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Temperature passively affects biological processes involved in plant growth. Therefore, it is challenging to study the dedicated temperature signalling pathways that orchestrate thermomorphogenesis, a suite of elongation growth‐based adaptations that enhance leaf‐cooling capacity. We screened a chemical library for compounds that restored hypocotyl elongation in the pif4‐2–deficient mutant background at warm temperature conditions in Arabidopsis thaliana to identify modulators of thermomorphogenesis. The small aromatic compound ‘Heatin’, containing 1‐iminomethyl‐2‐naphthol as a pharmacophore, was selected as an enhancer of elongation growth. We show that ARABIDOPSIS ALDEHYDE OXIDASES redundantly contribute to Heatin‐mediated hypocotyl elongation. Following a chemical proteomics approach, the members of the NITRILASE1‐subfamily of auxin biosynthesis enzymes were identified among the molecular targets of Heatin. Our data reveal that nitrilases are involved in promotion of hypocotyl elongation in response to high temperature and Heatin‐mediated hypocotyl elongation requires the NITRILASE1‐subfamily members, NIT1 and NIT2. Heatin inhibits NIT1‐subfamily enzymatic activity in vitro and the application of Heatin accordingly results in the accumulation of NIT1‐subfamily substrate indole‐3‐acetonitrile in vivo. However, levels of the NIT1‐subfamily product, bioactive auxin (indole‐3‐acetic acid), were also significantly increased. It is likely that the stimulation of hypocotyl elongation by Heatin might be independent of its observed interaction with NITRILASE1‐subfamily members. However, nitrilases may contribute to the Heatin response by stimulating indole‐3‐acetic acid biosynthesis in an indirect way. Heatin and its functional analogues present novel chemical entities for studying auxin biology.

Corrigendum of “Molecular and biochemical analysis of XDH from Phaseolus vulgaris suggest that uric acid protects the enzyme against the inhibitory effects of nitric oxide in nodules” [Plant Physiol. Biochem. 143 (2019) 364–374], Xanthine dehydrogenase (XDH) is essential for the assimilation of symbiotically fixed nitrogen in ureidic legumes. Uric acid, produced in the reaction catalyzed by XDH, is the precursor of the ureides, allantoin and allantoate, which are the main N-transporting molecules in these plants. XDH and uric acid have been reported to be involved in the response to stress, both in plants and animals. However, the physiological role of XDH under stressful conditions in ureidic legumes remains largely unexplored. In vitro assays showed that Phaseolus vulgaris XDH (PvXDH) can behave as a dehydrogenase or as an oxidase. Therefore, it could potentially protect against oxidative radicals or, in contrast, it could increase their production. In silico analysis of the upstream genomic region of XDH coding gene from P. vulgaris revealed the presence of several stress-related cis-regulatory elements. PvXDH mRNA and enzymatic activity in plants treated with stress-related phytohormones or subjected to dehydration and stressful temperatures showed several fold induction. However, PvXDH activity was in vivo and in vitro inhibited by nitric oxide in leaves but not in nodules. In extracts from RNAi PvXDH silenced nodules, with lower levels of uric acid, XDH activity was inhibited by SNP which indicates that uric acid produced by XDH in the nodules of this ureidic legume could help to protect XDH against the inhibitory effects of nitric oxide.

Corrigendum of “Molecular and biochemical analysis of XDH from Phaseolus vulgaris suggest that uric acid protects the enzyme against the inhibitory effects of nitric oxide in nodules” [Plant Physiol. Biochem. 143 (2019) 364–374], Xanthine dehydrogenase (XDH) is essential for the assimilation of symbiotically fixed nitrogen in ureidic legumes. Uric acid, produced in the reaction catalyzed by XDH, is the precursor of the ureides, allantoin and allantoate, which are the main N-transporting molecules in these plants. XDH and uric acid have been reported to be involved in the response to stress, both in plants and animals. However, the physiological role of XDH under stressful conditions in ureidic legumes remains largely unexplored. In vitro assays showed that Phaseolus vulgaris XDH (PvXDH) can behave as a dehydrogenase or as an oxidase. Therefore, it could potentially protect against oxidative radicals or, in contrast, it could increase their production. In silico analysis of the upstream genomic region of XDH coding gene from P. vulgaris revealed the presence of several stress-related cis-regulatory elements. PvXDH mRNA and enzymatic activity in plants treated with stress-related phytohormones or subjected to dehydration and stressful temperatures showed several fold induction. However, PvXDH activity was in vivo and in vitro inhibited by nitric oxide in leaves but not in nodules. In extracts from RNAi PvXDH silenced nodules, with lower levels of uric acid, XDH activity was inhibited by SNP which indicates that uric acid produced by XDH in the nodules of this ureidic legume could help to protect XDH against the inhibitory effects of nitric oxide.

Corrigendum of “Molecular and biochemical analysis of XDH from Phaseolus vulgaris suggest that uric acid protects the enzyme against the inhibitory effects of nitric oxide in nodules” [Plant Physiol. Biochem. 143 (2019) 364–374], Xanthine dehydrogenase (XDH) is essential for the assimilation of symbiotically fixed nitrogen in ureidic legumes. Uric acid, produced in the reaction catalyzed by XDH, is the precursor of the ureides, allantoin and allantoate, which are the main N-transporting molecules in these plants. XDH and uric acid have been reported to be involved in the response to stress, both in plants and animals. However, the physiological role of XDH under stressful conditions in ureidic legumes remains largely unexplored. In vitro assays showed that Phaseolus vulgaris XDH (PvXDH) can behave as a dehydrogenase or as an oxidase. Therefore, it could potentially protect against oxidative radicals or, in contrast, it could increase their production. In silico analysis of the upstream genomic region of XDH coding gene from P. vulgaris revealed the presence of several stress-related cis-regulatory elements. PvXDH mRNA and enzymatic activity in plants treated with stress-related phytohormones or subjected to dehydration and stressful temperatures showed several fold induction. However, PvXDH activity was in vivo and in vitro inhibited by nitric oxide in leaves but not in nodules. In extracts from RNAi PvXDH silenced nodules, with lower levels of uric acid, XDH activity was inhibited by SNP which indicates that uric acid produced by XDH in the nodules of this ureidic legume could help to protect XDH against the inhibitory effects of nitric oxide.

Aldehyde Oxidase (AO) is an enzyme involved in the metabolism of aldehydes and N-containing heterocyclic compounds. Many drug compounds contain heterocyclic moieties, and AO metabolism has lead to failure of several late-stage drug candidates. Therefore, it is important to take AO-mediated metabolism into account early in the drug discovery process, and thus, to have fast and reliable models to predict the site of metabolism (SOM). We have collected a dataset of 78 substrates of human AO with a total of 89 SOMs and 347 non-SOMs and determined atomic descriptors for each compound. The descriptors comprise NMR shielding and ESP charges from density functional theory (DFT), NMR chemical shift from ChemBioDraw, and Gasteiger charges from RDKit. Additionally, atomic accessibility was considered using 2D-SASA and relative span descriptors from SMARTCyp. Finally, stability of the product, the metabolite, was determined with DFT and also used as a descriptor. All descriptors have AUC larger than 0.75. In particular, descriptors related to the chemical shielding and chemical shift (AUC = 0.96) and ESP charges (AUC = 0.96) proved to be good descriptors. We recommend two simple methods to identify the SOM for a given molecule: 1) use ChemBioDraw to calculate the chemical shift or 2) calculate ESP charges or chemical shift using DFT. The first approach is fast but somewhat difficult to automate, while the second is more time-consuming, but can easily be automated. The two methods predict correctly 93% and 91%, respectively, of the 89 experimentally observed SOMs.

Aldehyde Oxidase (AO) is an enzyme involved in the metabolism of aldehydes and N-containing heterocyclic compounds. Many drug compounds contain heterocyclic moieties, and AO metabolism has lead to failure of several late-stage drug candidates. Therefore, it is important to take AO-mediated metabolism into account early in the drug discovery process, and thus, to have fast and reliable models to predict the site of metabolism (SOM). We have collected a dataset of 78 substrates of human AO with a total of 89 SOMs and 347 non-SOMs and determined atomic descriptors for each compound. The descriptors comprise NMR shielding and ESP charges from density functional theory (DFT), NMR chemical shift from ChemBioDraw, and Gasteiger charges from RDKit. Additionally, atomic accessibility was considered using 2D-SASA and relative span descriptors from SMARTCyp. Finally, stability of the product, the metabolite, was determined with DFT and also used as a descriptor. All descriptors have AUC larger than 0.75. In particular, descriptors related to the chemical shielding and chemical shift (AUC = 0.96) and ESP charges (AUC = 0.96) proved to be good descriptors. We recommend two simple methods to identify the SOM for a given molecule: 1) use ChemBioDraw to calculate the chemical shift or 2) calculate ESP charges or chemical shift using DFT. The first approach is fast but somewhat difficult to automate, while the second is more time-consuming, but can easily be automated. The two methods predict correctly 93% and 91%, respectively, of the 89 experimentally observed SOMs.

Neonicotinoids are the most important class of insecticides. Seven commercial neonicotinoids currently account for approximately 25% of the total insecticide market and are increasing in use as they replace other major classes such as organophosphates and methylcarbamates. Neonicotinoids are extensively metabolized in plants and mammals to produce over 100 metabolites. The overall goal of this study was to better understand three aspects of neonicotinoid metabolism in mice: the importance of aldehyde oxidase (AOX) in vivo, the fate of a new neonicotinoid, cycloxaprid (CYC), and the production of formaldehyde-generating intermediates from the hepatotoxicant, thiamethoxam (TMX).Neonicotinoids are metabolized in vitro by cytochrome P450s (CYPs) via oxidation reactions and by AOX on reduction of the nitroimino group. AOX metabolizes many xenobiotics in vitro but its importance in vivo is unknown relative to CYPs and other detoxification systems. Here we establish the relative importance of AOX and CYPs in vivo in neonicotinoid metabolism using the mouse model. AOX activity was reduced in mice by 45% with tungsten, 61% with hydralazine and 81% in AOX-deficient mice relative to controls and CYP activity was not affected. When mice were treated intraperitoneally with the major neonicotinoid imidacloprid (IMI), metabolism by CYP-oxidation reactions was not appreciably affected whereas the AOX-generated nitrosoguanidine metabolite was decreased by 30% with tungsten, 56% with hydralazine and 86% in the AOX-deficient mice. Another IMI nitroreduction metabolite, desnitro-IMI, was decreased by 55, 65, and 81% with tungsten, hydralazine and in the AOX-deficient mice, respectively. Thus, decreasing liver AOX activity by three quite different procedures gave a corresponding decrease for in vivo reductive metabolites in the liver of IMI-treated mice. Possible AOX involvement in IMI metabolism in insects was evaluated using AOX-expressing and AOX-deficient Drosophila, but no differen

Neonicotinoids are the most important class of insecticides. Seven commercial neonicotinoids currently account for approximately 25% of the total insecticide market and are increasing in use as they replace other major classes such as organophosphates and methylcarbamates. Neonicotinoids are extensively metabolized in plants and mammals to produce over 100 metabolites. The overall goal of this study was to better understand three aspects of neonicotinoid metabolism in mice: the importance of aldehyde oxidase (AOX) in vivo, the fate of a new neonicotinoid, cycloxaprid (CYC), and the production of formaldehyde-generating intermediates from the hepatotoxicant, thiamethoxam (TMX).Neonicotinoids are metabolized in vitro by cytochrome P450s (CYPs) via oxidation reactions and by AOX on reduction of the nitroimino group. AOX metabolizes many xenobiotics in vitro but its importance in vivo is unknown relative to CYPs and other detoxification systems. Here we establish the relative importance of AOX and CYPs in vivo in neonicotinoid metabolism using the mouse model. AOX activity was reduced in mice by 45% with tungsten, 61% with hydralazine and 81% in AOX-deficient mice relative to controls and CYP activity was not affected. When mice were treated intraperitoneally with the major neonicotinoid imidacloprid (IMI), metabolism by CYP-oxidation reactions was not appreciably affected whereas the AOX-generated nitrosoguanidine metabolite was decreased by 30% with tungsten, 56% with hydralazine and 86% in the AOX-deficient mice. Another IMI nitroreduction metabolite, desnitro-IMI, was decreased by 55, 65, and 81% with tungsten, hydralazine and in the AOX-deficient mice, respectively. Thus, decreasing liver AOX activity by three quite different procedures gave a corresponding decrease for in vivo reductive metabolites in the liver of IMI-treated mice. Possible AOX involvement in IMI metabolism in insects was evaluated using AOX-expressing and AOX-deficient Drosophila, but no differen

Background: A protective immune response against Hepatitis B infection can be obtained through the administration of a single viral polypeptide, the Hepatitis B surface antigen (HBsAg). Thus, the Hepatitis B vaccine is generated through the utilization of recombinant DNA technology, preferentially by using yeast-based expression systems. However, the polypeptide needs to assemble into spherical particles, so-called virus-like particles (VLPs), to elicit the required protective immune response. So far, no clear evidence has been presented showing whether HBsAg assembles in vivo inside the yeast cell into VLPs or later in vitro during down-stream processing and purification.Results: High level production of HBsAg was carried out with recombinant Pichia pastoris using the methanol inducible AOX1 expression system. The recombinant vaccine was isolated in form of VLPs after several down-stream steps from detergent-treated cell lysates. Search for the intracellular localization of the antigen using electron microscopic studies in combination with immunogold labeling revealed the presence of HBsAg in an extended endoplasmic reticulum where it was found to assemble into defined multi-layered, lamellar structures. The distance between two layers was determined as ~6 nm indicating that these lamellas represent monolayers of well-ordered HBsAg subunits. We did not find any evidence for the presence of VLPs within the endoplasmic reticulum or other parts of the yeast cell.Conclusions: It is concluded that high level production and intrinsic slow HBsAg VLP assembly kinetics are leading to retention and accumulation of the antigen in the endoplasmic reticulum where it assembles at least partly into defined lamellar structures. Further transport of HBsAg to the Golgi apparatus is impaired thus leading to secretory pathway disfunction and the formation of an extended endoplasmic reticulum which bulges into irregular cloud-shaped formations. As VLPs were not found within the cells i

Molybdenum (Mo) is an essential element for higher plants. Recent research indicated that Mo application enhanced cold-resistance of winter wheat. In order to improve our understanding of the mechanisms of cold resistance enhanced by molybdenum application in winter wheat, liquid nutrient culture experiments were conducted to investigate the effects of molybdenum on endogenesis hormone in winter wheat (Mo efficient cv. 97003 and Mo inefficient cv. 97014) under low temperature stress. The results showed that Mo application increased the AO activities, the contents of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the leaves of +Mo winter wheat under low temperature stress. No significant difference in the contents of GA3 and Z was detected between -Mo and +Mo winter wheat at the earlier stage of low temperature stress, whereas the contents of gibberellin (GA3) increased significantly, and zeatin (Z) decreased significantly in the leaves of +Mo winter wheat at the later stage of low temperature stress. Mo-deficiency also induced the drastic decrease of the ABA/GA in the leaves of winter wheat under low temperature stress. These results indicated that Mo regulated the ABA and IAA biosynthesis via AO in winter wheat and the response of ABA and IAA to Mo deficiency was prior to that of GA3 and ZT. The dramatic decrease in ABA and IAA levels in Mo deficient winter wheat may affect the homeostasis of endogenous hormones and Mo regulated cold resistance via the levels and balance of endogenous hormone in winter wheat.

Molybdenum (Mo) is an essential element for higher plants. Recent research indicated that Mo application enhanced cold-resistance of winter wheat. In order to improve our understanding of the mechanisms of cold resistance enhanced by molybdenum application in winter wheat, liquid nutrient culture experiments were conducted to investigate the effects of molybdenum on endogenesis hormone in winter wheat (Mo efficient cv. 97003 and Mo inefficient cv. 97014) under low temperature stress. The results showed that Mo application increased the AO activities, the contents of abscisic acid (ABA) and indole-3-acetic acid (IAA) in the leaves of +Mo winter wheat under low temperature stress. No significant difference in the contents of GA3 and Z was detected between -Mo and +Mo winter wheat at the earlier stage of low temperature stress, whereas the contents of gibberellin (GA3) increased significantly, and zeatin (Z) decreased significantly in the leaves of +Mo winter wheat at the later stage of low temperature stress. Mo-deficiency also induced the drastic decrease of the ABA/GA in the leaves of winter wheat under low temperature stress. These results indicated that Mo regulated the ABA and IAA biosynthesis via AO in winter wheat and the response of ABA and IAA to Mo deficiency was prior to that of GA3 and ZT. The dramatic decrease in ABA and IAA levels in Mo deficient winter wheat may affect the homeostasis of endogenous hormones and Mo regulated cold resistance via the levels and balance of endogenous hormone in winter wheat.

Lupinus is a legume with great agronomic potential due to the high protein content of its seeds and its positive effect on soil fertility. It is able to fix atmospheric nitrogen through the establishment of a symbiosis with soil bacteria of the genus Bradyrhizobium. The process is carried out in a special subclass of indeterminate nodules known as lupinoid nodules. The Bradyrhizobium-Lupinus symbiosis has particular characteristics, which makes it different from other Rhizobium-legume symbioses. The entry of the bacteria into the plant root occurs at the junction between the root hair base and the adjacent epidermic cell and only sporadic “infection threads” have been observed. The involvement of mitogen-activated protein kinases (MAPKs) and aldehyde oxidase in infection and nodule morphogenesis have been reported for the first time in this symbiosis. The presence of nitric oxide synthase activity in plants has been detected for the first time, in roots and nodules of Lupinus albus. The unique lupin nodule cortical structure has contributed to the visualization and elucidation of the operational mechanisms of the oxygen diffusion barrier. Nodule senescence takes place in a similar way to that of determinate nodules, starting in the central area of the nodule. This symbiosis is especially resistant to abiotic stresses such as herbicides, nitrate, salinity and heavy metals. This unusual tolerance has permitted the use of inoculated lupin plants for the re-vegetation of degraded areas and as a pioneer plant to fight soil erosion and to reclaim eroded soils. The capability to accumulate Cd, Zn and others heavy metals in the nodulated roots has resulted in the consideration of this symbiosis as a potential phytoremediator

Gene duplication is a primary source of molecular substrate for the emergence of evolutionary novelties. The chances for redundant gene sequences to evolve new functions are small compared with the probability that the copies become disabled by deleterious mutations. Functional divergence after gene duplication can result in two alternative evolutionary fates: one copy acquires a novel function (neofunctionalization), or each copy adopts part of the tasks of their parental gene (subfunctionalization). The relative prevalence of each outcome is unknown. Similarly unknown is the relative importance of positive selection versus random fixation of neutral mutations. Aldehyde oxidase (Ao) and xanthine dehydrogenase (Xdh) genes encode two complex members of the xanthine oxidase family of molybdo-flavoenzymes that carry different functions. Ao is known to have originated from a duplicate of an Xdh gene in eukaryotes, before the origin of multicellularity. We show that (i) Ao evolved independently twice from two different Xdh paralogs, the second time in the chordates, before the diversification of vertebrates; (h) after each duplication, the Ao duplicate underwent a period of rapid evolution during which identical sites across the two molecules, involving the flavin adenine dinucleotide and substrate-binding pockets, were subjected to intense positive Darwinian selection; and (iii) the second Ao gene likely endured two periods of redundancy, initially as a duplicate of Xdh and later as a functional equivalent of the old Ao, which is currently absent from the vertebrate genome. Caution is appropriate in structural genomics when using sequence similarity for assigning protein function.

Gene duplication is a primary source of molecular substrate for the emergence of evolutionary novelties. The chances for redundant gene sequences to evolve new functions are small compared with the probability that the copies become disabled by deleterious mutations. Functional divergence after gene duplication can result in two alternative evolutionary fates: one copy acquires a novel function (neofunctionalization), or each copy adopts part of the tasks of their parental gene (subfunctionalization). The relative prevalence of each outcome is unknown. Similarly unknown is the relative importance of positive selection versus random fixation of neutral mutations. Aldehyde oxidase (Ao) and xanthine dehydrogenase (Xdh) genes encode two complex members of the xanthine oxidase family of molybdo-flavoenzymes that carry different functions. Ao is known to have originated from a duplicate of an Xdh gene in eukaryotes, before the origin of multicellularity. We show that (i) Ao evolved independently twice from two different Xdh paralogs, the second time in the chordates, before the diversification of vertebrates; (h) after each duplication, the Ao duplicate underwent a period of rapid evolution during which identical sites across the two molecules, involving the flavin adenine dinucleotide and substrate-binding pockets, were subjected to intense positive Darwinian selection; and (iii) the second Ao gene likely endured two periods of redundancy, initially as a duplicate of Xdh and later as a functional equivalent of the old Ao, which is currently absent from the vertebrate genome. Caution is appropriate in structural genomics when using sequence similarity for assigning protein function.