SWATH-MS-based proteogenomic analysis reveals the involvement of alternative splicing in poplar upon lead stress.

Alternative splicing (AS) regulates gene expression and increases proteomic diversity for the fine tuning of stress responses in plants, but the exact mechanism through which AS functions in plant stress responses is not thoroughly understood. Here, we investigated how AS functions in poplar (Populus trichocarpa), a popular plant for bioremediation, in response to lead (Pb) stress. Using a proteogenomic analysis, we determine that Pb stress induced alterations in AS patterns that are characterized by an increased use of nonconventional splice sites and a higher abundance of Pb-responsive splicing factors (SFs) associated with Pb-responsive transcription factors. A strong Pb(II)-inducible chaperone protein, PtHSP70, that undergoes AS was further characterized. Overexpression of its two spliced isoforms, PtHSP70-AS1 and PtHSP70-AS2, in poplar and Arabidopsis significantly enhances the tolerance to Pb. Further characterization shows that both isoforms can directly bind to Pb(II), and PtHSP70-AS2 exhibits 10-fold higher binding capacities and a greater increase in expression under Pb stress, thereby reducing cellular toxicity through Pb(II) extrusion and conferring Pb tolerance. AS of PtHSP70 is found to be regulated by PtU1-70K, a Pb(II)-inducible core SF involved in 5'-splice site recognition. Because the same splicing pattern is also found in HSP70 orthologs in other plant species, AS of HSP70 may be a common regulatory mechanism to cope with Pb(II) toxicity. Overall, we have revealed a novel post-transcriptional machinery that mediates heavy metal tolerance in diverse plant species. Our findings offer new molecular targets and bioengineering strategies for phytoremediation and provide new insight for future directions in AS research.

Disruption of p-coumaroyl-CoA:monolignol transferases in rice drastically alters lignin composition.

Grasses are abundant feedstocks that can supply lignocellulosic biomass for production of cell-wall-derived chemicals. In grass cell walls, lignin is acylated with p-coumarate. These p-coumarate decorations arise from the incorporation of monolignol p-coumarate conjugates during lignification. A previous biochemical study identified a rice (Oryza sativa) BAHD acyltransferase (AT) with p-coumaroyl-CoA:monolignol transferase (PMT) activity in vitro. In this study, we determined that that enzyme, which we name OsPMT1 (also known as OsAT4), and the closely related OsPMT2 (OsAT3) harbor similar catalytic activity toward monolignols. We generated rice mutants deficient in either or both OsPMT1 and OsPMT2 by CRISPR/Cas9-mediated mutagenesis and subjected the mutants' cell walls to analysis using chemical and nuclear magnetic resonance methods. Our results demonstrated that OsPMT1 and OsPMT2 both function in lignin p-coumaroylation in the major vegetative tissues of rice. Notably, lignin-bound p-coumarate units were undetectable in the ospmt1 ospmt2-2 double-knockout mutant. Further, in-depth structural analysis of purified lignins from the ospmt1 ospmt2-2 mutant compared with control lignins from wild-type rice revealed stark changes in polymer structures, including alterations in syringyl/guaiacyl aromatic unit ratios and inter-monomeric linkage patterns, and increased molecular weights. Our results provide insights into lignin polymerization in grasses that will be useful for the optimization of bioengineering approaches for the effective use of biomass in biorefineries.

Altered Lignin Accumulation in Sorghum Mutated in Silicon Uptake Transporter SbLsi1.

Sorghum [Sorghum bicolor (L.) Moench] has been receiving attention as a feedstock for lignocellulose biomass energy. During the combustion process, the ash containing silicon (Si) can be produced, which causes problems in furnace maintenance. Hence, lowering Si content in the plants is crucial. Nevertheless, limiting Si supply to crops is difficult in practice because Si is abundant in soil. Previously, a Si uptake transporter (SbLsi1) has been identified, and the Si-depleted mutant has also been generated in the model sorghum variety BTx623. In this study, we aim to investigate the change induced by the mutation of SbLsi1 on the accumulation and the structure of lignin in cell walls. Through chemical and NMR analyses, we demonstrated that the lsi1 mutation resulted in a significant increase in lignin accumulation levels as well as a significant reduction in Si content. At least some of the modification was induced by transcriptional changes, as suggested by the upregulation of phenylpropanoid biosynthesis-related genes in the mutant plants. These findings derived from the model variety would be useful for the future development of practical cultivars with high biomass and less Si content for bioenergy applications.

Simultaneous suppression of lignin, tricin and wall-bound phenolic biosynthesis via the expression of monolignol 4-O-methyltransferases in rice.

Grass lignocelluloses feature complex compositions and structures. In addition to the presence of conventional lignin units from monolignols, acylated monolignols and flavonoid tricin also incorporate into lignin polymer; moreover, hydroxycinnamates, particularly ferulate, cross-link arabinoxylan chains with each other and/or with lignin polymers. These structural complexities make grass lignocellulosics difficult to optimize for effective agro-industrial applications. In the present study, we assess the applications of two engineered monolignol 4-O-methyltransferases (MOMTs) in modifying rice lignocellulosic properties. Two MOMTs confer regiospecific para-methylation of monolignols but with different catalytic preferences. The expression of MOMTs in rice resulted in differential but drastic suppression of lignin deposition, showing more than 50% decrease in guaiacyl lignin and up to an 90% reduction in syringyl lignin in transgenic lines. Moreover, the levels of arabinoxylan-bound ferulate were reduced by up to 50%, and the levels of tricin in lignin fraction were also substantially reduced. Concomitantly, up to 11 µmol/g of the methanol-extractable 4-O-methylated ferulic acid and 5-7 µmol/g 4-O-methylated sinapic acid were accumulated in MOMT transgenic lines. Both MOMTs in vitro displayed discernible substrate promiscuity towards a range of phenolics in addition to the dominant substrate monolignols, which partially explains their broad effects on grass phenolic biosynthesis. The cell wall structural and compositional changes resulted in up to 30% increase in saccharification yield of the de-starched rice straw biomass after diluted acid-pretreatment. These results demonstrate an effective strategy to tailor complex grass cell walls to generate improved cellulosic feedstocks for the fermentable sugar-based production of biofuel and bio-chemicals.

Lignocellulose molecular assembly and deconstruction properties of lignin-altered rice mutants.

Bioengineering approaches to modify lignin content and structure in plant cell walls have shown promise for facilitating biochemical conversions of lignocellulosic biomass into valuable chemicals. Despite numerous research efforts, however, the effect of altered lignin chemistry on the supramolecular assembly of lignocellulose and consequently its deconstruction in lignin-modified transgenic and mutant plants is not fully understood. In this study, we aimed to close this gap by analyzing lignin-modified rice (Oryza sativa L.) mutants deficient in 5-HYDROXYCONIFERALDEHYDE O-METHYLTRANSFERASE (CAldOMT) and CINNAMYL ALCOHOL DEHYDROGENASE (CAD). A set of rice mutants harboring knockout mutations in either or both OsCAldOMT1 and OsCAD2 was generated in part by genome editing and subjected to comparative cell wall chemical and supramolecular structure analyses. In line with the proposed functions of CAldOMT and CAD in grass lignin biosynthesis, OsCAldOMT1-deficient mutant lines produced altered lignins depleted of syringyl and tricin units and incorporating noncanonical 5-hydroxyguaiacyl units, whereas OsCAD2-deficient mutant lines produced lignins incorporating noncanonical hydroxycinnamaldehyde-derived units. All tested OsCAldOMT1- and OsCAD2-deficient mutants, especially OsCAldOMT1-deficient lines, displayed enhanced cell wall saccharification efficiency. Solid-state nuclear magnetic resonance (NMR) and X-ray diffraction analyses of rice cell walls revealed that both OsCAldOMT1- and OsCAD2 deficiencies contributed to the disruptions of the cellulose crystalline network. Further, OsCAldOMT1 deficiency contributed to the increase of the cellulose molecular mobility more prominently than OsCAD2 deficiency, resulting in apparently more loosened lignocellulose molecular assembly. Such alterations in cell wall chemical and supramolecular structures may in part account for the variations of saccharification performance of the OsCAldOMT1- and OsCAD2-deficient rice mutants.

Seed-coat protective neolignans are produced by the dirigent protein AtDP1 and the laccase AtLAC5 in Arabidopsis.

Lignans/neolignans are generally synthesized from coniferyl alcohol (CA) in the cinnamate/monolignol pathway by oxidation to generate the corresponding radicals with subsequent stereoselective dimerization aided by dirigent proteins (DIRs). Genes encoding oxidases and DIRs for neolignan biosynthesis have not been identified previously. In Arabidopsis thaliana, the DIR AtDP1/AtDIR12 plays an essential role in the 8-O-4' coupling in neolignan biosynthesis by unequivocal structural determination of the compound missing in the atdp1 mutant as a sinapoylcholine (SC)-conjugated neolignan, erythro-3-{4-[2-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-1-hydroxymethylethoxy]-3,5-dimethoxyphenyl}acryloylcholine. Phylogenetic analyses showed that AtDP1/AtDIR12 belongs to the DIR-a subfamily composed of DIRs for 8-8' coupling of monolignol radicals. AtDP1/AtDIR12 is specifically expressed in outer integument 1 cells in developing seeds. As a putative oxidase for neolignan biosynthesis, we focused on AtLAC5, a laccase gene coexpressed with AtDP1/AtDIR12. In lac5 mutants, the abundance of feruloylcholine (FC)-conjugated neolignans decreased to a level comparable to those in the atdp1 mutant. In addition, SC/FC-conjugated neolignans were missing in the seeds of mutants defective in SCT/SCPL19, an enzyme that synthesizes SC. These results strongly suggest that AtDP1/AtDIR12 and AtLAC5 are involved in neolignan biosynthesis via SC/FC. A tetrazolium penetration assay showed that seed coat permeability increased in atdp1 mutants, suggesting a protective role of neolignans in A. thaliana seeds.

Expression of laccase and ascorbate oxidase affects lignin composition in Arabidopsis thaliana stems.

Lignin is a phenolic polymer that is a major source of biomass. Oxidative enzymes, such as laccase and peroxidase, are required for lignin polymerisation. Laccase is a member of the multicopper oxidase family and has a high amino acid sequence similarity with ascorbate oxidase. However, the process of functional differentiation between the two enzymes remains poorly understood. In this study, the common ancestry sequence of laccase and ascorbate oxidase (AncMCO) was predicted via phylogenetic reconstruction, and its in vivo effect on lignin biosynthesis in Arabidopsis thaliana was assessed. The estimated AncMCO sequence conserved key residues that coordinate with copper ions, implying that the electron transfer system is likely to be conserved in AncMCO. However, multiple insertions/deletions corresponding to protein surface structures have been found between laccase, ascorbate oxidase, and AncMCO. The overexpression of canonical laccase (AtLAC4) and ascorbate oxidase (AtAAO1) in A. thaliana resulted in notable increases of syringyl/guaiacyl lignin unit ratio in stems, whereas, in contrast, the overexpression of AncMCO did not show any detectable change in lignin deposition. Transcriptomic analysis revealed that the AtAAO1-overexpressing line exhibited significant changes in the expression of a wide range of cell wall biosynthesis genes. These results highlight the importance of the molecular evolution of multicopper oxidase, which drives lignin biosynthesis during plant evolution.

Lignin-based barrier restricts pathogens to the infection site and confers resistance in plants.

Pathogenic bacteria invade plant tissues and proliferate in the extracellular space. Plants have evolved the immune system to recognize and limit the growth of pathogens. Despite substantial progress in the study of plant immunity, the mechanism by which plants limit pathogen growth remains unclear. Here, we show that lignin accumulates in Arabidopsis leaves in response to incompatible interactions with bacterial pathogens in a manner dependent on Casparian strip membrane domain protein (CASP)-like proteins (CASPLs). CASPs are known to be the organizers of the lignin-based Casparian strip, which functions as a diffusion barrier in roots. The spread of invading avirulent pathogens is prevented by spatial restriction, which is disturbed by defects in lignin deposition. Moreover, the motility of pathogenic bacteria is negatively affected by lignin accumulation. These results suggest that the lignin-deposited structure functions as a physical barrier similar to the Casparian strip, trapping pathogens and thereby terminating their growth.

Genome-edited rice deficient in two 4-COUMARATE:COENZYME A LIGASE genes displays diverse lignin alterations.

The 4-coumarate:coenzyme A ligase (4CL) is a key enzyme that contributes to channeling metabolic flux in the cinnamate/monolignol pathway, leading to the production of monolignols, p-hydroxycinnamates, and a flavonoid tricin, the major building blocks of lignin polymer in grass cell walls. Vascular plants often contain multiple 4CL genes; however, the contribution of each 4CL isoform to lignin biosynthesis remains unclear, especially in grasses. In this study, we characterized the functions of two rice (Oryza sativa L.) 4CL isoforms (Os4CL3 and Os4CL4) primarily by analyzing the cell wall chemical structures of rice mutants generated by CRISPR/Cas9-mediated targeted mutagenesis. A series of chemical and nuclear magnetic resonance analyses revealed that loss-of-function of Os4CL3 and Os4CL4 differently altered the composition of lignin polymer units. Loss of function of Os4CL3 induced marked reductions in the major guaiacyl and syringyl lignin units derived from both the conserved non-γ-p-coumaroylated and the grass-specific γ-p-coumaroylated monolignols, with more prominent reductions in guaiacyl units than in syringyl units. In contrast, the loss-of-function mutation to Os4CL4 primarily decreased the abundance of the non-γ-p-coumaroylated guaiacyl units. Loss-of-function of Os4CL4, but not of Os4CL3, reduced the grass-specific lignin-bound tricin units, indicating that Os4CL4 plays a key role not only in monolignol biosynthesis but also in the biosynthesis of tricin used for lignification. Further, the loss-of-function of Os4CL3 and Os4CL4 notably reduced cell-wall-bound ferulates, indicating their roles in cell wall feruloylation. Overall, this study demonstrates the overlapping but divergent roles of 4CL isoforms during the coordinated production of various lignin monomers.

Deficiency in flavonoid biosynthesis genes CHS, CHI, and CHIL alters rice flavonoid and lignin profiles.

Lignin is a complex phenylpropanoid polymer deposited in the secondary cell walls of vascular plants. Unlike most gymnosperm and eudicot lignins that are generated via the polymerization of monolignols, grass lignins additionally incorporate the flavonoid tricin as a natural lignin monomer. The biosynthesis and functions of tricin-integrated lignin (tricin-lignin) in grass cell walls and its effects on the utility of grass biomass remain largely unknown. We herein report a comparative analysis of rice (Oryza sativa) mutants deficient in the early flavonoid biosynthetic genes encoding CHALCONE SYNTHASE (CHS), CHALCONE ISOMERASE (CHI), and CHI-LIKE (CHIL), with an emphasis on the analyses of disrupted tricin-lignin formation and the concurrent changes in lignin profiles and cell wall digestibility. All examined CHS-, CHI-, and CHIL-deficient rice mutants were largely depleted of extractable flavones, including tricin, and nearly devoid of tricin-lignin in the cell walls, supporting the crucial roles of CHS and CHI as committed enzymes and CHIL as a noncatalytic enhancer in the conserved biosynthetic pathway leading to flavone and tricin-lignin formation. In-depth cell wall structural analyses further indicated that lignin content and composition, including the monolignol-derived units, were differentially altered in the mutants. However, regardless of the extent of the lignin alterations, cell wall saccharification efficiencies of all tested rice mutants were similar to that of the wild-type controls. Together with earlier studies on other tricin-depleted grass mutant and transgenic plants, our results reflect the complexity in the metabolic consequences of tricin pathway perturbations and the relationships between lignin profiles and cell wall properties.

Hydrogen Peroxide Treatment of Softwood-Derived Poly(Ethylene Glycol)-Modified Glycol Lignin at Room Temperature.

Recently, a large-scale production system of softwood-derived poly(ethylene glycol) (PEG)-modified glycol lignin (GL) was developed to produce high-quality lignin derivatives with substantially controlled chemical structures and attractive thermal properties. In this study, the further upgrading of GL properties with carboxy functionalization was demonstrated through the room-temperature hydrogen peroxide (H2O2) treatment with the mass ratio of H2O2 to GL, 1:1 and 1:3, for 7 d. The changes in the chemical structure, carboxy group content, molecular weight, and thermal properties of the insoluble portions of partially oxidized glycol lignins (OGLs) were then investigated. Nuclear magnetic resonance and thioacidolysis data revealed that the oxidative functionalization involved the cleavage of ß-O-4 linkages and the oxidative cleavage of guaiacyl aromatic rings into muconic acid-type structures. This was validated by attenuated total reflectance-Fourier transform infrared (ATR-FTIR) spectroscopy and potentiometric titration. Overall, the results suggested that the varying outcomes of carboxy group content (0.81-2.04 mmol/g OGL) after 7-d treatment depended on the type of the GL origin having varying amounts of the retained native lignin structure (e.g., ß-O-4 linkages), which were prepared from different source-wood-meal sizes and PEG molecular masses.

Possible mechanisms for the generation of phenyl glycoside-type lignin-carbohydrate linkages in lignification with monolignol glucosides.

The existence and formation of covalent lignin-carbohydrate (LC) linkages in plant cell walls has long been a matter of debate in terms of their roles in cell wall development and biomass use. Of the various putative LC linkages proposed to date, evidence of the native existence and formation mechanism of phenyl glycoside (PG)-type LC linkages in planta is particularly scarce. The present study aimed to explore previously overlooked mechanisms for the formation of PG-type LC linkages through the incorporation of monolignol glucosides, which are possible lignin precursors, into lignin polymers during lignification. Peroxidase-catalyzed lignin polymerization of coniferyl alcohol in the presence of coniferin and syringin in vitro resulted in the generation of PG-type LC linkages in synthetic lignin polymers, possibly via nucleophilic addition onto quinone methide (QM) intermediates formed during polymerization. Biomimetic lignin polymerization of coniferin via the ß-glucosidase/peroxidase system also resulted in the generation of PG-type as well as alkyl glycoside-type LC linkages. This occurred via non-enzymatic QM-involving reactions and also via enzymatic transglycosylations involving ß-glucosidase, which was demonstrated by in-depth structural analysis of the synthetic lignins by two-dimensional NMR. We collected heteronuclear single-quantum coherence (HSQC) NMR for native cell wall fractions prepared from pine (Pinus taeda), eucalyptus (Eucalyptus camaldulensis), acacia (Acacia mangium), poplar (Populus × eurarnericana) and bamboo (Phyllostachys edulis) wood samples, which exhibited correlations, albeit at low levels, that were well matched with those of the PG-type LC linkages in synthetic lignins incorporating monolignol glucosides. Overall, our results provide a molecular basis for feasible mechanisms for the generation of PG-type LC linkages from monolignol glucosides and further substantiates their existence in planta.

Localised laccase activity modulates distribution of lignin polymers in gymnosperm compression wood.

The woody stems of coniferous gymnosperms produce specialised compression wood to adjust the stem growth orientation in response to gravitropic stimulation. During this process, tracheids develop a compression-wood-specific S2 L cell wall layer with lignins highly enriched with p-hydroxyphenyl (H)-type units derived from H-type monolignol, whereas lignins produced in the cell walls of normal wood tracheids are exclusively composed of guaiacyl (G)-type units from G-type monolignol with a trace amount of H-type units. We show that laccases, a class of lignin polymerisation enzymes, play a crucial role in the spatially organised polymerisation of H-type and G-type monolignols during compression wood formation in Japanese cypress (Chamaecyparis obtusa). We performed a series of chemical-probe-aided imaging analysis on C. obtusa compression wood cell walls, together with gene expression, protein localisation and enzymatic assays of C. obtusa laccases. Our data indicated that CoLac1 and CoLac3 with differential oxidation activities towards H-type and G-type monolignols were precisely localised to distinct cell wall layers in which H-type and G-type lignin units were preferentially produced during the development of compression wood tracheids. We propose that, not only the spatial localisation of laccases, but also their biochemical characteristics dictate the spatial patterning of lignin polymerisation in gymnosperm compression wood.

OsMYB108 loss-of-function enriches p-coumaroylated and tricin lignin units in rice cell walls.

Breeding approaches to enrich lignins in biomass could be beneficial to improving the biorefinery process because lignins increase biomass heating value and represent a potent source of valuable aromatic chemicals. However, despite the fact that grasses are promising lignocellulose feedstocks, limited information is yet available for molecular-breeding approaches to upregulate lignin biosynthesis in grass species. In this study, we generated lignin-enriched transgenic rice (Oryza sativa), a model grass species, via targeted mutagenesis of the transcriptional repressor OsMYB108 using CRISPR/Cas9-mediated genome editing. The OsMYB108-knockout rice mutants displayed increased expressions of lignin biosynthetic genes and enhanced lignin deposition in culm cell walls. Chemical and two-dimensional nuclear magnetic resonance (NMR) analyses revealed that the mutant cell walls were preferentially enriched in γ-p-coumaroylated and tricin lignin units, both of which are typical and unique components in grass lignins. NMR analysis also showed that the relative abundances of major lignin linkage types were altered in the OsMYB108 mutants.

Lignin characterization of rice CONIFERALDEHYDE 5-HYDROXYLASE loss-of-function mutants generated with the CRISPR/Cas9 system.

The aromatic composition of lignin is an important trait that greatly affects the usability of lignocellulosic biomass. We previously identified a rice (Oryza sativa) gene encoding coniferaldehyde 5-hydroxylase (OsCAld5H1), which was effective in modulating syringyl (S)/guaiacyl (G) lignin composition ratio in rice, a model grass species. Previously characterized OsCAld5H1-knockdown rice lines, which were produced via an RNA-interference approach, showed augmented G lignin units yet contained considerable amounts of residual S lignin units. In this study, to further investigate the effect of suppression of OsCAld5H1 on rice lignin structure, we generated loss-of-function mutants of OsCAld5H1 using the CRISPR/Cas9-mediated genome editing system. Homozygous OsCAld5H1-knockout lines harboring anticipated frame-shift mutations in OsCAld5H1 were successfully obtained. A series of wet-chemical and two-dimensional NMR analyses on cell walls demonstrated that although lignins in the mutant were predictably enriched in G units all the tested mutant lines produced considerable numbers of S units. Intriguingly, lignin γ-p-coumaroylation analysis by the derivatization followed by reductive cleavage method revealed that enrichment of G units in lignins of the mutants was limited to the non-γ-p-coumaroylated units, whereas grass-specific γ-p-coumaroylated lignin units were almost unaffected. Gene expression analysis indicated that no homologous genes of OsCAld5H1 were overexpressed in the mutants. These data suggested that CAld5H is mainly involved in the production of non-γ-p-coumaroylated S lignin units, common in both eudicots and grasses, but not in the production of grass-specific γ-p-coumaroylated S units in rice.

Plant-specific Dof transcription factors VASCULAR-RELATED DOF1 and VASCULAR-RELATED DOF2 regulate vascular cell differentiation and lignin biosynthesis in Arabidopsis.

KEY MESSAGE: Plant-specific Dof transcription factors VDOF1 and VDOF2 are novel regulators of vascular cell differentiation through the course of a lifetime in Arabidopsis, with shifting their transcriptional target genes. Vascular system is one of critical tissues for vascular plants to transport low-molecular compounds, such as water, minerals, and the photosynthetic product, sucrose. Here, we report the involvement of two Dof transcription factors, named VASCULAR-RELATED DOF1 (VDOF1)/VDOF4.6 and VDOF2/VDOF1.8, in vascular cell differentiation and lignin biosynthesis in Arabidopsis. VDOF genes were expressed in vascular tissues, but the detailed expression sites were partly different between VDOF1 and VDOF2. Vein patterning and lignin analysis of VDOF overexpressors and double mutant vdof1 vdof2 suggested that VDOF1 and VDOF2 would function as negative regulators of vein formation in seedlings, and lignin deposition in inflorescence stems. Interestingly, effects of VDOF overexpression in lignin deposition were different by developmental stages of inflorescence stems, and total lignin contents were increased and decreased in VDOF1 and VDOF2 overexpressors, respectively. RNA-seq analysis of inducible VDOF overexpressors demonstrated that the genes for cell wall biosynthesis, including lignin biosynthetic genes, and the transcription factor genes related to stress response and brassinosteroid signaling were commonly affected by VDOF1 and VDOF2 overexpression. Taken together, we concluded that VDOF1 and VDOF2 are novel regulators of vascular cell differentiation through the course of a lifetime, with shifting their transcriptional target genes: in seedlings, the VDOF genes negatively regulate vein formation, while at reproductive stages, the VDOF proteins target lignin biosynthesis.

Convergent recruitment of 5'-hydroxylase activities by CYP75B flavonoid B-ring hydroxylases for tricin biosynthesis in Medicago legumes.

Tricin (3',5'-dimethoxylated flavone) is a predominant flavonoid amongst monocots but occurs only in isolated and unrelated dicot lineages. Although tricin biosynthesis has been intensively studied in monocots, it has remained largely elusive in tricin-accumulating dicots. We investigated a subgroup of cytochrome P450 (CYP) 75B subfamily flavonoid B-ring hydroxylases (FBHs) from two tricin-accumulating legumes, Medicago truncatula and alfalfa (Medicago sativa), by phylogenetic, molecular, biochemical and mutant analyses. Five Medicago cytochrome P450 CYP75B FBHs are phylogenetically distant from other legume CYP75B members. Among them, MtFBH-4, MsFBH-4 and MsFBH-10 were expressed in tricin-accumulating vegetative tissues. In vitro and in planta analyses demonstrated that these proteins catalyze 3'- and 5'-hydroxylations critical to tricin biosynthesis. A key amino acid polymorphism, T492G, at their substrate recognition site 6 domain is required for the novel 5'-hydroxylation activities. Medicago truncatula mtfbh-4 mutants were tricin-deficient, indicating that MtFBH-4 is indispensable for tricin biosynthesis. Our results revealed that these Medicago legumes had acquired the tricin pathway through molecular evolution of CYP75B FBHs subsequent to speciation from other nontricin-accumulating legumes. Moreover, their evolution is independent of that of grass-specific CYP75B apigenin 3'-hydroxylases/chrysoeriol 5'-hydroxylases dedicated to tricin production and Asteraceae CYP75B flavonoid 3',5'-hydroxylases catalyzing the production of delphinidin-based pigments.

Fibre-specific regulation of lignin biosynthesis improves biomass quality in Populus.

Lignin is a major component of cell wall biomass and decisively affects biomass utilisation. Engineering of lignin biosynthesis is extensively studied, while lignin modification often causes growth defects. We developed a strategy for cell-type-specific modification of lignin to achieve improvements in cell wall property without growth penalty. We targeted a lignin-related transcription factor, LTF1, for modification of lignin biosynthesis. LTF1 can be engineered to a nonphosphorylation form which is introduced into Populus under the control of either a vessel-specific or fibre-specific promoter. The transgenics with lignin suppression in vessels showed severe dwarfism and thin-walled vessels, while the transgenics with lignin suppression in fibres displayed vigorous growth with normal vessels under phytotron, glasshouse and field conditions. In-depth lignin structural analyses revealed that such cell-type-specific downregulation of lignin biosynthesis led to the alteration of overall lignin composition in xylem tissues reflecting the population of distinctive lignin polymers produced in vessel and fibre cells. This study demonstrates that fibre-specific suppression of lignin biosynthesis resulted in the improvement of wood biomass quality and saccharification efficiency and presents an effective strategy to precisely regulate lignin biosynthesis with desired growth performance.

The Structural Integrity of Lignin Is Crucial for Resistance against Striga hermonthica Parasitism in Rice.

Striga species are parasitic weeds that seriously constrain the productivity of food staples, including cereals and legumes, in Sub-Saharan Africa and Asia. In eastern and central Africa, Striga spp. infest as much as 40 million hectares of smallholder farmland causing total crop failure during severe infestation. As the molecular mechanisms underlying resistance are yet to be elucidated, we undertook a comparative metabolome study using the Striga-resistant rice (Oryza sativa) cultivar 'Nipponbare' and the susceptible cultivar 'Koshihikari'. We found that a number of metabolites accumulated preferentially in the Striga-resistant cultivar upon Striga hermonthica infection. Most apparent was increased deposition of lignin, a phenylpropanoid polymer mainly composed of p-hydroxyphenyl (H), guaiacyl (G), and syringyl (S) aromatic units, around the site of interaction in Nipponbare. The increased deposition of lignin was accompanied by induction of the expression of corresponding enzyme-encoding genes in the phenylpropanoid pathway. In addition, perturbing normal lignin composition by knocking down or overexpressing the genes that regulate lignin composition, i.e. p-COUMARATE 3-HYDROXYLASE or FERULATE 5-HYDROXYLASE, enhanced susceptibility of Nipponbare to S hermonthica infection. These results demonstrate that enhanced lignin deposition and maintenance of the structural integrity of lignin polymers deposited at the infection site are crucial for postattachment resistance against S hermonthica.

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.