Waterlogging constitutes a significant abiotic stress that profoundly impacts plant growth, development, and agricultural productivity. This stress typically results from excessive rainfall, flooding, or inadequate soil drainage, leading to oxygen deficiency in the soil and rhizosphere (Kreuzwieser and Rennenberg, 2014). Amidst global climate change, the frequency and severity of extreme weather events, such as powerful typhoons and torrential rains, are on the rise, leading to more frequent and severe waterlogging episodes (Zhou W. et al., 2020). This has emerged as a serious threat to various plant species. The influence of global climate change on waterlogging is considerable. Rising temperatures can modify precipitation patterns, resulting in more frequent and intense rainfall in some regions, while others may endure prolonged droughts followed by heavy rainfall (Tabari, 2020). These fluctuations, in conjunction with changes in soil moisture and temperature, can exacerbate waterlogging conditions. For instance, in agricultural regions prone to rhizosphere hypoxia, the risk of soil waterlogging is anticipated to rise due to these climatic alterations (Tian et al., 2021). Waterlogging can elicit a spectrum of deleterious effects on plants, including reduced photosynthesis, decreased stomatal conductance, lower leaf chlorophyll content, hormonal imbalances, and compromised water and mineral uptake (Loreti et al., 2016). These impacts highlight theocratical need for strategies to mitigate the effects of waterlogging on plant health and agricultural systems. Fruit trees, such as apple, peach, and citrus, are particularly vulnerable to waterlogging. The tolerance of fruit trees to hypoxic conditions varies by species and cultivar. It is also influenced by factors such as plant age, developmental stage, soil type, and season (Habibi et al., 2023). Waterlogging has become one of the primary abiotic stresses limiting fruit productivity globally.

Phytohormones play a critical role in the plant response to rhizosphere hypoxia and serve as key regulators to overcoming stress conditions. Melatonin, a naturally occurring compound, has attracted attention as a potential modulator of plant stress responses, including those to waterlogging (Jahan et al., 2021). This molecule is implicated in numerous physiological processes in plants, such as scavenging reactive oxygen species (ROS), augmenting antioxidant enzyme activity, and stimulating photosynthesis (Sati et al., 2023; Wang X. et al., 2024). Recent studies suggested that the application of exogenous melatonin can enhance plant tolerance to waterlogging by regulating their antioxidant metabolism, nitrogen uptake, and the composition of the rhizosphere microbial community. For instance, in apples, melatonin facilitates post-waterlogging recovery by modulating the structure and function of the rhizosphere microbiome, bolstering antioxidant capacity, and optimizing nitrogen absorption and utilization (Cao et al., 2024a; Cao et al., 2024b). The mitigating effects of melatonin pretreatment on the adverse impacts of waterlogging stress have also been observed in other species, such as wheat (Ma et al., 2022) and peach (Gu et al., 2021).

Transcriptome and proteome analyses have yielded invaluable insights into the molecular mechanisms by which plants respond to waterlogging stress. These investigations have identified a multitude of differentially expressed genes (DEGs) and associated pathways that are integral to key processes such as respiration, carbohydrate metabolism, photosynthesis, phytohormone metabolism, ROS metabolism, mineral uptake, and protein and amino acid metabolism under hypoxic conditions (Lin Y. et al., 2019; Zeng et al., 2019; Li et al., 2022). A thorough comprehension of the physiological, biochemical, and molecular responses of plants to waterlogging is essential for formulating strategies to enhance their tolerance to this abiotic stress. By delineating these responses and identifying potential targets for genetic improvement, significant advancements can be made in the development of more resilient plant varieties and sustainable agricultural practices. This is particularly pertinent in light of rising frequency of waterlogging events associated with climate change, underscoring the imperative for innovative strategies to safeguard agricultural sustainability and food security.

Peach trees are highly susceptible to waterlogging, a condition that leads to rhizosphere hypoxia and substantially impairs their growth and development. The response of peach to rhizosphere hypoxia is an intricate process regulated by the interplay of multiple genes. In our previous research, we investigated the optimal concentration of melatonin for peach seedlings subjected to waterlogging, revealing that a 200 μM treatment was most effective in enhancing survival for over 10 days under such stress conditions (Gu et al., 2021). The present study utilized transcriptomic and proteomic approaches to pinpoint key DEGs and metabolic pathways that are vital for the response to waterlogging, as well as the regulatory effects of melatonin priming. The capacity of melatonin to mitigate waterlogging stress and the underlying mechanisms warrant further investigation. The results of this study establish a fundamental framework and constitute a valuable genetic resource for subsequent inquiries into the regulatory mechanisms at play. This research furthers our understanding of the multifaceted role of phytomelatonin in the plant response to waterlogging, thereby facilitating the development of strategies to enhance the resilience of peach and other crops to this abiotic stress.

Abiotic stresses, such as extreme temperatures (cold and heat), water imbalances (drought and waterlogging), salinity, and heavy metal exposure, markedly impede plant growth and development. The peach, celebrated for its exquisite taste, nutritional benefits, and broad appeal, is extensively cultivated in China (Li and Wang, 2020). Nevertheless, peach trees are especially susceptible to waterlogging due to their shallow root systems, which are highly oxygen-dependent and profoundly impacted by the hypoxic conditions of waterlogged soils. Waterlogging represents a significant impediment to the peach industry in southern China (Tian et al., 2021). Elucidating the regulatory mechanisms behind peach seedling responses to waterlogging is vital for comprehending plant adaptation in rainy environments. This insight can be leveraged to develop waterlogging-tolerant Prunus germplasms, thereby improving peach yield under challenging conditions (Ateeq et al., 2023). In this research, we utilized RNA-seq and DIA proteomic analyses to pinpoint genes and proteins involved in the response to melatonin and waterlogging. By integrating multiomics data, we conducted a systematic examination of peach roots to establish correlations between mRNA and protein expression. As a result, we identified DEGs and DEPs associated with waterlogging responses in peach.

The number of DEGs in the MT vs CK was lower compared to the other two comparisons, with the WL vs CK exhibiting the highest number of DEGs, in agreement with the proteomic data. These results imply that melatonin pretreatment induced a relatively modest differential gene expression, whereas waterlogging, representing a more severe stressor, triggered a substantially larger number of DEGs. This suggests that melatonin pretreatment may have mitigated the necessity for a robust activation of stress-responsive genes in response to subsequent waterlogging stress. Recent transcriptomic studies have illuminated the involvement of various metabolic pathways, including ‘global and overview maps’, ‘carbohydrate metabolism’, ‘amino acid metabolism’, and ‘energy metabolism’, in the responses to waterlogging (Kuai et al., 2016; Zhang et al., 2022) as well as in response to other abiotic stresses (Zhou D. et al., 2020). In the current study, KEGG annotation identified that the DEGs were mainly grouped in pathways associated with ‘global and overview maps’, ‘carbohydrate metabolism’, and ‘environmental adaptation’. These observations imply that waterlogging may affect the metabolism of soluble sugars, organic acids, and phenols in peach.

Based on Venn diagram analysis, 92 annotated transcription factors were identified, including members of the AP2/ERF family, which are prominent activated by flooding in plants (Mustroph et al., 2009; Wang et al., 2021). Previous research has demonstrated that AP2/ERF transcription factors play critical roles in regulating fermentation and ROS signalling by modulating the expression of genes such as ADH, PDC, RBOHD, HRU1 (Wang K. et al., 2024). Additionally, members of the ERF subfamily within group VII, which are involved in hypoxia response, have been functionally analysed in Arabidopsis and rice (Gasch et al., 2016; Lin C. et al., 2019; Weits et al., 2021). In the present study, the three most highly expressed transcription factors, Prupe.8G264900, Prupe.2G272300, and Prupe.5G090700, were classified as ERF transcription factors ( Figure 3A ). Furthermore, Prupe.8G264900 (ERF071) was identified as an ERF VII member ( Supplementary Table S6 ) (Zhao et al., 2018), highlighting the significance of ERF VII members in regulating the response of peach to waterlogging. In Arabidopsis, ERF VIIs have been identified as regulators of hypoxia survival responses, mediating the increase activities of PDC and ADH, which facilitate the transition from aerobic to anaerobic metabolism (Gibbs et al., 2011). Studies indicate that the upregulation of specific ERFVII transcription factors can induce the activation of genes such as LOB41, ADH1/2, and PDC2, involved in alcoholic fermentation and scavenge ROS, thereby enhancing waterlogging tolerance in kiwifruit (Pan et al., 2019; Liu et al., 2022; Bai et al., 2023). Consequently, in response to waterlogging signals like ethylene and hypoxia, ERF VIIs rapidly accumulate and initiate a signalling pathway that promotes tolerance to waterlogging.

In addition to AP2/ERF family, WRKY transcription factors are crucial for integrating environmental cues and influencing plant growth under stress conditions. In our study, 14 out of the 17 WRKY genes were upregulated in response to waterlogging, indicating a significant role for WRKYs in the waterlogging response of peach. Previous research on Phoebe bournei has shown that WRKY members are highly responsive to waterlogging, with PbWRKY36 identified as a candidate gene potentially plays a pivotal role in enhancing resilience to waterlogging (Wang Z. et al., 2024). Other studies have demonstrated that WRKYs act as positive regulators of hypoxia responses, such as at the onset of waterlogging in sesame (Wang et al., 2021) and submergence in rice (Viana et al., 2018). Moreover, the heterologous expression of the sunflower transcription factor HaWRKY76 in Arabidopsis was shown to confer submergence tolerance with preserved carbohydrate and increased cell membrane stability (Raineri et al., 2015).

Together, the robust expression of ERFs and WRKYs revealed in our study that these transcription factors are involved in the response to waterlogging in peach. Previous research by Gao indicated that the WRKY22/33 and AP2-EREBP genes may be implicated in antioxidant defense and enhance waterlogging tolerance in kiwifruit (Gao et al., 2022). The transcriptional alterations induced by waterlogging are predominantly mediated by the ERF and WRKY transcription factors. Nonetheless, it is yet to be determined whether these two gene families interact within the same signalling pathways during waterlogging. Exploring the potential interplay between WRKYs and ERFs in the context of waterlogging responses is a subject that deserves additional experimental investigation.

The KEGG pathway enrichment analysis of coregulated genes indicated that DEGs under waterlogging stress and melatonin priming were significantly enriched in pathways associated to ‘glycolysis/gluconeogenesis’, ‘Starch and sucrose metabolism’, and ‘plant hormone signal transduction’ ( Figure 4A ; Supplementary Figure S1 ). Beyond several commonly regulated expression pathways, glycolysis/gluconeogenesis exhibited the most pronounced response to waterlogging. Typically, plants depend on glycolysis and ethanol fermentation as alternative energy sources during the energy deficit induced by waterlogging (Zhou W. et al., 2020; Habibi et al., 2023). The expression of most genes encoding key enzymes in the glycolysis/gluconeogenesis pathway was upregulated compared to the control, with marked increases in fold change. These expression patterns are align with previous findings that underscore the importance of glycolysis/gluconeogenesis in the transcriptional regulation and metabolic adaptation of sweet cherry (Michailidis et al., 2021) and Chinese bayberry (Jiao et al., 2023). Furthermore, the enhanced expression of specific energy metabolism pathways aids peach in better managing waterlogging (Ateeq et al., 2023). This adaptive strategy reinforces the plant’s ability to withstand waterlogging, thereby enhancing survival.

Due to ATP depletion under waterlogging stress, soluble sugars are transported to the roots to furnish substrates for glycolysis and anaerobic respiration. Glycolysis maintains a sufficient supply of carbohydrates, enabling plants to endure hypoxic conditions (Habibi et al., 2023). In our study, both gene transcripts and metabolites, including ADH, PDC, PK, 6-phosphofructokinase (PFK), diphosphate-dependent phosphofructokinase (PFP), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH), were significantly enriched in the glycolysis/gluconeogenesis and pyruvate metabolism pathways during waterlogging. These results suggest that the response of peach to waterlogging is closely associated with respiratory metabolism. Anaerobic metabolism is sustained through the activation of fermentative pathways, which support ATP production via the glycolytic route. Glycolysis can proceed with NADH oxidation through lactic acid and ethanol fermentation from pyruvate (Bailey-Serres and Voesenek, 2008). Nevertheless, the fermentation pathway can be detrimental to plant cells due to the production of lactic acid and acetaldehyde (van Dongen and Licausi, 2015). The onset of ethanol fermentation involves inhibition of lactate dehydrogenase (LDH) activity and the stimulation of PDC. Transcripts of four ADH genes (Prupe.8G018100, Prupe.8G018300, Prupe.8G018400, and Prupe.8G018600) and two PDC genes (Prupe.4G058300 and Prupe.6G280800) were upregulated by waterlogging. However, melatonin priming in peach led to reduced expression levels of ADH, PDC, and other fermentation-related genes, indicating a regulatory role for melatonin in modulating the anaerobic metabolism pathway before waterlogging.

Although the multifunctional roles of melatonin in plants have been extensively studied, comprehensive transcriptomic and proteomic profiling of Prunus genotypes under stress conditions remains scarce. In our previous work, we demonstrated that 200 μM melatonin effectively enhanced the tolerance of peach plantlets to waterlogging by reducing lipid membrane peroxidation while also enhancing the antioxidant system (Gu et al., 2021). Building on these findings, subsequent omics analyses can elucidate the underlying regulatory mechanisms. Transcriptomic sequencing of Clematis varieties under waterlogging was performed to examine the effects of melatonin on waterlogging tolerance, revealing that melatonin enhanced flooding tolerance in Clematis by improving photosynthetic efficiency and antioxidant enzyme activity (Chen et al., 2024). In tomato, appropriate melatonin application significantly enhanced tolerance to low night temperatures, as evidenced by transcriptomic and proteomic approaches (Yang et al., 2022). In drought-stressed wheat treated with 100 μM melatonin, integrative physiological, transcriptomic, and proteomic data analyses indicated that melatonin modulated JA related genes, transcription factors, and starch and sucrose metabolism genes, thereby enhancing wheat drought tolerance (Luo et al., 2023). In apple, melatonin positively contributes to the accumulation of plant dry weight by upregulating genes associated with N cycling and significantly augmented the abundance of beneficial bacteria in the rhizosphere, facilitating the recovery of apple following waterlogging (Cao et al., 2024a). In the current study, through the combination of transcriptomic and proteomic analyses, we identified that more stress-responsive genes, such as ERF071, PCO2, HSP, and ATG11, were upregulated by waterlogging at both the transcriptional and protein levels. However, under melatonin priming, the expression levels of these genes did not exceed those observed under waterlogging alone, and protein levels remained comparable. Melatonin pretreatment may alter the internal physiological equilibrium of plants, prompting the adoption of alternative coping strategies to waterlogging. Peach pretreated with melatonin may prioritize energy allocation for maintaining the stability of fundamental cell metabolism over the induction of numerous stress-related genes. This could involve protective mechanisms that prevent excessive activation of these genes or proteins.

Significant challenges relate to the scalability of melatonin priming in field applications. In a controlled laboratory milieu, the precise delivery of melatonin and the monitoring of its effects are relatively straightforward. However, scaling up to field-level operations introduces a layer of complexity. Variations in soil types, fluctuating weather patterns, and differences in plant populations can all influence the effectiveness of melatonin priming. Moreover, the financial implications of applying melatonin across extensive agricultural areas could be daunting, which might limit its broader implementation. Consequently, additional research is essential to address both the practical application of melatonin in field settings and the genetic improvement of melatonin-associated genes to enhance plants’ ability to cope with stress.

In summary, waterlogging stress adversely affects the membrane system and oxidative balance in peach, while melatonin facilitates growth recovery and modulates the waterlogging response regulation ( Figure 8 ). Transcriptomic and proteomic analyses indicated that the number of upregulated DEGs was fewer compared to downregulated ones following waterlogging, and exogenous melatonin prevented excessive activation of genes or proteins. Furthermore, transcription factors from the AP2/ERF and WRKY families were implicated in the response to waterlogging. The glycolysis/gluconeogenesis pathway has been identified as a pivotal pathway for peach to withstand waterlogging. The ERF VII family member ERF071 (Prupe.8G264900), ADH (Prupe.8G018100), and PCO (Prupe.7G011000) are promising candidates for manipulating the waterlogging response. Future research should aim to develop transgenic lines and perform biochemical assays to delineate their functions more precisely. Our findings provide actionable targets for breeding waterlogging-tolerant peach varieties and strategies to alleviate abiotic stress in horticulture.