Non-destructive assessment of cannabis quality during drying process using hyperspectral imaging and machine learning.

Cannabis sativa L. is an industrially valuable plant known for its cannabinoids, such as cannabidiol (CBD) and Δ9-tetrahydrocannabinol (THC), renowned for its therapeutic and psychoactive properties. Despite its significance, the cannabis industry has encountered difficulties in guaranteeing consistent product quality throughout the drying process. Hyperspectral imaging (HSI), combined with advanced machine learning technology, has been used to predict phytochemicals that presents a promising solution for maintaining cannabis quality control. We examined the dynamic changes in cannabinoid compositions under diverse drying conditions and developed a non-destructive method to appraise the quality of cannabis flowers using HSI and machine learning. Even when the relative weight and water content remained constant throughout the drying process, drying conditions significantly influenced the levels of CBD, THC, and their precursors. These results emphasize the importance of determining the exact drying endpoint. To develop HSI-based models for predicting cannabis quality indicators, including dryness, precursor conversion of CBD and THC, and CBD : THC ratio, we employed various spectral preprocessing methods and machine learning algorithms, including logistic regression (LR), support vector machine (SVM), k-nearest neighbor (KNN), random forest (RF), and Gaussian naïve Bayes (GNB). The LR model demonstrated the highest accuracy at 94.7-99.7% when used in conjunction with spectral pre-processing techniques such as multiplicative scatter correction (MSC) or Savitzky-Golay filter. We propose that the HSI-based model holds the potential to serve as a valuable tool for monitoring cannabinoid composition and determining optimal drying endpoint. This tool offers the means to achieve uniform cannabis quality and optimize the drying process in the industry.

AraDQ: an automated digital phenotyping software for quantifying disease symptoms of flood-inoculated Arabidopsis seedlings.

BACKGROUND: Plant scientists have largely relied on pathogen growth assays and/or transcript analysis of stress-responsive genes for quantification of disease severity and susceptibility. These methods are destructive to plants, labor-intensive, and time-consuming, thereby limiting their application in real-time, large-scale studies. Image-based plant phenotyping is an alternative approach that enables automated measurement of various symptoms. However, most of the currently available plant image analysis tools require specific hardware platform and vendor specific software packages, and thus, are not suited for researchers who are not primarily focused on plant phenotyping. In this study, we aimed to develop a digital phenotyping tool to enhance the speed, accuracy, and reliability of disease quantification in Arabidopsis. RESULTS: Here, we present the Arabidopsis Disease Quantification (AraDQ) image analysis tool for examination of flood-inoculated Arabidopsis seedlings grown on plates containing plant growth media. It is a cross-platform application program with a user-friendly graphical interface that contains highly accurate deep neural networks for object detection and segmentation. The only prerequisite is that the input image should contain a fixed-sized 24-color balance card placed next to the objects of interest on a white background to ensure reliable and reproducible results, regardless of the image acquisition method. The image processing pipeline automatically calculates 10 different colors and morphological parameters for individual seedlings in the given image, and disease-associated phenotypic changes can be easily assessed by comparing plant images captured before and after infection. We conducted two case studies involving bacterial and plant mutants with reduced virulence and disease resistance capabilities, respectively, and thereby demonstrated that AraDQ can capture subtle changes in plant color and morphology with a high level of sensitivity. CONCLUSIONS: AraDQ offers a simple, fast, and accurate approach for image-based quantification of plant disease symptoms using various parameters. Its fully automated pipeline neither requires prior image processing nor costly hardware setups, allowing easy implementation of the software by researchers interested in digital phenotyping of diseased plants.

Synthetic Studies toward 5,6,7,3',4'-Monomethoxytetrahydroxyflavones: Synthesis of Pedalitin.

During the synthetic studies toward 5,6,7,3',4'-monomethoxytetrahydroxyflavones, a concise pedalitin synthesis procedure was achieved. As previously reported, 6-hydroxy-2,3,4-trimethoxyacetophenone was prepared by Friedel-Crafts acylation of 1,4-dihydroxy-2,6-dimethoxybenzene with boron trifluoride diethyl etherate in acetic acid. When aldol condensation of 6-hydroxy-2,3,4-trimethoxyacetophenone 2b with vanillin was performed in basic conditions, it produced 2'-hydroxychalcone 3b, and, surprisingly, along with 3-hydroxyflavone 4 in a considerable amount. We propose that this oxidative cyclization is presumably due to the contribution of a quinone methide, likely to be subjected to aerobic oxidation. The chalcone was then subjected to oxidative cyclization with iodine in dimethyl sulfoxide to afford flavone 5 in good yield. To our delight, serial demethylation of the three methoxy groups at the 5-, 6-, and 3'-positions of 5 proceeded smoothly to produce pedalitin 1, under hydrogen bromide solution (30% in acetic acid). The crystal structures of 3-hydroxyflavone 4 and pedalitin tetraacetate 6 were unambiguously determined by X-ray crystallography.

Sanguisorba officinalis L. Ameliorates Hepatic Steatosis and Fibrosis by Modulating Oxidative Stress, Fatty Acid Oxidation, and Gut Microbiota in CDAHFD-Induced Mice.

Non-alcoholic fatty liver disease (NAFLD) is a leading cause of chronic liver diseases and encompasses non-alcoholic steatosis, steatohepatitis, and fibrosis. Sanguisorba officinalis L. (SO) roots have traditionally been used for their antioxidant properties and have beneficial effects on metabolic disorders, including diabetes and obesity. However, its effects on hepatic steatosis and fibrosis remain unclear. In this study, we explored the effects of a 95% ethanolic SO extract (SOEE) on NAFLD and fibrosis in vivo and in vitro. The SOEE was orally administered to C57BL/6J mice fed a choline-deficient, L-amino-acid-defined, high-fat diet for 10 weeks. The SOEE inhibited hepatic steatosis by modulating hepatic malondialdehyde levels and the expression of oxidative stress-associated genes, regulating fatty-acid-oxidation-related genes, and inhibiting the expression of genes that are responsible for fibrosis. The SOEE suppressed the deposition of extracellular matrix hydroxyproline and mRNA expression of fibrosis-associated genes. The SOEE decreased the expression of fibrosis-related genes in vitro by inhibiting SMAD2/3 phosphorylation. Furthermore, the SOEE restored the gut microbial diversity and modulated specific bacterial genera associated with NAFLD and fibrosis. This study suggests that SOEE might be the potential candidate for inhibiting hepatic steatosis and fibrosis by modulating oxidative stress, fatty acid oxidation, and gut microbiota composition.

Integrative analysis of metabolite and transcriptome reveals biosynthetic pathway and candidate genes for eupatilin and jaceosidin biosynthesis in Artemisia argyi.

Artemisia argyi (A. argyi) is a medicinal plant belonging to the Asteraceae family and Artemisia genus. Flavonoids abundant in A. argyi are associated with anti-inflammatory, anticancer, and antioxidative effects. Eupatilin and jaceosidin are representative polymethoxy flavonoids with medicinal properties significant enough to warrant the development of drugs using their components. However, the biosynthetic pathways and related genes of these compounds have not been fully explored in A. argyi. This study comprehensively analyzed the transcriptome data and flavonoids contents from four different tissues of A. argyi (young leaves, old leaves, trichomes collected from stems, and stems without trichomes) for the first time. We obtained 41,398 unigenes through the de-novo assembly of transcriptome data and mined promising candidate genes involved in the biosynthesis of eupatilin and jaceosidin using differentially expressed genes, hierarchical clustering, phylogenetic tree, and weighted gene co-expression analysis. Our analysis led to the identification of a total of 7,265 DEGs, among which 153 genes were annotated as flavonoid-related genes. In particular, we were able to identify eight putative flavone-6-hydroxylase (F6H) genes, which were responsible for providing a methyl group acceptor into flavone basic skeleton. Furthermore, five O-methyltransferases (OMTs) gene were identified, which were required for the site-specific O-methylation during the biosynthesis of eupatilin and jaceosidin. Although further validation would be necessary, our findings pave the way for the modification and mass-production of pharmacologically important polymethoxy flavonoids through genetic engineering and synthetic biological approaches.

The Impact of Light Wavelength and Darkness on Metabolite Profiling of Korean Ginseng: Evaluating Its Anti-Cancer Potential against MCF-7 and BV-2 Cell Lines.

Korean ginseng is a source of functional foods and medicines; however, its productivity is hindered by abiotic stress factors, such as light. This study investigated the impacts of darkness and different light wavelengths on the metabolomics and anti-cancer activity of ginseng extracts. Hydroponically-grown Korean ginseng was shifted to a light-emitting diodes (LEDs) chamber for blue-LED and darkness treatments, while white fluorescent (FL) light treatment was the control. MCF-7 breast cancer and lipopolysaccharide (LPS)-induced BV-2 microglial cells were used to determine chemo-preventive and neuroprotective potential. Overall, 53 significant primary metabolites were detected in the treated samples. The levels of ginsenosides Rb1, Rb2, Rc, Rd, and Re, as well as organic and amino acids, were significantly higher in the dark treatment, followed by blue-LED treatment and the FL control. The dark-treated ginseng extract significantly induced apoptotic signaling in MCF-7 cells and dose-dependently inhibited the NF-κB and MAP kinase pathways in LPS-induced BV-2 cells. Short-term dark treatment increased the content of Rd, Rc, Rb1, Rb2, and Re ginsenosides in ginseng extracts, which promoted apoptosis of MCF-7 cells and inhibition of the MAP kinase pathway in BV-2 microglial cells. These results indicate that the dark treatment might be effective in improving the pharmacological potential of ginseng.

Somatic Mutations in Fruit Trees: Causes, Detection Methods, and Molecular Mechanisms.

Somatic mutations are genetic changes that occur in non-reproductive cells. In fruit trees, such as apple, grape, orange, and peach, somatic mutations are typically observed as "bud sports" that remain stable during vegetative propagation. Bud sports exhibit various horticulturally important traits that differ from those of their parent plants. Somatic mutations are caused by internal factors, such as DNA replication error, DNA repair error, transposable elements, and deletion, and external factors, such as strong ultraviolet radiation, high temperature, and water availability. There are several methods for detecting somatic mutations, including cytogenetic analysis, and molecular techniques, such as PCR-based methods, DNA sequencing, and epigenomic profiling. Each method has its advantages and limitations, and the choice of method depends on the research question and the available resources. The purpose of this review is to provide a comprehensive understanding of the factors that cause somatic mutations, techniques used to identify them, and underlying molecular mechanisms. Furthermore, we present several case studies that demonstrate how somatic mutation research can be leveraged to discover novel genetic variations. Overall, considering the diverse academic and practical value of somatic mutations in fruit crops, especially those that require lengthy breeding efforts, related research is expected to become more active.

Translating CO[Formula: see text] variability in a plant growth system into plant dynamics.

Plant growth occurs owing to the continuous interactions between environmental and genetic factors, and the analysis of plant growth provides crucial information on plant responses. Recent agronomic and analytical methodologies for plant growth require various channels for capturing broader and more dynamic plant traits. In this study, we provide a method of non-invasive growth analyses by translating CO[Formula: see text] variability around a plant. We hypothesized that the cumulative coefficient of variation (CCV) of plant-driven ambient CO[Formula: see text] variation in a plant growth system could yield a numerical indicator that is connected to the plant growth dynamics. Using the system outside-plant growth system-plant coupled dynamic model, we found that the CCV could translate dynamic plant growth under environmental and biophysical constraints. Furthermore, we experimentally demonstrated the application of CCV by using non-airtight growth chamber systems. Our findings may enrich plant growth information channels and assist growers or researchers to analyze plant growth comprehensively.

Genetic Diversity and Association Analysis for Carotenoid Content among Sprouts of Cowpea (Vigna unguiculata L. Walp).

The development and promotion of biofortified foods plants are a sustainable strategy for supplying essential micronutrients for human health and nutrition. We set out to identify quantitative trait loci (QTL) associated with carotenoid content in cowpea sprouts. The contents of carotenoids, including lutein, zeaxanthin, and ß-carotene in sprouts of 125 accessions were quantified via high-performance liquid chromatography. Significant variation existed in the profiles of the different carotenoids. Lutein was the most abundant (58 ± 12.8 mg/100 g), followed by zeaxanthin (14.7 ± 3.1 mg/100 g) and ß-carotene (13.2 ± 2.9 mg/100 g). A strong positive correlation was observed among the carotenoid compounds (r ≥ 0.87), indicating they can be improved concurrently. The accessions were distributed into three groups, following their carotenoid profiles, with accession C044 having the highest sprout carotenoid content in a single cluster. A total of 3120 genome-wide SNPs were tested for association analysis, which revealed that carotenoid biosynthesis in cowpea sprouts is a polygenic trait controlled by genes with additive and dominance effects. Seven loci were significantly associated with the variation in carotenoid content. The evidence of variation in carotenoid content and genomic regions controlling the trait creates an avenue for breeding cowpea varieties with enhanced sprouts carotenoid content.

Nutrient Dosing Framework for an Emission-Free Urban Hydroponic Production.

The urban hydroponic production system is accelerating industrialization in step with the potentials for reducing environmental impact. In contrast, establishing sustainable fertilizer dosing techniques still lags behind the pace of expansion of the system. The reproducibility of root-zone nutrient dynamics in the system is poorly understood, and managing nutrients has so far primarily relied on periodic discharge or dumping of highly concentrated nutrient solutions. Here, we assayed root-zone nutrient concentration changes using three possible nutrient dosing types. Three Brassica species were hydroponically cultivated in a controlled environment to apply the nutrient absorption and transpiration parameters to the simulation analysis. We found that nutrient dosing based on total ion concentration could provide more reproducible root-zone nutrient dynamics. Our findings highlight the nutrient absorption parameter domain in management practice. This simplifies conventional nutrient management into an optimization problem. Collectively, our framework can be extended to fertilizer-emission-free urban hydroponic production.

Exposure to Salinity and Light Spectra Regulates Glucosinolates, Phenolics, and Antioxidant Capacity of Brassica carinata L. Microgreens.

The effect of salt treatment on Brassica carinata (BC) microgreens grown under different light wavelengths on glucosinolates (GLs) and phenolic compounds were evaluated. Quantifiable GLs were identified using ultra-high performance-quadrupole time of flight mass spectrometry. Extracts' ability to activate antioxidant enzymes (superoxide dismutase (SOD) and catalase (CAT)) was evaluated on human colorectal carcinoma cells (HCT116). Furthermore, BC compounds' ability to activate expression of nuclear transcription factor-erythroid 2 related factor (Nrf2) and heme-oxygenase-1 (HO-1) proteins was examined using specific antibodies on HCT116 cells. Sinigrin (SIN) was the abundant GLs of the six compounds identified and its content together with total aliphatic GLs increased in saline conditions. Fluorescent (FL) and blue plus red (B1R1) lights were identified as stable cultivation conditions for microgreens, promoting biomass and glucobrassicin contents, whereas other identified individual and total indole GLs behaved differently in saline and non-saline environments. Blue light-emitting diodes and FL light in saline treatments mostly enhanced SIN, phenolics and antioxidant activities. The increased SOD and CAT activities render the BC microgreens suitable for lowering oxidative stress. Additionally, activation of Nrf2, and HO-1 protein expression by the GLs rich extracts, demonstrate their potential to treat and prevent oxidative stress and inflammatory disorders. Therefore, effective salt treatments and light exposure to BC microgreens present an opportunity for targeted regulation of growth and accumulation of bioactive metabolites.

Postharvest Drying Techniques Regulate Secondary Metabolites and Anti-Neuroinflammatory Activities of Ganoderma lucidum.

Ganoderma lucidum extract is a potent traditional remedy for curing various ailments. Drying is the most important postharvest step during the processing of Ganoderma lucidum. The drying process mainly involves heat (36 h at 60 °C) and freeze-drying (36 h at -80 °C). We investigated the effects of different postharvest drying protocols on the metabolites profiling of Ganoderma lucidum using GC-MS, followed by an investigation of the anti-neuroinflammatory potential in LPS-treated BV2 microglial cells. A total of 109 primary metabolites were detected from heat and freeze-dried samples. Primary metabolite profiling showed higher levels of amino acids (17.4%) and monosaccharides (8.8%) in the heat-dried extracts, whereas high levels of organic acids (64.1%) were present in the freeze-dried samples. The enzymatic activity, such as ATP-citrate synthase, pyruvate kinase, glyceraldehyde-3-phosphatase dehydrogenase, glutamine synthase, fructose-bisphosphate aldolase, and D-3-phosphoglycerate dehydrogenase, related to the reverse tricarboxylic acid cycle were significantly high in the heat-dried samples. We also observed a decreased phosphorylation level of the MAP kinase (Erk1/2, p38, and JNK) and NF-κB subunit p65 in the heat-dried samples of the BV2 microglia cells. The current study suggests that heat drying improves the production of ganoderic acids by the upregulation of TCA-related pathways, which, in turn, gives a significant reduction in the inflammatory response of LPS-induced BV2 cells. This may be attributed to the inhibition of NF-κB and MAP kinase signaling pathways in cells treated with heat-dried extracts.

Golden Gate Cloning-Compatible DNA Replicon/2A-Mediated Polycistronic Vectors for Plants.

The expression of multiple proteins and high-throughput vector assembly system are highly relevant in the field of plant genetic engineering and synthetic biology. Deployment of the self-cleaving 2A peptide that mediates polycistronic gene expression has been an effective strategy for multigene expression, as it minimizes issues in coordinated transgene regulation and trait staking in plants. However, efficient vector assembly systems optimized for 2A peptide-mediated polycistronic expression are currently unavailable. Furthermore, it is unclear whether protein expression levels are influenced by the transgene position in the polycistronic expression cassette. In this article, we present Golden Gate cloning-compatible modular systems allowing rapid and flexible construction of polycistronic expression vectors applicable for plants. The genetic modules comprised 2A peptides (T2A and P2A)-linked tricistron expression cassette and its acceptor backbones, named pGO-DV1 and pGO-DV2. While both acceptor backbones were binary T-DNA vectors, pGO-DV2 was specially designed to function as a DNA replicon enhancing gene expression levels. Using the Golden Gate cloning, a set of six tricistronic vectors was constructed, whereby three transgenes encoding fluorescent proteins (mCherry, eYFP, and eGFP) were combinatorially placed along the expression cassette in each of the binary vectors. Transient expression of the construct in tobacco leaves revealed that the expression levels of three fluorescent proteins were comparable each other regardless of the gene positions in the tricistronic expression cassette. pGO-DV2-based constructs were able to increase protein expression level by up to 71%, as compared to pGO-DV1-based constructs.

Transcriptome analysis of Panax ginseng response to high light stress.

BACKGROUND: Ginseng (Panax ginseng Meyer) is an essential source of pharmaceuticals and functional foods. Ginseng productivity has been compromised by high light (HL) stress, which is one of the major abiotic stresses during the ginseng cultivation period. The genetic improvement for HL tolerance in ginseng could be facilitated by analyzing its genetic and molecular characteristics associated with HL stress. METHODS: Genome-wide analysis of gene expression was performed under HL and recovery conditions in 1-year-old Korean ginseng (P. ginseng cv. Chunpoong) using the Illumina HiSeq platform. After de novo assembly of transcripts, we performed expression profiling and identified differentially expressed genes (DEGs). Furthermore, putative functions of identified DEGs were explored using Gene Ontology terms and Kyoto Encyclopedia of Genes and Genome pathway enrichment analysis. RESULTS: A total of 438 highly expressed DEGs in response to HL stress were identified and selected from 29,184 representative transcripts. Among the DEGs, 326 and 114 transcripts were upregulated and downregulated, respectively. Based on the functional analysis, most upregulated and a significant number of downregulated transcripts were related to stress responses and cellular metabolic processes, respectively. CONCLUSION: Transcriptome profiling could be a strategy to comprehensively elucidate the genetic and molecular mechanisms of HL tolerance and susceptibility. This study would provide a foundation for developing breeding and metabolic engineering strategies to improve the environmental stress tolerance of ginseng.

Largely enhanced bioethanol production through the combined use of lignin-modified sugarcane and xylose fermenting yeast strain.

The recalcitrant structure of lignocellulosic biomass is a major barrier in efficient biomass-to-ethanol bioconversion processes. The combination of feedstock engineering via modification in the lignin synthesis pathway of sugarcane and co-fermentation of xylose and glucose with a recombinant xylose utilizing yeast strain produced 148% more ethanol compared to that of the wild type biomass and control strain. The lignin reduced biomass led to a substantially increased release of fermentable sugars (glucose and xylose). The engineered yeast strain efficiently co-utilized glucose and xylose for fermentation, elevating ethanol yields. In this study, it was experimentally demonstrated that the combined efforts of engineering both feedstock and microorganisms largely enhances the bioconversion of lignocellulosic feedstock to bioethanol. This strategy will significantly improve the economic feasibility of lignocellulosic biofuels production.

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.

A standardized extract of Rhynchosia volubilis Lour. exerts a protective effect on benzalkonium chloride-induced mouse dry eye model.

ETHNOPHARMACOLOGICAL RELEVANCE: In contrast to other leguminous plants generally used as food, Rhynchosia volubilis Loureiro, a small soybean with a black seed coat, has been used as a traditional oriental remedy for various human diseases in Eastern Asia. In this study, we demonstrated the protective effect of R. volubilis against dry eye disease. AIM OF THE STUDY: We aimed to investigate whether a standardized ethanol extract of R. volubilis (EERV) can protect the cornea in a benzalkonium chloride (BAC)-induced mouse dry eye model. MATERIALS AND METHODS: Experimental dry eye was induced by the instillation of 0.2% BAC on mouse cornea. A standardized ethanol extract of R. volubilis (EERV) was orally administered following BAC treatment. The positive control group was treated with commercial eye drops. Fluorescein staining, tear break-up time (BUT), and hematoxylin and eosin staining were evaluated on the ocular surface. Squamous metaplasia and apoptosis in the corneal epithelial layer were detected by immunostaining. Furthermore, the protein expression of cytochrome c, Bcl-2, and Bax was determined. RESULTS: EERV treatment significantly improved fluorescein scoring, BUT, and smoothness in the cornea compared to the vehicle group. In addition, EERV inhibited squamous metaplasia and apoptosis in the cornea. The expression of cytochrome c and Bax was upregulated, while that of Bcl-2 was downregulated in the vehicle group compared with that in the control group. However, EERV treatment inhibited the expression of cytochrome c and Bax, while that of Bcl-2 was improved. CONCLUSION: Standardized EERV could be a beneficial candidate for the treatment of dry eye disease.

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.

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.

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.