The conducting state of TRPA1 modulates channel lateral mobility.

We have studied Danio rerio (Zebrafish) TRPA1 channel using a method that combines single channel electrophysiological and optical recordings to evaluate lateral mobility and channel gating simultaneously in single channels. TRPA1 channel activation by two distinct chemical ligands: allyl isothiocyanate (AITC) and TRPswitch B, results in substantial reduction of channel lateral mobility at the plasma membrane. Incubation with the cholesterol sequestering agent methyl-ß-cyclodextrin (MßCD), prevents the reduction on lateral mobility induced by the two chemical agonists. This results strongly suggest that the open conformation of TRPA1 modulates channel lateral mobility probably by facilitating the insertion of the channel into cholesterol-enriched domains at the plasma membrane.

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.

Endoscopic En-Bloc Resection of Intra-Muscular Lipoma of the Flexor Digitorum Profundus: A Uniportal Technique.

Deep-seated lipomas can be intramuscular, intermuscular, and rarely, parosteal lipomas. Intramuscular lipoma can be divided into infiltrative, well-circumscribed, and mixed types. Marginal excision is the treatment of choice for well-circumscribed intramuscular lipoma, and histopathology eliminates diagnosis of well-differentiated liposarcoma. The purpose of this technical note is to describe the details of endoscopic en-bloc resection of intramuscular lipoma of the flexor digitorum profundus. This minimally invasive approach allows en-bloc resection of the lipoma for histopathological study, with minimal risk to the surrounding neurovascular structures.

Multifaceted roles of flavonoids mediating plant-microbe interactions.

Plant-microbe interactions dynamically affect plant growth, health, and development. The mechanisms underpinning these associations are-to a large extent-mediated by specialized host-derived secondary metabolites. Flavonoids are one of the most studied classes of such metabolites, regulating both plant development and the interaction with commensal microbes. Here, we provide a comprehensive review of the multiple roles of flavonoids in mediating plant-microbe interactions. First, we briefly summarize the general aspects of flavonoid synthesis, transport, and exudation in plants. Then, we review the importance of flavonoids regulating plant-microbe interactions and dynamically influencing the overall community assembly of plant-root microbiomes. Last, we highlight potential knowledge gaps in our understanding of how flavonoids determine the interactions between plants and commensal microbes. Collectively, we advocate the importance of advancing research in this area toward innovative strategies to effectively manipulate plant-microbiome composition, in this case, via flavonoid production and exudation in plant roots. Video Abstract.

Longitudinal in vivo imaging of adult Danionella cerebrum using standard confocal microscopy.

Danionella cerebrum is a new vertebrate model that offers an exciting opportunity to visualize dynamic biological processes in intact adult animals. Key advantages of this model include its small size, life-long optical transparency, genetic amenability and short generation time. Establishing a reliable method for longitudinal in vivo imaging of adult D. cerebrum while maintaining viability will allow in-depth image-based studies of various processes involved in development, disease onset and progression, wound healing, and aging in an intact live animal. Here, a method for both prolonged and longitudinal confocal live imaging of adult D. cerebrum using custom-designed and 3D-printed imaging chambers is described. Two transgenic D. cerebrum lines were created to test the imaging system, i.e. Tg(mpeg1:dendra2) and Tg(kdrl:mCherry-caax). The first line was used to visualize macrophages and microglia, and the second for spatial registration. By using this approach, differences in immune cell morphology and behavior during homeostasis as well as in response to a stab wound or two-photon-induced brain injury were observed in intact adult fish over the course of several days.

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.

Alternative Splicing and Its Roles in Plant Metabolism.

Plant metabolism, including primary metabolism such as tricarboxylic acid cycle, glycolysis, shikimate and amino acid pathways as well as specialized metabolism such as biosynthesis of phenolics, alkaloids and saponins, contributes to plant survival, growth, development and interactions with the environment. To this end, these metabolic processes are tightly and finely regulated transcriptionally, post-transcriptionally, translationally and post-translationally in response to different growth and developmental stages as well as the constantly changing environment. In this review, we summarize and describe the current knowledge of the regulation of plant metabolism by alternative splicing, a post-transcriptional regulatory mechanism that generates multiple protein isoforms from a single gene by using alternative splice sites during splicing. Numerous genes in plant metabolism have been shown to be alternatively spliced under different developmental stages and stress conditions. In particular, alternative splicing serves as a regulatory mechanism to fine-tune plant metabolism by altering biochemical activities, interaction and subcellular localization of proteins encoded by splice isoforms of various genes.

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.

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.

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.

Cell type specific gene expression profiling reveals a role for complement component C3 in neutrophil responses to tissue damage.

Tissue damage induces rapid recruitment of leukocytes and changes in the transcriptional landscape that influence wound healing. However, the cell-type specific transcriptional changes that influence leukocyte function and tissue repair have not been well characterized. Here, we employed translating ribosome affinity purification (TRAP) and RNA sequencing, TRAP-seq, in larval zebrafish to identify genes differentially expressed in neutrophils, macrophages, and epithelial cells in response to wounding. We identified the complement pathway and c3a.1, homologous to the C3 component of human complement, as significantly increased in neutrophils in response to wounds. c3a.1-/- zebrafish larvae have impaired neutrophil directed migration to tail wounds with an initial lag in recruitment early after wounding. Moreover, c3a.1-/- zebrafish larvae have impaired recruitment to localized bacterial infections and reduced survival that is, at least in part, neutrophil mediated. Together, our findings support the power of TRAP-seq to identify cell type specific changes in gene expression that influence neutrophil behavior in response to tissue damage.

TRPswitch-A Step-Function Chemo-optogenetic Ligand for the Vertebrate TRPA1 Channel.

Chemo-optogenetics has produced powerful tools for optical control of cell activity, but current tools suffer from a variety of limitations including low unitary conductance, the need to modify the target channel, or the inability to control both on and off switching. Using a zebrafish behavior-based screening strategy, we discovered "TRPswitch", a photoswitchable nonelectrophilic ligand scaffold for the transient receptor potential ankyrin 1 (TRPA1) channel. TRPA1 exhibits high unitary channel conductance, making it an ideal target for chemo-optogenetic tool development. Key molecular determinants for the activity of TRPswitch were elucidated and allowed for replacement of the TRPswitch azobenzene with a next-generation azoheteroarene. The TRPswitch compounds enable reversible, repeatable, and nearly quantitative light-induced activation and deactivation of the vertebrate TRPA1 channel with violet and green light, respectively. The utility of TRPswitch compounds was demonstrated in larval zebrafish hearts exogenously expressing zebrafish Trpa1b, where the heartbeat could be controlled using TRPswitch and light. Therefore, TRPA1/TRPswitch represents a novel step-function chemo-optogenetic system with a unique combination of high conductance, high efficiency, activity against an unmodified vertebrate channel, and capacity for bidirectional optical switching. This chemo-optogenetic system will be particularly applicable in systems where a large depolarization current is needed or sustained channel activation is desirable.

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.

Flavonoids are indispensable for complete male fertility in rice.

Flavonoids are essential for male fertility in some but not all plant species. In rice (Oryza sativa), the chalcone synthase mutant oschs1 produces flavonoid-depleted pollen and is male sterile. The mutant pollen grains are viable with normal structure, but they display reduced germination rate and pollen-tube length. Analysis of oschs1/+ heterozygous lines shows that pollen flavonoid deposition is a paternal effect and fertility is independent of the haploid genotypes (OsCHS1 or oschs1). To understand which classes of flavonoids are involved in male fertility, we conducted detailed analysis of rice mutants for branch-point enzymes of the downstream flavonoid pathways, including flavanone 3-hydroxylase (OsF3H; flavonol pathway entry enzyme), flavone synthase II (CYP93G1; flavone pathway entry enzyme), and flavanone 2-hydroxylase (CYP93G2; flavone C-glycoside pathway entry enzyme). Rice osf3h and cyp93g1 cyp93g2 CRISPR/Cas9 mutants, and cyp93g1 and cyp93g2 T-DNA insertion mutants showed altered flavonoid profiles in anthers, but only the osf3h and cyp93g1 cyp93g2 mutants displayed reduction in seed yield. Our findings indicate that flavonoids are essential for complete male fertility in rice and a combination of different classes (flavanones, flavonols, flavones, and flavone C-glycosides) appears to be important, as opposed to the essential role played primarily by flavonols that has been previously reported in several plant species.

Boholamide A, an APD-Class, Hypoxia-Selective Cyclodepsipeptide.

Calcium homeostasis is implicated in some cancers, leading to the possibility that selective control of calcium might lead to new cancer drugs. On the basis of this idea, we designed an assay using a glioblastoma cell line and screened a collection of 1000 unique bacterial extracts. Isolation of the active compound from a hit extract led to the identification of boholamide A (1), a 4-amido-2,4-pentadieneoate (APD)-class peptide. Boholamide A (1) applied in the nanomolar range induces an immediate influx of Ca2+ in glioblastoma and neuronal cells. APD-class natural products are hypoxia-selective cytotoxins that primarily target mitochondria. Like other APD-containing compounds, 1 is hypoxia selective. Since APD natural products have received significant interest as potential chemotherapeutic agents, 1 provides a novel APD scaffold for the development of new anticancer compounds.