Lisa Jackson’s research while affiliated with Noble Research Institute and other places

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

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

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.

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.

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., PNAS 109: 1772-1777, 2012). 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-hydroxy-guaiacyl units, the first report of such a polymer occurring naturally in plants, and seeds of some species contain no lignin at all. We discuss the implications of these findings for the mechanisms underlying the biosynthesis of these newly discovered lignin types. © 2012 The Authors. The Plant Journal © 2012 Blackwell Publishing Ltd.

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

Down-regulation of the enzyme hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in thale cress (Arabidopsis thaliana) and alfalfa (Medicago sativa) leads to strongly reduced lignin levels, reduced recalcitrance of cell walls to sugar release, but severe stunting of the plants. Levels of the stress hormone salicylic acid (SA) are inversely proportional to lignin levels and growth in a series of transgenic alfalfa plants in which lignin biosynthesis has been perturbed at different biosynthetic steps. Reduction of SA levels by genetically blocking its formation or causing its removal restores growth in HCT-down-regulated Arabidopsis, although the plants maintain reduced lignin levels. SA-mediated growth inhibition may occur via interference with gibberellic acid signaling or responsiveness. Our data place SA as a central component in growth signaling pathways that either sense flux into the monolignol pathway or respond to secondary cell-wall integrity, and indicate that it is possible to engineer plants with highly reduced cell-wall recalcitrance without negatively impacting growth.

As a major component of the cell wall, lignin plays an important role in plant development and defense response to pathogens, but negatively impacts biomass processability for biofuels. Silencing the target lignin genes for greater biomass processability should not significantly affect plant development and biomass yield but also must not compromise disease resistance. Here, we report experiments to identify a set of lignin genes that may be silenced without compromising disease resistance. We profiled the expression of 32 lignin biosynthetic candidate genes by qRT-PCR in 17 wheat tissues collected at three developmental stages. Twenty-one genes were expressed at a much higher level in stems compared to sheaths and leaf blades. Expression of seven these genes significantly correlated with lignin content. The co-expression patterns indicated that these 21 genes are under strong developmental regulation and may play a role in lignin biosynthesis. Profiling gene expression of same tissues challenged by two fungal pathogens, Fusarium graminearum and Puccina triticina indicated that expression of 17 genes was induced by F. graminearum. Only PAL1, a non-developmental-regulated gene, was induced by P. triticina. Thus, lignin biosynthetic pathway overlaps defense response to F. graminearum. Based on these criteria, 17 genes, F5H1, F5H2, 4CL2, CCR2, COMT1, and COMT2 in particular that do not overlap with disease resistance pathway, may be the targets for downregulation. KeywordsLignin–Wheat–Development–Defense response–Quantitative real time PCR

Cinnamoyl CoA reductases (CCR) convert hydroxycinnamoyl CoA esters to their corresponding cinnamyl aldehydes in monolignol biosynthesis. We identified two CCR genes in the model legume Medicago truncatula. CCR1 exhibits preference for feruloyl CoA, but CCR2 prefers caffeoyl and 4-coumaroyl CoAs, exhibits sigmoidal kinetics with these substrates, and is substrate-inhibited by feruloyl and sinapoyl CoAs. M. truncatula lines harboring transposon insertions in CCR1 exhibit drastically reduced growth and lignin content, whereas CCR2 knockouts grow normally with moderate reduction in lignin levels. CCR1 fully and CCR2 partially complement the irregular xylem gene 4 CCR mutation of Arabidopsis. The expression of caffeoyl CoA 3-O-methyltransferase (CCoAOMT) is up-regulated in CCR2 knockout lines; conversely, knockout of CCoAOMT up-regulates CCR2. These observations suggest that CCR2 is involved in a route to monolignols in Medicago whereby coniferaldehyde is formed via caffeyl aldehyde which then is 3-O-methylated by caffeic acid O-methyltransferase.

A series of transgenic lines of alfalfa (Medicago sativa) were generated in which either one of the two potentially terminal enzymes of the monolignol pathway, cinnamoyl CoA reductase (CCR) or cinnamyl alcohol dehydrogenase (CAD) was down-regulated by expression of antisense transgenes. Levels of CCR enzymatic activity were reduced to between 10% to 65% of the control level, and levels of CAD activity were similarly reduced to between 5% to 40% of the control. Biomass yields were reduced in the most strongly down-regulated lines for both transgenes, but many of the lines exhibited reduced lignin levels but normal biomass and flowering time. In vitro dry matter digestibility was increased for most transgenic lines compared to controls. Saccharification efficiency was determined by measuring the release of sugars from cell walls directly, or after sulfuric acid pre-treatment and subsequent digestion with a mixture of cellulase and cellobiase. Several CCR down-regulated lines had significantly enhanced saccharification efficiency with both pre-treated and untreated tissues, whereas CAD down-regulation had less impact on sugar release when compared to that from CCR lines with similar lignin contents. One CCR line with a 50–60% improvement in saccharification efficiency exhibited normal biomass production, indicating the potential for producing high yielding, improved feedstocks for bioethanol production through genetic modification of the monolignol pathway.