Transcriptomic Analysis Reveals the High-Oleic Acid Feedback Regulating the Homologous Gene Expression of Stearoyl-ACP Desaturase 2 (SAD2) in Peanuts

Peanuts with high oleic acid content are usually considered to be beneficial for human health and edible oil storage. In breeding practice, peanut lines with high monounsaturated fatty acids are selected using fatty acid desaturase 2 (FAD2), which is responsible for the conversion of oleic acid (C18:1) to linoleic acid (C18:2). Here, comparative transcriptomics were used to analyze the global gene expression profile of high- and normal-oleic peanut cultivars at six time points during seed development. First, the mutant type of FAD2 was determined in the high-oleic peanut (H176). The result suggested that early translation termination occurred simultaneously in the coding sequence of FAD2-A and FAD2-B, and the cultivar H176 is capable of utilizing a potential germplasm resource for future high-oleic peanut breeding. Furthermore, transcriptomic analysis identified 74 differentially expressed genes (DEGs) involved in lipid metabolism in high-oleic peanut seed, of which five DEGs encoded the fatty acid desaturase. Aradu.XM2MR belonged to the homologous gene of stearoyl-ACP (acyl carrier protein) desaturase 2 (SAD2) that converted the C18:0 into C18:1. Further subcellular localization studies indicated that FAD2 was located at the endoplasmic reticulum (ER), and Aradu.XM2MR was targeted to the plastid in Arabidopsis protoplast cells. To examine the dynamic mechanism of this finding, we focused on the peroxidase (POD)-mediated fatty acid (FA) degradation pathway. The fad2 mutant significantly increased the POD activity and H2O2 concentration at the early stage of seed development, implying that redox signaling likely acted as a messenger to connect the signaling transduction between the high-oleic content and Aradu.XM2MR transcription level. Taken together, transcriptome analysis revealed the feedback mechanism of SAD2 (Aradu.XM2MR) associated with FAD2 mutation during the seed developmental stage, which could provide a potential peanut breeding strategy based on identified candidate genes to improve the content of oleic acid.


Introduction
The peanut (Arachis hypogaea L.) is a vital mercantile crop and is widely cultivated in tropical and sub-tropical regions, with a global production of more than 40 million tons per year. It provides stable The modern peanut is an allotetraploid crop that is derived from multiple hybridization events between diploid ancestor Arachis ipaensis (BB) and diverse Arachis duranensis (AA) during its origination on the South American continent [13]. The peanut genome is approximately 2.7 GB in size, and the complete nucleotide sequence was recently released [14][15][16]. However, it is difficult to assemble the cultivated peanut genome due to the close genetic relationship between the two diploid species [17]. One strategy is to obtain the whole genome sequences of Arachis duranensis and Arachis ipaensis and use them as the reference to assemble the tetraploid genome [18]. Without the genome sequence of the cultivated peanut, assembled transcripts based on the diploid ancestor's subgenome are a complimentary resource for establishing a global gene atlas of Arachis hypogaea. To date, transcriptome-based high-throughput sequencing has generated large volumes of data associated with lipid metabolism in the peanut [19]. For instance, de novo transcriptome analysis not only reveals the expression patterns of lipid genes during the development of peanut seeds, but also sheds light on the molecular mechanisms of lipid metabolism and highlights rate-limiting enzymes for lipid storage in peanuts with different oil contents [20]. Furthermore, the genetically controlled pod-filling stage is a key point for the development of peanut seeds. Identification of differentially expressed genes involved in peanut oil synthesis would explain the genotypic variation in oil biosynthesis and pod-filling, and it can provide the genetic basis for understanding seed development and oil accumulation in the peanut [21].
Previous studies focused mainly on the identification of various alleles of fad2 in high-oleate peanuts, and less attention was given to identifying the genes associated with high OA formation. Here, transcriptome analysis was performed to analyze the differentially expressed genes at six different time-points during seed development in normal-and high-oleate cultivars L70 and H176 in order to understand differences in the lipid network between normal-and high-oleic peanut seeds. Our findings provide critical knowledge on the mechanisms involved in OA biosynthesis and regulation in high-oleic peanuts.

Changes of Oil Composition during the Developmental Stages of Peanut Seeds
We firstly documented the morphology of developing seeds at six different time points after the gynophore was elongated in the soil in high-and normal-oleic cultivars ( Figure 1C). At the early stage (20-30 days after flowering (DAF)), the seed sizes of both peanut cultivars were maintained a lower growth rate and gynophore rapidly swelled to form pods. During this period, the contents of total oil, OA, and LOA in cotyledons did not display an obvious difference between the two cultivars ( Figure 1D-F). At the second critical development stage , the sizes of seeds and pods continuously increased, and the thickness of pod inner white spongy tissues showed a slight decrease. The contents of total oil in both cultivars showed no difference ( Figure 1D), but a significant difference was observed in the contents of OA and LOA between high-and normal-oleic peanut cultivars. The relative proportion of OA increased rapidly ( Figure 1E), thus leading to the decreased percentage of LOA in high-oleic cultivar ( Figure 1F). In contrast, the relative contents of OA and LOA in the normal-oleic cultivar exhibited no difference at the time points. At the end of seed filling, the seeds gradually stopped growing until maturity, the original spongy tissues disappeared, and the peanut shell was dehydrated to become hard outer shell. In short, OA content in high-oleic cultivar reached the maximum level at the terminal stage of seed growth ( Figure 1E), and the relative contents of OA and LOA in normal-oleate cultivar were stable ( Figure 1E,F).
The cultivated peanut is a tetraploid species whose genome consists of two diploid subgenomes Arachis duranensis (AA) and Arachis ipaensis (BB). Therefore, AhFAD2 contains two homologous genes, AhFAD2-A and AhFAD2-B, with similar functions. In order to determine whether the AhFAD2 mutant can cause OA accumulation in the H176 cultivar, we subsequently cloned the full-length sequence (CDS, coding sequence) encoding AhFAD2 in two peanut cultivars ( Figure S2). Sequence alignment indicated four and 12 mutational sites located at AhFAD2-A and AhFAD2-B encoding region in the H176 cultivar, respectively. The mutation at the 375 bp position caused the original TGT (cysteine) conversion to TGA (stop codon), inducing an early translation termination in AhFAD2-A ( Figure 1G). Moreover, a single base A was inserted at the position of 442 bp to produce a stop codon (TAA) at the position of 495 bp in the AhFAD2-B encoding sequence ( Figure 1H), resulting in the termination of translation of AhFAD2-B. Therefore, the high concentration of OA in H176 cultivar was attributed to the genotype of fad2-a/fad2-b.

Transcriptome Assembly
In order to understand the underlying molecular differences between normal-and high-oleic peanuts, we selected six developmental stages of whole seeds for transcriptome sequencing. All of the transcriptome sequence was assembled by a genome-guided strategy, Trinity [22], and individual RNA-seq library data were aligned to Arachis. duranensis and Arachis. ipaensis genomes (www. Peanutbase.org). The average number of raw reads generated from three biological replicates of each sample was more than 20 million, the average numbers of mapped reads and uniquely mapped reads were far greater than 10 million, and the most mapped ratio of each library to A. duranensis and A. ipaensis genomes was approximately 50% (Table 1 and Table S1). After removed the short and redundant reads (Table S2), the number of obtained transcript assembly contigs (TAC) above 100 bp approximated 257,335, the average length of assembled contig was 1114 bp, and the contig N50 was 1778 bp. Further, the calculated GC content of each library suggested that the average GC content was in the range of 40-50% ( Figures S3 and S4). Therefore, the assembled transcriptome was able to support our subsequent analysis.

Transcriptome Assembly
In order to understand the underlying molecular differences between normal-and high-oleic peanuts, we selected six developmental stages of whole seeds for transcriptome sequencing. All of the transcriptome sequence was assembled by a genome-guided strategy, Trinity [22], and individual RNA-seq library data were aligned to Arachis. duranensis and Arachis. ipaensis genomes (www.Peanutbase.org). The average number of raw reads generated from three biological replicates of each sample was more than 20 million, the average numbers of mapped reads and uniquely mapped reads were far greater than 10 million, and the most mapped ratio of each library to A. duranensis and A. ipaensis genomes was approximately 50% (Table 1 and S1). After removed the short and redundant reads (Table S2), the number of obtained transcript assembly contigs (TAC) above 100 bp approximated 257,335, the average length of assembled contig was 1,114 bp, and the contig N50 was 1,778 bp. Further, the calculated GC content of each library suggested that the average GC content was in the range of 40-50% ( Figure S3,S4). Therefore, the assembled transcriptome was able to support our subsequent analysis.

Analysis of Biological Processes in High-Oleic Peanuts
Furthermore, after removing the duplicative gene accession number, we obtained a total of 2138 DEGs in seeds of high-oleic cultivar H176 in comparison with the normal-oleic cultivar L70 at the same developmental stage (Table S3 sheet 1 and Figure S5). The DEG distribution statistics demonstrated that there were 777, 162, 442, 402, 884, and 995 DEGs from stages 1 to stage 6, respectively ( Figure 2A). A total of 1,308 up-regulated and 2,354 down-regulated DEGs were identified at six stages in H176 vs. L176 cultivars (Table S4), indicating that FAD2 mutation could induce the high-oleic phenotype and suppress generous gene expression. The quantities of identified DEGs in the initial (20)(21)(22)(23)(24)(25)(26)(27)(28)(29)(30) and mature (60-70 DAF) phases were obviously larger than in the middle developmental stage (40)(41)(42)(43)(44)(45)(46)(47)(48)(49)(50), suggesting the involvement of complex transcription regulation events during these stages, particularly the conversion of stearoyl-ACP into OA-ACP in the seed maturity phase. Among the six stages, the middle developmental stage harbored 174 specifically detected DEGs, and 565 DEGs were specifically detected in the stage of early seed swelling, whereas the largest number of specific DEGs (811) was only identified during the period of seed maturity ( Figure 2B and Table S8). In total, 103 DEGs were shared in across three periods ( Figure 2B).

Expression Analysis of DEGs
A heatmap representing the differential expressions of a total of 2,138 DEGs (Table S3 sheet 1) in H176 and L70 cultivars was constructed. It revealed the dynamic changes in the transcriptomes during the developmental stages of high-oleic peanut seeds. In the heatmap, the original gene expression data were transformed into log2 fold change (Table S3 Sheet 2) and further cluster analysis revealed the significant differences in gene expressions between high-and normal-oleic peanut seeds ( Figure S7A). In order to characterize these differential expressions in detail, trend analysis was carried out to explore the expression patterns of all DEGs. Total DEGs were clustered into 20 profiles, of which six trend profiles, including 0, 11,12,17,19, and 18 showed significant enrichment (p < 0.05) with the colored block, and 14 profiles represented the enrichment of significant trends without color ( Figure S7B). The expression of 307 genes displayed a reducing trend during the whole seed development in profile 0 and the expression of 129 genes demonstrated an opposite trend in profile 19. The expressions of 140 and 71 genes showed similar trends of increasing at the early stages in profiles 17 and 18, respectively. In profile 11, the expression of 232 genes showed an initial increase but subsequent reduction at stage 2 (30 DAF) to stage 3 (40 DAF) in developing seeds. However, the expressions of 206 genes exhibited only increases during the period of initial and terminal stages in profile 12 (Table S7).

Dynamic Changes of DEGs Related to Lipid Metabolism in High-Oleic Peanut
Negative variation in FAD2 of H176 cultivar promoted OA accumulation, but suppressed the production of PUFAs (polyunsaturated fatty acids). Here, DEGs associated with lipid biosynthesis and metabolic pathways were identified. Specifically, a total of 74 DEGs were characterized as involved in lipid metabolic pathway at all development stages of seeds (Table 2). Further GO analysis suggested that at least 10 genes were classified into a series of primary processes, such as lipid metabolism and biosynthesis, cellular lipid metabolism, and FA biosynthesis and degradation. KEGG The functional interpretation and gene product attributes of these identified DEGs were classified using Gene Ontology (GO) categories. According to the GO analysis, three histograms displayed the top 20 categorized DEG biological processes, molecular functions, and cellular compartments ( Figure S6A). In terms of biological processes, the metabolic process was the major group, including cellular, primary, protein, and macromolecular metabolism. In terms of molecular functions, at least 200 DEGs were involved in nucleotide and protein binding, and more than 600 DEGs were involved in catalytic activity and binding groups in developing high-oleic peanut seeds. Additionally, most of the DEGs were divided into the top three groups, including the cell, the intracellular area, and the membrane, by cellular compartment analysis (Table S5). Furthermore, KEGG (kyoto encyclopedia of genes and genomes) pathway enrichment analysis was used to categorize function annotations for all annotated genes. We found that 2138 DEGs were mainly categorized in the top 30 pathways, and the most highly enriched biological pathways in high-oleic peanut seeds were plant hormone signal transduction, starch and sucrose metabolism, phenylpropanoid biosynthesis, and plant-pathogen interaction ( Figure S6B). In addition, 12 and six DEGs were specifically classified in the pathways for fatty acid metabolism and biosynthesis of unsaturated FAs, respectively (Table S6).

Expression Analysis of DEGs
A heatmap representing the differential expressions of a total of 2138 DEGs (Table S3 sheet 1) in H176 and L70 cultivars was constructed. It revealed the dynamic changes in the transcriptomes during the developmental stages of high-oleic peanut seeds. In the heatmap, the original gene expression data were transformed into log 2 fold change (Table S3 Sheet 2) and further cluster analysis revealed the significant differences in gene expressions between high-and normal-oleic peanut seeds ( Figure S7A). In order to characterize these differential expressions in detail, trend analysis was carried out to explore the expression patterns of all DEGs. Total DEGs were clustered into 20 profiles, of which six trend profiles, including 0, 11,12,17,19, and 18 showed significant enrichment (p < 0.05) with the colored block, and 14 profiles represented the enrichment of significant trends without color ( Figure S7B). The expression of 307 genes displayed a reducing trend during the whole seed development in profile 0 and the expression of 129 genes demonstrated an opposite trend in profile 19. The expressions of 140 and 71 genes showed similar trends of increasing at the early stages in profiles 17 and 18, respectively. In profile 11, the expression of 232 genes showed an initial increase but subsequent reduction at stage 2 (30 DAF) to stage 3 (40 DAF) in developing seeds. However, the expressions of 206 genes exhibited only increases during the period of initial and terminal stages in profile 12 (Table S7).

Dynamic Changes of DEGs Related to Lipid Metabolism in High-Oleic Peanut
Negative variation in FAD2 of H176 cultivar promoted OA accumulation, but suppressed the production of PUFAs (polyunsaturated fatty acids). Here, DEGs associated with lipid biosynthesis and metabolic pathways were identified. Specifically, a total of 74 DEGs were characterized as involved in lipid metabolic pathway at all development stages of seeds (Table 2). Further GO analysis suggested that at least 10 genes were classified into a series of primary processes, such as lipid metabolism and biosynthesis, cellular lipid metabolism, and FA biosynthesis and degradation. KEGG enrichment indicated that the FAD2 mutation-associated high-oleic content contributed greatly to the gene expression patterns of FA and glycerolipid synthetic or metabolic pathways ( Figure 3A and Table S9). From the holistic perspective, the heatmap displayed differences in those gene expression patterns between high-and normal-oleic groups according to the shaded color blocks ( Figure 3B). Moreover, trend analysis was performed to reveal the expression patterns of all lipid DEGs. Total DEGs were clustered into top 10 profiles, of which three trend profiles, including profiles 0, 7, and 9, were significantly enriched (p < 0.05) (colored block), and seven profiles represented the enrichment of significant trends (without color) ( Figure 3C). The expression of 14 genes displayed a declining trend during the whole seed development stage in profile 0, but the expressions of nine genes demonstrated an opposite trend in profile 9. In profile 7, the expression of nine genes exhibited an obvious increase during the initial and terminal stages (Table S10).
Moreover, to explore the existing annotation information of all lipid DEGs more deeply, the InterPro web-database (www.ebi.ac.uk/interpro) was used to analyze and explain the molecular functions of lipid genes. Based on the valid functional unscramble of InterPro, a majority of DEG-encoded proteins contained the conversed domain of acyltransferase, whose molecular functions were tightly linked to FA biosynthesis-metabolism, FA oxidation-reduction, FA elongation, lipid oxdiation, and the glycerolipid biosynthesis pathway. Interestingly, five DEGs were found to encode plant fatty acid desaturase, including Aradu.AV1HQ, Aradu.XM2MR, Araip.S3GXY, Aradu.PR2BP, and Araip.31Q5V (Table 2). Among these identified fatty acid desaturease genes, Aradu.XM2MR (SAD2) encodes a stearoyl-acyl carrier protein desaturase whose relative expression specifically increased at the early stage (20-30 DAF) but decreased at the terminal stage (60-70 DAF) during the development of high-oleic peanut (H176) seeds. On the other hand, the expression of other four fatty acid desaturase genes were abnormally down-regulated at distinct time points, and their expression levels were inhibited due to the fad2 mutation-induced OA accumulation. Therefore, we concluded that fad2 repressed the expressions of multiple members of fatty acid desaturase family based on transcriptome analysis.

Analysis of the Expression of FADs at the mRNA level
Additionally, to validate whether the difference in RNA-seq levels of lipid DEGs is truly reflecting the actual transcription level, quantitative real-time PCR (polymerase chain reaction) was conducted for detecting the expression levels of DEGs at six consecutive periods between H176 and L70 cultivars. Indeed, the relative expression levels of fatty acid desaturase family genes (Aradu.AV1HQ, Aradu.XM2MR, Araip.S3GXY, and Araip.31Q5V) were consistent with the dynamic changing trend of RNA-seq obtained RPKM (reads per kilobase per million) values (Figure 4 and S8), but the opposite results were obtained from the examination of Aradu.PR2BP along with seed development. In particular, the expression of Aradu.XM2MR presented a sudden increase at the early stage (20−30 DAF) and then a gradual drop until the seed maturity in the H176 cultivar. Therefore, we concluded that FAD2 mutant-induced high oleate content affected the expression patterns of SAD2 (Aradu.XM2MR) at the mRNA level.

Analysis of the Expression of FADs at the mRNA level
Additionally, to validate whether the difference in RNA-seq levels of lipid DEGs is truly reflecting the actual transcription level, quantitative real-time PCR (polymerase chain reaction) was conducted for detecting the expression levels of DEGs at six consecutive periods between H176 and L70 cultivars. Indeed, the relative expression levels of fatty acid desaturase family genes (Aradu.AV1HQ, Aradu.XM2MR, Araip.S3GXY, and Araip.31Q5V) were consistent with the dynamic changing trend of RNA-seq obtained RPKM (reads per kilobase per million) values (Figure 4 and Figure S8), but the opposite results were obtained from the examination of Aradu.PR2BP along with seed development. In particular, the expression of Aradu.XM2MR presented a sudden increase at the early stage (20-30 DAF) and then a gradual drop until the seed maturity in the H176 cultivar. Therefore, we concluded that FAD2 mutant-induced high oleate content affected the expression patterns of SAD2 (Aradu.XM2MR) at the mRNA level.

Subcellular Localization of FAD2 and SAD2 (Aradu.XM2MR)
FAD2 mutation induced high-oleic feedback mechanisms to repress the transcription of upstream SAD2 (Aradu.XM2MR); however, whether SAD2 (Aradu.XM2MR) is also localized to the endoplasmic reticulum (ER) as FAD2 remains unclear. We showed that the green signal from SAD2-GFP (green fluorescent protein) when overlaid with red chloroplast marker presented an obvious yellow signal in Arabidopsis protoplast cells, suggesting that SAD2 was localized to chloroplast in the peanut ( Figure 5A,B). Additionally, the conversed fatty acid desaturase domain of FAD2 ( Figure S9) indicated that it was likely localized to the endoplasmic reticulum like its homologous gene in Arabidopsis, but it remains unclear whether the FAD2 targets the ER in peanut. We transiently coexpressed the FAD2-GFP fusion protein and ER marker in Arabidopsis' protoplast cells. As expected, the green signal of the anchored organelle of FAD2-GFP was completely co-localized with red ER marker in Arabidopsis protoplast cell ( Figure 5C), which suggested that FAD2 was localized to the ER in peanut. Subcellular localization analysis demonstrated that critical enzymes of oleic acid biosynthesis were located at different organelles; upstream SAD2 regulated the conversion of stearoyl-ACP (C18:0-ACP) to oleoyl-ACP (C18:1-ACP) in the chloroplast, but the downstream FAD2 mutation caused defects of oleic acid (C18:1) conversion to linoleic acid (C18:2) in the ER.

Subcellular Localization of FAD2 and SAD2 (Aradu.XM2MR)
FAD2 mutation induced high-oleic feedback mechanisms to repress the transcription of upstream SAD2 (Aradu.XM2MR); however, whether SAD2 (Aradu.XM2MR) is also localized to the endoplasmic reticulum (ER) as FAD2 remains unclear. We showed that the green signal from SAD2-GFP (green fluorescent protein) when overlaid with red chloroplast marker presented an obvious yellow signal in Arabidopsis protoplast cells, suggesting that SAD2 was localized to chloroplast in the peanut ( Figure 5A,B). Additionally, the conversed fatty acid desaturase domain of FAD2 ( Figure S9) indicated that it was likely localized to the endoplasmic reticulum like its homologous gene in Arabidopsis, but it remains unclear whether the FAD2 targets the ER in peanut. We transiently co-expressed the FAD2-GFP fusion protein and ER marker in Arabidopsis' protoplast cells. As expected, the green signal of the anchored organelle of FAD2-GFP was completely co-localized with red ER marker in Arabidopsis protoplast cell ( Figure 5C), which suggested that FAD2 was localized to the ER in peanut. Subcellular localization analysis demonstrated that critical enzymes of oleic acid biosynthesis were located at different organelles; upstream SAD2 regulated the conversion of stearoyl-ACP (C18:0-ACP) to oleoyl-ACP (C18:1-ACP) in the chloroplast, but the downstream FAD2 mutation caused defects of oleic acid (C18:1) conversion to linoleic acid (C18:2) in the ER.
Further phylogenetic analysis by subjecting the homologous sequence of stearoyl-ACP desaturase 2 in peanut genome to BLAST (basic local alignment search tool) analysis suggested that SAD2 (Aradu.XM2MR) exhibited high similarity in evolution with ahay.7G97IV and ahay.H654N0 (Figure 6A), as well the conserved fatty acid desaturase domain (56-413 amino acids) in the Aradu.XM2MR protein sequence ( Figure 6B). Additionally, ectopic expression of Aradu.XM2MR in yeast cells increased the relative intensity of C18:1 in the LC-MS (liquid chromatograph mass spectrometer) profile ( Figure 6C and Figure S11), suggesting that the Aradu.XM2MR-encoded product could transform C18:0 to C18:1 like the original SAD2. Further phylogenetic analysis by subjecting the homologous sequence of stearoyl-ACP desaturase 2 in peanut genome to BLAST (basic local alignment search tool) analysis suggested that SAD2 (Aradu.XM2MR) exhibited high similarity in evolution with ahay.7G97IV and ahay.H654N0 ( Figure  6A), as well the conserved fatty acid desaturase domain (56−413 amino acids) in the Aradu.XM2MR protein sequence ( Figure 6B). Additionally, ectopic expression of Aradu.XM2MR in yeast cells increased the relative intensity of C18:1 in the LC-MS (liquid chromatograph mass spectrometer) profile ( Figure 6C and S11), suggesting that the Aradu.XM2MR-encoded product could transform C18:0 to C18:1 like the original SAD2.

The Enhanced Activity of Peroxidase (POD) in High-Oleic H176
Peroxidase (POD) plays an important role in the metabolism of fatty acids, which takes place in the peroxisome. Therefore, we extracted and examined the POD activity at six different time points during seed development. In high-oleic cultivar H176, the POD activity was up-regulated by two-fold at 30-40 DAF ( Figure 7A). However, with the seed maturation, POD activity was hardly detected in normal-and high-oleic cultivars. This finding suggested that high-oleic H176 induced up-regulation of POD activity before seed maturation in comparison with normal-oleic cultivar L70. Furthermore, the H 2 O 2 content in both peanut cultivars were examined, which presented a positive correlation with POD activity at 30-40 DAF during seed filling, and the concentration of H 2 O 2 in the high-oleic cultivar was higher than that in the normal cultivar L70 ( Figure 7B). Twelve DEGs of the POD family were identified in high-oleic H176, implying that the excessive accumulation of high-oleic acid triggered the POD-mediated β-oxidation ( Figure 7C). To understand whether the expression level of SAD2 was affected by ROS (reactive oxygen species), we utilized the exogenous H 2 O 2 to treat the normal-oleic cultivar (L70) seedlings. This result indicated that H 2 O 2 was capable of triggering the expression of SAD2, but high content of H 2 O 2 displayed an inhibitory effect ( Figure 8A). According to our study, the redox signaling caused by POD likely acted as a secondary messenger to regulate fatty acid metabolism.

The Enhanced Activity of Peroxidase (POD) in High-Oleic H176
Peroxidase (POD) plays an important role in the metabolism of fatty acids, which takes place in the peroxisome. Therefore, we extracted and examined the POD activity at six different time points during seed development. In high-oleic cultivar H176, the POD activity was up-regulated by twofold at 30−40 DAF ( Figure 7A). However, with the seed maturation, POD activity was hardly detected in normal-and high-oleic cultivars. This finding suggested that high-oleic H176 induced upregulation of POD activity before seed maturation in comparison with normal-oleic cultivar L70. Furthermore, the H2O2 content in both peanut cultivars were examined, which presented a positive correlation with POD activity at 30−40 DAF during seed filling, and the concentration of H2O2 in the high-oleic cultivar was higher than that in the normal cultivar L70 ( Figure 7B). Twelve DEGs of the POD family were identified in high-oleic H176, implying that the excessive accumulation of higholeic acid triggered the POD-mediated β-oxidation ( Figure 7C). To understand whether the expression level of SAD2 was affected by ROS (reactive oxygen species), we utilized the exogenous H2O2 to treat the normal-oleic cultivar (L70) seedlings. This result indicated that H2O2 was capable of triggering the expression of SAD2, but high content of H2O2 displayed an inhibitory effect ( Figure  8A). According to our study, the redox signaling caused by POD likely acted as a secondary messenger to regulate fatty acid metabolism.

Discussion
Transcriptome sequencing provides a systemic approach for studying gene expression and network interactions in different organs of developing peanut seeds. Herein, by generating a highquality transcriptome dataset of high-oleic and low-oleic peanut seeds, we obtained 74 specific DEGs (Table 2) involved in the biosynthesis or metabolic pathways of fatty acid, lipid, phospholipid, sphingolipid, and glycerolipid. Based on the analysis of the gathered transcriptome data of the FA synthesis pathway, we concluded that FAD2 mutation-caused high-oleic feedback regulates the expression patterns of upstream oleoyl-ACP (C18:1-ACP) synthetase gene SAD2 (Aradu.XM2MR) [23] at the mRNA level during the development stages of high-oleate peanut seeds. Meanwhile, fad2a/fad2-b variation with a high content of OA not only affected upstream SAD2 (Aradu.XM2MR) expression, but also directly changed the level of downstream linolenic acid (C18:3) synthase FAD8 [24] and linoleic acid (C18:2) synthetase delta−6 desaturase (FAD6) [25] (Figure S10 and Table S12). Chloroplastic enzyme FAD6 is responsible for the biosynthesis of 16:2 and 18:2 FAs from galactolipids, sulpholipids, and phosphatidylglycerol using ferredoxin as the electron donor, and its mutation in plants could result in a reduced level of unsaturated fatty acids, thus leading to susceptibility to photo-inhibition [26]. FAD8 encodes omega−3 fatty acid desaturase, and gene mutation could lead to the reduction in desaturation of storage and membrane lipids [27]. In conclusion, FAD2 mutation prevented the carbon flux of unsaturated fatty acid synthesis, thus correspondingly resulting in the down-regulated transcription of FAD6 and FAD8 ( Figure S10). These results allowed us to propose a strategy for improving the content of unsaturated FA in the peanut by manipulating the FA desaturase family of genes.
Our results are consistent with a previous comparative proteomics study to dissect the higholeate-suppressed peanut SAD2 activity [28]. Our finding provides new insight into understanding the mechanism of OA synthesis in the peanut, and showed that over-accumulated OA, as the downstream reaction product, repressed the transcription of upstream rate-limiting enzyme SAD2 (Aradu.XM2MR) along with the maturation of the seeds. Additionally, the majority of SAD enzymes

Discussion
Transcriptome sequencing provides a systemic approach for studying gene expression and network interactions in different organs of developing peanut seeds. Herein, by generating a high-quality transcriptome dataset of high-oleic and low-oleic peanut seeds, we obtained 74 specific DEGs (Table 2) involved in the biosynthesis or metabolic pathways of fatty acid, lipid, phospholipid, sphingolipid, and glycerolipid. Based on the analysis of the gathered transcriptome data of the FA synthesis pathway, we concluded that FAD2 mutation-caused high-oleic feedback regulates the expression patterns of upstream oleoyl-ACP (C18:1-ACP) synthetase gene SAD2 (Aradu.XM2MR) [23] at the mRNA level during the development stages of high-oleate peanut seeds. Meanwhile, fad2-a/fad2-b variation with a high content of OA not only affected upstream SAD2 (Aradu.XM2MR) expression, but also directly changed the level of downstream linolenic acid (C18:3) synthase FAD8 [24] and linoleic acid (C18:2) synthetase delta−6 desaturase (FAD6) [25] ( Figure S10 and Table S12). Chloroplastic enzyme FAD6 is responsible for the biosynthesis of 16:2 and 18:2 FAs from galactolipids, sulpholipids, and phosphatidylglycerol using ferredoxin as the electron donor, and its mutation in plants could result in a reduced level of unsaturated fatty acids, thus leading to susceptibility to photo-inhibition [26]. FAD8 encodes omega−3 fatty acid desaturase, and gene mutation could lead to the reduction in desaturation of storage and membrane lipids [27]. In conclusion, FAD2 mutation prevented the carbon flux of unsaturated fatty acid synthesis, thus correspondingly resulting in the down-regulated transcription of FAD6 and FAD8 ( Figure S10). These results allowed us to propose a strategy for improving the content of unsaturated FA in the peanut by manipulating the FA desaturase family of genes.
Our results are consistent with a previous comparative proteomics study to dissect the high-oleate-suppressed peanut SAD2 activity [28]. Our finding provides new insight into understanding the mechanism of OA synthesis in the peanut, and showed that over-accumulated OA, as the downstream reaction product, repressed the transcription of upstream rate-limiting enzyme SAD2 (Aradu.XM2MR) along with the maturation of the seeds. Additionally, the majority of SAD enzymes [29,30] are usually predicted to be located at the plastid (dark condition) and chloroplast (light condition), and are responsible for catalyzing the substrate stearoyl-ACP (C18:0-ACP) to form oleoyl-ACP (C18:1-ACP) in the de novo plant FA synthesis. Subsequently, acyl-ACP thioesterase (FATA) [31] suppresses the ACP domain and accelerates the conversion of acyl-ACP fatty acid to free fatty acid. Finally, the released acyl-oleic acid (Acyl-C18:1) is exported from the cytoplasmic plastid into the endoplasmic reticulum (ER) by the acetyl coenzyme A transport pathway in order to synthesize linoleic acid or promote the flux of the 18-carbon atom chain (C18) for conversion into other unsaturated FAs [32,33]. FAD2 is mainly anchored in ER and catalyzes the transformation of OA into LOA, whereas the allelic mutant displays defects in this conversion, thus inducing high-oleic content [34]. It is well known that the regulation of transcription is dependent on intra-nuclear transcription factors [35]. A hypothesis of the decreased SAD2 (Aradu.XM2MR) mRNA level in the high-oleic peanut cultivar is attributed to uncharted transcriptional repressors. The excessive accumulation of OA could trigger multiple signal pathways closely connected with a relationship triangle of chloroplast-ER-nucleus-mediated signaling transduction for coordinated inhibition of SAD2 (Aradu.XM2MR) expression ( Figure 8B).
Alternatively, mature plant seeds store a diversity of FAs that are re-disintegrated to supply nutrients for the growth of post-germinative seedlings [36]. Once the relative content of OA exceeds a normal threshold in total reserved compounds, the feedback signaling can be detected by the peanut, and the bio-synthetic network of OA will down-regulate upstream reaction via repressing SAD2 (Aradu.XM2MR) expression. Meanwhile, we showed evidence that over-accumulation of OA can trigger the degradation of fatty acid by β-oxidation in the peroxisome thus generating ROS-modulated signaling transduction between FAD2 and SAD2 (Aradu.XM2MR). Free fatty acids as substrates are imported into peroxisome by an ABC (ATP-binding cassette) transporter CTS (COMATOSE) [37,38]. In addition, 12 DEGs relevant to the function of POD were identified in high-oleic cultivar H176, and POD activity and H 2 O 2 content showed significant increases in the early period of seed development, which exhibited the same trend as the expression pattern of SAD2 (Aradu.XM2MR) in high-oleic H176. Accordingly, FAD2 mutation mediated over-accumulation of oleic acid likely triggered a redox signaling burst that correlated with the peroxidase-mediated FA degradation ( Figure 7C). Additionally, a series of DEG-encoded enzymes are involved in FA oxidation-reduction process, such as trans-2-enoyl-CoA reductase, acetyl-CoA carboxylase, sterol C4-methyl oxidase, lipoxygenase, and seed linoleate 9S lipoxygenase ( Table 2) [39][40][41][42].
Theoretically, the dynamic change of FAD2 should be easily detected in the transcription profile due to the novel allelic mutation, and the predictable change was found at each period of seed developmental in the high-oleic peanut cultivar H176. However, our previous results indicated that the FAD2 protein was maintained at a relatively stable level for OA accumulation cross the entire time during the developmental stages of high-oleic peanut seeds [28]. Herein, an interpretation of the relevant phenomena from transcriptome-seq was based on the finding that FAD2 mRNA (Araip.S3GXY) was not able to normally transcribe along with the high-oleic seed maturation, thereby leading to the significantly declined transcription level when compared with normal cultivar, and the FAD2 enzyme lost its activity due to premature termination ( Figure 1G,H). Furthermore, high-oleic acid still can affect the expression levels of some chloroplast enzymes involved in FA biosynthesis process. For instance, 3-ketoacyl-CoA synthase (KCS) 11 can catalyze the first step during the fatty acid elongation process [43], which is the condensation of C2 units to acyl-CoA, and the peak value of KCS11 expression presented at stage 3. The 3-oxoacyl ACP synthase 3 (FabH) regulates malonyl-ACP in the presence of acyl-CoA to generate 3-ketoacyl-CoA [44]. Both enzymes exhibited consistent expression patterns. FabZ-encoded dehydrogenase promotes the conversion of hydroxyacyl-ACP to enoyl-ACP [45], whose expression showed a two-fold increase at stage 3 and at the terminal phase (Table S12). Taken together, a comprehensive overview of FAD2 mutant-induced expression network is provided, and the transcriptome-identified DEGs offer a potential resource to uncover the mechanisms of the FA synthesis pathway in different organelles. Moreover, the possible functions of those lipid DEGs will be further elucidated in coming investigations for dissecting OA-associated FA biosynthesis in the peanut.

Plant Materials
Two peanut cultivars H176 (Kainong176) and L70 (Kainong70) (Figure 1A,B) with different OA contents in their mature seeds (Kainong176 > 70% of total oil, Kainong70 < 40% of total oil) were used in the present study [28]. High-oleic-content mutant lines (Kainong176 and Kainong70) were isolated from the population of sexual hybridization by using Kainong30 (♀) and Kaixuan01−6 (♂) at Kaifeng (Henan Province, China) ( Figure S1). These two cultivars were provided by Kaifeng Academy of Agriculture and Forestry, and they were re-cultivated to flowering in the field of Baiyun District of Guangzhou City (Guangdong Province, China) under normal growth conditions. Subsequently, the aerial pegs were labeled with a pink tag after flowering, and secondary labeling was conducted at the time when the elongated gynophore first touched the ground [28]. The developmental stages of those marked gynophores were precisely recorded as days after flowering (DAF) in high-and normal-oleic cultivars. Non-elongated first-marked gynophores were discarded. When the gynophores entered into the soil and developed into pods, we collected the seeds at different times (from 20 to 70 DAF). In order to improve consistency, we chose pods with similar phenotypes at the same developmental stage for RNA analysis. The harvested seeds were quickly frozen in liquid nitrogen, and their total FA and RNA were extracted for relevant experiments.

Analysis of OA, LOA, and Oil Content
Oil content and composition were analyzed using 5 g of seeds collected from each developmental stage. The fresh seeds of different stages were ground into a fine powder in liquid nitrogen. The total oil content was determined following Soxhlet extraction using n-hexane. The molecular composition of extracted FAs was determined by gas chromatography of the methyl ester of the FAs according to the National Standard of the People's Republic of China (GB/T 17377−2008). Briefly, 100 mg peanut seeds were frozen and ground into a fine powder in liquid N2. The powder was mixed with 1.5 mL chloroform/methanol (2:1, v/v) and 100 µL internal standard solution C17:0 (1 mg/mL). The mixture was extracted by ultrasound-assisted extraction for 30 min and centrifuged at 12,000 rpm for 6 min, and then 1 mL supernatant was transferred into a fresh tube. The FA extracting solution was mixed with 0.2 mL KCl (75%, w/v) solution and centrifuged at 12,000 rpm for 6 min, and then 400 µL substratum chloroform extract phase was transferred into a new glass tube and prepared for methanol esterification. The extracting solution was mixed with 2 mL sulfuric acid in methanol (5:95, v/v) and incubated in a water bath at 85 • C for 1.5 h. Subsequently, 1 mL H 2 O and 1 mL hexane were added into the tube, the mixture was centrifuged at 5000 rpm for 5 min, and 500 µL superstratum hexane extraction phase was used for gas chromatography (Cat#YLSB076). The individual FA contents were reported as relative percentages of OA and LOA in the extracted oil [46].

Total RNA Isolation, Library Construction, and Transcriptome Sequencing
Total RNA was extracted from 300 mg of seeds from both peanut cultivars using the Trizol Reagent (Invitrogen, Beijing, China). The concentration of purified RNA was determined using a NanoDrop UV-visible spectrophotometer (Thermo Fisher, Waltham, MA, USA). Total RNA was used for library construction following the High-Throughput Illumina Strand-Specific RNA Sequencing Library protocol. Each library was made with three individual peanut seed samples, and each library was subjected to analysis with six replicates. The mRNA for sequencing was purified from 5 µg total RNA using Agencourt Ampure-XP magnetic beads (Beckman Coulter, Brea, CA, USA), and the purified mRNA was fragmented to the average size of 300 bp. The first-strand cDNA was synthesized using the fragmented mRNA as the template with random hexamer primers. Second-strand cDNA synthesis was performed by using DNA polymerase I and RNase H. All RNA-Seq libraries were sequenced on a Illumina HiSeq™2000, and all the transcriptome raw data have been deposited in Sequence Read Archive (SRA) database with the BioProject Accession number: PRJNA480751 [47].

Bioinformatic Analysis of Transcriptome Data
RNA-library sequencing and transcript assembly were performed by Novogene Ltd. based on the Trinity [22] method. The Trinity method consisted of three software modules, Inchworm, Chrysalis, and Butterfly, that were applied sequentially to process large volumes of RNA-Seq reads. Assembled reads were mapped to the published genome of two diploid ancestors Arachis duranensis and Arachis ipaensis (www.Peanutbase.org). Reads per kilobase per million (RPKM) were calculated by adopting an in-house script according to the count table of assembly sequences output. The differentially expressed genes (DEGs) were identified by applying a cutoff log 2 (fold change) of >1 and <−1 for up-regulated and down-regulated genes, respectively, as well as p < 0.05. Gene Ontology analysis of DEGs was performed by AgriGO web-based tools. KEGG pathway enrichment was assigned to the DEGs using the online KEGG web server.

Real-Time PCR Analysis
Real-time PCR was carried out according to a previously described method [48]. Briefly, 1 µg of total RNA was reversely transcribed into cDNA using the PrimeScript RT Kit (Takara, Dalian, China) according to the manufacturer's instructions. The PCR reaction was conducted on a 20-µL scale using SYBR Premix ExTaq™ (TaKaRa, Dalian, China) on an ABI (Applied Biosystems) StepOne Plus system. The relative expression of target genes was calculated by the 2 −∆∆Ct method and shown as fold changes relative to the normal-oleate cultivar kainong70. Yellow leaf specific 8 gene (yls8) (Forward Primer: 5 -AACTGCTTAGCTGCTATTACC−3 , Reverse Primer: 5 -TCGCCAAATAACACGTTGCAT−3 ) and alcohol dehydrogenase class III (adh3) (Forward Primer: 5 -GACGCTTGGCGAGATCAACA−3 , Reverse Primer: 5 -AACCGGACAACCACCACATG−3 ) were selected as the internal controls (Table S11). Each measurement was carried out in triplicate with three replicates, and data were expressed as means ± SE.

Subcellular Localization Analysis
The normal full-length FAD2 [10] and Aradu.XM2MR cDNA without a stop codon were cloned between the cauliflower mosaic virus (CaMV) 35S promoter and the GFP gene. The resulting FAD2/Aradu.XM2MR-GFP fusion construct and different organelle markers were transiently co-expressed in Arabidopsis protoplasts by PEG induction [49]. Briefly, Arabidopsis protoplast cells were isolated from two-week-old Arabidopsis leaves. The full leaves were cut into approximately 0.5−1 mm strips, and incubated in 10 mL enzyme solution (1.5% Cellulase RS, 0.75% Macerozyme R−10, 0.6 M mannitol, 10 mM MES at pH 5.7, 10 mM CaCl 2 , and 0.1% BSA) for 4 h in the dark with gentle shaking (60 rpm). After washing twice with W5 solution (154 mM NaCl, 125 mM CaCl 2 , 5 mM KCl and 2 mM MES at pH 5.7) and centrifuging at 100 g at 4 • C for 2 min, the supernatant was discarded. Protoplast cells were resuspended in MMG solution (0.4 M mannitol, 15 mM MgCl 2 and 4 mM MES at pH 5.7). Then, 100 µL of the protoplast cell solution with 10 µg plasmid and 110 µL 40% PEG 4000 were incubated at 28 • C for 15 min in the dark, followed by the addition of 1 mL W5 and centrifugation at 150 g at 4 • C for 2 min. The supernatant was discarded. The collected protoplast cells were resuspended in 500 µL WI solution (0.5 mM mannitol, 4 mM MES at pH 5.7 and 20 mM KCl) and incubated at 28 • C for 12−18 h in dark. Next, the protoplast cells were collected by centrifugation at 300 g at 4 • C for 10 min and resuspended in 40 µL WI solution for confocal microscopy. Fluorescence was examined under a laser-scanning confocal microscope (Model LSM 780; Carl Zeiss, Jena, Germany).

POD Activity Assay
Fresh peanut seeds (300 mg) were frozen and ground in liquid nitrogen. The powder was mixed with 4 mL 0.05 mol/L PBS (pH 7.8) and transferred into a 5-mL tube. After thawing, the tubes were centrifuged at 8000 rpm/min for 15 min, and the supernatant containing the total peroxidase was collected. The POD activity was measured as the rate of decomposition of H 2 O 2 with guaiacol as the hydrogen donor by spectrophotometrically measuring the rate of color development at 436 nm due to guaiacol oxidation [50]. H 2 O 2 content was determined by following the method described by Sui et al. (2018) [47]. The concentration of H 2 O 2 was estimated by detecting the absorbance of the titanium-hydroperoxide complex at 415 nm after calculation with a standard curve plotted with H 2 O 2 standards [47].

Ectopic Expression of SAD2 (Aradu.XM2MR) in Yeast Cells and Fatty Acid Composition Analysis
The protocol of ectopic expression in yeast cells was reported by Chi et al. (2011) [23]. Briefly, the SAD2 CDS sequence was cloned into the vector pYES2, and the CK group (empty vector pYES2) and pYES2-SAD2 were co-transformed into the yeast strain INVSc1 by utilizing the lithium acetate method. Positive yeast clones were selected on a medium plate (SD/-Ura), and the positive yeast clones were cultivated at 30 • C for 36 h to examine the fatty acid composition. LC-MS analysis of the fatty acid composition was performed by ultra-performance liquid chromatography coupled with an electrospray ionization-quadrupole time-of-flight mass spectrometer system. This experiment and relevant data analysis were performed by Deepxomics Co., Ltd., Shenzhen, China.