Genome-Wide Identification and Expression Profiling of Aconitase Gene Family Members Reveals Their Roles in Plant Development and Adaptation to Diverse Stress in Triticum aestivum L.

Global warming is a serious threat to food security and severely affects plant growth, developmental processes, and, eventually, crop productivity. Respiratory metabolism plays a critical role in the adaptation of diverse stress in plants. Aconitase (ACO) is the main enzyme, which catalyzes the revocable isomerization of citrate to isocitrate in the Krebs cycle. The function of ACO gene family members has been extensively studied in model plants, for instance Arabidopsis. However, their role in plant developmental processes and various stress conditions largely remained unknown in other plant species. Thus, we identified 15 ACO genes in wheat to elucidate their function in plant developmental processes and different stress environments. The phylogenetic tree revealed that TaACO genes were classified into six groups. Further, gene structure analysis of TaACOs has shown a distinctive evolutionary path. Synteny analysis showed the 84 orthologous gene pairs in Brachypodium distachyon, Aegilops tauschii, Triticum dicoccoides, Oryza sativa, and Arabidopsis thaliana. Furthermore, Ka/Ks ratio revealed that most TaACO genes experienced strong purifying selection during evolution. Numerous cis-acting regulatory elements were detected in the TaACO promoters, which play a crucial role in plant development processes, phytohormone signaling, and are related to defense and stress. To understand the function of TaACO genes, the expression profiling of TaACO genes were investigated in different tissues, developmental stages, and stress conditions. The transcript per million values of TaACOs genes were retrieved from the Wheat Expression Browser Database. We noticed the differential expression of the TaACO genes in different tissues and various stress conditions. Moreover, gene ontology analysis has shown enrichment in the tricarboxylic acid metabolic process (GO:0072350), citrate metabolic process (GO:0006101), isocitrate metabolic process GO:0006102, carbohydrate metabolic (GO:0005975), and glyoxylate metabolic process (GO:0046487). Therefore, this study provided valuable insight into the ACO gene family in wheat and contributed to the further functional characterization of TaACO during different plant development processes and various stress conditions.


Introduction
The Krebs cycle, or citric acid cycle, is also known as the Tricarboxylic acid (TCA) cycle [1]. The TCA cycle produces ATP in almost all living organisms, which is a vital source the global demand for wheat has been rising and we will need to increase wheat production to feed the ever-growing world population [34]. However, wheat production is severely affected by diverse stresses [35][36][37]. Thus, publicly available genome sequence data provide the opportunity to carry out a genome-wide analysis of the ACO gene family in wheat. Here, we identified and analyzed the ACO gene family of wheat using different computational tools. The analysis of the TaACO gene family includes phylogenetic analysis, chromosomal distribution, gene structure, synteny relationship, and protein-protein interaction. Further, the expression profiling of TaACO genes were investigated in different tissues, developmental stages, and stress conditions. The expression profile of TaACO genes revealed the differential expression pattern of the TaACO genes in different tissues and various stress conditions. Therefore, our results provide valuable information about the ACO gene family in wheat and contribute to the further characterization of ACOs during plant development and response to diverse biotic and abiotic stress.

Genome-Wide Characterization and Evolutionary Analysis of TaACO Genes
In the present investigation, 15 ACO genes were identified in the wheat genome by using different computational tools (Table 1; Table S1).
Compared to other plants, this number is relatively higher than, for instance, A.thaliana, O.sativa, G.max, B. distachyon and S. bicolor (Table S2).
This may be due to the fact that wheat is hexaploid with a bulky genome size. TaACO gene family members contain protein lengths ranging from 477-1007 amino acids with molecular weights 51.92-109.32 kilodalton for TaACO10 and TaACO2. The isoelectric point was 5.67 and 6.6 for TaACO14 and TaACO7. We also correlated the molecular weight of TaACOs with their isoelectric point values to understand the dispersal of various TaACOs ( Figure S1). These results indicate that all TaACOs had a similar isoelectric points and molecular weights. The GRAVY values ranged from −0.073 to −0.227, suggesting that TaACO proteins are hydrophilic. Further, we also predicted the subcellular location of TaACO proteins; most of the TaACOs localized at chloroplast thylakoid membrane, while two TaACOs were situated in the mitochondrion, cytoplasm, and one TaACO protein was found at extracellular space (Table 1).

Chromosomal Location, Gene Duplication and Synteny Relationship of TaACO Genes
The identified TaACO genes were mapped on the wheat chromosome using the Phengram webserver. The TaACO genes were located on 11 chromosomes of wheat ( Figure 2 and Table 1). The B sub-genomes contain the maximum TaACO genes (6), followed by A sub-genomes (5) and D sub-genomes (4), respectively ( Figure S3A). Further, a single gene was found on the chromosomes 2B, 3A, 3D, 4B, 5A, 7A, and 7D, while two TaACO genes were mapped on the chromosomes 3B, 6A, 6B, and 6D ( Figure S3B).
Conversely, none of TaACO genes were mapped on the chromosome 1 (A, B & D), 2 (A &D), 4 (A & D), 5 (B & D), or 7 (B). Thus, these results indicated that the TaACO gene family members were unequally distributed on the wheat chromosomes. Further, we also scanned for the gene duplication in the ACO gene family in wheat. The phylogenetic tree of TaACOs has shown some gene duplications ( Figure S3 and S4). We observed that ten TaACO genes were involved in duplication events ( Figure 3 and Figure S4 and Table S4), which indicates that the increase of the ACO gene family in wheat was mainly caused via segmental and whole-genome duplication.
To determine the selection force of duplicated TaACO genes, we calculated the Ka/Ks values for the five pairs of TaACO genes ( Figure S4; Table S4). The Ka/Ks value was <1 for the five TaACO genes, pointing out that TaACO genes undergo a solid purifying selection with minor alterations, and subsequent duplication. Therefore, these results suggest the preserved evolution of TaACO genes. We also examined the synteny analysis of TaACO genes with model plants and other wheat relatives, for instance, O. sativa A. thaliana, Ae. tauschii, B. distachyon, and T. dicoccoides; the MCScanX was used to identify the orthologous pair genes between these plant genomes ( Figure 4 and Table S5).   We found 13, 15, 17, 10, and 29 orthologous genes between TaACOs with other ACOs in O. sativa A. thaliana, Ae. tauschii, B. distachyon, and T. dicoccoides, respectively. The results show that 4, 22, 11, and 20 TaACO genes were collinear with ACO genes in O. sativa A. thaliana, Ae. tauschii, and B. distachyon, respectively. Except for the TaACO1 gene, all had two orthologous pairs of TaACO genes, which may play critical functions in the evolution of TaACOs. Thus, these outcomes indicate that TaACO genes may result from other crop ortholog genes.

Gene Structure and Conserved Motif and Protein Tertiary Structure Analysis of TaACO Genes
We also examined the structural features of the TaACO genes ( Figure 5). Gene structure analysis showed that TaACO genes significantly differed in gene structure ( Figure 5 and Figure S5).
TaACO genes comprise 12-19 introns, for instance, TaACO1, TaACO2, TaACO3, and TaACO4 had the minimum twelve introns, while a maximum of nineteen introns were identified in the TaACO9, TaACO10, TaACO11, TaACO12, TaACO14, and TaACO15. To understand the functions of TaACO genes, we analyzed the conserved motif of TaACO protein by the Multiple Em for Motif Elicitation (MEME) website. Finally, we identified the ten conserved motifs in TaACO proteins ( Figure 6 and Figure S6). The TaACO gene family was discovered by the existence of aconitase domain (pfam PF 00330) and all fifteen TaACO proteins contain at least one aconitase motif (Table S6) which participates in isomerization of citrate to isocitrate in the TCA cycle. Additionally, the alignment of amino acid sequence also revealed that TaACO proteins shared a conserved aconitase domain ( Figures S7 and  S8A). Further, to understand the biological function of TaACO genes in wheat, the threedimensional (3D) structure of TaACO proteins were predicted by phyre2 online web tools ( Figure S8B). TaACO proteins consist of four globular domains with a linker fragment.

Prediction of Cis-Acting Regulatory Elements (CAREs) in TaACO Genes
To examine the precise functions of TaACO genes, 2000 base pair upstream sequences of TaACO genes were evaluated via PlantCARE online web server. This analysis has shown that TaACO promoters comprise different cis-elements related to plant hormones, developmental processes, defense and stress response ( Figure 7A and Table S7). TaACO genes contain the five phytohormone responsive elements, such as the gibberellin responsive element (GARE), the auxin-responsive element (AuxRE), the abscisic acid response element (ABRE), the salicylic acid response element (SARE), and the MeJA response element (MeJARE). The CAREs fit into light responsive, stress responsive, ABRE, and MeJARE, which were the ubiquitous presence in the promoter region of TaACO gene family members ( Figure 7B). Hence, these findings indicate that TaACOs play an essential role in plant development and response to diverse stresses. Further, TaACOs also comprise CARE related to meristem expression, zein metabolism, circadian control, endosperm expression, cell cycle regulation, and seed-specific regulation. The CAREs reported in the TaACO gene family demonstrated that TaACOs might be involved in various biological processes. Thus, these results provide crucial information to understand the intricate regulatory network of the TaACO gene family in response to plant hormones, multifactorial stress, and different developmental processes.

Gene Ontology Analysis of TaACO Genes
Gene ontology (GO) enrichment analysis help in elucidating the function of any genes by comparing amino acid sequence with the known function of other plant species genes. All TaACO gens were magnificently interpreted and assigned GO terms by AgriGO, and further confirmed by EggNOG database ( Figure S9; Tables S8 and S9) that reverted the similar outcomes as AgriGO. TaACO genes have shown enrichment in the tricarboxylic acid metabolic process (GO:0072350), citrate metabolic process (GO:0006101), isocitrate metabolic process GO:0006102, carbohydrate metabolic (GO:0005975), nucleoside phosphate metabolic process GO:0006753, glyoxylate metabolic process (GO:0046487), cellular respiration (GO:0045333), response to metal ion (GO:0010038), response to oxidative stress (GO:0006979), response to osmotic stress (GO:0006970) in the biological process category ( Figure S9A), while in the cellular class, TaACO has displayed enrichment in the cytoplasm (GO:0005737), intracellular organelle (GO:0043229), membranebounded organelle (GO:0043227), intracellular membrane-bounded organelle (GO:0043231) ( Figure S9B). In addition, the prediction of subcellular localization also returns similar results (Table 1). In the molecular function class, catalytic activity (GO:0003824) and binding (GO:0005488) were the predominant groups that participated in the TCA cycle ( Figure S9C). Thus, these findings indicate that TaACO genes play an important function in plant developmental processes.

Expression Profiling of TaACO Genes in Different Tissues, Developmental Stages, and Stress Conditions
To understand the role of TaACO genes, the expression profiling of TaACO genes were investigated in different tissues, developmental stages, and stress environments. The TPM values of TaACOs genes were downloaded from the Wheat Expression Browser. These TPM numbers were used to create heatmaps and carry out a principal component analysis ( Figure 8, Figures S10A,B and S11). To understand the expression patterns of TaACOs, five tissues were taken from three different developmental stages. TaACOs genes exhibited differential expression in the various tissues and different developmental stages ( Figure 8); for instance, the expression levels of TaACO4, TaACO5, and TaACO12 in leaf_z10, the expression levels of TaACO2, TaACO3, TaACO4, TaACO5, TaACO8, and TaACO12 in leaf_z23, and TaACO1, TaACO8 and TaACO10 were significantly elevated in leaf_z71.
The expression of TaACO15 in grain_z75 was raised. The expression of TaACO10, TaACO12, TaACO14, and TaACO15 were elevated in grain_z85. In addition, the expression of TaACO6, TaACO7, TaACO9, TaACO11, and TaACO13 was significantly raised in root_z10, and TaACO3 was upregulated in spike_z30. These findings demonstrated that the TaACO gene family might be implicated in various plant developmental processes.
Expression profiling of TaACO was also examined in various stress conditions including cold, drought, heat, septoria tritici blotch, powdery mildew, and stripe rust ( Figure S11). The expression level of TaACO1 was significantly elevated in PM24h, while expression of TaACO9, TaACO13, and TaACO15 was significantly induced in PM48h, and expression of TaACO2, TaACO4, and TaACO8 was slightly upregulated in PM72h. TaACO2, TaACO4, TaACO5, and TaACO8 have shown elevated levels in SR72h, whereas the expression of TaACO11 was highly raised in Zt14d. Further, the differential expression pattern was also noticed for TaACO genes in abiotic stress. The expression level of TaACO5, TaACO10, and TaACO12 was significantly elevated in cold. Similarly, the expression level of TaACO6 and TaACO7 were highly induced in heat stress after 6 h. TaACO14 and TaACO15 have displayed higher expression in drought stress after 6 h. TaACO6 and TaACO7 also showed elevated expression levels in combined drought and heat stress after 6 h ( Figure S11). Furthermore, the expression kinetics of TaACO genes were confirmed by quantitative reverse transcription PCR (qRT-PCR). qRT-PCR analysis revealed that an almost similar expression pattern was observed under abiotic stress conditions ( Figure 9). Collectively, these results have shown that TaACO genes may participate in different developmental processes and respond to biotic and abiotic stresses in wheat.

Protein-Protein Interaction Network Analysis of TaACO Genes
To study the protein-protein interactions (PPIs) between TaACOs and other proteins, a protein network was generated by the STRING webserver ( Figure 10 and Table S10).
According to the predicted results, we reported that TaACO interacted with 98 different wheat proteins. TaACO5 [38,39]. Citrate synthase (CS) catalyzes the condensation of oxaloacetate (OAA) and acetyl coenzyme A to produce citrate in the TCA cycle [40,41]. These results provide valuable information to understand the biological functions of TaACO genes.

Discussion
The aconitase gene family in plants plays an important role in the glyoxylate and TCA cycles. ACO gene family has also been identified and characterized in other crops including three genes in A. thaliana, eight in G. max, two in B. distachyon, six in Z. mays, four in S. bicolor, and four in C. clementina [1,21]. However, for the first time, we identified the ACO gene family in wheat. Several researchers have shown that ACO genes are also present in primitive plants and expanded during evolution [1]. In this work, we performed an in silico analysis and identified the 15 ACO genes in wheat ( Table 1). The phylogenetic analysis A. thaliana, G. max, T. aestivum, B. distachyon, S. bicolor, Z. mays, P. patens, and S. moellendorffii revealed that ACO genes were divided into six classes ( Figure 1). The phylogenetic tree has shown that groups I and II were specific to monocot, whereas group IV had dicot-specific ACOs, and group V contained primitive-landedspecific ACOs (Figure 1 and Figure S2). The origin of this kind of genes suggests specific functions in the monocots that play a critical role in morphological development [7,26,28,42]. However, ACO genes were dispersed into the well-known clade Arabidopsis, soybean, sorghum, maize, and wheat, suggesting that the ACOs gene may originate from a common ancestor. The TaACO gene family is expanded in the wheat and contained more ACO genes compared to the earlier described ACOs in A. thaliana, G. max, Z. mays, O. sativa, B. distachyon, S. bicolor, P. trichocarpa, and C. clementina [1,21]. Tandem, segmental, and whole-genome duplications are the major forces involved in the gene duplication processes and expanding the gene family in various plant species [43,44]. Further, we also scanned for the gene duplication analysis of ACO gene family members in wheat. The phylogenetic tree of TaACOs has shown some gene duplications ( Figure S4).
We found ten TaACO genes involved in the duplication process ( Figure 3 and Table S4). The duplicated gene pairs are TaACO8:TaACO12, TaACO14:TaACO15, TaACO6:TaACO7, and TaACO11:TaACO13, that indicate that the expansion of the ACO gene family in wheat was mainly caused via segmental duplication and whole genome duplication. Similarly, duplication event was reported in angiosperms, which play an important role in the expansion of the ACO genes. Segmental duplication was observed in Z. mays, G. max, and P. trichocarpa. Segmental gene duplication might be the main driving force of ACO gene family expansion in the Z. mays, G. max, and P. trichocarpa [1]. Similar results were observed in citrus [21]. Additionally, the Ka/Ks value was <1 for the five TaACO genes pointing out that TaACO genes undergo a strong purifying selection with minor alterations after duplication. Therefore, these results suggest the conserved evolution of TaACO genes. Furthermore, chromosomal mapping of TaACOs showed that a single gene was found on the chromosomes 2B, 3A, 3D, 4B, 5A, 7A, and 7D, while two TaACO genes were mapped on the chromosomes 3B, 6A, 6B, and 6D ( Figure S3A). Conversely, none of the TaACO genes were mapped on the chromosomes 1A, 1B, 1D, 2A, 2D, 4A, 4D, 5B, 5D, and 7B. Hence, these results showed that the 15 TaACOs were unequally dispersed on the wheat chromosomes (Figure 2 and Figure S3B). Likewise, results were reported in PIN-FORMED (PIN), brassinazole-resistant (BZR), and PERKs gene family in wheat [26][27][28]. Thus, the unequal distribution of TaACO genes on the 11 chromosomes of wheat suggests that genes disappear and add through segmental duplication or whole genome events, and there might be errors during genome sequencing and assembly. Moreover, we found 13, 15, 17, 10, and 29 orthologous genes between TaACOs with other ACOs in O. sativa A. thaliana, Ae. tauschii, B. distachyon, and T. dicoccoides, respectively. The results show that 4, 22, 11, and 20 TaACO genes were collinear with ACO genes in O. sativa A. thaliana, Ae. tauschii, and B. distachyon, respectively. Except for theTaACO1 gene, all had two orthologous pairs of TaACO genes, which may play key functions in the evolution of TaACOs. During the evolution, there have been changes in the upstream and/or exon regions of duplicated genes, which has caused a change in their expression pattern and function [45,46]. Therefore, these findings indicate that TaACO genes may result from other plant orthologous genes.
Gene structure analysis provided key clues to understanding gene function and evolution. Gene structure analysis revealed that TaACO genes greatly differed in gene structure. TaACO genes comprise 12-19 introns, for instance, TaACO1, TaACO2, TaACO3, TaACO4 had the minimum of twelve introns, while a maximum of nineteen introns were identified in the TaACO9, TaACO10, TaACO11, TaACO12, TaACO14, and TaACO15 ( Figure 5 and Figure S5). The size of the intron is one of the important factors that influence the gene size, for example, a notable variance in gene size was observed between the largest gene TaACO4 (9 kb) and the smallest gene TaACO6 (2 kb) owing to the total intron length (9 kb vs. 2 kb). Several researchers have demonstrated the importance of introns in the evolution of various genes in plants [47,48]. Many gene families contain lower, absent, and higher numbers of introns [49,50]. The number of exon and intron differences may be suitable for the documentation of evolutionary mechanisms [50]. In this work, all 15 TaACO genes had higher numbers and bigger introns during evolution in wheat. In this study, genes have shown the variable intron-exon structures presenting diverse functions. The different gene structures of TaACO could result in diverse functions due to selection pressure during wheat genome evolution. Furthermore, examination of motifs revealed that all TaACO proteins comprise the 10 diverse motifs (Figure 6 and Figure  S6). We noticed that seven motifs were found in all the TaACO proteins (Motif 2, 3, 4, 5, 6, 9, and 10). Hence, we speculated that the same group of genes in the phylogenetic tree might share similar functions, even in the different plant species. Moreover, multiple protein sequence alignment of TaACO with other ACO proteins exhibited that TaACO shared a conserved aconitase domain (Figures S7 and S8A). WOX and YABBY gene family was evolutionarily conserved in G. hirsutum [51]. Moreover, 3D structure analysis has shown that TaACO proteins comprise the four globular domains with a linker fragment ( Figure S8B). Consequently, this result will help in understanding and explaining the molecular basis of the isomerization of citrate to isocitrate in the TCA cycle.
CAREs present in the promoter section play a key role in the function and regulation of genes [52]. TaACO promoter regions comprise the different CARE elements related to phytohormones, related to growth and stress response ( Figure 7A and Table S7). Additionally, ten CAREs were detected in each of the TaACO promoter regions (Table S7). A total of eight CAREs were predicted which are related to light response such as G-box (CARE participates in light response), Sp1 (light responsive element), GT1-motif (light responsive element), MRE (MYB binding site involved in light responsiveness), Box 4 (part of a conserved DNA module involved in light responsiveness), Box II (part of a light responsive element), chs-Unit 1 m1 (part of a light responsive element), and chs-CMA1a (part of a light responsive element) [53,54]. Further, we also found four CAREs related to growth, for instance, O 2 -site (zein metabolism regulation), GCN4-motif (endosperm expression), and CAT-box (meristem expression) [55,56]. Additionally, we detected phytohormone CARE related, for example, MeJA-responsive element (CGTCA-motif), abscisic acid-responsive element (ABRE), salicylic acid responsiveness (TCA-element), and gibberellin-responsive element (TATC-box-motif). The light responsive, defense and stress responsive, ABRE, and MeJARE elements were prevalent in TaACOs promoters ( Figure 7B and Table S7). Furthermore, we also found that CAREs participated in the response to multifactorial stress, including low-temperature responsiveness (LTR) and defense and stress responsiveness (ARE and GC-motif). These results suggest that TaACO genes might be regulated by diverse developmental processes, phytohormone and different biotic and abiotic stress conditions. Many studies have demonstrated that light plays an essential role in plant development processes [57]. Light also modulates the expression level of aconitase genes [58]. The microarray analysis in Arabidopsis has shown light suppression of the mitochondrial aconitase [59]. This could lead to the decreased respiration of mitochondria and enhanced photosynthesis efficiency. Hence, these results suggest that TaACOs play an essential role in plant development and response to diverse stress. Further, TaACOs also comprise CARE related to meristem expression, zein metabolism, circadian control, endosperm expression, seed-specific, and cell cycle regulation. The CAREs reported in the TaACO gene family demonstrated that TaACOs may be involved in a variety of biological processes. Thus, these results provide crucial information to understand the intricate regulatory network of the TaACO gene family in response to plant hormones, multifactorial stress, and different developmental processes.
TaACOs genes exhibited differential expression in the various tissues (Figure 8), for instance, the expression level of TaACO4, TaACO5, and TaACO12 in leaf_z10, TaACO2, TaACO3, TaACO4, TaACO5, TaACO8, and TaACO12 in leaf_z23, and TaACO1, TaACO8 and TaACO10 were significantly elevated in leaf_z71. The temporal and spatial expression pattern of TaACO genes indicates that TaACO genes might have a function in different tissues and various developmental stages in wheat. In addition, our gene ontology results revealed a wide range of roles of TaACO genes in the biological processes ( Figure S9A-C). These results demonstrated that TaACO genes play an important role in different tissues and developmental stages. Similarly, the expression level of PtACO3 was elevated in the root tissues of poplar [1]. The expression of cytosolic and mitochondrial forms of aconitase were regulated by phytochrome in maize leaves [24]. The expression of AtACO2 and AtACO3 were highly raised in the cauline leaf tissues in Arabidopsis [1]. Likewise, two SmACO genes were differentially expressed in leaf, stem, and root tissues. ZmACO4 showed a higher expression compared to the other five ACO genes in maize [1]. Aconitase genes have been detected in mitochondria and cytosol. The functions of each isoform are diverse [60][61][62]. Eprintsev et al. [23] also reported the differential activity and expression of Aco1 and Aco4 in scutellum during the germination of maize seeds. Further, Aconitase knockdown and knockdown mutant of Drosophila showed a lethal effect in Drosophila, suggesting that Aconitase is important for viability [63]. We assumed that all key enzymes of the TCA cycle might be controlled by light via aconitase [64]. However, no direct experimental evidence of light governing the aconitase function has been found. Carrari and colleague [16] reported that the aconitase activity decreased in the ACO mutant of tomato; however, the photosynthesis rate was significantly increased in tomato. CcAco3 was constitutively expressed-although at low levels-along all phases of fruit pulp ripening in citrus [21]. Thus, these findings demonstrated that the TaACO gene family may be implicated in various plant developmental processes.
Many age-linked and mitochondrial disorders are related to raised expression levels of reactive oxygen species following decreased activity of ACO [65]. The differential expression pattern was also noticed for TaACO genes in abiotic stress ( Figure S11). The expression level of TaACO5, TaACO10, and TaACO12 was significantly elevated in cold. Similarly, the expression level of TaACO6 and TaACO7 were highly induced in heat stress after 6 h. TaACO14 and TaACO15 displayed higher expression in drought stress after 6 h. TaACO6 and TaACO7 also showed elevated expression levels in combined drought and heat stress after 6 h ( Figure S11). The elevated expression level of TaACO genes under cold, heat, and drought stress indicates that TaACO genes might be involved in stress tolerance in wheat by coordinating regulation among the TaACOs. Several studies demonstrated that ACO genes have been implicated in response to oxidative stress in plants [11,12,66]. ABA and MeJA play a crucial role in oxidative homeostasis in plants. The CARE analyses in this work also revealed that all TaACO genes contained CAREs response to different phytohormones, defense and stress responsiveness. In addition, Wang et al. [1] showed that numerous ACO genes, for instance, BdACO2, GmACO3, and OsACO1 had an elevated expression level in root tissues of B. distachyon, G. max, and O. sativa during stress conditions. The elevated expression of citrate triggers the expression of the oxidase, thereby inhibiting ROS formation in mitochondria [66]. Furthermore, the qRT-PCR analysis revealed that an almost similar expression pattern was observed under abiotic stress conditions ( Figure 9). Moreover, our PPI results also revealed that TaACO interacted with 98 different wheat proteins. TaACO5 could interact with 13 other wheat proteins, while TaACO6 and TaACO7 could interact with 15 other wheat proteins. Hence, the TaACO protein also interacts with other proteins and modulates the metabolism and signaling pathways in wheat. These results suggested that plant ACO genes might be participated in oxidative stress by responding to different metabolisms and phytohormones. Therefore, these results have shown that TaACO genes may participate in different developmental processes and respond to abiotic stresses in wheat. Collectively, these findings demonstrated that TaACOs might regulate a wide range of cellular processes, phytohormones signaling and response to various stress; of course, this is important and must be proved by experimental data in the near future. Therefore, these results provided foundational information to elucidate the precise function of TaACO genes in plant developmental processes, responses to different plant hormones, and diverse stresses.

Identification of ACO Genes in Wheat
To identify the ACO gene in wheat, ACO protein sequences of A.thaliana, O.sativa, G.max, B. distachyon, S. bicolor, P. patens, and S. moellendorffii were downloaded from the Phytozome (https://phytozome-next.jgi.doe.gov/ accessed on 15 October 2022). The twentyeight ACO protein sequences used as query against the T. aestivum proteome with an e-value of 10 −5 and bit-score>100 kept cut-off to find TaACO genes and, finally, the BLASTp result was tabulated. Further verified for the existence of aconitase domain using other databases: InterPro [67], HMMscan [68], National Center for Biotechnology Information CDD [69] and Simple Modular 132 Architecture Research Tool (SMART, http://smart.emblheidelberg.de/ accessed on 15 October 2022). Eventually, the protein sequences with aconitase domain were selected and named according to their positions on the chromosomes.

Biophysical Characteristics, Subcellular Localization Genomic Localization, Gene Duplication and Synteny Analysis
The biophysical features of TaACO proteins, including isoelectric point, lengths, and molecular weight, were evaluated by the ExPASy server [70] and isoelectric point calculator [71]. Subcellular localization was predicted using CELLO

Expression Analysis of TaACO Genes
Gene expression data of TaACO genes in different wheat tissues (leaf, stem, root, spike, and grain) and different stress conditions (cold, drought, heat, combined drought, and heat) were obtained from WheatExp database (http://www.wheat-expression.com/ accessed on 22 October 2022). The expression pattern was presented as a heatmap based on transcript per million (TPM), which was mapped by ClustVis [74] and principal component analysis (PCA) plots generated by TBtools [73].

Plant Growth, Stress Conditions, and Quantitative Reverse Transcription PCR Analysis
Wheat seeds were seeded in plastic pots and grown in a greenhouse, and then 10-dayold seedlings were acclimatized for 2 days in growth chamber environments. The wheat seedlings were exposed to drought and heat stress (37 • C) for 1 h and 6 h, while cold stress was for 3 days (4 • C). For the combined drought and heat stress, first, the wheat seedling was exposed to drought stress and then subjected to heat stress for 1 h and 6 h at 37 • C in an incubator. Controls were kept at 25 • C. The drought-, heat-, and cold-stressed wheat seedlings were collected for RNA isolation and stored at −80 • C. Total RNA was extracted from the control, drought-, heat-, and cold-stressed wheat seedlings as described by [75,76]. cDNA was synthesized using the iScriptTM cDNA synthesis kit (Bio-Rad, Hercules, CA, USA). Wheat actin (AB181991) was used as the control to standardize the gene expression data and the qRT-PCR was executed with Applied Biosystems 7500 Fast Real-Time PCR. Each qRT-PCR reaction was performed with three technical replicates and repeated three times. The fold change value was calculated based on mean 2-∆∆CT and finally fold change value was used to plot the graph [77,78]. All primers used in this study were tabulated in Table S11.

Conclusions
Wheat is major cereal crop and widely consumed staple food worldwide. However, global warming is becoming a severe threat to food security due to the constant climate changes, largely affecting plant growth and development, eventually the yield. This has raised a significant challenge for plant biologists to increase yield and improve wheat's quality and various stress tolerance. In this work, we identified 15 ACO genes in wheat genome. Phylogenetic tree revealed that TaACO genes classified into six groups. Further, gene structure analysis of TaACOs has shown a distinctive evolutionary path. Furthermore, Ka/Ks ratio revealed that most TaACO genes experienced strong purifying selection during evolution. Numerous cis-acting regulatory elements were detected in the TaACO promoters, which might play a crucial role in plant development processes, phytohormone signaling, and are related to defense and stress. Moreover, expression profile revealed that TaACO genes were differentially expressed in different tissues, developmental stages, and abiotic stress conditions. Therefore, our results provide valuable information about the ACO gene family in wheat and contribute to the further characterization of ACOs during plant development and response to diverse biotic and abiotic stress.

Supplementary Materials:
The following are available online at https://www.mdpi.com/article/ 10.3390/plants11243475/s1. Figure S1: The molecular mass (kDa) and isoelectric point of plots of TaACO genes. Figure S2: Dispersal of TaACOs in a different group of the phylogenetic tree. Figure S3: Chromosomal locations of identified ACO genes and on the three sub-genomes of (A) Dispersal of ACO genes in the A, B and D sub-genomes. (B) Dispersal of ACO genes on wheat chromosomes. Figure S4: Evolutionary analysis of TaACO genes. A phylogenetic tree was produced using MEGA7 with the NJ method and 1000 bootstrap replications. A black asterisk denotes the duplicated gene pairs. Figure S5: The different number of exons and introns found in the TaACOs gene family. Figure S6: Sequence logo of TaACO motif and height of each mountain indicate the conservation at this position, whereas the height of the different letters represents the frequency of the corresponding amino acids. Figure S7: Alignment of the TaACO proteins and preserved aconitase domain is indicated with red color line. Amino acid residues conserved in all proteins were shaded blue and similar amino acids were gray shaded. Dashes denote gaps that led to exploiting the alignment of the homologous region. Figure Table S1: TaACO genomic, conserved domain sequence, protein and promoter sequence. Table S2: The number of ACO genes found in the other crop species. Table S3: ACO proteins from Arabidopsis, rice, soybean, wheat, brachypodium, sorghum, selaginella, and physcomitrella used to produce phylogenetic tree. Table S4: Estimated nonsynonymous substitutions (Ka) and synonymous substitutions Ks ratio of duplicate gene pairs in TaACO gene family. Table S5: Orthologous relationships of TaACO genes with other ACO genes in B. distachyon, Ae. tauschii, T. dicoccoides, O. sativa, and A. thaliana. Table S6: Identified the aconitase domains in the TaACO proteins predicted using pfam with default parameters. Table S7: Identified the cis-regulatory elements in the TaACO promoter region. Table S8: Predicted significant Go term in the TaACO gene family by AgriGo analysis. Table S9: TaACO gene annotation using EggNOG database. Table S10: The PPI network between TaACO and other proteins in wheat. Table S11: qRT-PCR primers used for the TaACO genes in this study.