p38 MAPK orchestrates cross-tissue potassium homeostasis for survival

Domain: chemistry
Published: 2026-03-09
Editorial outcome: major_revision

Abstract
Potassium is vital for life, yet how potassium homeostasis is maintained at the tissue or organismal level under dietary scarcity remains poorly understood. Stress-activated signaling pathway p38 MAPK is implicated in immune response and aging, but its specific role in low potassium response is unclear. Here we show that a specific p38 MAPK–ATF-7 pathway orchestrates cross-tissue potassium homeostasis in Caenorhabditis elegans. It drives transcriptional upregulation of a crucial P-type ATPase pump CATP-3 specifically in the hypodermis, a process that integrates cell-autonomous mechanisms with non-autonomous ASI neuronal signals, thereby enhancing organismal survival during potassium deficiency. Notably, this regulation is distinct from canonical osmotic stress responses, revealing a specialized and conserved survival strategy. Analogous p38-mediated control of P-type ATPases occurs in yeast and mammalian cells, suggesting broad relevance. Our findings redefine potassium regulation as a cross-tissue process linked to lifespan, stress signaling, and innate immunity with potential implications for aging and age-related diseases.

Introduction
Potassium is vital for cellular and tissue function, primarily obtained through dietary intake in humans1. Inadequate intake, a persistent global issue2-4, can lead to potassium deficiency (PD), causing hypokalemia (<3.5 mmol/L) and life-threatening consequences5. Although the critical role of dietary potassium in regulating lifespan has been recognized in model organisms, studies investigating this relationship and the underlying mechanisms remain scarce6. Potassium ion (K+) homeostasis is regulated at the organismal level via dietary intake and renal excretion, and within cells by Na+/K+-ATPase and K+ channels through active and passive transport, respectively7. Yet, whether these processes are coordinated across tissues to mitigate PD stress remains elusive. Although low potassium upregulates Na+/K+-ATPase expression in specific cells, such as renal tubular cells8, 9, the extent of synchronized regulation across tissues or the whole organism is unclear. Such coordination could enhance survival under PD, suggesting an evolutionary drive to adapt to potassium scarcity. Elucidating these mechanisms holds promise for advancing our understanding of cross-tissue responses to PD.

The p38 family of mitogen-activated protein kinases (MAPKs), conserved across species, governs cellular responses to diverse stresses, including environmental insults, infections, tumorigenesis, and growth-related challenges10, 11. In Caenorhabditis elegans, p38 MAPK signaling underpins longevity during dietary restriction, modulated by nutrient availability12. This suggests p38 may orchestrate broader metabolic defenses, potentially including potassium regulation. Here, we harnessed C. elegans’s genetic and phenotypic strengths to uncover a p38 MAPK pathway that boosts PD survival. This pathway coordinates K+ homeostasis across tissues, acting both autonomously and nonautonomously to regulate innate immune response genes and a P-type ATPase potassium pump. These findings reveal that animals can detect and respond to potassium scarcity through a cross-tissue signaling network, rather than isolated cellular responses. The integration of sensory neuron input with ion transport in distal tissues reshapes our understanding of potassium homeostasis, linking it to lifespan regulation and immune-like defense programs. This work identifies a conserved, systemic strategy for coping with low potassium stress that may inform approaches to aging and age-related diseases in mammals.

Results
Potassium plays a key role in the survival of p38 mutants

To investigate responses to insufficient availability of external potassium in C. elegans, we developed a method for rapidly assessing their lifespan in liquid culture without E. coli OP50-1 (a streptomycin-resistant strain that is widely used as a standard food source for C. elegans in laboratories), thereby minimizing the confounding effects of food-derived potassium. This approach utilizes day 3 adult worms to avoid interference with growth, development, or reproduction (Fig. 1a), with all survival data recorded from adult day 3 onward. Initially, wild type (N2) worms in distilled water without salt supplementation exhibited a mean lifespan of approximately 19 days, comparable to that on standard nematode growth medium (NGM) plates with live OP50-1 (Fig. 1b, Supplementary Fig. 1a and Supplementary Data 1)13. This observed lifespan likely reflects the net effect of two counteracting factors: prolongation arising from bacterial food deprivation14-16 and a countervailing shortening induced by the absence of essential salts. Although p38 MAPK signaling is well known for its roles in longevity and innate immunity in C. elegans, prior studies have also implicated the NSY-1(ASK1 MAPKKK)–SEK-1(MKK3/MKK6 MAPKK)–PMK-1(p38 MAPK) cascade in ionic and osmotic stress adaptation, including its function within the OSR-1–CaMKII–NSY-1 pathway for hyperosmotic resistance and its regulation of sodium leak channel activity that might influence Na⁺/K⁺ homeostasis17-19. Together with our preliminary observations showing hypersensitivity of sek-1(km4) null mutants to electrolyte depletion, these findings prompted us to investigate whether the p38 MAPK pathway plays a critical role in sensing and responding to potassium-deficient stress. In contrast, loss of sek-1 only minimally affects survival in NGM media20, but results in death within 2 days in water (Fig. 1c and Supplementary Fig.1a). Thus, SEK-1 plays a crucial role in responding to low osmotic or electrolyte stress.

We also observed that NaCl supplementation in liquid culture had a less beneficial effect on the survival of wild type animals compared to KCl (Fig. 1b,d). High NaCl concentration (250 mM) shortened lifespan due to hyperosmotic stress, while KCl supplementation caused no such effect. Notably, KCl significantly extended the lifespan of wild type worms across tested concentrations (Fig. 1d). Furthermore, KCl, rather than NaCl, markedly rescued the survival of sek-1 and pmk-1 (p38 MAPK) null mutants in distilled water (Fig. 1b–e and Supplementary Fig.1a). This effect is consistent with a model in which the apparent rescue reflects a combination of a general lifespan-extending effect of KCl and the alleviation of the increased susceptibility of sek-1 and pmk-1 mutants to PD. In addition, supplementation with Ca2+ failed to improve the survival of sek-1 mutants under water conditions (Supplementary Fig. 1b), further supporting the idea that p38 signaling is particularly required for survival under conditions of limited exogenous K+, rather than upon exposure to Na+, Ca2+ or Cl–. Together, these results suggest that p38 signaling promotes survival by mitigating PD (100 mM NaCl without exogenous K+) stress and argue against a primary role for nonspecific osmotic imbalance.

We investigated the effects of ion concentrations on worm survival and found that elevated K+ levels were crucial for extending longevity in wild type worms (Supplementary Fig.1a). However, direct pairwise comparisons showed that replacing K⁺ with Na⁺ (50 mM K-phosphate buffer vs. 50 mM Na-phosphate buffer, or 50 mM KCl vs. 50 mM NaCl) dramatically reduced lifespan of sek-1 and pmk-1 mutants, whereas switching anions while keeping the cation constant (50 mM K-phosphate vs. 50 mM KCl, or 50 mM Na-phosphate vs. 100 mM NaCl) had minimal effect (Fig. 1f–h). We also examined whether culture conditions (solid vs. liquid) influenced survival under PD, found that sek-1 or pmk-1 mutants were specifically sensitive to PD under both conditions, whether worms were grown on solid agar plates or in liquid culture in the presence of E. coli OP50-1 (Supplementary Fig.1c,d).

Extracellular pH can influence membrane potential and ion flux by modulating proton-sensitive K⁺ channels and, consequently, voltage-gated ion channel activity; therefore, we tested whether altered H⁺ levels contribute to the survival defects observed under PD. Interestingly, wild type worms survived longer in low-pH (4.3) medium than in high-pH (9.3) medium, regardless of K+ or Na+. Nevertheless, sek-1 mutants exhibited a greater dependence on K+ than on pH (Supplementary Fig. 1e,f). In addition, because the p38 MAPK pathway plays a central role in innate immunity, we also assessed pathogen sensitivity to determine whether the PD phenotype might be secondary to immune defects. Neither Na+ nor K+ influenced pathogenic survival in either slow-killing or fast-killing assays in wild type and sek-1 animals (Supplementary Fig. 1g,h), indicating a distinct process between p38-mediated pathogenic resistance and PD survival.

Furthermore, KCl supplementation rescued sek-1(km4) survival in a strictly time-dependent manner: adding 50 mM KCl within the first 2 hours of transfer to PD medium (100 mM NaCl) fully restored lifespan to normal K+ levels, whereas additions at later time points (≥2 h) yielded only partial or no rescue (Fig. 1i). This indicates that p38 signaling mediates a rapid, early protective response that must be engaged within a narrow temporal window to prevent irreversible physiological damage during acute PD. Notably, under PD conditions, sek-1 mutants exhibited severe stress phenotypes, including reduced body length, head contraction, and bursting of the intestine and eggs, resulting in rapid death (Fig. 1j, Supplementary Fig. 1i and Supplementary Video 1–4). These phenotypes were likely due to muscle spasms, as evidenced by contracted and disorganized muscle fibers observed under microscopy. Finally, to exclude altered feeding behavior as a confounding factor, we measured pharyngeal pumping under the same liquid survival conditions. PD exposure did not significantly alter pharyngeal pumping rates in wild-type worms, indicating that potassium deficiency per se does not impair pumping behavior in a normal genetic background (Supplementary Fig. 1j). In contrast, pmk-1 mutants exhibited a significant reduction in pumping rates under PD conditions, consistent with decreased overall physiological activity or heightened stress susceptibility specific to this genotype.

Collectively, our findings demonstrate that SEK-1 is essential for survival under PD conditions in the absence of exogenous K+, underscoring its role in mitigating potassium stress. These systematic analyses demonstrate that survival of p38 pathway mutants under PD depends strictly and specifically on extracellular K⁺. This requirement is independent of accompanying anions (Cl⁻ or phosphate), phosphate itself, buffering capacity, pH, the presence of E. coli OP50-1, other cations present in standard growth media (including Ca2+ and Mg2+), as well as culture format (liquid or solid).

PD survival requires the p38–ATF-7 pathway

To further elucidate the role of p38 MAPK signaling in worm survival under PD conditions, we analyzed the entire pathway, comprising TIR-1 (TIR domain protein), NSY-1 (MAPKKK), SEK-1 (MAPKK), PMK-1 (p38 MAPK), and the transcription factor ATF-7 (ATF2/ATF7/CREB5) (Fig. 2a and Supplementary Table 1). These components are critical for innate immunity and longevity in C. elegans12, 21, 22. The loss-of-function mutants—including sek-1 (km4 and ag1, null) (Fig. 2b and Supplementary Data 1)20, sek-1 and pmk-1 double mutants (Fig. 2c), and nsy-1 (ok593 and ag3, null/strong loss-of-function) (Fig. 2d)—exhibited similarly shortened survival times compared to wild type animals under PD conditions, suggesting that these core p38 MAPK components are essential. TIR-1, an upstream regulator of the p38 cascade known for pathogen response23, showed differential effects: tir-1 mutants impairing immunity (qd4, tm3036, TIR-domain loss or deletion)24, had shorter lifespans, whereas the immunity-sparing tir-1 (ok1052)25 displayed survival comparable to wild type worms (Fig. 2e). These findings indicate that TIR-1 acts through the canonical NSY-1–SEK-1–PMK-1 cascade in potassium homeostasis.

Next, we investigated the role of ATF-7, a transcription factor downstream of the p38 MAPK cascade that regulates immunity genes (Fig. 2a)22. ATF-7 acts as both transcriptional activator (when phosphorylated by PMK-1) and repressor (when unphosphorylated) of target genes. Under PD conditions, a loss-of-function atf-7(lf) mutant significantly reduced survival, while a gain-of-function atf-7(gf) mutant, acting solely as a repressor, exhibited even shorter survival than atf-7(lf) (Fig. 2f), mirroring their effects on pathogen resistance22. The markedly stronger phenotype of atf-7(gf) therefore reveals that active repression of these targets by unphosphorylated ATF-7 is considerably more deleterious than simple absence of activation during PD stress, highlighting the critical need for precise p38-dependent phospho-regulation of ATF-7. Notably, atf-7(lf) partially mitigated the survival defect of sek-1 or pmk-1 mutants (Fig. 2g), consistent with the loss of ATF-7’s repressor function. Collectively, our data demonstrate that the TIR-1–p38 MAPK–ATF-7 pathway is essential for survival under PD, sharing mechanistic similarities with its established roles in immunity and dietary restriction induced longevity12, 22.

The ocr-2(yz5) and ocr-2(ak47) alleles are loss-of-function mutations in the TRPV channel OCR-2, whose activity in ADF neurons regulates tph-1 expression and serotonin biosynthesis, and is also required for innate immunity24, 26. The tph-1(n4622) allele is a presumed null deletion27. Notably, ocr-2(yz5), ocr-2(ak47), and tph-1(n4622) mutants displayed only modest reductions in survival under PD (Fig. 2h), indicating that serotonin biosynthesis contributes only minimally to the PD response.

In the context of lifespan determination in C. elegans, genes associated with chemical sensing, osmotic response, and cytoprotection might contribute to survival under PD28, 29. Surprisingly, deletion of some well-studied genes, including those involved in insulin/IGF-1 and mTOR signaling13, reduced survival less severely than sek-1 deletion (Supplementary Table 1 and Supplementary Fig. 2), suggesting that PD survival depends more critically on the p38/ATF-7 pathway. To identify suppressors of sek-1(km4) lethality under PD conditions, we performed an unbiased forward-genetic suppressor screen. From approximately 6 million F2 L1 sek-1(km4) mutants exposed to PD conditions, 120 survived, 58 reached adulthood, and 18 produced progenies. Mapping the six longest-surviving mutants revealed all carried loss-of-function mutations in atf-7, including one known allele (Fig. 2i). Their survival resembled that of atf-7(lf) and sek-1;atf-7 double mutants (Fig. 2j). Together, these findings indicate that the p38–ATF-7 pathway is specifically critical for responding to PD conditions.

PD induces expression of p38–ATF-7-regulated genes

The essential role of the p38–ATF-7 pathway in survival under PD suggests that its downstream target genes are critical for this process. To identify PD-responsive genes, we performed mRNA expression profiling in wild type worms exposed to PD (100 mM NaCl), high potassium (100 mM KCl), or NGM (51 mM NaCl, 25 mM potassium phosphate buffer, KPB) conditions under solid agar plates with OP50-1 for 4 hours. This represents the standard growth condition and was used to assess transcriptional responses under physiologically relevant, food-present conditions. Relative to NGM, PD induced 998 downregulated and 1,241 upregulated differentially expressed genes (DEGs), and compared to high potassium, 1,028 downregulated and 1,340 upregulated DEGs, with substantial overlap between these sets. In contrast, only 137 genes were downregulated and 133 upregulated between high potassium and NGM (Fig. 3a–c and Supplementary Data 2). The markedly fewer transcriptional changes under KCl-replete conditions do not indicate lack of specificity; rather, they reflect successful physiological rescue. In contrast, NaCl-only conditions maintain lethal potassium deficiency, driving a large-scale stress induced transcriptional program.

We analyzed PD-specific DEGs (892 upregulated, 636 downregulated) between PD and either high potassium or NGM conditions (Fig. 3d,e). PD-upregulated genes were enriched in the hypodermis and excretory system (Supplementary Fig. 3a) and associated with cuticle development, innate immunity, apoptosis, and potassium ion import (Fig. 3f). These included known immunity genes encoding Metridin-like ShK toxins and CUB-like domain proteins (Supplementary Fig. 3b), and 115 SEK-1-induced genes (Fig. 3g,h)12. In contrast, PD-downregulated genes showed no such enrichment (Supplementary Fig. 3d–f). Notably, ATF-7 regulated more PD-upregulated genes than downregulated ones, unlike other transcription factors with balanced regulation (Supplementary Fig. 3g). In addition, we performed mRNA expression profiling in wild type and pmk-1(km25) worms exposed to PD (100 mM NaCl) and high potassium (100 mM KCl) in liquid culture without OP50-1 for 4 hours (Fig. 3i,j), conditions that recapitulate the liquid survival assays. This experiment was specifically designed to isolate the effects of PD and ionic environment without confounding influences from bacterial metabolism or liquid-associated feeding behavior. PD induced the upregulation of 105 genes (Supplementary Data 2), many of which were enriched in the hypodermis, excretory system, and pathways associated with immune response (Supplementary Fig. 3c). Among these, 24 genes were dependent on PMK-1 for their upregulation, and a substantial fraction overlapped with the 115 genes previously identified as induced by SEK-1 (Fig. 3k,l). Together, our data suggest that many p38–ATF-7-dependent genes, known to protect against pathogens12, are also induced under PD conditions.

The p38–ATF-7 pathway target CATP-3 mediates PD survival

Many downstream genes of the p38–ATF-7 pathway are essential for immunity and dietary restriction induced longevity12, 22. To identify PD-responsive targets, we tested both the top PD-upregulated and SEK-1-upregulated genes for their roles in survival under PD conditions (Fig. 3h,l). RNA interference (RNAi) and mutation analyses showed that most had negligible effects individually. However, deletion of catp-3, a conserved P-type ATPase potassium pump (Supplementary Fig. 4a), led to rapid death under PD conditions, with phenotypes resembling those of p38–ATF-7 pathway mutants, including muscle spasms (Supplementary Fig. 3h–j; Supplementary Video 5,6). The catp-3 mutant was highly sensitive to PD, showing significantly reduced survival at potassium levels below 5 mM (Fig. 3m and Supplementary Data 1), yet exhibited wild type lifespan and pathogen resistance under standard conditions (Fig. 3n and Supplementary Fig. 3k). These data suggest that CATP-3 is specifically required for survival under PD conditions.

To confirm the Na+/K+-ATPase activity of CATP-3, we conducted whole-cell patch-clamp experiments in HEK293T cells overexpressing catp-3. Notably, these cells showed significantly reduced pump activity upon treatment with the Na+/K+-ATPase inhibitor ouabain compared to mock-transfected cells, indicating CATP-3 contributes to pump function (Fig. 3o,p). In C. elegans, catp-3 has several paralogs with conserved ATPase domains but distinct expression patterns (Supplementary Fig. 4a–d). However, only catp-3 was essential for survival under PD conditions (Supplementary Fig. 4e), suggesting that p38–ATF-7-mediated upregulation of catp-3 evolved to specifically protect against PD. A decrease in extracellular K⁺ typically hyperpolarizes the membrane potential, which would reduce excitability. However, prolonged reduction in extracellular K⁺ can lead to secondary depletion of intracellular K⁺ due to passive leaks and incomplete compensation by other transporters. Thus, the upregulation of catp-3 under PD conditions may enhance pump capacity to restore intracellular K⁺ levels, thereby stabilizing membrane potential, osmotic balance, and cellular viability under PD conditions. In contrast, 100 mM KCl does not impose PD and thus does not activate this compensatory transcriptional response, explaining why catp-3 is not upregulated in the high-K⁺ condition.

Hypodermal expression of CATP-3 mediates PD survival

To examine the expression pattern of catp-3, we generated a knock-in strain with an 858 bp mCherry sequence inserted before the catp-3 stop codon. Expression was predominantly observed in the hypodermis, a single epithelial layer encasing the animal (Fig. 4a,b), which regulates body size, shape, and mechanical coupling between cuticle, muscles, and neurons30. We also assessed a transgenic line overexpressing a catp-3::GFP translational fusion driven by the catp-3 promoter, confirming localization to hypodermal cells, including seam cells (Fig. 4c). These findings align with single-cell RNA sequencing data31 showing catp-3 expression primarily in hypodermal cells (Fig. 4d and Supplementary Fig. 4b,c). Together, these results suggest that CATP-3 may contribute to hypodermal function under PD conditions.

To explore CATP-3’s role in the hypodermis, we generated three extrachromosomal transgenic arrays. Ex1 and Ex2 express catp-3::GFP under its native promoter with (Ex1) or without (Ex2) co-injecting marker rol-6(su1006) plasmid, while Ex3 uses the hypodermis-specific semo-1 promoter32. The gain-of-function rol-6(su1006) allele produces a dominant Roller phenotype that allows easy identification of transgenic animals and does not affect lifespan under our conditions (Fig. 4e). All three transgenes rescued the reduced lifespan of catp-3 mutants under PD conditions (Fig. 4e–g and Supplementary Data 1). In contrast, catp-3 expression in muscle or ASI neurons had no significant effect on PD survival (Fig. 4h,i). These data demonstrate that catp-3 is required in the hypodermis for PD survival.

The p38–ATF-7 pathway transcriptionally regulates CATP-3 in response to PD

To investigate catp-3 transcriptional regulation by the p38–ATF-7 pathway, we examined catp-3 mRNA expression in mutants of this pathway. Mutations in p38 pathway genes reduced catp-3 expression (Fig. 5a). Notably, the atf-7(gf) mutation, but not atf-7(lf), suppressed catp-3 mRNA, mirroring their effects on PD survival (Fig. 2f). Consistent with prior findings12, activation of the pathway by RNAi knockdown of the phosphatase vhp-1 or overexpression of sek-1 and atf-7 showed a trend toward increased catp-3 mRNA expression, suggesting that p38–ATF-7 signaling may contribute to catp-3 transcriptional regulation (Fig. 5a).

Developmental gene expression timing critically influences metabolism, behavior, and aging in adulthood33-35. Given that catp-3 expression rises across the four larval stages (Fig. 5b,c), we tested whether p38 MAPK regulates this increase and its relevance to PD survival. Notably, RNAi targeting sek-1 and pmk-1 altered PD survival and catp-3 expression in a developmentally timed manner (Fig. 5d–h and Supplementary Data 1). Two-generation (F2) RNAi treatment produced the most severe phenotypes, consistent with sek-1 and pmk-1 mutants. The markedly stronger phenotypes with F2 sek-1 RNAi compared to single-generation (F1) RNAi reflects the expected difference in knockdown depth and developmental timing: F2 treatment depletes both maternal and zygotic SEK-1 from embryogenesis onward, whereas F1 treatment leaves substantial maternal protein intact, particularly in neurons (Supplementary Fig. 7e,f). Since catp-3 is expressed robustly beginning at L2, RNAi initiated at L2/L3 (24 h post-L1 synchronization) had less effect than at earlier stages, while L4-stage RNAi impacted survival or catp-3 expression only minimally (Fig. 5d–h). These results suggest that p38–ATF-7-induced catp-3 expression must initiate during larval development to ensure adult PD survival.

As fasting/feeding states are known to modulate p38/MAPK signaling12, we tested whether KCl supplementation induces a fed-like signaling state or suppresses the fasting response in C. elegans. Neither qPCR nor RNA-seq analyses revealed significant changes in the expression of canonical fasting-responsive genes (lgg-1, atgl-1, sod-3, and pha-4) between PD and KCl conditions (Supplementary Fig. 5a–d), indicating that potassium supplementation does not reverse the fasting transcriptional program.

To determine whether p38/MAPK signaling is dynamically regulated by potassium availability or constitutively required under PD, we examined the expression of two established PMK-1 downstream targets, sysm-1 and catp-3. While sysm-1 expression was primarily dependent on feeding status and showed minimal or no induction by PD, catp-3 was robustly induced under PD and rapidly suppressed upon KCl supplementation, both in the presence and absence of food (Supplementary Fig. 5e,f). These results indicate that p38/MAPK signaling is not globally activated during PD, but instead regulates a potassium-responsive subset of target genes, supporting a model in which p38 activity is required for survival without uniform stress-induced activation.

Given that p38 MAPK-mediated innate immunosenescence occurs across species36, we observed an age-dependent decline in catp-3 expression (Fig. 5i–k) and the levels of PD-induced catp-3 mRNA expression (Fig. 5l,m). Collectively, these data suggest that catp-3 is transcriptionally regulated by p38–ATF-7 signaling in response to PD.

p38 pathway signaling functions tissue-specifically to mediate PD survival

p38–ATF-7 pathway components are expressed across multiple tissues, including hypodermis, intestine, neurons, and muscles, as shown by fluorescence microscopy and single-cell sequencing (Supplementary Fig. 6)31. Prior studies indicate that SEK-1 acts in the intestine to mediate pathogen defense and oxidative stress response27, 37, and in multiple tissues for dietary restriction induced longevity12. Expecting hypodermal sek-1 expression to be essential for PD survival, we tested tissue-specific requirements. Expression of sek-1 under its native promoter fully rescued PD survival, but we could not establish transgenic lines with sufficient hypodermal sek-1 expression, possibly due to toxicity. Intestinal sek-1 expression partially rescued PD survival (Fig. 6a,c), though less effectively than its role in pathogen resistance (Fig. 6e and Supplementary Data 1)27. These data suggest that tissue-specific roles of p38–ATF-7 signaling differ between PD survival and pathogen defense.

ASI sensory neurons in the head detect environmental cues like chemoattractants, temperature, and food availability, regulating metabolism38. They are critical for dauer formation, satiety quiescence, and dietary restriction induced longevity39, including p38-dependent innate immunity to pathogens40. These roles suggest that ASI neurons might sense PD conditions. Remarkably, ASI- specific sek-1 expression effectively rescued the reduced lifespan of sek-1(km4) mutants under PD conditions, outperforming intestinal expression (Fig. 6a,b). This was confirmed in integrated transgenic lines (Fig. 6c). Thus, sek-1 expression in ASI neurons provides significant protection against PD stress, suggesting that these neurons may perceive PD and trigger a protective gene expression response.

Interestingly, sek-1 expression in the intestine and ASI neurons differentially regulated downstream genes. Intestinal SEK-1 markedly increased the expression of the three well-characterized PMK-1/ATF-7 immune target genes (sysm-1, C17H12.8, K08D8.5) while suppressing catp-3, whereas ASI-specific SEK-1 fully restored catp-3 expression but only partially increased immune gene levels (Fig. 6d). This aligns with ASI SEK-1 partially rescuing pathogen resistance, unlike catp-3 expression (Fig. 6e). Thus, p38 MAPK signaling for innate immunity and PD responses varies across tissues in its downstream gene regulation.

To clarify the tissue-specific roles of p38 MAPK signaling in PD survival, we performed sek-1 RNAi in neurons, hypodermis, intestine, and muscle. RNAi-deficient rde-1(ne219) mutants exposed to F2 sek-1 RNAi maintained normal PD survival (Fig. 6f). Notably, sek-1 RNAi in neurons and hypodermis significantly reduced PD survival (Fig. 6g,h), similar to wild type worms treated with F2 sek-1 RNAi (Fig. 5e). Although hypodermal SEK-1 rescue experiments were infeasible due to potential toxicity, hypodermal-specific RNAi confirms that p38–ATF-7 signaling in this tissue is critical for PD survival, consistent with CATP-3’s role. Interestingly, intestinal sek-1 RNAi also reduced PD survival (Fig. 6i), whereas muscle RNAi had no substantial effect (Fig. 6j). Consistent with the modest PD survival reduction seen with F1 sek-1 RNAi (L4 to day 3 adults) in wild type worms, sek-1 RNAi in neurons, hypodermis, intestine, and muscle showed minimal effects (Supplementary Fig. 7a–d). Notably, F1 sek-1 RNAi significantly reduced intestinal SEK-1 but not in ASI neurons (Supplementary Fig. 7e,f), where earlier RNAi initiation (e.g., L1) was required. Furthermore, sek-1 deletion blocked PD-induced catp-3 mRNA expression, an effect also seen with sysm-1 (a p38 target) but not catp-2 (a paralog) (Fig. 6k–m). In sek-1 mutants, PD-induced catp-3 expression remained absent despite endogenous or hypodermal catp-3 overexpression, whereas catp-3 mutants overexpressing catp-3 showed no such defect (Fig. 6k–m), confirming that sek-1 is required specifically for p38-induced gene expression in response to PD.

Together, these findings indicate that p38–ATF-7 signaling regulates PD-induced catp-3 expression via cell-autonomous mechanisms (primarily in the hypodermis) and non-cell-autonomous pathways (mainly via ASI and other neurons) (Supplementary Fig. 7g). We propose that CATP-3, an ATPase potassium pump, plays a critical role in excitable tissues (muscles and neurons) by maintaining intracellular K+ levels under PD conditions. However, CATP-3 expression in muscle or ASI neurons had no significant effect on PD survival (Fig. 4h,i), suggesting that CATP-3 functions primarily in the hypodermis to indirectly support muscle and neuronal function, possibly via hypodermal-mediated mechanisms that presumably include enhancement of K+ uptake. We speculate that the strong induction of catp-3 specifically under PD represents a physiologically appropriate compensatory response: severe extracellular K⁺ depletion collapses the K⁺ gradient, depolarizes excitable tissues, and triggers lethal muscle spasms and neuronal dysfunction (Fig. 1j, Supplementary Video 1–6). Upregulation of the hypodermal Na⁺/K⁺-ATPase CATP-3 increases pump density, thereby enhancing K⁺ reuptake capacity into the animal. Because the hypodermis is the major non-excitable epithelial barrier in C. elegans and directly contacts pseudocoelomic fluid and muscles, elevated hypodermal CATP-3 likely restores organismal K⁺ homeostasis by: re-establishing trans-hypodermal K⁺ transport; repolarizing adjacent muscle and neurons indirectly; preventing catastrophic ion leakage and volume dysregulation. In contrast, KCl-replete conditions (100 mM KCl) maintain normal extracellular K⁺, eliminating the need for compensatory pump upregulation—hence catp-3 expression remains basal (Supplementary Fig. 7g). This is directly analogous to the well-documented upregulation of renal Na⁺/K⁺-ATPase α1 and β1 subunits during dietary or pathological potassium deficiency in mammals.

Conservation of p38-mediated potassium pump regulation in mammalian and yeast

Our findings suggest that p38-mediated potassium pump expression in PD defense may be evolutionarily conserved. In mammals, however, p38 deficiency results in embryonic lethality41 and the more complex tissue environment could lead to differences in tissue-specific K+ homeostasis mechanisms compared to C. elegans, despite conserved regulatory pathways. To explore this, we used the C2C12 mouse myoblast cell line, a model widely employed in muscle research for its rapid differentiation into contractile myotubes42, to investigate p38’s role in K+ regulation.

C2C12 myoblast differentiation into muscle requires p38 MAPK signaling42. Under PD conditions (0 mM KCl), proliferation in growth medium (GM) was unaffected (Fig. 7a and Supplementary Fig. 8a), but K+ was essential for myotube formation in differentiation medium (DM) (Fig. 7b and Supplementary Fig. 8b). K+ withdrawal from differentiation medium disrupted myotube morphology within 24 h, causing atrophy and cell atrophy and cell death (Supplementary Fig. 8c). In Mapk14 (p38α) knockout cells (Mapk14−/−), p38 was dispensable for growth medium proliferation but essential, alongside K+, for differentiation (Fig. 7a,b and Supplementary Fig. 8d,e), mirroring p38’s role in C. elegans (Fig. 1). Similarly, the p38 inhibitor SB203580 had no effect on proliferation but prevented differentiation (Supplementary Fig. 8f).

To identify p38 target genes during C2C12 myoblast differentiation, we performed mRNA expression profiling in wild type and Mapk14−/− cells exposed to GM and DM (Fig. 7c). Compared to GM, DM upregulated 2,345 genes in wild type cells (Fig. 7d and Supplementary Data 2). Under DM conditions, 2,157 genes were upregulated in a p38-dependent manner when comparing Mapk14−/− to wild type cells, whereas only 1,337 genes were p38-dependent under GM conditions (Fig. 7d). These results indicate a more prominent role for p38 during cell differentiation. Among the 2,345 DM-upregulated genes in wild type cells, 371 were upregulated under either GM or DM conditions (Fig. 7e,h) and were associated with sarcomere and actin filament organization, skeletal muscle development, differentiation, and contraction, underscoring p38’s critical role in C2C12 myoblast differentiation (Fig. 7g and Supplementary Fig. 8h,i). These genes included several well-characterized transcription factors, such as Peg3 (C2H2-type zinc finger protein, involved in cell proliferation), Zbtb18 (C2H2-type zinc finger protein, involved in neuronal development), Mef2d (myocyte-specific enhancer factor 2, regulates muscle and neuronal differentiation), Mef2c (Myocyte Enhancer Factor 2C, involved in myogenesis), Myod1 (Myogenic Differentiation 1, promotes muscle cell differentiation and regeneration), and Myf5 (Myogenic Factor 5, drives muscle-specific gene transcription and differentiation), all of which act downstream of Atf7 or Atf2—two key transcription factors regulated by p38 MAPK signaling (Fig. 7f,j and Supplementary Data 2).

Additionally, only three DM-upregulated Na+/K+-ATPase genes (Atp1a1, Atp1a2, Atp1a3) were p38-dependent (Fig. 7i and Supplementary Fig. 8g). Notably, Atp1a2, a subunit associated with familial hemiplegic migraine type 2 (FHM2) and other neurological disorders, was predominantly expressed in neurons and skeletal muscle in mouse and human43-45, distinct from Atp1a1 and Atp1a3 (Supplementary Fig. 8j,k). Furthermore, siRNA-mediated knockdown of Mapk14 or Atp1a2 inhibited differentiation, confirming their essential roles (Fig. 7k–m). Thus, these findings suggest that p38 MAPK regulates transcriptional Na+/K+-ATPase expression in response to PD and differentiation, possibly involving other transcription factors via an Atf7-dependent manner, a mechanism likely conserved in mammals.

The budding yeast strain Saccharomyces cerevisiae BY4742, widely used in aging studies, harbors three P-type ATPase genes: ENA1, ENA2, and ENA5. These genes encode proteins, each comprising 1,091 amino acids with over 97% sequence identity (Supplementary Fig. 8n). ENA1 encodes a sodium P-type ATPase critical for sodium detoxification and serves as a direct downstream target of the bZip transcription factor SKO1 (the yeast homolog of ATF-7) in response to osmotic stress46, 47. Similar to observations in C. elegans and mammals, ENA1 expression is significantly higher than that of ENA2 and ENA5, and ENA2 expression is minimal under standard potassium conditions but is induced by either Hog1 (the yeast homolog of p38) or low potassium (Fig. 7n). Additionally, 18 strongly interacting transcription factors, which are Sko1-mediated downstream targets, are upregulated by Hog1 and likely contribute to regulating P-type ATPase gene expression (Supplementary Fig. 8l,m). Notably, wild type yeast cells exhibit larger cell sizes in water and slightly reduced sizes in hyperosmotic conditions (50 mM NaCl + 300 mM KCl). However, deletion of HOG1 or ENA1,2,5 abolishes these cell size changes, suggesting that Hog1 and the P-type ATPases play a role in modulating yeast cell size (Fig. 7m and Supplementary Fig. 8o). These alterations could also activate p38 signaling through osmotic, mechanical, or membrane tension sensors48, 49. Collectively, our results indicate that p38-mediated P-type ATPases regulation as a highly conserved mechanism across evolution orchestrates cellular response to low potassium conditions (Supplementary Fig. 8p).

Methods
Worms
Worms were maintained at 20°C on standard nematode growth medium (NGM) agar plates seeded with Escherichia coli OP50-1 (a streptomycin-resistant strain for worm food). Strains obtained from other sources were outcrossed to the laboratory N2 strain as indicated in Supplementary Table 1. The sek-1 transgenic strains were a gift of Nick Bishop and Leonard Guarente (MIT).

Microbe Strains
E. coli OP50-1 was cultured overnight in Luria-Bertani (LB) medium supplemented with 10 mg/L streptomycin at 37°C. Following incubation, 1 mL of the liquid culture was seeded onto standard nematode growth medium (NGM) plates (100 mm  15 mm petri dishes) and allowed to grow for 2 days at room temperature. For RNAi feeding experiments, HT115 bacteria harboring the pL4440 plasmid were used as an empty vector (EV) control. These RNAi cultures were grown overnight at 37°C in 50 mL conical tubes containing 5 mL of LB medium supplemented with 50 μg/mL carbenicillin. After overnight incubation, the cultures were diluted 1:5 in LB medium containing carbenicillin to allow for re-entry into the logarithmic growth phase (~6 hours). Subsequently, the cultures were centrifuged at 3,900 g for 10 minutes and concentrated to a final volume of 3 mL in LB medium supplemented with 0.2 g/L (1 mM) IPTG before being seeded onto NGM plates containing 50 μg/mL carbenicillin and 0.2 g/L IPTG. Growth-arrested bacteria for liquid survival assays experiments were prepared according to the protocol described as described previously12. Specifically, OP50-1 bacteria were cultured in 500 mL of LB medium supplemented with 10 mg/L streptomycin in a 2 L flask overnight at 37°C. The cultures were then centrifuged at 3,900 g for 10 minutes, and the bacterial pellets were resuspended in S basal (1 SB solution: 5.85 g/L NaCl, 1 g/L K HPO , 6 g/L KH PO , autoclaved) supplemented with 5 mg/L cholesterol, 50 mg/L ampicillin, 10 mg/L kanamycin, 1 mg/L tetracycline, and 50 mg/L nystatin to an OD600 of 3. The bacterial suspension was maintained at room temperature for approximately 7 hours before being stored at 4°C for up to one week. For pathogenesis experiments a single colony of P. aeruginosa PA14 was cultured in LB without antibiotics at 37°C.

Worm transgenic strain construction
Extrachromosomal arrays were generated by injecting transgene DNA (10 ng/mL) into the gonads of young adult animals, either with or without the rol-6 marker (pRF4) at 50 ng/mL. Unless otherwise noted, transgene DNA was created using a fusion PCR method. The catp-3p::GFP transcriptional fusion was produced by fusion PCR, amplifying a 3,797 bp catp-3 promoter and a 1,964 bp GFP+3’UTR from vector pPD95.67. The catp-3::GFP translational fusion was constructed by PCR amplification of a 10,010 bp fragment from catp-3 fosmid transgenes (C09H5.2, TransgeneOme), which included a 3,847 bp catp-3 promoter, a 3,700 bp catp-3 genomic region, a 1,044 bp 2xTY1::EGFP::3xFlag sequence, and a 1,419 bp 3’UTR. The semo-1p::catp-3, myo-3p::catp-3 and gpa-4p::catp-3 translational fusions were generated by PCR fusion. This involved amplifying a 2,983 bp semo-1 promoter, 2,991 bp myo-3 promoter, and 2,903 bp gpa-4 promoter, respectively, each fused with a 4,116 bp catp-3 gDNA including the 3’UTR from worm genomic DNA. The catp-3(syb843) mutation was introduced by CRISPR-Cas9 knock-in of an 858 bp mCherry sequence before the termination codon of catp-3, performed by SunyBiotech Co., Ltd (Fuzhou, China).

Liquid survival assays
All worms were maintained at 20°C for at least two generations prior to the initiation of the assays. Synchronized populations of L1-stage animals were obtained through hypochlorite treatment and subsequently allowed to develop at 20°C on standard NGM plates seeded with OP50-1. After two days, the animals were transferred to standard NGM agar plates supplemented with 5 mg/L cholesterol, 100 mg/L FUdR, and an antibiotic cocktail (50 mg/L ampicillin, 10 mg/L kanamycin, 1 mg/L tetracycline, 50 mg/L nystatin), and seeded with cold- and antibiotic-treated OP50-112. Three days later, the day 3 adult animals were washed twice in 100 mM NaCl (PD) or the indicated solution and then transferred to individual wells of a 96-well microplate containing 150 μL of 100 mM NaCl or the indicated solution, supplemented with 5 mg/L cholesterol, 20 mg/L FUdR, and the antibiotic cocktail but without OP50-1. Each well contained 10–20 worms, and the plates were maintained at 20°C in the dark without shaking. Worms exhibiting pallor, a transparent appearance, a straight or rigid body, or complete immobility were classified as dead. Unless otherwise indicated, all worm PD survival assays reported in this study were performed using this method.

To exclude the effects of other NGM components, a potassium-free NGM solution was prepared using 100 mM NaCl, 25 mM sodium phosphate buffer (SPB: 2.38 g/L NaH PO and 735 mg/L Na HPO ) and supplemented with 1 mM CaCl , 1 mM MgSO , 2.5 g/L peptone, 5 mg/L cholesterol, 100 mg/L FUdR, and the same antibiotic cocktail. Synchronized worms were washed off standard NGM plates with this solution, rinsed twice, and transferred to the potassium-free NGM media for subsequent assays.

Solid PD assays with OP50-1
To establish solid PD conditions with OP50-1, potassium was removed from both the agar plates and the bacterial food source. First, agar plates were prepared in a manner similar to standard NGM plates, except that NaCl and potassium phosphate buffer were replaced with either 100 mM NaCl, 100 mM KCl, or NGM solution (51 mM NaCl, 25 mM potassium phosphate buffer, KPB). All plates were supplemented with 1 mM CaCl , 1 mM MgSO , 5 mg/L cholesterol, 100 mg/L FUdR, and an antibiotic cocktail consisting of 50 mg/L ampicillin, 10 mg/L kanamycin, 1 mg/L tetracycline, and 50 mg/L nystatin. Second, overnight cultures of OP50-1 were washed twice with distilled water and resuspended in water containing 5 mg/L cholesterol and the same antibiotic cocktail at an optical density of OD = 3 in 50-mL conical tubes. The bacterial suspensions were maintained at room temperature for 7 hours and subsequently stored at 4°C for one week. Third, immediately prior to plate seeding, the supernatant was removed and bacteria were resuspended in 3 ml of fresh 100 mM NaCl, 100 mM KCl, or NGM solution (51 mM NaCl, 25 mM KPB) containing the same antibiotic regimen and 100 mg/L FUdR. This step generated a concentrated OP50-1 suspension (OD ≈ 50), which was then seeded onto the corresponding salt agar plates. Finally, synchronized worms at different developmental stages were washed off standard NGM plates using the corresponding 100 mM NaCl, 100 mM KCl, or NGM solution. After two rapid washes with the same solution, animals were transferred onto the matched salt agar plates for subsequent assays.

Worm microscopy and image analysis
For fluorescence analysis, animals were immobilized for 5 minutes in M9 buffer with 0.06% tetramisole, mounted on 2% agarose pads on glass slides, and imaged using ZEN 2012 software on a Zeiss Axio Imager M2 fluorescence microscope equipped with a Zeiss AxioCam HRc high resolution 13 MP color CCD microscope camera. Fluorescence intensity was quantified with NIH ImageJ software (Version 1.50). For assessments of movement behavior, body shape, muscle morphology, and other phenotypes, animals were observed without immobilization under the specified conditions. Videos of the animals' status were recorded using an Olympus IX51 inverted microscope equipped with fluorescence and phase contrast capabilities, utilizing cellSens standard 1.12 software.

Pathogenesis assays
The standard slow-killing assays (small lawn) using P. aeruginosa PA14 were performed as described previously12. Briefly, single PA14 colonies were inoculated into 3 ml LB broth and incubated overnight at 37°C. Subsequently, 5 µL of the overnight culture was spotted at the center of 35-mm NGM agar plates supplemented with 20 mg/L FUdR. For the fast-killing assay (big lawn), the agar plate composition mirrored that of the slow-killing assay, but 5 µL of PA14 culture was spotted onto each plate and spread to cover the entire agar surface, as previously reported27. The seeded plates were incubated overnight at 37°C, then equilibrated at room temperature overnight. Approximately 40 animals were transferred to each prepared plate, with five plates evaluated per strain. The assays were conducted at 20°C, and animals unresponsive to gentle prodding with a platinum wire were recorded as deceased.

Pharyngeal pumping assay
Pharyngeal pumping was quantified by counting the number of pharyngeal contractions under a Zeiss Stereo Discovery V12 microscope at 100× magnification. To minimize variability caused by irregular pumping rhythms, contractions were recorded over a continuous 3-min interval for each worm. For each strain and each treatment, 15 healthy worms were analyzed in a blinded manner.

EMS mutagenesis screening assay
Worm genetic screen procedure based on the classical method described previously58. Synchronized L4-stage sek-1(km4) worms are collected and suspended in a 0.05 M ethyl methane sulphonate (EMS) solution, followed by gentle shaking at 20°C for 4 hours to induce random mutations. After the incubation, the mutagenesis is terminated by treating the worms with 1% sodium thiosulfate, and the EMS is thoroughly removed through multiple washes. The worms are then transferred to NGM plates for a 3-days recovery and egg-laying, and then bleached to obtain a synchronized population of F2 eggs. The F2 generation was allowed to hatch and develop on agar plates containing 100 mM NaCl, seeded with E. coli OP50-1 as a food source. This selective step aimed to identify worms with altered potassium sensitivity. A subset of 120 L4-stage worms was isolated for further analysis. These worms were transferred to individual wells of a 24-well plate and allowed to lay eggs. From these, 58 adult worms that successfully laid eggs were selected for subsequent analyses. These 58 F2 adult worms were subjected to a standard liquid survival assay in 100 mM NaCl. Worms exhibiting a rescue phenotype, indicated by enhanced survival under PD conditions, were identified. These rescued worms underwent a second round of the liquid assay to measure survival duration. The six longest-surviving candidate mutants were then selected for further genetic mapping using deep sequencing, following previously established methods59. The ld09 allele introduces a premature stop codon (GAA→TAA), producing a truncated protein that lacks the C-terminal bZIP DNA-binding and leucine-zipper dimerization domains, predicting molecular null. The ld18 allele is a well-established loss-of-function mutation reported in previous studies22. The ld37 allele contains a 158-bp deletion within the coding region, which removes part of the functional domain and is also considered a loss-of-function allele. The ld42 and ld44 alleles encode conservative hydrophobic substitutions within the leucine zipper (Leu→Phe and Ala→Val, respectively), which likely perturb hydrophobic-core packing and affect DNA binding. Finally, the ld53 allele introduces a premature stop codon (CAA→TAA) upstream of the C-terminal bZIP domain, eliminating both DNA-binding and dimerization domain, thereby predicting null.

Worm RNA sequencing (RNA-seq) samples preparation
For solid agar plate assay, approximately 2,000 synchronized L4-stage wild type (N2) animals were washed twice in either 100 mM NaCl, 100 mM KCl, or NGM solution (51 mM NaCl, 25 mM KPB) and then transferred to agar plates supplemented with 100 mM NaCl, 100 mM KCl, or NGM solution (51 mM NaCl, 25 mM KPB). These plates were seeded with OP50-1 bacteria that had been treated with the corresponding solutions. After 4 hours, the animals were collected and flash-frozen in liquid nitrogen. Genes are upregulated (PD up) or downregulated (PD down) under PD (100 mM NaCl) conditions were identified by comparison with both the 100 mM KCl and NGM conditions in N2 animals (100 mM NaCl vs 100 mM KCl & 100 mM NaCl vs NGM).

For liquid assay, approximately 1,000 synchronized day 1 adult N2 or pmk-1(km25) animals were quickly washed twice in either 100 mM NaCl or 100 mM KCl solution and then transferred to 100 mM NaCl or 100 mM KCl solution without OP50-1. After 4 hours, the animals were collected and flash-frozen in liquid nitrogen.

RNA-seq
In this study, total RNA was extracted and purified from all samples using M5 HiPer Universal Plus RNA Mini Kit (MF167, Mei5 Biotech, Beijing, China). All RNA samples were sent to Novogene Co., Ltd (Beijing, China) for quality control (Sample QC). After RNA library preparation, cDNA libraries are sequenced on Illumina NovaSeq platforms using a paired-end 150 bp sequencing strategy. Approximately 6 Gb of clean data per sample was obtained, with a uniquely mapped read rate exceeding 75%. Three biological replicates were included.

Processing and analysis of RNA-Seq data
FASTQ files were aligned to C. elegans WBcel269, Mus musculus GRCm39 or Saccharomyces cerevisiae R64-1-1.dna.toplevel reference genome using STAR (version 2.7.1a) with a 2-pass alignment procedure60. Gene and transcript quantification was carried out using RSEM (version 1.3.3)61. Differential expression analysis was conducted in R (version 4.3.1) using the DESeq2 package (version 1.40.2)62. Differentially expressed genes were defined with an adjusted P (false discovery rate, FDR) < 0.05.

GO term and pathway annotation
The WormBase enrichment suite was used for tissue analysis, while DAVID (version 2021) functional annotations were employed for GO biological process (GOTERM_BP_DIRECT) and InterPro protein domain enrichment analysis. Heatmaps were generated from the counts using Morpheus (https://software.broadinstitute.org/morpheus). Genes network graphs were created using STRING.

PD-mediated genes analysis
Heatmaps and gene expression cluster plots were generated using Morpheus (Broad Institute). Functional annotations, including biological processes and human ortholog matching, were retrieved from WormBase (build WS269). The WormBase enrichment suite (build WS289) was used for tissue enrichment analysis, while DAVID functional annotations (version v2022q1) were employed for Gene Ontology and InterPro protein domain enrichment analysis63. InterPro categorizes sequences at the superfamily, family, and subfamily levels and predicts the presence of functional domains and important sites64. By searching for specific transcription factor binding sites within the promoter region of genes [-1000, +100] using the Gene Transcription Regulation Database (GTRD) (v21.12)65, we identified a total of 271 transcription factors and their target genes. We then conducted an over-representation analysis on the PD-mediated gene sets. This analysis mapped the PD-mediated genes to the target genes of the 271 transcription factors and identified statistically significant enrichment of transcription factors using a custom model on the g:Profiler platform66.

Quantitative RT-PCR (qRT-PCR)
For each treatment, approximately 200 synchronized animals were collected, and frozen in liquid nitrogen. Total RNA was extracted using TRIzol (T9424, Sigma-Aldrich), and purified with Direct-zol™ RNA Kits (R2050, Zymo Research). First-strand cDNA was synthesized in duplicate from each sample (11752050, Invitogen First-Strand Synthesis SuperMix). SYBR green (11760500, Invitogen) was used to perform qRT-PCR (ABI 7900). At least three biological replicates were examined for each sample. Gene expression fold change was calculated using the ΔΔCt method. Primer sequences are as follows: catp-3 Forward: 5′-TCA TAT AAC AGC GCA AGA CC-3′ and catp-3 Reverse: 5′-CCA TCC AAT TGT GTT GAT TGC-3′; catp-2 Forward: 5′-ACA ACA GTT ATA ATA GTG GCC G-3′ and catp-2 Reverse: 5′-GAG CTG GAT TTG AAG TGA TCG-3′; C17H12.8 Forward: 5′-CAT GTT GAA CGA TTT GCG A-3′ and C17H12.8 Reverse: 5′-GCA ATC GAC AGT GAA ATT CTC-3′; K08D8.5 Forward: 5′-TTT ATG CGA CTG CTG TTG C-3′ and K08D8.5 Reverse: 5′-CTC ACG TTT GCA TTG TAT GGA-3′; sysm-1 Forward: 5′-TAC ACT GCT TCA GAG TCG TG-3′ and sysm-1 Reverse: 5′-CGA CAA CCA CTT CTA ACA TCT G-3′; tba-1 Forward: 5′-CCT TCG TTC ACT GGT ACG TC-3′ and tba-1 Reverse: CAG CCA AGT CTT CAC GAG CC-3′; lgg-1 Forward: 5′-ACC CAG ACC GTA TTC CAG TG-3′ and lgg-1 Reverse: 5′-ACG AAG TTG GAT GCG TTT TC-3′; atgl-1 Forward: 5′-GAT CGA CCG ATG ATT TAT CGA G-3′ and atgl-1 Reverse: 5′-GAGCCAATCCACATTTGGTC-3′; sod-3 Forward: 5′-ATG GAC ACT ATT AAG CGC GA-3′ and sod-3 Reverse: 5′-GCC TTG AAC CGC AAT AGT G-3′; pha-4 Forward: 5′-TCA AAG AGG AGC CAG AGT CG-3' and pha-4 Reverse: 5′-ACA CTT GAA GCA GCC GAC GA-3'.

Gene tissue expression analysis
The heatmap shows relative gene expression (dot color) and proportion (dot size) of catp-3 and its paralogs across all neurons and other tissues in L4 stage wild type worms. This heatmap was generated from C. elegans Neuronal Gene Expression Network (CeNGEN)31. The proportion is percentage of cells expressing the gene within each neuron cluster. This result uses thresholded data from threshold 2, and therefore only contains data from neuronal cell types. The scaled TPM (transcripts per million) expression value is the z-score for the gene in that neuron. The z-score represents how many standard deviations the TPM expression of a given gene in a given neuron is from the mean TPM for that gene. It is calculated by subtracting the mean TPM expression for the gene across all neurons from the TPM expression of the gene in the given neuron, then dividing by the standard deviation of TPM values for that gene. For the purpose of identifying alignment lengths, protein sequences were obtained from the NCBI RefSeq database release 207. In scenarios where multiple isoforms were available, the longest isoform was utilized. Alignments of the sequences were carried out via EMBOSS water software utilizing the BLOSUM62 matrix and retaining all default configurations.

Mammalian cells
HEK293T cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO incubator at 37°C with controlled moisture. C2C12 mouse myoblast cell line was purchased from the American type culture collection (ATCC, CRL-1772). Cells were cultured and maintained in growth medium (GM) containing high glucose DMEM medium supplemented with 10% FBS and 1% penicillin–streptomycin at 37°C in 5% CO . GM was replaced every two-days and cells were subcultured when they reached 70%-90% of confluence. Cells were seeded at 6-well plates and proliferated for 2 days to reach 50%-60% confluency in GM. The culture mediums were replaced with differentiation medium (DM) containing the DMEM medium supplemented with 2% horse serum (16050130, Gibco). The DM medium was replaced every 24 h to stimulate myotube formation.

Mammalian cell culture, vector construction, and transfection
The CDS of capt-3 was codon-optimized and synthesized by GenScript Biotech Corp. (Jiangsu, China), and then cloned into pcDNA3.1(+) vector. Mammalian cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) supplemented with 10% fetal bovine serum in a 5% CO incubator at 37°C with controlled moisture. HEK293T cells were transfected using Lipofectamine™ 3000 Transfection Reagent Kit (Invitrogen). Plasmids for transfection were extracted from E. coli (DH5α) using QIAGEN Plasmid Mini Kit (Qiagen), and 2 μg plasmid DNA was added into each well of 6-well plates (Nunc) containing the cells (70%–80% confluent). The ratio of plasmids pcDNA3.1(+)-capt-3 and pEGFP-C1 for co-expression of capt-3 and EGFP was 10:1. To confirm that the cells were successfully transfected, green fluorescent signals were examined using an inverted fluorescence microscope (Lecia DMi8 Inverted Microscope) before patch clamp.

Whole-cell patch-clamp recording
The whole-cell patch-clamp experiments were performed using an Axopatch-200B patch-clamp setup (Axon Instruments, CA, USA) with a Digitata1550 digitizer (Axon Instruments, CA, USA). Clampex10.7 software (Axon Instruments, CA, USA) was used for data acquisition and Clampfit 10.7 was used for data analysis. Na+/K+ pump currents (Ip) were recorded in HEK293T cells transfected with capt-3 as previously described67. The bath solution contained 144 mM NaCl, 5.4 mM KCl, 1.8 mM CaCl , 2 mM BaCl , 5 mM NiCl , 10 mM HEPES, pH 7.4 (adjusted with NaOH); the pipette solution contained 110 mM CsCl, 40 mM NaCl, 10 mM NaOH, 3 mM MgCl , 6 mM EGTA, 10 mM HEPES, 10 mM Mg-ATP, pH 7.4 (adjusted with CsOH). The osmolarity was adjusted to 315 mOsm with glucose. The voltage was held at 0 mV while recording, and 1 mM ouabain (4995, Sigma-Aldrich) was added into the bath solution as indicated.

Microscopy analysis of C2C12 cell morphology
Cell morphology was recorded using an Olympus IX51 inverted microscope equipped with fluorescence and phase contrast capabilities, utilizing cellSens standard 1.12 software.

Generation of Mapk14−/− C2C12 cells
C2C12 Mapk14 knockout cells were constructed using the CRISPR/Cas9 genome-editing system. The GenCrispr NLS-Cas9-EGFNP Nuclease, which harbored the green fluorescent protein (GFP) sequence for visual verification of transfection (Z03393; Genscript, China), was transfected into C2C12 cells using Lipofectamine 2000 (11668019, Thermo Fisher IScientific) in 6-well plates. Clones were then isolated in accordance with the recommended protocol for the CRISPR/Cas9 system. Briefly, at 72 h after transfection, cells that successfully transfection of the Mapk14 sgRNA/Cas9 were screened by Fluorescence Activating Cell Sorter (FACSmelody, BD biosciences, America) and plated at ∼1 cell/well in 96-well plates. Eight clones were further subcultured until reaching high confluence in 6-well plates. Stable C2C12 cell lines showing successful knockout of Mapk14 were confirmed by DNA sequencing using forward and reverse primers (forward 5′-TGG GGC CTT TAC TTT GAC CA-3′ and reverse 5′-CCT CGC GAT GAT CTG ACA GC-3′).

siRNA transfection in C2C12 cells
siRNA was synthesized by GenePharma (Shanghai, China), and the siRNA sequences of each gene are as follows: Mapk14 sense 5′-GGU CAC UGG AGG AAU UCA ATT-3′ and Mapk14 antisense 5′-UUG AAU UCC UCC AGU GAC CTT-3′; Atp1a2 sense 5′-GAG ACC CGC AAU AUC UGU UUC TT-3′ and Atp1a2 antisense 5′-GAA ACA GAU AUU GCG GGU CUC TT-3′; NC (negative control) sense 5′-UUC UCC GAA CGU GUC ACG UTT-3′ and NC antisense 5′-ACG UGA CAC GUU CGG AGA ATT-3′. C2C12 cells in the logarithmic growth phase were subcultured to 24-well plates so that the cell density was about 20%, and the cells were cultured in GM until the cell density reached 30%. At this time, the C2C12 cells were transfected with siRNA for the first time. 2 μL Lipofectamine 2000 and 40 pmol siRNA were diluted and mixed using Opti-MEM in each well of 24 plates. After gently mixing the mixture, let it stand for 20 min, replace the new culture medium for C2C12, and add the siRNA -lipo2000 mixture so that the volume of each well is 500 μL. The cells were cultured for 18 h and siRNA was transfected again using the same method. After 12 h, the cell density reached about 90%. The growth medium containing siRNA was removed, and the cells were cultured using DM. DM was replaced every 2 days, and cell morphology changes were continuously recorded.

C2C12 cell RNA-seq analysis
C2C12 wild type cells and Mapk14 knockout (Mapk14−/−) cells in the logarithmic growth phase were subcultured to 12-well plates and cultured in GM until the cell density reached about 60%. Wild-type and Mapk14−/− C2C12 cells were divided into two groups, one group continued to be cultured in GM, and the other group was cultured in DM, with new culture medium replaced every 2 days. Cell morphology changes were continuously recorded, and cell RNA was extracted after 5 days. Genes are downregulated (p38 up) in Mapk14 knockout cells when compared to wild type cells in GM or DM conditions (Mapk14−/− vs WT), and upregulated by DM (DM up) when compared to GM condition in wild type cells (GM vs DM).

Yeast
The wild type yeast strain Saccharomyces cerevisiae BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 ura3Δ0) and its derivative single-gene deletion mutant strains, all in the BY4742 genetic background, were procured from Horizon Discovery (Cambridge, UK). Yeast cultures were grown in either yeast extract peptone dextrose (YPD) medium (Solarbio Life Sciences, Beijing, China) or synthetic defined (SD) medium. The SD medium was composed of 20 g/L D-glucose, 1.7 g/L yeast nitrogen base (YNB), 5 g/L ammonium sulfate (AS), and a 1 amino acid supplement (uracil 100 mg/L, L-histidine 100 mg/L, L-leucine 300 mg/L, L-lysine-HCl 150 mg/L, Adenine 80 mg/L, L-arginine 40 mg/L, L-aspartic acid 100 mg/L, L-glutamic acid 100 mg/L, L-methionine 80 mg/L, L-phenylalanine 50 mg/L, L-serine 400 mg/L, L-threonine 200 mg/L, L-tryptophan 200 mg/L, L-tyrosine 40 mg/L, L-valine 150 mg/L, L-isoleucine 60 mg/L). To eliminate potassium from the YNB, potassium salts were substituted with their corresponding sodium salts. Potassium concentrations in SD medium were adjusted to 1 mM (low) or 7 mM (standard) by supplementing with 3 M KCl.

Generation of ENA genes deletion strain
Triple disruption of the ENA1, ENA2, and ENA5 genes (ena1,2,5Δ) in the S. cerevisiae BY4742 background, resulting in a total deletion of 11,046 bp, was performed by SunyBiotech Co., Ltd. (Fuzhou, China). A streamlined CRISPR-Cas9 protocol for marker-free gene knockout was employed68, utilizing the pML104 vector for co-expression of Cas9 and a single-guide RNA (sgRNA). A 20-mer sgRNA sequence (5′-ACA GAA TTG AAA ATT TTC GAT GG-3′) with a 5′-NGG-3′ protospacer adjacent motif (PAM) was selected, and a 90-mer repair template (5′-TAA TTA AAG AAA AAA CTT CGT ACA CAG AAT TGA AAA TTT TCG – TAA GGA GGG CAA TTT ATT GAG TGC CTT TCC TTT TGC ATC CCT-3′) was designed to introduce mutations and disrupt the PAM sequence. The pML104 vector was digested with BclI and SwaI, ligated with hybridized sgRNA oligonucleotides, and transformed into E. coli for sequence verification. The validated plasmid and repair template were co-transformed into S. cerevisiae along with salmon sperm DNA as a carrier, followed by plating on SC-URA medium and incubation at 30°C for 2–3 days. Twelve to twenty-four colonies were screened by PCR using two primer pairs: Pair 1 (forward: 5′-AGA GCA GT A TTC CCG AAT TAT-3′, reverse: 5′-TGA CGA AGC TCA ATT AGT CG-3′) and Pair 2 (forward: 5′-AGG TCT TAG AGC AGT ATT CCC-3′, reverse: 5′-GCA TGG CAAI ACG AGA TTA TC-3′). Successful gene disruptions were confirmed by Sanger sequencing. The pML104 vector was subsequently removed via counter-selection on 5-fluoroorotic acid (5-FOA) medium, and plasmid loss was verified by PCR.

Yeast RNA-Seq analysis
Yeast strains (BY4747 and hog1Δ), preserved at -80°C, were streaked onto YPD agar plates and incubated at 30°C for 2 days or until visible colonies appeared. Single colonies were picked and inoculated into 1.0 mL of YPD liquid medium, and cultured for 2 days at 30°C in a shaking incubator at 220 rpm. The cultures were diluted 10-fold with water and stored at 4°C for 24 h. Subsequently, 5 µL of the diluted culture was inoculated into 1.0 mL of SD medium supplemented with either 1 mM or 7 mM KCl and incubated at 30°C for 2 days in a shaking incubator at 220 rpm69. For each sample, approximately 1 mL of yeast culture was collected and flash-frozen in liquid nitrogen for storage.

Yeast cell imaging
A 5 μL aliquot of water-diluted yeast culture was inoculated into 1.0 mL of YPD liquid medium and incubated for 2 days at 30°C in a shaking incubator at 220 rpm. The resulting culture was washed three times with water and resuspended in one of the following solutions: water, 50 mM NaCl, 50 mM NaCl + 5 mM KCl, or 50 mM NaCl + 300 mM KCl. The suspensions were stored at 4°C for 24 h. Subsequently, a small volume of each cell suspension was placed on a clean glass slide, spread evenly, and sealed with a coverslip. Imaging was performed using a Leica STELLARIS 5 confocal microscope in brightfield mode with a 100× oil immersion objective lens (NA 1.4). Images were acquired using the LAS X software and saved in uncompressed TIFF format. To avoid imaging bias, all images were captured under identical light intensity and exposure settings. Image analysis was conducted using ImageJ (1.54e) software. The following processing steps were applied sequentially: Process → Binary → Make Binary → Dilate → Fill Holes → Watershed, to segment individual cells. Particles with an area between 10–50 μm² and a circularity of 0.7–1.0 were selected for quantitative analysis. The ggridges plots were generated using OmicStudio (https://www.omicstudio.cn, version V1.0) to visualize the distribution of cell size measurements across the indicated experimental groups. Single-cell size data were imported into OmicStudio and displayed as ridge density plots based on kernel density estimation. Each ridge represents the probability density distribution of cell size for one group, with ridge transparency (alpha) set to 0.6, allowing comparison of distribution patterns, dispersion, and shifts in central tendency between groups.

Quantification and statistical analysis
The significance of comparisons between two groups was determined by unpaired two-tailed t tests as indicated. Differences among means were analyzed using one-way ANOVA for multiple comparisons using GraphPad Prism (version 9.5.0). When ANOVA analysis revealed significant differences, pair-wise comparisons were performed using Dunnett's test or Tukey post-hoc test as indicated. The P values (log-rank test) for lifespan comparison, mean and median lifespan were determined using JMP software (Pro 17.0.0). All experiments were repeated independently at least three times. All lifespan results shown in figure panels are representative of three independent experiments, with detailed in Supplementary Data 1. Information about statistical tests and n numbers is provided in the respective figures and figure legends. Sample sizes were chosen based on the reproducibility of the experiments and are consistent with standard practices in the field.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Summary\nThis manuscript examines how potassium homeostasis influences immune responses and aging, primarily using\nCaenorhabditis elegans, with additional studies in mouse myotube cells and yeast. The authors propose a mechanism\ninvolving the p38–MAPK–ATF-7 axis, integrated with ASI neuronal signals, to promote survival under potassium deficiency.\nThe work suggests a broader link between ionic homeostasis, immune modulation, and strategies to combat antibiotic-\nresistant pathogens. While potentially impactful and of broad interest, the manuscript has significant issues with\nexperimental design, interpretation, and presentation.\nMajor Points\n1. The manuscript is dense, poorly organized, and sometimes lacks logical flow. Experiments and interpretations are not\nalways clearly described, making it difficult to read. Figures should be reorganized, with some moved to supplementary\nmaterials or removed entirely if not essential. For example:\no Fig. 1d would fit better in Fig. 2.\no Fig. 2h–j adds little and could be removed.\no Fig. 7k could be simplified by removing redundant time points and instead quantifying proliferation and myotube formation\n(see point 13).\n2. There are no direct data supporting coordination between tissues in response to low potassium, so this conclusion cannot\nbe made.\n3. The rationale for some experiments is unclear. For instance, phosphate buffer experiments in Fig. 1h–j are poorly\nexplained, and their purpose is ambiguous. Were they conducted to control for Cl- concentrations?\n4. The culture protocol departs from standard C. elegans methods but is insufficiently described. It is unclear how worms\nsurvive with cholesterol as the only carbon source, whether they consume egg lysates, and how FUDR is functioning in\nthese conditions.\n5. Lifespan experiments were done in liquid culture, while RNA-seq was performed on plate-grown worms, introducing\nconfounding variables. These experiments need to be conducted under the same conditions.\n6. RNA-seq analysis is incomplete. C2C12 sequencing should include a 0 mM KCl condition, and worm datasets should\ninclude mutants in the p38 pathway (e.g., sek-1).\n7. KCl rescues survival more effectively than NaCl but induces fewer transcriptional changes. This discrepancy should be\nexplained, possibly in terms of stress levels.\n8. The comparison in Fig. 2h between NaCl and NGM is not appropriate. Comparisons should instead include KCl vs. NaCl\nand KCl vs. NGM (or water, if done in liquid culture).\n9. WT worms survive better at acidic pH, while sek-1 mutants survive better at alkaline pH. This striking result is not\ndiscussed.\n10. Some data are inconsistent with the authors’ claims. For example:\n• Fig. 5a: vhp-1 RNAi does not significantly change catp-3 expression.\n• Fig. 5d–f: RNAi of catp-3 would have been more appropriate than stage-specific RNAi of sek-1 and pmk-1.\n• Fig. 3j: Claims about lifespan, size, development, brood size, and pathogen resistance are not supported by the figure.\n11. C2C12 myotube formation and proliferation need to be quantified.\n12. The effects of atf-7 mutants are shown, but it is not explained how the point mutations affect ATF-7 function.\n13. The upregulation of catp-3 in NaCl versus KCl needs functional interpretation.\n14. Differential effects of sek-1 RNAi on F1 versus F2 progeny are unexplained.\n15. Several datasets and controls are missing:\n• Extended Data Fig. 1 lacks Cl⁻ controls for pH 4.3 and 9.3.\n• Fig. 1k omits wild-type worms in KCl.\n• Fig. 1 is missing fluorescence images for WT ± KCl/NaCl and sek-1 without KCl/NaCl.\n16. Presentation issues further complicate interpretation:\n• Many images lack scale bars.\n• Words and labels inside the graphs are often not explains.\n• Reuse of lifespan datasets is not stated. It is understood that this can facilitate comparisons but it needs to be clearly stated\nin the anuscript.\n• GO term enrichment is redundant and should be revised.\n• Extended Data Fig. 2 is disorganized and should be reordered either by pathway or alphabetically.\nMinor Points\n• Fig. 7m: A ggridges plot would better show the data distribution.\n• Line 12: Change to “that integrates.”\n• Line 108: Change to “had shorter lifespans.”\n• Unnecessary abbreviations should be eliminated to improve readability.", "reviewer_2": "This manuscript presents interesting findings showing that the p38 MAPK–ATF-7 pathway orchestrates cross-tissue\npotassium homeostasis for organismal survival under potassium deficiency (PD). The authors demonstrate that ASI sensory\nneurons communicate with the hypodermis in C. elegans and extend their observations to mammalian and yeast systems.\nMutant worms with disruptions in the p38 MAPK pathway exhibited markedly reduced survival in water lacking salts or\nnutrients. Supplementation with potassium chloride (KCl), but not sodium chloride (NaCl), rescued the sensitivity of these\nmutants, suggesting that the pathway is critical when potassium is limiting. An EMS suppressor screen further identified\nATF-7, a downstream transcription factor of p38 MAPK, as an important regulator of potassium homeostasis. RNA-seq\nanalysis also revealed CATP-3, a P-type ATPase, as a functional downstream target. Together, these data provide\npotentially novel insights into the molecular mechanisms of potassium homeostasis across species.\nMajor Comments\n1. The standard nematode growth medium (NGM) contains peptone, cholesterol, NaCl, CaCl2, MgSO4, and potassium\nphosphate buffer, which are essential for worm growth and survival. The authors describe PD conditions as water\nsupplemented with NaCl. However, this medium lacks many NGM components and is not specifically deficient only for\npotassium. The absence of other essential nutrients complicates the interpretation. A true PD condition should include all\nother NGM components except potassium. This limitation should be addressed directly.\n2. KCl supplementation substantially increased survival in both wild-type and sek-1 (and pmk-1) mutants, with a stronger\neffect on the mutants. However, the magnitude of this effect raises the possibility of non-specific lifespan extension by KCl.\nThe specificity of this rescue should be clarified. In particular, the effects of other salts such as CaCl2 and MgSO4 should be\nexamined or discussed.\n3. Line 59: “Initially, wild type (N2) worms in water without salt supplementation exhibited a mean lifespan of approximately\n19 days, comparable to that on standard nematode growth medium (NGM) plates with live OP50-1 (Fig. 1b, Extended Data\nFig. 1a and Extended Data Table 1)13, indicating normal aging without significant stress under baseline conditions.” I\ndisagree with this interpretation. The reported 19-day lifespan cannot be regarded as “normal aging without significant\nstress.” Instead, it likely represents a composite outcome of two opposing influences: lifespan extension due to food\ndeprivation (T. L. Kaeberlein et al., Aging Cell 5, 487–494 (2006); G. D. Lee et al., Aging Cell 5, 515–524 (2006)) and a\ndetrimental effect caused by salt deprivation. This sentence should therefore be rewritten to avoid implying physiological\nnormalcy under such conditions.\nMoreover, as this condition forms the foundation of the experimental paradigm used throughout the study, the interpretation\nof all subsequent results requires careful reconsideration. For instance, the shortened lifespan of p38 MAPK mutants in\nwater could be at least partly due to the established requirement of p38 MAPK signaling for lifespan extension under food\ndeprivation (a form of dietary restriction in C. elegans). This possibility should be experimentally tested through additional\nrigorous control experiments and discussed thoroughly in the revised manuscript.\nMinor Comments\nLine 56: Define E. coli OP50-1 for readers unfamiliar with C. elegans culture.\nLine 62: Provide rationale for testing sek-1 mutants and the p38 MAPK pathway.\nLine 63: Clarify the nature of the allele (loss-of-function, hypomorphic, or null).\nLine 78: NGM contains additional ions beyond NaCl and potassium phosphate; please clarify.\nLines 81–86: Explain why pH and pathogen sensitivity were tested.\nLine 87: Provide more detail about the “time-dependent manner” for clarity.\nLine 91: The phenotypes in Fig. 1k are unclear; the sizes of sek-1 worms with 100 mM NaCl appear similar to wild type or\nsek-1 with 100 mM KCl. Representative images and quantitative analysis are needed.\nLine 102: Citation numbers are italicized. Please correct formatting.\nLine 103: Describe the alleles used for sek-1, nsy-1, and tir-1. As tir-1 alleles can produce different phenotypes, more\nexplanation is necessary.\nLine 114: Both atf-7(lf) and atf-7(gf) shortened lifespan under PD. Please clarify the interpretation.\nLine 118: Fig. 1j does not include atf-7 data.\nLine 125: jkk-1, jnk-1, tph-1, and ocr-2 do not seem to be well-studied genes involved in insulin/IGF-1 and mTOR signaling.\nJustify their inclusion.\nLine 132: Fig. 1j does not include atf-7 data.\nLine 140: Clarify whether NGM (51 mM NaCl, 25 mM potassium phosphate buffer) is an appropriate control.\nLine 151: Fig. 3b does not show the genes described in the text.\nLine 160: They tested both the top PD-upregulated and SEK-1-regulated genes\nLine 167: Fig. 3j or Extended Data Fig. 3j does not contain the data described.\nLine 176: Fig. 4e should be Extended Data Fig. 4e.\nLine 190: Explain rol-6 for broader readers.\nLine 193: Justify why ASI neurons were tested.\nFig. 4e–i: Clarify whether rescue experiments used catp-3 mutants or wild-type worms; provide strain information in the\nfigure legend.\nLine 241: Explain the rationale for testing sysm-1, C17H12.8, and K08D8.5.\nFigures 4 and 5: The reporter is labeled as catp-3::RFP. For accuracy and specificity, this should be corrected to catp-\n3::mCherry.\nFigure 5d and e: The colors of orange line and the label do not match.\nFigure 6k-n: Explain the rationale for using NGM plate as a control.", "reviewer_3": "This study, \"p38 MAPK orchestrates cross-tissue potassium homeostasis for survival,\" uncovers a previously\nuncharacterized role for p38/MAPK signaling in promoting survival during potassium deficiency (PD) in C. elegans.\nThe authors present convincing evidence that this pathway functions through hypodermal upregulation of CATP-3, a P-type\nATPase potassium pump, and integrates both neuronal and peripheral signaling. They further extend their findings to\nmammalian cell lines and yeast models, demonstrating the conserved role of p38/MAPK-mediated regulation of P-type\nATPases. Overall, the study is novel, the data are well supported, and the manuscript is clearly written and well organized.\nThere are some considerations, listed below, that should be addressed for clarity and to rule out alternative hypotheses.\nItems to address:\n1. Fasting is known to activate p38/MAPK signaling. It would be informative to test whether KCl supplementation triggers\nfed-like signaling or suppresses the fasting response in the worms.\n2. Pharyngeal pumping in C. elegans relies on Na⁺/K⁺-ATPase activity. How do the conditions used in this manucript (e.g.,\nPD and KCl supplementation) affect pharyngeal pumping behavior?\n3. While p38/MAPK is required for survival under PD, it remains unclear whether it is actively induced during this condition\nand subsequently returns to basal levels upon KCl addition or is just required.\n4. RNA-seq samples were prepared in the presence of bacteria, whereas the survival assays were performed in the absence\nof bacteria. Do these differences result in distinct levels of potassium deprivation between the two conditions? And does the\np38-mediated survival still hold in these conditions?", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "We thank all three Reviewers for their thorough, constructive, and highly insightful comments. We have now\nperformed a substantial set of new experiments, redone the RNA-seq under conditions that exac\ntly match the liquid survival assays (no bacteria, liquid medium), introduced a properly c\nontrolled potassium-specific deficiency medium, and extensively reorganized and clarified\nthe manuscript. All changes are highlighted in red in the revised manuscript. A detailed Rep\norting Summary, Source Data file, and updated Data Availability statement are now provided.\nReviewer #1 (Remarks to the Author):\nSummary\nThis manuscript examines how potassium homeostasis influences immune responses and aging, primarily using\nCaenorhabditis elegans, with additional studies in mouse myotube cells and yeast. The authors propose a mechanism\ninvolving the p38–MAPK–ATF-7 axis, integrated with ASI neuronal signals, to promote survival under potassium deficiency.\nThe work suggests a broader link between ionic homeostasis, immune modulation, and strategies to combat antibiotic-\nresistant pathogens. While potentially impactful and of broad interest, the manuscript has significant issues with\nexperimental design, interpretation, and presentation.\nResponse: We sincerely thank Reviewer #1 for the careful, detailed, and highly constructive\ncritique. We appreciate the Reviewer’s recognition that the study is “potentially impactful and of\nbroad interest.” The concerns raised have been extremely valuable in identifying critical\nexperimental gaps and presentation issues. We have addressed every major and minor point with\nnew data where required (including entirely new RNA-seq datasets under conditions identical to\nthe survival assays, properly controlled potassium-specific deficiency medium, quantification of\nmyotube formation, and many others), extensively reorganized the manuscript, and clarified all\ninterpretations. All changes are highlighted in the revised manuscript.\nMajor Points\n1. The manuscript is dense, poorly organized, and sometimes lacks logical flow. Experiments and\ninterpretations are not always clearly described, making it difficult to read. Figures should be reorganized,\nwith some moved to supplementary materials or removed entirely if not essential. For example:\nResponse: We sincerely thank the Reviewer for highlighting the issues with density, organization,\nand logical flow. In response, we closely follow the comments and carefully revised the manuscript\nto clarify description of observation, interpret data more clearly, and reorganize figures.\nResponse: Thanks, we agree. As suggested, the schematic diagram of the p38–ATF-7 pathway\n(former Fig. 1d) has been moved and is now presented as new Fig. 2a.\nFig. 2h–j adds little and could be removed.\nResponse: Thanks for this specific suggestion. In the revised manuscript, we have removed the\ndetailed schematic of the screening procedure (former Fig. 2h) to eliminate redundancy in the main\nfigures, while retained the screening results (former Fig. 2i,j) because it constitutes unbiased\nforward-genetic evidence that loss of atf-7 function is the major suppressor of sek-1 lethality under\npotassium deficiency (6/6 longest-surviving mutants carried independent atf-7 alleles). This\nremains one of the strongest pieces of evidence that ATF-7 is the critical downstream effector of\np38 signaling in this context.\nFig. 7k could be simplified by removing redundant time points and instead quantifying proliferation and\nmyotube formation (see point 13).\nResponse: Thank you for this precise and constructive suggestion. In the revised manuscript, we\nhave removed the day 1 and day 3 time-course images from the main figure 7k. Instead, we now\nprovide rigorous and widely accepted quantitative readouts of myoblast differentiation that do not\nrely on proliferation counting in confluent cultures. We quantified the length (Fig. 7l) and dimeter\n(Fig. 7m) of myotubes per field after 4 days of differentiation with/without siRNA of Mapk14 or\nAtp1a2 in C2C12 cells. As shown below, knockdown of Atp1a2 phenocopies p38α loss, confirming\nthat p38-mediated transcriptional upregulation of this Na⁺/K⁺-ATPase subunit is functionally\nrequired for myogenic differentiation.\n2. There are no direct data supporting coordination between tissues in response to low potassium, so this\nconclusion cannot be made.\nResponse: Thanks for this important critique. We agree that identifying direct signals across\n2\ntissues would be perfect and further strengthen this conclusion. Nevertheless, our current data\nprovided evidence for inter-tissue coordination in response to low potassium.\nWe performed tissue-specific rescue of an upstream component of the p38 MAPK-CATP-3\naxis, sek-1, in gene sek-1(km4) null mutant. Restoring sek-1 in ASI neurons significantly and\nmarkedly extended lifespan under PD conditions compared with the sek-1 null mutant (Fig. 6b, c).\nAlthough tissue-specific sek-1 RNAi experiments showed that knockdown in multiple tissues,\nincluding ASI, significantly affected PD survival (Fig. 6f–j), the rescue experiments provided\nadditional insight into how tissues coordinate during PD survival.\nAs shown in Fig. 6d, intestine-specific rescue restored expression of immune genes; restoration\nof sek-1 exclusively in the ASI fully rescued the dysregulated expression of the downstream target\ncatp-3 (Fig. 6d), which has been proved to mainly express and function in hypodermis — a distant\ntissue from ASI (Fig. 5). Because sek-1 is not expressed in hypodermis but only in ASI under gpa-\n4 promoter conditions, observed change in catp-3 mRNA level must be achieved via neuron-derived\nsignals acting on distal tissues. Therefore, the observation that restoring sek-1 only in ASI is\nsufficient to normalize catp-3 expression in another tissue demonstrated functional inter-tissue\nregulation in PD survival.\nTogether, these strengthened data demonstrate that potassium deficiency is sensed (at least in\npart) by ASI neurons, which then transmit a non-cell-autonomous signal that transcriptionally\nactivates the critical potassium pump CATP-3 in the hypodermis. This establishes bona fide cross-\ntissue coordination and justifies the conclusion that p38 orchestrates an organism-level response\nto potassium scarcity.\n3. The rationale for some experiments is unclear. For instance, phosphate buffer experiments in Fig. 1h–\nj are poorly explained, and their purpose is ambiguous. Were they conducted to control for Cl- concentrations?\nResponse: We thank the Reviewer for pointing out the lack of clarity. In the revised manuscript,\nwe have completely rewritten this section and added new control experiments (potassium-free\nNGM solution with CaCl₂ and MgSO₄) to make the rationale and interpretation explicit. The\npurpose of the phosphate buffer experiments (Fig. 1h–j) was to rigorously exclude contributions\nfrom anions (Cl⁻ vs. H₂PO₄⁻/HPO₄²⁻), phosphate itself, buffering capacity, and pH, thereby\nestablishing that K⁺ is the sole cation required for survival of p38 pathway mutants under\npotassium deficiency.\nThis conclusion can be underscored by direct pairwise comparisons (Fig. 1b–h; Extended\nData Fig. 1a). For mutant strains, survival of sek-1 and pmk-1 mutants dramatically changed\nbetween 50 mM KPB and 50 mM SPB (19.4 d vs 5.2 d in sek-1 mutant; 29.6 d vs 10.8 d in pmk-1\nmutant). In parallel, the difference between 50 mM KCl and 50 mM NaCl was also huge in both\nsek-1 and pmk-1 mutants (18.5 d vs 1.6 d in sek-1 mutant; 25.8 d vs 4.7 d in pmk-1 mutant).\nMeanwhile, survival differences between 50 mM KPB and 50 mM KCl, or between 50 mM SPB\nand 50 mM NaCl were mirror.\nIn the revised manuscript, we edit and improve the text as “Collectively, these systematic\n3\nanalyses demonstrate that survival of p38 pathway mutants under PD depends strictly and\nspecifically on extracellular K⁺. This requirement is independent of accompanying anions (Cl⁻ or\nphosphate), phosphate itself, buffering capacity, pH, the presence of E. coli OP50-1, other cations\npresent in standard growth media (including Ca2+ and Mg2+), as well as culture format (liquid or\nsolid).”\n4. The culture protocol departs from standard C. elegans methods but is insufficiently described. It is\nunclear how worms survive with cholesterol as the only carbon source, whether they consume egg lysates,\nand how FUDR is functioning in these conditions.\nResponse: For the liquid survival assays of in the method section, we have clearly described the\ncomponents of liquid medium as “Three days later, the day 3 adult animals were washed twice in\n100 mM NaCl or the indicated solution and then transferred to individual wells of a 96-well\nmicroplate containing 150 μL of 100 mM NaCl or the indicated solution, supplemented with 5 mg/L\ncholesterol, 20 mg/L FUdR, and the antibiotic cocktail but without OP50-1.”\nEnergy source: the adult C. elegans can survive for days without any bacterial food, relying\nentirely on their internal energy reserves. And the 5 µg/mL cholesterol is not used as the only\ncarbon/energy source, but serves only as an essential sterol for membrane synthesis (a requirement\nthat worms cannot meet de novo).\nRole of FUdR: FUdR is included to prevent egg hatching so that there weren’t be newly\nhatched larvae, thereby, ensuring that survival curves reflect only the lifespan of the original adult\ncohort. FUdR can stop hatching because it can block thymidylate synthase in early embryos,\ndepriving them of thymidine triphosphate and causing replication-fork collapse within 2–3 cell\ndivisions; the resulting nuclear fragmentation lyses the developing cells inside the egg shell.\n5. Lifespan experiments were done in liquid culture, while RNA-seq was performed on plate-grown\nworms, introducing confounding variables. These experiments need to be conducted under the same\nconditions.\nResponse: Thank you for this critical point and have fully addressed it. In the revised manuscript,\na new worm RNA-seq data have been performed with new datasets generated under conditions\nidentical to the liquid survival assays (no bacteria, liquid medium) for each wild type and pmk-1\nmutant. For each strain, about 2,000 synchronized young adult worms were washed twice and\ncultured for 4 hours under 20°C in either 100 mM NaCl or 100 mM KCl. The animals were then\ncollected for the RNA extraction and later RNA sequencing. The new liquid-condition\ntranscriptome perfectly recapitulates and strengthens the original findings (Fig. 3i–l; Extended\nData Fig. 3c, see below): 105 genes significantly upregulated by potassium deficiency in wild type\nN2 worms (100 mM NaCl vs. 100 mM KCl, FDR < 0.05), including strong enrichment of\nhypodermal genes, innate immune effectors, and catp-3 (PMK-1-dependent). The transcriptional\nresponse described in the revised manuscript now directly reflects the physiological state\nexperienced during the survival assays, eliminating any confounding variables from solid agar,\nlive\nbacteria, or developmental staging.\n4\n6. RNA-seq analysis is incomplete. C2C12 sequencing should include a 0 mM KCl condition, and worm\ndatasets should include mutants in the p38 pathway (e.g., sek-1).\nResponse: Thanks for this important comment. As described above, we have generated a new\nRNA-seq in wild-type and pmk-1(km25) animals under identical liquid 100 mM NaCl vs. 100 mM\nKCl conditions. These data demonstrate that potassium-deficiency induced catp-3 is p38-\ndependent.\nBecause Mapk14-knockout C2C12 cells exhibit growth restriction under low-potassium\nconditions, they cannot undergo normal differentiation assays. Moreover, both potassium ions and\nthe Mapk14 gene are indispensable for C2C12 myogenic differentiation; the absence of either factor\nabolishes differentiation. Consequently, when wild-type and Mapk14-knockout C2C12 cells are\nexposed to combinations of normal- and low-potassium conditions, only wild-type cells in a normal-\npotassium environment are capable of differentiating, whereas the remaining three conditions fail\nto support differentiation. Therefore, even if transcriptomic sequencing were performed under\nthese conditions, it would not allow meaningful analysis of the role of Mapk14 in C2C12\ndifferentiation, nor would it illuminate the potassium-responsive mechanisms governed by Mapk14.\n7. KCl rescues survival more effectively than NaCl but induces fewer transcriptional changes. This\ndiscrepancy should be explained, possibly in terms of stress levels.\nResponse: Thanks for highlighting this important point. The apparent discrepancy is now\nexplicitly addressed in the revised manuscript. We fully understand that this apparent discrepancy\nmight seem puzzling, and the presentation in our original manuscript might contribute to this\n5\nimpression. Indeed, conditions containing KCl (KCl alone or KCl rescue) provided environments\nclose to the standard one, and worms were not under strong stress. In contrast, worms in NaCl\nexperienced severe potassium-deficiency (PD) stress, resulting in significantly shortened lifespan\nand transcriptional changes as adaptive responses to this adverse PD environment. Overall, long\nlifespan and relatively few transcriptional changes observed in the KCl-rescued groups, compared\nto the NaCl groups, directly reflects the severe stress imposed by potassium deprivation in the latter,\nwhereas KCl rescue restored the stress to a near-standard state and thereby alleviates the need for\na large-scale stress response. For better clarity, we have revised the relevant content to eliminate\nany potential confusion or misunderstanding. We now state clearly: “The markedly fewer\ntranscriptional changes under KCl-replete conditions do not indicate lack of specificity; rather,\nthey reflect successful physiological rescue. Rapid restoration of K⁺ immediately corrects the\nprimary defect—disrupted ionic gradients and excitability—obviating the need for sustained\nstress-responsive transcription. In contrast, NaCl-only conditions maintain lethal potassium\ndeficiency, driving a large-scale stress induced transcriptional program.”\n8. The comparison in Fig. 2h between NaCl and NGM is not appropriate. Comparisons should instead\ninclude KCl vs. NaCl and KCl vs. NGM (or water, if done in liquid culture).\nResponse: We thank the Reviewer for this comment. In Fig. 3h, we have included three pairwise\ncomparisons to address this concern: PD (100 mM NaCl) vs. high K⁺ (100 mM KCl), PD (100 mM\nNaCl) vs. standard NGM, and high K⁺ (100 mM KCl) vs. standard NGM. These contrasts provide\na comprehensive and balanced evaluation of the effects of NaCl, KCl, and the NGM control.\n9. WT worms survive better at acidic pH, while sek-1 mutants survive better at alkaline pH. This striking\nresult is not discussed.\nResponse: We appreciate the Reviewer for raising this point. However, the apparent “better”\nsurvival of sek-1 mutants under alkaline conditions is observed only in the 0.1 M KCl condition\n(15.4 d at pH 9.3 vs. 11.8 d at pH 4.3). As shown in Extended Data Table 1 (and summarized in the\nsupplemental table provided here), this difference is relatively small and is not consistently\nobserved across other ionic conditions. More importantly, the magnitude of this pH-dependent\nvariation is minor compared with the much more pronounced effect caused by the presence or\nabsence of potassium, which remains the dominant determinant of sek-1 mutant survival.\nTherefore, this limited inconsistency does not affect the central conclusion of our study that\npotassium availability is the predominant factor governing the survival of p38-pathway mutants.\n10. Some data are inconsistent with the authors’ claims. For example:\n• Fig. 5a: vhp-1 RNAi does not significantly change catp-3 expression.\nResponse: We thank the Reviewer for identifying this inaccuracy. In Fig. 5a, we used the\nhypomorphic allele vhp-1(sa366). We acknowledge that this mutant does not significantly alter\ncatp-3 expression, and we have revised the text to accurately reflect this result. The revised sentence\nin the main text now reads: “Consistent with prior findings, activation of the pathway by\noverexpression of sek-1 and atf-7 strongly induced catp-3 mRNA, demonstrating that p38–ATF-7\nsignaling is sufficient for catp-3 transcriptional upregulation.”\n• Fig. 5d–f: RNAi of catp-3 would have been more appropriate than stage-specific RNAi of sek-1 and\npmk-1.\nResponse: Thanks for this comment. The essential role of CATP-3 in PD survival has already been\nrigorously demonstrated in Figures 3 and 4. Therefore, the experiments in Fig. 5d–h were designed\nto address a different question—namely, how p38 signaling regulates catp-3 expression and PD\nsurvival, rather than re-examining the effect of catp-3 depletion itself.\nIn Fig. 5d–h, stage-specific RNAi of sek-1 and pmk-1 allowed us to directly assess how\nperturbing p38 signaling affects PD survival, as well as how reduced p38 activity (via sek-1 RNAi)\ninfluences catp-3 expression. Using catp-3 RNAi in this context would only reiterate the established\nrequirement of CATP-3 for PD survival, without providing insights into the regulatory relationship\nbetween the p38 pathway and catp-3.\nAdditionally, catp-3 RNAi is technically challenging: despite multiple attempts, we found that\ncatp-3 RNAi was ineffective for unknown reasons, and the high sequence similarity between catp-\n3 and catp-2 (Extended Data Fig. 4d) makes it difficult to ensure target specificity and interpret\nRNAi results with confidence. For these reasons, sek-1 and pmk-1 RNAi were the most appropriate\napproaches for dissecting how p38 signaling modulates catp-3 expression and PD survival.\n• Fig. 3j: Claims about lifespan, size, development, brood size, and pathogen resistance are not supported\nby the figure.\nResponse: We thank the Reviewer for catching this oversight. We adjust our description in the\nmain text. “The catp-3 mutant was highly sensitive to PD, showing significantly reduced survival\nat potassium levels below 5 mM (Fig. 3i), yet exhibited wild type lifespan and pathogen resistance\nunder standard conditions (Fig. 3j and Extended Data Fig. 3j).”\n11. C2C12 myotube formation and proliferation need to be quantified.\nResponse: Thank you for this precise and constructive suggestion. In the revised manuscript, we\nhave removed the Day 1 and Day 3 time-course images from the main Fig. 7k. Instead, we now\n7\nprovide rigorous and widely accepted quantitative measurements of C2C12 differentiation that do\nnot depend on proliferation counts in confluent cultures. Specifically, after 4 days of differentiation\nwith or without siMapk14 or siAtp1a2, we quantified two standard morphological indicators of\nmyogenic maturation: myotube length (Fig. 7l) and myotube diameter (Fig. 7m) per field. These\nquantitative analyses demonstrate that Atp1a2 knockdown phenocopies p38α loss, supporting the\nconclusion that p38α-dependent transcriptional upregulation of this Na⁺/K⁺-ATPase subunit is\nfunctionally required for proper myotube formation.\n12. The effects of atf-7 mutants are shown, but it is not explained how the point mutations affect ATF-7\nfunction.\nResponse: We thank the Reviewer for pointing out the need to clarify how the atf-7 point mutations\naffect ATF-7 function. We have added a detailed explanation of all atf-7 alleles used in the Figure\n2j legend in revised manuscript. Summary of allele effects (now explicitly stated):\natf-7(qd22gf): Prevents relieving of repressive activity → acts as a constitutive repressor\n(Shivers et al., PLoS Genet 2010).\nLoss-of-function alleles identified in our suppressor screen:\nld09: GAA→TAA premature stop codon → truncation lacking the entire bZIP DNA-binding\nand dimerization domain → predicted null.\nld18 (qd137): established strong loss-of-function allele (a deletion in exon 4).\nld37: 158-bp deletion removing most of the bZIP domain.\nld42 and ld44: two conservative hydrophobic substitutions (L→F and A→V, respectively) in\nthe bZIP region; these disrupt leucine-zipper packing and affect DNA binding.\nld53: CAA→TAA premature stop codon upstream of bZIP → truncation eliminating DNA-\nbinding and phosphorylation sites → null.\n13. The upregulation of catp-3 in NaCl versus KCl needs functional interpretation.\nResponse: Thank you for this comment. We have added a clear functional interpretation in the\nrevised manuscript. The strong induction of catp-3 specifically under potassium deficiency (100\nmM NaCl) represents a physiologically appropriate compensatory response: severe extracellular\nK⁺ depletion collapses the K⁺ gradient, depolarizes excitable tissues, and triggers lethal muscle\nspasms and neuronal dysfunction (Fig. 1k, Supplementary Videos). Upregulation of the\nhypodermal Na⁺/K⁺-ATPase CATP-3 increases pump density, thereby enhancing K⁺ reuptake\ncapacity into the animal. Because the hypodermis is the major non-excitable epithelial barrier in\nC. elegans and directly contacts pseudocoelomic fluid and muscles, elevated hypodermal CATP-3\nlikely restores organismal K⁺ homeostasis by: re-establishing trans-hypodermal K⁺ transport;\nrepolarizing adjacent muscle and neurons indirectly; preventing catastrophic ion leakage and\nvolume dysregulation.\nIn contrast, KCl-replete conditions (100 mM KCl) maintain normal extracellular K⁺,\neliminating the need for compensatory pump upregulation—hence catp-3 expression remains basal.\nThis is directly analogous to the well-documented upregulation of renal Na⁺/K⁺-ATPase α1 and β1\n8\nsubunits during dietary or pathological potassium deficiency in mammals.\n14. Differential effects of sek-1 RNAi on F1 versus F2 progeny are unexplained.\nResponse: Thanks for this comment. We have added a clear explanation and supporting\nquantitative data in the revised manuscript. The stronger phenotype with F2 (two-generation) sek-\ndepth and developmental timing: F2 RNAi: Continuous exposure from embryogenesis onward →\nnear-complete depletion of both maternal and zygotic SEK-1 protein. F1 RNAi: Only 3–4 days of\nexposure beginning at L4 → partial reduction of zygotic SEK-1, with substantial maternal protein\npersisting (especially in long-lived tissues such as ASI neurons). This is now directly demonstrated\nfluorescence quantification of SEK-1::GFP in intestinal and ASI-specific rescue strains (Extended\nData Fig. 6e–f). The revised text now states: “The markedly stronger phenotypes with two-\ngeneration (F2) sek-1 RNAi compared to single-generation (F1, L4-to-day-3) RNAi reflect greater\nknockdown depth: F2 treatment depletes both maternal and zygotic SEK-1 from embryogenesis\nonward, whereas F1 treatment leaves substantial maternal protein intact, particularly in neurons\n(Extended Data Fig. 6e–f).”\n15. Several datasets and controls are missing:\n• Extended Data Fig. 1 lacks Cl⁻ controls for pH 4.3 and 9.3.\nResponse: We appreciate why this point was raised. As noted in our response above, the paired\ncomparisons presented in Fig. 1b–h demonstrate that neither the identity of the anion (including\nCl⁻) nor the buffering capacity is a major determinant of PD survival. Accordingly, the effects of\npH observed in phosphate-based media are expected to be similar in chloride-based environments.\nTherefore, the existing data in Extended Data Fig. 1e,f are sufficient to support our conclusions\nregarding pH effects on PD survival.\n• Fig. 1k omits wild-type worms in KCl.\nResponse: The first panel in Fig. 1k shows wild-type worms in 0.1 M KCl.\n• Fig. 1 is missing fluorescence images for WT ± KCl/NaCl and sek-1 without KCl/NaCl.\nResponse: We understand that including panels for WT worms could provide a more complete\nvisual set. However, as shown in Fig. 1j and Extended Data Fig. 1i, WT worms exhibited no\nappreciable differences in body morphology or length under either KCl or NaCl conditions. In\ncontrast, significant phenotypic changes were observed only in sek-1 mutants under PD conditions.\nTherefore, we focused on presenting fluorescence images of sek-1 mutants to clearly illustrate the\nsensitivity of p38 pathway mutants to extracellular potassium, highlighting the critical role of K⁺\nin their survival.\n16. Presentation issues further complicate interpretation:\n• Many images lack scale bars.\nreviewed all figures in the manuscript, ensuring that scale bars have been added to all relevant\nimages.\n• Words and labels inside the graphs are often not explains.\nResponse: Thanks for this important comment. In the revised manuscript, we have completely\nrevised every figure and its legend to ensure that all labels, abbreviations, and symbols appearing\ninside graphs are fully explained either directly in the panel or in the legend.\n• Reuse of lifespan datasets is not stated. It is understood that this can facilitate comparisons but it needs\nto be clearly stated in the manuscript.\nResponse: We thank the Reviewer for raising this point. The lifespan datasets are included in\nExtended Data Table S1 and were cited in the original manuscript.\n• GO term enrichment is redundant and should be revised.\nResponse: We appreciate the Reviewer’s comment regarding potential redundancy in GO term\nenrichment panels. While several GO-enrichment analyses are presented, each provides distinct\nand important information. The top-ranked GO terms were obtained directly from WormBase or\nDAVID functional annotations (GOTERM_BP_DIRECT) without manual modification, ensuring\nan unbiased representation of the major biological processes. This approach allows us to highlight\nrelevant pathways activated or repressed under potassium deprivation while maintaining\nconsistency and transparency in data presentation.\n• Extended Data Fig. 2 is disorganized and should be reordered either by pathway or alphabetically.\nResponse: Thanks for this helpful suggestion. In the legend of Extended Data Fig. 2, we provide a\ntable introducing the genes and their functions, organized to reflect functional pathways rather\nthan alphabetical order. Specifically, genes are grouped as follows: sensory neuron–related genes\n(tax-2/tax-4, TRP family, tmc-1, trapa-1, gpa-3, ODR and CHE families); cellular stress response\ngenes (mitochondrial unfolded protein response, electron transport chain, heat shock response, ER\nstress response, antioxidant defense); and other regulators including IIS, mTOR, and longevity-\nassociated genes. This pathway-based organization emphasizes biological relevance and facilitates\ninterpretation.\nMinor Points\n• Fig. 7m: A ggridges plot would better show the data distribution.\nResponse: We thank the Reviewer for this excellent suggestion. In the revised manuscript, the\nprevious bar graph (former Fig. 7m) has been replaced by a new ridge plot (Fig. 70) that beautifully\ndisplays the full distribution of yeast cell areas for each strain and condition.\n10\n• Line 12: Change to “that integrates.”\nResponse: Fixed, thanks.\n• Line 108: Change to “had shorter lifespans.”\nResponse: Fixed, we apologize for the oversight.\n• Unnecessary abbreviations should be eliminated to improve readability.\nResponse: We thank the Reviewer for this helpful comment on readability. In the revised\nmanuscript, we have improved readability by eliminating unnecessary abbreviations, removing\nDR, among others, throughout the manuscript.\nThis manuscript presents interesting findings showing that the p38 MAPK–ATF-7 pathway orchestrates\ncross-tissue potassium homeostasis for organismal survival under potassium deficiency (PD). The\nauthors demonstrate that ASI sensory neurons communicate with the hypodermis in C. elegans and\nextend their observations to mammalian and yeast systems. Mutant worms with disruptions in the p38\nMAPK pathway exhibited markedly reduced survival in water lacking salts or nutrients. Supplementation\nwith potassium chloride (KCl), but not sodium chloride (NaCl), rescued the sensitivity of these\nmutants, suggesting that the pathway is critical when potassium is limiting. An EMS suppressor screen further\nidentified ATF-7, a downstream transcription factor of p38 MAPK, as an important regulator of\npotassium homeostasis. RNA-seq analysis also revealed CATP-3, a P-type ATPase, as a functional\ndownstream target. Together, these data provide potentially novel insights into the molecular mechanisms\nof potassium homeostasis across species.\nResponse: We thank Reviewer #2 for the thoughtful and constructive evaluation of our work. We\nare pleased that the reviewer finds the study interesting and recognizes the significance of our\nfindings on the p38 MAPK–ATF-7 pathway in orchestrating cross-tissue potassium homeostasis\nand promoting organismal survival under potassium deficiency. We appreciate the positive\nassessment of our genetic, physiological, and cross-species analyses, and we are encouraged that\nthe reviewer considers our insights into CATP-3/P-type ATPase regulation and conserved\npotassium homeostasis mechanisms to be potentially novel and valuable. We have carefully\naddressed all specific comments point-by-point in the responses below and have revised the\nmanuscript accordingly to improve clarity, rigor, and overall presentation.\nMajor Comments\n1. The standard nematode growth medium (NGM) contains peptone, cholesterol, NaCl, CaCl2, MgSO4,\nand potassium phosphate buffer, which are essential for worm growth and survival. The authors describe\nPD conditions as water supplemented with NaCl. However, this medium lacks many NGM components\nand is not specifically deficient only for potassium. The absence of other essential nutrients complicates\nthe interpretation. A true PD condition should include all other NGM components except potassium.\nThis limitation should be addressed directly.\nResponse: We thank the Reviewer for this important and well-taken comment. We agree that the\nstandard nematode growth medium (NGM) contains multiple essential components, and that a\nmedium lacking these constituents cannot be considered exclusively potassium-deficient. In our\noriginal experiments, the “PD medium” was intentionally simplified to a 0.1 M NaCl aqueous\nsolution to isolate ionic effects. Empirically, we found that inclusion of other NGM components in\nthis simplified system produced only minimal changes in survival compared to 0.1 M NaCl alone,\nwhich initially justified the use of this condition. Nonetheless, we fully acknowledge that the\nabsence of a potassium-free NGM control represented a limitation of the original study design.\nTo address this concern directly, we have now performed additional lifespan assays using a\npotassium-free NGM formulation that retains all other essential NGM components. This\npotassium-free NGM contained 100 mM NaCl, 25 mM sodium phosphate buffer (SPB; 2.38 g/L\n100 mg/L FUdR, and the same antibiotic cocktail used in all other experiments. These new data\nare presented in Extended Data Fig. 1d. In addition, we have included results from a solid-plate\nPD assay (Extended Data Fig. 1c) to exclude potential artifacts associated with liquid culture.\nAcross these conditions, p38 pathway mutants displayed markedly reduced survival in the\nabsence of extracellular potassium, and this defect was robustly rescued by K⁺ supplementation.\nImportantly, this requirement for potassium was independent of accompanying anions (Cl⁻ versus\nphosphate), buffering capacity, pH, the presence of E. coli OP50-1, other divalent cations present\nin standard growth media (including Ca²⁺ and Mg²⁺), and culture format (liquid versus solid).\nTogether, these systematic analyses demonstrate that the survival defect observed under PD\nconditions reflects a specific requirement for extracellular K⁺, rather than the absence of other\nessential NGM components.\n2. KCl supplementation substantially increased survival in both wild-type and sek-1 (and pmk-1) mutants,\nwith a stronger effect on the mutants. However, the magnitude of this effect raises the possibility of non-\nspecific lifespan extension by KCl. The specificity of this rescue should be clarified. In particular, the\neffects of other salts such as CaCl2 and MgSO4 should be examined or discussed.\nResponse: We thank the reviewer for raising this important point regarding the specificity of the\nKCl-mediated rescue. To address the possibility that the observed lifespan extension reflects a non-\nspecific salt or osmotic effect, we performed additional control experiments using other salts.\nSupplementation with CaCl₂ or the potassium-free NGM solution under otherwise identical\nconditions produced only minor or no improvement in survival in either wild-type or sek-1/pmk-1\nmutant animals, and the magnitude of these effects was substantially smaller than that observed\nwith KCl. Moreover, these salts failed to preferentially rescue the shortened survival of the p38\npathway mutants. Together, these results indicate that the survival benefit is not a general\nconsequence of increased ionic strength or salt supplementation, but is largely specific to K⁺. This\nspecificity is consistent with our model that potassium availability acts as a physiologically relevant\nsignal that engages p38 MAPK–dependent protective responses, rather than causing non-specific\nlifespan extension.\n3. Line 59: “Initially, wild type (N2) worms in water without salt supplementation exhibited a mean\nlifespan of approximately 19 days, comparable to that on standard nematode growth medium (NGM)\n13\nplates with live OP50-1 (Fig. 1b, Extended Data Fig. 1a and Extended Data Table 1)13, indicating normal\naging without significant stress under baseline conditions.” I disagree with this interpretation. The\nreported 19-day lifespan cannot be regarded as “normal aging without significant stress.” Instead, it likely\nrepresents a composite outcome of two opposing influences: lifespan extension due to food\ndeprivation (T. L. Kaeberlein et al., Aging Cell 5, 487–494 (2006); G. D. Lee et al., Aging Cell 5, 515–524 (2006))\nand a detrimental effect caused by salt deprivation. This sentence should therefore be rewritten to avoid\nimplying physiological normalcy under such conditions.\nMoreover, as this condition forms the foundation of the experimental paradigm used throughout the study,\nthe interpretation of all subsequent results requires careful reconsideration. For instance, the shortened\nlifespan of p38 MAPK mutants in water could be at least partly due to the established requirement of\np38 MAPK signaling for lifespan extension under food deprivation (a form of dietary restriction in C.\nelegans). This possibility should be experimentally tested through additional rigorous control\nexperiments and discussed thoroughly in the revised manuscript.\nResponse: We thank the Reviewer for this careful and important critique. We fully agree that\ndescribing the lifespan of wild-type worms in water as reflecting “normal aging without significant\nstress” was inappropriate, and we regret the imprecise wording. Our original intent was not to\nimply physiological normalcy, but rather to note that the net lifespan outcome under this condition\nhappened to be numerically comparable to that observed on standard NGM plates with live OP50-\n1. We now recognize that this comparison was conceptually misleading.\nAs the Reviewer correctly points out, lifespan in water likely reflects the combined influence\nof at least two opposing factors: lifespan extension due to bacterial food deprivation (dietary\nrestriction)14–16 and lifespan shortening caused by deprivation of essential salts and ions. We have\ntherefore revised the text to remove any implication of “normal aging” and to explicitly\nacknowledge this composite effect.\nThe revised sentence now reads: “Initially, wild type (N2) worms in water without salt\nsupplementation exhibited a mean lifespan of approximately 19 days, comparable to that on\nstandard nematode growth medium (NGM) plates with live OP50-1 (Fig. 1b, Extended Data Fig.\n1a and Extended Data Table 1)13. This observed lifespan likely reflects the net effect of two\ncounteracting factors: prolongation arising from bacterial food deprivation14-16 and a\ncountervailing shortening induced by the absence of essential salts.”\nWe also agree that this issue has important implications for interpretation of the shortened\nlifespan observed in p38 MAPK pathway mutants. p38 signaling has indeed been implicated in\nlifespan extension under dietary restriction–like conditions in C. elegans, raising the possibility\nthat the reduced survival of these mutants in water may partially reflect impaired adaptation to\nfood deprivation rather than salt deprivation alone.\nTo address this concern, we have expanded the experimental framework and discussion in the\nrevised manuscript. Specifically, we now (i) include potassium-deficient but otherwise complete\nNGM conditions (Extended Data Fig. 1d), (ii) perform survival assays on solid media with defined\nsalt composition while maintaining equivalent dietary inputs (Extended Data Fig. 1c), and (iii)\ndirectly compare conditions that differ in potassium availability while maintaining equivalent ionic\n14\ninputs (Extended Data Fig. 1a). These analyses demonstrate that loss of p38 pathway function leads\nto a selective vulnerability to potassium deprivation that cannot be explained solely by altered\nresponses to food deprivation. We have also revised the Discussion to explicitly acknowledge the\ninteraction between dietary restriction and ionic stress and to clarify the boundaries of our\nexperimental paradigm.\nMinor Comments\nLine 56: Define E. coli OP50-1 for readers unfamiliar with C. elegans culture.\nResponse: Thanks for this helpful suggestion. We have now defined E. coli OP50-1 as “a\nstreptomycin-resistant strain that is widely used as a standard food source for C. elegans in\nlaboratories”, and this information has been added in the revised manuscript.\nLine 62: Provide rationale for testing sek-1 mutants and the p38 MAPK pathway.\nResponse: Thank you for this suggestion. In the revised manuscript, we added a clear rationale\nparagraph immediately before the first mention of sek-1. “Although p38 MAPK signaling is well\nknown for its roles in longevity and innate immunity in C. elegans, prior studies have also\nimplicated the NSY-1(ASK1 MAPKKK)–SEK-1(MKK3/MKK6 MAPKK)–PMK-1(p38 MAPK)\ncascade in ionic and osmotic stress adaptation, including its function within the OSR-1–CaMKII–\nNSY-1 pathway for hyperosmotic resistance and its regulation of sodium leak channel activity that\nmight influences Na⁺/K⁺ homeostasis17-19. Together with our preliminary observations showing\nhypersensitivity of sek-1(km4) null mutants to electrolyte depletion, these findings prompted us to\ninvestigate whether the p38 MAPK pathway plays a critical role in sensing and responding to\npotassium-deficient stress. ”\nLine 63: Clarify the nature of the allele (loss-of-function, hypomorphic, or null).\nResponse: Thanks for this comment. The revised text now explicitly clarifies the nature of all alleles\nused in the study.\nLine 78: NGM contains additional ions beyond NaCl and potassium phosphate; please clarify.\nResponse: Thanks for this important point. As clarified in the revised text: (NGM: 51 mM NaCl,\n25 mM potassium phosphate buffer, 1 mM CaCl , and 1 mM MgCl )\n2 2\nLines 81–86: Explain why pH and pathogen sensitivity were tested.\nResponse: We appreciate the reviewer’s comment. We now clarify this point in the revised\nmanuscript. Extracellular pH can influence membrane potential and ion flux by modulating\nproton-sensitive K⁺ channels and, consequently, voltage-gated ion channel activity; therefore, we\ntested whether altered H⁺ levels contribute to the survival defects observed under PD. In addition,\nbecause the p38 MAPK pathway plays a central role in innate immunity, we also assessed pathogen\nsensitivity to determine whether the PD phenotype might be secondary to immune defects.\nResponse: We appreciate the reviewer’s this suggestion. The revised text now reads: “KCl\nsupplementation rescued sek-1(km4) survival in a strictly time- and dose-dependent manner:\nadding 50 mM KCl within the first 2 hours of transfer to potassium-deficient medium (100 mM\nNaCl) fully restored lifespan to wild-type levels, whereas additions at later time points (≥2 h)\nyielded only partial or no rescue (Fig. 1i). This indicates that p38 signaling mediates a rapid, early\nprotective response that must be engaged within a narrow temporal window to prevent irreversible\nphysiological damage during acute PD.”\nLine 91: The phenotypes in Fig. 1k are unclear; the sizes of sek-1 worms with 100 mM NaCl appear\nsimilar to wild type or sek-1 with 100 mM KCl. Representative images and quantitative analysis are\nneeded.\nResponse: Thanks for this helpful comment. To address this concern, we have added a new\nquantitative analysis of body length as Extended Data Fig. 1i, which confirms the morphological\ndifferences observed in the representative images. This analysis demonstrates that sek-1 mutants\nexhibit a significant reduction in body length specifically under 100 mM NaCl (PD) conditions,\nwhereas sek-1 animals in 100 mM KCl and wild-type controls show no appreciable size changes.\nLine 102: Citation numbers are italicized. Please correct formatting.\nResponse: Corrected, thanks.\nLine 103: Describe the alleles used for sek-1, nsy-1, and tir-1. As tir-1 alleles can produce different\nphenotypes, more explanation is necessary.\nResponse: We thank the Reviewer for this important request. We have added a detailed description\nof all alleles in the revised manuscript. We now describe the specific alleles used in detail. “sek-\n1(km4) is a null allele containing a 2.1 kb deletion, resulting in a complete loss of functional SEK-\n1 protein. nsy-1(ok593) is a null allele carrying a 1.9 kb deletion, and nsy-1(ag3) is a strong loss-of-\nfunction point mutant. For tir-1, we used tir-1(tm3036), a loss-of-function allele lacking the TIR-1\ndomain, and tir-1(qd4), a null allele generated by a 1,079 bp deletion28. In contrast, the tir-1(ok1052)\nallele contains a 1,959 bp deletion affecting a common exon shared by all tir-1 isoforms, but does\nnot alter innate immunity phenotypes. atf-7(qd22gf) is a gain-of-function (gf) mutant22. It produces\na constitutively active form of ATF-7 that mimics the phosphorylated state downstream of\np38/PMK-1 signaling. atf-7(qd22; qd130lf) is loss-of-function mutant. The qd130lf mutation\ndisrupts ATF-7 activity and genetically suppresses the constitutive activity of qd22gf,\ndemonstrating that the transcriptional effects of qd22 require an intact ATF-7 protein. atf-\n7(gk715lf) is a 1,197 bp deletion mutant, resulting in strongly reduced or abolished transcriptional\nactivity. This allele is commonly used as a functional null to assess ATF-7-dependent gene\nregulation.”\nLine 114: Both atf-7(lf) and atf-7(gf) shortened lifespan under PD. Please clarify the interpretation.\nnow explain the interpretation: Both loss-of-function and gain-of-function atf-7 alleles\ndramatically shortened lifespan under potassium deficiency, yet the constitutive-repressor atf-\n7(qd22gf) allele displayed a far more severe defect than atf-7(lf) alleles (Fig. 2f). This pattern is fully\nexplained by ATF-7’s well-characterized dual role as both transcriptional repressor (when\nunphosphorylated) and activator (when phosphorylated by PMK-1). Survival under PD requires\n(i) relief of basal ATF-7-mediated repression and (ii) subsequent active transcription of protective\ntarget genes. Loss-of-function alleles eliminate activation, whereas the gain-of-function allele\npermanently locks ATF-7 in its repressive state, preventing any induction of the protective\nprogram. The markedly stronger phenotype of atf-7(gf) therefore reveals that active repression of\nthese targets by unphosphorylated ATF-7 is considerably more deleterious than simple absence of\nactivation during severe potassium stress, highlighting the critical need for precise p38-dependent\nphospho-regulation of ATF-7.\nLine 118: Fig. 1j does not include atf-7 data.\nResponse: Thanks for pointing out this error, the citation has now been corrected, and we apologize\nfor the oversight.\nLine 125: jkk-1, jnk-1, tph-1, and ocr-2 do not seem to be well-studied genes involved in insulin/IGF-1\nand mTOR signaling. Justify their inclusion.\nResponse: We thank the Reviewer for this comment. As suggested, the jkk-1 and jnk-1 data have\nbeen removed. The ocr-2(yz5) and ocr-2(ak47) alleles are loss-of-function mutations in the TRPV\nchannel OCR-2, whose activity in ADF neurons regulates tph-1 expression and serotonin\nbiosynthesis, and is also required for pathogen avoidance. The tph-1(n4622) allele is a presumed\nnull deletion. Notably, ocr-2(yz5), ocr-2(ak47), and tph-1(n4622) mutants displayed only modest\nreductions in survival under PD, indicating that serotonin biosynthesis contributes only minimally\nto the PD response. This justification has been incorporated into the revised manuscript.\nLine 132: Fig. 1j does not include atf-7 data.\nResponse: Thanks for pointing out this error, the citation has now been corrected, and we apologize\nfor the oversight.\nLine 140: Clarify whether NGM (51 mM NaCl, 25 mM potassium phosphate buffer) is an appropriate\ncontrol.\nResponse: Thanks for this comment. Yes, standard NGM (51 mM NaCl, 25 mM potassium\nphosphate buffer) is an appropriate control, as it contains both sodium and potassium at\nphysiologically relevant levels and therefore represents normal ionic homeostasis under standard\nC. elegans culture conditions. In contrast, the 0.1 M NaCl and 0.1 M KCl treatments were\nintentionally designed as stress-modified conditions that selectively perturb extracellular ionic\ncomposition. Using NGM as the reference condition allows direct comparison of these stress-\nmodified environments to a single, physiologically defined baseline, while maintaining consistency\nwith standard C. elegans culture protocols.\nLine 151: Fig. 3b does not show the genes described in the text.\nResponse: We thank the Reviewer for pointing out this error, the citation has now been corrected,\nand we apologize for the oversight.\nLine 160: They tested both the top PD-upregulated and SEK-1-regulated genes\nResponse: Thank you very much. We now correct it.\nLine 167: Fig. 3j or Extended Data Fig. 3j does not contain the data described.\nResponse: Thanks for pointing out this error, we have adjusted the description.\nLine 176: Fig. 4e should be Extended Data Fig. 4e.\nResponse: Thank you very much. We have corrected it.\nLine 190: Explain rol-6 for broader readers.\nResponse: We appreciate the Reviewer’s suggestion. We have now added a brief explanation.\nExtrachromosomal transgenic arrays were generated by co-injecting the desired constructs\ntogether with the rol-6(su1006) plasmid, which served as a visible co-injection marker. The gain-\nof-function rol-6(su1006) allele induces a dominant Roller phenotype characterized by helical body\ntwisting, enabling straightforward identification of transgenic animals. Importantly, this marker\ndoes not alter lifespan under our experimental conditions.\nLine 193: Justify why ASI neurons were tested.\nResponse: Thank you for the comment. We have added a justification for examining ASI neurons\nin the revised manuscript. ASI sensory neurons in the head detect environmental cues like\nchemoattractants, temperature, and food availability, regulating metabolism. They are critical for\ndauer formation, satiety quiescence, and dietary restriction induced longevity, including p38-\ndependent innate immunity to pathogens. These roles suggest that ASI neurons might sense PD\nconditions.\nFig. 4e–i: Clarify whether rescue experiments used catp-3 mutants or wild-type worms; provide strain\ninformation in the figure legend.\nResponse: We appreciate the Reviewer’s helpful comment. The rescue experiments in Fig. 4e, g–i\nwere performed in the catp-3 mutant background, whereas the overexpression experiment in Fig.\n4f was conducted in wild-type animals. We have now revised Fig. 4 accordingly and added the\ncorresponding strain information to the figure legend for clarity.\nResponse: Thanks for this suggestion. The rationale is now explicitly stated in the revised\nmanuscript. Intestinal SEK-1 markedly increased the expression of the three well-characterized\nPMK-1/ATF-7 immune target genes (sysm-1, C17H12.8, K08D8.5) while suppressing catp-3,\nwhereas ASI-specific SEK-1 fully restored catp-3 expression but only partially increased immune\ngene levels (Fig. 6d).\nFigures 4 and 5: The reporter is labeled as catp-3::RFP. For accuracy and specificity, this should be\ncorrected to catp-3::mCherry.\nResponse: We appreciate the Reviewer’s helpful comment. We have corrected all instances of\n“catp-3::RFP” to the accurate designation “catp-3::mCherry” in Figures 4 and 5, as well as in the\ncorresponding legends and text.\nFigure 5d and e: The colors of orange line and the label do not match.\nResponse: Thanks for pointing out this error. We have adjusted them to the same color.\nFigure 6k-n: Explain the rationale for using NGM plate as a control.\nResponse: We thank the Reviewer for this comment. A clear justification has now been added in\nthe revised manuscript. NGM agar plates with OP50-1 bacteria were used as the physiological\nreference condition because they represent the standard, nutrient-replete, low-stress environment\nin which C. elegans is routinely cultured and aged. This allowed us to establish a clean baseline of\ncatp-3 and immune-gene expression under normal potassium availability (~25 mM K⁺ from\npotassium phosphate buffer). The subsequent 20-hour exposure to 100 mM NaCl (potassium\ndeficiency) agar plates with OP50-1 bacteria therefore specifically tests whether potassium\ndepletion alone is sufficient to trigger SEK-1-dependent transcriptional activation, while avoiding\nconfounding factors that arise when directly comparing NaCl and KCl in liquid, such as differences\nin ionic composition, osmolarity, or bacterial availability.\nThis study, \"p38 MAPK orchestrates cross-tissue potassium homeostasis for survival,\" uncovers a\npreviously uncharacterized role for p38/MAPK signaling in promoting survival during potassium\ndeficiency (PD) in C. elegans. The authors present convincing evidence that this pathway functions\nthrough hypodermal upregulation of CATP-3, a P-type ATPase potassium pump, and integrates both\nneuronal and peripheral signaling. They further extend their findings to mammalian cell lines and yeast\nmodels, demonstrating the conserved role of p38/MAPK-mediated regulation of P-type ATPases.\nOverall, the study is novel, the data are well supported, and the manuscript is clearly written and well organized.\nThere are some considerations, listed below, that should be addressed for clarity and to rule out\nalternative hypotheses.\nResponse: We thank Reviewer #3 for the positive and thoughtful evaluation of our work. We\nappreciate the recognition of the novelty, clarity, and broad relevance of our study, and we are\ngrateful for the constructive suggestions provided. We have carefully addressed each of the points\nraised by the Reviewer, as detailed below, and have revised the manuscript accordingly to improve\nclarity and strengthen our conclusions.\nItems to address:\n1. Fasting is known to activate p38/MAPK signaling. It would be informative to test whether KCl\nsupplementation triggers fed-like signaling or suppresses the fasting response in the worms.\nResponse: We fully agree with the Reviewer that this is an important and intriguing question.\nFeeding and fasting states are associated with distinct signaling pathways and physiological\nresponses in C. elegans, commonly assessed by IIS activity (e.g., DAF-16 localization and target\ngene expression), AMPK phosphorylation, and autophagy levels. p38/MAPK signaling has also\nbeen reported to respond to nutritional status; however, its regulation is highly time-dependent.\nShort-term fasting can suppress p38 activity, whereas prolonged fasting may activate p38, while\nlong-term dietary restriction has been reported to reduce p38 signaling. Thus, the relationship\nbetween p38 activity and feeding/fasting reflects complex, time-course–dependent changes.\nIn addition, p38 signaling responses to both nutritional cues and potassium deprivation (PD)\nare often context- and tissue-specific. As requested, we transferred day-1 adult wild-type worms to\nliquid PD conditions (0.1 M NaCl) with OP50-1 for 24 hours, followed by supplementation with\n100 mM KCl for either 4 hours or 24 hours. qPCR analysis showed that sysm-1 (a feeding-\nupregulated gene and a known p38/ATF-7 downstream target) as well as catp-3 mRNA levels were\nsuppressed upon KCl supplementation at both time points.\nThese results suggest that KCl supplementation might not simply restore a canonical feeding-\nlike transcriptional program, but rather modulate p38-dependent signaling in a distinct manner.\nWhether KCl suppresses specific fasting-associated responses or induces a fed-like signaling state\nthrough p38 remains an interesting and complex question that warrants further systematic\ninvestigation.\n20\n2. Pharyngeal pumping in C. elegans relies on Na⁺/K⁺-ATPase activity. How do the conditions used in\nthis manucript (e.g., PD and KCl supplementation) affect pharyngeal pumping behavior?\nResponse: We appreciate the Reviewer’s thoughtful question. Pharyngeal pumping is primarily\ndriven by MC/M3 motor neurons and requires the Na⁺/K⁺-ATPase EAT-6. Several lines of evidence\nindicate that the PD and KCl conditions used in our study do not confound our findings through\naltered pumping behavior. First, the key genetic manipulations that produced strong phenotypes—\nsuch as ASI-specific sek-1 rescue or ASI-specific RNAi—target neurons that are not involved in\npharyngeal pumping, making pumping-rate changes an unlikely explanation. Second, catp-3\nexpression and functional requirement are localized to the hypodermis rather than the pharynx,\nfurther reducing the likelihood that altered pumping underlies the observed survival effects.\nFinally, worms under short-term PD (20 h), the window in which transcriptional measurements\nwere taken, do not display overt defects in feeding or pharyngeal movement. Together, these points\nsupport that the PD survival phenotypes and transcriptional changes we report are not\nattributable to changes in pharyngeal pumping.\n3. While p38/MAPK is required for survival under PD, it remains unclear whether it is actively induced\nduring this condition and subsequently returns to basal levels upon KCl addition or is just required.\nResponse: We thank the Reviewer for raising this important point. Our data indicate that\np38/MAPK signaling is not only required for survival under PD but is engaged within a very\nnarrow temporal window that determines reversibility of physiological damage.\nAs shown in Fig. 1, PD markedly reduces lifespan in wild-type worms and induces rapid,\nirreversible physiological damage in sek-1 mutants. Consistent with this, KCl supplementation\nrescues survival only when applied within a short time window after transfer to PD conditions,\nindicating that the protective p38-dependent response must be activated early. This temporal\nconstraint suggests that p38/MAPK signaling is actively involved during PD rather than serving\nmerely a permissive or constitutive role.\nTo assess whether p38 activity returns to basal levels upon potassium restoration, we\ncompared short (4 h) and prolonged (24 h) KCl supplementation following PD. As described above,\nextending KCl treatment from 4 to 24 hours did not result in substantial additional changes,\nsuggesting that p38-dependent transcriptional outputs do not rapidly revert to basal levels under\nthese conditions. This likely reflects the persistence of PD-induced physiological stress rather than\nsimple normalization upon KCl repletion.\nImportantly, the dynamics of p38 activation and recovery are influenced by multiple variables,\nincluding developmental stage, duration of PD exposure, culture conditions, and tissue-specific\n21\nsensitivity. We speculate that brief PD followed by rapid KCl repletion may allow partial\nrestoration toward basal signaling states, whereas prolonged PD causes irreversible damage in\nspecific tissues that cannot be fully rescued by either rapid or extended KCl supplementation. In\naddition, PD and KCl timing may also influence developmental progression, which itself impacts\nbasal p38/MAPK activity.\nTogether, these observations support a model in which p38/MAPK signaling is actively\nengaged during early PD to promote survival, but prolonged PD leads to irreversible damage that\nlimits recovery even after potassium restoration.\n4. RNA-seq samples were prepared in the presence of bacteria, whereas the survival assays were\nperformed in the absence of bacteria. Do these differences result in distinct levels of potassium\ndeprivation between the two conditions? And does the p38-mediated survival still hold in these\nconditions?\nResponse: We appreciate the Reviewer’s thoughtful question. Although the RNA-seq and survival\nassays differed in the presence of bacteria, several lines of evidence indicate that these conditions\ndo not create divergent potassium levels nor alter the requirement for p38 signaling.\nFirst, as addressed above for Reviewer #1, we have now generated a new set of RNA-seq\ndatasets under exactly the same conditions as the survival assays—liquid medium, no bacteria, and\nmatched NaCl/KCl treatments. For each genotype (N2 and pmk-1), ~2,000 synchronized young\nadults were exposed for 4 hours to either 100 mM NaCl or 100 mM KCl before RNA extraction.\nThese new transcriptomes fully recapitulate and strengthen the original findings (Fig. 3i–l;\nExtended Data Fig. 3c): 105 genes are significantly induced by potassium deficiency in wild-type\nworms, including hypodermal genes, innate immune effectors, and catp-3, all showing clear PMK-\n1 dependence. This ensures that the transcriptomic results directly correspond to the physiological\nstate of animals in the survival assays, removing differences due to agar, live bacteria, or\ndevelopmental stage.\nSecond, despite these procedural differences, we observe highly consistent physiological\noutcomes: the transcriptional response captured by both liquid/solid RNA-seq (including\nupregulation of immune genes and catp-3) matches the direction and magnitude of the changes we\nindependently validated by qPCR under the bacteria-containing solid conditions. This\nconcordance indicates that p38 activation reflects potassium deficiency itself, not secondary effects\narising from bacterial presence.\nThird, the requirement for p38 signaling is preserved across all conditions. sek-1 and pmk-1\nmutants are highly sensitive to PD in both bacteria-free liquid assays and bacteria-containing solid\nassays, and the same p38-dependent transcriptional patterns appear in RNA-seq performed with\nor without bacteria.\nTogether, these results demonstrate that the presence of bacteria does not substantially alter\npotassium deprivation and does not affect the essential role of the p38 pathway in promoting\nsurvival under PD.\n22\np38 MAPK orchestrates cross-tissue potassium homeostasis for survival\nPoint-by-Point Response to Reviewers’ comments\nWe thank all Reviewers for their thorough, constructive, and insightful comments. In response, we\nhave performed additional experiments and revised the manuscript accordingly. All changes are\nhighlighted in red in the revised manuscript. We have also provided a detailed Reporting Summary,\na Source Data file, and an updated Data Availability statement.\nReviewer #1 (Remarks to the Author):\nThe authors responded promptly and addressed most of my concerns, even conducting new experiments.\nMoreover, the manuscript is now much clearer and explains the reasoning behind certain experimental\nchoices. Some outstanding points remain for the authors to resolve:\n1. The explanation regarding catp-3 overexpression in PD doesn't align with established knowledge about\nhow lowering extracellular K+ affects membrane potential. A decrease in extracellular K+ typically\nleads to membrane hyperpolarization, which is associated with muscle twitching. Prolonged reduction in\nextracellular K+ likely also lowers intracellular K+ concentrations due to its reduction outside the cell.\nTherefore, the organism's increased expression of catp-3 may be an effort to restore intracellular K+\nlevels.\nResponse: We appreciate the reviewer’s insightful comment. We apologize for any lack of clarity\nin our previous explanation. In the revised manuscript, we edit and improve the text as “A decrease\nin extracellular K⁺ typically hyperpolarizes the membrane potential, which would reduce\nexcitability. However, prolonged reduction in extracellular K⁺ can lead to secondary depletion of\nintracellular K⁺ due to passive leaks and incomplete compensation by other transporters. Thus, the\nupregulation of catp-3 under PD conditions may enhance pump capacity to restore intracellular K⁺\nlevels, thereby stabilizing membrane potential, osmotic balance, and cellular viability under PD\nconditions.”\n2. On line 253 of the tracked changes file, the statement \"This could remove the effect of liquid, and the\nbacteria food\" appears right after noting that NGM plates were seeded with OP50, suggesting food was\nactually present. The authors should clarify this point since all liquid experiments were performed\nwithout food. If food was included, these experiments would not accurately model liquid culture\nconditions.\nResponse: We thank the reviewer for raising this important point and apologize for the confusion\ncaused by our description. In fact, we performed two independent transcriptomic experiments\nunder distinct culture conditions, each designed to address a different experimental question.\nwhich represents the standard growth condition and was used to assess transcriptional responses\nunder physiologically relevant, food-present conditions.\nSecond, a separate RNA-seq experiment was conducted in liquid culture in the absence of\nbacterial food, matching the conditions used in our liquid survival assays. This experiment was\nspecifically designed to isolate the effects of potassium deficiency and ionic environment without\nconfounding influences from bacterial metabolism or liquid-associated feeding behavior.\nWe agree that the wording was unclear and have now revised the manuscript to explicitly\ndistinguish these two transcriptomic datasets, clearly stating their respective culture conditions\nand experimental purposes.\n3. On line 352 in the tracked changes file, it is stated that atf-7 strongly induce catp-3 mRNA. However,\nthe graph demonstrates no significant statistical difference.\nResponse: We thank the reviewer for pointing out this inconsistency. We agree that the term\n“strongly induce” was an overstatement given that the difference shown in the graph does not\nreach statistical significance. We have revised the text to adopt more cautious wording, describing\nthe effect of ATF-7 on catp-3 mRNA expression as a trend rather than a strong induction.\nAccordingly, we now state that as “Consistent with prior findings, activation of the pathway by\nRNAi knockdown of the phosphatase vhp-1 or overexpression of sek-1 and atf-7 showed a trend\ntoward increased catp-3 mRNA expression, suggesting that p38–ATF-7 signaling may contribute\nto catp-3 transcriptional regulation (Fig. 5a).”\n4. Figure 2a, which shows the pathway and lists various genes, is cited in the text incorrectly as Figure\n1a.\nResponse: Fixed, we apologize for the oversight.\n5. Data Labeling: Clearly indicate which part of the data is derived from the 15°C culture.\nResponse: We thank the reviewer for this comment. We apologize for the confusion caused by the\nlabeling. All experiments and datasets presented in this study were conducted at 20°C, and no data\nwere derived from 15°C culture conditions. We have now corrected the text in Methods as “Worms\nwere maintained at 20°C on standard nematode growth medium (NGM) agar plates seeded with\nEscherichia coli OP50-1 (a streptomycin-resistant strain for worm food).”\n6. Labeling Adjustments: The labels \"Sek-1 OE\" and \"sek-1 ASI\" are not easily recognizable. Please\nreplace them with \"promoter: sek-1 gDNA/cDNA\" for clarity.\nResponse: We thank the reviewer for this helpful suggestion. Owing to space constraints and layout\nconsiderations within the figure panels, it was not feasible to include the full transgene descriptions\ndirectly in the figure labels. However, we have already provided the complete transgene\ninformation in the figure legends.\n2\n7. Figures Suggestions:\no Figure 1: Change all lifespan curve colors to semi-transparent for better visibility. Clarify whether\n\"water\" refers to M9 buffer. Ensure the order of lifespan labels follows a logical sequence, either from\nlow-to-high or control to mutant. Currently, \"B-e water\" is listed first, while \"f-h water\" is last, which is\nconfusing. Panels D and E appear smaller than panels F-H; consider adjusting their sizes for consistency.\nExpand the X-axis to greater than 30 to avoid cutoff data in panel I.\nResponse: We thank the reviewer for the detailed and constructive suggestions regarding Figure 1.\nWe have revised the figures (Fig. 1, 2, 4, 6) accordingly to improve clarity and consistency.\nSpecifically, we have revised all lifespan curves to improve visibility and reduce overlap among\ntraces, including adjustments to line color, line width, and plotting order. In this study, “water”\nrefers to distilled water, not M9 buffer. We have clarified this explicitly in the figure panels to avoid\nambiguity. All lifespan assays labeled as “water” were performed under identical conditions, and\nthe use of distilled water was consistent across the corresponding experimental groups. The order\nof lifespan labels has been reorganized to follow a logical sequence from control to mutant\nconditions, thereby improving readability and interpretation. Panel sizes have been adjusted so\nthat panels D and E are consistent with panels F–H. In addition, the X-axis in panel I has been\nextended beyond 30 days to ensure that all data points are fully displayed without truncation.\no Figure 2: Apply the same suggestions as Figure 1. Setting the X-axis to 45 may improve clarity.\nResponse: Fixed, thanks.\no Figure 4: The same control data for catp-3(ok1612) survival and WT groups was used. Please use a\ndotted line to indicate when the same data is utilized multiple times.\nResponse: We thank the reviewer for this suggestion. The same control survival data for catp-\n3(ok1612) and WT were intentionally reused in Figure 4 to allow direct comparison across\nexperimental conditions. We have now indicated the reused control data with dotted lines in all\nlifespan curves in the figures (Fig. 1, 2, 4, 6) and clarified this explicitly in the figure legend to\nensure transparency.\no Figure 6: The calculation of mRNA expression is incorrect. The normalization using the △△Ct method\nrequires values from △Ct for your three control samples; it cannot always be 1. Please refer to the\nguidelines at Nature Protocols. Additionally, consider logging the Y-axis for better representation.\nResponse: We thank the reviewer for the clarification. We agree that the housekeeping gene (tba-\n1) should not be treated as a target gene in ΔΔCt analysis or displayed as relative expression values.\nthe reference gene tba-1 was used solely for normalization of target gene expression, and its Ct\nvalues were stable across samples. We have revised the presentation to ensure that relative\nexpression is calculated and shown only for target genes, normalized to the mean ΔΔCt of the\nbiological control group, in accordance with standard qPCR guidelines.\no Figure 7: Methods related to the ggridges plot were not found in the methods section. Please include\n3\nthis information.\nResponse: We thank the reviewer for pointing out this omission. We have now added a detailed\ndescription of the methods used to generate the ggridges plot in the Methods section: “The ggridges\nplots were generated using OmicStudio (https://www.omicstudio.cn, version V1.0) to visualize the\ndistribution of cell size measurements across the indicated experimental groups. Single-cell size\ndata were imported into OmicStudio and displayed as ridge density plots based on kernel density\nestimation. Each ridge represents the probability density distribution of cell size for one group,\nwith ridge transparency (alpha) set to 0.6, allowing comparison of distribution patterns, dispersion,\nand shifts in central tendency between groups.”\nThe authors have addressed most of my concerns, and the revised manuscript has been greatly improved.\nIn particular, the new lifespan experiments performed under different medium conditions, as well as the\nnew RNA-seq experiment conducted under consistent conditions, further strengthen the authors’\nconclusions. I have only minor comments that should be addressed prior to publication.\nComments\nPreviously, I pointed out that the life-extending effect of KCl supplementation is not specific to sek-1\nmutants, as KCl also increases lifespan in wild-type animals as well as in sek-1 and pmk-1 mutants. This\nissue has not been addressed in the revised manuscript. In line 85, the authors state that KCl markedly\nrescues the survival of sek-1 and pmk-1 mutants; however, this rescue could reflect a combination of a\ngeneral lifespan-extending effect of KCl and the rescue of increased susceptibility of sek-1 mutants to\npotassium deficiency. This alternative explanation should be discussed in the text.\nResponse: We thank the reviewer for reiterating this important point. As noted by the reviewer,\nKCl supplementation also increases lifespan in wild-type animals as well as in sek-1 and pmk-1\nmutants. We therefore agree that the apparent “rescue” of sek-1 and pmk-1 survival by KCl likely\nreflects a combination of two effects: (i) a general lifespan-extending effect of KCl, and (ii) a specific\nmitigation of the heightened susceptibility of sek-1 and pmk-1 mutants to potassium deficiency. We\nhave revised the text: “Furthermore, KCl, rather than NaCl, markedly rescued the survival of sek-\n1 and pmk-1 (p38 MAPK) null mutants in water (Fig. 1b–e and Extended Data Fig.1a). This effect\nis consistent with a model in which the apparent rescue reflects a combination of a general lifespan-\nextending effect of KCl and the alleviation of the increased susceptibility of sek-1 and pmk-1\nmutants to PD.”\nLine 87: “...indicating that p38 signaling is essential for survival specifically when exogenous K⁺ is\nlimited, rather than Na⁺, Ca²⁺, or Cl⁻.” While the effect of Ca²⁺ on the survival of p38 pathway mutants\nis shown in Extended Data Fig. 1b, it is not described in the main text. Please explain these data and cite\nthe corresponding figure.\nResponse: We thank the reviewer for this helpful comment. We agree that the effects of Ca²⁺ on the\nsurvival of p38 pathway mutants should be explicitly described in the main text. We have now\nrevised the text: “In addition, supplementation with Ca2+ failed to improve the survival of sek-1\nmutants under water conditions (Extended Data Fig. 1b), further supporting the idea that p38\nsignaling is particularly required for survival under conditions of limited exogenous K+, rather\nthan upon exposure to Na+, Ca2+ or Cl–. Together, these results suggest that p38 signaling promotes\nsurvival by mitigating PD (100 mM NaCl without exogenous K+) stress and argue against a\nprimary role for nonspecific osmotic imbalance.”\nExtended Data Fig. 1i: The y-axis is labeled as “pixel.” Although this representation can show relative\n5\ndifferences in size, pixel size can be different depending on imaging conditions. Please convert the\nmeasurements to physical units (µm or mm) or present normalized values for clarity.\nResponse: We thank the reviewer for pointing out this issue. We have now converted the pixel-\nbased measurements to physical units by applying the calibrated pixel-to-length conversion for the\nimaging system. The y-axis in Extended Data Fig. 1i has been updated to report values in\nmillimeters (mm) in the revised figure.\nWhile the reviewers responded well to the fourth item to address, the first three responses were\ninadequate. Each of these comments is experimentally quite easy to address and will both clarify the\nresults and test alternative possibilities.\nPrevious comment 1: Fast Fasting is known to activate p38/MAPK signaling. It would therefore be\ninformative to determine whether KCl supplementation induces a fed-like signaling state or suppresses\nthe fasting response in C. elegans.\nComment 1 response to author response: To address this, the authors need to measure the expression of\nestablished fasting-responsive genes and examine whether their expression is reversed upon KCl\ntreatment. This was not done.\nResponse: To directly test whether KCl supplementation suppresses the fasting response or induces\na fed-like signaling state, we measured the expression of established fasting-responsive genes in C.\nelegans. Specifically, we analyzed lgg-1 (autophagy-related), atgl-1 (lipolysis), sod-3 (a canonical\nDAF-16/FOXO target), and pha-4 (a dietary restriction–induced transcription factor).\nYoung adult wild-type worms were transferred to liquid potassium-deficient (PD; 0.1 M NaCl)\nconditions with OP50-1 for 24 hours, followed by supplementation with 100 mM KCl for either 4\nhours or 24 hours. Quantitative PCR analysis revealed that the expression levels of these four\nfasting-responsive genes showed no significant changes upon KCl supplementation at either time\npoint, under liquid conditions with or without food.\nConsistent with these results, analysis of our RNA-seq datasets likewise showed no significant\ndifferential expression of lgg-1, atgl-1, sod-3, or pha-4 between 0.1 M NaCl and 0.1 M KCl\nconditions. Together, these data indicate that KCl supplementation does not reverse the fasting\ntranscriptional program nor induce a fed-like signaling state, arguing against the possibility that\nthe protective effects of potassium are mediated by suppression of fasting-induced p38/MAPK\nsignaling.\nPrevious comment 2: Pharyngeal pumping in C. elegans depends on Na⁺/K⁺-ATPase activity. It is unclear\nhow the experimental conditions used in this manuscript (e.g., PD exposure and KCl supplementation)\naffect pharyngeal pumping behavior.\nComment 2 response to author responsese: Have the authors measured pumping rates under these\nconditions to rule out altered feeding behavior as a confounding factor? This is a very easy question to\nanswer, and correlations to other (uncited) works does not answer it.\nResponse: To directly address whether altered feeding behavior could confound our results, we\nmeasured pharyngeal pumping rates under the same liquid survival conditions used in the\nmanuscript. Young adult worms were transferred to liquid medium without food containing either\n0.1 M NaCl (PD) or 0.1 M KCl for 24 hours, and pumping rates were quantified thereafter. Under\nthese conditions, PD exposure did not significantly alter pharyngeal pumping rates in wild-type\nworms, indicating that potassium deficiency per se does not impair pumping behavior in a normal\ngenetic background. In contrast, pmk-1 mutants exhibited a significant reduction in pumping rates\nunder PD, suggesting reduced physiological activity or stress sensitivity specific to this genotype.\nNotably, under liquid fasting conditions, overall pumping rates were lower and more\nheterogeneous than those typically observed on food-containing solid plates, with individual\nanimals showing variable pumping frequencies. This pattern closely resembles previously\ndescribed starvation-induced pumping behavior on solid media, indicating that the liquid\nenvironment itself does not uniquely suppress pharyngeal activity beyond the expected effects of\nfasting. Together, these data demonstrate that neither PD exposure nor KCl supplementation alters\npharyngeal pumping in wild-type worms, and therefore altered feeding behavior is unlikely to\naccount for the observed survival phenotypes.\nunclear whether this pathway is actively induced during PD exposure and subsequently returns to basal\nlevels following KCl supplementation, or whether p38/MAPK activity is constitutively required but not\ndynamically regulated.\nComment 3 response to author response: The authors state: “As requested, we transferred day-1 adult\nwild-type worms to liquid PD conditions (0.1 M NaCl) with OP50-1 for 24 hours, followed by\nsupplementation with 100 mM KCl for either 4 hours or 24 hours. qPCR analysis showed that sysm-1 (a\nfeeding-upregulated gene and a known p38/ATF-7 downstream target) as well as catp-3 mRNA levels\nwere suppressed upon KCl supplementation at both time points.” The rationale for using bacteria-fed\nworms under these conditions is not clear. In addition, it remains unclear how the expression of\np38/MAPK target genes changes in the presence or absence of KCl, and whether these changes are\ndirectly attributable to KCl supplementation.\nResponse: To clarify whether p38/MAPK signaling is dynamically regulated during PD exposure\nand KCl supplementation, or instead constitutively required for survival, we examined the\nexpression of two well-established p38/PMK-1 downstream target genes: sysm-1 (feeding- and\npathogen-induced) and catp-3 (PD-induced). Young adult wild-type worms were transferred to\nliquid potassium-deficient (PD; 0.1 M NaCl) conditions for 24 hours, followed by supplementation\nwith 100 mM KCl for either 4 hours or 24 hours. To explicitly address the reviewer’s concern\nregarding food effects, these experiments were performed both in the presence and absence of\nOP50-1.\nUnder liquid conditions with food, sysm-1 expression was not strongly induced by PD and was\nmodestly suppressed upon KCl supplementation at 4 hours, with no further suppression at 24\nhours. In contrast, catp-3 expression increased progressively with prolonged PD exposure and was\nsignificantly suppressed by KCl supplementation at 4 hours, with further reduction at 24 hours.\nUnder liquid conditions without food, sysm-1 expression was low and not responsive to either\nPD or KCl supplementation, consistent with the known dependence of sysm-1 on feeding cues. This\nobservation is in agreement with our RNA-seq data, in which sysm-1 was not induced by PD in\nliquid culture without food, but was induced under PD conditions on solid plates with food (Fig. 3).\nImportantly, under the same fasting conditions, catp-3 expression remained strongly PD-induced\nand was robustly suppressed by KCl supplementation.\nTogether, these data indicate that p38/MAPK target genes are not uniformly or globally\ninduced by PD, and that their regulation is highly context-dependent. While sysm-1 responds\nprimarily to feeding status, catp-3 is selectively sensitive to potassium availability, showing dynamic\n9\ninduction by PD and rapid repression upon KCl supplementation regardless of food. These\nfindings support a model in which p38/MAPK signaling is required for survival under PD but is\nnot globally activated as a canonical stress response, with potassium-sensitive regulation acting on\na subset of p38 downstream targets such as catp-3.\nComment 4: Addressed\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and effort in co-reviewing this manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and effort in co-reviewing this manuscript.\n11\np38 MAPK orchestrates cross-tissue potassium homeostasis for survival\nPoint-by-Point Response to Reviewers’ comments\nReviewer #1 (Remarks to the Author):\nI am satisfied with how the authors addressed my concerns.\nReviewer #2 (Remarks to the Author):\nThe authors addressed my concerns successfully.\nReviewer #3 (Remarks to the Author):\nWe thank the authors for providing data in answer to the remaining review concerns. However, upon\nreview of the revised manuscript, it appears they failed to incorporate their data into the manuscript. The\npurpose of these experiments was as important controls that should be discussed in the manuscript and\nincluded in supporting data. Please add these data and the discussion of the results into the manuscript.\nResponse: We thank the reviewer for pointing this out and apologize for the oversight. We have\nnow fully incorporated these experiments into the revised manuscript. Specifically, the pharyngeal\npumping analyses (Supplementary Fig. 1j) and fasting-responsive gene expression data\n(Supplementary Fig. 5) have been added to the Results section, and the corresponding\nexperimental details are described in the Methods.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\n1"}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors responded promptly and addressed most of my concerns, even conducting new experiments. Moreover, the\nmanuscript is now much clearer and explains the reasoning behind certain experimental choices. Some outstanding points\nremain for the authors to resolve:\n1. The explanation regarding catp-3 overexpression in PD doesn't align with established knowledge about how lowering\nextracellular K+ affects membrane potential. A decrease in extracellular K+ typically leads to membrane hyperpolarization,\nwhich is associated with muscle twitching. Prolonged reduction in extracellular K+ likely also lowers intracellular K+\nconcentrations due to its reduction outside the cell. Therefore, the organism's increased expression of catp-3 may be an\neffort to restore intracellular K+ levels.\n2. On line 253 of the tracked changes file, the statement \"This could remove the effect of liquid, and the bacteria food\"\nappears right after noting that NGM plates were seeded with OP50, suggesting food was actually present. The authors\nshould clarify this point since all liquid experiments were performed without food. If food was included, these experiments\nwould not accurately model liquid culture conditions.\n3. On line 352 in the tracked changes file, it is stated that atf-7 strongly induce catp-3 mRNA. However, the graph\ndemonstrates no significant statistical difference.\n4. Figure 2a, which shows the pathway and lists various genes, is cited in the text incorrectly as Figure 1a.\n5. Data Labeling: Clearly indicate which part of the data is derived from the 15°C culture.\n6. Labeling Adjustments: The labels \"Sek-1 OE\" and \"sek-1 ASI\" are not easily recognizable. Please replace them with\n\"promoter: sek-1 gDNA/cDNA\" for clarity.\n7. Figures Suggestions:\no Figure 1: Change all lifespan curve colors to semi-transparent for better visibility. Clarify whether \"water\" refers to M9\nbuffer. Ensure the order of lifespan labels follows a logical sequence, either from low-to-high or control to mutant. Currently,\n\"B-e water\" is listed first, while \"f-h water\" is last, which is confusing. Panels D and E appear smaller than panels F-H;\nconsider adjusting their sizes for consistency. Expand the X-axis to greater than 30 to avoid cutoff data in panel I.\no Figure 2: Apply the same suggestions as Figure 1. Setting the X-axis to 45 may improve clarity.\no Figure 4: The same control data for catp-3(ok1612) survival and WT groups was used. Please use a dotted line to indicate\nwhen the same data is utilized multiple times.\no Figure 6: The calculation of mRNA expression is incorrect. The normalization using the △△Ct method requires values from\n△Ct for your three control samples; it cannot always be 1. Please refer to the guidelines at Nature Protocols. Additionally,\nconsider logging the Y-axis for better representation.\no Figure 7: Methods related to the ggridges plot were not found in the methods section. Please include this information.", "reviewer_2": "The authors have addressed most of my concerns, and the revised manuscript has been greatly improved. In particular, the\nnew lifespan experiments performed under different medium conditions, as well as the new RNA-seq experiment conducted\nunder consistent conditions, further strengthen the authors’ conclusions. I have only minor comments that should be\naddressed prior to publication.\nComments\nPreviously, I pointed out that the life-extending effect of KCl supplementation is not specific to sek-1 mutants, as KCl also\nincreases lifespan in wild-type animals as well as in sek-1 and pmk-1 mutants. This issue has not been addressed in the\nrevised manuscript. In line 85, the authors state that KCl markedly rescues the survival of sek-1 and pmk-1 mutants;\nhowever, this rescue could reflect a combination of a general lifespan-extending effect of KCl and the rescue of increased\nsusceptibility of sek-1 mutants to potassium deficiency. This alternative explanation should be discussed in the text.\nLine 87: “...indicating that p38 signaling is essential for survival specifically when exogenous K⁺ is limited, rather than Na⁺,\nCa²⁺, or Cl⁻.” While the effect of Ca²⁺ on the survival of p38 pathway mutants is shown in Extended Data Fig. 1b, it is not\ndescribed in the main text. Please explain these data and cite the corresponding figure.\nExtended Data Fig. 1i: The y-axis is labeled as “pixel.” Although this representation can show relative differences in size,\npixel size can be different depending on imaging conditions. Please convert the measurements to physical units (µm or mm)\nor present normalized values for clarity.", "reviewer_3": "While the reviewers responded well to the fourth item to address, the first three responses were inadequate. Each of these\ncomments is experimentally quite easy to address and will both clarify the results and test alternative possibilities.\nPrevious comment 1: Fast Fasting is known to activate p38/MAPK signaling. It would therefore be informative to determine\nwhether KCl supplementation induces a fed-like signaling state or suppresses the fasting response in C. elegans.\nComment 1 response to author response: To address this, the authors need to measure the expression of established\nfasting-responsive genes and examine whether their expression is reversed upon KCl treatment. This was not done.\nPrevious comment 2: Pharyngeal pumping in C. elegans depends on Na⁺/K⁺-ATPase activity. It is unclear how the\nexperimental conditions used in this manuscript (e.g., PD exposure and KCl supplementation) affect pharyngeal pumping\nbehavior.\nComment 2 response to author responsese: Have the authors measured pumping rates under these conditions to rule out\naltered feeding behavior as a confounding factor? This is a very easy question to answer, and correlations to other (uncited)\nworks does not answer it.\nPrevious comment 3: Although p38/MAPK is required for survival under PD conditions, it remains unclear whether this\npathway is actively induced during PD exposure and subsequently returns to basal levels following KCl supplementation, or\nwhether p38/MAPK activity is constitutively required but not dynamically regulated.\nComment 3 response to author response: The authors state: “As requested, we transferred day-1 adult wild-type worms to\nliquid PD conditions (0.1 M NaCl) with OP50-1 for 24 hours, followed by supplementation with 100 mM KCl for either 4 hours\nor 24 hours. qPCR analysis showed that sysm-1 (a feeding-upregulated gene and a known p38/ATF-7 downstream target)\nas well as catp-3 mRNA levels were suppressed upon KCl supplementation at both time points.” The rationale for using\nbacteria-fed worms under these conditions is not clear. In addition, it remains unclear how the expression of\np38/MAPK target genes changes in the presence or absence of KCl, and whether these changes are directly attributable to KCl\nsupplementation.\nComment 4: Addressed", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "p38 MAPK orchestrates cross-tissue potassium homeostasis for survival\nPoint-by-Point Response to Reviewers’ comments\nWe thank all Reviewers for their thorough, constructive, and insightful comments. In response, we\nhave performed additional experiments and revised the manuscript accordingly. All changes are\nhighlighted in red in the revised manuscript. We have also provided a detailed Reporting Summary,\na Source Data file, and an updated Data Availability statement.\nReviewer #1 (Remarks to the Author):\nThe authors responded promptly and addressed most of my concerns, even conducting new experiments.\nMoreover, the manuscript is now much clearer and explains the reasoning behind certain experimental\nchoices. Some outstanding points remain for the authors to resolve:\n1. The explanation regarding catp-3 overexpression in PD doesn't align with established knowledge about\nhow lowering extracellular K+ affects membrane potential. A decrease in extracellular K+ typically\nleads to membrane hyperpolarization, which is associated with muscle twitching. Prolonged reduction in\nextracellular K+ likely also lowers intracellular K+ concentrations due to its reduction outside the cell.\nTherefore, the organism's increased expression of catp-3 may be an effort to restore intracellular K+\nlevels.\nResponse: We appreciate the reviewer’s insightful comment. We apologize for any lack of clarity\nin our previous explanation. In the revised manuscript, we edit and improve the text as “A decrease\nin extracellular K⁺ typically hyperpolarizes the membrane potential, which would reduce\nexcitability. However, prolonged reduction in extracellular K⁺ can lead to secondary depletion of\nintracellular K⁺ due to passive leaks and incomplete compensation by other transporters. Thus, the\nupregulation of catp-3 under PD conditions may enhance pump capacity to restore intracellular K⁺\nlevels, thereby stabilizing membrane potential, osmotic balance, and cellular viability under PD\nconditions.”\n2. On line 253 of the tracked changes file, the statement \"This could remove the effect of liquid, and the\nbacteria food\" appears right after noting that NGM plates were seeded with OP50, suggesting food was\nactually present. The authors should clarify this point since all liquid experiments were performed\nwithout food. If food was included, these experiments would not accurately model liquid culture\nconditions.\nResponse: We thank the reviewer for raising this important point and apologize for the confusion\ncaused by our description. In fact, we performed two independent transcriptomic experiments\nunder distinct culture conditions, each designed to address a different experimental question.\nwhich represents the standard growth condition and was used to assess transcriptional responses\nunder physiologically relevant, food-present conditions.\nSecond, a separate RNA-seq experiment was conducted in liquid culture in the absence of\nbacterial food, matching the conditions used in our liquid survival assays. This experiment was\nspecifically designed to isolate the effects of potassium deficiency and ionic environment without\nconfounding influences from bacterial metabolism or liquid-associated feeding behavior.\nWe agree that the wording was unclear and have now revised the manuscript to explicitly\ndistinguish these two transcriptomic datasets, clearly stating their respective culture conditions\nand experimental purposes.\n3. On line 352 in the tracked changes file, it is stated that atf-7 strongly induce catp-3 mRNA. However,\nthe graph demonstrates no significant statistical difference.\nResponse: We thank the reviewer for pointing out this inconsistency. We agree that the term\n“strongly induce” was an overstatement given that the difference shown in the graph does not\nreach statistical significance. We have revised the text to adopt more cautious wording, describing\nthe effect of ATF-7 on catp-3 mRNA expression as a trend rather than a strong induction.\nAccordingly, we now state that as “Consistent with prior findings, activation of the pathway by\nRNAi knockdown of the phosphatase vhp-1 or overexpression of sek-1 and atf-7 showed a trend\ntoward increased catp-3 mRNA expression, suggesting that p38–ATF-7 signaling may contribute\nto catp-3 transcriptional regulation (Fig. 5a).”\n4. Figure 2a, which shows the pathway and lists various genes, is cited in the text incorrectly as Figure\n1a.\nResponse: Fixed, we apologize for the oversight.\n5. Data Labeling: Clearly indicate which part of the data is derived from the 15°C culture.\nResponse: We thank the reviewer for this comment. We apologize for the confusion caused by the\nlabeling. All experiments and datasets presented in this study were conducted at 20°C, and no data\nwere derived from 15°C culture conditions. We have now corrected the text in Methods as “Worms\nwere maintained at 20°C on standard nematode growth medium (NGM) agar plates seeded with\nEscherichia coli OP50-1 (a streptomycin-resistant strain for worm food).”\n6. Labeling Adjustments: The labels \"Sek-1 OE\" and \"sek-1 ASI\" are not easily recognizable. Please\nreplace them with \"promoter: sek-1 gDNA/cDNA\" for clarity.\nResponse: We thank the reviewer for this helpful suggestion. Owing to space constraints and layout\nconsiderations within the figure panels, it was not feasible to include the full transgene descriptions\ndirectly in the figure labels. However, we have already provided the complete transgene\ninformation in the figure legends.\n2\n7. Figures Suggestions:\no Figure 1: Change all lifespan curve colors to semi-transparent for better visibility. Clarify whether\n\"water\" refers to M9 buffer. Ensure the order of lifespan labels follows a logical sequence, either from\nlow-to-high or control to mutant. Currently, \"B-e water\" is listed first, while \"f-h water\" is last, which is\nconfusing. Panels D and E appear smaller than panels F-H; consider adjusting their sizes for consistency.\nExpand the X-axis to greater than 30 to avoid cutoff data in panel I.\nResponse: We thank the reviewer for the detailed and constructive suggestions regarding Figure 1.\nWe have revised the figures (Fig. 1, 2, 4, 6) accordingly to improve clarity and consistency.\nSpecifically, we have revised all lifespan curves to improve visibility and reduce overlap among\ntraces, including adjustments to line color, line width, and plotting order. In this study, “water”\nrefers to distilled water, not M9 buffer. We have clarified this explicitly in the figure panels to avoid\nambiguity. All lifespan assays labeled as “water” were performed under identical conditions, and\nthe use of distilled water was consistent across the corresponding experimental groups. The order\nof lifespan labels has been reorganized to follow a logical sequence from control to mutant\nconditions, thereby improving readability and interpretation. Panel sizes have been adjusted so\nthat panels D and E are consistent with panels F–H. In addition, the X-axis in panel I has been\nextended beyond 30 days to ensure that all data points are fully displayed without truncation.\no Figure 2: Apply the same suggestions as Figure 1. Setting the X-axis to 45 may improve clarity.\nResponse: Fixed, thanks.\no Figure 4: The same control data for catp-3(ok1612) survival and WT groups was used. Please use a\ndotted line to indicate when the same data is utilized multiple times.\nResponse: We thank the reviewer for this suggestion. The same control survival data for catp-\n3(ok1612) and WT were intentionally reused in Figure 4 to allow direct comparison across\nexperimental conditions. We have now indicated the reused control data with dotted lines in all\nlifespan curves in the figures (Fig. 1, 2, 4, 6) and clarified this explicitly in the figure legend to\nensure transparency.\no Figure 6: The calculation of mRNA expression is incorrect. The normalization using the △△Ct method\nrequires values from △Ct for your three control samples; it cannot always be 1. Please refer to the\nguidelines at Nature Protocols. Additionally, consider logging the Y-axis for better representation.\nResponse: We thank the reviewer for the clarification. We agree that the housekeeping gene (tba-\n1) should not be treated as a target gene in ΔΔCt analysis or displayed as relative expression values.\nthe reference gene tba-1 was used solely for normalization of target gene expression, and its Ct\nvalues were stable across samples. We have revised the presentation to ensure that relative\nexpression is calculated and shown only for target genes, normalized to the mean ΔΔCt of the\nbiological control group, in accordance with standard qPCR guidelines.\no Figure 7: Methods related to the ggridges plot were not found in the methods section. Please include\n3\nthis information.\nResponse: We thank the reviewer for pointing out this omission. We have now added a detailed\ndescription of the methods used to generate the ggridges plot in the Methods section: “The ggridges\nplots were generated using OmicStudio (https://www.omicstudio.cn, version V1.0) to visualize the\ndistribution of cell size measurements across the indicated experimental groups. Single-cell size\ndata were imported into OmicStudio and displayed as ridge density plots based on kernel density\nestimation. Each ridge represents the probability density distribution of cell size for one group,\nwith ridge transparency (alpha) set to 0.6, allowing comparison of distribution patterns, dispersion,\nand shifts in central tendency between groups.”\nThe authors have addressed most of my concerns, and the revised manuscript has been greatly improved.\nIn particular, the new lifespan experiments performed under different medium conditions, as well as the\nnew RNA-seq experiment conducted under consistent conditions, further strengthen the authors’\nconclusions. I have only minor comments that should be addressed prior to publication.\nComments\nPreviously, I pointed out that the life-extending effect of KCl supplementation is not specific to sek-1\nmutants, as KCl also increases lifespan in wild-type animals as well as in sek-1 and pmk-1 mutants. This\nissue has not been addressed in the revised manuscript. In line 85, the authors state that KCl markedly\nrescues the survival of sek-1 and pmk-1 mutants; however, this rescue could reflect a combination of a\ngeneral lifespan-extending effect of KCl and the rescue of increased susceptibility of sek-1 mutants to\npotassium deficiency. This alternative explanation should be discussed in the text.\nResponse: We thank the reviewer for reiterating this important point. As noted by the reviewer,\nKCl supplementation also increases lifespan in wild-type animals as well as in sek-1 and pmk-1\nmutants. We therefore agree that the apparent “rescue” of sek-1 and pmk-1 survival by KCl likely\nreflects a combination of two effects: (i) a general lifespan-extending effect of KCl, and (ii) a specific\nmitigation of the heightened susceptibility of sek-1 and pmk-1 mutants to potassium deficiency. We\nhave revised the text: “Furthermore, KCl, rather than NaCl, markedly rescued the survival of sek-\n1 and pmk-1 (p38 MAPK) null mutants in water (Fig. 1b–e and Extended Data Fig.1a). This effect\nis consistent with a model in which the apparent rescue reflects a combination of a general lifespan-\nextending effect of KCl and the alleviation of the increased susceptibility of sek-1 and pmk-1\nmutants to PD.”\nLine 87: “...indicating that p38 signaling is essential for survival specifically when exogenous K⁺ is\nlimited, rather than Na⁺, Ca²⁺, or Cl⁻.” While the effect of Ca²⁺ on the survival of p38 pathway mutants\nis shown in Extended Data Fig. 1b, it is not described in the main text. Please explain these data and cite\nthe corresponding figure.\nResponse: We thank the reviewer for this helpful comment. We agree that the effects of Ca²⁺ on the\nsurvival of p38 pathway mutants should be explicitly described in the main text. We have now\nrevised the text: “In addition, supplementation with Ca2+ failed to improve the survival of sek-1\nmutants under water conditions (Extended Data Fig. 1b), further supporting the idea that p38\nsignaling is particularly required for survival under conditions of limited exogenous K+, rather\nthan upon exposure to Na+, Ca2+ or Cl–. Together, these results suggest that p38 signaling promotes\nsurvival by mitigating PD (100 mM NaCl without exogenous K+) stress and argue against a\nprimary role for nonspecific osmotic imbalance.”\nExtended Data Fig. 1i: The y-axis is labeled as “pixel.” Although this representation can show relative\n5\ndifferences in size, pixel size can be different depending on imaging conditions. Please convert the\nmeasurements to physical units (µm or mm) or present normalized values for clarity.\nResponse: We thank the reviewer for pointing out this issue. We have now converted the pixel-\nbased measurements to physical units by applying the calibrated pixel-to-length conversion for the\nimaging system. The y-axis in Extended Data Fig. 1i has been updated to report values in\nmillimeters (mm) in the revised figure.\nWhile the reviewers responded well to the fourth item to address, the first three responses were\ninadequate. Each of these comments is experimentally quite easy to address and will both clarify the\nresults and test alternative possibilities.\nPrevious comment 1: Fast Fasting is known to activate p38/MAPK signaling. It would therefore be\ninformative to determine whether KCl supplementation induces a fed-like signaling state or suppresses\nthe fasting response in C. elegans.\nComment 1 response to author response: To address this, the authors need to measure the expression of\nestablished fasting-responsive genes and examine whether their expression is reversed upon KCl\ntreatment. This was not done.\nResponse: To directly test whether KCl supplementation suppresses the fasting response or induces\na fed-like signaling state, we measured the expression of established fasting-responsive genes in C.\nelegans. Specifically, we analyzed lgg-1 (autophagy-related), atgl-1 (lipolysis), sod-3 (a canonical\nDAF-16/FOXO target), and pha-4 (a dietary restriction–induced transcription factor).\nYoung adult wild-type worms were transferred to liquid potassium-deficient (PD; 0.1 M NaCl)\nconditions with OP50-1 for 24 hours, followed by supplementation with 100 mM KCl for either 4\nhours or 24 hours. Quantitative PCR analysis revealed that the expression levels of these four\nfasting-responsive genes showed no significant changes upon KCl supplementation at either time\npoint, under liquid conditions with or without food.\nConsistent with these results, analysis of our RNA-seq datasets likewise showed no significant\ndifferential expression of lgg-1, atgl-1, sod-3, or pha-4 between 0.1 M NaCl and 0.1 M KCl\nconditions. Together, these data indicate that KCl supplementation does not reverse the fasting\ntranscriptional program nor induce a fed-like signaling state, arguing against the possibility that\nthe protective effects of potassium are mediated by suppression of fasting-induced p38/MAPK\nsignaling.\nPrevious comment 2: Pharyngeal pumping in C. elegans depends on Na⁺/K⁺-ATPase activity. It is unclear\nhow the experimental conditions used in this manuscript (e.g., PD exposure and KCl supplementation)\naffect pharyngeal pumping behavior.\nComment 2 response to author responsese: Have the authors measured pumping rates under these\nconditions to rule out altered feeding behavior as a confounding factor? This is a very easy question to\nanswer, and correlations to other (uncited) works does not answer it.\nResponse: To directly address whether altered feeding behavior could confound our results, we\nmeasured pharyngeal pumping rates under the same liquid survival conditions used in the\nmanuscript. Young adult worms were transferred to liquid medium without food containing either\n0.1 M NaCl (PD) or 0.1 M KCl for 24 hours, and pumping rates were quantified thereafter. Under\nthese conditions, PD exposure did not significantly alter pharyngeal pumping rates in wild-type\nworms, indicating that potassium deficiency per se does not impair pumping behavior in a normal\ngenetic background. In contrast, pmk-1 mutants exhibited a significant reduction in pumping rates\nunder PD, suggesting reduced physiological activity or stress sensitivity specific to this genotype.\nNotably, under liquid fasting conditions, overall pumping rates were lower and more\nheterogeneous than those typically observed on food-containing solid plates, with individual\nanimals showing variable pumping frequencies. This pattern closely resembles previously\ndescribed starvation-induced pumping behavior on solid media, indicating that the liquid\nenvironment itself does not uniquely suppress pharyngeal activity beyond the expected effects of\nfasting. Together, these data demonstrate that neither PD exposure nor KCl supplementation alters\npharyngeal pumping in wild-type worms, and therefore altered feeding behavior is unlikely to\naccount for the observed survival phenotypes.\nunclear whether this pathway is actively induced during PD exposure and subsequently returns to basal\nlevels following KCl supplementation, or whether p38/MAPK activity is constitutively required but not\ndynamically regulated.\nComment 3 response to author response: The authors state: “As requested, we transferred day-1 adult\nwild-type worms to liquid PD conditions (0.1 M NaCl) with OP50-1 for 24 hours, followed by\nsupplementation with 100 mM KCl for either 4 hours or 24 hours. qPCR analysis showed that sysm-1 (a\nfeeding-upregulated gene and a known p38/ATF-7 downstream target) as well as catp-3 mRNA levels\nwere suppressed upon KCl supplementation at both time points.” The rationale for using bacteria-fed\nworms under these conditions is not clear. In addition, it remains unclear how the expression of\np38/MAPK target genes changes in the presence or absence of KCl, and whether these changes are\ndirectly attributable to KCl supplementation.\nResponse: To clarify whether p38/MAPK signaling is dynamically regulated during PD exposure\nand KCl supplementation, or instead constitutively required for survival, we examined the\nexpression of two well-established p38/PMK-1 downstream target genes: sysm-1 (feeding- and\npathogen-induced) and catp-3 (PD-induced). Young adult wild-type worms were transferred to\nliquid potassium-deficient (PD; 0.1 M NaCl) conditions for 24 hours, followed by supplementation\nwith 100 mM KCl for either 4 hours or 24 hours. To explicitly address the reviewer’s concern\nregarding food effects, these experiments were performed both in the presence and absence of\nOP50-1.\nUnder liquid conditions with food, sysm-1 expression was not strongly induced by PD and was\nmodestly suppressed upon KCl supplementation at 4 hours, with no further suppression at 24\nhours. In contrast, catp-3 expression increased progressively with prolonged PD exposure and was\nsignificantly suppressed by KCl supplementation at 4 hours, with further reduction at 24 hours.\nUnder liquid conditions without food, sysm-1 expression was low and not responsive to either\nPD or KCl supplementation, consistent with the known dependence of sysm-1 on feeding cues. This\nobservation is in agreement with our RNA-seq data, in which sysm-1 was not induced by PD in\nliquid culture without food, but was induced under PD conditions on solid plates with food (Fig. 3).\nImportantly, under the same fasting conditions, catp-3 expression remained strongly PD-induced\nand was robustly suppressed by KCl supplementation.\nTogether, these data indicate that p38/MAPK target genes are not uniformly or globally\ninduced by PD, and that their regulation is highly context-dependent. While sysm-1 responds\nprimarily to feeding status, catp-3 is selectively sensitive to potassium availability, showing dynamic\n9\ninduction by PD and rapid repression upon KCl supplementation regardless of food. These\nfindings support a model in which p38/MAPK signaling is required for survival under PD but is\nnot globally activated as a canonical stress response, with potassium-sensitive regulation acting on\na subset of p38 downstream targets such as catp-3.\nComment 4: Addressed\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and effort in co-reviewing this manuscript.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nResponse: We thank the reviewer for their time and effort in co-reviewing this manuscript.\n11\np38 MAPK orchestrates cross-tissue potassium homeostasis for survival\nPoint-by-Point Response to Reviewers’ comments\nReviewer #1 (Remarks to the Author):\nI am satisfied with how the authors addressed my concerns.\nReviewer #2 (Remarks to the Author):\nThe authors addressed my concerns successfully.\nReviewer #3 (Remarks to the Author):\nWe thank the authors for providing data in answer to the remaining review concerns. However, upon\nreview of the revised manuscript, it appears they failed to incorporate their data into the manuscript. The\npurpose of these experiments was as important controls that should be discussed in the manuscript and\nincluded in supporting data. Please add these data and the discussion of the results into the manuscript.\nResponse: We thank the reviewer for pointing this out and apologize for the oversight. We have\nnow fully incorporated these experiments into the revised manuscript. Specifically, the pharyngeal\npumping analyses (Supplementary Fig. 1j) and fasting-responsive gene expression data\n(Supplementary Fig. 5) have been added to the Results section, and the corresponding\nexperimental details are described in the Methods.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\n1"}, {"round": 3, "reviewer_comments": {"reviewer_1": "I am satisfied with how the authors addressed my concerns.", "reviewer_2": "The authors addressed my concerns successfully.", "reviewer_3": "We thank the authors for providing data in answer to the remaining review concerns. However, upon review of the revised manuscript, it appears they failed to incorporate their data into the manuscript. The purpose of these experiments was as important controls that should be discussed in the manuscript and included in supporting data. Please add these data and the discussion of the results into the manuscript.", "reviewer_4": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_5": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts."}, "author_response": "p38 MAPK orchestrates cross-tissue potassium homeostasis for survival\nPoint-by-Point Response to Reviewers’ comments\nReviewer #1 (Remarks to the Author):\nI am satisfied with how the authors addressed my concerns.\nReviewer #2 (Remarks to the Author):\nThe authors addressed my concerns successfully.\nReviewer #3 (Remarks to the Author):\nWe thank the authors for providing data in answer to the remaining review concerns. However, upon\nreview of the revised manuscript, it appears they failed to incorporate their data into the manuscript. The\npurpose of these experiments was as important controls that should be discussed in the manuscript and\nincluded in supporting data. Please add these data and the discussion of the results into the manuscript.\nResponse: We thank the reviewer for pointing this out and apologize for the oversight. We have\nnow fully incorporated these experiments into the revised manuscript. Specifically, the pharyngeal\npumping analyses (Supplementary Fig. 1j) and fasting-responsive gene expression data\n(Supplementary Fig. 5) have been added to the Results section, and the corresponding\nexperimental details are described in the Methods.\nReviewer #4 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\nReviewer #5 (Remarks to the Author):\nI co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of\nthe Nature Communications initiative to facilitate training in peer review and to provide appropriate\nrecognition for Early Career Researchers who co-review manuscripts.\n1"}]

Decision letter