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<p>Rutin, a bioactive flavonoid with potent antioxidant, anti-diabetic, and cardioprotective properties, is synthesized in buckwheat (<i>Fagopyrum sp.</i>) via the phenylpropanoid pathway initiated by the enzyme phenylalanine ammonia lyase (PAL). In this study, the PAL gene was cloned from a high-rutin Tartary buckwheat genotype, BWZ-90 (1403.22&#xa0;ppm rutin in seed endosperm), using the Gateway cloning system. Sequence analysis revealed multiple single-nucleotide polymorphisms (SNPs) and a unique 39-amino-acid insertion absent in low-rutin genotypes, suggesting a structural basis for enhanced catalytic efficiency and flavonoid biosynthesis. The cloned PAL gene was fused to a yellow fluorescent protein (YFP) tag and transiently expressed in <i>Nicotiana benthamiana</i> to determine subcellular localization. Confocal microscopy analysis of the YFP-tagged PAL fusion protein transiently expressed in <i>Nicotiana benthamiana</i> revealed that PAL is predominantly localized in the cytosol, consistent with previous reports for phenylpropanoid enzymes. In addition to cytosolic distribution, peripheral fluorescence signals were occasionally observed near the cell boundaries, suggesting a possible transient or peripheral association with membrane compartments. These findings indicate that while PAL primarily functions in the soluble fraction, dynamic spatial interactions with membrane-associated components cannot be excluded. Collectively, the results provide insights into the structural variation and subcellular behavior of PAL in relation to enhanced rutin accumulation. These findings establish a direct link between PAL gene variation, subcellular localization, and elevated rutin biosynthesis, offering valuable insights for metabolic engineering and marker-assisted breeding of nutraceutical-rich buckwheat cultivars. This article aligns with SDG 15 (Life on Land) of the UN Agenda for Sustainable Development.</p>

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Phenylalanine ammonia lyase as a molecular driver of rutin enrichment: evidence from cloning and localization studies in buckwheat

  • 期刊论文

  • 发表日期：

    2026年4月20日

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  • Mohsin Altaf Hajini,
  • Saika Bashir,
  • Mansoor Showkat,
  • Majid Rashid,
  • Parvaze Ahmad Sofi,
  • Tanveer Ali Dar,
  • Ajaz A. Malik,
  • Nitin Jain,
  • M. Ashraf Bhat,
  • Sajad Majeed Zargar

摘要

Rutin, a bioactive flavonoid with potent antioxidant, anti-diabetic, and cardioprotective properties, is synthesized in buckwheat (Fagopyrum sp.) via the phenylpropanoid pathway initiated by the enzyme phenylalanine ammonia lyase (PAL). In this study, the PAL gene was cloned from a high-rutin Tartary buckwheat genotype, BWZ-90 (1403.22 ppm rutin in seed endosperm), using the Gateway cloning system. Sequence analysis revealed multiple single-nucleotide polymorphisms (SNPs) and a unique 39-amino-acid insertion absent in low-rutin genotypes, suggesting a structural basis for enhanced catalytic efficiency and flavonoid biosynthesis. The cloned PAL gene was fused to a yellow fluorescent protein (YFP) tag and transiently expressed in Nicotiana benthamiana to determine subcellular localization. Confocal microscopy analysis of the YFP-tagged PAL fusion protein transiently expressed in Nicotiana benthamiana revealed that PAL is predominantly localized in the cytosol, consistent with previous reports for phenylpropanoid enzymes. In addition to cytosolic distribution, peripheral fluorescence signals were occasionally observed near the cell boundaries, suggesting a possible transient or peripheral association with membrane compartments. These findings indicate that while PAL primarily functions in the soluble fraction, dynamic spatial interactions with membrane-associated components cannot be excluded. Collectively, the results provide insights into the structural variation and subcellular behavior of PAL in relation to enhanced rutin accumulation. These findings establish a direct link between PAL gene variation, subcellular localization, and elevated rutin biosynthesis, offering valuable insights for metabolic engineering and marker-assisted breeding of nutraceutical-rich buckwheat cultivars. This article aligns with SDG 15 (Life on Land) of the UN Agenda for Sustainable Development.