Microbial impact to environmental toxicants Ni(II) and Co(II): Joint toxicity and cellular response in Paramecium.

Cobalt (Co) and Nickel (Ni) are increasingly found in our environment. We analysed their combined toxicity and uptake mechanisms in the early food chain by studying bacteria and the bacterivorous ciliate Paramecium as a primary consumer. We exposed both species to these metals to measure the toxicity, uptake and transfer of metals from bacteria to Paramecium. We found that Ni is more toxic than Co, and that toxicity increases for both metals when (i) food bacteria are absent and (ii) both metals are applied in combination. The cellular content in bacteria after exposure shows a concentration dependent bias for either Ni or Co. Comparing single treatment and joint exposure, bacteria show increased levels of both metals when these are both exposed. To imitate the basic level of the food chain, we fed these bacteria to paramecia. The cellular content shows a similar ratio of Nickel and Cobalt as in food bacteria. This is different to the direct application of both metals to paramecia, where Cobalt is enriched over Nickel. This indicates that bacteria can selectively pre-accumulate metals for introduction into the food chain. We also analysed the transcriptomic response of Paramecium to sublethal doses of Nickel and Cobalt to gain insight into their toxicity mechanisms. Gene ontology (GO) analysis indicates common deregulated pathways, such as ammonium transmembrane transport and ubiquitine-associated protein degradation. Many redox-related genes also show deregulation of gene expression, indicating cellular adaptation to increased RONS stress. This suggests that both metals may also target the same cellular pathways and this is consistent with the increased toxicity of both metals when used together. Our data reveal complex ecotoxicological pathways for these metals and highlights the different parameters for their fate in the ecosystem, in the food chain and their ecotoxicological risk after environmental contamination.

Nucleotide Limitation Results in Impaired Photosynthesis, Reduced Growth and Seed Yield Together with Massively Altered Gene Expression.

Nucleotide limitation and imbalance is a well-described phenomenon in animal research but understudied in the plant field. A peculiarity of pyrimidine de novo synthesis in plants is the complex subcellular organization. Here, we studied two organellar localized enzymes in the pathway, with chloroplast aspartate transcarbamoylase (ATC) and mitochondrial dihydroorotate dehydrogenase (DHODH). ATC knock-downs were most severely affected, exhibiting low levels of pyrimidine nucleotides, a low energy state, reduced photosynthetic capacity and accumulation of reactive oxygen species. Furthermore, altered leaf morphology and chloroplast ultrastructure were observed in ATC mutants. Although less affected, DHODH knock-down mutants showed impaired seed germination and altered mitochondrial ultrastructure. Thus, DHODH might not only be regulated by respiration but also exert a regulatory function on this process. Transcriptome analysis of an ATC-amiRNA line revealed massive alterations in gene expression with central metabolic pathways being downregulated and stress response and RNA-related pathways being upregulated. In addition, genes involved in central carbon metabolism, intracellular transport and respiration were markedly downregulated in ATC mutants, being most likely responsible for the observed impaired growth. We conclude that impairment of the first committed step in pyrimidine metabolism, catalyzed by ATC, leads to nucleotide limitation and by this has far-reaching consequences on metabolism and gene expression. DHODH might closely interact with mitochondrial respiration, as seen in delayed germination, which is the reason for its localization in this organelle.

Nucleotide Imbalance, Provoked by Downregulation of Aspartate Transcarbamoylase Impairs Cold Acclimation in Arabidopsis.

Aspartate transcarbamoylase (ATC) catalyzes the first committed step in pyrimidine de novo synthesis. As shown before, mutants with 80% reduced transcript and protein levels exhibit reduced levels of pyrimidine metabolites and thus nucleotide limitation and imbalance. Consequently, reduced photosynthetic capacity and growth, accompanied by massive transcriptional changes, were observed. Here, we show that nucleotide de novo synthesis was upregulated during cold acclimation of Arabidopsis thaliana (ecotype Columbia, Col-0) plants, but ATC knockdown mutants failed to acclimate to this condition as they did not accumulate neutral sugars and anthocyanins. A global transcriptome analysis revealed that most of the transcriptional changes observed in Col-0 plants upon cold exposure were also evident in ATC knockdown plants. However, several responses observed in cold-treated Col-0 plants could already be detected in knockdown plants when grown under standard conditions, suggesting that these mutants exhibited typical cold responses without prior cold stimulation. We believe that nucleotide signaling is involved in "cold-like priming" and "cold acclimation" in general. The observed transcript levels of genes involved in central carbon metabolism and respiration were an exception to these findings. These were upregulated in the cold but downregulated in warm-grown ATC mutants.