Genome-Wide Identification, Evolutionary And Expression Analyses of LEA Gene Family In Peanut (Arachis Hypogaea L.)

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RuoLan Huang, Dong Xiao, Xin Wang, Yi Shen, Jie Zhan, AiQing Wang, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-775523/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 30 Mar, 2022 Read the published version in BMC Plant Biology → Version 1 posted 8 You are reading this latest preprint version Abstract Background: Late embryogenesis abundant (LEA) proteins are a group of highly hydrophilic glycine-rich proteins, which accumulate in the late stage of seed maturation and are associated with many abiotic stresses. However, few peanut LEA genes had been reported, and the research on the number, location, structure, molecular phylogeny and expression of AhLEA s was very limited. Results: In this study, 126 LEA genes were identified in the peanut genome through genome-wide analysis and were further divided into eight groups. Sequence analysis showed that most of the AhLEA s (85.7 %) had no or only one intron. LEA genes were randomly distributed on 20 chromosomes. Compared with tandem duplication, segmental duplication played a more critical role in AhLEA s amplication, and 93 segmental duplication AhLEA s and 5 pairs of tandem duplication genes were identified. Synteny analysis showed that some AhLEA s genes come from a common ancestor, and genome rearrangement and translocation occurred among these genomes. Almost all promoters of LEA s contain ABRE, MYB recognition sites, MYC recognition sites, and ERE cis-acting elements, suggesting that the LEA genes were involved in stress response. Gene expression analyses revealed that most of the LEA s were expressed in the late stages of peanut embryonic development. LEA3 (AH16G06810.1, AH06G03960.1), and Dehydrin (AH07G18700.1, AH17G19710.1) were highly expressed in roots, stems, leaves and flowers. Moreover, 100 AhLEA s were involved in response to drought, low-temperature, or Al stresses. Some LEA s that were regulated by different abiotic stresses were also regulated by hormones including ABA, brassinolide, ethylene and salicylic acid. Interestingly, AhLEA s that were up-regulated by ethylene and salicylic acid showed obvious subfamily preferences. Conclusions: AhLEA s are involved in abiotic stress response, and segmental duplication plays an important role in the evolution and amplification of AhLEA s. The genome-wide identification, classification, evolutionary and expression analyses of the AhLEA gene family provide a foundation for further exploring the LEA genes’ function in response to abiotic stress in peanuts. Plant Molecular Biology and Genetics Plant Physiology and Morphology Arachis hypogaea L. Late embryogenesis abundant Expression profiles Abiotic stress Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 Figure 10 Figure 11 Figure 12 Figure 13 Background Plant in nature often encounters various abiotic stresses including drought, cold, high temperature, and salinity, which affect growth and development, reduce its yield and survival rate. Plants have evolved many mechanisms to cope with various environmental stresses. It is known that the late embryogenesis abundant (LEA) proteins play important roles in protecting cells under abiotic stresses, and many LEA s are induced by cold, drought, salinity, abscisic acid (ABA), and ethylene (Kentaro et al., 2014 ; Zegzouti et al., 1997 ; Park et al., 2003 ). Moreover, it has been confirmed that AdDHN1 , a member of the Dehydrin family, can improve the drought resistance of transgenic Arabidopsis, but it is more sensitive to nematodes (Mota et al., 2018), which indicated that some of the LEA s may respond to abiotic stress as well as biotic stress. LEA proteins are highly hydrophilic glycine-rich proteins, which accumulate largely in the later stage of seed maturation and fade away following germination (Battaglia and Covarrubias, 2013 ; Lan et al., 2013 ). As water-binding molecules, the role of LEA proteins is enhancing the stability of protein and membrane. Subcellular localization analysis has indicated that LEA proteins are mainly located in nuclear regions and the cytoplasm (Adrien et al., 2014). LEA proteins have been observed in the roots, leaves, buds, and seedlings, although they mainly appear in seeds of plants (Shao, 2005; Hundertmark et al., 2008). LEA protein families were identified in many plant species by genome-wide identification and analysis, such as Arabidopsis thaliana (Bies-Ethève et al., 2008 ), Populus trichocarpa (Lan et al., 2013 ), Camellia sinensis (Jin et al., 2019 ), Brassica napus (Liang et al., 2016 ), and Triticum aestivum (Liu et al., 2019 ). During the growth and development of plants, LEA proteins are considered to play important roles. It was reported that Medicago falcate LEA3 conferred multiple abiotic stress tolerance by involving the protection of catalase activity (Shi et al., 2020 ). A heterologous expression of a barley LEA3 protein gene, HVA1 , improved tolerance to water stress in rice and wheat (Sivamani et al., 2000 ; Xu et al., 1996 ). AtLEA5 protects yeast cells against oxidative stress (Mowla et al., 2010 ). Escherichia coli can grow in high salt and extreme temperature conditions due to the over-expression of soybean PM2 protein (LEA3) (Yun and Zheng, 2005 ; Yun et al., 2010 ). ABA can regulate the expression of many LEA proteins, and it was proved that the expression of LEA4 subfamily members was upregulated by exogenous ABA (Zamora-Briseño et al., 2016). Peanut is one of the main oils and cash crops all over the world. Peanut is a rainfed crop, but it is sensitive to water deficit stress in the flowering and pegging stages, which would impact the yield of peanuts (Bhogireddy et al., 2020 ). Also, Al stress inhibition of growth reduces peanut yield in acid soil (Qiu et al., 2019 ). To date, the function of the LEA gene family in peanuts has little been reported. In this study, we identified the LEA s in peanuts and analyzed the structure, evolution, and chromosome location of peanut LEA s. Our findings provide a foundation for the evolutionary and functional characterization of LEA gene families in peanut and other plant species. Results 2.1. Identification and characteristics of AhLEA gene in peanut By using the publicly available peanut genome sequence data, the genome-wide identification of LEA s in peanuts was based on sequence homology with 51 Arabidopsis LEA s (Hundertmark et al., 2008) (Table 1 and Additional file 1: Table S1). Proteins that contained a conserved LEA domain were screened by the NCBI-BLAST online tool. Eventually, 126 AhLEA s were identified. All of these genes were grouped with 51 AtLEA s by phylogenetic analyses. The AhLEA s were classified into eight subfamilies including LEA1 , LEA2 , LEA3 , LEA4 , LEA5 , PvLEA18 , SMP , and Dehydrin (Fig. 1 ). The LEA2 family was the largest, with 78 members. The LEA3 s and LEA5 s had 14 and 10 members, respectively. The LEA1 s had 8 members, SMP had 6 members and PvLEA18 had 4 members. The LEA4 and Dehydrin families had 3 members. The species-specific group ( AtM ) of Arabidopsis was absent in the peanut. Table 1 The classification of LEA proteins in Arachis hypogaea is based on Arabidopsis. In this study IPR ID Pfam ID Hundertmark et al. (2008) Arabidopsis A. hypogaea LEA1 IPR005513 PF03760 LEA 1 7 8 LEA2 IPR004864/IPR013990 PF03168 LEA 2 3 78 LEA3 IPR004926 PF03242 LEA 3 4 14 LEA4 IPR004238 PF02987 LEA 4 18 3 LEA5 IPR000389 PF00477 LEA 5 2 10 PvLEA18 IPR018930 PF10714 PvLEA18 3 4 SMP IPR007011 PF04927 SMP 6 6 Dehydrin IPR000167 PF00257 Dehydrin AtM 10 2 3 0 2.2. Chromosomal locations, gene duplication and synteny analysis of the AhLEA s The identified 126 AhLEA s were further located on the 20 chromosomes (Fig. 2 ). The largest number of AhLEA s was found on chromosome 12, fourteen genes, followed by chromosome 14 (eleven genes). The lowest loci density was observed on chromosome 8, with only two genes. Eight genes were located on chromosomes 13 and 15, seven genes were located on chromosome 6. Five chromosomes (chr1, chr2, chr3, chr4, and chr16) carried six AhLEA s and four chromosomes (chr5, chr7, chr19, and chr20) carried five AhLEA s. Chromosomes 9, 10, and 11 contained four AhLEA s, and chromosomes 17, 18 contained three AhLEA s. The AhLEA s were distributed unevenly among the 20 chromosomes in peanut. Different LEA subfamilies were located on different chromosomes. All chromosomes contained the LEA2 s, and all of the LEA s on chromosomes 3, 8, 10, 13, 18, and 20 belonged to the LEA2 s. The LEA1 s were distributed on chromosomes 1, 6, 9, 11, 16, and 19. The LEA3 s were mainly distributed on chromosomes 1, 2, 4, 6, 11, 12, 14, and 16. The LEA4 s were mainly found on chromosomes 6, 12, and 16. The LEA5 s were distributed on chromosomes 2, 12, 14, and 16. The PvLEA18 s were distributed on chromosomes 1, 5, 11, and 15. The SMP s were distributed on chromosomes 2, 6, 9, 12, and 19. The Dehydrin s were distributed on chromosomes 7, 15, and 17. Chromosomal location analysis of AhLEA s indicated that eight subfamilies were distributed unevenly in the genome. The generation and evolution of gene families may be caused by tandem duplication and segmental duplication (Mehan et al., 2004 ; Kent et al., 2003 ). To investigate the evolutionary relationships of the AhLEA gene family, we analyzed the duplication events of AhLEA s (Fig. 3 ). In this study, five pairs of tandem duplication and 93 pairs of segmental duplication were identified (Fig. 3 , Additional file 1: Table S2). Five tandem duplication pairs (AH12G24920.1: AH12G24910.1, AH12G24930.1: AH12G24910.1, AH15G25110.1: AH15G24880.1, AH15G25120.1: AH15G24880.1, AH15G25120.1: AH15G25110.1) belong to the LEA5 s and LEA2 s, and located on chromosomes 12 and 15. The segmental duplication genes were mainly distributed on chromosome 12. All members of the LEA1 s, SMP s, and PvLEA18 s were segmental duplication genes, followed by LEA2 s (79.5 %) and LEA3 s (71.4 %). The Ka/Ks values of all the tandem duplication gene pairs were less than 1. Except for four segmental duplication gene pairs whose Ka/Ks values could not be calculated, the Ka/Ks values of the most segmental duplication gene pairs were less than 1, and only two pairs (2.2 %) were more than 1. (Fig. 4 , Additional file 1: Table S2). The divergence time of tandem duplication events was mainly 0–10 million years ago (MYA), and 49.5 % (46/93) of segmental duplication events occurred between 0–5 MYA (Fig. 5 , Additional file 1: Table S2) To explore the evolutionary process of the peanut LEA genes, we performed synteny analysis among peanut, Arabidopsis, and soybean. AhLEA s showed a more syntenic to soybean than Arabidopsis (Fig. 6 , Additional file 1: Table S3). Thirteen orthologous pairs exhibited single gene correspondences between peanut and Arabidopsis, and five orthologous pairs exhibited single gene correspondences peanut and soybean. Five AhLEA s were associated with multiple AtLEA s, and fourteen AhLEA s were associated with GmLEA s. Additionally, there were nine cases that peanut segmental duplications that corresponded to a single Arabidopsis gene, and eleven cases that AhLEA s corresponded to a single soybean gene. Finally, some genes showed more-to-more correspondence, for example, AH02G02040.1/ AH12G02210.1/ AH04G26920.1/ AH14G31640.1-AT1G01470.1/ AT2G46140.1 and AH01G11560.1/ AH05G04840.1/ AH11G11350.1/ AH15G00880.1-Glyma05g22030.1/ Glyma17g17860.1. 2.3. Analysis of gene structure and protein motifs of LEA s in peanut To examine the structural characteristics of AhLEA s, an unrooted phylogenetic tree that combines the UTR-CDS structures and motifs were constructed based on the full lengths of the 126 peanut LEA gene sequences by using the Maximum-Likelihood method (Fig. 7 ). The majority of the AhLEA s contained zero or one intron, with 55 and 53, respectively. Sixteen genes had two introns. One gene, AH19G03360.1, contained three introns, and one gene, AH12G35940.1, contained seven introns. All the LEA1 s and Dehydrin s contained only one intron, and the main members of the LEA3 and LEA5 subfamilies had one intron. The majority of the LEA2 s had no intron. To identify the conserved protein motifs, the MEME ( http://meme-suite.org/tools/meme ) online software was used to predict putative motifs of these proteins, with a maximum number of the different motifs at 20. Motif analysis indicated that members of each subfamily had the group-specific conserved domain, and AhLEA s with closer evolutionary relationships had more similar motif numbers. MEME analysis revealed that most AhLEA s contained motif 3 and all the LEA4 s and LEA1 s had motif 13. The LEA2 s had the greatest number of motifs, which were approximately 7, while other subfamily members had 1 to 4 motifs. 2.4. Analysis of cis-acting elements in promoters of AhLEA s To investigate the cis-acting elements of AhLEA s, 2 kb upstream of the translation initiation sites of all the LEA genes were obtained from the peanut genome database. Many cis-acting regulatory elements that may be involved in the plant’s response to environmental stresses, including ABRE, WRE3, ERE, MYB recognition sites, MYC recognition sites, TC-rich repeats, STRE, and MRE, were detected (Fig. 8 ). The promoter of subfamily LEA2 contained the most cis-acting elements, followed by subfamily LEA3 , LEA5 , and LEA1 . The promoter of subfamily LEA4 , SMP , PvLEA18 , and Dehydrin contained the least elements. Among the identified cis-acting elements, ABRE (22.2 %), ERE (55.6 %), MYB recognition sites (65.9 %), and MYC recognition sites (70.6 %) cis-acting elements were over-represented. 2.5. Expression profiles of AhLEA s at different stages of embryo development To investigate the expression profiles of AhLEA s across different stages of embryo development and different tissues, the transcriptomic data of a cultivated variety ( A. hypogaea L.) in gene bank were further scrutinized ( http://peanutgr.fafu.edu.cn/Transcriptome.php ) (Fig. 9 , Additional file 1: Table S4). Not all AhLEA s were expressed at the four embryo development stages. Meanwhile, twenty-seven genes were not detected at any tested stages. Sixty-eight LEA s had different expression levels among the four stages. In the early embryo development stages, most LEA3 s were up-regulated. Among them, three LEA3 s (AH01G27080.1, AH01G27080.2, and AH11G30560.1) exhibited very high expression levels in the early stages, which showed up to 10-fold higher than those in the late stages. Nevertheless, AhLEA1 s, AhLEA4 s, and AhLEA5 s were up-regulated mainly in the late stages. Four genes including two LEA5 s (AH12G24910.1 and AH12G24920.1) and two LEA1 s (AH06G01030.1 and AH16G03650.1) exhibited very high expression levels in the late stages. Two genes of the Dehydrin s expressed at a high level in stages I, and Ⅱ, while another Dehydrin (AH17G19580.1) expressed at a high level in stages Ⅲ, and Ⅳ. The expression of most AhLEA2 s was not changed as embryo development, while the expression level one LEA2 (AH12G34850.1) in the early stages showed up to 26-fold higher than those in the later stages. As shown in Fig. 10 , the expression profiles of eight subfamilies, including LEA1 s, LEA2 s, LEA3 s, LEA4 s LEA5 s, SMP s, PvLEA18 s, and Dehydrin s, were similar in roots, stems, leaves, and flowers. Among them, the members of LEA2 s, LEA3 s, and Dehydrin s were expressed at a high level in all four tissues. Twenty-four LEA s were highly expressed in roots, 21 in stems, 15 in leaves, and 20 in flowers. Two Dehydrin s (AH07G18700.1 and AH17G19710.1) and two LEA3 s (AH16G06810.1 and AH06G03960.1) had the highest expression levels in the stem (Additional file 1: Table S5). 2.6. Expression profiles of AhLEA s in response to drought and low-temperature stresses. To investigate the transcriptional changes of the AhLEA s under cold and drought stresses, the expression profiles of these genes were examined by using transcriptomic data (Fig. 11 ). Under drought treatment, 28.6 % (36 out of 126) of the AhLEA s were more than 2-fold up-regulated compared with the control, while the expression levels of 21.4 % (27 out of 126) genes were down-regulated more than 2-fold. Among the 27 genes that down-regulated more than 2-fold, 24 genes belonged to the LEA2 subfamily. Two LEA3 s (AH01G27080.1, and AH01G27080.2) showed the highest expression levels under drought stress (Additional file 1: Table S6). Under low-temperature treatment, 28.6 % (36 out of 126) of the AhLEA s were more than 2-fold up-regulated compared with the control, while the expression levels of 14.3 % (18 out of 126) genes were down-regulated more than 2-fold. It was found that 21 genes of LEA2 s were up-regulated and 11 genes were down-regulated. It is noteworthy that all Dehydrin s were up-regulated under drought and low-temperature stresses. Interestingly, the genes expressed the highest under low-temperature stress were also two LEA3 subfamily genes (AH16G06810.1, AH06G03960.1). Besides, 60 % of LEA5 s genes were not detected under drought and low-temperature (Fig. 11 : Additional file 1: Table S6). 2.7. Expression profiles of AhLEA genes in response to hormone To understand the expression changes of the AhLEA s under drought and low-temperature stresses, the responses of 126 AhLEA s to four stress-related hormones (abscisic acid, brassinolide, ethylene, and salicylic acid) were investigated (Fig. 12 ). The expression profiles of these genes were examined by using transcriptomic data. After ABA treatment, 8 LEA s were induced more than 2-fold, while 19 LEA s were down-regulated more than 2-fold. After brassinolide treatment, 5 genes were up-regulated more than 2-fold, while and 31 genes were down-regulated more than 2-fold. The expression of 13 AhLEA s was up-regulated more than 2-fold after ethylene treatment, while 28 genes were down-regulated more than 2-fold. The expression of 10 AhLEA s was up-regulated more than 2-fold after salicylic acid treatment, while 16 genes were down-regulated more than 2-fold. Although the main AhLEA s were down-regulated by these four hormones, half of the LEA3 s (7 out of 14) were up-regulated more than 2-fold after ethylene treatment, all members of LEA4 s were induced by salicylic acid. Moreover, the expression of five AhLEA s was up-/down-regulated more than 2-fold by all four tested hormones. These genes included four LEA2 s (AH06G19190.1, AH16G23780.1, AH20G34490.1, and AH16G06810.1) which were down-regulated after hormone treatment and a PvLEA18 (AH11G11350.1) that was up-regulated (Additional file 1: Table S7). 2.8. Expression pattern of AhLEA s under Al stress To gain a broader understanding of the putative functions of peanut LEA s in response to Al stress, the expression profiles of these genes were examined by using the RNA-Seq data which was generated from the root tips of two peanut cultivars that exhibited different Al sensitivity and had already been deposited in NCBI (Xiao et al., 2021 ). ZH2 is known as an Al sensitive peanut cultivar and 99-1507 is proved as an Al tolerant peanut cultivar (Zhan et al., 2008). Here, a total of 50 AhLEA s were found to be aluminum stress-responsive genes (Fig. 13 , Additional file 1: Table S8). LEA2 s which included twenty-three DEGs had the most aluminum stress-responsive genes. All of the members in LEA4 s and Dehydrin s were aluminum stress-responsive genes, and both of these two subfamilies were composed of three genes. The aluminum stress-responsive genes accounted for 75 % (3 out of 4), 60 % (6 out of 10), 50 % (3 out of 6, and 4 out of 8), and 35.7 % (5 out of 14) of the members in PvLEA18 s, LEA5 s, SMP s (and LEA1 s), and LEA3 s, respectively. Five AhLEA s were significantly up-regulated after 8h of Al treatment in ZH2. Seventeen AhLEA s were significantly up-regulated after 8h of Al treatment in 99-1507, including 66 % of LEA4 s and Dehydrin s (2 out of 3) and 50 % of SMP s (3 out of 6). Nineteen AhLEA s were significantly up-regulated after 24 h of Al treatment in ZH2, including all members of LEA5 s and PvLEA18 s and half of LEA1 s (4 out of 8) and SMP s (3 out of 6). Nine AhLEA s were significantly up-regulated after 24 h of Al treatment in 99-1507. Twelve AhLEA s were down-regulated after 8h of Al treatment in ZH2, and all PvLEA18 s were down-regulated. Two AhLEA s were down-regulated after 8h of Al treatment in 99-1507. Seven AhLEA s were down-regulated after 24h of Al treatment in ZH2. Eight AhLEA s were down-regulated after 24h of Al treatment in 99-1507. Furthermore, we compared the DEGs in Al stress and the genes that were up-/down-regulated more than 2-fold under drought and low-temperature stresses. As shown in Fig S1, a total of 100 AhLEA s were regulated under drought, low temperature, and Al stress. Among these genes, 35 common AhLEA s were involved in the responses to low-temperature and drought stresses, 29 common AhLEA s that were involved in the responses to drought and Al stresses, and 22 common AhLEA s that were involved in the responses to low-temperature and Al stresses. Sixteen AhLEA s were overlaps among the three abiotic stresses (Additional file 2: Fig S1). Discussion 3.1. Identification of the LEA gene family In this study, 126 LEA genes were identified from whole peanut genome sequences. Based on the phylogenetic relationship with Arabidopsis, these 126 AhLEA s were distributed to eight groups. The number of peanut LEA s was twice that in Arabidopsis. According to the studies of the LEA family in other species, the number of LEA s may be related to the polyploidy of plants (Ibrahime et al., 2019 ). For example, many LEA genes were found in polyploids of upland cotton (Fang and Magwanga, 2018), Triticum aestivum (Shi et al., 2020 ), and Brassica napus (Liu et al., 2019 ). The LEA2 s had more members than other subfamilies in the majority of species so far studied (Ibrahime et al., 2019 ). The LEA2 s were the largest LEA subfamily in Citrus sinensis, Oryza sativa, Populus trichocarpa (Pedrosa et al., 2015 ), and upland cotton (Fang and Magwanga, 2018). Similarly, the AhLEA s mainly belong to the LEA2 s, which accounted for 61.9 % of the LEA genes. However, the LEA2 s were not found as a large subfamily in the previous works in Arabidopsis (Hundertmark and Hincha, 2008 ), Oryza sativa (Wang et al., 2007 ), and Populus trichocarpa (Lan et al., 2013 ). This result can be partly explained by the fact that improved annotation of higher plant genomes can be found on phytochrome (v10.2), and LEA2 is an unusual component of "a typical" LEA proteins because they are more hydrophobic. In addition, there were three pairs of tandem duplication in AhLEA s, which belong to the LEA2 s. This result supported the view that tandem duplications have contributed significantly to the expansion and diversity of the LEA2 s in most species (Artur et al., 2019 ). 3.2. Analysis of conserved domains and introns revealed that LEA s might be stress-response genes Motif analysis of the AhLEA s showed that members of each LEA group contained specific conserved motifs. Most members of the same subfamily have similar motifs, indicating an important role of these conserved motifs in the evolution of the LEA gene family. Big differences were found in the structure of different clades. For example, LEA1 s contained motifs 13 and 17, whereas LEA5 s contained motif 2 and 9, which indicated the complexity and group-specific of LEA protein function. The conserved motifs observed in each LEA group suggested that genes with the same motif might be amplified from genes within the same evolutionary clade or group. It has been reported that stress-responding genes usually contain fewer introns (Fang and Magwanga, 2018; Xie et al., 2015 ). Introns have harmful effects on gene expression by delaying transcription products (Lane et al., 2010). Moreover, introns can extend the length of new transcripts, resulting in additional energy consumption for transcription (Jeffares et al., 2008 ). Up to 85.7 % of the AhLEA s had zero or only one intron, which further suggested that AhLEA s were stress-related genes. 3.3. Segmental duplication plays an important role in the evolution and amplification of AhLEA s Gene duplication plays an important role in the evolution and amplification of gene families (Flagel and Wendel, 2009 ). In this study, 93 pairs of segmental duplication and 5 pairs of tandem duplication were identified, it could be inferred that segmental duplication and tandem duplication contribute to the common expansion of the AhLEA s family, but the former played a predominant role. This finding was similar to previous studies on Brassica napus and upland cotton ( Gossypium hirsutum ) (Liu et al., 2019 ; Fang and Magwanga, 2018) and consistent with our previous study on receptor-like protein kinase (RLK) in peanut (Wang et al., 2021 ). According to Ka/Ks estimation, 94.9 % of the duplication gene pairs of AhLEA s were less than 1, indicating the results of the purification selection. The Ka/Ks ratios of two gene pairs (AH01G27080.1 and AH11G30560.1, AH05G16640.1 and AH15G06250.1) were more than 1, which indicated that these genes were in a state of positive selection in peanuts. We calculated the divergence time, and the results showed that many duplication events appeared to have occurred during relatively recent key periods. For example, all tandem duplication events occurred at 0–10 MYA, and 49.5 % of segmental duplication occurred at 0–5 MYA. These results indicated that many AhLEA s were produced by the recent gene duplication events in peanuts. This may be related to the origin of cultivated peanut, through a single and recent polyploidization event, and then continuous selection in breeding work, forming a highly conserved genome (Cunha et al., 2008 ). The closer the species are, the greater the genome coverage of synteny fragments and the more genes they contain (Ye et al., 2020 ). Syntenic analysis showed that more homologous gene pairs were found between peanut and soybean. There were 13 single peanut-to-Arabidopsis LEA gene correspondences and 5 single peanut-to-soybean LEA gene correspondences. These results indicated that these genes come from a common ancestor. Among these genes, two soybean genes (Glyma11g02290.1 and Glyma09g30400.1) did not belong to the LEA family. The rest genes showed one-to-more, more-to-one and more-to-more correspondence, and most of the genes included in these cases appeared more than once. However, 15 of the 23 orthologs of AhLEA s in soybean (Glyma09g38980.1, Glyma19g37350.1, Glyma08g22050.1, Glyma12g09590.1, Glyma12g32090.1, Glyma13g38380.1, Glyma03g34670.1, Glyma10g07360.1, Glyma13g21240.1, Glyma19g37340.1, Glyma06g01170.1, Glyma07g06960.1, Glyma13g43610.1, Glyma09g30400.1, and Glyma20g35880.1) were not LEA genes, which implied that their genomes underwent multiple rounds of chromosomal rearrangement and fusions. Allotetraploid-cultivated peanut composed of A and B genomes and was generated from diploid A. duranesis (AA) and A. ipaensis (BB) (Zhuang et al., 2019 ). Taking into account the divergence time of the duplications, we inferred that the divergence of many AhLEA s duplications occurred after the divergence of peanut and Arabidopsis/soybean from their last common ancestor. Combined with the results of phylogenetic tree analysis, there were nine orthologs including nineteen peanut LEA genes (AH12G35940.1-AT2G36640.1, AH17G19580.1-AT2G21490.1, AH12G35940.1-AT3G22500.1, AH02G22690.1/ AH12G24910.1-AT3G51810.1, AH05G04840.1/ AH15G00880.1-AT2G23110.1, AH04G10170.1/ AH14G12410.1-AT4G15910.1, AH06G03960.1/ AH12G32330.1/ AH12G37270.1/ AH16G06810.1-AT1G02820.1/ AT4G02380.3, AH02G06810.1/ AH12G08270.2-AT1G03120.1/ AT3G22490.1, AH02G02040.1/ AH04G26920.1/ AH12G02210.1/ AH14G31640.1-AT2G46140.1/ AT1G01470.1) that could be clustered together in the phylogenetic tree and were also contained in the syntenic map. We speculate that the functions of these AhLEA s are more similar to their Arabidopsis homologs than the other AhLEA s in the phylogenetic tree and syntenic map. 3.4. MYB and MYC recognition sites may be involved in the response of AhLEA s to abiotic stress Many studies have shown that LEA s play an important role in abiotic stress. In this study, many cis-acting elements related to abiotic stress and plant hormones were identified, such as ABRE, ERE, MYB recognition sites, MYC recognition sites, and STRE. We found that the MYB and MYC recognition sites were presented in the most promoters of the AhLEA s. It is reported that MYBs and MYCs are transcription factors that participate in ABA-dependent signaling pathways to cope with abiotic stresses such as drought, salt, and low-temperature (Li et al., 2015 ; Boter, 2014). Consistently, the LEA s that contain MYB recognition sites and MYC recognition sites, including AH16G06810.1, and AH06G03960.1, were induced under ABA, salicylic acid, drought, and low-temperature stress. Besides, most of the LEA1 s, LEA5 s, SMP s, and Dehydrin s were highly expressed under aluminum stress, and these genes contained a large number of MYB and MYC recognition sites. Therefore, we speculated that the up-regulation of LEA s expression under aluminum stress might be regulated by MYB and MYC transcription factors. This provides a theoretical basis for further exploring the response regulation mechanism of LEA s containing cis-acting elements of MYB and MYC recognition sites under stress. In this study, many LEA s responses to abiotic stresses were found to be regulated by ABA. However, only five AhLEA s (AH03G11350.1, AH03G03840.1, AH02G02050.1, AH01G27080.1, AH01G27080.2) contained ABRE cis-acting elements. The promoter region of many AhLEA s had recognition sites for MYB and MYC. These results suggested that AhLEA s responded to abiotic stresses such as drought, low-temperature, and Al stress might be direct or indirect activated ABA-dependent signaling pathways. 3.5. Expression analysis revealed AhLEA s respond to different abiotic stresses It can obtain clues from gene expression patterns to explore the function of genes (Chen et al., 2019 ). We investigated the expression level of AhLEA s in different tissues, at different embryo development stages, under different abiotic stresses (drought, low-temperature, and Al treatment), and after different hormone treatments. In four different embryo development stages, there were sixty-eight differentially expressed genes. Consistent with previous studies (Liang et al., 2016 ) that LEA s were up-regulated as the embryo developed, most of the AhLEA s were expressed at a high level at stages Ⅲ, and Ⅳ. However, the majority of LEA3 s were highly expressed at an early stage, suggesting the potential roles of LEA3 s in the early embryo development stage. As shown in Fig. 9 , subfamily LEA2 was the biggest subfamily, but the expression levels of most LEA2 s at four embryo development stages were stable, suggesting that LEA2 s might not play important roles during embryo development. The expression level of most AhLEA s in the root, stem, leaf, and flower tissues was similar. The expression level of many AhLEA s was low, while there were still several genes of subfamily LEA2 , LEA3 , and Dehydrin that exhibited a high expression level in the four tissues. Two LEA3 s (AH16G06810.1, AH06G03960.1) were very highly expressed in different peanut tissues (Fig. 10 ). It was reported that the LEA3 s play an important role in plant growth, development, and response to abiotic stresses (Yu et al., 2019 ; Koubaa and Brini 2020 ), and these two genes might be suitable candidates to understand the role of LEA3 s in peanut. Under drought stress, 50 % of the AhLEA s were up-/down-regulated for more than 2-fold compared with control. Among them, LEA2 s contributed most genes, containing 10 up-regulated genes and 24 down-regulated genes. This is consistent with the fact that LEA2 s were the largest subfamily in peanuts. Among the genes that were down-regulated for more than 2-fold, most of them were LEA2 s. Additionally, four AhLEA1 s and three AhLEA3 s were induced more than 60-fold by drought stress, implying their potential roles in enhancing drought stress tolerance in peanuts. Under low-temperature stress, 36 AhLEA s were up-regulated more than 2-fold, while 18 genes were down-regulated more than 2-fold. LEA2 s also contributed to most genes. Twenty-one AhLEA2 s were up-regulated and eleven genes were down-regulated. Interestingly, the LEA2 s that down-regulated under drought stress was also down-regulated under low-temperature stress, which suggested that there was a common mechanism to regulate LEA2 s expression. Seventeen genes were up-regulated after 8h of Al treatment in 99-1507, and two of their (AH16G20700.1 and AH06G16990.1) were also up-regulated after 24h of Al treatment. In ZH2, only five AhLEA s were up-regulated after 8h of Al treatment, while sixteen AhLEA s were up-regulated after 24h of Al treatment. Interestingly, three SMP s (AH12G08270.1, AH12G08270.2, and AH02G06810.1) were up-regulated after 8h of Al treatment in both cultivars, suggesting that these genes might play important roles in Al tolerance in peanuts. Together, the Al-tolerant cultivar 99-1507 exhibited a rapid response to Al treatment, and the LEA s that induced rapidly should be studied in future work. As shown in Fig. S1, the majority of the 126 LEA s were induced under at least one stress condition. Sixty-three (50 %), fifty-four (42.9 %), and fifty (39.7 %) the AhLEA s were induced by drought, low-temperature, and Al stresses, respectively. Among these genes, sixteen were induced only under drought stress, fourteen were induced only under low-temperature, and sixteen were induced only under Al stress (Fig S1). These results implied that these genes play distinct roles in response to different abiotic stresses in peanuts. Some AhLEA s were regulated by different stress conditions. Three genes including two LEA5 s (AH12G24910.1 and AH12G24920.1) and one LEA1 (AH19G11740.1) were up-regulated greatly under both drought and Al stresses (Additional file 1: Table S6, Table S 8). The expression of LEA1 (AH19G11740.1) was induced more than 2-fold by ABA treatment. Two LEA3 s (AH01G27080.1 and AH11G30560.1) and one LEA4 (AH12G35940.1) were down-regulated under drought, low-temperature, and Al stresses. The expression of that two LEA3 s was significantly induced by ethylene, while LEA4 (AH12G35940.1) was down-regulated by ABA treatment. Two LEA2 s (AH02G02040.1 and AH12G02210.1) were up-regulated under drought, low-temperature, and Al stresses, and they were also up-regulated by ABA. Many AhLEA s that were regulated more than 2-fold by hormones such as abscisic acid, brassinolide, ethylene, and salicylic acid were found to be down-regulated. As revealed by table S7, these down-regulated genes showed no obvious subfamily preference. However, AhLEA s that were up-regulated more than 2-fold by ethylene and salicylic acid showed obvious subfamily preference. Seven AhLEA3 s were induced by ethylene. Five AhLEA3 s induced by ethylene were also involved in response to drought and low-temperature stresses. The expression level of AH12G37280.1 was increased up to 8.45-fold under low-temperature stress. AH12G32330.1 was up-regulated 3.5-fold under drought stress. Moreover, three AhLEA3 s (AH01G27080.1, AH01G27080.2, AH11G30560.1) were up-regulated greatly under both drought and low-temperature stresses. These results revealed the important roles of the AhLEA3 subfamily in the ethylene-mediated response under drought and low-temperature stresses. Additionally, all AhLEA4 s were induced by salicylic acid, and all AhLEA4 s were also regulated greatly under drought and low-temperature stresses. Among them, two genes (AH06G16990.1 and AH12G35940.1) were induced more than 6-fold under drought and low-temperature stresses, and one gene (AH16G20700.1) was down-regulated 3.5-fold under low-temperature stress, which implied that subfamily AhLEA4 played important roles in SA-mediated response under drought and low-temperature stresses in peanut. Taken together, these results suggested that common mechanisms might be initiated in peanuts to cope with different abiotic stresses. Hormones were involved in regulating LEA ’s expression under abiotic stresses. The role of hormones in regulating gene expression had a preference among AhLEA gene families. Conclusions In this study, 126 LEA genes in Arachis hypogaea were identified. They were divided into eight groups according to homologous in Arabidopsis thaliana . AhLEA s are randomly distributed on the chromosome, and most of them may be segmental duplication. The exon-intron and motif structures indicated that the LEA s’ family functions are highly conserved. Some cis-elements of abiotic stress response were also found in the upstream sequences of most AhLEA s. The comprehensive analysis of AhLEA s gene expression profiles showed that the LEA3 s, LEA4 s, and SMP s played an important role in abiotic stress response, and also showed the functional differences among other subfamilies. This study provided a reference for further exploring the mechanism of LEA s in response to abiotic stress in peanuts. Materials And Methods 5.1. Identification of LEA s in peanut To identify the AhLEA s, we used 51 LEA genes (Hundertmark et al., 2008) in Arabidopsis thaliana acquire Pfam ID (PF03760, PF03168, PF03242, PF02987, PF00477, PF10714, PF04927, PF00257) and InterPro ID (IPR005513, IPR004864, /IPR013990, IPR004926, IPR004238, IPR000389, IPR018930, IPR007011, IPR000167) from Peanut Base ( https://www.peanutbase.org/ ). By acquiring LEA peanut protein sequences based on InterPro ID search of Peanut Genome Resource (PGR) ( http://peanutgr.fafu.edu.cn/ ). NCBI’s Conserved Domains Database ( https://www.ncbi.nlm.nih.gov/cdd ) and PFAM ( http://pfam.xfam.org/ ) database were used to verify the presence of the LEA domains and finally obtained 126 AhLEA s. 5.2. Phylogenetic relationships, gene structures, conserved motifs, and chromosomal locations of the AhLEA s The phylogenetic tree was constructed by the maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0 software (Sudhir et al., 2016 ). Multiple Expectation Maximization for Motif Elicitation (MEME) ( http://meme-suite.org/tools/meme ) (Bailey et al., 2009 ) was used to identify the conserved protein motifs, with a maximum number of the different motif at 20. The exon-intron structures were identified using the TBtools software (Chen et al., 2020 ). The physical location of each AhLEA is determined by identifying the starting position of all genes on each chromosome, searching the local database of Peanut Genome Resources by BLAST. Using TBtools of Gene location visualize from GFF/GFF3 to draw chromosome mapping and tandem duplication pairs. 5.3. Promoter cis-element analysis Genomic data were obtained from Peanut Genome Resource (PGR) ( http://peanutgr.fafu.edu.cn/ ), and TBtools software was used to extract all LEA upstream 2kd promoter sequences. Transcriptional response elements of LEA gene promoters were predicted using the PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) (Higo et al., 1999 ). 5.4. Gene duplication and evolutionary analysis We used Virtual Machine to construct the tandem and segmental of the putative duplication of the AhLEA s and calculate the ratio of the nonsynonymous substitution rate (Ka) to the synonymous substitution rate (Ks) by the Simple Ka/Ks calculator (NG) of TBtools (Chen et al., 2020 ). LEA s clustered together within 100 kb, length of the alignable sequence covers > 75 % of longer gene and similarity of aligned regions > 75 % were regarded as tandem duplicated genes. The relationship between Ka/Ks ratio and value 1, Ka larger than Ks (or Ka/Ks > > 1), Ka equals Ks (Ka/Ks = 1), and Ka less than Ks (or Ka/Ks < < 1), which represent positive (or diversifying) selection, neutral evolution and negative (or purifying) selection, respectively. Divergence time was calculated with the formula T = Ks/2r, where r is 1.5 ×10 − 8 synonymous substitutions per site per year and it is the rate of divergence for nuclear genes from plants (Koch et al., 2000 ). We used Multiple Synteny Plot software (Chen et al., 2020 ) to explore the collinear relationship between the AhLEA and LEA genes from Arabidopsis thaliana and Glycine max . All the soybean LEA domain-containing protein sequences were downloaded from the Soybase Glyma.Wm82.a2.v1 ( http://www.soybase.org/ ). The NCBI’s Conserved Domains Database ( https://www.ncbi.nlm.nih.gov/cdd ) and PFAM ( http://pfam.xfam.org/ ) database were used to verify the presence of the LEA domains. The GmLEA s that were identified in the previous study were also screened (Li et al., 2011 ). After eliminating the invalid sequence, a total of 132 GmLEA s were identified. 5.5. Expression analysis of AhLEA s The blast was performed in the transcriptome of the PGR database using the protein sequences of 126 AhLEA s. RNA-Seq data were downloaded from PGR and used to generate the expression patterns of AhLEA s in different tissues (root, stem, leaf, and flower), different embryo development stages, and various abiotic stresses (cold, and drought), and different hormones treatment on leaves. Transcriptome data that were generated from peanut root tips under Al stress were used to generate the expression patterns of AhLEA s under Al stress. The data had been deposited in the database of the National Center for Biotechnology Information (NCBI) under accession number PRJNA525247 ( https://www.ncbi.nlm.nih.gov/sra/PRJNA525247 ). TBtools were used to generate heat maps and combine phylogenetic tree, gene, and protein structure (Chen et al., 2020 ). Abbreviations Al: Aluminum At : Arabidopsis thaliana Ah : Arachis hypogaea . L ABRE: ABA-responsive element ERE: Ethylene response element WRE3: Water response element MYB: Transcription factor MYC: Transcription factor TC-rich repeats: Cis-acting element involved in defense and stress responsiveness MRE: Metal responsive element STRE: Stress response element DEGs: Differentially expressed genes Declarations Ethics approval and consent to participate Not applicable. Consent for publication Not applicable. Competing interests: All authors declare no conflicting interest. Funding: This work was supported by the National Natural Science Foundation of China (Grant No. 31701356, 32060419) and College Students’ Innovative Entrepreneurial Training Plan Program (201910593083). Apart from providing financial support, funding bodies were not involved in the study design, data analyses, and interpretation of results or manuscript preparation. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. Authors’ contribution: RLH: Writing Original Draft, Data analysis, Conceptualization; XW and YS: Data analysis; LFH, AQW, and JZ: manuscript review; DX: Conceptualization, supervision, Writing—Review & Editing. All authors read and approved the final manuscript. Acknowledgments: We thank Mr Yun Xiong Zhao and Miss Xu Fang for providing the software needed for drawing and data collection. Availability of data and materials: The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. The following are all databases in this study and are open. Peanut Base ( https://www.peanutbase.org/ ) Peanut Genome Resource (PGR) ( http://peanutgr.fafu.edu.cn/ ) Transcriptome of Peanut Genome Resource (PGR) ( http://peanutgr.fafu.edu.cn/Transcriptome.php ) NCBI’s Conserved Domains Database ( https://www.ncbi.nlm.nih.gov/cdd ) PFAM ( http://pfam.xfam.org/ ) Expectation Maximization for Motif Elicitation (MEME) ( http://meme-suite.org/tools/meme ) PlantCARE database ( http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ ) Soybase Glyma.Wm82.a2.v1 ( http://www.soybase.org/ ) PRJNA525247 ( https://www.ncbi.nlm.nih.gov/sra/PRJNA525247 ) References Adrien C., Gal P., Martine N. et al. The Ubiquitous Distribution of Late Embryogenesis Abundant Proteins across Cell Compartments in Arabidopsis Offers Tailored Protection against Abiotic Stress. [J]. The Plant Cell, 2014(7): 7. DOI: 10.1105/tpc.114.127316 Artur, M. A. S., Zhao T., Ligterink W. et al. 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DOI: 10.1007/s00284-009-9552-2 Ye J., Yang X., Hu G. et al. Genome-Wide Investigation of Heat Shock Transcription Factor Family in Wheat (Triticum aestivum L.) and Possible Roles in Anther Development. [J]. International Journal of Molecular Sciences, 2020, 21(2). DOI: 10.3390/ijms21020608 Zamora-Briseño, J. A. and de Jiménez E. S. A LEA 4 protein up-regulated by ABA is involved in drought response in maize roots. [J]. Molecular Biology Reports, 2016. 43(4): 221–228. DOI: 10.1007/s11033-016-3963-5 Zegzouti H., Jones B., Marty C. et al. Er5, a tomato cDNA encoding an ethylene-responsive LEA-like protein: characterization and expression in response to drought, ABA and wounding. [J]. Plant Molecular Biology, 1997, 35(6): 847–854. DOI: 10.1023/A:1005860302313 Zhan J., Kou R. J., He L. F. et al. Effects of aluminum on morphological structure of peanut root tips. [J]. Chinese Journal of Oil Crop Sciences, 2008(01): 79–83. DOI: 10.3724/SP.J.1011.2008.00534 Zhuang W. J., Chen H., Yang M. et al. The genome of cultivated peanut provides insight into legume karyotypes, polyploid evolution, and crop domestication. [J]. Nature Genetics, 2019, 51(5): 865–876. DOI: 10.1038/s41588-019-0402-2 Additional Declarations No competing interests reported. Supplementary Files Additionalfile1.xlsx Additionalfile2.docx Cite Share Download PDF Status: Published Journal Publication published 30 Mar, 2022 Read the published version in BMC Plant Biology → Version 1 posted Editorial decision: Major revision 23 Sep, 2021 Reviews received at journal 13 Sep, 2021 Reviewers agreed at journal 27 Aug, 2021 Reviewers invited by journal 27 Aug, 2021 Editor assigned by journal 26 Aug, 2021 Editor invited by journal 26 Aug, 2021 Submission checks completed at journal 26 Aug, 2021 First submitted to journal 02 Aug, 2021 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-775523","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":48422626,"identity":"b3b92fec-b47f-41fe-9aca-f9788fd2af5c","order_by":0,"name":"RuoLan Huang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"RuoLan","middleName":"","lastName":"Huang","suffix":""},{"id":48422628,"identity":"ea3a1f72-2409-45fb-82d7-ca491972f276","order_by":1,"name":"Dong Xiao","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA20lEQVRIiWNgGAWjYDACZiDmAWJ+BJdYLZINRGthgGoxOECsFoPjzA8fvKm4Y7f5/BozCYYK68QG9rMH8GqRbGYzNpxz5lnythvP0iQYzqQnNvDkJeDVws/MYCbN23Y42ezG4WMSjG2HExskeAzwamFjZv8G1mI842CbBOM/IrTwM/OAbbEz4G8G2tJAhBbJZp5ioF8OJ0jcYEu2SDiWbtzGk4Nfi8H54xuBIXbYnr//jOGNDzXWsv3sZ/BrgQGgexIYGBJAviNKPRDYM/AfIFbtKBgFo2AUjDQAAMrAQkpaWQYrAAAAAElFTkSuQmCC","orcid":"","institution":"Guangxi University","correspondingAuthor":true,"prefix":"","firstName":"Dong","middleName":"","lastName":"Xiao","suffix":""},{"id":48422631,"identity":"9f84f986-1a22-4022-9d2d-baacb9dc7200","order_by":2,"name":"Xin Wang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Xin","middleName":"","lastName":"Wang","suffix":""},{"id":48422636,"identity":"77bf2987-2a89-4f0a-bbf9-10a3bafb772b","order_by":3,"name":"Yi Shen","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Yi","middleName":"","lastName":"Shen","suffix":""},{"id":48422640,"identity":"ac537c31-4fb3-4537-b6ab-ca729cc1b6db","order_by":4,"name":"Jie Zhan","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"Jie","middleName":"","lastName":"Zhan","suffix":""},{"id":48422642,"identity":"4727959f-6268-478b-80d9-3fb495bb4abb","order_by":5,"name":"AiQing Wang","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"AiQing","middleName":"","lastName":"Wang","suffix":""},{"id":48422645,"identity":"d87ccf2f-7db4-45e6-9a44-62a70c895658","order_by":6,"name":"LongFei He","email":"","orcid":"","institution":"Guangxi University","correspondingAuthor":false,"prefix":"","firstName":"LongFei","middleName":"","lastName":"He","suffix":""}],"badges":[],"createdAt":"2021-08-02 09:29:07","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-775523/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-775523/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s12870-022-03462-7","type":"published","date":"2022-03-30T09:04:08+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":12945239,"identity":"e416eebb-8a17-4f1e-9a03-e7f3bda00000","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":1234649,"visible":true,"origin":"","legend":"Phylogenetic relationships of the AhLEAs and AtLEAs. The Maximum Likelihood (ML) tree was generated using MEGA7 with 1000 bootstrap replicates. LEA gene families are distinguished by different colors.","description":"","filename":"fig1.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/eb315628bcd0770be2bf100d.png"},{"id":12945245,"identity":"117e6070-2a53-45fe-825f-e9252224eb88","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":168523,"visible":true,"origin":"","legend":"Chromosome distributions of the AhLEAs and gene duplication events. Distribution of 126 genes on chromosomes of peanut, the blue words represent pairs of tandem duplication genes.","description":"","filename":"fig2.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/0db2e65752be26e7754c5635.png"},{"id":12945331,"identity":"e1e9f71d-7d7d-4859-a1f3-3aa3e768b19e","added_by":"auto","created_at":"2021-08-31 18:19:45","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":934803,"visible":true,"origin":"","legend":"Duplication analysis of 126 AhLEAs. The rectangle on the outer ring represents peanut chromosome 00-20. The purple line on chromosomes 12, 15 represents tandem duplication gene pairs, and light orange lines on chromosomes represent segmental duplication gene pairs.","description":"","filename":"fig3.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/2a0e6a45576736cb8afddf5a.png"},{"id":12945330,"identity":"316807b9-fa7d-4b59-8f39-3b5bdbfc0996","added_by":"auto","created_at":"2021-08-31 18:19:45","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":33435,"visible":true,"origin":"","legend":"The distribution of Ka/Ks values in all tandem and segmental duplicated AhLEAs.","description":"","filename":"fig4.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/118889b996909c53dc8a9bd3.png"},{"id":12945238,"identity":"268640a5-6056-4f74-aac4-0255782ca654","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":22649,"visible":true,"origin":"","legend":"The distribution of divergence time (MYA) in all tandem and segmental duplicated AhLEAs.","description":"","filename":"fig5.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/61a2e7345cc78561cb538fc8.png"},{"id":12945332,"identity":"440cf45f-6323-406e-9c60-d2eeaab4d330","added_by":"auto","created_at":"2021-08-31 18:19:45","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":455923,"visible":true,"origin":"","legend":"Synteny analyses of AhLEAs to Arabidopsis and G. max. Gray lines in the background indicate collinear blocks within peanut and Arabidopsis, soybean genomes, while blue lines highlight syntenic LEA gene pairs, Red chromosome blocks represent tandem duplicated genes.","description":"","filename":"fig6.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/d0ec4c1b40028533eb4d4804.png"},{"id":12945250,"identity":"e756bad0-2b2c-49f8-93cd-ef34ffdee3c4","added_by":"auto","created_at":"2021-08-31 18:16:46","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":10325095,"visible":true,"origin":"","legend":"Phylogenetic relationships, gene structures, and compositions of the conserved protein motifs of the AhLEAs. Ⅰ: LEA2; Ⅱ: Dehydrin; Ⅲ: LEA3; Ⅳ: SMP; Ⅴ: LEA5; Ⅵ: PvLEA18; Ⅶ: LEA4; Ⅷ: LEA1; a: Phylogenetic relationships, b: conversed motif, c: UTR–CDS organization, black lines represent intron.","description":"","filename":"fig7.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/30a3abd1638eb7577ce52047.png"},{"id":12945240,"identity":"cba6fa88-ff5c-4285-9aef-902162a5ea70","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":48539,"visible":true,"origin":"","legend":"Distribution of major abiotic stress-responsive cis-elements in the promoter sequences of the 126 AhLEAs.","description":"","filename":"fig8.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/c923087a3871b2f382b135e7.png"},{"id":12945333,"identity":"9b3b2c46-65e9-4ecd-b8fc-0078ec4d82db","added_by":"auto","created_at":"2021-08-31 18:19:45","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":963461,"visible":true,"origin":"","legend":"A heatmap showing the hierarchical clustering of the expression levels of the 126 AhLEAs in the four embryo periods in peanut. Ⅰ: LEA2; Ⅱ: Dehydrin; Ⅲ: LEA3; Ⅳ: SMP; Ⅴ: LEA5; Ⅵ: PvLEA18; Ⅶ: LEA4; Ⅷ: LEA1.","description":"","filename":"fig9.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/4a36963fa075f3113f3e8e45.png"},{"id":12945512,"identity":"7f5fc5f4-e906-4e4d-a16f-a879106e99b8","added_by":"auto","created_at":"2021-08-31 18:22:45","extension":"png","order_by":10,"title":"Figure 10","display":"","copyAsset":false,"role":"figure","size":1875781,"visible":true,"origin":"","legend":"A heatmap showing the hierarchical clustering of the expression levels of the 126 AhLEAs in the roots, stems, leaves, and flowers of peanut. Ⅰ: LEA2; Ⅱ: Dehydrin; Ⅲ: LEA3; Ⅳ: SMP; Ⅴ: LEA5; Ⅵ: PvLEA18; Ⅶ: LEA4; Ⅷ: LEA1.","description":"","filename":"fig10.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/30a24e5f6f84b051890cd697.png"},{"id":12945247,"identity":"06a6163a-4d92-4eda-ad41-4fe1c7ce5065","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":11,"title":"Figure 11","display":"","copyAsset":false,"role":"figure","size":1706278,"visible":true,"origin":"","legend":"Expression profiles of the AhLEAs in peanut. Dynamic expression profiles of AhLEAs drought and low-temperature treatments using heatmap of hierarchical clustering. Ⅰ: LEA2; Ⅱ: Dehydrin; Ⅲ: LEA3; Ⅳ: SMP; Ⅴ: LEA5; Ⅵ: PvLEA18; Ⅶ: LEA4; Ⅷ: LEA1.","description":"","filename":"fig11.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/02e5cbfcbf423b599068dadd.png"},{"id":12945249,"identity":"143ba38b-c3bb-4ccd-99b1-5845e176d902","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":12,"title":"Figure 12","display":"","copyAsset":false,"role":"figure","size":977774,"visible":true,"origin":"","legend":"A heatmap showing the hierarchical clustering of the expression levels of the 126 AhLEAs under different hormone treatments in peanuts. Ⅰ: LEA2; Ⅱ: Dehydrin; Ⅲ: LEA3; Ⅳ: SMP; Ⅴ: LEA5; Ⅵ: PvLEA18; Ⅶ: LEA4; Ⅷ: LEA1.","description":"","filename":"fig12.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/87b8a185834ba12087054d6e.png"},{"id":12945241,"identity":"6df97457-56b1-4a4a-8024-75f40db378c1","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"png","order_by":13,"title":"Figure 13","display":"","copyAsset":false,"role":"figure","size":1008554,"visible":true,"origin":"","legend":"Expression profiles of Al-responsive AhLEAs in two varieties. The blue words represent not detected in the RNA-Seq dataset, and the orange words represent LEA genes were not expressed, the red words represent 50 differentially expressed genes. Ⅰ: LEA2; Ⅱ: Dehydrin; Ⅲ: LEA3; Ⅳ: SMP; Ⅴ: LEA5; Ⅵ: PvLEA18; Ⅶ: LEA4; Ⅷ: LEA1.","description":"","filename":"fig13.png","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/86c74d5e3b1f5e506553cf0a.png"},{"id":19766742,"identity":"d8f95df4-f9f2-4782-856f-9f0fbbefba65","added_by":"auto","created_at":"2022-03-30 09:04:17","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5831439,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/4fcea1ed-a661-41db-8256-5685db1a7ff8.pdf"},{"id":12945243,"identity":"c2ba1c0e-515b-465c-9906-c510c6563036","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"xlsx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":64424,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile1.xlsx","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/1acc779c8d31f11a2c5aa9f5.xlsx"},{"id":12945236,"identity":"86cf290f-c867-453d-a2f9-77cbd205a354","added_by":"auto","created_at":"2021-08-31 18:16:45","extension":"docx","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":38280,"visible":true,"origin":"","legend":"","description":"","filename":"Additionalfile2.docx","url":"https://assets-eu.researchsquare.com/files/rs-775523/v1/7b7de5b7ba91049b241d79df.docx"}],"financialInterests":"No competing interests reported.","formattedTitle":"\u003cp\u003eGenome-Wide Identification, Evolutionary And Expression Analyses of LEA Gene Family In Peanut (\u003cem\u003eArachis Hypogaea\u003c/em\u003e L.)\u003c/p\u003e","fulltext":[{"header":"Background","content":"\u003cp\u003ePlant in nature often encounters various abiotic stresses including drought, cold, high temperature, and salinity, which affect growth and development, reduce its yield and survival rate. Plants have evolved many mechanisms to cope with various environmental stresses. It is known that the late embryogenesis abundant (LEA) proteins play important roles in protecting cells under abiotic stresses, and many \u003cem\u003eLEA\u003c/em\u003es are induced by cold, drought, salinity, abscisic acid (ABA), and ethylene (Kentaro et al., \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e2014\u003c/span\u003e; Zegzouti et al., \u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e1997\u003c/span\u003e; Park et al., \u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e2003\u003c/span\u003e). Moreover, it has been confirmed that \u003cem\u003eAdDHN1\u003c/em\u003e, a member of the Dehydrin family, can improve the drought resistance of transgenic Arabidopsis, but it is more sensitive to nematodes (Mota et al., 2018), which indicated that some of the \u003cem\u003eLEA\u003c/em\u003es may respond to abiotic stress as well as biotic stress.\u003c/p\u003e \u003cp\u003eLEA proteins are highly hydrophilic glycine-rich proteins, which accumulate largely in the later stage of seed maturation and fade away following germination (Battaglia and Covarrubias, \u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e2013\u003c/span\u003e; Lan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e). As water-binding molecules, the role of LEA proteins is enhancing the stability of protein and membrane. Subcellular localization analysis has indicated that LEA proteins are mainly located in nuclear regions and the cytoplasm (Adrien et al., 2014). LEA proteins have been observed in the roots, leaves, buds, and seedlings, although they mainly appear in seeds of plants (Shao, 2005; Hundertmark et al., 2008).\u003c/p\u003e \u003cp\u003eLEA protein families were identified in many plant species by genome-wide identification and analysis, such as \u003cem\u003eArabidopsis thaliana\u003c/em\u003e (Bies-Eth\u0026egrave;ve et al., \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003ePopulus trichocarpa\u003c/em\u003e (Lan et al., \u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e2013\u003c/span\u003e), \u003cem\u003eCamellia sinensis\u003c/em\u003e (Jin et al., \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e2019\u003c/span\u003e), \u003cem\u003eBrassica napus\u003c/em\u003e (Liang et al., \u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e2016\u003c/span\u003e), and \u003cem\u003eTriticum aestivum\u003c/em\u003e (Liu et al., \u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). During the growth and development of plants, LEA proteins are considered to play important roles. It was reported that \u003cem\u003eMedicago falcate LEA3\u003c/em\u003e conferred multiple abiotic stress tolerance by involving the protection of catalase activity (Shi et al., \u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). A heterologous expression of a barley LEA3 protein gene, \u003cem\u003eHVA1\u003c/em\u003e, improved tolerance to water stress in rice and wheat (Sivamani et al., \u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e2000\u003c/span\u003e; Xu et al., \u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e1996\u003c/span\u003e). \u003cem\u003eAtLEA5\u003c/em\u003e protects yeast cells against oxidative stress (Mowla et al., \u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). \u003cem\u003eEscherichia coli\u003c/em\u003e can grow in high salt and extreme temperature conditions due to the over-expression of soybean PM2 protein (LEA3) (Yun and Zheng, \u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e2005\u003c/span\u003e; Yun et al., \u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e2010\u003c/span\u003e). ABA can regulate the expression of many LEA proteins, and it was proved that the expression of \u003cem\u003eLEA4\u003c/em\u003e subfamily members was upregulated by exogenous ABA (Zamora-Brise\u0026ntilde;o et al., 2016).\u003c/p\u003e \u003cp\u003ePeanut is one of the main oils and cash crops all over the world. Peanut is a rainfed crop, but it is sensitive to water deficit stress in the flowering and pegging stages, which would impact the yield of peanuts (Bhogireddy et al., \u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e2020\u003c/span\u003e). Also, Al stress inhibition of growth reduces peanut yield in acid soil (Qiu et al., \u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e2019\u003c/span\u003e). To date, the function of the \u003cem\u003eLEA\u003c/em\u003e gene family in peanuts has little been reported. In this study, we identified the \u003cem\u003eLEA\u003c/em\u003es in peanuts and analyzed the structure, evolution, and chromosome location of peanut \u003cem\u003eLEA\u003c/em\u003es. Our findings provide a foundation for the evolutionary and functional characterization of \u003cem\u003eLEA\u003c/em\u003e gene families in peanut and other plant species.\u003c/p\u003e"},{"header":"Results","content":"\u003cdiv class=\"Section2\" id=\"Sec3\"\u003e\n \u003ch2\u003e2.1. Identification and characteristics of \u003cem\u003eAhLEA\u003c/em\u003e gene in peanut\u003c/h2\u003e\n \u003cp\u003eBy using the publicly available peanut genome sequence data, the genome-wide identification of \u003cem\u003eLEA\u003c/em\u003es in peanuts was based on sequence homology with 51 Arabidopsis \u003cem\u003eLEA\u003c/em\u003es (Hundertmark et al., 2008) (Table 1 and Additional file 1: Table S1). Proteins that contained a conserved LEA domain were screened by the NCBI-BLAST online tool. Eventually, 126 \u003cem\u003eAhLEA\u003c/em\u003es were identified. All of these genes were grouped with 51 \u003cem\u003eAtLEA\u003c/em\u003es by phylogenetic analyses. The \u003cem\u003eAhLEA\u003c/em\u003es were classified into eight subfamilies including \u003cem\u003eLEA1\u003c/em\u003e, \u003cem\u003eLEA2\u003c/em\u003e, \u003cem\u003eLEA3\u003c/em\u003e, \u003cem\u003eLEA4\u003c/em\u003e, \u003cem\u003eLEA5\u003c/em\u003e, \u003cem\u003ePvLEA18\u003c/em\u003e, \u003cem\u003eSMP\u003c/em\u003e, and \u003cem\u003eDehydrin\u003c/em\u003e (Fig. \u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003e). The \u003cem\u003eLEA2\u003c/em\u003e family was the largest, with 78 members. The \u003cem\u003eLEA3\u003c/em\u003es and \u003cem\u003eLEA5\u003c/em\u003es had 14 and 10 members, respectively. The \u003cem\u003eLEA1\u003c/em\u003es had 8 members, \u003cem\u003eSMP\u003c/em\u003e had 6 members and \u003cem\u003ePvLEA18\u003c/em\u003e had 4 members. The \u003cem\u003eLEA4\u003c/em\u003e and \u003cem\u003eDehydrin\u003c/em\u003e families had 3 members. The species-specific group (\u003cem\u003eAtM\u003c/em\u003e) of Arabidopsis was absent in the peanut.\u003c/p\u003e\n\u003cp\u003eTable 1 The classification of LEA proteins in \u003cem\u003eArachis hypogaea\u003c/em\u003e is based on Arabidopsis.\u003c/p\u003e\n\u003ctable border=\"1\" cellpadding=\"0\" cellspacing=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003eIn this study\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePfam ID\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003eHundertmark et al. (2008)\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003eArabidopsis\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003eA. hypogaea\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR005513\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF03760\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA 1\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e7\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e8\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR004864/IPR013990\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF03168\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA 2\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e78\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR004926\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF03242\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA 3\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e14\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR004238\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF02987\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA 4\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e18\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR000389\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF00477\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eLEA 5\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003ePvLEA18\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR018930\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF10714\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003ePvLEA18\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e4\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eSMP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR007011\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF04927\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eSMP\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e6\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eDehydrin\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003e\u0026nbsp;\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"25.59414990859232%\"\u003e\n \u003cp\u003eIPR000167\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.625228519195613%\"\u003e\n \u003cp\u003ePF00257\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"15.904936014625228%\"\u003e\n \u003cp\u003e\u003cem\u003eDehydrin\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eAtM\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"13.162705667276052%\"\u003e\n \u003cp\u003e10\u003c/p\u003e\n \u003cp\u003e2\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd valign=\"top\" width=\"14.808043875685557%\"\u003e\n \u003cp\u003e3\u003c/p\u003e\n \u003cp\u003e0\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec4\"\u003e\n \u003ch2\u003e2.2. Chromosomal locations, gene duplication and synteny analysis of the \u003cem\u003eAhLEA\u003c/em\u003es\u003c/h2\u003e\n \u003cp\u003eThe identified 126 \u003cem\u003eAhLEA\u003c/em\u003es were further located on the 20 chromosomes (Fig. \u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003e). The largest number of \u003cem\u003eAhLEA\u003c/em\u003es was found on chromosome 12, fourteen genes, followed by chromosome 14 (eleven genes). The lowest loci density was observed on chromosome 8, with only two genes. Eight genes were located on chromosomes 13 and 15, seven genes were located on chromosome 6. Five chromosomes (chr1, chr2, chr3, chr4, and chr16) carried six \u003cem\u003eAhLEA\u003c/em\u003es and four chromosomes (chr5, chr7, chr19, and chr20) carried five \u003cem\u003eAhLEA\u003c/em\u003es. Chromosomes 9, 10, and 11 contained four \u003cem\u003eAhLEA\u003c/em\u003es, and chromosomes 17, 18 contained three \u003cem\u003eAhLEA\u003c/em\u003es. The \u003cem\u003eAhLEA\u003c/em\u003es were distributed unevenly among the 20 chromosomes in peanut.\u003c/p\u003e\n \u003cp\u003eDifferent \u003cem\u003eLEA\u003c/em\u003e subfamilies were located on different chromosomes. All chromosomes contained the \u003cem\u003eLEA2\u003c/em\u003es, and all of the \u003cem\u003eLEA\u003c/em\u003es on chromosomes 3, 8, 10, 13, 18, and 20 belonged to the \u003cem\u003eLEA2\u003c/em\u003es. The \u003cem\u003eLEA1\u003c/em\u003es were distributed on chromosomes 1, 6, 9, 11, 16, and 19. The \u003cem\u003eLEA3\u003c/em\u003es were mainly distributed on chromosomes 1, 2, 4, 6, 11, 12, 14, and 16. The \u003cem\u003eLEA4\u003c/em\u003es were mainly found on chromosomes 6, 12, and 16. The \u003cem\u003eLEA5\u003c/em\u003es were distributed on chromosomes 2, 12, 14, and 16. The \u003cem\u003ePvLEA18\u003c/em\u003es were distributed on chromosomes 1, 5, 11, and 15. The \u003cem\u003eSMP\u003c/em\u003es were distributed on chromosomes 2, 6, 9, 12, and 19. The \u003cem\u003eDehydrin\u003c/em\u003es were distributed on chromosomes 7, 15, and 17. Chromosomal location analysis of \u003cem\u003eAhLEA\u003c/em\u003es indicated that eight subfamilies were distributed unevenly in the genome.\u003c/p\u003e\n \u003cp\u003eThe generation and evolution of gene families may be caused by tandem duplication and segmental duplication (Mehan et al., \u003cspan class=\"CitationRef\"\u003e2004\u003c/span\u003e; Kent et al., \u003cspan class=\"CitationRef\"\u003e2003\u003c/span\u003e). To investigate the evolutionary relationships of the \u003cem\u003eAhLEA\u003c/em\u003e gene family, we analyzed the duplication events of \u003cem\u003eAhLEA\u003c/em\u003es (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). In this study, five pairs of tandem duplication and 93 pairs of segmental duplication were identified (Fig. \u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e, Additional file 1: Table S2). Five tandem duplication pairs (AH12G24920.1: AH12G24910.1, AH12G24930.1: AH12G24910.1, AH15G25110.1: AH15G24880.1, AH15G25120.1: AH15G24880.1, AH15G25120.1: AH15G25110.1) belong to the \u003cem\u003eLEA5\u003c/em\u003es and \u003cem\u003eLEA2\u003c/em\u003es, and located on chromosomes 12 and 15. The segmental duplication genes were mainly distributed on chromosome 12. All members of the \u003cem\u003eLEA1\u003c/em\u003es, \u003cem\u003eSMP\u003c/em\u003es, and \u003cem\u003ePvLEA18\u003c/em\u003es were segmental duplication genes, followed by \u003cem\u003eLEA2\u003c/em\u003es (79.5 %) and \u003cem\u003eLEA3\u003c/em\u003es (71.4 %). The Ka/Ks values of all the tandem duplication gene pairs were less than 1. Except for four segmental duplication gene pairs whose Ka/Ks values could not be calculated, the Ka/Ks values of the most segmental duplication gene pairs were less than 1, and only two pairs (2.2 %) were more than 1. (Fig. \u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003e, Additional file 1: Table S2). The divergence time of tandem duplication events was mainly 0\u0026ndash;10 million years ago (MYA), and 49.5 % (46/93) of segmental duplication events occurred between 0\u0026ndash;5 MYA (Fig. \u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e, Additional file 1: Table S2)\u003c/p\u003e\n \u003cp\u003eTo explore the evolutionary process of the peanut \u003cem\u003eLEA\u003c/em\u003e genes, we performed synteny analysis among peanut, Arabidopsis, and soybean. \u003cem\u003eAhLEA\u003c/em\u003es showed a more syntenic to soybean than Arabidopsis (Fig. \u003cspan class=\"InternalRef\"\u003e6\u003c/span\u003e, Additional file 1: Table S3). Thirteen orthologous pairs exhibited single gene correspondences between peanut and Arabidopsis, and five orthologous pairs exhibited single gene correspondences peanut and soybean. Five \u003cem\u003eAhLEA\u003c/em\u003es were associated with multiple \u003cem\u003eAtLEA\u003c/em\u003es, and fourteen \u003cem\u003eAhLEA\u003c/em\u003es were associated with \u003cem\u003eGmLEA\u003c/em\u003es. Additionally, there were nine cases that peanut segmental duplications that corresponded to a single Arabidopsis gene, and eleven cases that \u003cem\u003eAhLEA\u003c/em\u003es corresponded to a single soybean gene. Finally, some genes showed more-to-more correspondence, for example, AH02G02040.1/ AH12G02210.1/ AH04G26920.1/ AH14G31640.1-AT1G01470.1/ AT2G46140.1 and AH01G11560.1/ AH05G04840.1/ AH11G11350.1/ AH15G00880.1-Glyma05g22030.1/ Glyma17g17860.1.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec5\"\u003e\n \u003ch2\u003e2.3. Analysis of gene structure and protein motifs of \u003cem\u003eLEA\u003c/em\u003es in peanut\u003c/h2\u003e\n \u003cp\u003eTo examine the structural characteristics of \u003cem\u003eAhLEA\u003c/em\u003es, an unrooted phylogenetic tree that combines the UTR-CDS structures and motifs were constructed based on the full lengths of the 126 peanut \u003cem\u003eLEA\u003c/em\u003e gene sequences by using the Maximum-Likelihood method (Fig. \u003cspan class=\"InternalRef\"\u003e7\u003c/span\u003e). The majority of the \u003cem\u003eAhLEA\u003c/em\u003es contained zero or one intron, with 55 and 53, respectively. Sixteen genes had two introns. One gene, AH19G03360.1, contained three introns, and one gene, AH12G35940.1, contained seven introns. All the \u003cem\u003eLEA1\u003c/em\u003es and \u003cem\u003eDehydrin\u003c/em\u003es contained only one intron, and the main members of the \u003cem\u003eLEA3\u003c/em\u003e and \u003cem\u003eLEA5\u003c/em\u003e subfamilies had one intron. The majority of the \u003cem\u003eLEA2\u003c/em\u003es had no intron. To identify the conserved protein motifs, the MEME (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003c/span\u003e) online software was used to predict putative motifs of these proteins, with a maximum number of the different motifs at 20. Motif analysis indicated that members of each subfamily had the group-specific conserved domain, and \u003cem\u003eAhLEA\u003c/em\u003es with closer evolutionary relationships had more similar motif numbers. MEME analysis revealed that most \u003cem\u003eAhLEA\u003c/em\u003es contained motif 3 and all the \u003cem\u003eLEA4\u003c/em\u003es and \u003cem\u003eLEA1\u003c/em\u003es had motif 13. The \u003cem\u003eLEA2\u003c/em\u003es had the greatest number of motifs, which were approximately 7, while other subfamily members had 1 to 4 motifs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec6\"\u003e\n \u003ch2\u003e2.4. Analysis of cis-acting elements in promoters of \u003cem\u003eAhLEA\u003c/em\u003es\u003c/h2\u003e\n \u003cp\u003eTo investigate the cis-acting elements of \u003cem\u003eAhLEA\u003c/em\u003es, 2 kb upstream of the translation initiation sites of all the \u003cem\u003eLEA\u003c/em\u003e genes were obtained from the peanut genome database. Many cis-acting regulatory elements that may be involved in the plant\u0026rsquo;s response to environmental stresses, including ABRE, WRE3, ERE, MYB recognition sites, MYC recognition sites, TC-rich repeats, STRE, and MRE, were detected (Fig. \u003cspan class=\"InternalRef\"\u003e8\u003c/span\u003e). The promoter of subfamily \u003cem\u003eLEA2\u003c/em\u003e contained the most cis-acting elements, followed by subfamily \u003cem\u003eLEA3\u003c/em\u003e, \u003cem\u003eLEA5\u003c/em\u003e, and \u003cem\u003eLEA1\u003c/em\u003e. The promoter of subfamily \u003cem\u003eLEA4\u003c/em\u003e, \u003cem\u003eSMP\u003c/em\u003e, \u003cem\u003ePvLEA18\u003c/em\u003e, and \u003cem\u003eDehydrin\u003c/em\u003e contained the least elements. Among the identified cis-acting elements, ABRE (22.2 %), ERE (55.6 %), MYB recognition sites (65.9 %), and MYC recognition sites (70.6 %) cis-acting elements were over-represented.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec7\"\u003e\n \u003ch2\u003e2.5. Expression profiles of \u003cem\u003eAhLEA\u003c/em\u003es at different stages of embryo development\u003c/h2\u003e\n \u003cp\u003eTo investigate the expression profiles of \u003cem\u003eAhLEA\u003c/em\u003es across different stages of embryo development and different tissues, the transcriptomic data of a cultivated variety (\u003cem\u003eA. hypogaea\u003c/em\u003e L.) in gene bank were further scrutinized (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://peanutgr.fafu.edu.cn/Transcriptome.php\u003c/span\u003e\u003c/span\u003e) (Fig. \u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, Additional file 1: Table S4). Not all \u003cem\u003eAhLEA\u003c/em\u003es were expressed at the four embryo development stages. Meanwhile, twenty-seven genes were not detected at any tested stages. Sixty-eight \u003cem\u003eLEA\u003c/em\u003es had different expression levels among the four stages. In the early embryo development stages, most \u003cem\u003eLEA3\u003c/em\u003es were up-regulated. Among them, three \u003cem\u003eLEA3\u003c/em\u003es (AH01G27080.1, AH01G27080.2, and AH11G30560.1) exhibited very high expression levels in the early stages, which showed up to 10-fold higher than those in the late stages. Nevertheless, \u003cem\u003eAhLEA1\u003c/em\u003es, \u003cem\u003eAhLEA4\u003c/em\u003es, and \u003cem\u003eAhLEA5\u003c/em\u003es were up-regulated mainly in the late stages. Four genes including two \u003cem\u003eLEA5\u003c/em\u003es (AH12G24910.1 and AH12G24920.1) and two \u003cem\u003eLEA1\u003c/em\u003es (AH06G01030.1 and AH16G03650.1) exhibited very high expression levels in the late stages. Two genes of the \u003cem\u003eDehydrin\u003c/em\u003es expressed at a high level in stages I, and Ⅱ, while another \u003cem\u003eDehydrin\u003c/em\u003e (AH17G19580.1) expressed at a high level in stages Ⅲ, and Ⅳ. The expression of most \u003cem\u003eAhLEA2\u003c/em\u003es was not changed as embryo development, while the expression level one \u003cem\u003eLEA2\u003c/em\u003e (AH12G34850.1) in the early stages showed up to 26-fold higher than those in the later stages.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. \u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e, the expression profiles of eight subfamilies, including \u003cem\u003eLEA1\u003c/em\u003es, \u003cem\u003eLEA2\u003c/em\u003es, \u003cem\u003eLEA3\u003c/em\u003es, \u003cem\u003eLEA4\u003c/em\u003es \u003cem\u003eLEA5\u003c/em\u003es, \u003cem\u003eSMP\u003c/em\u003es, \u003cem\u003ePvLEA18\u003c/em\u003es, and \u003cem\u003eDehydrin\u003c/em\u003es, were similar in roots, stems, leaves, and flowers. Among them, the members of \u003cem\u003eLEA2\u003c/em\u003es, \u003cem\u003eLEA3\u003c/em\u003es, and \u003cem\u003eDehydrin\u003c/em\u003es were expressed at a high level in all four tissues. Twenty-four \u003cem\u003eLEA\u003c/em\u003es were highly expressed in roots, 21 in stems, 15 in leaves, and 20 in flowers. Two \u003cem\u003eDehydrin\u003c/em\u003es (AH07G18700.1 and AH17G19710.1) and two \u003cem\u003eLEA3\u003c/em\u003es (AH16G06810.1 and AH06G03960.1) had the highest expression levels in the stem (Additional file 1: Table S5).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec8\"\u003e\n \u003ch2\u003e2.6. Expression profiles of \u003cem\u003eAhLEA\u003c/em\u003es in response to drought and low-temperature stresses.\u003c/h2\u003e\n \u003cp\u003eTo investigate the transcriptional changes of the \u003cem\u003eAhLEA\u003c/em\u003es under cold and drought stresses, the expression profiles of these genes were examined by using transcriptomic data (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e). Under drought treatment, 28.6 % (36 out of 126) of the \u003cem\u003eAhLEA\u003c/em\u003es were more than 2-fold up-regulated compared with the control, while the expression levels of 21.4 % (27 out of 126) genes were down-regulated more than 2-fold. Among the 27 genes that down-regulated more than 2-fold, 24 genes belonged to the \u003cem\u003eLEA2\u003c/em\u003e subfamily. Two \u003cem\u003eLEA3\u003c/em\u003es (AH01G27080.1, and AH01G27080.2) showed the highest expression levels under drought stress (Additional file 1: Table S6).\u003c/p\u003e\n \u003cp\u003eUnder low-temperature treatment, 28.6 % (36 out of 126) of the \u003cem\u003eAhLEA\u003c/em\u003es were more than 2-fold up-regulated compared with the control, while the expression levels of 14.3 % (18 out of 126) genes were down-regulated more than 2-fold. It was found that 21 genes of \u003cem\u003eLEA2\u003c/em\u003es were up-regulated and 11 genes were down-regulated. It is noteworthy that all \u003cem\u003eDehydrin\u003c/em\u003es were up-regulated under drought and low-temperature stresses. Interestingly, the genes expressed the highest under low-temperature stress were also two \u003cem\u003eLEA3\u003c/em\u003e subfamily genes (AH16G06810.1, AH06G03960.1). Besides, 60 % of \u003cem\u003eLEA5\u003c/em\u003es genes were not detected under drought and low-temperature (Fig. \u003cspan class=\"InternalRef\"\u003e11\u003c/span\u003e: Additional file 1: Table S6).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec9\"\u003e\n \u003ch2\u003e2.7. Expression profiles of \u003cem\u003eAhLEA\u003c/em\u003e genes in response to hormone\u003c/h2\u003e\n \u003cp\u003eTo understand the expression changes of the \u003cem\u003eAhLEA\u003c/em\u003es under drought and low-temperature stresses, the responses of 126 \u003cem\u003eAhLEA\u003c/em\u003es to four stress-related hormones (abscisic acid, brassinolide, ethylene, and salicylic acid) were investigated (Fig. \u003cspan class=\"InternalRef\"\u003e12\u003c/span\u003e). The expression profiles of these genes were examined by using transcriptomic data. After ABA treatment, 8 \u003cem\u003eLEA\u003c/em\u003es were induced more than 2-fold, while 19 \u003cem\u003eLEA\u003c/em\u003es were down-regulated more than 2-fold. After brassinolide treatment, 5 genes were up-regulated more than 2-fold, while and 31 genes were down-regulated more than 2-fold. The expression of 13 \u003cem\u003eAhLEA\u003c/em\u003es was up-regulated more than 2-fold after ethylene treatment, while 28 genes were down-regulated more than 2-fold. The expression of 10 \u003cem\u003eAhLEA\u003c/em\u003es was up-regulated more than 2-fold after salicylic acid treatment, while 16 genes were down-regulated more than 2-fold. Although the main \u003cem\u003eAhLEA\u003c/em\u003es were down-regulated by these four hormones, half of the \u003cem\u003eLEA3\u003c/em\u003es (7 out of 14) were up-regulated more than 2-fold after ethylene treatment, all members of \u003cem\u003eLEA4\u003c/em\u003es were induced by salicylic acid. Moreover, the expression of five \u003cem\u003eAhLEA\u003c/em\u003es was up-/down-regulated more than 2-fold by all four tested hormones. These genes included four \u003cem\u003eLEA2\u003c/em\u003es (AH06G19190.1, AH16G23780.1, AH20G34490.1, and AH16G06810.1) which were down-regulated after hormone treatment and a \u003cem\u003ePvLEA18\u003c/em\u003e (AH11G11350.1) that was up-regulated (Additional file 1: Table S7).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec10\"\u003e\n \u003ch2\u003e2.8. Expression pattern of \u003cem\u003eAhLEA\u003c/em\u003es under Al stress\u003c/h2\u003e\n \u003cp\u003eTo gain a broader understanding of the putative functions of peanut \u003cem\u003eLEA\u003c/em\u003es in response to Al stress, the expression profiles of these genes were examined by using the RNA-Seq data which was generated from the root tips of two peanut cultivars that exhibited different Al sensitivity and had already been deposited in NCBI (Xiao et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). ZH2 is known as an Al sensitive peanut cultivar and 99-1507 is proved as an Al tolerant peanut cultivar (Zhan et al., 2008). Here, a total of 50 \u003cem\u003eAhLEA\u003c/em\u003es were found to be aluminum stress-responsive genes (Fig. \u003cspan class=\"InternalRef\"\u003e13\u003c/span\u003e, Additional file 1: Table S8). \u003cem\u003eLEA2\u003c/em\u003es which included twenty-three DEGs had the most aluminum stress-responsive genes. All of the members in \u003cem\u003eLEA4\u003c/em\u003es and \u003cem\u003eDehydrin\u003c/em\u003es were aluminum stress-responsive genes, and both of these two subfamilies were composed of three genes. The aluminum stress-responsive genes accounted for 75 % (3 out of 4), 60 % (6 out of 10), 50 % (3 out of 6, and 4 out of 8), and 35.7 % (5 out of 14) of the members in \u003cem\u003ePvLEA18\u003c/em\u003es, \u003cem\u003eLEA5\u003c/em\u003es, \u003cem\u003eSMP\u003c/em\u003es (and \u003cem\u003eLEA1\u003c/em\u003es), and \u003cem\u003eLEA3\u003c/em\u003es, respectively.\u003c/p\u003e\n \u003cp\u003eFive \u003cem\u003eAhLEA\u003c/em\u003es were significantly up-regulated after 8h of Al treatment in ZH2. Seventeen \u003cem\u003eAhLEA\u003c/em\u003es were significantly up-regulated after 8h of Al treatment in 99-1507, including 66 % of \u003cem\u003eLEA4\u003c/em\u003es and \u003cem\u003eDehydrin\u003c/em\u003es (2 out of 3) and 50 % of \u003cem\u003eSMP\u003c/em\u003es (3 out of 6). Nineteen \u003cem\u003eAhLEA\u003c/em\u003es were significantly up-regulated after 24 h of Al treatment in ZH2, including all members of \u003cem\u003eLEA5\u003c/em\u003es and \u003cem\u003ePvLEA18\u003c/em\u003es and half of \u003cem\u003eLEA1\u003c/em\u003es (4 out of 8) and \u003cem\u003eSMP\u003c/em\u003es (3 out of 6). Nine \u003cem\u003eAhLEA\u003c/em\u003es were significantly up-regulated after 24 h of Al treatment in 99-1507. Twelve \u003cem\u003eAhLEA\u003c/em\u003es were down-regulated after 8h of Al treatment in ZH2, and all \u003cem\u003ePvLEA18\u003c/em\u003es were down-regulated. Two \u003cem\u003eAhLEA\u003c/em\u003es were down-regulated after 8h of Al treatment in 99-1507. Seven \u003cem\u003eAhLEA\u003c/em\u003es were down-regulated after 24h of Al treatment in ZH2. Eight \u003cem\u003eAhLEA\u003c/em\u003es were down-regulated after 24h of Al treatment in 99-1507.\u003c/p\u003e\n \u003cp\u003eFurthermore, we compared the DEGs in Al stress and the genes that were up-/down-regulated more than 2-fold under drought and low-temperature stresses. As shown in Fig S1, a total of 100 \u003cem\u003eAhLEA\u003c/em\u003es were regulated under drought, low temperature, and Al stress. Among these genes, 35 common \u003cem\u003eAhLEA\u003c/em\u003es were involved in the responses to low-temperature and drought stresses, 29 common \u003cem\u003eAhLEA\u003c/em\u003es that were involved in the responses to drought and Al stresses, and 22 common \u003cem\u003eAhLEA\u003c/em\u003es that were involved in the responses to low-temperature and Al stresses. Sixteen \u003cem\u003eAhLEA\u003c/em\u003es were overlaps among the three abiotic stresses (Additional file 2: Fig S1).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Discussion","content":"\u003cdiv class=\"Section2\" id=\"Sec12\"\u003e\n \u003ch2\u003e3.1. Identification of the \u003cem\u003eLEA\u003c/em\u003e gene family\u003c/h2\u003e\n \u003cp\u003eIn this study, 126 \u003cem\u003eLEA\u003c/em\u003e genes were identified from whole peanut genome sequences. Based on the phylogenetic relationship with Arabidopsis, these 126 \u003cem\u003eAhLEA\u003c/em\u003es were distributed to eight groups. The number of peanut \u003cem\u003eLEA\u003c/em\u003es was twice that in Arabidopsis. According to the studies of the \u003cem\u003eLEA\u003c/em\u003e family in other species, the number of \u003cem\u003eLEA\u003c/em\u003es may be related to the polyploidy of plants (Ibrahime et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). For example, many \u003cem\u003eLEA\u003c/em\u003e genes were found in polyploids of \u003cem\u003eupland cotton\u003c/em\u003e (Fang and Magwanga, 2018), \u003cem\u003eTriticum aestivum\u003c/em\u003e (Shi et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), and \u003cem\u003eBrassica napus\u003c/em\u003e (Liu et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003eLEA2\u003c/em\u003es had more members than other subfamilies in the majority of species so far studied (Ibrahime et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). The \u003cem\u003eLEA2\u003c/em\u003es were the largest \u003cem\u003eLEA\u003c/em\u003e subfamily in \u003cem\u003eCitrus sinensis, Oryza sativa, Populus trichocarpa\u003c/em\u003e (Pedrosa et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e), and \u003cem\u003eupland cotton\u003c/em\u003e (Fang and Magwanga, 2018). Similarly, the \u003cem\u003eAhLEA\u003c/em\u003es mainly belong to the \u003cem\u003eLEA2\u003c/em\u003es, which accounted for 61.9 % of the \u003cem\u003eLEA\u003c/em\u003e genes. However, the \u003cem\u003eLEA2\u003c/em\u003es were not found as a large subfamily in the previous works in Arabidopsis (Hundertmark and Hincha, \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e), \u003cem\u003eOryza sativa\u003c/em\u003e (Wang et al., \u003cspan class=\"CitationRef\"\u003e2007\u003c/span\u003e), and \u003cem\u003ePopulus trichocarpa\u003c/em\u003e (Lan et al., \u003cspan class=\"CitationRef\"\u003e2013\u003c/span\u003e). This result can be partly explained by the fact that improved annotation of higher plant genomes can be found on phytochrome (v10.2), and LEA2 is an unusual component of \u0026quot;a typical\u0026quot; LEA proteins because they are more hydrophobic. In addition, there were three pairs of tandem duplication in \u003cem\u003eAhLEA\u003c/em\u003es, which belong to the \u003cem\u003eLEA2\u003c/em\u003es. This result supported the view that tandem duplications have contributed significantly to the expansion and diversity of the \u003cem\u003eLEA2\u003c/em\u003es in most species (Artur et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec13\"\u003e\n \u003ch2\u003e3.2. Analysis of conserved domains and introns revealed that \u003cem\u003eLEA\u003c/em\u003es might be stress-response genes\u003c/h2\u003e\n \u003cp\u003eMotif analysis of the \u003cem\u003eAhLEA\u003c/em\u003es showed that members of each \u003cem\u003eLEA\u003c/em\u003e group contained specific conserved motifs. Most members of the same subfamily have similar motifs, indicating an important role of these conserved motifs in the evolution of the \u003cem\u003eLEA\u003c/em\u003e gene family. Big differences were found in the structure of different clades. For example, \u003cem\u003eLEA1\u003c/em\u003es contained motifs 13 and 17, whereas \u003cem\u003eLEA5\u003c/em\u003es contained motif 2 and 9, which indicated the complexity and group-specific of LEA protein function. The conserved motifs observed in each \u003cem\u003eLEA\u003c/em\u003e group suggested that genes with the same motif might be amplified from genes within the same evolutionary clade or group. It has been reported that stress-responding genes usually contain fewer introns (Fang and Magwanga, 2018; Xie et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e). Introns have harmful effects on gene expression by delaying transcription products (Lane et al., 2010). Moreover, introns can extend the length of new transcripts, resulting in additional energy consumption for transcription (Jeffares et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e). Up to 85.7 % of the \u003cem\u003eAhLEA\u003c/em\u003es had zero or only one intron, which further suggested that \u003cem\u003eAhLEA\u003c/em\u003es were stress-related genes.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec14\"\u003e\n \u003ch2\u003e3.3. Segmental duplication plays an important role in the evolution and amplification of \u003cem\u003eAhLEA\u003c/em\u003es\u003c/h2\u003e\n \u003cp\u003eGene duplication plays an important role in the evolution and amplification of gene families (Flagel and Wendel, \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e). In this study, 93 pairs of segmental duplication and 5 pairs of tandem duplication were identified, it could be inferred that segmental duplication and tandem duplication contribute to the common expansion of the \u003cem\u003eAhLEA\u003c/em\u003es family, but the former played a predominant role. This finding was similar to previous studies on \u003cem\u003eBrassica napus\u003c/em\u003e and upland cotton (\u003cem\u003eGossypium hirsutum\u003c/em\u003e) (Liu et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Fang and Magwanga, 2018) and consistent with our previous study on receptor-like protein kinase (RLK) in peanut (Wang et al., \u003cspan class=\"CitationRef\"\u003e2021\u003c/span\u003e). According to Ka/Ks estimation, 94.9 % of the duplication gene pairs of \u003cem\u003eAhLEA\u003c/em\u003es were less than 1, indicating the results of the purification selection. The Ka/Ks ratios of two gene pairs (AH01G27080.1 and AH11G30560.1, AH05G16640.1 and AH15G06250.1) were more than 1, which indicated that these genes were in a state of positive selection in peanuts. We calculated the divergence time, and the results showed that many duplication events appeared to have occurred during relatively recent key periods. For example, all tandem duplication events occurred at 0\u0026ndash;10 MYA, and 49.5 % of segmental duplication occurred at 0\u0026ndash;5 MYA. These results indicated that many \u003cem\u003eAhLEA\u003c/em\u003es were produced by the recent gene duplication events in peanuts. This may be related to the origin of cultivated peanut, through a single and recent polyploidization event, and then continuous selection in breeding work, forming a highly conserved genome (Cunha et al., \u003cspan class=\"CitationRef\"\u003e2008\u003c/span\u003e).\u003c/p\u003e\n \u003cp\u003eThe closer the species are, the greater the genome coverage of synteny fragments and the more genes they contain (Ye et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). Syntenic analysis showed that more homologous gene pairs were found between peanut and soybean. There were 13 single peanut-to-Arabidopsis \u003cem\u003eLEA\u003c/em\u003e gene correspondences and 5 single peanut-to-soybean \u003cem\u003eLEA\u003c/em\u003e gene correspondences. These results indicated that these genes come from a common ancestor. Among these genes, two soybean genes (Glyma11g02290.1 and Glyma09g30400.1) did not belong to the \u003cem\u003eLEA\u003c/em\u003e family. The rest genes showed one-to-more, more-to-one and more-to-more correspondence, and most of the genes included in these cases appeared more than once. However, 15 of the 23 orthologs of \u003cem\u003eAhLEA\u003c/em\u003es in soybean (Glyma09g38980.1, Glyma19g37350.1, Glyma08g22050.1, Glyma12g09590.1, Glyma12g32090.1, Glyma13g38380.1, Glyma03g34670.1, Glyma10g07360.1, Glyma13g21240.1, Glyma19g37340.1, Glyma06g01170.1, Glyma07g06960.1, Glyma13g43610.1, Glyma09g30400.1, and Glyma20g35880.1) were not \u003cem\u003eLEA\u003c/em\u003e genes, which implied that their genomes underwent multiple rounds of chromosomal rearrangement and fusions. Allotetraploid-cultivated peanut composed of A and B genomes and was generated from diploid \u003cem\u003eA. duranesis\u003c/em\u003e (AA) and \u003cem\u003eA. ipaensis\u003c/em\u003e (BB) (Zhuang et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). Taking into account the divergence time of the duplications, we inferred that the divergence of many \u003cem\u003eAhLEA\u003c/em\u003es duplications occurred after the divergence of peanut and Arabidopsis/soybean from their last common ancestor. Combined with the results of phylogenetic tree analysis, there were nine orthologs including nineteen peanut \u003cem\u003eLEA\u003c/em\u003e genes (AH12G35940.1-AT2G36640.1, AH17G19580.1-AT2G21490.1, AH12G35940.1-AT3G22500.1, AH02G22690.1/ AH12G24910.1-AT3G51810.1, AH05G04840.1/ AH15G00880.1-AT2G23110.1, AH04G10170.1/ AH14G12410.1-AT4G15910.1, AH06G03960.1/ AH12G32330.1/ AH12G37270.1/ AH16G06810.1-AT1G02820.1/ AT4G02380.3, AH02G06810.1/ AH12G08270.2-AT1G03120.1/ AT3G22490.1, AH02G02040.1/ AH04G26920.1/ AH12G02210.1/ AH14G31640.1-AT2G46140.1/ AT1G01470.1) that could be clustered together in the phylogenetic tree and were also contained in the syntenic map. We speculate that the functions of these \u003cem\u003eAhLEA\u003c/em\u003es are more similar to their Arabidopsis homologs than the other \u003cem\u003eAhLEA\u003c/em\u003es in the phylogenetic tree and syntenic map.\u003c/p\u003e\n \u003ch2\u003e3.4. MYB and MYC recognition sites may be involved in the response of \u003cem\u003eAhLEA\u003c/em\u003es to abiotic stress\u003c/h2\u003e\n \u003cp\u003eMany studies have shown that \u003cem\u003eLEA\u003c/em\u003es play an important role in abiotic stress. In this study, many cis-acting elements related to abiotic stress and plant hormones were identified, such as ABRE, ERE, MYB recognition sites, MYC recognition sites, and STRE. We found that the MYB and MYC recognition sites were presented in the most promoters of the \u003cem\u003eAhLEA\u003c/em\u003es. It is reported that MYBs and MYCs are transcription factors that participate in ABA-dependent signaling pathways to cope with abiotic stresses such as drought, salt, and low-temperature (Li et al., \u003cspan class=\"CitationRef\"\u003e2015\u003c/span\u003e; Boter, 2014). Consistently, the \u003cem\u003eLEA\u003c/em\u003es that contain MYB recognition sites and MYC recognition sites, including AH16G06810.1, and AH06G03960.1, were induced under ABA, salicylic acid, drought, and low-temperature stress. Besides, most of the \u003cem\u003eLEA1\u003c/em\u003es, \u003cem\u003eLEA5\u003c/em\u003es, \u003cem\u003eSMP\u003c/em\u003es, and \u003cem\u003eDehydrin\u003c/em\u003es were highly expressed under aluminum stress, and these genes contained a large number of MYB and MYC recognition sites. Therefore, we speculated that the up-regulation of \u003cem\u003eLEA\u003c/em\u003es expression under aluminum stress might be regulated by MYB and MYC transcription factors. This provides a theoretical basis for further exploring the response regulation mechanism of \u003cem\u003eLEA\u003c/em\u003es containing cis-acting elements of MYB and MYC recognition sites under stress. In this study, many \u003cem\u003eLEA\u003c/em\u003es responses to abiotic stresses were found to be regulated by ABA. However, only five \u003cem\u003eAhLEA\u003c/em\u003es (AH03G11350.1, AH03G03840.1, AH02G02050.1, AH01G27080.1, AH01G27080.2) contained ABRE cis-acting elements. The promoter region of many \u003cem\u003eAhLEA\u003c/em\u003es had recognition sites for MYB and MYC. These results suggested that \u003cem\u003eAhLEA\u003c/em\u003es responded to abiotic stresses such as drought, low-temperature, and Al stress might be direct or indirect activated ABA-dependent signaling pathways.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec15\"\u003e\n \u003ch2\u003e3.5. Expression analysis revealed \u003cem\u003eAhLEA\u003c/em\u003es respond to different abiotic stresses\u003c/h2\u003e\n \u003cp\u003eIt can obtain clues from gene expression patterns to explore the function of genes (Chen et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e). We investigated the expression level of \u003cem\u003eAhLEA\u003c/em\u003es in different tissues, at different embryo development stages, under different abiotic stresses (drought, low-temperature, and Al treatment), and after different hormone treatments. In four different embryo development stages, there were sixty-eight differentially expressed genes. Consistent with previous studies (Liang et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e) that \u003cem\u003eLEA\u003c/em\u003es were up-regulated as the embryo developed, most of the \u003cem\u003eAhLEA\u003c/em\u003es were expressed at a high level at stages Ⅲ, and Ⅳ. However, the majority of \u003cem\u003eLEA3\u003c/em\u003es were highly expressed at an early stage, suggesting the potential roles of \u003cem\u003eLEA3\u003c/em\u003es in the early embryo development stage. As shown in Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e9\u003c/span\u003e, subfamily \u003cem\u003eLEA2\u003c/em\u003e was the biggest subfamily, but the expression levels of most \u003cem\u003eLEA2\u003c/em\u003es at four embryo development stages were stable, suggesting that \u003cem\u003eLEA2\u003c/em\u003es might not play important roles during embryo development.\u003c/p\u003e\n \u003cp\u003eThe expression level of most \u003cem\u003eAhLEA\u003c/em\u003es in the root, stem, leaf, and flower tissues was similar. The expression level of many \u003cem\u003eAhLEA\u003c/em\u003es was low, while there were still several genes of subfamily \u003cem\u003eLEA2\u003c/em\u003e, \u003cem\u003eLEA3\u003c/em\u003e, and \u003cem\u003eDehydrin\u003c/em\u003e that exhibited a high expression level in the four tissues. Two \u003cem\u003eLEA3\u003c/em\u003es (AH16G06810.1, AH06G03960.1) were very highly expressed in different peanut tissues (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e10\u003c/span\u003e). It was reported that the \u003cem\u003eLEA3\u003c/em\u003es play an important role in plant growth, development, and response to abiotic stresses (Yu et al., \u003cspan class=\"CitationRef\"\u003e2019\u003c/span\u003e; Koubaa and Brini \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e), and these two genes might be suitable candidates to understand the role of \u003cem\u003eLEA3\u003c/em\u003es in peanut.\u003c/p\u003e\n \u003cp\u003eUnder drought stress, 50 % of the \u003cem\u003eAhLEA\u003c/em\u003es were up-/down-regulated for more than 2-fold compared with control. Among them, \u003cem\u003eLEA2\u003c/em\u003es contributed most genes, containing 10 up-regulated genes and 24 down-regulated genes. This is consistent with the fact that \u003cem\u003eLEA2\u003c/em\u003es were the largest subfamily in peanuts. Among the genes that were down-regulated for more than 2-fold, most of them were \u003cem\u003eLEA2\u003c/em\u003es. Additionally, four \u003cem\u003eAhLEA1\u003c/em\u003es and three \u003cem\u003eAhLEA3\u003c/em\u003es were induced more than 60-fold by drought stress, implying their potential roles in enhancing drought stress tolerance in peanuts.\u003c/p\u003e\n \u003cp\u003eUnder low-temperature stress, 36 \u003cem\u003eAhLEA\u003c/em\u003es were up-regulated more than 2-fold, while 18 genes were down-regulated more than 2-fold. \u003cem\u003eLEA2\u003c/em\u003es also contributed to most genes. Twenty-one \u003cem\u003eAhLEA2\u003c/em\u003es were up-regulated and eleven genes were down-regulated. Interestingly, the \u003cem\u003eLEA2\u003c/em\u003es that down-regulated under drought stress was also down-regulated under low-temperature stress, which suggested that there was a common mechanism to regulate \u003cem\u003eLEA2\u003c/em\u003es expression.\u003c/p\u003e\n \u003cp\u003eSeventeen genes were up-regulated after 8h of Al treatment in 99-1507, and two of their (AH16G20700.1 and AH06G16990.1) were also up-regulated after 24h of Al treatment. In ZH2, only five \u003cem\u003eAhLEA\u003c/em\u003es were up-regulated after 8h of Al treatment, while sixteen \u003cem\u003eAhLEA\u003c/em\u003es were up-regulated after 24h of Al treatment. Interestingly, three \u003cem\u003eSMP\u003c/em\u003es (AH12G08270.1, AH12G08270.2, and AH02G06810.1) were up-regulated after 8h of Al treatment in both cultivars, suggesting that these genes might play important roles in Al tolerance in peanuts. Together, the Al-tolerant cultivar 99-1507 exhibited a rapid response to Al treatment, and the \u003cem\u003eLEA\u003c/em\u003es that induced rapidly should be studied in future work.\u003c/p\u003e\n \u003cp\u003eAs shown in Fig. S1, the majority of the 126 \u003cem\u003eLEA\u003c/em\u003es were induced under at least one stress condition. Sixty-three (50 %), fifty-four (42.9 %), and fifty (39.7 %) the \u003cem\u003eAhLEA\u003c/em\u003es were induced by drought, low-temperature, and Al stresses, respectively. Among these genes, sixteen were induced only under drought stress, fourteen were induced only under low-temperature, and sixteen were induced only under Al stress (Fig S1). These results implied that these genes play distinct roles in response to different abiotic stresses in peanuts.\u003c/p\u003e\n \u003cp\u003eSome \u003cem\u003eAhLEA\u003c/em\u003es were regulated by different stress conditions. Three genes including two \u003cem\u003eLEA5\u003c/em\u003es (AH12G24910.1 and AH12G24920.1) and one \u003cem\u003eLEA1\u003c/em\u003e (AH19G11740.1) were up-regulated greatly under both drought and Al stresses (Additional file 1: Table S6, Table S 8). The expression of \u003cem\u003eLEA1\u003c/em\u003e (AH19G11740.1) was induced more than 2-fold by ABA treatment. Two \u003cem\u003eLEA3\u003c/em\u003es (AH01G27080.1 and AH11G30560.1) and one \u003cem\u003eLEA4\u003c/em\u003e (AH12G35940.1) were down-regulated under drought, low-temperature, and Al stresses. The expression of that two \u003cem\u003eLEA3\u003c/em\u003es was significantly induced by ethylene, while \u003cem\u003eLEA4\u003c/em\u003e (AH12G35940.1) was down-regulated by ABA treatment. Two \u003cem\u003eLEA2\u003c/em\u003es (AH02G02040.1 and AH12G02210.1) were up-regulated under drought, low-temperature, and Al stresses, and they were also up-regulated by ABA.\u003c/p\u003e\n \u003cp\u003eMany \u003cem\u003eAhLEA\u003c/em\u003es that were regulated more than 2-fold by hormones such as abscisic acid, brassinolide, ethylene, and salicylic acid were found to be down-regulated. As revealed by table S7, these down-regulated genes showed no obvious subfamily preference. However, \u003cem\u003eAhLEA\u003c/em\u003es that were up-regulated more than 2-fold by ethylene and salicylic acid showed obvious subfamily preference. Seven \u003cem\u003eAhLEA3\u003c/em\u003es were induced by ethylene. Five \u003cem\u003eAhLEA3\u003c/em\u003es induced by ethylene were also involved in response to drought and low-temperature stresses. The expression level of AH12G37280.1 was increased up to 8.45-fold under low-temperature stress. AH12G32330.1 was up-regulated 3.5-fold under drought stress. Moreover, three \u003cem\u003eAhLEA3\u003c/em\u003es (AH01G27080.1, AH01G27080.2, AH11G30560.1) were up-regulated greatly under both drought and low-temperature stresses. These results revealed the important roles of the \u003cem\u003eAhLEA3\u003c/em\u003e subfamily in the ethylene-mediated response under drought and low-temperature stresses. Additionally, all \u003cem\u003eAhLEA4\u003c/em\u003es were induced by salicylic acid, and all \u003cem\u003eAhLEA4\u003c/em\u003es were also regulated greatly under drought and low-temperature stresses. Among them, two genes (AH06G16990.1 and AH12G35940.1) were induced more than 6-fold under drought and low-temperature stresses, and one gene (AH16G20700.1) was down-regulated 3.5-fold under low-temperature stress, which implied that subfamily \u003cem\u003eAhLEA4\u003c/em\u003e played important roles in SA-mediated response under drought and low-temperature stresses in peanut.\u003c/p\u003e\n \u003cp\u003eTaken together, these results suggested that common mechanisms might be initiated in peanuts to cope with different abiotic stresses. Hormones were involved in regulating \u003cem\u003eLEA\u003c/em\u003e\u0026rsquo;s expression under abiotic stresses. The role of hormones in regulating gene expression had a preference among \u003cem\u003eAhLEA\u003c/em\u003e gene families.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"Conclusions","content":"\u003cp\u003eIn this study, 126 \u003cem\u003eLEA\u003c/em\u003e genes in \u003cem\u003eArachis hypogaea\u003c/em\u003e were identified. They were divided into eight groups according to homologous in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e. \u003cem\u003eAhLEA\u003c/em\u003es are randomly distributed on the chromosome, and most of them may be segmental duplication. The exon-intron and motif structures indicated that the \u003cem\u003eLEA\u003c/em\u003es\u0026rsquo; family functions are highly conserved. Some cis-elements of abiotic stress response were also found in the upstream sequences of most \u003cem\u003eAhLEA\u003c/em\u003es. The comprehensive analysis of \u003cem\u003eAhLEA\u003c/em\u003es gene expression profiles showed that the \u003cem\u003eLEA3\u003c/em\u003es, \u003cem\u003eLEA4\u003c/em\u003es, and \u003cem\u003eSMP\u003c/em\u003es played an important role in abiotic stress response, and also showed the functional differences among other subfamilies. This study provided a reference for further exploring the mechanism of \u003cem\u003eLEA\u003c/em\u003es in response to abiotic stress in peanuts.\u003c/p\u003e"},{"header":"Materials And Methods","content":"\u003cdiv class=\"Section2\" id=\"Sec18\"\u003e\n \u003ch2\u003e5.1. Identification of \u003cem\u003eLEA\u003c/em\u003es in peanut\u003c/h2\u003e\n \u003cp\u003eTo identify the \u003cem\u003eAhLEA\u003c/em\u003es, we used 51 \u003cem\u003eLEA\u003c/em\u003e genes (Hundertmark et al., 2008) in \u003cem\u003eArabidopsis thaliana\u003c/em\u003e acquire Pfam ID (PF03760, PF03168, PF03242, PF02987, PF00477, PF10714, PF04927, PF00257) and InterPro ID (IPR005513, IPR004864, /IPR013990, IPR004926, IPR004238, IPR000389, IPR018930, IPR007011, IPR000167) from Peanut Base (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.peanutbase.org/\u003c/span\u003e\u003c/span\u003e). By acquiring LEA peanut protein sequences based on InterPro ID search of Peanut Genome Resource (PGR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://peanutgr.fafu.edu.cn/\u003c/span\u003e\u003c/span\u003e). NCBI\u0026rsquo;s Conserved Domains Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/span\u003e\u003c/span\u003e) and PFAM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003c/span\u003e) database were used to verify the presence of the \u003cem\u003eLEA\u003c/em\u003e domains and finally obtained 126 \u003cem\u003eAhLEA\u003c/em\u003es.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec19\"\u003e\n \u003ch2\u003e5.2. Phylogenetic relationships, gene structures, conserved motifs, and chromosomal locations of the \u003cem\u003eAhLEA\u003c/em\u003es\u003c/h2\u003e\n \u003cp\u003eThe phylogenetic tree was constructed by the maximum-likelihood method with 1000 bootstrap replicates in MEGA 7.0 software (Sudhir et al., \u003cspan class=\"CitationRef\"\u003e2016\u003c/span\u003e). Multiple Expectation Maximization for Motif Elicitation (MEME) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://meme-suite.org/tools/meme\u003c/span\u003e\u003c/span\u003e) (Bailey et al., \u003cspan class=\"CitationRef\"\u003e2009\u003c/span\u003e) was used to identify the conserved protein motifs, with a maximum number of the different motif at 20. The exon-intron structures were identified using the TBtools software (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). The physical location of each \u003cem\u003eAhLEA\u003c/em\u003e is determined by identifying the starting position of all genes on each chromosome, searching the local database of Peanut Genome Resources by BLAST. Using TBtools of Gene location visualize from GFF/GFF3 to draw chromosome mapping and tandem duplication pairs.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec20\"\u003e\n \u003ch2\u003e5.3. Promoter cis-element analysis\u003c/h2\u003e\n \u003cp\u003eGenomic data were obtained from Peanut Genome Resource (PGR) (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://peanutgr.fafu.edu.cn/\u003c/span\u003e\u003c/span\u003e), and TBtools software was used to extract all \u003cem\u003eLEA\u003c/em\u003e upstream 2kd promoter sequences. Transcriptional response elements of \u003cem\u003eLEA\u003c/em\u003e gene promoters were predicted using the PlantCARE database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/span\u003e\u003c/span\u003e) (Higo et al., \u003cspan class=\"CitationRef\"\u003e1999\u003c/span\u003e).\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec21\"\u003e\n \u003ch2\u003e5.4. Gene duplication and evolutionary analysis\u003c/h2\u003e\n \u003cp\u003eWe used Virtual Machine to construct the tandem and segmental of the putative duplication of the \u003cem\u003eAhLEA\u003c/em\u003es and calculate the ratio of the nonsynonymous substitution rate (Ka) to the synonymous substitution rate (Ks) by the Simple Ka/Ks calculator (NG) of TBtools (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e). \u003cem\u003eLEA\u003c/em\u003es clustered together within 100 kb, length of the alignable sequence covers\u0026thinsp;\u0026gt;\u0026thinsp;75 % of longer gene and similarity of aligned regions\u0026thinsp;\u0026gt;\u0026thinsp;75 % were regarded as tandem duplicated genes. The relationship between Ka/Ks ratio and value 1, Ka larger than Ks (or Ka/Ks\u0026thinsp;\u0026gt;\u0026thinsp;\u0026gt;\u0026thinsp;1), Ka equals Ks (Ka/Ks\u0026thinsp;=\u0026thinsp;1), and Ka less than Ks (or Ka/Ks\u0026thinsp;\u0026lt;\u0026thinsp;\u0026lt;\u0026thinsp;1), which represent positive (or diversifying) selection, neutral evolution and negative (or purifying) selection, respectively. Divergence time was calculated with the formula T\u0026thinsp;=\u0026thinsp;Ks/2r, where r is 1.5 \u0026times;10\u003csup\u003e\u0026minus;\u0026thinsp;8\u003c/sup\u003e synonymous substitutions per site per year and it is the rate of divergence for nuclear genes from plants (Koch et al., \u003cspan class=\"CitationRef\"\u003e2000\u003c/span\u003e). We used Multiple Synteny Plot software (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e) to explore the collinear relationship between the \u003cem\u003eAhLEA\u003c/em\u003e and \u003cem\u003eLEA\u003c/em\u003e genes from \u003cem\u003eArabidopsis thaliana\u003c/em\u003e and \u003cem\u003eGlycine max\u003c/em\u003e. All the soybean LEA domain-containing protein sequences were downloaded from the Soybase Glyma.Wm82.a2.v1 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://www.soybase.org/\u003c/span\u003e\u003c/span\u003e). The NCBI\u0026rsquo;s Conserved Domains Database (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/span\u003e\u003c/span\u003e) and PFAM (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://pfam.xfam.org/\u003c/span\u003e\u003c/span\u003e) database were used to verify the presence of the LEA domains. The \u003cem\u003eGmLEA\u003c/em\u003es that were identified in the previous study were also screened (Li et al., \u003cspan class=\"CitationRef\"\u003e2011\u003c/span\u003e). After eliminating the invalid sequence, a total of 132 \u003cem\u003eGmLEA\u003c/em\u003es were identified.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv class=\"Section2\" id=\"Sec22\"\u003e\n \u003ch2\u003e5.5. Expression analysis of \u003cem\u003eAhLEA\u003c/em\u003es\u003c/h2\u003e\n \u003cp\u003eThe blast was performed in the transcriptome of the PGR database using the protein sequences of 126 \u003cem\u003eAhLEA\u003c/em\u003es. RNA-Seq data were downloaded from PGR and used to generate the expression patterns of \u003cem\u003eAhLEA\u003c/em\u003es in different tissues (root, stem, leaf, and flower), different embryo development stages, and various abiotic stresses (cold, and drought), and different hormones treatment on leaves. Transcriptome data that were generated from peanut root tips under Al stress were used to generate the expression patterns of \u003cem\u003eAhLEA\u003c/em\u003es under Al stress. The data had been deposited in the database of the National Center for Biotechnology Information (NCBI) under accession number PRJNA525247 (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/PRJNA525247\u003c/span\u003e\u003c/span\u003e). TBtools were used to generate heat maps and combine phylogenetic tree, gene, and protein structure (Chen et al., \u003cspan class=\"CitationRef\"\u003e2020\u003c/span\u003e).\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eAl: Aluminum\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAt\u003c/em\u003e: \u003cem\u003eArabidopsis thaliana\u003c/em\u003e\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAh\u003c/em\u003e:\u003cem\u003e\u0026nbsp;Arachis hypogaea\u003c/em\u003e. L\u003c/p\u003e\n\u003cp\u003eABRE: ABA-responsive element\u003c/p\u003e\n\u003cp\u003eERE: Ethylene response element\u003c/p\u003e\n\u003cp\u003eWRE3: Water response element\u003c/p\u003e\n\u003cp\u003eMYB: Transcription factor\u003c/p\u003e\n\u003cp\u003eMYC: Transcription factor\u003c/p\u003e\n\u003cp\u003eTC-rich repeats: Cis-acting element involved in defense and stress responsiveness\u003c/p\u003e\n\u003cp\u003eMRE: Metal responsive element\u003c/p\u003e\n\u003cp\u003eSTRE: Stress response element\u003c/p\u003e\n\u003cp\u003eDEGs: Differentially expressed genes\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests:\u003c/strong\u003e All authors declare no conflicting interest. \u003cstrong\u003e\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding:\u003c/strong\u003e This work was supported by the National Natural Science Foundation of China (Grant No. 31701356, 32060419) and College Students\u0026rsquo; Innovative Entrepreneurial Training Plan Program (201910593083). Apart from providing financial support, funding bodies were not involved in the study design, data analyses, and interpretation of results or manuscript preparation. This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution:\u003c/strong\u003e RLH: Writing Original Draft, Data analysis, Conceptualization; XW and YS: Data analysis; LFH, AQW, and JZ: manuscript review; DX: Conceptualization, supervision, Writing\u0026mdash;Review \u0026amp; Editing. All authors read and approved the final manuscript.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgments:\u003c/strong\u003e We thank Mr Yun Xiong Zhao and Miss Xu Fang for providing the software needed for drawing and data collection.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials:\u003c/strong\u003e The datasets generated and analyzed during the current study are available from the corresponding author on reasonable request. The following are all databases in this study and are open.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePeanut Base (\u003ca href=\"https://www.peanutbase.org/\"\u003ehttps://www.peanutbase.org/\u003c/a\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePeanut Genome Resource (PGR) \u0026nbsp;(\u003ca href=\"http://peanutgr.fafu.edu.cn/\"\u003ehttp://peanutgr.fafu.edu.cn/\u003c/a\u003e)\u003c/p\u003e\n\u003cp\u003eTranscriptome of Peanut Genome Resource (PGR) (\u003ca href=\"http://peanutgr.fafu.edu.cn/Transcriptome.php\"\u003ehttp://peanutgr.fafu.edu.cn/Transcriptome.php\u003c/a\u003e)\u003c/p\u003e\n\u003cp\u003eNCBI\u0026rsquo;s Conserved Domains Database (\u003ca href=\"https://www.ncbi.nlm.nih.gov/cdd\"\u003ehttps://www.ncbi.nlm.nih.gov/cdd\u003c/a\u003e)\u0026nbsp;\u003c/p\u003e\n\u003cp\u003ePFAM (\u003ca href=\"http://pfam.xfam.org/\"\u003ehttp://pfam.xfam.org/\u003c/a\u003e) \u0026nbsp;\u003c/p\u003e\n\u003cp\u003eExpectation Maximization for Motif Elicitation (MEME) (\u003ca href=\"http://meme-suite.org/tools/meme\"\u003ehttp://meme-suite.org/tools/meme\u003c/a\u003e)\u003c/p\u003e\n\u003cp\u003ePlantCARE database (\u003ca href=\"http://bioinformatics.psb.ugent.be/webtools/plantcare/html/\"\u003ehttp://bioinformatics.psb.ugent.be/webtools/plantcare/html/\u003c/a\u003e)\u003c/p\u003e\n\u003cp\u003eSoybase Glyma.Wm82.a2.v1 (\u003ca href=\"http://www.soybase.org/\"\u003ehttp://www.soybase.org/\u003c/a\u003e)\u003c/p\u003e\n\u003cp\u003ePRJNA525247\u0026nbsp;(\u003ca href=\"https://www.ncbi.nlm.nih.gov/sra/PRJNA525247\"\u003ehttps://www.ncbi.nlm.nih.gov/sra/PRJNA525247\u003c/a\u003e)\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eAdrien C., Gal P., Martine N. et al. 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DOI: \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e10.1038/s41588-019-0402-2\u003c/span\u003e\u003c/span\u003e\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Arachis hypogaea L., Late embryogenesis abundant, Expression profiles, Abiotic stress","lastPublishedDoi":"10.21203/rs.3.rs-775523/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-775523/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eBackground: Late embryogenesis abundant (LEA) proteins are a group of highly hydrophilic glycine-rich proteins, which accumulate in the late stage of seed maturation and are associated with many abiotic stresses. However, few peanut \u003cem\u003eLEA\u003c/em\u003e genes had been reported, and the research on the number, location, structure, molecular phylogeny and expression of \u003cem\u003eAhLEA\u003c/em\u003es was very limited. \u003c/p\u003e\u003cp\u003eResults: In this study, 126 \u003cem\u003eLEA\u003c/em\u003e genes were identified in the peanut genome through genome-wide analysis and were further divided into eight groups. Sequence analysis showed that most of the \u003cem\u003eAhLEA\u003c/em\u003es (85.7 %) had no or only one intron. \u003cem\u003eLEA\u003c/em\u003e genes were randomly distributed on 20 chromosomes. Compared with tandem duplication, segmental duplication played a more critical role in \u003cem\u003eAhLEA\u003c/em\u003es amplication, and 93 segmental duplication \u003cem\u003eAhLEA\u003c/em\u003es and 5 pairs of tandem duplication genes were identified. Synteny analysis showed that some \u003cem\u003eAhLEA\u003c/em\u003es genes come from a common ancestor, and genome rearrangement and translocation occurred among these genomes. Almost all promoters of \u003cem\u003eLEA\u003c/em\u003es contain ABRE, MYB recognition sites, MYC recognition sites, and ERE cis-acting elements, suggesting that the \u003cem\u003eLEA\u003c/em\u003e genes were involved in stress response. Gene expression analyses revealed that most of the \u003cem\u003eLEA\u003c/em\u003es were expressed in the late stages of peanut embryonic development. \u003cem\u003eLEA3 \u003c/em\u003e(AH16G06810.1, AH06G03960.1), and \u003cem\u003eDehydrin\u003c/em\u003e (AH07G18700.1, AH17G19710.1) were highly expressed in roots, stems, leaves and flowers. Moreover, 100 \u003cem\u003eAhLEA\u003c/em\u003es were involved in response to drought, low-temperature, or Al stresses. Some \u003cem\u003eLEA\u003c/em\u003es that were regulated by different abiotic stresses were also regulated by hormones including ABA, brassinolide, ethylene and salicylic acid. Interestingly, \u003cem\u003eAhLEA\u003c/em\u003es that were up-regulated by ethylene and salicylic acid showed obvious subfamily preferences.\u003c/p\u003e\u003cp\u003eConclusions: \u003cem\u003eAhLEA\u003c/em\u003es are involved in abiotic stress response, and segmental duplication plays an important role in the evolution and amplification of \u003cem\u003eAhLEA\u003c/em\u003es. The genome-wide identification, classification, evolutionary and expression analyses of the \u003cem\u003eAhLEA\u003c/em\u003e gene family provide a foundation for further exploring the \u003cem\u003eLEA\u003c/em\u003e genes’ function in response to abiotic stress in peanuts.\u003c/p\u003e","manuscriptTitle":"Genome-Wide Identification, Evolutionary And Expression Analyses of LEA Gene Family In Peanut (Arachis Hypogaea L.)","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2021-08-31 18:16:43","doi":"10.21203/rs.3.rs-775523/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Major revision","date":"2021-09-23T06:51:14+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2021-09-13T04:38:06+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"21a20c42-4f4f-4c33-917e-4b9ca79ea936","date":"2021-08-27T07:55:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2021-08-27T05:30:46+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2021-08-27T02:09:13+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2021-08-27T01:45:06+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2021-08-27T01:41:27+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Plant Biology","date":"2021-08-02T09:17:44+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-plant-biology","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"pbio","sideBox":"Learn more about [BMC Plant Biology](http://bmcplantbiol.biomedcentral.com/)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/pbio/default.aspx","title":"BMC Plant Biology","twitterHandle":"BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"2c617f4d-6e29-4de2-b46e-64938a1dec3a","owner":[],"postedDate":"August 31st, 2021","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":6845034,"name":"Plant Molecular Biology and Genetics"},{"id":6845035,"name":"Plant Physiology and Morphology"}],"tags":[],"updatedAt":"2022-03-30T09:04:08+00:00","versionOfRecord":{"articleIdentity":"rs-775523","link":"https://doi.org/10.1186/s12870-022-03462-7","journal":{"identity":"bmc-plant-biology","isVorOnly":false,"title":"BMC Plant Biology"},"publishedOn":"2022-03-30 09:04:08","publishedOnDateReadable":"March 30th, 2022"},"versionCreatedAt":"2021-08-31 18:16:43","video":"","vorDoi":"10.1186/s12870-022-03462-7","vorDoiUrl":"https://doi.org/10.1186/s12870-022-03462-7","workflowStages":[]},"version":"v1","identity":"rs-775523","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-775523","identity":"rs-775523","version":["v1"]},"buildId":"B-jG_2CBjPDmsCi4Wdhf-","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}