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Biochemical Investigations of Five Recombinantly Expressed Tyrosinases Reveal Two Novel Mechanisms Impacting Carbon Storage in Wetland Ecosystems.

ABSTRACT: Wetlands are globally distributed ecosystems characterized by predominantly anoxic soils, resulting from water-logging. Over the past millennia, low decomposition rates of organic matter led to the accumulation of 20-30% of the world's soil carbon pool in wetlands. Phenolic compounds are critically involved in stabilizing wetland carbon stores as they act as broad-scale inhibitors of hydrolytic enzymes. Tyrosinases are oxidoreductases capable of removing phenolic compounds in the presence of O₂ by oxidizing them to the corresponding o-quinones. Herein, kinetic investigations (k_cat and K_m values) reveal that low-molecular-weight phenolic compounds naturally present within wetland ecosystems (including monophenols, diphenols, triphenols, and flavonoids) are accepted by five recombinantly expressed wetland tyrosinases (TYRs) as substrates. Investigations of the interactions between TYRs and wetland phenolics reveal two novel mechanisms that describe the global impact of TYRs on the wetland carbon cycle. First, it is shown that o-quinones (produced by TYRs from low-molecular-weight phenolic substrates) are capable of directly inactivating hydrolytic enzymes. Second, it is reported that o-quinones can interact with high-molecular-weight phenolic polymers (which inhibit hydrolytic enzymes) and remove them through precipitation. The balance between these two mechanisms will profoundly affect the fate of wetland carbon stocks, particularly in the wake of climate change.

SUBMITTER: Panis F

PROVIDER: S-EPMC10515480 | biostudies-literature | 2023 Sep

REPOSITORIES: biostudies-literature

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Biochemical Investigations of Five Recombinantly Expressed Tyrosinases Reveal Two Novel Mechanisms Impacting Carbon Storage in Wetland Ecosystems.

Panis Felix F Rompel Annette A

Environmental science & technology 20230901 37

Wetlands are globally distributed ecosystems characterized by predominantly anoxic soils, resulting from water-logging. Over the past millennia, low decomposition rates of organic matter led to the accumulation of 20-30% of the world's soil carbon pool in wetlands. Phenolic compounds are critically involved in stabilizing wetland carbon stores as they act as broad-scale inhibitors of hydrolytic enzymes. Tyrosinases are oxidoreductases capable of removing phenolic compounds in the presence of O<s ...[more]

PMID: 37656057

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Project description:Over the last millennia, wetlands have been sequestering carbon from the atmosphere via photosynthesis at a higher rate than releasing it and, therefore, have globally accumulated 550 × 1015 g of carbon, which is equivalent to 73% of the atmospheric carbon pool. The accumulation of organic carbon in wetlands is effectuated by phenolic compounds, which suppress the degradation of soil organic matter by inhibiting the activity of organic-matter-degrading enzymes. The enzymatic removal of phenolic compounds by bacterial tyrosinases has historically been blocked by anoxic conditions in wetland soils, resulting from waterlogging. Bacterial tyrosinases are a subgroup of oxidoreductases that oxidatively remove phenolic compounds, coupled to the reduction of molecular oxygen to water. The biochemical properties of bacterial tyrosinases have been investigated thoroughly in vitro within recent decades, while investigations focused on carbon fluxes in wetlands on a macroscopic level have remained a thriving yet separated research area so far. In the wake of climate change, however, anoxic conditions in wetland soils are threatened by reduced rainfall and prolonged summer drought. This potentially allows tyrosinase enzymes to reduce the concentration of phenolic compounds, which in turn will increase the release of stored carbon back into the atmosphere. To offer compelling evidence for the novel concept that bacterial tyrosinases are among the key enzymes influencing carbon cycling in wetland ecosystems first, bacterial organisms indigenous to wetland ecosystems that harbor a TYR gene within their respective genome (tyr+) have been identified, which revealed a phylogenetically diverse community of tyr+ bacteria indigenous to wetlands based on genomic sequencing data. Bacterial TYR host organisms covering seven phyla (Acidobacteria, Actinobacteria, Bacteroidetes, Firmicutes, Nitrospirae, Planctomycetes, and Proteobacteria) have been identified within various wetland ecosystems (peatlands, marshes, mangrove forests, bogs, and alkaline soda lakes) which cover a climatic continuum ranging from high arctic to tropic ecosystems. Second, it is demonstrated that (in vitro) bacterial TYR activity is commonly observed at pH values characteristic for wetland ecosystems (ranging from pH 3.5 in peatlands and freshwater swamps to pH 9.0 in soda lakes and freshwater marshes) and toward phenolic compounds naturally present within wetland environments (p-coumaric acid, gallic acid, protocatechuic acid, p-hydroxybenzoic acid, caffeic acid, catechin, and epicatechin). Third, analyzing the available data confirmed that bacterial host organisms tend to exhibit in vitro growth optima at pH values similar to their respective wetland habitats. Based on these findings, it is concluded that, following increased aeration of previously anoxic wetland soils due to climate change, TYRs are among the enzymes capable of reducing the concentration of phenolic compounds present within wetland ecosystems, which will potentially destabilize vast amounts of carbon stored in these ecosystems. Finally, promising approaches to mitigate the detrimental effects of increased TYR activity in wetland ecosystems and the requirement of future investigations of the abundance and activity of TYRs in an environmental setting are presented.

Project description:BackgroundReduced height-1 dwarfing alleles affect DELLA proteins belonging to a family of putative transcriptional regulators that modulate plant growth and development. The Arabidopsis thaliana genome encodes five DELLA proteins, whereas monocot plants, such as rice, barley, and wheat, each have a single DELLA protein. In wheat, wild-type Rht-B1a and Rht-D1a genes encode DELLA proteins and have many alleles that contain lesions. Among them, Rht-B1b and Rht-D1b are the most common mutant dwarfing alleles, which have played a key part in the creation of high-yielding wheat varieties. Despite their fundamental roles in plant biology, until now, DELLA proteins in wheat have been mainly researched regarding the phenotypic effect of defective Rht mutants on yield-related traits, without studies on the underlying mechanisms. The RHT-1 protein has yet to be detected in wheat tissues, owing to a lack of appropriate molecular tools for characterization of RHT function and protein interactions in signal transduction. This study is focused on the production of a polyclonal antibody to the wheat RHT-D1A protein.ResultsTo generate the anti-RHT-D1A antibody, we expressed and purified soluble 6xHis-tagged RHT-D1A. The purified recombinant RHT-D1A was injected into New Zealand white rabbits to generate polyclonal antiserum. The polyclonal anti-RHT-D1A antibody was purified by ammonium sulfate precipitation, followed by affinity chromatography on protein A-agarose beads. The purified polyclonal antibody was demonstrated to be effective in immunoblotting, western blot hybridization, and immunoprecipitation. In wheat seedling extracts, the polyclonal antibody recognized a protein with a molecular mass close to the predicted molecular weight of the endogenous RHT-D1A protein. We also demonstrated that RHT-D1A disappears in response to exogenous and endogenous gibberellic acid.ConclusionThe purified polyclonal antibody raised against the recombinant RHT-D1A protein is sufficiently specific and sensitive and could be a useful tool for future insights into upstream and downstream components of DELLA-regulatory mechanisms in wheat plants.

Project description:Extracellular matrix glycoprotein tenascin-C (TnC) is highly expressed in vertebrates during embryonic development and thereafter transiently in tissue niches undergoing extensive remodeling during regeneration after injury. TnC's different functions can be attributed to its multimodular structure represented by distinct domains and alternatively spliced isoforms. Upon central nervous system injury, TnC is upregulated and secreted into the extracellular matrix mainly by astrocytes. The goal of the present study was to elucidate the role of different TnC domains in events that take place after spinal cord injury (SCI). Astrocyte cultures prepared from TnC-deficient (TnC-/-) and wild-type (TnC+/+) mice were scratched and treated with different recombinantly generated TnC fragments. Gap closure, cell proliferation and expression of GFAP and cytokines were determined in these cultures. Gap closure in vitro was found to be delayed by TnC fragments, an effect mainly mediated by decreasing proliferation of astrocytes. The most potent effects were observed with fragments FnD, FnA and their combination. TnC-/- astrocyte cultures exhibited higher GFAP protein and mRNA expression levels, regardless of the type of fragment used for treatment. Application of TnC fragments induced also pro-inflammatory cytokine production by astrocytes in vitro. In vivo, however, the addition of FnD or Fn(D+A) led to a difference between the two genotypes, with higher levels of GFAP expression in TnC+/+ mice. FnD treatment of injured TnC-/- mice increased the density of activated microglia/macrophages in the injury region, while overall cell proliferation in the injury site was not affected. We suggest that altogether these results may explain how the reaction of astrocytes is delayed while their localization is restricted to the border of the injury site to allow microglia/macrophages to form a lesion core during the first stages of glial scar formation, as mediated by TnC and, in particular, the alternatively spliced FnD domain.

Dataset Information

Biochemical Investigations of Five Recombinantly Expressed Tyrosinases Reveal Two Novel Mechanisms Impacting Carbon Storage in Wetland Ecosystems.

Publications

Biochemical Investigations of Five Recombinantly Expressed Tyrosinases Reveal Two Novel Mechanisms Impacting Carbon Storage in Wetland Ecosystems.

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