Transcription factor LcNAC002 coregulates chlorophyll degradation and anthocyanin biosynthesis in litchi.

Chlorophyll degradation and anthocyanin biosynthesis, which often occur almost synchronously during fruit ripening, are crucial for vibrant coloration of fruits. However, the interlink point between their regulatory pathways remains largely unknown. Here, two litchi (Litchi chinensis Sonn.) cultivars with distinctively different coloration patterns during ripening, i.e. slow-reddening/stay-green 'Feizixiao' (FZX) vs rapid-reddening/degreening 'Nuomci' (NMC), were selected as the materials to study the key factors determining coloration. L. chinensis STAY-GREEN (LcSGR) was confirmed as the critical gene in pericarp chlorophyll loss and chloroplast breakdown during fruit ripening, as LcSGR directly interacted with pheophorbide a oxygenase (PAO), a key enzyme in chlorophyll degradation via the PAO pathway. L. chinensis NAM (No apical meristem), ATAF1/2(Arabidopsis transcription activation factor 1/2) and CUC2 (cup-shaped cotyledon 2) (LcNAC002) was identified as a positive regulator in the coloration of litchi pericarp. The expression of LcNAC002 was significantly higher in NMC than in FZX. Virus induced gene silencing (VIGS) of LcNAC002 significantly decreased the expression of LcSGR as well as L. chinensis MYELOBLASTOSIS1 (LcMYB1), and inhibited chlorophyll loss and anthocyanin accumulation. A dual-luciferase reporter (DLR) assay revealed that LcNAC002 significantly activates the expression of both LcSGR and LcMYB1. Furthermore, yeast one-hybrid (Y1H) and electrophoretic mobility shift assay (EMSA) results showed that LcNAC002 directly binds to the promoters of LcSGR and LcMYB1. These findings suggest that LcNAC002 is an important ripening-related transcription factor that interlinks chlorophyll degradation and anthocyanin biosynthesis by co-activating the expression of both LcSGR and LcMYB1.