text:
- Research article
- Open Access
Functional characterization of cinnamyl alcohol dehydrogenase and caffeic acid O-methyltransferase in Brachypodium distachyon
© Trabucco et al.; licensee BioMed Central Ltd. 2013
- Received: 20 May 2013
- Accepted: 11 June 2013
- Published: 31 July 2013
Lignin is a significant barrier in the conversion of plant biomass to bioethanol. Cinnamyl alcohol dehydrogenase (CAD) and caffeic acid O-methyltransferase (COMT) catalyze key steps in the pathway of lignin monomer biosynthesis. Brown midrib mutants in Zea mays and Sorghum bicolor with impaired CAD or COMT activity have attracted considerable agronomic interest for their altered lignin composition and improved digestibility. Here, we identified and functionally characterized candidate genes encoding CAD and COMT enzymes in the grass model species Brachypodium distachyon with the aim of improving crops for efficient biofuel production.
We developed transgenic plants overexpressing artificial microRNA designed to silence BdCAD1 or BdCOMT4. Both transgenes caused altered flowering time and increased stem count and weight. Downregulation of BdCAD1 caused a leaf brown midrib phenotype, the first time this phenotype has been observed in a C3 plant. While acetyl bromide soluble lignin measurements were equivalent in BdCAD1 downregulated and control plants, histochemical staining and thioacidolysis indicated a decrease in lignin syringyl units and reduced syringyl/guaiacyl ratio in the transgenic plants. BdCOMT4 downregulated plants exhibited a reduction in total lignin content and decreased Maule staining of syringyl units in stem. Ethanol yield by microbial fermentation was enhanced in amiR-cad1-8 plants.
These results have elucidated two key genes in the lignin biosynthetic pathway in B. distachyon that, when perturbed, may result in greater stem biomass yield and bioconversion efficiency.
- Lignin
- Lignin Biosynthesis
- Cinnamyl Alcohol Dehydrogenase
- Empty Vector Control
- COMT Gene
Plant biomass offers a sustainable, low-carbon-emitting source of biofuel feedstock to potentially alleviate both environmental and economic disadvantages of fossil fuel usage [1]. Fossil fuel combustion has resulted in elevated atmospheric CO2 levels that continue to rise, threatening air quality, wildlife habitat, and human health [2, 3]. A viable, cost-effective alternative is to replace and/or blend gasoline and diesel fuels with biofuel. The efficiency of existing approaches to generating biofuels can be improved through research into plant feedstock attributes, namely biomass yield and recalcitrance to conversion [4].
Plant cell walls are mostly comprised of cellulose, hemicellulose, and lignin. The composition and interaction among these three constituents largely dictate the amenability of a plant feedstock for conversion to simple sugars and then to biofuels. Cellulose and hemicellulose are important raw materials, for they are an abundant source of monomeric sugars that can be hydrolyzed by enzymes and fermented by microbes, while lignin cannot [5]. Due to the recalcitrance of plant cell walls, a strong acid, heat, or other chemical pretreatment is required to first break up cell wall fibers and access the polysaccharides for enzyme treatment. Lignin in particular can impede the pretreatment process by adhering to the hydrolytic enzymes used to saccharify cellulose and other polysaccharides, affecting their ability to work efficiently. Additionally, some lignin degradation products can inhibit subsequent fermentation steps by releasing aromatic compounds that inhibit enzymes used for converting sugars to ethanol [6]. Saccharification of Medicago sativa expressing antisense transcripts designed to silence lignin biosynthesis genes showed an inverse relationship between lignin content and sugar yield, revealing that lignin is indeed a significant obstacle to obtaining high yields of cell wall sugars [7]. Consequently, there has been strong interest in the engineering of bioenergy crops, namely grasses, that are more amenable to feedstock conversion, notably by the manipulation of lignin biosynthesis, in order to improve efficiency of the pretreatment process and obtain maximum fuel yield [8].
Lignin is a complex phenolic polymer, and despite its recalcitrant properties, it is important in providing structural support, hydrophobicity, and protection against pathogens [9]. It is comprised of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) units derived from the monolignol precursors p-coumaryl, coniferyl, and sinapyl alcohols, respectively. A series of ten enzymes is involved in the phenylpropanoid pathway for monolignol biosynthesis [9]. The eudicot Arabidopsis thaliana has served as a valuable tool to understand lignin biosynthesis [9]. However, there are major plant cell wall structural differences between eudicots and grasses including the type and abundance of lignocellulosic components, pectins, and proteins and the linkages between them [10, 11].
Cinnamyl-alcohol dehydrogenase (CAD) functions in one of the final steps of monolignol biosynthesis that catalyzes the reduction of cinnamyl aldehyde to cinnamyl alcohol prior to polymerization into the lignin polymer. The highly conserved Rossmann fold NAD(P)H/NAD(P)+ binding domain found in CAD monolignol-synthesizing proteins indicates the use of NADPH as a cofactor in the reduction reaction [12]. CAD tends to exist in multi-gene families with one gene being primarily responsible for lignin biosynthesis. The effect of CAD downregulation has been studied using a transgenic approach in several eudicot species including Nicotiana tabacum[13–16], M. sativa[17], Populus sp.[18–20] and Eucalyptus camaldulensis[21]. Reports of lignin modification by downregulation of CAD in grasses are limited to Panicum virgatum[22, 23], Zea mays[24], and Festuca arundinacea[25]. In general, the transgenic downregulation of CAD does not affect total lignin content; instead, the inhibition of monolignol biosynthesis leads to changes in lignin composition, such as incorporation of accumulated aldehyde precursors or novel units into the lignin polymer, changes that make biomass more digestible [26].
Caffeic acid O-methyltransferase (COMT) is an O-methyltransferase that tends to be broad in substrate affinity and can potentially act in various branches of the phenylpropanoid pathway. The highly conserved S-adenosyl methionine (SAM) binding domain in COMT proteins indicates the use of SAM as the methyl group donor to the hydroxyl group of a methyl acceptor molecule [27]. COMT is involved in the methylation of caffeic acid to ferulic acid, which is then hydroxylated at position five by ferulate-5-hydroxylase. The subsequent methylation by COMT at this position yields sinapic acid. Similarly, COMT catalyzes the 3-O-methylation of caffeal- and coniferal- aldehyde/alcohol precursors to G and S lignin [28, 29]. In Brachypodium distachyon, COMT has high affinity for a variety of substrates including flavonoid compounds, with the greatest activity with caffeic acid and caffealdehyde [30]. Transgenic downregulation of COMT has been reported in eudicots including M. sativa[31], N. tabacum[32, 33], and Populus sp.[18, 34, 35]. Reports of COMT-downregulated transgenic grasses are more limited, including P. virgatum[36], Z. mays[37], F.arundinacea[38] and Lolium perenne[39]. Perturbation of the COMT enzyme often results in the inhibition of S lignin formation and consequently the accumulation of 5-OH coniferyl alcohol that could not be synthesized into S lignin is instead converted into a G lignin monomer, resulting in incorporation of a novel 5-hydroxyguaiacyl lignin unit into the lignin polymer [33, 40]. Accordingly, COMT transgenics with altered lignin composition tend to be more digestible [26].
In the early 1920's, brown midrib (bmr) mutants were identified in Z. mays as displaying a brownish-red to tan pigmentation of the leaf midrib associated with reduced lignin content or altered lignin composition [41]. A similar discoloration was observed in lignified tissues in the stalk. Changes in development are also apparent, as exemplified by variation in flowering time [42]. While the bmr mutants tend to have improved digestibility, various unfavorable traits including decreased grain and stem yield, increased lodging, and increased disease susceptibility sometimes develop [40, 43, 44]. Mutants have been isolated in the C4 grasses Z. mays, S. bicolor, and Pennisetum glaucum. Four bmr loci in Z. mays, bm1, bm2, bm3 and bm4, represent spontaneous mutants that were first isolated almost a century ago; an additional locus, bm5, was identified later [40]. In S. bicolor, four brown midrib loci, bmr2, bmr6, bmr12, and bmr19, were isolated from mutagenized populations [45, 46]. Three brown midrib mutants have been isolated in P. glaucum, although they have not been as well characterized [47]. In Z. mays and S. bicolor, the bmr phenotype is associated with orthologous loci that encompass CAD and COMT genes. The bm1 mutation affects expression of ZmCAD2 and five alleles of the orthologous bmr6 have been characterized in S. bicolor[48–51]. The bm3 locus in Z. mays and bmr12 in S. bicolor correspond to orthologous COMT genes [52, 53]. The bmr mutants have the potential to act as viable bioenergy crops, as the visual phenotype seems to be an effective marker for impaired lignin biosynthesis associated with improved digestibility. A more complete understanding of the genes responsible for the phenotype will help provide novel breeding strategies and expand the resources of conversion-efficient plants.
Grasses are a key source of grain and forage and have recently gained in importance as feedstocks for the biofuel industry. Large perennial bioenergy crops such as P. virgatum and Miscanthus sp. have a relatively long generation time, complex genomes, and tall stature that make these species difficult research subjects. In order to better understand grasses, the small annual grass, B. distachyon, was used here as a research model from which we can translate results to the improvement of crops for efficient biofuel production [54]. It has many attributes of a model system including a small diploid and sequenced genome, rapid generation time, short stature, and it is easily transformable [54]. In this study, we used artificial microRNAs to disrupt the function of candidate CAD or COMT genes, with the objective of characterizing their role in lignin biosynthesis in B. distachyon.
Characterization and phylogenetic analysis of BdCAD and BdCOMTgene families
BdCAD1 and BdCOMT4are highly expressed in developing stem
Anatomical expression data were also considered in evaluating the multi-gene BdCAD and BdCOMT families for a role in lignification. We used microarray data to analyze gene expression of each member of the family in developing stems, roots, and leaves. Lignin biosynthesis genes are expected to be highly expressed in stems, where secondary cell walls are prevalent and lignification occurs, while remaining at relatively low levels in roots and especially leaves. Of the seven CAD genes identified in B. distachyon, BdCAD1 expression was greatest in stem tissue, exhibiting ten-fold higher transcript level than any of the other seven BdCAD genes (Figure 4C). Expression of BdCOMT4 was also greatest in the stem and was eighteen-fold greater than the expression of the other three BdCOMT genes (Figure 4D). As might be expected, both BdCAD1 and BdCOMT4 were expressed at a slightly lower level in roots, where lignin is also present, and at a significantly lower level in leaves, where there is not much lignin at all.
BdCAD1 and BdCOMT4transgenic plants
The velocity of CAD in crude protein extracts was determined for aboveground tissue of empty vector control and amiR-cad1 transgenic plants as the inflorescent first emerged from the flag leaf (Additional file 1: Figure S1). Sinapaldehyde was used as a substrate to evaluate total CAD activity that includes seven other putative CAD enzymes (Additional file 1: Figure S1). Although not statistically significant, total CAD activity was reduced by 6% in amiR-cad1-1 and 17% in amiR-cad1-8 plants relative to empty vector control.
Effects of downregulation of BdCAD1 and BdCOMT4on development
Transgenic plants have lignin-associated phenotypes
Since COMT is important in the production of the sinapyl alcohol precursor for S lignin, we expected BdCOMT4-downregulated plants would exhibit a more drastic change, specifically in S lignin, more so than in total G and H lignin content (Figure 1). The Maule reagent was used to observe S lignin amount and localization in stems; the reagent stain S lignin a dark red-purple color. A shift to yellow-brown is indicative of a decrease in S lignin. Empty vector control stem sections treated with the Maule reagent stained a red-purple color (Figure 8D). Examination of the amiR-cad1 stems revealed a reduction in red staining and a slight shift to yellow-orange color, notably in the epidermal cells and sclerenchyma fibers (Figure 8E). There was a striking decrease in staining of the amiR-comt4 stem sections, in which the majority of tissue stained pale brown in color, with slight purple coloration seen in the outer regions of the pith (Figure 8F). The dramatic shift in color was reflective of a severe loss of S lignin in amiR-comt4 stems compared to the empty vector control.
Effect of BdCAD1 or BdCOMT4downregulation on lignin composition
Lignin composition in CAD1- and COMT4- downregulated transgenic plants
Plant line1
S (μmol/g)2,3
G (μmol/g)2,3
H (μmol/g)2,3
Control 1
89.7 ± 2.3
69.5 ± 1.4
8.9 ± 0.1
87.7 ± 1.8
75.8 ± 1.8
10.0 ± 0.1
75.2 ± 2.0
75.0 ± 1.7
9.4 ± 0.2
Control 2
97.2 ± 5.6
65.7 ± 3.6
9.5 ± 0.5
86.6 ± 3.8
54.2 ± 1.4
8.9 ± 0.2
92.4 ± 1.9
60.0 ± 0.9
10.8 ± 0.2
88.5 ± 2.7
55.8 ± 1.3
9.1 ± 0.2
Biological conversion efficiency in transgenic plants
In this study, we used a candidate gene approach to identify the CAD and COMT genes involved in monolignol biosynthesis in B. distachyon. While well studied as key enzymes in the lignin pathway that influence forage quality, understanding of these enzymes is now of increased interest due to an apparently similar effect on biofuel feedstock quality [8]. To date, there have only been a few reports of CAD or COMT downregulation in transgenic monocots [36–39]. These species, such as F. arundinacae and L. perenne have proven to be challenging research subjects. We report here the effectiveness of artificial microRNA silencing in a species considerably more amenable to research, B. distachyon, from which gained knowledge can be contributed towards the optimization of bioenergy grasses.
In monocots such as Z. mays, S. bicolor and O. sativa, CAD tends to exist as a multi-gene family with one CAD primarily involved in monolignol biosynthesis [48, 49, 55]. In contrast, in the eudicot A. thaliana, the last step of monolignol biosynthesis is controlled synergistically by two genes, AtCAD-C and AtCAD-D[57]. Based on amino acid sequence and gene expression pattern, we identified BdCAD1 as a likely candidate for having a role in lignin biosynthesis. Albeit with significantly lower efficiency than BdCAD1, Bradi4g29770 can also use coniferyl aldehyde and coniferyl alcohol as substrates and may also have a role in monolignol biosynthesis [58]. Unlike the other BdCADs, BdCAD1 contains all sequence features characteristic of a zinc-dependent alcohol dehydrogenase and more specifically, appears to be a member of the medium-chain dehydrogenase/reductase superfamily. High similarity in sequence motifs and substrate binding position suggests that BdCAD1 shares the same function as the bona fide CADs [56]. Consistent with multi-gene CAD families in Z. mays, S. bicolor and O. sativa, one particular protein in B. distachyon shared a significantly higher degree of homology with other known CADs than any other family member [48, 49, 55]. Gene expression analysis revealed that BdCAD1 was the most highly expressed member of the gene family and that transcript abundance was particularly high in stem and root, where secondary cell walls are prevalent. Lignin-associated CAD expression was similarly high in stem and root tissues in P. virgatum, F. arundinacae, O. sativa, and S. bicolor[22, 38, 55].
The downregulation of BdCAD1 caused phenotypes characteristic of lignin deficiency without reducing plant biomass. The delay in flowering time of the amiR-cad1 plants is consistent with the phenotypes in five S. bicolor bmr mutants including CAD impaired bmr6[42, 59]. The variation in flowering time observed in lignin mutants from S. bicolor and B. distachyon reinforces the possibility of an evolutionarily conserved mechanism between cell wall biosynthesis and production of flowers [60]. Because lignin has a significant role in xylem function, it is possible that changes in lignin may alter development by perturbing water transport. However, this rationale seems unlikely considering that, while amiR-cad1 plants were developmentally delayed, mature transgenic plants were significantly larger than empty vector control plants (Figure 6D). An increase in aboveground stem biomass, even coupled with delayed flowering, is a favorable trait for a perennial energy crop, considering crop rotation will not be part of cultivation. While mutations in CAD can sometimes lead to pleiotropic effects of dwarfing, lodging, and a decrease in grain and/or biomass yield, these effects are mostly background-dependent [40]. Further understanding of gene-by-gene interactions that result in these deleterious effects will increase the efficiency of cultivar development.
We present here the brown midrib leaf phenotype for the first time in a C3 grass [40]. Genetic redundancy may explain why the phenotype has not been observed in mutant polyploid species such as T. aestivum and F. arundinacae; however, CAD mutants of diploid O. sativa do not exhibit a leaf brown midrib [25, 55, 61, 62]. It has been suggested that the brown midrib phenotype may present itself differently in various species. In O. sativa, a mutation in GOLD HULL AND INTERNODE 2, which encodes a CAD enzyme, caused a red-brown pigment in the hull, internode, and basal leaf sheath while the leaf midrib did not show the same discoloration [63]. Similarly, recent research in B. distachyon indicated that CAD mutants displayed the red-brown pigmentation in various tissues including nodes and flowers, but not in the leaf midrib [64]. We did not observe color differences in tissues other than leaf midrib. In other species, including Populus sp. and N. tabacum, transverse stem cross-sections of transgenic CAD-downregulated plants exhibited unusually red xylem [13, 16, 20]. On the other hand, no visible mutant phenotype was observed in CAD-RNAi Z. mays plants [13]. Here, the BdCAD1-downregulated plants phenotypically resembled Z. mays, S. bicolor, and P. glaucum leaf brown-midrib mutants. The brown-midrib phenotype may occur only when CAD activity is decreased beneath a certain threshold [65]. For example, in four lines of antisense-CAD transgenic tobacco with residual CAD activity ranging from 8-56%, the extent of CAD downregulation was correlated with the presence and pattern of reddish-brown xylem [16]. We measured relatively small changes in CAD activity, but at a developmental stage that had not yet exhibited the brown-midrib phenotype. Nonetheless, the amiR-cad1-8 plants which were most reduced in CAD activity were significantly more digestible when the plant had completely senesced. It is possible at a subsequent developmental stage characterized by greater lignin biosynthesis that diminished CAD activity would be more evident.
The quality of lignin was altered in amiR-cad1 plants, as indicated by a significant decrease in S units observed by thioacidolysis and in agreement with histochemical staining by the Maule reagent. Previous research in CAD-downregulated N. tabacum and CAD mutant B. distachyon showed that the most dramatic change in lignin composition in plants was a severe decrease in S lignin [16, 64]. Some CAD gene knockouts produce functional lignin through increased incorporation of cinnamyl aldehyde subunits into the lignin polymer [43]. Previous reports for Z. mays, N. tabacum, and Populus sp. demonstrated an incorporation of aldehydes into the lignin polymer, in which increased coniferyl aldehyde caused an increase in intensity of the Wiesner stain [13, 20]. On the contrary, the Wiesner stain in amiR-cad1 lines was less intense than control plants. A likely explanation is that the inhibition of S lignin synthesis still caused an accumulation of aldehydes, but specifically sinapyl aldehydes, which are not detected by Wiesner staining. This is consistent with decreased Wiesner staining and incorporation of 8-O-4-coupled sinapyl aldehyde in B. distachyon CAD mutant plants [64].
Consistent with reports in N. tabacum, M. sativa, E. camaldulensis, Populus sp., F. arundinacae, and Z. mays, amiR-cad1 plants were unchanged in the amount of acetyl bromide soluble lignin polymer, but thioacidolysis indicated changes in lignin monomer composition [13, 16–18, 20, 21, 24, 38]. The lignin in CAD downregulated plants was generally more reactive. This has been illustrated by improved pulping properties in Populus sp., forage digestibility in N. tabacum and F. arundinacae, saccharification in P. virgatum and B. distachyon, and digestibility in Z. mays[13, 16–18, 20, 22, 23, 25, 64]. Along the same lines, the modified lignin in amiR-cad1-8 improved biological conversion efficiency by a statistically significant 17%.
One protein among the four COMTs identified in B. distachyon, BdCOMT4 (Bradi3g16530), contained all of the signature features of a plant O-methyltransferase. Plant O-methyltransferases tend to have broad substrate specificity, and all nine substrate binding and positioning residues in BdCOMT4 are common to COMT proteins in other species. Similar to L. perenne, F. arundinacae, P. tremuloides, and M. sativa COMT genes [38, 66–68], BdCOMT4 was the most highly expressed COMT in stem, root, and leaf tissues than any of the three other B. distachyon family members. Similar to observations made in P. tremuloides, BdCOMT4 expression was relatively low in leaves compared to stems [68]. Phylogeny, amino acid sequence, and the abundance of transcript in lignified tissues concurrently support that BdCOMT4 is an O-methyltransferase involved in monolignol biosynthesis in B. distachyon.
The downregulation of COMT resulted in changes in various phenotypic traits. Mutants tended to flower earlier than the empty vector control, as seen for the lignin mutants bm1 in Z. mays and bmr7 in S. bicolor[42, 59]. In general, the brown midrib phenotype is not common in COMT-downregulated transgenic plants, with the only reports of a reddish-brown coloration of the leaf and internodes being in Z. mays[37, 69]. Although the cause of discoloration in plants with impaired CAD activity is often attributed to the incorporation of aldehydes into the lignin polymer, there is no obvious correlation between the phenotype and the activities of other enzymes of the monolignol biosynthesis pathway. Previous biochemical analysis has indicated that the brown coloration is not a result of accumulated carotenoids, anthocyanins, flavones, tannins, or flavonols, but could possibly be due to incorporation of other phenolic compounds into the lignin polymer [40]. Accumulation of novel 5-OH-G units has been observed in COMT-downregulated transgenic M. sativa and Z. mays, although a visual phenotype associated with this phenomenon has not been defined [33, 37, 69, 70].
In our transgenic B. distachyon, the perturbation of the COMT enzyme had a deleterious effect on the total quantity of lignin produced in the plant. Similarly, downregulated COMT mutants in Z. mays, F. arundinacae, L. perenne, S. bicolor, and Saccharum spp. were also reduced in total lignin [38, 39, 69, 71]. Staining with the Maule reagent revealed an obvious difference between control and amiR-comt4 transverse stem cross sections. We measured an increase in ethanol yield of up to 10% in amiR-comt4 lines, which is consistent with the characterization of COMT mutants in other species [33, 37, 38, 69, 71–73].
One of the more costly steps of producing liquid fuels from biomass on the biochemical platform is the pretreatment required to reduce biomass recalcitrance for the enzymatic and fermentation steps. As a result, there has been increasing effort to identify the factors behind biomass recalcitrance. In this study, modification of CAD and COMT expression induced changes in cell wall composition that improved amenability to conversion. A significant 17% increase in ethanol yield from plant biomass, as observed here by CAD downregulation, would increase industrial efficiency of processing such feedstock. Genetic modification of lignin biosynthesis may provide a means of improvement of biofuel crop conversion efficiency by reducing biomass pretreatment costs, thereby improving the bioethanol production process overall.
Phylogenetic analysis
Candidate CAD and COMT in B. distachyon were identified by amino acid homology with known proteins in other plant systems by BLAST search of the Phytozome v8.0 and NCBI databases [74, 75]. Multiple amino acid sequences were aligned using ClustalW and analyzed with the associated editing program JalView 2.0 [76]. A neighbor-joining tree with bootstrap 1000 was constructed with MEGA [77]. Sequence data from this article can be found in the GenBank/EMBL databases under the following accession numbers: Sbbmr6 (BAF42789.1), Zmbm1 (ACG45271.1), PviCAD2 (ADO01602.1), OsCAD2 (NP_001046132.1), FaCAD (AAK97809.1), SoCAD (O82056.1), TaCAD (ADI59734.1), SbCOMT (AAO43609.1), Zmbm3 (NP_001106047.1), PviCOMT (ADX98508.1), TaCM (ABP63535.1).
Microarray expression profiling
Expression patterns of the multi-gene CAD and COMT families were observed using microarray expression profiling of leaf, root, and stem tissue of B. rachypodium distachyon. Plants were in a growth chamber at 20°C with 20 h light:4 h dark cycles at a fluence rate of 220 μmol of photons.m-2.s-1 and relative humidity of 67–69. Additionally, for plate-grown plants, seeds were de-hulled and then imbibed in water for two hours with shaking. Then, seeds were treated with 70% ethanol for 20 seconds, rinsed with sterile water, then soaked in 1.3% sodium hypochlorite for 4 minutes at room temperature while shaking. Seeds were subsequently rinsed three times with sterile water and stored in the dark at 4°C for a minimum of 2 days in a sterile Petri dish with filter paper. Seedlings were grown for seven days on 0.5X Murashige and Skoog (MS) medium containing 0.7% bactoagar adjusted to a pH of 5.8 with KOH.
Approximately 30 days following germination, total leaf and stem were collected as the inflorescence emerged from the flag leaf. Leaves were collected from the stems with a curved-tip probe. Nodes and internodes from the second leaf junction to the internode below the inflorescence were placed in a tube cooled with liquid nitrogen. Seven-day-old whole seedlings were flash frozen in liquid nitrogen and then the roots were snapped off into a sterile culture tube. The six time points were collected over the course of one day at ZT2, 6, 10, 14, 18, and 22. Three plants were dissected for each time point and in triplicate for each tissue type. Samples were stored in liquid nitrogen or at −80°C until RNA extraction. Tissue was ground with mortars and pestles in liquid nitrogen. RNA was extracted using the Qiagen (Valencia, CA) Plant RNaeasy Kit according to the manufacturer's instructions. Labeled sense strand cDNA probes were synthesized using the Ambion WT expression kit.
Transcript abundance of three biological replicates of three-week-old leaves and stems, and seven day old roots was measured using the Affymetrix B. distachyon BradiAR1b520742 whole genome tiling array. The array contains ~6.5 M unique 25-mer oligonucleotide features, both the forward and reverse strand sequence. The complete genome sequence is tiled with an average of 30 bases between each array feature; 1.6 million features correspond to exons and introns and 4.9 million features between gene models. Version 1.0 genome annotation includes a total of 25,532 protein coding genes and 2,542 non-coding genes [74]. Approximately ~95% (~26,670) of the genes have at least five corresponding exon array features and from those a summary value was calculated for each gene model. The average number of array features corresponding to the CAD and COMT families is 38, ranging from 10 to 111. Probeset values were calculated using gcRMA. The Affymetrix BradiAR1b520742 GeneChip data (.CEL files) have been deposited at PLEXdb [Accession no: BD3].
Generation of transgenic plants
The WMD Version 3 web-based tool (http://wmd3.weigelworld.org) was used to design highly specific artificial microRNA (amiRNA) constructs to target the BdCAD1 (Bradi3g06480) and BdCOMT4 (Bradi3g16530) transcripts. This program selects a 21-mer sequence in the target gene from which an amiRNA can be produced. The gene aliases for BdCAD1 and BdCOMT4 were searched in the WMD-3 B. distachyon 1.0 genome database for the amiRNA sequence which would most likely hybridize to the target mRNA without affecting the rest of the genome. The native BdCAD1 transcript was targeted by the amiRNA sequence AAGCGCTTACTTCTCAGATCA, corresponding to part of the fourth exon, with hybridization energy of the target site in the target gene of −38.50 kcal/mol. The native BdCOMT4 transcript was targeted by the amiRNA sequence, ACGAAGCTCCTCGACTTATAA, corresponding to the second exon, with hybridization energy of −39.85 kcal/mol.
Modified polymerase chain reactions were performed as described in the protocols section of the WMD3 website to engineer the amiRNA sequences into the endogenous O. sativa microRNA precursor osa-MIR528 in the PNW55 vector [78]. All PCRs were performed with Phusion High-Fidelity DNA Polymerase (New England Biolabs, Ipswitch, MA) according to the manufacturer's instructions. AmiRNA precursors were then cloned into the Gateway compatible vector pENTR/D- TOPO (Invitrogen, Grand Island, NY). Following sequence confirmation, entry clones were recombined into the destination vector pOL001 using the LR Clonase II Plus enzyme (Invitrogen, Grand Island, NY). This vector confers hygromycin resistance with the HptII gene and will express the amiRNAs under the constitutive ubiquitin promoter UBQ from Z. mays. Electroporation was used to transform the amiRNAs into Agrobacterium tumefaciens strain AGL1.
Transgenes were integrated into the B. distachyon accession Bd21-3 genome by Agrobacterium-mediated transformation of embryogenic calli following the protocol described by Vogel and Hill [79]. Regenerated plants from BdCAD1 transformation events amiR-cad1-1 and amiR-cad1-8 along with regenerated BdCOMT4 plants from transformation events amiR-comt4-3, amiR-comt4-5, and amiR-comt4-7 were selected for characterization in the T2 generation. Control plants were obtained by transformation with the empty binary vector pOL001. Plants were grown in a growth chamber at 20°C with 20 h light: 4 h dark cycles at a fluence rate of 220 μmol of photons.m-2.s-1 and relative humidity of 67–69.
Transgenic and empty vector control plants were genotyped by PCR of leaf genomic DNA. Leaf tissue was frozen in liquid nitrogen and pulverized with 6.35 mm stainless steel beads (BioSpec, Bartlesville, OK) in a Retsch Mixer Mill MM400. Pulverized tissue was treated with 600 μl DNA Extraction Buffer (100 mM NaCl, 50 mM Tris, 25 mM EDTA, 1% SDS, 10 mM 2-mercaptoethanol) at 65°C for 10 minutes. Samples were then placed on ice, mixed with 250 μl 5 M potassium acetate, and incubated for 20 minutes. The solution was centrifuged in a tabletop centrifuge at 12,000 rpm for 10 minutes. The supernatant was transferred to tubes containing 600 μl 100% isopropanol, mixed, and centrifuged to pellet the DNA. The supernatant was discarded and the pellet rinsed with 300 μl 70% ethanol. The pellet was resuspended in 225 μl T10E5 (10 mM Tris, 5 mM EDTA), mixed with 25 μl 3 M sodium acetate, 500 μl 100% ethanol and centrifuged in a tabletop centrifuge at 10,000 rpm for 7 minutes. Supernatant was discarded and the pellet was rinsed with 70% ethanol and centrifuged an additional 7 minutes. The pellet was allowed to air dry and was then resuspended in 30 μl T10E1 (10 mMTris, 1 mM EDTA). All putative transgenic and empty vector control plants were tested for the presence of the HptII transgene by PCR of leaf genomic DNA with primer forward: 5′ AGAATCTCGTGCTTTCAGCTTCGA 3′ and primer reverse: 5′ TCAAGACCAATGCGGAGCATATAC 3′. Only confirmed positive transformants were analyzed in subsequent experiments.
Molecular characterization of transgenic plants
Transgenic and empty vector control plants were subjected to quantitative real-time PCR to assay for BdCAD1 and BdCOMT4 gene expression. Whole stem tissue was sampled as the inflorescence first emerged from the flag leaf, frozen in liquid nitrogen, and pulverized with 6.35 mm stainless steel beads (BioSpec, Bartlesville, OK) using Retsch Mixer Mill MM400. Total RNA was extracted from pulverized tissue using an RNeasy Plant Mini Kit (Qiagen, Valencia, CA) according to the manufacturer's instructions; RNA purification was analyzed with the NanoDrop1000 (ThermoScientific, Waltham, MA). RNA samples were reverse-transcribed into cDNA using the SuperScript III First-Strand Synthesis System (Invitrogen, Grand Island, NY). Primers for gene-specific real-time PCR were selected using the QuantPrime design tool [80]. Reactions were completed using a QuantiFast SYBR Green PCR Kit (Qiagen, Valencia, CA) in an Eppendorf Mastercycler ep realplex2. To assay for BdCAD1 expression, cDNA from amiR-CAD1 and empty-vector plants was amplified with BdCAD1-specific primers (forward: 5′AGGATAGAATGGGCAGCATCGC 3′; reverse: 5′ ATCTTCAGGGCCTGTCTTCCTGAG 3′). To assay for BdCOMT4 expression, cDNA from amiR-COMT4 and empty vector plant was amplified with BdCOMT4-specific primers (forward: 5′ TGGAGAGCTGGTACTACCTGAAG 3′; reverse: 5′ CGACATCCCGTATGCCTTGTTG 3′). Expression values were normalized with the real-time PCR signal for the housekeeping gene Bradi5g25870 with its gene specific primers (forward: 5′- TCAGCAGGGTGCTAATTCAGTTC 3′; reverse: 5′ CGACAGAGTTTAGCGGTCTTAGC 3′). The selected housekeeping gene Bradi5g25870 exhibits moderate expression levels and extremely low variance across numerous B. distachyon array experiments. All qRT-PCR reactions were performed in triplicates.
Enzymatic assays
CAD activity was determined by generally following previously published procedures [50, 81–83]. Approximately 150 mg of frozen plant tissue that had been ground as previously described and stored at −80°C was taken up in 700 μL buffer (100 mMTris-Cl, pH 7.5, 5 mM DTT, 5% ethylene glycol) and then sonicated to disrupt cell walls using a sonicator equipped with a micro-tip (Branson Digital Sonifier 450, Branson Ultrasonic Corp., Danbury CT). Sonicated extracts were centrifuged at 4°C for 10 min. at 14,000 RPM and the crude protein extract was placed into new 1.5 mL tubes and kept on ice. Each plant sample was extracted twice and enzyme activity was tested by monitoring absorbance changes on a microplate reader (BioTek Synergy HT, BioTek Instruments, Winooski, VT) at A340. All reactions were carried out in a volume of 200 μL and consisted of 100 mM MES at pH 6.5, 200 μM NADPH, 100 mMsinapyl aldehyde, and 10 μL of the crude protein extract. Absorbance changes were monitored for three minutes after addition of crude protein extract. Enzyme velocities were determined by fitting a line using linear least squares to the absorbance data; the slope of the line was used in velocity determinations. Protein concentrations were determined using the Pierce 660 nm protein assay (Pierce Biotechnology, Rockford, IL) and served to normalize the calculated velocity for each extract.
Histochemical staining of lignin
Cross sections of stems were manually dissected from the first internode of developmentally equivalent transgenic and control plants. The Wiesner staining method [84] was used to visualize total lignin content and localization in the stem. Sections were stained with 1% phloroglucinol for 2 minutes followed by a wash in 50% HCl and were mounted onto microscope slides for observation. The Maule reagent [85] was used to observe S lignin content and localization in stems. Sections were treated with 1% KMnO4 for 5 minutes and rinsed with water. Sections were then treated with 10% HCl for 2 minutes, rinsed with water, and mounted on microscope slides in 1% NH4OH. Images were captured using a Nikon Eclipse E200MV R microscope with a 3 PixeLINK 3 MP camera.
Lignin content and composition analysis
Senesced stem material was dried thoroughly following wash steps with 70% ethanol at 65°C for 1 hour, and following ethanol removal was rinsed with acetic acid and allowed to air dry. Stem was pulvarized into powder with metal beads in a Retsch Mixer Mill MM400. Acetyl-bromide-soluble lignin was quantified using the procedure described by Foster et al. [86]. Triplicate samples of 1.5 mg powdered stem material were used for AcBr lignin analysis. Lignin composition was evaluated by thioacidolysis and S, G, and H unit quantification was conducted as described by Foster et al. [86].
Digestibility analysis
Biological conversion efficiency was measured using the microbial system developed for Clostridium phytofermentans, which converts plant biomass to ethanol, following the protocol as described by Lee et al. [72]. Triplicate 20 mg samples, dried and prepared as described above, were incubated with C. phytofermentans in 96-well plates and supernatant ethanol concentration was measured with high-performance liquid chromatography (HPLC) with RI detection [87].
Statistical analysis
For each measurement, 4 to 17 independent plants were sampled. Analysis of variance and Dunnett's contrasts were performed in R v2.15.0.
We thank Ludmila Tyler, Elsbeth Walker, and Ana Caicedo for helpful discussion and critical reading of this manuscript. We acknowledge Pubudu Handakumbura for expert assistance with tissue culture.
This research was supported by the Office of Science (BER) Department of Energy Grant DE-FG02-08ER64700DE to SPH and TCM.
Authors' Affiliations
- Somerville C: Biofuels. Curr Biology. 2007, 17 (4): R115-R119. 10.1016/j.cub.2007.01.010.View ArticleGoogle Scholar
- Archer D, Eby M, Brovkin V, Ridgwell A, Cao L, Mikolajewicz U, Caldeira K, Matsumoto K, Munhoven G, Montenegro A, et al: Atmospheric lifetime of fossil fuel carbon dioxide. Annu Rev Earth Pl Sc. 2009, 37: 117-134. 10.1146/annurev.earth.031208.100206.View ArticleGoogle Scholar
- Luthi D, Le Floch M, Bereiter B, Blunier T, Barnola J-M, Siegenthaler U, Raynaud D, Jouzel J, Fischer H, Kawamura K, et al: High-resolution carbon dioxide concentration record 650,000-800,000 years before present. Nature. 2008, 453 (7193): 379-382. 10.1038/nature06949.View ArticleGoogle Scholar
- Lynd L, Laser M, Brandsby D, Dale B, Davison B, Hamilton R, Himmel M, Keller M, McMillan J, Sheehan J, et al: How biotech can transform biofuels. Nat Biotechnol. 2008, 26: 169-172. 10.1038/nbt0208-169.View ArticleGoogle Scholar
- Carroll A, Somerville C: Cellulosic biofuels. Annu Rev Plant Biol. 2009, 60 (1): 165-182. 10.1146/annurev.arplant.043008.092125.View ArticleGoogle Scholar
- Keating JD, Panganiban C, Mansfield SD: Tolerance and adaptation of ethanologenic yeasts to lignocellulosic inhibitory compounds. Biotechnol Bioeng. 2006, 93 (6): 1196-1206. 10.1002/bit.20838.View ArticleGoogle Scholar
- Chen F, Dixon R: Lignin modification improves fermentable sugar yields for biofuel production. Nat Biotechnol. 2007, 25: 759-761. 10.1038/nbt1316.View ArticleGoogle Scholar
- Vanholme R, Morreel K, Ralph J, Boerjan W: Lignin engineering. Curr Opin Plant Biol. 2008, 11: 278-10.1016/j.pbi.2008.03.005.View ArticleGoogle Scholar
- Bonawitz ND, Chapple C: The genetics of lignin biosynthesis: connecting genotype to phenotype. Ann Rev Genet. 2010, 44: 337-363. 10.1146/annurev-genet-102209-163508.View ArticleGoogle Scholar
- Vogel J: Unique aspects of the grass cell wall. Curr Opin Plant Biol. 2008, 11 (3): 301-307. 10.1016/j.pbi.2008.03.002.View ArticleGoogle Scholar
- Handakumbura PP, Hazen SP: Transcriptional regulation of grass secondary cell wall biosynthesis: playing catch-up with Arabidopsis thaliana. Front Plant Sci. 2012, 3:Google Scholar
- Rossmann MG, Moras D, Olsen KW: Chemical and biological evolution of a nucleotide-binding protein. Nature. 1974, 250 (5463): 194-199. 10.1038/250194a0.View ArticleGoogle Scholar
- Halpin C, Knight ME, Foxon GA, Campbell MM, Boudet AM, Boon JJ, Chabbert B, Tollier M-T, Schuch W: Manipulation of lignin quality by downregulation of Cinnamyl alcohol dehydrogenase. Plant J. 1994, 6 (3): 339-350. 10.1046/j.1365-313X.1994.06030339.x.View ArticleGoogle Scholar
- Bernard Vailhé MA, Besle JM, Maillot MP, Cornu A, Halpin C, Knight M: Effect of down-regulation of Cinnamyl alcohol dehydrogenase on cell wall composition and on degradability of tobacco stems. J Sci Food Agric. 1998, 76 (4): 505-514. 10.1002/(SICI)1097-0010(199804)76:4<505::AID-JSFA981>3.0.CO;2-M.View ArticleGoogle Scholar
- O'Connell A, Holt K, Piquemal J, Grima-Pettenati J, Boudet A, Pollet B, Lapierre C, Petit-Conil M, Schuch W, Halpin C: Improved paper pulp from plants with suppressed Cinnamoyl-CoA reductase or Cinnamyl alcohol dehydrogenase. Transgenic Res. 2002, 11 (5): 495-503. 10.1023/A:1020362705497.View ArticleGoogle Scholar
- Yahiaoui N, Marque C, Myton KE, Negrel J, Boudet AM: Impact of different levels of Cinnamyl alcohol dehydrogenase down-regulation on lignins of transgenic tobacco plants. Planta. 1997, 204 (1): 8-15. 10.1007/s004250050224.View ArticleGoogle Scholar
- Baucher M, Bernard-Vailhe M, Chabbert B, Besle J, Opsomer C, Van Montagu M, Botterman J: Down-regulation of Cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility. Plant Mol Biol. 1999, 39 (3): 437-447. 10.1023/A:1006182925584.View ArticleGoogle Scholar
- Lapierre C, Pollet B, Petit-Conil M, Toval G, Romero J, Pilate G, Leple J, Boerjan W, Ferret V, De Nadai V, et al: Structural alterations of lignins in transgenic poplars with depressed cinnamyl alcohol dehydrogenase or caffeic acid O-methyltransferase activity have an opposite impact on the efficiency of industrial kraft pulping. Plant Physiol. 1999, 119 (1): 153-164. 10.1104/pp.119.1.153.View ArticleGoogle Scholar
- Lapierre C, Pilate G, Pollet B, Mila I, Leple J-C, Jouanin L, Kim H, Ralph J: Signatures of cinnamyl alcohol dehydrogenase deficiency in poplar lignins. Phytochemistry. 2004, 65 (3): 313-321. 10.1016/j.phytochem.2003.11.007.View ArticleGoogle Scholar
- Baucher M, Chabbert B, Pilate G, Van Doorsselaere J, Tollier MT, Petit-Conil M, Cornu D, Monties B, Van Montagu M, Inze D, et al: Red xylem and higher lignin extractability by down-regulating a Cinnamyl alcohol dehydrogenase in poplar. Plant Physiol. 1996, 112 (4): 1479-1490.Google Scholar
- Valério L, Carter D, Rodrigues JC, Tournier V, Gominho J, Marque C, Boudet A-M, Maunders M, Pereira H, Teulières C: Down regulation of Cinnamyl alcohol dehydrogenase, a lignification enzyme, in Eucalyptus camaldulensis. Mol Breed. 2003, 12 (2): 157-167. 10.1023/A:1026070725107.View ArticleGoogle Scholar
- Fu C, Xiao X, Xi Y, Ge Y, Chen F, Bouton J, Dixon R, Wang Z-Y: Downregulation of Cinnamyl alcohol dehydrogenase (CAD) leads to improved saccharification efficiency in switchgrass. BioEnergy Res. 2011, 4: 153-164. 10.1007/s12155-010-9109-z.View ArticleGoogle Scholar
- Saathoff AJ, Sarath G, Chow EK, Dien BS, Tobias CM: Downregulation of Cinnamyl-alcohol dehydrogenase in switchgrass by RNA silencing results in enhanced glucose release after cellulase treatment. PLoS One. 2011, 6 (1): e16416-10.1371/journal.pone.0016416.View ArticleGoogle Scholar
- Fornale S, Capellades M, Encina A, Wang K, Irar S, Lapierre C, Ruel K, Joseleau J-P, Berenguer J, Puigdomenech P, et al: Altered lignin biosynthesis improves cellulosic bioethanol production in transgenic maize plants down-regulated for Cinnamyl alcohol dehydrogenase. Mol Plant. 2011, 5 (4): 817-830.View ArticleGoogle Scholar
- Chen L, Auh CK, Dowling P, Bell J, Chen F, Hopkins A, Dixon RA, Wang ZY: Improved forage digestibility of tall fescue (Festuca arundinacea) by transgenic down-regulation of cinnamyl alcohol dehydrogenase. Plant Biotechnol J. 2003, 1 (6): 437-449. 10.1046/j.1467-7652.2003.00040.x.View ArticleGoogle Scholar
- Li X, Weng J-K, Chapple C: Improvement of biomass through lignin modification. Plant J. 2008, 54 (4): 569-581. 10.1111/j.1365-313X.2008.03457.x.View ArticleGoogle Scholar
- Joshi CP, Chiang VL: Conserved sequence motifs in plant S-adenosyl-L-methionine-dependent methyltransferases. Plant Mol Biol. 1998, 37 (4): 663-674. 10.1023/A:1006035210889.View ArticleGoogle Scholar
- Whetten R, Sederoff R: Lignin biosynthesis. Plant Cell. 1995, 7 (7): 1001-1013.View ArticleGoogle Scholar
- Humphreys JM, Chapple C: Rewriting the lignin roadmap. Curr Opin Plant Biol. 2002, 5 (3): 224-229. 10.1016/S1369-5266(02)00257-1.View ArticleGoogle Scholar
- Wu X, Wu J, Luo Y, Bragg J, Anderson O, Vogel J, Gu YQ: Phylogenetic, molecular, and biochemical characterization of caffeic acid o-methyltransferase gene family in Brachypodium distachyon. Int J Plant Genomics. 2013, 2013: 12-View ArticleGoogle Scholar
- Guo D, Chen F, Wheeler J, Winder J, Selman S, Peterson M, Dixon RA: Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases. Transgenic Res. 2001, 10 (5): 457-464. 10.1023/A:1012278106147.View ArticleGoogle Scholar
- Ni W, Paiva NL, Dixon RA: Reduced lignin in transgenic plants containing a Caffeic acid O-methyltransferase antisense gene. Transgenic Res. 1994, 3 (2): 120-126. 10.1007/BF01974090.View ArticleGoogle Scholar
- Guo D, Chen F, Inoue K, Blount JW, Dixon RA: Downregulation of Caffeic acid 3-O-methyltransferase and Caffeoyl CoA 3-O-methyltransferase in transgenic alfalfa: impacts on lignin structure and implications for the biosynthesis of G and S lignin. Plant Cell. 2001, 13 (1): 73-88.View ArticleGoogle Scholar
- Van Doorsselaere J, Baucher M, Chognot E, Chabbert B, Tollier M-T, Petit-Conil M, Leplé J-C, Pilate G, Cornu D, Monties B, et al: A novel lignin in poplar trees with a reduced Caffeic acid/5-hydroxyferulic acid O-methyltransferase activity. Plant J. 1995, 8 (6): 855-864. 10.1046/j.1365-313X.1995.8060855.x.View ArticleGoogle Scholar
- Jouanin L, Goujon T, de Nadai V, Martin M-T, Mila I, Vallet C, Pollet B, Yoshinaga A, Chabbert B, Petit-Conil M, et al: Lignification in transgenic poplars with extremely reduced Caffeic acid O-methyltransferase activity. Plant Physiol. 2000, 123 (4): 1363-1374. 10.1104/pp.123.4.1363.View ArticleGoogle Scholar
- Fu C, Mielenz JR, Xiao X, Ge Y, Hamilton CY, Rodriguez M, Chen F, Foston M, Ragauskas A, Bouton J, et al: Genetic manipulation of lignin reduces recalcitrance and improves ethanol production from switchgrass. Proc Natl Acad Sci USA. 2011, 108: 3803-3803. 10.1073/pnas.1100310108.View ArticleGoogle Scholar
- Pichon M, Deswartes C, Gerentes D, Guillaumie S, Lapierre C, Toppan A, Barrière Y, Goffner D: Variation in lignin and cell wall digestibility in Caffeic acid O-methyltransferase down-regulated maize half-sib progenies in field experiments. Mol Breed. 2006, 18 (3): 253-261. 10.1007/s11032-006-9033-2.View ArticleGoogle Scholar
- Chen L, Auh C-K, Dowling P, Bell J, Lehmann D, Wang Z-Y: Transgenic down-regulation of Caffeic acid O-methyltransferase (COMT) led to improved digestibility in tall fescue (Festuca arundinacea). Funct Plant Biol. 2004, 31 (3): 235-245. 10.1071/FP03254.View ArticleGoogle Scholar
- Tu Y, Rochfort S, Liu Z, Ran Y, Griffith M, Badenhorst P, Louie GV, Bowman ME, Smith KF, Noel JP, et al: Functional analyses of caffeic acid O-methyltransferase and cinnamoyl-CoA-reductase genes from perennial ryegrass (Lolium perenne). Plant Cell. 2010, 22 (10): 3357-3373. 10.1105/tpc.109.072827.View ArticleGoogle Scholar
- Sattler S, Funnell-Harris D, Pedersen J: Brown midrib mutations and their importance to the utilization of maize, sorghum, and pearl millet lignocellulosic tissues. Plant Sci. 2010, 178: 229-238. 10.1016/j.plantsci.2010.01.001.View ArticleGoogle Scholar
- Jorgensen JR: Brown midrib in maize and its linkage relationships. J Am Soc Agron. 1931, 23: 549-557. 10.2134/agronj1931.00021962002300070005x.View ArticleGoogle Scholar
- Vermerris W, McIntyre LM: Time to flowering in brown midrib mutants of maize: an alternative approach to the analysis of developmental traits. Heredity. 1999, 83: 171-178. 10.1046/j.1365-2540.1999.00569.x.View ArticleGoogle Scholar
- Barriere Y, Ralph J, Mechin V, Guillaumie S, Grabber JH: Genetic and molecular basis of grass cell wall biosynthesis and degradability. II. Lessons from brown-midrib mutants. C R Biol. 2004, 327: 847-10.1016/j.crvi.2004.05.010.View ArticleGoogle Scholar
- Pedersen JF, Vogel KP, Funnell DL: Impact of reduced lignin on plant fitness. Crop Sci. 2005, 45 (3): 812-819. 10.2135/cropsci2004.0155.View ArticleGoogle Scholar
- Porter KS, Axtell JD, Lechtenberg VL, Colenbrander VF: Phenotype, fiber composition, and in vitro dry matter disappearance of chemically induced brown midrib (bmr) mutants of sorghum. Crop Sci. 1978, 18 (2): 205-208. 10.2135/cropsci1978.0011183X001800020002x.View ArticleGoogle Scholar
- Saballos A, Vermerris W, Rivera L, Ejeta G: Allelic association, chemical characterization and saccharification properties of brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench). BioEnerg Res. 2008, 1 (3): 193-204.View ArticleGoogle Scholar
- Cherney JH, Axtell JD, Hassen MM, Anliker KS: Forage quality characterization of a chemically induced brown-midrib mutant In pearl millet. Crop Sci. 1988, 28 (5): 783-787. 10.2135/cropsci1988.0011183X002800050012x.View ArticleGoogle Scholar
- Halpin C, Holt K, Chojecki J, Oliver D, Chabbert B, Monties B, Edwards K, Barakate A, Foxon GA: Brown-midrib maize (bm1) - a mutation affecting the cinnamyl alcohol dehydrogenase gene. Plant J. 1998, 14: 545-553. 10.1046/j.1365-313X.1998.00153.x.View ArticleGoogle Scholar
- Saballos A, Ejeta G, Sanchez E, Kang C, Vermerris W: A genomewide analysis of the cinnamyl alcohol dehydrogenase family in sorghum [Sorghum bicolor (L.) Moench] identifies SbCAD2 as the brown midrib6 gene. Genetics. 2009, 181 (2): 783-795.View ArticleGoogle Scholar
- Sattler SE, Saathoff AJ, Haas EJ, Palmer NA, Funnell-Harris DL, Sarath G, Pedersen JF: A nonsense mutation in a Cinnamyl alcohol dehydrogenase gene Is responsible for the sorghum brownmidrib6 phenotype. Plant Physiol. 2009, 150 (2): 584-595. 10.1104/pp.109.136408.View ArticleGoogle Scholar
- Gorthy S, Mayandi K, Faldu D, Dalal M: Molecular characterization of allelic variation in spontaneous brown midrib mutants of sorghum (Sorghum bicolor (L.) Moench). Mol Breed. 2013, 31 (4): 795-803. 10.1007/s11032-012-9834-4.View ArticleGoogle Scholar
- Vignols F, Rigau J, Torres M, Capellades M, Puigdomenech P: The brown midrib3 (bm3) mutation in maize occurs in the gene encoding caffeic acid O-methyltransferase. Plant Cell. 1995, 7 (4): 407-416.View ArticleGoogle Scholar
- Bout S, Vermerris W: A candidate-gene approach to clone the sorghum Brown midrib gene encoding Caffeic acid O-methyltransferase. MGG. 2003, 269 (2): 205-214.Google Scholar
- Brkljacic J, Grotewold E, Scholl R, Mockler T, Garvin DF, Vain P, Brutnell T, Sibout R, Bevan M, Budak H, et al: Brachypodium as a model for the grasses: today and the future. Plant Physiol. 2011, 157 (1): 3-13. 10.1104/pp.111.179531.View ArticleGoogle Scholar
- Tobias C, Chow E: Structure of the Cinnamyl-alcohol dehydrogenase gene family in rice and promoter activity of a member associated with lignification. Planta. 2005, 220 (5): 678-688. 10.1007/s00425-004-1385-4.View ArticleGoogle Scholar
- Youn B, Camacho R, Moinuddin SGA, Lee C, Davin LB, Lewis NG, Kang C: Crystal structures and catalytic mechanism of the Arabidopsis cinnamyl alcohol dehydrogenases AtCAD5 and AtCAD4. Org Biomol Chem. 2006, 4 (9): 1687-1697. 10.1039/b601672c.View ArticleGoogle Scholar
- Sibout R, Eudes A, Mouille G, Pollet B, Lapierre C, Jouanin L, Seguin A: Cinnamyl alcohol dehydrogenase-C and -D are the primary genes involved in lignin biosynthesis in the floral stem of Arabidopsis. Plant Cell. 2005, 17 (7): 2059-2076. 10.1105/tpc.105.030767.View ArticleGoogle Scholar
- Bukh C, Nord-Larsen PH, Rasmussen SK: Phylogeny and structure of the cinnamyl alcohol dehydrogenase gene family in Brachypodium distachyon. J Exp Bot. 2012, 63 (17): 6223-6236. 10.1093/jxb/ers275.View ArticleGoogle Scholar
- Vermerris W, Thompson KJ, McIntyre LM: The maize Brown midrib1 locus affects cell wall composition and plant development in a dose-dependent manner. Heredity. 2002, 88 (6): 450-457. 10.1038/sj.hdy.6800078.View ArticleGoogle Scholar
- Vermerris W, Thompson K, McIntyre L, Axtell J: Evidence for an evolutionarily conserved interaction between cell wall biosynthesis and flowering in maize and sorghum. BMC Evol Biol. 2002, 2 (1): 2-10.1186/1471-2148-2-2.View ArticleGoogle Scholar
- Ma Q-H: The expression of caffeic acid 3-O-methyltransferase in two wheat genotypes differing in lodging resistance. J Exp Bot. 2009, 60 (9): 2763-2771. 10.1093/jxb/erp132.View ArticleGoogle Scholar
- Hirano K, Aya K, Kondo M, Okuno A, Morinaka Y, Matsuoka M: OsCAD2 is the major CAD gene responsible for monolignol biosynthesis in rice culm. Plant Cell Rep. 2012, 31 (1): 91-101. 10.1007/s00299-011-1142-7.View ArticleGoogle Scholar
- Zhang K, Qian Q, Huang Z, Wang Y, Li M, Hong L, Zeng D, Gu M, Chu C, Cheng Z: GOLD HULL AND INTERNODE2 encodes a primarily multifunctional cinnamyl-alcohol dehydrogenase in rice. Plant Physiol. 2006, 140 (3): 972-983. 10.1104/pp.105.073007.View ArticleGoogle Scholar
- d'Yvoire MB, Bouchabke-Coussa O, Voorend W, Antelme S, Cézard L, Legée F, Lebris P, Legay S, Whitehead C, McQueen-Mason SJ, et al: Disrupting the cinnamyl alcohol dehydrogenase 1 gene (BdCAD1) leads to altered lignification and improved saccharification in Brachypodium distachyon. Plant J. 2012, 73: 496-508.Google Scholar
- Anterola AM, Lewis NG: Trends in lignin modification: a comprehensive analysis of the effects of genetic manipulations/mutations on lignification and vascular integrity. Phytochemistry. 2002, 61 (3): 221-294. 10.1016/S0031-9422(02)00211-X.View ArticleGoogle Scholar
- Inoue K, Sewalt VJH, Murray Ballance G, Ni W, Sturzer C, Dixon RA: Developmental expression and substrate specificities of slfalfa Caffeic acid 3-O-methyltransferase and Caffeoyl coenzyme A 3-O-methyltransferase in relation to lignification. Plant Physiol. 1998, 117 (3): 761-770. 10.1104/pp.117.3.761.View ArticleGoogle Scholar
- McAlister FM, Jenkins CLD, Watson JM: Sequence and expression of a stem-abundant caffeic acid O-methyltransferase cDNA from perennial ryegrass (Lolium perenne). Funct Plant Biol. 1998, 25 (2): 225-235.Google Scholar
- Bugos RC, Chiang VLC, Campbell WH: cDNA cloning, sequence analysis and seasonal expression of lignin-bispecific caffeic acid/5-hydroxyferulic acid O-methyltransferase of aspen. Plant Mol Biology. 1991, 17 (6): 1203-1215. 10.1007/BF00028736.View ArticleGoogle Scholar
- Piquemal J, Chamayou S, Nadaud I, Beckert M, Barriere Y, Mila I, Lapierre C, Rigau J, Puigdomenech P, Jauneau A, et al: Down-regulation of Caffeic acid O-methyltransferase in maize revisited using a transgenic approach. Plant Physiol. 2002, 130 (4): 1675-1685. 10.1104/pp.012237.View ArticleGoogle Scholar
- Lapierre CT, Tollier MT, Monties B: A new type of constitutive unit in lignins from the corn bm3 mutant. CR Acad Sci. 1988, 307: 723-728.Google Scholar
- Jung JH, Vermerris W, Gallo M, Fedenko JR, Erickson JE, Altpeter F: RNA interference suppression of lignin biosynthesis increases fermentable sugar yields for biofuel production from field-grown sugarcane. Plant Biotech J. 2013, 11 (6): 709-16. 10.1111/pbi.12061.View ArticleGoogle Scholar
- Lee SJ, Warnick TA, Pattathil S, Alvelo-Maurosa JG, Serapiglia MJ, McCormick H, Brown V, Young NF, Schnell DJ, Smart LB, et al: Biological conversion assay using Clostridium phytofermentans to estimate plant feedstock quality. Biotechnol Biofuels. 2012, 5 (1): 5-10.1186/1754-6834-5-5.View ArticleGoogle Scholar
- Dien B, Sarath G, Pedersen J, Sattler S, Chen H, Funnell-Harris D, Nichols N, Cotta M: Improved sugar conversion and ethanol yield for forage sorghum (Sorghum bicolor L. Moench) lines with reduced lignin contents. BioEnerg Res. 2009, 2 (3): 153-164. 10.1007/s12155-009-9041-2.View ArticleGoogle Scholar
- Initiative TIB: Genome sequencing and analysis of the model grass Brachypodium distachyon. Nature. 2010, 463 (7282): 763-768. 10.1038/nature08747.View ArticleGoogle Scholar
- Altschul S, Madden T, Schaffer A, Zhang J, Zhang Z, Miller W, Lipman D: Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res. 1997, 25 (17): 3389-3402. 10.1093/nar/25.17.3389.View ArticleGoogle Scholar
- Thompson JD, Higgins DG, Gibson TJ: CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. Nucl Acids Res. 1994, 22 (22): 4673-4680. 10.1093/nar/22.22.4673.View ArticleGoogle Scholar
- Tamura K, Peterson D, Peterson N, Stecher G, Nei M, Kumar S: MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. Mol Biol Evol. 2011, 28 (10): 2731-2739. 10.1093/molbev/msr121.View ArticleGoogle Scholar
- Warthmann N, Chen H, Ossowski S, Weigel D, Hervre P: Highly specific gene silencing by artificial miRNAs in rice. PLoS One. 2008, 3 (3): e1829-10.1371/journal.pone.0001829.View ArticleGoogle Scholar
- Vogel J, Hill T: High-efficiency Agrobacterium-mediated transformation of Brachypodium distachyon inbred line Bd21-3. Plant Cell Rep. 2008, 27 (3): 471-478. 10.1007/s00299-007-0472-y.View ArticleGoogle Scholar
- Arvidsson S, Kwasniewski M, Riano-Pachon D, Mueller-Roeber B: QuantPrime - a flexible tool for reliable high-throughput primer design for quantitative PCR. BMC Bioinforma. 2008, 9 (1): 465-10.1186/1471-2105-9-465.View ArticleGoogle Scholar
- Hawkins SW, Boudet AM: Purification and characterization of cinnamyl alcohol dehydrogenase isoforms from the periderm of Eucalyptus gunnii hook. Plant Physiol. 1994, 104 (1): 75-84.Google Scholar
- Mansell RL, Gross GG, Stöckigt J, Franke H, Zenk MH: Purification and properties of cinnamyl alcohol dehydrogenase from higher plants involved in lignin biosynthesis. Phytochemistry. 1974, 13 (11): 2427-2435. 10.1016/S0031-9422(00)86917-4.View ArticleGoogle Scholar
- Saathoff A, Tobias C, Sattler S, Haas E, Twigg P, Sarath G: Switchgrass contains two cinnamyl alcohol dehydrogenases involved in lignin formation. BioEnergy Res. 2011, 4 (2): 120-133. 10.1007/s12155-010-9106-2.View ArticleGoogle Scholar
- Wiesner J: Note über das verhalten des phloroglucins und einiger verwandter körper zur verholzten zellmembran. Sitzungsberichte der kaiserlichen akademie der wissenschaften. Math Nat Classe. 1978, 77: 60-66.Google Scholar
- Maule C: Das verhalten verholzter membranen gegen kalium-permanganat, eine holzreaktion neuer art. Beiträge zur wissenschaftlichen Botanik. 1901, 4: 166-185.Google Scholar
- Foster CE, Martin TM, Pauly M: Comprehensive compositional analysis of plant cell walls (lignocellulosic biomass) Part I: lignin. J Vis Exp. 2010, 37: e1745-Google Scholar
- Lee SJ, Warnick TA, Leschine SB, Hazen SP: A high-throughput biological conversion assay for determining lignocellulosic quality. Methods Mol Biol. 2013, 918: 341-349.View ArticleGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.


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5 Reasons Why AWS Rules
In this article, we will look at 5 reasons why AWS is the place to go for all your cloud services. Read on to find out more!
Cloud computing has evolved a lot over the years and has almost become a mainstream part of everyday society. From storing large amounts of data to having burst compute capacity at your fingertips and enterprise software applications available on demand any time anywhere, these are just some of the key benefits that clouds provide today. At the forefront of this new way of computing is Amazon Web Services (AWS).
AWS is a comprehensive public cloud computing platform made up of remote computing services called web services that make up the platform. Once a part of the e-commerce giant Amazon.com, it wasn't until 2006 that AWS became a separate entity of its own.
With data centers located in USA, Europe, Brazil, Singapore, Japan, China, and Australia, AWS operates globally to offer a variety of web-based products and services on an on-demand and pay-per-use basis. AWS provides a variety of mechanisms which end users can connect to and leverage its services. The most common form of interaction is the web-based dashboard, AWS Management Console.
5 reasons that keep AWS on top
AWS boasts an array of Infrastructure as a Service (IaaS) and Platform as a Service (PaaS) capabilities, with a diverse customer base and a strong network of software vendors who have integrated their solutions with AWS. Here is our list of top 5 reasons why AWS rules the cloud market:
Deployment Speed
We all have gone through the pain of long wait for provisioning a hosted web service. However, AWS shrinks this waiting time from hours to mere minutes. With multiple strategies for infrastructure provisioning ranging from Amazon EC2, Amazon EBS, and Amazon S3 to Amazon RDS, AWS offers promptness and quick availability as well as greater flexibility to deploy your website or application.
AWS also enables automation in its deployment services, namely:
- AWS Elastic Beanstalk
- AWS CloudFormation
- AWS OpsWorks
- AWS CodeCommit
- AWS CodePipeline
- AWS CodeDeploy
- Amazon EC2 Container Service
Scalable and Reliable
With AWS, you can scale your application with ease during periods of increased demand. AWS also prides itself on the fact that it is reliable and provides a better experience whilst keeping the costs under control.
AWS OpsWorks, Auto Scaling, and Elastic Load Balancing are some of the AWS tools can help you scale your business up or down on demand. AWS provides on-demand services giving you the opportunity to flex your resources as and when needed.
One of the best features of AWS is its flexibility. It lets you select from a plethora of services such as:
- Operating system
- Programming language
- Web application platform
- Database
AWS provides a virtual environment that lets you load the software and services required by your application. It simplifies the migration process for already existing applications and provides options for building new ones. With faster scalability, de-facto standard APIs, and mind-blowing deployment speed, you can have a completely customized ready-to-use solution in under 10 minutes.
On the Go Pricing
We all know that hosting a service on cloud sounds like a pretty massive investment, but this is resolved by AWS's pricing structure. AWS offers "pay for what you use". This means that you pay only for the resources you use. This is a refreshing approach from AWS, making it more user-friendly, and allowing you to choose your infrastructure and service needs with zero capital expenditure. This cost-effectiveness has really helped startups grow their business on AWS. Users can even try the AWS Free Tier.
With AWS Free Tier, you can enjoy hands-on experience with the AWS platform, products, and services for free. This no-cost option includes offers that expire after 1 year of signing up and others that never expire. This is one of the best ways for a new user to try out AWS.
Security is a core requirement for any application whether it is hosted in an on-premise data center or a cloud such as AWS. It is a fundamental service that protects your applications and data from a variety of cyber-attacks, security breaches, accidental or deliberate data deletions, theft, and much more.
The obvious doubt lingering in your mind while considering AWS must be is it really secure? Is it safe to use AWS? The answer is a big yes!
Apart from private subnets, multi-factor authentication (MFA), Isolate GovCloud, and encrypted data storage, AWS uses different layers of security to safeguard its resources:
- Physical data center security – The data centers are housed at non-disclosed locations and their entry is strictly controlled, managed, logged and audited on a regular basis
- Virtualization and OS security – AWS regularly patches and updates virtualization operating systems against a variety of cyber-attacks
- Regulatory compliance – AWS is certified for security and data protection in accordance with various industry and government requirements
AWS also enhances its security with IAM (Identity and Access Management). Using this authentication greatly reduces the risk.
If this article has got you hooked on AWS, you should definitely read my fourth book titled: AWS Administration - The Definitive Guide
This book is for those who want to learn and leverage the rich plethora of services provided by AWS. It guides you through some of the most popular AWS services, such as EC2, Elastic Beanstalk, EFS, CloudTrail, Redshift, EMR, Data Pipeline, and IoT using a simple, real-world, application-hosting example. Each chapter is designed to provide you with maximum information about each AWS service, coupled with easy to follow, hands-on steps, best practices, tips, and recommendations. By the end of the book, you will be able to create a highly secure, fault-tolerant, and scalable environment for your applications to run on.

subset: commoncrawl


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Budget 2007 is likely to deliver a mixed package for exporters with regard to service tax. While the finance ministry is in no mood to remove the 12% service tax on exporters for services availed within the country, it is seriously considering scrapping service tax on services enjoyed overseas.
The pressure on the finance ministry to do away with tax on overseas services is growing with a number of exporters all over the country legally contesting its imposition. The major beneficiaries of the removal of the tax would be the pharmaceuticals, textiles, chemicals and engineering goods sector as they have to utilise services of foreign agents much more than other sectors.
The official sources said that the finmin was looking at ways in which tax on overseas services could be excluded from the services tax net. The finance ministry has realised that it might not be legally tenable to continue with the tax as it has been challenged by exporters in courts throughout the country, the official said. The main arguement of exporters opposing the tax is that the government cannot impose a tax when the destination of services is outside India.
The incidence of service tax on overseas services which includes commission to foreign agents, participation in trade fairs and service maintenance contract for overseas services is as high as 18.2% of total export value of the pharmaceuticals sector. For the textiles sector, tax on overseas services amounts to 11% of the export value while for engineering and chemicals it is 8.3% and 7% respectively. The figures reveal the extent to which the comeptitveness of the identified sectors is eroded due to the imposition of the unwarrented tax, the official said.
The finance ministry has, however, outright rejected the commerce departments demand of totally exempting exporters from paying service tax. Its arguement for continuing with it is that the service tax paid on input services is neutralised under various schemes such as refund, rebate and duty drawback. The commerce department, however, claims that rebate or refund involves a cost and consumes time which exporters can ill-afford. Officials further point out that there is no provision to refund service tax on services in connection with export of non-excisable goods.


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- Home
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- Public Notices
On Dec. 4 and 5, the city of Lancaster hosted the annual Santa Train Express. Because of the generous contributions of the L&C Railroad, Lancaster County, Duke Energy, Comporium Communications and Mayor Ann Taylor of Heath Springs, the Santa Express served more than 600 people. Children of all ages enjoyed two days of taking the Lancaster & Chester Railroad train from the South Pole (Lancaster Depot) to the North Pole (Heath Springs) for a Christmas experience.
If you currently subscribe or have subscribed in the past to the The Lancaster News, then simply find your account number on your mailing label and enter it below.
Click the question mark below to see where your account ID appears on your mailing label.
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BluScenes' video strategy is mentioned on CIO.com's "How to Make Video Marketing Work for Your Business"
Posted on April 20, 2012 by Scenic Labs | 0 comments
CIO.com published an interesting article about the use of video to promote products. We provided some data about our own experience.
While part of the article focused on viral campaigns, we discussed how full-length trailers increased our direct sales and reduced returns because people knew what they were buying from the get-go.
Obviously, our model is not the Hollywood business model, because we don't have to worry about spoiling the plot when we post the entire disc.


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Mindboggle's open source brain morphometry platform takes in preprocessed T1-weighted MRI data, and outputs volume, surface, and tabular data containing label, feature, and shape information for further analysis. Mindboggle can be run on the command line as "mindboggle" and also exists as a cross-platform Docker container for convenience and reproducibility of results. The software runs on Linux and is written in Python 3 and Python-wrapped C++ code called within a Nipype pipeline framework. We have tested the software most extensively with Python 3.5.1 on Ubuntu Linux 14.04.
|December 19, 2018
A Klein, SS Ghosh, FS Bao, J Giard, Y Hame, E Stavsky, N Lee, B Rossa, M Reuter, EC Neto, A Keshavan. 2017. Mindboggling morphometry of human brains. PLoS Computational Biology 13(3): e1005350. doi:10.1371/journal.pcbi.1005350
Getting help¶
General questions about Mindboggle? Please post to NeuroStars with the tag "mindboggle". Found a bug, big or small? Please submit an issue on GitHub.
To run the Mindboggle jupyter notebook tutorial, first install the Mindboggle Docker container (see below) and enter the bash shell of the container from your $HOST (e.g., /Users/arno):
docker run --rm -ti -v $HOST:/home/jovyan/work -p 8888:8888 nipy/mindboggle
Then run the notebook from within the container:
jupyter notebook /opt/mindboggle/docs/mindboggle_tutorial.ipynb
We recommend installing Mindboggle and its dependencies as a cross-platform Docker container for greater convenience and reproducibility of results. All the examples below assume you are using this Docker container, with the path /home/jovyan/work/ pointing to your host machine. (Alternatively, one can create a Singularity image, or Mindboggle can be installed from scratch on a Linux machine using this script).
1. Install and run Docker on your (macOS, Linux, or Windows) host machine.
2. Download the Mindboggle Docker container (copy/paste the following in a terminal window):
docker pull nipy/mindboggle
Note 1: This contains FreeSurfer, ANTs, and Mindboggle, so it is currently over 6GB.*
Note 2: You may need to increase memory allocated by Docker to at least 5GB. For example: By default, Docker for Mac is set to use 2 GB runtime memory.
3. Recommended: download sample data. To try out the
mindboggle examples
below, download and unzip the directory of example input data
mindboggle_input_example.zip (455 MB).
For example MRI data to preprocess with FreeSurfer and ANTs software,
download and unzip
example_mri_data.zip (29 MB).
4. Recommended: set environment variables for clarity in the commands below (modify accordingly, except for DOCK – careful, this step is tricky!):
HOST=/Users/binarybottle # path on host to access input/output
DOCK=/home/jovyan/work # path to HOST from Docker container
IMAGE=$DOCK/example_mri_data/T1.nii.gz # input image on HOST
ID=arno # ID for brain image
Run one command¶
The Mindboggle Docker container can be run as a single command to process
a T1-weighted MR brain image through FreeSurfer, ANTs, and Mindboggle.
Skip to the next section if you wish to run
antsCorticalThickness.sh, and
mindboggle differently:
docker run --rm -ti -v $HOST:$DOCK nipy/mindboggle $IMAGE --id $ID
Outputs are stored in $DOCK/mindboggle123_output/ by default,
but you can set a different output path with
--out $OUT.
Run separate commands¶
If finer control is needed over the software in the Docker container, the following instructions outline how to run each command separately. Mindboggle currently takes output from FreeSurfer and optionally from ANTs. FreeSurfer version 6 or higher is recommended because by default it uses Mindboggle's DKT-100 surface-based atlas to generate corresponding labels on the cortical surfaces and in the cortical and non-cortical volumes (v5.3 generates these surface labels by default; older versions require "-gcs DKTatlas40.gcs" to generate these surface labels).
1. Enter the Docker container's bash shell to run
antsCorticalThickness.sh, and
mindboggle commands:
docker run --rm -ti -v $HOST:$DOCK nipy/mindboggle
Recommended: reset environment variables as above within the Docker container:
DOCK=/home/jovyan/work # path to HOST from Docker container IMAGE=$DOCK/example_mri_data/T1.nii.gz # input image on HOST ID=arno # ID for brain image
3. FreeSurfer generates labeled
cortical surfaces, and labeled cortical and noncortical volumes.
recon-all on a T1-weighted IMAGE file (and optionally a T2-weighted
image), and set the output ID name as well as the $FREESURFER_OUT output
recon-all -all -i $IMAGE -s $ID -sd $FREESURFER_OUT
4. ANTs provides brain volume extraction,
segmentation, and registration-based labeling.
generates transforms and segmentation files used by Mindboggle, and is run
on the same IMAGE file and ID as above, with $ANTS_OUT output directory.
TEMPLATE points to the OASIS-30_Atropos_template folder
already installed in the Docker container ("" splits the command for readability):
antsCorticalThickness.sh -d 3 -a $IMAGE -o $ANTS_OUT/$ID/ants \
-e $TEMPLATE/T_template0.nii.gz \
-t $TEMPLATE/T_template0_BrainCerebellum.nii.gz \
-m $TEMPLATE/T_template0_BrainCerebellumProbabilityMask.nii.gz \
-f $TEMPLATE/T_template0_BrainCerebellumExtractionMask.nii.gz \
-p $TEMPLATE/Priors2/priors%d.nii.gz \
-u 0
5. Mindboggle can be run on data preprocessed by
recon-all and
antsCorticalThickness.sh as above by setting:
OUT=$DOCK/mindboggled # output folder
Or it can be run on the mindboggle_input_example preprocessed data by setting:
OUT=$DOCK/mindboggled # output folder
Example Mindboggle commands:
To learn about Mindboggle's command options, type this in a terminal window:
mindboggle -h
Example 1: Run Mindboggle on data processed by FreeSurfer but not ANTs:
mindboggle $FREESURFER_SUBJECT --out $OUT
Example 2: Same as Example 1 with output to visualize surface data with roygbiv:
mindboggle $FREESURFER_SUBJECT --out $OUT --roygbiv
Example 3: Take advantage of ANTs output as well ("" splits for readability):
mindboggle $FREESURFER_SUBJECT --out $OUT --roygbiv \
--ants $ANTS_SUBJECT/antsBrainSegmentation.nii.gz
Example 4: Generate only volume (no surface) labels and shapes:
mindboggle $FREESURFER_SUBJECT --out $OUT \
--ants $ANTS_SUBJECT/antsBrainSegmentation.nii.gz \
Visualize output¶
To visualize Mindboggle output with roygbiv, start the Docker image with:
docker run --rm -ti -v $HOST:$DOCK -p 5000:5000 nipy/mindboggle
and then inside the image, run roygbiv on an output directory:
roygbiv $OUT/$ID
and open a browser to localhost:5000.
Right now, roygbiv only shows summarized data, but Anisha Keshavan is working on by-vertex visualizations (for the latter, try Paraview).
Appendix: processing¶
The following steps are performed by Mindboggle (with links to code on GitHub):
Create hybrid gray/white segmentation from FreeSurfer and ANTs output (combine_2labels_in_2volumes).
Fill hybrid segmentation with FreeSurfer- or ANTs-registered labels.
Compute volume shape measures for each labeled region:
- volume (volume_per_brain_region)
- thickness of cortical labels (thickinthehead, off by default)
Compute surface shape measures for every cortical mesh vertex:
- surface area
- travel depth
- geodesic depth
- mean curvature
- convexity (from FreeSurfer)
- thickness (from FreeSurfer)
Extract cortical surface features:
For each cortical surface label/sulcus, compute:
- area
- mean coordinates: means_per_label
- mean coordinates in MNI152 space
- Laplace-Beltrami spectrum
- Zernike moments
Compute statistics (
stats_per_labelin compute.py) for each shape measure in #4 for each label/feature:
- median
- median absolute deviation
- mean
- standard deviation
- skew
- kurtosis
- lower quartile
- upper quartile
Appendix: output¶
Example output data can be found on Mindboggle's examples site on osf.io. By default, output files are saved in $HOME/mindboggled/SUBJECT, where $HOME is the home directory and SUBJECT is a name representing the person's brain that has been scanned. Volume files are in NIfTI format, surface meshes in VTK format, and tables are comma-delimited. Each file contains integers that correspond to anatomical labels or features (0-24 for sulci). All output data are in the original subject's space. The following include outputs from most, but not all, optional arguments.
|number-labeled surfaces and volumes
|.vtk, .nii.gz
|surfaces with features: sulci, fundi
|surfaces with shape measures (per vertex)
|tables of shape measures (per label/feature/vertex)
mindboggled / $SUBJECT /
labels /
freesurfer_wmparc_labels_in_hybrid_graywhite.nii.gz: hybrid segmentation filled with FS labels
ants_labels_in_hybrid_graywhite.nii.gz: hybrid segmentation filled with ANTs + FS cerebellar labels
[left,right]_cortical_surface / freesurfer_cortex_labels.vtk: DKT cortical surface labels
features / [left,right]_cortical_surface /
folds.vtk: (unidentified) depth-based folds
sulci.vtk: sulci defined by DKT label pairs in depth-based folds
fundus_per_sulcus.vtk: fundus curve per sulcus – UNDER EVALUATION –
cortex_in_MNI152_space.vtk: cortical surfaces aligned to an MNI152 template
shapes / [left,right]_cortical_surface /
area.vtk: per-vertex surface area
mean_curvature.vtk: per-vertex mean curvature
geodesic_depth.vtk: per-vertex geodesic depth
travel_depth.vtk: per-vertex travel depth
freesurfer_curvature.vtk: FS curvature files converted to VTK
freesurfer_sulc.vtk: FS sulc (convexity) files converted to VTK
freesurfer_thickness.vtk: FS thickness files converted to VTK
tables /
volume_per_freesurfer_label.csv: volume per FS label
volumes_per_ants_label.csv: volume per ANTs label
thickinthehead_per_freesurfer_cortex_label.csv: FS cortex label thickness – UNDER EVALUATION –
thickinthehead_per_ants_cortex_label.csv: ANTs cortex label thickness – UNDER EVALUATION –
[left,right]_cortical_surface /
label_shapes.csv: per-label surface shape statistics
sulcus_shapes.csv: per-sulcus surface shape statistics
fundus_shapes.csv: per-fundus surface shape statistics – UNDER EVALUATION –
vertices.csv: per-vertex surface shape statistics


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Denise Neil
Chris Mann: Behind-the-scenes drama 'overshadowed the music' on 'The Voice'
Two days after he was named fourth place (or as I prefer to call it, third runner-up) on the season finale of the NBC singing competition "The Voice," Chris Mann was still trying to sort it all out.
Calling from Los Angeles on Thursday, Mann was subdued and a little hoarse from what sounded like 36 straight hours of post-show festivities.
The Wichita native, who survived weeks of competition and dozens of other talented contestants to make it into the final four, said he was both relieved to be done with the strenuous process and still grateful and in disbelief about how far he made it and how much exposure he got.
But he also sounded more than a little discouraged about all the tension and drama that erupted during the final days of the competition — and he wondered if it might have hurt his chances.
"It's been a really sort of strange couple of days, I'm not going to lie," Mann said.
The drama is sort of hard to understand if you didn't watch the show closely and follow along on the Hollywood gossip blogs. But in essence, Mann's celebrity coach, singer Christina Aguilera, developed a bit of a beef with contestant Tony Lucca, who appeared with her on "The Mickey Mouse Club" back when both were kids.
She was critical of him on the air, which angered Lucca's celebrity coach, singer Adam Levine.
The angered ones, Mann said, countered by choosing Jay-Z's song "99 Problems," which contains an unflattering reference to a woman, and it was obvious that the woman the song was directed at was Aguilera.
She objected to Lucca singing the song with its original lyrics, and Levine objected to the objection.
Bloggers picked up on the unflattering feud, and many theorized that it contributed to Mann and Lucca finishing last among the four finalists.
(Jermaine Paul, Mann's roommate during the show, won the whole thing, and Juliet Simms was second.)
"I was so happy with how I sang and who I was and the way I was presenting myself," Mann said. "But the drama that was going on really overshadowed the music. I think it cost me in the end to have been associated with it."
I told Chris that I always assumed such drama was staged to keep things exciting on reality shows. But Mann assured me that — at least in this case — the tension was all too real.
He insisted that he wasn't angry, though, and said he was excited to see how the exposure would translate into his post-"Voice" career.
"I'm not mad," he said. "I represented myself musically, and I wasn't using PR stunts to advance. I couldn't be more happy for Jermaine. He was my best friend on the show."
Mann's next move?
First, he's taking some much-needed vacation time in Las Vegas.
Sometime in the next two weeks, his new single "Roads" should be digitally released and available on iTunes. He recorded the song, an inspirational tune, before "The Voice" started.
He has several appearances scheduled, including a homecoming concert in Wichita as part of the LIVfest on June 16 at The Orpheum.
Most important, though: Mann has to choose a recording contract. His managers are in discussions with several labels. Now, it's just a matter of choosing which one will be best for his career, which will follow the script of his appearance on "The Voice."
Mann hopes to make a name for himself singing the big, operatic, classical songs that helped him stand out on the show.
He wants to move fast and promises to keep Wichita updated on what he decides to do.
"I was ready for it to be done, and I just can't believe all that we did," he said. "But I got really got sad on Tuesday night when I got home. I thought, 'Man, I can't believe it's over. This is the most fun I've ever had.' "


text:
Planning Policy
Planning Policy documents provide the framework to deliver sustainable development in Hart.
The Planning Policy pages provide information on current and emerging planning policy in Hart and the supporting evidence base. They are also home to a suite of Conservation Area appraisals.
National planning policies for England are set out by the government in the National Planning Policy Framework (NPPF). Local planning policies must be prepared to be consistent with the NPPF.
Local Plan
The Local Plan contains the policies that have statutory weight in the determination of planning applications. Information on the current and emerging Local Plan for Hart is available at www.hart.gov.uk/local-plan
Community Infrastructure Development CIL
The Community Infrastructure Levy (CIL) is a new standard charge which local authorities in England and Wales can charge on most types of new development in their area. Funds collected through CIL will be used to help fund infrastructure needed to support the development proposed in the Council's emerging Local Plan.
Further details on CIL will be set out on the website in due course.
Neighbourhood Plans
Neighbourhood planning was introduced as a new power to communities by the Localism Act 2011. A Neighbourhood Plan can establish general planning policies for the development and use of land in a neighbourhood.
Conservation Areas
This area provides details of all the Conservation Areas in Hart. For further advice please contact firstname.lastname@example.com
How to contact Planning Policy
Email: email@example.com
Phone: 01252 774118


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SOURCE: Colgate-Palmolive
Catch Up with Reality Show Star Trista Sutter and Find Out How To Help Local Schools
The original Bachelorette, Trista Sutter talks about how she manages dental health for her own children and how she is teaming up with Colgate, Walmart and the not-for-profit organziation Champions for Kids for the SIMPLE Giving campaign, which provides oral care products to needy children in local school districts. Trista also talks about what she's been up to since her season of The Bachelorette, including her new book.
As part of the campaign, Colgate will also be donating $100,000 of oral care products to help school districts in need. You can help too by simply purchasing and donating toothbrushes, toothpaste and hand soap at your local Walmart from March 22 through April 7. All donations will go to a school district in your area.
Client firstname.lastname@example.com


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Jiggs pBone – The Plastic Trombone
The new Jiggs pBone trombones might look like a toy, but they are anything but! Designed in conjunction with professional trombonist Jiggs Whigham and distributed by Conn-Selmer, makers of Bach, Conn, King & Martin, the Jiggs pBone is a true musical instrument designed to revolutionize how we think about trombones.
Additional Information
Plastic Body
The body is made from a durable ABS plastic that is available in many different colors with the slide tubes being made of fiberglass. The handslide uses brass stockings and the instrument will use any standard small shank mouthpiece (though a matching color plastic mouthpiece is also included). The instrument comes in a padded gig bag and carries a 6 month manufacturer's warranty.
Fiberglass Slides
The Fiberglass slides will not require traditional lubrication and the instrument will not be subject to the same types of damage and repair as a traditional brass instrument. Best of all is the price. At this price, these instruments are beyond affordable and will make a great instrument for a beginner, marching band player or a gig bone as well!
Jiggs pBone Trombone Specifications
|Body Material
|Slide Material
|Fiberglass (brass stockings)
|.500″ Small Bore
|800 grams (less than 2 lbs)
|Includes a matched color plastic mouthpiece. Also fits standard small shank mouthpieces
|Available Colors
|Red, Blue, Green, Yellow, Purple & White
|6 Month Defects Warranty
Join the bell and slide sections together and secure them simply with resistance. Insert the mouthpiece gently into the mouthpiece receiver. Before first use: Pour a small amount of lukewarm water (NEVER HOT) into slide section and work the slide, rinse and drain. Run lukewarm water through the bell section. Rinse and drain.
Be very careful when using mutes as twisting the mute in the bell may damage the seam. Don't try too hard with plunger or wah wah mutes as this will dislodge the bell from the slide!
Tuning Slide
Remove tuning slide. Apply a generous amount of tuning slide grease or petroleum jelly to each slide and spread over entire surface. Insert tuning slide completely into receivers and wipe off excess grease with a soft cloth.
Apply a small amount of slide cream to the stockings or apply oil/liquid slide lube to the inner slide top, allowing the oil/lube to run down to the stockings. Spray inner slides with water and work up and down to distribute the cream/oil/lube.
Kessler & Sons Music recommends Slide-O-Mix "Rapid Comfort" lubricant on the Jiggs pBone.
When new the slide may feel slow and scratchy. The slide will "run-in" with use as the brass stockings polish themselves and at the same time smooth down the interior of the outer slides. During this "running-in period" you will need to clean the inner slides frequently with a lint free cloth or baby-wipe. You should expect to find a dark deposit both on your cloth and in the condensation coming from the water key, this is normal and shows that the stockings are doing their job smoothing down the interior of the outer slide tubes. With use and over time this process will diminish and disappear as the slide becomes quicker, quieter and smoother to run. You may find that the slide needs less lube during the "running-in" period. If during this period, the slide slows it will almost certainly be a build up of the dark deposit so always give the slide a good clean should it slow down.
To maintain your instrument's finish, never clean with hot water, chemical agents or harsh detergents or solvents. Use only lukewarm water for cleaning purposes. The colored parts can be cleaned with a soft cloth. The glass fibre outer slides may be cleaned with warm soapy water and then lightly dressed with vegetable oil to restore the look of the surface.


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You might also be interested: 1. Is Tofurky (Tofu turkey) Healthy? 2. Is Tofurky Gluten Free? 3. Why is my Cornbread Dry? 4. How to Substitute Eggs in Cornbread?Christmas Appetizers
|Four Bean Salad
|Baked Onion Garlic Soup
|Pumpkin and Peanut Butter Soup
|Christmas Tree Popcorn Clusters
|Creamy Artichode Spinach Dip
|Beet Hummus
|Vegetarian Shepherd's Pie
|Vegetarian Meat Loaf
|Tofu Turkey (Vegan)
|Easy vegetable Pot Pie
|Brown BUtter Pumpkin Risotto
|Stuffed Acorn Squash with with Mixed rice pilaf
|Green Beans Casserole
|Oven Roasted Fingerling Potatoes
|Skillet Jalapeno & Cheddar Cornbread
|Mashed Sweet Potatoes with Gojji Berries
|Maple Cranberry Sauce with Walnuts
|Healthy Buttermilk Mashed Potatoes
|Sugar Cookies
|Christmas Cookies
|Gingerbread (Wo)Man Cookies
|Nutritious Gingersnaps
|Ginger Cookies
|Honey Walnut Pumpkin Bread
|White Dinner Rolls
|Quinoa Dinner Rolls
|Bulgur Dinner Rolls
|Cranberry Orange Bread
|Cinnamon Chocolate Brownies
|Coconut Tres Leches Hot Chocolate
|Moist Quinoa Chocolate Cake
|Apple Rosemary Pie
|Pineapple Upside Down Cake
|Ricotta Cheesecake
|Hummingbird Cake (Lightened)
|Beetroot Chocolate Brownies
|Classic Carrot Cake
|Brown Sugar Toffee Cake
|Wholegrain Pumpkin Pie
|Austrian Apple Strudel


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I asked this young man what type of items he liked to buy when he visits the flea market. " When you've been coming here as long as I have, you develop a taste for everything" he said. Bravo!
Take special note of the pink check shirt with pink and white stripe ribbon belt


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'04 Vince Carter
Premium / All-Star Weekend
General information
ThemeAll Star Weekend
Height6'6" (198cm)
Weight220lbs (99kg)
TeamToronto Raptors
FromNorth Carolina
Dynamic Duo97 Jason Kidd
PlaysP&R Ball Handler Mid Range 3 PT Guard Post Up
98 Overall
96 Outside scoring +2
- 96 Open shot mid +3
- 96 Contested shot mid +3
- 96 Off dribble shot mid +3
- 96 Open shot 3pt +2
- 96 Contested shot 3pt +2
- 96 Off dribble shot 3pt +2
- 98 Shot IQ
- 95 Free throw +3
- 98 OffensiveOff. consistency
94 Athleticism +1
- 96 Speed +2
- 96 Acceleration
- 99 Vertical
- 74 Strength
- 98 Stamina
- 95 Hustle
- 95 Overall durability
90 Inside scoring +5
- 98 Shot close
- 96 Standing layup +3
- 96 Driving layup +3
- 80 Standing dunk +19
- 99 Driving dunk
- 99 Contact dunk
- 97 Draw foul +2
- 75 Post control +5
- 46 Post hook +32
- 90 Post fadeaway +3
- 98 Hands
82 Playmaking +5
- 93 Ball control +2
- 82 Passing accuracy +13
- 70 Passing vision
- 85 Passing IQ +10
- 90 Speed with ball +4
84 Defending +9
- 85 On-ball defensedef. IQ +11
- 60 Low post defensedef. IQ +15
- 84 Pick & roll defensedef. IQ +12
- 85 Help defensedef. IQ +11
- 95 Lateral quickness +2
- 88 Pass perception +7
- 95 Reaction time
- 88 Steal +6
- 65 Block +15
- 90 Shot contest +7
- 85 DefensiveDef. consistency +10
70 Rebounding +10
- 62 Offensive rebound +16
- 72 Defensive rebound +8
- 75 Boxout +5
- 98 Potential
- 60 Intangibles
- 4043 Total attributesattr. +241
Comments Sort by
Greatest dunker of all time. Don't even get me started
cedi osman
when a reply gets more likes than the comment itself...Show 2 replies...
Reverse 360 windmill. Greatest dunk of all time. It's not just the dunk itself but the execution, the power, the grace, the style. How he starts screaming as he's in midair. SO SICK!!!
So true. My favorite dunk of his is the Honey Dip but the windmill 360...
Absolutely legendary
Idk the Aaron Gordon under the legs windmill over someone's head might be betterShow 5 replies...
@Kai2139 L
gordon's under the legs dunk over the mascot is the best of all time imo. that mascot is almost 7 feet tall, so when you add on the hoverboard and the diameter of the ball, gordon pretty much cleared 8 feet. he did an under the legs dunk 8 feet in the air
GDOAT (Greatest Dunker of All Time)
This man was dunking in Grade 6... let that sink in. Truly the GDOATShow 8 replies...
he is still dunking on NBA players at age 40... let that sink in. Truly the GDOAT
Fun fact Demar Derozan could dunk over his dad in grade 6. That's crazy
There is videos of Webber dunking at 12-13. It's remarkable the God given Athleticism some of these guys have.
fym god gave them athleticism, do you think god gave them thousands of hours of hard work?
It's natural talent on top of that hard work! LeBron James wouldn't be the same if he didn't have that amazing frame that he has. What if he was 5'9"? He would Still be an amazing talent. It's a figure of speech. I'm saying it's the work on top of that natural gifts that make them great.
Kobe is a 6'6" man with an insane body
Wilt was the prime example of natural athleticism
Shaq was able to move in an enamoring way despite being 300+ pounds
Anthony Davis is a monster athletically and developed a skill set to match it
Mark Eaton was a behemoth before he learned to become an amazing shot blocker
See what I mean bro?
this dunk earned a 50/50 at the dunk contest
Did Julius Erving fall off the face of the Earth or something?
jason richardson
Its over Ladies and Gentlemen
I said that with Kenny Smith's voice
one of the most iconic phrases smith ever uttered
100 Driving Dunk Tendencies
100 Flashy Dunk Tendencies
100 Old AF for the NBA Tendencies
Pretty good tendencies to me
Half-Man, Half-Holy Fucking Shit
41 and still going
age is just a number ;)
The Card that will lead my myteam to 82-0
Except I have 0% chance of having enough money to buy it
Rate my lineup? http://2kmtcentral.com/18/lineups/369283/op-sqaud
Rate lineup http://2kmtcentral.com/18/lineups/387387/tmac
HOF limitless?? No way 2k
Woo! Those outside scoring stats are so damn fire once I get sweaty when I saw this
This is a good card... No wait... it's..
Whoever gets this card is going to win EVERY. SINGLE. GAME.
Why does it he is a 97 when other says he 98
best pic
My boy just got this card for 85k
The dunk that put the crowd in silence
HOF LIMITLESS RANGEO!!! 96 THREE!!!!! 99 DUNKS and 100 DUNK TENDENCY!!!!!! IT'S TOO DEADDLY (not a gento quote)
Half man, Half amazing. 41 years old with a 43 in vert
vince carter more like vince farter lol got em deez nutsssssszszszszsszsz
vince carter is amazing because he literally put is whole arm down in the hoop
in the menu it says he is a 98 and then here he is a 97 can someone help me out here
Started at 97, now he's 98 with better defense stats
oh :(
Dear 2k, if you're gonna make these premium packs retardedly expensive can you atleast make it so we don't pull bronzes and silvers 15/20 cards. Still don't understand why they are 220,000 vc. Take tips from EA and make it so we get a 90ovr ATLEAST.
Don't think these are his final stats, has worse defense than his amethyst card and higher tier versions always suppose to be better in 2k18, don't make any sense
Should be a PD
Really satisfied by this card but... THAT GUY DOESN'T EVEN LOOKS LIKE VINCE CARTER !
2K please, make an effort so the current VC wouldn't be the only version to see a realistic face on the screen...
glad i saved up mt
99 fucking jump over a 7 foot tall center
its says 98 overall when i type his name
but it says 97 '
like bruh what?
The elbow dunks he put up were wet
lmao he so ovverated he so trash
Wait why the hell doesn't he have a dynamic duo with Tracy? This would make both him and Mcgrady 99's or on the verge of a 99 and with a shoe a 99
Hof limitless?? Cmon this game is the funniest so far, u re making it so bad, cant wait to see marc gasol with hof limitless and c&s badges ffs, this year, there is 15+- players with hof limitless, literally u can create a hof limitless range squad with some rly rly not great shooters, this game is so bad so far, start making it real for once in lifetime
but the thing is vince carter is actually a good shooter, also damn why you comparing him to marc gasol
He was a great shooter but he dosent deserve limitless range
why this dunk why not the 360
cause it's more iconic
99 driving dunk ohhh
In all seriousness does anyone else think that his dunks in the 2000 contest were extremely overrated and not impressive
I mean overrated maybe but they were still quite impressive
if he did those dunks in the 2016 dunk contest he would be easily forgotten. I myself can nearly do his elbow dunk and i'm not even athletic.Show 9 replies...
send video
How tall are you
I find it hard to believe anyone on this site is above the height of 5'10" and older than 13
Makes sense he is 6,6
I am also following you