Identification of the function of a key gene NnHCT1 in lignin synthesis in petioles of Nelumbo nucifera.

Leaf petiole or stem strength is an important agronomic trait affecting the growth of underground organs as a channel for material exchange and plays a vital role in the quality and yield of crops and vegetables. There are two different types of petioles in lotus, floating leaf petioles and vertical leaf petioles; however, the internal difference mechanism between these petioles is unclear. In this study, we investigated the differences between the initial vertical leaf petioles and the initial floating leaf petioles based on RNA sequencing (RNA-seq), and >2858 differentially expressed genes were annotated. These genes were chiefly enriched in phenylpropanoid biosynthesis, which is the source of the lignin and cellulose in petioles and stems. Lignin biology-related gene NnHCT1 was identified, and subsequent biological function validation demonstrated that the transient overexpression of NnHCT1 significantly increased the lignin and cellulose contents in lotus petioles and tobacco leaves. In contrast, silencing NnHCT1 through virus-induced gene silencing significantly reduced petiole lignin synthesis. Additionally, differentially up-regulated MYB family transcription factors were identified using RNA-seq. Yeast-one-hybrid and dual-luciferase reporter assays demonstrated that MYB4 could bind to the NnHCT1 promoter and up-regulate NnHCT1 expression. These findings demonstrate the significant potential of NnHCT1 to enhance lignin synthesis, thereby improving stem or petiole resistance to stunting and explaining the need for the study of differential petiole relationships in plants.

VAMP726 from maize and Arabidopsis confers pollen resistance to heat and UV radiation by influencing lignin content of sporopollenin.

Sporopollenin in the pollen cell wall protects male gametophytes from stresses. Phenylpropanoid derivatives, including guaiacyl (G) lignin units, are known to be structural components of sporopollenin, but the exact composition of sporopollenin remains to be fully resolved. We analyzed the phenylpropanoid derivatives in sporopollenin from maize and Arabidopsis by thioacidolysis coupled with nuclear magnetic resonance (NMR) and gas chromatography-mass spectrometry (GC-MS). The NMR and GC-MS results confirmed the presence of p-hydroxyphenyl (H), G, and syringyl (S) lignin units in sporopollenin from maize and Arabidopsis. Strikingly, H units account for the majority of lignin monomers in sporopollenin from these species. We next performed a genome-wide association study to explore the genetic basis of maize sporopollenin composition and identified a vesicle-associated membrane protein (ZmVAMP726) that is strongly associated with lignin monomer composition of maize sporopollenin. Genetic manipulation of VAMP726 affected not only lignin monomer composition in sporopollenin but also pollen resistance to heat and UV radiation in maize and Arabidopsis, indicating that VAMP726 is functionally conserved in monocot and dicot plants. Our work provides new insight into the lignin monomers that serve as structural components of sporopollenin and characterizes VAMP726, which affects sporopollenin composition and stress resistance in pollen.

Fusion gene 4CL-CCR promotes lignification in tobacco suspension cells.

KEY MESSAGE: The fusion gene 4CL-CCR promotes lignification and activates lignin-related MYB expression in tobacco but inhibits auxin-related gene expression and hinders the auxin absorption of cells. Given the importance of lignin polymers in plant growth and their industrial value, it is necessary to investigate how plants synthesize monolignols and regulate the level of lignin in cell walls. In our previous study, expression of the Populus tomentosa fusion gene 4CL-CCR significantly promoted the production of 4-hydroxycinnamyl alcohols. However, the function of 4CL-CCR in organisms remains poorly understood. In this study, the fusion gene 4CL-CCR was heterologously expressed in tobacco suspension cells. We found that the transgenic suspension cells exhibited lignification earlier. Furthermore, 4CL-CCR significantly reduced the content of phenolic acids and increased the content of aldehydes in the medium, which led to an increase in lignin deposition. Moreover, transcriptome results showed that the genes related to lignin synthesis, such as PAL, 4CL, CCoAOMT and CAD, were significantly upregulated in the 4CL-CCR group. The expression of genes related to auxin, such as ARF3, ARF5 and ARF6, was significantly downregulated. The downregulation of auxin affected the expression of transcription factor MYBs. We hypothesize that the upregulated genes MYB306 and MYB315 are involved in the regulation of cell morphogenesis and lignin biosynthesis and eventually enhance lignification in tobacco suspension cells. Our findings provide insight into the function of 4CL-CCR in lignification and how secondary cell walls are formed in plants.

Characterization of plant laccase genes and their functions.

Laccase is a copper-containing polyphenol oxidase found in different organisms. The multigene family that encodes laccases is widely distributed in plant genomes. Plant laccases oxidize monolignols to produce lignin which is important for plant growth and stress responses. Industrial applications of fungal and bacterial laccases are extensively explored and addressed. Recently many studies have focused on the significance of plant laccase, particularly in crop yield, and its functions in different environmental conditions. This review summarizes the transcriptional and posttranscriptional regulation of plant laccase genes and their functions in plant growth and development. It especially describes the responses of laccase genes to various stresses and their contributions to plant biotic and abiotic stress resistance. In-depth explanations and scientific advances will serve as foundations for research into plant laccase genes' function, mechanism, and possible applications.

Acibenzolar-S-methyl activates calcium signalling to mediate lignin synthesis in the exocarp of Docteur Jules Guyot pears.

'Docteur Jules Guyot' pears were immersed in acibenzolar-S-methyl (ASM) and 0.01 mol L-1 ethyl glycol tetra acetic acid (EGTA) to investigate the changes of Ca2+ receptor proteins and phenylpropanoid pathway. Results showed that ASM treatment increased the activities of phenylalanine ammonia-lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate coenzyme A ligase (4CL), polyphenol oxidase (PPO), and cinnamyl alcohol dehydrogenase (CAD) in the exocarp of pears, whereas EGTA pre-treatment inhibited the activities of these enzymes. ASM treatment also enhanced the transcription of PcPAL, PcC4H, Pc4CL, PcC3H, PcCOMT, PcCCoAOMT, PcCCR, PcPOD, PcCDPK1, PcCDPK2, PcCDPK5, PcCDPK11, PcCDPK13, PcCBL1, PcCBL9, PcCIPK14, and PcCML27 in pears. EGTA + ASM treatments inhibited the transcription of PcPAL, PcC4H, Pc4CL, PcC3H, PcCCR, PcF5H, PcCAD, PcCDPK11, PcCDPK26, PcCDPK32, PcCBL1, PcCIPK14, PcCIPK23, and PcCaM in the fruit. All these results indicated that ASM induced the gene expressions of Ca2+ receptor proteins, the key enzyme activities and gene expressions in phenylpropanoid pathway; Ca2+ mediated phenylpropane metabolism in pears after ASM treatment.

Insights into the Molecular Regulation of Lignin Content in Triploid Poplar Leaves.

After polyploidization, plants usually undergo some morphological and physiological changes, including the lignin content of polyploids usually becoming lower than that of diploids. However, the regulatory mechanism of the variation of lignin content in polyploid plants remains unclear. Therefore, in this research, we used full-sib poplar triploids and diploids to explore the molecular regulatory basis of lignin content in poplar triploid leaves through the determination of lignin content, the observation of xylem cells, and transcriptome sequencing. The results showed that the lignin content of triploid leaves was significantly lower than that of diploid leaves. The xylem cells of triploid leaves were significantly larger than those of diploids. Transcriptome sequencing data show that most lignin biosynthesis genes were significantly downregulated, and genes related to cell growth were mostly upregulated in triploid leaves compared with diploid leaves. In addition, co-expression network analysis showed that several transcription factors might be involved in the regulation of lignin biosynthesis. Consequently, the altered expression of genes related to lignin might lead to the reduced lignin content in triploids. These results provide a theoretical basis for further exploring the molecular mechanism of the variation of polyploid lignin content and the utilization of polyploid lignocellulosic resources.

Identification of a novel laccase gene EuLAC1 and its potential resistance against Botrytis cinerea.

In this study, a novel laccase gene, EuLAC1, was cloned from Eucommia ulmoides Oliver (E. ulmoides). An overexpression vector harboring the EuLAC1 was constructed and introduced into the tobacco (Nicotiana tabacum cv. Xanthi). The laccase activity, resistance to Botrytis cinerea (B. cinerea) and lignin level in wild-type and transgenic plants were thereafter investigated. Interestingly, the transgenic tobacco displayed a significantly higher laccase activity and resistance to gray mold as compared to the wild-type tobacco. Additionally, the lignin contents in the leaves and stems of the transgenic tobacco were significantly higher in comparison to the wild-type tobacco. Scanning electron microscopy was used to observe the cross sections of wild-type and transgenic tobacco stems and it was noted that the cell wall near the xylem catheter of the transgenic tobacco was substantially thicker and the outline clearer than that of the wild-type. Thus, the EuLAC1 gene can significantly increase laccase activity and lignin content in tobacco, leading to an increase in the physical defenses, thereby increasing tobacco resistance to gray mold.

Molecular cloning and characterization of two distinct caffeoyl CoA O-methyltransferases (CCoAOMTs) from the liverwort Marchantia paleacea.

Caffeoyl CoA O-methyltransferases (CCoAOMTs) catalyze the transfer of a methyl group from S-adenosylmethionine to a hydroxyl moiety of caffeoyl-CoA as part of the lignin biosynthetic pathway. CCoAOMT-like proteins also catalyze to a variety of flavonoids, coumarins, and phenylpropanoids. Several CCoAOMTs that prefer flavonoids as substrates have been characterized from liverworts. Here, we cloned two CCoAOMT genes, MpalOMT2 and MpalOMT3, from the liverwort Marchantia paleacea. MpalOMT3 has a second ATG codon downstream and the truncated version that lacks 11 amino acids was named MpalOMT3-Tr. Phylogenetic analysis placed MpalOMT3 at the root of the clade with true CCoAOMTs from vascular plants and placed MpalOMT2 between the CCoAOMT and CCoAOMT-like proteins. Recombinant OMTs methylated caffeoyl CoA, phenylpropanoids, and flavonoids containing two or three vicinal hydroxyl groups. MpalOMT3 showed higher catalytic activity for phenylpropanoids than MpalOMT2, but MpalOMT2 showed more promiscuous towards eriodictyol and myricetin. The lignin content in Arabidopsis thaliana stems increased with constitutive heterologous expression of MpalOMT3-Tr, but not MpalOMT2. Subcellular localization experiments indicated that the N-terminus of MpalOMT3 probably served as a chloroplast transit peptide and inhibited its enzymatic activity. Combining the phylogenetic analysis and functional characterization, we conclude that the liverwort M. paleacea harbors true CCoAOMT and CCoAOMT-like genes.

Homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of transgenic tobacco.

Flavonoids and lignin consist of a large number of secondarymetabolites which are derived from the phenylpropanoid pathway, and they act as a significant role in plant growth, development, and stress response. However, few reports have documented that how different subbranches of phenylpropanoid metablolic pathway mutually interact. In Arabidopsis, AtCPC (AtCAPRICE) is known to play a negative role in anthocyanin accumulation. Nonetheless, whether AtCPC could control the biosynthesis of lignin is largely unknown. Additionally, whether the RrFLS and RrANR, flavonol synthase and anthocyanidin reductase, from Rosa rugosa regulate different branches of phenylpropanoid pathway is unclear. Here, we performed a series of transgenic experiments with short life cycle tobacco and RNA-Seq analysis. Finally, a series of assays related to biological, physiological, and phenotypic characteristics were undertaken. Our results indicated that ectopic expression of AtCPC in tobacco not only decreased the flavonoid compound accumulation, but also up-regulated several lignin biosynthetic genes, and significantly increased the accumulation of lignin. Our results also revealed that although they respectively improved the flavonol and proanthocyanidin contents, the overexpression of RrFLS and RrANR plays positive roles in lignin biosynthesis in transgenic tobacco plants. Our findings provide a novel insight into the mechanism underlying homeostatic regulation of flavonoid and lignin biosynthesis in phenylpropanoid pathway of plants.

Exogenous chalcone synthase expression in developing poplar xylem incorporates naringenin into lignins.

Lignin, a polyphenolic polymer, is a major chemical constituent of the cell walls of terrestrial plants. The biosynthesis of lignin is a highly plastic process, as highlighted by an increasing number of noncanonical monomers that have been successfully identified in an array of plants. Here, we engineered hybrid poplar (Populus alba x grandidentata) to express chalcone synthase 3 (MdCHS3) derived from apple (Malus domestica) in lignifying xylem. Transgenic trees displayed an accumulation of the flavonoid naringenin in xylem methanolic extracts not inherently observed in wild-type trees. Nuclear magnetic resonance analysis revealed the presence of naringenin in the extract-free, cellulase-treated xylem lignin of MdCHS3-poplar, indicating the incorporation of this flavonoid-derived compound into poplar secondary cell wall lignins. The transgenic trees also displayed lower total cell wall lignin content and increased cell wall carbohydrate content and performed significantly better in limited saccharification assays than their wild-type counterparts.

Nucleoside 5'-Phosphoramidates Control the Phenylpropanoid Pathway in Vitis vinifera Suspension-Cultured Cells.

It is known that cells contain various uncommon nucleotides such as dinucleoside polyphosphates (NpnN's) and adenosine 5'-phosphoramidate (NH2-pA) belonging to nucleoside 5'-phosphoramidates (NH2-pNs). Their cellular levels are enzymatically controlled. Some of them are accumulated in cells under stress, and therefore, they could act as signal molecules. Our previous research carried out in Arabidopsis thaliana and grape (Vitis vinifera) showed that NpnN's induced the expression of genes in the phenylpropanoid pathway and favored the accumulation of their products, which protect plants against stress. Moreover, we found that NH2-pA could play a signaling role in Arabidopsis seedlings. Data presented in this paper show that exogenously applied purine (NH2-pA, NH2-pG) and pyrimidine (NH2-pU, NH2-pC) nucleoside 5'-phosphoramidates can modify the expression of genes that control the biosynthesis of both stilbenes and lignin in Vitis vinifera cv. Monastrell suspension-cultured cells. We investigated the expression of genes encoding for phenylalanine ammonia-lyase (PAL1), cinnamate-4-hydroxylase (C4H1), 4-coumarate:coenzyme A ligase (4CL1), chalcone synthase (CHS1), stilbene synthase (STS1), cinnamoyl-coenzyme A:NADP oxidoreductase (CCR2), and cinnamyl alcohol dehydrogenase (CAD1). Each of the tested NH2-pNs also induced the expression of the trans-resveratrol cell membrane transporter VvABCG44 gene and caused the accumulation of trans-resveratrol and trans-piceid in grape cells as well as in the culture medium. NH2-pC, however, evoked the most effective induction of phenylpropanoid pathway genes such as PAL1, C4H1, 4CL1, and STS1. Moreover, this nucleotide also induced at short times the accumulation of N-benzoylputrescine (BenPut), one of the phenylamides that are derivatives of phenylpropanoid and polyamines. The investigated nucleotides did not change either the lignin content or the cell dry weight, nor did they affect the cell viability throughout the experiment. The results suggest that nucleoside 5'-phosphoramidates could be considered as new signaling molecules.

Application of CRISPR/Cas9 Tools for Genome Editing in the White-Rot Fungus Dichomitus squalens.

Dichomitus squalens is an emerging reference species that can be used to investigate white-rot fungal plant biomass degradation, as it has flexible physiology to utilize different types of biomass as sources of carbon and energy. Recent comparative (post-) genomic studies on D. squalens resulted in an increasingly detailed knowledge of the genes and enzymes involved in the lignocellulose breakdown in this fungus and showed a complex transcriptional response in the presence of lignocellulose-derived compounds. To fully utilize this increasing amount of data, efficient and reliable genetic manipulation tools are needed, e.g., to characterize the function of certain proteins in vivo and facilitate the construction of strains with enhanced lignocellulolytic capabilities. However, precise genome alterations are often very difficult in wild-type basidiomycetes partially due to extremely low frequencies of homology directed recombination (HDR) and limited availability of selectable markers. To overcome these obstacles, we assessed various Cas9-single guide RNA (sgRNA) ribonucleoprotein (RNP) -based strategies for selectable homology and non-homologous end joining (NHEJ) -based gene editing in D. squalens. We also showed an induction of HDR-based genetic modifications by using single-stranded oligodeoxynucleotides (ssODNs) in a basidiomycete fungus for the first time. This paper provides directions for the application of targeted CRISPR/Cas9-based genome editing in D. squalens and other wild-type (basidiomycete) fungi.

Hevea brasiliensis coniferaldehyde-5-hydroxylase (HbCAld5H) regulates xylogenesis, structure and lignin chemistry of xylem cell wall in Nicotiana tabacum.

KEY MESSAGE: The HbCAld5H1 gene cloned from Hevea brasiliensis regulates the cambial activity, xylem differentiation, syringyl-guaiacyl ratio, secondary wall structure, lignification pattern and xylan distribution in xylem fibres of transgenic tobacco plants. Molecular characterization of lignin biosynthesis gene coniferaldehyde-5-hydroxylase (CAld5H) from Hevea brasiliensis and its functional validation was performed. Both sense and antisense constructs of HbCAld5H1 gene were introduced into tobacco through Agrobacterium-mediated genetic transformation for over expression and down-regulation of this key enzyme to understand its role affecting structural and cell wall chemistry. The anatomical studies of transgenic tobacco plants revealed the increase of cambial activity leading to xylogenesis in sense lines and considerable reduction in antisense lines. The ultra-structural studies showed that the thickness of secondary wall (S2 layer) of fibre had been decreased with non-homogenous lignin distribution in antisense lines, while sense lines showed an increase in S2 layer thickness. Maule color reaction revealed that syringyl lignin distribution in the xylem elements was increased in sense and decreased in antisense lines. The immunoelectron microscopy revealed a reduction in LM 10 and LM 11 labelling in the secondary wall of antisense tobacco lines. Biochemical studies showed a radical increase in syringyl lignin in sense lines without any significant change in total lignin content, while S/G ratio decreased considerably in antisense lines. Our results suggest that CAld5H gene plays an important role in xylogenesis stages such as cambial cell division, secondary wall thickness, xylan and syringyl lignin distribution in tobacco. Therefore, CAld5H gene could be considered as a promising target for lignin modification essential for timber quality improvement in rubber.

MicroRNA397b negatively regulates resistance of Malus hupehensis to Botryosphaeria dothidea by modulating MhLAC7 involved in lignin biosynthesis.

MicroRNA (miRNA)-mediated post-transcriptional regulation plays a vital role in the response of plants to pathogens. Although the microRNA397 family has been implicated in physiological processes as an important regulator, little is known about its function in the resistance of plants to pathogens. Here, Malus hupehensis miR397, which was induced by Botryosphaeria dothidea infection, was identified to directly target M. hupehensis Laccase7 (MhLAC7). The expression analysis of mature Mh-miR397 and MhLAC7 revealed their partly opposite expression patterns. The coexpression of Mh-miR397b in MhLAC7 overexpressing Nicotiana benthamiana suppressed the accumulation of exogenous MhLAC7 and endogenous NbLAC7, which led to decreased lignin content and reduced plant resistance to Botrytis cinerea. As reflected by increasing disease severity and pathogen growth, overexpression of miR397b in both the resistant M. hupehensis and susceptible M. domestica 'Gala' resulted in an increased sensitivity to B. dothidea infection, owing to reduced LAC7 expression and lignin content; however, the inhibition of miR397 had opposite effects. MicroRNA397 functions as a negative regulator in the resistance of Malus to B. dothidea by modulating the LAC7 expression and lignin biosynthesis.

Functional Characteristics of Caffeoyl Shikimate Esterase in Larix Kaempferi and Monolignol Biosynthesis in Gymnosperms.

Caffeoyl shikimate esterase (CSE) has been reported to be involved in lignin biosynthesis; however, studies of CSE in gymnosperms are lacking. In this study, CSE was successfully cloned from Larix kaempferi (LkCSE) based on Larix laricina transcriptome screening. LkCSE was likely to have catalytic activity based on homologous sequence alignment and phylogenetic analyses of CSEs from different species. In vitro assays with the recombinant enzyme validated the catalytic activity of LkCSE, indicating its function in converting caffeoyl shikimate into caffeate and shikimate. Additionally, the optimum reaction pH and temperature of LkCSE were determined to be 6.0 and 30 °C, respectively. The values of Km and Vmax of CSE for caffeoyl shikimate were 98.11 µM and 14.44 nM min-1, respectively. Moreover, LkCSE was observed to have tissue expression specificity and was abundantly expressed in stems and leaves, especially stems, which was 50 times higher than the expression levels of roots. Lastly, translational fusion assays using LkCSE fused with green fluorescent proteins (GFP) in tobacco leaves indicated that LkCSE was localized in the plasma membrane and endoplasmic reticulum (ER). These results revealed that CSE clearly functions in gymnosperms and it is possible for LkCSE to interact with other ER-resident proteins and regulate mass flux in the monolignol biosynthesis pathway.

Discovery of novel enzyme genes involved in the conversion of an arylglycerol-ß-aryl ether metabolite and their use in generating a metabolic pathway for lignin valorization.

Microbial conversions known as "biological funneling" have attracted attention for their ability to upgrade heterogeneous mixtures of low-molecular-weight aromatic compounds obtained by chemical lignin depolymerization. ß-hydroxypropiovanillone (HPV) and its analogs can be obtained by chemoselective catalytic oxidation of lignin using 2,3-dichloro-5,6-dicyano-1,4-benzoquinone/tert-butyl nitrite/O2, followed by cleavage of arylglycerol-ß-aryl ether with zinc. Sphingobium sp. strain SYK-6 can degrade HPV generated by the catabolism of arylglycerol-ß-aryl ether through 2-pyrone-4,6-dicarboxylate (PDC), a promising platform chemical. Therefore, production of PDC from HPV can be achieved using the HPV catabolic pathway. However, the pathway and genes involved in the catabolism of vanilloyl acetic acid (VAA) generated during HPV catabolism have not been investigated. In the present study, we isolated SLG_24960 (vceA), which encodes an enzyme that converts VAA into a coenzyme A (CoA) derivative of vanillate (vanilloyl-CoA) from SYK-6, by shotgun cloning. The analysis of a vceA mutant indicated that this gene is not required for VAA conversion in vivo, but it encodes a major enzyme catalyzing CoA-dependent VAA conversion in vitro. We also identified SLG_12450 (vceB), whose product can convert vanilloyl-CoA to vanillate. Enzyme genes besides vceA and vceB, which are necessary for the conversions of HPV to VAA and of vanillate to PDC, were introduced and expressed in Pseudomonas putida. The resulting engineered strain completely converted 1  mM HPV into PDC after 24  h. Our results suggest that the enzyme genes that are not required for the catabolic pathway in microorganisms but can be used for the conversion of target substrates are buried in microbial genomes. These genes are, thus, useful for designing metabolic pathways to produce value-added metabolites.

Alteration of S-adenosylhomocysteine levels affects lignin biosynthesis in switchgrass.

Methionine (Met) synthesized from aspartate is a fundamental amino acid needed to produce S-adenosylmethionine (SAM) that is an important cofactor for the methylation of monolignols. As a competitive inhibitor of SAM-dependent methylation, the effect of S-adenosylhomocysteine (SAH) on lignin biosynthesis, however, is still largely unknown in plants. Expression levels of Cystathionine γ-synthase (PvCGS) and S-adenosylhomocysteine hydrolase 1 (PvSAHH1) were down-regulated by RNAi technology, respectively, in switchgrass, a dual-purpose forage and biofuel crop. The transgenic switchgrass lines were subjected to studying the impact of SAH on lignin biosynthesis. Our results showed that down-regulation of PvCGS in switchgrass altered the accumulation of aspartate-derived and aromatic amino acids, reduced the content of SAH, enhanced lignin biosynthesis and stunted plant growth. In contrast, down-regulation of PvSAHH1 raised SAH levels in switchgrass, impaired the biosynthesis of both guaiacyl and syringyl lignins and therefore significantly increased saccharification efficiency of cell walls. This work indicates that SAH plays a crucial role in monolignol methylation in switchgrass. Genetic regulation of either PvCGS or PvSAHH1 expression in switchgrass can change intracellular SAH contents and SAM to SAH ratios and therefore affect lignin biosynthesis. Thus, our study suggests that genes involved in Met metabolism are of interest as new valuable targets for cell wall bioengineering in future.

Optimization of RT-PCR reactions in studies with genes of lignin biosynthetic route in Saccharum spontaneum

ABSTRACT Saccharum spontaneum has been used for the development of energy cane a crop aimed to be used for the production of second-generation ethanol, or lignocellulosic ethanol. Lignin is a main challenge in the conversion of cell wall sugars into ethanol. In our studies to isolate the genes the lignin biosynthesis in S. spontaneum we have had great difficulty in RT-PCR reactions. Thus, we evaluated the effectiveness of different additives in the amplification of these genes. While COMT and CCoAOMT genes did not need any additives for other genes there was no amplification (HCT, F5H, 4CL and CCR) or the yield was very low (CAD and C4H). The application of supplementary cDNA was enough to overcome the non-specificity and low yield for C4H and C3H, while the addition of 0.04% BSA + 2% formamide was effective to amplify 4CL, CCR, F5H and CCR. HCT was amplified only by addition of 0.04% BSA + 2% formamide + 0.1 M trehalose and amplification of PAL was possible with addition of 2% of DMSO. Besides optimization of expression assays, the results show that additives can act independently or synergistically.

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.

Selection Signatures in Four Lignin Genes from Switchgrass Populations Divergently Selected for In Vitro Dry Matter Digestibility.

Switchgrass is undergoing development as a dedicated cellulosic bioenergy crop. Fermentation of lignocellulosic biomass to ethanol in a bioenergy system or to volatile fatty acids in a livestock production system is strongly and negatively influenced by lignification of cell walls. This study detects specific loci that exhibit selection signatures across switchgrass breeding populations that differ in in vitro dry matter digestibility (IVDMD), ethanol yield, and lignin concentration. Allele frequency changes in candidate genes were used to detect loci under selection. Out of the 183 polymorphisms identified in the four candidate genes, twenty-five loci in the intron regions and four loci in coding regions were found to display a selection signature. All loci in the coding regions are synonymous substitutions. Selection in both directions were observed on polymorphisms that appeared to be under selection. Genetic diversity and linkage disequilibrium within the candidate genes were low. The recurrent divergent selection caused excessive moderate allele frequencies in the cycle 3 reduced lignin population as compared to the base population. This study provides valuable insight on genetic changes occurring in short-term selection in the polyploid populations, and discovered potential markers for breeding switchgrass with improved biomass quality.