Curing Behavior of Sucrose with p-Toluenesulfonic Acid.

With respect to the fossil resources shortage, the development of bio-based wood adhesives is an important research topic in wood science. There has been research on using sucrose for bio-based adhesives. However, a high acid catalyst content and a high hot-pressing temperature are required when manufacturing particleboards. In this study, to explore the possibility of p-toluenesulfonic acid (PTSA) as a promising acid catalyst for sucrose-based adhesives, the curing behavior of sucrose with PTSA (Suc-PTSA) was clarified. The thermal analysis results showed that the thermal properties of sucrose decreased significantly with the addition of PTSA. Based on the results of the insoluble matter rate, the optimal mixture ratio and heating conditions were determined to be 95:5 and 180 °C for 10 min, respectively. According to the results of FT-IR, the heat-treated Suc-PTSA contained furan compounds. In the context of the dynamic viscoelasticity, the onset temperature at which the storage modulus (E') begins to rise was significantly lower than those of the other sucrose-based adhesives. PTSA has the potential to cure sucrose more efficiently and at lower temperatures than previous sucrose-based adhesives, making it a promising acid catalyst for sucrose.

Chemical Structures of Adhesive and Interphase Parts in Sucrose/Citric Acid Type Adhesive Wood-Based Molding Derived from Japanese Cedar (Cryptomeria japonica).

In sucrose/citric acid based wood adhesive, the detailed bonding mechanism has still been unknown. Here, we investigated the detailed chemical structures of this adhesive wood (Japanese cedar)-based molding by using heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR). NMR peaks associated with the furan-type structure appeared, suggesting that the furan compound was formed from sucrose and converted to a furan polymer during the adhesive process and that some of the furan structures in the polymers were ester-bonded with citric acid. The secondary forces between the furan polymers and wood components were thought to contribute to the adhesive effect. In our analysis of the interphase structure, primary hydroxyl groups of both polysaccharides and of lignin substructures were found to be esterified with citric acid. Additionally, some of the glycosidic bonds in polysaccharides were cleaved during the acidic condition produced by citric acid. The above results provided evidence of the polymerization of sucrose-derived 5-HMF, the esterification of wood components, and the degradation of polysaccharides during the molding process. Citric acid functioned as a clamp between the obtained furan polymer and the wood components. The sucrose/citric acid based wood adhesive can be defined as a hybrid-type wood adhesive, involving both secondary forces and chemical bonding interactions.

Bond Structures between Wood Components and Citric Acid in Wood-Based Molding.

Citric acid-based wood adhesive is considered a chemical-bonding wood adhesive. However, the detailed structures of the bonds between wood components and citric acid remain unknown. Here, we examine the chemical bonding structures between citric acid and wood by heteronuclear single quantum coherence-nuclear magnetic resonance (HSQC-NMR) analysis of wood-based molding using Japanese cedar (Cryptomeria japonica) and citric acid. In the HSQC-NMR spectrum of the wood molding, some esterified C/H correlation peaks appeared. The primary hydroxyl groups of polysaccharides, such as cellulose and galactoglucomannan, and the primary hydroxyl groups of the ß-O-4 and ß-5 substructures in lignin were found to be esterified with citric acid. In contrast, the secondary hydroxyl groups, except for xylan, barely reacted because of the steric hindrance. Additionally, the C/H correlation peak volumes of the reducing ends of mannan and xylan in the anomeric region increased after molding. It was clarified that the glycosidic bonds in the hemicelluloses were cleaved under the acidic molding condition with citric acid. The HSQC-NMR analysis revealed that the esterification of hemicellulose and lignin, and degradation of hemicellulose, proceeded under the molding condition. These results will promote understanding of the adhesive mechanism of citric acid-based wood adhesive and of the properties of the molding.

Investigation of Synthesis Mechanism, Optimal Hot-Pressing Conditions, and Curing Behavior of Sucrose and Ammonium Dihydrogen Phosphate Adhesive.

In this study, a further investigation was carried out on the synthesis mechanism, optimal manufacturing conditions, and curing behavior of a sucrose-ammonium dihydrogen phosphate (SADP) adhesive. The results of 13C nuclear magnetic resonance (NMR) spectroscopy confirmed that SADP was composed of 5-hydroxymethylfurfural (5-HMF), deoxyfructosazine (DOF), amino compounds, Schiff base, monosaccharides, and oligosaccharide. The optimal hot-pressing conditions were a hot-pressing temperature of 170 °C, a hot-pressing time of 7 min, and a spread rate of 120 g/m2. The wet shear strength of plywood bonded at optimal manufacturing conditions met the requirements of China National Standard (GB/T 9846-2015). Thermal analysis and insoluble mass proportion measurements showed that the main curing behavior of the SADP adhesive occurred at curing temperatures higher than 145 °C, and more than 50% insoluble mass was formed when the heating time was longer than 5 min. Fourier-transform infrared spectroscopy (FT-IR) indicated that cross-linking of the cured adhesive was promoted by prolonging the heating time. In addition, pyrolysis gas chromatography and mass spectrometry (Py-GC/MS) confirmed that the cured SADP adhesive was composed of furan and nitrogen-containing compounds.

Investigation and Characterization of Synthesis Conditions on Sucrose-ammonium Dihydrogen Phosphate (SADP) Adhesive: Bond Performance and Chemical Transformation.

Sucrose is one of the most abundantly available renewable chemicals in the world, and it is expected to be utilized as a raw material for wood-based material products. Herein, a novel adhesion system that was based on sucrose and ammonium dihydrogen phosphate (ADP) was synthesized into an adhesive with 80% solid content, and this eco-friendly was utilized on the fabrication of plywood. The effects of the synthesis conditions on the plywood bond performance and synthesis mechanism were investigated. The optimal synthesis conditions were as follows: the mass proportion between sucrose and ADP was 90/10, the synthesis temperature was 90 °C, and the synthesis time was 3 h. The bonding performance of the plywood that was bonded by optimal SADP adhesive satisfied the GB/T 9846-2015 standard. The chemical analysis was performance tested by using High-Performance Liquid Chromatography (HPLC), Attenuated Total Reflection-Fourier Transform Infrared Spectra (ATR-FTIR), and Pyrolysis Gas Chromatography and Mass Spectrometry (Py-GC/MS) to understand the chemical transformation during the synthesis process. The chemical analysis results confirmed that the hydrolysis and conversation reaction of sucrose occurred in the synthesized SADP adhesive, and ADP promoted the pyrolysis efficiency of sucrose.

Further Exploration of Sucrose-Citric Acid Adhesive: Investigation of Optimal Hot-Pressing Conditions for Plywood and Curing Behavior.

In previous research, sucrose and citric acid were used to synthesize an eco-friendly plywood adhesive. Herein, further research was performed to determine the optimal hot-pressing conditions and curing behavior of a sucrose-citric acid (SC) adhesive. The results of dry and wet shear strength measurements showed that the optimal hot-pressing temperature, hot-pressing time, and spread rate of plywood samples bonded by the SC adhesive were 190 °C, 7 min, and 140 g/m2, respectively. When plywood was bonded at the optimal hot-pressing conditions, the wet shear strength met the requirements of the China National Standard GB/T 9846-2015. Thermal analysis showed that the thermal degradation and endothermic reaction temperatures of the SC 25/75 adhesive were lower than either sucrose or citric acid individually. In addition, the insoluble mass proportion increased with the heating temperature and time. The Pyrolysis Gas Chromatography and Mass Spectrometr (Py-GC/MS) analysis confirmed that the SC adhesive was cured by the reaction between furan compounds, saccharide, and citric acid, and the resulting polymer appeared to be joined by ether linkages.

Further Exploration of Sucrose-Citric Acid Adhesive: Synthesis and Application on Plywood.

The development of eco-friendly adhesives is a major research direction in the wood-based material industry. Previous research has already demonstrated the mixture of sucrose and citric acid could be utilized as an adhesive for the manufacture of particleboard. Herein, based on the chemical characteristics of sucrose, a synthesized sucrose-citric acid (SC) adhesive was prepared, featuring suitable viscosity and high solid content. The investigation of synthesis conditions on the bond performance showed that the optimal mass proportion between sucrose and citric acid was 25/75, the synthesis temperature was 100 °C, and the synthesis time was 2 h. The wet shear strength of the plywood bonded with SC adhesive, which was synthesized at optimal conditions and satisfied the China National Standard GB/T 9846-2015. The synthesis mechanism was studied by both 13C NMR analysis and HPLC, and the chemical composition manifesting caramelization reaction occurred during the synthesis process. The results of ATR FT-IR indicated the formation of a furan ring, carbonyl, and ether groups in the cured insoluble matter of the SC adhesive, which indicated dehydration condensation as the reaction mechanism between sucrose and citric acid.

Synthesis and Characterization of Sucrose and Ammonium Dihydrogen Phosphate (SADP) Adhesive for Plywood.

The development of eco-friendly adhesives for wood composite products has been a major topic in the field of wood science and product engineering. Although the research on tannin-based and soybean protein-based adhesives has already reached, or at least nears, industrial implementation, we also face a variety of remaining challenges with regards to the push for sustainable adhesives. First, petroleum-derived substances remain a pre-requisite for utilization of said adhesive systems, and also the viscosity of these novel adhesives continues to limit its ability to serve as a drop-in substitute. Within this study, we focus upon the development of an eco-friendly plywood adhesive that does not require any addition of petroleum derived reagents, and the resultant liquid adhesive has both high solid contents as well as a manageably low viscosity at processing temperatures. Specifically, a system based on sucrose and ammonium dihydrogen phosphate (ADP) was synthesized into an adhesive with ~80% solid content and with viscosities ranging from 480-1270 mPa·s. The bonding performance of all adhesive-bound veneer specimens satisfied GB/T 9846-2015 standard at 170 °C hot pressing temperature. To better explain the system's efficiency, in-depth chemical analysis was performed in an effort to understand the chemical makeup of the cured adhesives as well as the components over the time course of curing. Several new structures involving the fixation of nitrogen speak to a novel adhesive molecular network. This research provides a possibility of synthesizing an eco-friendly wood adhesive with a high solid content and a low viscosity by renewable materials, and this novel adhesive system has the potential to be widely utilized in the wood industry.

A Novel Eco-Friendly Wood Adhesive Composed by Sucrose and Ammonium Dihydrogen Phosphate.

Development of a bio-based wood adhesive is a significant goal for several wood-based material industries. In this study, a novel adhesive based upon sucrose and ammonium dihydrogen phosphate (ADP) was formulated in hopes of furthering this industrial goal through realization of a sustainable adhesive with mechanical properties and water resistance comparable to the synthetic resins used today. Finished particleboards exhibited excellent mechanical properties and water resistance at the revealed optimal adhesive conditions. In fact, the board properties fulfilled in principle the requirements of JIS A 5908 18 type standard, however this occured at production conditions for the actual state of development as reported here, which are still different to usual industrial conditions. Thermal analysis revealed addition of ADP resulted in decreases to the thermal thresholds associated with degradation and curing of sucrose. Spectral results of FT-IR elucidated that furanic ring chemistry was involved during adhesive curing. A possible polycondensation reaction pathway was proposed from this data in an attempt to explain why the adhesive exhibited such favorable bonding properties.

Effects of Sulfuric Acid on the Curing Behavior and Bonding Performance of Tannin⁻Sucrose Adhesive.

The development of biomaterials-based adhesives is one of the main research directions for the wood-based material industry. In previous research, tannin and sucrose were used as adhesive to manufacture particleboard. However, the reaction conditions need to be optimized. In this study, sulfuric acid was added to the tannin⁻sucrose adhesive as a catalyst to improve the curing process. Thermal analysis, insoluble mass proportion, FT-IR, and solid state 13C NMR were used to investigate the effects of sulfuric acid on the curing behavior of tannin and sucrose. Thermal analysis showed weight loss and endotherm temperature reduced from 205 and 215 to 136 and 138 °C, respectively, by adding sulfuric acid. In case of the adhesive with pH = 1.0, the insoluble mass proportion achieved 81% at 160 °C, which was higher than the reference at 220 °C. FT-IR analysis of the uncured adhesives showed that adding sulfuric acid leads to hydrolysis of sucrose; then, glucose and fructose converted to 5-hydroxymehthylfurfural (HMF) and levulinic acid. Dimethylene ether bridges were observed by FT-IR analysis of the cured adhesives. The results of solid state 13C NMR spectrum indicated that 5-HMF participated in the curing process and formed methylene bridges with the C8 position of the resorcinol A-rings of tannin, whereas dimethylene ether bridges were detected as a major chemical chain of the polymer. Lab particleboards were produced using 20 wt % resin content at 180 °C and 10 min press time; the tannin⁻sucrose adhesive modified with sulfuric acid to pH = 1.0 exhibited better performance than the unmodified tannin⁻sucrose adhesive; the properties of the boards fulfilled the requirement of Japanese Industrial Standard (JIS) A5908 type 15.

Indole-3-acetic acid-induced oxidative burst and an increase in cytosolic calcium ion concentration in rice suspension culture.

Indole-3-acetic acid (IAA) is the major natural auxin involved in the regulation of a variety of growth and developmental processes such as division, elongation, and polarity determination in growing plant cells. It has been shown that dividing and/or elongating plant cells accompanies the generation of reactive oxygen species (ROS) and a number of reports have suggested that hormonal actions can be mediated by ROS through ROS-mediated opening of ion channels. Here, we surveyed the link between the action of IAA, oxidative burst, and calcium channel activation in a transgenic cells of rice expressing aequorin in the cytosol. Application of IAA to the cells induced a rapid and transient generation of superoxide which was followed by a transient increase in cytosolic Ca(2+) concentration ([Ca(2+)]c). The IAA-induced [Ca(2+)]c elevation was inhibited by Ca(2+) channel blockers and a Ca(2+) chelator. Furthermore, ROS scavengers effectively blocked the action of IAA on [Ca(2+)]c elevation.

The E3 Ligase APIP10 Connects the Effector AvrPiz-t to the NLR Receptor Piz-t in Rice.

Although nucleotide-binding domain, leucine-rich repeat (NLR) proteins are the major immune receptors in plants, the mechanism that controls their activation and immune signaling remains elusive. Here, we report that the avirulence effector AvrPiz-t from Magnaporthe oryzae targets the rice E3 ligase APIP10 for degradation, but that APIP10, in return, ubiquitinates AvrPiz-t and thereby causes its degradation. Silencing of APIP10 in the non-Piz-t background compromises the basal defense against M. oryzae. Conversely, silencing of APIP10 in the Piz-t background causes cell death, significant accumulation of Piz-t, and enhanced resistance to M. oryzae, suggesting that APIP10 is a negative regulator of Piz-t. We show that APIP10 promotes degradation of Piz-t via the 26S proteasome system. Furthermore, we demonstrate that AvrPiz-t stabilizes Piz-t during M. oryzae infection. Together, our results show that APIP10 is a novel E3 ligase that functionally connects the fungal effector AvrPiz-t to its NLR receptor Piz-t in rice.

A chloroplast-localized protein LESION AND LAMINA BENDING affects defence and growth responses in rice.

Understanding how plants allocate their resources to growth or defence is of long-term importance to the development of new and improved varieties of different crops. Using molecular genetics, plant physiology, hormone analysis and Next-Generation Sequencing (NGS)-based transcript profiling, we have isolated and characterized the rice (Oryza sativa) LESION AND LAMINA BENDING (LLB) gene that encodes a chloroplast-targeted putative leucine carboxyl methyltransferase. Loss of LLB function results in reduced growth and yield, hypersensitive response (HR)-like lesions, accumulation of the antimicrobial compounds momilactones and phytocassanes, and constitutive expression of pathogenesis-related genes. Consistent with these defence-associated responses, llb shows enhanced resistance to rice blast (Magnaporthe oryzae) and bacterial blight (Xanthomonas oryzae pv. oryzae). The lesion and resistance phenotypes are likely to be caused by the over-accumulation of jasmonates (JAs) in the llb mutant including the JA precursor 12-oxo-phytodienoic acid. Additionally, llb shows an increased lamina inclination and enhanced early seedling growth due to elevated brassinosteroid (BR) synthesis and/or signalling. These findings show that LLB functions in the chloroplast to either directly or indirectly repress both JA- and BR-mediated responses, revealing a possible mechanism for controlling how plants allocate resources for defence and growth.

An OsCEBiP/OsCERK1-OsRacGEF1-OsRac1 module is an essential early component of chitin-induced rice immunity.

OsCEBiP, a chitin-binding protein, and OsCERK1, a receptor-like kinase, are plasma membrane (PM) proteins that form a receptor complex essential for fungal chitin-driven immune responses in rice. The signaling events immediately following chitin perception are unclear. Investigating the spatiotemporal regulation of the rice small GTPase OsRac1, we find that chitin induces rapid activation of OsRac1 at the PM. Searching for OsRac1 interactors, we identified OsRacGEF1 as a guanine nucleotide exchange factor for OsRac1. OsRacGEF1 interacts with OsCERK1 and is activated when its C-terminal S549 is phosphorylated by the cytoplasmic domain of OsCERK1 in response to chitin. Activated OsRacGEF1 is required for chitin-driven immune responses and resistance to rice blast fungus infection. Further, a protein complex including OsCERK1 and OsRacGEF1 is transported from the endoplasmic reticulum to the PM. Collectively, our results suggest that OsCEBiP, OsCERK1, OsRacGEF1, and OsRac1 function as key components of a "defensome" critically engaged early during chitin-induced immunity.

The bHLH Rac Immunity1 (RAI1) Is Activated by OsRac1 via OsMAPK3 and OsMAPK6 in Rice Immunity.

The Rac/Rop GTPase OsRac1 plays an essential role in rice immunity. However, the regulatory genes acting downstream of OsRac1 are largely unknown. We focused on the RAI1 gene, which is up-regulated in suspension cells expressing a constitutively active form of OsRac1. RAI1 encodes a putative basic helix-loop-helix transcription factor. A microarray analysis of cells transformed with an inducible RAI1 construct showed increased expression of PAL1 and OsWRKY19 genes after induction, suggesting that these genes are regulated by RAI1. This was confirmed using RAI1 T-DNA activation-tagged and RNA interference lines. The PAL1 and OsWRKY19 genes were also up-regulated by sphingolipid and chitin elicitors, and the RAI1 activation-tagged plants had increased resistance to a rice blast fungus. These results indicated that RAI1 is involved in defense responses in rice. RAI1 interacted with OsMAPK3 and OsMAPK6 proteins in vivo and in vitro. Also, RAI1 was phosphorylated by OsMAPK3/6 and OsMKK4-dd in vitro. Overexpression of OsMAPK6 and/or OsMAPK3 together with OsMKK4-dd increased PAL1 and OsWRKY19 expression in rice protoplasts. Therefore, the regulation of PAL1 and OsWRKY19 expression by RAI1 could be controlled via an OsMKK4-OsMAPK3/6 cascade. Co-immunoprecipitation assays indicated that OsMAPK3 and OsRac1 occur in the same complex as OsMAPK6. Taken together, our results indicate that RAI1 could be regulated by OsRac1 through an OsMAPK3/6 cascade. In this study, we have identified RAI1 as the first transcription factor acting downstream of OsRac1. This work will help us to understand the immune system regulated by OsRac1 in rice and its orthologs in other plant species.

Negative feedback regulation of microbe-associated molecular pattern-induced cytosolic Ca2+ transients by protein phosphorylation.

Microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) often induce rises in cytosolic free Ca(2+) concentration ([Ca(2+)](cyt)) and protein phosphorylation. Though they are postulated to play pivotal roles in plant innate immunity, their molecular links and the regulatory mechanisms remain largely unknown. To investigate the regulatory mechanisms for MAMP-induced Ca(2+) mobilization, we have established a transgenic rice (Oryza sativa) cell line stably expressing apoaequorin, and characterized the interrelationship among MAMP-induced changes in [Ca(2+)](cyt), production of reactive oxygen species (ROS) and protein phosphorylation. Oligosaccharide and sphingolipid MAMPs induced Ca(2+) transients mainly due to plasma membrane Ca(2+) influx, which were dramatically suppressed by a protein phosphatase inhibitor, calyculin A (CA). Hydrogen peroxide and hypo-osmotic shock triggered similar [Ca(2+)](cyt) elevations, which were not affected by CA. MAMP-induced protein phosphorylation, which is promoted by CA, has been shown to be required for ROS production and MAPK activation, while it negatively regulates MAMPs-induced Ca(2+) mobilization and may play a crucial role in temporal regulation of [Ca(2+)](cyt) signature.

Contribution of salicylic acid glucosyltransferase, OsSGT1, to chemically induced disease resistance in rice plants.

Systemic acquired resistance (SAR), a natural disease response in plants, can be induced chemically. Salicylic acid (SA) acts as a key endogenous signaling molecule that mediates SAR in dicotyledonous plants. However, the role of SA in monocotyledonous plants has yet to be elucidated. In this study, the mode of action of the agrochemical protectant chemical probenazole was assessed by microarray-based determination of gene expression. Cloning and characterization of the most highly activated probenazole-responsive gene revealed that it encodes UDP-glucose:SA glucosyltransferase (OsSGT1), which catalyzes the conversion of free SA into SA O-beta-glucoside (SAG). We found that SAG accumulated in rice leaf tissue following treatment with probenazole or 2,6-dichloroisonicotinic acid. A putative OsSGT1 gene from the rice cultivar Akitakomachi was cloned and the gene product expressed in Escherichia coli was characterized, and the results suggested that probenazole-responsive OsSGT1 is involved in the production of SAG. Furthermore, RNAi-mediated silencing of the OsSGT1 gene significantly reduced the probenazole-dependent development of resistance against blast disease, further supporting the suggestion that OsSGT1 is a key mediator of development of chemically induced disease resistance. The OsSGT1 gene may contribute to the SA signaling mechanism by inducing up-regulation of SAG in rice plants.

RACK1 functions in rice innate immunity by interacting with the Rac1 immune complex.

A small GTPase, Rac1, plays a key role in rice (Oryza sativa) innate immunity as part of a complex of regulatory proteins. Here, we used affinity column chromatography to identify rice RACK1 (for Receptor for Activated C-Kinase 1) as an interactor with Rac1. RACK1 functions in various mammalian signaling pathways and is involved in hormone signaling and development in plants. Rice contains two RACK1 genes, RACK1A and RACK1B, and the RACK1A protein interacts with the GTP form of Rac1. Rac1 positively regulates RACK1A at both the transcriptional and posttranscriptional levels. RACK1A transcription was also induced by a fungal elicitor and by abscisic acid, jasmonate, and auxin. Analysis of transgenic rice plants and cell cultures indicates that RACK1A plays a role in the production of reactive oxygen species (ROS) and in resistance against rice blast infection. Overexpression of RACK1A enhances ROS production in rice seedlings. RACK1A was shown to interact with the N terminus of NADPH oxidase, RAR1, and SGT1, key regulators of plant disease resistance. These results suggest that RACK1A functions in rice innate immunity by interacting with multiple proteins in the Rac1 immune complex.

Characterization of an elicitor-induced rice WRKY gene, OsWRKY71.

Expression of OsWRKY71, a rice WRKY gene, was induced by biotic elicitors and pathogen infection. It was also found that OsWRKY71 has features characteristic of a transcriptional repressor. Microarray analysis revealed that several elicitor-induced defense-related genes were upregulated in rice cells overexpressing OsWRKY71. These results indicate that the activation of defense-related genes by OsWRKY71 was probably indirect.

Effects of a bile acid elicitor, cholic acid, on the biosynthesis of diterpenoid phytoalexins in suspension-cultured rice cells.

An elicitor of rice defense responses was recently isolated from human feces and was identified as cholic acid (CA). Pathogen infection in rice leaves induces phytocassanes and momilactones, both of which are major diterpenoid phytoalexins in rice, whereas CA mainly induces phytocassanes. We established a high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry protocol for the rapid and accurate quantification of phytocassanes and momilactones. Using this method, we showed that CA preferentially induced the formation of phytocassanes in suspension-cultured rice cells, while a fungal chitin oligosaccharide elicitor induced that of both phytocassanes and momilactones. We further investigated the effects of CA on the expression of diterpene cyclase genes involved in phytoalexin biosynthesis. CA induced the transcription of the genes OsCPS2 (OsCyc2) and OsKSL7 (OsDTC1), which are involved in phytocassane biosynthesis, to a greater extent than the genes OsCPS4 (OsCyc1) and OsKSL4, which are involved in momilactone biosynthesis. OsCPS2 was particularly strongly induced, suggesting that it is one of the main mechanisms by which CA induces high levels of phytocassanes.