ROS Consumers or Producers? Interpreting Transcriptomic Data by AlphaFold Modeling Provides Insights into Class III Peroxidase Functions in Response to Biotic and Abiotic Stresses

Abstract

Participating in both biotic and abiotic stress responses, plant-specific class III peroxidases (PERs) show promise as candidates for crop improvement. The multigenic PER family is known to take part in diverse functions, such as lignin formation and defense against pathogens. Traditionally linked to hydrogen peroxide (H₂O₂) consumption, PERs can also produce reactive oxygen species (ROS), essential in tissue development, pathogen defense and stress signaling. The amino acid sequences of both orthologues and paralogues of PERs are highly conserved, but discovering correlations between sequence differences and their functional diversity has proven difficult. By combining meta-analysis of transcriptomic data and sequence alignments, we discovered a correlation between three key amino acid positions and gene expression in response to biotic and abiotic stresses. Phylogenetic analysis revealed evolutionary pressure on these amino acids toward stress responsiveness. Using AlphaFold modeling, we found unique interdomain and protein-heme interactions involving those key amino acids in stress-induced PERs. Plausibly, these structural interactions may act as "gate keepers" by preventing larger substrates from accessing the heme and thereby shifting PER function from consumption to the production of ROS.

Keywords: AlphaFold; Arabidopsis; ROS signaling; class III peroxidases; phosphate deficiency; plant stress response.

Figure 1

(a–c) Hierarchically clustered heatmaps generated with Genevestigator reveal differential gene expression of Arabidopsis PERs. Upregulated genes are indicated by red, and downregulated by green boxes. Pairs of letters above each gene indicate amino acids in two positions as explained in the text. (a) Root response to P_i deficiency, showing an almost opposite pattern of differential gene expression in short-term (1 h, 6 h, and 24 h; top 3 rows), compared to long-term P_i deficiency (bottom row). (b) Root responses to various pathogens (biotic stresses). Biotic stresses include infection with Sclerotinia sclerotiorum, Alternaria brassicicola, Plectosphaerella cucumerina, Phytophthora infestans, Pseudomonas syringae, Liriomyza huidobrensis, and Hyaloperonospora arabidopsidis. (c) Root responses to salt stress. (d) Venn diagrams visualizing shared upregulated and downregulated genes in response to biotic stresses, short-term P_i deficiency, and salt stress.

Figure 2

(a) MSA of common stress-upregulated (top 13 sequences) and downregulated (bottom 16 sequences) PER amino acid sequences reveals a difference between both groups at the two Alpha Buttons. The amino acid position is shaded in red for upregulated and green for downregulated PER genes. In cyan, three highly conserved active site residues are also indicated. (b) The same MSA revealed another conserved difference at the end of the first of two beta strands, at a position we termed the Beta Button, shaded as for the Alpha Buttons. (c) Support for the significance of the two Alpha Buttons: a position-specific scoring matrix, part of an InterProScan search, revealed both Alpha Button positions (marked by a black rectangle) as likely having a role in the active site and in heme binding.

Figure 3

Maximum likelihood tree based on the 73 PER coding RNA sequences. The four main clades are distinguished by color and labeled 1−4. Approximate likelihood-ratio values are shown in red near the corresponding nodes. The particular amino acid at the Alpha1 and Alpha2 Buttons and the Beta Button are noted at the end of each protein name, using the single letter code. Note that Supplementary Figure S2 shows all three buttons in a rectangular phylogenetic tree for better legibility. The insert shows gene expression as log2 fold change under short-term (24 h) P_i deficiency, with ±0.5 log2 fold differentially expressed genes, highlighted by color shading. Circled in magenta are PER62 and PER71, which we postulate have convergently evolved to function like members of clade 3.

Figure 4

(a) Cartoon view of PER53 based on the crystal structure [17], with bound heme (ball and stick model) located between the distal domain (red), the proximal domain (blue), and the beta domain (yellow and green). The heme propionate groups, each ending with a pair of oxygen atoms (red balls), emerge from the γ-edge of the heme (to the right in the side view, toward the viewer in the front view). (b) “Front” views into the “mouth” of PER AlphaFold models and PER53 crystal structure with coloring and orientation as in Figure 4a (black rectangle), except here the green highlights are Alpha1 (α1), Alpha2 (α2) and Beta (β) Button residues (indicated by arrows). In the left column (PER53 and PER33,) Alpha2 is occluded from view, and the mouth is less open. In the right column (PER01 and PER44), the mouth is more open, as evidenced by more of the bound heme being visible. We hypothesize that the less open mouth restricts access to the heme for larger substrates while allowing access for small molecules such as H₂O₂, H₂O, •OH, O₂•⁻, etc.