Yuki Tobimatsu

Disruption of Mediator rescues the stunted growth of a lignin-deficient Arabidopsis mutant

Lignin is a phenylpropanoid-derived heteropolymer important for the strength and rigidity of the plant secondary cell wall. Genetic disruption of lignin biosynthesis has been proposed as a means to improve forage and bioenergy crops, but frequently results in stunted growth and developmental abnormalities, the mechanisms of which are poorly understood. Here we show that the phenotype of a lignin-deficient Arabidopsis mutant is dependent on the transcriptional co-regulatory complex, Mediator. Disruption of the Mediator complex subunits MED5a (also known as REF4) and MED5b (also known as RFR1) rescues the stunted growth, lignin deficiency and widespread changes in gene expression seen in the phenylpropanoid pathway mutant ref8, without restoring the synthesis of guaiacyl and syringyl lignin subunits. Cell walls of rescued med5a/5b ref8 plants instead contain a novel lignin consisting almost exclusively of p-hydroxyphenyl lignin subunits, and moreover exhibit substantially facilitated polysaccharide saccharification. These results demonstrate that guaiacyl and syringyl lignin subunits are largely dispensable for normal growth and development, implicate Mediator in an active transcriptional process responsible for dwarfing and inhibition of lignin biosynthesis, and suggest that the transcription machinery and signalling pathways responding to cell wall defects may be important targets to include in efforts to reduce biomass recalcitrance.

A polymer of caffeyl alcohol in plant seeds

Lignins are complex phenylpropanoid polymers mostly associated with plant secondary cell walls. Lignins arise primarily via oxidative polymerization of the three monolignols, p-coumaryl, coniferyl, and sinapyl alcohols. Of the two hydroxycinnamyl alcohols that represent incompletely methylated biosynthetic products (and are not usually considered to be monolignols), 5-hydroxyconiferyl alcohol is now well established as incorporating into angiosperm lignins, but incorporation of caffeyl alcohol has not been shown. We report here the presence of a homopolymer of caffeyl alcohol in the seed coats of both monocot and dicot plants. This polymer (C-lignin) is deposited to high concentrations in the seed coat during the early stages of seed development in the vanilla orchid (Vanilla planifolia), and in several members of the Cactaceae. The lignin in other parts of the Vanilla plant is conventionally biosynthesized from coniferyl and sinapyl alcohols. Some species of cacti contain only C-lignin in their seeds, whereas others contain only classical guaiacyl/syringyl lignin (derived from coniferyl and sinapyl alcohols). NMR spectroscopic analysis revealed that the Vanilla seed-coat polymer was massively comprised of benzodioxane units and was structurally similar to the polymer synthesized in vitro by peroxidase-catalyzed polymerization of caffeyl alcohol. CD spectroscopy did not detect any optical activity in the seed polymer. These data support the contention that the C-lignin polymer is produced in vivo via combinatorial oxidative radical coupling that is under simple chemical control, a mechanism analogous to that theorized for classical lignin biosynthesis.

Laccases Direct Lignification in the Discrete Secondary Cell Wall Domains of Protoxylem

The discrete localization of laccases to secondary cell walls directs lignification in protoxylem. Plants precisely control lignin deposition in spiral or annular secondary cell wall domains during protoxylem tracheary element (TE) development. Because protoxylem TEs function to transport water within rapidly elongating tissues, it is important that lignin deposition is restricted to the secondary cell walls in order to preserve the plasticity of adjacent primary wall domains. The Arabidopsis (Arabidopsis thaliana) inducible VASCULAR NAC DOMAIN7 (VND7) protoxylem TE differentiation system permits the use of mutant backgrounds, fluorescent protein tagging, and high-resolution live-cell imaging of xylem cells during secondary cell wall development. Enzymes synthesizing monolignols, as well as putative monolignol transporters, showed a uniform distribution during protoxylem TE differentiation. By contrast, the oxidative enzymes LACCASE4 (LAC4) and LAC17 were spatially localized to secondary cell walls throughout protoxylem TE differentiation. These data support the hypothesis that precise delivery of oxidative enzymes determines the pattern of cell wall lignification. This view was supported by lac4lac17 mutant analysis demonstrating that laccases are necessary for protoxylem TE lignification. Overexpression studies showed that laccases are sufficient to catalyze ectopic lignin polymerization in primary cell walls when exogenous monolignols are supplied. Our data support a model of protoxylem TE lignification in which monolignols are highly mobile once exported to the cell wall, and in which precise targeting of laccases to secondary cell wall domains directs lignin deposition.

Loss of function of cinnamyl alcohol dehydrogenase 1 leads to unconventional lignin and a temperature-sensitive growth defect in Medicago truncatula

There is considerable debate over the capacity of the cell wall polymer lignin to incorporate unnatural monomer units. We have identified Tnt1 retrotransposon insertion mutants of barrel medic (Medicago truncatula) that show reduced lignin autofluorescence under UV microscopy and red coloration in interfascicular fibers. The phenotype is caused by insertion of retrotransposons into a gene annotated as encoding cinnamyl alcohol dehydrogenase, here designated M. truncatula CAD1. NMR analysis indicated that the lignin is derived almost exclusively from coniferaldehyde and sinapaldehyde and is therefore strikingly different from classical lignins, which are derived mainly from coniferyl and sinapyl alcohols. Despite such a major alteration in lignin structure, the plants appear normal under standard conditions in the greenhouse or growth chamber. However, the plants are dwarfed when grown at 30 °C. Glycome profiling revealed an increased extractability of some xylan and pectin epitopes from the cell walls of the cad1-1 mutant but decreased extractability of others, suggesting that aldehyde-dominant lignin significantly alters cell wall structure.

Coexistence but Independent Biosynthesis of Catechyl and Guaiacyl/Syringyl Lignin Polymers in Seed Coats

This study provides evidence that several dicot plants (Euphorbiaceae and Cleomaceae) produce two distinctively different types of lignin polymers (i.e., catechyl and guaiacyl/syringyl lignins) in a spatio-temporally specific manner within the walls of their seed coat cells. The findings highlight the flexible and elaborately controlled assembly of lignin polymers in plant cell walls. Lignins are phenylpropanoid polymers, derived from monolignols, commonly found in terrestrial plant secondary cell walls. We recently reported evidence of an unanticipated catechyl lignin homopolymer (C lignin) derived solely from caffeyl alcohol in the seed coats of several monocot and dicot plants. We previously identified plant seeds that possessed either C lignin or traditional guaiacyl/syringyl (G/S) lignins, but not both. Here, we identified several dicot plants (Euphorbiaceae and Cleomaceae) that produce C lignin together with traditional G/S lignins in their seed coats. Solution-state NMR analyses, along with an in vitro lignin polymerization study, determined that there is, however, no copolymerization detectable (i.e., that the synthesis and polymerization of caffeyl alcohol and conventional monolignols in vivo is spatially and/or temporally separated). In particular, the deposition of G and C lignins in Cleome hassleriana seed coats is developmentally regulated during seed maturation; C lignin appears successively after G lignin within the same testa layers, concurrently with apparent loss of the functionality of O-methyltransferases, which are key enzymes for the conversion of C to G lignin precursors. This study exemplifies the flexible biosynthesis of different types of lignin polymers in plants dictated by substantial, but poorly understood, control of monomer supply by the cells.

Manipulation of Guaiacyl and Syringyl Monomer Biosynthesis in an Arabidopsis Cinnamyl Alcohol Dehydrogenase Mutant Results in Atypical Lignin Biosynthesis and Modified Cell Wall Structure

The phenotypes of Arabidopsis plants with altered aldehyde contents and guaiacyl-to-syringyl ratios provide insights into the effects of lignin composition on plant growth and cell wall degradability. Modifying lignin composition and structure is a key strategy to increase plant cell wall digestibility for biofuel production. Disruption of the genes encoding both cinnamyl alcohol dehydrogenases (CADs), including CADC and CADD, in Arabidopsis thaliana results in the atypical incorporation of hydroxycinnamaldehydes into lignin. Another strategy to change lignin composition is downregulation or overexpression of ferulate 5-hydroxylase (F5H), which results in lignins enriched in guaiacyl or syringyl units, respectively. Here, we combined these approaches to generate plants enriched in coniferaldehyde-derived lignin units or lignins derived primarily from sinapaldehyde. The cadc cadd and ferulic acid hydroxylase1 (fah1) cadc cadd plants are similar in growth to wild-type plants even though their lignin compositions are drastically altered. In contrast, disruption of CAD in the F5H-overexpressing background results in dwarfism. The dwarfed phenotype observed in these plants does not appear to be related to collapsed xylem, a hallmark of many other lignin-deficient dwarf mutants. cadc cadd, fah1 cadc cadd, and cadd F5H-overexpressing plants have increased enzyme-catalyzed cell wall digestibility. Given that these CAD-deficient plants have similar total lignin contents and only differ in the amounts of hydroxycinnamaldehyde monomer incorporation, these results suggest that hydroxycinnamaldehyde content is a more important determinant of digestibility than lignin content.

Novel seed coat lignins in the Cactaceae: structure, distribution and implications for the evolution of lignin diversity

We have recently described a hitherto unsuspected catechyl lignin polymer (C-lignin) in the seed coats of Vanilla orchid and in cacti of one genus, Melocactus (Chen et al., Proc. Natl. Acad. Sci. USA. 2012, 109, 1772-1777.). We have now determined the lignin types in the seed coats of 130 different cactus species. Lignin in the vegetative tissues of cacti is of the normal guaiacyl/syringyl (G/S) type, but members of most genera within the subfamily Cactoidae possess seed coat lignin of the novel C-type only, which we show is a homopolymer formed by endwise β-O-4-coupling of caffeyl alcohol monomers onto the growing polymer resulting in benzodioxane units. However, the species examined within the genera Coryphantha, Cumarinia, Escobaria and Mammillaria (Cactoideae) mostly had normal G/S lignin in their seeds, as did all six species in the subfamily Opuntioidae that were examined. Seed coat lignin composition is still evolving in the Cactaceae, as seeds of one Mammillaria species (M. lasiacantha) possess only C-lignin, three Escobaria species (E. dasyacantha, E. lloydii and E. zilziana) contain an unusual lignin composed of 5-hydroxyguaiacyl units, the first report of such a polymer that occurs naturally in plants, and seeds of some species contain no lignin at all. We discuss the implications of these findings for the mechanisms that underlie the biosynthesis of these newly discovered lignin types.

Syringyl lignin production in conifers: Proof of concept in a Pine tracheary element system.

Significance This study shows that metabolic engineering can be used to imbue pine tracheary elements with an ability to synthesize sinapyl alcohol, a lignin monomer not normally used for lignification in conifers such as pine. The dynamic nature of the lignification process enables pines to incorporate this monolignol, allowing them to produce hardwood-like lignins that are known to facilitate refining processes such as biofuel production and chemical pulping. The potential to improve the refining of conifer-derived biomass through lignin manipulations is important, as even small improvements in yield can lead to significant environmental and economic benefits in such processes. Conifers (softwoods) naturally lack syringyl units in their lignins, rendering lignocellulosic materials from such species more difficult to process than syringyl-rich hardwood species. Using a transformable Pinus radiata tracheary element (TE) system as an experimental platform, we investigated whether metabolic engineering can be used to create syringyl lignin in conifers. Pyrolysis-GC/MS and 2D-NMR analysis of P. radiata TE cultures transformed to express ferulate 5-hydroxylase (F5H) and caffeic acid O-methyltransferase (COMT) from Liquidambar styraciflua confirmed the production and incorporation of sinapyl alcohol into the lignin polymer. Transformation with F5H was sufficient for the production of syringyl lignin in TEs, but cotransformation with COMT improved its formation. In addition, lower levels of the pathway intermediate 5-hydroxyconiferyl alcohol were evidenced in cotransformation experiments, indicating that the introduction of the COMT overcame the inefficiency of the native pine methyltransferases for supporting sinapyl alcohol production.Our results provide the proof of concept that it is possible to generate a lignin polymer that contains syringyl units in softwood species such as P. radiata, suggesting that it might be possible to retain the outstanding fiber properties of softwoods while imbuing them with the lignin characteristics of hardwoods that are more favorable for industrial processing.

Hydroxycinnamate Conjugates as Potential Monolignol Replacements: In vitro Lignification and Cell Wall Studies with Rosmarinic Acid

The plasticity of lignin biosynthesis should permit the inclusion of new compatible phenolic monomers, such as rosmarinic acid (RA) and analogous catechol derivatives, into cell-wall lignins that are consequently less recalcitrant to biomass processing. In vitro lignin polymerization experiments revealed that RA readily underwent peroxidase-catalyzed copolymerization with monolignols and lignin oligomers to form polymers with new benzodioxane inter-unit linkages. Incorporation of RA permitted extensive depolymerization of synthetic lignins by mild alkaline hydrolysis, presumably by cleavage of ester intra-unit linkages within RA. Copolymerization of RA with monolignols into maize cell walls by in situ peroxidases significantly enhanced alkaline lignin extractability and promoted subsequent cell wall saccharification by fungal enzymes. Incorporating RA also improved cell wall saccharification by fungal enzymes and by rumen microflora even without alkaline pretreatments, possibly by modulating lignin hydrophobicity and/or limiting cell wall cross-linking. Consequently, we anticipate that bioengineering approaches for partial monolignol substitution with RA and analogous plant hydroxycinnamates would permit more efficient utilization of plant fiber for biofuels or livestock production.

Visualization of plant cell wall lignification using fluorescence‐tagged monolignols

Lignin is an abundant phenylpropanoid polymer produced by the oxidative polymerization of p-hydroxycinnamyl alcohols (monolignols). Lignification, i.e., deposition of lignin, is a defining feature of secondary cell wall formation in vascular plants, and provides an important mechanism for their disease resistance; however, many aspects of the cell wall lignification process remain unclear partly because of a lack of suitable imaging methods to monitor the process in vivo. In this study, a set of monolignol analogs γ-linked to fluorogenic aminocoumarin and nitrobenzofuran dyes were synthesized and tested as imaging probes to visualize the cell wall lignification process in Arabidopsis thaliana and Pinus radiata under various feeding regimens. In particular, we demonstrate that the fluorescence-tagged monolignol analogs can penetrate into live plant tissues and cells, and appear to be metabolically incorporated into lignifying cell walls in a highly specific manner. The localization of the fluorogenic lignins synthesized during the feeding period can be readily visualized by fluorescence microscopy and is distinguishable from the other wall components such as polysaccharides as well as the pre-existing lignin that was deposited earlier in development.