The extent of multiallelic, co-editing of LIGULELESS1 in highly polyploid sugarcane tunes leaf inclination angle and enables selection of the ideotype for biomass yield.

Sugarcane (Saccharum spp. hybrid) is a prime feedstock for commercial production of biofuel and table sugar. Optimizing canopy architecture for improved light capture has great potential for elevating biomass yield. LIGULELESS1 (LG1) is involved in leaf ligule and auricle development in grasses. Here, we report CRISPR/Cas9-mediated co-mutagenesis of up to 40 copies/alleles of the putative LG1 in highly polyploid sugarcane (2n = 100-120, x = 10-12). Next generation sequencing revealed co-editing frequencies of 7.4%-100% of the LG1 reads in 16 of the 78 transgenic lines. LG1 mutations resulted in a tuneable leaf angle phenotype that became more upright as co-editing frequency increased. Three lines with loss of function frequencies of ~12%, ~53% and ~95% of lg1 were selected following a randomized greenhouse trial and grown in replicated, multi-row field plots. The co-edited LG1 mutations were stably maintained in vegetative progenies and the extent of co-editing remained constant in field tested lines L26 and L35. Next generation sequencing confirmed the absence of potential off targets. The leaf inclination angle corresponded to light transmission into the canopy and tiller number. Line L35 displaying loss of function in ~12% of the lg1 NGS reads exhibited an 18% increase in dry biomass yield supported by a 56% decrease in leaf inclination angle, a 31% increase in tiller number, and a 25% increase in internode number. The scalable co-editing of LG1 in highly polyploid sugarcane allows fine-tuning of leaf inclination angle, enabling the selection of the ideotype for biomass yield.

Engineering triacylglycerol accumulation in duckweed (Lemna japonica).

Duckweeds are amongst the fastest growing of higher plants, making them attractive high-biomass targets for biofuel feedstock production. Their fronds have high rates of fatty acid synthesis to meet the demand for new membranes, but triacylglycerols (TAG) only accumulate to very low levels. Here we report on the engineering of Lemna japonica for the synthesis and accumulation of TAG in its fronds. This was achieved by expression of an estradiol-inducible cyan fluorescent protein-Arabidopsis WRINKLED1 fusion protein (CFP-AtWRI1), strong constitutive expression of a mouse diacylglycerol:acyl-CoA acyltransferase2 (MmDGAT), and a sesame oleosin variant (SiOLE(*)). Individual expression of each gene increased TAG accumulation by 1- to 7-fold relative to controls, while expression of pairs of these genes increased TAG by 7- to 45-fold. In uninduced transgenics containing all three genes, TAG accumulation increased by 45-fold to 3.6% of dry weight (DW) without severely impacting growth, and by 108-fold to 8.7% of DW after incubation on medium containing 100 µm estradiol for 4 days. TAG accumulation was accompanied by an increase in total fatty acids of up to three-fold to approximately 15% of DW. Lipid droplets from fronds of all transgenic lines were visible by confocal microscopy of BODIPY-stained fronds. At a conservative 12 tonnes (dry matter) per acre and 10% (DW) TAG, duckweed could produce 350 gallons of oil/acre/year, approximately seven-fold the yield of soybean, and similar to that of oil palm. These findings provide the foundation for optimizing TAG accumulation in duckweed and present a new opportunity for producing biofuels and lipidic bioproducts.

dCas9-3xSRDX-mediated transcriptional repression in sugarcane.

KEY MESSAGE: Targeting dCas9 fused with the 3xSRDX effector to the 5'UTR leads to strong repression of magnesium chelatase in highly polyploid sugarcane.

Metabolic engineering of energycane to hyperaccumulate lipids in vegetative biomass.

BACKGROUND: The metabolic engineering of high-biomass crops for lipid production in their vegetative biomass has recently been proposed as a strategy to elevate energy density and lipid yields for biodiesel production. Energycane and sugarcane are highly polyploid, interspecific hybrids between Saccharum officinarum and Saccharum spontaneum that differ in the amount of ancestral contribution to their genomes. This results in greater biomass yield and persistence in energycane, which makes it the preferred target crop for biofuel production. RESULTS: Here, we report on the hyperaccumulation of triacylglycerol (TAG) in energycane following the overexpression of the lipogenic factors Diacylglycerol acyltransferase1-2 (DGAT1-2) and Oleosin1 (OLE1) in combination with RNAi suppression of SUGAR-DEPENDENT1 (SDP1) and Trigalactosyl diacylglycerol1 (TGD1). TAG accumulated up to 1.52% of leaf dry weight (DW,) a rate that was 30-fold that of non-modified energycane, in addition to almost doubling the total fatty acid content in leaves to 4.42% of its DW. Pearson's correlation analysis showed that the accumulation of TAG had the highest correlation with the expression level of ZmDGAT1-2, followed by the level of RNAi suppression for SDP1. CONCLUSIONS: This is the first report on the metabolic engineering of energycane and demonstrates that this resilient, high-biomass crop is an excellent target for the further optimization of the production of lipids from vegetative tissues.

Towards oilcane: preliminary field evaluation of metabolically engineered sugarcane with hyper-accumulation of triacylglycerol in vegetative tissues.

We recently generated oilcane, a metabolically engineered sugarcane with hyper-accumulation of energy dense triacylglycerol in vegetative tissues. Refinement of this strategy in high biomass crops like sugarcane may result in elevated lipid yields that exceed traditional oilseed crops for biodiesel production. This is the first report of agronomic performance, stable co-expression of lipogenic factors, and TAG accumulation in transgenic sugarcane under field conditions. Co-expression of WRI1; DGAT1, OLE1, and RNAi suppression of PXA1 was stable during the 2-year field evaluation and resulted in TAG accumulation up to 4.4% of leaf DW. This TAG accumulation was 70-fold higher than in non-transgenic sugarcane and more than 2-fold higher than previously reported for the same line under greenhouse conditions. TAG accumulation correlated highest with the expression of WRI1. However, constitutive expression of WRI1 was negatively correlated with biomass accumulation. Transgenic lines without WRI1 expression accumulated TAG up to 1.6% of leaf DW and displayed no biomass yield penalty in the plant cane. These findings confirm sugarcane as a promising platform for the production of vegetative lipids and will be used to inform strategies to maximize future biomass and lipid yields. The main conclusion is that constitutive expression of WRI1 in combination with additional lipogenic factors (DGAT1-2, OLE1, PXA1) in sugarcane under field conditions leads to hyper-accumulation of TAG and reduces biomass yield. Supplementary Information: The online version contains supplementary material available at 10.1007/s11032-022-01333-5.

Generation of a selectable marker free, highly expressed single copy locus as landing pad for transgene stacking in sugarcane.

KEY MESSAGE: A selectable marker free, highly expressed single copy locus flanked by insulators was created as landing pad for transgene stacking in sugarcane. These events displayed superior transgene expression compared to single-copy transgenic lines lacking insulators. Excision of the selectable marker gene from transgenic sugarcane lines was supported by FLPe/FRT site-specific recombination. Sugarcane, a tropical C4 grass in the genus Saccharum (Poaceae), accounts for nearly 80% of sugar produced worldwide and is also an important feedstock for biofuel production. Generating transgenic sugarcane with predictable and stable transgene expression is critical for crop improvement. In this study, we generated a highly expressed single copy locus as landing pad for transgene stacking. Transgenic sugarcane lines with stable integration of a single copy nptII expression cassette flanked by insulators supported higher transgene expression along with reduced line to line variation when compared to single copy events without insulators by NPTII ELISA analysis. Subsequently, the nptII selectable marker gene was efficiently excised from the sugarcane genome by the FLPe/FRT site-specific recombination system to create selectable marker free plants. This study provides valuable resources for future gene stacking using site-specific recombination or genome editing tools.

Advancing Crop Transformation in the Era of Genome Editing.

Plant transformation has enabled fundamental insights into plant biology and revolutionized commercial agriculture. Unfortunately, for most crops, transformation and regeneration remain arduous even after more than 30 years of technological advances. Genome editing provides novel opportunities to enhance crop productivity but relies on genetic transformation and plant regeneration, which are bottlenecks in the process. Here, we review the state of plant transformation and point to innovations needed to enable genome editing in crops. Plant tissue culture methods need optimization and simplification for efficiency and minimization of time in culture. Currently, specialized facilities exist for crop transformation. Single-cell and robotic techniques should be developed for high-throughput genomic screens. Plant genes involved in developmental reprogramming, wound response, and/or homologous recombination should be used to boost the recovery of transformed plants. Engineering universal Agrobacterium tumefaciens strains and recruiting other microbes, such as Ensifer or Rhizobium, could facilitate delivery of DNA and proteins into plant cells. Synthetic biology should be employed for de novo design of transformation systems. Genome editing is a potential game-changer in crop genetics when plant transformation systems are optimized.

TALEN-mediated targeted mutagenesis of more than 100 COMT copies/alleles in highly polyploid sugarcane improves saccharification efficiency without compromising biomass yield.

Sugarcane is the world's most efficient feedstock for commercial production of bioethanol due to its superior biomass production and accumulation of sucrose in stems. Integrating first- and second-generation ethanol conversion processes will enhance the biofuel yield per unit area by utilizing both sucrose and cell wall-bound sugars for fermentation. RNAi suppression of the lignin biosynthetic gene caffeic acid O-methyltransferase (COMT) has been demonstrated to improve bioethanol production from lignocellulosic biomass. Genome editing has been used in a number of crops for creation of loss of function phenotypes but is very challenging in sugarcane due to its highly polyploid genome. In this study, a conserved region of COMT was targeted with a single-transcription activator-like effector nuclease (TALEN) pair for multi-allelic mutagenesis to modify lignin biosynthesis in sugarcane. Field-grown TALEN-mediated COMT mutants showed up to 19.7% lignin reduction and significantly decreased syringyl to guaiacyl (S/G) ratio resulting in an up to 43.8% improved saccharification efficiency. Biomass production of COMT mutant lines with superior saccharification efficiency did not differ significantly from the original cultivar under replicated field conditions. Sanger sequencing of cloned COMT amplicons (1351-1657 bp) revealed co-editing of 107 of the 109 unique COMT copies/alleles in vegetative progeny of line CB6 using a single TALEN pair. Line CB6 combined altered cell wall composition and drastically improved saccharification efficiency with good agronomic performance. These findings confirm the feasibility of co-mutagenesis of a very large number of target alleles/copies for improvement in crops with complex genomes.

In planta production and characterization of a hyperthermostable GH10 xylanase in transgenic sugarcane.

Sugarcane (Saccharum sp. hybrids) is one of the most efficient and sustainable feedstocks for commercial production of fuel ethanol. Recent efforts focus on the integration of first and second generation bioethanol conversion technologies for sugarcane to increase biofuel yields. This integrated process will utilize both the cell wall bound sugars of the abundant lignocellulosic sugarcane residues in addition to the sucrose from stem internodes. Enzymatic hydrolysis of lignocellulosic biomass into its component sugars requires significant amounts of cell wall degrading enzymes. In planta production of xylanases has the potential to reduce costs associated with enzymatic hydrolysis but has been reported to compromise plant growth and development. To address this problem, we expressed a hyperthermostable GH10 xylanase, xyl10B in transgenic sugarcane which displays optimal catalytic activity at 105 °C and only residual catalytic activity at temperatures below 70 °C. Transgene integration and expression in sugarcane were confirmed by Southern blot, RT-PCR, ELISA and western blot following biolistic co-transfer of minimal expression cassettes of xyl10B and the selectable neomycin phosphotransferase II. Xylanase activity was detected in 17 transgenic lines with a fluorogenic xylanase activity assay. Up to 1.2% of the total soluble protein fraction of vegetative progenies with integration of chloroplast targeted expression represented the recombinant Xyl10B protein. Xyl10B activity was stable in vegetative progenies. Tissues retained 75% of the xylanase activity after drying of leaves at 35 °C and a 2 month storage period. Transgenic sugarcane plants producing Xyl10B did not differ from non-transgenic sugarcane in growth and development under greenhouse conditions. Sugarcane xylan and bagasse were used as substrate for enzymatic hydrolysis with the in planta produced Xyl10B. TLC and HPLC analysis of hydrolysis products confirmed the superior catalytic activity and stability of the in planta produced Xyl10B with xylobiose as a prominent degradation product. These findings will contribute to advancing consolidated processing of lignocellulosic sugarcane biomass.

TALEN mediated targeted mutagenesis of the caffeic acid O-methyltransferase in highly polyploid sugarcane improves cell wall composition for production of bioethanol.

Sugarcane (Saccharum spp. hybrids) is a prime crop for commercial biofuel production. Advanced conversion technology utilizes both, sucrose accumulating in sugarcane stems as well as cell wall bound sugars for commercial ethanol production. Reduction of lignin content significantly improves the conversion of lignocellulosic biomass into ethanol. Conventional mutagenesis is not expected to confer reduction in lignin content in sugarcane due to its high polyploidy (x = 10-13) and functional redundancy among homo(eo)logs. Here we deploy transcription activator-like effector nuclease (TALEN) to induce mutations in a highly conserved region of the caffeic acid O-methyltransferase (COMT) of sugarcane. Capillary electrophoresis (CE) was validated by pyrosequencing as reliable and inexpensive high throughput method for identification and quantitative characterization of TALEN mediated mutations. Targeted COMT mutations were identified by CE in up to 74 % of the lines. In different events 8-99 % of the wild type COMT were converted to mutant COMT as revealed by pyrosequencing. Mutation frequencies among mutant lines were positively correlated to lignin reduction. Events with a mutation frequency of 99 % displayed a 29-32 % reduction of the lignin content compared to non-transgenic controls along with significantly reduced S subunit content and elevated hemicellulose content. CE analysis displayed similar peak patterns between primary COMT mutants and their vegetative progenies suggesting that TALEN mediated mutations were faithfully transmitted to vegetative progenies. This is the first report on genome editing in sugarcane. The findings demonstrate that targeted mutagenesis can improve cell wall characteristics for production of lignocellulosic ethanol in crops with highly complex genomes.

Precision breeding for RNAi suppression of a major 4-coumarate:coenzyme A ligase gene improves cell wall saccharification from field grown sugarcane.

Sugarcane (Saccharum spp. hybrids) is a major feedstock for commercial bioethanol production. The recent integration of conversion technologies that utilize lignocellulosic sugarcane residues as well as sucrose from stem internodes has elevated bioethanol yields. RNAi suppression of lignin biosynthetic enzymes is a successful strategy to improve the saccharification of lignocellulosic biomass. 4-coumarate:coenzyme A ligase (4CL) is a key enzyme in the biosynthesis of phenylpropanoid metabolites, such as lignin and flavonoids. Identifying a major 4CL involved in lignin biosynthesis among multiple isoforms with functional divergence is key to manipulate lignin biosynthesis. In this study, two full length 4CL genes (Sh4CL1 and Sh4CL2) were isolated and characterized in sugarcane. Phylogenetic, expression and RNA interference (RNAi) analysis confirmed that Sh4CL1 is a major lignin biosynthetic gene. An intragenic precision breeding strategy may facilitate the regulatory approval of the genetically improved events and was used for RNAi suppression of Sh4CL1. Both, the RNAi inducing cassette and the expression cassette for the mutated ALS selection marker consisted entirely of DNA sequences from sugarcane or the sexually compatible species Sorghum bicolor. Field grown sugarcane with intragenic RNAi suppression of Sh4CL1 resulted in reduction of the total lignin content by up to 16.5 % along with altered monolignol ratios without reduction in biomass yield. Mature, field grown, intragenic sugarcane events displayed 52-76 % improved saccharification efficiency of lignocellulosic biomass compared to wild type (WT) controls. This demonstrates for the first time that an intragenic approach can add significant value to lignocellulosic feedstocks for biofuel and biochemical production.

Metabolic engineering of sugarcane to accumulate energy-dense triacylglycerols in vegetative biomass.

Elevating the lipid content in vegetative tissues has emerged as a new strategy for increasing energy density and biofuel yield of crops. Storage lipids in contrast to structural and signaling lipids are mainly composed of glycerol esters of fatty acids, also known as triacylglycerol (TAG). TAGs are one of the most energy-rich and abundant forms of reduced carbon available in nature. Therefore, altering the carbon-partitioning balance in favour of TAG in vegetative tissues of sugarcane, one of the highest yielding biomass crops, is expected to drastically increase energy yields. Here we report metabolic engineering to elevate TAG accumulation in vegetative tissues of sugarcane. Constitutive co-expression of WRINKLED1 (WRI1), diacylglycerol acyltransferase1-2 (DGAT1-2) and oleosin1 (OLE1) and simultaneous cosuppression of ADP-glucose pyrophosphorylase (AGPase) and a subunit of the peroxisomal ABC transporter1 (PXA1) in transgenic sugarcane elevated TAG accumulation in leaves or stems by 95- or 43-fold to 1.9% or 0.9% of dry weight (DW), respectively, while expression or suppression of one to three of the target genes increased TAG levels by 1.5- to 9.5-fold. Accumulation of TAG in vegetative progeny plants was consistent with the results from primary transgenics and contributed to a total fatty acid content of up to 4.7% or 1.7% of DW in mature leaves or stems, respectively. Lipid droplets were visible within mesophyll cells of transgenic leaves by confocal fluorescence microscopy. These results provide the basis for optimizations of TAG accumulation in sugarcane and other high yielding biomass grasses and will open new prospects for biofuel applications.

Advances in genomics and genome editing for improving strawberry (Fragaria ×ananassa).

The cultivated strawberry, Fragaria ×ananassa, is a recently domesticated fruit species of economic interest worldwide. As such, there is significant interest in continuous varietal improvement. Genomics-assisted improvement, including the use of DNA markers and genomic selection have facilitated significant improvements of numerous key traits during strawberry breeding. CRISPR/Cas-mediated genome editing allows targeted mutations and precision nucleotide substitutions in the target genome, revolutionizing functional genomics and crop improvement. Genome editing is beginning to gain traction in the more challenging polyploid crops, including allo-octoploid strawberry. The release of high-quality reference genomes and comprehensive subgenome-specific genotyping and gene expression profiling data in octoploid strawberry will lead to a surge in trait discovery and modification by using CRISPR/Cas. Genome editing has already been successfully applied for modification of several strawberry genes, including anthocyanin content, fruit firmness and tolerance to post-harvest disease. However, reports on many other important breeding characteristics associated with fruit quality and production are still lacking, indicating a need for streamlined genome editing approaches and tools in Fragaria ×ananassa. In this review, we present an overview of the latest advancements in knowledge and breeding efforts involving CRISPR/Cas genome editing for the enhancement of strawberry varieties. Furthermore, we explore potential applications of this technology for improving other Rosaceous plant species.

RNA interference suppression of lignin biosynthesis increases fermentable sugar yields for biofuel production from field-grown sugarcane.

The agronomic performance, cell wall characteristics and enzymatic saccharification efficiency of transgenic sugarcane plants with modified lignin were evaluated under replicated field conditions. Caffeic acid O-methyltransferase (COMT) was stably suppressed by RNAi in the field, resulting in transcript reduction of 80%-91%. Along with COMT suppression, total lignin content was reduced by 6%-12% in different transgenic lines. Suppression of COMT also altered lignin composition by reducing syringyl units and p-coumarate incorporation into lignin. Reduction in total lignin by 6% improved saccharification efficiency by 19%-23% with no significant difference in biomass yield, plant height, stalk diameter, tiller number, total structural carbohydrates or brix value when compared with nontransgenic tissue culture-derived or transgenic control plants. Lignin reduction of 8%-12% compromised biomass yield, but increased saccharification efficiency by 28%-32% compared with control plants. Biomass from transgenic sugarcane lines that have 6%-12% less lignin requires approximately one-third of the hydrolysis time or 3- to 4-fold less enzyme to release an equal or greater amount of fermentable sugar than nontransgenic plants. Reducing the recalcitrance of lignocellulosic biomass to saccharification by modifying lignin biosynthesis is expected to greatly benefit the economic competitiveness of sugarcane as a biofuel feedstock.

Targeted mutagenesis with sequence-specific nucleases for accelerated improvement of polyploid crops: Progress, challenges, and prospects.

Many of the world's most important crops are polyploid. The presence of more than two sets of chromosomes within their nuclei and frequently aberrant reproductive biology in polyploids present obstacles to conventional breeding. The presence of a larger number of homoeologous copies of each gene makes random mutation breeding a daunting task for polyploids. Genome editing has revolutionized improvement of polyploid crops as multiple gene copies and/or alleles can be edited simultaneously while preserving the key attributes of elite cultivars. Most genome-editing platforms employ sequence-specific nucleases (SSNs) to generate DNA double-stranded breaks at their target gene. Such DNA breaks are typically repaired via the error-prone nonhomologous end-joining process, which often leads to frame shift mutations, causing loss of gene function. Genome editing has enhanced the disease resistance, yield components, and end-use quality of polyploid crops. However, identification of candidate targets, genotyping, and requirement of high mutagenesis efficiency remain bottlenecks for targeted mutagenesis in polyploids. In this review, we will survey the tremendous progress of SSN-mediated targeted mutagenesis in polyploid crop improvement, discuss its challenges, and identify optimizations needed to sustain further progress.

Multi-allelic gene editing in an apomictic, tetraploid turf and forage grass (Paspalum notatum Flüggé) using CRISPR/Cas9.

Polyploidy is common among grasses (Poaceae) and poses challenges for conventional breeding. Genome editing technology circumvents crossing and selfing, enabling targeted modifications to multiple gene copies in a single generation while maintaining the heterozygous context of many polyploid genomes. Bahiagrass (Paspalum notatum Flüggé; 2n=4x=40) is an apomictic, tetraploid C4 species that is widely grown in the southeastern United States as forage in beef cattle production and utility turf. The chlorophyll biosynthesis gene magnesium chelatase (MgCh) was selected as a rapid readout target for establishing genome editing in tetraploid bahiagrass. Vectors containing sgRNAs, Cas9 and nptII were delivered to callus cultures by biolistics. Edited plants were characterized through PCR-based assays and DNA sequencing, and mutagenesis frequencies as high as 99% of Illumina reads were observed. Sequencing of wild type (WT) bahiagrass revealed a high level of sequence variation in MgCh likely due to the presence of at least two copies with possibly eight different alleles, including pseudogenes. MgCh mutants exhibited visible chlorophyll depletion with up to 82% reductions in leaf greenness. Two lines displayed progression of editing over time which was linked to somatic editing. Apomictic progeny of a chimeric MgCh editing event were obtained and allowed identification of uniformly edited progeny plants among a range of chlorophyll depletion phenotypes. Sanger sequencing of a highly edited mutant revealed elevated frequency of a WT allele, probably due to frequent homology-directed repair (HDR). To our knowledge these experiments comprise the first report of genome editing applied in perennial, warm-season turf or forage grasses. This technology will accelerate bahiagrass cultivar development.

Intron-mediated enhancement of DIACYLGLYCEROL ACYLTRANSFERASE1 expression in energycane promotes a step change for lipid accumulation in vegetative tissues.

BACKGROUND: Metabolic engineering for hyperaccumulation of lipids in vegetative tissues is a novel strategy for enhancing energy density and biofuel production from biomass crops. Energycane is a prime feedstock for this approach due to its high biomass production and resilience under marginal conditions. DIACYLGLYCEROL ACYLTRANSFERASE (DGAT) catalyzes the last and only committed step in the biosynthesis of triacylglycerol (TAG) and can be a rate-limiting enzyme for the production of TAG. RESULTS: In this study, we explored the effect of intron-mediated enhancement (IME) on the expression of DGAT1 and resulting accumulation of TAG and total fatty acid (TFA) in leaf and stem tissues of energycane. To maximize lipid accumulation these evaluations were carried out by co-expressing the lipogenic transcription factor WRINKLED1 (WRI1) and the TAG protect factor oleosin (OLE1). Including an intron in the codon-optimized TmDGAT1 elevated the accumulation of its transcript in leaves by seven times on average based on 5 transgenic lines for each construct. Plants with WRI1 (W), DGAT1 with intron (Di), and OLE1 (O) expression (WDiO) accumulated TAG up to a 3.85% of leaf dry weight (DW), a 192-fold increase compared to non-modified energycane (WT) and a 3.8-fold increase compared to the highest accumulation under the intron-less gene combination (WDO). This corresponded to TFA accumulation of up to 8.4% of leaf dry weight, a 2.8-fold or 6.1-fold increase compared to WDO or WT, respectively. Co-expression of WDiO resulted in stem accumulations of TAG up to 1.14% of DW or TFA up to 2.08% of DW that exceeded WT by 57-fold or 12-fold and WDO more than twofold, respectively. Constitutive expression of these lipogenic "push pull and protect" factors correlated with biomass reduction. CONCLUSIONS: Intron-mediated enhancement (IME) of the expression of DGAT resulted in a step change in lipid accumulation of energycane and confirmed that under our experimental conditions it is rate limiting for lipid accumulation. IME should be applied to other lipogenic factors and metabolic engineering strategies. The findings from this study may be valuable in developing a high biomass feedstock for commercial production of lipids and advanced biofuels.

Microbiome differences in sugarcane and metabolically engineered oilcane accessions and their implications for bioenergy production.

Oilcane is a metabolically engineered sugarcane (Saccharum spp. hybrid) that hyper-accumulates lipids in its vegetable biomass to provide an advanced feedstock for biodiesel production. The potential impact of hyper-accumulation of lipids in vegetable biomass on microbiomes and the consequences of altered microbiomes on plant growth and lipid accumulation have not been explored so far. Here, we explore differences in the microbiome structure of different oilcane accessions and non-modified sugarcane. 16S SSU rRNA and ITS rRNA amplicon sequencing were performed to compare the characteristics of the microbiome structure from different plant compartments (leaf, stem, root, rhizosphere, and bulk soil) of four greenhouse-grown oilcane accessions and non-modified sugarcane. Significant differences were only observed in the bacterial microbiomes. In leaf and stem microbiomes, more than 90% of the entire microbiome of non-modified sugarcane and oilcane was dominated by similar core taxa. Taxa associated with Proteobacteria led to differences in the non-modified sugarcane and oilcane microbiome structure. While differences were observed between multiple accessions, accession 1566 was notable in that it was consistently observed to differ in its microbial membership than other accessions and had the lowest abundance of taxa associated with plant-growth-promoting bacteria. Accession 1566 is also unique among oilcane accessions in that it has the highest constitutive expression of the WRI1 transgene. The WRI1 transcription factor is known to contribute to significant changes in the global gene expression profile, impacting plant fatty acid biosynthesis and photomorphogenesis. This study reveals for the first time that genetically modified oilcanes associate with distinct microbiomes. Our findings suggest potential relationships between core taxa, biomass yield, and TAG in oilcane accessions and support further research on the relationship between plant genotypes and their microbiomes.

RNAi suppression of lignin biosynthesis in sugarcane reduces recalcitrance for biofuel production from lignocellulosic biomass.

Sugarcane is a prime bioethanol feedstock. Currently, sugarcane ethanol is produced through fermentation of the sucrose, which can easily be extracted from stem internodes. Processes for production of biofuels from the abundant lignocellulosic sugarcane residues will boost the ethanol output from sugarcane per land area. However, unlocking the vast amount of chemical energy stored in plant cell walls remains expensive primarily because of the intrinsic recalcitrance of lignocellulosic biomass. We report here the successful reduction in lignification in sugarcane by RNA interference, despite the complex and highly polyploid genome of this interspecific hybrid. Down-regulation of the sugarcane caffeic acid O-methyltransferase (COMT) gene by 67% to 97% reduced the lignin content by 3.9% to 13.7%, respectively. The syringyl/guaiacyl ratio in the lignin was reduced from 1.47 in the wild type to values ranging between 1.27 and 0.79. The yields of directly fermentable glucose from lignocellulosic biomass increased up to 29% without pretreatment. After dilute acid pretreatment, the fermentable glucose yield increased up to 34%. These observations demonstrate that a moderate reduction in lignin (3.9% to 8.4%) can reduce the recalcitrance of sugarcane biomass without compromising plant performance under controlled environmental conditions.

Production of hyperthermostable GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants enables complete hydrolysis of methylglucuronoxylan to fermentable sugars for biofuel production.

Overcoming the recalcitrance in lignocellulosic biomass for efficient hydrolysis of the polysaccharides cellulose and hemicellulose to fermentable sugars is a research priority for the transition from a fossilfuel-based economy to a renewable carbohydrate economy. Methylglucuronoxylans (MeGXn) are the major components of hemicellulose in woody biofuel crops. Here, we describe efficient production of the GH10 xylanase Xyl10B from Thermotoga maritima in transplastomic plants and demonstrate exceptional stability and catalytic activities of the in planta produced enzyme. Fully expanded leaves from homotransplastomic plants contained enzymatically active Xyl10B at a level of 11-15% of their total soluble protein. Transplastomic plants and their seed progeny were morphologically indistinguishable from non-transgenic plants. Catalytic activity of in planta produced Xyl10B was detected with poplar, sweetgum and birchwood xylan substrates following incubation between 40 and 90 °C and was also stable in dry and stored leaves. Optimal yields of Xyl10B were obtained from dry leaves if crude protein extraction was performed at 85 °C. The transplastomic plant derived Xyl10B showed exceptional catalytic activity and enabled the complete hydrolysis of MeGXn to fermentable sugars with the help of a single accessory enzyme (α-glucuronidase) as revealed by the sugar release assay. Even without this accessory enzyme, the majority of MeGXn was hydrolyzed by the transplastomic plant-derived Xyl10B to fermentable xylose and xylobiose.