Comparative evaluation of <Emphasis Type="Italic">Populus</Emphasis> variants total sugar release and structural features following pretreatment and digestion by two distinct biological systems

Background

Populus natural variants have been shown to realize a broad range of sugar yields during saccharification, however, the structural features responsible for higher sugar release from natural variants are not clear. In addition, the sugar release patterns resulting from digestion with two distinct biological systems, fungal enzymes and Clostridium thermocellum, have yet to be evaluated and compared. This study evaluates the effect of structural features of three natural variant Populus lines, which includes the line BESC standard, with respect to the overall process of sugar release for two different biological systems.

Results

Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar release from hydrothermal pretreatment combined with either enzymatic hydrolysis or Clostridium thermocellum fermentation compared to the Populus natural variant, BESC standard. However, C. thermocellum outperformed the fungal cellulases yielding 96.0, 95.5, and 85.9% glucan plus xylan release from SKWE 24-2, BESC 876, and BESC standard, respectively. Among the feedstock properties evaluated, cellulose accessibility and glycome profiling provided insights into factors that govern differences in sugar release between the low recalcitrant lines and the BESC standard line. However, because this distinction was more apparent in the solids after pretreatment than in the untreated biomass, pretreatment was necessary to differentiate recalcitrance among Populus lines. Glycome profiling analysis showed that SKWE 24-2 contained the most loosely bound cell wall glycans, followed by BESC 876, and BESC standard. Additionally, lower molecular weight lignin may be favorable for effective hydrolysis, since C. thermocellum reduced lignin molecular weight more than fungal enzymes across all Populus lines.

Conclusions

Low recalcitrant Populus natural variants, SKWE 24-2 and BESC 876, showed higher sugar yields than BESC standard when hydrothermal pretreatment was combined with biological digestion. However, C. thermocellum was determined to be a more robust and effective biological catalyst than a commercial fungal cellulase cocktail. As anticipated, recalcitrance was not readily predicted through analytical methods that determined structural properties alone. However, combining structural analysis with pretreatment enabled the identification of attributes that govern recalcitrance, namely cellulose accessibility, xylan content in the pretreated solids, and non-cellulosic glycan extractability.

     

Development of a rapid,low-cost protoplast transfection system for switchgrass (<Emphasis Type="Italic">Panicum virgatum</Emphasis> L.)

Key message

A switchgrass protoplast system was developed, achieving a cost reduction of ~1000-fold, a threefold increase in transformation efficiency, and a fourfold reduction in required DNA quantity compared to previous methods.

Abstract

In recent years, there has been a resurgence in the use of protoplast systems for rapid screening of gene silencing and genome-editing targets for siRNA, miRNA, and CRISPR technologies. In the case of switchgrass (Panicum virgatum L.), to achieve economic feasibility for biofuel production, it is necessary to develop plants with decreased cell wall recalcitrance to reduce processing costs. To achieve this goal, transgenic plants have been generated with altered cell wall chemistry; however, with limited success owing to the complexity of cell walls. Because of the considerable cost, time, and effort required to screen transgenic plants, a protoplast system that can provide data at an early stage has potential to eliminate low performing candidate genes/targets prior to the creation of transgenic plants. Despite the advantages of protoplast systems, protoplast isolation in switchgrass has proven costly, requiring expensive lab-grade enzymes and high DNA quantities. In this paper, we describe a low-cost protoplast isolation system using a mesophyll culture approach and a cell suspension culture. Results from this work show a cost reduction of ~1000-fold compared to previous methods of protoplast isolation in switchgrass, with a cost of $0.003 (USD) per reaction for mesophyll protoplasts and $0.018 for axenic cell culture-derived protoplasts. Further, the efficiency of protoplast transformation was optimized threefold over previous methods, despite a fourfold reduction in DNA quantity. The methods developed in this work remove the cost barrier previously limiting high-throughput screening of genome-editing and gene silencing targets in switchgrass, paving the way for more efficient development of transgenic plants.

     

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.     

A precise and consistent assay for major wall polymer features that distinctively determine biomass saccharification in transgenic rice by near-infrared spectroscopy

Background

The genetic modification of plant cell walls has been considered to reduce lignocellulose recalcitrance in bioenergy crops. As a result, it is important to develop a precise and rapid assay for the major wall polymer features that affect biomass saccharification in a large population of transgenic plants. In this study, we collected a total of 246 transgenic rice plants that, respectively, over-expressed and RNAi silenced 12 genes of the OsGH9 and OsGH10 family that are closely associated with cellulose and hemicellulose modification. We examined the wall polymer features and biomass saccharification among 246 transgenic plants and one wild-type plant. The samples presented a normal distribution applicable for statistical analysis and NIRS modeling.

Results

Among the 246 transgenic rice plants, we determined largely varied wall polymer features and the biomass enzymatic saccharification after alkali pretreatment in rice straws, particularly for the fermentable hexoses, ranging from 52.8 to 95.9%. Correlation analysis indicated that crystalline cellulose and lignin levels negatively affected the hexose and total sugar yields released from pretreatment and enzymatic hydrolysis in the transgenic rice plants, whereas the arabinose levels and arabinose substitution degree (reverse xylose/arabinose ratio) exhibited positive impacts on the hexose and total sugars yields. Notably, near-infrared spectroscopy (NIRS) was applied to obtain ten equations for predicting biomass enzymatic saccharification and seven equations for distinguishing major wall polymer features. Most of the equations exhibited high R ²/R ² _cv/R ² _ev and RPD values for a perfect prediction capacity.

Conclusions

Due to large generated populations of transgenic rice lines, this study has not only examined the key wall polymer features that distinctively affect biomass enzymatic saccharification in rice but has also established optimal NIRS models for a rapid and precise screening of major wall polymer features and lignocellulose saccharification in biomass samples. Importantly, this study has briefly explored the potential roles of a total of 12 OsGH9 and OsGH10 genes in cellulose and hemicellulose modification and cell wall remodeling in transgenic rice lines. Hence, it provides a strategy for genetic modification of plant cell walls by expressing the desired OsGH9 and OsGH10 genes that could greatly improve biomass enzymatic digestibility in rice.

     

RNAi downregulation of three key lignin genes in sugarcane improves glucose release without reduction in sugar production

Background

Sugarcane is a subtropical crop that produces large amounts of biomass annually. It is a key agricultural crop in many countries for the production of sugar and other products. Residual bagasse following sucrose extraction is currently underutilized and it has potential as a carbohydrate source for the production of biofuels. As with all lignocellulosic crops, lignin acts as a barrier to accessing the polysaccharides, and as such, is the focus of transgenic efforts. In this study, we used RNAi to individually reduce the expression of three key genes in the lignin biosynthetic pathway in sugarcane. These genes, caffeoyl-CoA O-methyltransferase (CCoAOMT), ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT), impact lignin content and/or composition.

Results

For each RNAi construct, we selected three events for further analysis based on qRT-PCR results. For the CCoAOMT lines, there were no lines with a reduction in lignin content and only one line showed improved glucose release. For F5H, no lines had reduced lignin, but one line had a significant increase in glucose release. For COMT, one line had reduced lignin content, and this line and another released higher levels of glucose during enzymatic hydrolysis. Two of the lines with improved glucose release (F5H-2 and COMT-2) also had reduced S:G ratios.

Conclusions

Along with improvements in bagasse quality for the production of lignocellulosic-based fuels, there was only one line with reduction in juice sucrose extraction, and three lines with significantly improved sucrose production, providing evidence that the alteration of sugarcane for improved lignocellulosic ethanol production can be achieved without negatively impacting sugar production and perhaps even enhancing it.

     

Down-regulation of the caffeic acid <Emphasis Type="Italic">O</Emphasis>-methyltransferase gene in switchgrass reveals a novel monolignol analog

Background

Down-regulation of the caffeic acid 3-O-methyltransferase EC 2.1.1.68 (COMT) gene in the lignin biosynthetic pathway of switchgrass (Panicum virgatum) resulted in cell walls of transgenic plants releasing more constituent sugars after pretreatment by dilute acid and treatment with glycosyl hydrolases from an added enzyme preparation and from Clostridium thermocellum. Fermentation of both wild-type and transgenic switchgrass after milder hot water pretreatment with no water washing showed that only the transgenic switchgrass inhibited C. thermocellum. Gas chromatography–mass spectrometry (GCMS)-based metabolomics were undertaken on cell wall aqueous extracts to determine the nature of the microbial inhibitors.

Results

GCMS confirmed the increased concentration of a number of phenolic acids and aldehydes that are known inhibitors of microbial fermentation. Metabolomic analyses of the transgenic biomass additionally revealed the presence of a novel monolignol-like metabolite, identified as trans-3, 4-dimethoxy-5-hydroxycinnamyl alcohol (iso-sinapyl alcohol) in both non-pretreated, as well as hot water pretreated samples. iso-Sinapyl alcohol and its glucoside were subsequently generated by organic synthesis and the identity of natural and synthetic materials were confirmed by mass spectrometric and NMR analyses. The additional novel presence of iso-sinapic acid, iso-sinapyl aldehyde, and iso-syringin suggest the increased activity of a para-methyltransferase, concomitant with the reduced COMT activity, a strict meta-methyltransferase. Quantum chemical calculations were used to predict the most likely homodimeric lignans generated from dehydration reactions, but these products were not evident in plant samples.

Conclusions

Down-regulation of COMT activity in switchgrass resulted in the accumulation of previously undetected metabolites resembling sinapyl alcohol and its related metabolites, but that are derived from para-methylation of 5-hydroxyconiferyl alcohol, and related precursors and products; the accumulation of which suggests altered metabolism of 5-hydroxyconiferyl alcohol in switchgrass. Given that there was no indication that iso-sinapyl alcohol was integrated in cell walls, it is considered a monolignol analog. Diversion of substrates from sinapyl alcohol to free iso-sinapyl alcohol, its glucoside, and associated upstream lignin pathway changes, including increased phenolic aldehydes and acids, are together associated with more facile cell wall deconstruction, and to the observed inhibitory effect on microbial growth. However, iso-sinapyl alcohol and iso-sinapic acid, added separately to media, were not inhibitory to C. thermocellum cultures.

     

Field‐grown transgenic switchgrass (Panicum virgatum L.) with altered lignin does not affect soil chemistry,microbiology, and carbon storage potential

Cell wall recalcitrance poses a major challenge on cellulosic biofuel production from feedstocks such as switchgrass (Panicum virgatum L.). As lignin is a known contributor of recalcitrance, transgenic switchgrass plants with altered lignin have been produced by downregulation of caffeic acid O‐methyltransferase (COMT). Field trials of COMT‐downregulated plants previously demonstrated improved ethanol conversion with no adverse agronomic effects. However, the rhizosphere impacts of altering lignin in plants are unknown. We hypothesized that changing plant lignin composition may affect residue degradation in soils, ultimately altering soil processes. The objective of this study was to evaluate effects of two independent lines of COMT‐downregulated switchgrass plants on soils in terms of chemistry, microbiology, and carbon cycling when grown in the field. Over the first two years of establishment, we observed no significant differences between transgenic and control plants in terms of soil pH or the total concentrations of 19 elements. An analysis of soil bacterial communities via high‐throughput 16S rRNA gene amplicon sequencing revealed no effects of transgenic plants on bacterial diversity, richness, or community composition. We also did not observe a change in the capacity for soil carbon storage: There was no significant effect on soil respiration or soil organic matter. After five years of establishment, δ¹³C of plant roots, leaves, and soils was measured and an isotopic mixing model used to estimate that 11.2 to 14.5% of soil carbon originated from switchgrass. Switchgrass‐contributed carbon was not significantly different between transgenic and control plants. Overall, our results indicate that over the short term (two and five years), lignin modification in switchgrass through manipulation of COMT expression does not have an adverse effect on soils in terms of total elemental composition, bacterial community structure and diversity, and capacity for carbon storage.     

The Growth of Transgenic Tobacco Plants with Estradiol-Induced Expression of Tomato Xyloglucan Endotransglucosylase Gene <Emphasis Type="Italic">tXET-B2</Emphasis> under Stress Conditions

The role of xyloglucan endotransglycosylases in the regulation and promotion of plant growth in response to such widespread stress factors as drought, salinization, and hypothermia remains poorly understood. The tXET-B2 (SlXTH10) gene encodes one of the xyloglucan endotransglycosylases of tomato, which is most closely related in the nucleotide sequence to the AtXTH15 and AtXTH16 genes of Arabidopsis thaliana. At present, the specific functions of the tXET-B2 gene, as well as of its homologs, AtXTH15 and AtXTH16, remain obscure. To study the role of tXET-B2 in the regulation of growth and adaptation to abiotic stress factors, transgenic tobacco plants with estradiol-inducible expression of the tomato tXET-B2 gene were generated. Overexpression of this gene promoted tobacco root growth in a medium containing 50 mM NaCl. Under drought conditions, exogenous treatment with estradiol resulted in a considerable increase in fresh and dry weight in many of the studied transgenic lines. Under normal conditions, as well as under salinization and hypothermia stress, such positive effect was detected only for some transgenic lines. The obtained data point to the possibility of using genetically engineered constructs of the tXET-B2 gene to correct growth parameters of transgenic plants under the influence of stress factors.     

The targeting of starch binding domains from starch synthase III to the cell wall alters cell wall composition and properties

Key message

Starch binding domains of starch synthase III from Arabidopsis thaliana (SBD123) binds preferentially to cell wall polysaccharides rather than to starch in vitro. Transgenic plants overexpressing SBD123 in the cell wall are larger than wild type. Cell wall components are altered in transgenic plants. Transgenic plants are more susceptible to digestion than wild type and present higher released glucose content. Our results suggest that the transgenic plants have an advantage for the production of bioethanol in terms of saccharification of essential substrates.

Abstract

The plant cell wall, which represents a major source of biomass for biofuel production, is composed of cellulose, hemicelluloses, pectins and lignin. A potential biotechnological target for improving the production of biofuels is the modification of plant cell walls. This modification is achieved via several strategies, including, among others, altering biosynthetic pathways and modifying the associations and structures of various cell wall components. In this study, we modified the cell wall of A. thaliana by targeting the starch-binding domains of A. thaliana starch synthase III to this structure. The resulting transgenic plants (E8-SDB123) showed an increased biomass, higher levels of both fermentable sugars and hydrolyzed cellulose and altered cell wall properties such as higher laxity and degradability, which are valuable characteristics for the second-generation biofuels industry. The increased biomass and degradability phenotype of E8-SBD123 plants could be explained by the putative cell-wall loosening effect of the in tandem starch binding domains. Based on these results, our approach represents a promising biotechnological tool for reducing of biomass recalcitrance and therefore, the need for pretreatments.

     

Reaction wood – a key cause of variation in cell wall recalcitrance in willow

Background

The recalcitrance of lignocellulosic cell wall biomass to deconstruction varies greatly in angiosperms, yet the source of this variation remains unclear. Here, in eight genotypes of short rotation coppice willow (Salix sp.) variability of the reaction wood (RW) response and the impact of this variation on cell wall recalcitrance to enzymatic saccharification was considered.

Results

A pot trial was designed to test if the ‘RW response’ varies between willow genotypes and contributes to the differences observed in cell wall recalcitrance to enzymatic saccharification in field-grown trees. Biomass composition was measured via wet chemistry and used with glucose release yields from enzymatic saccharification to determine cell wall recalcitrance. The levels of glucose release found for pot-grown control trees showed no significant correlation with glucose release from mature field-grown trees. However, when a RW phenotype was induced in pot-grown trees, glucose release was strongly correlated with that for mature field-grown trees. Field studies revealed a 5-fold increase in glucose release from a genotype grown at a site exposed to high wind speeds (a potentially high RW inducing environment) when compared with the same genotype grown at a more sheltered site.

Conclusions

Our findings provide evidence for a new concept concerning variation in the recalcitrance to enzymatic hydrolysis of the stem biomass of different, field-grown willow genotypes (and potentially other angiosperms). Specifically, that genotypic differences in the ability to produce a response to RW inducing conditions (a ‘RW response’) indicate that this RW response is a primary determinant of the variation observed in cell wall glucan accessibility. The identification of the importance of this RW response trait in willows, is likely to be valuable in selective breeding strategies in willow (and other angiosperm) biofuel crops and, with further work to dissect the nature of RW variation, could provide novel targets for genetic modification for improved biofuel feedstocks.

     

Mechanism of lignin inhibition of enzymatic biomass deconstruction

Background

The conversion of plant biomass to ethanol via enzymatic cellulose hydrolysis offers a potentially sustainable route to biofuel production. However, the inhibition of enzymatic activity in pretreated biomass by lignin severely limits the efficiency of this process.

Results

By performing atomic-detail molecular dynamics simulation of a biomass model containing cellulose, lignin, and cellulases (TrCel7A), we elucidate detailed lignin inhibition mechanisms. We find that lignin binds preferentially both to the elements of cellulose to which the cellulases also preferentially bind (the hydrophobic faces) and also to the specific residues on the cellulose-binding module of the cellulase that are critical for cellulose binding of TrCel7A (Y466, Y492, and Y493).

Conclusions

Lignin thus binds exactly where for industrial purposes it is least desired, providing a simple explanation of why hydrolysis yields increase with lignin removal.

     

Transgenic citrus expressing synthesized <Emphasis Type="Italic">cecropin</Emphasis> B genes in the phloem exhibits decreased susceptibility to Huanglongbing

Key message

Expression of synthesized cecropin B genes in the citrus phloem, where Candidatus Liberibacter asiaticus resides, significantly decreased host susceptibility to Huanglongbing.

Abstract

Huanglongbing (HLB), associated with Candidatus Liberibacter asiaticus bacteria, is the most destructive disease of citrus worldwide. All of the commercial sweet orange cultivars lack resistance to this disease. The cationic lytic peptide cecropin B, isolated from the Chinese tasar moth (Antheraea pernyi), has been shown to effectively eliminate bacteria. In this study, we demonstrated that transgenic citrus (Citrus sinensis Osbeck) expressing the cecropin B gene specifically in the phloem had a decreased susceptibility to HLB. Three plant codon-optimized synthetic cecropin B genes, which were designed to secrete the cecropin B peptide into three specific sites, the extracellular space, the cytoplasm, and the endoplasmic reticulum, were constructed. Under the control of the selected phloem-specific promoter GRP1.8, these constructs were transferred into the citrus genome. All of the cecropin B genes were efficiently expressed in the phloem of transgenic plants. Over more than a year of evaluation, the transgenic lines exhibited reduced disease severity. Bacterial populations in transgenic lines were significantly lower than in the controls. Two lines, in which bacterial populations were significantly lower than in others, showed no visible symptoms. Thus, we demonstrated the potential application of the phloem-specific expression of an antimicrobial peptide gene to protect citrus plants from HLB.

     

Mutant <Emphasis Type="Italic">TP53</Emphasis> G245C and R273H promote cellular malignancy in esophageal squamous cell carcinoma

Background

TP53 gene mutations occur in more than 50% of human cancers and the vast majority of these mutations in human cancers are missense mutations, which broadly occur in DNA binding domain (DBD) (Amino acids 102–292) and mainly reside in six “hotspot” residues. TP53 G245C and R273H point mutations are two of the most frequent mutations in tumors and have been verified in several different cancers. In the previous study of the whole genome sequencing (WGS), we found some mutations of TP53 DBD in esophageal squamous cell carcinoma (ESCC) clinical samples. We focused on two high-frequent mutations TP53 p.G245C and TP53 p.R273H and investigated their oncogenic roles in ESCC cell lines, p53-defective cell lines H1299 and HCT116 p53?/?.

Results

MTS and colony formation assays showed that mutant TP53 G245C and R273H increased cell vitality and proliferation. Flow cytometry results revealed inhibition of ultraviolet radiation (UV)- and ionizing radiation (IR)- induced apoptosis and disruption of TP53-mediated cell cycle arrest after UV, IR and Nocodazole treatment. Transwell assays indicated that mutant TP53 G245C and R273H enhanced cell migration and invasion abilities. Moreover, western blot revealed that they were able to suppress the expression of TP53 downstream genes in the process of apoptosis and cell cycle arrest induced by UV, which suggests that these two mutations can influence apoptosis and growth arrest might be due, at least in part, to down-regulate the expression of P21, GADD45α and PARP.

Conclusions

These results indicate that mutant TP53 G245C and R273H can lead to more aggressive phenotypes and enhance cancer cell malignancy, which further uncover TP53 function in carcinogenesis and might be useful in clinical diagnosis and therapy of TP53 mutant cancers.

     

Control of brown patch (<Emphasis Type="Italic">Rhizoctonia solani</Emphasis>) in tall fescue (<Emphasis Type="Italic">Festuca arundinacea</Emphasis> Schreb.) by host induced gene silencing

Key message

Transgenic tall fescue plants expressing RNAi constructs of essential genes of Rhizoctonia solani were resistant to R. solani.

Abstract

Tall fescue (Festuca arundinacea Schreb.) is an important turf and forage grass species widely used for home lawns and on golf courses in North Carolina and other transition zone states in the US. The most serious and frequently occurring disease of tall fescue is brown patch, caused by a basidiomycete fungus, Rhizoctonia solani. This research demonstrates resistance to brown patch disease achieved by the application of host induced gene silencing. We transformed tall fescue with RNAi constructs of four experimentally determined “essential” genes from R. solani (including genes encoding RNA polymerase, importin beta-1 subunit, Cohesin complex subunit Psm1, and a ubiquitin E3 ligase) to suppress expression of those genes inside the fungus and thus inhibit fungal infection. Four gene constructs were tested, and 19 transgenic plants were obtained, among which 12 plants had detectable accumulation of siRNAs of the target genes. In inoculation tests, six plants displayed significantly improved resistance against R. solani. Lesion size was reduced by as much as 90 %. Plants without RNAi accumulation did not show resistance. To our knowledge, this is the first case that RNAi constructs of pathogen genes introduced into a host plant can confer resistance against a necrotrophic fungus.