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Iron-dependent reprogramming of damage-associated peptide receptor signaling coordinates immunity and phosphate stress adaptation

Natsuki Tsuchida, Tae‐Hong Lee, Maxmilyand Leiwakabessy, Kota YamashitaiD, Kei HirumaiD, Kentaro Okada, Taishi Hirase, Shigetaka YasudaiD, Yuniar Devi UtamiiD, Salvatore CosentinoiD, Min Fey ChekiD, H ArigaiD, Miki FujitaiD, Taishi UmezawaiD, Yusuke SaijoiD

Abstract

Plant adaptation to inorganic phosphate (Pi) limitation entails extensive developmental and metabolic reprogramming, collectively termed the phosphate starvation response (PSR). How plants balance nutrient acquisition with immune competence remains unresolved. Here, we show that PSR selectively remodels pattern-recognition receptor (PRR) signaling in Arabidopsis thaliana. Membrane proteomics reveals increased abundance of the Pep receptor PEPR1 under Pi deficiency, whereas most receptor kinases, including the microbial PRRs FLS2 and CERK1, are reduced. Immunoblotting further shows increased PEPR2 abundance. Consistently, Pep-triggered responses are strongly potentiated under Pi depletion, while flagellin- and chitin-induced responses are not, indicating selective PEPR sensitization rather than global immune suppression. This sensitization requires the PSR-associated ferroxidases LPR1/LPR2, NADPH oxidases RBOHD/RBOHF, and transcription factor WRKY33, linking iron-dependent redox remodeling to immune amplification. Beyond inducible defense, PEPRs promote basal PSR transcriptional reprogramming and growth under Pi limitation. Loss of PEPR1/PEPR2 compromises bacterial resistance and alters root-associated microbiota assembly under nutrient deficiency. Together, PEPRs exhibit context-dependent dual functionality, promoting either immunity or nutrient stress adaptation, potentially in response to distinct PROPEP-derived ligands, thereby supporting nutrient acquisition without compromising immune vigilance. These findings provide a framework for how nutritional status selectively remodels PRR signaling to coordinate immunity with nutrient stress adaptation.

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Claude Opus 5.5 · xhigh effort · 8 Nov 2026 · from the full text of version 2 (25 Aug 2026) · CC BY 4.0 · version 3 was posted after this summary

Phosphate starvation in Arabidopsis potentiates Pep-PEPR signaling, but not flagellin or chitin responses, via LPR1/LPR2 iron-redox, RBOHD/RBOHF and WRKY33; basal PEPR activity also supports phosphate-starvation responses.

Earlier studies found phosphate starvation attenuates FLS2 immunity through PHR1 or PHT1, read as a global nutrient-immunity trade-off, leaving open how plants keep pathogen resistance while adapting to low phosphate. This work instead shows receptor- and output-selective remodeling of pattern-recognition signaling and casts PEPR receptors as dual-function hubs for defense and nutrient adaptation, tuned by iron and PROPEP-derived forms.

Key results

  1. ShownLow phosphate reshapes the plasma membrane receptor landscape selectively: PEPR1 and RLK7 are kept or raised and PEPR2 rises, while FLS2, CERK1 and many other receptor kinases decline, a pattern not explained by transcript levels.

    Label-free proteomics of seedlings at 10 µM vs 625 µM Pi identified 116 plasma membrane receptors (n = 3; Student's t-test, *p < 0.05, ***p < 0.0001); immunoblots confirmed FLS2 decrease and PEPR2-FLAG increase (n = 3-4); PEPR1, PEPR2, FLS2 and CERK1 mRNAs all rose at low Pi; only PEPR1 has a PHR1-binding site. · Pi deficiency differentially alters the…; Figure legends

  2. ShownPhosphate deficiency potentiates Pep1 signaling in an output-selective way: MAPK activation and PEPR-dependent transcription are amplified, whereas the ROS burst and PEPR1-BAK1 complex formation are unchanged, and chitin, flg22 and SCOOP12 responses are not enhanced.

    Among the top 150 genes per elicitor, many Pep1-induced genes rose further at low Pi while most chitin genes were attenuated; 1,511 (+Pi) and 1,288 (-Pi) PEPR-dependent Pep1 genes (log2FC > 1, FDR < 0.05) showed larger induction at low Pi; CYP71A12/PDF1.2a enhanced (n = 3, **p < 0.01, ***p < 0.0001); persisted in bak1-4; MAPK phosphorylation stronger (n = 2-4). · Pi deficiency potentiates PEPR signaling through…; External Pi signaling via LPR1/LPR2 promotes…; Figure legends

  3. ShownImmune potentiation depends on an iron-redox module: LPR1/LPR2 ferroxidases, RBOHD/RBOHF NADPH oxidases and WRKY33 are required, excess iron or acidic pH boosts Pep1 responses, and LPR1/LPR2-dependent genes are enriched for jasmonic acid pathways.

    Excess Fe enhanced and Fe deficiency attenuated Pep1-induced CYP71A12/PDF1.2a and MAPK at low Pi, not flg22 or chitin; lpr1 lpr2, rbohD rbohF and wrky33 reduced Pep1 marker induction (n = 3); phr1 phl1 effects mostly at 2 h, lpr1 lpr2 persisted at 10 h; WRKY33/WRKY25 motifs enriched; low Pi plus excess Fe boosted Pep1- but not flg22-induced WRKY33-HA. · External Pi signaling via LPR1/LPR2 promotes…; LPR1/LPR2-dependent iron-ROS signaling…; WRKY33 contributes to PEPR-dependent immune…; Figure legends

  4. ShownWithout exogenous Pep1, basal PEPR signaling supports the phosphate starvation response: pepr1 pepr2 shows weaker induction of starvation genes, including PHR1/PHL1 targets for Pi uptake, and PEPR overexpression increases shoot biomass at low Pi.

    RNA-seq showed weaker induction of the low Pi-induced gene cluster in non-elicited pepr1 pepr2 than WT; WT and pepr1 pepr2 shoot biomass were similar, but PEPR1-FLAG or PEPR2-FLAG in pepr1 pepr2 raised shoot fresh weight after 10 days at 10 µM Pi (n = 12; Tukey p < 0.05); exogenous Pep1 instead suppressed these genes. · PEPR1/PEPR2 promote PSR and shape root…; Figure legends

  5. ShownFull-length PROPEP6, which resists processing into a mature Pep, binds PEPR1 and acts as a reversible, PEPR1-selective brake on Pep1 signaling, while endogenous PROPEPs restrain immunity and contribute to low-Pi root growth inhibition and PHT1;4 induction.

    PROPEP6 suppressed Pep1-induced ROS in N. benthamiana (n = 8) and recombinant PROPEP6 did so in WT but not pepr1 pepr2 leaves; it reduced PEPR1-BAK1 but not PEPR2-BAK1 association, overcome by high Pep1; higher-order propep mutants, but not propep6, had stronger Pep1 responses; propep6 eased root inhibition like the octuple mutant. · Non-canonical PROPEP-derived forms modulate PEPR…; Figure legends

Methods, data and reproducibility

Label-free plasma membrane proteomics of Arabidopsis seedlings at 625 µM vs 10 µM Pi, plus root RNA-seq and qRT-PCR after Pep1, flg22, chitin and other peptide elicitors in PSR, iron-redox and signaling mutants; MAPK, ROS and co-IP assays, N. benthamiana transient expression, CRISPR propep mutants, Pto DC3000 infection and root 16S rRNA profiling.

  • Plasma membrane-enriched fractions of Col-0 seedlings grown at 625 µM vs 10 µM Pi, label-free LC-MS/MS (116 receptor proteins identified) · Arabidopsis thaliana · n = 3 biological replicates · jPOST preview 1230333443699fda85f12b0 (access key 7188)
  • Root RNA-seq after Pep1 or chitin treatment under Pi-sufficient and Pi-deficient conditions · Arabidopsis thaliana · DDBJ PRJDB37679 / PRJDB35626 (dataset-to-experiment mapping not stated)
  • Root RNA-seq time course (2 h and 10 h Pep1) in WT, phr1 phl1, lpr1 lpr2 and pepr1 pepr2 seedlings at +Pi and -Pi · Arabidopsis thaliana · DDBJ PRJDB37679 / PRJDB35626
  • 16S rRNA V4 amplicon sequencing (Illumina MiSeq) of root microbiota from WT and PSR/PEPR mutants grown in nutrient-deficient soil · soil bacterial communities on Arabidopsis roots · n = 4-5 biological replicates · DRA012727
  • IP-MS of PEPR1 immunoprecipitates (anti-FLAG beads) from seedlings at +Pi or -Pi, with or without Pep1 · Arabidopsis thaliana · jPOST preview 1230333443699fda85f12b0 (access key 7188; LC-MS/MS raw data)
  • Transient expression of Arabidopsis PEPR1, PEPR2 and PROPEPs, together with GFP-detected BAK1, in leaves for ROS and co-immunoprecipitation assays · Nicotiana benthamiana · n = 8 biological replicates (ROS assays)
  • Pto DC3000 leaf infection of WT and pepr1 pepr2 plants in nutrient-deficient soil, with or without Pep1 pretreatment · Arabidopsis thaliana; Pseudomonas syringae pv. tomato DC3000 · n = 8-9 biological replicates
  • Low-Pi growth assays of PEPR1-FLAG, PEPR2-FLAG, PROPEP6-mCitrine and CRISPR propep lines (shoot fresh weight, primary root length) · Arabidopsis thaliana · n = 12; 21-23; 18-24; 10-12 biological replicates
Methods & tools
Proteome Discoverer 2.5 (SEQUEST HT, Percolator, label-free quantification), Araport11 protein database, Easy-nLC 1200 with Orbitrap Exploris 480 and FAIMS Pro, NEBNext Ultra II RNA Library Prep Kit for Illumina, qRT-PCR (ΔΔCt method, ACTIN2 reference), Claident (clsplitseq demultiplexing), DADA2 (R package dada2), SINA v1.2.11 with SILVA SSURef NR99 release 132, QIIME2 on Galaxy Europe (Bray-Curtis, PERMANOVA, 999 permutations), CRISPR/Cas9 multiplex sgRNA vectors (pUC-AtU6-26_gtg, pGHO-YC9), L-012 luminol ROS assay and H2DCFDA confocal staining, ImageJ band quantification; ggplot2
Data & accessions
https://repository.jpostdb.org/preview/1230333443699fda85f12b0
Reagents & environment
Proteome Discoverer 2.5 (SEQUEST HT, Araport11, FDR <1%); R dada2 (truncLen 220/150, maxEE 2); SINA v1.2.11 with SILVA SSURef NR99 release 132; QIIME2 via Galaxy Europe; MiSeq Reagent Kit v3 (300-bp paired-end).
Reproducibility gaps
No analysis code shared; RNA-seq run IDs left as placeholders (DRR to DRR) and RNA-seq methods truncated; proteomics via a jPOST preview link needing an access key; which PRJDB project holds which experiment is not stated.
Limitations (6)
  • The specific endogenous PROPEP-derived forms acting in planta are undefined, and whether basal PEPR support of PSR stems from low-level constitutive signaling, ligand-independent activity or alternative PROPEP forms is unknown. · stated by the authors
  • WRKY33 is implicated in output-selective amplification, but how it connects mechanistically to LPR1/LPR2-dependent iron-redox signaling remains unresolved. · stated by the authors
  • Root microbiota differences were community-level only: no individual ASV or genus differed after multiple-testing correction, so effects reflect many modest coordinated shifts rather than specific taxa. · stated by the authors
  • Stronger MAPK and transcriptional outputs at low Pi did not translate into greater Pep1-induced protection against Pto DC3000, which was similar under both Pi regimes. · stated by the authors
  • Pathway genetics (lpr1 lpr2, rbohD rbohF, wrky33, iron, pH) rest mainly on two qRT-PCR markers (CYP71A12, PDF1.2a; n = 3), and PEPR1 selectivity of PROPEP6 is largely shown in N. benthamiana, where PROPEP6 processing differed from Arabidopsis. · noted in this summary
  • The abstract says PEPR loss compromises bacterial resistance, but although Fig. 2e includes pepr1 pepr2 (n = 8-9), the results text describes only Pep1-pretreatment protection; infection and microbiota (n = 4-5) work used one nutrient-deficient soil mix. · noted in this summary

Who should read:Read this if you study plant nutrient-immunity crosstalk, phytocytokine receptors such as PEPR1/PEPR2, or how phosphate and iron status reshape pattern-triggered immunity and root microbiota in Arabidopsis.

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