Identification and Expression Profile of NCED Genes in Arachis hypogaea L. during Drought Stress

Peanut (Arachis hypogaea L.) is an important crop that provides essential proteins and oils for human and animal consumption. 9-cis-epoxycarotenoid dioxygenase (NCED) have been found can play a vital role in abscisic acid (ABA) biosynthesis and may be a response to drought stress. Until now, in Arachis hypogaea, no information about the NCED gene family has been reported and the importance of NCED-related drought tolerance is unclear. In this study, eight NCED genes in Arachis hypogaea, referred to as AhNCEDs, are distributed across eight chromosomes, with duplication events in AhNCED1 and AhNCED2, AhNCED3 and AhNCED4, and AhNCED6 and AhNCED7. Comparative analysis revealed that NCED genes are highly conserved among plant species, including Pisum sativum, Phaseolus vulgaris, Glycine max, Arabidopsis thaliana, Gossypium hirsutum, and Oryza sativa. Further promoter analysis showed AhNCEDs have ABA-related and drought-inducible elements. The phenotyping of Arachis hypogaea cultivars NH5 and FH18 demonstrated that NH5 is drought-tolerant and FH18 is drought-sensitive. Transcriptome expression analysis revealed the differential regulation of AhNCEDs expression in both NH5 and FH18 cultivars under drought stress. Furthermore, compared to the Arachis hypogaea cultivar FH18, the NH5 exhibited a significant upregulation of AhNCED1/2 expression under drought. To sum up, this study provides an insight into the drought-related AhNCED genes, screened out the potential candidates to regulate drought tolerance and ABA biosynthesis in Arachis hypogaea.


Introduction
Drought is one of the most serious abiotic stresses, can cause a series of injury symptoms in plants and decline the global crop yields [1,2].In order to adapt to drought stress, plants have improved their physiological and biochemical metabolism, forming a complex and effective signal network, including ABA biosynthesis pathway [3].
The peanut (Arachis hypogaea L.), a widely cultivated crop, is an important source of dietary protein and oils [17,18].The growth and development of Arachis hypogaeas are easily affected by drought stress, especially during the seedling stage [19].In addition, drought is particularly detrimental to Arachis hypogaea kernel production [20][21][22][23][24].Under drought conditions, Arachis hypogaea plants exhibit various physiological and morphological alterations such as leaf curling, wilting, and senescence [25].These changes reflect adaptive strategies to minimise water loss and cope with the hydric stress [25].
Until now, although the diversity, distribution, and regulation of the NCED gene family have been investigated in some plant species like Populus davidiana, Gossypium hirsutum, and Oryza sativa and its participation in drought resistance has been explicated [13][14][15], there is limited information on Arachis hypogaea.In order to explore the drought-related NCED in Arachis hypogaea, this study used bioinformatic methods to identify Arachis hypogaea NCED genes and explored their basic characteristics.Additionally, the expression patterns of AhNCED genes related to drought stresses between drought-tolerant and droughtsensitive Arachis hypogaea varieties were also investigated.Finally, this study emphasised the immediate response of drought-related AhNCED genes, screened out the potential candidate to regulate drought tolerance and ABA biosynthesis.

Identification and Characterisation of AhNCEDs
To identify the NCED genes in the Arachis hypogaea genome, NCED genes from Pisum sativum, Phaseolus vulgaris, Glycine max, Arabidopsis thaliana, Gossypium hirsutum, and Oryza sativa were used as queries to BLAST against the Peanut Genome Resource (PGR) database.A total of eight AhNCED genes were identified.The protein sequences of AhNCEDs range from 546 to 642 amino acids, with molecular weights in the range of 6.16~7.14kD (Table 1).In addition, the theoretical pI of the AhNCEDs varies from 5.66 to 8.49, indicating different charges under different pH conditions (Table 1).Specifically, the average GRAVY value of these proteins was negative, suggesting that they were hydrophilic proteins (Table 1).The instability coefficients of the AhNCEDs range from 31.72 to 43.00, with a maximum of 43.00 for the most unstable NCED protein, indicating that most of them are stable proteins (Table 1).In addition, the gene subfamilies and relationships were further investigated.A phylogenetic tree was constructed using NCED protein sequences from Pisum sativum (PsNCED), Phaseolus vulgaris (PvNCED), Gossypium max (GmNCED), Arabidopsis thaliana (AtNCED), Gossypium hirsutum (GhNCED), and Oryza sativa (OsNCED) (Figure 1).The analysis revealed that NCED proteins in these species could be classified into five subgroups: I, II, III, IV, and V. Based on the evolutionary analysis of protein sequences (Figure 1), these eight AhNCEDs were also classified into four subgroups, labelled as I, III, IV, and V, with four, two, one, and one members, respectively (Table 1).Genes in the same subgroup exhibited high sequence similarity and close genetic relationships, indicating the presence of homologous structures.Notably, the selected proteins displayed a relatively high bootstrap value, particularly in the Fabaceae family, indicating their high conservation among these species (Figure 1).Consequently, the bootstrap values close to 100 in the phylogenetic analysis indicate a high level of confidence in the evolutionary relationship between AhNCED1 and AhNCED2 and their close relatives PsNCED1, PvNCED1, and GmNCED1-2 within the Fabaceae family (Figure 1).Furthermore, A. thaliana in the Brassicaceae family, G. hirsutum in the Malvaceae family, and O. sativa in the Poaceae family also exhibited a relatively high bootstrap value in these five subgroups, suggesting that the NCED gene belongs to an ancient gene family that is conserved across different plant species during their evolutionary differentiation (Figure 1).

Analysis of Conserved Motifs and Domains of AhNCEDs
Consistent patterns in the quantity and assortment of conserved motifs were observed within each AhNCED protein subgroup.AhNCED1 and AhNCED2 from subgroup I shared eight motifs, AhNCED3 and AhNCED4 from subgroup I shared ten motifs, while AhNCED6 and AhNCED7 from subgroup III each contained five identical motifs (Figure 2).Additionally, the other four AhNCED proteins each displayed a set of seven distinct motifs, specifically motifs 1-5 and motif 8, with AhNCED8 being the exception as it lacks motif 6 (Figure 2).The subgroups' shared and distinct motif patterns suggest significant structural conservation and specific functions within the AhNCEDs.All AhNCED proteins possess the RPE65 domain, indicating that AhNCEDs are highly conserved across species from different families (Figure 2).The exclusive presence of the RPE65 domain in the NCED family underscores its critical role in the functionality and structural integrity of NCED proteins.The RPE65 domain predominantly occupies positions within the NCED protein sequence (Figure 2), highlighting its importance for the activity and stability of the NCED enzyme.The multiple sequence alignment of AhNCED domains revealed numerous conserved amino acids, particularly histidine (H), glycine (G), and threonine (T), signifying a high degree of conservation in the domain sequences of AhNCEDs (Figure 3).

Gene Structure Prediction of AhNCEDs
By comparing with gene structure maps, it is evident that NCED genes in different subgroups within the same species exhibit significant differences in the number and

Gene Structure Prediction of AhNCEDs
By comparing with gene structure maps, it is evident that NCED genes in different subgroups within the same species exhibit significant differences in the number and length of their exons and introns (Figure 4).Conversely, genes within the same subgroup share similar lengths and have comparable numbers and lengths of introns and exons (Figure 4), resulting in identical gene types.For instance, the subgroups I and II consist of only one or two exons (Figure 4).Interestingly, genes in the same subfamily, such as AhNCED1, AhNCED2, PsNCED1, PvNCED1, and GmNCED1, possess only a single exon, highlighting a unique structural characteristic within this group (Figure 4).All NCED genes in subgroup III contain 14 exons (Figure 4).The NCED genes in subgroup III contain five or six exons, while the NCED gene in subgroup IV comprises 14 exons (Figure 4).Subgroup V includes thirteen NCED genes, with eight genes containing 13 exons, four genes containing 12 exons (OsNCED7, AhNCED8, GhNCED12, GhNCED13), and one gene containing 10 exons (PvNCED4) (Figure 4).Therefore, the gene structures of the same subgroup exhibit similarities, including the number and length of introns and exons.AhNCED1, AhNCED2, PsNCED1, PvNCED1, and GmNCED1, possess only a single exon, highlighting a unique structural characteristic within this group (Figure 4).All NCED genes in subgroup Ⅲ contain 14 exons (Figure 4).The NCED genes in subgroup Ⅲ contain five or six exons, while the NCED gene in subgroup Ⅳ comprises 14 exons (Figure 4).Subgroup V includes thirteen NCED genes, with eight genes containing 13 exons, four genes containing 12 exons (OsNCED7, AhNCED8, GhNCED12, GhNCED13), and one gene containing 10 exons (PvNCED4) (Figure 4).Therefore, the gene structures of the same subgroup exhibit similarities, including the number and length of introns and exons.

Stress-Related Cis-Elements in the Promoters of AhNCEDs
Cis-acting elements located in the promoter regions of genes initiate and regulate gene expression.The analysis revealed that the 2000 bp sequence upstream of the translational start site of each AhNCEDs contains 163 cis-acting elements related to hormones and stress responses (Figure 5; Table S2).The potential cis-elements of AhNCEDs are implicated in multiple responses to hormones salicylic acid (SA), methyl jasmonate (MeJA), gibberellin (GA), abscisic acid (ABA), auxin (IAA), and stress (light, anaerobic, defence and stress, drought, low-temperature) (Figure 5).Interestingly, nearly all the promoters of Arachis hypogaeaNCED genes contained cis-acting elements related to plant hormones and stress responsiveness (Figure 5).Additionally, a large number of SA-related (4.91%), MeJA-related (6.13%), GA-related (3.07%), ABA-related (19.63%), and IAA-related (1.84%) cis-acting elements are found in the AhNCEDs promoters (Figure S2).Furthermore, AhNCED3-4 and AhNCED8 contained drought-inducible elements, each harbouring four, four and one such element(s), respectively (Figure 5; Table S2).Moreover, a total of 32 ABA-related elements were identified, with AhNCED1, AhNCED2, AhNCED3, AhNCED4, AhNCED6, and AhNCED7 containing the highest numbers of these elements, which were eight, six, five, four, four, and three elements, respectively (Figure 5; Table S2).Further details on the elements and functions of individual genes are listed in Supplementary Table S2.

Stress-Related Cis-Elements in the Promoters of AhNCEDs
Cis-acting elements located in the promoter regions of genes initiate and regulate gene expression.The analysis revealed that the 2000 bp sequence upstream of the translational start site of each AhNCEDs contains 163 cis-acting elements related to hormones and stress responses (Figure 5; Table S2).The potential cis-elements of AhNCEDs are implicated in multiple responses to hormones salicylic acid (SA), methyl jasmonate (MeJA), gibberellin (GA), abscisic acid (ABA), auxin (IAA), and stress (light, anaerobic, defence and stress, drought, low-temperature) (Figure 5).Interestingly, nearly all the promoters of Arachis hypogaeaNCED genes contained cis-acting elements related to plant hormones and stress responsiveness (Figure 5).Additionally, a large number of SA-related (4.91%), MeJA-related (6.13%), GA-related (3.07%), ABA-related (19.63%), and IAA-related (1.84%) cis-acting elements are found in the AhNCEDs promoters (Figure S2).Furthermore, AhNCED3-4 and AhNCED8 contained drought-inducible elements, each harbouring four, four and one such element(s), respectively (Figure 5; Table S2).Moreover, a total of 32 ABA-related elements were identified, with AhNCED1, AhNCED2, AhNCED3, AhNCED4, AhNCED6, and AhNCED7 containing the highest numbers of these elements, which were eight, six, five, four, four, and three elements, respectively (Figure 5; Table S2).Further details on the elements and functions of individual genes are listed in Supplementary Table S2.

Chromosomal Localisation of AhNCEDs
For chromosomal location analysis, 38 NCED genes are located on 23 chromosomes, with 12 (Gossypium hirsutum), 3 (Arabidopsis thaliana), and 8 (Arachis hypogae) chromosomes, respectively.The chromosome analysis of the NCED gene family across different species revealed an uneven distribution of genes across various chromosomes (Figure 6).For instance, eight AhNCED genes were found on seven chromosomes, with Chr10 containing the highest number of AhNCED genes (Figure 6).Interestingly, AhNCED6 and AhNCED8 were closely clustered on Chr10, indicating possible tandem repeat gene duplication (Figure 6).The AtNCED genes exhibited a relatively uniform distribution on chromosomes one, three, and four, whereas in Gossypium hirsutum, NCED genes are distributed inconsistently across different chromosomes (Figure 6).A total of fifteen GhNCED genes were identified on twelve chromosomes (Figure 6).Furthermore, the NCED genes in these three species are distributed on three (Arabidopsis thaliana), seven (Arachis hypogae), and twelve (Gossypium hirsutum) chromosomes, with a number that approaches half of the chromosome complement, indicating the widespread distribution of NCED genes in the species.

Chromosomal Localisation of AhNCEDs
For chromosomal location analysis, 38 NCED genes are located on 23 chromosomes, with 12 (Gossypium hirsutum), 3 (Arabidopsis thaliana), and 8 (Arachis hypogae) chromosomes, respectively.The chromosome analysis of the NCED gene family across different species revealed an uneven distribution of genes across various chromosomes (Figure 6).For instance, eight AhNCED genes were found on seven chromosomes, with Chr10 containing the highest number of AhNCED genes (Figure 6).Interestingly, AhNCED6 and AhNCED8 were closely clustered on Chr10, indicating possible tandem repeat gene duplication (Figure 6).The AtNCED genes exhibited a relatively uniform distribution on chromosomes one, three, and four, whereas in Gossypium hirsutum, NCED genes are distributed inconsistently across different chromosomes (Figure 6).A total of fifteen GhNCED genes were identified on twelve chromosomes (Figure 6).Furthermore, the NCED genes in these three species are distributed on three (Arabidopsis thaliana), seven (Arachis hypogae), and twelve (Gossypium hirsutum) chromosomes, with a number that approaches half of the chromosome complement, indicating the widespread distribution of NCED genes in the species.

Collinearity Analysis of AhNCEDs
Collinearity analysis provides valuable information for identifying homologous gene pairs between species.Using Tbtools-II [31], a multicollinearity analysis of the NCED gene family was performed in the model plant Arabidopsis thaliana, the Fabaceae family A. hypogaea, and the drought-tolerant cotton Gossypium hirsutum (Figure 7).Five collinear genes were found between Arachis hypogaea and Arabidopsis thaliana and eighteen pairs between Arachis hypogaea and Gossypium hirsutum (Figure 7).The number of NCED genes was higher in polyploid species (Arachis hypogaea and Gossypium hirsutum) than in diploid species (Arabidopsis thaliana), indicating that polyploidisation may have contributed to gene family expansion.Most NCED genes had one-to-one orthologs between species, but some had multiple orthologs, indicating that they originated from a common ancestor.Collinearity analysis revealed the occurrence of tandem duplications in Arachis hypogaea in the collinearity plots (Figure 7), suggesting a potential tandem repeat gene duplication event in AhNCED1-2 and AhNCED3-4 as well as AhNCED6-7.For instance, AhNCED2 (on Chr07) and AhNCED1 (on Chr18) correspond to AtNCED4, GhNCED6, and GhNCED7; AhNCED3 (on Chr03) corresponds with AtNCED2, GhNCED1, GhNCED3, and GhNCED5.In a similar manner, AhNCED4 (on Chr13) corresponds with AtNCED2, GhNCED1, GhNCED3, and GhNCED5; AhNCED6 (on Chr10) and AhNCED7 (on Chr20) both correspond with GhNCED15 (Figures 7 and S3).The sequence alignment confirmed the three pairs of genes initially identified as potential tandem repeats to be three distinct sets of Gossypium hirsutum has 52 chromosomes, with NCED genes on 13 chromosomes.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gh: Gossypium hirsutum.

Collinearity Analysis of AhNCEDs
Collinearity analysis provides valuable information for identifying homologous gene pairs between species.Using Tbtools-II [31], a multicollinearity analysis of the NCED gene family was performed in the model plant Arabidopsis thaliana, the Fabaceae family A. hypogaea, and the drought-tolerant cotton Gossypium hirsutum (Figure 7).Five collinear genes were found between Arachis hypogaea and Arabidopsis thaliana and eighteen pairs between Arachis hypogaea and Gossypium hirsutum (Figure 7).The number of NCED genes was higher in polyploid species (Arachis hypogaea and Gossypium hirsutum) than in diploid species (Arabidopsis thaliana), indicating that polyploidisation may have contributed to gene family expansion.Most NCED genes had one-to-one orthologs between species, but some had multiple orthologs, indicating that they originated from a common ancestor.Collinearity analysis revealed the occurrence of tandem duplications in Arachis hypogaea in the collinearity plots (Figure 7), suggesting a potential tandem repeat gene duplication event in AhNCED1-2 and AhNCED3-4 as well as AhNCED6-7.For instance, AhNCED2 (on Chr07) and AhNCED1 (on Chr18) correspond to AtNCED4, GhNCED6, and GhNCED7; AhNCED3 (on Chr03) corresponds with AtNCED2, GhNCED1, GhNCED3, and GhNCED5.In a similar manner, AhNCED4 (on Chr13) corresponds with AtNCED2, GhNCED1, GhNCED3, and GhNCED5; AhNCED6 (on Chr10) and AhNCED7 (on Chr20) both correspond with GhNCED15 (Figure 7 and Figure S3).The sequence alignment confirmed the three pairs of genes initially identified as potential tandem repeats to be three distinct sets of duplicated genes (Figure S3).The collinearity analysis suggests that the NCED gene family underwent conservation and diversification during the evolution of Arachis hypogaea, Arabidopsis thaliana, and Gossypium hirsutum.
Int. J. Mol.Sci.2022, 23, x FOR PEER REVIEW 10 of 21 duplicated genes (Figure S3).The collinearity analysis suggests that the NCED gene family underwent conservation and diversification during the evolution of Arachis hypogaea, Arabidopsis thaliana, and Gossypium hirsutum.

Prediction of AhNCED Proteins Structures
Utilizing VP14 for protein structure simulation, it was observed that the majority of NCED proteins fold into a seven-bladed β-propeller structure, accompanied by four αhelices forming an α-helical domain at the top of the β-propeller (Figure 8).This structure is characterised by conserved features composed of three phenylalanine (Phe) amino acids, with some regions formed by two amino acids (Figures 8 and S4).

Prediction of AhNCED Proteins Structures
Utilizing VP14 for protein structure simulation, it was observed that the majority of NCED proteins fold into a seven-bladed β-propeller structure, accompanied by four α-helices forming an α-helical domain at the top of the β-propeller (Figure 8).This structure is characterised by conserved features composed of three phenylalanine (Phe) amino acids, with some regions formed by two amino acids (Figures 8 and S4).

Expression Profile of AhNCEDs under Drought Stress
The transcriptome data (NCBI database SRA accession number: PRJNA657965) were studied by treating with 20% PEG6000 at time points of 0 h, 4 h, 8 h, and 24 h [32].It is clear that the expression levels of AhNCED1 and AhNCED2 were significantly increased, exhibiting a fold change under 20% PEG6000-induced drought stress (Tables S2 and S3).Specifically, the expression levels of AhNCED1 and AhNCED2 gradually decreased as the treatment time extended (Figure 9).The expression levels of AhNCED6 and AhNCED8 reached their maximum at 4 h, AhNCED3 and AhNCED4 peaked at 8 h, and AhNCED5 peaked at 0 h.These results suggest that AhNCED1 and AhNCED2 may significantly re-

Expression Profile of AhNCEDs under Drought Stress
The transcriptome data (NCBI database SRA accession number: PRJNA657965) were studied by treating with 20% PEG6000 at time points of 0 h, 4 h, 8 h, and 24 h [32].It is clear that the expression levels of AhNCED1 and AhNCED2 were significantly increased, exhibiting a fold change under 20% PEG6000-induced drought stress (Tables S2 and S3).Specifically, the expression levels of AhNCED1 and AhNCED2 gradually decreased as the treatment time extended (Figure 9).The expression levels of AhNCED6 and AhNCED8 reached their maximum at 4 h, AhNCED3 and AhNCED4 peaked at 8 h, and AhNCED5 peaked at 0 h.These results suggest that AhNCED1 and AhNCED2 may significantly respond to plant drought stress, exhibiting temporal variations in their regulation.

Phenotyping of Two Arachis Hypogaea Varieties under Drought Stress
After 10 days of drought treatment, the drought-tolerant Arachis hypogaea varie NH5 exhibited minor stress symptoms compared to the control group, which was treate with distilled water.These symptoms included slight leaf curling, indicating its relative strong drought resistance (Figure 10A,B).
In contrast, the drought-sensitive Arachis hypogaea variety FH18 showed significa stress symptoms after the drought treatment compared to the control group.These symp toms were characterised by notable leaf curling and wilting, with leaves becoming dr and brittle and changing colour from green to brown (Figure 10C,D).These changes su gest a poor adaptation of FH18 to drought conditions.About the overall plants, the 10-day drought treatment led both Arachis hypogaea v rieties to exhibit phenotypic changes, including a reduction in overall size and thinn stems (Figure 10E-H).However, the stress symptoms of FH18 were more severe tha those observed in NH5, further emphasising the significant differences in drought r sistance between them.From the results of Heatmaps A and B, AhNCED1 and AhNCED2 displayed similar expression patterns under various levels of drought stress, consistently showing higher expression levels.The overall expression levels of AhNCED3 and AhNCED4, and AhNCED6 and AhNCED8 were relatively high.In contrast, AhNCED5 genes exhibited overall low expression levels, with no significant differences between the two Arachis hypogaea cultivars (Figure 9).The expression patterns and levels of different genes varied between the two Arachis hypogaea cultivars (NH5 and FH18).

Phenotyping of Two Arachis Hypogaea Varieties under Drought Stress
After 10 days of drought treatment, the drought-tolerant Arachis hypogaea variety NH5 exhibited minor stress symptoms compared to the control group, which was treated with distilled water.These symptoms included slight leaf curling, indicating its relatively strong drought resistance (Figure 10A,B).

Expression Analysis of AhNCEDs in Arachis Hypogaea Varieties under Drought Stress
Owing to the fact that the two genes are in tandem repeats and exhibit a high degree of sequence similarity, it is not feasible to design fluorescent quantitative PCR primers that can distinguish between them.Consequently, the symbol '/' is employed to denote the two individual genes.The quantitative PCR results and heatmap expression profiles were broadly consistent, indicating that AhNCED1/2 exhibited the most significant expression at all eight-time points, markedly higher than the other six genes (Figure 11b,c).AhNCED3/4 and AhNCED5 displayed moderate expression levels, while AhNCED6/7 and AhNCED8 showed lower expression levels (Figure 11d,e).
Under drought stress, both NH5 (drought-tolerant) and FH18 (drought-sensitive) varieties showed synchronous expression patterns.The AhNCED1/2 genes demonstrated notable upregulated expression over the 10 days, whereas AhNCED3/4 only showed increased expression within the first 8 h, peaked and then declined (Figure 11b).The expression of AhNCED5 generally decreased over time, while AhNCED6/7 and AhNCED8 peaked at 4 h and then fell to an almost non-detectable level (Figure 11c-e).
In both the NH5 (drought-tolerant) and FH18 (drought-sensitive) varieties, the relative expression levels of different genes simultaneously showed consistency across the two varieties.The expression levels in NH5 were initially lower than in FH18 but became higher over time (Figure 11a).For AhNCED1-4 at 0, 4, and 8 h, the expression levels in NH5 were lower than in FH18, but at the other five time points (1, 3, 5, 7, and 10 days), NH5 exhibited higher expression than FH18 (Figure 11a,b).For AhNCED5 and AhNCED8, in the first four time points (0 h, 4 h, 8 h, 1 d), NH5 had relatively lower expression than FH18 but higher expression in the latter four time points (Figure 11c,e).For AhNCED6/7, in the first two time points (0 h, 4 h), NH5 showed relatively higher expression than FH18 but lower expression in the subsequent six time points (Figure 11d).
After the fifth day, different expression levels of the AhNCED1/2 gene were observed in two Arachis hypogaea cultivars.In NH5 (drought-tolerant), the expression levels at 5 days, 7 days, and 10 days were approximately 2.55, 3.77, and 8.04 times higher, respectively, compared to the 0 h timepoint (standard water) (Figure 11a).In contrast, FH18 (drought-sensitive) showed about 1.05, 1.30, and 1.98 times the expression levels at 5 days, 7 days, and 10 days, respectively, compared to 0 h (standard water) (Figure 11a).In contrast, the drought-sensitive Arachis hypogaea variety FH18 showed significant stress symptoms after the drought treatment compared to the control group.These symptoms were characterised by notable leaf curling and wilting, with leaves becoming dry and brittle and changing colour from green to brown (Figure 10C,D).These changes suggest a poor adaptation of FH18 to drought conditions.About the overall plants, the 10-day drought treatment led both Arachis hypogaea varieties to exhibit phenotypic changes, including a reduction in overall size and thinner stems (Figure 10E-H).However, the stress symptoms of FH18 were more severe than those observed in NH5, further emphasising the significant differences in drought resistance between them.

Expression Analysis of AhNCEDs in Arachis Hypogaea Varieties under Drought Stress
Owing to the fact that the two genes are in tandem repeats and exhibit a high degree of sequence similarity, it is not feasible to design fluorescent quantitative PCR primers that can distinguish between them.Consequently, the symbol '/' is employed to denote the two individual genes.The quantitative PCR results and heatmap expression profiles were broadly consistent, indicating that AhNCED1/2 exhibited the most significant expression at all eight-time points, markedly higher than the other six genes (Figure 11b,c).AhNCED3/4 and AhNCED5 displayed moderate expression levels, while AhNCED6/7 and AhNCED8 showed lower expression levels (Figure 11d,e).
Under drought stress, both NH5 (drought-tolerant) and FH18 (drought-sensitive) varieties showed synchronous expression patterns.The AhNCED1/2 genes demonstrated notable upregulated expression over the 10 days, whereas AhNCED3/4 only showed increased expression within the first 8 h, peaked and then declined (Figure 11b).The expression of AhNCED5 generally decreased over time, while AhNCED6/7 and AhNCED8 peaked at 4 h and then fell to an almost non-detectable level (Figure 11c-e).

Discussion
A wealth of research has demonstrated the ubiquity of the NCED gene family in plants, which are highly conserved across diverse lineages [21].An unrooted phylogenetic tree was constructed to study the distribution of these genes, which was then classified into five subgroups (I-V) (Figures 1 and S1).The number of motifs, the number of exons in a gene, and the gene lengths were similar within the same subgroup (Figures 2 and 4).Additionally, all NCED genes only contain the RPE65 domain.The correspondence between AhNCED1 and AhNCED2, AhNCED3 and AhNCED4, and AhNCED6 and AhNCED7 suggests that these genes share a common ancestor and have been retained throughout evolution (Figure 7).These gene pairs belonged to the same subgroup in the phylogenetic tree (Figure 1), and exhibited high similarity regarding their gene structures, conserved motifs, and domains (Figures 2 and 4), indicating that their evolution has been conserved.
Tandem repeat gene duplication occurs in Arachis hypogaea.Gene replication refers to a gene duplicating in the genome, which can occur through various mechanisms such as haploid genome polyploidisation, chromosome recombination, and gene transposition In both the NH5 (drought-tolerant) and FH18 (drought-sensitive) varieties, the relative expression levels of different genes simultaneously showed consistency across the two varieties.The expression levels in NH5 were initially lower than in FH18 but became higher over time (Figure 11a).For AhNCED1-4 at 0, 4, and 8 h, the expression levels in NH5 were lower than in FH18, but at the other five time points (1, 3, 5, 7, and 10 days), NH5 exhibited higher expression than FH18 (Figure 11a,b).For AhNCED5 and AhNCED8, in the first four time points (0 h, 4 h, 8 h, 1 d), NH5 had relatively lower expression than FH18 but higher expression in the latter four time points (Figure 11c,e).For AhNCED6/7, in the first two time points (0 h, 4 h), NH5 showed relatively higher expression than FH18 but lower expression in the subsequent six time points (Figure 11d).
After the fifth day, different expression levels of the AhNCED1/2 gene were observed in two Arachis hypogaea cultivars.In NH5 (drought-tolerant), the expression levels at 5 days, 7 days, and 10 days were approximately 2.55, 3.77, and 8.04 times higher, respectively, compared to the 0 h timepoint (standard water) (Figure 11a).In contrast, FH18 (drought-sensitive) showed about 1.05, 1.30, and 1.98 times the expression levels at 5 days, 7 days, and 10 days, respectively, compared to 0 h (standard water) (Figure 11a).

Discussion
A wealth of research has demonstrated the ubiquity of the NCED gene family in plants, which are highly conserved across diverse lineages [21].An unrooted phylogenetic tree was constructed to study the distribution of these genes, which was then classified into five subgroups (I-V) (Figure 1 and Figure S1).The number of motifs, the number of exons in a gene, and the gene lengths were similar within the same subgroup (Figures 2 and 4).Additionally, all NCED genes only contain the RPE65 domain.The correspondence between AhNCED1 and AhNCED2, AhNCED3 and AhNCED4, and AhNCED6 and AhNCED7 suggests that these genes share a common ancestor and have been retained throughout evolution (Figure 7).These gene pairs belonged to the same subgroup in the phylogenetic tree (Figure 1), and exhibited high similarity regarding their gene structures, conserved motifs, and domains (Figures 2 and 4), indicating that their evolution has been conserved.
Tandem repeat gene duplication occurs in Arachis hypogaea.Gene replication refers to a gene duplicating in the genome, which can occur through various mechanisms such as haploid genome polyploidisation, chromosome recombination, and gene transposition [33,34].This process enhances genome diversity and complexity, providing resources for gene evolution [35,36].Based on the phylogenetic analysis, It was observed that Ah-NCED1 and AhNCED2, AhNCED3 and AhNCED4 as well as AhNCED6 and AhNCED7, clustered together (Figure 1), and were found to be homologous to Arabidopsis thaliana and Gossypium hirsutum in the collinearity analysis (Figure 7 and Figure S3).These results strongly support the occurrence of tandem repeat gene duplication events in AhNCED1-2 and AhNCED3-4 as well as AhNCED6-7.NCED gene replication is not exclusive to Arachis hypogaea, as it has also been observed in other plants, such as upland Gossypium hirsutum and Arabidopsis thaliana [12].Moreover, duplication events can effectively improve plants' ability to adapt to various environments [37], and NCED gene duplication is likely to enhance drought tolerance in Arachis hypogaea.Interestingly, four NCED genes (GhNCED3, GhNCED13, GhNCED5, GhNCED15) were present on ChrD05 and ChrD06 of Gossypium hirsutum, respectively, with duplicated homologs in Arachis hypogaea (Figure 7).Therefore, the presence of these genes on the two chromosomes in Gossypium hirsutum is likely due to genome duplication events, as Gossypium hirsutum and Arachis hypogaea may share a common ancestor, and these genes have been retained during evolution.Based on the analysis of conserved structural domains, sequence alignment results, and collinearity analysis, the analysis revealed that AhNCED contained only one RPE65 domain and three pairs of tandemly duplicated genes (Figures 2c, 3 and 7; Figure S3).This suggests that a gene family contraction may have occurred in Arachis hypogaea during its evolutionary process.Gene duplication events in AhNCED genes enhance plant adaptability and offer valuable traits for agricultural biotechnology to improve resilience to climate stress.
The presence of ABA-related and drought-inducibility elements in the promoters of NCED genes provides further evidence for their crucial roles in drought response processes.Hormone signalling, particularly ABA, MeJA, and SA, plays a vital role in plant stress resistance [38].The induction of ABA biosynthesis by NCED in response to drought is observed in many other plants, including Populus davidiana, Gossypium hirsutum, and Oryza sativa [13][14][15].In A. hypogaea, AhNCED1 and AhNCED2 possess the highest number of ABA-related elements, witsix eight and 6 elements, respectively (Figure 5; Table S2).In addition, AhNCED1 contains two MeJA elements and one SA element, while AhNCED2 contains one SA element (Table S2).ABA can interact with hormones such as MeJA and SA [16].Therefore, the expression levels of AhNCED1 and AhNCED2 were significantly upregulated under 20% PEG6000-induced drought stress in the NH5 (drought-tolerant) and FH18 (drought-sensitive) cultivars (Figure 9).These genes exhibit high expression levels in the heatmaps, confirming their essential roles in defence responses to various stresses and plant hormones (Figure 9).Understanding the interaction between AhNCED genes and ABA synthesis can aid in developing crops with superior drought tolerance, addressing the increasing global frequency of drought conditions.
The AhNCEDs responds to drought stress, exhibiting differential expression patterns among its members and across cultivars.Specifically, we observed temporal differences in the expression levels of the NCED genes.Notably, AhNCED1-2 showed a gradual decrease in expression (Figure 9), yet its expression exhibited a high fold change in drought-tolerant NH5 and drought-sensitive cultivar FH18 (Tables S3 and S4).Interestingly, the fold changes in NH5 were less pronounced than those in FH18, suggesting that NH5 may not require as high expression levels of these genes to cope with drought stress.This pattern mirrors observations in other plant species, where drought-tolerant cultivars often exhibit lower expression levels than drought-sensitive ones.For example, the StDRO1 gene in potato and the asparagine synthase and AMPD genes in rice display similar trends [39,40].Due to the identical amino acid sequences in the promoter regions, approximately 300 bp upstream, of AhNCED1/2, AhNCED3/4, and AhNCED6/7, three pairs of similar primers were designed (Table S4).Based on qRT-PCR results, a significant finding of our study is the temporal dynamics of gene expression in NH5 compared to FH18.Initially, AhNCED genes in NH5 showed lower expression levels than in FH18.However, as the stress period progressed, NH5 demonstrated a relative increase in expression levels, particularly for AhNCED1/2 (Figure 11).This pattern, where NH5 starts with lower expression but eventually surpasses FH18, indicates a delayed but robust activation of drought response mechanisms in the drought-tolerant variety (Figure 11).
This study underscores the crucial role of AhNCED1 and AhNCED2 in Huayu2 ′ 's drought tolerance, with significant upregulation observed under drought conditions (Figures 9b and 11).These two genes exhibited the highest expression increase among the AhNCEDs, particularly in NH5, highlighting their crucial role in enhancing drought resistance (Figures 9b and 11).In contrast, the drought-sensitive FH18 showed more moderate gene expression changes (Figures 9b and 11).Post 10-day drought treatment, phenotypic analysis revealed minor stress symptoms in NH5, like slight leaf curling, aligning with its strong genetic drought resistance (Figure 10A,B).In contrast, FH18, a drought-sensitive variety, exhibited severe stress symptoms, including leaf curling, wilting, and discolouration (Figure 10C,D), correlating with lower AhNCED1/2 expression.Both varieties showed phenotypic changes like reduced size and thinner stems (Figure 10E-H), but FH18 symptoms were more severe, highlighting the distinct drought resistance between the varieties.These results emphasise that AhNCED1 and AhNCED2 are important in drought tolerance and shed light on the genetic basis of drought resistance in Arachis hypogaea, supporting efforts to genetically improve crops for greater resilience against climate change.

Plant Material and Treatments
The seeds of Arachis hypogaea cultivars (NH5 and FH18, also named Huayu22 and-Huayu23, respectively) was gifted by Ms. Lina Yang from the College of Geography and Oceanography at Minjiang University, Fuzhou, Fujian Province, China.Arachis hypogaea seeds were sowed and cultivated in the greenhouse at a 23 ± 2 • C, under long-day conditions featuring a 16 h light/8 h dark cycle, with a light intensity of 300 µmol m −2 s −1 .After 21 days, Arachis hypogaea seedlings were chosen for water interruption treatment and foliar application of 20% PEG6000.Samples were collected at different time points (0 h, 4 h, 8 h, 24 h, 3 d, 5 d, 7 d, 10 d) [32].Then, the samples were either used immediately or shock-frozen in liquid nitrogen and stored at −80 • C.

Phylogenetic Analysis of NCED Protein Sequences
The domain architecture of NCED protein sequences was analysed using the National Center for Biotechnology Information Conserved Domain Database (https://www.ncbi.nlm.nih.gov/cdd,accessed on 20 December 2023).All amino acid sequences lacking the required conserved structure were removed from the original sequence, resulting in a comprehensive list of NCED gene family members.Based on protein domain prediction results, a total of 48 protein sequences, including 8 AhNCED, 1 PsNCED, 5 PvNCED, 5 GmNCED, 7 AtNCED, 15 GhNCED and 7 OsNCED protein sequences, were retrieved from the NCBI database.Multiple sequence alignment of these 48 NCED protein sequences was performed using IQ-tree (http://www.iqtree.org/,accessed on 21 December 2023), and a maximum likelihood (ML) phylogenetic tree was constructed using the IQ-tree with JTT+R5 model (best-fit model) and 1000 bootstrap test.A Pisum sativum NCED protein sequence was selected as an outgroup to determine the evolutionary relationship between the NCED genes.The NCED genes were named based on their position on the phylogenetic tree.The resulting phylogenetic tree was visualised using the Interactive Tree of Life (iTOL) web tool [41].

Protein Motif and Domain Analysis and Multiple Sequence Alignments of AhNCEDs
The conserved domains of NCED proteins were detected using CDD while identifying NCED genes.Full-length protein sequences were analysed using the MEME web to identify conserved motifs.All the gene structures of NCED genes were identified and visualised using TBtools-II [31] based on the genome annotation gff3 files.To identify conserved motifs in NCED genes, the MEME v5.5.1 web server was employed using Zoop for site distribution and setting the maximum number of motifs to 8. Finally, the phylogenetic tree, conserved motifs, and conserved domains were combined and visualised using the Gene Location Visualise Advanced of TBtools-II (South China Agricultural University, Guangzhou, China) [31].The software also envisioned and merged genetic results and phylogenetic trees.Multiple sequence alignments were constructed using MEGA software using the ClustalW algorithm [42] and manually verified using the ESPript 3.0 web tool (http://espript.ibcp.fr/ESPript/ESPript/,accessed on 20 December 2023).Identifying the RPE65 domain and annotating the multiple sequences were conducted as previously described [29].

Evolutionary Analysis of NCED Genes
The genome annotation GFF3 files of Pisum sativum, Phaseolus vulgaris, Glycine max, Arabidopsis thaliana, Gossypium hirsutum, and Oryza sativa were downloaded from the NCBI database.And the genome annotation GFF3 files of A. hypogaea were downloaded from the Peanut Genome Resource (PGR) database (http://peanutgr.fafu.edu.cn/Download.php,accessed on 20 December 2023).TBtools-II [31] was used to analyse the structure of NCED genes in each species in combination with the evolutionary tree.The NCBI GenBank database (https://www.ncbi.nlm.nih.gov/genbank/,accessed on 20 December 2023) was also used to retrieve the exon quantity of the NCED genes.Adobe Illustrator software v26.0.3 (64-bit) was used for visualisation.

Cis-Elements Analysis of AhNCEDs Promoters
To identify stress-related cis-acting regulatory elements in the promoter sequences, the 2000 bp upstream regions (from the translation starting sites) of the NCED genes were extracted and examined using the PlantCARE web tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/,accessed on 21 December 2023).Predictions were made for ten stress-related cis-elements associated with the hormones salicylic acid (SA), methyl jasmonate (MeJA), gibberellin (GA), abscisic acid (ABA), auxin (IAA), and stress (light, anaerobic, defence and stress, drought, low-temperature).The PsNCED1 gene was also included as an outgroup to expand the analysis.Multiple sequence alignment of the eight Arachis hypogaea NCED protein sequences was performed using IQ-tree (http: //www.iqtree.org/,accessed on 21 December 2023), and a maximum likelihood (ML) phylogenetic tree with statistical support was constructed using the VT+I model (best-fit model) and 1000 bootstrap test.The cis-acting elements and the phylogenetic tree were then used to generate a figure using TBtools-II [31], which visualises the relationship between the NCED gene family and the stress-related cis-acting regulatory elements in the promoter sequences.

Chromosomal Localisation of AhNCEDs
The Gene Density Profile function of TBtools-II was utilised with a bin size of 100,000 and the genome annotation GFF3 files to obtain the gene density information of Arachis hypogaea, Arabidopsis thaliana, and Gossypium hirsutum.The gene location visualisation function of TBtools-II [31] was used to determine the chromosomal locations of NCED genes in these three species, with the genome annotation GFF3 files.The results were then plotted on the chromosome location of NCED genes, and the chromosome blanks were filled with gene density information.

Synteny Analysis of AhNCEDs
The genome annotation files of Arachis hypogaea, Arabidopsis thaliana, and Gossypium hirsutum were obtained from the NCBI database (https://www.ncbi.nlm.nih.gov/assembly/,accessed on 20 December 2023).To explore the synteny relationship of NCED genes among different species, synteny analysis was performed using the One Step MCScanX module of TBtools-II [31], with an E-value threshold of 10 −10 and five retained blast hits.Collinear gene pairs were identified using the BLASTP algorithm with an E-value threshold of 10 −10 , and the results were visualised using the Multiple Synteny Plot module of TBtools-II [31].The Clustal Omega website (https://www.ebi.ac.uk/Tools/msa/clustalo/, accessed on 22 December 2023) was used to perform multiple sequence alignment of nucleotide sequences of the tandem repeat sequences.

Modelling of AhNCEDs Proteins
Using PDB: VP14 (GeneBank database number: AAB62181.2) as a template and employing SWISS-MODEL (https://swissmodel.expasy.org/interactive,accessed on 23 December 2023), a homology model of NCED was constructed, and tertiary structure predictions were conducted for the NCED protein sequences identified in the transcriptome [43].Based on the target-template alignment, the highest-quality templates were determined and selected for model construction [43].The protein structures, along with the pocket amino acids, were visualised using PyMOL (https://pymol.org/2/,accessed on 23 December 2023).

Heatmap of AhNCEDs Gene Expression Patterns
Furthermore, two A. hypogaea varieties, NH5 and FH18 were sourced from 23 main commercial Arachis hypogaea cultivars for physiological characterisation and transcriptomic analysis [32].The SRA data for NH5 and FH18 under drought stress were obtained from the NCBI database (SRA accession number: PRJNA657965), and the FPKM (fragments per kilobase million) values were extracted for AhNCED gene expression analysis [32].The heat map values of AhNCEDs were visualised using the online website Omicshare (http://www.omicshare.com,accessed on 23 December 2023).

RNA Extraction and Gene Expression Analysis
The second leaves of NH5 and FH18 were subjected to drought stress using 20% PEG6000.The experiment spanned eight time points: 0, 4, 8, 24 h, and 3, 5, 7, and 10 days.Each time point was replicated three times to ensure statistical reliability.The protocol extracted RNA from the Arachis hypogaea tissues using the Papure Plant RNA kit (Magen Biotech, Guangzhou, China).RNA integrity was confirmed by agarose gel electrophoresis, and quantification was performed using a Thermo NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Waltham, MA, USA).Subsequently, RNA was reverse-transcribed into cDNA using HiScript II Q RT SuperMix for qPCR (+gDNA wiper) (Vazyme, Nanjing, China).The qPCR primers were listed in Table S4 and were designed using the Primer3Plus website (https://www.primer3plus.com/index.html,accessed on 23 December 2023).qPCR was conducted on a Bio-Rad CFX96 Real-Time PCR System (BioRad, Hercules, CA, USA), following this program: an initial denaturation at 95 • C for 3 min, followed by 40 cycles of 95 • C for 10 s and 60 • C for 30 s. Actin was the internal reference gene; each sample was analysed in triplicate.Data were processed using the 2 −∆∆Ct method [44].
Furthermore, parts of the NH5 and FH18 plants were subjected to a 10-day wholeplant drought stress treatment using 20% PEG6000 (Sangon Biotech, Shanghai, China).After this period, leaves were collected to observe phenotypic changes.Additionally, morphological changes in NH5 and FH18 were assessed and compared between the control group (standard water) and the drought stress group (subjected to a 10-day whole-plant drought treatment using 20% PEG6000).

Statistical Analysis
Statistical analyses were conducted utilizing SPSS software (version 26.0, SPSS Institute, USA).Comparative evaluations of mean values derived from the various treatments were executed via Student's t-test.Significant disparities are denoted by asterisks, with *, **, and *** signifying p values less than 0.05, 0.01, and 0.001, respectively, indicating increasing levels of statistical significance.

Conclusions
This research identified eight NCED genes in Arachis hypogaea, analysed their phylogenetic relationships, conserved protein motifs and domains, gene structures, cis-acting elements, and chromosomal localisation and carried out synteny analysis.The presence of ABA-related and drought-inducible elements in the promoters of AhNCEDs underscores their crucial roles in drought response.Notably, tandem repeat gene duplication in Arachis hypogaea, particularly in AhNCED1 and AhNCED2, AhNCED3 and AhNCED4, and AhNCED6 and AhNCED7, suggests an evolutionary adaptation.Moreover, the research revealed that AhNCED1 and AhNCED2, markedly upregulated in both NH5 (droughttolerant) and FH18 (drought-sensitive) cultivars, play a vital role in the rapid drought response of Arachis hypogaea.Additionally, compared to the FH18, the NH5 variety exhibits a delayed yet robust activation of drought response mechanisms, which was particularly evident in the AhNCED1 and AhNCED2.In summary, this research provides valuable insights into the NCED genes of Arachis hypogaea, contributing to future efforts to enhance the agronomic traits of Arachis hypogaea plants.

Supplementary Materials:
The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25105564/s1, Figure S1.Phylogenetic relationship of NCEDs. Figure S2.Different bar lengths in the chart represent the number of various functional cis-acting elements.Figure S3.Three pairs of tandem duplicated AhNCEDs. Figure S4.Protein structure modelling of the AhNCED3-8 proteins.Table S1.Cis-acting elements of AhNCEDs.Table S2.Fold change of AhNCEDs expression in drought-tolerant variety (NH5) under drought stress treatment.Table S3.Fold change of AhNCEDs expression in drought-sensitive variety (FH18) under different time points of drought stress treatment.Table S4.Primers for quantitative PCR.

Figure 2 .
Figure 2. Phylogenetic relationship, conserved motifs, and domains of the NCED proteins.The motifs and domains are arranged schematically based on the pattern of the phylogenetic tree.(a) A maximum-likelihood phylogenetic tree was constructed using IQ-tree software v.1.6.8 with the JTT+R5 model and 1000 bootstrap replicates.(b) Conserved motifs of NCED proteins were identified using the MEME web server.Different coloured boxes highlight the eight different motifs.(c) Domain structure of NCED proteins.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gm: Glycine max; Ps: Pisum sativum; Pv: Phaseolus vulgaris; Os: Oryza sativa.

Figure 2 .
Figure 2. Phylogenetic relationship, conserved motifs, and domains of the NCED proteins.The motifs and domains are arranged schematically based on the pattern of the phylogenetic tree.(a) A maximum-likelihood phylogenetic tree was constructed using IQ-tree software v.1.6.8 with the JTT+R5 model and 1000 bootstrap replicates.(b) Conserved motifs of NCED proteins were identified using the MEME web server.Different coloured boxes highlight the eight different motifs.(c) Domain structure of NCED proteins.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gm: Glycine max; Ps: Pisum sativum; Pv: Phaseolus vulgaris; Os: Oryza sativa.

21 Figure 3 .
Figure 3.The RPE65 domain in AhNCED proteins.Sequence alignment of eight AhNCED proteins with four species (Arabidopsis thaliana, Morus alba, Oryza sativa, Phaseolus vulgaris) known for their NCED genes [26-30].The identified RPE65 domains are in black parentheses.Dots represent the positions of the numbers in the protein sequence.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Ma: Morus alba; Os: Oryza sativa.

Figure 3 .
Figure 3.The RPE65 domain in AhNCED proteins.Sequence alignment of eight AhNCED proteins with four species (Arabidopsis thaliana, Morus alba, Oryza sativa, Phaseolus vulgaris) known for their NCED genes [26-30].The identified RPE65 domains are in black parentheses.Dots represent the positions of the numbers in the protein sequence.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Ma: Morus alba; Os: Oryza sativa.

Figure 4 .
Figure 4. Phylogenetic relationship and gene structure of the NCED genes.(a) Five subgroups based on the tree shape of rootless evolutionary trees were divided.(b) The number of exons in the NCED genes.(c) The coding sequence and intronic regions are depicted using yellow and green boxes.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gm: Glycine max; Ps: Pisum sativum; Pv: Phaseolus vulgaris; Os: Oryza sativa.

Figure 4 .
Figure 4. Phylogenetic relationship and gene structure of the NCED genes.(a) Five subgroups based on the tree shape of rootless evolutionary trees were divided.(b) The number of exons in the NCED genes.(c) The coding sequence and intronic regions are depicted using yellow and green boxes.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gm: Glycine max; Ps: Pisum sativum; Pv: Phaseolus vulgaris; Os: Oryza sativa.

Figure 5 .
Figure 5. Distribution of hormone responsiveness or stress-related cis-acting regulatory elements in the promoter of AhNCEDs (2000 bp upstream region).Ah: Arachis hypogaea.

Figure 5 .
Figure 5. Distribution of hormone responsiveness or stress-related cis-acting regulatory elements in the promoter of AhNCEDs (2000 bp upstream region).Ah: Arachis hypogaea.

Figure 7 .
Figure 7.The collinearity relationship among NCED genes in Arachis hypogaea, Arabidopsis thaliana, and Gossypium hirsutum.The chromosomes of each species are represented by pink (Arachis hypogaea), green (Arabidopsis thaliana), and blue (Gossypium hirsutum) colours.Purple lines indicate the syntenic NCED gene pairs between the species, and the highlighted gene names are labelled on the chromosomes.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gh: Gossypium hirsutum.

Figure 7 .
Figure 7.The collinearity relationship among NCED genes in Arachis hypogaea, Arabidopsis thaliana, and Gossypium hirsutum.The chromosomes of each species are represented by pink (Arachis hypogaea), green (Arabidopsis thaliana), and blue (Gossypium hirsutum) colours.Purple lines indicate the syntenic NCED gene pairs between the species, and the highlighted gene names are labelled on the chromosomes.Ah: Arachis hypogaea; At: Arabidopsis thaliana; Gh: Gossypium hirsutum.

Figure 8 .
Figure 8.Protein structure simulation diagrams of the 9-cis-epoxycarotenoid dioxygenase 1 and 2 in Arachis hypogaea (AhNCED1 and AhNCED2).Blue denotes alpha helices, while purple indicates beta sheets.The red areas represent the active sites of the proteins.Amino acids in the active site pockets are depicted in both stick and sphere formats.These diagrams highlight the structural features of the NCED enzymes in two phenotypically distinct Arachis hypogaea cultivars, the droughttolerant NH5 and the drought-sensitive FH18, which share the same genotype.Ah: Arachis hypogaea.

Figure 8 .
Figure 8.Protein structure simulation diagrams of the 9-cis-epoxycarotenoid dioxygenase 1 and 2 in Arachis hypogaea (AhNCED1 and AhNCED2).Blue denotes alpha helices, while purple indicates beta sheets.The red areas represent the active sites of the proteins.Amino acids in the active site pockets are depicted in both stick and sphere formats.These diagrams highlight the structural features of the NCED enzymes in two phenotypically distinct Arachis hypogaea cultivars, the drought-tolerant NH5 and the drought-sensitive FH18, which share the same genotype.Ah: Arachis hypogaea.

Figure 9 .
Figure 9. Transcriptome expression analysis of AhNCED genes in Arachis hypogaea cultivars und drought stress: (a) in the drought-tolerant cultivar NH5, and (b) in the drought-sensitive cultiv FH18.This heatmap displays the differential expression of AhNCED genes over time followin drought exposure.The x-axis represents the time after drought exposure in hours (h), while the axis shows the gene names.Expression levels are indicated by a colour scale ranging from −2 (lo expression, shown in blue) to +2 (high expression, shown in red).Ah: Arachis hypogaea.

Figure 9 .
Figure 9. Transcriptome expression analysis of AhNCED genes in Arachis hypogaea cultivars under drought stress: (a) in the drought-tolerant cultivar NH5, and (b) in the drought-sensitive cultivar FH18.This heatmap displays the differential expression of AhNCED genes over time following drought exposure.The x-axis represents the time after drought exposure in hours (h), while the y-axis shows the gene names.Expression levels are indicated by a colour scale ranging from −2 (low expression, shown in blue) to +2 (high expression, shown in red).Ah: Arachis hypogaea.

Figure 10 .
Figure 10.Phenotyping of Arachis hypogaea leaves and entire plants under normal and 10-day drought conditions.Drought-tolerant variety (NH5) grown under normal conditions (A,E), and drought treatment (B,F).Drought-sensitive variety (FH18) grown under normal conditions (C,G), and drought treatment (D,H).

Figure 10 .
Figure 10.Phenotyping of Arachis hypogaea leaves and entire plants under normal and 10-day drought conditions.Drought-tolerant variety (NH5) grown under normal conditions (A,E), and drought treatment (B,F).Drought-sensitive variety (FH18) grown under normal conditions (C,G), and drought treatment (D,H).

Table 1 .
Characteristics of the eight AhNCEDs in Arachis hypogaea L. The protein sequences of NCEDs from Arachis hypogaea were used as reference sequences to target and blast the candidate NCEDs in the Peanut Genome Resource (PGR) database (http://peanutgr.fafu.edu.cn/Download.php,accessed on 20 December 2023).Ah: Arachis hypogaea.

Table 1 .
Characteristics of the eight AhNCEDs in Arachis hypogaea L. The protein sequences of NCEDs from Arachis hypogaea were used as reference sequences to target and blast the candidate NCEDs in the Peanut Genome Resource (PGR) database (http://peanutgr.fafu.edu.cn/Download.php,accessed on 20 December 2023).Ah: Arachis hypogaea.