2.1. Identification and characteristics of AhLEA gene in peanut
By using the publicly available peanut genome sequence data, the genome-wide identification of LEAs in peanuts was based on sequence homology with 51 Arabidopsis LEAs (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 AhLEAs were identified. All of these genes were grouped with 51 AtLEAs by phylogenetic analyses. The AhLEAs 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 LEA3s and LEA5s had 14 and 10 members, respectively. The LEA1s 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 AhLEAs
The identified 126 AhLEAs were further located on the 20 chromosomes (Fig. 2). The largest number of AhLEAs 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 AhLEAs and four chromosomes (chr5, chr7, chr19, and chr20) carried five AhLEAs. Chromosomes 9, 10, and 11 contained four AhLEAs, and chromosomes 17, 18 contained three AhLEAs. The AhLEAs were distributed unevenly among the 20 chromosomes in peanut.
Different LEA subfamilies were located on different chromosomes. All chromosomes contained the LEA2s, and all of the LEAs on chromosomes 3, 8, 10, 13, 18, and 20 belonged to the LEA2s. The LEA1s were distributed on chromosomes 1, 6, 9, 11, 16, and 19. The LEA3s were mainly distributed on chromosomes 1, 2, 4, 6, 11, 12, 14, and 16. The LEA4s were mainly found on chromosomes 6, 12, and 16. The LEA5s were distributed on chromosomes 2, 12, 14, and 16. The PvLEA18s were distributed on chromosomes 1, 5, 11, and 15. The SMPs were distributed on chromosomes 2, 6, 9, 12, and 19. The Dehydrins were distributed on chromosomes 7, 15, and 17. Chromosomal location analysis of AhLEAs 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 AhLEAs (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 LEA5s and LEA2s, and located on chromosomes 12 and 15. The segmental duplication genes were mainly distributed on chromosome 12. All members of the LEA1s, SMPs, and PvLEA18s were segmental duplication genes, followed by LEA2s (79.5 %) and LEA3s (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. AhLEAs 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 AhLEAs were associated with multiple AtLEAs, and fourteen AhLEAs were associated with GmLEAs. Additionally, there were nine cases that peanut segmental duplications that corresponded to a single Arabidopsis gene, and eleven cases that AhLEAs 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 LEAs in peanut
To examine the structural characteristics of AhLEAs, 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 AhLEAs 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 LEA1s and Dehydrins contained only one intron, and the main members of the LEA3 and LEA5 subfamilies had one intron. The majority of the LEA2s 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 AhLEAs with closer evolutionary relationships had more similar motif numbers. MEME analysis revealed that most AhLEAs contained motif 3 and all the LEA4s and LEA1s had motif 13. The LEA2s 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 AhLEAs
To investigate the cis-acting elements of AhLEAs, 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 AhLEAs at different stages of embryo development
To investigate the expression profiles of AhLEAs 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 AhLEAs were expressed at the four embryo development stages. Meanwhile, twenty-seven genes were not detected at any tested stages. Sixty-eight LEAs had different expression levels among the four stages. In the early embryo development stages, most LEA3s were up-regulated. Among them, three LEA3s (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, AhLEA1s, AhLEA4s, and AhLEA5s were up-regulated mainly in the late stages. Four genes including two LEA5s (AH12G24910.1 and AH12G24920.1) and two LEA1s (AH06G01030.1 and AH16G03650.1) exhibited very high expression levels in the late stages. Two genes of the Dehydrins 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 AhLEA2s 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 LEA1s, LEA2s, LEA3s, LEA4s LEA5s, SMPs, PvLEA18s, and Dehydrins, were similar in roots, stems, leaves, and flowers. Among them, the members of LEA2s, LEA3s, and Dehydrins were expressed at a high level in all four tissues. Twenty-four LEAs were highly expressed in roots, 21 in stems, 15 in leaves, and 20 in flowers. Two Dehydrins (AH07G18700.1 and AH17G19710.1) and two LEA3s (AH16G06810.1 and AH06G03960.1) had the highest expression levels in the stem (Additional file 1: Table S5).
2.6. Expression profiles of AhLEAs in response to drought and low-temperature stresses.
To investigate the transcriptional changes of the AhLEAs 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 AhLEAs 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 LEA3s (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 AhLEAs 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 LEA2s were up-regulated and 11 genes were down-regulated. It is noteworthy that all Dehydrins 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 LEA5s 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 AhLEAs under drought and low-temperature stresses, the responses of 126 AhLEAs 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 LEAs were induced more than 2-fold, while 19 LEAs 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 AhLEAs was up-regulated more than 2-fold after ethylene treatment, while 28 genes were down-regulated more than 2-fold. The expression of 10 AhLEAs was up-regulated more than 2-fold after salicylic acid treatment, while 16 genes were down-regulated more than 2-fold. Although the main AhLEAs were down-regulated by these four hormones, half of the LEA3s (7 out of 14) were up-regulated more than 2-fold after ethylene treatment, all members of LEA4s were induced by salicylic acid. Moreover, the expression of five AhLEAs was up-/down-regulated more than 2-fold by all four tested hormones. These genes included four LEA2s (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 AhLEAs under Al stress
To gain a broader understanding of the putative functions of peanut LEAs 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 AhLEAs were found to be aluminum stress-responsive genes (Fig. 13, Additional file 1: Table S8). LEA2s which included twenty-three DEGs had the most aluminum stress-responsive genes. All of the members in LEA4s and Dehydrins 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 PvLEA18s, LEA5s, SMPs (and LEA1s), and LEA3s, respectively.
Five AhLEAs were significantly up-regulated after 8h of Al treatment in ZH2. Seventeen AhLEAs were significantly up-regulated after 8h of Al treatment in 99-1507, including 66 % of LEA4s and Dehydrins (2 out of 3) and 50 % of SMPs (3 out of 6). Nineteen AhLEAs were significantly up-regulated after 24 h of Al treatment in ZH2, including all members of LEA5s and PvLEA18s and half of LEA1s (4 out of 8) and SMPs (3 out of 6). Nine AhLEAs were significantly up-regulated after 24 h of Al treatment in 99-1507. Twelve AhLEAs were down-regulated after 8h of Al treatment in ZH2, and all PvLEA18s were down-regulated. Two AhLEAs were down-regulated after 8h of Al treatment in 99-1507. Seven AhLEAs were down-regulated after 24h of Al treatment in ZH2. Eight AhLEAs 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 AhLEAs were regulated under drought, low temperature, and Al stress. Among these genes, 35 common AhLEAs were involved in the responses to low-temperature and drought stresses, 29 common AhLEAs that were involved in the responses to drought and Al stresses, and 22 common AhLEAs that were involved in the responses to low-temperature and Al stresses. Sixteen AhLEAs were overlaps among the three abiotic stresses (Additional file 2: Fig S1).