymorphism (SNP)-phenotype associations in a Populus trichocarpa population distributed from California, USA to British Columbia, Canada. We used whole-genome resequencing data of 882 trees with more than 6.78 million SNPs, coupled with multitrait association to detect polymorphisms with potentially pleiotropic effects. Candidate genes were validated with functional data. Broad-sense heritability (H2 ) ranged from 0.30 to 0.56 for morphological traits and 0.08 to 0.36 for physiological traits. In total, 4 and 20 gene models were detected using the single-trait and multitrait association methods, respectively. Several of these associations were corroborated by additional lines of evidence, including co-expression networks, metabolite analyses, and direct confirmation of gene function through RNAi. Multitrait association identified many more significant associations than single-trait association, potentially revealing pleiotropic effects of individual genes. This approach can be particularly useful for challenging physiological traits such as water-use efficiency or complex traits such as leaf morphology, for which we were able to identify credible candidate genes by combining multitrait association with gene co-expression and co-methylation data.

Correction to: Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

[This corrects the article DOI: 10.1186/s13068-019-1353-7.].

Multiple levers for overcoming the recalcitrance of lignocellulosic biomass.

Background: The recalcitrance of cellulosic biomass is widely recognized as a key barrier to cost-effective biological processing to fuels and chemicals, but the relative impacts of physical, chemical and genetic interventions to improve biomass processing singly and in combination have yet to be evaluated systematically. Solubilization of plant cell walls can be enhanced by non-biological augmentation including physical cotreatment and thermochemical pretreatment, the choice of biocatalyst, the choice of plant feedstock, genetic engineering of plants, and choosing feedstocks that are less recalcitrant natural variants. A two-tiered combinatoric investigation of lignocellulosic biomass deconstruction was undertaken with three biocatalysts (Clostridium thermocellum, Caldicellulosiruptor bescii, Novozymes Cellic® Ctec2 and Htec2), three transgenic switchgrass plant lines (COMT, MYB4, GAUT4) and their respective nontransgenic controls, two Populus natural variants, and augmentation of biological attack using either mechanical cotreatment or cosolvent-enhanced lignocellulosic fractionation (CELF) pretreatment. Results: In the absence of augmentation and under the conditions tested, increased total carbohydrate solubilization (TCS) was observed for 8 of the 9 combinations of switchgrass modifications and biocatalysts tested, and statistically significant for five of the combinations. Our results indicate that recalcitrance is not a trait determined by the feedstock only, but instead is coequally determined by the choice of biocatalyst. TCS with C. thermocellum was significantly higher than with the other two biocatalysts. Both CELF pretreatment and cotreatment via continuous ball milling enabled TCS in excess of 90%. Conclusion: Based on our results as well as literature studies, it appears that some form of non-biological augmentation will likely be necessary for the foreseeable future to achieve high TCS for most cellulosic feedstocks. However, our results show that this need not necessarily involve thermochemical processing, and need not necessarily occur prior to biological conversion. Under the conditions tested, the relative magnitude of TCS increase was augmentation > biocatalyst choice > plant choice > plant modification > plant natural variants. In the presence of augmentation, plant modification, plant natural variation, and plant choice exhibited a small, statistically non-significant impact on TCS.

Downregulation of pectin biosynthesis gene GAUT4 leads to reduced ferulate and lignin-carbohydrate cross-linking in switchgrass.

Knockdown (KD) expression of GAlactUronosylTransferase 4 (GAUT4) in switchgrass improves sugar yield and ethanol production from the biomass. The reduced recalcitrance of GAUT4-KD transgenic biomass is associated with reduced cell wall pectic homogalacturonan and rhamnogalacturonan II content and cross-linking, and the associated increases in accessibility of cellulose to enzymatic deconstruction. To further probe the molecular basis for the reduced recalcitrance of GAUT4-KD biomass, potential recalcitrance-related factors including the physicochemical properties of lignin and hemicellulose are investigated. We show that the transgenic switchgrass have a lower abundance of ferulate and lignin-carbohydrate complex cross-linkages, reduced amounts of residual arabinan and xylan in lignin-enriched fractions after enzymatic hydrolysis, and greater coalescence and migration of lignin after hydrothermal pretreatment in comparison to the wild-type switchgrass control. The results reveal the roles of both decreased lignin-polymer and pectin cross-links in the reduction of recalcitrance in PvGAUT4-KD switchgrass.

Functional Analysis of Cellulose Synthase CesA4 and CesA6 Genes in Switchgrass (Panicum virgatum) by Overexpression and RNAi-Mediated Gene Silencing.

Switchgrass (Panicum virgatum L.) is a leading lignocellulosic bioenergy feedstock. Cellulose is a major component of the plant cell walls and the primary substrate for saccharification. Accessibility of cellulose to enzymatic breakdown into fermentable sugars is limited by the presence of lignin in the plant cell wall. In this study, putatively novel switchgrass secondary cell wall cellulose synthase PvCesA4 and primary cell wall PvCesA6 genes were identified and their functional role in cellulose synthesis and cell wall composition was examined by overexpression and knockdown of the individual genes in switchgrass. The endogenous expression of PvCesA4 and PvCesA6 genes varied among including roots, leaves, stem, and reproductive tissues. Increasing or decreasing PvCesA4 and PvCesA6 expression to extreme levels in the transgenic lines resulted in decreased biomass production. PvCesA6-overexpressing lines had reduced lignin content and syringyl/guaiacyl lignin monomer ratio accompanied by increased sugar release efficiency, suggesting an impact of PvCesA6 expression levels on lignin biosynthesis. Cellulose content and cellulose crystallinity were decreased, while xylan content was increased in PvCesA4 and PvCesA6 overexpression or knockdown lines. The increase in xylan content suggests that the amount of non-cellulosic cell wall polysaccharide was modified in these plants. Taken together, the results show that the manipulation of the cellulose synthase genes alters the cell wall composition and availability of cellulose as a bioprocessing substrate.

Sugar release and growth of biofuel crops are improved by downregulation of pectin biosynthesis.

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.

Working towards recalcitrance mechanisms: increased xylan and homogalacturonan production by overexpression of GAlactUronosylTransferase12 (GAUT12) causes increased recalcitrance and decreased growth in Populus.

BACKGROUND: The development of fast-growing hardwood trees as a source of lignocellulosic biomass for biofuel and biomaterial production requires a thorough understanding of the plant cell wall structure and function that underlie the inherent recalcitrance properties of woody biomass. Downregulation of GAUT12.1 in Populus deltoides was recently reported to result in improved biomass saccharification, plant growth, and biomass yield. To further understand GAUT12.1 function in biomass recalcitrance and plant growth, here we report the effects of P. trichocarpa GAUT12.1 overexpression in P. deltoides. RESULTS: Increasing GAUT12.1 transcript expression by 7-49% in P. deltoides PtGAUT12.1-overexpression (OE) lines resulted in a nearly complete opposite biomass saccharification and plant growth phenotype to that observed previously in PdGAUT12.1-knockdown (KD) lines. This included significantly reduced glucose, xylose, and total sugar release (12-13%), plant height (6-54%), stem diameter (8-40%), and overall total aerial biomass yield (48-61%) in 3-month-old, greenhouse-grown PtGAUT12.1-OE lines compared to controls. Total lignin content was unaffected by the gene overexpression. Importantly, selected PtGAUT12.1-OE lines retained the recalcitrance and growth phenotypes upon growth for 9 months in the greenhouse and 2.8 years in the field. PtGAUT12.1-OE plants had significantly smaller leaves with lower relative water content, and significantly reduced stem wood xylem cell numbers and size. At the cell wall level, xylose and galacturonic acid contents increased markedly in total cell walls as well as in soluble and insoluble cell wall extracts, consistent with increased amounts of xylan and homogalacturonan in the PtGAUT12.1-OE lines. This led to increased cell wall recalcitrance, as manifested by the 9-15% reduced amounts of recovered extractable wall materials and 8-15% greater amounts of final insoluble pellet in the PtGAUT12.1-OE lines compared to controls. CONCLUSIONS: The combined phenotype and chemotype data from P. deltoides PtGAUT12.1-OE and PdGAUT12.1-KD transgenics clearly establish GAUT12.1 as a recalcitrance- and growth-associated gene in poplar. Overall, the data support the hypothesis that GAUT12.1 synthesizes either an HG-containing primer for xylan synthesis or an HG glycan required for proper xylan deposition, anchoring, and/or architecture in the wall, and the possibility of HG and xylan glycans being connected to each other by a base-sensitive covalent linkage.

Agronomic performance of Populus deltoides trees engineered for biofuel production.

BACKGROUND: One of the major barriers to the development of lignocellulosic feedstocks is the recalcitrance of plant cell walls to deconstruction and saccharification. Recalcitrance can be reduced by targeting genes involved in cell wall biosynthesis, but this can have unintended consequences that compromise the agronomic performance of the trees under field conditions. Here we report the results of a field trial of fourteen distinct transgenic Populus deltoides lines that had previously demonstrated reduced recalcitrance without yield penalties under greenhouse conditions. RESULTS: Survival and productivity of the trial were excellent in the first year, and there was little evidence for reduced performance of the transgenic lines with modified target gene expression. Surprisingly, the most striking phenotypic effects in this trial were for two empty-vector control lines that had modified bud set and bud flush. This is most likely due to somaclonal variation or insertional mutagenesis. Traits related to yield, crown architecture, herbivory, pathogen response, and frost damage showed few significant differences between target gene transgenics and empty vector controls. However, there were a few interesting exceptions. Lines overexpressing the DUF231 gene, a putative O-acetyltransferase, showed early bud flush and marginally increased height growth. Lines overexpressing the DUF266 gene, a putative glycosyltransferase, had significantly decreased stem internode length and slightly higher volume index. Finally, lines overexpressing the PFD2 gene, a putative member of the prefoldin complex, had a slightly reduced volume index. CONCLUSIONS: This field trial demonstrates that these cell wall modifications, which decreased cell wall recalcitrance under laboratory conditions, did not seriously compromise first-year performance in the field, despite substantial challenges, including an outbreak of a stem boring insect (Gypsonoma haimbachiana), attack by a leaf rust pathogen (Melampsora spp.), and a late frost event. This bodes well for the potential utility of these lines as advanced biofuels feedstocks.

Comparison of four glycosyl residue composition methods for effectiveness in detecting sugars from cell walls of dicot and grass tissues.

BACKGROUND: The effective use of plant biomass for biofuel and bioproduct production requires a comprehensive glycosyl residue composition analysis to understand the different cell wall polysaccharides present in the different biomass sources. Here we compared four methods side-by-side for their ability to measure the neutral and acidic sugar composition of cell walls from herbaceous, grass, and woody model plants and bioenergy feedstocks. RESULTS: Arabidopsis, Populus, rice, and switchgrass leaf cell walls, as well as cell walls from Populus wood, rice stems, and switchgrass tillers, were analyzed by (1) gas chromatography-mass spectrometry (GC-MS) of alditol acetates combined with a total uronic acid assay; (2) carbodiimide reduction of uronic acids followed by GC-MS of alditol acetates; (3) GC-MS of trimethylsilyl (TMS) derivatives; and (4) high-pressure, anion-exchange chromatography (HPAEC). All four methods gave comparable abundance ranking of the seven neutral sugars, and three of the methods were able to quantify unique acidic sugars. The TMS, HPAEC, and carbodiimide methods provided comparable quantitative results for the specific neutral and acidic sugar content of the biomass, with the TMS method providing slightly greater yield of specific acidic sugars and high total sugar yields. The alditol acetate method, while providing comparable information on the major neutral sugars, did not provide the requisite quantitative information on the specific acidic sugars in plant biomass. Thus, the alditol acetate method is the least informative of the four methods. CONCLUSIONS: This work provides a side-by-side comparison of the efficacy of four different established glycosyl residue composition analysis methods in the analysis of the glycosyl residue composition of cell walls from both dicot (Arabidopsis and Populus) and grass (rice and switchgrass) species. Both primary wall-enriched leaf tissues and secondary wall-enriched wood/stem tissues were analyzed for mol% and mass yield of the non-cellulosic sugars. The TMS, HPAEC, and carbodiimide methods were shown to provide comparable quantitative data on the nine neutral and acidic sugars present in all plant cell walls.

Transgenic switchgrass (Panicum virgatum L.) targeted for reduced recalcitrance to bioconversion: a 2-year comparative analysis of field-grown lines modified for target gene or genetic element expression.

Transgenic Panicum virgatum L. silencing (KD) or overexpressing (OE) specific genes or a small RNA (GAUT4-KD, miRNA156-OE, MYB4-OE, COMT-KD and FPGS-KD) was grown in the field and aerial tissue analysed for biofuel production traits. Clones representing independent transgenic lines were established and senesced tissue was sampled after year 1 and 2 growth cycles. Biomass was analysed for wall sugars, recalcitrance to enzymatic digestibility and biofuel production using separate hydrolysis and fermentation. No correlation was found between plant carbohydrate content and biofuel production pointing to overriding structural and compositional elements that influence recalcitrance. Biomass yields were greater for all lines in the second year as plants establish in the field and standard amounts of biomass analysed from each line had more glucan, xylan and less ethanol (g/g basis) in the second- versus the first-year samples, pointing to a broad increase in tissue recalcitrance after regrowth from the perennial root. However, biomass from second-year growth of transgenics targeted for wall modification, GAUT4-KD, MYB4-OE, COMT-KD and FPGS-KD, had increased carbohydrate and ethanol yields (up to 12% and 21%, respectively) compared with control samples. The parental plant lines were found to have a significant impact on recalcitrance which can be exploited in future strategies. This summarizes progress towards generating next-generation bio-feedstocks with improved properties for microbial and enzymatic deconstruction, while providing a comprehensive quantitative analysis for the bioconversion of multiple plant lines in five transgenic strategies.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance.

BACKGROUND: The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. RESULTS: A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. CONCLUSIONS: A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

Downregulation of GAUT12 in Populus deltoides by RNA silencing results in reduced recalcitrance, increased growth and reduced xylan and pectin in a woody biofuel feedstock.

BACKGROUND: The inherent recalcitrance of woody bioenergy feedstocks is a major challenge for their use as a source of second-generation biofuel. Secondary cell walls that constitute the majority of hardwood biomass are rich in cellulose, xylan, and lignin. The interactions among these polymers prevent facile accessibility and deconstruction by enzymes and chemicals. Plant biomass that can with minimal pretreatment be degraded into sugars is required to produce renewable biofuels in a cost-effective manner. RESULTS: GAUT12/IRX8 is a putative glycosyltransferase proposed to be involved in secondary cell wall glucuronoxylan and/or pectin biosynthesis based on concomitant reductions of both xylan and the pectin homogalacturonan (HG) in Arabidopsis irx8 mutants. Two GAUT12 homologs exist in Populus trichocarpa, PtGAUT12.1 and PtGAUT12.2. Knockdown expression of both genes simultaneously has been shown to reduce xylan content in Populus wood. We tested the proposition that RNA interference (RNAi) downregulation of GAUT12.1 alone would lead to increased sugar release in Populus wood, that is, reduced recalcitrance, based on the hypothesis that GAUT12 synthesizes a wall structure required for deposition of xylan and that cell walls with less xylan and/or modified cell wall architecture would have reduced recalcitrance. Using an RNAi approach, we generated 11 Populus deltoides transgenic lines with 50 to 67% reduced PdGAUT12.1 transcript expression compared to wild type (WT) and vector controls. Ten of the eleven RNAi lines yielded 4 to 8% greater glucose release upon enzymatic saccharification than the controls. The PdGAUT12.1 knockdown (PdGAUT12.1-KD) lines also displayed 12 to 52% and 12 to 44% increased plant height and radial stem diameter, respectively, compared to the controls. Knockdown of PdGAUT12.1 resulted in a 25 to 47% reduction in galacturonic acid and 17 to 30% reduction in xylose without affecting total lignin content, revealing that in Populus wood as in Arabidopsis, GAUT12 affects both pectin and xylan formation. Analyses of the sugars present in sequential cell wall extracts revealed a reduction of glucuronoxylan and pectic HG and rhamnogalacturonan in extracts from PdGAUT12.1-KD lines. CONCLUSIONS: The results show that downregulation of GAUT12.1 leads to a reduction in a population of xylan and pectin during wood formation and to reduced recalcitrance, more easily extractable cell walls, and increased growth in Populus.

Deletion of a gene cluster encoding pectin degrading enzymes in Caldicellulosiruptor bescii reveals an important role for pectin in plant biomass recalcitrance.

BACKGROUND: A major obstacle, and perhaps the most important economic barrier to the effective use of plant biomass for the production of fuels, chemicals, and bioproducts, is our current lack of knowledge of how to efficiently and effectively deconstruct wall polymers for their subsequent use as feedstocks. Plants represent the most desired source of renewable energy and hydrocarbons because they fix CO2, making their use carbon neutral. Their biomass structure, however, is a barrier to deconstruction, and this is often referred to as recalcitrance. Members of the bacterial genus Caldicellulosiruptor have the ability to grow on unpretreated plant biomass and thus provide an assay for plant deconstruction and biomass recalcitrance. RESULTS: Using recently developed genetic tools for manipulation of these bacteria, a deletion of a gene cluster encoding enzymes for pectin degradation was constructed, and the resulting mutant was reduced in its ability to grow on both dicot and grass biomass, but not on soluble sugars. The plant biomass from three phylogenetically diverse plants, Arabidopsis (a herbaceous dicot), switchgrass (a monocot grass), and poplar (a woody dicot), was used in these analyses. These biomass types have cell walls that are significantly different from each other in both structure and composition. While pectin is a relatively minor component of the grass and woody dicot substrates, the reduced growth of the mutant on all three biomass types provides direct evidence that pectin plays an important role in biomass recalcitrance. Glycome profiling of the plant material remaining after growth of the mutant on Arabidopsis biomass compared to the wild-type revealed differences in the rhamnogalacturonan I, homogalacturonan, arabinogalactan, and xylan profiles. In contrast, only minor differences were observed in the glycome profiles of the switchgrass and poplar biomass. CONCLUSIONS: The combination of microbial digestion and plant biomass analysis provides a new and important platform to identify plant wall structures whose presence reduces the ability of microbes to deconstruct plant walls and to identify enzymes that specifically deconstruct those structures.

Aspen pectate lyase PtxtPL1-27 mobilizes matrix polysaccharides from woody tissues and improves saccharification yield.

BACKGROUND: Wood cell walls are rich in cellulose, hemicellulose and lignin. Hence, they are important sources of renewable biomass for producing energy and green chemicals. However, extracting desired constituents from wood efficiently poses significant challenges because these polymers are highly cross-linked in cell walls and are not easily accessible to enzymes and chemicals. RESULTS: We show that aspen pectate lyase PL1-27, which degrades homogalacturonan and is expressed at the onset of secondary wall formation, can increase the solubility of wood matrix polysaccharides. Overexpression of this enzyme in aspen increased solubility of not only pectins but also xylans and other hemicelluloses, indicating that homogalacturonan limits the solubility of major wood cell wall components. Enzymatic saccharification of wood obtained from PL1-27-overexpressing trees gave higher yields of pentoses and hexoses than similar treatment of wood from wild-type trees, even after acid pretreatment. CONCLUSIONS: Thus, the modification of pectins may constitute an important biotechnological target for improved wood processing despite their low abundance in woody biomass.