Mitochondria play a critical role in eukaryotes by hosting some of the most vital biochemical pathways essential for cellular function. These organelles are central to the production of adenosine triphosphate (ATP) through the electron transport chain, which serves as the primary energy source for the cell. Beyond energy production, mitochondria are involved in cellular signaling, stress responses, reactive oxygen species (ROS) regulation, biosynthesis of essential biomolecules, photorespiration, and programmed cell death (Qin et al., 2021; Rasool et al., 2022). Mitochondrial functions are also critical targets for abiotic stresses, which are major environmental factors that limit plant growth and productivity (Munns and Millar, 2023). Furthermore, mitochondria are crucial in plant cell adaptation to abiotic stresses, such as drought and salinity, which induce oxidative stress at the cellular level (Xiong et al., 2017). Energy-dissipating mechanisms regulate the production of ROS in plant mitochondria, and respiration plays a pivotal role in synthesizing ascorbate, an antioxidant critical for detoxifying ROS (Rasool et al., 2022).

Global climate change exacerbates various abiotic stress conditions, including droughts, floods, and salinity (Munns and Millar, 2023). Drought is one of the most detrimental environmental stresses, significantly limiting plant growth and threatening global agricultural production by causing substantial yield losses in major crops such as wheat (Trenberth et al., 2014). Wheat, a staple crop consumed by nearly half of the world’s population, is particularly vulnerable to drought stress, severely impacting its productivity (Rasool et al., 2022). Various biotic and abiotic stress factors, including drought, challenge wheat and other crops, affecting their agronomic performance and productivity (Xiong et al., 2017; Pandey et al., 2017).

In higher plants, RNA editing is a post-transcriptional modification primarily occurring in mitochondrial- and chloroplast-encoded transcripts, where cytidine residues are converted into uridine. This editing process ensures the homology of encoded proteins and is crucial for gene expression (Qin et al., 2021). RNA editing is observed in coding sequences and untranslated regions, tRNAs, and rRNAs, with significant implications for protein function and cellular stress responses (Edera et al., 2018; Xiong et al., 2017). RNA editing in plant mitochondria is linked to regulating environmental stress responses, including oxidative stress caused by abiotic factors such as drought and salinity (Rasool et al., 2022).

The phosphorylating NADH dehydrogenase (Complex I) and non-phosphorylating NAD(P)H dehydrogenases are vital components of the NAD(P)H oxidation pathways in plant mitochondria (Xiong et al., 2017). The NAD protein complex plays a crucial role in cellular respiration, particularly in the oxidative phosphorylation pathway, which involves a series of redox reactions that generate the proton gradient necessary for ATP synthesis (Ghifari and Murcha, 2020). These reactions occur primarily in the inner mitochondrial membrane, facilitated by large protein complexes within the electron transport chain (Ramadan, 2020).

Research has shown that RNA editing of NADH dehydrogenase subunits, such as NAD9, occurs in plant mitochondria and is associated with varying stress responses. For example (Xiong et al., 2017), identified 90 RNA editing sites in nad9 transcripts in six mitochondrial genes across two rice varieties, many of which were stress-responsive. Such findings underscore the importance of RNA editing in mitochondrial function and stress adaptation, particularly under drought conditions (Rasool et al., 2022).

The present study aims to investigate nad9 RNA editing patterns in wheat in response to drought stress and compare these patterns between drought-tolerant and sensitive wheat cultivars. This research aims to enhance our understanding of how RNA editing contributes to plant adaptation to environmental stresses, potentially guiding the development of more resilient crop varieties.

Identifying RNA editing sites provides novel insights into regulating the Triticum aestivum nad9 gene, with potential implications for understanding plant responses to stress. RNA editing is crucial for ensuring the correct expression of functional proteins, particularly within mitochondria and chloroplasts. Prior research has established a link between abiotic stress and RNA editing, underscoring the importance of this mechanism in maintaining cellular homeostasis (Yuan and Liu, 2012). NADH dehydrogenase 9 (NAD9) is a mitochondrially encoded subunit of plant respiratory chain complex I (Maldonado et al., 2022). The relationship between RNA editing and plant responses to environmental stress has been extensively studied (Rodrigues et al., 2017; Xiong et al., 2017), highlighting a potential role in stress regulation (Yuan and Liu, 2012). Partial editing of mitochondrial nad9 transcripts, which encode the NADH dehydrogenase subunit 9, has been observed in several plant species, including potatoes (Grohmann et al., 1994). In this study, we examined RNA editing of the nad9 gene in response to drought stress in two wheat cultivars, one sensitive and the other tolerant. In the drought-tolerant cultivar Giza168, 22 RNA editing sites were identified, predominantly involving C-to-T transitions ( Figure 1 ; Supplementary Tables S2 , S4 ), consistent with common plant editing mechanisms (Mohammed et al., 2022). Although other types of editing have been reported (Ramadan et al., 2023), the mechanisms underlying these processes remain largely unknown.

Drought stress significantly affected 14 of the 22 editing sites (C178, C208, C308, C386, C517, C553, C592, C604, C673, G807, C820, T837, T855, and C861); however, seven of these sites resulted in synonymous amino acid changes, suggesting an unexplored involvement of tRNA in these processes. A similar pattern was observed in the sensitive cultivar Gemmiza 10, where seven of the 19 identified sites were significantly affected by drought stress (C178, C592, C622, C662, C820, T855, and C861), with synonymous amino acids detected at three sites (C178, T855, and C861). Three critical editing sites distinguished the tolerant cultivar Giza168 from the sensitive cultivar Gemmiza10: C553, where editing leads to a synonymous amino acid change (arginine), but is absent in the sensitive cultivar; C673, which is fully edited in the tolerant cultivar but not in the sensitive one; and C820, where editing increases under stress in the tolerant plants but decreases in the sensitive ones. These findings suggest that drought stress may negatively impact several factors, such as the PPR protein responsible for editing these sites, which may be absent or dysfunctional in sensitive plants (Laluk et al., 2011; Lu et al., 2022; Sun et al., 2018).

Generally, RNA editing directly impacts proteins’ primary, secondary, and tertiary structures. At the primary structure level, amino acids such as arginine 185 (R) to glycine (G), threonine 202 (T) to leucine (L), proline 225 (P) to serine (S), and arginine 274 (R) to cysteine (C) are altered due to drought stress. In the sensitive cultivar, changes such as threonine-60 (T) to leucine (L), serine-221 (S) to phenylalanine (F), and aspartic acid-256 (D) to tyrosine (Y) were observed ( Supplementary Figures S1 , S2 ). Our result, like most RNA editing sites in the mitochondrial nad and atp genes, resulted in the production of hydrophobic amino acids, which may enhance the affinity of the resulting subunits for the mitochondrial inner membrane (Ibrahim et al., 2023; Rusman et al., 2023).

Drought stress also induces changes in secondary structures, particularly affecting the conformation of alpha helices and beta sheets by altering the amino acid composition. Overall, the protein profiles (beta sheets and alpha helix) of both cultivars were highly similar under control conditions. However, significant differences emerged under stress, both at the 2-hour and 12-hour time points ( Supplementary Figures S5 - S7 ). In the drought-tolerant cultivar Giza168, beta-sheet formation was modified, with one segment becoming two due to an R185G substitution, an increase in beta-sheet length due to a T202L substitution, and the disappearance of alpha helices due to an R274C substitution, however, the P225S substitution preserved the protein loop structure ( Supplementary Figure S3 ). In contrast, the sensitive cultivar Gemmiza10 did not exhibit a specific pattern in response to editing, except for an increase in length in some protein regions, such as regions in amino acids 39-94, 212-214, etc. Our study suggests that RNA editing plays a significant role in enhancing plant resilience to drought. If RNA editing fails to improve protein efficiency, the plant may become more susceptible to abiotic stress. These conclusions align with other studies exploring the relationship between RNA editing and environmental stress adaptation (Qin et al., 2021; Ramadan et al., 2023; Xiong et al., 2017).

The predicted secondary structure of the 98 amino acids from multiple sequence alignment underscores the variability and conjectured secondary structure of the 98 N-terminal amino acids, indicating structural plasticity that may facilitate regulatory protein interactions and posttranslational modifications. This level of regulatory sophistication, inferred from sequence variability, indicates the evolutionary adaptations of Triticum aestivum, which could profoundly affect the organism’s metabolic efficiency, stress adaptability, and agricultural yield. These insights augment our understanding of the regulatory intricacies governing plant mitochondrial proteins and highlight the potential of these posttranslational modifications as targets for enhancing crop resilience in an era of climatic uncertainty. The elucidation of the structure of plant mitochondrial complex I lagged that of chloroplasts until the complete complex I structure was recently resolved in Arabidopsis thaliana via cryo-electron microscopy (cryo-EM) (Klusch et al., 2021). The observed variability in the initial 98 amino acids among the different plant species suggested that the regulatory function of these genes’ merits further investigation. The absence of the first 98 amino acids in the Arabidopsis structure, present in Triticum aestivum Gemmiza10 and Giza168, presents a unique opportunity to explore species-specific adaptations.

An examination of the N-terminal region of the NAD9 protein in Triticum aestivum revealed a complex regulatory environment characterized by posttranslational modifications. Identified phosphorylation sites for GSK3 kinase suggest that phosphorylation events could significantly influence the functions of NAD9. Concurrently, SPOP-binding motifs hint at targeted protein degradation pathways that may be crucial for controlling protein turnover. The ubiquitination mechanism, often a precursor to proteasomal degradation, is hypothesized to play a critical role in regulating mitochondrial function, particularly the specific responses to environmental stress observed in Triticum aestivum. These regulatory mechanisms underscore the versatility of the NAD9 protein and its potential importance in plants’ metabolic regulation and ecological response. The coexistence of these regulatory motifs within the NAD9 sequence exemplifies the complexity of cellular control systems, particularly in organisms where metabolic adaptability is essential. The GSK3 (MOD_GSK3_1) motif suggests a modulatory effect on the activity of the NAD9 protein, indicating its involvement in diverse signaling pathways that govern mitochondrial metabolic processes. Previous studies have shown that GSK3 regulates various cellular functions, including metabolism and energy production (Cohen and Frame, 2001; Jope and Johnson, 2004). Given the pivotal function of complex I in cellular energy production (Hirst, 2013), these phosphorylation sites are posited to be critical for the delicate modulation of metabolic activity, which is essential for wheat growth and development. The phosphorylation of mitochondrial proteins by GSK3 has been implicated in regulating ATP production and maintaining mitochondrial integrity (Rusman et al., 2023), further supporting the significance of these sites in the NAD9 protein.

The RNA editing-induced NAD9 protein structural changes of the Giza168 cultivar could significantly impact the protein’s stability by introducing a sulfur-containing side chain ( Figure 9 ). These RNA editing events collectively suggest a posttranscriptional modification pattern that may play a critical role in the structural and functional adaptation of the NAD9 protein in response to temporal cues in Triticum aestivum. Replacing amino acids within the protein sequence can alter the protein’s physicochemical properties, potentially affecting complex I’s assembly and function in the mitochondria. The S103F amino acid change in Gemmiza10, potentially alters its interaction with the mitochondrial membrane or affect the protein’s stability. Similarly, P136 T may influence the protein’s regulation and interaction with other mitochondrial components ( Figure 10 ). This editing event could be critical for modulating the enzyme’s activity in response to cellular energy needs.

Our findings extend the understanding of plant mitochondrial dynamics, highlighting how posttranslational modifications orchestrate energy production and contribute to a plant’s ability to withstand fluctuating conditions. Such insights promise to advance wheat crop resilience, offering a foundation for future research to optimize plant performance in response to global agricultural challenges.

This study examined RNA editing in the NAD9 in response to drought stress in wheat cultivars, focusing on tolerant and sensitive varieties. The study identified 22 RNA editing sites in the drought-tolerant cultivar Giza168, with 14 sites influenced by drought stress and seven exhibiting synonymous amino acids. Additionally, three critical editing sites were found to differentiate the tolerant cultivar Giza168 from the sensitive cultivar Gemmiza10. Drought stress altered the primary and secondary structures of nad9 due to changes in alpha helices and beta sheets, with the tolerant cultivar Giza168 experiencing alterations in beta-sheet length and alpha-helix number. In contrast, the sensitive cultivar Gemmiza10 did not exhibit a specific pattern in response to editing, except for an increase in length in some regions. These findings suggest that RNA editing plays a crucial role in enhancing plant drought tolerance, and failure to improve protein efficiency through RNA editing may increase plant susceptibility to abiotic stress.