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Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies - PubMed

  • ️Fri Jan 01 2021

Proteomics reveals synergy between biomass degrading enzymes and inorganic Fenton chemistry in leaf-cutting ant colonies

Morten Schiøtt et al. Elife. 2021.

Abstract

The symbiotic partnership between leaf-cutting ants and fungal cultivars processes plant biomass via ant fecal fluid mixed with chewed plant substrate before fungal degradation. Here we present a full proteome of the fecal fluid of Acromyrmex leaf-cutting ants, showing that most proteins function as biomass degrading enzymes and that ca. 85% are produced by the fungus and ingested, but not digested, by the ants. Hydrogen peroxide producing oxidoreductases were remarkably common in the proteome, inspiring us to test a scenario in which hydrogen peroxide reacts with iron to form reactive oxygen radicals after which oxidized iron is reduced by other fecal-fluid enzymes. Our biochemical assays confirmed that these so-called Fenton reactions do indeed take place in special substrate pellets, presumably to degrade plant cell wall polymers. This implies that the symbiotic partnership manages a combination of oxidative and enzymatic biomass degradation, an achievement that surpasses current human bioconversion technology.

Keywords: Acromyrmex echinatior; GMC oxidoreductases; ecology; evolutionary biology; hydroxyl radicals; symbiosis.

Plain language summary

Colonies of tropical leaf-cutting ants live in underground nests where a fungus grows that feeds them. The ants, in turn, provide the fungus with the freshly-cut leaf fragments it needs for nutrition. The relationship between the ants and the fungus, in which they live close together and help one another survive, is known as symbiosis. It is an ancient, extremely well integrated relationship, in which neither species can survive without the other. However, the details of how the ants and the fungus work together to break down the leaf fragments so they can be used for nutrition are not well understood. When the ants eat the fungus, they do not digest its enzymes (the proteins that accelerate chemical reactions in a cell). Instead, the fungal enzymes travel through the ants’ gut and into their fecal liquid, which gets deposited on the fresh-cut leaves when the ants collect them. The ants then make temporary pellets out of the new leaf fragments before providing them to the fungus. To better understand how each species contributes to the breakdown of the leaf fragments, Schiøtt and Boomsma identified all the proteins present in the fecal fluid of the ants. Once they had a complete list of about 100 proteins, they determined which of them were produced by the fungus and which by the ant. Schiøtt and Boomsma observed that certain combinations of fungal and ant enzymes could trigger a Fenton reaction – a chemical reaction that efficiently begins the breakdown of the tough walls around plant cells. This reaction is so aggressive that it is rarely found in nature, but it could help explain the high efficiency of the fungus and the ants symbiotically processing leaf fragments. But could a Fenton reaction actually proceed in the ants’ nest without hurting the ants or affecting the rest of the fungal garden? The evidence obtained suggested that the temporary pellets made by the ants serve to isolate the reaction, so the aggressive chemistry takes place away from the ants and detached from the fungal gardens. Schiøtt and Boomsma showed that the symbiotic relationship between the ants and the fungus has led to a sustainable and efficient way of breaking down plant materials to use them for nutrition. The Fenton reaction is economically important in many industries, including bioethanol production, the detergent industry, and food production. Emulating the methods used by leaf-cutting ants, which have been fine-tuned by millions of years of natural selection, may allow humans to develop more efficient technologies for breaking down organic compounds.

© 2021, Schiøtt and Boomsma.

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Conflict of interest statement

MS, JB No competing interests declared

Figures

Figure 1.
Figure 1.. Statistics of the fecal fluid proteome of Acromyrmex echinatior.

(A) Venn diagrams, constructed using the web application Venny 2.1 (

http://bioinfogp.cnb.csic.es/tools/venny/index.html

) showing the overlap of protein profiles identified in the four fecal fluid samples obtained from colonies Ae263, Ae322, Ae356, and Ae372. (B) Pie chart showing the abundances of proteins across colonies assigned to six functional categories based on the label-free quantification (LFQ) values provided by MaxQuant.

Figure 2.
Figure 2.. Hypothesized reaction scheme for the generation of hydroxyl radicals when ant fecal fluid interacts with chewed leaf fragments in temporary substrate pellets, based on the presence of enzymes in the total fecal fluid proteome (Supplementary file 1).

Fungal enzymes produced in gongylidia of the symbiotic garden-cultivar that the ants ingest pass unharmed through the gut to end up in the fecal fluid (Boyd and Martin, 1975b; Kooij et al., 2014b; Schiøtt et al., 2010; De Fine Licht et al., 2013; Kooij et al., 2016). After droplets of fecal fluid are deposited and become exposed to oxygen, the fungal oxidoreductases produce hydrogen peroxide while aryl alcohols are converted to aryl aldehydes. The hydrogen peroxide then reacts with reduced iron (Fe2+) to produce hydroxyl radicals (OH•) in a Fenton reaction, which aggressively breaks down cell walls of the plant substrate. Oxidized iron (Fe3+) can then be reduced again by ant-encoded glucose dehydrogenase, using glucose released via plant cell wall decomposition as electron donor. The leaf substrate is initially concentrated in green pellets of ca. 3 mm diameter distributed across the top of fungus gardens, which turn black in a few hours when subjected to Fenton-mediated degradation (inset image). Compounds ultimately derived from the fungal symbiont are in red text and compounds directly produced by the ants in blue.

Figure 3.
Figure 3.. Gene tree of the five fungal glucose-methanol-choline (GMC) oxidoreductases identified in the ant fecal fluid (present study) and in representative other basidomycete fungi, all assigned to functional groups based on a previous study (Ferreira et al., 2015).

All fungal GMC oxidoreductases from the fecal fluid (Protein IDs 9, 23, 86, 178, and 191 in Table 1; red text) clustered among the known aryl-alcohol oxidases. Other closely related functional groups are glucose oxidases, methanol oxidases, pyranose-2 oxidases, and cellulose dehydrogenases, which were retrieved in Phanerochaete chrysosporium (PHACH), Rhodonia placenta (RHOPL), Trametes versicolor (TRAVE), and Phlebia brevispora (PHLBR), but not in fecal fluid of the leaf-cutting ant A. echinatior. Note that the ant-encoded glucose dehydrogenase is also a GMC oxidoreductase, but would sequence-wise not fit into this phylogeny of fungal proteins. Numbers are aLRT SH-like support values for nodes. The scale bar represents 0.5 substitutions per site.

Figure 4.
Figure 4.. Bioassays to demonstrate that inorganic.

Fenton chemistry must be taking place when ant fecal fluid is exposed to oxygen while being deposited on leaf pulp pellets chewed by the ants, testing the key interactions hypothesized in Figure 2. (A) Bar plot showing the concentrations (means ± SE across six colonies) of hydrogen peroxide in fecal fluid after adding potential substrates (glucose, methanol, glyoxal, or veratryl alcohol) of glucose-methanol-choline (GMC) oxidoreductases with or without the hydrogen peroxide degrading enzyme catalase. One-way ANOVA showed a highly significant overall effect of treatments: F6,36 = 14.597, p=2.863e-08, and pairwise post hoc t-tests on matching samples from the same ant colonies (corrected for multiple testing with the Holm–Bonferroni method) confirmed both the enhancing effects of veratryl alcohol and glyoxal and the inhibiting effect of catalase (p-values in plot unless non-significant) (NS). (B) Deoxyribose assays showing that ant fecal fluid has the capacity to produce hydroxyl radicals (means ± SE across six colonies). Phenanthroline is known to work as an iron chelator and significantly reduced degradation of 2-deoxy-D-ribose while the solvent (methanol) of phenanthroline did not. Paired t-tests followed the same protocol as in the A-panel except that the overall ANOVA was omitted because there were only three means to compare. (C) A Ferrozine assay (means ± SE across six colonies) showing the capacity of ant fecal fluid to reduce Fe3+ to Fe2+, confirming that addition of glucose increases the rate of iron reduction. Statistics as in the B-panel.

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