Citrus NAC senescence‐associated factor 1 regulates post‐harvest fruit lignification via activation of 4CL1

Post-harvest lignification drives fruit quality deterioration and represents a senescence program evolutionarily distinct from the well-studied softening process, yet the underlying mechanism remains largely unclear. Hence, this study aimed to identify and functionally verify NAC transcription factors that govern lignin deposition during citrus juice sac granulation, a major physiological disorder occurring in citrus fruit during senescence. Through comprehensive analysis of 116 NAC genes in Citrus sinensis, we identified CitNSF1 (Citrus NAC senescence-associated factor 1). CitNSF1, which is not homologous to reported lignin-associated NAC transcription factors, localizes to the nucleus and exhibits transactivational activity. Transcriptomics analysis revealed unique induction of CitNSF1 during juice sac granulation, contrasting with transcriptional silence or depression of homologs to lignin-associated NAC genes in multiple growing seasons. Transient overexpression of CitNSF1 in various systems, including Nicotiana benthamiana leaves, Citrus grandis, and C. sinensis pericarp, and C. grandis juice sacs significantly accelerated lignin accumulation. Stable overexpression of CitNSF1 in Arabidopsis thaliana also enhanced lignification. Dual-luciferase reporter assay revealed that CitNSF1 activates Cit4CL1 transcription; yeast one-hybrid assay and electrophoretic mobility-shift assay revealed that CitNSF1 directly binds to the Cit4CL1 promoter via the SNBE motif. Additionally, overexpression of Cit4CL1 in C. grandis pericarp and A. thaliana activated lignin biosynthesis. We conclude that CitNSF1, an evolutionarily distinct NAC factor, directly activates Cit4CL1 to drive post-harvest lignification during citrus juice sac granulation, revealing a previously unexplored regulatory pathway in fruit senescence.