Rice (Oryza sativa L.) is one of the most important staple crops that feeds more than half of the world’s population. Thailand, a major player in global rice production, ranks as the sixth-largest rice producer, following China, India, Indonesia, Bangladesh, and Vietnam (FAO, 2017). Farming practices in Thailand and other countries have significantly evolved over the years, with mechanization becoming increasingly central. The use of heavy machinery, such as power tillers, tractors, and harvesters, has become widespread in agricultural operations, including soil preparation, sowing, and harvesting (FAO, 2017). While these advancements have boosted agricultural productivity, they have also led to unintended consequences, particularly soil compaction, which poses a significant challenge to crop growth and yield (Hassan et al., 2007). Soil compaction, a condition where soil particles are pressed together, reducing macropore spaces, is a major constraint in modern agriculture. It negatively impacts crop production by restricting root growth, limiting soil water and nutrient availability, and decreasing soil porosity (Arocena, 2000; Khan et al., 2012). Additionally, soil compaction hinders gas exchange between plant roots and the surrounding environment, further exacerbating plant stress. In this context, plants naturally produce various volatile phytohormones, such as ethylene, which can diffuse through air spaces between soil aggregates in noncompacted soil. However, soil compaction restricts ethylene diffusion in soil micropores, leading to inhibited root elongation and reduced overall root growth, which negatively impacts the plant’s ability to access water and nutrients (Pandey et al., 2021; Arocena, 2000). Elevated ethylene levels in compacted soils can trigger stress responses that further restrict root development, thereby compromising plant growth and yield (Lin et al., 2010).

Ethylene, a gaseous phytohormone synthesized from methionine in the presence of oxygen, is integral to plant stress responses. It is produced in most plant tissues and serves as a key regulator of various physiological processes, particularly under stress conditions (Lin et al., 2010). Ethylene Response Factors (ERFs) are central to ethylene’s role in stress response, acting as regulatory hubs that interact with other stress-related signals, such as abscisic acid and (ABA) and jasmonate (Müller and Munné-Bosch, 2015). These interactions are critical in modulating plant responses to diverse abiotic stresses, including salinity, drought, low temperature, heat, and variations in light availability. The ethylene signaling pathway is a complex network involving several key components, including receptors, EIN2, and transcription factors like EIN3 (Ethylene insensitive 3)/EIL1 (Ethylene insensitive-Like1). These components activate ERFs, which, in turn, regulate the expression of genes involved in stress and downstream hormone signaling pathway. Notably, rice mutant lines with alterations in EIN3/EIL1 maintain normal primary root growth even under compacted soil conditions, similar to wild-type plants in non-compacted soils (Pandey et al., 2021). This resilience is likely due to the disrupted ethylene signaling in these mutants, which prevents the typical ethylene-induced inhibition of root elongation in response to soil compaction.

The regulation of root growth and development in rice is a complex process influenced by multiple phytohormones, including auxin, ABA, cytokinin, and ethylene. These hormones are involved in various aspects of root growth and development, from biosynthesis to signal transduction, and play a crucial role in how plants respond and acclimate to environmental stresses (Dello Ioio et al., 2007; De Smet and Jürgens, 2007; McSteen, 2010). Ethylene, in particular, interacts with auxin by modulating its biosynthesis and transport, highlighting its role as a mediator in the regulation of root development (Le et al., 2001). Given the critical role of ethylene in plant stress response, manipulating its pathway offers a promising strategy for improving crop production, especially under adverse conditions. For instance, in maize, mutations in the ACC synthase gene, a key enzyme in ethylene biosynthesis, have been shown to delay leaf senescence and maintain photosynthesis during drought, leading to increased grain yield under stress (Habben et al., 2014). However, breeding for ethylene-responsive traits in crops remains challenging, particularly in non-GMO contexts. Nevertheless, utilizing diverse plant collections and innovative screening protocols can provide valuable genetic resources for breeding programs aimed at enhancing tolerance to unfavorable environments. In rice, developing ethylene-insensitive varieties could be a potential strategy to improve growth and production, particularly in compacted soils where water and nutrient extraction are critical for survival. This approach is especially relevant for countries like Thailand, where traditional breeding methods are preferred over genetic modification. By leveraging the genetic diversity found in local or landrace rice accessions, which often contain adaptive traits to harsh environments, breeders can identify and incorporate valuable alleles into commercial cultivars.

In this study, we aimed to investigate the root length response of 220 rice accessions under controlled and ethylene-treated conditions. By employing a genome-wide association study (GWAS), we sought to identify the genomic regions and key genes regulating root length and ethylene sensitivity in rice. The findings from this study are expected to contribute to the development of new rice varieties with enhanced root penetration ability and improved adaptation to soil compaction and other abiotic stresses.

Root plays a vital role in rice growth and performance under both normal and stress conditions, serving as the primary interface between the plant and its environment. The growth, development, and adaptive responses of rice are regulated by a complex network of internal and external factors, including phytohormones such as ethylene, auxin and cytokinin. The hormones can act synergistically or antagonistically to modulate plant growth, particularly under stress conditions (He et al., 2005; Ueda et al., 2020; Yamauchi et al., 2020). Ethylene, often referred to as the stress hormones, is a key mediator in plant adaptive responses, interacting with other hormones to regulate growth under adverse conditions (Huang et al., 2020; Ma et al., 2013; Yang et al., 2015). Roots are particularly sensitive to changes in ethylene concentration, which can lead to reduced root length and increased radial cell expansion (Vaseva et al., 2018). Additionally, ethylene has been shown to influence root hair growth and development (Feng et al., 2017) and plays a role in the plant’s response to soil compaction by reducing root length and increasing root thickness in rice, a process mediated by abscisic acid (ABA) and auxin (Huang et al., 2022). Transgenic rice plants overproducing ethylene exhibit lower 1000-grain weight and faster leaf senescence compared to those with reduced ethylene production, indicating the significant impact of ethylene on overall plant development and yield (Ma et al., 2013). In the ethylene signaling pathway, the ethylene gas triggers the translocation of CONSTITUTIVE TRIPLE RESPONSE1 (CTR1) from the endoplasmic reticulum to the nucleus. This process involves the phosphorylation of ETHYLENE-INSENSITIVE2 (EIN2) by CTR1, which inhibits EIN2 from signaling in the absence of ethylene (Ju et al., 2012; Jun et al., 2004). In Arabidopsis, plants with enhanced nuclear-localized CTR1 have shown to improve tolerance to drought and salinity stress (Park et al., 2023). These findings suggest that manipulating ethylene sensitivity could be a valuable strategy for improving crop resilience under unfavorable environmental conditions, such as those exacerbated by climate change, while maintaining productivity.

In rice, ethylene’s role in root response to soil compaction has been further elucidated through studies involving rice mutants with varying levels of ethylene sensitivity. For instance, ethylene-insensitive mutants exhibited significantly longer root than those with high ethylene sensitivity under soil compaction, highlighting the genetic basis of ethylene responsiveness (Pandey et al., 2021). In this study, 220 rice accessions were collected from many areas in Thailand in order to have a diverse genetic source to study GWAS. According to Kinship and PCA analysis, our germplasm showed the low levels of relatedness within the rice indica panel (Aesomnuk et al., 2021). Thus, this diversity panel can be used in GWAS analysis. Importantly, the higher genetic diversity of germplasm, the more genetic variation in target trait, leading to successfully identify the crucial gene underlying the target trait (Quero et al., 2018). This will help plant breeder to enhance breeding programs to genetically improve target traits (Mourad et al., 2020). Our genome-wide association study (GWAS) using 220 indica rice accessions identified a QTL, qRLR10, associated with difference in root length reduction (RLR) in response to ethylene. Within this QTL region, 20 candidate genes were identified. The subsequent gene-based haplotype analysis revealed that specific haplotypes within three of these genes—Os10g0124700, Os10g0126600, and Os10g0128200—are significantly associated with variations in RLR. This association underscores the potential functional importance of these genes in the regulation of ethylene responsiveness.

The gene Os10g0124700 (OsFbox508, OsFBX344), which encodes an F-box protein, showed the highest expression in rice roots before the flowering stage according to the strand-specific RNA-Seq database in RAP-DB (Wang et al., 2015). F-box proteins are components of the SCF (SKP1-CUL1-F-box protein) complex, which functions as an E3 ubiquitin ligase, tagging target proteins for degradation. This pathway is crucial for regulating various physiological processes, including growth, development, and stress responses (Gagne et al., 2002; Lechner et al., 2006; Dreher and Callis, 2007; Zhang et al., 2008; Guo et al., 2018; Xu et al., 2021). F-box proteins are likely involved in the fine-tuning of ethylene responses by regulation the stability of key component signaling pathway. For example, in Arabidopsis, the F-box protein EBF1/EBF2 targets the ethylene-responsive transcription factor EIN3 for degradation, thus modulating the ethylene response. Given the association of Os10g0124700 with variations in RLR, it is plausible that this gene plays a similar role in rice. Os10g0126600 (OsFbox512, OsFBX348) is another F-box domain-containing protein, which has been linked to drought resistance in Shanlan upland rice through its involvement with long non-coding RNA (lncRNA) (Wang et al., 2015). F-box proteins are known to integrate with signaling pathways, including those mediated by phytohormones like ethylene, auxin, and ABA (Koops et al., 2011; Sasaki et al., 2003; Wang et al., 2009; Xu et al., 2021; Yan et al., 2011). The association of Os10g126600 with RLR suggests its involvement in the regulation of root architecture in response to ethylene, potentially through interactions with other hormones and stress-related pathways. In drought conditions, ethylene production is often elevated, and the ability of roots to adapt to this stress is critical for plant survival. The role of Os10g0126600 in drought resistance suggests that it may be crucial for modulating root growth under conditions where ethylene levels are altered, helping the plant to balance growth and survival (Yang et al., 2022). This gene’s involvement in RLR further supports its importance in the adaptive response of rice roots to environmental stresses, likely through the modulation of hormone signaling networks. Os10g0128200 (OsFbox517, OsFBX353) has also been identified as a gene associated with RLR, though its specific role in ethylene signaling and root development is less characterized compared to the other two genes. However, its significant association with root length reduction suggest that it may be involved in key regulatory process that control root growth in response to ethylene.

Although not all identified candidate genes showed significant differences in RLR among haplotypes, they may still play roles in root growth and ethylene response. Evidence suggests crosstalk among plant hormones, with ethylene inhibiting rice root growth in compacted soil via ABA and auxin mechanisms (Huang et al., 2022). For example, OsSNDP1 (Os10g0122600), involved in root hair elongation, might contribute to root responses under compacted soil conditions (Huang et al., 2013; Kong et al., 2024). OsFbx352 (Os10g0127900) influences ABA biosynthesis and catabolism, affecting root architecture under stress (Song et al., 2012). OsCAF1-15 (Os10g0124200) is involved in stress hormone responses, suggesting a role in ethylene signaling under compaction (Liang et al., 2009). Additional functional studies on these genes, as well as research on root growth in compacted soil conditions, will be necessary to clearly understand the roles of these genes in ethylene signaling and root development. Understanding the specific functions of these genes could provide valuable insights into the genetic basis of stress resilience in rice, with potential applications in breeding programs aimed at improving crop performance under adverse environmental conditions.

In this study, we identified a QTL on chromosome 10, qRLR10, associated with root length reduction (RLR) in response to ethylene using GWAS in 220 indica rice accessions. Within this QTL region, a cluster of genes encoding F-box proteins were identified as candidate genes, with three of these F-box genes considered particularly promising. These findings highlight the crucial role of ethylene in root development and stress adaptation, and the identification of these candidate genes lays the groundwork for future research and crop improvement strategies.