Targeting hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase for lignin modification in Brachypodium distachyon.

BACKGROUND: Hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) is a central enzyme of the so-called "esters" pathway to monolignols. As originally envisioned, HCT functions twice in this pathway, to form coumaroyl shikimate and then, in the "reverse" direction, to convert caffeoyl shikimate to caffeoyl CoA. The discovery of a caffeoyl shikimate esterase (CSE) that forms caffeic acid directly from caffeoyl shikimate calls into question the need for the reverse HCT reaction in lignin biosynthesis. Loss of function of HCT gives severe growth phenotypes in several dicot plants, but less so in some monocots, questioning whether this enzyme, and therefore the shikimate shunt, plays the same role in both monocots and dicots. The model grass Brachypodium distachyon has two HCT genes, but lacks a classical CSE gene. This study was therefore conducted to evaluate the utility of HCT as a target for lignin modification in a species with an "incomplete" shikimate shunt. RESULTS: The kinetic properties of recombinant B. distachyon HCTs were compared with those from Arabidopsis thaliana, Medicago truncatula, and Panicum virgatum (switchgrass) for both the forward and reverse reactions. Along with two M. truncatula HCTs, B. distachyon HCT2 had the least kinetically unfavorable reverse HCT reaction, and this enzyme is induced when HCT1 is down-regulated. Down regulation of B. distachyon HCT1, or co-down-regulation of HCT1 and HCT2, by RNA interference led to reduced lignin levels, with only modest changes in lignin composition and molecular weight. CONCLUSIONS: Down-regulation of HCT1, or co-down-regulation of both HCT genes, in B. distachyon results in less extensive changes in lignin content/composition and cell wall structure than observed following HCT down-regulation in dicots, with little negative impact on biomass yield. Nevertheless, HCT down-regulation leads to significant improvements in biomass saccharification efficiency, making this gene a preferred target for biotechnological improvement of grasses for bioprocessing.

ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1) releases latent defense signals in stems with reduced lignin content.

There is considerable interest in engineering plant cell wall components, particularly lignin, to improve forage quality and biomass properties for processing to fuels and bioproducts. However, modifying lignin content and/or composition in transgenic plants through down-regulation of lignin biosynthetic enzymes can induce expression of defense response genes in the absence of biotic or abiotic stress. Arabidopsis thaliana lines with altered lignin through down-regulation of hydroxycinnamoyl CoA:shikimate/quinate hydroxycinnamoyl transferase (HCT) or loss of function of cinnamoyl CoA reductase 1 (CCR1) express a suite of pathogenesis-related (PR) protein genes. The plants also exhibit extensive cell wall remodeling associated with induction of multiple cell wall-degrading enzymes, a process which renders the corresponding biomass a substrate for growth of the cellulolytic thermophile Caldicellulosiruptor bescii lacking a functional pectinase gene cluster. The cell wall remodeling also results in the release of size- and charge-heterogeneous pectic oligosaccharide elicitors of PR gene expression. Genetic analysis shows that both in planta PR gene expression and release of elicitors are the result of ectopic expression in xylem of the gene ARABIDOPSIS DEHISCENCE ZONE POLYGALACTURONASE 1 (ADPG1), which is normally expressed during anther and silique dehiscence. These data highlight the importance of pectin in cell wall integrity and the value of lignin modification as a tool to interrogate the informational content of plant cell walls.

Elicitors and defense gene induction in plants with altered lignin compositions.

A reduction in the lignin content in transgenic plants induces the ectopic expression of defense genes, but the importance of altered lignin composition in such phenomena remains unclear. Two Arabidopsis lines with similar lignin contents, but strikingly different lignin compositions, exhibited different quantitative and qualitative transcriptional responses. Plants with lignin composed primarily of guaiacyl units overexpressed genes responsive to oomycete and bacterial pathogen attack, whereas plants with lignin composed primarily of syringyl units expressed a far greater number of defense genes, including some associated with cis-jasmone-mediated responses to aphids; these plants exhibited altered responsiveness to bacterial and aphid inoculation. Several of the defense genes were differentially induced by water-soluble extracts from cell walls of plants of the two lines. Glycome profiling, fractionation and enzymatic digestion studies indicated that the different lignin compositions led to differential extractability of a range of heterogeneous oligosaccharide epitopes, with elicitor activity originating from different cell wall polymers. Alteration of lignin composition affects interactions with plant cell wall matrix polysaccharides to alter the sequestration of multiple latent defense signal molecules with an impact on biotic stress responses.

Metabolic flux towards the (iso)flavonoid pathway in lignin modified alfalfa lines induces resistance against Fusarium oxysporum f. sp. medicaginis.

Downregulation of lignin in alfalfa (Medicago sativa L.) is associated with increased availability of cell wall polysaccharides in plant cells. We tested transgenic alfalfa plants downregulated for Caffeoyl-CoA O-methyltransferase (CCoAOMT) against an economically important fungal disease of alfalfa, Fusarium wilt caused by Fusarium oxysporum f. sp. medicaginis, and found it more resistant to this disease. Transcriptomic and metabolomic analyses indicated that the improved disease resistance against Fusarium wilt is due to increased accumulation and/or spillover of flux towards the (iso)flavonoid pathway. Some (iso)flavonoids and their pathway intermediate compounds showed strong accumulation in CCoAOMT downregulated plants after F. oxysporum f. sp. medicaginis inoculation. The identified (iso)flavonoids, including medicarpin and 7,4'-dihydroxyflavone, inhibited the in vitro growth of F. oxysporum f. sp. medicaginis. These results suggested that the increased accumulation and/or shift/spillover of flux towards the (iso)flavonoid pathway in CCoAOMT downregulated plants is associated with induced disease resistance.

Development and use of a switchgrass (Panicum virgatum L.) transformation pipeline by the BioEnergy Science Center to evaluate plants for reduced cell wall recalcitrance.

BACKGROUND: The mission of the BioEnergy Science Center (BESC) was to enable efficient lignocellulosic-based biofuel production. One BESC goal was to decrease poplar and switchgrass biomass recalcitrance to biofuel conversion while not affecting plant growth. A transformation pipeline (TP), to express transgenes or transgene fragments (constructs) in these feedstocks with the goal of understanding and decreasing recalcitrance, was considered essential for this goal. Centralized data storage for access by BESC members and later the public also was essential. RESULTS: A BESC committee was established to codify procedures to evaluate and accept genes into the TP. A laboratory information management system (LIMS) was organized to catalog constructs, plant lines and results from their analyses. One hundred twenty-eight constructs were accepted into the TP for expression in switchgrass in the first 5 years of BESC. Here we provide information on 53 of these constructs and the BESC TP process. Eleven of the constructs could not be cloned into an expression vector for transformation. Of the remaining constructs, 22 modified expression of the gene target. Transgenic lines representing some constructs displayed decreased recalcitrance in the field and publications describing these results are tabulated here. Transcript levels of target genes and detailed wall analyses from transgenic lines expressing six additional tabulated constructs aimed toward modifying expression of genes associated with wall structure (xyloglucan and lignin components) are provided. Altered expression of xyloglucan endotransglucosylase/hydrolases did not modify lignin content in transgenic plants. Simultaneous silencing of two hydroxycinnamoyl CoA:shikimate hydroxycinnamoyl transferases was necessary to decrease G and S lignin monomer and total lignin contents, but this reduced plant growth. CONCLUSIONS: A TP to produce plants with decreased recalcitrance and a LIMS for data compilation from these plants were created. While many genes accepted into the TP resulted in transgenic switchgrass without modified lignin or biomass content, a group of genes with potential to improve lignocellulosic biofuel yields was identified. Results from transgenic lines targeting xyloglucan and lignin structure provide examples of the types of information available on switchgrass lines produced within BESC. This report supplies useful information when developing coordinated, large-scale, multi-institutional reverse genetic pipelines to improve crop traits.

Combining enhanced biomass density with reduced lignin level for improved forage quality.

To generate a forage crop with increased biomass density that retains forage quality, we have genetically transformed lines of alfalfa (Medicago sativa L.) expressing antisense constructs targeting two different lignin pathway biosynthetic genes with a construct for down-regulation of a WRKY family transcription factor that acts as a repressor of secondary cell wall formation in pith tissues. Plants with low-level expression of the WRKY dominant repressor construct produced lignified cell walls in pith tissues and exhibited enhanced biomass and biomass density, with an increase in total sugars in the cell wall fraction; however, lines with high expression of the WRKY dominant repressor construct exhibited a very different phenotype, with loss of interfascicular fibres associated with repression of the NST1 transcription factor. This latter phenotype was not observed in transgenic lines in which the WRKY transcription factor was down-regulated by RNA interference. Enhanced and/or ectopic deposition of secondary cell walls was also seen in corn and switchgrass expressing WRKY dominant repressor constructs, with enhanced biomass in corn but reduced biomass in switchgrass. Neutral detergent fibre digestibility was not impacted by WRKY expression in corn. Cell walls from WRKY-DR-expressing alfalfa plants with enhanced secondary cell wall formation exhibited increased sugar release efficiency, and WRKY dominant repressor expression further increased sugar release in alfalfa down-regulated in the COMT, but not the HCT, genes of lignin biosynthesis. These results suggest that significant enhancements in forage biomass and quality can be achieved through engineering WRKY transcription factors in both monocots and dicots.

Activation of miR165b represses AtHB15 expression and induces pith secondary wall development in Arabidopsis.

Secondary cell-wall thickening takes place in sclerenchyma cells, but not in surrounding parenchyma cells. The molecular mechanism of switching on and off secondary wall synthesis in various cell types is still elusive. Here, we report the identification of a dominant mutant stp-2d showing secondary wall thickening in pith cells (STP). Immunohistochemistry assays confirmed accumulation of secondary cell walls in the pith cells of the stp-2d mutant. Activation of microRNA 165b (miR165b) expression is responsible for the STP phenotype, as demonstrated by transgenic over-expression experiments. The expression of three class III HD-ZIP transcription factor genes, including AtHB15, was repressed in the stp-2d mutant. Transgenic over-expression of a mutant form of AtHB15 that is resistant to miR165-mediated cleavage reversed the stp-2d mutant phenotype to wild-type, indicating that AtHB15 represses secondary wall development in pith. Characterization of two athb15 mutant alleles further confirmed that functional AtHB15 is necessary for retaining primary walls in parenchyma pith cells. Expression analyses of cell-wall synthetic genes and wall-related transcription factors indicated that a transcriptional pathway is involved in AtHB15 function. These results provide insight into the molecular mechanism of secondary cell-wall development.

Lignin modification leads to increased nodule numbers in alfalfa.

Reduction of lignin levels in the forage legume alfalfa (Medicago sativa) by down-regulation of the monolignol biosynthetic enzyme hydroxycinnamoyl coenzyme A:shikimate hydroxycinnamoyl transferase (HCT) results in strongly increased digestibility and processing ability of lignocellulose. However, these modifications are often also associated with dwarfing and other changes in plant growth. Given the importance of nitrogen fixation for legume growth, we evaluated the impact of constitutively targeted lignin modification on the belowground organs (roots and nodules) of alfalfa plants. HCT down-regulated alfalfa plants exhibit a striking reduction in root growth accompanied by an unexpected increase in nodule numbers when grown in the greenhouse or in the field. This phenotype is associated with increased levels of gibberellins and certain flavonoid compounds in roots. Although HCT down-regulation reduced biomass yields in both the greenhouse and field experiments, the impact on the allocation of nitrogen to shoots or roots was minimal. It is unlikely, therefore, that the altered growth phenotype of reduced-lignin alfalfa is a direct result of changes in nodulation or nitrogen fixation efficiency. Furthermore, HCT down-regulation has no measurable effect on carbon allocation to roots in either greenhouse or 3-year field trials.

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.

Salicylic acid mediates the reduced growth of lignin down-regulated plants.

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.

Integrative analysis of transgenic alfalfa (Medicago sativa L.) suggests new metabolic control mechanisms for monolignol biosynthesis.

The entanglement of lignin polymers with cellulose and hemicellulose in plant cell walls is a major biological barrier to the economically viable production of biofuels from woody biomass. Recent efforts of reducing this recalcitrance with transgenic techniques have been showing promise for ameliorating or even obviating the need for costly pretreatments that are otherwise required to remove lignin from cellulose and hemicelluloses. At the same time, genetic manipulations of lignin biosynthetic enzymes have sometimes yielded unforeseen consequences on lignin composition, thus raising the question of whether the current understanding of the pathway is indeed correct. To address this question systemically, we developed and applied a novel modeling approach that, instead of analyzing the pathway within a single target context, permits a comprehensive, simultaneous investigation of different datasets in wild type and transgenic plants. Specifically, the proposed approach combines static flux-based analysis with a Monte Carlo simulation in which very many randomly chosen sets of parameter values are evaluated against kinetic models of lignin biosynthesis in different stem internodes of wild type and lignin-modified alfalfa plants. In addition to four new postulates that address the reversibility of some key reactions, the modeling effort led to two novel postulates regarding the control of the lignin biosynthetic pathway. The first posits functionally independent pathways toward the synthesis of different lignin monomers, while the second postulate proposes a novel feedforward regulatory mechanism. Subsequent laboratory experiments have identified the signaling molecule salicylic acid as a potential mediator of the postulated control mechanism. Overall, the results demonstrate that mathematical modeling can be a valuable complement to conventional transgenic approaches and that it can provide biological insights that are otherwise difficult to obtain.

Selective lignin downregulation leads to constitutive defense response expression in alfalfa (Medicago sativa L.).

• Downregulation of hydroxycinnamoyl CoA: shikimate hydroxycinnamoyl transferase (HCT) in alfalfa (Medicago sativa) reduces lignin levels and improves forage quality and saccharification efficiency for bioethanol production. However, the plants have reduced stature. It was previously reported that HCT-down-regulated Arabidopsis have impaired auxin transport, but this has recently been disproved. • To address the basis for the phenotypes of lignin-modified alfalfa, we measured auxin transport, profiled a range of metabolites including flavonoids and hormones, and performed in depth transcriptome analyses. • Auxin transport is unaffected in HCT antisense alfalfa despite increased flavonoid biosynthesis. The plants show increased cytokinin and reduced auxin levels, and gibberellin levels and sensitivity are both reduced. Levels of salicylic, jasmonic and abscisic acids are elevated, associated with massive upregulation of pathogenesis and abiotic stress-related genes and enhanced tolerance to fungal infection and drought. • We suggest that HCT downregulated alfalfa plants exhibit constitutive activation of defense responses, triggered by release of bioactive cell wall fragments and production of hydrogen peroxide as a result of impaired secondary cell wall integrity.

Genome-wide analysis of phenylpropanoid defence pathways.

Phenylpropanoids can function as preformed and inducible antimicrobial compounds, as well as signal molecules, in plant-microbe interactions. Since we last reviewed the field 8 years ago, there has been a huge increase in our understanding of the genes of phenylpropanoid biosynthesis and their regulation, brought about largely by advances in genome technology, from whole-genome sequencing to massively parallel gene expression profiling. Here, we present an overview of the biosynthesis and roles of phenylpropanoids in plant defence, together with an analysis of confirmed and predicted phenylpropanoid pathway genes in the sequenced genomes of 11 plant species. Examples are provided of phylogenetic and expression clustering analyses, and the large body of underlying genomic data is provided through a website accessible from the article.

Inhibition of auxin transport from the ovary or from the apical shoot induces parthenocarpic fruit-set in tomato mediated by gibberellins.

Fruit-set in tomato (Solanum lycopersicum) depends on gibberellins and auxins (GAs). Here, we show, using the cv MicroTom, that application of N-1-naphthylphthalamic acid (NPA; an inhibitor of auxin transport) to unpollinated ovaries induced parthenocarpic fruit-set, associated with an increase of indole-3-acetic acid (IAA) content, and that this effect was negated by paclobutrazol (an inhibitor of GA biosynthesis). NPA-induced ovaries contained higher content of GA(1) (an active GA) and transcripts of GA biosynthetic genes (SlCPS, SlGA20ox1, and -2). Interestingly, application of NPA to pollinated ovaries prevented their growth, potentially due to supraoptimal IAA accumulation. Plant decapitation and inhibition of auxin transport by NPA from the apical shoot also induced parthenocarpic fruit growth of unpollinated ovaries. Application of IAA to the severed stump negated the plant decapitation effect, indicating that the apical shoot prevents unpollinated ovary growth through IAA transport. Parthenocarpic fruit growth induced by plant decapitation was associated with high levels of GA(1) and was counteracted by paclobutrazol treatment. Plant decapitation also produced changes in transcript levels of genes encoding enzymes of GA biosynthesis (SlCPS and SlGA20ox1) in the ovary, quite similar to those found in NPA-induced fruits. All these results suggest that auxin can have opposing effects on fruit-set, either inducing (when accumulated in the ovary) or repressing (when transported from the apical shoot) that process, and that GAs act as mediators in both cases. The effect of NPA application and decapitation on fruit-set induction was also observed in MicroTom lines bearing introgressed DWARF and SELF-PRUNING wild-type alleles.

An NAC transcription factor orchestrates multiple features of cell wall development in Medicago truncatula.

To identify genes controlling secondary cell wall biosynthesis in the model legume Medicago truncatula, we screened a Tnt1 retrotransposon insertion mutant population for plants with altered patterns of lignin autofluorescence. From more than 9000 R1 plants screened, four independent lines were identified with a total lack of lignin in the interfascicular region. The mutants also showed loss of lignin in phloem fibers, reduced lignin in vascular elements, failure in anther dehiscence and absence of phenolic autofluorescence in stomatal guard cell walls. Microarray and PCR analyses confirmed that the mutations were caused by the insertion of Tnt1 in a gene annotated as encoding a NAM (no apical meristem)-like protein (here designated Medicago truncatula NAC SECONDARY WALL THICKENING PROMOTING FACTOR 1, MtNST1). MtNST1 is the only family member in Medicago, but has three homologs (AtNST1-AtNST3) in Arabidopsis thaliana, which function in different combinations to control cell wall composition in stems and anthers. Loss of MtNST1 function resulted in reduced lignin content, associated with reduced expression of most lignin biosynthetic genes, and a smaller reduction in cell wall polysaccharide content, associated with reduced expression of putative cellulose and hemicellulose biosynthetic genes. Acid pre-treatment and cellulase digestion released significantly more sugars from cell walls of nst1 mutants compared with the wild type. We discuss the implications of these findings for the development of alfalfa (Medicago sativa) as a dedicated bioenergy crop.

Gibberellin homeostasis in tobacco is regulated by gibberellin metabolism genes with different gibberellin sensitivity.

Gibberellins are phytohormones that regulate growth and development of plants. Gibberellin homeostasis is maintained by feedback regulation of gibberellin metabolism genes. To understand this regulation, we manipulated the gibberellin pathway in tobacco and studied its effects on the morphological phenotype, gibberellin levels and the expression of endogenous gibberellin metabolism genes. The overexpression of a gibberellin 3-oxidase (biosynthesis gene) in tobacco (3ox-OE) induced slight variations in phenotype and active GA(1) levels, but we also found an increase in GA(8) levels (GA(1) inactivation product) and a conspicuous induction of gibberellin 2-oxidases (catabolism genes; NtGA2ox3 and -5), suggesting an important role for these particular genes in the control of gibberellin homeostasis. The effect of simultaneous overexpression of two biosynthesis genes, a gibberellin 3-oxidase and a gibberellin 20-oxidase (20ox/3ox-OE), on phenotype and gibberellin content suggests that gibberellin 3-oxidases are non-limiting enzymes in tobacco, even in a 20ox-OE background. Moreover, the expression analysis of gibberellin metabolism genes in transgenic plants (3ox-OE, 20ox-OE and hybrid 3ox/20ox-OE), and in response to application of different GA(1) concentrations, showed genes with different gibberellin sensitivity. Gibberellin biosynthesis genes (NtGA20ox1 and NtGA3ox1) are negatively feedback regulated mainly by high gibberellin levels. In contrast, gibberellin catabolism genes which are subject to positive feedback regulation are sensitive to high (NtGA2ox1) or to low (NtGA2ox3 and -5) gibberellin concentrations. These two last GA2ox genes seem to play a predominant role in gibberellin homeostasis under mild gibberellin variations, but not under large gibberellin changes, where the biosynthesis genes GA20ox and GA3ox may be more important.

Flowering in tobacco needs gibberellins but is not promoted by the levels of active GA1 and GA4 in the apical shoot.

Flowering of Nicotiana tabacum cv Xhanti depends on gibberellins because gibberellin-deficient plants, due to overexpression of a gibberellin 2-oxidase gene (35S:NoGA2ox3) or to treatment with the gibberellin biosynthesis inhibitor paclobutrazol, flowered later than wild type. These plants also showed inhibition of the expression of molecular markers related to floral transition (NtMADS-4 and NtMADS-11). To investigate further the role of gibberellin in flowering, we quantified its content in tobacco plants during development. We found a progressive reduction in the levels of GA1 and GA4 in the apical shoot during vegetative growth, reaching very low levels at floral transition and beyond. This excludes these two gibberellins as flowering-promoting factors in the apex. The evolution of active gibberellin content in apical shoots agrees with the expression patterns of gibberellin metabolism genes: two encoding gibberellin 20-oxidases (NtGA20ox1 = Ntc12, NtGA20ox2 = Ntc16), one encoding a gibberellin 3-oxidase (NtGA3ox1 = Nty) and one encoding a gibberellin 2-oxidase (NtGA2ox1), suggesting that active gibberellins are locally synthesized. In young apical leaves, GA1 and GA4 content and the expression of gibberellin metabolism genes were rather constant. Our results support that floral transition in tobacco, in contrast to that in Arabidopsis, is not regulated by the levels of GA1 and GA4 in apical shoots, although reaching a threshold in gibberellin levels may be necessary to allow meristem competence for flowering.