Identification key genes involved in lignin and flavonoid accumulation during Tilia tuan seed maturation

Tilia tuan Szyszyl. (T. tuan) is a timber tree species with important ecological and commercial value. However, its highly lignified pericarp results in a low seed germination rate and a long dormancy period. In addition, it is unknown whether there is an interaction between the biosynthesis of flavonoids and lignin as products of the phenylpropanoid pathway during seed development. To explore the molecular regulatory mechanism of lignin and flavonoid biosynthesis, T. tuan seeds were harvested at five stages (30, 60, 90, 120, and 150 days after pollination) for lignin and flavonoid analyses. The results showed that lignin accumulated rapidly in the early and middle stages (S1, S3, and S4), and rapid accumulation of flavonoids during the early and late stages (S1 and S5). High-throughput RNA sequencing analysis of developing seeds identified 50,553 transcripts., including 223 phenylpropanoid biosynthetic pathway genes involved in lignin accumulation grouped into three clusters, and 106 flavonoid biosynthetic pathway genes (FBPGs) grouped into two clusters. Subsequent WGCNA and time-ordered gene co-expression network (TO-GCN) analysis revealed that 24 TFs (e.g., TtARF2and TtWRKY15) were involved in lignin and flavonoid biosynthesis regulation. The transcriptome data were validated by qRT-PCR to analyze the expression profiles of key enzyme-coding genes. This study revealed that there existed a competitive relationship between the flavonoid biosynthesis pathway and the lignin biosynthesis pathway, both of which are downstream pathways of phenylpropane that provide a foundation for the further exploration of molecular mechanisms underlying lignin and flavonoid accumulation in T. tuanseeds.