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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.

The major cell wall pectic glycan homogalacturonan (HG) is crucial for plant growth, development, and reproduction. HG synthesis occurs in the Golgi and is catalyzed by members of the galacturonosyltransferase (GAUT) family with GAUT1 being the archetypal and best studied family member. In Arabidopsis suspension culture cells and tobacco leaves, the Golgi localization of Arabidopsis GAUT1 has been shown to require protein-protein interactions with its homolog GAUT7. Here we show that in pollen tubes GAUT5 and GAUT6, homologs of GAUT7, also target GAUT1 to the Golgi apparatus. Pollen tube germination and elongation in double homozygous knock-out mutants (gaut5 gaut6,gaut5 gaut7, andgaut6 gaut7) are moderately impaired, whereasgaut5(-/-)gaut6(-/-)gaut7(+/-)triple mutant is severely impaired and male infertile. Amounts and distributions of methylesterified HG in the pollen tube tip were severely distorted in the double and heterozygous triple mutants. A chimeric protein comprising GAUT1 and a non-cleavable membrane anchor domain was able to partially restore pollen tube germination and elongation and to reverse male sterility in the triple mutant. These results indicate that GAUT5, GAUT6, and GAUT7 are required for synthesis of native HG in growing pollen tubes and have critical roles in pollen tube growth and male fertility in Arabidopsis.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13068-017-0991-x) contains supplementary material, which is available to authorized users.

This chapter focuses on the effects of salt stress on the light reactions of photosynthesis and some defense mechanisms, such as osmoprotectants and the antioxidant defense system. Salt stress strongly affects photosynthesis, which is the most fundamental and intricate physiological process in all green plants. A high salt concentration inhibits the growth of plants, reducing the fresh the dry weight of roots, stems, and leaves. Chlorophyll a, chlorophyll b and carotenoids are the main photosynthetic pigments, and they play an important role in photosynthesis, as the changes in their content affect the photosynthetic activity. The lipids in biological membranes, including the thylakoid membranes, are very important not only for the formation of the lipid bilayer but also for the structure and the function of membrane complexes. Osmoprotectants are small, electrically neutral, non-toxic molecules at molar concentrations; they are highly soluble organic compounds that efficiently maintain osmotic balance and stabilize proteins and membranes under salt, drought, or other stresses.

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.

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.

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.

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. Electronic supplementary material The online version of this article (10.1186/s13068-019-1353-7) contains supplementary material, which is available to authorized users.

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.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0934-6) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (10.1186/s13068-017-1002-y) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-017-0866-1) contains supplementary material, which is available to authorized users.

Summary 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.

[EN] Introduction Most aerial plant organs are covered by a cuticle, which largely consists of cutin and wax. Cuticular waxes are mixtures of dozens of compounds, mostly very-long-chain aliphatics that are easily extracted by solvents. Over the last four decades, diverse cuticular wax analysis protocols have been developed, most of which are complex and time-consuming, and need to be adapted for each plant species or organ. Plant genomics and breeding programs often require mid-throughput metabolic phenotyping approaches to screen large numbers of individuals and obtain relevant biological information. Objectives To generate a fast, simple and user-friendly methodology able to capture most wax complexity independently of the plant, cultivar and organ. Methods Here we present a simple GC-MS method for screening relatively small wax amounts, sampled by short extraction with a versatile, uniform solvent. The method will be tested and validated in leaves and fruits from three different crop species: tomato (Solanum lycopersicum), apple (Malus domestica) and hybrid aspen (Populus tremula x tremuloides). Results Consistent results were obtained in tomato cultivar M82 across three consecutive years (2010-2012), two organs (leaf and fruit), and also in two different tomato (M82 and MicroTom) and apple (Golden Delicious and Granny Smith) cultivars. Our results on tomato wax composition match those reported previously, while our apple and hybrid aspen analyses provide the first comprehensive cuticular wax profile of these species. Conclusion This protocol allows standardized identification and quantification of most cuticular wax components in a range of species., Research at the IBMCP was supported by MINECO Grant BIO2013-42193-R and from EC H2020 TRADITOM SFS7a-2014- (contract 634561) to Antonio Granell and by FPU-MECD personal Grant to Josefina Patricia Fernandez Moreno (AP-2007-01905). Research at the Weizmann Institute of Sciences was supported by the Israel Science Foundation (ISF) personal Grant to Asaph Aharoni (ISF Grant No. 646/11). We also thank COST FA1106 Quality Fruit for funding networking activities.

The thermoluminescence (TL) parameters in the intact leaves and the thylakoids isolated from leaves of NaCl treated seedlings showed different patterns of change. NaCl treatment brings about a destabilization of QA and QB, leading to a decrease in Q and B bands in the leaves. However, the Q and B band intensity of isolated thylakoids increased in NaCl-treated seedlings. The differences in the TL intensities are described as the action of NaCl on the density of quinones per unit leaf area and on chlorophyll units in isolated thylakoids. Copyright © 2002 John Wiley & Sons, Ltd.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-015-0218-y) contains supplementary material, which is available to authorized users.

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. Electronic supplementary material The online version of this article (doi:10.1186/s13068-014-0147-1) contains supplementary material, which is available to authorized users.

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.

The thermoluminescence (TL) parameters in intact leaves and thylakoids isolated from leaves showed a different pattern of change during leaf ageing. Ageing of leaves brought about a decrease in the B-band and a simultaneous increase in the Q-band. Thylakoids showed only a decrease in the B-band. The TL bands further show that there is generation of an endogenous electron transport inhibitor during leaf ageing.

In plants external stimuli are perceived through a cascade of signals and signal transduction pathways. Protein phosphorylation and de-phosphorylation is one of the most important transduction paths for the perception of signals in plants. The highest concentrations of plant phospho-proteins are located in chloroplasts. This facilitates the protection of thylakoid membranes from stress-induced damage and augments adaptive strategies in plants. In this review, the protein kinases associated with phosphorylation of thylakoid membrane protein, and the adaptive changes in thylakoid membrane architecture and developmental cues are given. The presence of membrane bound kinases in thylakoid membranes have evolutionary implications for the signal transduction pathways and the photosynthetic gene expression for thylakoid membrane protein dynamics.

Chlorophyll (Chl) b is synthesized by oxidation of a methyl group on the B ring of the porphyrin molecule to a formyl group by chlorophyllide (Chlide) a oxygenase (CAO). The overexpression of Arabidopsis thaliana full length CAO (AtCAO) in tobacco (Nicotiana tabacum) resulted in an increased Chl synthesis and a decreased Chl a/b ratio in low-light-grown (LL) as well as in high-light-grown (HL) tobacco plants, where the effect was more pronounced. In HL-plants, increased [Chl b] resulted in efficient capture of solar energy and enhanced (40%–80%) electron transport rates at both limiting and saturating light intensities.

Chlorophyll b is synthesized by the oxidation of a methyl group on the B ring of a tetrapyrrole molecule to a formyl group by chlorophyllide a oxygenase (CAO). The full-length CAO from Arabidopsis (Arabidopsis thaliana) was overexpressed in tobacco (Nicotiana tabacum) that grows well at light intensities much higher than those tolerated by Arabidopsis. This resulted in an increased synthesis of glutamate semialdehyde, 5-aminolevulinic acid, magnesium-porphyrins, and chlorophylls. Overexpression of CAO resulted in increased chlorophyll b synthesis and a decreased chlorophyll a/b ratio in low light-grown as well as high light-grown tobacco plants; this effect, however, was more pronounced in high light. The increased potential of the protochlorophyllide oxidoreductase activity and chlorophyll biosynthesis compensated for the usual loss of chlorophylls in high light. Increased chlorophyll b synthesis in CAO-overexpressed plants was accompanied not only by an increased abundance of light-harvesting chlorophyll proteins but also of other proteins of the electron transport chain, which led to an increase in the capture of light as well as enhanced (40%–80%) electron transport rates of photosystems I and II at both limiting and saturating light intensities. Although the quantum yield of carbon dioxide fixation remained unchanged, the light-saturated photosynthetic carbon assimilation, starch content, and dry matter accumulation increased in CAO-overexpressed plants grown in both low- and high-light regimes. These results demonstrate that controlled up-regulation of chlorophyll b biosynthesis comodulates the expression of several thylakoid membrane proteins that increase both the antenna size and the electron transport rates and enhance carbon dioxide assimilation, starch content, and dry matter accumulation.

Chlorophyllide a oxygenase (CAO) that converts chlorophyllide a to chlorophyllide b was overexpressed in tobacco to increase chlorophyll (Chl) b biosynthesis and alter the Chl a/b ratio. Transgenic plants along with their wild-type cultivars were grown in low and high light intensities. In low light there was 20% increase in chlorophyll b contents in transgenic plants, which resulted in 16% reduction in the Chl a/b ratio. In high light, total Chl contents were 31% higher in transgenic plants than those of wild type. The increase in Chl a was 19% and that of Chl b was 72% leading to 31% decline of Chl a/b ratio. The increase in Chl b contents was accompanied by enhanced CAO expression that was highly pronounced in low light. As compared to low light, in high light Lhcb1 and Chl a/b transcripts abundance was significantly increased in transgenic plants suggesting a close relationship between Chl b synthesis and cab gene expression. However, there was a small increase in expression of LHCII proteins, which did not correspond to 72% increase in Chl b content in transgenic line, implying that LHCPII has the ability to bind more Chl b molecules.

The three major components of plant biomass, cellulose, hemicellulose and lignin, are highly recalcitrant and deconstruction involves thermal and chemical pretreatment. Microbial conversion is a possible solution, but few anaerobic microbes utilize both cellulose and hemicellulose and none are known to solubilize lignin. Herein, we show that the majority (85%) of insoluble switchgrass biomass that had not been previously chemically treated was degraded at 78 °C by the anaerobic bacterium Caldicellulosiruptor bescii. Remarkably, the glucose/xylose/lignin ratio and physical and spectroscopic properties of the remaining insoluble switchgrass were not significantly different than those of the untreated plant material. C. bescii is therefore able to solubilize lignin as well as the carbohydrates and, accordingly, lignin-derived aromatics were detected in the culture supernatants. From mass balance analyses, the carbohydrate in the solubilized switchgrass quantitatively accounted for the growth of C. bescii and its fermentation products, indicating that the lignin was not assimilated by the microorganism. Immunoanalyses of biomass and transcriptional analyses of C. bescii showed that the microorganism when grown on switchgrass produces enzymes directed at key plant cell wall moieties such as pectin, xyloglucans and rhamnogalacturonans, and that these and as yet uncharacterized enzymes enable the degradation of cellulose, hemicellulose and lignin at comparable rates. This unexpected finding of simultaneous lignin and carbohydrate solubilization bodes well for industrial conversion by extremely thermophilic microbes of biomass that requires limited or, more importantly, no chemical pretreatment.