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Oxidation and reduction of sulfite by chloroplasts and formation of sulfite addition compounds.

After exposing intact chloroplasts isolated from spinach (Spinacia oleracea L. cv Yates) and capable of photoreducing CO(2) at high rates to different concentrations of radioactive sulfite in the light or in the dark, (35)SO(2) and H(2) (35)S were removed from the acidified suspensions in a stream of nitrogen. Remaining activity could be fractionated into sulfate, organic sulfides, and sulfite addition compounds. When chloroplast suspensions contained catalase, superoxide dismutase and O-acetylserine, the oxidation of sulfite to sulfate was slower in the light than the reductive formation of sulfides that exhibited a maximum rate of about 2 micromoles per milligram chlorophyll per hour, equivalent to about 1% of maximum carbon assimilation. Botht the oxidative and the reductive detoxification of sulfite were very slow in the dark. Oxidation was somewhat, but not much, accelerated in the light in the absence of O-acetylserine, which caused a dramatic decrease in the formation of organic sulfides and an equally dramatic increase in the concentration of sulfite addition compounds whose formation was light-dependent. The sulfite addition compounds were not identified. Addition compounds did not accumulate in the dark. In the light, the electron transport inhibitor 3-(3,4-dichlorophenyl)-1,1-dimethylurea, diuron, decreased not only the reduction, but also the oxidation of sulfite and the formation of addition compounds.

Journal Article↗

Metabolism of 2'-carboxyarabinitol in leaves.

Results presented here indicate that 2'-carboxyarabinitol (CA) is the in vivo precursor and product of 2'-carboxyarabinitol 1-phosphate (CA1P) metabolism in leaves. When [2-(14)C]CA was fed in the light to leaves of five species known to be highly active in CA1P metabolism (Phaseolus vulgaris, Lycopersicon esculentum, Helianthus annuus, Petunia hybrida, and Beta vulgaris), [(14)C]CA1P was formed in the dark. Reillumination of a Phaseolus leaf caused this [(14)C]CA1P to be rapidly metabolized to [(14)C]CA (t((1/2)) = 1 min). The epimer 2'-carboxyribitol could not substitute for CA in the dark synthesis of CA1P, and CA in the anionic form was a better substrate than CA in the lactone form. In leaves of Phaseolus vulgaris, the active CA pool size used in the dark synthesis of CA1P is between about 70 and 110 nanomoles per milligram of chlorophyll. The photosynthetic electron transport inhibitor diuron did not affect the dark synthesis of [(14)C]CA1P, but did greatly reduce the rate of its subsequent light degradation (t((1/2)) = approximately 10 min). Dark synthesis of [(14)C]CA1P was inhibited by dithiothreitol and NaF. From the present data, we suggest that CA1P and CA participate in a metabolic substrate cycle in vivo.

Journal Article↗

Molecular and biophysical analysis of herbicide-resistant mutants of Chlamydomonas reinhardtii: structure-function relationship of the photosystem II D1 polypeptide.

Plants and green algae can develop resistance to herbicides that block photosynthesis by competing with quinones in binding to the chloroplast photosystem II (PSII) D1 polypeptide. Because numerous herbicide-resistant mutants of Chlamydomonas reinhardtii with different patterns of resistance to such herbicides are readily isolated, this system provides a powerful tool for examining the interactions of herbicides and endogenous quinones with the photosynthetic membrane, and for studying the structure-function relationship of the D1 protein with respect to PSII electron transfer. Here we report the results of DNA sequence analysis of the D1 gene from four mutants not previously characterized at the molecular level, the correlation of changes in specific amino acid residues of the D1 protein with levels of resistance to the herbicides atrizine, diuron, and bromacil, and the kinetics of fluorescence decay for each mutant, which show that changes at two different amino acid residues dramatically slow PSII electron transfer. Our analyses, which identify a region of 57 amino acids of the D1 polypeptide involved in herbicide binding and which define a D1 binding niche for the second quinone acceptor, QB of PSII, provide a strong basis of support for structural and functional models of the PSII reaction center.

Amino Acid Sequence↗

Sclerodermatous syndrome after occupational exposure to herbicides--response to systemic steroids.

We report a case of generalized cutaneous sclerosis associated with muscle and oesophageal involvement in a patient exposed to herbicides containing bromocil, diuron and aminotriazole. Treatment with oral prednisolone resulted in some improvement in the cutaneous signs, particularly over the face, trunk and proximal limbs. The considerable exposure to chemicals, time course and unusual pattern of organ involvement suggested a diagnosis of occupational scleroderma.

Herbicides↗

Mitochondrial cytochrome b genes with a six-nucleotide deletion or single-nucleotide substitutions confer resistance to antimycin A in the yeast Kluyveromyces lactis.

Extrachromosomal mutants resistant to antimycin, from the yeast Kluyveromyces lactis, have been isolated, genetically characterized, and assigned to two specific genetic loci (Brunner et al., 1987). In the present work the cytochrome b nucleotide sequence from six of these mutants was determined. Five mutants had single point mutations, corresponding to transversions. In one mutant, a six-base-pair deletion, beginning at nucleotide 689, was observed. The amino acid sequence derived from the coding strand showed that, in three independent antimycin-resistant mutants, a change of asparagine 31 into lysine took place (two of these mutants are also resistant to diuron). Two other mutants showed a change from lysine 228 into isoleucine (or methionine). Leucine 230, isoleucine 231, and threonine 232, were lost in the deletion mutant and were replaced by serine.

Amino Acid Sequence↗

Functional and structural changes of the photosystem II complex induced by high irradiance in cyanobacterial cells.

A gradual disintegration of the photosystem II (PSII) complex, initiated by a release of the chlorophyll-protein CP43, was identified during low-temperature illumination of Synechococcus cells. This process was slower compared to the decline of the PSII primary charge separation activity, and much slower than the photoinactivation of oxygen evolution. All three processes were slowed down in the presence of diuron. The results indicate that when the PSII repair was blocked, the inactivation of charge separation activity and the release of CP43 preceded the degradation of the D1 protein. In contrast, a much faster degradation of D1 connected to its rapid exchange was triggered by inactivation of oxygen evolution, and no disassembly of PSII was needed. We propose the existence of two different mechanisms of D1 degradation in the cells of the thermophilic cyanobacterium Synechococcus elongatus.

Cyanobacteria↗

Biochemical and functional properties of photosystem II in agranal membranes from maize mesophyll and bundle sheath chloroplasts.

We have studied the occurrence and organization of photosystem II (PSII) in bundle sheath thylakoids and stroma lamellae from maize. As shown by non-denaturing lauryl beta-D- iminopropionidate (Deriphat)/PAGE, PSII exists in a dimeric form in grana membranes. In bundle sheath and stroma lamellae, however, only a monomeric form was found. Based on immunotitration data, we estimated the stoichiometry of the individual components of the PSII core complex and antenna systems. In stroma lamellae, all PSII antenna complexes had a stoichiometry similar to that in grana membranes, with the exception of light-harvesting complex II (LHCII) that was somewhat over-represented, while the minor antenna complexes CP26 and CP29 were under-represented. In bundle sheath, the amount of LHCII was approximately eight times higher than expected with respect to D1. The 33-kDa protein of the oxygen-evolving enhancer polypeptides was not detectable nor was the ferredoxin-NADP+ reductase, thus strongly suggesting that no significant linear electron transport occurs in bundle sheath thylakoids. Fluorescence induction data suggest that most of the PSII reaction centers in bundle sheath and stroma lamellae sustain electron transport towards a secondary acceptor pool. Stromal PSII centers are only weakly inhibited by 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), whereas, unexpectedly, dichlorobenzoquinone and methyl viologen had a pronounced inhibitory effect of the QA- reoxidation. An additional specificity of these centers is the slow rate (50-ms range) of the QA to QB electron transfer. The amplitude of variable fluorescence found in stroma lamellae can only account for a small fraction (1-2%) of the variable fluorescence of whole thylakoids. This suggests that stromal PSII cannot be solely responsible for the slow beta-phase of the induction kinetics.

Chlorophyll↗

Molecular identification and expression study of differentially regulated genes in the Pacific oyster Crassostrea gigas in response to pesticide exposure.

The effects of pesticide contamination on the metabolism of marine molluscs are poorly documented. We investigated the response of a marine bivalve, the Pacific oyster, Crassostrea gigas, using a suppression subtractive hybridization method to identify up- and down-regulated genes after a 30-day exposure period to herbicides (a cocktail of atrazine, diuron and isoproturon, and to the single herbicide glyphosate). A total of 137 unique differentially expressed gene sequences was identified, as well as their associated physiological process. The expression of 18 of these genes was analyzed by RT-PCR under laboratory experimental conditions. The metabolic functions they are associated with include xenobiotic detoxification, energy production, immune system response and transcription. This study provides a preliminary basis for studying the response of marine bivalves to long-term herbicide exposure in terms of regulated gene expression and characterizes new potential genetic markers of herbicide contamination.

Acetylcholinesterase↗

Multifrequency cross-correlation phase fluorometry of chlorophyll a fluorescence in thylakoid and PSII-enriched membranes.

We present here a comparative study on the decay of chlorophyll (Chl) a fluorescence yield in thylakoid membranes and photosystem II (PSII)-enriched samples, measured with multifrequency cross-correlation phase fluorometry. These measurements confirm the general conclusions of Van Mieghem et al. (Biochim. Biophys. Acta 1100, 198-206, 1992), obtained with a flash method, on the effects of reduction of the primary quinone acceptor (QA) on Chl a fluorescence yield of PSII. Different states of the reaction centers of PSII were produced by: (1) pretreatment with sodium dithionite and methyl viologen followed by laser illumination: the doubly reduced QA (QAH2) centers; (2) with laser illumination or pretreatment with diuron: QA- centers; and (3) the addition of micromolar concentration of dichlorobenzoquinone (DCBQ): oxidized QA centers. The data were analyzed with Lorentzian distribution as well as with multiexponential fluorescence decay functions. The analysis with Lorentzian distribution function showed that upon formation of QA-, the major lifetime distribution peak shifted to longer lifetimes: from 0.25 ns to 1.66 ns (pea thylakoid membranes) and from 0.24 ns to 1.31 ns (core PSII). However, when QAH2 was formed, the lifetime distribution peaks shifted back to shorter lifetimes (0.57-0.77 ns) both in thylakoids and PSII membranes. Multiexponential analysis showed three lifetime components: fast (40-400 ps), middle (300-1500 ps) and slow (5-25 ns). When QA- was formed in PSII centers, the amplitude of the fast component decreased, but both the amplitude and the lifetime of the middle component increased severalfold.(ABSTRACT TRUNCATED AT 250 WORDS)

Chlorophyll↗

Enrichment and molecular characterization of a bacterial culture that degrades methoxy-methyl urea herbicides and their aniline derivatives.

Soil treated with linuron for more than 10 years showed high biodegradation activity towards methoxy-methyl urea herbicides. Untreated control soil samples taken from the same location did not express any linuron degradation activity, even after 40 days of incubation. Hence, the occurrence in the field of a microbiota having the capacity to degrade a specific herbicide was related to the long-term treatment of the soil. The enrichment culture isolated from treated soil showed specific degradation activity towards methoxy-methyl urea herbicides, such as linuron and metobromuron, while dimethyl urea herbicides, such as diuron, chlorotoluron, and isoproturon, were not transformed. The putative metabolic intermediates of linuron and metobromuron, the aniline derivatives 3, 4-dichloroaniline and 4-bromoaniline, were also degraded. The temperature of incubation drastically affected degradation of the aniline derivatives. Whereas linuron was transformed at 28 and 37 degrees C, 3,4-dichloroaniline was transformed only at 28 degrees C. Monitoring the enrichment process by reverse transcription-PCR and denaturing gradient gel electrophoresis (DGGE) showed that a mixture of bacterial species under adequate physiological conditions was required to completely transform linuron. This research indicates that for biodegradation of linuron, several years of adaptation have led to selection of a bacterial consortium capable of completely transforming linuron. Moreover, several of the putative species appear to be difficult to culture since they were detectable by DGGE but were not culturable on agar plates.

Aniline Compounds↗

Isolation from agricultural soil and characterization of a Sphingomonas sp. able to mineralize the phenylurea herbicide isoproturon.

A soil bacterium (designated strain SRS2) able to metabolize the phenylurea herbicide isoproturon, 3-(4-isopropylphenyl)-1,1-dimethylurea (IPU), was isolated from a previously IPU-treated agricultural soil. Based on a partial analysis of the 16S rRNA gene and the cellular fatty acids, the strain was identified as a Sphingomonas sp. within the alpha-subdivision of the proteobacteria. Strain SRS2 was able to mineralize IPU when provided as a source of carbon, nitrogen, and energy. Supplementing the medium with a mixture of amino acids considerably enhanced IPU mineralization. Mineralization of IPU was accompanied by transient accumulation of the metabolites 3-(4-isopropylphenyl)-1-methylurea, 3-(4-isopropylphenyl)-urea, and 4-isopropyl-aniline identified by high-performance liquid chromatography analysis, thus indicating a metabolic pathway initiated by two successive N-demethylations, followed by cleavage of the urea side chain and finally by mineralization of the phenyl structure. Strain SRS2 also transformed the dimethylurea-substituted herbicides diuron and chlorotoluron, giving rise to as-yet-unidentified products. In addition, no degradation of the methoxy-methylurea-substituted herbicide linuron was observed. This report is the first characterization of a pure bacterial culture able to mineralize IPU.

Agriculture↗

In vivo regulation of glutamine synthetase activity in the marine chlorophyll b-containing cyanobacterium Prochlorococcus sp. strain PCC 9511 (oxyphotobacteria).

The physiological regulation of glutamine synthetase (GS; EC 6.3.1.2) in the axenic Prochlorococcus sp. strain PCC 9511 was studied. GS activity and antigen concentration were measured using the transferase and biosynthetic assays and the electroimmunoassay, respectively. GS activity decreased when cells were subjected to nitrogen starvation or cultured with oxidized nitrogen sources, which proved to be nonusable for Prochlorococcus growth. The GS activity in cultures subjected to long-term phosphorus starvation was lower than that in equivalent nitrogen-starved cultures. Azaserine, an inhibitor of glutamate synthase, provoked an increase in enzymatic activity, suggesting that glutamine is not involved in GS regulation. Darkness did not affect GS activity significantly, while the addition of diuron provoked GS inactivation. GS protein determination showed that azaserine induces an increase in the concentration of the enzyme. The unusual responses to darkness and nitrogen starvation could reflect adaptation mechanisms of Prochlorococcus for coping with a light- and nutrient-limited environment.

Adaptation, Physiological↗

Effect of herbicides on cellulose decomposition by Sporocytophaga myxococcoides.

The effects of 14 herbicides on cellulose decomposition by Sporocytophaga myxococcoides were studied by growing the organism in two different liquid cellulose-mineral salts media in the presence of the test agent and then quantitatively determining the residual cellulose. In the first medium, containing acid-hydrolyzed cellulose, anthrone reagent was used to determine residual carbohydrate. In the second medium, Gooch Pyrex fritted-glass crucibles were used to determine the amount of residual Whatman powdered cellulose. The herbicides used were Atrazine, Simazine, Dacthal, Diuron, Amiben, Banvel-D, Banvel-T,2,3,6-TBA, Dicryl, Maleic Hydrazide, Stam F-34, Zytron, Fenac, and Dalapon. Of these, Zytron appeared to be the most inhibitory to the test bacterium, and was the only compound exhibiting inhibition at a commonly used field rate of application (2 ppm).

Bacteria↗

Effect of selected herbicides on bacterial growth rates.

Specific growth rate constants were used to evaluate the effects of selected herbicides on Erwinia carotovora, Pseudomonas fluorescens, and Bacillus sp. Comparison of growth rate constants permitted the identification of either stimulatory or inhibitory effects of these substances. E. carotovora was inhibited by 6,7-dihydrodipyrido(1,2-a:2'-c)pyrazinediium (diquat) and 4-hydroxy-3,5-diiodobenzonitrile (ioxynil) at 25 mug/ml; 1,1'-dimethyl-4,4'-bipyridinium (paraquat) at 50 mug/ml; and pentachlorophenol (PCP) at 10 mug/ml. P. fluorescens was inhibited by paraquat and PCP at 25 mug/ml and by 4-amino-3,5,6-trichloropicolinic acid (picloram) at 50 mug/ml. Stimulation of P. fluorescens was observed with 4-(methylsulfonyl)-2,6-dinitro-N,N-dipropylaniline (nitralin) at 25 mug/ml. The Bacillus species was inhibited by diquat (25 mug/ml), ioxynil (10 mug/ml), and paraquat and PCP (5 mug/ml). No significant effect of 2-chloro-4-(ethylamino)-6-(isopropylamino)-s-triazine (atrazine), 3-(3,4-dichlorophenyl)-1,1-dimethylurea (diuron), alpha,alpha,alpha-trifluoro-2,6-dinitro-N,N-dipropyl-p-toluidine (trifluralin), or 1,1-dimethyl-3-(alpha,alpha,alpha-trifluoro-m-tolyl)urea (fluometuron) on growth rates of the bacteria was observed at 25 and 50 mug/ml.

Bacillus↗

Degradation of chlorbromuron and related compounds by the fungus Rhizoctonia solani.

The ability of the soil fungus Rhizoctonia solani to degrade phenyl-substituted urea herbicides was investigated. The fungus was able to transform chlorbromuron [3-(3-chloro-4-bromophenyl)-1-methyl-1-methoxyurea] to the demethylated product [3-(3-chloro-4-bromophenyl)-1-methoxyurea], which was isolated and identified. Evidence was obtained that further degradation of chlorbromuron occurred. Several other phenylurea compounds (chloroxuron, diuron, fenuron, fluometuron, linuron, metobromuron, neburon, and siduron) were also metabolized by the fungus, indicating that R. solani may possess a generalized ability to attack this group of herbicides.

Aniline Compounds↗

Mathematical modeling of controlled-release systems of herbicides using lignins as matrices. A review.

The herbicides applied in soils can be easily lost, owing to leaching, volatilization, and bio- and photodegradation. Controlled-release systems using polymeric matrices claim to solve these problems. The movement of the herbicides in the soil is also an important phenomenon to be studied in order to evaluate the loss processes. The development of mathematical models is a relevant requirement for simulation and optimization of such systems. This study reviews mathematical models as an initial step for modeling data obtained for controlled-release systems of herbicides (diuron, 2,4-dichlorophenoxyacetic acid, and ametryn) using sugarcane bagasse lignin as a polymeric matrix. The release kinetic studies were carried out using several acceptor systems including a water bath, soil, and soil-packed columns. Generally, these models take into account phenomena such as unsteady-state mass transfer by diffusion (Fick's law) and convection, consumption by several processes, and partitioning processes, resulting in partial differential equations with respect to time and space variables.

Delayed-Action Preparations↗

Molecular modelling studies on the binding of phenylurea inhibitors to the D 1 protein of photosystem II.

A hypothetical molecular model of part of the D 1 protein of photosystem II, based on the analogous portion of the L subunit of the Rhodopseudomonas viridis reaction centre, has been used to study the binding of an extended hydrophobic phenylurea inhibitor (N,N-dimethyl-carbamoyl)4-amino-4'-chloro-trans-stilbene) (I) to the QB site. The inhibitor was fitted by eye into a cleft in the site, and a limited part of the inhibitor/D 1 complex was energy minimized. The gross orientation of the inhibitor placed the dimethylurea moiety towards the predicted binding domain of the plastoquinone head group, and the stilbene moiety directed along the quinone isoprenoid side chain binding domain, suggesting a similar pathway of approach of the two molecules from the membrane into the binding site. Binding interactions of the inhibitor included hydrogen bonds to the side chain hydroxyl of ser 264 and the peptide carbonyl group of ala 251, with the side chain hydroxyl of ser 268 as an alternative ligand. Numerous hydrophobic contacts were also possible. Although phenylureas do not bind to reaction centres of Rp. viridis, many of the binding interactions to D 1 could also be detected in Rp. viridis. However, the beta-CH2, and delta-CO2- groups of glu 212 in Rp. viridis are located in the corresponding region of D 1 occupied by the dimethylurea moiety of the inhibitor in our model of its binding to D 1. This may explain why diuron (DCMU) does not bind to Rp. viridis reaction centres.

Amino Acid Sequence↗

Mutational analysis of the PsbL protein of photosystem II in the cyanobacterium Synechocystis sp. PCC 6803.

The psbL gene is a member of the psbEFLJ gene cluster in the cyanobacterium Synechocystis sp. PCC 6803 and higher plants. psbL, a 4.5 kDa protein encoded by this gene, is a component of the photosystem II complex. The amino acid sequence of this protein indicates that it has a single membrane-spanning alpha-helical domain. We have used a targeted mutagenesis technique to delete the coding region of the psbL gene in Synechocystis 6803. The resultant mutant strain T345 did not show any PSII-mediated oxygen evolution activity and, as a result, could not grow under photoautotrophic conditions. However, it had normal PSI activity. The chlorophyll to phycobilin ratio in the T345 cells was significantly lower than that in the wild type cells. Fluorescence emission spectra (77 K) of the mutant cells showed the absence of a 695 nm band that usually originates from the PSII complex. Binding assays with radioactive diuron demonstrated that the mutant cells did not have any herbicide binding activity. However, immunostaining experiments showed that both the D 1 (the herbicide binding protein) and the D2 proteins of the PSII reaction center were present at > 25% of their normal levels in the thylakoid membranes of the T345 mutant cells. Our data indicate that the PsbL protein is essential for the normal functioning of PSII.

Amino Acid Sequence↗