A natriuretic peptide from Arabidopsis thaliana (AtPNP-A) can modulate catalase 2 activity

Abstract

Analogues of vertebrate natriuretic peptides (NPs) present in plants, termed plant natriuretic peptides (PNPs), comprise a novel class of hormones that systemically affect salt and water balance and responses to plant pathogens. Several lines of evidence indicate that Arabidopsis thaliana PNP (AtPNP-A) affects cellular redox homeostasis, which is also typical for the signaling of its vertebrate analogues, but the molecular mechanism(s) of this effect remains elusive. Here we report identification of catalase 2 (CAT2), an antioxidant enzyme, as an interactor of AtPNP-A. The full-length AtPNP-A recombinant protein and the biologically active fragment of AtPNP-A bind specifically to CAT2 in surface plasmon resonance (SPR) analyses, while a biologically inactive scrambled peptide does not. In vivo bimolecular fluorescence complementation (BiFC) showed that CAT2 interacts with AtPNP-A in chloroplasts. Furthermore, CAT2 activity is lower in homozygous atpnp-a knockdown compared with wild type plants, and atpnp-a knockdown plants phenocopy CAT2-deficient plants in their sensitivity to elevated H₂O₂, which is consistent with a direct modulatory effect of the PNP on the activity of CAT2 and hence H₂O₂ homeostasis. Our work underlines the critical role of AtPNP-A in modulating the activity of CAT2 and highlights a mechanism of fine-tuning plant responses to adverse conditions by PNPs.

Figure 1

Biologically active peptide containing the active region of AtPNP-A protein binds to CAT2. (a) Domain organization of AtPNP-A and the amino acid sequences of C-terminally biotinylated peptides used in affinity chromatography-based experiments—a peptide containing the active site of AtPNP-A (indicated as pAtPNP-A) or the corresponding scrambled peptide (indicated as pScr). Cysteine residues forming a disulfide bond, characteristic to natriuretic peptide (NP)-like molecules, are underlined. SP, signal peptide. (b) Assaying biological activity of the AtPNP-A peptide (pAtPNP-A) and purified recombinant protein (rAtPNP-A). A. thaliana (Col-0) mesophyll cell protoplasts suspended in 0.4 M mannitol were treated with either water or 100 nM pScr (negative controls) or with 100 nM pAtPNP-A or with 1 μg mL⁻¹ of rAtPNP-A protein for 20 min at room temperature. In each treatment, 50 randomly selected protoplasts with diameter > 20 µm were included in quantitative analysis (scale bar = 20 µm). Protoplast volume was measured and the data obtained from an exemplar experiment are plotted. Columns with different superscript (a and b) indicate significantly different results (mean ± SD, one way ANOVA followed by Tukey–Kramer multiple comparison test, n = 50, P < 0.0001). (c) Exemplar MS/MS spectrum of a unique tryptic peptide of CAT2 (At4g35090) protein.

Figure 2

AtPNP-A directly interacts with Arabidopsis CAT2 and bovine liver CAT in vitro. (a) Molecular docking of AtPNP-A and CAT2. Surface model depicts predicted docking of AtPNP-A (blue), with its active region (cyan), and CAT2 monomer (tan). The structures of AtPNP-A as well as CAT2 monomer were predicted using the iterative threading assembly refinement (I-TASSER; version 5.1; https://zhanglab.ccmb.med.umich.edu/I-TASSER/) method. Protein–protein docking was performed using ClusPro (version 2.0; https://cluspro.bu.edu/publications.php). The models were analyzed and visualized using UCSF Chimera (version 1.10.2; https://www.cgl.ucsf.edu/chimera/). (b) Exemplar sensorgrams depicting referenced binding response of pAtPNP-A or pScr with CAT2 recombinant protein immobilized on the active surface of the NTA sensor chip. Reference surface of the NTA chip was not modified, according to the manufacturer’s instructions, and did not carry the recombinant protein. In both analyses the ligand was immobilized at the same level (app. 4,500 RU), analytes are injected at the same concentration and conditions of runs kept constant. (c) Exemplar sensorgrams depicting referenced binding response in kinetic analysis of binding between pAtPNP-A (3.78 μM and consecutive two-fold dilutions, as in Supplementary Table S2) and bovine liver CAT immobilized on the active surface of the CM5 sensor chip. Reference surface of the NTA chip was not modified, according to the manufacturer’s instructions, and did not carry any protein.

Figure 3

AtPNP-A directly interacts with CAT2 to modulate its enzymatic activity in vitro. (a) Zymogram depicting changes in the enzymatic activity of CAT isoforms extracted from wild type (WT) or cat2-2 knockout mutant seedlings in response to 1 nM pAtPNP-A or pScr. Densitometric semi-quantification of bands corresponding to CAT2, normalized to the loading control (dark-coloured band on zymogram) for WT samples treated with 1 nM pAtPNP-A or pScr (mean ± SD, Student’s t-test, n = 3, P < 0.05). Different superscript (a and b) indicates significantly different results. (b) Total CAT activity in protein extracted from WT seedlings assayed with Amplex Red catalase assay kit in the presence of 1 nM pAtPNP-A, rAtPNP-A, or pScr. Different superscript (a, b, and c) indicates significantly different results (mean ± SD, one-way ANOVA, followed by Tukey–Kramer multiple comparison test, n = 3, P < 0.05). (c) Enzymatic activity of rCAT2 in the presence of 100 nM pAtPNP-A, rAtPNP-A, or pScr. Different superscript (a and b) indicates significantly different results from three independent experiments (mean ± SD, one-way ANOVA followed by Tukey–Kramer multiple comparison test, n = 3, P < 0.01).

Figure 4

AtPNP-A interacts with CAT2 in vivo. (a)–(f) BiFC reveals AtPNP-A (indicated as P) and CAT2 (indicated as C) interact in the chloroplasts of tobacco leaf protoplasts. Exemplar merged images of protoplasts isolated from a leaf infiltrated with different combinations of N- or C-terminally tagged CAT2 and AtPNP-A are shown. (g) BiFC/Red fluorescence chloroplast analysis with mean ± SEM in pink. Normalized BiFC fluorescence is significantly higher in chloroplasts from protoplasts expressing NeYFP-CAT2, indicated as NY-C, and CeYFP-AtPNP-A, indicated as CY-P (mean ± SEM, one-way ANOVA followed by Sidak’s post-hoc test, n = 30, ****P < 0.0001).

Figure 5

The atpnp-a mutant plants phenocopy CAT2-deficient plants in their ability to cope with H₂O₂ stress. (a) Total CAT enzymatic activities in equal amounts of protein extracted from leaves of 4 week-old WT and atpnp-a mutant seedlings. The graph shows data from three independent experiments (mean ± SD, Student’s t-test, n = 3, * P = 0.0409). (b) Zymogram of CAT isoform activities in protein extracts from atpnp-a or WT seedlings separated in 8% native PAGE and stained specifically for CAT activity. Coomassie brilliant blue (CBB) shows equal loading. Full-length gels are presented in Supplementary Fig. S5a. (c) Germination of WT, cat2-2, and atpnp-a seeds 14 days after sowing on MS agar supplemented with 3 mM H₂O₂. (d) Quantification of germination (as shown in section (c)) by the presence of green cotyledons. The graph shows data from three independent experiments (mean ± SD, one-way ANOVA followed by Tukey–Kramer multiple comparison test, n = 300, ** P < 0.01, *** P < 0.001).