Antioxidant and UV-blocking activity of PVA/tannin-based bioplastics in food packaging application.

In this study, bioplastics with antioxidant and UV protection properties based on tannin and PVA were created for packaging uses. Using a hot water extraction method at various extraction temperatures (60-100 °C), tannins were removed from the bark of Acacia mangium. Tannins with the best antioxidant activity were extracted at 80 °C. In order to create bioplastic formulations (PVA/Tannins), the extract is then employed. The non-heating bioplastic method's preparation (M3) stage produced the highest levels of antioxidant activity. Therefore, subsequent tests were conducted using the non-heating method (M3). On the opacity, UV protective activity, antioxidant capacity, mechanical strength, thermal stability, and water vapor permeability of the resultant bioplastics, the impact of tannin concentration (0.1-0.5 g) was examined. The findings of the experiments demonstrate that PVA/Tannin bioplastics are less transparent than pure PVA. The PVA/tannin bioplastics that are formed, on the whole, show strong antioxidant and UV protection action. Comparing PVA/Tannin bioplastics to pure PVA also revealed a small improvement in thermal stability and tensile strength. In PVA bioplastics with resistant tannins, moisture content was marginally greater even at low tannin concentrations (0.1 g). Based on the findings, bioplastics made from PVA and the tannin A. mangium have the potential to be used to create packaging that is UV and active antioxidant resistant. It can be applied as the second (inner) layer of the primary packaging to protect food freshness and nutrition due to their antioxidant activity.

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.

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.

Regioselective stilbene O-methylations in Saccharinae grasses.

O-Methylated stilbenes are prominent nutraceuticals but rarely produced by crops. Here, the inherent ability of two Saccharinae grasses to produce regioselectively O-methylated stilbenes is reported. A stilbene O-methyltransferase, SbSOMT, is first shown to be indispensable for pathogen-inducible pterostilbene (3,5-bis-O-methylated) biosynthesis in sorghum (Sorghum bicolor). Phylogenetic analysis indicates the recruitment of genus-specific SOMTs from canonical caffeic acid O-methyltransferases (COMTs) after the divergence of Sorghum spp. from Saccharum spp. In recombinant enzyme assays, SbSOMT and COMTs regioselectively catalyze O-methylation of stilbene A-ring and B-ring respectively. Subsequently, SOMT-stilbene crystal structures are presented. Whilst SbSOMT shows global structural resemblance to SbCOMT, molecular characterizations illustrate two hydrophobic residues (Ile144/Phe337) crucial for substrate binding orientation leading to 3,5-bis-O-methylations in the A-ring. In contrast, the equivalent residues (Asn128/Asn323) in SbCOMT facilitate an opposite orientation that favors 3'-O-methylation in the B-ring. Consistently, a highly-conserved COMT is likely involved in isorhapontigenin (3'-O-methylated) formation in wounded wild sugarcane (Saccharum spontaneum). Altogether, our work reveals the potential of Saccharinae grasses as a source of O-methylated stilbenes, and rationalize the regioselectivity of SOMT activities for bioengineering of O-methylated stilbenes.

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.

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.

Pathogen-induced autophagy regulates monolignol transport and lignin formation in plant immunity.

The evolutionary plant-pathogen arms race has equipped plants with the immune system that can defend against pathogens. Pattern-triggered immunity and effector-triggered immunity are two major branches of innate immunity that share immune responses, including oxidative bursts, transcriptional reprogramming, and cell wall modifications such as lignin deposition. In a previous study, we reported that lignin rapidly accumulates in pathogen-infected Arabidopsis leaves and acts as a mechanical barrier, spatially restricting pathogens and cell death. Lignin deposition into the cell wall is a three-step process: monolignol biosynthesis, transport, and polymerization. While monolignol biosynthesis and polymerization are relatively well understood, the mechanism of monolignol transport remains unclear. In this study, we show that macroautophagy/autophagy modulates pathogen-induced lignin formation. Lignification and other immune responses were impaired in autophagy-defective atg (autophagy-related) mutants. In microscopy analyses, monolignols formed punctate structures in response to pathogen infection and colocalized with autophagic vesicles. Furthermore, autophagic activity and lignin accumulation were both enhanced in dnd1 (defense, no death 1) mutant with elevated disease resistance but no cell death and crossing dnd1-1 with atg mutants resulted in a lignin deficit, further supporting that lignin formation requires autophagy. Collectively, these findings demonstrate that lignification, particularly monolignol transport, is achieved through autophagic membrane trafficking in plant immunity.Abbreviations: ABC transporter: ATP-binding cassette transporter; ACD2/AT4G37000: accelerated cell death 2; ATG: autophagy-related; C3'H/AT2G40890: p-coumaroyl shikimate 3-hydroxylase; C4H/AT2G30490: cinnamate 4-hydroxylase; CA: coniferyl alcohol; CaMV: cauliflower mosaic virus; CASP: Casparian strip membrane domain protein; CASPL: CASP-like protein; CBB: Coomassie Brilliant Blue; CCoAOMT1/AT4G34050: caffeoyl-CoA O-methyltransferase 1; CCR1/AT1G15950: cinnamoyl-CoA reductase 1; CFU: colony-forming unit; COMT1/AT5G54160: caffeic acid O-methyltransferase 1; Con A: concanamycin A; DMAC: dimethylaminocoumarin; DND1/AT5G15410: defense, no death 1; CNGC2: cyclic nucleotide-gated channel 2; ER: endoplasmic reticulum; ESB1/AT2G28670/DIR10: enhanced suberin 1; ETI: effector-triggered immunity; EV: extracellular vesicle; F5H/AT4G36220: ferulate-5-hydroxylase; Fluo-3 AM: Fluo-3 acetoxymethyl ester; GFP: green fluorescent protein; HCT/AT5G48930: p-hydroxycinnamoyl-CoA:quinate/shikimate p-hydroxycinnamoyltransferase; HR: hypersensitive response; LAC: laccase; LTG: LysoTracker Green; LSD1/AT4G200380: lesion stimulating disease 1; PAL1/AT2G37040: phenylalanine ammonia-lyase 1; PAMP: pathogen-associated molecular patterns; PCD: programmed cell death; PE: phosphatidylethanolamine; PRX: peroxidase; Pst DC3000: Pseudomonas syringe pv. tomato DC3000; PTI: pattern-triggered immunity; SA: salicylic acid; SD: standard deviation; SID2/AT1G7410: SA induction-deficient 2; UGT: UDP-glucosyltransferase; UPLC: ultraperformance liquid chromatography; UPS: unconventional protein secretion; V-ATPase: vacuolar-type H+-translocating ATPase.

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.

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.

Limiting silicon supply alters lignin content and structures of sorghum seedling cell walls.

Sorghum has been recognized as a promising energy crop. The composition and structure of lignin in the cell wall are important factors that affect the quality of plant biomass as a bioenergy feedstock. Silicon (Si) supply may affect the lignin content and structure, as both Si and lignin are possibly involved in plant mechanical strength. However, our understanding regarding the interaction between Si and lignin in sorghum is limited. Therefore, in this study, we analyzed the lignin in the cell walls of sorghum seedlings cultured hydroponically with or without Si supplementation. Limiting the Si supply significantly increased the thioglycolic acid lignin content and thioacidolysis-derived syringyl/guaiacyl monomer ratio. At least part of the modification may be attributable to the change in gene expression, as suggested by the upregulation of phenylpropanoid biosynthesis-related genes under -Si conditions. The cell walls of the -Si plants had a higher mechanical strength and calorific value than those of the +Si plants. These results provide some insights into the enhancement of the value of sorghum biomass as a feedstock for energy production by limiting Si uptake.

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.

Nitrogen deficiency results in changes to cell wall composition of sorghum seedlings.

Sorghum [Sorghum bicolor (L.) Moench] has been gaining attention as a feedstock for biomass energy production. While it is obvious that nitrogen (N) supply significantly affects sorghum growth and biomass accumulation, our knowledge is still limited regarding the effect of N on the biomass quality of sorghum, such as the contents and structures of lignin and other cell wall components. Therefore, in this study, we investigated the effects of N supply on the structure and composition of sorghum cell walls. The cell walls of hydroponically cultured sorghum seedlings grown under sufficient or deficient N conditions were analyzed using chemical, two-dimensional nuclear magnetic resonance, gene expression, and immunohistochemical methods. We found that the level of N supply considerably affected the cell wall structure and composition of sorghum seedlings. Limitation of N led to a decrease in the syringyl/guaiacyl lignin unit ratio and an increase in the amount and alteration of tissue distribution of several hemicelluloses, including mixed linkage (1 → 3), (1 → 4)-ß-D-glucan, and arabinoxylan. At least some of these cell wall alterations could be associated with changes in gene expression. Nitrogen status is thus one of the factors affecting the cell wall properties of sorghum seedlings.

New Insights on Structures Forming the Lignin-Like Fractions of Ancestral Plants.

In the present work, lignin-like fractions were isolated from several ancestral plants -including moss (Hypnum cupressiforme and Polytrichum commune), lycophyte (Selaginella kraussiana), horsetail (Equisetum palustre), fern (Nephrolepis cordifolia and Pteridium aquilinum), cycad (Cycas revoluta), and gnetophyte (Ephedra fragilis) species- and structurally characterized by pyrolysis-gas chromatography-mass spectrometry (Py-GC/MS) and two-dimensional nuclear magnetic resonance (2D-NMR) spectroscopy. Py-GC/MS yielded marker compounds characteristic of lignin units, except in the H. cupressiforme, P. commune and E. palustre "lignins," where they were practically absent. Additional structural information on the other five samples was obtained from 2D-NMR experiments displaying intense correlations signals of guaiacyl (G) units in the fern and cycad lignins, along with smaller amounts of p-hydroxyphenyl (H) units. Interestingly, the lignins from the lycophyte S. kraussiana and the gnetophyte E. fragilis were not only composed of G- and H-lignin units but they also incorporated significant amounts of the syringyl (S) units characteristic of angiosperms, which appeared much later in plant evolution, most probably due to convergent evolution. The latter finding is also supported by the abundance of syringol derivatives after the Py-GC/MS analyses of these two samples. Regarding lignin structure, ß-O-4' alkyl-aryl ethers were the most abundant substructures, followed by condensed ß-5' phenylcoumarans and ß-ß' resinols (and dibenzodioxocins in the fern and cycad lignins). The highest percentages of alkyl-aryl ether structures correlated with the higher S/G ratio in the S. Kraussiana and E. fragilis lignin-like fractions. More interestingly, apart from the typical monolignol-derived lignin units (H, G and S), other structures, assigned to flavonoid compounds never reported before in natural lignins (such as amentoflavone, apigenin, hypnogenol B, kaempferol, and naringenin), could also be identified in the HSQC spectra of all the lignin-like fractions analyzed. With this purpose, in vitro synthesized coniferyl-naringenin and coniferyl-apigenin dehydrogenation polymers were used as standards. These flavonoids were abundant in H. cupressiforme appearing as the only constituents of the moss lignin-like fraction (including 84% of dimeric hypnogenol B) and their abundance decreased in those of S. Kraussiana (with amentoflavone and naringenin representing 14% of the total aromatic units), and the two ancient gymnosperms (0.4-1.2%) and ferns (0-0.7%).

Tricin Biosynthesis and Bioengineering.

Tricin (3',5'-dimethoxyflavone) is a specialized metabolite which not only confers stress tolerance and involves in defense responses in plants but also represents a promising nutraceutical. Tricin-type metabolites are widely present as soluble tricin O-glycosides and tricin-oligolignols in all grass species examined, but only show patchy occurrences in unrelated lineages in dicots. More strikingly, tricin is a lignin monomer in grasses and several other angiosperm species, representing one of the "non-monolignol" lignin monomers identified in nature. The unique biological functions of tricin especially as a lignin monomer have driven the identification and characterization of tricin biosynthetic enzymes in the past decade. This review summarizes the current understanding of tricin biosynthetic pathway in grasses and tricin-accumulating dicots. The characterized and potential enzymes involved in tricin biosynthesis are highlighted along with discussion on the debatable and uncharacterized steps. Finally, current developments of bioengineering on manipulating tricin biosynthesis toward the generation of functional food as well as modifications of lignin for improving biorefinery applications are summarized.

Monolignol acyltransferase for lignin p-hydroxybenzoylation in Populus.

Plant lignification exhibits notable plasticity. Lignin in many species, including Populus spp., has long been known to be decorated with p-hydroxybenzoates. However, the molecular basis for such structural modification remains undetermined. Here, we report the identification and characterization of a Populus BAHD family acyltransferase that catalyses monolignol p-hydroxybenzoylation, thus controlling the formation of p-hydroxybenzoylated lignin structures. We reveal that Populus acyltransferase PHBMT1 kinetically preferentially uses p-hydroxybenzoyl-CoA to acylate syringyl lignin monomer sinapyl alcohol in vitro. Consistently, disrupting PHBMT1 in Populus via CRISPR-Cas9 gene editing nearly completely depletes p-hydroxybenzoates of stem lignin; conversely, overexpression of PHBMT1 enhances stem lignin p-hydroxybenzoylation, suggesting PHBMT1 functions as a prime monolignol p-hydroxybenzoyltransferase in planta. Altering lignin p-hydroxybenzoylation substantially changes the lignin solvent dissolution rate, indicative of its structural significance on lignin physiochemical properties. Identification of monolignol p-hydroxybenzoyltransferase offers a valuable tool for tailoring lignin structure and physiochemical properties and for engineering the industrially important platform chemical in woody biomass.

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.

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.

Identifying transcription factors that reduce wood recalcitrance and improve enzymatic degradation of xylem cell wall in Populus.

Developing an efficient deconstruction step of woody biomass for biorefinery has been drawing considerable attention since its xylem cell walls display highly recalcitrance nature. Here, we explored transcriptional factors (TFs) that reduce wood recalcitrance and improve saccharification efficiency in Populus species. First, 33 TF genes up-regulated during poplar wood formation were selected as potential regulators of xylem cell wall structure. The transgenic hybrid aspens (Populus tremula × Populus tremuloides) overexpressing each selected TF gene were screened for in vitro enzymatic saccharification. Of these, four transgenic seedlings overexpressing previously uncharacterized TF genes increased total glucan hydrolysis on average compared to control. The best performing lines overexpressing Pt × tERF123 and Pt × tZHD14 were further grown to form mature xylem in the greenhouse. Notably, the xylem cell walls exhibited significantly increased total xylan hydrolysis as well as initial hydrolysis rates of glucan. The increased saccharification of Pt × tERF123-overexpressing lines could reflect the improved balance of cell wall components, i.e., high cellulose and low xylan and lignin content, which could be caused by upregulation of cellulose synthase genes upon the expression of Pt × tERF123. Overall, we successfully identified Pt × tERF123 and Pt × tZHD14 as effective targets for reducing cell wall recalcitrance and improving the enzymatic degradation of woody plant biomass.

The Arabidopsis R2R3 MYB Transcription Factor MYB15 Is a Key Regulator of Lignin Biosynthesis in Effector-Triggered Immunity.

Lignin, a major component of the secondary cell wall, is important for plant growth and development. Moreover, lignin plays a pivotal role in plant innate immunity. Lignin is readily deposited upon pathogen infection and functions as a physical barrier that limits the spread of pathogens. In this study, we show that an Arabidopsis MYB transcription factor MYB15 is required for the activation of lignin biosynthesis genes such as PAL, C4H, 4CL, HCT, C3'H, COMT, and CAD, and consequently lignin formation during effector-triggered immune responses. Upon challenge with the avirulent bacterial pathogen Pst DC3000 (AvrRpm1), lignin deposition and disease resistance were reduced in myb15 mutant plants. Furthermore, whereas invading pathogens, together with hypersensitive cell death, were restricted to the infection site in wild-type leaves, they spread beyond the infected area in myb15 mutants. The exogenous supply of the lignin monomer coniferyl alcohol restored lignin production and rescued immune defects in myb15 plants. These results demonstrate that regulation at the transcriptional level is key to pathogen-induced lignification and that MYB15 plays a central role in this process.

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.