Transcription Factor FcrNAC22 Regulates Chlorophyll Catabolic Genes to Accelerate De-Greening in Kumquat Fruit.

Citrus fruit de-greening, a critical process for quality and marketability, is governed by chlorophyll degradation, yet its regulatory mechanisms remain poorly understood. Here, we identify FcrNAC22, a NAC transcription factor (TF) in kumquat (Fortunella crassifolia), as a pivotal regulator of chlorophyll catabolism activated in response to de-greening cues. FcrNAC22 functions as a transcriptional activator induced by red light, abscisic acid (ABA), and ethephon, with both its mRNA and protein levels peaking at the fruit colour-breaker stage. The overexpression of FcrNAC22 in Nicotiana benthamiana leaves, tomato (Solanum esculentum), and kumquat fruits expedited chlorophyll breakdown and upregulated the expression of chlorophyll catabolic genes (CCGs). In contrast, the interference with FcrNAC22 expression in kumquat fruits impeded chlorophyll degradation and suppressed the transcription of CCGs. Protein-DNA interaction assays verified that FcrNAC22 directly binds to and activates the promoters of chloroplast-localized STAY-GREEN (FcrSGR), chlorophyllase (FcrCLH), pheophytinase (FcrPPH), pheophorbide a oxygenase (FcrPAO), and NON-YELLOW COLORING1 (FcrNYC1), which explains the de-greening phenotypes witnessed in the aforementioned transgenic FcrNAC22 lines. These findings not only reveal FcrNAC22 as a crucial integrator of environmental and developmental signals, but also provide a theoretical basis for manipulating fruit de-greening in citrus and related species.