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<p>Insect pests are responsible for significant yield losses across various crops. To mitigate the adverse effects of insect pressures on crop yields, it is essential to implement sustainable insect resistance strategies. Spinach (<i>Spinacia oleracea</i> L.), a prominent leafy vegetable, produces 20-hydroxyecdysone (20E), which offers protection against insect attacks and also have beneficial effects on human health. The detailed structure of 20E is known, however, the complete biosynthetic pathway is still elusive. This study showed a comprehensive genome-wide identification, phylogenetic analysis and functional characterization of <i>cycloartenol synthase</i> involved in the biosynthesis of 20E in spinach. Phylogenetic analysis of the four newly identified <i>oxidosqualene cyclases</i> (<i>OSCs</i>) from <i>S. oleracea</i> indicates that these <i>OSCs</i> have undergone lineage-specific duplication events and exhibit a clear orthologous relationship. Artificial microRNA (amiRNA)-mediated silencing showed down regulation of <i>S. oleracea cycloartenol synthase</i> (<i>SoCAS</i>) in the silenced plants. Liquid chromatography mass spectroscopy (LC–MS/MS) analysis revealed a corresponding decrease in related metabolites, including cycloartenol (7.93 fold)<b>,</b> lathosterol (9.45-fold) and 20E (7.77-fold) as compared to non-infiltrated control plants. Furthermore, the overexpression of a codon-optimized full-length <i>SoCAS</i> gene resulted in a marked increase in the accumulation of cycloartenol (30.37-fold), cycloartenol acetate (6.49-fold), and campesterol (8.11-fold) in <i>N. benthamiana</i>, as well as cycloartenol (31.05-fold), lathosterol (20.08-fold) and 20E (21.09-fold) in <i>S. oleracea</i> as compared with non-infiltrated control plants. This study provides a new insight on the role of <i>OSC</i> in the production of cycloartenol and the 20E biosynthesis pathway intermediates in spinach, which could be utilized for the genetic improvement of plants that are resistant to herbivorous insects.</p>

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Identification and functional characterization of the cycloartenol synthase gene involved in the biosynthesis of the insect molting hormone 20-hydroxyecdysone from Spinacia oleracea L.

  • 期刊论文

  • 发表日期：

    2025年9月22日

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  • Harshad A. Shirke,
  • Arati P. Vasav,
  • Ashwini M. Darshetkar,
  • Rucha C. Godbole,
  • Swapnil B. Kadam,
  • Swaranjali S. Patil,
  • Vikas B. Naikawadi,
  • P. B. Kavi Kishor,
  • Tukaram D. Nikam,
  • Vitthal T. Barvkar

摘要

Insect pests are responsible for significant yield losses across various crops. To mitigate the adverse effects of insect pressures on crop yields, it is essential to implement sustainable insect resistance strategies. Spinach (Spinacia oleracea L.), a prominent leafy vegetable, produces 20-hydroxyecdysone (20E), which offers protection against insect attacks and also have beneficial effects on human health. The detailed structure of 20E is known, however, the complete biosynthetic pathway is still elusive. This study showed a comprehensive genome-wide identification, phylogenetic analysis and functional characterization of cycloartenol synthase involved in the biosynthesis of 20E in spinach. Phylogenetic analysis of the four newly identified oxidosqualene cyclases (OSCs) from S. oleracea indicates that these OSCs have undergone lineage-specific duplication events and exhibit a clear orthologous relationship. Artificial microRNA (amiRNA)-mediated silencing showed down regulation of S. oleracea cycloartenol synthase (SoCAS) in the silenced plants. Liquid chromatography mass spectroscopy (LC–MS/MS) analysis revealed a corresponding decrease in related metabolites, including cycloartenol (7.93 fold), lathosterol (9.45-fold) and 20E (7.77-fold) as compared to non-infiltrated control plants. Furthermore, the overexpression of a codon-optimized full-length SoCAS gene resulted in a marked increase in the accumulation of cycloartenol (30.37-fold), cycloartenol acetate (6.49-fold), and campesterol (8.11-fold) in N. benthamiana, as well as cycloartenol (31.05-fold), lathosterol (20.08-fold) and 20E (21.09-fold) in S. oleracea as compared with non-infiltrated control plants. This study provides a new insight on the role of OSC in the production of cycloartenol and the 20E biosynthesis pathway intermediates in spinach, which could be utilized for the genetic improvement of plants that are resistant to herbivorous insects.