Soybeans (Glycine max (L.) Merr.) are critical resources for vegetable oil production for human and animal consumption as well as biofuel synthesis. Soybean oils are primarily composed of triacylglycerols (TAGs) with a certain mixture of fatty acid moieties. In general, the composition is approximately 62% polyunsaturated fatty acids (PUFAs), 23% monounsaturated fatty acids (MUFAs) and 15% saturated fatty acids (SFAs). The relatively high concentration of PUFAs linolenic acid (C18:3) and linoleic acid (C18:2) heavily contribute to oxidative rancidity, flavour reversion and short shelf-life of soybean oil (Warner and Gupta, 2005). In contrast, enhanced MUFAs oleic acid (C18:1) content in vegetable oils can produce significant health benefits, as well as improved oxidative stability, which is essential for both food usage and biodiesel and other renewable resource syntheses (Haslam et al., 2013; Knothe, 2005).

Phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT) serves as a catalyst for the interconversion of phosphatidylcholine (PC) and diacylglycerol (DAG). This, in turn enriches the PC-modified fatty acids in the DAG pool before TAG formation. Emerging studies revealed that a knockout or knockdown of PDCT genes enhances MUFAs levels while decreasing PUFAs among Arabidopsis (Lu et al., 2009), Crambe abyssinica (Guan et al., 2015) and Thlaspi arvense (Jarvis et al., 2021). Apart from its significant role in FA content modulation, there is little information on the biochemical characteristics of PDCT in major field crops including soybean.

Herein, we identified two close homologues, namely, GmPDCT1 and GmPDCT2 of the reported AtPDCT (At3g15820) in the soybean genome (Figure 1a, Table S1). These homologues shared comparable gene composition and conserved motifs (Figure S1). Moreover, both homologues abundantly expressed proteins in the cytosol when ectopically expressed in the Arabidopsis mesophyll protoplasts (Figure 1b). In addition, the GmPDCT1 and GmPDCT2 genes were strongly upregulated in the mid-late stage developing seeds (Figure 1a, Table S2), which corroborated with the PDCTs expression profiles in other plants (Lu et al., 2009; Wickramarathna et al., 2015). To further elucidate the significance of GmPDCTs in the FAs composition of soybean seed oil, we generated two sgRNAs within the common GmPDCTs region, and generated CRISPR/Cas9 vectors for knock out experimentation (Figure 1c,d). Following the Agrobacterium tumefaciens-mediated transformation of soybean cultivar DN50, 15 positive T0 lines were acquired, among which, 7 (46.7%) simultaneously edited both GmPDCT1 and GmPDCT2 (Figure S2, Table S3). In all target sites, we observed single nucleotide insertion/deletion and relatively large deletions (7–128 bps) via PCR and Sanger sequencing (Figure 1e,f). In a majority of cases, the mutations produced premature translation termination, and this mutation was stably inherited in T1–T2 generations (Figures S2–S4). Lastly, no mutation events occurred across all potential off-target sites (Table S4).

We selected three T2 homozygous mutants for additional investigations, and GmPDCT1 and GmPDCT2 transcripts in m3-1-1, m6-2-1 and m6-2-2 were strongly downregulated, relative to wildtype (WT) plants (Figure 1g). We employed western blot analysis using the PDCT polyclonal antibody to further confirm the GmPDCT protein knockout in mutant lines (Figure 1h). There were no discernible differences in seed size, seed weight or visible growth phenotypes of mutant, CK or WT lines (Figure S5, Table S5). In addition, the cotyledon cell structure, and total protein and oil contents in the mutant seeds remained unaltered, relative to WT (Figure 1i,j). However, the gmpdct1gmpdct2 mutants displayed marked elevations in MUFA content, along with a considerable decrease in PUFAs content. In mutant seeds, the mean C18:1 content was 2.49 folds that of WT, and the C18:2 and C18:3 contents were strongly diminished by 38.69% and 97.47%, respectively (Figure 1j, Table S6).

To elucidate changes in lipid metabolism in the gmpdct1gmpdct2 double mutants, we further assessed the lipid profiles of developing seeds using electrospray ionization tandem mass spectrometry (ESI-MS/MS). Relative to WT, the MUFAs-containing TAGs were strongly elevated by 57.01% in the m3-1-1 and by 33.13% in the m6-2-1 mutants, respectively. In contrast, the TAG containing PUFAs C18:3 were strongly diminished, followed by C18:2 (Figure 1k, Table S7). Both DAG and PC exhibited a shift toward a more monounsaturated molecular species in the mutant lines (Figure 1k), as evidenced by the elevated 16:0/18:1 and 18:1/18:1 contents, with simultaneous decreases in the 16:0/18:2, 16:0/18:3, and 18:2/18:2 contents (Tables S8 and S9). It was previously reported that the TAG derived from PC-based DAG is laden with PUFAs, and the TAG derived from de novo DAG using glycerol-3-phosphate (G3P) acylation (Kennedy pathway) is rich in MUFAs (Eskandari et al., 2013). Herein, our TAG, DAG, and PC from GmPDCTs-deficient mutants revealed a steep increase in C18:1, along with a concomitant decrease in C18:2 and C18:3, suggesting that the de novo DAG network was upregulated while the PC-derived DAG was suppressed, resulting in very low incorporation of PC modified-PUFAs into TAG (Figure 1l). Based on these evidences, the PC to DAG turnover was potentially produced as a result of the catalytic action of PDCT in soybean developing seeds.

In conclusion, herein, we successfully produced elevated-MUFAs soybean using GmPDCTs knockout via a CRISPR-Cas9 genome editing system. Our discovery of GmPDCTs provides numerous opportunities to modify plant oil composition for enhanced nutrition and shelf stability.

大豆 ( Glycine max (L.) Merr.) 是生产供人类和动物消费的植物油以及合成生物燃料的重要资源。大豆油主要由三酰甘油 (TAG) 和一定的脂肪酸部分混合物组成。一般来说，其组成约为 62% 多不饱和脂肪酸 (PUFA)、23% 单不饱和脂肪酸 (MUFA) 和 15% 饱和脂肪酸 (SFA)。相对较高浓度的 PUFA 亚麻酸 (C18:3) 和亚油酸 (C18:2) 会严重导致大豆油的氧化酸败、风味回复和保质期缩短（Warner 和 Gupta， 2005 ）。相比之下，植物油中增强的 MUFA 油酸 (C18:1) 含量可以产生显着的健康益处，并提高氧化稳定性，这对于食品使用、生物柴油和其他可再生资源合成至关重要（Haslam等， 2013 ）诺特， 2005 ）。

磷脂酰胆碱：二酰甘油磷酸胆碱转移酶（PDCT）充当磷脂酰胆碱（PC）和二酰甘油（DAG）相互转化的催化剂。这反过来又在 TAG 形成之前丰富了 DAG 池中的 PC 修饰脂肪酸。新兴研究表明，拟南芥(Lu et al ., 2009 )、海甘蓝(Guan et al ., 2015 ) 和Thlaspi arvense (Jarvis et al ., 2021 ) 中PDCT基因的敲除或敲低可提高 MUFA 水平，同时降低 PUFA。除了在 FA 含量调节中发挥重要作用外，关于 PDCT 在包括大豆在内的主要大田作物中的生化特性的信息很少。

在此，我们在大豆基因组中鉴定了两个密切的同源物，即报道的 AtPDCT (At3g15820) 的GmPDCT1和GmPDCT2 （图 1a，表 S1）。这些同源物具有相似的基因组成和保守基序（图 S1）。此外，当在拟南芥叶肉原生质体中异位表达时，两种同源物在细胞质中大量表达蛋白质（图1b）。此外， GmPDCT1和GmPDCT2基因在种子发育中后期强烈上调（图1a，表S2），这证实了其他植物中的PDCT表达谱（Lu等， 2009 ；Wickramarathna等， 2015 ）。为了进一步阐明GmPDCT在大豆籽油 FA 组成中的重要性，我们在共同的GmPDCT区域内生成了两个 sgRNA，并生成了用于敲除实验的 CRISPR/Cas9 载体（图 1c，d）。通过根癌农杆菌介导的大豆品种DN50的转化，获得了15个阳性T0系，其中7个（46.7%）同时编辑了GmPDCT1和GmPDCT2 （图S2，表S3）。在所有目标位点中，我们通过PCR 和 Sanger 测序观察到单核苷酸插入/缺失和相对较大的缺失（7-128 bps）（图 1e，f）。在大多数情况下，突变会导致翻译提前终止，并且这种突变在 T1-T2 代中稳定遗传（图 S2-S4）。最后，所有潜在的脱靶位点均未发生突变事件（表 S4）。

我们选择了三个T2纯合突变体进行额外的研究，相对于野生型（WT）植物， m3-1-1 、 m6-2-1和m6-2-2中的GmPDCT1和GmPDCT2转录物被强烈下调（图1g）。我们使用 PDCT 多克隆抗体进行蛋白质印迹分析，以进一步确认突变系中的 GmPDCT 蛋白敲除（图 1h）。突变体、CK 或 WT 系的种子大小、种子重量或可见生长表型没有明显差异（图 S5、表 S5）。此外，相对于 WT，突变种子中的子叶细胞结构以及总蛋白质和油含量保持不变（图 1i，j）。然而， gmpdct1gmpdct2突变体的 MUFA 含量显着升高，同时 PUFA 含量显着降低。在突变种子中，平均 C18:1 含量是 WT 的 2.49 倍，C18:2 和 C18:3 含量分别大幅减少 38.69% 和 97.47%（图 1j，表 S6）。

为了阐明gmpdct1gmpdct2双突变体中脂质代谢的变化，我们使用电喷雾电离串联质谱 (ESI-MS/MS) 进一步评估了发育种子的脂质谱。相对于 WT， m3-1-1突变体中含有 MUFA 的 TAG 显着升高了 57.01%， m6-2-1突变体中则显着升高了 33.13%。相比之下，含有 PUFA C18:3 的 TAG 急剧减少，其次是 C18:2（图 1k，表 S7）。 DAG 和 PC 在突变系中都表现出向更单不饱和分子种类的转变（图 1k），如 16:0/18:1 和 18:1/18:1 含量升高所证明，同时 16 :0/18:2、16:0/18:3 和 18:2/18:2 内容（表 S8 和 S9）。之前有报道称，源自基于 PC 的 DAG 的 TAG 富含 PUFA，而使用 3-磷酸甘油 (G3P) 酰化（Kennedy 途径）从头DAG 衍生的 TAG 富含 MUFA（Eskandari等人， 2013 ）。在此，我们来自GmPDCTs缺陷突变体的 TAG、DAG 和 PC 显示 C18:1 急剧增加，同时 C18:2 和 C18:3 随之减少，表明从头DAG 网络上调，而 PC-衍生的 DAG 被抑制，导致 PC 修饰的 PUFA 掺入 TAG 的量非常低（图 1l）。基于这些证据，PC 到 DAG 的转换可能是由于 PDCT 在大豆种子发育中的催化作用而产生的。

总之，本文中，我们通过CRISPR-Cas9 基因组编辑系统使用GmPDCT敲除成功生产了高 MUFA 大豆。我们对GmPDCT的发现提供了许多改变植物油成分以增强营养和货架稳定性的机会。