The genomic sequences of a cultivated peanut A. hypogaea cv. ShiTouQi were downloaded from Peanut Genome Resouce (http://peanutgr.fafu.edu.cn/index.php). The peanut oleosin genes were identified using two tools: BLASTP search and HMMER. The protein sequences of Arabidopsis thaliana oleosin genes, obtained from TAIR (https://www.arabidopsis.org/), were used as queries for BLASTP searches against the annotated peanut proteins and to identify peanut oleosin homologs. The conserved oleosin domain (PF01277) was then employed to identify the proteins through HMMER program (Finn et al., 2011). By integrating the sequences derived from both methods, peanut oleosin proteins were identified.

The gene structure information was retrieved from Peanut Genome Resouce (http://peanutgr.fafu.edu.cn/index.php). The chromosome location, gene structure and conserved domain were visualized using MG2C (https://qiaoyundeng.github.io/) and TBtools (Chao et al., 2021). The predicted molecular weights and theoretical isoelectric points (PI) were calculated using Expasy - ProtParam (https://web.expasy.org/protparam/). The conserved domains were identified using Pfam (http://pfam.xfam.org/).

The oleosin protein sequences of rapeseed (Brassica napus L.) were retrieved from BnTIR (https://yanglab.hzau.edu.cn/BnTIR). The oleosin protein sequences of Arachis duranensis and Arachis ipaensis were obtained from Peanut Base (https://www.peanutbase.org/). Multiple sequence alignments of the oleosin protein sequences were performed using ClustalW software (Higgins and Sharp, 1988). A phylogenetic tree was constructed using the neighbor-joining method in the MEGA X, employing 1000 bootstrap replicates (Kumar et al., 2018). The evolutionary tree was visualized using the online tool iTOL (https://itol.embl.de/itol.cgi).

The collinearity within and between A. hypogaea, A. duranensis and A. ipaensis was established using MCScanX (http://chibba.pgml.uga.edu/duplication/) incorporated into TBtools. The collinear relationships of oleisin genes were drawn using Circos (Krzywinski et al., 2009).

The promoter sequences of peanut oleosin genes were extracted from the ShiTouQi genome using TBtools, encompassing a 2000bp DNA sequence upstream of the ATG start codon. The cis-elements in the promoter regions were predicted using New PLACE websites (https://www.dna.affrc.go.jp/PLACE/?action=newplace). The positions of cis-elements were visualized using TBtools.

The multi-tissue transcriptome data used in this study were downloaded from Peanut Genome Resouce (http://peanutgr.fafu.edu.cn/index.php). Additionally, transcriptome data of leaves subjected to drought and cold stress, as well as those treated with plant hormone, were also obtained from this database. The heat maps were generated using TBtools.

To validate the expression profile, roots, leaves, stems, flowers, and developing seeds at 20, 30, 40, 50, and 60 days after pollination (DAP) of Zhonghua 12 were collected and five development stages as described previously (Huai et al., 2020). Total RNA was extracted using RNAprep Pure Plant Kit (DP 441, TIANGEN, China). First strand cDNA was syntheisized from 1 µg RNA using HiScript III 1st Strand cDNA Synthesis Kit (R 312-02, Vazyme). Quantitative real-time PCR (RT-qPCR) was performed according to the instructions of ChamQ Universal SYBR qPCR Master Mix (Vazyme, China). The primers for RT-qPCR were designed using NCBI ( Supplementary Table S3 ). The relative expression levels of genes were calculated by the 2^−△△CT method. The experiment was performed using three biological replicates.

Total RNA was extracted from developing seeds of Zhonghua 12 at 40 days after pollination using TRIzol reagent (DP 424, TIANGEN, China), following the manufacturer’s protocol. Reverse transcription was performed using HiScript IV 1st Strand cDNA Synthesis Kit (R412-01, Vazyme, China) as described by the manufacturer. The AhOle11 (AH16G32210) gene was cloned using the primers: 5’-ATGTCTGATCAAACAAGGACA-3’ and 5’- TCAATACCCTTGTGTGCCCTC-3’.

The AhOle11 gene without a stop codon were amplified by PCR, and inserted between the cauliflower mosaic virus (CaMV) 35S promoter and the green fluorescent protein (GFP) gene. The resulting plasmids were designated as pHBT-AhOle11-GFP. The empty vector pHBT-GFP was analyzed as a control. The AtOle5 (AT3G27660) from Arabidopsis was fused with the red fluorescent protein (RFP) gene. The p35S:AtOle5-RFP construct was used as an oil body marker. The pHBT-GFP and pHBT-AhOle11-GFP were each transiently co-expressed with the oil body marker in Arabidopsis protoplasts by PEG transformation, respectively (Yoo et al., 2007). Fluorescence signals were detected using a confocal laser scanning microscope (Nikon C2-ER, Japan). GFP was excited at 488nm, while RFP was excited at 561 nm.

The AhOle11 gene was amplified via PCR and inserted into the pBinGlyRed2 vector, which contains a DsRed2 driven by 35S promoter. As a result, the AhOle11 was positioned between the seed-specific soybean glycinin-1 promoter and its 3’UTR.The recombinant plasmid was introduced into Agrobacterium tumefaciens strain GV3101 and transformed into Arabidopsis by the floral dip method. DsRed-positive seeds were identified using a green LED flashlight with a red camera filter lens (Huai et al., 2023).

The oil content and fatty acid composition of mature DsRed-positive Arabidopsis seeds were analyzed as previously described (Huai et al., 2018). In brief, 20 mg grounded seeds were placed into a glass tube. Subsequently, 1.5ml of 2.5% sulfuric acid-methanol solution, 0.35 ml of toluene and 0.3 ml of C15:0 (internal standard) solution in methanol (10 mg/ml) were added. Fatty acids were transmethylated at 90°C for 30–45 min. After cooling to room temperature, 1 ml H₂O and 1 ml hexane were added into the tube. The supernatant was filtered through a 0.45 μm microporous membrane and transferred into an autosampler vial. The fatty acid contents were determined by gas chromatograph (GC) using an Agilent 7890B with flame ionization detection and the DB-23 column. The concentration of fatty acids methyl esters (FAMEs) was quantified based on the ratio of different FAMEs peak areas relative to the internal standardization (C15:0). The fatty acid composition is expressed as absolute concentrations (mg/g). Three biological replicates per line were analyzed in this experiment. Statistically significant differences were analyzed using Student’s t-test by the SPSS SPSS19.0; The phenotypes was performed on GraphPad Prism 8.0.

Fixation of seeds and lipid staining were performed as previously described with modifications (Cai et al., 2015; Zhang et al., 2019). Mature seeds were imbibed in distilled water for 20~30 min for seed coat removal. Then embryos were immediately immersed in formalin-acetic acid-alcohol (FAA) fixative (formalin: acetic acid: 50% ethanol = 1:1:18 v/v) for at least 24 h. The fixed tissues were trimmed using a scalpel and subsequently immersed in 15% sucrose solution at 4°C until they sank. They were then transferred to a 30% sucrose solution at 4°C and incubated until they sedimented. The fixed tissues were embedded with optimal cutting temperature compound (OCT) compound. Cryosections (8~10 µm) were prepared using a Cryostat Microtome (Thermo, CRYOSTAR NX50, USA). The cryosections were stained with BODIPY 493/503 for 20 min and subsequently washed with PBS (pH 7.4). Thereafter, the sections were re-stained with 4’,6-diamidino-2-phenylindole (DAPI) for 10 min in the dark, followed by additional washes with PBS (pH 7.4). Image observation for BODIPY and DAPI was performed using a confocal laser scanning microscope (Nikon C2-ER, Japan). DAPI was excited at 330-380nm, while BODIPY was excited at 488 nm.

A total of 12 oleosin genes were identified in the peanut genome and were designated as AhOle1~AhOle12 based on their chromosomal locations ( Table 1 ). The coding sequences (CDSs) of peanut oleosin genes ranged from 414 to 918 bp in length, encoding proteins consisting of 137 to 305 amino acids, with molecular weights from 14.3 to 31.7 kDa ( Table 1 ). All AhOle genes were intronless throughout their entire open reading frames ( Figure 1A ). Furthermore, all AhOles contained solely the oleosin domain ( Figure 1B ).

The AhOle genes were unevenly distributed across 9 chromosomes in peanut, an allotetraploid comprising A and B subgenomes. Five AhOle genes was detected on Chr01, Chr05, Chr08 and Chr10 in subgenome A, whereas seven AhOle genes was located on Chr11, Chr14 Chr15, Chr16 and Chr20 in subgenome B. Notably, Chr05, Chr08, Chr10, Chr15, Chr16 and Chr20 each contained only a single AhOle gene, whereas Chr01, Chr11 and Chr14 each harbored two such genes ( Figure 2 ).

An un-rooted phylogenetic tree was constructed in MEGA X based on the protein sequences sourced from A. hypogaea (12 AhOles), A. duranensis (6 AdOles), A. ipanesis (7 AiOles), A. thaliana (17 AtOles) and B. napus (48 BnOles). The oleosin proteins were divided into four groups: U, SL, SH and T lineages. AhOle was classified into U, SL and SH lineages, but no AhOle was detected in T lineage ( Figure 3A ). AhOle2 and AhOle4 were grouped into the U lineage, while AhOle5 and AhOle12 were grouped into the SL lineage. Eight AhOles were grouped into the SH lineage, including AhOle1, AhOle3, AhOle6, AhOle7, AhOle8, AhOle9, AhOle10 and AhOle11 ( Figure 3A ). These results indicate that only three common oleosin proteins are present in peanut: U, SL and SH oleosins.

Through intragenomic comparison analysis, three collinear gene pairs in A. hypogaea were obtained ( Figure 3B ). AhOle genes on Chr01, Chr05 and Chr10 were syntenic to AhOle genes on corresponding Chr11, 15 and 20. The AhOle2 and AhOle4 in subgenome A were not paired, as well as four AhOle genes in subgeome B (AhOle7, AhOle8, AhOle9 and AhOle11) ( Figure 3B ).

To achieve a deeper understanding of the phylogenetic mechanisms occurring in the peanut oleosin family, the synteny maps of the oleosin genes in the A. hypogaea (AABB) genome and their homologous genes in two ancestral species A. duranensis (AA) and A. ipaensis (BB) were constructed. Through intergenomic comparison analysis, four gene pairs between A. hypogaea and A. duranensis and five gene pairs between A. hypogaea and A. ipaensis were identified ( Figure 3C ). The syntenic genes of AdOle1 and AdOle5 in A. duranensis were not found in A. hypogaea, as well as AiOle3 and AiOle6 in A. ipaensis. In addition, no syntenic genes of AhOle5, AhOle8 and AhOle9 were identified in A. duranensis and A. ipaensis ( Figure 3C ). AhOle8 and AhOle9 were found to be homologous gene pairs, potentially resulting from tandem duplication events ( Figure 3A ).

To elucidate the roles of AhOle genes during different growth and developmental stages in peanuts, the expression patterns of these genes were analyzed using transcriptome data from the reference A. hypogaea cv. ShiTouQi. A heat-map of AhOle genes was created to demonstrate their expression profile ( Figure 4 ). AhOle8 and AhOle9 were not expressed in any of the tested tissues, whereas other AhOle genes exhibit high expression. Among them, the expression levels of AhOle4, AhOle5, AhOle11 and AhOle12 were found to be the highest during seed development. The expression level of AhOle3 and AhOle10 in developing seeds were observed moderately high, followed by AhOle1, AhOle6 and AhOle2. AhOle7 was exclusively expressed at the initial and final stages of seed development ( Figure 4 ).

To validate the transcriptome data, the expression levels of SH (AhOle1/6, AhOle3/10, and AhOle11), SL (AhOle5/12), and U (AhOle2) Oleosin genes were further investigated in Zhonghua12 tissues. The results indicated that eight genes exhibited significantly different expression patterns ( Supplementary Figure S1 ). AhOle1/6 are expressed in stems, leaves, and seeds. AhOle2 and AhOle11 are expressed in all the tissues, and transcription abundance is the highest in seeds. AhOle3/10 exhibited predominant expression in seeds and displayed a pattern of increasing expression level along with seed development ( Supplementary Figure S1A ). The expression of AhOle5/12 is significantly higher in the later stage of seeds and leaves than in other tissues ( Supplementary Figure S1A ). In addition, comparison analysis of AhOle1/6, AhOle2, AhOle3/10, AhOle5/12, and AhOle11 expression throughout peanut seed developmental stage showed higher transcription accumulation during 30 and 60 DAP, with AhOle11 demonstrating the highest expression abundance among those genes ( Supplementary Figure S1B ). This result indicates that qPCR analysis of AhOle gene expression patterns was consistent with transcriptome data.

Oleosin genes have been reported to be regulated in response to various abiotic stresses. The cis-elements within promoters play a crucial role in modulating gene transcription. To investigate the potential cis-elements involved in response of peanut to abiotic stresses, the upstream 2000-bp regions of AhOle genes were analyzed. A diverse array of cis-element was identified, including those associated with development, phytohormone responses, and stress tolerance ( Figure 5A ). For instance, the LTR element (CCGAAA), which is linked to low temperature tolerance, was identified in promoters of AhOle2, AhOle7, AhOle8 and AhOle11. The MYB binding sites (TAACTG) implicated in drought inducibility was found in all AhOle gene promoters. The ABRE element (AAGAA-motif), which responds to abscisic acid (ABA), was present in the promoters of AhOle1, AhOle2, AhOle3, AhOle5, AhOle6, AhOle7, AhOle8, AhOle10, AhOle11 and AhOle12. The TGACG-motif and CGTCA-motif, both responsive to jasmonic acid (JA), were identified in the promoters of AhOle2, AhOle5, AhOle6, AhOle7, AhOle8, AhOle10, AhOle11 and AhOle12. The TCA-element, which respond to salicylic acid (SA), was detected in the promoters of AhOle1, AhOle2, AhOle3, AhOle6, AhOle8, AhOle11 and AhOle12. The GARE-motif, associated with gibberellin (GA) response, was present in the promoters of AhOle2, AhOle3, AhOle4, AhOle5, AhOle6, AhOle7, AhOle10, AhOle11 and AhOle12. The ERELEE4-motif, involved in ethylene response, was found in the promoters of AhOle4, AhOle5, AhOle6, AhOle8, AhOle10, AhOle11 and AhOle12 ( Figure 5A ).

The transcriptome data of peanut leaves subjected to drought and cold stress, as well as those treated with plant hormone, were downloaded and analyzed ( Figure 5B ). The expression levels of AhOle1, AhOle4 and AhOle6 were significantly up-regulated under drought stress, while those of AhOle8 and AhOle9 were significantly down-regulated. Under cold stress, the expression level of AhOle11 was significantly up-regulated, whereas those of AhOle4, AhOle8 and AhOle9 were significantly down-regulated. Following treatment with paclobutrazol, the expression level of AhOle5, AhOle10 and AhOle11 were significantly up-regulated. Additionally, the expression level of AhOle5 and AhOle11 were significantly up-regulated after treated with salicylic acid ( Figure 5B ).

The expression of AhOle8 was down-regulated in response to both drought and cold stresses, along with three MYB elements and one LTR element in its promoter. The expression of AhOle11 was up-regulated upon exposure to salicylic acid, with a TCA-element detected in its promoter. Meanwhile, the expression levels of AhOle5, AhOle10, and AhOle11 were up-regulated following treatments with abscisic acid, salicylic acid, and ethephon, respectively, which is consistent with the predicted cis-elements. These findings suggested that AhOle genes play an important role in peanut development and responses to abiotic stress.

The AhOle11 gene was selected for further functional analysis, as it belongs to the SH lineage and exhibited the highest expression level during seed development ( Figures 3 , 4 ). To investigate the subcellular localization of AhOle11, it was fused with GFP and subsequently co-expressed with an oil body marker in Arabidopsis protoplasts. As anticipated, the green fluorescence from the empty vector was observed throughout the entire cell. In contrast, the green fluorescence of AhOle11 was completely co-localized with the red fluorescence of the oil body marker ( Figure 6 ). These results indicated that AhOle11 was located within the oil body, where oleosin exerts its function.

To further confirm the function of AhOle11 in plants, the AhOle11 gene was overexpressed in the Arabidopsis. A total of ten AhOle11-overexpressing lines were obtained. The oil content and fatty acid composition of DsRed positive seeds from three homozygous T₃ lines (OX-1, OX-7, and OX-8) were determined by gas chromatography. The oil contents in Arabidopsis WT seeds varied between 31.2% and 32.9%, whereas the oil content in AhOle11-overexpressing lines ranged from 33.7% to 40.8% ( Figure 7A ). The oil contents in seeds of AhOle11-overexpressing lines were significantly higher than that in WT seeds. Among the overexpressing lines, the OX-8 line exhibited the highest oil content (38.4%~40.8%), which represents a 22.4% increase compared to the control ( Figure 7A ). Then the oil bodies in seeds of WT and overexpressing lines were analyzed. Compared to WT, a significant higher oil body density was observed in the overexpressing lines, whereas no significant difference in oil body size was observed between them ( Figure 7D ). The result visibly illustrated that AhOle11-overexpressing lines contained higher level of oil accumulation in seed more than WT.

In addition, the absolute quantification of fatty acid levels was examined in lines overexpressing AhOle11. Compared to WT, significant increases in fatty acid contents were observed in the following: C18:1, C20:1 and C20:2 in OX-1; C16:0, C18:3, C20:1, C20:2 and C22:1 in OX-7; as well as C18:0, C18:1, C18:2, C18:3, C20:0, C20:1 and C22:1 in OX-8 ( Figure 7C ). In terms of the relative fatty acid compositions, the contents of C20:1 in OX-1 and C18:1 in OX-8 were significantly elevated, while the contents of C18:2 in OX-1 and C16:0 as well as C16:1 in OX-8 were significantly reduced ( Figure 7B ). In summary, overexpression of AhOle11 led to a significant increase in total fatty acid production, while the overall fatty acid composition remained largely consistent.