Rapid, Microscale, Acetyl Bromide-Based Method for High-Throughput Determination of Lignin Content in Arabidopsis thaliana

Chang, Xue Feng; Chandra, Richard P.; Berleth, Thomas; Beatson, Rodger P.

doi:10.1021/jf800775f

Cited by 97 publications

(70 citation statements)

References 30 publications

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“…Extracts were separated by HPLC using the same method described above for HCEs, and metabolites were quantified using authentic standards obtained from Sigma-Aldrich. Lignin content was determined using the acetyl bromide method as described by Fukushima and Hatfield (2004), using the extinction coefficient 23.35 g L 21 cm 21 as determined by Chang et al, (2008).…”

Section: Cell Wall-bound Secondary Metabolite Analysismentioning

confidence: 99%

Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

et al. 2015

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Phenylpropanoids are phenylalanine-derived specialized metabolites and include important structural components of plant cell walls, such as lignin and hydroxycinnamic acids, as well as ultraviolet and visible light-absorbing pigments, such as hydroxycinnamate esters (HCEs) and anthocyanins. Previous work has revealed a remarkable degree of plasticity in HCE biosynthesis, such that most Arabidopsis (Arabidopsis thaliana) mutants with blockages in the pathway simply redirect carbon flux to atypical HCEs. In contrast, the ferulic acid hydroxylase1 (fah1) mutant accumulates greatly reduced levels of HCEs, suggesting that phenylpropanoid biosynthesis may be repressed in response to the loss of FERULATE 5-HYDROXYLASE (F5H) activity. Here, we show that in fah1 mutant plants, the activity of HCE biosynthetic enzymes is not limiting for HCE accumulation, nor is phenylpropanoid flux diverted to the synthesis of cell wall components or flavonol glycosides. We further show that anthocyanin accumulation is also repressed in fah1 mutants and that this repression is specific to tissues in which F5H is normally expressed. Finally, we show that repression of both HCE and anthocyanin biosynthesis in fah1 mutants is dependent on the MED5a/5b subunits of the transcriptional coregulatory complex Mediator, which are similarly required for the repression of lignin biosynthesis and the stunted growth of the phenylpropanoid pathway mutant reduced epidermal fluorescence8. Taken together, these observations show that the synthesis of HCEs and anthocyanins is actively repressed in a MEDIATOR-dependent manner in Arabidopsis fah1 mutants and support an emerging model in which MED5a/5b act as central players in the homeostatic repression of phenylpropanoid metabolism.

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Section: Cell Wall-bound Secondary Metabolite Analysismentioning

confidence: 99%

Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

et al. 2015

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“…These data point to a reduction in lignin content in the cell walls of the MOMT transgenic lines. To assess the quantitative alteration in lignin deposition as a consequence of the expression of MOMT4, we measured the total lignin content of 10-week-old T2 generation transgenic plants using the acetyl bromide method (Iiyama and Wallis, 1990;Chang et al, 2008). Lignin content in all MOMT4 transgenic plants was reduced, and the maximum reduction reached;24% in transgenic line MOMT4-3, compared with the control plants ( Table 2).…”

Section: Expressing Momt4 In Arabidopsis Lowers Lignin Content In Thementioning

confidence: 99%

“…The wall-bound phenolics were extracted with 2 N NaOH in the dark at 37°C for 16 h and analyzed as described (Gou et al, 2008). Total lignin was quantified by the acetyl bromide method (Chang et al, 2008). To analyze the monomeric composition of lignin, we followed the thioacidolysis method (Rolando et al, 1992).…”

Section: Analysis Of Wall-bound Phenolics and Ligninmentioning

confidence: 99%

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

et al. 2012

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Although the practice of protein engineering is industrially fruitful in creating biocatalysts and therapeutic proteins, applications of analogous techniques in the field of plant metabolic engineering are still in their infancy. Lignins are aromatic natural polymers derived from the oxidative polymerization of primarily three different hydroxycinnamyl alcohols, the monolignols. Polymerization of lignin starts with the oxidation of monolignols, followed by endwise cross-coupling of (radicals of) a monolignol and the growing oligomer/polymer. The para-hydroxyl of each monolignol is crucial for radical generation and subsequent coupling. Here, we describe the structure-function analysis and catalytic improvement of an artificial monolignol 4-O-methyltransferase created by iterative saturation mutagenesis and its use in modulating lignin and phenylpropanoid biosynthesis. We show that expressing the created enzyme in planta, thus etherifying the para-hydroxyls of lignin monomeric precursors, denies the derived monolignols any participation in the subsequent coupling process, substantially reducing lignification and, ultimately, lignin content. Concomitantly, the transgenic plants accumulated de novo synthesized 4-O-methylated soluble phenolics and wall-bound esters. The lower lignin levels of transgenic plants resulted in higher saccharification yields. Our study, through a structure-based protein engineering approach, offers a novel strategy for modulating phenylpropanoid/lignin biosynthesis to improve cell wall digestibility and diversify the repertories of biologically active compounds.

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“…Simple phenolic content (monomeric phenolic compounds) was determined by the Folin-Ciocalteu method (51). Lignin content was determined by the rapid microscale acetyl bromide-based method (15). Carbon and nitrogen concentrations were determined using a Costech Elemental Analyzer model 4010 (Analytical Technologies, Inc., Valencia, CA).…”

Section: Methodsmentioning

confidence: 99%

Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO ₂

Kelly

Bansal

Winkelman

et al. 2010

Appl Environ Microbiol

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Elevated atmospheric CO 2 can cause increased carbon fixation and altered foliar chemical composition in a variety of plants, which has the potential to impact forested headwater streams because they are detritusbased ecosystems that rely on leaf litter as their primary source of organic carbon. Fungi and bacteria play key roles in the entry of terrestrial carbon into aquatic food webs, as they decompose leaf litter and serve as a source of nutrition for invertebrate consumers. This study tested the hypothesis that changes in leaf chemistry caused by elevated atmospheric CO 2 would result in changes in the size and composition of microbial communities colonizing leaves in a woodland stream. Three tree species, Populus tremuloides, Salix alba, and Acer saccharum, were grown under ambient (360 ppm) or elevated (720 ppm) CO 2 , and their leaves were incubated in a woodland stream. Elevated-CO 2 treatment resulted in significant increases in the phenolic and tannin contents and C/N ratios of leaves. Microbial effects, which occurred only for P. tremuloides leaves, included decreased fungal biomass and decreased bacterial counts. Analysis of fungal and bacterial communities on P. tremuloides leaves via terminal restriction fragment length polymorphism (T-RFLP) and clone library sequencing revealed that fungal community composition was mostly unchanged by the elevated-CO 2 treatment, whereas bacterial communities showed a significant shift in composition and a significant increase in diversity. Specific changes in bacterial communities included increased numbers of alphaproteobacterial and cytophaga-flavobacter-bacteroides (CFB) group sequences and decreased numbers of betaproteobacterial and firmicutes sequences, as well as a pronounced decrease in overall Gram-positive bacterial sequences.

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Rapid, Microscale, Acetyl Bromide-Based Method for High-Throughput Determination of Lignin Content in Arabidopsis thaliana

Cited by 97 publications

References 30 publications

Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO ₂

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Rapid, Microscale, Acetyl Bromide-Based Method for High-Throughput Determination of Lignin Content in Arabidopsis thaliana

Cited by 97 publications

References 30 publications

Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

Loss of ferulate 5-hydroxylase leads to Mediator-dependent inhibition of soluble phenylpropanoid biosynthesis in Arabidopsis

An Engineered Monolignol 4-O-Methyltransferase Depresses Lignin Biosynthesis and Confers Novel Metabolic Capability in Arabidopsis

Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO 2

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Alteration of Microbial Communities Colonizing Leaf Litter in a Temperate Woodland Stream by Growth of Trees under Conditions of Elevated Atmospheric CO ₂