Genome and transcriptomics provide insights on stipular spine morphogenesis in Robinia pseudoacacia

Robinia pseudoacacia (black locust) is a widely introduced and extensively cultivated species notably for its specialized thorn-like structure, known as the stipular spine. Here, combining Oxford Nanopore high-accuracy long-read sequencing and high-throughput chromatin conformation capture (Hi-C) scaffolding, a de novo, chromosome-level assembly of the R. pseudoacacia genome is presented, with a total size of 681.6 Mb and an N50 of 1.1 Mb. Observations showed that fiber cells beneath the epidermis of stipular spines undergo extensive lignification. The total lignin content is significantly higher in stipular spines (48.57%-63.71%) than in stem xylem (24.57%-27.99%), with syringyl (S-type) lignin being the predominant form in both, accounting for 69.79%-73.27% of the total lignin. By leveraging this genome, the transcriptomic and time-ordered gene co-expression network (TO-GCN) analyses uncovered core regulatory networks underlying stipular spine lignification, in which NAC transcription factors RopNST1/2 regulate secondary cell wall thickening and lignin biosynthesis during stipular spine hardening and the monolignol biosynthetic pathway enzyme genes RopCCoAOMT3, RopHCT13b/79/48/14b/20/86/9a, and RopCCR3b/16/24/5a act downstream during lignin biosynthesis. Collectively, these results provide cytological and molecular insights into the hardness of black locust stipular spines, and the high-quality reference genome offers a valuable resource for genomic and evolutionary studies in this species.