Identification of xylan arabinosyl 2-O-xylosyltransferases catalyzing the addition of 2-O-xylosyl residue onto arabinosyl side chains of xylan in grass species.

Grass xylan, the major hemicellulose in both primary and secondary cell walls, is heavily decorated with α-1,3-linked arabinofuranosyl (Araf) residues that may be further substituted at O-2 with xylosyl (Xyl) or Araf residues. Although xylan 3-O-arabinosyltransferases (XATs) catalyzing 3-O-Araf addition onto xylan have been characterized, glycosyltransferases responsible for the transfer of 2-O-Xyl or 2-O-Araf onto 3-O-Araf residues of xylan to produce the Xyl-Araf and Araf-Araf disaccharide side chains remain to be identified. In this report, we showed that a rice GT61 member, named OsXAXT1 (xylan arabinosyl 2-O-xylosyltransferase 1) herein, was able to mediate the addition of Xyl-Araf disaccharide side chains onto xylan when heterologously co-expressed with OsXAT2 in the Arabidopsis gux1/2/3 (glucuronic acid substitution of xylan 1/2/3) triple mutant that lacks any glycosyl substitutions. Recombinant OsXAXT1 protein expressed in human embryonic kidney 293 cells exhibited a xylosyltransferase activity catalyzing the addition of Xyl from UDP-Xyl onto arabinosylated xylooligomers. Consistent with its function as a xylan arabinosyl 2-O-xylosyltransferase, CRISPR-Cas9-mediated mutations of the OsXAXT1 gene in transgenic rice plants resulted in a reduction in the level of Xyl-Araf disaccharide side chains in xylan. Furthermore, we revealed that XAXT1 close homologs from several other grass species, including switchgrass, maize, and Brachypodium, possessed the same functions as OsXAXT1, indicating functional conservation of XAXTs in grass species. Together, our findings establish that grass XAXTs are xylosyltransferases catalyzing Xyl transfer onto O-2 of Araf residues of xylan to form the Xyl-Araf disaccharide side chains, which furthers our understanding of genes involved in xylan biosynthesis.

Pigmentation and Flavonoid Metabolite Diversity in Immature 'Fuji' Apple Fruits in Response to Lights and Methyl Jasmonate.

Artificial pigmentation of apple fruits has been intensely evaluated to generate less pigmented red apples, which are profitable because of the changes in fruit quality. In this study, we analyzed the diversity of flavonoids and the patterns of flavonoid metabolic gene expression under light irradiation with or without methyl jasmonate (MeJA) treatment in immature (S1) and color-turning (S2) staged 'Fuji' apples. Further, we assessed the metabolic regulation at the gene level between anthocyanin and flavonol in light-responsive apple skins. UV-B exposure within 3 days was found to significantly stimulate anthocyanin accumulation in apple skin compared to other light exposure. S1 skin was more sensitive to UV-B and MeJA treatment, in the aspect of indaein accumulation. The enhancement of apple pigmentation following treatment with adequate levels of UV-B and MeJA was maximized at approximately 72 h. Red (range from 4.25 to 17.96 µg·g-1 DW), blue (range from 4.59 to 9.17 µg·g-1 DW) and UV-A (range from 3.98 to 19.12 µg·g-1 DW) lights contributed to the induction of idaein content. Most genes related to the flavonoid pathways increased their expression under UV-B exposure, including the gene expression of the transcription factor, MdMYB10, a well-known upstream factor of flavonoid biosynthesis in apples. The boosted upregulation of MdMYB10, MdCHS, MdF3H MdLDOX, and MdUFGT genes due to MeJA in UV-B was found and may contribute the increase of idaein. UV-A and UV-B caused higher quercetin glycoside content in both S1 and S2 apple skins than longer wavelengths, resulting in significant increases in quercetin-3-O-galactoside and quercetin-3-O-glucoside. These results suggest that the application of adequate UV-B with MeJA in less-pigmented postharvest apples will improve apple color quality within a short period.

CRISPR-Knockout of CSE Gene Improves Saccharification Efficiency by Reducing Lignin Content in Hybrid Poplar.

Caffeoyl shikimate esterase (CSE) has been shown to play an important role in lignin biosynthesis in plants and is, therefore, a promising target for generating improved lignocellulosic biomass crops for sustainable biofuel production. Populus spp. has two CSE genes (CSE1 and CSE2) and, thus, the hybrid poplar (Populus alba × P. glandulosa) investigated in this study has four CSE genes. Here, we present transgenic hybrid poplars with knockouts of each CSE gene achieved by CRISPR/Cas9. To knockout the CSE genes of the hybrid poplar, we designed three single guide RNAs (sg1-sg3), and produced three different transgenic poplars with either CSE1 (CSE1-sg2), CSE2 (CSE2-sg3), or both genes (CSE1/2-sg1) mutated. CSE1-sg2 and CSE2-sg3 poplars showed up to 29.1% reduction in lignin deposition with irregularly shaped xylem vessels. However, CSE1-sg2 and CSE2-sg3 poplars were morphologically indistinguishable from WT and showed no significant differences in growth in a long-term living modified organism (LMO) field-test covering four seasons. Gene expression analysis revealed that many lignin biosynthetic genes were downregulated in CSE1-sg2 and CSE2-sg3 poplars. Indeed, the CSE1-sg2 and CSE2-sg3 poplars had up to 25% higher saccharification efficiency than the WT control. Our results demonstrate that precise editing of CSE by CRISPR/Cas9 technology can improve lignocellulosic biomass without a growth penalty.

RSL Class II Transcription Factors Guide the Nuclear Localization of RHL1 to Regulate Root Hair Development.

Root hairs are important for absorption of nutrients and water from the rhizosphere. The Root Hair Defective-Six Like (RSL) Class II family of transcription factors is expressed preferentially in root hairs and has a conserved role in root hair development in land plants. We functionally characterized the seven members of the RSL Class II subfamily in the rice (Oryza sativa) genome. In root hairs, six of these genes were preferentially expressed and four were strongly expressed. Phenotypic analysis of each mutant revealed that Os07g39940 plays a major role in root hair formation, based on observations of a short root hair phenotype in those mutants. Overexpression (OX) for each of four family members in rice resulted in an increase in the density and length of root hairs. These four members contain a transcription activation domain and are targeted to the nucleus. They interact with rice Root Hairless1 (OsRHL1), a key regulator of root hair development. When heterologously expressed in epidermal cells of Nicotiana benthamiana leaves, OsRHL1 was predominantly localized to the cytoplasm. When coexpressed with each of the four RSL Class II members, however, OsRLH1 was translocated to the nucleus. Transcriptome analysis using Os07g39940-OX plants revealed that 86 genes, including Class III peroxidases, were highly up-regulated. Furthermore, reactive oxygen species levels in the root hairs were increased in Os07g39940-OX plants but were drastically reduced in the os07g39940 and rhl1 mutants. Our results demonstrate that RSL Class II members function as essential regulators of root hair development in rice.

Physiological Importance of Pectin Modifying Genes During Rice Pollen Development.

Although cell wall dynamics, particularly modification of homogalacturonan (HGA, a major component of pectin) during pollen tube growth, have been extensively studied in dicot plants, little is known about how modification of the pollen tube cell wall regulates growth in monocot plants. In this study, we assessed the role of HGA modification during elongation of the rice pollen tube by adding a pectin methylesterase (PME) enzyme or a PME-inhibiting catechin extract (Polyphenon 60) to in vitro germination medium. Both treatments led to a severe decrease in the pollen germination rate and elongation. Furthermore, using monoclonal antibodies toward methyl-esterified and de-esterified HGA epitopes, it was found that exogenous treatment of PME and Polyphenon 60 resulted in the disruption of the distribution patterns of low- and high-methylesterified pectins upon pollen germination and during pollen tube elongation. Eleven PMEs and 13 PME inhibitors (PMEIs) were identified by publicly available transcriptome datasets and their specific expression was validated by qRT-PCR. Enzyme activity assays and subcellular localization using a heterologous expression system in tobacco leaves demonstrated that some of the pollen-specific PMEs and PMEIs possessed distinct enzymatic activities and targeted either the cell wall or other compartments. Taken together, our findings are the first line of evidence showing the essentiality of HGA methyl-esterification status during the germination and elongation of pollen tubes in rice, which is primarily governed by the fine-tuning of PME and PMEI activities.

Effects of elastic band exercise on subjects with rounded shoulder posture and forward head posture.

[Purpose] This study performed to investigate the effect of elastic band exercise program on the posture of subjects with rounded shoulder and forward head posture. [Subjects and Methods] The body length, forward shoulder angle, craniovertebral angle, and cranial rotation angle of participants (n=12) were measured before and after the exercise program. Furthermore, the thicknesses of the pectoralis major, rhomboid major, and upper trapezius were measured using an ultrasonographic imaging device. The exercises program was conducted with elastic bands, with 15 repetitions per set and 3 sets in total. [Results] The length of the pectoralis major, forward shoulder angle, and craniovertebral angle showed significant changes between before and after the exercise program, whereas the changes in the other measurements were not significant. The thickness of the upper trapezius showed a significant increase between before and after the elastic band exercise. [Conclusion] These findings suggest that the elastic band exercise program used in the study is effective for lengthening the pectoralis major and correcting rounded shoulder and forward head posture.

Isoflavone accumulation and the metabolic gene expression in response to persistent UV-B irradiation in soybean sprouts.

This study investigated the effects of persistent ultraviolet B (UV-B) irradiation on isoflavone accumulation in soybean sprouts. Three malonyl isoflavones were increased by UV-B. Malonylgenistin specifically accumulated upon UV-B exposure, whereas the other isoflavones were significantly increased under both dark conditions and UV-B exposure. The results of isoflavone accumulation to UV-B irradiation time were observed as following: acetyl glycitin rapidly increased and then gradually decreased; malonyl daidzin and malonyl genistin were highly accumulated within an intermediate period; genistein and daidzin were gradually maximized; daidzin, glycitin, genistein, and malonyl glycitin did not increase; and glycitin, acetyl daidzin, and acetyl genistin exhibited trace amounts. Transcriptional analysis of isoflavonoid biosynthetic genes demonstrated that most metabolic genes were highly activated in response to UV-B 24 and UV-B 36 treatments. In particular, it was found that GmCHS6, GmCHS7, and GmCHS8 genes among the eight known genes encoding chalcone synthase were specifically related to UV-B response.

Significant Performance and Stability Improvements of Low-Temperature IGZO TFTs by the Formation of In-F Nanoparticles on an SiO2 Buffer Layer.

We report the performance improvement of low-temperature coplanar indium-gallium-zinc-oxide (IGZO) thin-film transistors (TFTs) with a maximum process temperature of 230 °C. We treated F plasma on the surface of an SiO2 buffer layer before depositing the IGZO semiconductor by reactive sputtering. The field-effect mobility increases from 3.8 to 9.0 cm2 V-1·s-1, and the threshold voltage shift (ΔVth) under positive-bias temperature stress decreases from 3.2 to 0.2 V by F-plasma exposure. High-resolution transmission electron microscopy and atom probe tomography analysis reveal that indium fluoride (In-F) nanoparticles are formed at the IGZO/buffer layer interface. This increases the density of the IGZO and improves the TFT performance as well as its bias stability. The results can be applied to the manufacturing of low-temperature coplanar oxide TFTs for oxide electronics, including information displays.

Antibacterial poly (3,4-ethylenedioxythiophene):poly(styrene-sulfonate)/agarose nanocomposite hydrogels with thermo-processability and self-healing.

Recently, Near-infrared (NIR)-induced photothermal killing of pathogenic bacteria has received considerable attention due to the increase in antibiotic resistant bacteria. In this paper, we report a simple aqueous solution-based strategy to construct an effective photothermal nanocomposite composed of poly(3,4-ethylenedioxythiophene):poly(styrene-sulfonate) (PEDOT:PSS) and agarose with thermo-processability, light triggered self-healing, and excellent antibacterial activity. Our experiments revealed that PEDOT:PSS/agarose was easily coated on both a 2D glass substrate and 3D cotton structure. Additionally, PEDOT:PSS/agarose can be designed into free-standing objects of diverse shape as well as restored through an NIR light-induced self-healing effect after damage. Taking advantage of strong NIR light absorption, PEDOT:PSS/agarose exhibited a sharp temperature increase of 24.5 °C during NIR exposure for 100 s. More importantly, we demonstrated that the temperature increase on PEDOT:PSS/agarose via photothermal conversion resulted in the rapid and effective killing of nearly 100% of the pathogenic bacteria within 2 min of NIR irradiation.

Integrative analysis of pectin methylesterase (PME) and PME inhibitors in tomato (Solanum lycopersicum): Identification, tissue-specific expression, and biochemical characterization.

Although previous studies have demonstrated that the degree of demethylesterification of pectin polysaccharides is modulated during tomato fruit ripening, its involvement in vegetative organ development has been seldom investigated. As a first step in understanding the importance of pectin modification during vegetative stages, we used chemical, biochemical, and molecular approaches to analyze PMEs and PMEIs in tomato plants. We found that tomato cell walls isolated from vegetative tissues as well as the fruit contain substantial quantities of pectin, and different degrees of methylesterification were evident in different tissues. Our chemical study was further substantiated by immunolocalization analysis, which showed that selective removal of pectin-bound methyl groups is required for proper organ development and growth. In the tomato genome, there exists 79 PMEs and 48 PMEIs with temporally and spatially regulated expression. As a case study, we showed that two tomato PMEIs (SolycPMEI13 and SolycPMEI14) exhibited PMEI activities. This is the first report regarding the genome-wide identification and expression profiling of PME/PMEIs in tomato and the first chemical evidence of the differential degrees of pectin methylesterification in vegetative and reproductive tissues. Taken together, our findings provide an important tool to unravel the molecular and physiological functions of tomato PME and PMEI in further study.

Rice pectin methylesterase inhibitor28 (OsPMEI28) encodes a functional PMEI and its overexpression results in a dwarf phenotype through increased pectin methylesterification levels.

Pectin methylesterases (PMEs, EC 3.1.1.11) belonging to carbohydrate esterase family 8 cleave the ester bond between a galacturonic acid and an methyl group and the resulting change in methylesterification level plays an important role during the growth and development of plants. Optimal pectin methylesterification status in each cell type is determined by the balance between PME activity and post-translational PME inhibition by PME inhibitors (PMEIs). Rice contains 49 PMEIs and none of them are functionally characterized. Genomic sequence analysis led to the identification of rice PMEI28 (OsPMEI28). Recombinant OsPMEI28 exhibited inhibitory activity against commercial PME protein with the highest activities detected at pH 8.5. Overexpression of OsPMEI28 in rice resulted in an increased level of cell wall bound methylester groups and differential changes in the composition of cell wall neutral monosaccharides and lignin content in culm tissues. Consequently, transgenic plants overexpressing OsPMEI28 exhibited dwarf phenotypes and reduced culm diameter. Our data indicate that OsPMEI28 functions as a critical structural modulator by regulating the degree of pectin methylesterification and that an impaired status of pectin methylesterification affects physiochemical properties of the cell wall components and causes abnormal cell extensibility in rice culm tissues.

Treadmill exercise facilitates synaptic plasticity on dopaminergic neurons and fibers in the mouse model with Parkinson's disease.

Exercise for patients with Parkinson's disease (PD) helps to alleviate clinical symptoms such as tremor, balance instability, gait dysfunction, and rigidity. However, molecular mechanism about effect of exercise is poorly unknown. In this study, we investigated effect of exercise in synapse and dendritic spine of nigrostriatal dopaminergic neurons on mice with PD. The C57BL/6J male mice (n=40) were divided by sham group, sham-exercise treated group, 1-Methyl-4-phenyl-l,2,3,6-tetrahydropyridine (MPTP) treated group, and MPTP-exercise treated group. For exercise treatment, the mice were put on the treadmill to run for 8m/min, 30min/day, and 5 times/week for 2 weeks. Coordination ability was checked by rota rod test. Expression of tyrosine hydroxylase (TH), synaptophysin, and post-synaptic density protein 95 (PSD-95) was confirmed at substantia nigra pars compacta (SNpc) or striatum using western blotting, or immunohistochemistry. To check dendritic spine in striatum, we used Golgi staining. The results revealed that MPTP treated group displayed poor coordination ability compared with sham group. However, MPTP-exercise treated group showed good coordination ability compared with MPTP treated group. As well as, we also found that MPTP-exercise group increases expression of synaptophysin, PSD-95, TH, and dendritic spine in nigrostriatal dopaminergic neurons and fibers than MPTP treated group (p<0.05). Our findings suggest that exercise may give beneficial effects to patients with PD by facilitating synaptic plasticity and increasing dendritic spines.

Molecular and biochemical characterization of rice pectin methylesterase inhibitors (OsPMEIs).

Cell wall modifications such as partial degradation and depolymerization by cell wall hydrolases are normal cellular processes and are required for the functionalities of different cell types. Pectin, one of the major cell wall polysaccharides, is predominantly found in primary cell walls and middle lamellae and is subjected to in muro modification, primarily by cell wall-localized pectin methylesterases (PMEs). Molecular biochemical studies have demonstrated that enzymatic activities of PMEs are governed by multiple pectin methylesterase inhibitors (PMEIs), which consequently control the pectin methylesterification status. Although a few studies in Arabidopsis have shown the importance of this PMEI-mediated regulation in the biophysical properties of cell walls, little is known about the molecular physiological functions of rice PMEIs. We found 49 members of the PMEI family in the rice genome. Analysis of their transcript levels by quantitative real-time PCR and meta expression analysis showed that they are regulated spatially and temporally, as well as in response to diverse stresses. Quantification of cell wall-bound methylesters indicated that the degree of pectin methylesterification is developmentally regulated; in particular, higher PMEI activities were detected in cell wall proteins prepared from young leaves. Furthermore, an activity assay demonstrated that two recombinant OsPMEI proteins (OsPMEI8 and 12) were able to inhibit the enzymatic activity of a commercial PME protein. Subcellular localization indicated that OsPMEI8 is targeted to the middle lamella and OsPMEI12 is localized in the plasma membrane and nucleus. Taken together, our findings provide the first molecular and biochemical evidence for functional characterization of PMEIs in rice growth and development.

Genome-wide identification and expression analysis of rice pectin methylesterases: Implication of functional roles of pectin modification in rice physiology.

Pectin, which is enriched in primary cell walls and middle lamellae, is an essential polysaccharide in all higher plants. Homogalacturonans (HGA), a major form of pectin, are synthesized and methylesterified by enzymes localized in the Golgi apparatus and transported into the cell wall. Depending on cell type, the degree and pattern of pectin methylesterification are strictly regulated by cell wall-localized pectin methylesterases (PMEs). Despite its importance in plant development and growth, little is known about the physiological functions of pectin in rice, which contains 43 different types of PME. The presence of pectin in rice cell walls has been substantiated by uronic acid quantification and immunodetection of JIM7 monoclonal antibodies. We performed PME activity assays with cell wall proteins isolated from different rice tissues. In accordance with data from Arabidopsis, the highest activity was observed in germinating tissues, young culm, and spikelets, where cells are actively elongating. Transcriptional profiling of OsPMEs by real-time PCR and meta-analysis indicates that PMEs exhibit spatial- and stress-specific expression patterns during rice development. Based on in silico analysis, we identified subcellular compartments, isoelectric point, and cleavage sites of OsPMEs. Our findings provide an important tool for further studies seeking to unravel the functional importance of pectin modification during plant growth and abiotic and biotic responses of grass plants.

Synchrony of neuronal oscillations controlled by GABAergic reversal potentials.

GABAergic synapse reversal potential is controlled by the concentration of chloride. This concentration can change significantly during development and as a function of neuronal activity. Thus, GABA inhibition can be hyperpolarizing, shunting, or partially depolarizing. Previous results pinpointed the conditions under which hyperpolarizing inhibition (or depolarizing excitation) can lead to synchrony of neural oscillators. Here we examine the role of the GABAergic reversal potential in generation of synchronous oscillations in circuits of neural oscillators. Using weakly coupled oscillator analysis, we show when shunting and partially depolarizing inhibition can produce synchrony, asynchrony, and coexistence of the two. In particular, we show that this depends critically on such factors as the firing rate, the speed of the synapse, spike frequency adaptation, and, most important, the dynamics of spike generation (type I versus type II). We back up our analysis with simulations of small circuits of conductance-based neurons, as well as large-scale networks of neural oscillators. The simulation results are compatible with the analysis: for example, when bistability is predicted analytically, the large-scale network shows clustered states.