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Functional verification of the key gene NtLPAT involved in lipid biosynthesis in tobacco

JI Bai-Lu1,3,**,SUN Yi-Wen1,**,LIU Wan-Feng2,QIAN Ya-Xin1,3,JIANG Cai-Hong1,GENG Rui-Mei1,LIU Dan1,CHENG Li-Rui1,YANG Ai-Guo1,HUANG Li-Yu3,LI Xiao-Xu2,PU Wen-Xuan2,GAO Jun-Ping2,*,ZHANG Qiang4,*,WEN Liu-Ying1,*   

  1. 1 Tobacco Research Institute, Chinese Academy of Agricultural Sciences, Qingdao 266101, Shandong, China; 2 China Tobacco Hunan Industrial Co., Ltd., Changsha 410007, Hunan, China; 3 School of Agriculture, Yunnan University, Kunming 650500, Yunnan, China; 4 Tobacco Institute of Shaanxi Province, Xi’an 710061, Shaanxi, China
  • Received:2024-12-24 Revised:2025-06-04 Accepted:2025-06-04 Published:2025-06-16
  • Supported by:
    This study was supported by the Agricultural Science and Technology Innovation Program of CAAS (ASTIP-TRIC01), the Natural Science Foundation of Shandong Province, China (ZR2023MC139), the Natural Science Foundation of Tobacco Genome Project of State Tobacco Monopoly Administration (110202201010(JY-17)), and the Technology Project of the Shaanxi Provincial Company of China National Tobacco Corporation (KJ-2023-02). 

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Abstract:

Lysophosphatidyl transferase (LPAT) is a key enzyme in the lipid biosynthesis pathway, catalyzing the transfer of a fatty acyl group from fatty acyl-CoA (Acyl-CoA) to lysophosphatidic acid (LPA) to produce phosphatidic acid (PA). However, the functional role of LPAT in tobacco remains largely unexplored. In this study, we cloned the NtLPAT gene from the tobacco cultivar K326 and generated an NtLPAT knockout mutant (ntlpat) using CRISPR/Cas9 technology. The ntlpat mutant was evaluated for agronomic traits, disease resistance, and phenotypic appearance. In addition, lipidomic and transcriptomic analyses were conducted to assess the impact of NtLPAT loss of function. Our results showed that NtLPAT expression was induced by infection with Ralstonia solanacearum and Phytophthora parasitica. The ntlpat mutant exhibited reduced plant height but enhanced resistance to cucumber mosaic virus (CMV) and bacterial wilt. Lipidomic analysis revealed altered glyceride metabolism in the mutant: triacylglycerol (TAG) levels were significantly decreased, while the contents of two major glycerol glycolipids—monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), both critical components of the thylakoid membrane—were increased. Additionally, changes were observed in sphingolipid and phosphatidylinositol compositions. Transcriptomic analysis indicated that genes involved in photosynthesis, carbon fixation, sphingolipid biosynthesis, and phosphatidylinositol signaling pathways were reprogrammed in ntlpat. These findings suggest that NtLPAT possesses acyltransferase activity, regulates de novo TAG biosynthesis, and plays a role in cellular signaling pathways, thereby affecting tobacco growth and resistance to CMV and R. solanacearum. This study provides valuable genetic resources and an experimental basis for tobacco breeding improvement.

Key words: lysophosphatidyl transferase, tobacco, gene knock-out, lipidomics, transcriptome

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