Genome-Wide Identification, Characterization, and Expression Analysis of Long-Chain Acyl-CoA Synthetases in Carya illinoinensis under Different Treatments

As crucial enzymes in the lipid metabolic network, long-chain acyl-CoA synthases (LACSs) are members of the acyl-activated enzyme superfamily and play a crucial role in epidermal wax synthesis, plant lipid anabolic metabolism, and stress tolerance. In this study, 11 pecan LACS genes were identified and categorized into five groups and located on nine chromosomes. The significant degree of conservation in the AtLACS and CiLACS protein sequences was demonstrated by multiple sequence alignment and conserved motif analysis. Cis-acting element analysis identified numerous stress-responsive and hormone-inducible elements in the promoter regions of CiLACS genes. The expression levels of CiLACS9 and CiLACS9-1 were considerably up-regulated under salt and drought stress, according to the qRT-RCR study. Treatment with ABA also led to increased expression levels of CiLACS1, CiLACS1-1, CiLACS2, and CiLACS9-1. Notably, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1 exhibited peak expression levels at 135 days after anthesis and are likely to have been crucial in the accumulation of seed kernel oil. Moreover, the CiLACS9 gene was shown to be located in the cytoplasm. These findings offer a theoretical framework for clarifying the roles of LACS genes in the processes of pecan kernel oil synthesis and response to abiotic stressors.


Introduction
The acetyl coenzyme A synthetase (ACS) superfamily, which may be further subdivided into short-chain, medium-chain, long-chain, and extra-long-chain lipid coenzyme A synthases based on the length of their carbon chains [1], includes long-chain acyl-CoA synthetases (LACSs). They are crucial in the production of plant lipids. The synthesis of lipids in plant epidermal waxes and seeds requires the participation of the substrate lipid coenzyme A [2], LACS catalyzes the formation of lipid coenzyme A from free fatty acids in a two-step reaction, in which the free fatty acids react with ATP in the presence of Mg 2+ to form the adenosylated intermediate acyl-AMP, which then binds to the thiolipid bond of CoA to release lipid coenzyme A and AMP [3]. The AMP-binding domain in LACS, which is highly conserved and specific to the AMP-binding superfamily, and a conserved ACS signaling motif in the AMP-binding superfamily both play important roles in this activation process [4,5]. LACS can contribute significantly to plant stress tolerance and fatty acid anabolic metabolism thanks to these particular structural configurations.
Several members of the LACS family in plants were found in various subcellular compartments, including the endoplasmic reticulum, peroxisomes, and the chloroplast outer

Genome-Wide Identification and Characterization of LACS Genes in Pecan
Eleven LACS genes were selected from the whole pecan genome; they were renamed (CiLACS1~CiLACS9-1) based on their similarity to Arabidopsis, and their amino acid sequences were examined. According to the findings, the CiLACS genes encode amino acid residues with a theoretical range of 613 to 730, the theoretical relative molecular masses of the encoded proteins range from 67.60 to 880.16 kDa, the theoretical isoelectric points are 5.96~8. 36, and the theoretical instability coefficients are 27.79~40.36 (Table S1). All others had instability coefficients below 40, except for CiLACS2, which had an instability coefficient of 40.36. According to the results of the predicted subcellular localization, six CiLACS proteins were located in the chloroplast, one was located in the nucleus, and four were located in the cytoplasm.
LACS protein sequences from pecan and seven additional species (Arabidopsis, rape, Chinese hickory, soybean, poplar, and rice) were used to create phylogenetic trees, and LACS members of these species were renamed in accordance with their affinities to Arabidopsis (Table S2). The results revealed that the CiLACS proteins were more evenly distributed among the five subclades of LACS proteins, which formed a total of five subclades ( Figure 1). Of them, three belonged to subclade 5, two to subclade 1, one to subclade 1, two to subclade 2, two to subclade 3, and three to subclade 4. These CiLACSs, however, demonstrated a stronger affinity for pecan. quences were examined. According to the findings, the CiLACS genes encode amino residues with a theoretical range of 613 to 730, the theoretical relative molecular ma of the encoded proteins range from 67.60 to 880.16 kDa, the theoretical isoelectric po are 5.96~8.36, and the theoretical instability coefficients are 27.79~40.36 (Table S1). All ers had instability coefficients below 40, except for CiLACS2, which had an instability efficient of 40.36. According to the results of the predicted subcellular localization, six LACS proteins were located in the chloroplast, one was located in the nucleus, and were located in the cytoplasm.
LACS protein sequences from pecan and seven additional species (Arabidopsis, r Chinese hickory, soybean, poplar, and rice) were used to create phylogenetic trees, LACS members of these species were renamed in accordance with their affinities to bidopsis (Table S2). The results revealed that the CiLACS proteins were more evenly tributed among the five subclades of LACS proteins, which formed a total of five clades ( Figure 1). Of them, three belonged to subclade 5, two to subclade 1, one to subc 1, two to subclade 2, two to subclade 3, and three to subclade 4. These CiLACSs, howe demonstrated a stronger affinity for pecan.

Multiple Sequence Alignment, Gene Structure, and Conserved Motif Analysis of the CiLACS Genes
Eleven pecans and nine Arabidopsis had high homology and high conservatio their LACS amino acid sequences ( Figure 2). The AMP-binding domain (red box) and

Multiple Sequence Alignment, Gene Structure, and Conserved Motif Analysis of the CiLACS Genes
Eleven pecans and nine Arabidopsis had high homology and high conservation in their LACS amino acid sequences ( Figure 2). The AMP-binding domain (red box) and the ACS signaling domain (green box), two commonly conserved structural domains of the LACS family, are shared by nearly all members of the CiLACS family. variation between subfamilies ( Figure 3A). In subclades 1, 2, and 3, there are 19 exons and 18 to 20 introns, while in subclade 4, there are 23 exons and 22 introns, and in subclade 5, there are 11 exons and 10 to 11 introns. The CiLACS proteins are highly conserved throughout evolutionary history, as shown by a motif analysis of the CiLACS protein (Figure 3B), which revealed that each CiLACS contains an AMP-binding domain (motif 3) and the ACS signaling domain (motif 1).   Gene structure and motif analyses of the CiLACS genes were carried out to examine the evolutionary conservation of these genes. The results revealed that CiLACS has roughly the same number of exons and introns within the same subfamily, with some variation between subfamilies ( Figure 3A). In subclades 1, 2, and 3, there are 19 exons and 18 to 20 introns, while in subclade 4, there are 23 exons and 22 introns, and in subclade 5, there are 11 exons and 10 to 11 introns. The CiLACS proteins are highly conserved throughout evolutionary history, as shown by a motif analysis of the CiLACS protein ( Figure 3B), which revealed that each CiLACS contains an AMP-binding domain (motif 3) and the ACS signaling domain (motif 1). Gene structure and motif analyses of the CiLACS genes were carried out to examine the evolutionary conservation of these genes. The results revealed that CiLACS has roughly the same number of exons and introns within the same subfamily, with some variation between subfamilies ( Figure 3A    The horizontal axis of (A) indicates the length of the protein sequence, and the horizontal axis of (B) indicates the length of the promoter sequence.

Chromosomal Localization and Colinearity Analyses of CiLACS Genes
Using the MCScanX program, which is based on the genomic database, the intraspecific homology of 11 CiLACS genes and the interspecific homology of 9 Arabidopsis LACS genes were examined to better understand the potential functions of CiLACS genes. The results are shown in Figure 4. All of the identified CiLACS genes were found to be located on the nine pecan chromosomes. Each chromosome had one to two CiLACS genes, with chromosomes 6 and 8 having the most. The pecan LACS family also contained a total of six colinear pairs and fifteen colinear pairs with the model plant Arabidopsis, with each member of the family sharing at least one homologous pair of genes with Arabidopsis (Table S3). on the nine pecan chromosomes. Each chromosome had one to two CiLACS gen chromosomes 6 and 8 having the most. The pecan LACS family also contained six colinear pairs and fifteen colinear pairs with the model plant Arabidopsis, w member of the family sharing at least one homologous pair of genes with Arabid ble S3).
Ka and Ks were further calculated to analyze the traits of CiLACS family m during evolution. Ka/Ks can be used to explore the selective pressure on gene evolution; a value of Ka/Ks > 1 denotes positive selection, a value of Ka/Ks < 1 negative selection, and a value of Ka/Ks = 1 denotes neutral selection. The replicati of the pecan LACS family had Ka/Ks values between 0.1 and 0.2, which suggests CiLACS genes underwent significant negative selection during evolution (Table

Tissue-Specific Expression Analysis of the CiLACS Genes
Understanding the biological functions of genes is aided by tissue-specific of those genes. Analysis of transcriptome data based on pecan in six different tiss ure 5) to determine the tissue expression levels of LACS genes revealed that Ci was expressed at high levels in all tissues except seeds; CiLACS8 was expressed levels in all tissues, especially in roots and seeds; CiLACS1-1 was expressed at hi in flowers and fruits; CiLACS6-1 and CiLACS9 were expressed at high levels leaves, and seeds; and CiLACS1 and CiLACS9-1 were highly expressed in flow fruits ( Figure 6). Ka and Ks were further calculated to analyze the traits of CiLACS family members during evolution. Ka/Ks can be used to explore the selective pressure on genes during evolution; a value of Ka/Ks > 1 denotes positive selection, a value of Ka/Ks < 1 denotes negative selection, and a value of Ka/Ks = 1 denotes neutral selection. The replication genes of the pecan LACS family had Ka/Ks values between 0.1 and 0.2, which suggests that the CiLACS genes underwent significant negative selection during evolution (Table S4).

Tissue-Specific Expression Analysis of the CiLACS Genes
Understanding the biological functions of genes is aided by tissue-specific analysis of those genes. Analysis of transcriptome data based on pecan in six different tissues ( Figure 5) to determine the tissue expression levels of LACS genes revealed that CiLACS4-1 was expressed at high levels in all tissues except seeds; CiLACS8 was expressed at high levels in all tissues, especially in roots and seeds; CiLACS1-1 was expressed at high levels in flowers and fruits; CiLACS6-1 and CiLACS9 were expressed at high levels in fruits, leaves, and seeds; and CiLACS1 and CiLACS9-1 were highly expressed in flowers and fruits ( Figure 6).

Cis-Element Analysis in the Promoter Regions of the CiLACS Genes in Pecan
Potential cis-acting elements on the CiLACS gene promoter regions were analyzed to forecast the putative functions of the CiLACS genes in response to various abiotic stresses. The findings (Figure 7) demonstrated that all 11 LACS gene promoter regions contained

Cis-Element Analysis in the Promoter Regions of the CiLACS Genes in Pecan
Potential cis-acting elements on the CiLACS gene promoter regions were analyzed to forecast the putative functions of the CiLACS genes in response to various abiotic stresses. The findings (Figure 7) demonstrated that all 11 LACS gene promoter regions contained numerous hormone-and environment-like response elements, with the highest concentration among the hormone-like response elements (ABA, methyl jasmonate, salicylic acid, etc.) being that of ABA response elements. Additionally, there are numerous light response elements in the CiLACS gene promoter region.

Cis-Element Analysis in the Promoter Regions of the CiLACS Genes in Pecan
Potential cis-acting elements on the CiLACS gene promoter regions were analyzed to forecast the putative functions of the CiLACS genes in response to various abiotic stresses. The findings (Figure 7) demonstrated that all 11 LACS gene promoter regions contained numerous hormone-and environment-like response elements, with the highest concentration among the hormone-like response elements (ABA, methyl jasmonate, salicylic acid, etc.) being that of ABA response elements. Additionally, there are numerous light response elements in the CiLACS gene promoter region.  Cis-element analysis in the CiLACS gene promoter regions. Three types of cis-acting elements were identified, including hormone-responsive elements, elements related to resistance to harsh environments and stresses, and elements related to plant growth and development.

Expression Analysis of CiLACS Genes under Different Stress Conditions and during Different Periods of Fruit Development
We examined the levels of CiLACS expression in pecan under drought, NaCl stress, and ABA induction to examine the response of 11 CiLACS genes to abiotic stress and hormone induction (Figures 8-10), and the findings revealed that the majority of CiLACS genes responded to the various treatments. CiLACS4, CiLACS4-1, CiLACS6-1, and CiLACS9 expression levels were significantly up-regulated during the drought treatment (p < 0.05), peaking 15 days after treatment, whereas CiLACS1, CiLACS1-1, CiLACS2, and CiLACS7 expression levels were down-regulated as the drought treatment progressed. CiLACS9 and CiLACS9-1 expression levels peaked on day 16 after treatment, while CiLACS1, CiLACS1-1, and CiLACS4-1 expression levels were down-regulated in salt stress. The expression levels of the other genes peaked on day 8 after NaCl treatment before declining. Several CiLACS genes, including CiLACS1, CiLACS1-1, CiLACS2, CiLACS7, and CiLACS9-1, had increased expression levels in response to ABA treatment. In particular, the expression level of CiLACS1 was 70-fold higher at 24 h of treatment than it was at 0 h. pression levels of the other genes peaked on day 8 after NaCl treatment before declining. Several CiLACS genes, including CiLACS1, CiLACS1-1, CiLACS2, CiLACS7, and CiLACS9-1, had increased expression levels in response to ABA treatment. In particular, the expression level of CiLACS1 was 70-fold higher at 24 h of treatment than it was at 0 h.    We also examined the expression of CiLACS in pecan fruits at five different times to further understand the expression of LACS genes during oil synthesis in pecan fruits. The outcomes confirmed what was anticipated ( Figure 11). Nearly all of the CiLACS genes were up-regulated; CiLACS1, CiLACS1-1, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9- We also examined the expression of CiLACS in pecan fruits at five different times to further understand the expression of LACS genes during oil synthesis in pecan fruits. The outcomes confirmed what was anticipated ( Figure 11). Nearly all of the CiLACS genes were up-regulated; CiLACS1, CiLACS1-1, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1, as well as CiLACS6, CiLACS6-1, and CiLACS7, reached their peak expression levels 135 days after flowering before being down-regulated. The expression levels of CiLACS6, CiLACS6-1, CiLACS7, and CiLACS8 were still up-regulated 120 days after flowering and peaked 165 and 180 days after flowering.

Subcellular Localization of CiLACS9s in Tobacco Leaves
LACSs play a role in many biological processes involving fatty acids, and understanding their subcellular localization can shed light on their functional functions [13,24]. In this study, EGFP fluorescence of CiLACS9 fusions was mainly detected in the cytoplasm and wrapped around the periphery of the nucleus, as shown by subcellular co-localization experiments under confocal laser microscopy ( Figure 12).

Discussion
With the advancement of high-throughput sequencing technology in recent years, an increasing number of researchers have been studying some important gene families linked to particular plant traits and biological functions at the whole-genome level. Preliminary research has been conducted on the structure and function of LACS genes that have been isolated from plants including Arabidopsis, rice, wheat, and kale-type oilseed rape. Although LACS genes have been demonstrated to be crucial for plant lipid synthesis and stress resistance [25], there is currently a lack of comprehensive reports on the identification and functional studies of this gene family in pecan. In this study, we identified 11 members of the CiLACS gene family in pecan and divided them into five subfamilies (Figure 1). Multiple sequence alignment of CiLACS and AtLACS showed that CiLACS protein sequences have AMP-binding (PF00501) and ACS signaling domains typical of the LACS family ( Figure 2). Notably, AMP binding plays an important role in lipid anabolic metabolism, which was consistent with previous results in Arabidopsis [26]. Interestingly, the conserved motifs of CiLACS family members within the same subfamily showed high similarity ( Figure 3A,B). Subfamilies I, II, and III also showed high similarity in the type, number, and position of motifs, with subfamily IV being slightly different from the other subfamilies, having motif 9 in addition to motif 8 but not motif 6. Additionally, the number of introns-exons in the CiLACS family is relatively consistent across subclades, suggesting that LACS genes are evolutionarily conserved and LACS genes within subclades may have similar functions.
Gene duplication plays a significant role in the amplification of gene families during evolution, and adaptation to the environment through the family members acquired in response to selection pressure [20]. Chromosomal localization showed that ( Figure 4) the 11 CiLACS genes were distributed on nine chromosomes, with CiLACS2 and CiLACS9 on the same chromosome and CiLACS1-1 and CiLACS7 on the same chromosome. The different chromosomal distributions suggest that pecan LACS genes have undergone genetic variation during evolution. Colinearity and Ka/Ks (Table S4) analyses showed that there were 15 colinear gene pairs between pecan LACS and Arabidopsis LACS, and six colinear gene pairs in pecan LACS, and these genes were all derived from fragment duplication, indicating that fragment duplication played a central role in the evolution of CiLACS (Figure 5). Surprisingly, the Ka/Ks values of the CiLACS colinear gene pairs were all less than 1, suggesting that CiLACS family genes may have been subject to strong negative selection

Discussion
With the advancement of high-throughput sequencing technology in recent years, an increasing number of researchers have been studying some important gene families linked to particular plant traits and biological functions at the whole-genome level. Preliminary research has been conducted on the structure and function of LACS genes that have been isolated from plants including Arabidopsis, rice, wheat, and kale-type oilseed rape. Although LACS genes have been demonstrated to be crucial for plant lipid synthesis and stress resistance [25], there is currently a lack of comprehensive reports on the identification and functional studies of this gene family in pecan. In this study, we identified 11 members of the CiLACS gene family in pecan and divided them into five subfamilies (Figure 1). Multiple sequence alignment of CiLACS and AtLACS showed that CiLACS protein sequences have AMP-binding (PF00501) and ACS signaling domains typical of the LACS family ( Figure 2). Notably, AMP binding plays an important role in lipid anabolic metabolism, which was consistent with previous results in Arabidopsis [26]. Interestingly, the conserved motifs of CiLACS family members within the same subfamily showed high similarity ( Figure 3A,B). Subfamilies I, II, and III also showed high similarity in the type, number, and position of motifs, with subfamily IV being slightly different from the other subfamilies, having motif 9 in addition to motif 8 but not motif 6. Additionally, the number of introns-exons in the CiLACS family is relatively consistent across subclades, suggesting that LACS genes are evolutionarily conserved and LACS genes within subclades may have similar functions.
Gene duplication plays a significant role in the amplification of gene families during evolution, and adaptation to the environment through the family members acquired in response to selection pressure [20]. Chromosomal localization showed that ( Figure 4) the 11 CiLACS genes were distributed on nine chromosomes, with CiLACS2 and CiLACS9 on the same chromosome and CiLACS1-1 and CiLACS7 on the same chromosome. The different chromosomal distributions suggest that pecan LACS genes have undergone genetic variation during evolution. Colinearity and Ka/Ks (Table S4) analyses showed that there were 15 colinear gene pairs between pecan LACS and Arabidopsis LACS, and six colinear gene pairs in pecan LACS, and these genes were all derived from fragment duplication, indicating that fragment duplication played a central role in the evolution of CiLACS ( Figure 5). Surprisingly, the Ka/Ks values of the CiLACS colinear gene pairs were all less than 1, suggesting that CiLACS family genes may have been subject to strong negative selection during evolution (Table S4). During gene evolution, repetition time may lead to the accumulation of degenerative mutations, whereas negative selection may favor the evolution of the family genes by eliminating the deleterious variants that emerge, which also suggests that the CiLACS family genes are functionally conserved.
It has been discovered that the promoter regions of CiLACS genes also contain a number of regulatory elements associated with hormone response and stress, including ABA, MeJA, drought, and low-temperature response elements, the presence of which may be crucial in the ability of CiLACS to enhance resistance in pecan [27]. Previous studies have demonstrated that LACS proteins are involved in the activation of long-chain fatty acids and produce lipid acyl-coenzyme A, which is crucial for plant growth and development. AtLACS1, AtLACS2, and AtLACS4 are crucial for the production of keratin and waxes that make up the epidermis in Arabidopsis. Among them, double mutants of AtLACS1 and AtLACS2 have decreased epidermal wax content and increased epidermal permeability, while mutants of Arabidopsis AtLACS1 and AtLACS4 have 35% and 73% of the wax content of the wild type, respectively [9]. The physiological and metabolic regulation of epidermal wax in Arabidopsis helps plants cope with drought, salt, and ABA treatment [28][29][30]. These studies suggested that the mechanism of the response of LACS genes to environmental stress may be due to their regulatory role in wax synthesis. Here, we investigated the expression patterns of CiLACS family genes in different pecan tissues and under different treatments ( Figure 6). Comparison of CiLACS expression levels in different tissues revealed that most CiLACS genes showed high expression levels in flowers and leaves, and it was hypothesized that these genes might be involved in the synthesis of epidermal waxes in flowers and leaves. The expression levels of CiLACS4-1, CiLACS6-1, CiLACS8, and CiLACS9 were higher in most tissues, especially in fruits and mature seeds, and it is speculated that these genes may be involved in plant seed oil synthesis. Furthermore, the CiLACS genes showed different expression patterns under different treatments. Fifteen days after the no-watering treatment, the expression levels of the genes CiLACS4, CiLACS4-1, CiLACS6, CiLACS6-1, and CiLACS9 were consistently up-regulated; in particular, the relative expression of CiLACS6-1 on day 15 was 10 times higher than that of CK. Under salt stress, the expression levels of CiLACS6, CiLACS7, CiLACS9, and CiLACS9-1 peaked 8 and 16 days after treatment. Under ABA treatment, the relative expression of CiLACS1, CiLACS1-1, and CiLACS2 peaked at 24 h of treatment, and CiLACS7 and CiLACS9-1 peaked at 12 h post-treatment; in particular, the relative expression of CiLACS1 was more than 60-fold higher at 24 h than it was at 0 h. These results indicate that these genes play a crucial role in pecan in response to ABA, drought, and salt treatments.
Fatty acids are produced in the plastid of the plant's body, where they are activated to lipoyl CoA before entering the endoplasmic reticulum to take part in TAG synthesis. Previous studies revealed that AtLACS1 and AtLACS2 were located in the endoplasmic reticulum, and the AtLACS1, AtLACS2 double mutant caused a reduction in the production of leaf epidermal wax [9]. BnLACS4 was also localized in the endoplasmic reticulum and may be involved in lipid synthesis in kale-type oilseed rape [14]. AtLACS9 was designated in the chloroplast envelope. Associated with LACS activity in chloroplasts [13]. OsLACS9 in rice was localized in the plastids of onion epidermal cells, and its mutation resulted in the accumulation of large amounts of starch granules in chloroplasts [19]. The subcellular localization of CiLACS9 in this study showed that CiLACS9 was located in the cytoplasm, wrapped around the nucleus, and formed a network-like structure. Previous studies have shown that LACS genes are involved in plant seed oil synthesis. In this study, the expression patterns of CiLACS genes during pecan seed kernel development from five different developmental stages were analyzed, and it was found that the expression levels of CiLACS1, CiLACS1-1, CiLACS4, CiLACS4-1, CiLACS9, and CiLACS9-1 peaked 135 days after flowering (high oil accumulation stage), while CiLACS6, CiLACS6-1, CiLACS7, and CiLACS8 peaked 165 days after flowering (stable oil synthesis period). These CiLACS genes displayed different expression patterns in different stages of kernel development, indicating that they were crucial for lipid synthesis and metabolism at various stages of kernel development.

Genome-Wide Identification of the LACS Gene in Eight Different Plant Species
The well-studied sequences of nine Arabidopsis (Arabidopsis thaliana) LACS proteins [1] were downloaded from The Arabidopsis Information Resource (TAIR, http://www.Ar abidopsis.org/index.jsp (accessed on 6 November 2022)). To identify the LACS family members from the entire pecan (Carya illinoinensis) genome, we downloaded the Hidden Markov Model (HMM) file of the AMP domain (AMP-binding: PF00501) from Pfam (http://pfam.xfam.org/ (accessed on 6 November 2022)) [31] and used as a query to investigate LACS in pecan using HMMER software (version 3.1b2) (www.hmmer.org (accessed on 6 November 2022)). The candidate LACS proteins were further examined by BLASTP analysis [32] in the NCBI database (https://blast.ncbi.nlm.nih.gov/Blast.cgi (accessed on 6 November 2022)), and redundant sequences were removed from the list. Furthermore, the same identification method was used to identify the LACS in Chinese hickory (Carya cathayensis), rice (Oryza sativa), poplar (Populus trichocarpa), walnut (Juglans regia), rape (Brassica napus), and soybean (Glycine max).

Multiple Sequence Alignment and Phylogenetic Analysis
The multiple sequence alignment of the 11 CiLACS proteins and 9 AtLACS proteins was carried out using DNAMAN software (version 7.0) with default parameters (http://dnaman.software.informer.com/ (accessed on 9 November 2022)). The evolutionary relationships among the LACS protein sequences of pecan and seven other species were established by constructing a phylogenetic tree via the neighbor-joining method (NJ method) with the following settings: bootstrap method, 1000 replications; Poisson model and complete deletion, using MEGA software (version 7.0) [34]. The phylogenetic tree consisted of several species, including Arabidopsis thaliana, Chinese hickory, rice, poplar, walnut, rape, and soybean. The resulting tree was visualized via the online website Evolview (https://evolgenius.info/ (accessed on 9 November 2022)) [35].

Analysis of Gene Structure and Conserved Motifs
Gene structure information of pecan LACSs was retrieved by surveying the General Feature Format (GFF) file from GigaDB (http://gigadb.org/dataset/100571 (accessed on 12 November 2022)) [36]. The information about the gene structure diagram of the 11 CiLACS genes was generated using TBtools software (https://github.com/CJ-Chen/TBt ools (accessed on 12 November 2022)) [37]. Conserved motifs of CiLACS protein sequence were identified using the MEME program (http://meme-suite.org/tools/meme (accessed on 12 November 2022)) [38] with the search number of 10 and the optimum motif length of between 6 and 100 residues.

Chromosome Localization and Gene Duplication Event Analysis among CiLACSs and AtLACSs
The chromosomal distributions of 11 CiLACS genes and 9 AtLACS genes were visualized using the Circos program in TBtools software. Colinearity analysis of the CiLACS and AtLACS was performed using the MCScanX program in TBtools software. Similarly, colinearity analysis between CiLACS genes was also carried out by this method. To detect the selection pressure of the duplication events, the CDSs of the CiLACS and AtLACS were aligned using TBtools software, and the synonymous substitution (Ks) and nonsynonymous substitution (Ka) of tandem and segmental duplication events were calculated using MEGA software (version 7.0) [39]. Finally, the selection pressure was evaluated using the Ka/Ks ratios.

Tissue-Specific Expression Analysis of CiLACS Gene Family
To further characterize the different expression patterns of the CiLACSs in various organs, the transcriptome data were downloaded from accession numbers GSE179336 and PRJNA799663 that were previously generated by our group and include root, leaf, mature pistillate and staminate flower, young fruit, and seed. The expression levels of all CiLACS genes were quantified based on their fragments per kilobase per million of reads mapped (FPKM) values using RSEM software [40]. The results, log 2 expression values of the 11 CiLACS genes, were used for heat-map generation using TBtools software (version 7.0).

Cis-Acting Element Analysis
We downloaded the entire pecan genome sequence from the Phytozome database (https://phytozome-next.jgi.doe.gov/ (accessed on 15 November 2022) and extracted the 2000 bp genomic sequence upstream of the transcription start site of these 11 CiLACS genes. Then, the cis-acting elements were predicted using PlantCARE software (version 7.0, https://phytozome-next.jgi.doe.gov/ accessed on 15 November 2022) [41]. The hormone response-related elements in the 2000 bp upstream regions of CiLACS genes were visualized using TBtools software (version 7.0).

Plant Material and Sample Collection
The material used in this experiment was obtained from the pecan experimental base of Nanjing Forestry University (119 • 9 6 E, 31 • 52 45 N). For the drought treatment, the annual grafted pecan variety "Pawnee" was moved to the climatic chamber of No. 403, Biotechnology Building, Nanjing Forestry University, in August 2020, and three groups of three plants were put up. The incubation conditions were 14 h of light at 24 • C, 10 h of darkness at 22 • C, 60~70% humidity, watering twice a week, and a 15-day drought treatment (no watering) after one month. Leaves were collected on days 3, 6, 9, 12, and 15 of the drought treatment. For the ABA and NaCI treatments, annual pecan seedlings were transported to the climatic chamber of Nanjing Forestry University, Biotechnology Building, No. 403, in June 2022, in groups of three plants, with three groups. The humidity ranged from 70% to 80%, and the temperature ranged from 25 to 22 degrees. Watering was performed every 2 days, and ABA and NaCI treatments were administered after one week. Leaves were collected on days 8, 16, and 24 after NaCI treatment and at hours 3, 6, 12, and 24 after ABA treatment. Pecan fruit was harvested from six healthy 9-year-old plants of pecan variety "Pawnee" in 2022 on days 120, 135, 150, 165, and 180 after flowering; it was stored on ice, with the skins were peeled off and the seed kernels retained.
Three biological replicates were set up for each sample collected. The collected leaves and seed kernels were immersed in liquid nitrogen for quick freezing, and then they were kept in a −80 • C refrigerator.

Subcellular Localizations of the CiLACS9 Protein
To determine the subcellular localizations of the CiLACS9s and the design of primer sequences for cloning CiLACS9 using snapgene software (version 7.0, Table S5), the fulllength sequences of the CiLACS9s were inserted into 35S::EGFP vectors, and the resulting constructs were transformed into the Agrobacterium strain GV3101 [42]. Then, the leaves of Nicotiana benthamiana were used as the host for colocalization experiments involving the Agrobacterium-mediated transient expression of the target gene. After injection of the plasmid carrying the target gene into the leaves, the plants were grown normally for 2 days after incubation in the dark at 22 • C for 24 h. The leaves were cut out and placed in DAPI dye for 10 min, and then the lower epidermis of the leaves was observed using a laser scanning confocal microscope (Zeiss LSM710). Excitation wavelengths for EGFP and DAPI were 488 nm and 405 nm.

RNA Collection and Real-Time Quantitative PCR (qRT-PCR)
All collected samples were ground into powder in liquid nitrogen, and total RNA was extracted according to the instructions for the RNA extraction kit (Vazyme, Nanjing, China). cDNA was subsequently synthesized by reverse transcription using a reverse transcription kit (Vazyme, Nanjing, China), stored in a −20 • C refrigerator, and set aside. Primers for 11 CiLACS genes were designed using the online website Primer Quest Tool (https://sg.idtdna.com/PrimerQuest/Home/Index (accessed on 20 September 2022) and were synthesized by Beijing Prime Tech Biotechnology Co. (Beijing, China) (Table S6).

Statistical Analysis
Statistical analysis was performed using IBM SPSS25 software (SPSS Inc., Chicago, IL, USA). One-way analysis of variance (one-way ANOVA) and multiple comparisons (LSD Duncan) were selected for significance analysis (p < 0.05).

Conclusions
In conclusion, we discovered 11 LACS genes in pecan and divided them into five subgroups. The evolutionary relationships, gene structures and conserved structural domains, chromosomal positions, duplication events, and expression patterns of the CiLACS family of genes were investigated. The findings imply that genes from the CiLACS family may be crucial for pecan oil synthesis and stress resistance. These discoveries significantly advance our knowledge of LACS genes in pecans and lay the groundwork for future investigations into the role of LACS family genes in pecans and other species.

Institutional Review Board Statement:
The experimental material in this study was plant-based and did not involve human or animal studies; therefore, ethical review and approval were waived for this study.
Informed Consent Statement: Written informed consent has been obtained from the patient(s) to publish this paper. Data Availability Statement: Not applicable.

Conflicts of Interest:
The authors declare no conflict of interest.