Genetic basis for the biodegradation of a polyether-polyurethane-acrylic copolymer by a landfill microbial community inferred by metagenomic deconvolution analysis.

Plastic accumulation in the world amounts to approximately 8300 million tons. Polyurethanes (PU) account for 7.7 % of total plastics production worldwide, and their diverse chemical composition makes them highly recalcitrant to biodegradation. Several works have reported polyurethane-degrading microbial communities. However, it is still necessary to learn more about the chemical, biochemical, and genetic bases linked to the polyurethanolytic phenotype and the microbial taxonomic determinants responsible for metabolizing the PU polymer and its associated chemical additives. To shed light on this problem, we applied physical, chemical, biochemical, metagenomic, and bioinformatic analyses to explore the biodegradation capability and related biochemical and genetic determinants of the BP6 microbial community that can grow in PolyLack, a commercial coating containing a polyether polyurethane acrylate (PE-PU-A) copolymer and several additives, as sole carbon source. We observed complete additives (isopropanol, N-methyl-2-pyrrolidone, 2-butoxyethanol, alkyl glycol ethers) biodegradation and the appearance of released polymer components (toluene diisocyanate (TDI) and methylene diphenyl diisocyanate (MDI) derivatives), and multiple degradation products since early cultivation times. The Hi-C metagenomic analysis identified a complex microbiome with 35 deconvolved Metagenome-Assembled Genomes (MAGs) - several new species - and biodegradation markers that suggest the coexistence of hydrolytic, oxidative, and reductive metabolic strategies for degrading the additives and the PU copolymer. This work also provides evidence of the metabolic capability the BP6 community has for biodegrading polyether polyurethane foams. Based on these analyses, we propose a novel metabolic pathway for 4,4'-methylenedianiline (MDA), an initial biodegradation intermediate of MDI-based PU, encoded in the complex BP6 community metagenome and suggest that this community is a potential biotechnological tool for PU bio-recycling.

Synthesis, characterization and biodegradation studies of polyurethanes: Effect of unsaturation on biodegradability.

The presence of unsaturation in the main chain of the polymer promotes the biodegradation process. To elucidate this hypothesis, one unsaturated polyurethane (PUU) and another saturated polyurethane (PUS) were synthesized and then biodegraded, and evidence was found to support this hypothesis. The polyurethanes were synthesized by a polycondensation reaction with yields up to 97%. It is important to note that both polyurethanes were constituted only by the recalcitrant hard segment and showed low crystallinity and molecular weight. Spectroscopic, thermal, and chromatographic techniques were used for physical and structural characterization. Both polyurethanes were biodegraded by the BP8 microbial community and the Cladosporium tenuissimum A3.I.1 fungus during a two-month period. A postbiodegradation characterization revealed the detriment of properties in both materials, indicating successful biodegradation. As a general trend, more efficient biodegradation was observed by the Cladosporium tenuissimum fungus A3.I.1 than by the BP8 microbial community. Specifically, with the fungus, the infrared analysis showed a decrease in the characteristic bands as well as the appearance of new carboxylic acid signals (approximately 1701 cm-1), suggesting the enzymatic cleavage of the urethane group. By comparison to polyurethanes, PUU showed superior biodegradation; using the fungus, a 51% decrease in molecular weight (Mw) for PUU was achieved, in contrast with 36% achieved for PUS. Despite the low crystallinity and molecular weight, the determining factor in biodegradation was the presence of unsaturations along the main chain. Thus, a more efficient oxidative attack is carried out by microorganisms on double bonds. The novel PUU showed similar biodegradation to the known polyester-type PU with highly hydrolysable groups. Consequently, PUU represents a green alternative to conventional polyurethanes and is a key material to achieve biorecycling.

Concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities during Impranil biodegradation by Alicycliphilus denitrificans BQ1.

The concerted action of commercial esterases, proteases and amidases has been demonstrated to be relevant in polyurethane (PU) degradation by in vitro experiments. However, the spatial and temporal dynamics of these activities during PU biodegradation by PU-degrading bacteria have not been addressed. Here, we examined the capability of Alicycliphilus denitrificans BQ1 to biodegrade the polyester (PS)-PU Impranil, analyzed the temporal and spatial coordination between the extracellular and cytoplasmic esterase and urethane-cleaving activities, and their independent and combined effects on Impranil biodegradation. A. denitrificans BQ1 grew in Impranil, and its clearing was correlated with the cleavage of ester and urethane groups since early times, with decrements of some Impranil compounds and the appearance of biodegradation products. While extracellular esterase was active at early times with its maximum at 18 h, urethanase appeared at this time and increased up to the end of the analysis (48 h), with the cytoplasmic activities behaving similarly but with lower levels than the extracellular ones. Both enzymatic activities exhibited distinct substrate specificity depending on their cellular localization and cultivation times, suggesting they cleave differentially located groups. As the urethane cleavage occurred since early times, when no urethane-cleaving activity was detected, different proteins should be acting at early and late times. In vitro experiments with independent or combined cellular protein fractions supported the previous deduction and confirmed the concerted action of extracellular and cytoplasmic esterase and urethane-cleaving activities. A two-stage process for Impranil degradation by A. denitrificans BQ1 is proposed.

Exploring the polyurethanolytic activity and microbial composition of landfill microbial communities.

The microbial composition of polyurethane degrading communities has been barely addressed, and it is unknown if microenvironmental conditions modify its composition, affecting its biodegradative capacity. The polyurethanolytic activity and taxonomic composition of five microbial communities, selected by enrichment in the polyether-polyurethane-acrylic (PE-PU-A) coating PolyLack®, from deteriorated PU foams collected at different microenvironments in a municipal landfill (El Bordo Poniente, BP) were explored. All BP communities grew similarly in PolyLack® as the sole carbon source, although BP1, BP4, and BP5 showed better performance than BP2 and BP7. FTIR spectroscopy showed that ester, urethane, ether, aromatic and aliphatic groups, and the acrylate component were targets of the biodegradative activity. Extracellular esterase activity was higher at 5 days of cultivation and decreased at 21 days, while urease activity showed the opposite. Microbial composition analysis, assessed by 16S rDNA V3 region PCR-DGGE, revealed a preponderance of Rhizobiales and Micrococcales. The reported PU-degrading genera Paracoccus, Acinetobacter, and Pseudomonas were identified. In contrast, Advenella, Bordetella, Microbacterium, Castellaniella, and Populibacterium, some of them xenobiotics degraders, can be considered potentially PU-degrading genera. Correspondence analysis identified independent groups for all communities, except the BP4 and BP5. Although partial taxonomic redundancy was detected, unique OTUs were identified, e.g., three members of the Weeksellaceae family were present only in the BP4/BP5 group. These results suggest that the microenvironmental conditions where the landfill microbial communities were collected shaped their taxonomical composition, impacting their PE-PU biodegradative capacities. These BP communities represent valuable biological material for the treatment of PU waste and other xenobiotics. KEY POINTS: • Landfill microbial communities display slightly different capacities for growing in polyether-polyurethane-acrylic. • Ester, urethane, ether, aromatic, aliphatic, and acrylate groups were attacked. • Esterase activity was more significant at early culture times while urease activity at latter. • Landfill microenvironments shape partial taxonomical redundancy in the communities. • Best communities' performance seems to be related to unique members' composition.

Current status on the biodegradability of acrylic polymers: microorganisms, enzymes and metabolic pathways involved.

Acrylic polymers (AP) are a diverse group of materials with broad applications, frequent use, and increasing demand. Some of the most used AP are polyacrylamide, polyacrylic acid, polymethyl methacrylates, and polyacrylonitrile. Although no information for the production of all AP types is published, data for the most used AP is around 9 MT/year, which gives an idea of the amount of waste that can be generated after products' lifecycles. After its lifecycle ends, the fate of an AP product will depend on its chemical structure, the environmental setting where it was used, and the regulations for plastic waste management existing in the different countries. Even though recycling is the best fate for plastic polymer wastes, few AP can be recycled, and most of them end up in landfills. Because of the pollution crisis the planet is immersed, setting regulations and developing technological strategies for plastic waste management are urgent. In this regard, biotechnological approaches, where microbial activity is involved, could be attractive eco-friendly strategies. This mini-review describes the broad AP diversity, their properties and uses, and the factors affecting their biodegradability, underlining the importance of standardizing biodegradation quantification techniques. We also describe the enzymes and metabolic pathways that microorganisms display to attack AP chemical structure and predict some biochemical reactions that could account for quaternary carbon-containing AP biodegradation. Finally, we analyze strategies to increase AP biodegradability and stress the need for more studies on AP biodegradation and developing stricter legislation for AP use and waste control. KEY POINTS: • Acrylic polymers (AP) are a diverse and extensively used group of compounds. • The environmental fates and health effects of AP waste are not completely known. • Microorganisms and enzymes involved in AP degradation have been identified. • More biodegradation studies are needed to develop AP biotechnological treatments.

Biodegradation of polyacrylic and polyester polyurethane coatings by enriched microbial communities.

Microbial communities are more effective in degrading natural polymers and xenobiotics than pure cultures. Biodegradation of polyacrylic and polyurethane polymers by bacterial and fungal strains has been addressed, but limited information about their biodegradation by microbial communities exists. The aim of this work was to evaluate the ability of three enriched microbial communities (BP1h, BP3h, and BP7h), selected from deteriorated foam pieces collected in a landfill, to biodegrade the polyacrylic component of the 2K-PU coating Bayhydrol® A2470 and the polyester polyurethane coating NeoRez™ R-9637. Two communities were further selected to quantify extracellular esterase, protease, and urease activities, to identify their taxonomic composition, and to analyze the ability of their isolated members to grow in those polymers. The growth of the three communities was larger in polyester polyurethane than in polyacrylic and their biodegradative activities affected ester, urethane, ether, aromatic, and aliphatic groups of the compounds present in the coatings. From all the communities growing in polyacrylic or in polyester polyurethane, two and five different types of colonies were isolated, respectively. In polyacrylic, extracellular esterase and protease activities were at their maximum level at 7 days of culture, whereas in polyester polyurethane, protease and urease were greatest at 21 days. All the isolated community members were identified as xenobiotics degraders. The complete communities grew better in media with the polymers than the isolated members. This is one of the few studies reporting biodegradation of synthetic polymers by microbial communities and serves as basis for developing synthetic consortia with enhanced degradative abilities.

Degradation of Recalcitrant Polyurethane and Xenobiotic Additives by a Selected Landfill Microbial Community and Its Biodegradative Potential Revealed by Proximity Ligation-Based Metagenomic Analysis.

Polyurethanes (PU) are the sixth most produced plastics with around 18-million tons in 2016, but since they are not recyclable, they are burned or landfilled, generating damage to human health and ecosystems. To elucidate the mechanisms that landfill microbial communities perform to attack recalcitrant PU plastics, we studied the degradative activity of a mixed microbial culture, selected from a municipal landfill by its capability to grow in a water PU dispersion (WPUD) as the only carbon source, as a model for the BP8 landfill microbial community. The WPUD contains a polyether-polyurethane-acrylate (PE-PU-A) copolymer and xenobiotic additives (N-methylpyrrolidone, isopropanol and glycol ethers). To identify the changes that the BP8 microbial community culture generates to the WPUD additives and copolymer, we performed chemical and physical analyses of the biodegradation process during 25 days of cultivation. These analyses included Nuclear magnetic resonance, Fourier transform infrared spectroscopy, Thermogravimetry, Differential scanning calorimetry, Gel permeation chromatography, and Gas chromatography coupled to mass spectrometry techniques. Moreover, for revealing the BP8 community structure and its genetically encoded potential biodegradative capability we also performed a proximity ligation-based metagenomic analysis. The additives present in the WPUD were consumed early whereas the copolymer was cleaved throughout the 25-days of incubation. The analysis of the biodegradation process and the identified biodegradation products showed that BP8 cleaves esters, C-C, and the recalcitrant aromatic urethanes and ether groups by hydrolytic and oxidative mechanisms, both in the soft and the hard segments of the copolymer. The proximity ligation-based metagenomic analysis allowed the reconstruction of five genomes, three of them from novel species. In the metagenome, genes encoding known enzymes, and putative enzymes and metabolic pathways accounting for the biodegradative activity of the BP8 community over the additives and PE-PU-A copolymer were identified. This is the first study revealing the genetically encoded potential biodegradative capability of a microbial community selected from a landfill, that thrives within a WPUD system and shows potential for bioremediation of polyurethane- and xenobiotic additives-contamitated sites.

Novel Metabolic Pathway for N-Methylpyrrolidone Degradation in Alicycliphilus sp. Strain BQ1.

The molecular mechanisms underlying the biodegradation of N-methylpyrrolidone (NMP), a widely used industrial solvent that produces skin irritation in humans and is teratogenic in rats, are unknown. Alicycliphilus sp. strain BQ1 degrades NMP. By studying a transposon-tagged mutant unable to degrade NMP, we identified a six-gene cluster (nmpABCDEF) that is transcribed as a polycistronic mRNA and encodes enzymes involved in NMP biodegradation. nmpA and the transposon-affected gene nmpB encode an N-methylhydantoin amidohydrolase that transforms NMP to γ-N-methylaminobutyric acid; this is metabolized by an amino acid oxidase (NMPC), either by demethylation to produce γ-aminobutyric acid (GABA) or by deamination to produce succinate semialdehyde (SSA). If GABA is produced, the activity of a GABA aminotransferase (GABA-AT), not encoded in the nmp gene cluster, is needed to generate SSA. SSA is transformed by a succinate semialdehyde dehydrogenase (SSDH) (NMPF) to succinate, which enters the Krebs cycle. The abilities to consume NMP and to utilize it for growth were complemented in the transposon-tagged mutant by use of the nmpABCD genes. Similarly, Escherichia coli MG1655, which has two SSDHs but is unable to grow in NMP, acquired these abilities after functional complementation with these genes. In wild-type (wt) BQ1 cells growing in NMP, GABA was not detected, but SSA was present at double the amount found in cells growing in Luria-Bertani medium (LB), suggesting that GABA is not an intermediate in this pathway. Moreover, E. coli GABA-AT deletion mutants complemented with nmpABCD genes retained the ability to grow in NMP, supporting the possibility that γ-N-methylaminobutyric acid is deaminated to SSA instead of being demethylated to GABA.IMPORTANCEN-Methylpyrrolidone is a cyclic amide reported to be biodegradable. However, the metabolic pathway and enzymatic activities for degrading NMP are unknown. By developing molecular biology techniques for Alicycliphilus sp. strain BQ1, an environmental bacterium able to grow in NMP, we identified a six-gene cluster encoding enzymatic activities involved in NMP degradation. These findings set the basis for the study of new enzymatic activities and for the development of biotechnological processes with potential applications in bioremediation.

Biodegradative Activities of Selected Environmental Fungi on a Polyester Polyurethane Varnish and Polyether Polyurethane Foams.

UNLABELLED: Polyurethane (PU) is widely used in many aspects of modern life because of its versatility and resistance. However, PU waste disposal generates large problems, since it is slowly degraded, there are limited recycling processes, and its destruction may generate toxic compounds. In this work, we isolated fungal strains able to grow in mineral medium with a polyester PU (PS-PU; Impranil DLN) or a polyether PU (PE-PU; Poly Lack) varnish as the only carbon source. Of the eight best Impranil-degrading strains, the six best degraders belonged to the Cladosporium cladosporioides complex, including the species C. pseudocladosporioides, C. tenuissimum, C. asperulatum, and C. montecillanum, and the two others were identified as Aspergillus fumigatus and Penicillium chrysogenum The best Impranil degrader, C. pseudocladosporioides strain T1.PL.1, degraded up to 87% after 14 days of incubation. Fourier transform infrared (FTIR) spectroscopy analysis of Impranil degradation by this strain showed a loss of carbonyl groups (1,729 cm(-1)) and N-H bonds (1,540 and 1,261 cm(-1)), and gas chromatography-mass spectrometry (GC-MS) analysis showed a decrease in ester compounds and increase in alcohols and hexane diisocyanate, indicating the hydrolysis of ester and urethane bonds. Extracellular esterase and low urease, but not protease activities were detected at 7 and 14 days of culture in Impranil. The best eight Impranil-degrading fungi were also able to degrade solid foams of the highly recalcitrant PE-PU type to different extents, with the highest levels generating up to 65% of dry-weight losses not previously reported. Scanning electron microscopy (SEM) analysis of fungus-treated foams showed melted and thinner cell wall structures than the non-fungus-treated ones, demonstrating fungal biodegradative action on PE-PU. IMPORTANCE: Polyurethane waste disposal has become a serious problem. In this work, fungal strains able to efficiently degrade different types of polyurethanes are reported, and their biodegradative activity was studied by different experimental approaches. Varnish biodegradation analyses showed that fungi were able to break down the polymer in some of their precursors, offering the possibility that they may be recovered and used for new polyurethane synthesis. Also, the levels of degradation of solid polyether polyurethane foams reported in this work have never been observed previously. Isolation of efficient polyurethane-degrading microorganisms and delving into the mechanisms they used to degrade the polymer provide the basis for the development of biotechnological processes for polyurethane biodegradation and recycling.

Protein phosphorylation regulates in vitro spinach chloroplast petD mRNA 3'-untranslated region stability, processing, and degradation.

RNA-binding proteins (RNPs) participate in diverse processes of mRNA metabolism, and phosphorylation changes their binding properties. In spinach chloroplasts, 24RNP and 28RNP are associated with polynucleotide posphorylase forming a complex on charge of pre-mRNA 3'-end maturation. Here, we tested the hypothesis that the phosphorylation status of 24RNP and 28RNP, present in a spinach chloroplast mRNA 3'-UTR processing extract (CPE), controls the transition between petD precursor stabilization, 3'-UTR processing, and RNA degradation in vitro. The CPE processed or stabilized petD precursor depending on the ATP concentration present in an in vitro 3'-UTR processing (IVP) assay. These effects were also observed when ATP was pre-incubated and removed before the IVP assay. Moreover, a dephosphorylated (DP)-CPE degraded petD precursor and recovered 3'-UTR processing or stabilization activities in an ATP concentration dependent manner. To determine the role 24/28RNP plays in regulating these processes a 24/28RNP-depleted (Δ24/28)CPE was generated. The Δ24/28CPE degraded the petD precursor, but when it was reconstituted with recombinant non-phosphorylated (NP)-24RNP or NP-28RNP, the precursor was stabilized, whereas when Δ24/28CPE was reconstituted with phosphorylated (P)-24RNP or P-28RNP, it recovered 3'-UTR processing, indicating that 24RNP or 28RNP is needed to stabilize the precursor, have a redundant role, and their phosphorylation status regulates the transition between precursor stabilization and 3'-UTR processing. A DP-Δ24/28CPE reconstituted or not with NP-24/28RNP degraded petD precursor. Pre-incubation of DP-Δ24/28CPE with NP-24/28RNP plus 0.03 mM ATP recovered 3'-UTR processing activity, and its reconstitution with P-24/28RNP stabilized the precursor. However, pre-incubation of DP-Δ24/28CPE with 0.03 mM ATP, and further reconstitution with NP-24/28RNP or P-24/28RNP produced precursor stability instead of RNA degradation, and RNA processing instead of precursor stability, respectively. Moreover, in vitro phosphorylation of CPE showed that 24RNP, 28RNP, and other proteins may be phosphorylated. Altogether, these results reveal that phosphorylation of 24RNP, 28RNP, and other unidentified CPE proteins mediates the in vitro interplay between petD precursor stability, 3'-UTR processing, and degradation, and support the idea that protein phosphorylation plays an important role in regulating mRNA metabolism in chloroplast.

Purification of an Arabidopsis chloroplast extract with in vitro RNA processing activity on psbA and petD 3'-untranslated regions.

The mature 3'-end of many chloroplast mRNAs is generated by the processing of the 3'-untranslated region (3'-UTR), which is a mechanism that involves the removal of a segment located downstream an inverted repeat sequence that forms a stem-loop structure. Nuclear-encoded chloroplast RNA binding proteins associate with the stem-loop to process the 3'-UTR or to influence mRNA stability. A spinach chloroplast processing extract (CPE) has been previously generated and used to in vitro dissect the biochemical mechanism underlying 3'-UTR processing. Being Arabidopsis thaliana an important genetic model, the development of a CPE allowing to correlate 3'-UTR processing activity with genes encoding proteins involved in this process, would be of great relevance. Here, we developed a purification protocol that generated an Arabidopsis CPE able to correctly process a psbA 3'-UTR precursor. By UV crosslinking, we characterized the protein patterns generated by the interaction of RNA binding proteins with Arabidopsis psbA and petD 3'-UTRs, finding that each 3'-UTR bound specific proteins. By testing whether Arabidopsis CPE proteins were able to bind spinach ortholog 3'-UTRs, we also found they were bound by specific proteins. When Arabidopsis CPE 3'-UTR processing activity on ortholog spinach 3'-UTRs was assessed, stable products appeared: for psbA, a smaller size product than the expected mature 3'-end, and for petD, low amounts of the expected product plus several others of smaller sizes. These results suggest that the 3'-UTR processing mechanism of these chloroplast mRNAs might be partially conserved in Arabidopsis and spinach.

Germination behavior, biochemical features and sequence analysis of the RACK1/arcA homolog from Phaseolus vulgaris.

Partial peptide sequence of a 36 kDa protein from common bean embryo axes showed 100% identity with a reported beta-subunit of a heterotrimeric G protein from soybean. Analysis of the full sequence showed 96.6% identity with the reported soybean G(beta)-subunit, 86% with RACK1B and C from Arabidopsis and 66% with human and mouse RACK1, at the amino acid level. In addition, it showed 85.5, 85 and 83% identities with arcA from Solanum lycopersicum, Arabidopsis (RACK1A) and Nicotiana tabacum, respectively. The amino acid sequence displayed seven WD40 domains and two sites for activated protein kinase C binding. The protein showed a constant expression level but the mRNA had a maximum at 32 h post-imbibition. Western immunoblotting showed the protein in vegetative plant tissues, and in both microsomal and soluble fractions from embryo axes. Synthetic auxin treatment during germination delayed the peak of RACK1 mRNA expression to 48 h but did not affect the protein expression level while the polar auxin transport inhibitor, naphtylphtalamic acid had no effect on either mRNA or protein expression levels. Southern blot and genomic DNA amplification revealed a small gene family with at least one member without introns in the genome. Thus, the RACK1/arcA homolog from common bean has the following features: (1) it is highly conserved; (2) it is both soluble and insoluble within the embryo axis; (3) it is encoded by a small gene family; (4) its mRNA has a peak of expression at the time point of germination stop and (5) its expression is only slightly affected by auxin but unaffected by an auxin transport blocker.

Characterization of the polyurethanolytic activity of two Alicycliphilus sp. strains able to degrade polyurethane and N-methylpyrrolidone.

Two bacterial strains (BQ1 and BQ8) were isolated from decomposed soft foam. These were selected for their capacity to grow in a minimal medium (MM) supplemented with a commercial surface-coating polyurethane (PU) (Hydroform) as the carbon source (MM-PUh). Both bacterial strains were identified as Alicycliphilus sp. by comparative 16S rRNA gene sequence analysis. Growth in MM-PUh showed hyperbolic behavior, with BQ1 producing higher maximum growth (17.8 +/- 0.6 mg.ml(-1)) than BQ8 (14.0 +/- 0.6 mg.ml(-1)) after 100 h of culture. Nuclear magnetic resonance, Fourier transform infrared (IR) spectroscopy, and gas chromatography-mass spectrometry analyses of Hydroform showed that it was a polyester PU type which also contained N-methylpyrrolidone (NMP) as an additive. Alicycliphilus sp. utilizes NMP during the first stage of growth and was able to use it as the sole carbon and nitrogen source, with calculated K(s) values of about 8 mg.ml(-1). Enzymatic activities related to PU degradation (esterase, protease, and urease activities) were tested by using differential media and activity assays in cell-free supernatants of bacterial cultures in MM-PUh. Induction of esterase activity in inoculated MM-PUh, but not that of protease or urease activities, was observed at 12 h of culture. Esterase activity reached its maximum at 18 h and was maintained at 50% of its maximal activity until the end of the analysis (120 h). The capacity of Alicycliphilus sp. to degrade PU was demonstrated by changes in the PU IR spectrum and by the numerous holes produced in solid PU observed by scanning electron microscopy after bacterial culture. Changes in the PU IR spectra indicate that an esterase activity is involved in PU degradation.

Cytokinin promotes catalase and ascorbate peroxidase activities and preserves the chloroplast integrity during dark-senescence.

Increased oxidative stress displayed during dark-senescence of wheat leaves (Triticum aestivum L.) is caused not only by the increased levels of radicals but also by a loss of antioxidant capacity. Mature leaves were incubated in 6-benzylaminopurine (BAP 10(-4)M) or water (control) during 6d in the dark. The senescence-delaying effect of BAP was associated with the retention of the chloroplast structure, 60% of the initial content of chlorophyll (Chl) and 77% of the initial content of protein. BAP reduced the degradation of the light-harvesting chlorophyll a/b binding protein (LHCP-2), and the large (LSU) and small subunits (SSU) of Rubisco. Our results indicated that the presence of the NADPH:protochlorophyllide oxidoreductase (POR, EC.1.6.99.1) was not promoted by the cytokinin, leading to the conclusion that BAP maintains the level of Chl, preventing its degradation, rather than inducing Chl biosynthesis. The internal structure of chloroplasts was maintained in BAP-treated leaves for up to 6d, with well-organized grana thylakoids and small plastoglobuli; in contrast, chloroplasts of control leaves deteriorated rapidly from day 4 with disorganized internal membranes, and more and larger plastoglobuli. BAP increased the activities of catalase (CAT, EC 1.11.1.6) and ascorbate peroxidase (APX, EC 1.11.1.11) and reduced the level of H(2)O(2) in the delayed-senescence tissue. The present research indicates that BAP reduces levels of reactive oxygen species (ROS), and enhances the activity of antioxidant enzymes (CAT, APX). Our results suggest that BAP protects the cell membranes and the photosynthetic machinery from oxidative damage during delay of senescence in the dark.

Sulfur assimilation and glutathione metabolism under cadmium stress in yeast, protists and plants.

Glutathione (gamma-glu-cys-gly; GSH) is usually present at high concentrations in most living cells, being the major reservoir of non-protein reduced sulfur. Because of its unique redox and nucleophilic properties, GSH serves in bio-reductive reactions as an important line of defense against reactive oxygen species, xenobiotics and heavy metals. GSH is synthesized from its constituent amino acids by two ATP-dependent reactions catalyzed by gamma-glutamylcysteine synthetase and glutathione synthetase. In yeast, these enzymes are found in the cytosol, whereas in plants they are located in the cytosol and chloroplast. In protists, their location is not well established. In turn, the sulfur assimilation pathway, which leads to cysteine biosynthesis, involves high and low affinity sulfate transporters, and the enzymes ATP sulfurylase, APS kinase, PAPS reductase or APS reductase, sulfite reductase, serine acetyl transferase, O-acetylserine/O-acetylhomoserine sulfhydrylase and, in some organisms, also cystathionine beta-synthase and cystathionine gamma-lyase. The biochemical and genetic regulation of these pathways is affected by oxidative stress, sulfur deficiency and heavy metal exposure. Cells cope with heavy metal stress using different mechanisms, such as complexation and compartmentation. One of these mechanisms in some yeast, plants and protists is the enhanced synthesis of the heavy metal-chelating molecules GSH and phytochelatins, which are formed from GSH by phytochelatin synthase (PCS) in a heavy metal-dependent reaction; Cd(2+) is the most potent activator of PCS. In this work, we review the biochemical and genetic mechanisms involved in the regulation of sulfate assimilation-reduction and GSH metabolism when yeast, plants and protists are challenged by Cd(2+).

Chromium toxicity in plants.

Due to its wide industrial use, chromium is considered a serious environmental pollutant. Contamination of soil and water by chromium (Cr) is of recent concern. Toxicity of Cr to plants depends on its valence state: Cr(VI) is highly toxic and mobile whereas Cr(III) is less toxic. Since plants lack a specific transport system for Cr, it is taken up by carriers of essential ions such as sulfate or iron. Toxic effects of Cr on plant growth and development include alterations in the germination process as well as in the growth of roots, stems and leaves, which may affect total dry matter production and yield. Cr also causes deleterious effects on plant physiological processes such as photosynthesis, water relations and mineral nutrition. Metabolic alterations by Cr exposure have also been described in plants either by a direct effect on enzymes or other metabolites or by its ability to generate reactive oxygen species which may cause oxidative stress. The potential of plants with the capacity to accumulate or to stabilize Cr compounds for bioremediation of Cr contamination has gained interest in recent years.

Mercury pretreatment selects an enhanced cadmium-accumulating phenotype in Euglena gracilis.

Pre-treatment of heterotrophic cultures of Euglena gracilis with 1.5 microM HgCl(2) for at least 60 generations resulted in a cell population that showed both increased resistance to Cd(2+) and ability to accumulate it, when compared to non-Hg(2+)-pretreated Euglena. These Hg(2+)-enhanced capacities were evident in cells cultured in the dark in a medium with lactate, but not in cells cultured with glutamate plus malate. After culturing with 0.1 mM CdCl(2) through three consecutive transfers, the mercury-pretreated cells still grew and maintained high levels of glutathione-related metabolites, while the non-Hg(2+)-pretreated cells died. Cultures of Hg(2+)-pretreated cells, after transfer to media with or without cadmium, did not alter either their enhanced Cd(2+) accumulation or their increased production of glutathione-related metabolites. These observations suggested that the Hg(2+)-pretreated population underwent a permanent change that improved its Cd(2+) resistance. Several factors that contributed to the improved capacities included: (a) higher cellular malate, cysteine and glutathione levels induced by Hg(2+) before and after Cd(2+) exposure; and (b) increased storage of Cd(2+) in mitochondria along with increased intramitochondrial citrate, cysteine, and glutathione levels. These characteristics suggested that this Cd(2+) hyper-accumulating strain of E. gracilis might be a suitable candidate for Cd(2+)-bioremediation of polluted water systems.

Cadmium accumulation in the chloroplast of Euglena gracilis.

Intracellular distribution of Cd, cysteine, glutathione, and Cd-induced thiol peptides in Euglena gracilis cultured under photoheterotrophic conditions was studied. After 3 days of culture with 0.2 mM CdCl2, 62% of the Cd accumulated by cells was equally distributed between the cytosolic and chloroplastic fractions. However, after 8 days, metal content increased in the crude chloroplastic fraction to 40% of total and decreased to 19% in the cytosol; in Percoll-purified chloroplasts the estimated content of Cd raised to 62%. Accumulation of Cd in chloroplasts could be mediated by a transporter of free Cd2+, since uptake of added CdCl2 in isolated chloroplasts exhibited a hyperbolic type of kinetics with a Km of 57 &mgr;M and Vmax of 3.7 nmol (mg protein)-1 min-1. The contents of cysteine and glutathione markedly increased in both chloroplasts (7-19 times) and cytosol (4-9 times) by exposure to Cd2+, although they were always higher in the cytosol. Thiol-containing peptides induced by Cd were mainly located in the cytosol after 3 days, and in the chloroplasts after 8 days of culture. The data suggested that Cd was compartmentalized into chloroplasts in a process that may involve the transport of free Cd and the participation of thiol-peptides.