Transcriptome-based analysis of lignin accumulation in the regulation of fruit stone development and endocarp hardening in Chinese jujube

• RNA-Seq analysis of three jujube cultivars across key endocarp developmental stages revealed dynamic gene expression networks critical for regulating stone formation in Chinese jujube. • Lignin accumulation in the endocarp, regulated by ZjF6H1-3 , ZjPOD genes, and bHLH transcription factors, is pivotal for stone formation in jujube. • This study offers novel molecular insights and a framework for breeding stoneless jujube cultivars through targeted manipulation of lignin-related genes and transcriptional networks. Stone fruits, also known as drupes, have evolved an extremely hard wood-like shell called a stone to protect the seeds. Recently, the market value of stoneless cultivars has risen dramatically, which highlights the need to cultivate stoneless fruit. Therefore, determining the underlying mechanism of fruit stone development is urgently needed. By employing the stone-containing jujube cultivar ‘Youhe’ and two stoneless Chinese jujube cultivars, ‘Wuhefeng’ and ‘Daguowuhe’, we comprehensively studied the mechanism of fruit stone development in jujube. Anatomical analysis and lignin staining revealed that the stone cultivar ‘Youhe’ jujube exhibited much greater lignin accumulation in the endocarp than the two stoneless cultivars. Lignin accumulation may be the key factor in fruit stone formation. By analyzing the transcriptome data and identifying differentially expressed genes (DEGs), 49 overlapping DEGs were identified in the comparisons of ‘Youhe’ jujube vs. ‘Wuhefeng’ jujube and ‘Youhe’ jujube vs. ‘Daguowuhe’ jujube. ZjF6H1-3 and ZjPOD , which are involved in lignin synthesis, were identified among these DEGs. The overexpression and silencing of ZjF6H1-3 and ZjPOD in wild jujube seedlings further confirmed their roles in lignin synthesis. In addition, two bHLH transcription factors were included in the 49 overlapping DEGs, and bHLH transcription factor binding motifs were found in the promoters of ZjF6H1-3 and ZjPOD , indicating that bHLH transcription factors are also involved in lignin synthesis and stone formation in Chinese jujube. This study provides new insights into the molecular networks underlying fruit stone formation and can serve as an important reference for the molecular design and breeding of stoneless fruit cultivars of jujube and fruit trees.