Expression profiling of NF-Y gene family in a domestic wheat cultivar (Triticum aestivum L. cv. Keumkang) under waterlogging stressABSTRACT
Enhancing wheat’s ability to withstand waterlogging stress is critical for improving productivity. Transcription factors, particularly the NF-Y family, play key roles in plant growth, development, and abiotic stress responses. The NF-Y family in plants is composed of three subunits: NF-YA, NF-YB, and NF-YC, which are involved in regulating growth and stress adaptation. In wheat, several NF-Y genes have been identified for their roles in stress response, though their expression patterns under waterlogging stress remain unclear. In this study, RNA-Seq data (PRJNA943453) were analyzed to investigate the expression of TaNF-YA, TaNF-YB, and TaNF-YC genes in waterlogged wheat roots. Results revealed that a subset of TaNF-Y genes, including TaNF-YB2, TaNF-YB3, TaNF-YB5, TaNF-YB11, and TaNF-YC7 were significantly up-regulated in response to waterlogging. These findings suggested that TaNF-Y transcription factors play an important role in wheat’s adaptation to waterlogging stress. The upregulation of these genes under waterlogging conditions indicates their potential for enhancing wheat’s tolerance to transient low oxygen environments. This study provides valuable insights into the role of TaNF-Y genes in stress response, offering potential strategies for improving wheat resilience under waterlogging conditions.
Key words
abiotic stress; NF-Y transcription factor; waterlogging; wheat
Introduction
Wheat (Triticum aestivum L.) is a critical global cereal crop, with its production directly influencing food supply, dietary structure, and agricultural trade (Daniel and Hartman 2024). However, wheat is highly susceptible to waterlogging stress, which occurs due to excessive rainfall and poor soil drainage, particularly during its long growing period. Waterlogging severely limits wheat yield and quality, posing a significant challenge to global food security, especially as climate change intensifies such events (Huang et al. 2024). Waterlogging stress leads to oxygen depletion (hypoxia) in the soil, impacting wheat growth and development, particularly during sensitive stages like stem elongation. The identification of waterlogging-tolerant wheat varieties is crucial for improving crop resilience and ensuring sustainable agricultural practices. Plants have evolved various adaptive strategies to cope with waterlogging, including changes in root architecture, regulation of osmotic potential, and the formation of adventitious roots that enhance oxygen uptake (Huang et al. 2024). Additionally, waterlogging-induced aerenchyma and lignin formation help maintain root function under hypoxic conditions. Understanding these physiological and molecular responses to waterlogging stress will provide valuable insights for wheat breeding programs aimed at improving resistance to this growing challenge, ensuring stable yields in the face of unpredictable climate changes (Li et al. 2024).
The nuclear factor-Y (NF-Y) transcription factors are essential regulators of various biological processes in plants, including growth, development, and stress responses. Composed of three subunits–NF-YA, NF-YB, and NF-YC–, NF-Y proteins function as heterotrimeric complexes that bind to CCAAT motifs in the promoters of target genes, thereby modulating gene expression (Li et al. 2022). In plants, NF-Y subunits are encoded by multiple genes, with each subunit playing a specific role in regulating critical processes such as gametogenesis, embryogenesis, seed development, and abiotic stress responses (Luan et al. 2022). NF-Y transcription factors have been shown to influence root development, including adventitious root formation and root meristem activity, crucial for adapting to environmental stress. For instance, in rice (Manghwar et al. 2024), barley (Mao et al. 2020), and wheat (Pan et al. 2020), NF-Y genes have been implicated in stress tolerance, with specific subunits enhancing drought resistance, flowering time regulation, and stress-responsive gene expression. In wheat, NF-Y genes like TaNF-YA10 and TaNF-YB2 are critical for regulating drought tolerance and spike development. Despite extensive studies on NF-Y genes in model plants, their roles in wheat’s response to waterlogging stress remain underexplored (Pan et al. 2020). Understanding the regulatory mechanisms of NF-Y genes in wheat under waterlogging conditions could provide valuable insights into improving wheat’s resilience to this important abiotic stress.
To better understand the functional roles of TaNF-Y genes in wheat’s response to waterlogging, several experiments were conducted, including subcellular localization, yeast two-hybrid (Y2H) assays, and bimolecular fluorescence complementation (BiFC). Subcellular localization revealed that TaNF-YA, TaNF-YB, and TaNF-YC proteins are primarily located in the nucleus, suggesting their involvement in regulating gene expression during stress responses. Additionally, through transcriptome analysis, five specific NF-Y genes–TaNF-YB2, TaNF-YB3, TaNF-YB5, TaNF-YB11, and TaNF-YC7–were identified as key players in wheat’s response to waterlogging stress. These results not only highlight the importance of the NF-Y gene family in plant stress responses but also provide insights into the molecular mechanisms underlying waterlogging tolerance in wheat.
By elucidating the role of transcription factor-mediated adaptation, this study offers valuable gene resources and theoretical support for wheat genetic improvement and water-saving cultivation practices. Ultimately, these findings contribute to a deeper understanding of the molecular pathways involved in wheat’s response to waterlogging stress, paving the way for future breeding efforts aimed at enhancing waterlogging tolerance in wheat.
Materials and methods
Plant materials and growth condition
Korean wheat cultivar Keumkang (IT213100) seeds were provided by Professor Chul Soo Park (Jeonbuk National University, South Korea). The seeds were germinated on moist filter paper in a Petri dish at 23°C under long-day (16-h light:8-h dark) conditions with 120 μmol·m-2·s-1 photosynthetically active radiation. Germinated seedlings were transferred to plastic pots filled with tap water, and for the waterlogging treatment, they were incubated in a stagnant solution containing 0.1% (w/v) dissolved agar. At the third-leaf stage, roots from both waterlogged and untreated (control) seedlings were harvested at 0, 4, 8, and 24 h after treatment.
For the subcellular localization pattern analysis, Arabidopsis Columbia ecotype (Col-0) was used. The plants were grown either on soil (Sunshine Mix1) or on Murashige and Skoog (MS) medium basal salt mixture (Duchefa Biochemie) plates at 23°C under long-day conditions (16-hour light, 8-hour dark) with a light intensity of 120 μmol·m-2·s-1 and humidity of 50–60%. For seed sterilization, seeds grown on half-strength MS plates were sequentially washed with 70% ethanol + 0.5% triton X, then with 95% ethanol. After sterilization, seeds were sown on half-strength MS medium and stratified at 4°C in the dark for two days. Seeds planted in soil were incubated in 0.1% Phyto Agar (Duchefa Biochemie) for two days at 4°C before being transferred to growth rooms and MLR-352H growth chambers (PHC Corporation, Biomedical Division).
For bimolecular fluorescence complementation analysis, Nicotiana benthamiana seeds were germinated and grown in soil at 23°C under long-day conditions (16-h light/8-h dark) at a light intensity of 120 μmol·m-2·s-1 with humidity of 50 to 60%.
Subcellular localization patterns
Three-week-old Arabidopsis Columbia ecotype (Col-0) plants were grown in soil, and true leaves were used for protoplast isolation. Leaves were carefully cut 0.5 mm from the midrib using a sharp razor blade. Approximately 20 leaf segments were transferred to an enzyme solution (1.5% cellulose, 0.4% macerozyme, 400 mM mannitol, 8 mM CaCl2, 5 mM MES-KOH, and 0.1% bovine serum albumin) and incubated for 8 h at room temperature on a shaker (50–70 rpm) in the dark. The enzyme solution turned green, indicating protoplast release. The solution was filtered through a 0.75-μM mesh filter and diluted with an equal volume of W5 solution (154 mM NaCl, 125 mM CaCl2, 5 mM KCl, pH 5.7, 1.5 mM MES-KOH). Protoplasts were centrifuged at 100 × g for 5 minutes, and the supernatant was discarded. The pellet was resuspended in 5 mL W5 solution and centrifuged on a 21% sucrose density gradient. The protoplasts were then washed, and their concentration was determined using a hemocytometer. DNA (30 μg) was added to the protoplasts in MMG solution, followed by incubation for 7 minutes. After washing the protoplasts with W5 solution, they were incubated in WI solution (0.5 M mannitol, 4 mM MES-KOH, 20 mM KCl) for 16–18 h at 23°C. Protoplasts were then observed using a Carl Zeiss super resolution confocal laser scanning microscope (LSM 880 with Airyscan) to detect green fluorescent protein (GFP) and red fluorescent protein (RFP) fusion proteins.
Yeast two hybrid assay
To prepare competent yeast cells, 50 μL of cells were thawed on ice. High-quality plasmid DNA (100 ng) and carrier DNA (5 μL of 10 μg/μL yeast maker carrier DNA, Invitrogen) were mixed with the cells. The carrier DNA was denatured by heating at 98°C for 5 minutes, then rapidly cooled in an ice bath. The plasmid and carrier DNA mixture was added to the competent cells and mixed with 500 μL of PEG/LiAc. After incubation at 30°C for 30 minutes with gentle mixing every 10 minutes, 20 μL of DMSO was added. The mixture was heat-shocked in a 42°C water bath for 15 minutes with gentle mixing every 5 minutes. The cells were then centrifuged, resuspended in 1 mL YPDA medium, and incubated at 30°C for 1 h. After another centrifugation, the pellet was resuspended in 200 μL of 0.9% NaCl and plated on SD/-Leu-Trp medium. The plates were incubated at 30°C for three to five days.
To culture yeast cells, a single colony was inoculated into 5 mL SD/-Leu-Trp medium and grown at 30°C with shaking until the OD600 reached 1.0–1.5. The cells were aliquoted into microtubes, centrifuged, and resuspended in SD/-Leu-Trp and SD/–Ade–His–Leu–Trp media. Serial dilutions (10-1, 10-2, 10-3) were spotted on SD/-Leu-Trp and SD/–Ade–His–Leu–Trp+X-alpha Gal plates and incubated at 30°C, monitoring growth.
Bimolecular fluorescence complementation
Agrobacterium competent cells were thawed on ice, and 1 μg of cloned plasmid DNA in a binary vector was added to the cells and gently mixed. The mixture was incubated on ice for 5 minutes, followed by a 5-minute treatment in liquid nitrogen and a final 5-minute incubation at 37°C. Afterwards, 1 mL of Luria-Bertani (LB) medium was added to the mixture, which was incubated at 30°C with shaking at 250 rpm for 2–4 h. Healthy Arabidopsis Col-0 plants were grown until flowering. Agrobacterium strain GV3101 carrying the gene of interest on a pCHF3 binary vector was cultured in liquid LB medium with 100 μg/mL spectinomycin and 25 μg/mL rifampicin at 28°C. Two days later, 2 mL of cultured Agrobacterium was centrifuged, resuspended in 5% sucrose solution, and mixed with 0.05% Silwet L-77. The Agrobacterium solution was used to dip the above-ground parts of the plants, which were then placed in a tray with high humidity and kept in a dark growth room overnight. The plants were subsequently grown under normal conditions. Protoplasts were observed using a super resolution confocal laser scanning microscope (Carl Zeiss LSM 880 with Airyscan), with YFP and mChe fusion proteins excited at 488 and 568 nm to detect green and red fluorescence, respectively. The protoplast images were captured and processed using ZEN 3.3 blue edition software.
RESULTS AND DISCUSSION
Wheat seedlings exposed to waterlogging for seven or 10 days exhibited clear growth inhibition compared to those maintained under normal aerobic conditions (Fig. 1). The expression patterns of TaNF-YA, TaNF-YB, and TaNF-YC genes in wheat roots subjected to waterlogging stress, based on RNA-seq data, revealed a distinct temporal response. At the 4-h mark, a decrease in gene expression was observed for these transcription factors. This initial reduction can be interpreted as a part of the plant’s early stress response to waterlogging-induced hypoxia (Sharmin et al. 2020, Feng et al. 2024). During the early stages of waterlogging, wheat roots experience oxygen deficiency, which may trigger a rapid protective mechanism aimed at conserving energy and stabilizing cellular functions. The downregulation of these genes might represent a temporary adaptive strategy to minimize cellular damage under acute stress. This response is consistent with the concept of a stress defense phase, in which plants suppress non-essential processes to prioritize survival under adverse conditions (Shen et al. 2020). The early reduction in gene expression could be also due to the immediate metabolic reprogramming required to cope with the sudden onset of low-oxygen stress, a critical factor in waterlogging tolerance (Tian et al. 2024).
Waterlogging treatment on wheat roots. Wheat seedlings were subjected to waterlogging stress by transferring them to a stagnant solution containing 0.1% dissolved agar. The roots of the seedlings were harvested at various time points (0, 4, 8, and 24 h) after the initiation of waterlogging treatment. This setup was used to study the temporal expression changes of TaNF-YA, TaNF-YB, and TaNF-YC genes in response to waterlogging stresS
However, as the waterlogging stress continued, the expression of TaNF-YA, TaNF-YB, and TaNF-YC genes increased over time, reflecting a shift toward a more adaptive and recovery-oriented response. Following the initial suppression, the upregulation of these genes at later time points suggests that wheat roots begin to activate compensatory mechanisms to adapt to the prolonged hypoxic environment. The increase in gene expression likely indicates the activation of pathways involved in stress tolerance, such as those regulating antioxidant defense, metabolic adjustment, and recovery from oxygen deprivation (Luan et al. 2022). This dynamic expression pattern points to the role of NF-Y transcription factors in mediating wheat’s adaptation to low-oxygen conditions, especially under prolonged waterlogging stress (Yamauchi et al. 2019). The observed increase in gene expression may reflect the plant’s effort to repair cellular damage and sustain vital processes during the extended period of hypoxia. Overall, the temporal pattern of expression, characterized by an initial decrease followed by subsequent recovery, highlights the complex, time-dependent nature of wheat’s molecular response to waterlogging and the crucial role of NF-Y transcription factors in this adaptive process. These paragraphs convey the analysis in a clear, scientific manner, emphasizing both the early response and the subsequent adaptive mechanisms of wheat under waterlogging stress (Yu et al. 2019).
Subcellular localization studies aimed to identify the cellular compartments in which these proteins function. Given NF-Y proteins are known to act as transcription factors, their localization in the nucleus would imply a role in regulating stress-related gene expression. Conversely, localization in other compartments might suggest additional functions in signaling or protein interactions. Constructs 35S::TaNF-YB2:GFP, 35S::TaNF-YB3:GFP, 35S::TaNF-YB5:GFP, 35S::TaNF-YB11:GFP, and 35S::TaNF-YC7:GFP were introduced into Arabidopsis protoplasts, with the nucleus-localized ARR2:RFP as a control. While TaNF-YC7 is localized to the cytoplasm, TaNF-YB2, TaNF-YB3, TaNF-YB5, and TaNF-YB11 showed both nuclear and cytoplasmic localization, indicating diverse functional roles (Fig. 2 and Table 1).
Subcellular localization patterns of TaNF-YB2, TaNF-YB3, TaNF-YB5, TaNF-YB11 and TaNF-YC7 proteins in Arabidopsis protoplasts. ARR2:RFP was used as a nucleus-localized protein. Subcellular localization of TaNF-YB2, TaNF-YB3, TaNF-YB5, TaNF-YB11, and TaNF-YC7 in Arabidopsis protoplasts. Scale bars = 10 μm.
These findings align with studies on NF-Y proteins in other species, such as Arabidopsis (Yu et al. 2019) and rice (Yamauchi et al. 2017), in which NF-Y subunits are primarily localized in the nucleus to regulate stress responses. However, some variations in localization patterns, as seen in Zea mays (Andrews et al. 1993), suggest that NF-Y proteins may also have roles outside transcription regulation. Understanding these localization dynamics is key to deciphering how these proteins contribute to plant stress tolerance, particularly under waterlogging conditions.
The yeast two-hybrid (Y2H) assay was conducted to investigate the interactions between TaNF-YB2 (Fig. 3a), TaNF-YB3 (Fig. 3b), TaNF-YB5 (Fig. 3c), TaNF-YB11 (Fig. 3d), and TaNF-YC7 (Fig. 3e) with various TaNF-YC and TaNF-YB proteins. The analysis revealed that TaNF-YB3 interacts with TaNF-YC1, TaNF-YC2, and TaNF-YC6 (Fig. 3b), while TaNF-YB5 interacts with TaNF-YC2, TaNF-YC6, and TaNF-YC8B (Fig. 3c). Additionally, TaNF-YB11 specifically interacts with TaNF-YC2 (Fig. 3d), and TaNF-YC7 interacts with TaNF-YB1 and TaNF-YB9 (Fig. 3e). As a positive control, SNAC7 was found to interact with TaNF-YB2. These findings provide valuable insights into the protein-protein interactions that contribute to the formation of NF-Y heterotrimeric complexes in wheat (Fig. 3 and Table 2). The Y2H results suggest that TaNF-YB and TaNF-YC subunits interact in specific combinations, which may play a critical role in regulating gene expression during stress responses and developmental processes.
Y2H analysis. The empty pGBKT7 and SNAC7 was used as controls in SD/-Leu-Trp medium. The yeast cells were grown on SD/-Leu-Trp medium plate and and SD/–Ade–His–Leu–Trp selection medium. (a) TaNF-YC1, TaNF-YC2, TaNF-YC3, TaNF-YC4, TaNF-YC5, TaNF-YC6, TaNF-YC7, TaNF-YC8B, TaNF-YC8D andTaNF-YC14were cloned into pGBKT7 vector, TaNF-YB2 was cloned into pGADT7 vector. (b) TaNF-YB3 was cloned into pGADT7 vector, TaNF-YC1, TaNF-YC2, TaNF-YC3, TaNF-YC4, TaNF-YC5, TaNF-YC6, TaNF-YC7, TaNF-YC8B, TaNF-YC8Dand TaNF-YC14were cloned into pGBKT7 vector. (c) TaNF-YB5was cloned into pGADT7 vector, TaNF-YC1, TaNF-YC2, TaNF-YC3, TaNF-YC4, TaNF-YC5, TaNF-YC6, TaNF-YC7, TaNF-YC8B, TaNF-YC8D and TaNF-YC14 were cloned into pGBKT7 vector. (d) TaNF-YB11was cloned into pGADT7 vector, TaNF-YC1, TaNF-YC2, TaNF-YC3, TaNF-YC4, TaNF-YC5, TaNF-YC6, TaNF-YC7, TaNF-YC8B, TaNF-YC8D and TaNF-YC14 were cloned into pGBKT7 vector. (e) TaNF-YC7 was cloned into pGADT7 vector, TaNF-YB1, TaNF-YB2, TaNF-YB3, TaNF-YB4, TaNF-YB5, TaNF-YB6, TaNF-YB9, TaNF-YB10, TaNF-YB11, TaNF-Dr1A and TaNF-Dr1B were cloned into pGBKT7 vector.
NF-Y complexes are known to influence various physiological processes, including stress tolerance, root development, and flowering time (Bailey-Serres and Chang 2005). The identification of interactions among TaNF-Y subunits highlights their potential to work together in enhancing the plant’s resilience to environmental stresses like waterlogging and drought (Benjamini et al. 2001). Additionally, these interactions could involve other regulatory components, such as additional transcription factors or signaling molecules, further enriching our understanding of the molecular mechanisms governing plant stress responses (Borrego-Benjumea et al. 2021).
The interaction between TaNF-YB3 and TaNF-YC1, TaNF-YB3 and TaNF-YC2, TaNF-YB5 and TaNF-YC8, TaNF-YB10 and TaNF-YC7, and TaNF-Dr1B and TaNF-YC7 was further confirmed using BiFC assays in Nicotiana benthamiana leaves (Fig. 4 and Table 3). Co-expression of TaYC1-cYFP and TaYB3-nYFP resulted in yellow fluorescent protein (YFP) signals in both the nucleus and cytoplasm, confirming the interaction between TaNF-YB3 and TaNF-YC1 (Fig. 4a). However, no YFP signal was observed with other combinations, such as TaYC1-cYFP and nYFP or TaYB3-nYFP and cYFP, indicating that the interaction is specific. The interaction between TaNF-YB3 and TaNF-YC2 was not observed, suggesting that these two proteins may not form a functional complex under the experimental conditions (Fig. 4b).
Bimolecular fluorescence complementation (BiFC) analysis between TaNF-YB protein and TaNF-YC protein. Agrobacterium tumefaciens strains containingTaNF-YB3 and TaNF-YC1, TaNF-YB3 and TaNF-YC2, TaNF-YB5 and TaNF-YC8, TaNF-YB10and TaNF-YC7, TaNF-Dr1B and TaNF-YC7 were further confirmed by BiFC assays in the leaves of Nicotiana benthamiana. The Agrobacterium suspensions were then injected into the abaxial surface of tobacco leaves (6 weeks old) using a needleless syringe. The leaves injected with Agrobacterium tumefaciens were kept in the dark for 48–60 h. Tobacco leaves injected with the empty vector controls nYFP and cYFP served as negative controls. The red color is the chlorophyll autofluorescence.
Similarly, co-expression of TaYC8-cYFP and TaYB5-nYFP resulted in YFP signals in the nucleus, confirming the interaction between TaNF-YB5 and TaNF-YC8 (Fig. 4c). Other combinations did not show fluorescence signals. Additionally, co-expression of TaYC7-cYFP and TaYB10-nYFP in tobacco leaves resulted in YFP signals in the nucleus and cytoplasm, confirming the interaction between these two proteins (Fig. 4d). Lastly, co-expression of TaYC7-cYFP and TaNF-Dr1B-nYFP showed YFP signals in the cytoplasm, indicating a specific interaction between TaNF-YC7 and TaNF-Dr1B (Fig. 4e). These results confirmed the interactions among the selected NF-Y subunits and provide further insights into their potential functional roles in wheat stress responses.
In this study, wheat was subjected to waterlogging treatment, and RNA samples were collected at 4, 8, and 24-h post-treatment for RNA-seq analysis. Seven libraries were used in the RNA-seq experiment. However, due to logistical constraints, biological replicates were not included in the experimental design. The absence of biological replicates introduces several limitations that should be carefully considered when interpreting the results.
First, biological replicates are essential for capturing the biological variability between independent samples and ensuring the reliability and generalizability of the findings. Without biological replicates, it is challenging to determine whether the observed changes in gene expression represent true biological trends or are merely the result of technical noise or sample-specific variations (Kim et al. 2024). The lack of replicates may lead to overinterpretation or underestimation of gene expression changes, potentially affecting the robustness and reproducibility of the conclusions.
Second, biological replicates enhance the statistical power of the analysis by allowing for the quantification of variability within the samples. In the absence of biological replicates, the RNA-seq data may lack the necessary statistical reliability, affecting the identification of differentially expressed genes. Typically, three biological replicates improve the statistical efficacy of differential expression analysis, making the results more reliable and accurate (Jahan et al. 2024). Without sufficient replicates, the risk of false positives and false negatives increases, as the gene expression data from a single library may be influenced by sample-specific characteristics or sequencing errors, thus undermining the accuracy of the subsequent analysis.
Nevertheless, this study provides valuable preliminary data on the gene expression changes in wheat under waterlogging stress. These results offer important insights, particularly for identifying and characterizing genes related to waterlogging tolerance. However, future studies should ensure the inclusion of biological replicates to enhance the statistical reliability of the findings and provide more accurate and comprehensive understanding of the molecular mechanisms involved in wheat’s response to waterlogging stress (Huang et al. 2024).
This study analyzed the gene expression changes in wheat roots under waterlogging stress using RNA-seq technology and revealed the expression profiles of several waterlogging-responsive genes. The results demonstrated that as the duration of waterlogging stress increased, the expression of waterlogging-responsive genes in wheat roots gradually enhanced, indicating that waterlogging stress induced the expression of genes involved in root morphology changes and carbon-nitrogen metabolism at different time points. These findings are consistent with previous studies in other crops, such as Arabidopsis, barley, maize, and rice, which have reported that waterlogging stress regulates the expression of specific genes, facilitating plants’ adaptation to waterlogging (Feng et al. 2024).
As a model plant, Arabidopsis (Kim et al. 2024) has been extensively studied for its gene expression responses under waterlogging stress, providing valuable insights. Studies have shown that multiple NF-Y transcription factor family members in Arabidopsis are significantly upregulated under waterlogging stress, particularly NF-YA, NF-YB, and NF-YC subunits, which enhance stress tolerance through heterotrimer formation and regulation of downstream gene transcription (Kim et al. 2024). In line with this, the present study observed a significant increase in the expression levels of TaNF-YB and TaNF-YC members in wheat roots under waterlogging stress, suggesting that these NF-Y family members may regulate stomatal movement, cell protective enzymes, and other stress-related pathways to enhance wheat’s tolerance to waterlogging.
Similar findings have been reported in barley (Luan et al. 2022) and maize (Pan et al. 2020), in which NF-Y transcription factors play a crucial role in waterlogging stress responses. In barley, NF-YB and NF-YC family members have been shown to regulate oxidative stress by modulating antioxidant enzyme activity, thereby protecting cells from reactive oxygen species-induced damage during waterlogging stress (Feng et al. 2022, Luan et al. 2022). Likewise, in maize, NF-YB and NF-YC subunits also respond to waterlogging stress by interacting with NF-YA to regulate genes involved in stress responses (Pan et al. 2020, Wang et al. 2021). These results corroborate our findings that the wheat NF-Y family regulates reactive oxygen species balance and enhances antioxidant enzyme activity, contributing to improving stress tolerance during waterlogging.
Moreover, the transcriptomic analysis revealed that many genes associated with root morphology changes and carbon-nitrogen metabolism were differentially expressed under waterlogging stress. This is consistent with the fact that waterlogging stress induces oxygen deficiency in plant roots, which affects root respiratory metabolism. To cope with this stress, plants modify root growth and enhance water and nutrient absorption by regulating genes involved in root development and metabolism (Andrews et al. 1994). Similarly, rice has been shown to modify root morphology under waterlogging stress by activating genes involved in starch and amino acid metabolism, which helps enhance root development under anaerobic conditions (Nguyen et al. 2018, Watanabe et al. 2017). These results provide theoretical support for understanding how wheat adjusts root morphology and metabolism in response to waterlogging.
Further high-throughput transcriptomic analysis revealed that the number of differentially expressed genes increased significantly at 8 and 24 h post-treatment, with more upregulated genes detected than at 4 h. This suggests that the induction of waterlogging-responsive genes requires more time, and at earlier time points, the responses may not be as prominent. Similar patterns have been observed in maize and rice, in which the expression of waterlogging-responsive genes increases over time (Park et al. 2020, Sharmin et al. 2020). These findings indicate that waterlogging stress induces a time-dependent gene expression response in plants, leading to gradual activation of stress-responsive genes as the stress intensifies.
The transcriptome data also highlighted significant involvement of genes related to carbon and nitrogen metabolism under waterlogging stress. Waterlogging stress leads to reduced oxygen availability in roots, disrupting aerobic metabolism. In response, plants regulate key metabolic processes to optimize energy utilization and material transport under anaerobic conditions (Broughton et al. 2015, Yamauchi et al. 2016). This has been observed in other crops such as rice and wheat, in which waterlogging stress modulates metabolic pathways to improve stress adaptation.
CONCLUSION
This study provides valuable preliminary data on the molecular mechanisms underlying wheat’s response to waterlogging stress, especially focusing on the NF-Y transcription factor family. Future research should further explore the functional roles of these transcription factors through molecular mechanisms to provide a more comprehensive understanding of their regulatory roles in wheat’s adaptation to waterlogging stress and their potential application in breeding for waterlogging tolerance.
ACKNOWLEDGMENTS
We would like to thank Professor Liu for providing wheat seeds.
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How to cite: Chen, M., Lee, J. H. and Liu, H. (2025). Expression profiling of NF-Y gene family in a domestic wheat cultivar (Triticum aestivum L. cv. Keumkang) under waterlogging stress. Bragantia, 84, e20240302. https://doi.org/10.1590/1678-4499.20240302
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FUNDING
BK21 FOUR Program by Jeonbuk National University ResearchChina Scholarship CouncilGrant no.: 202208260064
DATA AVAILABILITY STATEMENT
The data are available in a data repository https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA943453.
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Edited by
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Section Editor:
Carlos Alberto Scapim https://orcid.org/0000-0002-7047-9606
YFP: yellow fluorescent protein.