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Global climate change, especially heatwaves and aridity, is a major threat to agricultural production and food security. This requires common efforts from the scientific community to find effective solutions to better understand and protect the plant's vulnerabilities to high temperatures. The current study demonstrates the potential of cellooligosaccharides (COS), which are native and oxidized signaling molecules released by lytic polysaccharide monooxygenases (LPMO) enzymes during cell wall degradation by microbial pathogens. The extracellular perception of COS leads to the activation of damage-triggered immunity, often protecting the plant against biotic stress. However, how these signaling molecules affect abiotic stress tolerance is poorly understood. Here, we show that native COS and oxidized COS (oxiCOS) perception increase the transcript levels of several HEAT SHOCK FACTORS (HSFs) and HEAT SHOCK PROTEINS (HSPs) genes in Arabidopsis plants. However, only oxiCOS treatment triggers ethylene priming and increases thermotolerance. Furthermore, the function of the transcription factor HSFA2 is required for these processes. Altogether, our results indicate that the perception of Damage-Associated Molecular Patterns (DAMPs) may improve tolerance to adverse abiotic conditions, like exposure to high temperatures.
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Proteínas de Arabidopsis , Arabidopsis , Oligosacáridos/metabolismo , Termotolerancia , Arabidopsis/metabolismo , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Proteínas de Unión al ADN/metabolismo , Etilenos/metabolismo , Regulación de la Expresión Génica de las Plantas , Factores de Transcripción del Choque Térmico/genética , Factores de Transcripción del Choque Térmico/metabolismo , Respuesta al Choque Térmico/genética , Calor , Oxidación-Reducción , Proteínas de Plantas/metabolismo , Termotolerancia/genéticaRESUMEN
DspA/E is a type three effector injected by the pathogenic bacterium Erwinia amylovora inside plant cells. In non-host Arabidopsis thaliana, DspA/E inhibits seed germination, root growth, de novo protein synthesis and triggers localized cell death. To better understand the mechanisms involved, we performed EMS mutagenesis on a transgenic line, 13-1-2, containing an inducible dspA/E gene. We identified three suppressor mutants, two of which belonged to the same complementation group. Both were resistant to the toxic effects of DspA/E. Metabolome analysis showed that the 13-1-2 line was depleted in metabolites of the TCA cycle and accumulated metabolites associated with cell death and defense. TCA cycle and cell-death associated metabolite levels were respectively increased and reduced in both suppressor mutants compared to the 13-1-2 line. Whole genome sequencing indicated that both suppressor mutants displayed missense mutations in conserved residues of Glycolate oxidase 2 (GOX2), a photorespiratory enzyme that we confirmed to be localized in the peroxisome. Leaf GOX activity increased in leaves infected with E. amylovora in a DspA/E-dependent manner. Moreover, the gox2-2 KO mutant was more sensitive to E. amylovora infection and displayed reduced JA-signaling. Our results point to a role for glycolate oxidase in type II non-host resistance and to the importance of central metabolic functions in controlling growth/defense balance.
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Arabidopsis , Erwinia amylovora , Oxidorreductasas de Alcohol/metabolismo , Arabidopsis/metabolismo , Proteínas Bacterianas/metabolismo , Erwinia amylovora/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiologíaRESUMEN
Current environmental and climate changes are having a pronounced influence on the outcome of plant-pathogen interactions, further highlighting the fact that abiotic stresses strongly affect biotic interactions at various levels. For instance, physiological parameters such as plant architecture and tissue organization together with primary and specialized metabolism are affected by environmental constraints, and these combine to make an individual plant either a more or less suitable host for a given pathogen. In addition, abiotic stresses can affect the timely expression of plant defense and pathogen virulence. Indeed, several studies have shown that variations in temperature, and in water and mineral nutrient availability affect the expression of plant defense genes. The expression of virulence genes, known to be crucial for disease outbreak, is also affected by environmental conditions, potentially modifying existing pathosystems and paving the way for emerging pathogens. In this review, we summarize our current knowledge on the impact of abiotic stress on biotic interactions at the transcriptional level in both the plant and the pathogen side of the interaction. We also perform a metadata analysis of four different combinations of abiotic and biotic stresses, which identifies 197 common modulated genes with strong enrichment in Gene Ontology terms related to defense . We also describe the multistress-specific responses of selected defense-related genes.
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Regulación de la Expresión Génica de las Plantas , Estrés Fisiológico , Expresión Génica , Genes de Plantas , Enfermedades de las Plantas/genética , Plantas/genéticaRESUMEN
Stabilisers, such as surfactants, polymers and polyaromatic molecules, offer an effective way to produce graphene dispersions in water by Liquid Phase Exfoliation (LPE) without degrading the properties of graphene. In particular, pyrene derivatives provide better exfoliation efficiency than traditional surfactants and polymers. A stabiliser is expected to be relatively soluble in order to disperse hydrophobic graphene in water. Here, we show that exfoliation can also be achieved with insoluble pyrene stabilisers if appropriately designed. In particular, bis-pyrene stabilisers (BPSs) functionalised with pyrrolidine provide a higher exfoliation efficiency and percentage of single layers compared to traditional pyrene derivatives under the same experimental conditions. This is attributed to the enhanced interactions between BPS and graphene, provided by the presence of two pyrene binding groups. This approach is therefore attractive not only to produce highly concentrated graphene, but also to use graphene to disperse insoluble molecules in water. The enhanced adsorption of BPS on graphene, however, is reflected in higher toxicity towards human epithelial bronchial immortalized cells, limiting the use of this material for biomedical applications.
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The use of highly polluting chemicals for plant and crop protection is one of the components of the negative environmental impact of agricultural activities. In the present paper, an environmentally friendly alternative to pesticide application has been studied, based on the so-called electrochemically activated solutions (ECAS). Experiments have been carried out, by applying ECAS having different contents of active ingredients, on tobacco plants at a laboratory scale and on apple trees at fruit garden scale. The results, accumulated during a couple of years, have shown that properly selected dilute solutions of chlorides, once activated by an electrochemical treatment, exhibit a very effective protecting action of plants, irrespective of their nature. Extension of the research has shown that the observed effect is the result of two distinct factors: the expected anti-microbial action of the electrochemically synthesized oxidants, and an unexpected priming of immune plant defenses, which is clearly due to the treatment with ECAS. Interestingly, the repetition of ECAS application triggers an even stronger activation of defense genes. No oxidative damages, due to the use of the activated solutions, could be detected.
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Antiinfecciosos/farmacología , Cloruros/farmacología , Desinfectantes/farmacología , Malus/inmunología , Nicotiana/inmunología , Inmunidad de la Planta/efectos de los fármacos , Electroquímica , Tecnología Química Verde , Malus/efectos de los fármacos , Malus/genética , Malus/microbiología , Oxidantes/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Soluciones , Nicotiana/efectos de los fármacos , Nicotiana/genética , Nicotiana/microbiologíaRESUMEN
2-Dimensional (2D) materials are attracting strong interest in printed electronics because of their unique properties and easy processability, enabling the fabrication of devices with low cost and mass scalable methods such as inkjet printing. For the fabrication of fully printed devices, it is of fundamental importance to develop a printable dielectric ink, providing good insulation and the ability to withstand large electric fields. Hexagonal boron nitride (h-BN) is typically used as a dielectric in printed devices. However, the h-BN film thickness is usually above 1 µm, hence limiting the use of h-BN in low-voltage applications. Furthermore, the h-BN ink is composed of nanosheets with broad lateral size and thickness distributions, due to the use of liquid-phase exfoliation (LPE). In this work, we investigate anatase TiO2 nanosheets (TiO2-NS), produced by a mass scalable bottom-up approach. We formulate the TiO2-NS into a water-based and printable solvent and demonstrate the use of the material with sub-micron thickness in printed diodes and transistors, hence validating the strong potential of TiO2-NS as a dielectric for printed electronics.
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BACKGROUND: Nevoid melanoma (NeM) is a rare variant of melanoma resembling melanocytic nevus. The aim of the study was to systematically review the dermoscopic features of NeM. METHODS: A hospital-based retrospective study was conducted. Dermoscopic features of NeMs diagnosed through excisional biopsy between January 2015 1, and March 1, 2021, were compared to superficial spreading melanomas (SSMs) matched by Breslow's thickness. Then, a literature search was performed. Electronic searches on PubMed database via Medline were conducted to retrieve any manuscript reporting detailed dermoscopic features of histopathologically confirmed NeM. RESULTS: A total of 60 malignant melanomas (MM) comprising 20 NeM and 40 SSM were collected. Twelve out of 20 (60%) NeM showed a nevus-like appearance, including reticular and globular patterns, and in 35% of these cases it was detected because of dermoscopic changes. Then, a total of seven original manuscripts were retrieved from the literature review, comprising 56 cases overall. NeM showed nevus-like pattern in 53% of the cases, multicomponent pattern in 21% and amelanotic in 9%. Enlargement, irregularly distributed dots/globules, irregular pigmentation, and atypical vascular pattern were found in NeM with nevus-like appearance. NeM with multicomponent pattern were characterized by irregular pigmentation, blue-white veil, irregular dots and atypical vascular pattern. Amelanotic NeM is rare and show atypical vascular pattern and milia-like cysts. CONCLUSIONS: Dermoscopy of NeM is challenging as it frequently shows a nevus-like pattern, but clues and detection of dermoscopic changes may help to identify it.
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Quiste Epidérmico , Melanoma Amelanótico , Nevo Pigmentado , Nevo , Neoplasias Cutáneas , Dermoscopía , Humanos , Melanoma , Melanoma Amelanótico/patología , Nevo/patología , Nevo Pigmentado/diagnóstico , Estudios Retrospectivos , Neoplasias Cutáneas/diagnóstico , Melanoma Cutáneo MalignoRESUMEN
Lytic Polysaccharide MonoOxygenases display great variability towards cellulose ultrastructure while performing oxidative functionalization of the polymers. Aiming at employing AA9-LPMOs for isolation of cellulose nano-crystals (CNCs), the ratio between functionalization/crystalline degradation became a crucial parameter. Here are reported the constraints posed by the substrate ultrastructure on the activity of seven different AA9 LPMOs representative of various regioselectivity and substrate affinity: TtAA9E, TaAA9A, PcAA9D, MtAA9A, MtAA9D, MtAA9I-CBM and MtAA9J. The substrate crystallinity and dry matter loading greatly affected the seven AA9s in an enzyme-specific manner, impacting their efficiency for CNCs functionalization purposes. X-ray diffraction pattern analyses were used to assess the cracking efficacy of the enzymatic treatment to de-crystallize CNCs, revealing that those AA9s with minor efficiency in releasing oligosaccharides resulted instead the most disruptive towards the crystal lattice and in reducing the particle sizes. These non-catalytic effects were found comparable with the one caused by the expansin BsEXLX1 enzyme.
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Celulosa , Oxigenasas de Función Mixta , Celulosa/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oxidación-Reducción , Polímeros , PolisacáridosRESUMEN
Two-dimensional (2D) anatase titanium dioxide (TiO2) is expected to exhibit different properties as compared to anatase nanocrystallites, due to its highly reactive exposed facets. However, access to 2D anatase TiO2 is limited by the non-layered nature of the bulk crystal, which does not allow use of top-down chemical exfoliation. Large efforts have been dedicated to the growth of 2D anatase TiO2 with high reactive facets by bottom-up approaches, which relies on the use of harmful chemical reagents. Here, we demonstrate a novel fluorine-free strategy based on topochemical conversion of 2D 1T-TiS2 for the production of single crystalline 2D anatase TiO2, exposing the {001} facet on the top and bottom and {100} at the sides of the nanosheet. The exposure of these faces, with no additional defects or doping, gives rise to a significant activity enhancement in the hydrogen evolution reaction, as compared to commercially available Degussa P25 TiO2 nanoparticles. Because of the strong potential of TiO2 in many energy-based applications, our topochemical approach offers a low cost, green and mass scalable route for production of highly crystalline anatase TiO2 with well controlled and highly reactive exposed facets.
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Lytic polysaccharide monooxygenases (LPMOs) are industrially important enzymes able to enhance the enzymatic lignocellulose saccharification in synergism with classical glycoside hydrolases. Fungal LPMOs have been classified as AA9, AA11, and AA13-16 families showing a diverse specificity for substrates such as soluble and insoluble beta-glucans, chitin, starch, and xylan, besides cellulose. These enzymes are still not fully characterized, and for example this is testify by their mechanism of oxidation regularly reviewed multiple times in the last decade. Noteworthy is that despite the extremely large abundance in the entire Tree of Life, our structural and functional knowledge is based on a restricted pool of LPMO, and probably one of the main reason reside in the challenging posed by their heterologous expression. Notably, the lack of a simple cloning protocol that could be universally applied to LPMO, hinders the conversion of the ever-increasing available genomic information to actual new enzymes. Here, we provide an easy and fast protocol for cloning, expression, and purification of active LPMOs in the following architecture: natural signal peptide, LPMO enzyme, TEV protease site, and His6-Tag. For this purpose, a commercial methanol inducible expression vector was initially modified to allow the LPMO expression containing the above characteristics. Gibson assembly, a one-step isothermal DNA assembly, was adopted for the direct assembly of intron-less or intron-containing genes and the modified expression vector. Moreover, His6-tagged LPMO constructs can be submitted to TEV proteolysis for removal of the questionable C-terminal His6-Tag, obtaining a close-to-native form of LPMO. We successfully applied this method to clone, express, and purify six LPMOs from AA9 family with different regioselectivities. The proposed protocol, provided as step-by-step, could be virtually applied in many laboratories thanks to the choice of popular and commons materials.
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Proteínas Fúngicas , Oxigenasas de Función Mixta , Clonación Molecular , Proteínas Fúngicas/genética , Humanos , Oxigenasas de Función Mixta/genética , Polisacáridos , XilanosRESUMEN
Nanocellulose (NC) is getting ahead as a renewable, biodegradable and biocompatible biomaterial. The NCs for this study were recovered from industrial cotton waste (CFT) by acid hydrolysis (HNC) and by 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) mediated oxidation (ONC). They were functionalized by radical based glycidyl methacrylate (GMA) grafting providing crystalline HNC-GMA and ONC-GMA, and by allylation (ALL) providing amorphous HNC-ALL and ONC-ALL. HNC, ONC and their derivatives were chemically and morphologically characterized. Crystalline NCs were found capable to adsorb, from diluted water solution (2 × 10-3 M), the antibiotics vancomycin (VC), ciprofloxacin (CP), amoxicillin (AM) and the disinfectant chlorhexidine (CHX), while amorphous NCs did not show any significant adsorption properties. Adsorption capability was quantified by measuring the concentration change in function of the contact time. The adsorption kinetics follow the pseudo-second order model and show complex adsorption mechanisms investigated by an intraparticle diffusion model and interpreted by structure-property relationships. ONC and ONC-GMA loaded with VC, and HNC and HNC-GMA loaded with CP were not colonized by Staphylococcus aureus and by Klebsiella pneumonia and suggested long lasting release capability. Our results can envisage developing CFT derived NCs for environmental applications (water remediation) and for biomedical applications (antibacterial NC). Among the future developments, it could also be of interest to take advantage of acidic, glycidyl and allyl groups' reactivity to provide other NCs from the NC object of this study.
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Lytic Polysaccharide Monooxygenases (LPMOs) are powerful redox enzymes able to oxidatively cleave recalcitrant polysaccharides. Widely conserved across biological kingdoms, LPMOs of the AA9 family are deployed by phytopathogens to deconstruct cellulose polymers. In response, plants have evolved sophisticated mechanisms to sense cell wall damage and thus self-triggering Damage Triggered Immunity responses. Here, we show that Arabidopsis plants exposed to LPMO products triggered the innate immunity ultimately leading to increased resistance to the necrotrophic fungus Botrytis cinerea. We demonstrated that plants undergo a deep transcriptional reprogramming upon elicitation with AA9 derived cellulose- or cello-oligosaccharides (AA9_COS). To decipher the specific effects of native and oxidized LPMO-generated AA9_COS, a pairwise comparison with cellobiose, the smallest non-oxidized unit constituting cellulose, is presented. Moreover, we identified two leucine-rich repeat receptor-like kinases, namely STRESS INDUCED FACTOR 2 and 4, playing a crucial role in signaling the AA9_COS-dependent responses such as camalexin production. Furthermore, increased levels of ethylene, jasmonic and salicylic acid hormones, along with deposition of callose in the cell wall was observed. Collectively, our data reveal that LPMOs might play a crucial role in plant-pathogen interactions.
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Arabidopsis/inmunología , Botrytis/inmunología , Celulosa/metabolismo , Oxigenasas de Función Mixta/metabolismo , Oligosacáridos/metabolismo , Enfermedades de las Plantas/inmunología , Arabidopsis/metabolismo , Arabidopsis/microbiología , Resistencia a la Enfermedad , Perfilación de la Expresión Génica , Regulación de la Expresión Génica de las Plantas , Oxigenasas de Función Mixta/fisiología , Oligosacáridos/fisiología , Enfermedades de las Plantas/microbiología , Sordariales/metabolismoRESUMEN
A new and diverse family of pyrene derivatives was synthesised via palladium-catalysed C-H ortho-arylation of pyrene-1-carboxylic acid. The strategy affords easy access to a broad scope of 2-substituted and 1,2-disubstituted pyrenes. The C1-substituent can be easily transformed into carboxylic acid, iodide, alkynyl, aryl or alkyl functionalities. This approach gives access to arylated pyrene ammonium salts, which outperformed their non-arylated parent compound during aqueous Liquid Phase Exfoliation (LPE) of graphite and compare favourably to state-of-the-art sodium pyrene-1-sulfonate PS1. This allowed the production of concentrated and stable suspensions of graphene flakes in water.
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Proline accumulation occurs in plants following the exposure to a wide array of stress conditions, as well as during numerous physiological and adaptive processes. Increasing evidence also supports the involvement of proline metabolism in the plant response to pathogen attack. This requires that the biosynthetic pathway is triggered by components of numerous and different signal transduction chains. Indeed, several reports recently described activation of genes coding for enzymes of the glutamate pathway by transcription factors (TFs) belonging to various families. Here, we summarize some of these findings with special emphasis on rice, and show the occurrence of a plethora of putative TF binding sites in the promoter of such genes.
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Disease has an effect on crop yields, causing significant losses. As the worldwide demand for agricultural products increases, there is a need to pursue the development of new methods to protect crops from disease. One mechanism of plant protection is through the activation of the plant immune system. By exogenous application, 'plant activator molecules' with elicitor properties can be used to activate the plant immune system. These defence-inducing molecules represent a powerful and often environmentally friendly tool to fight pathogens. We show that the secondary bile acid deoxycholic acid (DCA) induces defence in Arabidopsis and reduces the proliferation of two bacterial phytopathogens: Erwinia amylovora and Pseudomonas syringae pv. tomato. We describe the global defence response triggered by this new plant activator in Arabidopsis at the transcriptional level. Several induced genes were selected for further analysis by quantitative reverse transcription-polymerase chain reaction. We describe the kinetics of their induction and show that abiotic stress, such as moderate drought or nitrogen limitation, does not impede DCA induction of defence. Finally, we investigate the role in the activation of defence by this bile acid of the salicylic acid biosynthesis gene SID2, of the receptor-like kinase family genes WAK1-3 and of the NADPH oxidase-encoding RbohD gene. Altogether, we show that DCA constitutes a promising molecule for plant protection which can induce complementary lines of defence, such as callose deposition, reactive oxygen species accumulation and the jasmonic acid and salicylic acid signalling pathways.
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Arabidopsis/inmunología , Arabidopsis/microbiología , Ácido Desoxicólico/farmacología , Enfermedades de las Plantas/inmunología , Enfermedades de las Plantas/microbiología , Pseudomonas syringae/fisiología , Arabidopsis/efectos de los fármacos , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sequías , Regulación de la Expresión Génica de las Plantas/efectos de los fármacos , Cinética , Enfermedades de las Plantas/genética , Hojas de la Planta/efectos de los fármacos , Hojas de la Planta/genética , Hojas de la Planta/microbiología , Pseudomonas syringae/efectos de los fármacos , Pseudomonas syringae/crecimiento & desarrollo , Estrés Fisiológico/efectos de los fármacos , Estrés Fisiológico/genética , Transcriptoma/efectos de los fármacos , Transcriptoma/genéticaRESUMEN
While intracellular proline accumulation in response to various stress conditions has been investigated in great detail, the biochemistry and physiological relevance of proline degradation in plants is much less understood. Moreover, the second and last step in proline catabolism, the oxidation of δ(1)-pyrroline-5-carboxylic acid (P5C) to glutamate, is shared with arginine catabolism. Little information is available to date concerning the regulatory mechanisms coordinating these two pathways. Expression of the gene coding for P5C dehydrogenase was analyzed in rice by real-time PCR either following the exogenous supply of amino acids of the glutamate family, or under hyperosmotic stress conditions. The rice enzyme was heterologously expressed in E. coli, and the affinity-purified protein was thoroughly characterized with respect to structural and functional properties. A tetrameric oligomerization state was observed in size exclusion chromatography, which suggests a structure of the plant enzyme different from that shown for the bacterial P5C dehydrogenases structurally characterized to date. Kinetic analysis accounted for a preferential use of NAD(+) as the electron acceptor. Cations were found to modulate enzyme activity, whereas anion effects were negligible. Several metal ions were inhibitory in the micromolar range. Interestingly, arginine also inhibited the enzyme at higher concentrations, with a mechanism of uncompetitive type with respect to P5C. This implies that millimolar levels of arginine would increase the affinity of P5C dehydrogenase toward its specific substrate. Results are discussed in view of the involvement of the enzyme in either proline or arginine catabolism.
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The majority of plant species accumulate high intracellular levels of proline to cope with hyperosmotic stress conditions. Proline synthesis from glutamate is tightly regulated at both the transcriptional and the translational levels, yet little is known about the mechanisms for post-translational regulation of the enzymatic activities involved. The gene coding in rice (Oryza sativa L.) for δ(1)-pyrroline-5-carboxylate (P5C) reductase, the enzyme that catalyzes the second and final step in this pathway, was isolated and expressed in Escherichia coli. The structural and functional properties of the affinity-purified protein were characterized. As for most species, rice P5C reductase was able to use in vitro either NADH or NADPH as the electron donor. However, strikingly different effects of cations and anions were found depending on the pyridine nucleotide used, namely inhibition of NADH-dependent activity and stimulation of NADPH-dependent activity. Moreover, physiological concentrations of proline and NADP(+) were strongly inhibitory for the NADH-dependent reaction, whereas the NADPH-dependent activity was mildly affected. Our results suggest that only NADPH may be used in vivo and that stress-dependent variations in ion homeostasis and NADPH/NADP(+) ratio could modulate enzyme activity, being functional in promoting proline accumulation and potentially also adjusting NADPH consumption during the defense against hyperosmotic stress. The apparent molecular weight of the native protein observed in size exclusion chromatography indicated a high oligomerization state. We also report the first crystal structure of a plant P5C reductase at 3.40-Šresolution, showing a decameric quaternary assembly. Based on the structure, it was possible to identify dynamic structural differences among rice, human, and bacterial enzymes.
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An amino-trisulfonate tetraphenylporphyrin was conjugated to carboxylate cellulose nanocrystals giving rise to a new nanomaterial with interesting binding properties.