Mitochondrial nad3 gene RNA editing in Withania somnifera: Implications for protein structure and stability

1. Introduction

RNA editing refers to the process whereby RNA sequences are altered following transcription. This alteration may involve the addition, deletion, or alteration of specific nucleotides (Shikanai, 2015, Ruchika and Nakamura, 2022). This modification is crucial for regulating protein expression across various organisms (Ruchika and Nakamura, 2022). It has been observed in a wide range of organisms, including animals, viruses, and reduced eukaryotic cells, indicating that RNA modification may confer evolutionary advantages (Liu et al., 2024). Initial evidence of RNA alteration emerged from studies that elucidated the molecular mechanisms of this process, which was subsequently termed “RNA editing” (Shikanai, 2015). The methods used to find out how to edit RNA have resulted in the creation of a dynamic transcriptome library that allows scientists to view different RNA transcripts simultaneously and in real-time (Karagianni et al., 2024). Several gene sequences produce different transcripts through the process of RNA editing (Ruchika and Nakamura, 2022). In general, the literature categorizes RNA editing into two main types: guide-based editing, which includes A-I and C-U editing, and non-guide-based editing, comprising C-U, U-C, and U-A editing (Petersen et al., 2013, Ramadan et al., 2024). Numerous studies underscore the importance of RNA editing in adapting to environmental changes and maintaining cellular homeostasis (Ramadan et al., 2024). RNA editing occurs not only in organelles but also specifically within the mitochondria and plastids of land plants, exhibiting unique characteristics (Hao et al., 2021). The higher plant species composition, including both angiosperms and gymnosperms, includes spermatophytes, where extensive RNA editing occurs, and bryophytes, which display minimal or no RNA editing (Huang et al., 2022). Evidence suggests that RNA editing in land plants originated independently several times in both mitochondria and plastids, leading to varied distributions of RNA editing across different species within these organelles (Hao et al., 2021).

The overall pattern of RNA editing has shown significant diversification, primarily attributed to the acquisition of new introns (Ruchika and Nakamura, 2022). This increased transcript diversity resulting from RNA editing provides a valuable foundation for exploring the phylogeny and evolution of land plants. Land plant RNA editing has developed tissue-specific patterns, dual cytidines, and shows parallel evolution concerning the directions of first to third codon sites, along with conserved protein origins that may reflect the evolutionary history of RNA editors (Ichinose and Sugita, 2016, Pereira et al., 2024, Duan et al., 2023). Consequently, it allows for the reconstruction of phylogenetic relationships among major land plant lineages and deepens our understanding of early diversification (Dong et al., 2023). Moreover, special nucleotide substitution profiles of RNA editing have been associated with distinctive evolutionary adaptations in several genera spanning diverse taxonomic classifications, thus illuminating the environmental challenges encountered by these plant lineages throughout their evolution (Li et al., 2023). The marked divergence of plastid genomes is characterized by the patchy occurrence of RNA editing, extending the phylogenetic distribution of these mechanisms to encompass the entire Saxifragales clade (including Paeoniaceae) (Mulligan et al., 2007, Tang et al., 2024). Comparative analyses of RNA editing profiles further reveal conserved patterns of interspecific adaptive evolution across embryophytes (Edera et al., 2018, Hao et al., 2021). Under stress conditions, signals like abscisic acid (ABA) and reactive oxygen species (ROS) upregulate PPR proteins and associated factors, enhancing editing activity (Xiao et al., 2018, Wang and Tan, 2025, Luo et al., 2022). This regulation is supported by crosstalk between the nucleus and mitochondria, enabling stress-induced transcriptional adjustments to optimize mitochondrial RNA editing. During environmental light, editing fine-tunes mitochondrial Atp4 transcripts to balance energy allocation in Calotropis procera (Ramadan et al., 2024). Collectively, these findings highlight RNA editing as a dynamic, adaptive mechanism that underpins plant survival in challenging environments.

The nad3 gene is an important part of mitochondrial Complex I in the electron transport chain (ETC). It helps make ATP by promoting redox processes in complexes I, III, IV, and V (Čermáková et al., 2021). The NAD complex is necessary for oxidative phosphorylation, which helps cells breathe through electron carriers like NADH and FADH2. These carriers are mostly made in the tricarboxylic acid (TCA) cycle. These carriers help move electrons along the ETC. This creates a proton gradient across the mitochondrial membrane, which in turn generates ATP (Arnold and Finley, 2023). This cohesive process highlights the interrelationships between mitochondrial pathways in energy metabolism.

The inhibition of the RNA editing site at position 250 of the nad3 gene has been shown to lead to decreased mitochondrial function and varied impacts on energy metabolism in Arabidopsis (Yuan and Liu, 2012). This suggests that differences in reversion patterns could contribute to changes in energy metabolism, potentially influencing developmental rate and timing. The importance of this RNA editing is highlighted by its evolutionary conservation across numerous species (Ichinose and Sugita, 2016). Thus, environmental pressures might affect editing at these sites, potentially stabilizing them into certain sequences and transcripts that could promote adaptive changes (Ramadan et al., 2024).

RNA editing at nad3 predominantly involves the conversion of cytidines to uridines, as recognized by reverse transcriptase and presumably the ribosome (Schuster et al., 1990, Yu et al., 2025, Gerke et al., 2020). A reverse alteration was observed in the cytochrome b locus, where a genomically encoded T was changed to C in the cDNA sequence (Ichinose and Sugita, 2016). The nad3 locus transcripts in the mitochondria of the higher plant Oenothera contain several altered cytosines, now recognized as uridines. Sixteen distinct nucleotide changes were found in the nad3 coding region of Oenothera mitochondria (Schuster et al., 1990) and fifteen sites in Carthamus tinctorius (Yamini et al., 2008, Wu et al., 2023b). The significance of nad3 editing in drought tolerance was examined at eleven locations in the nad3 gene of the desert plant Calotropis procera (Yuan and Liu, 2012, Ramadan, 2014).

To identify and characterize RNA editing sites, researchers have developed various experimental methods and bioinformatics tools, including high-throughput sequencing of transcripts and reverse transcription PCR (Ramadan et al., 2023, Satam et al., 2023). High-throughput sequencing techniques have proven to be efficient, specific, sensitive, and robust in identifying and detecting frequent edits within transcripts (Mohammed et al., 2022). Additionally, widely accepted computational tools for RNA editing detection have been established; these tools are designed to classify computationally predicted sites to facilitate their manual verification (Wu et al., 2023a).

This study examines RNA editing sites in the mitochondrial nad3 gene of medicinal plant Withania somnifera (Khalid et al., 2024) under salinity stress. By analyzing RNA-seq data and modeling tertiary protein structures, we identify salinity-induced changes in editing patterns and their structural consequences for the NAD3 protein. These findings highlight whether RNA editing in nad3 is common in all plants, as previous investigations into abiotic stress adaptation or not.

2. Materials and Methods

3. Results

3.1 Characterization of ithania somnifera nad3 gene and CDS: Nucleotide sequence variations and amino acid structural changes

The genomic DNA of the mitochondrial nad3 gene, designated with Accession number OR451222, was successfully extracted from Withania somnifera and incorporated transcript data from two distinct conditions: a control sample, identified by Accession number OR451223, and a sample subjected to salinity stress. To pinpoint transcripts of the nad3 gene, a substantial number of paired-end reads were utilized; specifically, 128,140,192 reads were analyzed from the control sample, while for the sample subjected to one week of salt stress at a concentration of 100 mM NaCl, 117,692,344 reads were processed.

A detailed examination of the nad3 genomic sequence, along with two cDNA sequences (Table S2) exposed to different treatments (specifically control and salinity), revealed a total of sixteen C-to-U editing sites (C5, C44, C61, C62, C146, C208, C209, C215, C230, C247, C251, C266, C275, C317, C344, and C349) (Fig. 1).

Table S2

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Fig. 1.

RNA editing positions for the nad3 gene obtained from RNA-seq data, compared with the DNA sequence (wild type). The data from the replicates are presented as means with standard deviations (black bars). Statistically significant differences are indicated with ** at P < 0.01.

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ANOVA revealed highly significant differences (p < 0.01) of RNA editing in this plant, but no significant difference between Control and Salt Stress conditions across all tested positions, with F-values ranging from 15,757 to 364,809. The calculated effect sizes (η²) indicated strong treatment effects, with η² values ranging from 0.74 to 0.99.

3.2 Validation of nad3 gene editing and conserved domain analysis

To confirm the identified RNA editing loci in the nad3 gene and assess the reliability of RNA-seq as a characterization technique, the editing sites were verified using qRT-PCR. The investigation assessed and evaluated the extent of nad3 editing (C5, C44, C61, C62, C146, C208, C209, C215, C230, C247, C251, C266, C275, C317, C344, C349) across two distinct experimental conditions (control and salinity). The findings from this analysis are illustrated in Fig. 2, which depicts the levels at each editing position under the respective treatments. The primary objective of this experiment was to validate the precision of the bioinformatics data by juxtaposing it with the results obtained from qRT-PCR.

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Fig. 2.

qRT-PCR confirmation of nad3 gene editing sites. The data from the replicates are presented as means with standard deviations (black bars). Statistically significant differences are indicated with ** at P < 0.01.

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The examination of the domain was conducted on the identified NADH dehydrogenase subunits, with their respective Accession numbers identified through the utilization of the InterPro Database. The NADH dehydrogenase subunit 3 was found to have the accession numbers pfam00507, PTHR11058, and IPR000440 (Fig. 3).

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Fig. 3.

Conserved domain analysis for the obtained protein sequence of NADH dehydrogenase subunit 3.

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3.3 Secondary structure of edited and non-edited nad3 subunit

The secondary structure of NADH dehydrogenase subunit 3 showed variation in the size of beta sheets before and after RNA editing, like the region from amino acid 73 to 78. Also, release new beta-sheets after editing in regions 80 to 83. However, the same number of beta sheets (six) was observed in the nad3 subunit before and after editing (Fig. 4).

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Fig. 4.

The quantity of secondary structure domains present in NADH dehydrogenase subunit 3 before and subsequent to RNA editing is documented. The beta sheets are denoted in red.

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3.4 Protein edits resulted from the RNA edits event(s)

Due to the similarity of salt stress RNA editing with the control, we will continue with the control NAD3 protein in this section. The RNA edits effectively changed 14 amino acids despite the salt’s stress. The edits noticed were S2L, P15T, P21L, S49F, P70F, P72 T, S77F, P83S, P84 T, P89 T, S92F, S106F, S115L, and R117W (Fig. 5). The NAD3 protein domain secondary structure analysis comprises three main alpha helices. All of them interact with the NADH‐dehydrogenase subunit 1 (NAD1) domain of NAD complex I. Two helices are aligned, and the third is connected through a long flexible loop.

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Fig. 5.

Secondary Structure and Sequence Alignment of NAD3_Wt (Wild-Type DNA) and Edited Proteins (NAD3_Ctrl and NAD3_Salt). (a) NAD3_Wt Protein. (b) NAD3_Control (Ctrl) Protein Secondary Structure. The Wild-Type protein sequence is shown along with its secondary structure elements. Orange coils represent alpha-helices, while the purple lines represent beta-strands. The black line denotes the protein backbone.

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3.5 Edited and non-edited protein modeling and their thermal stability

The observed changes were used to build up the protein models. The models’ quality was good regarding residues in the most favored regions, which were above 90 for all three models, and the Ramachandran plots aligned with it (Fig. 6). The overall models align perfectly with the secondary structure prediction (Fig. 5).

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Fig. 6.

Comparison of Structural and Stability Analysis of NAD3_Wt and Edited Proteins. Panel (a) WT (Wild-Type) Protein Analysis and Panel (b) Edited Protein Analysis. Top Left Graph: The plot represents the raw stability scores (green dots) and averaged stability scores (blue line) for each Wt/Ctrl protein residue. The stability scores are plotted against the residue positions. Bottom Left Ramachandran Plot: The Ramachandran plot shows the distribution of phi (Φ) and psi (ψ) angles for the Wt/Ctrl protein residues. The plot highlights allowed (yellow) regions and disallowed conformations (Yamini et al., 2008). Bottom Left Structure: The cartoon represents the Wt (deep purple)/Ctrl (aqua blue) protein structure, highlighting its secondary structural elements.

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The protein stability was computationally measured for NAD3_Ctrl compared to the NAD3_Wt with a negative Gibbs free energy (ΔΔG) value for both the sum ΔΔG (-7.8 kcal/mol) and the prediction ΔΔG (-1.4 kcal/mol), indicating that the edits, on average, stabilize the protein (Fig. 7). However, the sum ΔΔG suggests a more substantial stabilization effect compared to the prediction ΔΔG. This discrepancy might arise due to how individual edits interact with each other or because of compensatory effects within the protein structure. The average distance of 21.95 Å indicates significant structural rearrangements. While the stabilization metrics are positive, the substantial conformational changes could affect the protein’s function, mainly if these edits affect regions critical for activity or interactions.

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Fig. 7.

DynaMut Analysis of Protein Stability and Flexibility upon Multiple Edits. The top section lists the specific edits analyzed: A S2L; A P15T; A P21L; A S49F; A P70F; A P72T; A S77F; A P83S; A P84T; A P89T; A S92F; A S106F; A S115L; A R117W. The cartoon representation shows the protein’s secondary structure with magenta alpha-helices and white loops. Dashed orange lines highlight Hydrogen Bonds, while dashed green lines highlight the hydrophobic interactions. The stick representation of side chains illustrates where edits have been introduced, with different colors indicating various types of interactions and residues affected.

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3.6 Docking analysis of edited and non-edited NAD3-NAD1 complex with quinine

The effects of RNA edits on the binding affinity and interaction profile of the NAD3-NAD1 complex with quinine were examined. Docking results were analyzed for the wild-type (virtual translated protein from DNA) and control versions of the complex, with structures and interaction profiles visualized in Fig. 8. The wild-type NAD3-NAD1 complex exhibited a binding affinity of -7.0 kcal/mol with quinine (Fig. 8a). Fundamental interactions involved residues PHE225, ALA226, and LEU229, which formed van der Waals and alkyl interactions with quinine. The binding pocket was well-defined, with quinine fitting adequately into the cavity formed by the interaction residues.

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Fig. 8.

Docking analysis of NAD3-NAD1 Complex with Quinine. (a) Wild-type NAD3 (pink)-NAD1 (cyan) complex showing quinine (blue) with a binding affinity of -7.0 kcal/mol. Key interactions involve residues PHE225, ALA226, and LEU229. (b) Control NAD3-NAD1 complex with RNA edits, including LEU21 (purple), showing a binding affinity of -7.3 kcal/mol. LEU21 establishes significant van der Waals interactions, enhancing binding stability. (c) Superimposition of wild-type (blue) and control (purple) complexes with an RMSD of 3.9078, indicating significant conformational changes. Interaction maps highlight hydrogen bonds and van der Waals forces, with notable contributions from LEU21 in the control complex.

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In contrast, the control version of the NAD3-NAD1 complex, which incorporated specific RNA edits, showed a slightly improved binding affinity of -7.3 kcal/mol (Fig. 8b). The interaction profile for the control complex highlighted similar residues (PHE225, ALA226, LEU229) and additional interactions involving ASP228 and GLY230, suggesting a more stabilized binding conformation for quinine. One notable interaction observed in the control complex was with LEU21. This residue, introduced through RNA editing, established a crucial van der Waals interaction with quinine, contributing significantly to the overall binding affinity. The presence of LEU21 in the control complex underscores the impact of RNA edits in enhancing specific interactions within the binding pocket. Structural superimposition of the wild-type and control complex revealed substantial conformational changes at the binding site, with an RMSD of 3.9078 (Fig. 8c). These structural alterations are likely responsible for the observed differences in binding affinity and interaction profiles. Detailed interaction maps (bottom panels of Fig. 8a and b) revealed that the RNA edits introduced new hydrogen bonds and alkyl interactions in the control complex, which were absent in the wild-type. Specifically, the interaction involving LEU21 in the control complex added a novel van der Waals interaction, enhancing the overall interaction network and binding stability.

4. Discussion

The utilization of Alkekengi officinarum mitochondrial nad3 gene sequences as a template for homologous nad3 gene reconstruction in Withania somnifera demonstrates methodological efficacy, attributable to the phylogenetic proximity of these species within the Solanaceae family. High-throughput RNA sequencing yielded substantial transcriptomic datasets, comprising 128,140,192 and 117,692,344 reads for control and one-week salt-stress experimental groups, respectively. These datasets facilitated robust identification of nad3 transcripts in W. somnifera, thereby elucidating molecular mechanisms associated with mitochondrial protein restoration and contributing to a broader understanding of plant mitochondrial evolution.

Critical analysis of RNA editing sites within these transcripts reveals post-transcriptional regulatory mechanisms governing nad3 expression in W. somnifera. Such findings hold implications for understanding adaptive protein modification strategies in plants, particularly those enhancing enzymatic efficiency under physiological constraints. RNA editing, a conserved process essential for organellar protein functionality (Yuan and Liu, 2012, Castandet and Araya, 2011), has been empirically linked to environmental stress adaptation. For instance, Arabidopsis models demonstrate that impaired RNA editing correlates with dysregulated ROS accumulation and compromised dehydration tolerance (Yuan and Liu, 2012). These observations underscore RNA editing’s role in maintaining cellular homeostasis under abiotic stress, suggesting analogous mechanisms may operate in W. somnifera to mediate salt stress responses.

This investigation advances understanding of RNA editing’s evolutionary significance in plant mitochondrial genomes while providing a framework for further inquiry into stress-responsive molecular adaptations in medicinal species. Future research may explore causal relationships between specific editing events and functional protein diversification in fluctuating environmental contexts. Numerous plant species have been observed to possess distinct editing locations for the nad3 gene. Specifically, Arabidopsis demonstrates nine editing sites, onion reveals thirteen, cucumber possesses ten, wild carrot features eighteen, soybean comprises twelve, and sunflower displays eleven (Edera et al., 2018). This study established that heteroplasmy is not present and identified 16 specific locations that generate editing in nad3 gene of Withania somnifera, namely C5, C44, C61, C62, C146, C208, C209, C215, C230, C247, C251, C266, C275, C317, C344, and C349 (Fig. 2, Table S2). This research uncovered several significant findings regarding RNA editing in this desert plant. Salt stress did not affect any of the identified editing sites of Withania somnifera nad3 gene, although it is reported in other plants (Yuan and Liu, 2012, Ramadan, 2014, Ramadan, 2020). We suggest the absence of the salt effect may be due to the absence of the leading editing site C250, which was observed to be related to salt tolerance in previous investigations (Yuan and Liu, 2012; Ramadan, 2014; Ramadan, 2020). In addition, RNA editing in the nad3 gene is a mechanism intended to restore conserved protein functions, possibly evolving to reverse previous edits. This finding underscores the significance of RNA editing as a critical biological process. Additionally, no synonymous amino acid changes were observed, contrasting with earlier studies (Edera et al., 2018).

The secondary structure of NADH dehydrogenase subunit 3 revealed that RNA editing has a profound impact, resulting in alterations to the dimensions and configuration of beta sheets. RNA editing was observed to restore or elongate beta sheets in specific regions, such as the 80-83 residue region (Fig. 4), and merge or elongate others (e.g., residues 7-21) compared to the corresponding DNA sequences. These findings align with our previous results from the study of the Ccmfn gene (Ramadan et al., 2023), suggesting that RNA editing plays a significant role in the structural integrity and functional optimization of proteins. The DynaMut analysis indicated that the combined edits generally stabilized the protein, as evidenced by the negative ΔΔG values. However, the average distance metric suggests significant conformational changes. The interaction map further illustrates how specific edits influence hydrogen bonding and hydrophobic interactions, contributing to protein stability.

The RNA editing events resulted in significant changes to the amino acid sequence of the NAD3 protein, with 14 amino acid substitutions observed despite the salt stress conditions. These edits were primarily located within the three central alpha-helices of the NAD3 protein, all interacting with the NAD1 domain in the NAD complex I. The structural analysis demonstrated that RNA editing in mitochondrial genes such as nad3 significantly impacts protein function and stability, highlighting the functional importance of these edits (Xiao et al., 2018, Yang et al., 2012). Stability scores and Ramachandran plots indicate that the edits generally stabilize the protein, with the control NAD3_Ctrl showing a negative Gibbs free energy (ΔΔG) value of -7.8 kcal/mol for the sum ΔΔG and -1.4 kcal/mol for the prediction ΔΔG. This suggests that, on average, the edits enhance stability, although significant structural rearrangements (average distance of 21.95 Å) were observed, which could impact protein function. This is consistent with previous studies demonstrating that specific RNA edits can enhance protein stability (Yuan and Liu, 2012, Wang et al., 2015).

The RNA edits implemented in the control complex significantly altered the binding pocket of the NAD3-NAD1 complex. These changes were reflected in the binding affinity and interaction profile with quinine. In this context, the RNA edits have optimized the binding site for quinine within the NAD3-NAD1 complex, resulting in increased interactions. Quinine is known to interact with mitochondrial proteins, including components of the ETC (Pereira Jr and Kowaltowski, 2021). The RNA edits described in the paper can significantly alter the binding pocket of mitochondrial protein complexes, including the NAD3-NAD1 protein complex (Maldonado et al., 2022). The increased number of interactions, including the notable contribution of LEU21, and the slight improvement in binding affinity suggest that the RNA edits enhance the stability and specificity of quinine binding. These structural alterations could affect how small molecules like quinine interact with these complexes. RNA editing can fine-tune the structural configuration of these complexes, potentially enhancing or reducing the affinity for various ligands (Maldonado et al., 2022). Therefore, the increased interactions and improved binding affinity of quinine observed in edited complexes might be explained by the specific RNA-induced structural changes that make the binding site more favorable for quinine interaction.

The increased binding affinity of quinine observed in RNA-edited complexes underscores the potential for RNA editing to enhance or alter the interaction between mitochondrial protein complexes and small molecules. Furthermore, the adaptogenic properties of Withania somnifera compounds, known for their mitochondrial-protective effects, may be synergistically enhanced when interacting with RNA-edited versions of the NAD3-NAD1 complex. This suggests a novel usage of RNA editing could be leveraged to optimize mitochondrial function and improve the efficacy of plant-based compounds in mitigating mitochondrial dysfunction. The detailed interaction maps highlighted new hydrogen bonds and alkyl interactions introduced by RNA edits in the control complex, absent in the wild-type, with LEU21 playing a pivotal role in these enhanced interactions. Previous research has similarly shown how RNA editing can influence ligand binding and protein-ligand interactions (Gray, 2009).

This study investigates the complex interactions between quinine, bioactive compounds from Withania somnifera, and the NAD3-NAD1 complex. Our findings indicate that RNA editing significantly alters the structural conformation of the NAD3-NAD1 complex, potentially enhancing its binding affinity for specific ligands such as quinine. These structural changes may be an adaptive response due to salt stress, optimizing the complex’s interaction profile to maintain mitochondrial function under challenging conditions. The protective role of Withania somnifera is highlighted by its bioactive compounds, which are known to support mitochondrial integrity. The potential synergistic effects between these compounds and RNA-edited versions of the NAD3-NAD1 complex suggest that Withania somnifera may enhance mitochondrial stability and function. This engagement may play a vital role in reducing the harmful impacts on plant mitochondria while maintaining the overall health and productivity of the plants.

5. Conclusions

This study extensively examines RNA editing in Withania somnifera’s mitochondrial nad3 gene, demonstrating its importance in protein structure, stability, and function. A consistent set of 16 RNA editing sites across control and stress conditions emphasizes the importance of this post-transcriptional process in protein function and stability. RNA editing altered the amino acid sequence and structural conformation of NAD3, specifically its beta sheets and alpha helices. These structural alterations stabilized the protein and caused conformational shifts that could affect its function. The increased binding affinity and interaction profile with quinine show that RNA editing can change protein-ligand interactions, which may affect plant adaptability. This study suggests that RNA editing is necessary for mitochondrial protein function and may allow plants to evolve protein function. This finding expands the biotechnology uses of RNA editing due to its stability and selectivity. Future research should examine how RNA editing across gene networks affects plant resilience and productivity under diverse environmental conditions.