Knock-out of GhPDCT with the CRISPR/Cas9 system increases the oleic acid content in cottonseed oil

Cotton is a pivotal economic crop for natural textile fibers that also serves as an important source of edible oil (Long et al. 2023). Cottonseed oil contains approximately 14% oleic acid and 59% linoleic acid. An increase in monounsaturated fatty acids, particularly oleic acid, enhances the oxidative stability and nutritional value of edible oil (Chen et al. 2021). Currently, the demand for edible oil in China is increasing in terms of both production and nutrition. Improving cottonseed oil’s storability and nutritional value is crucial for the comprehensive utilization of cotton. However, cottonseed has long been regarded as a by-product in the cotton industry, so research on improving the content and quality of cottonseed oil has lagged compared to other crop attributes. Phosphatidylcholine: diacylglycerol cholinephospho-transferase (PDCT) is the gate-keeping enzyme for the conversion between phosphatidylcholine and diacylglycerol (Lu et al. 2009). Studies in multiple plants have revealed increases in monounsaturated fatty acids in seeds with PDCT knock-out. To clone the PDCTs of upland cotton (Gossypium hirsutum), the protein sequences of PDCT from Arabidopsis (Lu et al. 2009), oilseed rape (Brassica napus; Bai et al. 2020), soybean (Glycine max; Li et al. 2023), peanut (Arachis hypogaea), and sesame (Sesamum indicum) were used as references for BLAST searches in CottonMD (https://yanglab.hzau.edu.cn/CottonMD; Yang et al. 2023). Four PDCT homologs in cotton were obtained and named GhPDCT1 (Gh_D06G1990), GhPDCT2 (Gh_A06G1621), GhPDCT3 (Gh_A05G3864), and GhPDCT4 (Gh_D05G1178) (Fig. 1-A). The sequence similarities between the four GhPDCTs and AtPDCT are 58.47, 60.13, 45.18, and 58.61%, respectively. Further, the phylogenetic analysis revealed that the GhPDCTs are clustered with the PDCTs of Brassica napus (Fig. 1-A). The heatmap of GhPDCTs in cotton tissues was built using released transcriptome data. The results showed that GhPDCT3 and GhPDCT4 had very little expression in all tissues (Fig. 1-B). GhPDCT2 was expressed in roots, stems, leaves and ovules at different developmental stages, but at relatively low levels. GhPDCT1 shared similar basal expression with GhPDCT2, but the transcript level of GhPDCT1 in ovules was significantly higher than that of GhPDCT2. Notably, the expression of GhPDCT1 was sharply up-regulated in ovules at 20 and 25 days post anthesis (DPA). The expression pattern of GhPDCT1 was further verified by RT-qPCR, which indicated that GhPDCT1 was up-regulated in the late stage of ovule development and peaked around 25 DPA. Previous reports highlighted the rapid accumulation of oil content in cotton seeds at 20–30 DPA (Zhao et al. 2018). Therefore, GhPDCT1 is considered the key candidate for regulating the seed oil content of cotton (Fig. 1-B). Sequence analysis showed that GhPDCT1/2 and GmPDCT1/2 contain similar conserved motifs, as well as a C-terminal PAP2_3 domain (Fig. 1-C). The GmPDCT1 and GmPDCT2 in soybean were both found to be located in the cytosol (Li et al. 2023). To study the subcellular localization of GhPDCT, a GFP-PDCT1 fusion protein was expressed in the protoplasts of cotton cotyledons (Hu et al. 2022), and the RFP-labeled transcription factor GoPGF (Zhang et al. 2024) was co-expressed to mark the nucleus. Observations with a laser scanning confocal microscope showed the green fluorescence of GFP-PDCT1 expressed in the cytoplasm (Fig. 1-D). Knock-out of GhPDCT was achieved with the optimized CRISPR/Cas9 system of cotton (Wang et al. 2018). Due to the high similarity (94.2%) of the coding sequences of GhPDCT1 and GhPDCT2, two sgRNAs respectively targeting two different sites of the 1st exon were designed for the simultaneous mutagenesis of GhPDCT1 and GhPDCT2 (Fig. 1-E). The G. hirsutum L. line ‘Jin668’ was used to produce the GhPDCT1/2 mutant of cotton (ghpdct) with Agrobacterium-mediated transformation (Zhu et al. 2023). The DNA of the ghpdct mutant was extracted for Hi-TOM sequencing, and the offspring of ghpdct-5 with the full mutation were planted for further studies. As shown in Fig. 1-F, ghpdct-5 has a 1 nt deletion at target 2 of GhPDCT2 (A subgenome). In addition, two types of mutations were found in GhPDCT1 (D subgenome), one with a 1 nt insertion at target 1, and the other with a 1 nt insertion and a 2 nt deletion at target 1. The wild type (WT) and ghpdct were planted in the field and a phenotypic study was conducted during the whole growing period. No obvious differences in plant growth were observed between WT and ghpdct. For example, the plant height, fiber length, seed weight of WT and ghpdct showed no statistically significant differences (Fig. 1-G–I). The fatty acids in seeds of WT and ghpdct were measured by gas chromatography-mass spectrometry (GC-MS) (Fig. 1-J). Oleic acid (OA, C18:1) accounted for an average of 14.46% of the total fatty acids in seeds of WT, and 16.49% in seeds of ghpdct, which indicates the up-regulation of oleic acid in the ghpdct mutant. Conversely, linoleic acid (LA, C18:2) was reduced in seeds of ghpdct (52.83%) compared to seeds of WT (59.98%). In addition, knockout of GhPDCT increased the seed content of palmitic acid (PA, C16:0) from 21.24% in WT to 25.85% in ghpdct, and the content of stearic acid (SA, C18:0) increased from 1.70% in WT seeds to 2.39% in ghpdct seeds. These results indicated that the GhPDCT mutation alters the balance of monounsaturated and polyunsaturated fatty acids in cotton seeds, with minimal impacts on growth and development beyond seed oil metabolism. In conclusion, we have produced the ghpdct mutant of cotton using the CRISPR/Cas9 system. Knock-out of GhPDCT1/2 affects the conversion between phosphatidylcholine and diacylglycerol in cottonseeds, and changes the contents of oleic acid, linoleic acid, palmitic acid, and stearic acid. We obtained a new germplasm with a higher oleic acid content in cottonseed oil, which can be applied to enhance the economic and nutritional value of cotton as an oil crop, thereby contributing to the industrial upgrading of cotton.