Lignin engineering

Lignins are aromatic polymers that are present mainly in secondarily thickened plant cell walls. Several decades of research have elucidated the main biosynthetic routes toward the monolignols and demonstrated that lignin amounts can be engineered and that plants can cope with large shifts in p-hydroxyphenyl/guaiacyl/syringyl (H/G/S) lignin compositional ratios. It has also become clear that lignins incorporate many more units than the three monolignols described in biochemistry textbooks. Together with the theory that lignin polymerization is under chemical control, observations hint at opportunities to design lignin structure to the needs of agriculture. An increasing number of examples illustrates that lignin engineering can improve the processing efficiency of plant biomass for pulping, forage digestibility and biofuels. Systems approaches, in which the plant's response to engineering of a single gene in the pathway is studied at the organismal level, are beginning to shed light on the interaction of lignin biosynthesis with other metabolic pathways and processes.     

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

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.     

The Minor Wall-Networks between Monolignols and Interlinked-Phenolics Predominantly Affect Biomass Enzymatic Digestibility in Miscanthus

Plant lignin is one of the major wall components that greatly contribute to biomass recalcitrance for biofuel production. In this study, total 79 representative Miscanthus germplasms were determined with wide biomass digestibility and diverse monolignol composition. Integrative analyses indicated that three major monolignols (S, G, H) and S/G ratio could account for lignin negative influence on biomass digestibility upon NaOH and H₂SO₄ pretreatments. Notably, the biomass enzymatic digestions were predominately affected by the non-KOH-extractable lignin and interlinked-phenolics, other than the KOH-extractable ones that cover 80% of total lignin. Furthermore, a positive correlation was found between the monolignols and phenolics at p<0.05 level in the non-KOH-extractable only, suggesting their tight association to form the minor wall-networks against cellulases accessibility. The results indicated that the non-KOH-extractable lignin-complex should be the target either for cost-effective biomass pretreatments or for relatively simply genetic modification of plant cell walls in Miscanthus.     

Downregulation of caffeoyl-CoA O-methyltransferase (CCoAOMT) by RNA interference leads to reduced lignin production in maize straw

Lignin is a major cell wall component of vascular plants that provides mechanical strength and hydrophobicity to vascular vessels. However, the presence of lignin limits the effective use of crop straw in many agroindustrial processes. Here, we generated transgenic maize plants in which the expression of a lignin biosynthetic gene encoding CCoAOMT, a key enzyme involved in the lignin biosynthesis pathway was downregulated by RNA interference (RNAi). RNAi of CCoAOMT led to significantly downregulated expression of this gene in transgenic maize compared with WT plants. These transgenic plants exhibited a 22.4% decrease in Klason lignin content and a 23.3% increase in cellulose content compared with WT plants, which may reflect compensatory regulation of lignin and cellulose deposition. We also measured the lignin monomer composition of the RNAi plants by GC-MS and determined that transgenic plants had a 57.08% higher S/G ratio than WT plants. In addition, histological staining of lignin with Wiesner reagent produced slightly more coloration in the xylem and sclerenchyma than WT plants. These results provide a foundation for breeding maize with low-lignin content and reveal novel insights about lignin regulation via genetic manipulation of CCoAOMT expression.     

Basic peroxidases: The gateway for lignin evolution?

Lignins are cell wall phenolic heteropolymers which result from the oxidative coupling of three monolignols, p-coumaryl, coniferyl and sinapyl alcohol, in a reaction mediated by peroxidases. The most distinctive variation in the monomer composition of lignins in vascular plants is that found between the two main groups of seed plants. Thus, while gymnosperms lignins are typically composed of G units, with a minor proportion of H units, angiosperms lignins are largely composed of similar levels of G and S units. The presence of S units in angiosperm lignins raises certain concerns in relation with the step of lignin assembly due to the inability of most peroxidases to oxidize syringyl moieties. Zinnia elegans is currently used as a model for lignification studies: – first because of the simplicity and duality of the lignification pattern shown by hypocotyls and stems, in which hypocotyl lignins are typical of angiosperms, while young stem lignins partially resemble those occurring in gymnosperms. Secondly, because of the nature of the peroxidase isoenzyme complement, which is almost completely restricted to the presence of a basic peroxidase isoenzyme, which is capable of oxidizing both coniferyl and sinapyl alcohol, as well as both coniferyl and sinapyl aldehyde. In fact, the versatility of this enzyme is such that the substrate preference covers the three p-hydroxybenzaldehydes and the three p-hydroxycinnamic acids. The basic pI nature of this peroxidase is not an exceptional frame point in this system since basic peroxidases are differentially expressed during lignification in other model systems, show unusual and unique biochemical properties as regards the oxidation of syringyl moieties, and their down-regulation in transgenic plants leads to a reduction in lignin (G+S) levels. Basic peroxidase isoenzymes capable of oxidizing syringyl moieties are already present in basal gymnosperms, an observation that supports the idea that these enzymes were probably present in an ancestral plant species, pre-dating the early radiation of seed plants. It also suggests that the evolutionary gain of the monolignol branch which leads to the biosynthesis of sinapyl alcohol, and of course to syringyl lignins, was not only possible but also favored because the enzymes responsible for its polymerization had evolved previously. In this scenario, it is not surprising that these enzymes responsible for lignin construction appeared early in the evolution of land plants, and have been largely conserved during plant evolution. Abreviations: 4CL –p-hydroxycinnamate CoA ligase; C3H –p-coumarate-3-hydroxylase; C4H – cinnamate-4-hydroxylase; p-CA –p-coumaric acid; CAD – coniferyl alcohol dehydrogenase; CAld5H – coniferylaldehyde-5-hydroxylase; CCR –p-hydroxycinnamoyl-CoA reductase; CoI – compound I; CoII – compound II; G – guaiacyl unit; H –p-hydroxyphenyl unit; PAL – phenylalanine ammonia-lyase; S – syringyl unit.     

On the role of cell wall lignin in determining the fineness of jute fibre

Though finer quality fibre is of great demand in the industry, a reasonable biological assessment of the factors controlling jute fibre fineness is lacking. Our aim was to relate lignin synthesis and accumulation in the secondary wall of the fibre cells with fibre fineness by anatomical and physiological evidences. Several jute genotypes including a low lignin mutant, dlpf (INGR No. 04107) and its lignin sufficient parent (JRC 212) were grown under different growth conditions. Their cell wall morphology and cellulose, hemicellulose and lignin content of fibre were estimated. The fineness of the extracted fibre was examined gravimetrically as well as by air-flow method on individual plant basis to relate it with their chemical constituents. Effect of incident light and some plant growth regulators on glucan and lignin biosynthetic enzymes as well as fibre fineness was determined. Positive relationship between cell wall thickness and lignin and negative relationship between fibre fineness and lignin of jute fibre were established. Application of the GA biosynthetic inhibitor helped to reduce lignin synthesis and to increase fibre fineness. Genotypes with thinner cell wall and lesser lignin may be utilized in breeding for improving the fibre fineness of jute. Field application of GA biosynthetic inhibitors, like daminozide, is recommended to reduce the cell wall thickness of lignin sufficient high yielding jute varieties.     

Down-regulation of cinnamyl alcohol dehydrogenase in transgenic alfalfa (Medicago sativa L.) and the effect on lignin composition and digestibility

To improve the digestibility of the forage crop alfalfa (Medicago sativa L.), cinnamyl alcohol dehydrogenase (CAD), which catalyses the last step in the biosynthesis of the lignin monomers, was down-regulated by using an antisense approach. A subset of six transgenic lines with reduced CAD activity and control lines were analysed when grown in the greenhouse and in the field. The down-regulation of the CAD enzyme was associated with a red coloration of the stem. The lignin quantity remained unchanged, but the lignin composition, as determined by thioacidolysis, was altered. The highest reduction of CAD activity was associated with a lower syringyl/guaiacyl (S/G) ratio and a lower S+G yield, mainly because of a decreased amount of S units. An increase in in situ disappearance of dry matter and of cell wall residue was detected in one of the transgenic lines grown in the greenhouse, and for two of the lines grown in the field the rate of disappearance of dry matter slightly improved. Furthermore, these two lines had a higher solubility in alkali as shown by the lower yield of saponified residue. This study opens perspectives for improving forage crop digestibility by the modulation of enzymes involved in lignin biosynthesis.     

Unique Features of Two Potassium Channels,OsKAT2 and OsKAT3, Expressed in Rice Guard Cells

Potassium is the most abundant cation and a myriad of transporters regulate K⁺ homeostasis in plant. Potassium plays a role as a major osmolyte to regulate stomatal movements that control water utility of land plants. Here we report the characterization of two inward rectifying shaker-like potassium channels, OsKAT2 and OsKAT3, expressed in guard cell of rice plants. While OsKAT2 showed typical potassium channel activity, like that of Arabidopsis KAT1, OsKAT3 did not despite high sequence similarity between the two channel proteins. Interestingly, the two potassium channels physically interacted with each other and such interaction negatively regulated the OsKAT2 channel activity in CHO cell system. Furthermore, deletion of the C-terminal domain recovered the channel activity of OsKAT3, suggesting that the C-terminal region was regulatory domain that inhibited channel activity. Two homologous channels with antagonistic interaction has not been previously reported and presents new information for potassium channel regulation in plants, especially in stomatal regulation.     

Degradation of lignin β‐aryl ether units in Arabidopsis thaliana expressing LigD,LigF and LigG from Sphingomonas paucimobilis SYK‐6

Lignin is a major polymer in the secondary plant cell wall and composed of hydrophobic interlinked hydroxyphenylpropanoid units. The presence of lignin hampers conversion of plant biomass into biofuels; plants with modified lignin are therefore being investigated for increased digestibility. The bacterium Sphingomonas paucimobilis produces lignin‐degrading enzymes including LigD, LigF and LigG involved in cleaving the most abundant lignin interunit linkage, the β‐aryl ether bond. In this study, we expressed the LigD, LigF and LigG (LigDFG) genes in Arabidopsis thaliana to introduce postlignification modifications into the lignin structure. The three enzymes were targeted to the secretory pathway. Phenolic metabolite profiling and 2D HSQC NMR of the transgenic lines showed an increase in oxidized guaiacyl and syringyl units without concomitant increase in oxidized β‐aryl ether units, showing lignin bond cleavage. Saccharification yield increased significantly in transgenic lines expressing LigDFG, showing the applicability of our approach. Additional new information on substrate specificity of the LigDFG enzymes is also provided.     

Ectopic deposition of lignin in the pith of stems of two Arabidopsis mutants

The biosynthesis of lignin in vascular plants is regulated both developmentally and environmentally. In the inflorescence stems of Arabidopsis, lignin is mainly deposited in the walls of xylem cells and interfascicular fiber cells during normal plant growth and development. The mechanisms controlling the spatial deposition of lignin remain unknown. By screening ethyl methanesulfonate-mutagenized populations of Arabidopsis, we have isolated two allelic elp1 (ectopic deposition of lignin in pith) mutants with altered lignin deposition patterns. In elp1 stems, lignin was ectopically deposited in the walls of pith parenchyma cells in addition to its normal deposition in the walls of xylem and fiber cells. Lignin appeared to be deposited in patches of parenchyma cells in the pith of both young and mature elp1 stems. The ectopic deposition of lignin in the pith of elp1 stems was accompanied by an increase in the activities of enzymes in the lignin biosynthetic pathway and with the ectopic expression of caffeoyl coenzyme A O-methyltransferase in pith cells. These results indicate that the ELP1 locus is involved in the repression of the lignin biosynthetic pathway in the pith. Isolation of the elp1 mutants provides a novel means with which to study the molecular mechanisms underlying the spatial control of lignification.     

Heterotrimeric G protein signalling in the plant kingdom

In animals, heterotrimeric G proteins, comprising α-, β-and γ-subunits, perceive extracellular stimuli through cell surface receptors, and transmit signals to ion channels, enzymes and other effector proteins to affect numerous cellular behaviours. In plants, G proteins have structural similarities to the corresponding molecules in animals but transmit signals by atypical mechanisms and effector proteins to control growth, cell proliferation, defence, stomate movements, channel regulation, sugar sensing and some hormonal responses. In this review, we summarize the current knowledge on the molecular regulation of plant G proteins, their effectors and the physiological functions studied mainly in two model organisms: Arabidopsis thaliana and rice (Oryza sativa). We also look at recent progress on structural analyses, systems biology and evolutionary studies.     

Improvement of in-rumen digestibility of alfalfa forage by genetic manipulation of lignin O-methyltransferases

Lignin inhibits forage digestibility by ruminant animals, and lignin levels and the proportion of dimethylated syringyl (S) lignin monomers increase with progressive maturity in stems of forage crops. We generated transgenic alfalfa (Medicago sativa L.) with reduced lignin content and altered lignin composition. Down-regulation of caffeic acid 3-O-methyltransferase (COMT) reduces lignin content, accompanied by near total loss of S lignin, whereas down-regulation of caffeoyl coenzyme A 3-O-methyltransferase (CCoAOMT) reduces lignin content without reduction in S lignin. These changes are not accompanied by altered ratios of cell wall polysaccharides. Analysis of rumen digestibility of alfalfa forage in fistulated steers revealed improved digestibility of forage from COMT down-regulated plants, but a greater improvement in digestibility following down-regulation of CCoAOMT. The results indicate that both lignin content and composition affect digestibility of alfalfa forage, and reveal a new strategy for forage quality improvement by genetic manipulation of CCoAOMT expression.     

Effects on Lignin Structure of Coumarate 3-Hydroxylase Downregulation in Poplar

The lignin structural ramifications of coumarate 3-hydroxylase (C3H) downregulation have not been addressed in hardwoods. Such information is required to accompany an assessment of the digestibility and bioenergy performance characteristics of poplar, in particular. Structurally rich 2D NMR methods were applied to the entire lignin fraction to delineate lignin p-hydroxyphenyl:guaiacyl:syringyl (H:G:S) levels and linkage distribution changes (and to compare with traditional degradative analyses). C3H downregulation reduced lignin levels by half and markedly increased the proportion of H units relative to the normally dominant G and S units. Relative stem H unit levels were up by ???100-fold to ???31?%, almost totally at the expense of G units; differences in the lignin interunit linkage distributions were more subtle. The H level in the most drastically C3H-downregulated transgenic poplar falls well beyond the H:G:S compositional bounds of normal angiosperms. The response observed here, in poplar, differs markedly from that reported for alfalfa where the S:G ratio remained almost constant even at substantial H levels, highlighting the often differing responses among plant species.     

Convergent Evolution of Syringyl Lignin Biosynthesis via Distinct Pathways in the Lycophyte Selaginella and Flowering Plants

Phenotypic convergence in unrelated lineages arises when different organisms adapt similarly under comparable selective pressures. In an apparent example of this process, syringyl lignin, a fundamental building block of plant cell walls, occurs in two major plant lineages, lycophytes and angiosperms, which diverged from one another more than 400 million years ago. Here, we show that this convergence resulted from independent recruitment of lignin biosynthetic cytochrome P450-dependent monooxygenases that route cell wall monomers through related but distinct pathways in the two lineages. In contrast with angiosperms, in which syringyl lignin biosynthesis requires two phenylpropanoid meta-hydroxylases C3′H and F5H, the lycophyte Selaginella employs one phenylpropanoid dual meta-hydroxylase to bypass several steps of the canonical lignin biosynthetic pathway. Transgenic expression of the Selaginella hydroxylase in Arabidopsis thaliana dramatically reroutes its endogenous lignin biosynthetic pathway, yielding a novel lignin composition not previously identified in nature. Our findings demonstrate a unique case of convergent evolution via distinct biochemical strategies and suggest a new way to genetically reconstruct lignin biosynthesis in higher plants.