CsNIP2 and CsNIP16 facilitated Al-F transport and hyperaccumulation in tea plants (Camellia sinensis)

Although Al-F complexes (e.g., AlF²⁺, AlF₂⁺) exist in the tea apoplast and symplast, their root uptake mechanisms and translocation to mature leaves remain elusive. Here, we identified two plasma membrane-localized Nodulin 26-like Intrinsic Proteins (NIPs), CsNIP2 and CsNIP16, as crucial mediators of Al-F complex transport. CsNIP2 is predominantly expressed in roots and enriched in the phloem-epidermic part tissues, whereas CsNIP16 is predominantly expressed in stems and abundantly in xylem tissues. Heterologous expression in yeast revealed that both transporters facilitate Al-F uptake, significantly increasing cellular sensitivity to Al and F. Furthermore, targeted knockdown of CsNIP2 and CsNIP16 in tea shoots via antisense oligonucleotides (asODN) and virus-induced gene silencing (VIGS) impaired the vascular translocation and leaf accumulation of Al-F complexes. Overexpression of CsNIP2 in Arabidopsis promoted uptake and root-shoot transport of F, suggesting that transport function of F or its complex by CsNIP2. Al/F stress-induced CsMYB152 and CsNST1 were involved in transcriptional regulation of CsNIP2 transcription: the root-specific repressor CsMYB152 inhibited CsNIP2 promoter activity to prevent excessive Al-F influx, while the cell wall regulator CsNST1 activated CsNIP2 to promote root-to-shoot translocation and coupled detoxification. These findings uncover a coordinated F and Al-F transport network by CsNIP2 and CsNIP16, providing a mechanistic framework for Al-F hyperaccumulation in tea plants. The study could provide molecular tools for breeding new tea varieties with reduced Al and F contents in leaves and tea drinks.