Laccase is necessary and nonredundant with peroxidase for lignin polymerization during vascular development in Arabidopsis.

The evolution of lignin biosynthesis was critical in the transition of plants from an aquatic to an upright terrestrial lifestyle. Lignin is assembled by oxidative polymerization of two major monomers, coniferyl alcohol and sinapyl alcohol. Although two recently discovered laccases, LAC4 and LAC17, have been shown to play a role in lignin polymerization in Arabidopsis thaliana, disruption of both genes only leads to a relatively small change in lignin content and only under continuous illumination. Simultaneous disruption of LAC11 along with LAC4 and LAC17 causes severe plant growth arrest, narrower root diameter, indehiscent anthers, and vascular development arrest with lack of lignification. Genome-wide transcript analysis revealed that all the putative lignin peroxidase genes are expressed at normal levels or even higher in the laccase triple mutant, suggesting that lignin laccase activity is necessary and nonredundant with peroxidase activity for monolignol polymerization during plant vascular development. Interestingly, even though lignin deposition in roots is almost completely abolished in the lac11 lac4 lac17 triple mutant, the Casparian strip, which is lignified through the activity of peroxidase, is still functional. Phylogenetic analysis revealed that lignin laccase genes have no orthologs in lower plant species, suggesting that the monolignol laccase genes diverged after the evolution of seed plants.

Early lignin pathway enzymes and routes to chlorogenic acid in switchgrass (Panicum virgatum L.).

Studying lignin biosynthesis in Panicum virgatum (switchgrass) has provided a basis for generating plants with reduced lignin content and increased saccharification efficiency. Chlorogenic acid (CGA, caffeoyl quinate) is the major soluble phenolic compound in switchgrass, and the lignin and CGA biosynthetic pathways potentially share intermediates and enzymes. The enzyme hydroxycinnamoyl-CoA: quinate hydroxycinnamoyltransferase (HQT) is responsible for CGA biosynthesis in tobacco, tomato and globe artichoke, but there are no close orthologs of HQT in switchgrass or in other monocotyledonous plants with complete genome sequences. We examined available transcriptomic databases for genes encoding enzymes potentially involved in CGA biosynthesis in switchgrass. The protein products of two hydroxycinnamoyl-CoA shikimate/quinate hydroxycinnamoyltransferase (HCT) genes (PvHCT1a and PvHCT2a), closely related to lignin pathway HCTs from other species, were characterized biochemically and exhibited the expected HCT activity, preferring shikimic acid as acyl acceptor. We also characterized two switchgrass coumaroyl shikimate 3'-hydroxylase (C3'H) enzymes (PvC3'H1 and PvC3'H2); both of these cytochrome P450s had the capacity to hydroxylate 4-coumaroyl shikimate or 4-coumaroyl quinate to generate caffeoyl shikimate or CGA. Another switchgrass hydroxycinnamoyl transferase, PvHCT-Like1, is phylogenetically distant from HCTs or HQTs, but exhibits HQT activity, preferring quinic acid as acyl acceptor, and could therefore function in CGA biosynthesis. The biochemical features of the recombinant enzymes, the presence of the corresponding activities in plant protein extracts, and the expression patterns of the corresponding genes, suggest preferred routes to CGA in switchgrass.

Syringyl lignin biosynthesis is directly regulated by a secondary cell wall master switch.

Lignin is a major component of plant secondary cell walls and is derived from p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) monolignols. Among higher plants, S lignin is generally considered to be restricted to angiosperms, which contain the S lignin-specific cytochrome P450-dependent monooxygenase, ferulic acid/coniferaldehyde/coniferyl alcohol 5-hydoxylase (F5H). The transcription factor MYB58 directly regulates expression of monolignol pathway genes except for F5H. Here we show that F5H expression is directly regulated by the secondary cell wall master switch NST1/SND1, which is known to regulate expression of MYB58. Deletion of NST1 expression in Medicago truncatula leads to a loss of S lignin associated with a more than 25-fold reduction of F5H expression but only around a 2-fold reduction in expression of other lignin pathway genes. A detailed phylogenetic analysis showed that gymnosperms lack both F5H and orthologs of NST1/SND1. We propose that both F5H and NST1 appeared at a similar time after the divergence of angiosperms and gymnosperms, with F5H possibly originating as a component of a defense mechanism that was recruited to cell wall biosynthesis through the evolution of NST1-binding elements in its promoter.

Caldicellulosiruptor core and pangenomes reveal determinants for noncellulosomal thermophilic deconstruction of plant biomass.

Extremely thermophilic bacteria of the genus Caldicellulosiruptor utilize carbohydrate components of plant cell walls, including cellulose and hemicellulose, facilitated by a diverse set of glycoside hydrolases (GHs). From a biofuel perspective, this capability is crucial for deconstruction of plant biomass into fermentable sugars. While all species from the genus grow on xylan and acid-pretreated switchgrass, growth on crystalline cellulose is variable. The basis for this variability was examined using microbiological, genomic, and proteomic analyses of eight globally diverse Caldicellulosiruptor species. The open Caldicellulosiruptor pangenome (4,009 open reading frames [ORFs]) encodes 106 GHs, representing 43 GH families, but only 26 GHs from 17 families are included in the core (noncellulosic) genome (1,543 ORFs). Differentiating the strongly cellulolytic Caldicellulosiruptor species from the others is a specific genomic locus that encodes multidomain cellulases from GH families 9 and 48, which are associated with cellulose-binding modules. This locus also encodes a novel adhesin associated with type IV pili, which was identified in the exoproteome bound to crystalline cellulose. Taking into account the core genomes, pangenomes, and individual genomes, the ancestral Caldicellulosiruptor was likely cellulolytic and evolved, in some cases, into species that lost the ability to degrade crystalline cellulose while maintaining the capacity to hydrolyze amorphous cellulose and hemicellulose.

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.

Bioinformatics Analysis of Plant Cell Wall Evolution.

In the past hundreds of millions of years, from green algae to land plants, cell walls have developed into a highly complex structure that is essential for plant growth and survival. Plant cell wall diversity and evolution can be directly investigated by chemically profiling polysaccharides and lignins in the cell walls of diverse plants and algae. With the increasingly low cost and high throughput of DNA sequencing technologies, cell wall evolution can also be studied by bioinformatics analysis of the occurrence of cell wall synthesis-related enzymes in the genomes and transcriptomes of different species. This chapter presents a bioinformatics workflow running on a Linux platform to process genomic data for such gene occurrence analysis. As a case study, cellulose synthase (CesA) and CesA-like (Csl) protein families are mined for in two newly sequenced organisms: the charophyte green alga Klebsormidium flaccidum (renamed as Klebsormidium nitens) and the fern Lygodium japonicum.

Pinoresinol reductase 1 impacts lignin distribution during secondary cell wall biosynthesis in Arabidopsis.

Pinoresinol reductase (PrR) catalyzes the conversion of the lignan (-)-pinoresinol to (-)-lariciresinol in Arabidopsis thaliana, where it is encoded by two genes, PrR1 and PrR2, that appear to act redundantly. PrR1 is highly expressed in lignified inflorescence stem tissue, whereas PrR2 expression is barely detectable in stems. Co-expression analysis has indicated that PrR1 is co-expressed with many characterized genes involved in secondary cell wall biosynthesis, whereas PrR2 expression clusters with a different set of genes. The promoter of the PrR1 gene is regulated by the secondary cell wall related transcription factors SND1 and MYB46. The loss-of-function mutant of PrR1 shows, in addition to elevated levels of pinoresinol, significantly decreased lignin content and a slightly altered lignin structure with lower abundance of cinnamyl alcohol end groups. Stimulated Raman scattering (SRS) microscopy analysis indicated that the lignin content of the prr1-1 loss-of-function mutant is similar to that of wild-type plants in xylem cells, which exhibit a normal phenotype, but is reduced in the fiber cells. Together, these data suggest an association of the lignan biosynthetic enzyme encoded by PrR1 with secondary cell wall biosynthesis in fiber cells.

Self-assembled monolayers with different chemical group substrates for the study of MCF-7 breast cancer cell line behavior.

The interactions between cancer cells and the extracellular matrix (ECM) are important with respect to a number of cell behavoirs, yet remain unclear. In this study, self-assembled monolayers with different terminal chemical groups (hydroxyl (-OH), carboxyl (-COOH), animo (-NH2), mercapto (-SH), and methyl (-CH3)) were employed as substrates for the culture of MCF-7 cells to examine effects on cell behavior. Cell spreading was investigated by scanning electron microscopy, tallin expression by immunofluorescence, proliferation rate by counting cell numbers, cell cycle by flow cytometry, metabolism by high-performance liquid chromatography and cell migration by live cell imaging. Annexin V-FITC (fluorescein isothiocyanate) and JC-1 assays were performed to determine cell apoptosis and mitochondrial membrane potential, respectively. Our results demonstrate the varied behaviors of MCF-7 cells in response to different chemical groups. Specifically, NH2 and COOH terminal functional groups promote proliferation, the production of lactic acid and mobility of MCF-7 cells; SH and OH terminal groups enhance the expression and distribution of tallin but result in weak cell proliferation, metabolism, spreading and mobility. These results are meaningful for uncovering the interactions between the ECM and cancer cells; they are potentially useful for designing novel cancer treatment strategies.

Classification of 'Anaerocellum thermophilum' strain DSM 6725 as Caldicellulosiruptor bescii sp. nov.

The thermophilic, cellulolytic, anaerobic bacterium 'Anaerocellum thermophilum' strain Z-1320 was isolated from a hot spring almost two decades ago and deposited in the German Collection of Microorganisms and Cell Cultures (DSMZ) as DSM 6725. The organism was classified as representing a new genus, 'Anaerocellum', primarily on its growth physiology, cell-wall type and morphology. The results of recent physiological studies and of phylogenetic and genome sequence analyses of strain DSM 6725 of 'A. thermophilum' obtained from the DSMZ showed that its properties differed from those originally described for strain Z-1320. In particular, when compared with strain Z-1320, strain DSM 6725 grew at higher temperatures and had an expanded range of growth substrates. Moreover, the 16S rRNA gene sequence of strain DSM 6725 fell within the Caldicellulosiruptor clade. It is therefore suggested that 'Anaerocellum thermophilum' should be classified as a member of the genus Caldicellulosiruptor, for which the name Caldicellulosiruptor bescii sp. nov. is proposed (type strain DSM 6725(T)=ATCC BAA-1888(T)). C. bescii sp. nov. DSM 6725(T) is the most thermophilic cellulose-degrading organism known. The strain was able to grow up to 90 degrees C (pH 7.2) and degraded crystalline cellulose and xylan as well as untreated plant biomass, including potential bioenergy plants such as poplar and switchgrass.