New Phytologist | Jiangxi Agricultural University and others reveal the CaHY5-CaBBX2-CaACS8 cascade module that regulates capsaicin biosynthesis by light signals
Capsaicin is a unique alkaloid found in chili peppers, giving them their spicy flavor and possessing significant value in food, medicine, and industry. Whether and how light, as a key environmental signal, regulates capsaicin biosynthesis remains unclear. Recently, a research team from Jiangxi Agricultural University and other institutions revealed the molecular mechanism by which light signals positively regulate capsaicin biosynthesis through the CaHY5-CaBBX2-CaACS8 transcriptional cascade module. The related research findings, titled “Light regulates capsaicinoid biosynthesis via the CaHY5-CaBBX2-CaACS8 module in pepper,” were published in the academic journal New Phytologist.
This study first performed metabolomic and transcriptomic analyses on pepper fruits subjected to bagging and shading treatment (dark adaptation) and normal light exposure (light adaptation). The results showed that capsaicin and/or dihydrocapsaicin content was significantly reduced in dark-adapted fruits, and the expression of key structural genes in capsaicin biosynthesis (such as CaKAS, CaACS8, CaPAL3, and CaAT3) was downregulated, indicating that light is indispensable for capsaicin biosynthesis.
Transcriptomic analysis revealed that the expression of the core light signaling transcription factor CaHY5 was significantly reduced in dark-adapted fruits. Silencing CaHY5 using viral-induced gene silencing significantly decreased capsaicin and dihydrocapsaicin content in pepper fruits, and suppressed the expression of capsaicin synthesis structural genes; while transient overexpression of CaHY5 significantly promoted capsaicin accumulation and structural gene expression, indicating that CaHY5 is a positive regulator of light-induced capsaicin synthesis.
To reveal the downstream target genes of CaHY5, this study used DAP-seq technology to identify the motifs (G-box and ACE-box) that CaHY5 directly binds to, and screened for two direct target genes: the B-box transcription factor CaBBX2 and the key enzyme gene CaACS8 for capsaicin synthesis. Yeast one-hybrid assays, dual-luciferase reporter assays, and electrophoretic mobility shift assays confirmed that CaHY5 directly binds to the G-box elements in the promoters of CaBBX2 and CaACS8, activating their transcription.
Further research revealed that CaBBX2 also directly binds to the T/G-box elements in the promoter of CaACS8, activating its expression. Silencing either CaBBX2 or CaACS8 led to a decrease in capsaicin content, while overexpression promoted capsaicin accumulation. Epistasis analysis showed that CaBBX2 acts downstream of CaHY5, and CaACS8 acts downstream of CaBBX2, forming a transcriptional cascade regulatory pathway of CaHY5→CaBBX2→CaACS8. Interestingly, no protein-protein interaction between CaHY5 and CaBBX2 was detected in the yeast two-hybrid assay, suggesting that they are synergistically regulated through a transcriptional cascade rather than protein-protein interaction.
In summary, this study reveals for the first time the molecular mechanism by which light signals positively regulate capsaicin biosynthesis through the CaHY5-CaBBX2-CaACS8 transcriptional cascade module: light activates CaHY5 expression, CaHY5 directly activates the transcription of CaBBX2 and CaACS8, and the activated CaBBX2 further enhances CaACS8 expression, thereby amplifying capsaicin synthesis in a cascade. This discovery not only elucidates the signaling pathway of light-regulated capsaicin synthesis but also provides important theoretical basis and gene targets for improving chili spiciness through light environment regulation and molecular breeding.
