Gene regulatory networks for lignin biosynthesis in switchgrass (Panicum virgatum).

Cell wall recalcitrance is the major challenge to improving saccharification efficiency in converting lignocellulose into biofuels. However, information regarding the transcriptional regulation of secondary cell wall biogenesis remains poor in switchgrass (Panicum virgatum), which has been selected as a biofuel crop in the United States. In this study, we present a combination of computational and experimental approaches to develop gene regulatory networks for lignin formation in switchgrass. To screen transcription factors (TFs) involved in lignin biosynthesis, we developed a modified method to perform co-expression network analysis using 14 lignin biosynthesis genes as bait (target) genes. The switchgrass lignin co-expression network was further extended by adding 14 TFs identified in this study, and seven TFs identified in previous studies, as bait genes. Six TFs (PvMYB58/63, PvMYB42/85, PvMYB4, PvWRKY12, PvSND2 and PvSWN2) were targeted to generate overexpressing and/or down-regulated transgenic switchgrass lines. The alteration of lignin content, cell wall composition and/or plant growth in the transgenic plants supported the role of the TFs in controlling secondary wall formation. RNA-seq analysis of four of the transgenic switchgrass lines revealed downstream target genes of the secondary wall-related TFs and crosstalk with other biological pathways. In vitro transactivation assays further confirmed the regulation of specific lignin pathway genes by four of the TFs. Our meta-analysis provides a hierarchical network of TFs and their potential target genes for future manipulation of secondary cell wall formation for lignin modification in switchgrass.

Transcriptome analysis of secondary cell wall development in Medicago truncatula.

BACKGROUND: Legumes are important to humans by providing food, feed and raw materials for industrial utilizations. Some legumes, such as alfalfa, are potential bioenergy crops due to their high biomass productivity. Global transcriptional profiling has been successfully used to identify genes and regulatory pathways in secondary cell wall thickening in Arabidopsis, but such transcriptome data is lacking in legumes. RESULTS: A systematic microarray assay and high through-put real time PCR analysis of secondary cell wall development were performed along stem maturation in Medicago truncatula. More than 11,000 genes were differentially expressed during stem maturation, and were categorized into 10 expression clusters. Among these, 279 transcription factor genes were correlated with lignin/cellulose biosynthesis, therefore representing putative regulators of secondary wall development. The b-ZIP, NAC, WRKY, C2H2 zinc finger (ZF), homeobox, and HSF gene families were over-represented. Gene co-expression network analysis was employed to identify transcription factors that may regulate the biosynthesis of lignin, cellulose and hemicellulose. As a complementary approach to microarray, real-time PCR analysis was used to characterize the expression of 1,045 transcription factors in the stem samples, and 64 of these were upregulated more than 5-fold during stem maturation. Reverse genetics characterization of a cellulose synthase gene in cluster 10 confirmed its function in xylem development. CONCLUSIONS: This study provides a useful transcriptome and expression resource for understanding cell wall development, which is pivotal to enhance biomass production in legumes.

The trans-acting short interfering RNA3 pathway and no apical meristem antagonistically regulate leaf margin development and lateral organ separation, as revealed by analysis of an argonaute7/lobed leaflet1 mutant in Medicago truncatula.

Leaf shape elaboration and organ separation are critical for plant morphogenesis. We characterized the developmental roles of lobed leaflet1 by analyzing a recessive mutant in the model legume Medicago truncatula. An ortholog of Arabidopsis thaliana argonaute7 (AGO7), Mt-AGO7/lobed leaflet1, is required for the biogenesis of a trans-acting short interfering RNA (ta-siRNA) to negatively regulate the expression of auxin response factors in M. truncatula. Loss of function in AGO7 results in pleiotropic phenotypes in different organs. The prominent phenotype of the ago7 mutant is lobed leaf margins and more widely spaced lateral organs, suggesting that the trans-acting siRNA3 (TAS3) pathway negatively regulates the formation of boundaries and the separation of lateral organs in M. truncatula. Genetic interaction analysis with the smooth leaf margin1 (slm1) mutant revealed that leaf margin formation is cooperatively regulated by the auxin/SLM1 (ortholog of Arabidopsis PIN-formed1) module, which influences the initiation of leaf margin teeth, and the TAS3 ta-siRNA pathway, which determines the degree of margin indentation. Further investigations showed that the TAS3 ta-siRNA pathway and no apical meristem (ortholog of Arabidopsis cup-shaped cotyledon) antagonistically regulate both leaf margin development and lateral organ separation, and the regulation is partially dependent on the auxin/SLM1 module.

A genomics approach to deciphering lignin biosynthesis in switchgrass.

It is necessary to overcome recalcitrance of the biomass to saccharification (sugar release) to make switchgrass (Panicum virgatum) economically viable as a feedstock for liquid biofuels. Lignin content correlates negatively with sugar release efficiency in switchgrass, but selecting the right gene candidates for engineering lignin biosynthesis in this tetraploid outcrossing species is not straightforward. To assist this endeavor, we have used an inducible switchgrass cell suspension system for studying lignin biosynthesis in response to exogenous brassinolide. By applying a combination of protein sequence phylogeny with whole-genome microarray analyses of induced cell cultures and developing stem internode sections, we have generated a list of candidate monolignol biosynthetic genes for switchgrass. Several genes that were strongly supported through our bioinformatics analysis as involved in lignin biosynthesis were confirmed by gene silencing studies, in which lignin levels were reduced as a result of targeting a single gene. However, candidate genes encoding enzymes involved in the early steps of the currently accepted monolignol biosynthesis pathway in dicots may have functionally redundant paralogues in switchgrass and therefore require further evaluation. This work provides a blueprint and resources for the systematic genome-wide study of the monolignol pathway in switchgrass, as well as other C4 monocot species.

A deep transcriptomic analysis of pod development in the vanilla orchid (Vanilla planifolia).

BACKGROUND: Pods of the vanilla orchid (Vanilla planifolia) accumulate large amounts of the flavor compound vanillin (3-methoxy, 4-hydroxy-benzaldehyde) as a glucoside during the later stages of their development. At earlier stages, the developing seeds within the pod synthesize a novel lignin polymer, catechyl (C) lignin, in their coats. Genomic resources for determining the biosynthetic routes to these compounds and other flavor components in V. planifolia are currently limited. RESULTS: Using next-generation sequencing technologies, we have generated very large gene sequence datasets from vanilla pods at different times of development, and representing different tissue types, including the seeds, hairs, placental and mesocarp tissues. This developmental series was chosen as being the most informative for interrogation of pathways of vanillin and C-lignin biosynthesis in the pod and seed, respectively. The combined 454/Illumina RNA-seq platforms provide both deep sequence coverage and high quality de novo transcriptome assembly for this non-model crop species. CONCLUSIONS: The annotated sequence data provide a foundation for understanding multiple aspects of the biochemistry and development of the vanilla bean, as exemplified by the identification of candidate genes involved in lignin biosynthesis. Our transcriptome data indicate that C-lignin formation in the seed coat involves coordinate expression of monolignol biosynthetic genes with the exception of those encoding the caffeoyl coenzyme A 3-O-methyltransferase for conversion of caffeoyl to feruloyl moieties. This database provides a general resource for further studies on this important flavor species.

Engineering of molecularly imprinted cavity within 3D covalent organic frameworks: An innovation for enhanced extraction and removal of microcystins.

The scarcity of selective adsorbents for efficient extraction and removal of microcystins (MCs) from complex samples greatly limits the precise detection and effective control of MCs. Three-dimensional covalent organic frameworks (3D COFs), characterized by their large specific surface areas and highly ordered rigid structure, are promising candidates, but suffer from lack of specific recognition. Herein, we design to engineer molecularly imprinted cavities within 3D COFs via molecularly imprinted technology, creating a novel adsorbent with exceptional selectivity, kinetics and capacity for the efficient extraction and removal of MCs. As proof-of-concept, a new CC bond-containing 3D COF, designated JNU-7, is designed and prepared for copolymerization with methacrylic acid, the pseudo template L-arginine and ethylene dimethacrylate to yield the JNU-7 based molecularly imprinted polymer (JNU-7-MIP). The JNU-7-MIP exhibits a great adsorption capacity (156 mg g-1) for L-arginine. Subsequently, the JNU-7-MIP based solid-phase extraction coupled with high performance liquid chromatography-mass spectrometry achieves low detection limit of 0.008 ng mL-1, wide linear range of 0.025-100 ng mL-1, high enrichment factor of 186, rapid extraction of 10 min, and good recoveries of 92.4%-106.5% for MC-LR. Moreover, the JNU-7-MIP can rapidly remove the MC-LR from 1 mg L-1 to levels (0.26-0.35 µg L-1) lower than the WHO recommended limit for drinking water (1 µg L-1). This work reveals the considerable potential of 3D COF based MIPs as promising adsorbents for the extraction and removal of contaminants in complex real samples.

Functional characterization of the switchgrass (Panicum virgatum) R2R3-MYB transcription factor PvMYB4 for improvement of lignocellulosic feedstocks.

• The major obstacle for bioenergy production from switchgrass biomass is the low saccharification efficiency caused by cell wall recalcitrance. Saccharification efficiency is negatively correlated with both lignin content and cell wall ester-linked p-coumarate: ferulate (p-CA : FA) ratio. In this study, we cloned and functionally characterized an R2R3-MYB transcription factor from switchgrass and evaluated its potential for developing lignocellulosic feedstocks. • The switchgrass PvMYB4 cDNAs were cloned and expressed in Escherichia coli, yeast, tobacco and switchgrass for functional characterization. Analyses included determination of phylogenetic relations, in situ hybridization, electrophoretic mobility shift assays to determine binding sites in target promoters, and protoplast transactivation assays to demonstrate domains active on target promoters. • PvMYB4 binds to the AC-I, AC-II and AC-III elements of monolignol pathway genes and down-regulates these genes in vivo. Ectopic overexpression of PvMYB4 in transgenic switchgrass resulted in reduced lignin content and ester-linked p-CA : FA ratio, reduced plant stature, increased tillering and an approx. threefold increase in sugar release efficiency from cell wall residues. • We describe an alternative strategy for reducing recalcitrance in switchgrass by manipulating the expression of a key transcription factor instead of a lignin biosynthetic gene. PvMYB4-OX transgenic switchgrass lines can be used as potential germplasm for improvement of lignocellulosic feedstocks and provide a platform for further understanding gene regulatory networks underlying switchgrass cell wall recalcitrance.

Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.).

• Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity.

Switchgrass (Panicum virgatum) possesses a divergent family of cinnamoyl CoA reductases with distinct biochemical properties.

The down-regulation of enzymes of the monolignol pathway results in reduced recalcitrance of biomass for lignocellulosic ethanol production. Cinnamoyl CoA reductase (CCR) catalyzes the first step of the phenylpropanoid pathway specifically dedicated to monolignol biosynthesis. However, plants contain multiple CCR-like genes, complicating the selection of lignin-specific targets. This study was undertaken to understand the complexity of the CCR gene family in tetraploid switchgrass (Panicum virgatum) and to determine the biochemical properties of the encoded proteins. Four switchgrass cDNAs (most with multiple variants) encoding putative CCRs were identified by phylogenetic analysis, heterologously expressed in Escherichia coli, and the corresponding enzymes were characterized biochemically. Two cDNAs, PvCCR1 and PvCCR2, encoded enzymes with CCR activity. They are phylogenetically distinct, differentially expressed, and the corresponding enzymes exhibited different biochemical properties with regard to substrate preference. PvCCR1 has higher specific activity and prefers feruloyl CoA as substrate, whereas PvCCR2 prefers caffeoyl and 4-coumaroyl CoAs. Allelic variants of each cDNA were detected, but the two most diverse variants of PvCCR1 encoded enzymes with similar catalytic activity. Based on its properties and expression pattern, PvCCR1 is probably associated with lignin biosynthesis during plant development (and is therefore a target for the engineering of improved biomass), whereas PvCCR2 may function in defense.

[Comparison of dentinal microcracks formation in severely curved root canal with different Ni-Ti instruments].

PURPOSE: This study was aimed to compare the incidence of dentinal microcracks produced by 3 kinds of Ni-Ti instruments during root canal procedures in severely curved canals. METHODS: Two hundred and forty extracted human molars with mesial roots of 25° to 40° curvatures were selected and divided into A, B, C group, with 80 teeth in each group according to root curvature of 25°-30°ï¼ˆexcluding 25°ï¼‰, 30°-35°,35°-40°. Each of them was prepared with K file to 15#. Then, each group was divided into 4 sub-groups (n=20), one was as control, the others were prepared with WaveOne, ProTaper Next and M3-Pro, respectively. After preparation, all roots were stained with 1% methylene blue for 24 hours. The roots were then sectioned at the most curved plane and 2mm below and above the most curved plane with alow-speed saw under cold water. Stereomicroscope was used to inspect dentinal microcracks and differences between each group were analyzed using Chi-square test with SPSS 20.0 software package. RESULTS: Microcracks were observed in the group of WaveOne, ProTaper Next and M3-Pro. WaveOne system induced more dentinal microcracks compared with ProTaper Next and M3-Pro system (P<0.05), and there was no significant difference between ProTaper Next and M3-Pro system (P>0.05). The number of dentinal microcracks in WaveOne, ProTaper Next and M3-Pro group increased with the increase of root curvature. Except Waveone in group A (25°-30°) and group C (35°-40°), the occurrence of dentinal microcracks in two groups had significant difference (P<0.05), there was no significant difference among other groups (P>0.05). CONCLUSIONS: Compared to WaveOne, ProTaper Next and M3-Pro are more suitable for severely curved canal preparation.

Overexpression of AtLOV1 in Switchgrass alters plant architecture, lignin content, and flowering time.

BACKGROUND: Switchgrass (Panicum virgatum L.) is a prime candidate crop for biofuel feedstock production in the United States. As it is a self-incompatible polyploid perennial species, breeding elite and stable switchgrass cultivars with traditional breeding methods is very challenging. Translational genomics may contribute significantly to the genetic improvement of switchgrass, especially for the incorporation of elite traits that are absent in natural switchgrass populations. METHODOLOGY/PRINCIPAL FINDINGS: In this study, we constitutively expressed an Arabidopsis NAC transcriptional factor gene, LONG VEGETATIVE PHASE ONE (AtLOV1), in switchgrass. Overexpression of AtLOV1 in switchgrass caused the plants to have a smaller leaf angle by changing the morphology and organization of epidermal cells in the leaf collar region. Also, overexpression of AtLOV1 altered the lignin content and the monolignol composition of cell walls, and caused delayed flowering time. Global gene-expression analysis of the transgenic plants revealed an array of responding genes with predicted functions in plant development, cell wall biosynthesis, and flowering. CONCLUSIONS/SIGNIFICANCE: To our knowledge, this is the first report of a single ectopically expressed transcription factor altering the leaf angle, cell wall composition, and flowering time of switchgrass, therefore demonstrating the potential advantage of translational genomics for the genetic improvement of this crop.