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Seed-specific expression of phosphatidate phosphohydrolases increases soybean oil content and seed weight

Biotechnology for Biofuels and Bioproducts volume 18, Article number: 23 (2025) Cite this article

Abstract

Background

Soybean is a major oil crop and a primary protein source for livestock, and soybean oil is the most common input for biodiesel. Identifying genes that enhance soybean yield and oil content is crucial for breeding programs. Phosphatidic acid (PA) phosphohydrolase (PAH), which dephosphorylates PA to diacylglycerol (DAG), plays a critical role in lipid synthesis, and yet their potential in improving agronomic traits of oil crops remains unexplored.

Results

This study shows that seed-specific expression of AtPAH1/2 enhances PA turnover into DAG and triacylglycerol (TAG) accumulation in soybean seeds. PAH overexpression upregulated the expression of DAG acyltransferase (DGAT) but suppressed phospholipid: DAG acyltransferase (PDAT). In addition, seed-specific expression of AtPAH1/2 increases soybean seed size and weight. Furthermore, analysis of the variation of the soybean PAHs in 4414 soybean accessions indicated that the advantageous effects of GmPAHs on oil content and seed weight were selected during domestication.

Conclusion

These findings suggest that targeting PAHs represents a promising strategy for enhancing soybean seed oil content and yield in current cultivars and landraces soybeans.

Background

Soybeans are a crucial global source of vegetable proteins and oils, accounting for approximately 22% of world fat and oil production. Soybean oil is the most common source of biodiesel, accounting for over 40% of the total feedstock used to make biomass-based diesel. However, soybean seeds currently contain only around 20% oil, indicating significant potential for improvement compared to other oilseed crops, such as sunflower (40–50%) and rapeseed (35–50%) [1]. Genes involved in fatty acid synthesis have been extensively investigated to enhance plant seed oil content [2, 3]. Phosphatidic acid (PA) phosphohydrolases (PAH) catalyze an Mg2+-dependent dephosphorylation of PA to diacylglycerol (DAG) that can be converted to triacylglycerol (TAG) by either DAG acyltransferase (DGAT) or phospholipid: DAG acyltransferase (PDAT). DAG also serves as a precursor for the synthesis of membrane phospholipids, such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by DAG-choline or -ethanolamine phosphotransferase. Additionally, PA contributes to the synthesis of phosphatidylglycerol (PG), phosphatidylinositols (PI), and phosphatidylserines (PS) via the cytidine diphosphate diacylglycerol (CDP-DAG) pathway [4]. Therefore, PAH plays a central role in regulating the levels of membrane phospholipids and storage lipids, affecting various aspects of plant growth, development, and stress response [5,6,7,8].

PAH is localized at the cytosolic and membrane fractions of cells [9]. In yeast and mammalian cells, PAH1 and PAH2 serve distinct physiological roles. PAH1, encoded by phosphatidate phosphatase 1, is involved in de novo lipid synthesis, whereas PAH2 participates in lipid signaling [10,11,12,13,14]. In Arabidopsis, PAH1 and PAH2 play redundant, important roles in phospholipid biosynthesis [7]. Studies have shown that pah1pah2 double-mutants exhibit increased phospholipid content due to enhanced net PC biosynthesis and reduced TAG synthesis [5]. Mutants lacking PAH displayed elevated PA levels and retarded growth phenotypes in both plants and animals [7, 15,16,17]. Under phosphate-deficient conditions, pah1/pah2 exhibited retarded plant growth and glycolipid synthesis compared to phosphate-sufficient conditions [17]. Overall, these findings suggest that PAH plays a significant role in regulating cell size and proliferation under normal and stressful conditions.

Recent findings have revealed a positive correlation between seed oil content and seed size, suggesting a shared genetic control over these traits [18,19,20,21]. The shape and size of membrane-bound organelles undergo dynamic and remarkable changes throughout the cell cycle and developmental stages, facilitated by lipid bilayer components [22, 23]. In yeast, the PAH homolog plays a regulatory role in the growth of nuclear and ER membranes during the cell cycle [16]. Additionally, PAH is implicated in cell cycle regulation through its modulation of TAG or phospholipid levels, with its activity repressed by the cell cycle regulator cyclin-dependent kinase A-1 (CDKA,1) [10, 24,25,26,27]. However, the impact of PAH on crop organ size remains poorly understood. In this study, we introduced Arabidopsis PAH1 and PAH2 (AtPAH1/2) genes driven by a seed-specific promoter into soybean to investigate the role of PAHs in seed oil accumulation and seed development. Our results indicate that seed-specific expression of AtPAH genes influences lipid metabolism and seed size.

Results

Overexpression of PAHs increased TAG accumulation in soybean seeds

To investigate the role of PAH in soybean seeds, we introduced AtPAH1 and AtPAH2 under the control of the seed-specific promoters of glycinin and β-conglycinin, respectively (Fig. 1A). Genomic DNA analysis confirmed the insertion of AtPAH1 and AtPAH2 into the soybean genome across three independent transgenic lines (Supplemental Fig. 1). To assess gene expression during seed development, RT-qPCR was performed on stable transgenic soybean lines (Fig. 1B). The expression levels of AtPAH1 and AtPAH2 varied among the lines, with the highest expression observed in transgenic line 2 (OE-2). During seed development, AtPAH1, driven by the glycinin promoter, displayed higher expression in the early stages of developing seeds, such as 10 and 20 days after flowering. In contrast, AtPAH2, driven by the β-conglycinin promoter, exhibited higher levels of expression at 30 and 45 days after flowering (Fig. 1C).

Fig. 1
figure 1

Expression of AtPAHs in soybean with seed-specific promoters. A Construction of AtPAHs overexpression in soybean. AtPAH1 is driven by the glycinin promoter and AtPAH2 is promoted by the β-conglycinin promoter. B Transcript level of AtPAHs in developing seeds. Data are means ± SD (n = 3). Differences between WT and overexpression lines were analyzed, *P < 0.05; **P < 0.01 using Student’s t-test. C Transcription level of AtPAH1 and AtPAH2 in transgenic developing soybean seeds. RNA was extracted from T5 generation developing seeds at 10, 20, 30, and 45 days after flowering. Data are mean ± SD (n = 3). ANOVA (analysis of variance) was employed to assess differences in AtPAHs gene expression across various developmental stages. A p-value of < 0.05 was considered statistically significant

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Given that PAH catalyzes a pivotal step in glycerolipid synthesis by converting PA to DAG, a precursor for TAG production, we examined whether seed-specific expression of AtPAH1/2 influenced DAG and TAG accumulation in soybean seeds. Lipids were extracted from developmental seeds (10, 20, 30, and 45 days after flowering) and mature seeds (60 days after flowering) (Fig. 2C). TAG levels in developing (45 d) and mature AtPHA1/2-expressed seeds (219.8 ± 3.3 and 329.6 ± 2.7 mg per g, respectively) were 20.9% and 6.7% higher compared to wild-type (WT) seeds (181.7 ± 10.7 and 308.8 ± 4.2 mg per g, respectively) (Fig. 2A). DAG content was elevated in AtPAH-expressed lines at 10, 20, and 30 days, and the level went down at 45 days, which could mean efficient DAG turnover to TAG in older seeds. We assessed the transcription level of the key TAG biosynthesis genes, GmDGAT1A (Glyma.13g106100) and GmPDAT1B (Glyma.13g108100), which are known to have the highest expression in soybean seeds [28]. The results showed that the expression levels of GmDGAT1A began rising at 20 days and peaked at 30 days. The expression levels of GmDGAT1A were higher in the developing seeds of AtPAH1/2-expressed soybean at 30 days and 45 days compared to WT (Fig. 2D). On the other hand, GmPDAT1B expression was lower in AtPAH1/2-expressed seeds than WT (Fig. 2E). Additionally, a time-course analysis of fatty acid composition indicated no significant changes in fatty acid composition in AtPAH1/2-expressed seeds (Supplemental Fig. 2).

Fig. 2
figure 2

Increased oil content in AtPAHs-expressed soybean seeds. A TAG contents in AtPAHs-expressed or WT developing and mature soybean seeds. Values are means ± SD (n = 5). B DAG content in WT and AtPAHs overexpression developing seeds. Data are means ± SD (n = 5). C Appearances of WT and AtPAHs overexpression developing seeds. Bar = 1cm. D TAG synthesis genes GmDGAT1A and GmPDAT1B expression level in WT and transgenic soybean seeds. Data are means ± SD (n = 3). Student’s t-test for significant difference, *p < 0.05; **p < 0.01

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Polar lipid composition changed in developing seeds

To explore the effect of ectopic expression of AtPAH1/2 on polar lipid metabolism in transgenic lines, lipid extraction was conducted from seeds harvested at 10, 20, 30, and 45 days after flowering, followed by mass spectrometry profiling. Significant alterations in various membrane lipid classes were detected in developing seeds compared to WT controls (Fig. 3). All three AtPAH1/2-expressed soybean lines displayed significant increases in monogalactosyldiacylglycerol (MGDG), PG, and PS in 20-day-old developing seeds compared to WT. At the 20-day-old seeds, the level of digalactosyldiacylglycerol (DGDG) and PE tended to increase in all three AtPAH1/2-expressed lines, but only the line OE-2 that had the highest AtPAH1/2 expression showed a significant increase. In 30-day-old seeds, the PE level increased in OE-1 and OE-3 (Fig. 3). By comparison, the PC level showed no significant difference between WT and AtPAH1/2-expressed soybean lines. The PC level decreased as seeds matured (Fig. 3). In contrast, all three AtPAH1/2-expressed soybean lines showed a marked decrease in PA levels, the substrate for PAH, at the early stages of seed development compared to WT (Fig. 3). These results suggest that the ectopic expression of AtPAH1/2 in soybean seeds promotes the conversion of PA into polar lipid and DAG.

Fig. 3
figure 3

Effect of AtPAHs expression on membrane glycerolipid levels in developing seeds. Total lipids were extracted and analyzed using ESI–MS/MS. Data are means ± SD (n = 5). Significance compared with WT at 10, 20, 30, and 45 days after flowering. *p < 0.05; **p < 0.01 (Student’s t-test)

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Seeds expressing AtPAH1/2 enhanced polar lipid turnover to TAG

To further investigate the impact of ectopic expression of AtPAH1/2 on lipid metabolism in seeds, we analyzed lipid biosynthesis in developing seeds using [3H]-acetate pulse-chase labeling. Developing seeds at 19–20 days after flowering from WT, OE-1, and OE-2 lines were labeled with [3H]-acetate for one hour and then chased for 0, 1, 6, and 12 h. The results revealed a progressive decrease in the concentration of 3H in DAG and PC throughout the chase period. Conversely, levels of TAG and PE, which both utilize DAG as substrate, showed an increase (Fig. 4). Concurrently, the radioactivity percentage in MGDG and DGDG exhibited a gradual rise, peaking at the 6-h chase period (Fig. 4).

Fig. 4
figure 4

In vivo labeling lipids of developing seeds with [3H]-acetic acid. Developing seeds at 19 to 20 days after flowering were incubated with [3H]-acetic acid for 1 h and subjected to lipid extraction at 0, 1, 6, and 12 h, respectively. Values are means ± SD (n = 5). Significance compared with WT, *p < 0.05; ** p < 0.01 (Student’s t-test)

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In transgenic lines, 3H concentration in PA initially rose but declined by the 12-h mark. By comparison, the PA level remained elevated in WT and higher than that in AtPAH1/2-expressed lines. Notably, transgenic plants exhibited a higher DAG content than WT before the chase, and the labeled DAG level became similar at 6 h after the chase (Fig. 4). TAG content in transgenic seeds remained higher than in WT. Compared to WT seeds, AtPAH1/2-OE seeds had a lower level of PA but a higher level of DAG at earlier stages, consistent with the effect of PAH conversion to DAG. The lack of difference of DAG at later stages between AtPAH1/2-OE and WT plants could result from the increased conversion of DAG to TAG (Fig. 4). The lower PA labeling than DAG could result from the lower PA than DAG levels in seeds. Overall, these labeling patterns support that AtPAH1/2-expressed soybean seeds increase the conversion of PA to DAG and subsequently to TAG compared to WT seeds (Fig. 4).

Seed-specific expression of AtPAH1/2 increased seed weight in soybean

The Arabidopsis pah1/pha2 double-mutants displayed an abnormal growth phenotype with slower growth rates than WT [7]. To investigate the impact of seed-specific expression of AtPAH1/2, we evaluated seed size and weight. Results showed that AtPAH1/2-expressed soybean seeds were larger in ten-seed width (7.3 ± 0.05 cm) and ten-seed length (8.4 ± 0.07 cm) by 5.0% and 6.7%, respectively, compared to WT soybean seeds (6.9 ± 0.12 width and 7.86 ± 0.14 length) (Fig. 5A, B). Consistent with the increase in seed size, the hundred-seed weight of the AtPAH1/2 lines was 11.4% heavier than that of WT (Fig. 5C). Moreover, the transgenic plants displayed a marked increase in the number of seeds per plant (Fig. 5D). To probe the mechanisms underlying seed size effect by AtPAH1/2, we examined the expression level of BIG SEEDS1 (BS1), a conserved gene known to regulate seed size by inhibiting primary cell proliferation [29]. Transcriptional analysis revealed a notable decrease in GmBS1 expression in 20-day-old seeds of three independent transgenic soybean plants (Fig. 5E). Additionally, genes specific to the S-phase of cell division, cyclin D3 (GmCYCD3) and GmHISTONE4, were significantly upregulated in these transgenic plants (Fig. 5E). These data could mean that enhanced cell proliferation during seed development could play a role in the larger seed size, but the effect of the AtPHA1/2 overexpression on cell size and proliferation requires further investigation.

Fig. 5
figure 5

Enhanced seed size and weight in AtPAHs expressing soybean. A Seed width is measured from the left to the right, with the seed umbilicus being oriented to the right. B Seed length is measured from the left to right, with the seed umbilicus facing upwards. Bars = 1cm. Comparisons of seed width (A, right), seed length (B, right), seed weight (C), and seed number per plant D of WT and AtPAHs overexpression line. Values are means ± SD (n = 10). E Expression level of seed size-related genes in 20d soybean seeds. Data are means ± SD (n = 3). P < 0.05 is indicated by *, and P < 0.01 is indicated by **, determined by Student’s t-tests

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GmPAH1 effects on seed size and oil content have been selected through domestication

The functionality of the lipid biosynthesis gene tends to be conserved across different species. In soybean, there are three PAH1 homologous genes: Glyma.10g046400, Glyma.13g134500, and Glyma.19g175600 (Supplemental Fig. 3). Protein sequence alignment revealed that Glyma.10g046400 and Glyma.13g134500 exhibit a high degree of similarity (94% identity) with each other, while their similarity with Glyma.19g175600 is lower, at 73% and 74% identity, respectively. To investigate the impact of PAH homologous on seed size (100-seed weight) and seed oil content in soybean, genetic variation was explored using the soybean multi-omics database (soyMD) [30]. Analysis of nucleotide polymorphisms in the PAH1 homolog Glyma.10g046400 revealed nine haplotypes (hap). The oil content and 100-seed weight of the hap0 to hap7 genotypes were considerably higher than those of the hap8 genotype, with 139 SNPs displayed p-values of 9.78e−20 and 7.735e−11, respectively (Fig. 6A, B). We studied the distribution of various haplotypes in cultivars, landraces, and wild populations since domestication is the process by which humans apply artificial selection to a wild soybean (Glycine soja). Allele-frequency analysis of 4,414 accessions from various sub-populations showed that both cultivars and landraces have hap0 through hap7. On the other hand, hap8 was only present in wild soybeans and not in cultivars or landraces (Fig. 6C). Further geographical distribution analysis of the nine haplotypes revealed that hap8 is exclusively distributed in Asia, the origin of soybean (Fig. 6D).

Fig. 6
figure 6

Haplotype analysis of Glyma.10g046400 in the soybean natural variation population. A Total oil content (%) of each soybean accession across nine haplotypes of Glyma.10g046400 on Chr10:4155215.4164211 ± 2 kb. B One hundred seed weight (g) of each soybean accession across the nine haplotypes of Glyma.10g046400. C Allele frequencies of the nine haplotypes of Glyma.10g046400 in cultivar, wild, and landrace sub-populations. Numbers represent the percentage of accessions of each haplotype in the three sub-populations. D Geographical distribution of Glyma.10g046400 gene across different haplotypes. The pie chart areas represent the proportion of each haplotype in various regions. The colors corresponding to the haplotypes are shown to the right of panel A

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Haplotype analysis of another homolog, Glyma.13g134500, showed that hap0 and hap1 have higher oil content and 100-seed weight than hap2, with 97 SNPs showing p-values of 9.629e−22 and 3.837e−4, respectively (Fig. 7A, B). Hap2 was only found in wild soybeans (Fig. 7C) and is distributed in Asia (Fig. 7D). Meanwhile, Glyma.19g175600 exhibited haplotype differences in 100-seed weight (p-value = 6.404e−3) but not in total oil content (p-value = 0.1075) (Fig. 7E, F). The receptor soybean cultivar Jack used in this study contained the beneficial PAH haplotypes of both Glyma.10g046400 and Glyma.13g134500. These results suggest that Glyma.10g046400 and Glyma.13g134500 affect seed oil content and yield traits simultaneously and have been selected during the domestication and breeding of wild soybeans to cultivar or landrace varieties. In other words, the GmPAH genotype found in existing cultivars and landraces is a superior variant characterized by elevated oil content and increased seed weight. This implies that increasing oil content and seed weight in current cultivars and landraces through PAH genotype screening may not be feasible. Instead, using seed-specific promoters to increase the expression of PAH genes in soybean, as shown in the present study, presents a potential avenue to enhance oil content and seed weight in soybean cultivars.

Fig. 7
figure 7

Haplotype analysis of Glyma.13g134500 and Glyma.19g175600 in soybean population. A Total fat content (%) of each soybean accession across four haplotypes of Glyma.13g134500 (Chr13:24696109.24705068 ± 2 kb). B One hundred seed weight (g) of each soybean accession across the four haplotypes of Glyma.13g134500. C Allele frequencies of the five haplotypes of Glyma.13g134500 in cultivar, wild, and landrace sub-populations. Numbers represent the percentage of accessions of each haplotype in the three sub-populations. D Geographical distribution of Glyma.13g134500 gene across different haplotypes. The pie chart areas represent the proportion of each haplotype in various regions. The color of Glyma.13g134500 associated with the haplotypes is shown in panel C of the upper section. E Total fat content (%) of each soybean accession across three haplotypes of Glyma.19g175600 (Chr19:43561808.43569019 ± 2 kb). F One hundred seed weight (g) for each soybean accession in the three haplotypes of Glyma.19g175600. The colors of Glyma.19g175600 corresponding to the haplotypes are displayed to the right of panel E

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Discussion

Enhancing crop productivity and ensuring global food security are critical goals for sustaining adequate human nutrition. Transgenic technologies offer an efficient approach to boost crop productivity, protection, and quality. Numerous genes from various plant species have proven invaluable for crop enhancement. However, the use of constitutive expression promoters in transgenic plants can lead to abnormal morphology, including retarded development and sterility. [31, 32]. In response to these challenges, seed-specific promoters have been utilized to enhance seed oil accumulation and improve fatty acid composition. The promoters of Napins, which are vital seed storage proteins in Brassica species, have been utilized to drive the expression of leafy cotyledon1 (BnLEC1) and LEC1-like (BnL1L) genes, resulting in a significant increase of up to 20% in seed oil content without adverse effects on major agronomic traits [33]. Another study compared the effect of the β-conglycinin promoter-driven seed-specific vs the Cauliflower Mosaic Virus (CaMV) 35S promoter-driven constitutive expression of fatty acid desaturase 3 (FAD3) and found a 1.6-fold higher increase in fatty acid content with the seed-specific promoter compared to the CaMV 35S promoter [34]. Moreover, overexpression target genes in transgenic plants can introduce genetic diversity beyond a single species or genus, opening avenues for introducing specific traits from other species.

Cultivated soybeans, including landraces and cultivars, were domesticated from wild soybeans in China approximately 5,000 years ago [35]. Traits associated with advantageous genes and loci were selected during the transition from wild types to landraces, and these genes may not be used for further improvement in molecular design breeding. In this study, we identified favorable PAH genotypes selected through domestication and breeding and specifically expressed the Arabidopsis PAH gene in soybean seeds, resulting in increased seed oil content and seed weight (Fig. 2A, Fig. 5C). This suggests that tissue-specific expression of exogenous genes can further improve agronomic traits of crops based on previously selected beneficial genotypes. Therefore, leveraging transgenic approaches, alongside the identification and incorporation of beneficial genes from diverse organisms, holds substantial promise for significantly improving crop traits, particularly seed yield and quality in soybeans.

PAH is an evolutionarily conserved enzyme found in both plants and animals. PAH plays a crucial role in maintaining lipid homeostasis by regulating cellular levels of PA to DAG. Mutation in PAH retarded growth in Arabidopsis and mice [7, 53]. So, this study chose seed-specific expression of PAH1/2 to minimize PAH effects on other tissues. In soybean seeds, ectopic expression of AtPAHs not only increased TAG content, but also elevated glycolipid levels. This suggests that increased cellular pools of DAG can be utilized for the synthesis of both storage and membrane glycerolipids, consistent with similar findings in Arabidopsis [7]. While AtPAH expression increased TAG content in developing and mature seeds, there were no significant differences observed in the fatty acid composition of the TAGs.

The level of AtPAH1 ectopic expression was way higher than that of AtPAH2. In addition, AtPAH1 and 2 displayed different patterns of expression during the seed development, with AtPAH1 expressed highly in the early, whereas AtPAH2 highly in later stages (Fig. 1C). These disparities in expression could result from the use of different promoters as AtPAH1 was driven by the glycinin promoter whereas AtPAH2 was under the control of the β-conglycinin promoter. The current experimental design involved co-expression of both genes simultaneously and thus could not attribute the observed seed phenotype to the OE of single AtPAH1 or AtPAH2.

Genes involved in oil synthesis also have been reported to influence seed size and weight [36,37,38,39,40]. [19, 21]), potentially due to an increased supply of the source repository. Ectopic expression of AtPAH1/2 in soybean seeds upregulated GmDGAT1A RNA levels, enhancing DAG synthesis and subsequent TAG production by boosting GmDGAT1A activity. Previous Arabidopsis studies have shown that regulating carbon flux into TAGs can increase seed oil content and seed weight [39]. Analysis of dividing cells has revealed that lipids, including PA and TAG, contribute to the structural integrity during cell division. Knockdown of DGAT and other lipid metabolic enzymes results in cell division defects [41, 42]. In our study, ectopic expression of AtPAH1/2 in soybean seeds increased seed size, indicating an impact on cell proliferation during seed development. On the other hand, phospholipids also play a crucial role as cell membrane components during cell division, supporting changes in the plasma membrane. Notably, around 20 days after flowering, corresponding to rapid seed enlargement, the disparity in polar lipids content between soybeans expressing AtPAH1/2 and WT soybeans becomes most prominent. Moreover, both the substrate and the product involved in the PAH-catalyzed reaction serve as signaling lipids. Lipids can act directly as ligands or assist in assembling proteins into signaling platforms, which is crucial for cellular signaling processes. Given that PAH affects levels of PA and DAG in developing soybean seeds, investigating the potential contribution of these lipid signaling molecules to cell size regulation would be valuable.

Conclusion

Here, we identified that the superior GmPAH genotype, which enhances oil content and 100-seed weight, has been selected during the domestication of soybeans. We further showed that seed-specific expression of AtPAHs in cultivated soybeans enhanced seed oil content and seed weight. The ectopic expression of AtPAHs increased the conversion of PA to DAG that is further acylated to TAG via DGAT. Additionally, the expression of AtPAHs promoted the synthesis of phospholipids and glycolipids during seed development. These results indicate the potential application to use the seed-specific expression of PAHs to improve seed oil and crop yield even in the present superior genotypes.

Materials and methods

Plant materials and growth conditions

Soybean transformation was performed using an improved Agrobacterium-mediated method from the Plant Transformation Core (PTC) at the University of Missouri [43,44,45]. Agrobacterium-mediated soybean plants were selected based on glufosinate ammonium (Basta) resistance and the DsRed marker for selection. Positive plants were subsequently confirmed using gene-specific primers via PCR (Supplemental Fig. 1). Three independent lines from the T5 generation were used for subsequent experiments. Soybean wild-type Jack ecotype and three transgenic lines were grown in 2.5-gallon pots filled with Berger 7–35% soil mix in a greenhouse. The plants were cultivated under controlled conditions with 14 h of light and 10 h of darkness, maintaining temperatures at 26 °C during the day and 24 °C at night. Supplemental lighting was used when sunlight levels fell below 400W/m2 between 6 and 8 am, with humidity maintained at 40%. Additionally, a shade curtain automatically closed to 50% when sunlight exceeded 900W/m2, and fully closed at 100% when levels surpassed 1000W/m2.

Construction of plant transformation vectors

The genomic sequences of PAH1 and PAH2 from Arabidopsis were amplified using the following primer pairs: AtPAH1F (5'-GCGGAATTCATGAGTTTGGTTGGAAG-3') and AtPAH1R (5'-GCGGAATTCTCATTCAACCTCTTCTAT-3') for AtPHA1 (At3g09560), and AtPAH2F (5’-GCGGCGGCCGCATGAATGCCGTCGG-3’) and AtPAH2R (5’-GCGGCGGCCGCTCACATAAGCGATG-3’) for AtPAH2 (At5g42870). The AtPAH1 PCR product was then ligated into the pJC-Gly-DsRed binary vector at the EcoRI site, which includes the seed-specific glycinin promoter, the Basta resistance gene, and the DsRed marker. The AtPAH2 DNA fragment was cloned into the NotI site of the Beta-ConSoyHyg plasmid, containing the β-conglycinin promoter. Subsequently, the DNA segment comprising the β-conglycinin promoter, AtPAH2 genomic sequence, and terminator was excised from the Beta-ConSoyHyg plasmid using AscI, which has a compatible sticky end with MluI, and then inserted into the MluI site of the pJC-Gly-DsRed vector containing AtPAH1. The resultant constructs, named pJC-Gly-DsRed-AtPAH1/2, contained the genomic sequence of AtPAH1 under the control of the Gly-promoter, while AtPAH2 was driven by the β-conglycinin promoter. These constructs were then transformed into the GV3101 strain of Agrobacterium tumefaciens for soybean plant transformation.

RNA extraction and real-time PCR analyses

Total RNA was extracted from soybean seeds at various developmental stages using an RNA isolation kit (Qiagen) according to the manufacturer's instructions. Genomic DNA contamination was eliminated using RNase-free RQ1 DNase (Promega), and RNA concentration was assessed with a NanoPhotometer (Implen). Subsequently, cDNA synthesis was performed using a qScript cDNA Synthesis Kit (Quanta Biosciences) starting with 1 μg of total RNA. Transcript amplification utilized gene-specific primers, as detailed in Supplemental Table 1. PCR conditions involved initial denaturation at 94 °C for 3 min, followed by 40 cycles of 94 °C for 30 s, annealing at 58 °C for 30 s, and extension at 72 °C for 30 s, employing the iQ5 Real-time PCR system (BioRad) with SYBR Green. We processed the RT-qPCR data using the 2(-Delta Delta C(T)) method [46]. Expression levels of target genes were normalized to the internal control, soybean ACTIN.

Lipid extraction and profiling

Total lipids were extracted from developing seeds following a previously established protocol with minor adjustments [47]. Briefly, seeds at various developmental stages were isolated from pods and immediately immersed in 3 mL of preheated (75 °C) isopropanol containing 0.01% butylated hydroxytoluene (BHT), then incubated for 15 min. Next, 1.5 mL of chloroform and 0.6 mL of water were added, and the mixture was shaken for 1.5 h at 220 rpm. The resulting lipid extract was transferred to a new glass tube. The seeds were sliced into approximately 1-mm-thin pieces and placed in another glass tube. These slices were incubated with 3 mL of chloroform/methanol (2/1, v/v) in a shaking incubator for one hour. This chloroform/methanol extraction step was repeated five times. The combined lipid extracts were washed with 1 mL of 1 M KCl solution and 2 mL of water and then dried under a stream of nitrogen gas. Chloroform was added to dissolve the dried lipids, with the amount calculated based on the dry weight of the tissue. Phospholipids, galactolipids, and diacylglycerol were analyzed using an electrospray ionization triple quadrupole mass spectrometer, and lipid molecular species were quantified by comparison with internal standards, as previously described [48].

For fatty acid analysis, approximately 30 mg of seed powder was accurately weighed and placed into glass tubes with Teflon cover. For each tube, 1.5 mL of 2.5% H2SO4 in methanol with 0.01% BHT, 400 µL of toluene, and 200 µL of a 2 mg/mL internal standard of 17:0 fatty acid were added. The tubes were then heated at 90 °C for 1 h to esterify the fatty acids into methyl esters. The mixture was subsequently extracted with 1.8 mL of water and 1 mL of hexane. The upper hexane phase containing the fatty acid methyl esters was collected for analysis using gas chromatography (Shimadzu GC-17A, Kyoto, Japan), as described [49].

[3H] Acetate chase labeling

Soybean pods were collected 19 or 20 days after flowering. Seeds extracted from these pods were directly placed onto the culture medium composed of 5 mM MES, 0.5% sucrose, 1/2 Murashige and Skoog salts, pH5.8, kept on ice. Thirty embryos from each line were dissected and pre-incubated at room temperature for 40 min under a light intensity of 40 µmole m−2 s −1, with continuous shaking. Radiolabeling was initiated by adding 10 mL of culture medium containing 20 µCi/mL [3H] -acetate (5 mCi/ml in ethanol, American Radiolabeled Chemicals). The samples were then incubated for 1 h at room temperature with gentle agitation. After labeling, the embryos were washed twice with fresh culture medium, and the chase period was started. At specified time points, embryos were collected and transferred to preheated (75 °C) isopropanol containing 0.01% butylated hydroxytoluene for lipid extraction. Lipid components were extracted and separated using thin-layer chromatography (TLC), and the levels of 3H in glycolipid (MGDG, DGDG), phospholipid (PA, PC, PE), and glycerolipid (DAG, TAG) were quantified using a scintillation counter (Perkin Elmer). Phospholipids and glycolipids were separated on TLC plates using the following developing solvents: chloroform/methanol/water (65/25/4, v/v/v) and hexane/diethyl ether/acetic acid (75/25/1, v/v/v), respectively [50,51,52].

Soybean multi-omics database (soyMD) analysis of GmPAHs

The variation of GmPAH in 4414 resequenced soybean accessions was analyzed using the soybean multi-omics database (SoyMD) [30]. The single-locus model was employed to analyze the haplotypes of three GmPHA genes and their 2-kb flanking regions in relation to total oil content and one-hundred seed weight. The module on allele frequency in sub-population, geographical distribution, and phenotypic value was plotted based on the soyMD website to illustrate the functional role of GmPHAs.

Availability of data and materials

No datasets were generated or analysed during the current study.

Abbreviations

PA:

Phosphatidic acid

DAG:

Diacylglycerol

TAG:

Triacylglycerol

DGAT:

DAG acyltransferase

PDAT:

Phospholipid: DAG acyltransferase

PAH:

Phosphatidic acid phosphohydrolases

PC:

Phosphatidylcholine

PE:

Phosphatidylethanolamine

PG:

Phosphatidylglycerol

PI:

Phosphatidylinositols

PS:

Phosphatidylserines

CDP-DAG:

Cytidine diphosphate diacylglycerol

TLC:

Thin-layer chromatography

MGDG:

Monogalactosyldiacylglycerol

DGDG:

Digalactosyldiacylglycerol

hap:

Haplotype

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Acknowledgements

The authors gratefully acknowledge the support from Des Lee Collaborative Vision and Agriculture and Food Research Initiative (AFRI) award no. [2016-67013-24429/project accession number 1007600 and 2020-67013-30908/project accession number 1022148] from the USDA National Institute of Food and Agriculture. B.C. was supported in part by the National Natural Science Foundation of China (32200200), China Postdoctoral Science Foundation (GZC20240402).

Funding

This work was supported by grants from Agriculture and Food Research Initiative (AFRI) award no. [2016-67013-24429/project accession number 1007600 and 2020-67013-30908/project accession number 1022148] from the USDA National Institute of Food and Agriculture. B.C. was supported in part by the National Natural Science Foundation of China (32200200), China Postdoctoral Science Foundation (GZC20240402).

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Authors and Affiliations

  1. State Key Laboratory of Crop Stress Adaptation and Improvement, School of Life Sciences, Henan University, Zhengzhou, 450046, China

    Beibei Chen

  2. Department of Biology, University of Missouri-St. Louis, St. Louis, MO, 63121, USA

    Beibei Chen, Jianwu Li, Shuaibing Yao, Geliang Wang & Xuemin Wang

  3. Donald Danforth Plant Science Center, St. Louis, MO, 63132, USA

    Beibei Chen, Jianwu Li, Shuaibing Yao, Geliang Wang & Xuemin Wang

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  1. Beibei Chen
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  5. Xuemin Wang
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Contributions

B.C. designed and performed most of the experiments and wrote and revised the manuscript. J.L. did transgenic soybean screening. S.Y. helped analyze lipid data and edit the manuscript. G.W. performed AtPAH vector construction. X.W. proposed, designed, and supervised the study and revised the manuscript. All authors read and approved the final manuscript.

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Correspondence to Beibei Chen or Xuemin Wang.

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Chen, B., Li, J., Yao, S. et al. Seed-specific expression of phosphatidate phosphohydrolases increases soybean oil content and seed weight. Biotechnol. Biofuels Bioprod. 18, 23 (2025). https://doiorg.publicaciones.saludcastillayleon.es/10.1186/s13068-025-02620-x

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Biotechnology for Biofuels and Bioproducts

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