Genome-Wide Identification of Arachis hypogaea LEC1s, FUS3s, and WRIs and Co-Overexpression of AhLEC1b, AhFUS3b, AhWRI1a and AhWRI1d Increased Oil Content in Arabidopsis Seeds

Abstract

Peanut (Arachis hypogaea) is an important oil and economic crop widely cultivated worldwide. Increasing the oil yield is a major objective for oilseed crop improvement. Plant LEAFY COTYLEDON1s (LEC1s), FUSCA3s (FUS3s), and WRINKLED1s (WRI1s) are known master regulators of seed development and oil biosynthesis. While previous studies in peanut have primarily focused on two AhLEC1s and one AhWRI1 genes, this study identified a broader set of regulators, including two AhLEC1s, two AhFUS3s, nine AhWRI1s, two AhWRI2s, and four AhWRI3s from the variety HY917. The analyses of phylogenetic trees, gene structures, conserved domains, sequence alignment and identity, and collinearity revealed that they were highly similar to their homologs in other plants. Expression profiling demonstrated that two AhLEC1s, two AhFUS3s, and three AhWRI1s (AhWRI1a/b/c) were specifically expressed in developing seeds, suggesting critical roles in seed development, whereas AhWRI1d, AhWRI1f, and AhWRI1g showed high expression in root nodules, pointing to potential functions in symbiosis and nodulation. Furthermore, co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d in Arabidopsis significantly enhanced seed oil content and thousand-seed weight, but also led to reduced germination rate, plant height, and silique length. The findings allow for the extensive evaluation of AhLEC1s, AhFUS3s, and AhWRIs gene families, establishing a useful foundation for future research into their multiple roles in peanut development.

1. Introduction

Seed storage reserves in many plants primarily constitute starch, vegetable oil, and storage proteins. Plants synthesize and accumulate vegetable oils in seeds not only as major carbon and energy reservoirs to support germination and seedling development, but also as important resources for the human diet and renewable industrial feedstock [1,2,3]. The continuous growth of the global population has led to a rapid increase in food demand, posing a serious challenge to global food security and thus incentivizing tremendous research efforts focusing on enhancing plant oil biosynthesis [1,4].

In plants, seed oil accumulation is precisely regulated by intricate multilevel regulatory networks, with transcriptional regulation playing a central role in controlling oil biosynthesis [5]. Several key transcription factors, such as LEAFY COTYLEDON1 (LEC1), LEAFY COTYLEDON1-LIKE (L1L), FUSCA3 (FUS3), and WRINKLED1 (WRI1), have been identified as critical regulators of seed oil accumulation [6,7,8,9].

AtLEC1 and AtL1L are two HAP3 subunits of CCAAT-binding transcription factor [10,11,12]. Induced expression of AtLEC1 leads to a broad upregulation of genes involved in fatty acid (FA) metabolism, significantly promoting the accumulation of oil and major FA species [7]. Overexpression of ZmLEC1 enhances the oil content in transgenic maize, Arabidopsis thaliana, and Camelina sativa [13,14]. Similarly, overexpression of oilseed crop LEC1 homologs, including BnLEC1 and BnL1L in Brassica napus, GmLEC1 (Glyma.07G268100) in Glycine max, and AhNF-YB1 and AhNF-YB10 in Arachis hypogaea, also significantly increases total seed oil content in transgenic seeds [15,16,17]. In addition, LEC1 activation induces ectopic embryogenesis in Arabidopsis vegetative tissues [7,10], and OsNF-YB7, a rice homolog LEC1, functions as a key regulator of seed maturation but also inhibits embryo greening during seed development [18,19,20].

FUS3, a member of the B3-domain transcription factor family [21], serves as a master regulator of seed development, establishing and maintaining embryonic identity. It also modulates hormonal responses during late embryogenesis and germination [22,23,24]. FUS3 promotes seed oil accumulation by upregulating genes involved in photosynthesis, FA biosynthesis, and triacylglycerol assembly [8,25,26,27], and boosts the accumulation of seed storage proteins, such as 2S and 12S [24,28,29,30].

The WRI1/2/3/4 proteins belong to the APETALA2-ethylene-responsive element-binding protein (AP2/EREBP) family [31,32,33]. WRI1 can directly target many genes of glycolysis and fatty acid biosynthesis [34,35]. Mutations in AtWRI1 result in a wrinkled seed phenotype and significantly reduced FA levels in Arabidopsis seeds [9,31,36], whereas overexpression of AtWRI1 or its homologs markedly increases oil content in both seeds and vegetative tissues [37,38,39,40,41]. Overexpression of AtWRI3 or AtWRI4, but not AtWRI2, can rescue the low oil accumulation and wrinkled phenotype of the wri1-4 mutant [33]. Deficiencies in AtWRI2, AtWRI3, or AtWRI4 do not affect seed oil accumulation [33].

Peanut (A. hypogaea), one of the major oil crops in the world [42], is an allotetraploid (AABB) crop that possibly originated from the hybridization of two diploid progenitors, Arachis duranensis (AA) and Arachis ipaensis (BB) [43,44]. Functional analyses in polyploid oilseeds like peanut are complicated by gene redundancy from genome duplication. However, comprehensive analyses of the copy number, evolutionary relationships, gene structures, conserved domains, physicochemical properties, chromosome distributions, gene synteny, subcellular localization, cis-element distributions in promoters, and spatiotemporal expression patterns will allow for an extensive evaluation of redundant genes and build a useful foundation with which to dissect their functions in biochemical and physiological processes. Now these comprehensive analyses of AhLEC1s, AhFUS3s, and AhWRIs in A. hypogaea have not yet been performed. Although the roles of individual transcription factors LEC1, FUS3, and WRI1 have been explored in plants, there are currently no academic reports of these genes stacking to enhance seed oil content. Therefore, we try simultaneously to alter the expression of these genes and test empirically whether combinations provide a better effect.

In this study, we cloned and identified two AhLEC1s, two AhFUS3s, nine AhWRI1s, two AhWRI2s, and four AhWRI3s from the cultivated peanut variety HY917. We analyzed their evolutionary relationships, exon/intron gene structures, conserved domains, physicochemical properties, chromosome distributions, gene synteny, subcellular localization, and cis-element distributions in their promoters. Based on published transcriptome data, AhLEC1a/b, AhFUS3a/b, and AhWRI1a/b/c were specifically expressed in developing seeds. In contrast, AhWRI1d, AhWRI1f, and AhWRI1g were highly expressed in roots and nodules. AhWRI1d and AhWRI1e were upregulated under water-deficit conditions both with and without ABA, whereas AhWRI3c and AhWRI3d were repressed under drought or low-temperature treatments. Co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d in Arabidopsis increased the mature seed oil content and thousand-seed weight, but decreased the germination rate, plant height, and silique length. These findings provide valuable information for the peanut master regulators AhLEC1s, AhFUS3s, and AhWRIs, suggesting that they may play multiple important roles during peanut development.

2. Results

2.1. Cloning and Identification of AhLEC1s, AhFUS3s, and AhWRIs in A. hypogaea

To identify candidate members of LEC1s, FUS3s, and WRIs in A. hypogaea, protein sequences of A. thaliana homologs, including AtLEC1, AtL1L, AtFUS3, AtWRI1, AtWRI2, AtWRI3, and AtWRI4, were retrieved from The Arabidopsis Information Resource (TAIR, http://www.arabidopsis.org/; accessed on 10 May 2023) [45] and used as queries for BLAST searches against the following peanut genomic resources: Peanut Genome Resource (PGR, http://peanutgr.fafu.edu.cn/; accessed on 15 May 2023) [46], PeanutBase (https://www.peanutbase.org/; Tifrunner.gnm2.ann2.PVFB (https://data.legumeinfo.org/Arachis/hypogaea/annotations/Tifrunner.gnm2.ann2.PVFB/) and BaileyII.gnm1.ann1.PQM7 (https://data.legumeinfo.org/Arachis/hypogaea/annotations/BaileyII.gnm1.ann1.PQM7/); accessed on 15 May 2023) [47,48], Arachis hypogaeaLine8 v1.3 (https://phytozome-next.jgi.doe.gov/info/AhypogaeaLine8_v1_3; accessed on 10 October 2024) [49,50], and the allotetraploid wild peanut Arachis monticola genome (http://gigadb.org/dataset/100453; accessed on 18 May 2023) [51] (

Table 1. Members of LEC1, FUS3, and WRIs in A. hypogaea, A. duranensis, A. ipaensis, and A. monticola.

A. thaliana	A. hypogaea					A. monticola	A. duranensis	A. ipaensis
	Gene Name	PGR	Tifrunner	BaileyII	Line8 v1.3
AtLEC1 AtL1L	AhLEC1a	AH01G29730	Ah01g388600	chr01G4145	AhLine8.01G229500	EVM0044897	—	—
	AhLEC1b	AH11G27150	Ah11g380300	chr11G3925	AhLine8.11G179600	EVM0020102	—	—
AtFUS3	AhFUS3a	AH06G23930	Ah06g316900	chr06G3370	AhLine8.06G179300	EVM0043936	Aradu.EI7Y8	—
	AhFUS3b	AH16G29570	Ah16g401200	chr16G3980	AhLine8.16G197300	EVM0071796	—	Araip.3K0PR
AtWRI1	AhWRI1a	AH08G25830	Ah08g309800	chr08G3455	AhLine8.08G210300	EVM0023005	Aradu.EJI6Q	—
	AhWRI1b	AH18G30790	Ah18g415600	chr18G4478	AhLine8.18G216000	—	—	—
	AhWRI1c	AH15G12290	Ah15g212800	chr15G2176	AhLine8.15G128400	—	—	Araip.SZ63C
	AhWRI1d	—	Ah10g334400	IDmodified-mrna-2114	AhLine8.10G185300	EVM0064695	Aradu.387PF	—
	AhWRI1e	AH20G31430	Ah20g425400	chr20G4248	AhLine8.20G202100	EVM0058654	—	Araip.L05NW
	AhWRI1f	AH04G29530	Ah04g393900	chr04G4066	AhLine8.04G202100	EVM0014666	Aradu.G1CDJ	—
			Ah04g396000
			Ah04g394100
	AhWRI1g	AH14G34420	Ah14g462800	IDmodified-mrna-77	AhLine8.14G213200	—	—	Araip.34E5E
	AhWRI1h	AH03G41970	Ah03g520600	chr03G5733	AhLine8.03G308300	EVM0009822	—	Araip.N6C0B
			Ah03g520700
	AhWRI1i	AH13G44600	Ah13g549900	chr13G6111	AhLine8.13G320200	EVM0034741	Aradu.EN58B	—
AtWRI2	AhWRI2a	AH06G02150	Ah06g106000	chr06G1256	AhLine8.06G083900	—	Aradu.0KI78	—
					AhLine8.06G084200
	AhWRI2b	AH16G04650	Ah16g055500	chr16G566	AhLine8.16G038500	EVM0053674	—	Araip.6J28H
						EVM0007664
AtWRI3 AtWRI4	AhWRI3a	AH01G23420	Ah01g316300	IDmodified-mrna-7040	AhLine8.01G177800	EVM0007256	Aradu.A4KG4	—
	AhWRI3b	AH11G34510	Ah11g474800	chr11G4916	AhLine8.11G236600	—	—	—
	AhWRI3c	AH09G00410	Ah09g004900	IDmodified-mrna-2659	AhLine8.09G003900	—	Aradu.P6UBG	—
	AhWRI3d	AH19G01070	Ah19g015300	chr19G149	AhLine8.19G012100	EVM0074703	—	Araip.S3MIZ

The accession numbers of LEC1, FUS3, and WRI members in A. hypogaea, A. duranensis, A. ipaensis, and A. monticola were derived from the PGR [46], PeanutBase [47,48,52], Arachis hypogaeaLine8 v1.3 [49,50], and an allotetraploid wild peanut A. monticola genome [51], respectively.

and Table S1). Based on the retrieved nucleotide sequences, primers were designed (Table S3) to clone and identify the coding sequences (CDSs) of putative AhLEC1s, AhFUS3s, and AhWRIs from the cultivated peanut variety HY917 (Table S2). In addition, homologs of LEC1s, FUS3s, and WRIs were also identified in nine additional plant species (Tables S1 and S4). All putative CDS, genomic DNA, and protein sequences used in this study were obtained from their respective genome databases and are presented in Tables S5–S7.

Two putative LEC1s were detected in each of the four A. hypogaea genome annotations and in A. monticola, whereas no LEC1 homologs were found in A. duranensis and A. ipaensis (Table 1). From HY917, two AhLEC1 genes were successfully cloned and annotated as AhLEC1a and AhLEC1b (Table 1 and Table S2). Their CDSs contained two exons, similarly to most LEC1s in other plants (Figure 1A and Table S8). Both AhLEC1a and AhLEC1b, along with their homologs, contained a conserved CBFD_NFYB_HMF domain (pfam00808; Figure 1A). Phylogenetic analysis showed that both AhLEC1s were more closely related to AtL1L than to AtLEC1 (Figure 1A). Physicochemical property analysis demonstrated the molecular weight (MW) and theoretical isoelectric point (pI) of AhLEC1a and AhLEC1b are 25.29–25.44 kDa and 6.11–6.17, respectively (Table S8). Their aliphatic index and the grand average of hydropathicity (GRAVY) were predicted to be 60.88–61.16 and −0.735–−0.728 (Table S8), respectively, indicating high hydrophilicity, while their instability index was shown to be 36.10–37.40 (Table S8), suggesting high stability.

Two putative FUS3s were identified in each of the four A. hypogaea genomes annotations and in the A. monticola genome. These were cloned from HY917 and named AhFUS3a and AhFUS3b (Table 1 and Table S2). In contrast, only one putative FUS3 was detected in A. duranensis and A. ipaensis, respectively (Table 1). All of these genes have six exons and five introns, and their encoded proteins contain a B3 DNA-binding domain (cd10017), similar to those in other plant species (Figure 1B and Table S8). Phylogenetic analysis showed that both AhFUS3s are more closely related to their homologs from leguminous species (G. max, Glycine soja, and Medicago truncatula), than to those of other plants (Figure 1B).

Physicochemical property analysis indicated the molecular weight and theoretical pI of AhFUS3a and AhFUS3b are 42.24–42.31 kDa and 5.58, respectively (Table S8). Their aliphatic index and GRAVY were predicted to be 69.63–69.66 and −0.500–−0.491 (Table S8), respectively, indicating high hydrophilicity. Their instability index was shown to be 54.17–55.65 (Table S8), suggesting high instability.

Compared with the four A. hypogaea genomes, nine putative AhWRI1s and two putative AhWRI2s in A. hypogaea were successfully cloned from HY917 and designated as AhWRI1a to AhWRI1i and AhWRI2a to AhWRI2b, respectively (Table 1 and Table S2). Phylogenetic analysis indicated that AtWRI3 and AtWRI4 are more closely related to each other than to AtWRI1 or AtWRI2 (Figure 1C and Figure S1A). Accordingly, four genes in A. hypogaea that showed close homology to both AtWRI3 and AtWRI4 were annotated as AhWRI3a, AhWRI3b, AhWRI3c, and AhWRI3d (Table 1). In addition, genome surveys identified six WRI1s, two WRI2s, and two WRI3s in A. monticola, four WRI1s, one WRI2, and two WRI3s in A. duranensis, and four WRI1s, one WRI2, and one WRI3 in A. ipaensis (Table 1). Gene structure analysis revealed that all nine AhWRI1s and four AhWRI3s contained eight exons, whereas their homologs in other plant species exhibited variable exon numbers, ranging from six to eight exons (Figure 1C and Figure S1B and Table S8). Both AhWRI2s consistently contained nine exons, in contrast to the putative WRI2s in other plants, which range from seven to nine exons (Figure 1C and Figure S1B and Table S8). Notably, all AhWRI1s, AhWRI2s, and AhWRI3s were found to carry a 9 bp micro-exon (Figure 1C) consistent with previous reports for AtWRI1 [32]. Domain analysis showed that All AhWRI1s and AhWRI3s proteins contain two AP2 domains (smart00380 and cl00033 for AP2 and AP2 superfamily, respectively; Figure 1C and Figure S1C), similarly to their homologs in other plants [31,32,33]. In contrast, both AhWRI2s and other plant WRI2s possess only one AP2 domain (smart00380; Figure 1C and Figure S1C). It is worth noting that three putative WRI1s (EVM0034741.1, Araip.N6C0B.1, and Aradu.EN58B.1) and four WRI3s (Araip.S3MIZ.1, Aradu.P6UBG.1, EVM0007256.1, and Aradu.A4KG4.1) from the previously reported genomes of A. monticola, A. duranensis, and A. ipaensis were found to lack one or both AP2 domains (Figure 1C). Similarly, one putative WRI2 in A. monticola (EVM0007664.1) had also lacked the AP2 domain (Figure 1C). The genes in these reported genomes may be partial and thus there is need for additional cloning to identify the full length. Physicochemical property analysis indicated the molecular weight and theoretical pI of AhWRIs were 40.71–47.04 kDa and 6.37–9.45, respectively (Table S8). Their aliphatic index and GRAVY were predicted to be 55.24–64.45 and −1.019–−0.735 (Table S8), respectively, indicating high hydrophilicity. Their instability index was shown to be 41.41–65.85 (Table S8), suggesting high instability.

2.2. Protein Alignment and Identity Analysis

Protein alignment showed that the central B (CBFD_NFYB_HMF) domains of AhLEC1a and AhLEC1b demonstrated strong similarity to those of other plant LEC1s, as seen in the conserved central domain for both proteins (Figure 2A) as well as the full-length proteins (Figure S2). In contrast, the amino-terminal A or carboxyl-terminal C domains exhibited low sequence similarity among AhLEC1s and other LEC1 homologs in (Figure S2). Analyzing the identities of conserved domains (Table S9), 83.3–100.0% similarity was demonstrated among AhLEC1a, AhLEC1b, AtLEC1, and AtL1L; however, AhLEC1a and AhLEC1b showed 51.3–51.6% similarity with AtLEC1 and 59.6–60.6% similarity with AtL1L based on full-length proteins, while that between AhLEC1a and AhLEC1b was 95.6% for full-length proteins. The putative DNA-binding region, putative subunit association regions, and secondary structures (α-helices and coils) were observed in the highly conserved B domains as other NF-YBs or HAP3s (Figure 2A) [10,12,56,57]. Furthermore, AhLEC1a and AhLEC1b contain amino acid residues conserved in AtLEC1 and AtL1L B domains but divergent in other AtNF-YBs (Figure 2A) [11,12]. Notably, both proteins contained a conserved residue Asp (D) within the central B domain, which is essential for AtLEC1 function [12] (Figure 2A).

Protein alignment of FUS3 homologs indicated that two AhFUS3s, like other FUS3s, contain a conserved B3 domain, flanked by non-conserved N- and C-terminal region (Figure 2B and Figure S3). Full-length FUS3 proteins from A. hypogaea, A. monticola, A. duranensis, and A. ipaensis share 60.1–60.5% identity with AtFUS3, while their B3 domains share 75.9–76.7% identity. Among FUS3s of A. hypogaea, A. monticola, A. duranensis, and A. ipaensis sequence identity reached 97.1–100% for full-length proteins and 97.4–100% for conserved domains (Table S10). An aspartic acid residue (D) in the B3 domain, equivalent to D75 in HvFUS3 [58], was observed across all species (Figure 2B). The N-terminal regions of two AhFUS3s contain a putative B2 domain (Figure S3) with a potential nuclear localization signal (NLS; Table S8). The three serine sites (55-SSS-57) in AtFUS3, phosphorylated by AKIN10 to promote degradation [59,60], were not conserved among other FUS3 homologs (Figure S3). Two putative PEST sequences, associated with protein turnover in response to ABA and GA signaling [61,62], were identified in the C-terminal domain of two AhFUS3s, while one or two putative PEST sequences were also detected in other plant FUS3s (Table S8).

As in other WRIs in previous studies [33,40,63,64,65,66,67], all AhWRI1s and AhWRI3s contained two typical AP2/ERF DNA-binding domains, whereas two AhWRI2s possessed only one AP2 domain. All isoforms exhibited considerable sequence variation in their N- and C-terminal regions (Figure 2C,D and Figures S4–S6). AhWRI1a, AhWRI1b, and AhWRI1c showed high mutual similarity (87.4–97.5% and 93.9–100.0% for the full-length proteins and conserved regions, respectively), lower identity with other AhWRI1s (40.5–43.7% and 75.5–81.6%, respectively; Table S11). The remaining AhWRI1s (AhWRI1d–i) shared 55.5–99.5% (full-length) and 90.2–100.0% (conserved region) identity among themselves (Table S11). AhWRI3s have higher identity (93.9–100.0%) with each other than with AhWRI1s (77.3–80.4%) in conserved regions (Table S11). Both AhWRI2s have 69.6% and 100.0% identity with AtWRI2 for the full-length proteins and conserved regions, respectively (Table S12).

The tripeptide VYL encoded by the conserved micro-exon was identified in all AhWRI1s, AhWRI2s, and AhWRI3s (Figure 2C,D and Figures S4–S6) as previously reported in other WRIs [32,39,63,68,69,70,71]. The 14-3-3 proteins, which are phosphopeptide-binding proteins, were found to potentially bind to the first AP2 domain of AtWRI1 and increase its stability, while the 14-3-3 binding motif (AtWRI1^78-92) in AtWRI1 was overlapped with that for BPM [72]. A putative motif for binding both 14-3-3 and BPM was also identified within the first AP2 domain of peanut and other plant WRI1 and WRI3 proteins (Figure 2D).

2.3. Chromosomal Distribution and Collinearity Analysis

To uncover the chromosomal location of LEC1s, FUS3s, WRI1s, WRI2s, and WRI3s, we visualized their physical positions on the corresponding chromosomes of A. hypogaea, A. duranensis, A. ipaensis, and A. monticola (Figure 3). In A. hypogaea, with 40 total chromosomes, two AhLEC1s were identified on chr01 and chr11; two AhFUS3s on chr06 and chr16; nine AhWRI1s on chr03, chr04, chr08, chr10, chr13, chr14, chr15, chr18, and chr20; two AhWRI2s on chr06 and chr16; and four AhWRI3s on chr01, chr11, chr09, and chr19 (Figure 3A). Clearly, for A. duranensis and A. ipaensis, it would instead be 20 total chromosomes. In A. duranensis, one putative FUS3 was identified on Aradu.A06; four putative WRI1s on Aradu.A03, Aradu.A04, Aradu.A08, and Aradu.A10; one putative AhWRI2 on Aradu.A06; and two putative WRI3s on Aradu.A01 and Aradu.A09 (Figure 3B). In A. ipaensis, one putative FUS3 was identified on Araip.B06; four putative WRI1s on Araip.B03, Araip.B04, Araip.B05, and Araip.B10; one putative AhWRI2 on Araip.B06; and one putative WRI3 on Araip.B09 (Figure 3C). In A. monticola, two putative LEC1s were identified on A.mon-A01 and A.mon-B01; two putative FUS3s on A.mon-A06 and A.mon-B06; six putative WRI1s on A.mon-A04, A.mon-A08, A.mon-A10, A.mon-B03, A.mon-B03, and A.mon-B10; two putative AhWRI2s, in close proximity to each other, on A.mon-B06; and two putative AhWRI3s on A.mon-A01 and A.mon-B09 (Figure 3D).

We analyzed the collinear relationship of AhLEC1s, AhFUS3s, AhWRI1s, AhWRI2s, and AhWRI3s (Figure 4A). The results showed that AhFUS3a, AhWRI1a, AhWRI2a, AhWRI3a, and AhWRI3c had collinear relationships with AhFUS3b, AhWRI1b, AhWRI2b, AhWRI3b and AhWRI3d, respectively, and that AhWRI1d, AhWRI1e, AhWRI1f, AhWRI1g, AhWRI1h, and AhWRI1i shared the same collinear relationship. Conversely, AhLEC1a had no collinear relationship with AhLEC1b; AhWRI1a and AhWRI1b had no collinear relationships with the other seven AhWRI1s; AhWRI1c had no collinear relationships with the other eight AhWRI1s; and AhWRI3a and AhWRI3b had no collinear relationships with AhWRI3c and AhWRI3d, respectively.

To investigate the collinearity of LEC1, FUS3, WRI1, WRI2, and WRI3 genes across species, we performed analysis among A. thaliana, A. hypogaea, G. max, M. truncatula, Theobroma cacao, and Helianthus annuus (Figure 4B). For LEC1-type genes, AtL1L, AhLEC1a/AhLEC1b, Glyma.07G268100/Glyma.17G005600, Medtr4g133952, and Thecc.06G241100/Thecc.07G013400 shared conserved synteny. Glyma.20G000600 was collinear with Medtr1g039040, and Medtr5g095860 showed collinearity with Thecc.06G241100 and Thecc.07G013400. Thecc.06G241100 was also collinear with HanXRQChr16g0515761. Additionally, Glyma.07G268100 and Glyma.17G005600 were collinear with Medtr2g026710, which is phylogenetically closer to AtNF-YB3 (AT4G14540) than to AtLEC1 or AtL1L. All examined FUS3 orthologs across the six species exhibited conserved collinearity. In contrast, AtWRI1 did not show collinearity with any WRI1 homologs from the other species. Multiple AhWRI1 genes (AhWRI1d–i) shared synteny with Glyma.09G240400, Glyma.18G256000, Glyma.18G125200, and Glyma.08G297000. Other collinear relationships included Glyma.15G221600 and Glyma.08G227700 with Medtr8g044040 and Thecc.10G130200; Glyma.09G240400 and Glyma.18G256000 with Medtr7g009410 and Medtr6g011490; Glyma.18G125200 with Medtr8g468920, Medtr7g009410, and Medtr6g011490; Glyma.08G297000 with Medtr8g468920 and Medtr6g011490; Thecc.02G354400 and Thecc.04G075600 with Medtr8g468920, Medtr7g009410, Medtr6g011490, and HanXRQChr02g0056411; Thecc.04G075600 with HanXRQChr09g0265501. For WRI2s, AtWRI2 exhibited conserved synteny with AhWRI2a/AhWRI2b, Glyma.02G185200/Glyma.03G136100/Glyma.19G138000, Medtr7g091390, and Thecc.05G148300. For WRI3s, AtWRI3 was collinear with AhWRI3c/AhWRI3d, Glyma.07G021000/Glyma.08G220800, Medtr4g007770, Thecc.06G047900, and HanXRQChr09g0274201/HanXRQChr16g0499481. Additionally, AhWRI3a/AhWRI3b shared synteny with Glyma.02G207100/Glyma.17G070800, Medtr4g130270, and Thecc.09G154700. AtWRI4 showed no collinearity with any WRI3 homologs from the examined species.

2.4. Transcriptional Profiles of AhLEC1s, AhFUS3s, and AhWRIs

To investigate the expression patterns of AhLEC1s, AhFUS3s, AhWRI1s, AhWRI2s, and AhWRI3s across various tissues and under diverse abiotic stresses and phytohormone treatments, their transcriptional profiles were extracted from PGR (accessed on 11 June 2024; Figure 5A,B) [46], PeanutBase (accessed on 11 June 2024; Table S13) [73,74], and our RNA-seq data from six seed developmental stages of four sister lines (P19-19, P19-61, P19-57, and HY917; Figure 5C and Figure S7 and Table S14).

According to PGR (Figure 5A), AhLEC1a and AhLEC1b were specifically expressed in embryos (Embryo-I/II/III/IV) and seed coats (Testa-I/II). AhFUS3a and AhFUS3b showed high specific expression in embryos (Embryo-I/III/IV), with lower expression in seed coat. AhWRI1a, AhWRI1b, and AhWRI1c were specifically expressed in embryos (Embryo-I/III/IV), while AhWRI1f and AhWRI1g were specifically highly expressed in root nodules. In contrast, AhWRI1e, AhWRI1h, and AhWRI1i were barely detectable across tissues. AhWRI2a and AhWRI2b were widely expressed in multiple tissues. AhWRI3a and AhWRI3b were barely detectable across tissues, whereas AhWRI3c and AhWRI3d were highly expressed in florescences. In addition, the expression of AhWRI2a, AhWRI2b, AhWRI3c, and AhWRI3d gradually increased during the development of pericarp.

According to PeanutBase (Figure 5B), AhLEC1a, AhLEC1b, AhFUS3a, AhFUS3b, AhWRI1a, AhWRI1b, and AhWRI1c were highly expressed in developing seeds (Pattee seed 5–8). While AhWRI1d, AhWRI1f, and AhWRI1g were specifically highly expressed in roots and nodules. AhWRI2a and AhWRI2b were broadly expressed across tissues, while AhWRI3c and AhWRI3d were barely detected in seeds. Conversely, AhWRI1e, AhWRI1h, AhWRI1i, AhWRI3a, and AhWRI3b were expressed at very low or nearly undetectable levels in all examined tissues.

Based on our RNA-seq data across the six seed development stages of four sister lines (Figure 5C and Figure S7), AhLEC1a, AhLEC1b, and AhWRI1c were highly expressed in early seed development stages; AhFUS3a, AhFUS3b, and AhWRI1a were most lowly expressed in the final stage; AhWRI1h, AhWRI1i, AhWRI2a, and AhWRI2b were lowly expressed across all stages; and AhWRI1d, AhWRI1e, AhWRI1f, AhWRI1g, AhWRI3a, AhWRI3b, AhWRI3c, and AhWRI3d were weakly expressed or undetectable throughout seed development.

Under abiotic stresses and phytohormone treatments, AhWRI1d and AhWRI1e were highly induced under water-deficit conditions, both with and without ABA. In contrast, AhWRI1g expression was repressed under the same conditions, and AhWRI1i was down-regulated under water-deficit without ABA (Table S13A). AhWRI2a was induced under water-deficit with ABA, while that of AhWRI2b was up-regulated under water-deficit both with and without ABA (Table S13A). Moreover, their expressions were induced under drought but repressed under abscisic acid, brassinolide, ethephon, or paclobutrazol treatments (Table S13B). Expressions of AhWRI3c and AhWRI3d were repressed under water-deficit conditions (with or without ABA; Table S13A) as well as under drought or low-temperature stress (Table S13B).

2.5. Putative Cis-Elements in the Promoters of AhLEC1s, AhFUS3s, and AhWRIs and miRNA Regulating AhLEC1s, AhFUS3s, and AhWRIs

To investigate the potential function and regulatory mechanisms of AhLEC1s, AhFUS3s, and AhWRIs, we analyzed putative cis-elements in their putative promoter regions. These regions, defined as the 2000 bp upstream of the start codon, were extracted from the Arachis hypogaeaLine8 v1.3 genome (accessed on 10 October 2024; Table S15) and analyzed using PlantCARE [76]. For comparison, putative promoter sequences of AtLEC1, AtL1L, AtFUS3, AtWRI1, AtWRI2, AtWRI3, and AtWRI4, were derived from TAIR (Figure 6 and Table S15). The identified putative cis-elements were classified into fifteen functional categories based on their annotations, including MeJA responsiveness, meristem expression, abscisic acid responsiveness, light responsiveness, anaerobic induction, circadian control, defense and stress responsiveness, low-temperature responsiveness, gibberellin responsiveness, and drought inducibility (Figure 6 and Table S16).

AtLEC1, AtL1L, and two AhLEC1s contained cis-acting elements associated with light responsiveness, meristem expression, anaerobic induction, and gibberellin responsiveness. Similarity was found in the distribution of cis-acting elements between AtLEC1 and AtL1L and between two AhLEC1s. Additionally, AtLEC1 and AtL1L contained cis-acting elements involved in abscisic acid responsiveness, auxin responsiveness, low-temperature responsiveness, drought inducibility, circadian control, and endosperm expression.

AtFUS3 and two AhFUS3s contained cis-acting elements related to light responsiveness, MeJA-responsiveness, anaerobic induction, meristem expression, abscisic acid responsiveness, and defense and stress responsiveness. Two AhFUS3s demonstrated more similarity in their cis-element distribution than AtFUS3. They also contained elements involved in low-temperature responsiveness.

AtWRI1 and nine AhWRI1s all contained light responsive cis-acting elements. AhWRI1a and AhWRI1b, AhWRI1f and AhWRI1g, and AhWRI1h and AhWRI1i demonstrated similarity in their cis-element distribution. AtWRI2 and two AhWRI2s contained elements related to light responsiveness, MeJA responsiveness, low-temperature responsiveness, and anaerobic induction. Two AhWRI2s showed greater similarity to each other in their cis-element distribution than to AtWRI2 and also contained elements involved in gibberellin responsiveness, defense and stress responsiveness, drought inducibility, and zein metabolism regulation. AtWRI3, AtWRI4, and four AhWRI3s contained cis-acting elements associated with light responsiveness, abscisic acid responsiveness, MeJA responsiveness and defense and stress responsiveness. Similar distribution patterns were observed between AhWRI3a and AhWRI3b and between AhWRI3c and AhWRI3d. All predicted cis-acting elements require functional validation to confirm their roles. We also predicted ahy-miRNAs targeting AhLEC1s, AhFUS3s, and AhWRIs using the psRNATarget website (www.zhaolab.org/psRNATarget/, accessed on 22 March 2025) [77]. The results showed that only two miRNAs (ahy-miR3509-5p and ahy-miR3520-5p) putatively target AhWRI1s, while no miRNAs were predicted to target AhLEC1s, AhFUS3s, AhWRI2s, and AhWRI3s (Table S17). In contrast, eleven, three, three, twelve, five, one, and two miRNAs were predicted to target AtLEC1, AtL1L, AtFUS3, AtWRI1, AtWRI2, AtWRI3, and AtWRI4, respectively, in A. thaliana (Table S17).

2.6. Subcellular Localization of AhLEC1s, AhFUS3s, and AhWRIs

All AhLEC1, AhFUS3, and AhWRI proteins were predicted to localize in the nucleus using ProtComp 9.0 in softberry (Table S8), consistent with their homologs previously reported in other plants [20,39,59,78,79,80,81,82]. Analysis with cNLS Mapper [83] indicated that AhLEC1s lack putative NLSs, whereas all AhFUS3s shared one same putative NLS. Most AhWRIs were predicted to contain one putative NLS, except for AhWRI1a/b/c and AhWRI3c/d (Table S8). It has been previously demonstrated that the NLS of AtWRI1 (AtWRI1^33–41) is necessary and sufficient for its nuclear localization [84]. To experimentally validate their subcellular localization, we selected four highly expressed and/or stress-responsive isoforms: AhLEC1b, AhFUS3b, and AhWRI1a (highly expressed in seeds; Figure 5A,B) and AhWRI1d (upregulated under drought; Table S13A). Although all were predicted to be nuclear localized, we aimed to experimentally validate whether variations in their putative NLSs influence actual protein localization. Each was fused to GFP and expressed under the 35S promoter in Arabidopsis protoplasts, along with a nuclear mCherry marker. Confocal microscopy revealed that all four fusion proteins co-localized exclusively with the nuclear marker, confirming their nuclear localization (Figure 7), consistent with bioinformatic predictions.

2.7. Co-Overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d Increased Fatty Acids Accumulation and Thousand-Seed Weight, but Decreased Germination Rate, Plant Height, and Silique Length

AhLEC1b, AhFUS3b, and AhWRI1a were selected as representative isoforms due to their highest expression levels during seed development (Figure 5A,B), while AhWRI1d was chosen for its high expression in roots and nodules (Figure 5B) and upregulation under drought stress (Table S13A). Using these four selected genes, a tandem gene cassette was constructed with the following arrangement: P_35S::AhLEC1b::T_NOS::P_MAS::AhFUS3b::CaMV poly(A) signal::P_NOS::AhWRI1a::CaMV poly(A) signal::P_35S::AhWRI1d::T_PE9. This cassette was cloned into the pCAMBIA2300 vector to generate the co-overexpression construct pLFW (Figure 8A). The pLFW vector was then introduced into A. thaliana Col-0 via Agrobacterium tumefaciens-mediated transformation. Six independent transgenic lines were selected using kanamycin resistance and confirmed by PCR amplification with three pairs of primers specific to AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d (Table S3 and Figure S8). qRT-PCR was also performed to detect their relative expression in transgenic lines. And the results showed that these four genes were highly expressed in transgenic lines, compared to in wild type (WT) plants with lowly expression or not detected (Figure S8).

To determine the effect of co-overexpression of these four genes on storage oil accumulation, four transgenic lines (LFW-OX#1, LFW-OX#3, LFW-OX#4, and LFW-OX#5) were used for detecting fatty acid profiling in via gas chromatography. Most measured fatty acid species exhibited increased levels in transgenic seeds (Figure 8B). Specifically, the contents of 16:0, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, 20:2, and total fatty acids were significantly elevated by 13.48–19.81%, 20.14–28.54%, 11.79–38.10%, 9.18–26.60%, 11.31–18.17%, 20.05–22.57%, 21.28–32.26%, 11.20–18.09%, and 14.22–26.18%, respectively, compared to the wild type. Furthermore, the levels of unsaturated fatty acids, saturated fatty acids, C18, and C20 fatty acids increased significantly by 10.16–27.29%, 12.54–22.93%, 9.75–26.62%, and 12.15–30.13%, respectively (Figure S9A). The ratios of unsaturated to saturated fatty acids in LFW-OX#3 and C18 to C20 fatty acids in LFW-OX#1, LFW-OX#3, and LFW-OX#5 also increased significantly (Figure S9B).

The thousand-seed weight of mature seeds from the four transgenic lines increased significantly by 8.74–19.20% (Figure 8C). Plant height, silique length, and seed germination were measured only in lines LFW-OX#3 and LFW-OX#5. The co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d in both lines resulted in reductions of 16.91–35.48% in plant height, 11.63–19.53% in silique length, and 9.48–11.62% in seed germination, respectively (Figure 8D–F).

3. Discussion

A. hypogaea is an allotetraploid (AABB) species that possibly originated from the hybridization of two diploid progenitors, namely A. duranensis (AA) and A. ipaensis (BB) [43,44]. In this study, two AhLEC1s, two AhFUS3s, nine AhWRI1s, two AhWRI2s, and four AhWRI3s were cloned and identified from A. hypogaea. Systematic analyses of phylogenetic trees, gene structures, conserved domains, physicochemical properties, chromosome locations, gene synteny, subcellular localization, and promoter cis-element distributions were performed. Expression patterns were further investigated using RNA-seq data. Co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d enhanced fatty acids accumulation and thousand-seed weight, but reduced germination rate, plant height, and silique length. These results provide an extensive evaluation of AhLEC1s, AhFUS3s, and AhWRIs, and establish a useful foundation for further function analysis of their roles in biochemical and physiological processes.

3.1. Gene Duplication and Functional Diversification of AhLEC1s in A. hypogaea

In A. thaliana, AtLEC1 and AtL1L are recognized as LEC1-type NF-YB transcription factors [11,12], with their orthologs subsequently identified in diverse plant species, including H. annuus [85], T. cacao [86], Oryza sativa [20,87], B. napus [7,15], and G. max [16,88]. In this study, two AhLEC1 genes were identified in peanut (Table 1). Through comprehensive phylogenetic analysis, sequence alignment, identity matrix assessment, and gene synteny examination, both genes were shown to be more closely related to AtL1L than to AtLEC1 (Figure 1A, Figure 2A and Figure 4, and Figure S2 and Table S9). These findings suggest that AhLEC1a and AhLEC1b are likely orthologous to AtL1L, similar to the previously characterized PcL1L [11], rather than to AtLEC1. Interestingly, no LEC1-type NF-YBs were detected in the two diploid progenitors, A. duranensis and A. ipaensis (Table 1 and Figure 3B,C), implying possible evolutionary loss in these lineages.

Structurally, NF-YB subunits consist of three domains: the amino-terminal A domain, central B domain and carboxyl-terminal C domain [10,89]. Sequence analysis revealed that the central B domains of AhLEC1a and AhLEC1b are highly conserved compared to those of LEC1-type NY-YBs from other plants, whereas the A and C domains show considerable sequence divergence (Figure 2A and Figure S2 and Table S9). The conserved B domain and the conserved residue Asp (D) (indicated by a red star in Figure 2A) are required for AtLEC1 function [12,90]. These observations collectively indicate that the central B domains may play key roles in the molecular function of AhLEC1a and AhLEC1b.

LEC1-type NF-YBs in plants are typically expressed specifically in seeds during embryogenesis, but are generally absent or present only at very low levels in vegetative tissues [13,16,17,20,85,91]. Functionally, they regulate multiple processes in seed development, including the accumulation of storage compounds [16,17,20,82,92], acquisition of desiccation tolerance [16,19], preparation for dormancy [19], and suppression of premature germination [17]. Consistent with these reports, transcriptome analyses in this study revealed that both AhLEC1a and AhLEC1b are specifically and highly expressed in peanut seeds (Figure 5A,B), with notably strong expression during early seed development (Figure 5C), corroborating a recent study [17].

Cis-acting element analysis revealed two AhLEC1s contain the putative motifs associated with light responsiveness, meristem expression, anaerobic induction, and gibberellin responsiveness (Figure 6 and Table S16). However, their expression remained low or undetectable under abiotic stress or phytohormone treatments, similar to normal conditions (Table S13). In Arabidopsis, repression of AtLEC1 in vegetative tissues is mediated by distal upstream regulatory sequences, as shown by constitutive expression in the lec1-d^tnp mutant with a 3256 kb deletion in the upstream region [93]. Similarly, deletion of distal promoter regions in AhLEC1s led to ectopic expression in vegetative tissues, resembling the lec1-d^tnp phenotype [94,95], indicating that proximal promoter regions are necessary for seed-specific expression. Although AtLEC1 is activated by AtLEC2 via RY motifs in coordination with AtWRI1 [96], no such RY motifs were detected in the promoters of AtL1L or AhLEC1s. Furthermore, both PICKLE (PKL, a CHD3 chromatin remodeling factor) and VIVIPAROUS ABI3-LIKE (VAL) proteins act epigenetically to repress AtLEC1 expression during vegetative development [97]. Thus, the mechanism enabling specific high expression of AhLEC1s in seeds requires further investigation.

Overall, the above imply strongly both AhLEC1s potentially play key roles in peanut seed development, similar to LEC1-type NF-YBs in other plants [10,12,85,88,98,99,100]. This is supported by functional studies showing that both genes participate in embryogenesis, embryo development, and reserve deposition in cotyledons, albeit with partial redundancy [17]. Specifically, expression of AhNF-YB10 (AhLEC1b) via the AtLEC1 promoter nearly fully rescued embryonic defects in lec1-2 mutant, whereas AhNF-YB1 (AhLEC1a) only partially complemented these defects [17]. Furthermore, overexpression of AhLEC1a reduced germination rates and seed longevity, a phenotype not observed with AhLEC1b [17], indicating functional divergence between the two paralogs.

3.2. Gene Duplication and Functional Diversification of AhFUS3s in A. hypogaea

In plants, at least one FUS3 has been identified as members of B3 transcription factor family in seed development and maturation [21,24,27,58,81,101]. In this study, two AhFUS3s, designated AhFUS3a and AhFUS3b (Table 1), were shown to contain one conserved B3 domain, similarly to other plants (Figure 1B, Figure 2B and Figure S3). Genomic analysis further revealed that single FUS3 orthologs are present in the diploid progenitors A. duranensis and A. ipaensis (Table 1 and Figure 3B,C), indicating that the two AhFUS3s in tetraploid peanut originated from the respective ancestral genomes. Phylogenetically, AhFUS3a and AhFUS3b cluster closely with FUS3 proteins from other legume species, including G. max, G. soja, and M. truncatula (Figure 1B), suggesting that both AhFUS3s have conserved functions similar to their orthologs in other plants, especially in legume species.

The FUS3 protein contains one conserved B3 domain flanked by divergent N- and C-terminal regions, all of which contribute to its regulatory functions. The B3 domain mediates sequence-specific DNA-binding to RY motifs in target gene promoters [102]. For example, a D75N mutation in the B3 domain of HvFUS3 abolished its binding to RY elements in seed storage protein genes [58]. In this study, the B3 domains of two AhFUS3s showed high sequence identity to those of other FUS3s, and the aspartate residue equivalent to HvFUS3 D75 is strictly conserved (Figure 2 and Figure S3), indicating that both AhFUS3s are likely functional in RY motif recognition and binding.

The N-terminal region of FUS3 is involved in post-translational modifications and protein-protein interactions [58,59,103]. In Arabidopsis, AKIN10 interacts with and phosphorylates the N-terminus of AtFUS3 at three serine residues (55-SSS-57), enhancing its stability [59,60]. Although these serine residues are not fully conserved across species (Figure S3), the N-terminal region remains critical for interactions with regulatory partners, such as the E3 ubiquitin ligase AIP2 (which promotes FUS3 degradation) [103] HvBLZ2 in barley [58], and GmNDPK1 in soybean [27].

The C-terminal region of FUS3 plays a key role in hormone sensing and transcriptional activation. In Arabidopsis, ABA and GA signaling converge on a PEST sequence in the C-terminus to positively or negatively regulate AtFUS3 protein stability, respectively [61,62]. In this study, two putative PEST sequences were found in the C-terminal domain of both AhFUS3s, a feature shared with other plant FUS3 proteins (Table S8). This region is also essential for transcriptional activation function [29,58,81], as truncation of the C-terminus in AtFUS3 results in only partial functional complementation [29,62]. These findings suggest that the C-terminal domains of both AhFUS3s are likely important for protein stability, activation capacity, and hormonal regulation.

Expression analyses indicate that FUS3 genes are primarily associated with seed development. AtFUS3 is highly expressed during mid-embryogenesis [21,104], and GmFUS3 in soybean is specifically expressed in pods and seeds [27]. In this study, both AhFUS3s genes in peanut showed high specific expression in developing seeds (Figure 5), implying a principal role in seed maturation.

The regulation of FUS3 is complex and involves multiple transcription factors. In Arabidopsis, AtFUS3 is directly activated by AtLEC2, AtLEC1, and AtWRKY TFs through RY, CCAAT, and W-box elements, respectively [96,98,105,106,107], but repressed by AtTT2 and AtTT8 via MWAMC fragments and E-boxes, respectively, during seed development [108,109]. Promoter analysis of AhFUS3a and AhFUS3b identified two RY motifs and three CCAAT boxes, respectively (Table S16), suggesting potential regulation by LEC1 and LEC2 homologs for their high seed-specific expression. LTR and ABRE elements were also detected in both promoters (Figure 6 and Table S16). Although poplar PeFUS3 is activated by PeABF2 binding to ABRE under osmotic stress [101], neither AhFUS3 genes showed elevated expression in leaves under abiotic stress or hormone treatments (Table S13). This indicates that these stress-related cis-elements may not drive significant transcription under the conditions tested or may require specific developmental or environmental contexts.

3.3. Gene Duplication and Functional Diversification of AhWRIs in A. hypogaea

In Arabidopsis, four WRI paralogs (WRI1-WRI4) belong to the AP2/EREBP family [31,32,33], and homologs have since been identified in numerous species [39,41,66,67,78,79,110,111,112,113,114,115,116,117]. In peanut, we identified fifteen WRI genes: nine AhWRI1s, two AhWRI2s, and four AhWRI3s (Table 1). Consistent with WRIs in other species [33,40,63,64,65,66,67,78], all AhWRI1s and AhWRI3s contain two AP2 domains, whereas two AhWRI2s owned only one (Figure 1C, Figure 2C,D and Figures S1 and S4–S6). Both AP2 domains are essential for DNA-binding in R. communis RcWRI1 [41], and in AtWRI1, the first AP2 domain or the segment between the two AP2 domains mediates interaction with LEC2 [96], suggesting they play similar roles in AhWRI1 functions.

Several functional domains were identified in AhWRIs. All contained the conserved “VYL” motif within the first AP2 domain (Figure 2C,D and Figures S4–S6), as with other previously reported WRIs [32,39,63,68,69,70,71]. Although alternative splicing can lead to VYL-less isoforms in some species [38,39,63,70] (Figure 2C,D and Figures S4–S6), the VYL-containing variant represents the major and functional transcript in plants [63,70]. While the VYL motif is essential for the function of AtWRI1 and GmWRI1b [39,70,117], its requirement is not universal, as evidenced by functional variants in RcWRI1 [63] and OsWRI1-1 [117]. Therefore, the functional importance of the VYL motif in AhWRIs remains to be determined. The 14-3-3/BPM-binding motif (corresponding to AtWRI1^78–92) is critical for protein stability, mediating either degradation via BPM-proteasome pathways or stabilization through 14-3-3 interaction [72,118]. This suggests that the putative 14-3-3/BPM-binding motifs in AhWRI1s and AhWRI3s may play similar roles in regulating their protein stability. A putative NLS in AtWRI1 (AtWRI1^33-41) mediates its nuclear localization [84]. Most AhWRIs were predicted to contain one NLS, except for AhWRI1a/b/c and AhWRI3c/d AhWRI1a/b/c and AhWRI3c/d (Table S8). Consistent with bioinformatic predictions (ProtComp 9.0, Table S8), both AhWRI1a and AhWRI1d were experimentally confirmed to be nuclear localized (Figure 7).

The C-terminal acidic regions are required for transactivation in AtWRI1 and GhWRI1 [32,78], whereas in AtWRI4, the N-terminal region containing the first AP2 domain is sufficient for transcriptional activation, and the C-terminal region with the second AP2 domain is dispensable [80]. Although not required for transactivation, the C-terminal IDR3-PEST motif in AtWRI1 modulates its protein stability upon phosphorylation [119]. Additionally, AtMED15 interacts directly with the non-C-terminal acidic region of AtWRI1 to facilitate transcriptional activation [120]. Therefore, the functions of the C-terminal acidic regions in AhWRI1/3s and their contribution to transactivation activity require further investigation.

WRI1s are highly expressed in seeds and show co-expression with fatty acid biosynthetic genes across species [31,39,40,41,78,110,113,117]. Loss of WRI1 function reduces seed oil content [9,31,36,66,121], while its overexpression enhances oil accumulation in seeds and vegetative tissues [37,38]. In peanut, AhWRI1a, AhWRI1b, and AhWRI1c exhibit seed-specific expression (Figure 5A,B), consistent with a recent report on AhWRI1a (Ahy_A08g040760) [122]. Overexpression of the AhWRI1a (GG genotype) in Arabidopsis increased seed oil content and unsaturated FA levels [122], suggesting that AhWRI1a, AhWRI1b, and AhWRI1c may act redundantly in oil accumulation. Their promoters contain multiple RY and CCAAT elements (Table S16), implying potential regulation by LEC2 and NF-Y transcription factors, similar to the activation of AtWRI1 by AtLEC2 binding to RY motif [36,96], and Elaeis guineensis EgWRI1-1 by EgNF-Y complexes binding specifically to CCAAT-boxes [123].

Beyond their role in oil biosynthesis, plant WRI1 homologs exhibit pleiotropic effects in various processes, including root nodulation in soybean [39], arbuscular mycorrhiza symbiosis in M. truncatula [79], auxin homeostasis [124], and cutin and wax biosynthesis [33,125]. For instance, GmWRI1a/b redundantly regulate nodulation and seed filling in soybean through plastidic glycolysis, lipid synthesis, and hormone signaling [39]. In peanut, AhWRI1d, AhWRI1f, and AhWRI1g were highly expressed in nodules (Figure 5A,B), suggesting that they may play key roles in root nodulation, despite showing less phylogenetic proximity to GmWRI1a/b [39] than AhWRI1a/b/c (Figure S11). In M. truncatula, MtWRI5s are involved in arbuscular mycorrhizal symbiosis via the regulation of genes involved in the biosynthesis of fatty acids and phosphate uptake in arbuscule-containing cells [79]. Phylogenetic analysis revealed close relationships between certain AhWRI1s and MtWRI5 genes: AhWRI1d/e with MedtrWRI5c, AhWRI1f/g with MedtrWRI5b, and AhWRI1h/i with MedtrWRI5a (Figure S11). Collectively, these findings suggest that AhWRI1d, AhWRI1f, and AhWRI1g may function in carbon partitioning during both root nodulation and arbuscular mycorrhiza symbiosis in peanut.

AtWRI2 is broadly expressed, with elevated levels in drying siliques and mature seeds [33,80] (Figure S10). It is unable to complement the wri1 mutant phenotype, and its loss does not affect seed oil content [33]. In contrast, Persea americana (avocado) PaWRI2 is highly expressed during fruit development and enhances triacylglycerol accumulation in transient overexpression assays [67]. In peanut, both AhWRI2a and AhWRI2b showed broad tissue expression (Figure 5A–C), were induced by drought but repressed by ABA, brassinolide, ethephon, and paclobutrazol (Table S13A,B). Their promoters contain multiple cis-acting elements associated with MeJA, anaerobic induction, gibberellin response, defense and stress signaling, and drought, suggesting functional roles beyond fatty acid synthesis.

AtWRI3 and AtWRI4 are ubiquitously expressed, with highest levels in vegetative tissues and flowers [33] (Figure S10). Although capable of partially rescuing the wri1 mutant, they are not essential for seed oil accumulation [33], but contribute to cutin biosynthesis in floral tissues, preventing organ fusion [33]. Furthermore, AtWRI4 is salt-induced and regulates cuticular wax biosynthesis [80]. Homologs in other species exhibit diverse expression patterns: CeWRI4 (Cyperus esculentus) is abundant in leaves and roots [65], RcWRI3 is pollen-specific [41], and MdWRI4 (Malus × domestica) is pericarp-specific and stress-responsive [64]. Heterologous expression of CeWRI4 and MdWRI4 in Arabidopsis improves stress tolerance by enhancing cuticular wax deposition [64,65]. In this study, AhWRI3a/b showed very low expression across tissues (Figure 5A–C), while AhWRI3c/d were more widely expressed (Figure 5A,B), particularly in florescences (Figure 5A), but not in seeds (Figure 5C). In addition, AhWRI3c/d were suppressed under drought and low-temperature conditions. This suggests possible roles in cuticular wax formation, florescence development, and stress responses, which merit further investigation.

3.4. Co-Overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d Alter Key Major Agronomic Traits

Previous studies have demonstrated that overexpression of individual regulators such as LEC1, FUS3, and WRI1 homologs increases oil/FA content and often modifies FA composition in seeds or seedlings [37,38,97,102,126]. Specifically, transgenic expression of AhNF-YB1/10 (AhLEC1a/b) and AhWRI1a (GG genotype) in Arabidopsis increased seed oil content by 6.2–29.1% and approximately 4.7%, respectively [17,122]. In our study, co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d in Arabidopsis significantly increased total FA content by 14.22–26.18% (Figure 8B), an effect comparable to that of AhNF-YB1/10 expressions and greater than that achieved with AhWRI1a (GG).

Transgenic expression of AhNF-YB1/10 elevated the contents of major FAs, with notable increases in C18:1n9c, C18:2n6c, C18:3n3, C20:1, and C22:1n9 [17]. Similarly, AhWRI1a (GG) overexpression raised unsaturated FA levels [122]. In our study, co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d increased both saturated and unsaturated FAs (16:0, 18:0, 18:1, 18:2, 18:3, 20:0, 20:1, and 20:2), though only line LFW-OX#3 significantly increased the ratio of unsaturated to saturated FAs (Figure 8B and Figure S9B).

Thousand-seed weight in the four transgenic lines increased significantly by 8.74–19.20%, compared to the 3.31–27.81% increase in AhNF-YB1/10-overexpressing plants [17]. Although thousand-seed weight was not quantitatively reported, AhWRI1a-OE produced longer seeds [122]. Consistent with reports that LEC1 or WRI1 overexpression enhances seed weight [17,40,71,78,92,111,127,128]. Together, these findings suggest that AhLEC1b, AhWRI1a, and AhWRI1d may cooperatively regulate both FA composition and seed weight.

Overexpression of LEC1 and FUS3 homologs often lead to adverse agronomic traits, including decreased plant height, shorter siliques, and lower seed germination rates [13,17,24,129]. For example, overexpression of ZmLEC1 driven by two embryo-preferred promoters (P_OLE and P_EAP1) decreased plant height and germination [13]. However, these negative effects can be mitigated by using suitable promoters [14,15,92]. Notably, both AhLEC1s were expressed under the truncated NapA promoter P211 showed no obvious defects throughout the life cycle, whereas AtLEC1 promoter-driven AhLEC1a significantly reduced germination rate and seed longevity [17]. In contrast, AhWRI1a (GG) overexpression promoted larger rosette leaves, early flowering, larger pods, and longer seeds [122]. FUS3 is known to influence plant architecture and germination by modulating ABA and GA levels [27,61,102]. In our study, co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d reduced plant height, silique length, and germination rate (Figure 8D–F), suggesting that AhLEC1b and AhFUS3b may function in a coordinated manner to regulate these traits.

These results demonstrate that co-overexpression of these regulatory genes markedly influences key agricultural traits, yet the molecular mechanisms underlying the trade-offs among FA composition, seed weight, plant architecture, and germination efficiency remain unclear and warrant further study. Previous studies highlight the importance of selecting appropriate promoters to maximize target gene expression while minimizing adverse phenotypic effects [14,15,17,92,127,130]. For instance, seed-specific co-expression of AtWRI1, AtDGAT1, and RNAi-AtSDP1 significantly increase seed oil content without impairing seed vigor or plant growth [130]. Therefore, future work will utilize suitable seed-specific promoters to co-express these peanut genes, reducing the risk of undesirable traits. Additionally, the biological functions of AhLEC1, AhFUS3, and AhWRI genes require systematic characterization in peanut and/or Arabidopsis.

4. Materials and Methods

4.1. Plant Materials and Growth Conditions

At the Laixi Experimental Station of Shandong Peanut Research Institute, the roots, stems, leaves, root nodules, and developing pods of peanut cultivar HY917, grown under natural conditions, were collected and pooled for cloning members of AhLEC1, AhFUS3 and AhWRI gene families. Additionally, developing seeds of four peanut sister lines (HY917, P19-19, P19-61, and P19-57) were sampled, immediately frozen in liquid nitrogen, and stored at −80 °C for subsequent analysis. These seeds were categorized into six stages based on kernel morphology according to Pattee et al. [75] with three biological replicates each containing more than 10 seeds (Figure S7).

All transgenic and wild-type (Col-0) Arabidopsis plants were cultivated concurrently under controlled environmental conditions: 16-h of light/8-h dark photoperiods, 23 °C day/21 °C night temperatures, and 60% relative humidity. The light intensity, measured at the mid-canopy level, was maintained at 160 µmol m⁻² s⁻¹.

4.2. BLASTing and Cloning of LEC1, FUS3, and WRI Family Members in A. hypogaea

Protein sequences of AtLEC1 (AT1G21970), AtL1L (AT5G47670), AtFUS3 (AT3G26790), AtWRI1 (AT3G54320), AtWRI2 (AT2G41710), AtWRI3 (AT1G16060), and AtWRI4 (AT1G79700) were retrieved from The Arabidopsis Information Resource (TAIR; accessed on 10 May 2023) [45] and used as queries to identify homologous sequences through BLASTP analysis (default parameters) against the genomes of the following species: A. hypogaea (accessed on 10 May 2023) [46,47,48,49], A. duranensis (accessed on 10 May 2023) [52], A. ipaensis (accessed on 10 May 2023) [52], A. monticola (accessed on 18 May 2023) [51], G. max (accessed on 21 September 2024) [131], G. soja (accessed on 21 September 2024) [131], M. truncatula (accessed on 21 September 2024) [132], H. annuus (accessed on 21 September 2024) [133], Gossypium hirsutum (accessed on 23 September 2024) [134], R. communis (accessed on 23 September 2024) [135], and T. cacao (accessed on 23 September 2024) [136] (see Table S1). The CDSs of putative AhLEC1s, AhFUS3s, and AhWRIs (Table S7) were cloned from HY917 using primers (Table S2) designed based on their predicted nucleotide sequences from A. hypogaea genome databases. The CDS, genomic DNA and protein sequences of putative LEC1, FUS3, and WRI homologs from other plant species were downloaded from their respective genome databases (Tables S1 and S3–S6).

4.3. Construction of Plant Expression Vectors and Genetic Transformation

For subcellular localization analysis, AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d were individually cloned into the plant expression vector pAN580 (P_35S::MCS::GFP). The empty vector pAN580 was used as a control. Each construct was co-transformed with a nuclear-localized marker (P35S::NLS::mCherry) into Arabidopsis protoplasts as previously described [137]. Images were captured under bright field, GFP, and mCherry fluorescence channels using a confocal laser-scanning microscopy (Zeiss LSM 510 META; Carl Zeiss Microscopy GmbH, Jena, Germany).

A tandem gene cassette, P_35S::AhLEC1b::T_NOS::P_MAS::AhFUS3b::CaMV poly(A) signal::P_NOS::AhWRI1a::CaMV poly(A) signal::P_35S::AhWRI1d::T_PE9, was designed, synthesized, and inserted into the binary expression vector pCAMBIA2300 by Shaanxi Jiyingjia Biotechnology Co., Ltd., Xi’an, China (Figure 8A). The resulting construct was introduced into A. tumefaciens C58, and then transformed into A. thaliana (wild-type Col-0) via the floral dip method [138]. Putative transgenic seeds were surface-sterilized with 10% NaClO and 0.5% Triton X100 for 5 min, rinsed four times with sterile distilled water, and plated on half-strength MS medium containing 50 mg L⁻¹ Kanamycin. Resistant plants were then transferred to square pots filled with a 1:1 (v/v) mixture of peat-based compost and vermiculite and grown under the conditions as described above.

4.4. Analysis of Phylogenetic Tree, Conserved Domain, Physicochemical Properties, Sequence Alignments, Gene Structures, Identity, and PEST Motifs

Protein sequences were used to construct phylogenetic trees with MEGA11 software using the Maximum Likelihood method and the Jones Taylor Thornton (JTT) model with 2000 bootstrap replicates [53], to identify conserved domains using Batch CD-Search in the Conserved Domain Database (CDD) of NCBI (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi, accessed on 22 October 2024) [55] under automatic search mode, and to predict the theoretical molecular weight (MW) and isoelectric point (pI) using the ExPASy ProtParam tool (https://web.expasy.org/protparam/, accessed on 22 October 2024). Multiple sequence alignments were generated using CLC Sequence Viewer 6.8.1. Identity analyses of both conserved regions and full-length protein sequences were conducted with MegAlign software (Sequence Distances algorithm) in DNASTAR (Lasergene.v7.1 suite). Gene structures were illustrated based on genomic DNAs and CDSs using the Gene Structure Display Serve (GSDS2.0; https://gsds.gao-lab.org/, accessed on 20 October 2024) [54]. Subcellular location was predicted using ProtComp v.9.0 in softberry (http://www.softberry.com/; accessed on 20 December 2024). Nuclear localization signals (NLSs) were identified with cNLS Mapper (https://nls-mapper.iab.keio.ac.jp/cgi-bin/NLS_Mapper_form.cgi, accessed on 22 January 2025) [83]. Putative PEST motifs were detected using the epestfind tool (https://emboss.bioinformatics.nl/cgi-bin/emboss/epestfind, accessed on 22 May 2025).

4.5. Chromosomal Location and Gene Synteny Analysis

Chromosome locations of LEC1s, FUS3s, and WRIs in A. hypogaea, A. duranensis, A. ipaensis, and A. monticola were acquired from the GFF genome files downloaded from their respective genome databases (Table S1). The positions were visualized on chromosomes using on the chromosomes using Gene Location Visualized from GTF/GFF in TBtools (v2.142) [139]. Gene synteny analyses were performed using the MCScanX algorithm implemented TBtools (v2.142) [139].

4.6. Analysis of Expression Patterns

The transcriptional patterns of AhLEC1s, AhFUS3s, and AhWRIs in different tissues across various development stages of peanut were obtained from the Peanut Genome Resource (PGR, accessed on 11 June 2024) and PeanutBase (accessed on 11 June 2024) [73,74]. Expression values were normalized by log₂(FPKM + 1) for PGR and log₂(TPM + 1) for PeanutBase, respectively, and visualized as HeatMaps using TBtools (v2.142) [139]. The transcriptional profiles of these genes under various stresses and phytohormone treatments were also retrieved from both databases.

Expression levels of AhLEC1s, AhFUS3s, and AhWRIs were analyzed across six stages of seed development of four peanut sister lines (P19-19, P19-61, P19-57, and HY917) based on our RNA-seq data (unpublished). Total RNA was extracted from 72 tissue samples using E.Z.N.A.^® Plant RNA Kit (Cat#R6827; Omega Bio-tek, Norcross, GA, USA) following the manufacturer’s protocol. Contaminating genomic DNA was removed using RNase-Free DNase I Set (Cat#E1091; Omega Bio-tek, Norcross, GA, USA). cDNA libraries were constructed with the NEBNext^® Ultra^TM RNA II Library Prep Kit for Illumina^® (NEB #E7770; New England Biolabs (NEB), Ipswich, MA, USA) and sequenced on an Illumina NovaSeq 6000 platform (2 × 150 bp paired-end reads) at Wuhan Benagen Technology Company Limited. (Wuhan, China), according to the manufacturer’s instructions. Raw paired-end reads were processed with fastp [140], and data quality was evaluated with FastQC (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/; accessed on 12 October 2023). Clean reads were aligned to the peanut reference genome (Tifrunner.gnm2.ann1.4K0L; https://data.legumeinfo.org/Arachis/hypogaea/annotations/Tifrunner.gnm2.ann1.4K0L/; accessed on 18 October 2023) using STAR [141]. The resulting alignments were sorted indexed, and quantified using RSEM [142] to obtain unique read counts per gene. Gene expression levels were normalized and reported as FPKM (Fragments Per Kilobase per Million mapped fragments). The FPKM values or AhLEC1s, AhFUS3s, and AhWRIs are provided in Table S14.

Expression data of AtLEC1, AtL1L, AtFUS3, AtWRI1, AtWRI2, AtWRI3, and AtWRI4 were obtained from publicly available Arabidopsis microarray datasets through the Arabidopsis eFP browser (https://bar.utoronto.ca/efp/cgi-bin/efpWeb.cgi; accessed on 12 June 2024) [143].

4.7. Analyses of Cis-Acting Elements and miRNAs Targeting AhLEC1s, AhFUS3s, and AhWRIs

Cis-acting elements were predicted using the putative promoter regions (2000 bp upstream of the start-codon) of LEC1s, FUS3s, and WRIs from Arachis hypogaeaLine8 v1.3 and and A. thaliana Araport11 using PlantCARE (https://bioinformatics.psb.ugent.be/webtools/plantcare/html/; accessed on 12 October 2024) [76], and the results were then visualized with TBtools (v2.142) [139]. The CDSs of LEC1s, FUS3s, and WRIs from HY917 and A. thaliana were submitted to psRNATarget (https://www.zhaolab.org/psRNATarget/, accessed on 22 March 2025) [77] to identify potential targeting miRNAs under default parameters.

4.8. Analysis of the Content and Composition of Fatty Acids in Seeds

The mature seeds used for FA analysis were harvested from the lower part of the main stem of plants grown in different pots arranged randomly in one of three blocks. Seed FAs were extracted and analyzed as previously reported [144]. In detail, the total FAs were converted to FA methyl esters (FAMEs) in 1 M HCl-methanol at 80 °C for 2 h. FAMEs were analyzed via by GC–MS (GC-QQQ, 7890A-7001B, Agilent Technologies, Santa Clara, CA, USA) equipped with an HP-FFAP capillary column (30 m × 0.25 mm ID, 0.25 μm film thickness). Helium was used as the carrier gas at a flow rate of 1.0 mL/min with a split ratio of 5:1. The injection volume was 1.0 μL, and the injection temperatures were held at 250 °C. The temperature of the column oven was programmed from 40 °C (held for 5 min) to 250 °C (held for 5 min) at 8 °C/min. Mass spectrometry conditions were as follows: the energy of EI ionization source was 70 eV, the ion source temperature was 230 °C, the quadrupole temperature was 150 °C, the interface temperature was 250 °C, and the scan range was 30–400 m/z. Heptadecanoic acid (C17:0; Sigma-Aldrich, Saint Louis, MO, USA) was added as internal standard prior to extraction. Analyses were performed in biological triplicates.

4.9. Measurement of Thousand-Seed Weight, Plant Height, and Silique Length, and Warm Germination Test

Mature seeds used for thousand-seed weight analysis were collected from the lower portion of the main stems. The seeds were dried in open tubes in an oven for one week and then weighed using an electronic microbalance. Plant height and the silique length of main stem were measured on mature plants using 50 cm and 10 cm rulers, respectively.

For the warm germination test, mature seeds were evenly arranged in a row on a moist filter paper, covered with another moist filter paper, and then rolled up. The roll was wrapped in waxed paper and placed in a large beaker containing 2.5 cm (1 inch) of water at the bottom. The beaker was incubated in a growth chamber at 25 °C. Germination rates were assessed seven days after seeding.

4.10. Quantitative RT–PCR (qRT–PCR) Analysis of Gene Expression

The total RNA was extracted from Arabidopsis siliques at 15 days after flowering (DAF) using the RNAprep Pure Plant Kit (Cat#DP441, TIANGEN BIOTECH Co., Ltd., Beijing, China). First-strand cDNA was synthesized from 1 μg of total RNA using the FastKing RT Kit (with gDNase) (Cat#KR116, TIANGEN BIOTECH Co., Ltd., Beijing, China), according to the manufacturer’s instructions. Each cDNA sample was diluted 10–15-fold in sterile water prior to qRT–PCR analysis.

qRT–PCR was carried out on a CFX Connect^TM Real-Time PCR system (Bio–Rad, Hercules, CA, USA) with EvaGreen 2× qPCR MasterMix (MasterMix-S, Abm, Vancouver, BC, Canada), following the manufacturer’s protocol. The expression levels of the target genes were normalized using Arabidopsis AtActin as an internal control. Relative expression level was calculated by 2^−ΔΔCt. The primers used are listed in Table S3. All reactions were performed in biological triplicates.

4.11. Statistical Analysis

All the data are presented as the mean ± standard deviation at least three replicates. Statistical analyses were conducted in Excel 2016 and GraphPad Prism 6.02. Significant differences were determined by one-way analysis of variance (ANOVA) followed by Dunnett’s post hoc test. All bar charts were generated using GraphPad Prism 6.02.

5. Conclusions

In this study, two AhLEC1s, two AhFUS3s, nine AhWRI1s, two AhWRI2s, and four AhWRI3s were cloned from A. hypogaea HY917 and comprehensively analyzed using bioinformatics and expression profiling. The results revealed that AhLEC1a/b, AhFUS3a/b, and AhWRI1a/b/c, which are specifically expressed in developing peanut seeds, likely play critical roles in seed development, while AhWRI1d, AhWRI1f, and AhWRI1g, highly expressed in root nodules, may be involved in nodulation. Co-overexpression of AhLEC1b, AhFUS3b, AhWRI1a, and AhWRI1d in Arabidopsis significantly increased seed fatty acid content and thousand-seed weight. However, these improvements were accompanied by adverse agronomic traits, including reduced plant height, shorter siliques, and lower germination rates, highlighting a trade-off between lipid metabolism and plant growth. In summary, AhLEC1, AhFUS3, and AhWRI genes may play crucial roles in regulating oil synthesis and seed development in peanuts. Future work should prioritize using seed-specific promoters to drive the expression of these genes, thereby minimizing negative effects on plant growth. Further functional characterization in both peanut and Arabidopsis will be necessary to fully exploit their potential for improving oil traits in crops.