Iron sequestration in brown-rot fungi by oxalate and the production of reactive oxygen species (ROS)

ABSTRACT Fungi dominate the recycling of carbon sequestered in woody biomass. This process of organic turnover was first evolved among “white rot” fungi that degrade lignin to access carbohydrates and later evolved multiple times toward more efficient strategies to selectively target carbohydrates—“brown rot.” The brown rot adaption was often explained by mechanisms to deploy reactive oxygen species (ROS) to oxidatively attack wood structures. However, its genetic basis remains unclear, especially in the context of gene contractions of conventional carbohydrate-active enzymes (CAZYs) relative to white rot ancestors. Here, we hypothesized that these apparent gains in brown rot efficiency despite gene losses were due, in part, to upregulation of the retained genes. We applied comparative transcriptomics to multiple species of both rot types grown across a wood wafer to create a gradient of progressive decay and to enable tracking temporal gene expression. Dozens of “decay-stage-dependent” ortho-genes were isolated, narrowing a pool of candidate genes with time-dependent regulation unique to brown rot fungi. A broad comparison of the expression timing of CAZY families indicated a temporal regulatory shift of lignocellulose-oxidizing genes toward early stages in brown rot compared to white rot, enabling the segregation of oxidative treatment ahead of hydrolysis. These key brown rot ROS-generating genes with iron ion binding functions were isolated. Moreover, transcription energy was shifted to be invested on the retained GHs in brown rot fungi to strengthen carbohydrate conversion. Collectively, these results support the hypothesis that gene regulation shifts played a pivotal role in brown rot adaptation. IMPORTANCE Fungi dominate the turnover of wood, Earth’s largest pool of aboveground terrestrial carbon. Fungi first evolved this capacity by degrading lignin to access and hydrolyze embedded carbohydrates (white rot). Multiple lineages, however, adapted faster reactive oxygen species (ROS) pretreatments to loosen lignocellulose and selectively extract sugars (brown rot). This brown rot “shortcut” often coincided with losses (>60%) of conventional lignocellulolytic genes, implying that ROS adaptations supplanted conventional pathways. We used comparative transcriptomics to further pursue brown rot adaptations, which illuminated the clear temporal expression shift of ROS genes, as well as the shift toward synthesizing more GHs in brown rot relative to white rot. These imply that gene regulatory shifts, not simply ROS innovations, were key to brown rot fungal evolution. These results not only reveal an important biological shift among these unique fungi, but they may also illuminate a trait that restricts brown rot fungi to certain ecological niches.

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Oxidative Damage Control during Decay of Wood by Brown Rot Fungus Using Oxygen Radicals

Applied and Environmental Microbiology ◽

10.1128/aem.01937-18 ◽

2018 ◽

Vol 84 (22) ◽

Cited By ~ 3

Author(s):

Jesus D. Castaño ◽

Jiwei Zhang ◽

Claire E. Anderson ◽

Jonathan S. Schilling

Keyword(s):

Reactive Oxygen Species ◽

Cell Walls ◽

Reactive Oxygen ◽

Brown Rot ◽

White Rot ◽

Ros Generation ◽

Oxygen Species ◽

Content Type ◽

Brown Rot Fungi ◽

Brown Rot Fungus

ABSTRACTBrown rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that brown rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that brown rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grewPostia placentadirectionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that H2O2and Fe2+concentrations in the extracellular environment were conducive to HO˙ production in early (H2O2:Fe2+ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producerTrichoderma reesei. Collectively, this indicates thatP. placentauses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood.IMPORTANCEWood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white rot) or by using Fenton-generated reactive oxygen species (ROS) to “loosen” wood cell walls, enabling selective sugar extraction (brown rot). Although white rot lignin-degrading pathways are well characterized, there are many uncertainties in brown rot fungal mechanisms. Our study addressed a key uncertainty in how brown rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (H2O2, Fe2+), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.

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Laccase and Its Role in Production of Extracellular Reactive Oxygen Species during Wood Decay by the Brown Rot Basidiomycete Postia placenta

Applied and Environmental Microbiology ◽

10.1128/aem.02929-09 ◽

2010 ◽

Vol 76 (7) ◽

pp. 2091-2097 ◽

Cited By ~ 69

Author(s):

Dongsheng Wei ◽

Carl J. Houtman ◽

Alexander N. Kapich ◽

Christopher G. Hunt ◽

Daniel Cullen ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Protein Identification ◽

Reactive Oxygen ◽

Wood Decay ◽

Brown Rot ◽

Order Rate Constant ◽

Spectrometric Analysis ◽

Alternative Mechanism ◽

Oxygen Species ◽

Postia Placenta

ABSTRACT Brown rot basidiomycetes initiate wood decay by producing extracellular reactive oxygen species that depolymerize the structural polysaccharides of lignocellulose. Secreted fungal hydroquinones are considered one contributor because they have been shown to reduce Fe3+, thus generating perhydroxyl radicals and Fe2+, which subsequently react further to produce biodegradative hydroxyl radicals. However, many brown rot fungi also secrete high levels of oxalate, which chelates Fe3+ tightly, making it unreactive with hydroquinones. For hydroquinone-driven hydroxyl radical production to contribute in this environment, an alternative mechanism to oxidize hydroquinones is required. We show here that aspen wood undergoing decay by the oxalate producer Postia placenta contained both 2,5-dimethoxyhydroquinone and laccase activity. Mass spectrometric analysis of proteins extracted from the wood identified a putative laccase (Joint Genome Institute P. placenta protein identification number 111314), and heterologous expression of the corresponding gene confirmed this assignment. Ultrafiltration experiments with liquid pressed from the biodegrading wood showed that a high-molecular-weight component was required for it to oxidize 2,5-dimethoxyhydroquinone rapidly and that this component was replaceable by P. placenta laccase. The purified laccase oxidized 2,5-dimethoxyhydroquinone with a second-order rate constant near 104 M−1 s−1, and measurements of the H2O2 produced indicated that approximately one perhydroxyl radical was generated per hydroquinone supplied. Using these values and a previously developed computer model, we estimate that the quantity of reactive oxygen species produced by P. placenta laccase in wood is large enough that it likely contributes to incipient decay.

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A Fungal Secretome Adapted for Stress Enabled a Radical Wood Decay Mechanism

mBio ◽

10.1128/mbio.02040-21 ◽

2021 ◽

Author(s):

Jesus Castaño ◽

Jiwei Zhang ◽

Mowei Zhou ◽

Chia-Feng Tsai ◽

Joon Yong Lee ◽

...

Keyword(s):

Reactive Oxygen Species ◽

Cell Walls ◽

Critical Role ◽

Wood Decay ◽

Brown Rot ◽

Oxygen Species ◽

Decay Mechanism ◽

Brown Rot Fungi ◽

Carbon Recycling ◽

Fungal Secretome

Brown rot fungi play a critical role in carbon recycling and are of industrial interest. These fungi typically use reactive oxygen species (ROS) to indiscriminately “loosen” wood cell walls at the outset of decay.

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Significant levels of extracellular reactive oxygen species produced by brown rot basidiomycetes on cellulose

FEBS Letters ◽

10.1016/s0014-5793(02)03589-5 ◽

2002 ◽

Vol 531 (3) ◽

pp. 483-488 ◽

Cited By ~ 60

Author(s):

Roni Cohen ◽

Kenneth A Jensen ◽

Carl J Houtman ◽

Kenneth E Hammel

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Brown Rot ◽

Oxygen Species

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Mitochondria as a source of reactive oxygen species

Biochemical Journal ◽

10.1042/bj20090056 ◽

2009 ◽

pp. c3 ◽

Cited By ~ 1

Author(s):

Helena M. Cochemé ◽

Michael P. Murphy

Keyword(s):

Reactive Oxygen Species ◽

Reactive Oxygen ◽

Oxygen Species

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Clinical aspects of reactive oxygen and nitrogen species

Biochemical Society Symposium ◽

10.1042/bss0710121 ◽

2004 ◽

Vol 71 ◽

pp. 121-133 ◽

Cited By ~ 25

Author(s):

Ascan Warnholtz ◽

Maria Wendt ◽

Michael August ◽

Thomas Münzel

Keyword(s):

Oxidative Stress ◽

Reactive Oxygen Species ◽

Risk Factor ◽

Endothelial Dysfunction ◽

Vascular Tissue ◽

Rapid Development ◽

Reactive Oxygen ◽

Clinical Aspects ◽

No Production ◽

Oxygen Species

Endothelial dysfunction in the setting of cardiovascular risk factors, such as hypercholesterolaemia, hypertension, diabetes mellitus and chronic smoking, as well as in the setting of heart failure, has been shown to be at least partly dependent on the production of reactive oxygen species in endothelial and/or smooth muscle cells and the adventitia, and the subsequent decrease in vascular bioavailability of NO. Superoxide-producing enzymes involved in increased oxidative stress within vascular tissue include NAD(P)H-oxidase, xanthine oxidase and endothelial nitric oxide synthase in an uncoupled state. Recent studies indicate that endothelial dysfunction of peripheral and coronary resistance and conductance vessels represents a strong and independent risk factor for future cardiovascular events. Ways to reduce endothelial dysfunction include risk-factor modification and treatment with substances that have been shown to reduce oxidative stress and, simultaneously, to stimulate endothelial NO production, such as inhibitors of angiotensin-converting enzyme or the statins. In contrast, in conditions where increased production of reactive oxygen species, such as superoxide, in vascular tissue is established, treatment with NO, e.g. via administration of nitroglycerin, results in a rapid development of endothelial dysfunction, which may worsen the prognosis in patients with established coronary artery disease.

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