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S‐adenosyl‐l ‐methionine (SAM) is the major methyl donor in cells and it is also used for the biosynthesis of polyamines and the plant hormone ethylene. During climacteric ripening of tomato (Solanum lycopersicum ‘Bonaparte’), ethylene production rises considerably which makes it an ideal object to study SAM involvement. We examined in ripening fruit how a 1‐MCP treatment affects SAM usage by the three major SAM‐associated pathways. The 1‐MCP treatment inhibited autocatalytic ethylene production but did not affect SAM levels. We also observed that 1‐(malonylamino)cyclopropane‐1‐carboxylic acid formation during ripening is ethylene dependent. SAM decarboxylase expression was also found to be upregulated by ethylene. Nonetheless polyamine content was higher in 1‐MCP‐treated fruit. This leads to the conclusion that the ethylene and polyamine pathway can operate simultaneously. We also observed a higher methylation capacity in 1‐MCP‐treated fruit. During fruit ripening substantial methylation reactions occur which are gradually inhibited by the methylation product S‐adenosyl‐l ‐homocysteine (SAH). SAH accumulation is caused by a drop in adenosine kinase expression, which is not observed in 1‐MCP‐treated fruit. We can conclude that tomato fruit possesses the capability to simultaneously consume SAM during ripening to ensure a high rate of ethylene and polyamine production and transmethylation reactions. SAM usage during ripening requires a complex cellular regulation mechanism in order to control SAM levels.  相似文献   

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The FERONIA (FER) signaling pathway is known to have diverse roles in Arabidopsis thaliana, such as growth, reproduction, and defense, but how this receptor kinase is involved in various biological processes is not well established. In this work, we applied multiple mass spectrometry techniques to identify metabolites involved in the FER signaling pathway and to understand their biological roles. A direct infusion Fourier transform ion cyclotron resonance (FT‐ICR)‐MS approach was used for initial screening of wild‐type and feronia (fer) mutant plant extracts, and Arabidopsides were found to be significantly enriched in the mutant. As Arabidopsides are known to be induced by wounding, further experiments on wounded and non‐wounded leaf samples were carried out to investigate these oxylipins as well as related phytohormones using a quadrupole‐time‐of‐flight (Q‐TOF) MS by direct injection and LC‐MS/MS. In a root growth bioassay with Arabidopside A isolated from fer mutants, the wild‐type showed significant root growth inhibition compared with the fer mutant. Our results therefore implicated Arabidopsides, and Arabidopside A specifically, in FER functions and/or signaling. Finally, matrix‐assisted laser desorption/ionization MS imaging (MALDI‐MSI) was used to visualize the localization of Arabidopsides, and we confirmed that Arabidopsides are highly abundant at wounding sites in both wild‐type and fer mutant leaves. More significantly, five micron high‐spatial resolution MALDI‐MSI revealed that Arabidopsides are localized to the chloroplasts where many stress signaling molecules are made.  相似文献   

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Plant heterotrimeric G proteins modulate numerous developmental stress responses. Recently, receptor‐like kinases (RLKs) have been implicated as functioning with G proteins and may serve as plant G‐protein‐coupled‐receptors. The RLK FERONIA (FER), in the Catharantus roseus RLK1‐like subfamily, is activated by a family of polypeptides called rapid alkalinization factors (RALFs). We previously showed that the Arabidopsis G protein β subunit, AGB1, physically interacts with FER, and that RALF1 regulation of stomatal movement through FER requires AGB1. Here, we investigated genetic interactions of AGB1 and FER in plant salinity response by comparing salt responses in the single and double mutants of agb1 and fer. We show that AGB1 and FER act additively or synergistically depending on the conditions of the NaCl treatments. We further show that the synergism likely occurs through salt‐induced ROS production. In addition, we show that RALF1 enhances salt toxicity through increasing Na+ accumulation and decreasing K+ accumulation rather than by inducing ROS production, and that the RALF1 effect on salt response occurs in an AGB1‐independent manner. Our results indicate that RLK epistatic relationships are not fixed, as AGB1 and FER display different genetic relationships to RALF1 in stomatal versus salinity responses.  相似文献   

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Whether long interspersed nuclear element‐1 (LINE‐1) hypomethylation induced by reactive oxygen species (ROS) was mediated through the depletion of S‐adenosylmethionine (SAM) was investigated. Bladder cancer (UM‐UC‐3 and TCCSUP) and human kidney (HK‐2) cell lines were exposed to 20 μM H2O2 for 72 h to induce oxidative stress. Level of LINE‐1 methylation, SAM and homocysteine (Hcy) was measured in the H2O2‐exposed cells. Effects of α‐tocopheryl acetate (TA), N‐acetylcysteine (NAC), methionine, SAM and folic acid on oxidative stress and LINE‐1 methylation in the H2O2‐treated cells were explored. Viabilities of cells treated with H2O2 were not significantly changed. Intracellular ROS production and protein carbonyl content were significantly increased, but LINE‐1 methylation was significantly decreased in the H2O2‐treated cells. LINE‐1 methylation was restored by TA, NAC, methionine, SAM and folic acid. SAM level in H2O2‐treated cells was significantly decreased, while total glutathione was significantly increased. SAM level in H2O2‐treated cells was restored by NAC, methionine, SAM and folic acid; while, total glutathione level was normalized by TA and NAC. Hcy was significantly decreased in the H2O2‐treated cells and subsequently restored by NAC. In conclusion, in bladder cancer and normal kidney cells exposed to H2O2, SAM and Hcy were decreased, but total glutathione was increased. Treatments with antioxidants (TA and NAC) and one‐carbon metabolites (SAM, methionine and folic acid) restored these changes. This pioneer finding suggests that exposure of cells to ROS activates glutathione synthesis via the transsulfuration pathway leading to deficiency of Hcy, which consequently causes SAM depletion and eventual hypomethylation of LINE‐1. Copyright © 2015 John Wiley & Sons, Ltd.  相似文献   

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Arsenic (As) biomethylation is an important component of the As biogeochemical cycle that can influence As toxicity and mobility in the environment. Biomethylation of As is catalyzed by the enzyme arsenite (As[III]) S‐adenosylmethionine methyltransferase (ArsM). To date, all identified ArsM orthologs with As(III) methylation activities have four conserved cysteine residues, which are thought to be essential for As(III) methylation. Here, we isolated an As(III)‐methylating bacterium, Bacillus sp. CX‐1, and identified a gene encoding a S‐adenosylmethionine methyltranserase termed BlArsM with low sequence similarities (≤ 39%) to other ArsMs. BlArsM has six cysteine residues (Cys10, Cys11, Cys145, Cys193, Cys195 and Cys268), three of which (Cys10, Cys145 and Cys195) align with conserved cysteine residues found in most ArsMs. BlarsM is constitutively expressed in Bacillus sp. CX‐1. Heterologous expression of BlarsM conferred As(III) resistance. Purified BlArsM methylated both As(III) and methylarsenite (MAs[III]), with a final product of dimethylarsenate (DMAs[V]). When all six cysteines were individually altered to serine residues, only C145S and C195S derivatives lost the ability to methylate As(III) and MAs(III). The derivative C10S/C11S/C193S/C268S was still active. These results suggest that BlArsM is a novel As(III) S‐adenosylmethionine methyltransferase requiring only two conserved cysteine residues. A model of As(III) methylation by BlArsM is proposed.  相似文献   

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MiaB is a member of the methylthiotransferase subclass of the radical S‐adenosylmethionine (SAM) superfamily of enzymes, catalyzing the methylthiolation of C2 of adenosines bearing an N6‐isopentenyl (i6A) group found at position 37 in several tRNAs to afford 2‐methylthio‐N6‐(isopentenyl)adenosine (ms2i6A). MiaB uses a reduced [4Fe–4S]+ cluster to catalyze a reductive cleavage of SAM to generate a 5′‐deoxyadenosyl 5′‐radical (5′‐dA?)—a required intermediate in its reaction—as well as an additional [4Fe–4S]2+ auxiliary cluster. In Escherichia coli and many other organisms, re‐reduction of the [4Fe–4S]2+ cluster to the [4Fe–4S]+ state is accomplished by the flavodoxin reducing system. Most mechanistic studies of MiaBs have been carried out on the enzyme from Thermotoga maritima (Tm), which lacks the flavodoxin reducing system, and which is not activated by E. coli flavodoxin. However, the genome of this organism encodes five ferredoxins (TM0927, TM1175, TM1289, TM1533, and TM1815), each of which might donate the requisite electron to MiaB and perhaps to other radical SAM enzymes. The genes encoding each of these ferredoxins were cloned, and the associated proteins were isolated and shown to support turnover by Tm MiaB. In addition, TM1639, the ferredoxin‐NADP+ oxidoreductase subunit α (NfnA) from Tm was overproduced and isolated and shown to provide electrons to the Tm ferredoxins during Tm MiaB turnover. The resulting reactions demonstrate improved coupling between formation of the 5′‐dA? and ms2i6A production, indicating that only one hydrogen atom abstraction is required for the reaction.  相似文献   

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Vibrio vulnificus contains two coproporphyrinogen III oxidases (CPOs): O2‐dependent HemF and O2‐independent HemN. The growth of the hemF mutant HF1 was similar to wild‐type cells at pH 7.5 under 2% O2 conditions where HemN was active and had a half‐life of 64 min. However, HF1 did not grow when the medium pH decreased to pH 5.0, where oxidative stress affects endogenous S‐adenosylmethionine (SAM) levels. The growth of HF1 was restored not only by elevating the expression of MnSOD but also through the exogenous addition of SAM. For HF1 to grow under these SAM‐limiting conditions, a mutation arose in hemN, encoding HemNY74F. Refolding of the denatured enzymes in vitro revealed that the apparent binding affinity of HemNY74F for the cofactor SAM1, which coordinates the 4Fe‐4S cluster, was approximately sixfold higher than that of HemN. The Km of HemNY74F for the co‐substrate SAM2, which provides radicals for CPO reactions, was threefold lower than that of HemN. Thus, affinities for both SAM1 and SAM2 were higher with the Y74F mutation. Taken together, when SAM is limiting, HemN is apparently nonfunctional, and heme synthesis is continued by HemF.  相似文献   

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The AIDS‐associated lung pathogen Pneumocystis is classified as a fungus although Pneumocystis has several distinct features such as the absence of ergosterol, the major sterol of most fungi. The Pneumocystis carinii S‐adenosylmethionine:sterol C24‐methyltransferase (SAM:SMT) enzyme, coded by the erg6 gene, transfers either one or two methyl groups to the C‐24 position of the sterol side chain producing both C28 and C29 24‐alkylsterols in approximately the same proportions, whereas most fungal SAM:SMT transfer only one methyl group to the side chain. The sterol compositions of wild‐type Sacchromyces cerevisiae, the erg6 knockout mutant (Δerg6), and Δerg6 expressing the P. carinii or the S. cerevisiae erg6 gene were analyzed by a variety of chromatographic and spectroscopic procedures to examine functional complementation in the yeast expression system. Detailed sterol analyses were obtained using high performance liquid chromatography and proton nuclear magnetic resonance spectroscopy (1H‐NMR). The P. carinii SAM:SMT in the Δerg6 restored its ability to produce the C28 sterol ergosterol as the major sterol, and also resulted in low levels of C29 sterols. This indicates that while the P. carinii SAM:SMT in the yeast Δerg6 cells was able to transfer a second methyl group to the side chain, the action of Δ24(28)‐sterol reductase (coded by the erg4 gene) in the yeast cells prevented the formation and accumulation of as many C29 sterols as that found in P. carinii.  相似文献   

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S‐adenosyl‐l ‐methionine (SAM) synthetase is the key enzyme involved in the biosynthesis of SAM, which serves as a common precursor for polyamines (PAs) and ethylene. A SAM synthetase cDNA (SlSAMS1) was introduced into the tomato genome using the Agrobacterium tumefaciens transformation method. Transgenic plants overexpressing SlSAMS1 exhibited a significant increase in tolerance to alkali stress and maintained nutrient balance, higher photosynthetic capacity and lower oxidative stress compared with WT lines. Both in vivo and in vitro experiments indicated that the function of SlSAMS1 mainly depended on the accumulation of Spd and Spm in the transgenic lines. A grafting experiment showed that rootstocks from SlSAMS1‐overexpressing plants provided a stronger root system, increased PAs accumulation, essential elements absorption, and decreased Na+ absorption in the scions under alkali stress. As a result, fruit set and yield were significantly enhanced. To our knowledge, this is the first report to provide evidence that SlSAMS1 positively regulates tomato tolerance to alkali stress and plays a major role in modulating polyamine metabolism, resulting in maintainability of nutrient and ROS balance.  相似文献   

12.
Histone lysine methylation by histone lysine methyltransferases (HKMTs) has been implicated in regulation of gene expression. While significant progress has been made to understand the roles and mechanisms of animal HKMT functions, only a few plant HKMTs are functionally characterized. To unravel histone substrate specificity, degree of methylation and catalytic activity, we analyzed Arabidopsis Trithorax‐like protein (ATX), Su (var)3‐9 h omologs protein (SUVH), Su(var)3‐9 related protein (SUVR), ATXR5, ATXR6, and E(Z) HKMTs of Arabidopsis, maize and rice through sequence and structure comparison. We show that ATXs may exhibit methyltransferase specificity toward histone 3 lysine 4 (H3K4) and might catalyse the trimethylation. Our analyses also indicate that most SUVH proteins of Arabidopsis may bind histone H3 lysine 9 (H3K9). We also predict that SUVH7, SUVH8, SUVR1, SUVR3, ZmSET20 and ZmSET22 catalyse monomethylation or dimethylation of H3K9. Except for SDG728, which may trimethylate H3K9, all SUVH paralogs in rice may catalyse monomethylation or dimethylation. ZmSET11, ZmSET31, SDG713, SDG715, and SDG726 proteins are predicted to be catalytically inactive because of an incomplete S‐adenosylmethionine (SAM) binding pocket and a post‐SET domain. E(Z) homologs can trimethylate H3K27 substrate, which is similar to the Enhancer of Zeste homolog 2 of humans. Our comparative sequence analyses reveal that ATXR5 and ATXR6 lack motifs/domains required for protein‐protein interaction and polycomb repressive complex 2 complex formation. We propose that subtle variations of key residues at substrate or SAM binding pocket, around the catalytic pocket, or presence of pre‐SET and post‐SET domains in HKMTs of the aforementioned plant species lead to variations in class‐specific HKMT functions and further determine their substrate specificity, the degree of methylation and catalytic activity.  相似文献   

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Methylation of inorganic arsenic is a central process in the organoarsenical biogeochemical cycle. Members of every kingdom have ArsM As(III) S‐adenosylmethionine (SAM) methyltransferases that methylates inorganic As(III) into mono‐ (MAs(III)), di‐ (DMAs(III)) and tri‐ (TMAs(III)) methylarsenicals. Every characterized ArsM to date has four conserved cysteine residues. All four cysteines are required for methylation of As(III) to MAs(III), but methylation of MAs(III) to DMAs(III) requires only the two cysteines closest to the C‐terminus. Fungi produce volatile and toxic arsines, but the physiological roles of arsenic methylation and the biochemical basis is unknown. Here they demonstrate that most fungal species have ArsM orthologs with only three conserved cysteine residues. The genome of Aspergillus fumigatus has four arsM genes encoding ArsMs with only the second, third and fourth conserved cysteine residues. AfArsM1 methylates MAs(III) but not As(III). Heterologous expression of AfarsM1 in an Escherichia coli conferred resistance to MAs(III) but not As(III). The existence of ArsMs with only three conserved cysteine residues suggest that the ability to methylate MAs(III) may be an evolutionary step toward enzymes capable of methylating As(III), the result of a loss of function mutation in organisms with infrequent exposure to inorganic As(III) or as a resistance mechanism for MAs(III).  相似文献   

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Polar growth of root hairs is critical for plant survival and requires fine‐tuned Rho of plants (ROP) signaling. Multiple ROP regulators participate in root hair growth. However, protein S‐acyl transferases (PATs), mediating the S‐acylation and membrane partitioning of ROPs, are yet to be found. Using a reverse genetic approach, combining fluorescence probes, pharmacological drugs, site‐directed mutagenesis and genetic analysis with related root‐hair mutants, we have identified and characterized an Arabidopsis PAT, which may be responsible for ROP2 S‐acylation in root hairs. Specifically, functional loss of PAT4 resulted in reduced root hair elongation, which was rescued by a wild‐type but not an enzyme‐inactive PAT4. Membrane‐associated ROP2 was significantly reduced in pat4, similar to S‐acylation‐deficient ROP2 in the wild type. We further showed that PAT4 and SCN1, a ROP regulator, additively mediate the stability and targeting of ROP2. The results presented here indicate that PAT4‐mediated S‐acylation mediates the membrane association of ROP2 at the root hair apex and provide novel insights into dynamic ROP signaling during plant tip growth.  相似文献   

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The membranous quinone/quinol pool is essential to the majority of life forms and has been widely used as an important biomarker in microbial taxonomy. In the anaerobic world, the most important quinones are menaquinone (MK) and a methylated form of MK, designated methylmenaquinone (MMK), which is anticipated to serve specifically in low‐potential electron transport chains involved in anaerobic respiration. However, it has remained unclear how MMK is generated. Here, we show that a novel enzyme homologous to class C radical SAM methyltransferases (RSMTs) synthesizes MMK using MK as substrate. Such enzymes, termed either MenK or MqnK, are present in MMK‐producing bacteria (and some archaea) that possess either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). An mqnK deletion mutant of the model Epsilonproteobacterium Wolinella succinogenes was unable to produce MMK6 but its formation was restored upon genomic complementation using either the native mqnK gene or menK from the human gut bacterium Adlercreutzia equolifaciens or Shewanella oneidensis. Moreover, any of the menK genes enabled Escherichia coli cells to produce MMK8 and a methylated form of 2‐demethylmenaquinone8 (DMK8). The results expand the knowledge on quinone synthesis and demonstrate an unprecedented function for a class C RSMT‐type enzyme in primary cell metabolism.  相似文献   

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Nitric oxide (NO), a new addition to plant hormones, affects numerous processes in planta. It is produced as a part of stress response, but its signaling is poorly understood. S‐nitrosylation, a PTM, is currently the most investigated modification of NO. Recent studies indicate significant modulation of metabolome by S‐nitrosylation, as the identified targets span major metabolic pathways and regulatory proteins. Identification of S‐nitrosylation targets is necessary to understand NO signaling. By combining biotin switch technique and MS, 20 S‐nitrosylated proteins including four novel ones were identified from Brassica juncea. Further, to know if the abiotic stress‐induced NO evolution contributes to S‐nitrosothiols (SNO), the cellular NO reservoirs, SNO content was measured by Saville method. Low temperature (LT)‐stress resulted in highest (1.4‐fold) SNO formation followed by drought, high temperature and salinity. LT induced differentially nitrosylated proteins were identified as photosynthetic, plant defense related, glycolytic and signaling associated. Interestingly, both the subunits of ribulose‐1,5‐bisphosphate carboxylase/oxygenase (Rubisco) showed an increase as well as a decrease in nitrosylation by LT. Inactivation of Rubisco carboxylase by LT is well documented but the mechanism is not known. Here, we show that LT‐induced S‐nitrosylation is responsible for significant (~40%) inactivation of Rubisco. This in turn could explain cold stress‐induced photosynthetic inhibition.  相似文献   

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